Preterm Cord Blood Contains a Higher Proportion of Immature Hematopoietic Progenitors Compared to Term Samples.

PubMed

Podestà, Marina; Bruschettini, Matteo; Cossu, Claudia; Sabatini, Federica; Dagnino, Monica; Romantsik, Olga; Spaggiari, Grazia Maria; Ramenghi, Luca Antonio; Frassoni, Francesco

2015-01-01

Cord blood contains high number of hematopoietic cells that after birth disappear. In this paper we have studied the functional properties of the umbilical cord blood progenitor cells collected from term and preterm neonates to establish whether quantitative and/or qualitative differences exist between the two groups. Our results indicate that the percentage of total CD34+ cells was significantly higher in preterm infants compared to full term: 0.61% (range 0.15-4.8) vs 0.3% (0.032-2.23) p = 0.0001 and in neonates <32 weeks of gestational age (GA) compared to those ≥32 wks GA: 0.95% (range 0.18-4.8) and 0.36% (0.15-3.2) respectively p = 0.0025. The majority of CD34+ cells co-expressed CD71 antigen (p<0.05 preterm vs term) and grew in vitro large BFU-E, mostly in the second generation. The subpopulations CD34+CD38- and CD34+CD45- resulted more represented in preterm samples compared to term, conversely, Side Population (SP) did not show any difference between the two group. The absolute number of preterm colonies (CFCs/10microL) resulted higher compared to term (p = 0.004) and these progenitors were able to grow until the third generation maintaining an higher proportion of CD34+ cells (p = 0.0017). The number of colony also inversely correlated with the gestational age (Pearson r = -0.3001 p<0.0168). We found no differences in the isolation and expansion capacity of Endothelial Colony Forming Cells (ECFCs) from cord blood of term and preterm neonates: both groups grew in vitro large number of endothelial cells until the third generation and showed a transitional phenotype between mesenchymal stem cells and endothelial progenitors (CD73, CD31, CD34 and CD144)The presence, in the cord blood of preterm babies, of high number of immature hematopoietic progenitors and endothelial/mesenchymal stem cells with high proliferative potential makes this tissue an important source of cells for developing new cells therapies.

Effects of blocking developmental cell death on sexually dimorphic calbindin cell groups in the preoptic area and bed nucleus of the stria terminalis

PubMed Central

2012-01-01

Background Calbindin-D28 has been used as a marker for the sexually dimorphic nucleus of the preoptic area (SDN-POA). Males have a distinct cluster of calbindin-immunoreactive (ir) cells in the medial preoptic area (CALB-SDN) that is reduced or absent in females. However, it is not clear whether the sex difference is due to the absolute number of calbindin-ir cells or to cell position (that is, spread), and the cellular mechanisms underlying the sex difference are not known. We examined the number of cells in the CALB-SDN and surrounding regions of C57Bl/6 mice and used mice lacking the pro-death gene, Bax, to test the hypothesis that observed sex differences are due to cell death. Methods Experiment 1 compared the number of cells in the CALB-SDN and surrounding regions in adult males, females, and females injected with estradiol benzoate on the day of birth. In experiment 2, cell number in the CALB-SDN and adjacent regions were compared in wild-type and Bax knockout mice of both sexes. In addition, calbindin-ir cells were quantified within the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), a nearby region that is larger in males due to Bax-dependent cell death. Results Males had more cells in the CALB-SDN as well as in surrounding regions than did females, and estradiol treatment of females at birth masculinized both measures. Bax deletion had no effect on cell number in the CALB-SDN or surrounding regions but increased calbindin-ir cell number in the BNSTp. Conclusions The sex difference in the CALB-SDN of mice results from an estrogen-dependent difference in cell number with no evidence found for greater spread of cells in females. Blocking Bax-dependent cell death does not prevent sex differences in calbindin-ir cell number in the BNST or CALB-SDN but increases calbindin-ir cell number in the BNSTp of both sexes. PMID:22336348

Effects of blocking developmental cell death on sexually dimorphic calbindin cell groups in the preoptic area and bed nucleus of the stria terminalis.

PubMed

Gilmore, Richard F; Varnum, Megan M; Forger, Nancy G

2012-02-15

Calbindin-D28 has been used as a marker for the sexually dimorphic nucleus of the preoptic area (SDN-POA). Males have a distinct cluster of calbindin-immunoreactive (ir) cells in the medial preoptic area (CALB-SDN) that is reduced or absent in females. However, it is not clear whether the sex difference is due to the absolute number of calbindin-ir cells or to cell position (that is, spread), and the cellular mechanisms underlying the sex difference are not known. We examined the number of cells in the CALB-SDN and surrounding regions of C57Bl/6 mice and used mice lacking the pro-death gene, Bax, to test the hypothesis that observed sex differences are due to cell death. Experiment 1 compared the number of cells in the CALB-SDN and surrounding regions in adult males, females, and females injected with estradiol benzoate on the day of birth. In experiment 2, cell number in the CALB-SDN and adjacent regions were compared in wild-type and Bax knockout mice of both sexes. In addition, calbindin-ir cells were quantified within the principal nucleus of the bed nucleus of the stria terminalis (BNSTp), a nearby region that is larger in males due to Bax-dependent cell death. Males had more cells in the CALB-SDN as well as in surrounding regions than did females, and estradiol treatment of females at birth masculinized both measures. Bax deletion had no effect on cell number in the CALB-SDN or surrounding regions but increased calbindin-ir cell number in the BNSTp. The sex difference in the CALB-SDN of mice results from an estrogen-dependent difference in cell number with no evidence found for greater spread of cells in females. Blocking Bax-dependent cell death does not prevent sex differences in calbindin-ir cell number in the BNST or CALB-SDN but increases calbindin-ir cell number in the BNSTp of both sexes.

A study on relationship among apoptosis rates, number of peripheral T cell subtypes and disease activity in rheumatoid arthritis.

PubMed

Ji, Lanlan; Geng, Yan; Zhou, Wei; Zhang, Zhuoli

2016-02-01

Rheumatoid arthritis is characterized by type 17 helper T cell (Th17)/regulatory T cell (Treg) imbalance. The objective of this article is to study whether insufficient apoptosis contributes to the imbalance of Th17/Treg in rheumatoid arthritis. Twenty-one rheumatoid arthritis patients and eight healthy volunteers were involved in this study. The percentage of CD4(+) interleukin (IL)-17(+) T cells and CD4(+) transcription factor-forkhead box protein 3 (Foxp3)(+) T cells were measured by flow cytometry, and active caspase-3 labeling was used to detect early apoptosis. The number of T cell subtypes in peripheral blood between the two groups was compared, as well as the apoptotic ratio. Neither the number of Th17 nor Treg cells was significantly different between rheumatoid arthritis patients and healthy controls. However, the number of regulatory T cells positively correlated with erythrocyte sedimentation rate, Disease Activity Score of 28 joints and rheumatoid factor. For the apoptosis of T cell subtypes, the percentage of apoptotic Th17 cells was higher in peripheral blood of rheumatoid arthritis patients compared to controls. Furthermore, peripheral Th17 cells were more sensitive to apoptosis than Treg cells, but there was no difference between rheumatoid arthritis patients and controls. It seemed that there was no relationship between the number and apoptosis ratio of peripheral Th17/Treg cells. But the number of Treg cells positively correlated with disease activity. Furthermore, Th17 cells are more sensitive to apoptosis after freezing, especially in RA patients. This serendipitous finding may provide new areas for the further study of these two cell populations. © 2013 Asia Pacific League of Associations for Rheumatology and Wiley Publishing Asia Pty Ltd.

[A comparison of three different needles used for spinal anesthesia in terms of squamous epithelial cell transport risk].

PubMed

Çiğdem, Ünal Kantekin; Sevinç, Şahin; Esef, Bolat; Süreyya, Öztürk; Muzaffer, Gencer; Akif, Demirel

To investigate the differences in the number of squamous epithelial cells carried to the spinal canal by three different types of spinal needle tip of the same size. Patients were allocated into three groups (Group I, Group II, Group III). Spinal anesthesia was administered to Group I (n=50) using a 25G Quincke needle, to Group II (n=50) using a 25G pencil point spinal needle, and to Group III (n=50) using a non-cutting atraumatic needle with special bending. The first and third drops of cerebral spinal fluid (CSF) samples were taken from each patient and each drop was placed on a slide for cytological examination. Nucleated and non-nucleated squamous epithelial cells on the smear preparations were counted. There was statistically significant difference between the groups in respect to the number of squamous epithelial cells in the first drop (p<0.05). Group III had lower number of squamous epithelial cells in the first drop compared to that of Group I and Group II. Mean while Group I had higher number of squamous epithelial cells in the third drop compared to the other groups. The number of squamous epithelial cells in the first and third drops was statistically similar in each group respectively (p>0.05 for each group). In this study of different needle tips, it was seen that with atraumatic needle with special bending a significantly smaller number of cells were transported when compared to the Quincke tip needles, and with pencil point needles. Copyright © 2016 Sociedade Brasileira de Anestesiologia. Publicado por Elsevier Editora Ltda. All rights reserved.

An estimation of the number of cells in the human body.

PubMed

Bianconi, Eva; Piovesan, Allison; Facchin, Federica; Beraudi, Alina; Casadei, Raffaella; Frabetti, Flavia; Vitale, Lorenza; Pelleri, Maria Chiara; Tassani, Simone; Piva, Francesco; Perez-Amodio, Soledad; Strippoli, Pierluigi; Canaider, Silvia

2013-01-01

All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 10(12) and 10(16) and it is widely mentioned without a proper reference. To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72 × 10(13). Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

CELL RESPIRATION STUDIES : II. A COMPARATIVE STUDY OF THE OXYGEN CONSUMPTION OF BLOOD FROM NORMAL INDIVIDUALS AND PATIENTS WITH INCREASED LEUCOCYTE COUNTS (SEPSIS; CHRONIC MYELOGENOUS LEUCEMIA).

PubMed

Daland, G A; Isaacs, R

1927-06-30

1. The oxygen consumption of blood of normal individuals, when the hemoglobin is saturated with oxygen, is practically zero within the limits of experimental error of the microspirometer used. 2. The oxygen consumed in a microspirometer by the blood of patients with chronic myelogenous leucemia with a high white blood cell count, and of one with leucocytosis from sepsis, was proportional to the number of adult polymorphonuclear neutrophils in the blood. 3. No correlation could be made between the rate of oxygen absorption and the total number of white blood cells in the blood, or the total number of immature cells, or the number of red blood cells, or the amount of oxyhemoglobin. 4. The blood of patients with chronic myelogenous leucemia continued to use oxygen in the microspirometer longer than that of normal individuals, and the hemoglobin, in the leucemic bloods, became desaturated even though exposed to air. 5. In blood in which the bulk. of the cells were immature and the mature cells few, the oxygen consumption was lower than in blood in which the mature cells predominated. The rate of oxygen consumption of the immature cells was relatively low as compared to the mature. 6. The slower rate of oxygen absorption by the immature leucocytes in chronic myelogenous leucemia as compared to the mature cells, places them, in accord with Warburg's reports, in the class of the malignant tissues in this respect rather than in the group of young or embryonic cells.

Number of decidual natural killer cells & macrophages in pre-eclampsia

PubMed Central

Milosevic-Stevanovic, Jelena; Krstic, Miljan; Radovic-Janosevic, Dragana; Popovic, Jasmina; Tasic, Marija; Stojnev, Slavica

2016-01-01

Background & objectives: The process of human placentation is complex and still not well understood. This study was aimed to examine the relationship between clinical features of pre-eclampsia and degree of trophoblastic invasion after its immunohistochemical visualization in the context of possible alterations in the number of natural killer (NK) cells and macrophages in the decidua. Methods: This prospective study included a study group comprising 30 pregnant women with pre-eclampsia delivered by caesarean section and a control group comprising 20 healthy pregnant women also delivered by caesarean section. Samples of placental bed obtained during caesarean section were analyzed after immunohistochemical labelling CD56+ NK cells, CD68+ macrophages and cytokeratin 7 trophoblastic cells. Results: In pre-eclampsia, there was a significantly lower number of CD56+ NK cells in the decidua (P<0.001) and a higher number of CD68+ macrophages (P<0.001) compared to control group. In the subgroup of pre-eclampsia with intrauterine growth retardation (IUGR), a significantly greater number of NK cells (P<0.05) was recorded, as well as an increased number of macrophages, but not significantly compared to pre-eclampsia without IUGR. There was no significant difference in the distribution of these cells in the decidua in relation to the severity of pre-eclampsia. CD56+ NK cells were significantly less (P<0.05) and macrophages were more (P<0.05) in the group with poor trophoblastic invasion. Interpretation & conclusions: Alterations in the number of immune cells in relation to the degree of trophoblastic invasion indicated their role in aetiopathogenesis of pre-eclampsia, while the direct association between their number and severity of pre-eclampsia was not confirmed. PMID:28474619

Peripheral vestibular pathology in Mondini dysplasia.

PubMed

Kaya, Serdar; Hızlı, Ömer; Kaya, Fatıma Kübra; Monsanto, Rafael DaCosta; Paparella, Michael M; Cureoglu, Sebahattin

2017-01-01

In this study, our objective was to histopathologically analyze the peripheral vestibular system in patients with Mondini dysplasia. Comparative human temporal bone study. We assessed the sensory epithelium of the human vestibular system with a focus on the number of type I and type II hair cells, as well as the total number of hair cells. We compared those numbers in our Mondini dysplasia group versus our control group. The loss of type I and type II hair cells in the cristae of the superior, lateral, and posterior semicircular canals, as well as in the saccular and utricular macula, was significantly higher in our Mondini dysplasia group than in our control group. The total number of hair cells significantly decreased in the cristae of the superior, lateral, and posterior semicircular canals, as well as in the saccular and utricular macula, in our Mondini dysplasia group. Loss of vestibular hair cells can lead to vestibular dysfunction in patients with Mondini dysplasia. NA Laryngoscope, 127:206-209, 2017. © 2016 The American Laryngological, Rhinological and Otological Society, Inc.

[Distribution of lymphocyte subpopulations and plasma cells in the colonic mucosa of children with ulcerative colitis].

PubMed

Arató, A; Savilahti, E; Tainio, V M

1990-09-02

The distribution of lymphocyte subpopulations and plasma cells of the colonic and rectal mucosae were studied in eight children with ulcerative colitis and 12 healthy controls. In four patients the examinations were also carried out 3 months after the beginning of treatment. No difference in the number of intraepithelial lymphocytes was found between the patients and controls. The majority of these cells were T-cells, and among them the suppressor/cytotoxic cells were preponderant. In the lamina propria of both untreated and treated patients the numbers of T-cells, helper T-cells, and B-cells were elevated compared to controls. In the patients the number of IgG-containing cells was three times that of the controls; the number of IgE positive cells was also elevated. The numbers of IgA- and IgM-containing cells were not different from that of the controls. The results suggest that in ulcerative colitis the place of primary immunological processes inside the large bowel mucosa is the lamina propria.

Flow Cytometry of Human Primary Epidermal and Follicular Keratinocytes

PubMed Central

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-01-01

Objective: The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Methods: Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. Results: On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. Conclusion: The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis. PMID:18350110

Flow cytometry of human primary epidermal and follicular keratinocytes.

PubMed

Gragnani, Alfredo; Ipolito, Michelle Zampieri; Sobral, Christiane S; Brunialti, Milena Karina Coló; Salomão, Reinaldo; Ferreira, Lydia Masako

2008-02-19

The aim of this study was to characterize using flow cytometry cultured human primary keratinocytes isolated from the epidermis and hair follicles by different methods. Human keratinocytes derived from discarded fragments of total skin and scalp hair follicles from patients who underwent plastic surgery in the Plastic Surgery Division at UNIFESP were used. The epidermal keratinocytes were isolated by using 3 different methods: the standard method, upon exposure to trypsin for 30 minutes; the second, by treatment with dispase for 18 hours and with trypsin for 10 minutes; and the third, by treatment with dispase for 18 hours and with trypsin for 30 minutes. Follicular keratinocytes were isolated using the standard method. On comparing the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with dispase for 18 hours and with trypsin for 30 minutes, it was observed that the first group presented the largest number of viable cells, the smallest number of cells in late apoptosis and necrosis with statistical significance, and no difference in apoptosis. When we compared the group treated with dispase for 18 hours and with trypsin for 10 minutes with the group treated with trypsin, the first group presented the largest number of viable cells, the smallest number of cells in apoptosis with statistical significance, and no difference in late apoptosis and necrosis. When we compared the results of the group treated with dispase for 18 hours and with trypsin for 10 minutes with the results for follical isolation, there was a statistical difference in apoptosis and viable cells. The isolation method of treatment with dispase for 18 hours and with trypsin for 10 minutes produced the largest number of viable cells and the smallest number of cells in apoptosis/necrosis.

[A comparative study on inducing non-homologous mesenchymal stem cells to differentiate into neural stem cells using non-homologous cerebrospinal fluid].

PubMed

Ren, Chao; Liu, Xiaoyun; Wan, Meirong; Geng, Deqin; Ge, Wei; Li, Jinmei; Zhang, Weiwei

2013-12-01

In order to set up a base for stem cells to be widely used in clinical medicine, we tried to optimize, in this study, the technique that induces human mesenchymal stem cells (hMSCs) to differentiate into neural stem cells by using cerebrospinal fluid (CSF) from the different groups. After the induction, presence of neural stem cells was confirmed with microscope observation, flow cytometry analysis, immunohistochemistry and fluorescent immunohistochemistry. At the same time, we also compared and analysed the data of the number of stem cells when it totally met the requirements for clinical treatment and the days required. At last, we confirmed that hMSCs could be induced to differentiate into neural stem cells, and that the number of cells totally met the requirements for clinical treatment. But there were some differences both in the number of cells and the days required. Among the groups, the group that marrow mesenchymal stem cells from patients own induced by CSF from healthy volunteers used the shortest time and the quantity of the cells was significantly higher than those of the others.

Analysis of CD57+ natural killer cells and CD8+ T lymphocytes in periapical granulomas and radicular cysts.

PubMed

Silva, Luiz Arthur Barbosa da; Sá, Maria Alice Ramalho; Melo, Rafaela Albuquerque; Pereira, Joabe Dos Santos; Silveira, Éricka Janine Dantas da; Miguel, Márcia Cristina da Costa

2017-12-18

The aim of this study was to compare the number of CD57+ natural killer (NK) cells and CD8+ T lymphocytes between periapical granulomas (PGs) and radicular cysts (RCs). Twenty-fives cases of PGs and 25 of RCs were submitted to histological analysis and immunohistochemistry using anti-CD57 and anti-CD8 biomarkers. Positive cells were counted in 10 fields (400× magnification) and the median value was calculated for each case. Statistical tests were used to evaluate differences in the number of CD57+ NK cells and CD8+ T lymphocytes according to type of lesion, intensity of the infiltrate and thickness of the lining epithelium. The number of CD57+ NK cells and CD8+ T lymphocytes was higher in PGs than in RCs (p = 0.129 and p = 0.541, respectively). Comparison of the number of CD57+ NK cells in atrophic and hyperplastic epithelium revealed a larger number of cells in the atrophic epithelium (p = 0.042). A larger number of CD57+ NK cells and CD8+ T lymphocytes were observed in grade III infiltrates compared to grade I/II (p = 0.145 and p = 0.725, respectively). CD8+ T lymphocytes were more prevalent than CD57+ NK cells in most cases when PGs and RCs were analyzed separately or in combination (p < 0.0001). CD57+ NK cells and CD8+ T lymphocytes play a key role in antiviral defense and the presence of these cells supports evidence suggesting the participation of these microorganisms in the pathogenesis of PGs and RCs. The response mediated by CD8+ T lymphocytes was more frequent, indicating greater participation of the adaptive immunity in these chronic lesions.

Extraction of the number of peroxisomes in yeast cells by automated image analysis.

PubMed

Niemistö, Antti; Selinummi, Jyrki; Saleem, Ramsey; Shmulevich, Ilya; Aitchison, John; Yli-Harja, Olli

2006-01-01

An automated image analysis method for extracting the number of peroxisomes in yeast cells is presented. Two images of the cell population are required for the method: a bright field microscope image from which the yeast cells are detected and the respective fluorescent image from which the number of peroxisomes in each cell is found. The segmentation of the cells is based on clustering the local mean-variance space. The watershed transformation is thereafter employed to separate cells that are clustered together. The peroxisomes are detected by thresholding the fluorescent image. The method is tested with several images of a budding yeast Saccharomyces cerevisiae population, and the results are compared with manually obtained results.

[Response of HeLa cells to mitomycine C. III. The analysis of nucleoli of mother and daughter cells].

PubMed

Petrov, Iu P; Neguliaev, Iu A; Tsupkina, N V

2014-01-01

The comparative analysis of the number of nucleoli in cells of the established HeLa-M line was carried out before and after exposure to mitomycin C in a concentration of 10 μg/ml for 2 h. Using time-lapse microscopy, nucleoli in mother and their respective daughter cells were computed. It has been shown that the average number of nucleoli per cell is generally higher in daughter cells than in mother cells, and a standard deviation, on the contrary, decreases. An average number of nucleoli in daughter cells, whose mother cells had been treated with mitomycin C, was higher than in corresponding cells of control group. The separate analysis has been performed for the cells having from 1 to 4 nucleoli. Nonrandom complete coincidence of the number of nucleoli in mather and daughter cells has been typicaly shown for about 1/7 of the total cell population. Mitomycin C reduces this value of about 1.5 times.

Induction of micronuclei and apoptosis in natural killer cells compared to T lymphocytes after gamma-irradiation.

PubMed

Louagie, H; Philippé, J; Vral, A; Cornelissen, M; Thierens, H; De Ridder, L

1998-02-01

To investigate the chromosomal damage caused by gamma-irradiation in T lymphocytes and natural killer (NK) cells and compare this with apoptosis induction in both lymphocyte subsets. Apoptosis induction by gamma-irradiation in T lymphocytes and NK cells was quantified using the annexin V flow cytometric assay. The cytokinesis-block micronucleus (MN) assay was used to evaluate the induced cytogenetic damage. For the MN assays on NK cells, gamma-irradiated peripheral blood mononuclear cells were cultured and stimulated with interleukin 15 (IL-15). Afterwards the NK cells (characterized by the CD3-/CD56+ phenotype) were separated with the FACSort flow cytometer and the number of MN in the sorted binuclear cells was scored. Doses of 1 and 2 Gy gamma-irradiation were applied. Higher numbers of MN in NK cells were found compared with the MN yield in T lymphocytes. In contrast, NK cells were less than T lymphocytes prone to apoptosis after gamma-irradiation. The results support the view that cytogenetic damage and apoptosis after gamma-irradiation are not necessarily correlated.

Induction of Protective Immunity against Eimeria tenella, Eimeria maxima, and Eimeria acervulina Infections Using Dendritic Cell-Derived Exosomes

PubMed Central

Gallego, Margarita; Lee, Sung Hyen; Lillehoj, Hyun Soon; Quilez, Joaquin; Lillehoj, Erik P.; Sánchez-Acedo, Caridad

2012-01-01

This study describes a novel immunization strategy against avian coccidiosis using exosomes derived from Eimeria parasite antigen (Ag)-loaded dendritic cells (DCs). Chicken intestinal DCs were isolated and pulsed in vitro with a mixture of sporozoite-extracted Ags from Eimeria tenella, E. maxima, and E. acervulina, and the cell-derived exosomes were isolated. Chickens were nonimmunized or immunized intramuscularly with exosomes and subsequently noninfected or coinfected with E. tenella, E. maxima, and E. acervulina oocysts. Immune parameters compared among the nonimmunized/noninfected, nonimmunized/infected, and immunized/infected groups were the numbers of cells secreting Th1 cytokines, Th2 cytokines, interleukin-16 (IL-16), and Ag-reactive antibodies in vitro and in vivo readouts of protective immunity against Eimeria infection. Cecal tonsils, Peyer's patches, and spleens of immunized and infected chickens had increased numbers of cells secreting the IL-16 and the Th1 cytokines IL-2 and gamma interferon, greater Ag-stimulated proliferative responses, and higher numbers of Ag-reactive IgG- and IgA-producing cells following in vitro stimulation with the sporozoite Ags compared with the nonimmunized/noninfected and nonimmunized/infected controls. In contrast, the numbers of cells secreting the Th2 cytokines IL-4 and IL-10 were diminished in immunized and infected chickens compared with the nonimmunized/noninfected and the nonimmunized/infected controls. Chickens immunized with Ag-loaded exosomes and infected in vivo with Eimeria oocysts had increased body weight gains, reduced feed conversion ratios, diminished fecal oocyst shedding, lessened intestinal lesion scores, and reduced mortality compared with the nonimmunized/infected controls. These results suggest that successful field vaccination against avian coccidiosis using exosomes derived from DCs incubated with Ags isolated from Eimeria species may be possible. PMID:22354026

Increased Skin Inflammation and Blood Vessel Density in Human and Experimental Diabetes

PubMed Central

Tellechea, Ana; Kafanas, Antonios; Leal, Ermelindo C; Tecilazich, Francesco; Kuchibhotla, Sarada; Auster, Michael E; Kontoes, Iraklis; Paolino, Jacqueline; Carvalho, Eugenia; Nabzdyk, Leena Pradhan; Veves, Aristidis

2013-01-01

Systemic inflammation is associated with impaired wound healing in diabetic patients. Using immunohistochemistry techniques, the authors investigated changes in skin inflammation and skin blood vessels in human and experimental diabetes. Comparing to the non-DM human subjects, the total number of inflammatory cells per biopsy and the number of inflammatory cells around blood vessels, a strong indication of inflammation, were higher in DM subjects irrespective of their risk for developing diabetic foot ulcer. Inflammatory cell infiltration was robustly increased in all diabetic animal models compared to their non-diabetic controls. The number and density of blood vessels and CD31 positive proliferating endothelial cells around pre-existing skin vessels was also higher in the DM patients. However, there were no differences in the skin blood flow between the non-DM and DM subjects. The number of skin blood vessels was also increased in the DM animals; however, these differences were less obvious than the ones observed for inflammatory cells. We conclude that skin inflammation and skin blood vessel density is increased in diabetic human subjects and in rodent and rabbit models of diabetes. PMID:23446362

Effects of spaceflight on rat peripheral blood leukocytes and bone marrow progenitor cells

NASA Technical Reports Server (NTRS)

Ichiki, A. T.; Gibson, L. A.; Jago, T. L.; Strickland, K. M.; Johnson, D. L.; Lange, R. D.; Allebban, Z.

1996-01-01

The white blood cell (WBC) elements and the bone marrow myeloid progenitor cell populations were analyzed to ascertain adaptation to micro-gravity and subsequent readaptation to 1 G in rats flown on the 14-day Spacelab Life Sciences-2 (SLS-2) mission. Bone marrow cells were harvested from one group of rats killed inflight (FD13) and blood was drawn from three other groups at various times. The WBC level was normal on FD14 with the exception of neutrophilia. On FD13, numbers of colony-forming units-granulocyte (CFU-G), CFU-GM, and CFU-M from flight animals were decreased compared with ground controls when incubated with recombinant rat interleukin-3 (rrIL-3) alone or in combination with recombinant human erythropoietin (rhEpo). On recovery (R + 0), flight rats had decreased numbers of total leukocytes and absolute numbers of lymphocytes and monocytes with elevated neutrophils compared with control rats. They had lower numbers of CD4, CD8, CD2, CD3, and B cells in the peripheral blood but no differences in spleen lymphocytes.

Cyclophosphamide induces bone marrow to yield higher numbers of precursor dendritic cells in vitro capable of functional antigen presentation to T cells in vivo

PubMed Central

Salem, Mohamed L.; El-Naggar, Sabry A.; Cole, David J.

2009-01-01

We have shown recently that cyclophosphamide (CTX) treatment induced a marked increase in the numbers of immature dendritic cells (DCs) in blood, coinciding with enhanced antigen-specific responses of the adoptively transferred CD8+ T cells. Because this DC expansion was preceded by DC proliferation in bone marrow (BM), we tested whether BM post CTX treatment can generate higher numbers of functional DCs. BM was harvested three days after treatment of C57BL/6 mice with PBS or CTX and cultured with GM-CSF/IL-4 in vitro. Compared with control, BM from CTX-treated mice showed faster generation and yielded higher numbers of DCs with superior activation in response to toll-like receptor (TLR) agonists. Vaccination with peptide-pulsed DCs generated from BM from CTX-treated mice induced comparable adjuvant effects to those induced by control DCs. Taken together, post CTX BM harbors higher numbers of DC precursors capable of differentiating into functional DCs, which be targeted to create host microenvironment riches in activated DCs upon treatment with TLR agonists. PMID:20036354

Somatic mutations and affinity maturation are impaired by excessive numbers of T follicular helper cells and restored by Treg cells or memory T cells.

PubMed

Preite, Silvia; Baumjohann, Dirk; Foglierini, Mathilde; Basso, Camilla; Ronchi, Francesca; Fernandez Rodriguez, Blanca M; Corti, Davide; Lanzavecchia, Antonio; Sallusto, Federica

2015-11-01

We previously reported that Cd3e-deficient mice adoptively transferred with CD4(+) T cells generate high numbers of T follicular helper (Tfh) cells, which go on to induce a strong B-cell and germinal center (GC) reaction. Here, we show that in this system, GC B cells display an altered distribution between the dark and light zones, and express low levels of activation-induced cytidine deaminase. Furthermore, GC B cells from Cd3e(-/-) mice accumulate fewer somatic mutations as compared with GC B cells from wild-type mice, and exhibit impaired affinity maturation and reduced differentiation into long-lived plasma cells. Reconstitution of Cd3e(-/-) mice with regulatory T (Treg) cells restored Tfh-cell numbers, GC B-cell numbers and B-cell distribution within dark and light zones, and the rate of antibody somatic mutations. Tfh-cell numbers and GC B-cell numbers and dynamics were also restored by pre-reconstitution of Cd3e(-/-) mice with Cxcr5(-/-) Treg cells or non-regulatory, memory CD4(+) T cells. Taken together, these findings underline the importance of a quantitatively regulated Tfh-cell response for an efficient and long-lasting serological response. © 2015 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparison of Mast Cells and Inflammatory Cells within Periapical Lesions and Comparison of Degranulated Mast Cells Between Fibrous and Inflamed Area in Radicular Cysts: An Immunohistochemical Study.

PubMed

Shiromany, Aseem; Sood, Rahul; Akifuddin, Syed; Sidhu, Gagandeep Kaur; Khan, Nadia; Singla, Kapil

2014-12-01

The role of mast cells as the key effector of allergic inflammation, anaphylactic inflammatory reactions and in the pathogenesis of chronic inflammation, is well-known. The present study is adopted to compare mast cells and inflammatory cells within periapical granuloma and cysts and localize the mast cells and quantify their number in the periapical cysts so as to propose a role of mast cells in the pathogenesis of this lesion. Biopsy specimens of 30 periapical lesions were stained with hematoxylin-eosin, and immunohistochemical Mast Cell Tryptase from Bio SB (IHC detection system kit) antibody. The tryptase positive mast cells and mononuclear inflammatory cells were counted in 10 consecutive high power fields (100X) using the binocular microscope from Motic attached to a computer with Motic Advanced Images 3.2 software. Comparative microscopic analysis indicated that periapical cyst shows more percentage of mast cells and less percentage of inflammatory cell than periapical granuloma (comparison of mean and standard deviation of total number of mast cells and inflammatory cells, mast cells 3.15±1.39 in the granuloma group and 4.43±1.91in the cyst group, inflammatory cells, 67.11±1.2 in the granuloma group and 52.66±0.8 in the cyst group). Numerous degranulated mast cells were observed in the fibrous wall than the inflammatory infiltrate of the periapical cysts. The mean and standard deviation of degranulated mast cells between the inflammatory and fibrous zone within the cyst group, being 0.95±1.10 and1.68±1.34 respectively. The values varied significantly between the two zones. The number of inflammatory cells in the cyst group is less than periapical granuloma and total number of mast cells in the cyst group is more as compared to periapical granuloma. The degranulated cells were quantified and they were higher in the fibrous area of the cysts than the inflammatory zone. This study could support the fact that the various mediators released on degranulation play a role in the connective tissue remodeling, chronicity and expansion of the periapical lesion.

The effects of simulated hypogravity on murine bone marrow cells

NASA Technical Reports Server (NTRS)

Lawless, Desales

1989-01-01

Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

CELL RESPIRATION STUDIES

PubMed Central

Daland, Geneva A.; Isaacs, Raphael

1927-01-01

1. The oxygen consumption of blood of normal individuals, when the hemoglobin is saturated with oxygen, is practically zero within the limits of experimental error of the microspirometer used. 2. The oxygen consumed in a microspirometer by the blood of patients with chronic myelogenous leucemia with a high white blood cell count, and of one with leucocytosis from sepsis, was proportional to the number of adult polymorphonuclear neutrophils in the blood. 3. No correlation could be made between the rate of oxygen absorption and the total number of white blood cells in the blood, or the total number of immature cells, or the number of red blood cells, or the amount of oxyhemoglobin. 4. The blood of patients with chronic myelogenous leucemia continued to use oxygen in the microspirometer longer than that of normal individuals, and the hemoglobin, in the leucemic bloods, became desaturated even though exposed to air. 5. In blood in which the bulk. of the cells were immature and the mature cells few, the oxygen consumption was lower than in blood in which the mature cells predominated. The rate of oxygen consumption of the immature cells was relatively low as compared to the mature. 6. The slower rate of oxygen absorption by the immature leucocytes in chronic myelogenous leucemia as compared to the mature cells, places them, in accord with Warburg's reports, in the class of the malignant tissues in this respect rather than in the group of young or embryonic cells. PMID:19869329

Satellite cell response to concurrent resistance exercise and high-intensity interval training in sedentary, overweight/obese, middle-aged individuals.

PubMed

Pugh, Jamie K; Faulkner, Steve H; Turner, Mark C; Nimmo, Myra A

2018-02-01

Sarcopenia can begin from the 4-5th decade of life and is exacerbated by obesity and inactivity. A combination of resistance exercise (RE) and endurance exercise is recommended to combat rising obesity and inactivity levels. However, work continues to elucidate whether interference in adaptive outcomes occur when RE and endurance exercise are performed concurrently. This study examined whether a single bout of concurrent RE and high-intensity interval training (HIIT) alters the satellite cell response following exercise compared to RE alone. Eight sedentary, overweight/obese, middle-aged individuals performed RE only (8 × 8 leg extensions at 70% 1RM), or RE + HIIT (10 × 1 min at 90% HR max on a cycle ergometer). Muscle biopsies were collected from the vastus lateralis before and 96 h after the RE component to determine muscle fiber type-specific total (Pax7 + cells) and active (MyoD + cells) satellite cell number using immunofluorescence microscopy. Type-I-specific Pax7 + (P = 0.001) cell number increased after both exercise trials. Type-I-specific MyoD + (P = 0.001) cell number increased after RE only. However, an elevated baseline value in RE + HIIT compared to RE (P = 0.046) was observed, with no differences between exercise trials at 96 h (P = 0.21). Type-II-specific Pax7 + and MyoD + cell number remained unchanged after both exercise trials (all P ≥ 0.13). Combining a HIIT session after a single bout of RE does not interfere with the increase in type-I-specific total, and possibly active, satellite cell number, compared to RE only. Concurrent RE + HIIT may offer a time-efficient way to maximise the physiological benefits from a single bout of exercise in sedentary, overweight/obese, middle-aged individuals.

Use of HL cells for improved isolation and passage of Chlamydia pneumoniae.

PubMed Central

Cles, L D; Stamm, W E

1990-01-01

We compared growth of the recently discovered respiratory pathogen Chlamydia pneumoniae in McCoy, HeLa 229, BHK-21, and HL cells. When cells were not pretreated with DEAE-dextran, HL cells had significantly higher mean numbers of inclusion-forming units (IFUs) on initial inoculation than the other cell lines. When cells were pretreated with DEAE-dextran, HeLa 229 and HL cells had equivalent mean numbers of IFUs on initial inoculation. HL cells had strikingly higher mean numbers of IFUs in passage than HeLa 229, BHK-21, or McCoy cells. In addition, HL cells did not require pretreatment with DEAE-dextran and could be used from 2 to 4 days after seeding. We conclude that HL cells are an excellent cell culture system for laboratory propagation of C. pneumoniae and may be a more sensitive cell line for initial isolation. PMID:2191013

Distinctive profiles of tumor-infiltrating immune cells and association with intensity of infiltration in colorectal cancer.

PubMed

Wu, Yugang; Yuan, Lei; Lu, Qicheng; Xu, Haiyan; He, Xiaozhou

2018-03-01

Tumor-infiltrating immune cells are heterogeneous and consist of characteristic compartments, including T helper (Th)1 and regulatory T (Treg) cells that exhibit distinctive biological functions. The present study investigated the profile of infiltrating immune cells from surgically removed tumor tissues from patients with colorectal cancer. The characteristic transcription factors of Th1 and Th2 cells, Treg cells, Th17 cells and T follicular helper (Tfh) cells were analyzed. The results demonstrated that a marked increased number of Treg cells presented in tumor infiltrates when compared with non-tumor adjacent tissues. An increased number of Th1 and Tfh cells existed in tumor infiltrates compared with non-tumorous adjacent tissues, while the infiltration of Th17 and Th2 cells was similar between tumor and non-tumor adjacent tissues. Furthermore, there were an increased number of Treg cells in tumors with low infiltration compared with those with high infiltration. The expression of CXC motif chemokine (CXC) receptor 3, CXC ligand (CXCL)L9 and CXCL10 was significantly increased on infiltrating T cells in tumors with high infiltration as compared with those with low infiltration. Macrophages exhibited a dominant M2 phenotype in tumor infiltrates of colorectal cancer, whereas a balanced M1 and M2 phenotype presented in macrophages from the peripheral blood. In vitro stimulation of macrophages isolated from tumor tissue of colorectal cancer with granulocyte macrophage colony-stimulating factor and lipopolysaccharide did not drive to an inflammatory phenotype. The results provide insights into the pattern of immune cell infiltration in Chinese patients with colorectal cancer. It may be beneficial that patients with colorectal cancer are screened for the defined profile along with the expression of CXCL9 and CXCL10 in order to achieve better efficacy in clinical applications of immune-based therapy, including anti-programmed cell death protein 1 therapy.

[Neoplastic transformation of mouse fibroblasts under the influence of high-energy protons and gamma-rays].

PubMed

Voskanian, K Sh

2004-01-01

Oncoginic transformations of mouse fibroblasts C3H10T1/2 after exposure to proton energies 150 and 584 MeV were compared with fibroblast effects of gamma-radiation. Prior to exposure, cell populations (2.7 x 10(3) cells/cm2) were inoculated in plastic vials with the surface area of 75 cm2 and cultivated 11 days. Survivability was determined by comparing the number of cell colonies in irradiated and non-irradiated (control) vials. Transformation rate was calculated by dividing the total transformation focus number by the number of survived cells in a vial. Rate of oncogenic transformations after gamma- and proton (584 MeV) irradiation was essentially identical, i.e. the parameter grew rapidly at the doses < 1 Gy and slowed down at the doses > 1 Gy. In the dose interval between 1 and 5 Gy, transformation rate for proton energy 150 MeV was found low compared with gamma-radiation and proton energy 584 MeV. It is hypothesized that the different transformation rate after exposure to proton energy 150 MeV is linked with the high linear energy transfer as compared with the proton energy of 584 MeV and gamma-radiation.

Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach

PubMed Central

Kubínová, Zuzana

2014-01-01

Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use. PMID:24336344

Targeting survivin with prodigiosin isolated from cell wall of Serratia marcescens induces apoptosis in hepatocellular carcinoma cells.

PubMed

Yenkejeh, R A; Sam, M R; Esmaeillou, M

2017-04-01

Abnormal activation of the Wnt/β-catenin signaling pathway increases survivin expression that is involved in hepatocarcinogenesis. Therefore, downregulation of survivin may provide an attractive strategy for treatment of hepatocellular carcinoma. In this regard, little is known about the anticancer effects of prodigiosin isolated from cell wall of Serratia marcescens on the survivin expression and induction of apoptosis in hepatocellular carcinoma cells. Human hepatocellular carcinoma (HepG2) cells were treated with 100-, 200-, 400-, and 600-nM prodigiosin after which morphology of cells, cell number, growth inhibition, survivin expression, caspase-3 activation, and apoptotic rate were evaluated by inverted microscope, hemocytometer, MTT assay, RT-PCR, fluorometric immunosorbent enzyme assay, and flow cytometric analysis, respectively. Prodigiosin changed morphology of cells to apoptotic forms and disrupted cell connections. This compound significantly increased growth inhibition rate and decreased metabolic activity of HepG2 cells in a dose- and time-dependent manner. After 24-, 48-, and 72-h treatments with prodigiosin at concentrations ranging from 100 nM to 600 nM, growth inhibition rates were measured to be 1.5-10%, 24-47.5%, and 55.5-72.5%, respectively, compared to untreated cells. At the same conditions, metabolic activities were measured to be 91-83%, 74-53%, and 47-31% for indicated concentrations of prodigiosin, respectively, compared to untreated cells. We also found that treatment of HepG2 cells for 48 h decreased significantly cell number and survivin expression and increased caspase-3 activation in a dose-dependent manner. Specifically, treatment with 600-nM prodigiosin resulted in 77% decrease in cell number, 88.5% decrease in survivin messenger RNA level, and 330% increase in caspase-3 activation level compared to untreated cells. An increase in the number of apoptotic cells (late apoptosis) ranging from 36.9% to 97.4% was observed with increasing prodigiosin concentrations. From our data, prodigiosin is an attractive compound that turns the profile of high-level survivin expression in hepatocellular carcinoma cells into that of normal cells and may provide a novel approach to the hepatocellular carcinoma-targeted therapy.

Comparative study on morphologic changes and cell attachment of periodontitis-affected root surfaces following conditioning with CO2 and Er:YAG laser irradiations.

PubMed

Belal, Mahmoud Helmy; Watanabe, Hisashi

2014-10-01

Clinical application of lasers in periodontal therapy has continued to expand in last decades; however there are still some controversies. The present study aimed to compare the conditioning effects of the carbon dioxide (CO2) or erbium-doped: yttrium, aluminum and garnet (Er:YAG) laser on periodontally diseased root surfaces following scaling and root planing (SRP) in terms of the alteration of morphologies as well as the attachment of periodontal ligament cells. Forty-five periodontally affected root specimens were prepared and randomly assigned into three groups: I control (untreated diseased), II. SRP+CO2 laser (pulsed, noncontact mode), and III. SRP+Er:YAG laser (slight contact mode). After treatment, five specimens in each group were used for surface topographic examination. The remaining 10 specimens in each group were incubated with human periodontal ligament cell suspension. All the specimens were finally evaluated by scanning electron microscopy. The control specimens showed the lowest number of cultured cells, mostly in oval shape, with no tightly attached cells. The CO2 lased specimens showed a significant increase in the number of attached cells compared with controls, but demonstrated some major thermal alterations on the surfaces. The Er:YAG lased specimens showed the significantly highest number of attached cells, mostly in flat form, and did not show distinct thermal damage. The present study suggests that compared with the CO2 laser, the Er:YAG laser may constitute a more useful conditioning tool for enhancing periodontal cell attachment to periodontally diseased root surfaces, with fewer undesirable thermal side effects.

Magnetic beads (Dynabead) toxicity to endothelial cells at high bead concentration: implication for tissue engineering of vascular prosthesis.

PubMed

Tiwari, A; Punshon, G; Kidane, A; Hamilton, G; Seifalian, A M

2003-10-01

Magnetic beads (Dynabeads) have been used for the purification of endothelial cells. One application for this procedure may be for single-stage seeding of bypass grafts. The number of endothelial cells (EC) isolated is crucial and therefore to increase the number of cells extracted, a higher number of Dynabeads per cell may need to be used. The effect of large numbers of CD31 Dynabeads on cell proliferation/metabolism is unknown. We undertook this study using CD31-coated Dynabeads and EC from human umbilical vein. EC were coated at concentrations of 4, 10, or 50 beads per cell. The cells were cultured for 6 days with control being normal EC. Cellular proliferation was assessed by trypsinization of cells and metabolism assessed with an Alamar blue viability assay. In a further experiment a compliant polyurethane graft was single-stage seeded with both coated Dynabeads and normal EC. The results showed that using a higher number of beads per cell resulted in a reduction in cell proliferation and a reduction in cell metabolism. The total number of Dynabeads-coated cells in culture compared to controls (%) by day 6 were 30.7 +/- 2.56, 41.3 +/- 9.8 and 59.2 +/- 7.3 for 50, 10, and 4 beads per cell, respectively. The corresponding results for Alamar blue were 43.7 +/- 1.2, 61.8 +/- 1.4, and 72.1 +/- 4.3. The seeded grafts showed reduced metabolism with the Dynabeads-coated EC. In conclusion, high numbers of beads per cell have a late detrimental effect on cell proliferation and metabolism. Therefore for single-stage seeding lower numbers of Dynabeads will need to be used with resultant reduction in the number of available EC.

Modulation of osteoblast attachment and growth in vitro by inertial forces

NASA Astrophysics Data System (ADS)

Kacena, Melissa Ann

1999-11-01

Spaceflight exploration and associated experiments show that human bones lose in density during inertial unloading, due principally to their demineralization. This research project examines the effect of gravity on osteoblast attachment and function in various inertial environments. Chicken calvarial osteoblasts were cultured under the following inertial conditions: spaceflight, simulated shuttle launch accelerations and vibrations, centrifugation, clino-rotation, and inversion. Cultures exposed to these conditions were compared with cultures grown in the laboratory as static 1G controls. Electron and light microscopy revealed the number of total osteoblasts attached to their substrate. Biochemical assays discerned changes in viable cell number, alkaline phosphatase levels, and mineralization. Immunohistochemical assays were used to investigate differences in cytoskeletal and extracellular matrix protein concentrations in the cultures, the percentage of proliferative cells, and cell viability. Compared to controls, spaceflight results indicated that the number of attached osteoblast cells was reduced. Launch simulation data indicated that the associated accelerations and vibrations may contribute to the reduction of attached osteoblasts in spaceflight cultures. Following centrifugation, the number of attached cells was unaltered; however, immunostaining of actin, fibronectin, and vinculin did show alterations in cultures exposed to hypergravity. Confluent cultures that were right side up, inverted, and clino-rotated contained a comparable number of attached cells and functioned similarly on the basis of measured alkaline phosphatase and bound calcium content. Sparse clino-rotated or inverted cultures showed an immediate response of diminished viable osteoblast numbers, but this effect disappeared with time and all remaining attached cells functioned similarly (APase and bound calcium). On the basis of these data osteoblast attachment and function in confluent cultures is minimally, if at all, affected by alterations in inertial environments. However, in sparse cultures about half as many cells are found attached initially. The remaining attached cells appear to multiply and function normally. These results suggest that the effects of spaceflight on bone are thus not likely to be caused by direct intrinsic effects of gravity on single osteoblasts that can be simulated in laboratory experiments in vitro experiments.

Cellular internalization of polycation-coated microparticles and its dependence on their zeta potential

NASA Astrophysics Data System (ADS)

Kato, Noritaka; Kondo, Ryosuke

2018-03-01

By applying microparticles to HeLa cells, the number of particles adhered on the cell and that of the ones internalized in the cells were evaluated. Three-dimensional tomographic images of the cells with the particles were obtained by multiphoton excitation laser scanning microscopy, and the adhered and internalized particles were counted separately. When the surface charge of the particles was reversed from negative to positive by coating the particles with polycations, both numbers significantly increased owing to the electrostatic attraction between the cells and the polycation-coated particles. Four different positively charged particles were prepared using four different polycations, and the numbers of adhered and internalized particles were compared. Our results suggest that these numbers depended on the zeta potential rather than the molecular structure of the polycation.

Evaluation of Immune Responses Mediated by Listeria-Stimulated Human Dendritic Cells: Implications for Cancer Vaccine Therapy

DTIC Science & Technology

2015-09-01

Award Number: W81XWH-11-1-0384 TITLE: Evaluation of Immune Responses Mediated by Listeria-Stimulated Human Dendritic Cells : Implications for...Immune Responses Mediated by Listeria-Stimulated Human Dendritic Cells : Implications for Cancer Vaccine Therapy 5b. GRANT NUMBER CA100463 5c...Listeria monocytogenes (Lm) on human dendritic cells (DCs) to optimize Lm-based DC cancer vaccines. The project aims are: 1) Compare the activation and

A robust method to analyze copy number alterations of less than 100 kb in single cells using oligonucleotide array CGH.

PubMed

Möhlendick, Birte; Bartenhagen, Christoph; Behrens, Bianca; Honisch, Ellen; Raba, Katharina; Knoefel, Wolfram T; Stoecklein, Nikolas H

2013-01-01

Comprehensive genome wide analyses of single cells became increasingly important in cancer research, but remain to be a technically challenging task. Here, we provide a protocol for array comparative genomic hybridization (aCGH) of single cells. The protocol is based on an established adapter-linker PCR (WGAM) and allowed us to detect copy number alterations as small as 56 kb in single cells. In addition we report on factors influencing the success of single cell aCGH downstream of the amplification method, including the characteristics of the reference DNA, the labeling technique, the amount of input DNA, reamplification, the aCGH resolution, and data analysis. In comparison with two other commercially available non-linear single cell amplification methods, WGAM showed a very good performance in aCGH experiments. Finally, we demonstrate that cancer cells that were processed and identified by the CellSearch® System and that were subsequently isolated from the CellSearch® cartridge as single cells by fluorescence activated cell sorting (FACS) could be successfully analyzed using our WGAM-aCGH protocol. We believe that even in the era of next-generation sequencing, our single cell aCGH protocol will be a useful and (cost-) effective approach to study copy number alterations in single cells at resolution comparable to those reported currently for single cell digital karyotyping based on next generation sequencing data.

Single-cell copy number variation detection

PubMed Central

2011-01-01

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data. PMID:21854607

A carbon nanotube-polymer composite for T-cell therapy

NASA Astrophysics Data System (ADS)

Fadel, Tarek R.; Sharp, Fiona A.; Vudattu, Nalini; Ragheb, Ragy; Garyu, Justin; Kim, Dongin; Hong, Enping; Li, Nan; Haller, Gary L.; Pfefferle, Lisa D.; Justesen, Sune; Harold, Kevin C.; Fahmy, Tarek M.

2014-08-01

Clinical translation of cell therapies requires strategies that can manufacture cells efficiently and economically. One promising way to reproducibly expand T cells for cancer therapy is by attaching the stimuli for T cells onto artificial substrates with high surface area. Here, we show that a carbon nanotube-polymer composite can act as an artificial antigen-presenting cell to efficiently expand the number of T cells isolated from mice. We attach antigens onto bundled carbon nanotubes and combined this complex with polymer nanoparticles containing magnetite and the T-cell growth factor interleukin-2 (IL-2). The number of T cells obtained was comparable to clinical standards using a thousand-fold less soluble IL-2. T cells obtained from this expansion were able to delay tumour growth in a murine model for melanoma. Our results show that this composite is a useful platform for generating large numbers of cytotoxic T cells for cancer immunotherapy.

Rosuvastatin reduces atherosclerotic lesions and promotes progenitor cell mobilisation and recruitment in apolipoprotein E knockout mice.

PubMed

Schroeter, Marco R; Humboldt, Tim; Schäfer, Katrin; Konstantinides, Stavros

2009-07-01

Statins enhance incorporation of bone marrow-derived cells into experimental neointimal lesions. However, the contribution of progenitor cells to progression of spontaneous atherosclerotic plaques, and the possible modulatory role of statins in this process, remain poorly understood. We compared the effects of rosuvastatin (1 and 10mg/kg BW) and pravastatin (10mg/kg) on progenitor cell mobilisation, recruitment into atherosclerotic plaques, and lesion growth. Statins were administered over 8 weeks to apolipoprotein E knockout mice on atherogenic diet. In addition, mice were lethally irradiated, followed by transplantation of bone marrow from LacZ transgenic mice. Rosuvastatin reduced lesion area and intima-to-media ratio at the brachiocephalic artery compared to vehicle, while both parameters were not significantly altered by pravastatin. Rosuvastatin also augmented endothelialisation (P<0.05) and reduced the smooth muscle cells (SMC) content (P=0.042) of lesions. Numbers of c-kit, sca-1 and flk-1, sca-1 double-positive progenitor cells were significantly increased in rosuvastatin compared to control-treated mice, both in the bone marrow and the peripheral blood. Similarly, the number of spleen-derived acLDL, lectin double-positive progenitor cells (P=0.001) and colony-forming units (P=0.0104) was significantly increased in mice treated with rosuvastatin compared to vehicle alone. In the bone marrow, increased Akt and p42/44 MAP kinase phosphorylation and upregulated SDF1alpha mRNA expression were observed. Importantly, rosuvastatin treatment also increased the plasma levels of c-kit ligand (P=0.003), and the number of c-kit-positive cells within atherosclerotic lesions (P=0.041). Our findings suggest that rosuvastatin reduces the size of atherosclerotic plaques, and this effect appears to involve progenitor cell mobilisation and recruitment into vascular lesions.

Tracking of Mesenchymal Stem Cells with Fluorescence Endomicroscopy Imaging in Radiotherapy-Induced Lung Injury

NASA Astrophysics Data System (ADS)

Perez, Jessica R.; Ybarra, Norma; Chagnon, Frederic; Serban, Monica; Lee, Sangkyu; Seuntjens, Jan; Lesur, Olivier; El Naqa, Issam

2017-01-01

Mesenchymal stem cells (MSCs) have potential for reducing inflammation and promoting organ repair. However, limitations in available techniques to track them and assess this potential for lung repair have hindered their applicability. In this work, we proposed, implemented and evaluated the use of fluorescence endomicroscopy as a novel imaging tool to track MSCs in vivo. MSCs were fluorescently labeled and injected into a rat model of radiation-induced lung injury via endotracheal (ET) or intravascular (IV) administration. Our results show that MSCs were visible in the lungs with fluorescence endomicroscopy. Moreover, we developed an automatic cell counting algorithm to quantify the number of detected cells in each condition. We observed a significantly higher number of detected cells in ET injection compared to IV and a slight increase in the mean number of detected cells in irradiated lungs compared to control, although the latter did not reach statistical significance. Fluorescence endomicroscopy imaging is a powerful new minimally invasive and translatable tool that can be used to track and quantify MSCs in the lungs and help assess their potential in organ repair.

Mobilization of hematopoietic progenitor cells with granulocyte colony stimulating factors for autologous transplant in hematologic malignancies: a single center experience

PubMed Central

Gabús, Raul; Borelli, Gabriel; Ferrando, Martín; Bódega, Enrique; Citrín, Estela; Jiménez, Constanza Olivera; Álvarez, Ramón

2011-01-01

Background In 2006 the Hematology Service of Hospital Maciel published its experience with peripheral blood progenitor cell harvesting for autologous stem cell transplantation using Filgen JP (Clausen Filgrastim). After mobilization with a mean filgrastim dose of 78 mcg/Kg, 4.7 x 106 CD34+ cells/Kg were obtained by apheresis. Age above 50, multiple myeloma as underlying disease and a malignancy that was not in remission were identified as frequent characteristics among patients showing complex mobilization. Objective The aim of this study was to compare stem cell mobilization using different brands of filgrastim. Methods One hundred and fifty-seven mobilizations performed between 1997 and 2006 were analyzed. This retrospective analysis comparative two groups of patients: those mobilized with different brands of filgrastim (Group A) and those who received Filgen JP (Clausen Filgrastim) as mobilizing agent (Group B). A cluster analysis technique was used to identify four clusters of individuals with different behaviors differentiated by age, total dose of filgrastim required, number of apheresis and harvested CD34+ cells. Results The mean total dose of filgrastim administered was 105 mcg/Kg, the median number of apheresis was 2 procedures and the mean number of harvested stem cells was 4.98 x 106 CD34+ cells/Kg. No significant differences were observed between Groups A and B regarding the number of apheresis, harvested CD34+ cells and number of mobilization failures, however the total dose of filgrastim was significantly lower in Group B. Conclusions Among other factors, the origin of the cytokine used as mobilizing agent is an element to be considered when evaluating CD34+ cell mobilization results. PMID:23049356

Mobilization of hematopoietic progenitor cells with granulocyte colony stimulating factors for autologous transplant in hematologic malignancies: a single center experience.

PubMed

Gabús, Raul; Borelli, Gabriel; Ferrando, Martín; Bódega, Enrique; Citrín, Estela; Jiménez, Constanza Olivera; Alvarez, Ramón

2011-01-01

In 2006 the Hematology Service of Hospital Maciel published its experience with peripheral blood progenitor cell harvesting for autologous stem cell transplantation using Filgen JP (Clausen Filgrastim). After mobilization with a mean filgrastim dose of 78 mcg/Kg, 4.7 x 10(6) CD34(+) cells/Kg were obtained by apheresis. Age above 50, multiple myeloma as underlying disease and a malignancy that was not in remission were identified as frequent characteristics among patients showing complex mobilization. The aim of this study was to compare stem cell mobilization using different brands of filgrastim. One hundred and fifty-seven mobilizations performed between 1997 and 2006 were analyzed. This retrospective analysis comparative two groups of patients: those mobilized with different brands of filgrastim (Group A) and those who received Filgen JP (Clausen Filgrastim) as mobilizing agent (Group B). A cluster analysis technique was used to identify four clusters of individuals with different behaviors differentiated by age, total dose of filgrastim required, number of apheresis and harvested CD34(+) cells. The mean total dose of filgrastim administered was 105 mcg/Kg, the median number of apheresis was 2 procedures and the mean number of harvested stem cells was 4.98 x 10(6) CD34(+) cells/Kg. No significant differences were observed between Groups A and B regarding the number of apheresis, harvested CD34(+) cells and number of mobilization failures, however the total dose of filgrastim was significantly lower in Group B. Among other factors, the origin of the cytokine used as mobilizing agent is an element to be considered when evaluating CD34(+) cell mobilization results.

CRADA No. BNL-C-97-10 between BNL and Cotton, Inc. Final abstract and final report [Final Report of Research carried out under DOE CRADA No. BNL-C-97-10 - "Prediction of Yield in Cotton"

DOE Office of Scientific and Technical Information (OSTI.GOV)

None, None

The objectives of this work were to determine if the numbér of fiber cell initials varied genetically and to compare the number of initials with that of mature fibers obtained at harvest time. The method used to count the number of fiber cell initials is direct, simple, quick and done while the plant is growing. In contrast, the currently used commercial process is indirect and needs large amount mature fibers gathered at harvest time. However, all current work on cotton yield is based on fiber numbers obtained by the indirect commercial process. Consequently, it was necessary to compare results obtainedmore » from the two methods using the same plants as the source of material. The results show that the number of fiber initials per ovule differed significantly (P>0.05) for seven cultivars in 1995 and 1996. AIso, a 1997 study shows the number of fiber initials varied by 15% over boll positions and environments, with rankings among cultivars generally consistent across boll positions and sampling times. Finally, although there were differences among cultivars for initial fiber cell number, all cultivars had nearly the same number of mature lint fibers per seed. This last finding is significant. It indicates that the rate of fiber cell initiation varies among cultivars; the lower the rate, the greater the difference between the number of initials and the number of mature fiber cells. If the rate of fiber initiation is relatively high, the number of initials and mature fibers differs by about 11%; if it is low, the difference is as high as 31%. Cotton breeders may be able to use genetic differences for the number of fiber initials and/or the rate of fiber cell initiation in crop improvement programs.« less

High-content image analysis (HCIA) assay has the highest correlation with direct counting cell suspension compared to the ATP, WST-8 and Alamar blue assays for measurement of cytotoxicity.

PubMed

Tahara, Haruna; Matsuda, Shun; Yamamoto, Yusuke; Yoshizawa, Hiroe; Fujita, Masaharu; Katsuoka, Yasuhiro; Kasahara, Toshihiko

2017-11-01

Various cytotoxicity assays measuring indicators such as enzyme activity, dye uptake, or cellular ATP content are often performed using 96-well microplates. However, recent reports show that cytotoxicity assays such as the ATP assay and MTS assay underestimate cytotoxicity when compounds such as anti-cancer drugs or mutagens induce cell hypertrophy whilst increasing intracellular ATP content. Therefore, we attempted to evaluate the reliability of a high-content image analysis (HCIA) assay to count cell number in a 96-well microplate automatically without using a cell-number indicator. We compared cytotoxicity results of 25 compounds obtained from ATP, WST-8, Alamar blue, and HCIA assays with those directly measured using an automatic cell counter, and repeating individual experiments thrice. The number of compounds showing low correlation in cell viability measured using cytotoxicity assays compared to automatic cell counting (r 2 <0.8, at least 2 of 3 experiments) were follows: ATP assay; 7; WST-8 assay, 2; Alamar blue assay, 3; HCIA cytotoxicity assay, 0. Compounds for which correlation was poor in 3 assays, except the HCIA assay, induced an increase in nuclear and cell size. However, correlation between cell viability measured by automatic cell counter and the HCIA assay was strong regardless of nuclear and cell size. Additionally, correlation coefficients between IC 50 values obtained from automatic cell counter and from cytotoxicity assays were as follows: ATP assay, 0.80; WST-8 assay, 0.84; Alamar blue assay, 0.84; and HCIA assay, 0.98. From the above, we showed that the HCIA cytotoxicity assay produces similar data to the automatic cell counter and is highly accurate in measuring cytotoxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

PubMed

Schmidt, J; Zyba, V; Jung, K; Rinke, S; Haak, R; Mausberg, R F; Ziebolz, D

2016-01-01

This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

Phenotypic differences in leucocyte populations among healthy preterm and full-term newborns.

PubMed

Quinello, C; Silveira-Lessa, A L; Ceccon, M E J R; Cianciarullo, M A; Carneiro-Sampaio, M; Palmeira, P

2014-07-01

The immune system of neonates has been considered functionally immature, and due to their high susceptibility to infections, the aim of this study was to analyse the phenotypic differences in leucocyte populations in healthy preterm and full-term newborns. We evaluated the absolute numbers and frequencies of dendritic cells (DCs) and DC subsets, monocytes and T and B lymphocytes and subsets in the cord blood of healthy moderate and very preterm (Group 1), late preterm (Group 2) and full-term (Group 3) newborns and in healthy adults, as controls, by flow cytometry. The analyses revealed statistically higher absolute cell numbers in neonates compared with adults due to the characteristic leucocytosis of neonates. We observed a lower frequency of CD80(+) myeloid and plasmacytoid DCs in Group 1 and reduced expression of TLR-4 on myeloid DCs in all neonates compared with adults. TLR-2(+) monocytes were reduced in Group 1 compared with Groups 2 and 3, and TLR-4(+) monocytes were reduced in Groups 1 and 2 compared with Group 3. The frequencies and numbers of naïve CD4(+) T and CD19(+) B cells were higher in the three groups of neonates compared with adults, while CD4(+) effector and effector memory T cells and CD19(+) memory B cells were elevated in adults compared with neonates, as expected. Our study provides reference values for leucocytes in cord blood from term and preterm newborns, which may facilitate the identification of immunological deficiencies in protection against extracellular pathogens. © 2014 John Wiley & Sons Ltd.

Sex Differences in Stress and Group Housing Effects on the Number of Newly Proliferated Cells and Neuroblasts in Middle-Aged Dentate Gyrus.

PubMed

Tzeng, Wen-Yu; Wu, Hsin-Hua; Wang, Ching-Yi; Chen, Jin-Chung; Yu, Lung; Cherng, Chianfang G

2016-01-01

Sex differences in stress and coping responses have been frequently documented in aged people, while whether such differences in aged people may appear at the middle age are unknown. This study was undertaken to study the impact of acute stress and social interaction on early neurogenesis in the dentate gyrus (DG) and hippocampus-related memory in two sexes of middle-aged mice. The number of newly proliferated cells, neuroblasts in DG, the object recognition and location memory in 9-month-old male and female C57BL/6N mice were assessed under baseline conditions as well as following an acute stressor regimen and group housing. Three conspecific companions, serving as "the housing group," were used to model the social interaction throughout the stressor regimen. Males had lower numbers of newly proliferated cells and neuroblasts under baseline conditions as compared to females. The stressor regimen caused rapid decreases in the number of newly proliferated cells and neuroblasts in female DG but no obvious changes were observed in male DG. Group housing, regardless of companions' age, prevented the stress-induced decreases in the number of newly proliferated cells and neuroblasts in female DG. In contrast, the presence of young or age-matched companions potentiated the stress effect in males by decreasing the number of newly proliferated cells and neuroblasts. Finally, neither the stressor regimen nor group housing affected mouse performances in the object recognition and location memory in either sex. These findings, taken together, provide evidence to support a notion that middle-aged females appear to demonstrate more stress susceptibility on early neurogenesis in DG as compared to middle-aged males, although the hippocampus-related memory performances are comparable and not affected by stress in these males and females. Experiencing stress, middle-aged females are more prone to benefit from social interaction as compared to middle-aged males in this regard. We suggest, accordingly, that involving social interaction may afford a therapeutic advance in preventing stress-produced decreases in early neurogenesis in middle-aged females' DG.

Involvement of Trigeminal Transition Zone and Laminated Subnucleus Caudalis in Masseter Muscle Hypersensitivity Associated with Tooth Inflammation

PubMed Central

Shimizu, Kohei; Matsumoto, Kunihito; Noma, Noboru; Matsuura, Shingo; Ohara, Kinuyo; Komiya, Hiroki; Watase, Tetsuro; Ogiso, Bunnai; Tsuboi, Yoshiyuki; Shinoda, Masamichi; Hatori, Keisuke; Nakaya, Yuka; Iwata, Koichi

2014-01-01

A rat model of pulpitis/periapical periodontitis was used to study mechanisms underlying extraterritorial enhancement of masseter response associated with tooth inflammation. Periapical bone loss gradually increased and peaked at 6 weeks after complete Freund’s adjuvant (CFA) application to the upper molar tooth pulp (M1). On day 3, the number of Fos-immunoreactive (IR) cells was significantly larger in M1 CFA rats compared with M1 vehicle (veh) rats in the trigeminal subnucleus interpolaris/caudalis transition zone (Vi/Vc). The number of Fos-IR cells was significantly larger in M1 CFA and masseter (Mass) capsaicin applied (M1 CFA/Mass cap) rats compared with M1 veh/Mass veh rats in the contralateral Vc and Vi/Vc. The number of phosphorylated extracellular signal-regulated kinase (pERK)-IR cells was significantly larger in M1 CFA/Mass cap and M1 veh/Mass cap rats compared to Mass-vehicle applied rats with M1 vehicle or CFA in the Vi/Vc. Pulpal CFA application caused significant increase in the number of Fos-IR cells in the Vi/Vc but not Vc on week 6. The number of pERK-IR cells was significantly lager in the rats with capsaicin application to the Mass compared to Mass-vehicle treated rats after pulpal CFA- or vehicle-application. However, capsaicin application to the Mass did not further affect the number of Fos-IR cells in the Vi/Vc in pulpal CFA-applied rats. The digastric electromyographic (d-EMG) activity after Mass-capsaicin application was significantly increased on day 3 and lasted longer at 6 weeks after pulpal CFA application, and these increase and duration were significantly attenuated by i.t. PD98059, a MEK1 inhibitor. These findings suggest that Vi/Vc and Vc neuronal excitation is involved in the facilitation of extraterritorial hyperalgesia for Mass primed with periapical periodontitis or acute pulpal-inflammation. Furthermore, phosphorylation of ERK in the Vi/Vc and Vc play pivotal roles in masseter hyperalgesia after pulpitis or periapical periodontitis. PMID:25279551

Immune cell populations within the duodenal mucosa of dogs with enteropathies.

PubMed

German, A J; Hall, E J; Day, M J

2001-01-01

The mucosal immune system may play a critical role in the pathogenesis of small intestinal enteropathies. The aim of the current study was to assess mucosal immune cell populations in dogs with inflammatory bowel disease (IBD), idiopathic antibiotic-responsive diarrhea (ARD), and adverse reactions to food (FR). Endoscopic biopsies were performed of the duodenum of dogs with these conditions and from a group of dogs without enteric disease. Additional control samples were collected after death from other dogs that did not have evidence of enteric disease. Immunohistochemistry and computer-aided morphometry were used to assess the distribution of immune cell subsets in both lamina propria and intestinal epithelium. Compared with controls, dogs with ARD had increased numbers of lamina propria immunoglobulin (Ig) A- plasma cells and CD4+ cells. More marked alterations were noted in dogs with IBD, with significant increases in lamina propria IgG+ plasma cells, T cells (CD3+), CD4+ cells, macrophages, and neutrophils, but with reduced mast cell numbers. Increased intraepithelial CD3+ T cells were also present in the dogs with IBD, compared with controls. However, lamina propria and epithelial populations were unaltered in dogs with FR when compared with controls. The altered mucosal immune cell populations observed in dogs with ARD or IBD may reflect an underlying immunologic pathogenesis in these disorders.

Identification of metabolic profiling of cell culture of licorice compared with its native one.

PubMed

Man, Shuli; Guo, Songbo; Gao, Wenyuan; Wang, Juan; Zhang, Liming; Li, Xinglin

2013-04-01

Glycyrrhiza uralensis has long been used as a flavoring and sweetening agent in food products. In the last ten years, suspensions of Glycyrhiza cells have been successfully established. However, there is no report of full metabolic profiling research on these cells. To identify their composition we used HPLC-DAD coupled with ESI(+/-)-MS (n) to compare the constituents of cultured Glycyrhiza (CG) cells with those the native cells (NG). We identified 60 compounds including flavonoids, phenols, and triterpenoids. Among these compounds, 42 occurred both in NG and CG, nine were present in NG only and nine were present in CG alone. The number of the triterpenoid aglycones without glycones in CG was smaller than that in NG. The number of flavanone, isoflavone, isoflavan, and benzenoid compounds was also smaller in CG than that in NG, whereas the number of pterocarpans was much higher. Although differences existed between CG and NG, the extract of CG was similar to that of NG. With the development of cell suspension culture-based biotransformation, cell culture of Glycyrrhiza has the potential to be more profitable than field cultivation in some areas.

Semi-synthetic salinomycin analogs exert cytotoxic activity against human colorectal cancer stem cells.

PubMed

Klose, Johannes; Kattner, Sarah; Borgström, Björn; Volz, Claudia; Schmidt, Thomas; Schneider, Martin; Oredsson, Stina; Strand, Daniel; Ulrich, Alexis

2018-01-01

Salinomycin, a polyether antibiotic, is a well-known inhibitor of human cancer stem cells. Chemical modification of the allylic C20 hydroxyl of salinomycin has enabled access to synthetic analogs that display increased cytotoxic activity compared to the native structure. The aim of this study was to investigate the activity of a cohort of C20-O-acyl analogs of salinomycin on human colorectal cancer cell lines in vitro. Two human colorectal cancer cell lines (SW480 and SW620) were exposed to three C20-O-acylated analogs and salinomycin. The impact of salinomycin and its analogs on tumor cell number, migration, cell death, and cancer stem cell specifity was analyzed. Exposure of human colorectal cancer cells to the C20-O-acylated analogs of salinomycin resulted in reduced tumor cell number and impaired tumor cell migration at lower concentrations than salinomycin. When used at higher (micromolar) concentrations, these effects were accompanied by induction of apoptotic cell death. Salinomycin analogs further expose improved activity against cancer stem cells compared to salinomycin. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparison of Mast Cells and Inflammatory Cells within Periapical Lesions and Comparison of Degranulated Mast Cells Between Fibrous and Inflamed Area in Radicular Cysts: An Immunohistochemical Study

PubMed Central

Sood, Rahul; Akifuddin, Syed; Sidhu, Gagandeep Kaur; Khan, Nadia; Singla, Kapil

2014-01-01

Objective: The role of mast cells as the key effector of allergic inflammation, anaphylactic inflammatory reactions and in the pathogenesis of chronic inflammation, is well-known. The present study is adopted to compare mast cells and inflammatory cells within periapical granuloma and cysts and localize the mast cells and quantify their number in the periapical cysts so as to propose a role of mast cells in the pathogenesis of this lesion. Materials and Methods: Biopsy specimens of 30 periapical lesions were stained with hematoxylin–eosin, and immunohistochemical Mast Cell Tryptase from Bio SB (IHC detection system kit) antibody. The tryptase positive mast cells and mononuclear inflammatory cells were counted in 10 consecutive high power fields (100X) using the binocular microscope from Motic attached to a computer with Motic Advanced Images 3.2 software. Results: Comparative microscopic analysis indicated that periapical cyst shows more percentage of mast cells and less percentage of inflammatory cell than periapical granuloma (comparison of mean and standard deviation of total number of mast cells and inflammatory cells, mast cells 3.15±1.39 in the granuloma group and 4.43±1.91in the cyst group, inflammatory cells, 67.11±1.2 in the granuloma group and 52.66±0.8 in the cyst group). Numerous degranulated mast cells were observed in the fibrous wall than the inflammatory infiltrate of the periapical cysts. The mean and standard deviation of degranulated mast cells between the inflammatory and fibrous zone within the cyst group, being 0.95±1.10 and1.68±1.34 respectively. The values varied significantly between the two zones. Conclusion: The number of inflammatory cells in the cyst group is less than periapical granuloma and total number of mast cells in the cyst group is more as compared to periapical granuloma. The degranulated cells were quantified and they were higher in the fibrous area of the cysts than the inflammatory zone. This study could support the fact that the various mediators released on degranulation play a role in the connective tissue remodeling, chronicity and expansion of the periapical lesion. PMID:25654034

In Search of Cellular Immunophenotypes in the Blood of Children with Autism

PubMed Central

Ashwood, Paul; Corbett, Blythe A.; Kantor, Aaron; Schulman, Howard; Van de Water, Judy; Amaral, David G.

2011-01-01

Background Autism is a neurodevelopmental disorder characterized by impairments in social behavior, communication difficulties and the occurrence of repetitive or stereotyped behaviors. There has been substantial evidence for dysregulation of the immune system in autism. Methods We evaluated differences in the number and phenotype of circulating blood cells in young children with autism (n = 70) compared with age-matched controls (n = 35). Children with a confirmed diagnosis of autism (4–6 years of age) were further subdivided into low (IQ<68, n = 35) or high functioning (IQ≥68, n = 35) groups. Age- and gender-matched typically developing children constituted the control group. Six hundred and forty four primary and secondary variables, including cell counts and the abundance of cell surface antigens, were assessed using microvolume laser scanning cytometry. Results There were multiple differences in immune cell populations between the autism and control groups. The absolute number of B cells per volume of blood was over 20% higher for children with autism and the absolute number of NK cells was about 40% higher. Neither of these variables showed significant difference between the low and high functioning autism groups. While the absolute number of T cells was not different across groups, a number of cellular activation markers, including HLA-DR and CD26 on T cells, and CD38 on B cells, were significantly higher in the autism group compared to controls. Conclusions These results support previous findings that immune dysfunction may occur in some children with autism. Further evaluation of the nature of the dysfunction and how it may play a role in the etiology of autism or in facets of autism neuropathology and/or behavior are needed. PMID:21573236

Efficient Manufacturing of Therapeutic Mesenchymal Stromal Cells Using the Quantum Cell Expansion System

PubMed Central

Hanley, Patrick J.; Mei, Zhuyong; Durett, April G.; Cabreira-Harrison, Marie da Graca; Klis, Mariola; Li, Wei; Zhao, Yali; Yang, Bing; Parsha, Kaushik; Mir, Osman; Vahidy, Farhaan; Bloom, Debra; Rice, R. Brent; Hematti, Peiman; Savitz, Sean I; Gee, Adrian P.

2014-01-01

Background The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host-disease, diabetes, ischemic cardiomyopathy, and Crohn's disease has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells needs to be cost effective, safe, and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures, and require prolonged culture times. Methods We evaluated the Quantum Cell Expansion system for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results to a flask-based method currently in clinical trials. Results After only two passages, we were able to expand a mean of 6.6×108 MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts, and adipocytes. The Quantum was able to generate the target cell number of 2.0×108 cells in an average of 9-fewer days and in half the number of passages required during flask-based expansion. We estimated the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. Discussion The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared to flasks. In the Quantum, the risk of contamination is substantially reduced due to the substantial decrease in open procedures. PMID:24726657

Hippocampal neurogenesis and cortical cellular plasticity in Wahlberg's epauletted fruit bat: a qualitative and quantitative study.

PubMed

Gatome, Catherine W; Mwangi, Deter K; Lipp, Hans-Peter; Amrein, Irmgard

2010-01-01

Species-specific characteristics of neuronal plasticity emerging from comparative studies can address the functional relevance of hippocampal or cortical plasticity in the light of ecological adaptation and evolutionary history of a given species. Here, we present a quantitative and qualitative analysis of neurogenesis in young and adult free-living Wahlberg's epauletted fruit bats. Using the markers for proliferating cell nuclear antigen (PCNA), bromodeoxyuridine (BrdU), doublecortin (DCX) and polysialic acid neural cell adhesion molecule (PSA-NCAM), our findings in the hippocampus, olfactory bulb and cortical regions are described and compared to reports in other mammals. Expressed as a percentage of the total number of granule cells, PCNA- and BrdU-positive cells accounted for 0.04 in young to 0.01% in adult animals; DCX-positive cells for 0.05 (young) to 0.01% (adult); PSA-NCAM-positive cells for 0.1 (young) to 0.02% (adult), and pyknotic cells for 0.007 (young) to 0.005% (adult). The numbers were comparable to other long-lived, late-maturing mammals such as primates. A significant increase in the total granule cell number from young to adult animals demonstrated the successful formation and integration of new cells. In adulthood, granule cell number appeared stable and was surprisingly low in comparison to other species. Observations in the olfactory bulb and rostral migratory stream were qualitatively similar to descriptions in other species. In the ventral horn of the lateral ventricle, we noted prominent expression of DCX and PSA-NCAM forming a temporal migratory stream targeting the piriform cortex, possibly reflecting the importance of olfaction to these species. Low, but persistent hippocampal neurogenesis in non-echolocating fruit bats contrasted the findings in echolocating microbats, in which hippocampal neurogenesis was largely absent. Together with the observed intense cortical plasticity in the olfactory system of fruit bats we suggest a differential influence of sensory modalities on hippocampal and cortical plasticity in this mammalian order. Copyright © 2010 S. Karger AG, Basel.

The effects of pulsed electromagnetic field (PEMF) on osteoblast-like cells cultured on titanium and titanium-zirconium surfaces.

PubMed

Atalay, Belir; Aybar, Buket; Ergüven, Mine; Emes, Yusuf; Bultan, Özgür; Akça, Kivanç; Yalçin, Serhat; Baysal, Uğur; Işsever, Halim; Çehreli, Murat Cavit; Bilir, Ayhan

2013-11-01

Commercially pure Ti, together with Ti Ni, Ti-6Al-4V, and Ti-6Al-7Nb alloys, are among the materials currently being used for this purpose. Titanium-zirconium (TiZr) has been developed that allows SLActive surface modification and that has comparable or better mechanical strength and improved biocompatibility compared with existing Ti alloys. Furthermore, approaches have targeted making the implant surface more hydrophilic, as with the Straumann SLActive surface, a modification of the SLA surface. The aim of this study is to evaluate the effects of pulsed electromagnetic field (PEMF) to the behavior of neonatal rat calvarial osteoblast-like cells cultured on commercially pure titanium (cpTi) and titanium-zirconium alloy (TiZr) discs with hydrophilic surface properties. Osteoblast cells were cultured on titanium and TiZr discs, and PEMF was applied. Cell proliferation rates, cell numbers, cell viability rates, alkaline phosphatase, and midkine (MK) levels were measured at 24 and 72 hours. At 24 hours, the number of cells was significantly higher in the TiZr group. At 72 hours, TiZr had a significantly higher number of cells when compared to SLActive, SLActive + PEMF, and machine surface + PEMF groups. At 24 hours, cell proliferation was significantly higher in the TiZr group than SLActive and TiZr + PEMF group. At 72 hours, TiZr group had higher proliferation rate than machine surface and TiZr + PEMF. Cell proliferation in the machine surface group was lower than both SLActive + PEMF and machine surface + PEMF. MK levels of PEMF-treated groups were lower than untreated groups for 72 hours. Our findings conclude that TiZr surfaces are similar to cpTi surfaces in terms of biocompatibility. However, PEMF application has a higher stimulative effect on cells cultured on cpTi surfaces when compared to TiZr.

Characterization of winemaking yeast by cell number-size distribution analysis through flow field-flow fractionation with multi-wavelength turbidimetric detection.

PubMed

Zattoni, Andrea; Melucci, Dora; Reschiglian, Pierluigi; Sanz, Ramsés; Puignou, Lluís; Galceran, Maria Teresa

2004-10-29

Yeasts are widely used in several areas of food industry, e.g. baking, beer brewing, and wine production. Interest in new analytical methods for quality control and characterization of yeast cells is thus increasing. The biophysical properties of yeast cells, among which cell size, are related to yeast cell capabilities to produce primary and secondary metabolites during the fermentation process. Biophysical properties of winemaking yeast strains can be screened by field-flow fractionation (FFF). In this work we present the use of flow FFF (FlFFF) with turbidimetric multi-wavelength detection for the number-size distribution analysis of different commercial winemaking yeast varieties. The use of a diode-array detector allows to apply to dispersed samples like yeast cells the recently developed method for number-size (or mass-size) analysis in flow-assisted separation techniques. Results for six commercial winemaking yeast strains are compared with data obtained by a standard method for cell sizing (Coulter counter). The method here proposed gives, at short analysis time, accurate information on the number of cells of a given size, and information on the total number of cells.

Combination of the angiotensin-converting enzyme inhibitor perindopril and the diuretic indapamide activate postnatal vasculogenesis in spontaneously hypertensive rats.

PubMed

You, Dong; Cochain, Clément; Loinard, Céline; Vilar, José; Mees, Barend; Duriez, Micheline; Lévy, Bernard I; Silvestre, Jean-Sébastien

2008-06-01

Cardiovascular risk factors are associated with reduction in both the number and function of vascular progenitor cells. We hypothesized that 1) hypertension abrogates postnatal vasculogenesis, and 2) antihypertensive treatment based on the combination of perindopril (angiotensin-converting enzyme inhibitor) and indapamide (diuretic) may counteract hypertension-induced alteration in progenitor cell-related effects. Postischemic neovascularization was significantly lower in untreated spontaneously hypertensive rats (SHRs) compared with Wistar Kyoto (WKY) rats (p < 0.05). Treatment of SHRs with perindopril and the combination of perindopril/indapamide reduced the blood pressure levels and normalized vessel growth in ischemic area. Cotreatment with perindopril and indapamide increased vascular endothelial growth factor and endothelial nitric-oxide synthase protein contents, two key proangiogenic factors. It is interesting to note that 14 days after bone marrow mononuclear cell (BM-MNC) transplantation, revascularization was significantly lower in ischemic SHRs receiving BM-MNCs isolated from SHRs compared with those receiving BM-MNCs isolated from WKY rats (p < 0.05). Alteration in proangiogenic potential of SHR BM-MNCs was probably related to the reduction in their ability to differentiate into endothelial progenitor cells in vitro. Furthermore, the number of circulating endothelial progenitor cells (EPCs) was reduced by 3.1-fold in SHRs compared with WKY rats (p < 0.001). Treatments with perindopril or perindopril/indapamide restored the ability of BM-MNCs to differentiate in vitro into EPCs, increased the number of circulating EPCs, and re-established BM-MNC proangiogenic effects. Therefore, hypertension is associated with a decrease in the number of circulating progenitor cells and in the BM-MNC proangiogenic potential, probably leading to vascular complications in this setting. The combination of perindopril and indapamide counteracts hypertension-induced alterations in progenitor cell-related effects and restores blood vessel growth.

Disease progression in recurrent glioblastoma patients treated with the VEGFR inhibitor axitinib is associated with increased regulatory T cell numbers and T cell exhaustion.

PubMed

Du Four, Stephanie; Maenhout, Sarah K; Benteyn, Daphné; De Keersmaecker, Brenda; Duerinck, Johnny; Thielemans, Kris; Neyns, Bart; Aerts, Joeri L

2016-06-01

Recurrent glioblastoma is associated with a poor overall survival. Antiangiogenic therapy results in a high tumor response rate but has limited impact on survival. Immunotherapy has emerged as an efficient treatment modality for some cancers, and preclinical evidence indicates that anti-VEGF(R) therapy can counterbalance the immunosuppressive tumor microenvironment. We collected peripheral blood mononuclear cells (PBMC) of patients with recurrent glioblastoma treated in a randomized phase II clinical trial comparing the effect of axitinib with axitinib plus lomustine and analyzed the immunophenotype of PBMC, the production of cytokines and expression of inhibitory molecules by circulating T cells. PBMC of 18 patients were collected at baseline and at 6 weeks after initiation of study treatment. Axitinib increased the number of naïve CD8(+) T cells and central memory CD4(+) and CD8(+) T cells and reduced the TIM3 expression on CD4(+) and CD8(+) T cells. Patients diagnosed with progressive disease on axitinib had a significantly increased number of regulatory T cells and an increased level of PD-1 expression on CD4(+) and CD8(+) T cells. In addition, reduced numbers of cytokine-producing T cells were found in progressive patients as compared to patients responding to treatment. Our results suggest that axitinib treatment in patients with recurrent glioblastoma has a favorable impact on immune function. At the time of acquired resistance to axitinib, we documented further enhancement of a preexisting immunosuppression. Further investigations on the role of axitinib as potential combination partner with immunotherapy are necessary.

Bacterial lysate increases the percentage of natural killer T cells in peripheral blood and alleviates asthma in children.

PubMed

Lu, Yanming; Li, Yaqin; Xu, Lingyun; Xia, Min; Cao, Lanfang

2015-01-01

To assess the efficacy of conventional treatment combined with bacterial lysate [OM-85 Broncho-Vaxom (BV)] in the prevention of asthma in children as well as its influence on the number of natural killer T (NKT) cells and their cytokine production. Sixty children diagnosed with asthma were divided into either a BV-treated group (with oral OM-85 BV) or a conventional inhaled corticosteroid (ICS) group. The numbers of NKT cells and CD4+ NKT cells were measured in the peripheral blood by flow cytometry. The levels of IFN-γ, IL-4, and IL-10 after the blood cells had been cultured with an NKT cell agonist were detected by ELISA. After therapy, asthma attacks were significantly decreased compared with before therapy in both groups. However, after therapy, respiratory tract infections were reduced compared with before therapy in the BV-treated group only. Additionally, the frequency of asthma attacks and use of antibiotics in the BV-treated group were lower than in the ICS group. With BV treatment, the numbers of peripheral blood NKT cells and CD4+ NKT cells were higher after therapy than before therapy. After therapy, the ratio of IFN-γ/IL-4 and IL-10 levels were increased in the BV-treated group, whereas IL-4 was reduced in the BV-treated group compared with the ICS group. BV combined with conventional asthma treatment can prevent recurrent respiratory tract infections and suppress the severity of asthma attacks, possibly by altering the rates and cytokines of NKT cells. © 2015 S. Karger AG, Basel

Clinical scale rapid expansion of lymphocytes for adoptive cell transfer therapy in the WAVE® bioreactor

PubMed Central

2012-01-01

Background To simplify clinical scale lymphocyte expansions, we investigated the use of the WAVE®, a closed system bioreactor that utilizes active perfusion to generate high cell numbers in minimal volumes. Methods We have developed an optimized rapid expansion protocol for the WAVE bioreactor that produces clinically relevant numbers of cells for our adoptive cell transfer clinical protocols. Results TIL and genetically modified PBL were rapidly expanded to clinically relevant scales in both static bags and the WAVE bioreactor. Both bioreactors produced comparable numbers of cells; however the cultures generated in the WAVE bioreactor had a higher percentage of CD4+ cells and had a less activated phenotype. Conclusions The WAVE bioreactor simplifies the process of rapidly expanding tumor reactive lymphocytes under GMP conditions, and provides an alternate approach to cell generation for ACT protocols. PMID:22475724

In vitro culture of individual mouse preimplantation embryos: the role of embryo density, microwells, oxygen, timing and conditioned media.

PubMed

Kelley, Rebecca L; Gardner, David K

2017-05-01

Single embryo culture is suboptimal compared with group culture, but necessary for embryo monitoring, and culture systems should be improved for single embryos. Pronucleate mouse embryos were used to assess the effect of culture conditions on single embryo development. Single culture either before or after compaction reduced cell numbers (112.2 ± 3.1; 110.2 ± 3.5) compared with group culture throughout (127.0 ± 3.4; P < 0.05). Reduction of media volume from 20 µl to 2 µl increased blastocyst cell numbers in single embryos cultured in 5% oxygen (84.4 ± 3.2 versus 97.8 ± 2.8; P < 0.05), but not in 20% oxygen (55.2 ± 2.9 versus 57.1 ± 2.8). Culture in microwell plates for the EmbryoScope and Primo Vision time-lapse systems changed cleavage timings and increased inner cell mass cell number (24.1 ± 1.0; 23.4 ± 1.2) compared with a 2 µl microdrop (18.4 ± 1.0; P < 0.05). Addition of embryo-conditioned media to single embryos increased hatching rate and blastocyst cell number (91.5 ± 4.7 versus 113.1 ± 4.4; P < 0.01). Single culture before or after compaction is therefore detrimental; oxygen, media volume and microwells influence single embryo development; and embryo-conditioned media may substitute for group culture. Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

77 FR 29353 - Agency Information Collection Activities; Submission for Office of Management and Budget Review...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-05-17

... Request; Experimental Study on Comparing Data Obtained From Landline Telephone and Cell Phone Surveys... [email protected] . All comments should be identified with the OMB control number 0910-New and title ``Experimental Study on Comparing Data Obtained From Landline Telephone and Cell Phone...

Influence of physical activity on the immune system in breast cancer patients during chemotherapy.

PubMed

Schmidt, Thorsten; Jonat, Walter; Wesch, Daniela; Oberg, Hans-Heinrich; Adam-Klages, Sabine; Keller, Lisa; Röcken, Christoph; Mundhenke, Christoph

2018-03-01

Physical activity can impact the immune system in different ways, e.g. by alteration of the humoral and cellular immune response. Physical activity at medium intensity enhances numbers of cytotoxic T cells, NK cells and macrophages in healthy people. The aim of this study was to compare the effects of endurance and resistance training on the immune system in breast cancer patients during adjuvant chemotherapy. In a prospective, controlled and randomized intervention exploratory trial, 12-week supervised endurance or resistance training were compared with usual care twice a week. Endpoints were the absolute numbers of the immune cells such as CD3 + T lymphocytes including CD4 + - and CD8 + , αβ T cells, γδT cells, CD3 - /CD16 + /56 + NK cells and CD19 + B cells, before and after 12 weeks of treatment. Cell numbers were analyzed using fluorescence-activated cell sorting. Despite different physical interventions in all groups immune cell count decreased in CD3 T cells including TCR αβ and CD4 T cells, NK cells and CD19 B cells 12 weeks after initiation of chemotherapy and start of the physical intervention program, while the reduction of γδ T cells and CD8 T cells is less prominent in the RT and UC group. Chemotherapy led to a decrease in nearly all measured immune cells. In this study, physical intervention with endurance or resistance training did not suppress cellular immunity any further. Larger multicenter trials are needed to evaluate the exact impact of sports intervention on immune cell subpopulations.

Whole body proton irradiation causes acute damage to bone marrow hematopoietic progenitor and stem cells in mice.

PubMed

Chang, Jianhui; Wang, Yingying; Pathak, Rupak; Sridharan, Vijayalakshmi; Jones, Tamako; Mao, Xiao Wen; Nelson, Gregory; Boerma, Marjan; Hauer-Jensen, Martin; Zhou, Daohong; Shao, Lijian

2017-12-01

Exposure to proton irradiation during missions in deep space can lead to bone marrow injury. The acute effects of proton irradiation on hematopoietic stem and progenitor cells remain undefined and thus were investigated. We exposed male C57BL/6 mice to 0.5 and 1.0 Gy proton total body irradiation (proton-TBI, 150 MeV) and examined changes in peripheral blood cells and bone marrow (BM) progenitors and LSK cells 2 weeks after exposure. 1.0 Gy proton-TBI significantly reduced the numbers of peripheral blood cells compared to 0.5 Gy proton-TBI and unirradiated animals, while the numbers of peripheral blood cell counts were comparable between 0.5 Gy proton-TBI and unirradiated mice. The frequencies and numbers of LSK cells and CMPs in BM of 0.5 and 1.0 Gy irradiated mice were decreased in comparison to those of normal controls. LSK cells and CMPs and their progeny exhibited a radiation-induced impairment in clonogenic function. Exposure to 1.0 Gy increased cellular apoptosis but not the production of reactive oxygen species (ROS) in CMPs two weeks after irradiation. LSK cells from irradiated mice exhibited an increase in ROS production and apoptosis. Exposure to proton-TBI can induce acute damage to BM progenitors and LSK cells.

BMS 493 Modulates Retinoic Acid-Induced Differentiation During Expansion of Human Hematopoietic Progenitor Cells for Islet Regeneration.

PubMed

Elgamal, Ruth M; Bell, Gillian I; Krause, Sarah C T; Hess, David A

2018-06-06

Cellular therapies are emerging as a novel treatment strategy for diabetes. Thus, the induction of endogenous islet regeneration in situ represents a feasible goal for diabetes therapy. Umbilical cord blood-derived hematopoietic progenitor cells (HPCs), isolated by high aldehyde dehydrogenase activity (ALDH hi ), have previously been shown to reduce hyperglycemia after intrapancreatic (iPan) transplantation into streptozotocin (STZ)-treated nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. However, these cells are rare and require ex vivo expansion to reach clinically applicable numbers for human therapy. Therefore, we investigated whether BMS 493, an inverse retinoic acid receptor agonist, could prevent retinoic acid-induced differentiation and preserve islet regenerative functions during expansion. After 6-day expansion, BMS 493-treated cells showed a twofold increase in the number of ALDH hi cells available for transplantation compared with untreated controls. Newly expanded ALDH hi cells showed increased numbers of CD34 and CD133-positive cells, as well as a reduction in CD38 expression, a marker of hematopoietic cell differentiation. BMS 493-treated cells showed similar hematopoietic colony-forming capacity compared with untreated cells, with ALDH hi subpopulations producing more colonies than low aldehyde dehydrogenase activity subpopulations for expanded cells. To determine if the secreted proteins of these cells could augment the survival and/or proliferation of β-cells in vitro, conditioned media (CM) from cells expanded with or without BMS 493 was added to human islet cultures. The total number of proliferating β-cells was increased after 3- or 7-day culture with CM generated from BMS 493-treated cells. In contrast to freshly isolated ALDH hi cells, 6-day expansion with or without BMS 493 generated progeny that were unable to reduce hyperglycemia after iPan transplantation into STZ-treated NOD/SCID mice. Further strategies to reduce retinoic acid differentiation during HPC expansion is required to expand ALDH hi cells without the loss of islet regenerative functions.

Immunohistochemical expression of mast cell tryptase in giant cell fibroma and inflammatory fibrous hyperplasia of the oral mucosa.

PubMed

Santos, Pedro Paulo de Andrade; Nonaka, Cassiano Francisco Weege; Pinto, Leão Pereira; de Souza, Lélia Batista

2011-03-01

This study analysed the immunohistochemical expression of mast cell tryptase in giant cell fibromas (GCFs). In addition, the possible interaction of mast cells with stellate giant cells, as well as their role in fibrosis and tumour progression, was investigated. For this purpose, the results were compared with cases of inflammatory fibrous hyperplasia (IFH) and normal oral mucosa. Thirty cases of GCF, 30 cases of IFH and 10 normal mucosa specimens used as control were selected. Immunoreactivity of mast cells to the anti-tryptase antibody was analysed quantitatively in the lining epithelium and in connective tissue. In the epithelial component (p=0.250) and connective tissue (p=0.001), the largest mean number of mast cells was observed in IFHs and the smallest mean number in GCFs. In connective tissue, the mean percentage of degranulated mast cells was higher in GCFs than in IFHs and normal mucosa specimens (p<0.001). Analysis of the percentage of degranulated mast cells in areas of fibrosis and at the periphery of blood vessels also showed a larger mean number in GCFs compared to IFHs and normal mucosa specimens (p<0.001). The percent interaction between mast cells and stellate giant cells in GCFs was 59.62%. In conclusion, although mast cells were less numerous in GCFs, the cells exhibited a significant interaction with stellate giant cells present in these tumours. In addition, the results suggest the involvement of mast cells in the induction of fibrosis and modulation of endothelial cell function in GCFs. Crown Copyright © 2010. Published by Elsevier Ltd. All rights reserved.

Laminin-111 improves skeletal muscle stem cell quantity and function following eccentric exercise.

PubMed

Zou, Kai; De Lisio, Michael; Huntsman, Heather D; Pincu, Yair; Mahmassani, Ziad; Miller, Matthew; Olatunbosun, Dami; Jensen, Tor; Boppart, Marni D

2014-09-01

Laminin-111 (α1, β1, γ1; LM-111) is an important component of the extracellular matrix that is required for formation of skeletal muscle during embryonic development. Recent studies suggest that LM-111 supplementation can enhance satellite cell proliferation and muscle function in mouse models of muscular dystrophy. The purpose of this study was to determine the extent to which LM-111 can alter satellite and nonsatellite stem cell quantity following eccentric exercise-induced damage in young adult, healthy mice. One week following injection of LM-111 or saline, mice either remained sedentary or were subjected to a single bout of downhill running (EX). While one muscle was preserved for evaluation of satellite cell number, the other muscle was processed for isolation of mesenchymal stem cells (MSCs; Sca-1+CD45-) via FACS at 24 hours postexercise. Satellite cell number was approximately twofold higher in LM-111/EX compared with all other groups (p<.05), and the number of satellite cells expressing the proliferation marker Ki67 was 50% to threefold higher in LM-111/EX compared with all other groups (p<.05). LM-111 also increased the quantity of embryonic myosin heavy chain-positive (eMHC+) fibers in young mice after eccentric exercise (p<.05). Although MSC percentage and number were not altered, MSC proinflammatory gene expression was decreased, and hepatocyte growth factor gene expression was increased in the presence of LM-111 (p<.05). Together, these data suggest that LM-111 supplementation provides a viable solution for increasing skeletal muscle stem cell number and/or function, ultimately allowing for improvements in the regenerative response to eccentric exercise. ©AlphaMed Press.

Biological evaluation of partially stabilized zirconia added HA/HDPE composites with osteoblast and fibroblast cell lines.

PubMed

Yari Sadi, Amir; Shokrgozar, Mohammad Ali; Homaeigohar, Seyed Shahin; Khavandi, Alireza

2008-06-01

In the present study, the biocompatibility of partially stabilized zirconia (PSZ) added hydroxyapatite (HA)--high density polyethylene (HDPE) composites was evaluated by proliferation and cell attachment assays on two osteoblast cell lines (G-292, Saos-2) and a type of fibroblast cell isolated from bone tissue namely HBF in different time intervals. Cell-material interactions on the surface of the composites were observed by scanning electron microscopy (SEM). The effect of composites on the behavior of osteoblast and fibroblast cells was compared with those of HDPE and Tissue Culture Poly Styrene (TPS) (as negative control) samples. Results showed that the composite samples supported a higher proliferation rate of osteoblast cells in the presence of composite samples as compared to the HDPE and TPS samples after 3, 7 and 14 days of incubation period. It was showed that an equal or in some cases an even higher proliferation rate of G-292 and Saos-2 osteoblast cells on composite samples in compare to negative controls in culture period (P < 0.05). The number of adhered cells on the composite samples was equal and in some cases higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods (P < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were seen to be undergoing cell division.

Cytotoxicity of zinc oxide (ZnO) nanoparticles is influenced by cell density and culture format.

PubMed

Heng, Boon Chin; Zhao, Xinxin; Xiong, Sijing; Ng, Kee Woei; Boey, Freddy Yin-Chiang; Loo, Joachim Say-Chye

2011-06-01

A parameter that has often been overlooked in cytotoxicity assays is the density and confluency of mammalian cell monolayers utilized for toxicology screening. Hence, this study investigated how different cell seeding densities influenced their response to cytotoxic challenge with ZnO nanoparticles. Utilizing the same volume (1 ml per well) and concentration range (5-40 μg/ml) of ZnO nanoparticles, contradictory results were observed with higher-density cell monolayers (BEAS-2B cells) obtained either by increasing the number of seeded cells per well (50,000 vs. 200,000 cells per well of 12-well plate) or by seeding the same numbers of cells (50,000) within a smaller surface area (12-well vs. 48-well plate, 4.8 vs. 1.2 cm(2), respectively). Further experiments demonstrated that the data may be skewed by inconsistency in the mass/number of nanoparticles per unit area of culture surface, as well as by inconsistent nanoparticle to cell ratio. To keep these parameters constant, the same number of cells (50,000 per well) were seeded on 12-well plates, but with the cells being seeded at the edge of the well for the experimental group (by tilting the plate) to form a dense confluent monolayer, as opposed to a sparse monolayer for the control group seeded in the conventional manner. Utilizing such an experimental set-up for the comparative evaluation of four different cell lines (BEAS-2B, L-929, CRL-2922 and C2C12), it was observed that the high cell density monolayer was consistently more resistant to the cytotoxic effects of ZnO nanoparticles compared to the sparse monolayer for all four different cell types, with the greatest differences being observed above a ZnO concentration of 10 μg/ml. Hence, the results of this study demonstrate the need for the standardization of cell culture protocols utilized for toxicology screening of nanoparticles, with respect to cell density and mass/number of nanoparticles per unit area of culture surface.

Evaluation of umbilical cord blood CD34+ hematopoietic stem cells expansion with inhibition of TGF-β receptorII in co-culture with bone marrow mesenchymal stromal cells.

PubMed

Sohrabi Akhkand, Saman; Amirizadeh, Naser; Nikougoftar, Mahin; Alizadeh, Javad; Zaker, Farhad; Sarveazad, Arash; Joghataei, Mohammad Taghi; Faramarzi, Mahmood

2016-08-01

Umbilical cord blood (UCB) is an important source of hematopoietic stem cells (HSCs). However, low number of HSCs in UCB has been an obstacle for adult hematopoietic stem cell transplantation. The expansion of HSCs in culture is one approach to overcome this problem. In this study, we investigated the expansion of UCB-HSCs by using human bone marrow mesenchymal stromal cells (MSCs) as feeder layer as well as inhibiting the TGF-β signaling pathway through reduction of TGF-βRII expression. CD34(+) cells were isolated from UCB and transfected by SiRNA targeting TGF-βRII mRNA. CD34(+) cells were expanded in four culture media with different conditions, including 1) expansion of CD34(+) cells in serum free medium containing growth factors, 2) expansion of cells transfected with SiRNA targeting TGF-βRII in medium containing growth factors, 3) expansion of cells in presence of growth factors and MSCs, 4) expansion of cells transfected with SiRNA targeting TGF-βRII on MSCs feeder layer in medium containing growth factors. These culture conditions were evaluated for the number of total nucleated cells (TNCs), CD34 surface marker as well as using CFU assay on 8th day after culture. The fold increase in CD34(+) cells, TNCs, and colony numbers (71.8±6.9, 93.2±10.2 and 128±10, respectively) was observed to be highest in fourth culture medium compared to other culture conditions. The difference between number of cells in four culture media in 8th day compared to unexpanded cells (0day) before expansion was statistically significant (P<0.05). The results showed that transfection of CD34(+) cells with SiRNA targeting TGF-βRII and their co-culture with MSCs could considerably increase the number of progenitors. Therefore, this method could be useful for UCB-HSCs expansion. Copyright © 2016 Elsevier Ltd. All rights reserved.

Reprogramming of Adult Peripheral Blood Cells into Human Induced Pluripotent Stem Cells as a Safe and Accessible Source of Endothelial Cells.

PubMed

Simara, Pavel; Tesarova, Lenka; Rehakova, Daniela; Farkas, Simon; Salingova, Barbara; Kutalkova, Katerina; Vavreckova, Eva; Matula, Pavel; Matula, Petr; Veverkova, Lenka; Koutna, Irena

2018-01-01

New approaches in regenerative medicine and vasculogenesis have generated a demand for sufficient numbers of human endothelial cells (ECs). ECs and their progenitors reside on the interior surface of blood and lymphatic vessels or circulate in peripheral blood; however, their numbers are limited, and they are difficult to expand after isolation. Recent advances in human induced pluripotent stem cell (hiPSC) research have opened possible avenues to generate unlimited numbers of ECs from easily accessible cell sources, such as the peripheral blood. In this study, we reprogrammed peripheral blood mononuclear cells, human umbilical vein endothelial cells (HUVECs), and human saphenous vein endothelial cells (HSVECs) into hiPSCs and differentiated them into ECs. The phenotype profiles, functionality, and genome stability of all hiPSC-derived ECs were assessed and compared with HUVECs and HSVECs. hiPSC-derived ECs resembled their natural EC counterparts, as shown by the expression of the endothelial surface markers CD31 and CD144 and the results of the functional analysis. Higher expression of endothelial progenitor markers CD34 and kinase insert domain receptor (KDR) was measured in hiPSC-derived ECs. An analysis of phosphorylated histone H2AX (γH2AX) foci revealed that an increased number of DNA double-strand breaks upon reprogramming into pluripotent cells. However, differentiation into ECs restored a normal number of γH2AX foci. Our hiPSCs retained a normal karyotype, with the exception of the HSVEC-derived hiPSC line, which displayed mosaicism due to a gain of chromosome 1. Peripheral blood from adult donors is a suitable source for the unlimited production of patient-specific ECs through the hiPSC interstage. hiPSC-derived ECs are fully functional and comparable to natural ECs. The protocol is eligible for clinical applications in regenerative medicine, if the genomic stability of the pluripotent cell stage is closely monitored.

ROS-dependent Atg4 upregulation mediated autophagy plays an important role in Cd-induced proliferation and invasion in A549 cells.

PubMed

Lv, Wei; Sui, Linlin; Yan, Xiaona; Xie, Huaying; Jiang, Liping; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Kong, Ying; Cao, Jun

2018-01-05

Cadmium (Cd) is a toxic heavy metal that is widely used in industry and agriculture. In this study the role of autophagy in Cd-induced proliferation, migration and invasion was investigated in A549 cells. Exposure to Cd (2 μM) significantly increased reactive oxygen species (ROS) production, induced autophagy and enhanced cell growth, migration and invasion in A549 cells. Western blot analysis showed that the expression of autophagy-related proteins, LC3-II, Beclin-1 and Atg4 and invasion-related protein MMP-9 were upregulated in Cd-treated cells. N-acetyl cysteine (NAC) markedly prevented Cd-induced proliferation of A549 cells and the increasing protein level of LC3-II and Atg4. Blocking Atg4 expression by siRNA strongly reduced Beclin-1 and LC3-II protein expression and the number of autophagosome positive cells induced by Cd. Furthermore, Atg4 siRNA increased the number of cells at G0/G1 phase, reduced the number of S and G2/M phase cells, and inhibited Cd-induced cell growth significantly compared with that of Cd-treated Control siRNA cells. 3-MA pretreatment increased the percentage of G0/G1 phase cells, decreased S phase and G2/M phase percentage, and inhibited Cd-induced cell growth remarkably compared with that of only Cd-treated cells. Knocking down Atg4 reduced the number of cells that migrated and invaded through the Matrigel matrix significantly and led to a significant decrease of MMP-9 expression. In addition, in lung tissues of Cd-treated BALB/c mice, the increased expression of LC3-II, Beclin-1 and Atg4 were observed. Taken together, our results demonstrated that ROS-dependent Atg4-mediated autophagy plays an important role in Cd-induced cell growth, migration and invasion in A549 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

CXCL2 synthesized by oral squamous cell carcinoma is involved in cancer-associated bone destruction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Oue, Erika; Section of Maxillofacial Surgery, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University; Global Center of Excellence

Highlights: Black-Right-Pointing-Pointer Oral cancer cells synthesize CXCL2. Black-Right-Pointing-Pointer CXCL2 synthesized by oral cancer is involved in osteoclastogenesis. Black-Right-Pointing-Pointer CXCL2-neutralizing antibody inhibited osteoclastogenesis induced by oral cancer cells. Black-Right-Pointing-Pointer We first report the role of CXCL2 in cancer-associated bone destruction. -- Abstract: To explore the mechanism of bone destruction associated with oral cancer, we identified factors that stimulate osteoclastic bone resorption in oral squamous cell carcinoma. Two clonal cell lines, HSC3-C13 and HSC3-C17, were isolated from the maternal oral cancer cell line, HSC3. The conditioned medium from HSC3-C13 cells showed the highest induction of Rankl expression in the mouse stromal cellmore » lines ST2 and UAMS-32 as compared to that in maternal HSC3 cells and HSC3-C17 cells, which showed similar activity. The conditioned medium from HSC3-C13 cells significantly increased the number of osteoclasts in a co-culture with mouse bone marrow cells and UAMS-32 cells. Xenograft tumors generated from these clonal cell lines into the periosteal region of the parietal bone in athymic mice showed that HSC3-C13 cells caused extensive bone destruction and a significant increase in osteoclast numbers as compared to HSC3-C17 cells. Gene expression was compared between HSC3-C13 and HSC3-C17 cells by using microarray analysis, which showed that CXCL2 gene was highly expressed in HSC3-C13 cells as compared to HSC3-C17 cells. Immunohistochemical staining revealed the localization of CXCL2 in human oral squamous cell carcinomas. The increase in osteoclast numbers induced by the HSC3-C13-conditioned medium was dose-dependently inhibited by addition of anti-human CXCL2-neutralizing antibody in a co-culture system. Recombinant CXCL2 increased the expression of Rankl in UAMS-32 cells. These results indicate that CXCL2 is involved in bone destruction induced by oral cancer. This is the first report showing the role of CXCL2 in cancer-associated bone destruction.« less

The Rise of Allogeneic Natural Killer Cells As a Platform for Cancer Immunotherapy: Recent Innovations and Future Developments

PubMed Central

Veluchamy, John P.; Kok, Nina; van der Vliet, Hans J.; Verheul, Henk M. W.; de Gruijl, Tanja D.; Spanholtz, Jan

2017-01-01

Natural killer (NK) cells are critical immune effector cells in the fight against cancer. As NK cells in cancer patients are highly dysfunctional and reduced in number, adoptive transfer of large numbers of cytolytic NK cells and their potential to induce relevant antitumor responses are widely explored in cancer immunotherapy. Early studies from autologous NK cells have failed to demonstrate significant clinical benefit. In this review, the clinical benefits of adoptively transferred allogeneic NK cells in a transplant and non-transplant setting are compared and discussed in the context of relevant NK cell platforms that are being developed and optimized by various biotech industries with a special focus on augmenting NK cell functions. PMID:28620386

Diet-induced obesity, exogenous leptin-, and MADB106 tumor cell challenge affect tissue leukocyte distribution and serum levels of cytokines in F344 rats.

PubMed

Behrendt, Patrick; Buchenauer, Tobias; Horn, Rüdiger; Brabant, Georg; Jacobs, Roland; Bode, Felix; Stephan, Michael; Nave, Heike

2010-08-01

The adipocyte-derived catabolic protein leptin alters cell-mediated immunity and cytokine crosstalk. This may provide new insights into the altered immune response, seen in obese individuals. Therefore, we determined the tissue distribution of immune cells in diet-induced obese (dio) and normal weight F344 rats challenged with MADB106 tumor cells or leptin. Immune cell distribution in blood (by FACS analysis) and tissues (NK cells in spleen and liver, immunohistologically) as well as pro-inflammatory cytokines (IL-6, TNF-α; by flow cytometry) were investigated in 28 normal weight and 28 dio rats (n = 4-6/group). Pro-inflammatory cytokines were increased 3-fold for IL-6 and 7-fold for TNF-α in obese animals. Higher numbers of blood monocytes and NK cells were found in obese as compared to normal weight animals. In dio rats challenged with leptin and MADB106 tumor cells, monocyte numbers were decreased as compared to the obese control animals. Immunohistochemistry revealed an altered NK cell distribution in a compartment-, treatment-, and bodyweight-specific manner. In conclusion, our data reveal a distinct distribution pattern of monocytes and NK cells in dio rats as compared to normal weight littermates and an additional modulatory effect of a leptin- and MADB106 tumor cell challenge.

Impact of age and gender interaction on circulating endothelial progenitor cells in healthy subjects.

PubMed

Rousseau, Alexandra; Ayoubi, Fida; Deveaux, Christel; Charbit, Beny; Delmau, Catherine; Christin-Maitre, Sophie; Jaillon, Patrice; Uzan, Georges; Simon, Tabassome

2010-02-01

To assess the level of circulating endothelial progenitor cells (CEPC) in cycling women compared with men and menopausal women. Controlled clinical study. Healthy, nonsmoking volunteers. Twelve women, aged 18-40 years, with regular menstrual cycles, 12 menopausal women, and two groups of 12 age-matched men were recruited. Women did not receive any hormone therapy. Collection of 20 mL of peripheral blood. The number of CEPC, defined as (Lin-/7AAD-/CD34+/CD133+/KDR+) cells per 10(6) mononuclear cells (MNC), was measured by flow cytometry. The number of CEPC was significantly higher in cycling women than in age-matched men and menopausal women (26.5 per 10(6) MNC vs. 10.5 per 10(6) MNC vs. 10 per 10(6) MNC, respectively). The number of CEPC was similar in menopausal women, age-matched, and young men. The number of CEPC is influenced by an age-gender interaction. This phenomenon may explain in part the better vascular repair and relative cardiovascular protection in younger women as compared with age-matched men. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

Bach2 Controls Homeostasis of Eosinophils by Restricting the Type-2 Helper Function of T Cells.

PubMed

Sato, Yuki; Kato, Hiroki; Ebina-Shibuya, Risa; Itoh-Nakadai, Ari; Okuyama, Ryuhei; Igarashi, Kazuhiko

2017-03-01

Bach2 is a transcription factor which represses its target genes and plays important roles in the differentiation of B and T lymphoid cells. Bach2-deficient (KO) mice develop severe pulmonary alveolar proteinosis, which is associated with increased numbers of granulocytes and T cells. Bach2 is essential for the regulation of T cells, but its role in the regulation of granulocytes is not clear. Here, we observed increased numbers of eosinophils but not neutrophils in the bone marrow, spleen, peripheral blood, and bronchoalveolar lavage fluids of Bach2 KO mice compared with those of wild-type (WT) mice. Upon co-transplantation of the bone marrow cells from CD45.2 Bach2 KO and CD45.1/CD45.2 double-positive WT mice to irradiated WT CD45.1/CD45.2 mice, the reconstituted numbers of eosinophils were similar between Bach2 KO and WT cells. These results showed that the deficiency of Bach2 in eosinophils did not directly drive the differentiation of eosinophils. To investigate the effect of Bach2 KO CD4 + T cells upon eosinophils, we analyzed Rag2/Bach2-double deficient (dKO) mice which lack lymphocytes including CD4 + T cells. Rag2/Bach2 dKO mice did not show any increase in the numbers of eosinophils. Importantly, Bach2 KO mice showed an increase of interleukin-5 (Il-5) in the sera compared with WT mice. These results suggest that up-regulated functions of CD4 + T cells including secretion of Il-5 resulted in proliferation and/or migration to peripheral tissues of eosinophils in Bach2 KO mice. We propose that Bach2 controls homeostasis of eosinophils via restricting the production of Il-5 in CD4 + T cells.

Urinary and faecal N-methylhistamine concentrations do not serve as markers for mast cell activation or clinical disease activity in dogs with chronic enteropathies.

PubMed

Anfinsen, Kristin P; Berghoff, Nora; Priestnall, Simon L; Suchodolski, Jan S; Steiner, Jörg M; Allenspach, Karin

2014-12-21

This study sought to correlate faecal and urinary N-methylhistamine (NMH) concentrations with resting versus degranulated duodenal mast cell numbers in dogs with chronic enteropathies (CE), and investigate correlations between intestinal mast cell activation and clinical severity of disease as assessed by canine chronic enteropathy clinical activity index (CCECAI), and between urinary and faecal NMH concentrations, mast cell numbers, and histopathological scores. Twenty-eight dogs with CE were included. Duodenal biopsies were stained with haematoxylin and eosin (H&E), toluidine blue, and by immunohistochemical labelling for tryptase. Duodenal biopsies were assigned a histopathological severity score, and duodenal mast cell numbers were counted in five high-power fields after metachromatic and immunohistochemical staining. Faecal and urinary NMH concentrations were measured by gas chromatography-mass spectrometry. There was no correlation between the CCECAI and faecal or urinary NMH concentrations, mast cell numbers, or histopathological score - or between faecal or urinary NMH concentration and mast cell numbers. Post hoc analysis revealed a statistically significant difference in toluidine blue positive mast cells between two treatment groups (exclusion diet with/without metronidazole versus immunosuppression (IS)), with higher numbers among dogs not requiring IS. Faecal and urinary NMH concentrations and duodenal mast cell numbers were not useful indicators of severity of disease as assessed by the CCECAI or histological evaluation. The number of duodenal mast cells was higher in dogs that did not need IS, i.e. in dogs responding to an exclusion diet (with/without metronidazole), than in dogs requiring IS. Further studies comparing the role of mast cells in dogs with different forms of CE are needed.

Rapid acquisition of mean Raman spectra of eukaryotic cells for a robust single cell classification.

PubMed

Schie, Iwan W; Kiselev, Roman; Krafft, Christoph; Popp, Jürgen

2016-11-14

Raman spectroscopy has previously been used to identify eukaryotic and prokaryotic cells. While prokaryotic cells are small in size and can be assessed by a single Raman spectrum, the larger size of eukaryotic cells and their complex organization requires the acquisition of multiple Raman spectra to properly characterize them. A Raman spectrum from a diffraction-limited spot at an arbitrary location within a cell results in spectral variations that affect classification approaches. To probe whole cells with Raman imaging at high spatial resolution is time consuming, because a large number of Raman spectra need to be collected, resulting in low cell throughput and impairing statistical analysis due to low cell numbers. Here we propose a method to overcome the effects of cellular heterogeneity by acquiring integrated Raman spectra covering a large portion of a cell. The acquired spectrum represents the mean macromolecular composition of a cell with an exposure time that is comparable to acquisition of a single Raman spectrum. Data sets were collected from T lymphocyte Jurkat cells, and pancreatic cell lines Capan1 and MiaPaca2. Cell classification by support vector machines was compared for single spectra, spectra of images and integrated Raman spectra of cells. The integrated approach provides better and more stable prediction for individual cells, and in the current implementation, the mean macromolecular information of a cell can be acquired faster than with the acquisition of individual spectra from a comparable region. It is expected that this approach will have a major impact on the implementation of Raman based cell classification.

High-Yield Method for Dispersing Simian Kidneys for Cell Cultures

PubMed Central

de Oca, H. Montes; Probst, P.; Grubbs, R.

1971-01-01

A technique for dispersion of animal tissue cells is described. The proposed technique is based on the concomitant use of trypsin and disodium ethylenediamine tetraacetate (EDTA). The use of the two dispersing agents (trypsin and disodium EDTA) markedly enhances cell yield as compared with the standard cell dispersion methods. Moreover, significant reduction in the amount of time required for complete tissue dispersal, presence of a very low number of nonviable cells, less cell clumping, and more uniform monolayer formation upon cultivation compare favorably with the results usually obtained with the standard trypsinization technique. Images PMID:4993235

A replacement for islet equivalents with improved reliability and validity.

PubMed

Huang, Han-Hung; Ramachandran, Karthik; Stehno-Bittel, Lisa

2013-10-01

Islet equivalent (IE), the standard estimate of isolated islet volume, is an essential measure to determine the amount of transplanted islet tissue in the clinic and is used in research laboratories to normalize results, yet it is based on the false assumption that all islets are spherical. Here, we developed and tested a new easy-to-use method to quantify islet volume with greater accuracy. Isolated rat islets were dissociated into single cells, and the total cell number per islet was determined by using computer-assisted cytometry. Based on the cell number per islet, we created a regression model to convert islet diameter to cell number with a high R2 value (0.8) and good validity and reliability with the same model applicable to young and old rats and males or females. Conventional IE measurements overestimated the tissue volume of islets. To compare results obtained using IE or our new method, we compared Glut2 protein levels determined by Western Blot and proinsulin content via ELISA between small (diameter≤100 μm) and large (diameter≥200 μm) islets. When normalized by IE, large islets showed significantly lower Glut2 level and proinsulin content. However, when normalized by cell number, large and small islets had no difference in Glut2 levels, but large islets contained more proinsulin. In conclusion, normalizing islet volume by IE overestimated the tissue volume, which may lead to erroneous results. Normalizing by cell number is a more accurate method to quantify tissue amounts used in islet transplantation and research.

Oral squamous cell carcinoma in the background of oral submucous fibrosis is a distinct clinicopathological entity with better prognosis.

PubMed

Gadbail, Amol Ramchandra; Chaudhary, Minal; Gawande, Madhuri; Hande, Alka; Sarode, Sachin; Tekade, Satyajit Ashok; Korde, Sheetal; Zade, Prajakta; Bhowate, Rahul; Borle, Rajiv; Patil, Swati

2017-07-01

The aim of this study was to compare the clinicopathological features of oral squamous cell carcinoma in the background of oral submucous fibrosis (OSCC-OSMF) and oral squamous cell carcinoma (OSCC). A total of 217 cases of OSCC were retrieved from achieves for the analysis. OSCC-OSMF cases were segregated on the basis of history and clinicopathological parameters. The study included 217 patients of which 112 had OSCC and 105 OSCC-OSMF. OSCC-OSMFs were younger compared with OSCC. Overall oral cancer was noted predominantly in males compared to females. The number of OSCC-OSMF was more in clinical TNM stage I and stage II as compared to OSCC, whereas the number of OSCC was more in stage III and stage IV compared to OSCC-OSMF. Histological presentation of well-differentiated squamous cell carcinoma was significantly more in OSCC-OSMF compared to OSCC, whereas moderately differentiated squamous cell carcinoma was significantly more in OSCC compared to OSCC-OSMF. Regional lymph node metastasis was significantly higher in OSCC compared to OSCC-OSMF. Three-year disease-free survival rate was significantly higher in OSCC-OSMF compared to OSCC. The OSCC-OSMF was found to be a clinicopathologically distinct entity with a better grade of tumor differentiation, less incidence of nodal metastases, and early detection (early clinical TNM stage) compared to OSCC. All these factors probably contribute to a better prognosis and increased 3-year disease-free survival in OSCC-OSMF patients. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Peripheral natural killer cytotoxicity and CD56(pos)CD16(pos) cells increase during early pregnancy in women with a history of recurrent spontaneous abortion.

PubMed

Emmer, P M; Nelen, W L; Steegers, E A; Hendriks, J C; Veerhoek, M; Joosten, I

2000-05-01

For diagnostic purposes we assessed peripheral natural killer (NK) cell cytotoxicity and NK and T cell numbers to assess their putative predictive value in recurrent spontaneous abortion (RSA). A total of 43 women with subsequent pregnancy, 37 healthy controls and 39 women successfully partaking in an in-vitro fertilization (IVF) procedure, were included in the study. We show that before pregnancy, levels of NK cytotoxicity and numbers of both single CD56(pos) and double CD56(pos)CD16(pos) cells were similar between RSA women and controls. But notably, within the RSA group, NK cell numbers of <12% were strongly associated with a subsequent pregnancy carried to term. Supplementation of folic acid led to an increase of single CD56(pos) cells, but cytotoxic function appeared unaffected. The expression pattern of killer inhibitory receptors on CD56(pos) cells was not different between patients and controls. A longitudinal study revealed that, compared with controls, in RSA women higher numbers of double CD56(pos)CD16(pos) cells were present during early pregnancy, paralleled by an increase in cytotoxic NK cell reactivity. The single CD56(pos) population decreased in number. In conclusion, the analysis of peripheral NK cell characteristics appears a suitable diagnostic tool in RSA. Immunomodulation aimed at NK cell function appears a promising therapeutic measure.

Circulating natural killer T cells in patients with asthma.

PubMed

Ikegami, Yasuhiko; Yokoyama, Akihito; Haruta, Yoshinori; Hiyama, Keiko; Kohno, Nobuoki

2004-01-01

Recent studies suggest that therapies targeted at depletion or limiting of natural killer (NK) T cells may be a possible strategy for the treatment of asthma. In the present study, we measured the number of circulating V alpha24+ NKT cells in 32 asthmatic patients and compared these patients with 29 nonatopic healthy controls. We investigated the relationships between NKT cell number and clinical variables such as the number of eosinophils, the circulating level of IgE, and the severity of asthma. In addition, we also investigated the ability of NKT cells to proliferate in response to alpha-galactosyl ceramide (alpha-GalCer) in vitro. The V alpha24+ NKT cell counts of asthmatic patients were significantly lower than those of healthy controls. There were no significant differences observed in asthmatic patients among the subgroups in terms of atopic status and severity. There was no significant correlation between the number of NKT cells and clinical variables. The proliferative response to alpha-GalCer of the patients and controls was not significantly different, indicating no intrinsic proliferative defect of NKT cells in asthma. These results suggest that the number of circulating NKT cells was already decreased in patients with asthma. Further study, such as the evaluation of lung NKT cells, will be needed to determine the role of NKT cells in patients with asthma.

[Effect of electroacupuncture on differentiation and proliferation of hippocampal nerve stem cells in splenic asthenia pedo-rats].

PubMed

Zhuo, Yuan-yuan; Yang, Zhuo-xin; Wu, Jia-man

2011-10-01

To observe the effect of electroacupuncture (EA) on the differentiation and proliferation of nerve stem cells in the hippocampal dentate gyrus (DG) in splenic asthenia pedo-rats so as to study its central mechanism. A total of 72 SD male rats were randomly assigned to normal control group (n=24), model group (n=24) and EA group (n=24) which were further divided into 7 d, 14 d, 28 d and 49 d time-points (n=6). Splenic asthenia model was established by intraperitoneal injection of reserpine and gavage of Dahuang (Radix et Rhizoma Rhei) fluid. EA was applied to bilateral "Zusanli" (ST 36) and "Sanyinjiao" (SP 6) for 20 min, once daily for 7, 14, 28 and 49 days respectively. Brdu, Nestin, glial fibrillary acidic protein (GFAP), and neuron-specific enolase (NSE) expression in the DG of hippocampus were detected by immunohistochemistry double staining. Compared with the normal control group, the numbers of Brdu, Brdu/GFAP, Brdu/NSE Immunoreactive (IR) positive cells in the DG of hippocampus on day 7 and 14, and that of Brdu/Nestin IR-positive cells on day 7 were decreased considerably in the model group (P < 0.05). Compared to the model group, the numbers of hippocampal Brdu IR-positive cells at the 4 time-points, Brdu/Nestin and Brdu/GFAP on day 7, 14 and 49, and Brdu/NSE on day 7, 14 and 28 were increased significantly in the EA group (P < 0.05). No significant differences were found between the model and control groups in the numbers of hippocampal Brdu and Brdu/NSE IR-positive cells on day 28 and 49, in the number of Brdu/Nestin IR-positive cells on day 14 and 49, in the number of Brdu/GFAP IR-positive cells on day 28; and between the EA and model groups in the numbers of Brdu/Nestin and Brdu/GFAP IR-positive cells on day 28, and in the number of Brdu/NSE IR-positive cells on day 49 (P > 0.05). EA of ST 36 and SP 6 can effectively suppress splenic asthenia syndrome-induced decrease of the numbers of Brdu, Brdu/GFAP, Brdu/Nestin and Brdu/NSE IR-positive cells in the DG of hippocampus at the early stage in the splenic asthenia rats, which may contribute to its effect in improving splenic asthenia symptoms in clinic by promoting the proliferation and differentiation of some nerve stem cells in the hippocampus.

Increasing Stem Cell Dose Promotes Posttransplant Immune Reconstitution.

PubMed

Xu, Ning; Shen, Sylvie; Dolnikov, Alla

2017-04-01

Umbilical cord blood (UCB) transplantation can provide a successful therapeutic option for patients that have no suitable related donor. UCB transplantation is often limited by the relatively small hematopoietic stem cell (HSC) numbers in UCB especially for adult recipients. Early neutrophil and platelet engraftment correlates with the stem cell numbers in UCB transplant. Compared to other HSC sources, immune reconstitution following UCB transplant is slower and complicated by increased frequency of opportunistic infections. The effect of HSC numbers in UCB transplant on immune reconstitution was not thoroughly examined. Using immunocompromised mice transplanted with purified UCB CD34+ stem cells, we have demonstrated that increasing the numbers of CD34+ cells in the transplant promotes hematopoietic and immune reconstitution. At early stages posttransplant, high stem cell dose generated relatively more B cells, while lower dose generated more myeloid and T cells. Thus, the size of the stem cell graft appears to modulate the differentiation potential of infused stem cells. In addition, increasing stem cell dose in the transplant improved CD8+ T cell development and delayed late memory T cell skewing in expense of naive T cells highlighting the importance of HSC dose to maintain the pool of naive T cells able to develop strong immune responses. Transplantation of ex vivo expanded CD34+ cells did not promote, but rather delayed immune reconstitution suggesting the loss of primitive lymphoid precursor cells during ex vivo expansion.

Homozygously deleted gene DACH1 regulates tumor-initiating activity of glioma cells

PubMed Central

Watanabe, Akira; Ogiwara, Hideki; Ehata, Shogo; Mukasa, Akitake; Ishikawa, Shumpei; Maeda, Daichi; Ueki, Keisuke; Ino, Yasushi; Todo, Tomoki; Yamada, Yasuhiro; Fukayama, Masashi; Saito, Nobuhito; Miyazono, Kohei; Aburatani, Hiroyuki

2011-01-01

Loss or reduction in function of tumor suppressor genes contributes to tumorigenesis. Here, by allelic DNA copy number analysis using single-nucleotide polymorphism genotyping array and mass spectrometry, we report homozygous deletion in glioblastoma multiformes at chromosome 13q21, where DACH1 gene is located. We found decreased cell proliferation of a series of glioma cell lines by forced expression of DACH1. We then generated U87TR-Da glioma cells, where DACH1 expression could be activated by exposure of the cells to doxycycline. Both ex vivo cellular proliferation and in vivo growth of s.c. transplanted tumors in mice are reduced in U87TR-Da cells with DACH1 expression (U87-DACH1-high), compared with DACH1-nonexpressing U87TR-Da cells (U87-DACH1-low). U87-DACH1-low cells form spheroids with CD133 and Nestin expression in serum-free medium but U87-DACH1-high cells do not. Compared with spheroid-forming U87-DACH1-low cells, adherent U87-DACH1-high cells display lower tumorigenicity, indicating DACH1 decreases the number of tumor-initiating cells. Gene expression analysis and chromatin immunoprecipitation assay reveal that fibroblast growth factor 2 (FGF2/bFGF) is transcriptionally repressed by DACH1, especially in cells cultured in serum-free medium. Exogenous bFGF rescues spheroid-forming activity and tumorigenicity of the U87-DACH1-high cells, suggesting that loss of DACH1 increases the number of tumor-initiating cells through transcriptional activation of bFGF. These results illustrate that DACH1 is a distinctive tumor suppressor, which does not only suppress growth of tumor cells but also regulates bFGF-mediated tumor-initiating activity of glioma cells. PMID:21750150

Ratio of inner cell mass and trophoblastic cells in demi- and intact pig embryos.

PubMed

Tao, T; Reichelt, B; Niemann, H

1995-07-01

Pig morulae, early blastocysts and blastocysts were microsurgically bisected to produce zona-free demi-embryos or remained nonbisected with or without zona pellucida, and the presence of inner cell mass cells was determined using a differential fluorochrome staining technique. After 24 h of in vitro culture, all demi-embryos were classified into three categories, based on morphological criteria: 1, excellent; 2, fair; and 3, degenerated. The average number of total cells and inner cell mass cells in intact embryos cultured without zona pellucida for 24 h was higher (P < 0.05) than that for those with zona pellucida in morulae and early blastocysts. The percentage of demi-embryos without inner cell mass cells in these different morphological categories was 18.7%, 22.2% and 29.8% for morulae, respectively; 3.8%, 16.7% and 30.8% for early blastocysts, respectively; and 3.7%, 32.0% and 36.4% for blastocysts, respectively. The percentage of demi-embryos without inner cell mass cells was lower (P < 0.01) in demi-embryos classified in category 1 compared with category 3 in early blastocysts and in category 1 compared with categories 2 and 3 in blastocysts. Significant differences in the total number of cells and the number of inner cell mass cells were apparent among the three morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts. The ratio of total cells to inner cell mass cells was similar among intact pig embryos and the different morphological categories of demi-embryos derived from morulae, early blastocysts and blastocysts, with the exception of that between demi-blastocysts of category 1 and the other groups.(ABSTRACT TRUNCATED AT 250 WORDS)

The changes of stage distribution of seminiferous epithelium cycle and its correlations with Leydig cell stereological parameters in aging men.

PubMed

Huang, Rui; Zhu, Wei-Jie; Li, Jing; Gu, Yi-Qun

2014-12-01

To evaluate the changes of stage distribution of seminiferous epithelium cycle and its correlations with Leydig cell stereological parameters in aging men. Point counting method was used to analyze the stereological parameters of Leydig cells. The stage number of seminiferous epithelium cycle was calculated in the same testicular tissue samples which were used for Leydig cell stereological analysis. The aging group had shown more severe pathological changes as well as higher pathologic scores than the young group. Compared with the control group, the volume density (VV) and surface density (NA) of Leydig cells in the aging group were increased significantly. The stage number of seminiferous epithelium cycle in the aging group was decreased coincidently compared to the young group. Leydig cell Vv in the young group has a positive relationship with stages I, II, III, V and VI of seminiferous epithelium cycle, and Leydig cell NA and numerical density (NV) were positively related to stage IV. However, only the correlation between NV and stage II was found in the aging group. The stage number of seminiferous epithelium cycle was decreased in aging testes. Changes in the stage distribution in aging testes were related to the Leydig cell stereological parameters which presented as a sign of morphological changes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Comparison of mesenchymal stem cells obtained by suspended culture of synovium from patients with rheumatoid arthritis and osteoarthritis.

PubMed

Kohno, Yuji; Mizuno, Mitsuru; Ozeki, Nobutake; Katano, Hisako; Otabe, Koji; Koga, Hideyuki; Matsumoto, Mikio; Kaneko, Haruka; Takazawa, Yuji; Sekiya, Ichiro

2018-03-09

Mobilization of mesenchymal stem cells (MSCs) from the synovium was revealed using a "suspended synovium culture model" of osteoarthritis (OA). The pathology of rheumatoid arthritis (RA) differs from that of OA. We investigated whether mobilization of MSCs from the synovium also occurred in RA, and we compared the properties of synovial MSCs collected from suspended synovium culture models of RA and OA. Human synovium was harvested during total knee arthroplasty from the knee joints of patients with RA (n = 8) and OA (n = 6). The synovium was suspended in a bottle containing culture medium and a culture dish at the bottom. Cells were harvested from the dish and analyzed. No significant difference was observed between RA and OA in the harvested cell numbers per g of synovium. However, the variation in the number of cells harvested from each donor was greater for RA than for OA. The harvested cells were multipotent and no difference was observed in the cartilage pellet weight between RA and OA. The surface epitopes of the cells in RA and OA were similar to those of MSCs. Mobilization of MSCs from the synovium was demonstrated using a suspended synovium culture model for RA. The harvested cell numbers, chondrogenic potentials, and surface epitope profiles were comparable between the RA and OA models.

A role for autophagic protein beclin 1 early in lymphocyte development.

PubMed

Arsov, Ivica; Adebayo, Adeola; Kucerova-Levisohn, Martina; Haye, Joanna; MacNeil, Margaret; Papavasiliou, F Nina; Yue, Zhenyu; Ortiz, Benjamin D

2011-02-15

Autophagy is a highly regulated and evolutionarily conserved process of cellular self-digestion. Recent evidence suggests that this process plays an important role in regulating T cell homeostasis. In this study, we used Rag1(-/-) (recombination activating gene 1(-/-)) blastocyst complementation and in vitro embryonic stem cell differentiation to address the role of Beclin 1, one of the key autophagic proteins, in lymphocyte development. Beclin 1-deficient Rag1(-/-) chimeras displayed a dramatic reduction in thymic cellularity compared with control mice. Using embryonic stem cell differentiation in vitro, we found that the inability to maintain normal thymic cellularity is likely caused by impaired maintenance of thymocyte progenitors. Interestingly, despite drastically reduced thymocyte numbers, the peripheral T cell compartment of Beclin 1-deficient Rag1(-/-) chimeras is largely normal. Peripheral T cells displayed normal in vitro proliferation despite significantly reduced numbers of autophagosomes. In addition, these chimeras had greatly reduced numbers of early B cells in the bone marrow compared with controls. However, the peripheral B cell compartment was not dramatically impacted by Beclin 1 deficiency. Collectively, our results suggest that Beclin 1 is required for maintenance of undifferentiated/early lymphocyte progenitor populations. In contrast, Beclin 1 is largely dispensable for the initial generation and function of the peripheral T and B cell compartments. This indicates that normal lymphocyte development involves Beclin 1-dependent, early-stage and distinct, Beclin 1-independent, late-stage processes.

Exercise in Adulthood after Irradiation of the Juvenile Brain Ameliorates Long-Term Depletion of Oligodendroglial Cells.

PubMed

Bull, Cecilia; Cooper, Christiana; Lindahl, Veronica; Fitting, Sylvia; Persson, Anders I; Grandér, Rita; Alborn, Ann-Marie; Björk-Eriksson, Thomas; Kuhn, H Georg; Blomgren, Klas

2017-10-01

Cranial radiation severely affects brain health and function, including glial cell production and myelination. Recent studies indicate that voluntary exercise has beneficial effects on oligodendrogenesis and myelination. Here, we hypothesized that voluntary running would increase oligodendrocyte numbers in the corpus callosum after irradiation of the juvenile mouse brain. The brains of C57Bl/6J male mice were 6 Gy irradiated on postnatal day 9 during the main gliogenic developmental phase, resulting in a loss of oligodendrocyte precursor cells. Upon adulthood, the mice were injected with bromodeoxyuridine and allowed to exercise on a running wheel for four weeks. Cell proliferation and survival, Ascl1 + oligodendrocyte precursor and Olig2 + oligodendrocyte cell numbers as well as CC1 + mature oligodendrocytes were quantified using immunohistology. Radiation induced a reduction in the number of Olig2 + oligodendrocytes by nearly 50% without affecting production or survival of new Olig2 + cells. Ascl1 + cells earlier in the oligodendroglial cell lineage were also profoundly affected, with numbers reduced by half. By three weeks of age, Olig2 + cell numbers had not recovered, and this was paralleled by a volumetric loss in the corpus callosum. The deficiency of Olig2 + oligodendrocytes persisted into adulthood. Additionally, the depletion of Ascl1 + progenitor cells was irreversible, and was even more pronounced at 12 weeks postirradiation compared to day 2 postirradiation. Furthermore, the overall number of CC1 + mature oligodendrocytes decreased by 28%. The depletion of Olig2 + cells in irradiated animals was reversed by 4 weeks of voluntary exercise. Moreover, voluntary exercise also increased the number of Ascl1 + progenitor cells in irradiated animals. Taken together, these results demonstrate that exercise in adulthood significantly ameliorates the profound and long-lasting effects of moderate exposure to immature oligodendrocytes during postnatal development.

The role of colonic mast cells and myenteric plexitis in patients with diverticular disease.

PubMed

Bassotti, Gabrio; Villanacci, Vincenzo; Nascimbeni, Riccardo; Antonelli, Elisabetta; Cadei, Moris; Manenti, Stefania; Lorenzi, Luisa; Titi, Amin; Salerni, Bruno

2013-02-01

Gut mast cells represent an important cell population involved in intestinal homeostasis and inflammatory processes. However, their possible role has not to date been investigated in colonic diverticular disease. This study aims to evaluate colonic mast cells in patients undergoing surgery for diverticular disease. Surgical resection samples from 27 patients undergoing surgery for diverticular disease (12 emergency procedures for severe disease and 15 elective procedures) were evaluated. The number of mast cells was assessed in the various layers by means of a specific antibody (tryptase) and compared with those evaluated in ten controls. In patients with mast cells degranulation, double immunohistochemistry, also assessing nerve fibres, was carried out. In addition, the presence of myenteric plexitis was sought. Compared with controls, the number of mast cells in diverticular patients was significantly increased, both as an overall figure and in the various layers of the large bowel. In patients in whom mast cells degranulation was present, these were always closed to nerve fibres. No differences were found between the two subgroups of patients with respect to the number and distribution of mast cells; however, all patients undergoing emergency surgery (but none of those undergoing elective procedures) had myenteric plexitis, represented by lymphocytic infiltration in 67 % and eosinophilic infiltration in 33 % of cases. Patients with diverticular disease display an increase of mast cells in the large bowel. The presence of myenteric plexitis in those with complicated, severe disease, suggest that this could represent a histopathologic marker of more aggressive disease.

Human Mesenchymal Stem Cell Spheroids in Fibrin Hydrogels Exhibit Improved Cell Survival and Potential for Bone Healing

PubMed Central

Murphy, Kaitlin C.; Fang, Sophia Y.; Leach, J. Kent

2014-01-01

Mesenchymal stem cells (MSC) have great therapeutic potential for the repair of nonhealing bone defects due to their proliferative capacity, multilineage potential, trophic factor secretion, and lack of immunogenicity. However, a major barrier to the translation of cell-based therapies into clinical practice is ensuring their survival and function upon implantation into the defect site. We hypothesized that forming MSC into more physiologic 3-dimensional spheroids, rather than employing dissociated cells from 2-dimensional monolayer culture, would enhance their survival when exposed to a harsh microenvironment while maintaining their osteogenic potential. MSC spheroids were formed using the hanging drop method with increasing cell numbers. Compared to larger spheroids, the smallest spheroids which contained 15,000 cells exhibited increased metabolic activity, reduced apoptosis, and the most uniform distribution of proliferating cells. Spheroids were then entrapped in fibrin gels and cultured in serum-free media and 1% oxygen. Compared to identical numbers of dissociated MSC in fibrin gels, spheroids exhibited significantly reduced apoptosis and secreted up to 100-fold more VEGF. We also observed that fibrin gels containing spheroids and those containing an equivalent number of dissociated cells exhibited similar expression levels of early and late markers of osteogenic differentiation. These data demonstrate that MSC spheroids exhibit greater resistance to apoptosis and enhanced proangiogenic potential, while maintaining similar osteogenic potential to dissociated MSC entrapped in a clinically relevant biomaterial, supporting the use of MSC spheroids in cell-based approaches to bone repair. PMID:24781147

Ecabet sodium alleviates neomycin-induced hair cell damage.

PubMed

Rah, Yoon Chan; Choi, June; Yoo, Myung Hoon; Yum, Gunhwee; Park, Saemi; Oh, Kyoung Ho; Lee, Seung Hoon; Kwon, Soon Young; Cho, Seung Hyun; Kim, Suhyun; Park, Hae-Chul

2015-12-01

Ecabet sodium (ES) is currently applied to some clinical gastrointestinal disease primarily by the inhibition of the ROS production. In this study, the protective role of ES was evaluated against the neomycin-induced hair cell loss using zebrafish experimental animal model. Zebrafish larvae (5-7 dpf), were treated with each of the following concentrations of ES: 5, 10, 20, 40, and 80 μg/mL for 1 h, followed by 125 μM neomycin for 1h. The positive control group was established by 125 μM neomycin-only treatment (1h) and the negative control group with no additional chemicals was also established. Hair cells inside four neuromasts ( SO1, SO2, O1, OC1) were assessed using fluorescence microscopy (n = 10). Hair cell survival was calculated as the mean number of viable hair cells for each group. Apoptosis and mitochondrial damage were investigated using special staining (TUNEL and DASPEI assay, respectively), and compared among groups. Ultrastructural changes were evaluated using scanning electron microscopy. Pre-treatment group with ES increased the mean number of viable hair cells as a dose-dependent manner achieving almost same number of viable hair cells with 40 μM/ml ES treatment (12.98 ± 2.59 cells) comparing to that of the negative control group (14.15 ± 1.39 cells, p = 0.72) and significantly more number of viable hair cells than that of the positive control group (7.45 ± 0.91 cells, p < 0.01). The production of reactive oxygen species significantly increased by 183% with 125 μM neomycin treatment than the negative control group and significantly decreased down to 105% with the pre-treatment with 40 μM/ml ES (n = 40, p = 0.04). A significantly less number of TUNEL-positive cells (reflecting apoptosis, p < 0.01) and a significantly increased DASPEI reactivity (reflecting viable mitochondria, p < 0.01) were observed in 40 μM/ml ES pre-treatment group. Our data suggest that ES could protect against neomycin-induced hair cell loss possibly by reducing apoptosis, mitochondrial damages, and the ROS generation. Copyright © 2015 Elsevier Inc. All rights reserved.

Do bacterial cell numbers follow a theoretical Poisson distribution? Comparison of experimentally obtained numbers of single cells with random number generation via computer simulation.

PubMed

Koyama, Kento; Hokunan, Hidekazu; Hasegawa, Mayumi; Kawamura, Shuso; Koseki, Shigenobu

2016-12-01

We investigated a bacterial sample preparation procedure for single-cell studies. In the present study, we examined whether single bacterial cells obtained via 10-fold dilution followed a theoretical Poisson distribution. Four serotypes of Salmonella enterica, three serotypes of enterohaemorrhagic Escherichia coli and one serotype of Listeria monocytogenes were used as sample bacteria. An inoculum of each serotype was prepared via a 10-fold dilution series to obtain bacterial cell counts with mean values of one or two. To determine whether the experimentally obtained bacterial cell counts follow a theoretical Poisson distribution, a likelihood ratio test between the experimentally obtained cell counts and Poisson distribution which parameter estimated by maximum likelihood estimation (MLE) was conducted. The bacterial cell counts of each serotype sufficiently followed a Poisson distribution. Furthermore, to examine the validity of the parameters of Poisson distribution from experimentally obtained bacterial cell counts, we compared these with the parameters of a Poisson distribution that were estimated using random number generation via computer simulation. The Poisson distribution parameters experimentally obtained from bacterial cell counts were within the range of the parameters estimated using a computer simulation. These results demonstrate that the bacterial cell counts of each serotype obtained via 10-fold dilution followed a Poisson distribution. The fact that the frequency of bacterial cell counts follows a Poisson distribution at low number would be applied to some single-cell studies with a few bacterial cells. In particular, the procedure presented in this study enables us to develop an inactivation model at the single-cell level that can estimate the variability of survival bacterial numbers during the bacterial death process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Maternal hCG concentrations in early IVF pregnancies: associations with number of cells in the Day 2 embryo and oocytes retrieved.

PubMed

Tanbo, T G; Eskild, A

2015-12-01

Do number of cells in the transferred cleavage stage embryo and number of oocytes retrieved for IVF influence maternal hCG concentrations in early pregnancies? Compared with transfer of a 2-cell embryo, transfer of a 4-cell embryo results in higher hCG concentrations on Day 12 after transfer, and more than 20 oocytes retrieved were associated with low hCG concentrations. Maternal hCG concentration in very early pregnancy varies considerably among women, but is likely to be an indicator of time since implantation of the embryo into the endometrium, in addition to number and function of trophoblast cells. We followed 1047 pregnancies after IVF/ICSI from oocyte retrieval until Day 12 after embryo transfer. Women were recruited in Norway during the years 2005-2013. Successful pregnancies after transfer of one single embryo that had been cultured for 2 days were included. Maternal hCG was quantified on Day 12 after embryo transfer by chemiluminescence immunoassay, which measures intact hCG and the free β-hCG chain. Information on a successful pregnancy, defined as birth after >16 weeks, was obtained by linkage to the Medical Birth Registry of Norway. Transfer of a 4-cell embryo resulted in higher maternal hCG concentrations compared with transfer of a 2-cell embryo (134.8 versus 87.8 IU/l, P < 0.05). A high number of oocytes retrieved (>20) was associated with low hCG concentrations (P < 0.05). The factors studied explain a limited part of the total variation of hCG concentrations in early pregnancy. Although embryo transfer was performed at the same time after fertilization, we do not know the exact time of implantation. A further limitation to our study is that the number of pregnancies after transfer of a 2-cell embryo was small (27 cases). Number of cells in the transferred embryo and number of oocytes retrieved may influence the conditions and timing for embryo implantation in different ways and thereby influence maternal hCG concentrations. Such knowledge may be important for interpretation of hCG concentrations in early pregnancy. © The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Effect of topical rebamipide on goblet cells in the lid wiper of human conjunctiva

PubMed Central

Kase, Satoru; Shinohara, Toshiya; Kase, Manabu; Ishida, Susumu

2017-01-01

It has been demonstrated that topical administration of rebamipide, which is an antiulcer agent, increases the mucin level of the tear film and ameliorates ocular surface conditions such as lid wiper epitheliopathy. The aim of the present study was to analyze the changes in goblet cell number, cell proliferation, and epidermal growth factor receptor (EGFR) induced by topical rebamipide addition to the lid wiper of humans. A total of 30 eyelid tissue samples were obtained during involutional entropion surgeries, fixed in paraformaldehyde, embedded in paraffin and divided into two groups: Rebamipide or non-rebamipide. The tissues in the rebamipide group were obtained from patients who had a medical history of topical rebamipide use prior to surgery. The number of goblet cells was counted under light microscopy. A total of 22 eyelid tissue samples were further examined using immunohistochemistry with anti-Ki-67 and anti-EGFR antibodies to evaluate cell proliferation and EGFR expression, respectively. Histologically, the lid wiper and palpebral conjunctiva were clearly identified in the tissues. The number of goblet cells was significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0367). There was no significant difference in lid wiper cell proliferation between the rebamipide and non-rebamipide groups. Immunohistochemistry revealed that EGFR levels in the lid wiper epithelial cells were significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0237). These results suggest that topical rebamipide application increases the number of goblet cells in the lid wiper, which in turn upregulates the expression of EGFR. These findings may be clinically relevant and provide a therapeutic basis for the treatment of ocular disease such as dry eye and lid wiper epitheliopathy. PMID:28587435

Effect of topical rebamipide on goblet cells in the lid wiper of human conjunctiva.

PubMed

Kase, Satoru; Shinohara, Toshiya; Kase, Manabu; Ishida, Susumu

2017-06-01

It has been demonstrated that topical administration of rebamipide, which is an antiulcer agent, increases the mucin level of the tear film and ameliorates ocular surface conditions such as lid wiper epitheliopathy. The aim of the present study was to analyze the changes in goblet cell number, cell proliferation, and epidermal growth factor receptor (EGFR) induced by topical rebamipide addition to the lid wiper of humans. A total of 30 eyelid tissue samples were obtained during involutional entropion surgeries, fixed in paraformaldehyde, embedded in paraffin and divided into two groups: Rebamipide or non-rebamipide. The tissues in the rebamipide group were obtained from patients who had a medical history of topical rebamipide use prior to surgery. The number of goblet cells was counted under light microscopy. A total of 22 eyelid tissue samples were further examined using immunohistochemistry with anti-Ki-67 and anti-EGFR antibodies to evaluate cell proliferation and EGFR expression, respectively. Histologically, the lid wiper and palpebral conjunctiva were clearly identified in the tissues. The number of goblet cells was significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0367). There was no significant difference in lid wiper cell proliferation between the rebamipide and non-rebamipide groups. Immunohistochemistry revealed that EGFR levels in the lid wiper epithelial cells were significantly higher in the rebamipide group compared with the non-rebamipide group (P=0.0237). These results suggest that topical rebamipide application increases the number of goblet cells in the lid wiper, which in turn upregulates the expression of EGFR. These findings may be clinically relevant and provide a therapeutic basis for the treatment of ocular disease such as dry eye and lid wiper epitheliopathy.

Cysteinyl Leukotriene 1 Receptor Expression Associated With Bronchial Inflammation in Severe Exacerbations of COPD

PubMed Central

Zhu, Jie; Bandi, Venkata; Qiu, Shengyang; Figueroa, David J.; Evans, Jilly F.; Barnes, Neil; Guntupalli, Kay K.

2012-01-01

Background: Cysteinyl leukotriene 1 (CysLT1) receptor expression is known to be increased in the airway mucosa of patients with asthma, especially during exacerbations; however, nothing is known of its expression in COPD. Methods: We applied immunohistochemistry and in situ hybridization to endobronchial biopsies to determine inflammatory cell CysLT1 receptor protein and mRNA expression in the following: (1) 15 nonsmoker control subjects (NSC), (2) 16 smokers with moderate to severe COPD in its stable phase (S-COPD), and (3) 15 smokers with COPD hospitalized for a severe exacerbation (SE-COPD). Results: The total number of bronchial mucosal inflammatory cells (CD45+) and those expressing CysLT1 receptor protein were significantly greater in SE-COPD (CysLT1 receptor protein: median [range] = 139 [31-634]) as compared with S-COPD (32 [6-114]) or NSC (16 [4-66]) (P < .001 for both). CysLT1 receptor gene expression showed similar differences. A greater proportion of CD451 cells expressed CysLT1 receptor protein in SE-COPD (median [range] = 22% [8-81]) compared with S-COPD (10% [4-32]) (P < .03) or NSC (7% [1-19]) (P < .002). In SE-COPD, the relative frequencies of CysLT1 receptor-expressing cells were as follows: tryptase1 mast cells > CD681 monocytes/macrophage > neutrophils > CD201 B lymphocytes = EG21 eosinophils. Moreover, there were positive correlations between the numbers of cells expressing CysLT1 receptor protein and the numbers of CD451 cells (r = 0.78; P < .003) and tryptase1 mast cells (r = 0.62; P < .02). Conclusions: Bronchial mucosal CysLT1 receptor-positive inflammatory cells are present in the bronchial mucosa in COPD in greatest number in those experiencing a severe exacerbation. PMID:22871757

Clinical significance of detecting circulating tumor cells in colorectal cancer using subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH)

PubMed Central

Shen, Zhen; Jing, Yan; Lu, Haibo; Li, Heng; Yang, Xiaoye; Cui, Xiangbin; Li, Yuqing; Lou, Zheng; Liu, Peng; Zhang, Cun; Zhang, Wei

2017-01-01

Circulating tumor cells (CTC) are useful in early detection of colorectal cancer. This study described a newly developed platform, integrated subtraction enrichment and immunostaining-fluorescence in situ hybridization (SE-iFISH), to assess CTCs in colorectal cancer. CTCs were detected by SE-iFISH in 40 of 44 preoperative colorectal cancer patients, and yielded a sensitivity of 90.9%, which was significantly higher than CellSearch system (90.9% vs. 43.2%, P=0.033). No significant association was found between tumor stage, survival and preoperative CTC number. CTCs were detected in 10 colorectal cancer patients one week after surgery; seven patients with decreased CTC numbers (compared with preoperative CTC number) were free of recurrence; whereas two of the three patients with increased CTC numbers had tumor recurrence. Moreover, CTCs were detected in 34 colorectal cancer patients three months after surgery; patients with CTC<2 at three months after surgery had significantly longer Progression Free Survival than those with CTC>=2 (P=0.019); patients with decreased CTC number (compared with preoperative CTC number) had significantly longer Progression Free Survival than those with increased CTC number (P=0.003). In conclusion, CTCs could be detected in various stages of colorectal cancer using SE-iFISH. Dynamic monitoring of CTC numbers could predict recurrence and prognosis. PMID:28423493

Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing

PubMed Central

Suo, Biao; Yang, Hua; Wang, Yuexia; Lv, Haipeng; Li, Zhen; Xu, Chao; Ai, Zhilu

2018-01-01

When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food. PMID:29774015

Nanostructured Polyaniline Coating on ITO Glass Promotes the Neurite Outgrowth of PC 12 Cells by Electrical Stimulation.

PubMed

Wang, Liping; Huang, Qianwei; Wang, Jin-Ye

2015-11-10

A conducting polymer polyaniline (PANI) with nanostructure was synthesized on indium tin oxide (ITO) glass. The effect of electrical stimulation on the proliferation and the length of neurites of PC 12 cells was investigated. The dynamic protein adsorption on PANI and ITO surfaces in a cell culture medium was also compared with and without electrical stimulation. The adsorbed proteins were characterized using SDS-PAGE. A PANI coating on ITO surface was shown with 30-50 nm spherical nanostructure. The number of PC 12 cells was significantly greater on the PANI/ITO surface than on ITO and plate surfaces after cell seeding for 24 and 36 h. This result confirmed that the PANI coating is nontoxic to PC 12 cells. The electrical stimulation for 1, 2, and 4 h significantly enhanced the cell numbers for both PANI and ITO conducting surfaces. Moreover, the application of electrical stimulation also improved the neurite outgrowth of PC 12 cells, and the number of PC 12 cells with longer neurite lengths increased obviously under electrical stimulation for the PANI surface. From the mechanism, the adsorption of DMEM proteins was found to be enhanced by electrical stimulation for both PANI/ITO and ITO surfaces. A new band 2 (around 37 kDa) was observed from the collected adsorbed proteins when PC 12 cells were cultured on these surfaces, and culturing PC 12 cells also seemed to increase the amount of band 1 (around 90 kDa). When immersing PANI/ITO and ITO surfaces in a DMEM medium without a cell culture, the number of band 3 (around 70 kDa) and band 4 (around 45 kDa) proteins decreased compared to that of PC 12 cell cultured surfaces. These results are valuable for the design and improvement of the material performance for neural regeneration.

Genetic profiling of putative breast cancer stem cells from malignant pleural effusions.

PubMed

Tiran, Verena; Stanzer, Stefanie; Heitzer, Ellen; Meilinger, Michael; Rossmann, Christopher; Lax, Sigurd; Tsybrovskyy, Oleksiy; Dandachi, Nadia; Balic, Marija

2017-01-01

A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of rare cancer (stem) cells.

Platelet-rich plasma differs according to preparation method and human variability.

PubMed

Mazzocca, Augustus D; McCarthy, Mary Beth R; Chowaniec, David M; Cote, Mark P; Romeo, Anthony A; Bradley, James P; Arciero, Robert A; Beitzel, Knut

2012-02-15

Varying concentrations of blood components in platelet-rich plasma preparations may contribute to the variable results seen in recently published clinical studies. The purposes of this investigation were (1) to quantify the level of platelets, growth factors, red blood cells, and white blood cells in so-called one-step (clinically used commercial devices) and two-step separation systems and (2) to determine the influence of three separate blood draws on the resulting components of platelet-rich plasma. Three different platelet-rich plasma (PRP) separation methods (on blood samples from eight subjects with a mean age [and standard deviation] of 31.6 ± 10.9 years) were used: two single-spin processes (PRPLP and PRPHP) and a double-spin process (PRPDS) were evaluated for concentrations of platelets, red and white blood cells, and growth factors. Additionally, the effect of three repetitive blood draws on platelet-rich plasma components was evaluated. The content and concentrations of platelets, white blood cells, and growth factors for each method of separation differed significantly. All separation techniques resulted in a significant increase in platelet concentration compared with native blood. Platelet and white blood-cell concentrations of the PRPHP procedure were significantly higher than platelet and white blood-cell concentrations produced by the so-called single-step PRPLP and the so-called two-step PRPDS procedures, although significant differences between PRPLP and PRPDS were not observed. Comparing the results of the three blood draws with regard to the reliability of platelet number and cell counts, wide variations of intra-individual numbers were observed. Single-step procedures are capable of producing sufficient amounts of platelets for clinical usage. Within the evaluated procedures, platelet numbers and numbers of white blood cells differ significantly. The intra-individual results of platelet-rich plasma separations showed wide variations in platelet and cell numbers as well as levels of growth factors regardless of separation method.

The induction of menadione stress tolerance in the marine microalga, Dunaliella viridis, through cold pretreatment and modulation of the ascorbate and glutathione pools.

PubMed

Madadkar Haghjou, Maryam; Colville, Louise; Smirnoff, Nicholas

2014-11-01

The effect of cold pretreatment on menadione tolerance was investigated in the cells of the marine microalga, Dunaliella viridis. In addition, the involvement of ascorbate and glutathione in the response to menadione stress was tested by treating cell suspensions with l-galactono-1,4-lactone, an ascorbate precursor, and buthionine sulfoximine, an inhibitor of glutathione synthesis. Menadione was highly toxic to non cold-pretreated cells, and caused a large decrease in cell number. Cold pretreatment alleviated menadione toxicity and cold pretreated cells accumulated lower levels of reactive oxygen species, and had enhanced antioxidant capacity due to increased levels of β-carotene, reduced ascorbate and total glutathione compared to non cold-pretreated cells. Cold pretreatment also altered the response to l-galactono-1,4-lactone and buthionine sulfoximine treatments. Combined l-galactono-1,4-lactone and menadione treatment was lethal in non-cold pretreated cells, but in cold-pretreated cells it had a positive effect on cell numbers compared to menadione alone. Overall, exposure of Dunaliella cells to cold stress enhanced tolerance to subsequent oxidative stress induced by menadione. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Pathologic Changes of the Peripheral Vestibular System Secondary to Chronic Otitis Media.

PubMed

da Costa Monsanto, Rafael; Erdil, Mehmet; Pauna, Henrique F; Kwon, Geeyoun; Schachern, Patricia A; Tsuprun, Vladimir; Paparella, Michael M; Cureoglu, Sebahattin

2016-09-01

To evaluate the histopathologic changes of dark, transitional, and hair cells of the vestibular system in human temporal bones from patients with chronic otitis media. Comparative human temporal bone study. Otopathology laboratory. To compare the density of vestibular dark, transitional, and hair cells in temporal bones with and without chronic otitis media, we used differential interference contrast microscopy. In the chronic otitis media group (as compared with the age-matched control group), the density of type I and type II hair cells was significantly decreased in the lateral semicircular canal, saccule, and utricle (P < .05). The density of type I cells was also significantly decreased in the chronic otitis media group in the posterior semicircular canal (P = .005), but that of type II cells was not (P = .168). The mean number of dark cells was significantly decreased in the chronic otitis media group in the lateral semicircular canal (P = .014) and in the posterior semicircular canal (P = .002). We observed no statistically significant difference in the density of transitional cells between the 2 groups (P > .1). The findings of our study suggest that the decrease in the number of vestibular sensory cells and dark cells could be the cause of the clinical symptoms of imbalance of some patients with chronic otitis media. © American Academy of Otolaryngology—Head and Neck Surgery Foundation 2016.

Toxicity of copper oxide nanoparticles in lung epithelial cells exposed at the air-liquid interface compared with in vivo assessment.

PubMed

Jing, Xuefang; Park, Jae Hong; Peters, Thomas M; Thorne, Peter S

2015-04-01

The toxicity of spark-generated copper oxide nanoparticles (CuONPs) was evaluated in human bronchial epithelial cells (HBEC) and lung adenocarcinoma cells (A549 cells) using an in vitro air-liquid interface (ALI) exposure system. Dose-response results were compared to in vivo inhalation and instillation studies of CuONPs. Cells were exposed to filtered, particle-free clean air (controls) or spark-generated CuONPs. The number median diameter, geometric standard deviation and total number concentration of CuONPs were 9.2 nm, 1.48 and 2.27×10(7)particles/cm(3), respectively. Outcome measures included cell viability, cytotoxicity, oxidative stress and proinflammatory chemokine production. Exposure to clean air (2 or 4h) did not induce toxicity in HBEC or A549 cells. Compared with controls, CuONP exposures significantly reduced cell viability, increased lactate dehydrogenase (LDH) release and elevated levels of reactive oxygen species (ROS) and IL-8 in a dose-dependent manner. A549 cells were significantly more susceptible to CuONP effects than HBEC. Antioxidant treatment reduced CuONP-induced cytotoxicity. When dose was expressed per area of exposed epithelium there was good agreement of toxicity measures with murine in vivo studies. This demonstrates that in vitro ALI studies can provide meaningful data on nanotoxicity of metal oxides. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of Hematopoietic Stem Cell Mobilization Rates with Early Plerixafor Administration for Adult Stem Cell Transplantation.

PubMed

Stover, Jessica T; Shaw, J Ryan; Kuchibhatla, Maragatha; Horwitz, Mitchell E; Engemann, Ashley M

2017-08-01

The addition of plerixafor to high-dose colony-stimulating growth factor has been shown to improve stem cell mobilization rates in autologous transplant patients with multiple myeloma and non-Hodgkin lymphoma. This study evaluates the change in administration time of plerixafor to determine if cell mobilization rates are similar between the US Food and Drug Administration-approved administration time of 11 hours before apheresis and an earlier administration time of 16 hours before apheresis. Medical records of patients age ≥ 18 years undergoing autologous stem cell transplantation requiring the use of plerixafor after at least 4 days of granulocyte colony-stimulating factor therapy to complete stem cell mobilization from January 1, 2010 through September 30, 2014 were retrospectively reviewed. The primary outcome was CD34 + cell mobilization success rates when plerixafor was administered 11 ± 2 hours (standard administration group) compared with 16 ± 2 hours before cell apheresis (early administration group), as defined as collection of  ≥2 × 10 6 CD34 + cells/kg. Secondary outcomes included the number of plerixafor therapy days required to collect a total of ≥2 × 10 6 CD34 + cells/kg, the number of apheresis cycles required to achieve ≥2 × 10 6 CD34 + cells/kg, the median CD34 + cells/kg collected in each apheresis session, and the rates of reported adverse events that occurred in the standard administration time group compared with the early administration time group. Of the 197 patients included, 114 patients received plerixafor 11 ± 2 hours before apheresis and 83 patients received plerixafor 16 hours ± 2 hours before apheresis. Ninety-four percent of patients in the early administration group achieved successful stem cell mobilization compared with 81.6% in the standard administration group (P = .0111). The median number of plerixafor days to reach the collection goal of  ≥2 × 10 6 CD34 + cells/kg was 1 day for each group (P = .323), and the median number of apheresis days to reach the collection goal was 2 days for the standard administration group compared with 1 day for the early administration group (P = .0156). Most adverse events were similar between the 2 groups except for fever, which occurred in 4.8% of the patients in the early administration group and none of the patients in the standard administration group. This study demonstrates plerixafor effectively mobilizes peripheral blood stem cells when given at an early administration time of 16 hours before apheresis compared with standard administration of 11 hours before apheresis. However, further prospective studies could strengthen these results. Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Cultured smooth muscle cells of the human vesical sphincter are more sensitive to histamine than are detrusor smooth muscle cells.

PubMed

Neuhaus, Jochen; Oberbach, Andreas; Schwalenberg, Thilo; Stolzenburg, Jens-Uwe

2006-05-01

To compare histamine receptor expression in cultured smooth muscle cells from the human detrusor and internal sphincter using receptor-specific agonists. Smooth muscle cells from the bladder dome and internal sphincter were cultured from 5 male patients undergoing cystectomy for bladder cancer therapy. Calcium transients in cells stimulated with carbachol, histamine, histamine receptor 1 (H1R)-specific heptanecarboxamide (HTMT), dimaprit (H2R), and R-(alpha)-methylhistamine (H3R) were measured by calcium imaging. Histamine receptor proteins were detected by Western blot analysis and immunocytochemistry. H1R, H2R, and H3R expression was found in tissue and cultured cells. Carbachol stimulated equal numbers of detrusor and sphincter cells (60% and 51%, respectively). Histamine stimulated significantly more cells than carbachol in detrusor (100%) and sphincter (99.34%) cells. Calcium responses to carbachol in detrusor and sphincter cells were comparable and did not differ from those to histamine in detrusor cells. However, histamine and specific agonists stimulated more sphincter cells than did carbachol (P <0.001), and the calcium increase was greater in sphincter cells than in detrusor cells. Single cell analysis revealed comparable H2R responses in detrusor and sphincter cells, but H1R and H3R-mediated calcium reactions were significantly greater in sphincter cells. Histamine very effectively induces calcium release in smooth muscle cells. In sphincter cells, histamine is even more effective than carbachol regarding the number of reacting cells and the intracellular calcium increase. Some of the variability in the outcome of antihistaminic interstitial cystitis therapies might be caused by the ineffectiveness of the chosen antihistaminic or unintentional weakening of sphincteric function.

Treadmill exercise after social isolation increases the levels of NGF, BDNF, and synapsin I to induce survival of neurons in the hippocampus, and improves depression-like behavior

PubMed Central

Hong, Young-Pyo; Lee, Hyo-Chul; Kim, Hyun-Tae

2015-01-01

[Purpose] We investigated the effects of 8 weeks of treadmill exercise on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and synapsin I protein expression and on the number of 5-bromo-2'-deoxyuridine-5'-mono-phosphate (BrdU)-positive cells in the dentate gyrus of the hippocampus in socially isolated rats. Additionally, we examined the effects of exercise on the number of serotonin (5-HT)- and tryptophan hydroxylase (TPH)-positive cells in the raphe nuclei and on depression behaviors induced by social isolation. [Methods] Forty male Sprague-Dawley rats were divided into four groups: (1) group housing and control group (GCG, n = 10); (2) group housing and exercise group (GEG, n = 10); (3) isolated housing and control group (ICG, n = 10); and (4) isolated housing and exercise group (IEG, n = 10). After 1 week of housing under the normal condition of 3 animals per cage, rats were socially isolated via transfer to individual cages for 8 weeks. Rats were then subjected to treadmill exercise for 5 days per week for 8 weeks during which time the speed of the treadmill was gradually increased. [Results] Compared to the GCG, levels of NGF, BDNF, and synapsin I were significantly decreased in the ICG and significantly increased in the IEG (p < 0.001 respectively). Significantly more BrdU-positive cells in the GEG were present as compared to the GCG and ICG, and more BrdU-positive cells were found in the IEG as compared to the ICG (p < 0.001). 5-HT-positive cells in the GEG were significantly increased compared to the GCG and ICG, and more of these cells were found in the IEG as compared to the ICG (p < 0.01). TPH-positive cells in the GEG were significantly increased compared to those in the GCG and ICG (p < 0.05). In the forced swim test, immobility time was significantly increased in the ICG and significantly decreased in the IEG as compared to the ICG (p < 0.01). [Conclusion] These results showed that regular treadmill exercise following social isolation not only increased the levels of NGF, BDNF, and synapsin I to induce survival of neurons in the hippocampus but also improved depression by increasing the number of serotonergic cells in the raphe nuclei. PMID:25960950

Effect of high pressure homogenization on aqueous phase solvent extraction of lipids from Nannochloris Oculata microalgae

DOE PAGES

Samarasinghe, Nalin; Fernando, Sandun; Faulkner, William B.

2012-12-01

The ability to extract lipids from high-moisture Nannochloris Oculata algal biomass disrupted with high pressure homogenization was investigated. During the first phase, the effect of high pressure homogenization (system pressure and number of passes) on disrupting aqueous algae (of different concentrations and degree of stress) was investigated. Secondly, the effect of degree of cell wall disruption on the amount of lipids extracted with three solvents, namely: hexane, dichloromethane and chloroform, were compared. Studies reveled that high pressure homogenization is effective on cell disruption while the amount of system pressure being the most significant factor affecting the degree of cell breakage.more » Although the number of passes had some impact, the level of disruption seemed to level-off after a certain number of passes. The study revealed that slightly polar solvents (such as chloroform and dichloromethane) performed better in aqueous-phase lipid extractions as compared to hexane. Also, it was revealed that it was not necessary to disrupt the algal cells completely to achieve appreciable levels of lipid yields. In fact, conditions that exerted only 20% of the cells to completely disrupt, allowed sufficient damage to liberate most of the lipids contained in the remainder of the cells.« less

Identification of IL11RA and MELK amplification in gastric cancer by comprehensive genomic profiling of gastric cancer cell lines

PubMed Central

Calcagno, Danielle Queiroz; Takeno, Sylvia Santomi; Gigek, Carolina Oliveira; Leal, Mariana Ferreira; Wisnieski, Fernanda; Chen, Elizabeth Suchi; Araújo, Taíssa Maíra Thomaz; Lima, Eleonidas Moura; Melaragno, Maria Isabel; Demachki, Samia; Assumpção, Paulo Pimentel; Burbano, Rommel Rodriguez; Smith, Marília Cardoso

2016-01-01

AIM To identify common copy number alterations on gastric cancer cell lines. METHODS Four gastric cancer cell lines (ACP02, ACP03, AGP01 and PG100) underwent chromosomal comparative genome hybridization and array comparative genome hybridization. We also confirmed the results by fluorescence in situ hybridization analysis using the bacterial artificial chromosome clone and quantitative real time PCR analysis. RESULTS The amplification of 9p13.3 was detected in all cell lines by both methodologies. An increase in the copy number of 9p13.3 was also confirmed by fluorescence in situ hybridization analysis. Moreover, the interleukin 11 receptor alpha (IL11RA) and maternal embryonic leucine zipper kinase (MELK) genes, which are present in the 9p13.3 amplicon, revealed gains of the MELK gene in all the cell lines studied. Additionally, a gain in the copy number of IL11RA and MELK was observed in 19.1% (13/68) and 55.9% (38/68) of primary gastric adenocarcinoma samples, respectively. CONCLUSION The characterization of a small gain region at 9p13.3 in gastric cancer cell lines and primary gastric adenocarcinoma samples has revealed MELK as a candidate target gene that is possibly related to the development of gastric cancer. PMID:27920471

Effect of high pressure homogenization on aqueous phase solvent extraction of lipids from Nannochloris Oculata microalgae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Samarasinghe, Nalin; Fernando, Sandun; Faulkner, William B.

The ability to extract lipids from high-moisture Nannochloris Oculata algal biomass disrupted with high pressure homogenization was investigated. During the first phase, the effect of high pressure homogenization (system pressure and number of passes) on disrupting aqueous algae (of different concentrations and degree of stress) was investigated. Secondly, the effect of degree of cell wall disruption on the amount of lipids extracted with three solvents, namely: hexane, dichloromethane and chloroform, were compared. Studies reveled that high pressure homogenization is effective on cell disruption while the amount of system pressure being the most significant factor affecting the degree of cell breakage.more » Although the number of passes had some impact, the level of disruption seemed to level-off after a certain number of passes. The study revealed that slightly polar solvents (such as chloroform and dichloromethane) performed better in aqueous-phase lipid extractions as compared to hexane. Also, it was revealed that it was not necessary to disrupt the algal cells completely to achieve appreciable levels of lipid yields. In fact, conditions that exerted only 20% of the cells to completely disrupt, allowed sufficient damage to liberate most of the lipids contained in the remainder of the cells.« less

Neuroprotective Effect of Exogenous Melatonin on Dopaminergic Neurons of the Substantia Nigra in Ovariectomized Rats

PubMed Central

Mehraein, Fereshteh; Talebi, Reza; Jameie, Behnamedin; Joghataie, Mohammad Taghi; Madjd, Zahra

2011-01-01

Background: Melatonin has receptors in substantia nigra pars compacta (SNc) and regulates development of dopaminergic (DA) neurons. This study was undertaken to determine ability of melatonin to protect SNc dopaminergic neuron loss induced by estrogen deficiency in ovariectomized rats. Methods: Female rats were randomized into four groups of seven each: control, ethanol sham, ovariectomy (ovx) and ovx with melatonin (ovx + m). In ovx, ovaries were removed. Ovx + m group was intraperitoneally injected with melatonin for 10 days, while the ethanol sham group received only ethanol. All rats were perfused with 4% paraformaldehyde, midbrains removed, fixed and paraffin embedded, then processed for Nissl and tyrosine hydroxylase staining (IHC). Ten sections of SNc in Nissl and IHC staining were analyzed in each animal, Nissl stained and tyrosine hydroxylase (TH) immunoreactive cells were counted in five experimental groups randomly. Data was analyzed using SPSS by ANOVA and t-test. Differences were considered significant for P<0.05. Results: There was less cell number in ovx compared to control and ethanol sham groups significantly (P<0.001). The ovx + m group had more cells than the ovx group in the SNc significantly (P<0.001). Furthermore, there was significant decrease of TH positive cell number in the ovx group compared to control and ethanol sham groups (P<0.05). The number of TH immunoreactive cells was higher in ovx + m compared to the ovx group (P<0.05). Conclusion: These findings can be compared with human and used in clinical application for prevention of DA neuron death of SNc after ovariectomy. PMID:21725499

Effects of chemotherapy on immune responses in dogs with cancer.

PubMed

Walter, Claudia U; Biller, Barbara J; Lana, Susan E; Bachand, Annette M; Dow, Steven W

2006-01-01

Chemotherapy is assumed to be immunosuppressive; yet to the authors' knowledge, the effects of common chemotherapy protocols on adaptive immune responses in dogs with cancer have not been fully evaluated. Therefore, a study was conducted to evaluate the effects of 2 common chemotherapy protocols on T- and B-cell numbers and humoral immune responses to de novo vaccination in dogs with cancer. Twenty-one dogs with cancer (12 with lymphoma, 9 with osteosarcoma) were enrolled in a prospective study to assess effects of doxorubicin versus multi-drug chemotherapy on adaptive immunity. Numbers of circulating T and B cells were assessed by flow cytometry, and antibody responses to de novo vaccination were assessed before, during, and after chemotherapy. The T- and B-cell numbers before treatment also were compared with those of healthy, age-matched, control dogs. Prior to treatment, dogs with cancer had significantly fewer (P < .05) CD4+ T cells and CD8+ T cells than did healthy dogs. Doxorubicin treatment did not cause a significant decrease in T- or B-cell numbers, whereas treatment with combination chemotherapy caused a significant and persistent decrease in B-cell numbers. Antibody titers after vaccination were not significantly different between control and chemotherapy-treated dogs. These findings suggest that chemotherapy may have less impact on T-cell numbers and ability to mount antibody responses in dogs with cancer than was previously anticipated, though dogs with lymphoma or osteosarcoma appear to be relatively T-cell deficient before initiation of chemotherapy.

Reduced response to Epstein–Barr virus antigens by T-cells in systemic lupus erythematosus patients

PubMed Central

Draborg, Anette Holck; Jacobsen, Søren; Westergaard, Marie; Mortensen, Shila; Larsen, Janni Lisander; Houen, Gunnar; Duus, Karen

2014-01-01

Objective Epstein–Barr virus (EBV) has for long been associated with systemic lupus erythematosus (SLE). In this study, we investigated the levels of latent and lytic antigen EBV-specific T-cells and antibodies in SLE patients. Methods T cells were analyzed by flow cytometry and antibodies were analyzed by enzyme-linked immunosorbent assay. Results SLE patients showed a significantly reduced number of activated (CD69) T-cells upon ex vivo stimulation with EBV nuclear antigen (EBNA) 1 or EBV early antigen diffuse (EBV-EA/D) in whole blood samples compared with healthy controls. Also, a reduced number of T-cells from SLE patients were found to produce interferon-γ upon stimulation with these antigens. Importantly, responses to a superantigen were normal in SLE patients. Compared with healthy controls, SLE patients had fewer EBV-specific T-cells but higher titres of antibodies against EBV. Furthermore, an inverse correlation was revealed between the number of lytic antigen EBV-specific T-cells and disease activity of the SLE patients, with high-activity SLE patients having fewer T-cells than low-activity SLE patients. Conclusions These results indicate a limited or a defective EBV-specific T-cell response in SLE patients, which may suggest poor control of EBV infection in SLE with an immune reaction shift towards a humoral response in an attempt to control viral reactivation. A role for decreased control of EBV as a contributing agent in the development or exacerbation of SLE is proposed. PMID:25396062

Distemper virus encephalitis exerts detrimental effects on hippocampal neurogenesis.

PubMed

von Rüden, E-L; Avemary, J; Zellinger, C; Algermissen, D; Bock, P; Beineke, A; Baumgärtner, W; Stein, V M; Tipold, A; Potschka, H

2012-08-01

Despite knowledge about the impact of brain inflammation on hippocampal neurogenesis, data on the influence of virus encephalitis on dentate granule cell neurogenesis are so far limited. Canine distemper is considered an interesting model of virus encephalitis, which can be associated with a chronic progressing disease course and can cause symptomatic seizures. To determine the impact of canine distemper virus (CDV) infection on hippocampal neurogenesis, we compared post-mortem tissue from dogs with infection with and without seizures, from epileptic dogs with non-viral aetiology and from dogs without central nervous system diseases. The majority of animals with infection and with epilepsy of non-viral aetiology exhibited neuronal progenitor numbers below the age average in controls. Virus infection with and without seizures significantly decreased the mean number of neuronal progenitor cells by 43% and 76% as compared to age-matched controls. Ki-67 labelling demonstrated that hippocampal cell proliferation was neither affected by infection nor by epilepsy of non-viral aetiology. Analysis of CDV infection in cells expressing caspase-3, doublecortin or Ki-67 indicated that infection of neuronal progenitor cells is extremely rare and suggests that infection might damage non-differentiated progenitor cells, hamper neuronal differentiation and promote glial differentiation. A high inter-individual variance in the number of lectin-reactive microglial cells was evident in dogs with distemper infection. Statistical analyses did not reveal a correlation between the number of lectin-reactive microglia cells and neuronal progenitor cells. Our data demonstrate that virus encephalitis with and without seizures can exert detrimental effects on hippocampal neurogenesis, which might contribute to long-term consequences of the disease. The lack of a significant impact of distemper virus on Ki-67-labelled cells indicates that the infection affected neuronal differentiation and survival of newborn cells rather than hippocampal cell proliferation. © 2011 The Authors. Neuropathology and Applied Neurobiology © 2011 British Neuropathological Society.

Oestrus synchronization treatment induces histomorphological changes on the uterine tube epithelium of the gilt.

PubMed

Juárez-Mosqueda, M L; Anzaldúa Arce, S R; Palma Lara, I; García Dalmán, C; Cornejo Cortés, M A; Córdova Izquierdo, A; Villaseñor Gaona, H; Trujillo Ortega, M E

2015-12-01

The aim of this study was to determine the histomorphological changes that occurred in response to two treatments for oestrus synchronization in three different regions of the gilt's uterine tubes epithelium: the ampulla (AMP), ampulla-isthmic junction (AIJ) and isthmus (IST). Nine prepuberal gilts were divided into three groups (n = 3): (1) eCG 400 IU and hCG 200 IU (eCG/hCG), (2) progesterone agonist (P4) and (3) control group. The number of secretory cells (stained with periodic acid-Schiff reaction or PAS-positive cells) decreased in the AMP in the P4 treated group when compared to the control group, whereas, no difference was observed in the number of PAS-negative cells in the AMP of the three groups. A significant decrease in the number of PAS-positive cells was observed in the AIJ and IST of the P4 treated group when compared to the eCG/hCG and control groups. An increase in the number of PAS-negative cells was observed in the AIJ and IST in the P4 treated group. The epithelium height in the AMP and AIJ was increased in the eCG/hCG group when compared to the control and P4 groups. In this last group, we observed a reduced height compared with the other two groups for the AIJ. In the IST, there were no significant changes in the epithelium height of the control or the other two groups (eCG/hCG and P4). The epithelial cells of the P4 treated group had the least amount of cytoplasmic granules and the lowest intensity of PAS staining in the AMP, AIJ and IST. Animals treated with eCG/hCG showed an intermediate number of cytoplasmic granules and intensity in all regions evaluated. These data show that P4 treatment for synchronization induces a significant (P < 0.001) decrease of PAS-positive cells and staining intensity of cytoplasmic granules in the different regions studied and an increased number of PAS-negative cells in the AIJ and IST epithelium. Moreover, eCG/hCG treatment increased the height of the epithelium in the AMP and AIJ, while in this last region, the P4 treatment decreased the epithelium height. These results show that synchronization treatments with P4 and in a smaller proportion with eCG/hCG can modify the amount of PAS-positive and PAS-negative cells, and the epithelium height. This has influence in the secretory activity of the epithelium and possibly alters the fluid microenvironment of the gilt's uterine tube. The biological impact of regional variations in the epithelial cells of the gilt's uterine tube needs further investigation to understand the implications that the reproductive processes can have in the uterine tube. © 2014 Blackwell Verlag GmbH.

Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis.

PubMed

Kalmárová, M; Smirnov, E; Kovácik, L; Popov, A; Raska, I

2008-01-01

It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.

Positioning of the NOR-Bearing Chromosomes in Relation to Nucleoli in Daughter Cells after Mitosis

PubMed Central

KALMÁROVÁ, M.; SMIRNOV, E.; KOVÁČIK, L.; POPOV, A.; RAŠKA, I.

2008-01-01

Summary It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis. PMID:18597585

Human mesenchymal stem cells promote CD34+ hematopoietic stem cell proliferation with preserved red blood cell differentiation capacity.

PubMed

Lau, Show Xuan; Leong, Yin Yee; Ng, Wai Hoe; Ng, Albert Wee Po; Ismail, Ida Shazrina; Yusoff, Narazah Mohd; Ramasamy, Rajesh; Tan, Jun Jie

2017-06-01

Studies showed that co-transplantation of mesenchymal stem cells (MSCs) and cord blood-derived CD34 + hematopoietic stem cells (HSCs) offered greater therapeutic effects but little is known regarding the effects of human Wharton's jelly derived MSCs on HSC expansion and red blood cell (RBC) generation in vitro. This study aimed to investigate the effects of MSCs on HSC expansion and differentiation. HSCs were co-cultured with MSCs or with 10% MSCs-derived conditioned medium, with HSCs cultured under standard medium served as a control. Cell expansion rates, number of mononuclear cell post-expansion and number of enucleated cells post-differentiation were evaluated. HSCs showed superior proliferation in the presence of MSC with mean expansion rate of 3.5 × 10 8  ± 1.8 × 10 7 after day 7 compared to the conditioned medium and the control group (8.9 × 10 7  ± 1.1 × 10 8 and 7.0 × 10 7  ± 3.3 × 10 6 respectively, P < 0.001). Although no significant differences in RBC differentiation were observed between groups at passage IV, the number of enucleated cell was greater compared to earlier passages, indicating successful RBC differentiation. Cord blood-derived CD34 + HSCs can be greatly expanded by co-culturing with MSCs without affecting the RBC differentiation capability, suggesting the importance of direct MSC-HSCs contact in HSC expansion and RBC differentiation. © 2017 International Federation for Cell Biology.

Airway responsiveness to mannitol in asthma is associated with chymase-positive mast cells and eosinophilic airway inflammation.

PubMed

Sverrild, A; Bergqvist, A; Baines, K J; Porsbjerg, C; Andersson, C K; Thomsen, S F; Hoffmann, H J; Gibson, P; Erjefält, J S; Backer, V

2016-02-01

Airway hyperresponsiveness (AHR) to inhaled mannitol is associated with indirect markers of mast cell activation and eosinophilic airway inflammation. It is unknown how AHR to mannitol relates to mast cell phenotype, mast cell function and measures of eosinophilic inflammation in airway tissue. We compared the number and phenotype of mast cells, mRNA expression of mast cell-associated genes and number of eosinophils in airway tissue of subjects with asthma and healthy controls in relation to AHR to mannitol. Airway hyperresponsiveness to inhaled mannitol was measured in 23 non-smoking, corticosteroid-free asthmatic individuals and 10 healthy controls. Mast cells and eosinophils were identified in mucosal biopsies from all participants. Mast cells were divided into phenotypes based on the presence of chymase. mRNA expression of mast cell-associated genes was measured by real-time PCR. The proportion of submucosal MCTC was higher in asthmatic individuals with AHR to mannitol compared with asthmatic individuals without AHR (median: 40.3% vs. 18.7%, P = 0.03). Increased submucosal MCTC numbers were associated with increased levels of mRNA for thymic stromal lymphopoietin (TSLP) and CPA3 in asthmatics. Reactivity to mannitol correlated significantly with eosinophils in submucosa (r(s): 0.56, P = 0.01). Airway hyperresponsiveness to inhaled mannitol is associated with an altered submucosal mast cell profile in asthmatic individuals. This mast cell profile is associated with increased levels of TSLP and CPA3. The degree of AHR to mannitol is correlated with the degree of eosinophilic inflammation in the airway submucosa. © 2015 John Wiley & Sons Ltd.

Antileukotriene Reverts the Early Effects of Inflammatory Response of Distal Parenchyma in Experimental Chronic Allergic Inflammation

PubMed Central

Gobbato, Nathália Brandão; de Souza, Flávia Castro Ribas; Fumagalli, Stella Bruna Napolitano; Lopes, Fernanda Degobbi Tenório Quirino dos Santos; Prado, Carla Máximo; Martins, Milton Arruda; Tibério, Iolanda de Fátima Lopes Calvo; Leick, Edna Aparecida

2013-01-01

Aims. Compare the effects of montelukast or dexamethasone in distal lung parenchyma and airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods. GP have inhaled ovalbumin (OVA group-2x/week/4weeks). After the 4th inhalation, GP were treated with montelukast or dexamethasone. After 72 hours of the 7th inhalation, GP were anesthetised, and lungs were removed and submitted to histopathological evaluation. Results. Montelukast and dexamethasone treatments reduced the number of eosinophils in airway wall and distal lung parenchyma compared to OVA group (P < 0.05). On distal parenchyma, both treatments were effective in reducing RANTES, NF-κB, and fibronectin positive cells compared to OVA group (P < 0.001). Montelukast was more effective in reducing eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (P < 0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (P < 0.001). On airway walls, montelukast and dexamethasone were effective in reducing IGF-I, RANTES, and fibronectin positive cells compared to OVA group (P < 0.05). Dexamethasone was more effective in reducing the number of eotaxin and NF-κB positive cells than Montelukast (P < 0.05). Conclusions. In this animal model, both treatments were effective in modulating allergic inflammation and remodeling distal lung parenchyma and airway wall, contributing to a better control of the inflammatory response. PMID:24151607

Comparative Therapeutic Effects of Minocycline Treatment and Bone Marrow Mononuclear Cell Transplantation following Striatal Stroke

PubMed Central

Souza, Celice C.; da Silva, Michelle Castro; Lopes, Rosana Telma; Cardoso, Marcelo M.; Santos, Adriano Guimarães; dos Santos, Ijair Rogério

2017-01-01

We explored the comparative effects of minocycline treatment and intrastriatal BMMC transplantation after experimental striatal stroke in adult rats. Male Wistar adult rats were divided as follows: saline-treated (N = 5), minocycline-treated (N = 5), and BMMC-transplanted (N = 5) animals. Animals received intrastriatal microinjections of 80 pmol of endothelin-1 (ET-1). Behavioral tests were performed at 1, 3, and 7 days postischemia. Animals were treated with minocycline (50 mg/kg, i.p.) or intrastriatal transplants of 106 BMMCs at 24 h postischemia. Animals were perfused at 7 days after ischemic induction. Coronal sections were stained with cresyl violet for gross histopathological analysis and immunolabeled for the identification of neuronal bodies (NeuN), activated microglia/macrophages (ED1), and apoptotic cells (active caspase-3). BMMC transplantation and minocycline reduced the number of ED1+ cells (p < 0.05, ANOVA-Tukey), but BMMC afforded better results. Both treatments afforded comparable levels of neuronal preservation compared to control (p > 0.05). BMMC transplantation induced a higher decrease in the number of apoptotic cells compared to control and minocycline treatment. Both therapeutic approaches improved functional recovery in ischemic animals. The results suggest that BMMC transplantation is more effective in modulating microglial activation and reducing apoptotic cell death than minocycline, although both treatments are equally efficacious on improving neuronal preservation. PMID:28713482

Comparative Therapeutic Effects of Minocycline Treatment and Bone Marrow Mononuclear Cell Transplantation following Striatal Stroke.

PubMed

Souza, Celice C; da Silva, Michelle Castro; Lopes, Rosana Telma; Cardoso, Marcelo M; de Souza, Lucas Lacerda; Santos, Adriano Guimarães; Dos Santos, Ijair Rogério; Franco, Edna C S; Gomes-Leal, Walace

2017-01-01

We explored the comparative effects of minocycline treatment and intrastriatal BMMC transplantation after experimental striatal stroke in adult rats. Male Wistar adult rats were divided as follows: saline-treated ( N = 5), minocycline-treated ( N = 5), and BMMC-transplanted ( N = 5) animals. Animals received intrastriatal microinjections of 80 pmol of endothelin-1 (ET-1). Behavioral tests were performed at 1, 3, and 7 days postischemia. Animals were treated with minocycline (50 mg/kg, i.p.) or intrastriatal transplants of 106 BMMCs at 24 h postischemia. Animals were perfused at 7 days after ischemic induction. Coronal sections were stained with cresyl violet for gross histopathological analysis and immunolabeled for the identification of neuronal bodies (NeuN), activated microglia/macrophages (ED1), and apoptotic cells (active caspase-3). BMMC transplantation and minocycline reduced the number of ED1+ cells ( p < 0.05, ANOVA-Tukey), but BMMC afforded better results. Both treatments afforded comparable levels of neuronal preservation compared to control ( p > 0.05). BMMC transplantation induced a higher decrease in the number of apoptotic cells compared to control and minocycline treatment. Both therapeutic approaches improved functional recovery in ischemic animals. The results suggest that BMMC transplantation is more effective in modulating microglial activation and reducing apoptotic cell death than minocycline, although both treatments are equally efficacious on improving neuronal preservation.

Evaluation of the proliferation and differentiation behaviors of mesenchymal stem cells with partially converted borate glass containing different amounts of strontium in vitro.

PubMed

Zhu, Yi; Ouyang, Yuanming; Chang, Yi; Luo, Congfeng; Xu, Jun; Zhang, Changqing; Huang, Wenhai

2013-04-01

The objective of this study was to examine the proliferation and differentiation behaviors of different compositions of strontium-containing (from 0-12 mol%) borate glasses with mesenchymal stem cells (MSCs). The Cell Counting Kit-8 (CCK-8) assay revealed that after three days of culturing, the 6Sr group had the highest cell growth rate. Analysis of cell morphology revealed that cells proliferated well near the particles of the samples in all the groups on day 3. On day 7, cells in the 6Sr group demonstrated a higher proliferation rate than other 4 groups under the microscope. When performing the Live-Dead staining experiment, the 6Sr group had the least number of dead cells. Total DNA qualification indicated that the 6Sr group had a statistically higher concentration compared with the remaining groups. It was found that on day 7, compared with the 0Sr group, the core binding factor α1 (Cbfa1) mRNA expression level was significantly higher in the 6Sr, 9Sr and 12Sr groups. On day 14, compared with the 0Sr group, the bone sialoprotein (BSP) mRNA level was significantly higher in the 6Sr group. Additionally, on day 21, the 6Sr and 9Sr groups demonstrated higher osteocalcin (OCN) mRNA expression levels compared with the 0Sr group. In the alkaline phosphatase (ALP) activity test, on day 21, the 6Sr group presented a higher activity than the 0Sr group. Further, the number of mineralized nodules per unit in MSCs was measured by Alizarin Red S staining. The results showed that the 6Sr and 9Sr groups had the greatest number of mineralized nodules. Therefore, it could be concluded that borate glasses containing strontium oxide of 0, 3, 6, 9 and 12 mol% demonstrate a significant level of proliferation when interacting with MSCs. The borate glass containing 6 mol% strontium oxide had the greatest level of proliferation when cultured with MSCs. The borate glass containing 6 and 9 mol% strontium oxide facilitated an improved bone formation ability compared with the remaining two compositions.

P16INK4a Positive Cells in Human Skin Are Indicative of Local Elastic Fiber Morphology, Facial Wrinkling, and Perceived Age

PubMed Central

Waaijer, Mariëtte E. C.; Gunn, David A.; Adams, Peter D.; Pawlikowski, Jeff S.; Griffiths, Christopher E. M.; van Heemst, Diana; Slagboom, P. Eline; Westendorp, Rudi G. J.; Maier, Andrea B.

2016-01-01

Senescent cells are more prevalent in aged human skin compared to young, but evidence that senescent cells are linked to other biomarkers of aging is scarce. We counted cells positive for the tumor suppressor and senescence associated protein p16INK4a in sun-protected upper-inner arm skin biopsies from 178 participants (aged 45–81 years) of the Leiden Longevity Study. Local elastic fiber morphology, facial wrinkles, and perceived facial age were compared to tertiles of p16INK4a counts, while adjusting for chronological age and other potential confounders. The numbers of epidermal and dermal p16INK4a positive cells were significantly associated with age-associated elastic fiber morphologic characteristics, such as longer and a greater number of elastic fibers. The p16INK4a positive epidermal cells (identified as primarily melanocytes) were also significantly associated with more facial wrinkles and a higher perceived age. Participants in the lowest tertile of epidermal p16INK4a counts looked 3 years younger than those in the highest tertile, independently of chronological age and elastic fiber morphology. In conclusion, p16INK4a positive cell numbers in sun-protected human arm skin are indicative of both local elastic fiber morphology and the extent of aging visible in the face. PMID:26286607

Expression of cdk4 and p16 in Oral Lichen Planus.

PubMed

Goel, Sinny; Khurana, Nita; Marwah, Akanksha; Gupta, Sunita

2015-01-01

The purpose of this study was to evaluate the expression of cdk4 and p16, the proteins implicated in hyperproliferation and arrest in oral lichen planus and to compare their expression in erosive and non-erosive oral lichen planus and with normal mucosa and oral squamous cell carcinoma. Analysis of cdk4 and p16 expression was done in 43 erosive oral lichen planus (EOLP) and 17 non-erosive oral lichen planus (NOLP) cases, 10 normal mucosa and 10 oral squamous cell carcinoma (OSCC) cases with immunohistochemistry. This study demonstrated a significantly increased expression of cytoplasmic cdk4 (80% cases, cells stained - 19.6%), and cytoplasmic p16 (68.3% cases, cells stained - 16.4%) in oral lichen planus (OLP) compared to normal mucosa. cdk4 was much higher in OSCC in both cytoplasm and nuclei compared to normal mucosa. Also, while comparing OLP with positive control, significant difference was noted for cdk4 and p16, with expression being more in OSCC. While comparing EOLP with NOLP; significant differences were seen for cdk4 cytoplasmic staining only, for number of cases with positive staining as well as number of cells stained. Overexpression of cytoplasmic cdk4 and p16 was registered in oral lichen planus, however considerably lower than in squamous cell carcinoma. Erosive oral lichen planus demonstrated overexpression of cytoplasmic cdk4 and premalignant nature compared to non-erosive lesion. Therefore there is an obvious possibility for cytoplasmic expression of cdk4 and p16 to predict malignant potential of oral lichen planus lesions.

The Effects of Aloe Vera on TNF-a Levels, the Percentage of Nk Cells and Th 17 Cells in Rat That Received Izoniazid and Rifampycin.

PubMed

Mawarti, Herin; Rajin, Mukhamad; Asumta, Zulfikar

2017-10-01

The present study was undertaken to investigate the hepatoprotective effect of Aloe vera against side effect of antituberculosis drug. Twenty-five rats will be divided into five groups, namely the control group (without any treatment), the group of rats treated with anti-tuberculosis drugs, and a group of rats were treated antituberculosis drugs and got Aloe vera extract at a dose of 40; 80; and 120 mg/kg body weight. Antituberculosis drugs are isoniazid and rifampicin a dose of 50 mg/kg body weight. Antituberculosis treated group showed significantly increase levels of TNF-a, the percentage of NK cells and the number of Th17 cells compared with the control group ( p < 0.05). All doses of Aloe vera reduce levels of TNF-a compared with the antituberculosis group ( p < 0.05), although it has not yet reached levels comparable to the control group ( p > 0.05). Aloe vera at first and the third dose lower the number of NK cells compared to the antituberculosis group, although it has not yet reached a significant difference ( p > 0.05). The first dose of Aloe vera was significantly decreased the percentage of Th17 cells compared to the antituberculosis drug group ( p < 0.05), although it has not yet reached levels comparable to the control group ( p > 0.05). It was concluded that administration of Aloe vera can suppress the production of TNF-a and the percentage of Th17 cells as a result of antituberculosis drug administration. Thus, Aloe vera can be a useful alternative to natural materials in the successful treatment of tuberculosis through the inhibition of side effect.

The infiltration of experimentally induced lung metastases of colon carcinoma CC531 by adoptively transferred interleukin-2-activated natural killer cells in Wag rats.

PubMed

Kuppen, P J; Basse, P H; Goldfarb, R H; Van De Velde, C J; Fleuren, G J; Eggermont, A M

1994-02-15

The number of IL-2-activated natural killer (A-NK) cells reaching the tumor site in vivo may be crucial for their anti-tumor effect following adoptive immunotherapy. We investigated in a syngeneic rat model the infiltration of established lung metastases by adoptively transferred A-NK cells. The Wag rat colon carcinoma CC531 was injected via a tail vein to induce pulmonary metastases. Syngeneic A-NK cells were labeled with the fluorescent dye rhodamine (TRITC) and next injected via a tail vein in rats bearing day-12 lung tumors. The number of A-NK cells in tumor and in normal tissue per rat was counted in sections after administration of A-NK cells. At all time points tested, a significant linear relationship between the cross-section area of the tumor and the number of infiltrating cells was observed, but small tumor areas became fully infiltrated earlier than larger areas. At 24 hr after injection, approximately 10% of the injected cells were found in the tumor tissue and the average A-NK-cell-to-tumor-cell ratio was estimated to be 1:3. A-NK cells were found in the liver too, although the number of cells per mm2 tissue was low compared with the pulmonary tumor tissue. Very low numbers of A-NK cells were found in kidney, adrenal gland, spleen, and blood. We conclude that, in this syngeneic rat model, adoptively transferred A-NK cells are able to find and specifically infiltrate pulmonary metastases in a time-dependent fashion.

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells.

PubMed

Joshi, Nikhil S; Cui, Weiguo; Dominguez, Claudia X; Chen, Jonathan H; Hand, Timothy W; Kaech, Susan M

2011-10-15

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.

Method of oocyte activation affects cloning efficiency in pigs.

PubMed

Whitworth, Kristin M; Li, Rongfeng; Spate, Lee D; Wax, David M; Rieke, August; Whyte, Jeffrey J; Manandhar, Gaurishankar; Sutovsky, Miriam; Green, Jonathan A; Sutovsky, Peter; Prather, Randall S

2009-05-01

The following experiments compared the efficiency of three fusion/activation protocols following somatic cell nuclear transfer (SCNT) with porcine somatic cells transfected with enhanced green fluorescent protein driven by the chicken beta-actin/rabbit beta-globin hybrid promoter (pCAGG-EGFP). The three protocols included electrical fusion/activation (NT1), electrical fusion/activation followed by treatment with a reversible proteasomal inhibitor MG132 (NT2) and electrical fusion in low Ca(2+) followed by chemical activation with thimerosal/dithiothreitol (NT3). Data were collected at Days 6, 12, 14, 30, and 114 of gestation. Fusion rates, blastocyst-stage mean cell numbers, recovery rates, and pregnancy rates were calculated and compared between protocols. Fusion rates were significantly higher for NT1 and NT2 compared to NT3 (P < 0.05). There was no significant difference in mean nuclear number. Pregnancy rate for NT2 was 100% (n = 19) at all stages collected and was significantly higher than NT1 (71.4%, n = 28; P < 0.05), but was not significantly higher than NT3 (82.6%, n = 23; P < 0.15). Recovery rates were calculated based on the number of embryos, conceptuses, fetuses, or piglets present at the time of collection, divided by the number of embryos transferred to the recipient gilts. Recovery rates between the three groups were not significantly different at any of the stages collected (P > 0.05). All fusion/activation treatments produced live, pCAGG-EGFP positive piglets from SCNT. Treatment with MG132 after fusion/activation of reconstructed porcine embryos was the most effective method when comparing the overall pregnancy rates. The beneficial effect of NT2 protocol may be due to the stimulation of proteasomes that infiltrate donor cell nucleus shortly after nuclear transfer. (c) 2008 Wiley-Liss, Inc.

The Endocytic Adaptor Eps15 Controls Marginal Zone B Cell Numbers

PubMed Central

Pozzi, Benedetta; Amodio, Stefania; Lucano, Caterina; Sciullo, Anna; Ronzoni, Simona; Castelletti, Daniela; Adler, Thure; Treise, Irina; Betsholtz, Ingrid Holmberg; Rathkolb, Birgit; Busch, Dirk H.; Wolf, Eckhard; Fuchs, Helmut; Gailus-Durner, Valérie; de Angelis, Martin Hrabě; Betsholtz, Christer; Casola, Stefano; Di Fiore, Pier Paolo; Offenhäuser, Nina

2012-01-01

Eps15 is an endocytic adaptor protein involved in clathrin and non-clathrin mediated endocytosis. In Caenorhabditis elegans and Drosophila melanogaster lack of Eps15 leads to defects in synaptic vesicle recycling and synapse formation. We generated Eps15-KO mice to investigate its function in mammals. Eps15-KO mice are born at the expected Mendelian ratio and are fertile. Using a large-scale phenotype screen covering more than 300 parameters correlated to human disease, we found that Eps15-KO mice did not show any sign of disease or neural deficits. Instead, altered blood parameters pointed to an immunological defect. By competitive bone marrow transplantation we demonstrated that Eps15-KO hematopoietic precursor cells were more efficient than the WT counterparts in repopulating B220+ bone marrow cells, CD19− thymocytes and splenic marginal zone (MZ) B cells. Eps15-KO mice showed a 2-fold increase in MZ B cell numbers when compared with controls. Using reverse bone marrow transplantation, we found that Eps15 regulates MZ B cell numbers in a cell autonomous manner. FACS analysis showed that although MZ B cells were increased in Eps15-KO mice, transitional and pre-MZ B cell numbers were unaffected. The increase in MZ B cell numbers in Eps15 KO mice was not dependent on altered BCR signaling or Notch activity. In conclusion, in mammals, the endocytic adaptor protein Eps15 is a regulator of B-cell lymphopoiesis. PMID:23226392

Iron loading in HFE p.C282Y homozygotes found by population screening: relationships to HLA-type and T-lymphocyte subsets.

PubMed

Thorstensen, Ketil; Kvitland, Mona A; Irgens, Wenche Ø; Åsberg, Arne; Borch-Iohnsen, Berit; Moen, Torolf; Hveem, Kristian

2017-11-01

Iron loading in p.C282Y homozygous HFE hemochromatosis subjects is highly variable, and it is unclear what factors cause this variability. Finding such factors could aid in predicting which patients are at highest risk and require closest follow-up. The degree of iron loading has previously been associated with certain HLA-types and with abnormally low CD8 + cell counts in peripheral blood. In 183 Norwegian, p.C282Y homozygotes (104 men, 79 women) originally found through population screening we determined HLA type and measured total T-lymphocytes, CD4 + and CD8 + cells, and compared this with data on iron loading. In p.C282Y homozygous men, but not in homozygous women, we found that the presence of two HLA-A*03 alleles increased the iron load on average by approximately 2-fold compared to p.C282Y homozygous men carrying zero or one A*03 allele. On the other hand, the presence of two HLA-A*01 alleles, in male subjects, apparently reduced the iron loading. In p.C282Y homozygous individuals, the iron loading was increased if the CD8 + cell number was below the 25 percentile or if the CD4 + cell number was above the 75 percentile. This effect appeared to be additive to the effect of the number of HLA-A*03 alleles. Our data indicate that homozygosity for the HLA-A*03 allele significantly increases the risk of excessive iron loading in Norwegian p.C282Y homozygous male patients. In addition, low CD8 + cell number or high CD4 + cell number further increases the risk of excessive iron loading.

In vitro biological evaluation of beta-TCP/HDPE--A novel orthopedic composite: a survey using human osteoblast and fibroblast bone cells.

PubMed

Homaeigohar, S Sh; Shokrgozar, M A; Khavandi, A; Sadi, A Yari

2008-02-01

Beta-tricalcium phosphate reinforced high density polyethylene (beta-TCP/HDPE) was prepared to simulate bone composition and to study its capacity to act as bone tissue. This material was produced by replacing the mineral component and collagen soft tissue of the bone with beta-TCP and HDPE, respectively. The biocompatibility of the composite samples with different volume fractions of TCP (20, 30 and 40 vol %) was examined in vitro using two osteoblast cell lines G-292 and Saos-2, and also a type of fibroblast cell isolated from bone tissue, namely human bone fibroblast (HBF) by proliferation, and cell adhesion assays. Cell-material interaction with the surface of the composite samples was examined by scanning electron microscopy (SEM). The effect of beta-TCP/HDPE on the behavior of osteoblast and fibroblast cells was compared with those of composite and negative control samples; polyethylene (PE) and tissue culture polystyrene (TPS), respectively. In general, the results showed that the composite samples containing beta-TCP as reinforcement supported a higher rate of proliferation by various bone cells after 3, 7, and 14 days of incubation compared to the composite control sample. Furthermore, more osteoblast cells were attached to the surface of the composite samples when compared to the composite control samples after the above incubation periods (p < 0.05), while in the case of HBF an equal or even higher number of cells adhered to PE was observed. The number of adhered osteoblast cells was almost equal and in some days even higher than the number of adhered cells on negative control sample, while in the case of fibroblast this difference was significantly higher than TPS (p < 0.05). Adhered cells presented a normal morphology by SEM and many of the cells were observed to be undergoing cell division. These findings indicate that beta-TCP/HDPE composites are biocompatible, nontoxic, and act to stimulate proliferation and adhesion of the cells, whether osteoblast or fibroblast. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res, 2008.

Influence of Melanosome Dynamics on Melanoma Drug Sensitivity

PubMed Central

Chen, Kevin G.; Leapman, Richard D.; Zhang, Guofeng; Lai, Barry; Valencia, Julio C.; Cardarelli, Carol O.; Vieira, Wilfred D.; Hearing, Vincent J.

2009-01-01

Background Malignant melanomas are intrinsically resistant to many conventional treatments, such as radiation and chemotherapy, for reasons that are poorly understood. Here we propose and test a model that explains drug resistance or sensitivity in terms of melanosome dynamics. Methods The growth and sensitivity to cisplatin of MNT-1 cells, which are melanotic and enriched with mature stage III and IV melanosomes, and SK-MEL-28 cells, which have only immature stage I and II melanosomes, were compared using clonogenic assays. Differences in pigmentation, melanosome stages, melanosome number, and cellular structures in different cell lines in response to various treatments were examined by electron microscopy. The relative numbers of melanosomes of different stages were compared after treatment with 1-phenyl-2-thiourea. The relationship between drug transporter function and endogenous melanogenic toxicity was assessed by treating cells with the cyclosporin analog PSC-833 and by assessing vacuole formation and cell growth inhibition. All statistical tests were two-sided. Results Endogenous melanogenic cytotoxicity, produced by damaged melanosomes, resulted in pronounced cell growth inhibition in MNT-1 cells compared with amelanotic SK-MEL-28 cells. The sensitivity to CDDP of MNT-1 cells was 3.8-fold higher than that of SK-MEL-28 cells (mean IC50 for SK-MEL-28 and MNT-1 = 2.13 μM and 0.56 μM, respectively; difference = 1.57 μM, 95% confidence interval = 1.45 to 1.69; P = .0017). After treatment with 6.7 μM CDDP for 72 hours, the number of stage II-III melanosomes in surviving MNT-1 cells was 6.8-fold that of untreated cells. Modulation of MNT-1 cells to earlier-stage (II, II-III, III) melanosomes by treatment with the tyrosinase inhibitor 1-phenyl-2-thiourea dramatically increased CDDP resistance. Furthermore, PSC-833 principally suppressed MNT-1 melanotic cell growth via an elevation of autophagosome-like vacuolar structures, possibly by inhibiting melanosome membrane transporters. Conclusions Melanosome dynamics (including their biogenesis, density, status, and structural integrity) regulate the drug resistance of melanoma cells. Manipulation of melanosome functions may be an effective way to enhance the therapeutic activity of anticancer drugs against melanoma. PMID:19704071

Influence of melanosome dynamics on melanoma drug sensitivity.

PubMed

Chen, Kevin G; Leapman, Richard D; Zhang, Guofeng; Lai, Barry; Valencia, Julio C; Cardarelli, Carol O; Vieira, Wilfred D; Hearing, Vincent J; Gottesman, Michael M

2009-09-16

Malignant melanomas are intrinsically resistant to many conventional treatments, such as radiation and chemotherapy, for reasons that are poorly understood. Here we propose and test a model that explains drug resistance or sensitivity in terms of melanosome dynamics. The growth and sensitivity to cisplatin of MNT-1 cells, which are melanotic and enriched with mature stage III and IV melanosomes, and SK-MEL-28 cells, which have only immature stage I and II melanosomes, were compared using clonogenic assays. Differences in pigmentation, melanosome stages, melanosome number, and cellular structures in different cell lines in response to various treatments were examined by electron microscopy. The relative numbers of melanosomes of different stages were compared after treatment with 1-phenyl-2-thiourea. The relationship between drug transporter function and endogenous melanogenic toxicity was assessed by treating cells with the cyclosporin analog PSC-833 and by assessing vacuole formation and cell growth inhibition. All statistical tests were two-sided. Endogenous melanogenic cytotoxicity, produced by damaged melanosomes, resulted in pronounced cell growth inhibition in MNT-1 cells compared with amelanotic SK-MEL-28 cells. The sensitivity to CDDP of MNT-1 cells was 3.8-fold higher than that of SK-MEL-28 cells (mean IC(50) for SK-MEL-28 and MNT-1 = 2.13 microM and 0.56 microM, respectively; difference = 1.57 microM, 95% confidence interval = 1.45 to 1.69; P = .0017). After treatment with 6.7 microM CDDP for 72 hours, the number of stage II-III melanosomes in surviving MNT-1 cells was 6.8-fold that of untreated cells. Modulation of MNT-1 cells to earlier-stage (II, II-III, III) melanosomes by treatment with the tyrosinase inhibitor 1-phenyl-2-thiourea dramatically increased CDDP resistance. Furthermore, PSC-833 principally suppressed MNT-1 melanotic cell growth via an elevation of autophagosome-like vacuolar structures, possibly by inhibiting melanosome membrane transporters. Melanosome dynamics (including their biogenesis, density, status, and structural integrity) regulate the drug resistance of melanoma cells. Manipulation of melanosome functions may be an effective way to enhance the therapeutic activity of anticancer drugs against melanoma.

Antigen-specific tolerance inhibits autoimmune uveitis in pre-sensitized animals by deletion and CD4+CD25+ T-regulatory cells.

PubMed

Matta, Bharati; Jha, Purushottam; Bora, Puran S; Bora, Nalini S

2010-02-01

The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

GeneCount: genome-wide calculation of absolute tumor DNA copy numbers from array comparative genomic hybridization data

PubMed Central

Lyng, Heidi; Lando, Malin; Brøvig, Runar S; Svendsrud, Debbie H; Johansen, Morten; Galteland, Eivind; Brustugun, Odd T; Meza-Zepeda, Leonardo A; Myklebost, Ola; Kristensen, Gunnar B; Hovig, Eivind; Stokke, Trond

2008-01-01

Absolute tumor DNA copy numbers can currently be achieved only on a single gene basis by using fluorescence in situ hybridization (FISH). We present GeneCount, a method for genome-wide calculation of absolute copy numbers from clinical array comparative genomic hybridization data. The tumor cell fraction is reliably estimated in the model. Data consistent with FISH results are achieved. We demonstrate significant improvements over existing methods for exploring gene dosages and intratumor copy number heterogeneity in cancers. PMID:18500990

Toxicity of copper on the growth of marine microalgae Pavlova sp. and its chlorophyll-a

NASA Astrophysics Data System (ADS)

Purbonegoro, T.; Suratno; Puspitasari, R.; Husna, N. A.

2018-02-01

Marine microalgae is the primary producer at the base of the marine food chain. Their sensitivity to metal contamination provides important information for predicting the environmental impact of pollution. Toxicity testing using marine microalgae Pavlova sp. was carried out to assess the toxicity of copper on the growth and chlorophyll-a content. Results of this study show that adverse effects were observed by the increase of copper concentration. Cell number began to decrease at the lowest concentration (13 μg/L) and reduced drastically at 98 μg/L. Minimum cell number was observed at the highest concentration (890 μg/L). The inhibition concentration (IC50) value of copper for Pavlova sp. was 51.46 μg/L and at concentrations >29 μgL-1 the chlorophyll-a content decreased dramatically compared to the control. A variation in cell size and morphology was also observed at the higher concentration by the increase in the cell size and loss of setae compared to normal cells.

DNA methylation and copy number variation analyses of human embryonic stem cell-derived neuroprogenitors after low-dose decabromodiphenyl ether and/or bisphenol A exposure.

PubMed

Du, L; Sun, W; Li, X M; Li, X Y; Liu, W; Chen, D

2018-05-01

The polybrominated diphenyl ether flame retardants decabromodiphenyl ether (BDE-209) and bisphenol A (BPA) are environmental contaminants that can cross the placenta and exert toxicity in the developing fetal nervous system. Copy number variants (CNVs) play a role in a number of genetic disorders and may be implicated in BDE-209/BPA teratogenicity. In this study, we found that BDE-209 and/or BPA exposure decreased neural differentiation efficiency of human embryonic stem cells (hESCs), although there was a >90% induction of neuronal progenitor cells (NPCs) from exposed hESCs. However, the mean of CNV numbers in the NPCs with BDE-209 + BPA treatment was significantly higher compared to the other groups, whereas DNA methylation was lower and DNA methyltransferase(DNMT1 and DNMT3A) expression were significantly decreased in all of the BDE-209 and/or BPA treatment groups compared with the control groups. The number of CNVs in chromosomes 3, 4, 11, 22, and X in NPCs with BDE-209 and/or BPA exposure was higher compared to the control group. In addition, CNVs in chromosomes 7, 8, 14, and 16 were stable in hESCs and hESCs-derived NPCs irrespective of BDE-209/BPA exposure, and CNVs in chromosomes 20 q11.21 and 16 p13.11 might be induced by neural differentiation. Thus, BDE-209/BPA exposure emerges as a potential source of CNVs distinct from neural differentiation by itself. BDE-209 and/or BPA exposure may cause genomic instability in cultured stem cells via reduced activity of DNA methyltransferase, suggesting a new mechanism of human embryonic neurodevelopmental toxicity caused by this class of environmental toxins.

Minimal volume vitrification of epididymal spermatozoa results in successful in vitro fertilization and embryo development in mice

PubMed Central

Horta, Fabrizzio; Alzobi, Hamida; Jitanantawittaya, Sutthipat; Catt, Sally; Chen, Penny; Pangestu, Mulyoto; Temple-Smith, Peter

2017-01-01

This study compared three cryopreservation protocols on sperm functions, IVF outcomes, and embryo development. Epididymal spermatozoa cryopreserved using slow-cooling (18% w/v raffinose, RS-C) were compared with spermatozoa vitrified using 0.25 M sucrose (SV) or 18% w/v raffinose (RV). The motility, vitality, and DNA damage (TUNEL assay) of fresh control (FC) spermatozoa were compared with post-thawed or warmed RS-C, RV, and SV samples. Mouse oocytes (n = 267) were randomly assigned into three groups for insemination: RV (n = 102), RS-C (n = 86), and FC (n = 79). The number and the proportion of two-cell embryos and blastocysts from each treatment were assessed. Sperm motility (P < 0.01) and vitality (P < 0.05) were significantly reduced after vitrification compared with slow-cooled spermatozoa. However, DNA fragmentation was significantly reduced in spermatozoa vitrified using sucrose (15 ± 1.8% [SV] vs 26 ± 2.8% [RV] and 27 ± 1.2% [RS-C]; P < 0.01). Although the number of two-cell embryos produced by RS-C, RV, and FC spermatozoa was not significantly different, the number of blastocysts produced from two-cell embryos using RV spermatozoa was significantly higher than FC spermatozoa (P = 0.0053). This simple, small volume vitrification protocol and standard insemination method allows successful embryo production from small numbers of epididymal spermatozoa and may be applied clinically to circumvent the need for ICSI, which has the disadvantage of bypassing sperm selection. PMID:27427551

Clinical characteristics of vaginal discharge in bacterial vaginosis diagnosed by Nugent's criteria.

PubMed

Hapsari, E D; Hayashi, M; Matsuo, H

2006-01-01

To determine which clinical signs have an important contribution in bacterial vaginosis (BV) diagnosed by Nugent's criteria. This was a cross-sectional study of 58 women undergoing vaginal examination at Hayashi women's clinic, Hyogo Prefecture. Characteristics of vaginal discharge (color, amount, odor, and pH level) and the number of clue cells were compared among normal, intermediate, and BV groups. BV was diagnosed by using Nugent's criteria. The incidence of BV in our study population was 25.9%. Compared to the non-BV group, women in the BV group were found more often to have yellowish color and a moderate amount of discharge (66.67% vs 33.33%, NS; 55.56% vs 44.44%, NS), odorous discharge (100% vs 0%, p < 0.05) and furthermore, pH level and the number of clue cells were significantly higher. Odor, pH level and the number of clue cells in the vaginal discharge were helpful clinical signs for early detection of BV diagnosed by Nugent's criteria.

Bone marrow analysis of immune cells and apoptosis in patients with systemic lupus erythematosus.

PubMed

Park, J W; Moon, S Y; Lee, J H; Park, J K; Lee, D S; Jung, K C; Song, Y W; Lee, E B

2014-09-01

To examine the immune cell profile in the bone marrow of systemic lupus erythematosus (SLE) patients and to assess its clinical relevance. Sixteen bone marrow samples from 14 SLE patients were compared with seven healthy control samples. The numbers of immune cells and apoptotic cells in the bone marrow were examined by immunohistochemistry. The association between immune cell subsets and clinical features was investigated. CD4+ T cells, macrophages and plasma cells were more common in the bone marrow of SLE patients than in healthy controls (p=0.001, p=0.004 and p<0.001, respectively). Greater numbers of CD4+ T cells and macrophages were associated with high-grade bone marrow damage. The percentage of apoptotic cells in bone marrow of SLE patients was significantly higher than that in controls (p<0.001) and was positively correlated with the number of plasmacytoid dendritic cells (p=0.013). Increased number of plasma cells along with high interleukin-6 expression was correlated with anti-double stranded DNA antibody levels and the SLE disease activity index (p=0.031 and 0.013, respectively). Bone marrow from SLE patients showed a distinct immune cell profile and increased apoptosis. This, coupled with a correlation with disease activity, suggests that the bone marrow may play a critical role in the pathogenesis of SLE. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Flow cytometry analysis of inflammatory cells isolated from the sciatic nerve and DRG after chronic constriction injury in mice.

PubMed

Liu, Liping; Yin, Yan; Li, Fei; Malhotra, Charvi; Cheng, Jianguo

2017-06-01

Cellular responses to nerve injury play a central role in the pathogenesis of neuropathic pain. However, the analysis of site specific cellular responses to nerve injury and neuropathic pain is limited to immunohistochemistry staining with numerous limitations. We proposed to apply flow cytometry to overcome some of the limitations and developed two protocols for isolation of cells from small specimens of the sciatic nerve and dorsal root ganglion (DRG) in mice. RESULTS AND COMPARASION WITH EXISTING: methods We found that both the non-enzymatic and enzymatic approaches were highly effective in harvesting a sufficient number of cells for flow cytometry analysis in normal and pathological conditions. The total number of cells in the injury site of the sciatic and its DRGs increased significantly 14days after chronic constriction injury (CCI) of the sciatic nerve, compared to sham surgery control or the contralateral control. The enzymatic approach yielded a significantly higher total number of cells and CD45 negative cells, suggesting that this approach allows for harvest of more resident cells, compared to the non-enzymatic method. The percentage of CD45 + /CD11b + cells was significantly increased in the sciatic nerve but not in the DRG. These results were consistent with both protocols. We thus offer two simple and effective protocols that allow for application of flow cytometry to the investigation of cellular and molecular mechanisms of neuropathic pain. Copyright © 2017 Elsevier B.V. All rights reserved.

Micronuclei and nuclear anomalies in Mexico's indigenous population.

PubMed

Lazalde-Ramos, Blanca Patricia; Zamora-Pérez, Ana Lourdes; Sosa-Macías, Martha; Galaviz-Hernández, Carlos; Zúñiga-González, Guillermo Moisés

2017-01-01

To determine the number of micronuclei and nuclear anomalies in Mexico's indigenous population. One hundred twenty indigenous individuals were evaluated, including thirty from the ethnicities Cora, Huichol, Tarahumara and Tepehuano. The number of micronuclei (MN) and any nuclear abnormality (NA) in oral mucosa cells, including cells with nuclear buds, binucleated cells, cells with karyolysis, karyorrhetic, condensed chromatin and pyknotic cells were determined for each participant. Tepehuano and Tarahumaras showed the greatest damage to DNA. The Tepehuano group presented the highest number of MN and NA, this being a significant difference (p < 0.05) compared with the rest of the studied groups. This group also presented the highest herbicide exposure (46.7%). In relation to the smoking and drinking habits, these were more frequent in the Tarahumara group (33.3 and 50% respectively). The ethnic diversity, habits and customs may influence the DNA nuclear integrity in the Amerindian groups.

p53 Mutation suppresses adult neurogenesis in medaka fish (Oryzias latipes)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Isoe, Yasuko; Okuyama, Teruhiro; Taniguchi, Yoshihito

2012-07-13

Highlights: Black-Right-Pointing-Pointer Progenitor migration is accompanied by an increase in their numbers in the adult brain. Black-Right-Pointing-Pointer p53 Mutation suppressed an increase in the number of the migrated progenitors. Black-Right-Pointing-Pointer The decreased progenitor number is not due to enhanced cell death. Black-Right-Pointing-Pointer p53 Mutation did not affect proliferation of stem cells. -- Abstract: Tumor suppressor p53 negatively regulates self-renewal of neural stem cells in the adult murine brain. Here, we report that the p53 null mutation in medaka fish (Oryzias latipes) suppressed neurogenesis in the telencephalon, independent of cell death. By using 5-bromo-29-deoxyuridine (BrdU) immunohistochemistry, we identified 18 proliferation zonesmore » in the brains of young medaka fish; in situ hybridization showed that p53 was expressed selectively in at least 12 proliferation zones. We also compared the number of BrdU-positive cells present in the whole telencephalon of wild-type (WT) and p53 mutant fish. Immediately after BrdU exposure, the number of BrdU-positive cells did not differ significantly between them. One week after BrdU-exposure, the BrdU-positive cells migrated from the proliferation zone, which was accompanied by an increased number in the WT brain. In contrast, no significant increase was observed in the p53 mutant brain. Terminal deoxynucleotidyl transferase (dUTP) nick end-labeling revealed that there was no significant difference in the number of apoptotic cells in the telencephalon of p53 mutant and WT medaka, suggesting that the decreased number of BrdU-positive cells in the mutant may be due to the suppression of proliferation rather than the enhancement of neural cell death. These results suggest that p53 positively regulates neurogenesis via cell proliferation.« less

The number and function of circulating CD34+CD133+ progenitor cells decreased in stable coronary artery disease but not in acute myocardial infarction

PubMed Central

Kondo, Takahisa; Shintani, Satoshi; Maeda, Kengo; Hayashi, Mutsuharu; Inden, Yasuya; Numaguchi, Yasushi; Sugiura, Kaichiro; Morita, Yasuhiro; Kitamura, Tomoya; Kamiya, Haruo; Sone, Takahito; Ohno, Miyoshi; Murohara, Toyoaki

2010-01-01

Objective Circulating CD34+CD133+ cells are one of the main sources of circulating endothelial progenitor cells (EPCs). Age is inversely related to the number and function of CD34+CD133+ progenitor cells in stable coronary artery disease (CAD), but the relationship remains unclear in acute myocardial infarction (AMI). The authors aimed to clarify how ageing affects the number and function of mobilised CD34+CD133+ progenitor cells in AMI. Design and results Circulating CD34+CD133+ progenitor cells were measured by flow cytometry. Measurements were made at admission for CAD, or on day 7 after the onset of AMI. In stable CAD (n=131), circulating CD34+CD133+ cells decreased with age (r=−0.344, p<0.0001). In AMI, circulating CD34+CD133+ cells did not correlate with age (n=50), and multivariate analysis revealed that the decreased number of circulating CD34+CD133+ cells was associated with male sex and higher peak creatinine kinase. The ability to give rise to functional EPCs, which show good migratory and tube-forming capabilities, deteriorated among stable CAD subjects (n=10) compared with AMI subjects (N=6). Conclusions In stable CAD, the number and function of circulating CD34+CD133+ progenitor cells decreased with age, whereas those mobilised and circulating in AMI did not. PMID:27325937

Effect of cryopreservation and in vitro culture of bovine fibroblasts on histone acetylation levels and in vitro development of hand-made cloned embryos

USGS Publications Warehouse

Chacon, L.; Gomez, M.C.; Jenkins, J.A.; Leibo, S.P.; Wirtu, G.; Dresser, B.L.; Pope, C.E.

2011-01-01

In this study, the relative acetylation levels of histone 3 in lysine 9 (H3K9ac) in cultured and cryopreserved bovine fibroblasts was measured and we determined the influence of the epigenetic status of three cultured (C1, C2 and C3) donor cell lines on the in vitro development of reconstructed bovine embryos. Results showed that cryopreservation did not alter the overall acetylation levels of H3K9 in bovine fibroblasts analysed immediately after thawing (frozen/thawed) compared with fibroblasts cultured for a period of time after thawing. However, reduced cleavage rates were noted in embryos reconstructed with fibroblasts used immediately after thawing. Cell passage affects the levels of H3K9ac in bovine fibroblasts, decreasing after P1 and donor cells with lower H3K9ac produced a greater frequency of embryo development to the blastocyst stage. Cryopreservation did not influence the total cell and ICM numbers, or the ICM/TPD ratios of reconstructed embryos. However, the genetic source of donor cells did influence the total number of cells and the trophectoderm cell numbers, and the cell passage influenced the total ICM cell numbers. ?? Copyright Cambridge University Press 2010.

Development of a two-step protocol for culture expansion of human annulus fibrosus cells with TGF-β1 and FGF-2.

PubMed

Chou, Po-Hsin; Wang, Shih-Tien; Ma, Hsiao-Li; Liu, Chien-Lin; Chang, Ming-Chau; Lee, Oscar Kuang-Sheng

2016-07-12

Different biologic approaches to treat disc regeneration, including growth factors (GFs) application, are currently under investigation. Human annulus fibrosus (hAF) repair or regeneration is one of the key elements for maintenance and restoration of nucleus pulposus function. However, so far there is no effective treatment for this purpose. The aim of the present study was to investigate the response of hAF cells to different combinations of GFs, and develop a protocol for efficient culture expansion. hAF cells were harvested from degenerated disc tissues during surgical intervertebral disc removal, and hAF cells were expanded in a monolayer. The experiments were categorized based on different protocols with transforming growth factor (TGF-β1) and fibroblast growth factor (FGF-2) culture for 14 days: group 1 had no GFs (control group); group 2 received TGF-β1; group 3 received FGF-2; group 4 received both GFs; and group 5 (two-step) received both GFs for the first 10 days and TGF-β1 only for the next 4 days. Cell proliferation, collagen, and noncollagen extracellular matrix (ECM) production and genes expression were compared among these groups. At days 3, 7 and 10 of cultivation, groups 4 and 5 had significantly more cell numbers and faster cell proliferation rates than groups 1, 2, and 3. At 14 days of cultivation, significantly more cell numbers were observed in groups 3 and 4 than in group 5. The group 4 had the most cell numbers and the fastest proliferation rate at 14 days of cultivation. After normalization for cell numbers, group 5 (two-step) produced the most collagen and noncollagen ECM at 10 and 14 days of cultivation among the five groups. In group 5, ECM gene expression was significantly upregulated. High expression of matrix metalloproteinase-1 was upregulated with FGF-2 on the different days as compared to the other groups. Annulus fibrosus cell phenotypes were only marginally retained under the different protocols based on quantitative polymerase chain reaction results. Taken together, the two-step protocol was the most efficient among these different protocols with the most abundant ECM production after normalization for cell numbers for culture expansion of hAF cells. The protocol may be useful in further cell therapy and tissue engineering approaches for disc regeneration.

Improved priming for mobilization of and optimal timing for harvest of peripheral blood stem cells.

PubMed

Knudsen, L M; Gaarsdal, E; Jensen, L; Nielsen, K J; Nikolaisen, K; Johnsen, H E

1996-08-01

The time of stem cell harvest and the mobilization regimen may play important roles in terms of achieving adequate numbers of stem cells by leukapheresis. To optimize the timing of leukapheresis, we have determined simultaneously the number of CD34+ cells in the peripheral blood as well as in the leukapheresis product of 214 apheresis procedures performed in 66 unselected patients with malignant hematologic diseases and solid tumors. A significant correlation between the number of CD34+ cells in peripheral blood and the leukapheresis product (R = 0.8) was found. The presence of more than 20 x 10(3)/ml blood CD34+ cells gave a sufficient yield (> or = 1.0 x 10(6) CD34+ cells/kg) in 81% of the cases. In an attempt to compare two priming regimens, we performed leukapheresis twice in 12 patients with stable disease. In the first sequence, stem cells were mobilized with rhG-CSF (10 micrograms/kg/day) alone and, in the second sequence, with cyclophosphamide (4 g/m2) plus rhG-CSF. A significantly higher yield of CD34+ cells and a better correlation between CD34+ cells in the peripheral blood and the leukapheresis product were found after priming with high-dose cyclophosphamide plus rhG-CSF, compared with priming with rhG-CSF alone. In a multivariate analysis, three factors were found to correlate with the yield of CD34+ cells, namely prior chemotherapy, bone marrow function, and the mobilization regimen. The use of cyclophosphamide priming improves CD34+ mobilization, and the introduction of blood CD34+ level optimizes the timing for harvest of stem cells, which should be performed early during treatment of malignancies.

Comparison of the peripheral blood leukocyte population between Japanese Black and Holstein calves.

PubMed

Ohtsuka, Hiromichi; Ono, Maiko; Saruyama, Yumi; Mukai, Machiko; Kohiruimaki, Masayuki; Kawamura, Seiichi

2011-02-01

Japanese black (JB) calves have greater susceptibility to infectious diseases compared to Holstein (Hol) calves. In order to clarify the differences in cellular immune status between JB and Hol calves, the leukocyte population and lymphocyte proliferative ability were analyzed. In total 200 healthy calves, 1 day to 14 weeks of age, were examined: 105 JB and 95 Hol calves. Lower numbers in peripheral blood and percentage in peripheral blood mononuclear cells of CD3(+)TcR1-N12(+) T cells and major histocompatibility complex class-II(+)CD14(-) B cells were observed in the JB compared to the Hol. The percentage of TcR1-N12(+)CD25(+) T cell in the JB was significantly lower than that of the Hol at 4-6, and 8-10 weeks. Interleukin (IL)-2 sensitivity in the JB was lower than that in the Hol, and significant differences were observed in age groups of 6-8 weeks and 10-14 weeks. These findings indicated that the lower numbers of γδ T cells and B cells in the JB compared to the Hol might be associated with the specificity of the immune systems in JB calves. © 2011 The Authors. Journal compilation © 2011 Japanese Society of Animal Science.

Preliminary study of the role of gastrointestinal endocrine cells in the maintenance of villous structure following X-irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wyatt, M.G.; Hume, S.P.; Carr, K.E.

The mechanism of gastrointestinal villous damage following ionizing irradiation is complex. Various compartments within the gastrointestinal tract have in turn been considered important for the maintenance of normal villous structure. To date, however, evidence for a single overriding regulator of epithelial well-being is lacking. In this study, the role of the gastro-intestinal (enteroendocrine) cells is explored and comparison made between endocrine cell number and villous structure. Experiments were organized using both control and irradiated groups of mice. Two time points (1 and 3 days) and three radiation doses (6, 10 and 18Gy) were employed. A simple method for endocrine cellmore » identification and subsequent quantification is described. Endocrine cell number was then compared with villous surface detail, as seen with a scanning electron microscope (SEM). Results indicated a decrease in the endocrine cell number at all three radiation doses. Whereas at low doses endocrine cell recovery occurred between 1 and 3 days, at medium and high doses further decline was noticed. A similar pattern was seen when considering villous surface structure. It is suggested that both scanning electron microscopy and endocrine cell number provide a more sensitive indicator of gastrointestinal radiation damage than do current crypt counting techniques. In addition, a link between endocrine cell number and villous structure is proposed.« less

Three-dimensional telomere architecture of esophageal squamous cell carcinoma: comparison of tumor and normal epithelial cells.

PubMed

Sunpaweravong, S; Sunpaweravong, P; Sathitruangsak, C; Mai, S

2016-05-01

Telomeres are repetitive nucleotide sequences (TTAGGG)n located at the ends of chromosomes that function to preserve chromosomal integrity and prevent terminal end-to-end fusions. Telomere loss or dysfunction results in breakage-bridge-fusion cycles, aneuploidy, gene amplification and chromosomal rearrangements, which can lead to genomic instability and promote carcinogenesis. Evaluating the hypothesis that changes in telomeres contribute to the development of esophageal squamous cell carcinoma (ESCC) and to determine whether there are differences between young and old patients, we compared the three-dimensional (3D) nuclear telomere architecture in ESCC tumor cells with that of normal epithelial cells obtained from the same patient. Patients were equally divided by age into two groups, one comprising those less than 45 years of age and the other consisting of those over 80 years of age. Tumor and normal epithelial cells located at least 10 cm from the border of the tumor were biopsied in ESCC patients. Hematoxylin and eosin staining was performed for each sample to confirm and identify the cancer and normal epithelial cells. This study was based on quantitative 3D fluorescence in situ hybridization (Q-FISH), 3D imaging and 3D analysis of paraffin-embedded slides. The 3D telomere architecture data were computer analyzed using 100 nuclei per slide. The following were the main parameters compared: the number of signals (number of telomeres), signal intensity (telomere length), number of telomere aggregates, and nuclear volume. Tumor and normal epithelial samples from 16 patients were compared. The normal epithelial cells had more telomere signals and higher intensities than the tumor cells, with P-values of P < 0.0001 and P = 0.0078, respectively. There were no statistically significant differences in the numbers of telomere aggregates or the nuclear volumes between the tumor and normal epithelial cells. Secondary analyses examined the effects of age on 3D telomere architecture and found no statistically significant differences in any parameter tested between the young and old patients in either the tumor or epithelial cells. The 3D nuclear telomeric signature was able to detect differences in telomere architecture between the ESCC and normal epithelial tissues. However, there were no differences observed between the young and old patients. © 2015 International Society for Diseases of the Esophagus.

[Reparative regeneration of rat skin under influence of hollow cathode lamp (HCL) with manganese and copper line spectrum emission].

PubMed

Mel'nikova, V I; Izvol'skaia, M S; Voronova, S N; Sharipova, M M; Rukin, E M; Zakharova, L A

2010-01-01

Influence of local light exposure by hollow cathode lamp with typical manganese and copper (HCL-Mn, Cu) line emission spectrum on posttraumatic regeneration rate of rat skin has been investigated. We performed the comparative analysis of the morphology and the differentiation ability of rat skin on the 15th and 24th days after full-thickness skin wound had been inflicted on rat dorsums. On the 15th day after injury, the experimental group (daily 30 s exposure for two weeks) showed scab loss, re-epithelialization, and hair regrowth, in contrast to the control rats, where scabs were still observed on the 24th day. Histological analysis revealed that in contrast to the control group the treatment with HCL-Mn, Cu resulted in the increased number of hair follicles and sebaceous glands, the decreased number of blood vessels and horizontal orientation of collagen fibers. The immunohistochemistry for OX-62 revealed that the number of dermal dendritic cells in the experimental groups was maximal on the 15th day, and then decreased to the 24th day after injury. The number of dermal dendritic cells was significantly lower in the control group. The immunohistochemistry for pan-keratins in the control animals revealed a high number of cells expressing different types of keratins, distributed in the main part of the epidermis on the 15th day after surgery, whereas in the experimental group the number of such cells was significantly lower and the cells were concentrated more close to the external part of the epidermis. The number of cells stained for keratin 19 was higher in the experimental group on the 15th day after surgery, whereas this number decreased in this group on the 24th day after surgery as compared to the control group. Thus, typical manganese and copper line spectrum emission emitted by hollow cathode lamp stimulates innate immunity, accelerates restoration of derma, skin epithelium and other skin derivates, and stimulates wound healing in general.

Manipulating memory CD8 T cell numbers by timed enhancement of IL-2 signals1

PubMed Central

Kim, Marie T.; Kurup, Samarchith P.; Starbeck-Miller, Gabriel R.; Harty, John T.

2016-01-01

Due to the growing burden of tumors and chronic infections, manipulating CD8 T cell responses for clinical use has become an important goal for immunologists. Here, we show that dendritic cell (DC) immunization coupled with relatively early (days 1–3) or late (days 4–6) administration of enhanced IL-2-signals both increase peak effector CD8 T cell numbers, but only early IL-2 signals enhance memory numbers. IL-2 signals delivered at relatively late time points drive terminal differentiation, marked Bim mediated contraction and do not increase memory T cell numbers. In contrast, early IL-2 signals induce effector cell metabolic profiles more conducive to memory formation. Of note, down-regulation of CD80 and CD86 was observed on DCs in vivo following early IL-2 treatment. Mechanistically, early IL-2 treatment enhanced CTLA-4 expression on regulatory T (Treg) cells, and CTLA-4 blockade alongside IL-2 treatment in vivo prevented the decrease in CD80 and CD86, supporting a cell-extrinsic role of CTLA-4 in down-regulating B7-ligand expression on DCs. Finally, DC immunization followed by early IL-2 treatment and αCTLA-4 blockade resulted in lower memory CD8 T cell numbers compared to the DC + early IL-2 treatment group. These data suggest that curtailed signaling through the B7-CD28 co-stimulatory axis during CD8 T cell activation limits terminal differentiation and preserves memory CD8 T cell formation and thus, should be considered in future T cell vaccination strategies. PMID:27439516

Ex-vivo expansion of red blood cells: How real for transfusion in humans?

PubMed Central

Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

2013-01-01

Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5 × 1012 cells vs 107 cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality. PMID:22177597

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples.

PubMed

Sivaganesan, Mano; Siefring, Shawn; Varma, Manju; Haugland, Richard A

2011-12-01

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from different studies by this approach, either a consistent source of calibrator cells must be used or the estimates must account for any differences in target sequence recoveries from different sources of calibrator cells. In this report we describe two methods for estimating target sequence recoveries from whole cell calibrator samples based on qPCR analyses of their serially diluted DNA extracts and most probable number (MPN) calculation. The first method employed a traditional MPN calculation approach. The second method employed a Bayesian hierarchical statistical modeling approach and a Monte Carlo Markov Chain (MCMC) simulation method to account for the uncertainty in these estimates associated with different individual samples of the cell preparations, different dilutions of the DNA extracts and different qPCR analytical runs. The two methods were applied to estimate mean target sequence recoveries per cell from two different lots of a commercially available source of enumerated Enterococcus cell preparations. The mean target sequence recovery estimates (and standard errors) per cell from Lot A and B cell preparations by the Bayesian method were 22.73 (3.4) and 11.76 (2.4), respectively, when the data were adjusted for potential false positive results. Means were similar for the traditional MPN approach which cannot comparably assess uncertainty in the estimates. Cell numbers and estimates of recoverable target sequences in calibrator samples prepared from the two cell sources were also used to estimate cell equivalent and target sequence quantities recovered from surface water samples in a comparative Ct method. Our results illustrate the utility of the Bayesian method in accounting for uncertainty, the high degree of precision attainable by the MPN approach and the need to account for the differences in target sequence recoveries from different calibrator sample cell sources when they are used in the comparative Ct method. Published by Elsevier B.V.

Model of Exploratory Search for Mating Partners by Fission Yeast

NASA Astrophysics Data System (ADS)

Hurwitz, Daniel; Bendezu, Felipe; Martin, Sophie; Vavylonis, Dimitrios

2014-03-01

During conditions of nitrogen starvation, the model eukaryote S. pombe (fission yeast) undergoes sexual sporulation. Because fission yeast are non-motile, contact between opposite mating types during spore formation is accomplished by polarizing growth, via the Rho GTP-ase Cdc42, in each mating type towards the selected mate, a process known as shmooing. Recent findings showed that cells pick one of their neighboring compatible mates by randomizing the position of the Cdc42 complex about the cell membrane, such that the complex is stabilized near areas of high concentration of the opposite mating type pheromone. We developed Monte Carlo simulations to model partner finding in populations of mating cells and in small cell clusters. We assume that pheromones are secreted at the site of Cdc42 accumulation and that the Cdc42 dwell time increases in response to increasing pheromone concentration. We measured the number of cells that succeed in successful reciprocal pairing, the number of cells that were unable to find a partner, and the number of cells that picked a partner already engaged with another cell. For optimal cell pairing, we find the pheromone concentration decay length is around 1 micron, of order the cell size. We show that non-linear response of Cdc42 dwell time to pheromone concentration improves the number of successful pairs for a given spatial cell distribution. We discuss how these results compare to non-exploratory pairing mechanisms.

The Distinctive Sensitivity to Microgravity of Immune Cell Subpopulations

NASA Astrophysics Data System (ADS)

Chen, Hui; Luo, Haiying; Liu, Jing; Wang, Peng; Dong, Dandan; Shang, Peng; Zhao, Yong

2015-11-01

Immune dysfunction in astronauts is well documented after spaceflights. Microgravity is one of the key factors directly suppressing the function of immune system. However, it is unclear which subpopulations of immune cells including innate and adaptive immune cells are more sensitive to microgravity We herein investigated the direct effects of modeled microgravity (MMg) on different immune cells in vitro. Mouse splenocytes, thymocytes and bone marrow cells were exposed to MMg for 16 hrs. The survival and the phenotypes of different subsets of immune cells including CD4+T cells, CD8+T cells, CD4+Foxp3+ regulatory T cells (Treg), B cells, monocytes/macrophages, dendritic cells (DCs), natural killer cells (NK) were determined by flow cytometry. After splenocytes were cultured under MMg for 16h, the cell frequency and total numbers of monocytes, macrophages and CD4+Foxp3+T cells were significantly decreased more than 70 %. MMg significantly decreased the cell numbers of CD8+ T cells, B cells and neutrophils in splenocytes. The cell numbers of CD4+T cells and NK cells were unchanged significantly when splenocytes were cultured under MMg compared with controls. However, MMg significantly increased the ratio of mature neutrophils to immature neutrophils in bone marrow and the cell number of DCs in splenocytes. Based on the cell survival ability, monocytes, macrophages and CD4+Foxp3+Treg cells are most sensitive to microgravity; CD4+T cells and NK cells are resistant to microgravity; CD8+T cells and neutrophils are impacted by short term microgravity exposure. Microgravity promoted the maturation of neutrophils and development of DCs in vitro. The present studies offered new insights on the direct effects of MMg on the survival and homeostasis of immune cell subsets.

5-Fluorouracil may enrich cancer stem cells in canine mammary tumor cells in vitro.

PubMed

Zhou, Bin; Jin, Yipeng; Zhang, Di; Lin, Degui

2018-05-01

Mammary gland carcinomas are the most common neoplasms in women and unsterilized female dogs. Owing to the existence of cancer stem cells (CSCs), chemotherapy is not able to cure these types of diseases completely. A number of studies have demonstrated that CSCs are resistant to chemotherapeutic drugs, but whether canine mammary tumor cells that have acquired resistance to 5-fluorouracil (5-FU) exhibited properties of CSCs remains unknown. The aim of the present study was to investigate whether 5-fluorouracil-resistant canine mammary tumor cells exhibited properties of CSCs. CSCs were analyzed using western blot assays, ultra-low attachment sphere cultures, flow cytometry and migration (wound healing and Transwell) assays. The results indicated that, compared with parental cells, proteins associated with the Wnt/β-catenin signaling pathway and aldehyde dehydrogenase 1 were overexpressed, the number and size of spheres in the 5-FU-resistant cells were increased, the ratio of CD44 + /CD24 -/low cells was increased and the migratory ability was improved in vitro compared with the 5-FU-susceptible cells. In conclusion, stimulation with chemotherapeutic drugs including 5-FU is a good method for increasing the proportion of canine mammary tumor stem cells in vitro , which may provide further understanding of chemotherapeutic methods and CSCs.

Distribution of intrahepatic T, NK and CD3(+)CD56(+)NKT cells alters after liver transplantation: Shift from innate to adaptive immunity?

PubMed

Werner, Jens M; Lang, Corinna; Scherer, Marcus N; Farkas, Stefan A; Geissler, Edward K; Schlitt, Hans J; Hornung, Matthias

2011-07-01

The liver is an immunological organ containing a large number of T, NK and NKT cells, but little is known about intrahepatic immunity after LTx. Here, we investigated whether the distribution of T, NK and CD3(+)CD56(+)NKT cells is altered in transplanted livers under different circumstances. Core biopsies of transplanted livers were stained with antibodies against CD3 and CD56. Several cell populations including T (CD3(+)CD56(-)), NK (CD3(-)CD56(+)) and NKT cells (CD3(+)CD56(+)) were studied by fluorescence microscopy. Cell numbers were analyzed in relation to the time interval after LTx, immunosuppressive therapy and stage of acute graft rejection (measured with the rejection activity index: RAI) compared to tumor free liver tissue from patients after liver resection due to metastatic disease as control. Recruitment of CD3(+)CD56(+)NKT cells revealed a significant decrease during high RAI scores in comparison to low and middle RAI scores (RAI 7-9: 0.03±0.01/HPF vs. RAI 4-6: 0.1±0.005/HPF). CD3(+)CD56(+)NKT cells were also lower during immunosuppressive therapy with tacrolimus (0.03±0.01/HPF) than with cyclosporine (0.1±0.003/HPF), cyclosporine/MMF (0.1±0.003/HPF) or sirolimus (0.1±0.01/HPF) treatment. Intrahepatic T cell numbers increased significantly 50days after LTx compared to control liver tissue (4.5±0.2/HPF vs. 1.9±0.1/HPF). In contrast, NK cells (0.3±0.004/HPF) were significantly fewer in all biopsies after LTx compared to the control (0.7±0.04/HPF). These data indicate significant alterations in the hepatic recruitment of T, NK and CD3(+)CD56(+)NKT cells after LTx. The increase in T cells and the decrease in NK and CD3(+)CD56(+)NKT cells suggest a shift from innate to adaptive hepatic immunity in the liver graft. Copyright © 2011 Elsevier B.V. All rights reserved.

Effects of Forskolin on Trefoil factor 1 expression in cultured ventral mesencephalic dopaminergic neurons.

PubMed

Jensen, P; Ducray, A D; Widmer, H R; Meyer, M

2015-12-03

Trefoil factor 1 (TFF1) belongs to a family of secreted peptides that are mainly expressed in the gastrointestinal tract. Notably, TFF1 has been suggested to operate as a neuropeptide, however, its specific cellular expression, regulation and function remain largely unknown. We have previously shown that TFF1 is expressed in developing and adult rat ventral mesencephalic tyrosine hydroxylase-immunoreactive (TH-ir) dopaminergic neurons. Here, we investigated the expression of TFF1 in rat ventral mesencephalic dopaminergic neurons (embryonic day 14) grown in culture for 5, 7 or 10 days in the absence (controls) or presence of either glial cell line-derived neurotrophic factor (GDNF), Forskolin or the combination. No TFF1-ir cells were identified at day 5 and only a few at day 7, whereas TH was markedly expressed at both time points. At day 10, several TFF1-ir cells were detected, and their numbers were significantly increased after the addition of GDNF (2.2-fold) or Forskolin (4.1-fold) compared to controls. Furthermore, the combination of GDNF and Forskolin had an additive effect and increased the number of TFF1-ir cells by 5.6-fold compared to controls. TFF1 expression was restricted to neuronal cells, and the percentage of TH/TFF1 co-expressing cells was increased to the same extent in GDNF and Forskolin-treated cultures (4-fold) as compared to controls. Interestingly, the combination of GDNF and Forskolin resulted in a significantly increased co-expression (8-fold) of TH/TFF1, which could indicate that GDNF and Forskolin targeted different subpopulations of TH/TFF1 neurons. Short-term treatment with Forskolin resulted in an increased number of TFF1-ir cells, and this effect was significantly reduced by the MEK1 inhibitor PD98059 or the protein kinase A (PKA) inhibitor H89, suggesting that Forskolin induced TFF1 expression through diverse signaling pathways. In conclusion, distinct populations of cultured dopaminergic neurons express TFF1, and their numbers can be increased by factors known to influence survival and differentiation of dopaminergic cells. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells.

PubMed

Burns, Jorge S; Harkness, Linda; Aldahmash, Abdullah; Gautier, Laurent; Kassem, Moustapha

2017-12-01

Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as "Limited" or "Significant". Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures. Copyright © 2017. Published by Elsevier B.V.

Psychosocial factors and T lymphocyte counts in Brazilian peacekeepers.

PubMed

Silva, Angela M Monteiro da; Speranza, Francisco A B; Ishii, Solange Kiyoko; Hirata, Raphael; Mattos-Guaraldi, Ana Luíza; Milagres, Lucimar Gonçalves

2015-02-01

To investigate the associations between psychosocial factors and peripheral blood CD4 and CD8 T lymphocyte numbers in Brazilian peacekeepers. Venous blood was collected from 759 peacekeepers who had just returned from a peace mission in Haiti. Among the 759 soldiers, 642 individuals completed the psychosocial measures. CD4 and CD8 T lymphocyte counts were measured by flow cytometry using a commercially available kit. Psychosocial factors, including military peace force stressors, clinical stress, anxiety and depression, were recorded. As a reference for T lymphocyte numbers, we measured T lymphocyte counts in 75 blood donors from the Instituto de Biologia do Exército, Rio de Janeiro. The median numbers of CD4 and CD8 T lymphocytes in the blood donors were 819 cells/µl and 496 cells/µl, respectively, with a CD4:CD8 ratio of 1.6. Significantly (p<0.05) lower CD4 T cell counts (759 cells/µl) were recorded for peacekeepers, with similar CD8 levels (548 cells/µl) and smaller CD4:CD8 ratios (1.3, p<0.001) compared to blood donors. These differences were due to a group of 14 military personnel with CD4 and CD8 medians of 308 and 266 cells/µl, respectively. Only one (7.1%) of these 14 individuals was diagnosed with clinical stress compared with 13.5% of the individuals with normal levels of CD4 T lymphocytes. One individual out of 628 (0.16%) had a Lipp's Stress Symptom Inventory score of 3, indicating near exhaustion. The prevalence of psychological disorders was low and there were no associations with CD4 or CD8 T cell numbers.

Psychosocial factors and T lymphocyte counts in Brazilian peacekeepers

PubMed Central

Monteiro da Silva, Angela M; Speranza, Francisco A B; Ishii, Solange Kiyoko; Hirata, Raphael; Mattos-Guaraldi, Ana Luíza; Milagres, Lucimar Gonçalves

2015-01-01

OBJECTIVE: To investigate the associations between psychosocial factors and peripheral blood CD4 and CD8 T lymphocyte numbers in Brazilian peacekeepers. METHODS: Venous blood was collected from 759 peacekeepers who had just returned from a peace mission in Haiti. Among the 759 soldiers, 642 individuals completed the psychosocial measures. CD4 and CD8 T lymphocyte counts were measured by flow cytometry using a commercially available kit. Psychosocial factors, including military peace force stressors, clinical stress, anxiety and depression, were recorded. As a reference for T lymphocyte numbers, we measured T lymphocyte counts in 75 blood donors from the Instituto de Biologia do Exército, Rio de Janeiro. RESULTS: The median numbers of CD4 and CD8 T lymphocytes in the blood donors were 819 cells/µl and 496 cells/µl, respectively, with a CD4:CD8 ratio of 1.6. Significantly (p<0.05) lower CD4 T cell counts (759 cells/µl) were recorded for peacekeepers, with similar CD8 levels (548 cells/µl) and smaller CD4:CD8 ratios (1.3, p<0.001) compared to blood donors. These differences were due to a group of 14 military personnel with CD4 and CD8 medians of 308 and 266 cells/µl, respectively. Only one (7.1%) of these 14 individuals was diagnosed with clinical stress compared with 13.5% of the individuals with normal levels of CD4 T lymphocytes. One individual out of 628 (0.16%) had a Lipp's Stress Symptom Inventory score of 3, indicating near exhaustion. CONCLUSION: The prevalence of psychological disorders was low and there were no associations with CD4 or CD8 T cell numbers. PMID:25789525

Intermittent PTH treatment can delay the transformation of mature osteoblasts into lining cells on the periosteal surfaces.

PubMed

Jang, Mi-Gyeong; Lee, Ji Yeon; Yang, Jae-Yeon; Park, Hyojung; Kim, Jung Hee; Kim, Jung-Eun; Shin, Chan Soo; Kim, Seong Yeon; Kim, Sang Wan

2016-09-01

Mature osteoblasts have three fates: as osteocytes, quiescent lining cells, or osteoblasts that undergo apoptosis. However, whether intermittent parathyroid hormone (PTH) can modulate the fate of mature osteoblasts in vivo is uncertain. We performed a lineage-tracing study using an inducible gene system. Dmp1-CreERt2 mice were crossed with Rosa26R reporter mice to obtain targeted mature osteoblasts and their descendants, lining cells or osteocytes, which were detected using X-gal staining. Rosa26R:Dmp1-CreERt2(+) mice were injected with 0.25 mg 4-OH-tamoxifen (4-OHTam) on postnatal days 5, 7, 9, 16, and 23. In a previous study, at 22 days after the last 4-OHTam, most LacZ+ cells on the periosteal surface were inactive lining cells. On day 25 (D25), the mice were challenged with an injection of human PTH (1-34, 80 μg/kg) or vehicle daily for 10 (D36) or 20 days (D46). We evaluated the number and thickness of LacZ+ osteoblast descendants in the calvaria and tibia. In the vehicle group, the number and thickness of LacZ+ osteoblast descendants at both D36 and D46 significantly decreased compared to D25, which was attenuated in the PTH group. In line with these results, PTH inhibited the decrease in the number of LacZ+/osteocalcin-positive cells compared to vehicle at both D36 and D46. As well, the serum levels of sclerostin decreased, as did the protein expression of sclerostin in the cortical bone. These results suggest that intermittent PTH treatment can increase the number of periosteal osteoblasts by preventing mature osteoblasts from transforming into lining cells in vivo.

Sub-fertile sperm cells exemplify telomere dysfunction.

PubMed

Biron-Shental, Tal; Wiser, Amir; Hershko-Klement, Anat; Markovitch, Ofer; Amiel, Aliza; Berkovitch, Arie

2018-01-01

The purpose of this study was to evaluate telomere homeostasis in sub-fertile compared to fertile human sperm. This observational, comparative study included 16 sub-fertile men who required intracytoplasmic sperm injection and 10 fertile men. At least 100 sperm cells from each participant were assessed. Main outcome measures were telomere length and telomere aggregates. Telomerase RNA component (TERC) copy number and telomere capture were assessed using fluorescence in situ hybridization technique and human telomerase reverse transcriptase (hTERT) using immunohistochemistry. Clinical backgrounds were similar. The percentage of sperm cells with shorter telomeres was higher among the sub-fertile compared to the fertile participants (3.3 ± 3.1 vs. 0.6 ± 1.2%, respectively; P < 0.005). The percentage of cells with telomere aggregates was significantly higher in the sub-fertile group (15.12 ± 3.73 vs. 4.73 ± 3.73%; P < 0.005). TERC gene copy number was similar between groups. The percentage of cells that were positive for hTERT was lower in the sub-fertile group (3.81 ± 1.27 vs. 8.42 ± 1.80%; P < 0.005). Telomere capture rates were higher among the sub-fertile sperm cells (P < 0.005). Sub-fertile sperm cells have short telomeres that are elongated by the alternative pathway of telomere capture. Dysfunctional telomeres may affect sperm fertilizability.

Mesenchymal stromal cell-mediated neuroprotection and functional preservation of retinal ganglion cells in a rodent model of glaucoma.

PubMed

Mead, Ben; Hill, Lisa J; Blanch, Richard J; Ward, Kelly; Logan, Ann; Berry, Martin; Leadbeater, Wendy; Scheven, Ben A

2016-04-01

Glaucoma is a leading cause of irreversible blindness involving loss of retinal ganglion cells (RGC). Mesenchymal stromal cells (MSC) have shown promise as a paracrine-mediated therapy for compromised neurons. It is, however, unknown whether dental pulp stem cells (DPSC) are effective as a cellular therapy in glaucoma and how their hypothesized influence compares with other more widely researched MSC sources. The present study aimed to compare the efficacy of adipose-derived stem cells, bone marrow-derived MSC (BMSC) and DPSC in preventing the loss of RGC and visual function when transplanted into the vitreous of glaucomatous rodent eyes. Thirty-five days after raised intraocular pressure (IOP) and intravitreal stem cell transplantation, Brn3a(+) RGC numbers, retinal nerve fibre layer thickness (RNFL) and RGC function were evaluated by immunohistochemistry, optical coherence tomography and electroretinography, respectively. Control glaucomatous eyes that were sham-treated with heat-killed DPSC had a significant loss of RGC numbers, RNFL thickness and function compared with intact eyes. BMSC and, to a greater extent, DPSC provided significant protection from RGC loss and RNFL thinning and preserved RGC function. The study supports the use of DPSC as a neuroprotective cellular therapy in retinal degenerative disease such as glaucoma. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

* Tissue-Specific Extracellular Matrix Enhances Skeletal Muscle Precursor Cell Expansion and Differentiation for Potential Application in Cell Therapy.

PubMed

Zhang, Deying; Zhang, Yong; Zhang, Yuanyuan; Yi, Hualin; Wang, Zhan; Wu, Rongpei; He, Dawei; Wei, Guanghui; Wei, Shicheng; Hu, Yun; Deng, Junhong; Criswell, Tracy; Yoo, James; Zhou, Yu; Atala, Anthony

2017-08-01

Skeletal muscle precursor cells (MPCs) are considered a key candidate for cell therapy in the treatment of skeletal muscle dysfunction due to injury, disease, or age. However, expansion of a sufficient number of functional skeletal muscle cells in vitro from a small tissue biopsy has been challenging due to changes in phenotypic expression of these cells under traditional culture conditions. Thus, the aim of the study was to develop a better culture system for the expansion and myo-differentiation of MPCs that could further be used for therapy. For this purpose, we developed an ideal method of tissue decellularization and compared the ability of different matrices to support MPC growth and differentiation. Porcine-derived skeletal muscle and liver and kidney extracellular matrix (ECM) were generated by decellularization methods consisting of distilled water, 0.2 mg/mL DNase, or 5% fetal bovine serum. Acellular matrices were further homogenized, dissolved, and combined with a hyaluronic acid-based hydrogel decorated with heparin (ECM-HA-HP). The cell proliferation and myogenic differentiation capacity of human MPCs were assessed when grown on gel alone, ECM, or each ECM-HA-HP substrate. Human MPC proliferation was significantly enhanced when cultured on the ECM-HA-HP substrates compared to the other substrates tested, with the greatest proliferation on the muscle ECM-HA-HP (mECM-HA-HP) substrate. The number of differentiated myotubes was significantly increased on the mECM-HA-HP substrate compared to the other gel-ECM substrates, as well as the numbers of MPCs expressing specific myogenic cell markers (i.e., myosin, desmin, myoD, and myf5). In conclusion, skeletal mECM-HA-HP as a culture substrate provided an optimal culture microenvironment potentially due to its similarity to the in vivo environment. These data suggest a potential use of skeletal muscle-derived ECM gel for the expansion and differentiation of human MPCs for cell-based therapy for skeletal muscle dysfunction.

Kinetics of the immune response profile in guinea pigs after vaccination with Mycobacterium bovis BCG and infection with Mycobacterium tuberculosis.

PubMed

Grover, Ajay; Taylor, Jennifer; Troudt, JoLynn; Keyser, Andrew; Arnett, Kimberly; Izzo, Linda; Rholl, Drew; Izzo, Angelo

2009-11-01

The guinea pig model of tuberculosis is used extensively in assessing novel vaccines, since Mycobacterium bovis BCG vaccination effectively prolongs survival after low-dose aerosol infection with virulent M. tuberculosis. To better understand how BCG extends time to death after pulmonary infection with M. tuberculosis, we examined cytokine responses postvaccination and recruitment of activated T cells and cytokine response postinfection. At 10 weeks postvaccination, splenic gamma interferon (IFN-gamma) mRNA was significantly elevated compared to the levels at 5 weeks in ex vivo stimulation assays. At 15, 40, 60, and 120 days postinfection, T-cell activation (CD4+ CD62Llow and CD8+ CD62Llow) and mRNA expression of IFN-gamma, tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-10, IL-12, and eomesodermin were assessed. Our data show that at day 40, BCG-vaccinated guinea pigs had significantly increased levels of IFN-gamma mRNA expression but decreased TNF-alpha mRNA expression in their lungs compared to the levels in nonvaccinated animals. At day 120, a time when nonvaccinated guinea pigs succumbed to infection, low levels of IFN-gamma mRNA were observed even though there were increasing levels of IL-1, IL-12, and IL-10, and the numbers of activated T cells did not differ from those in BCG-vaccinated animals. BCG vaccination conferred the advantage of recruiting greater numbers of CD4+ CD62Llow T cells at day 40, although the numbers of CD8+ CD62Llow T cells were not elevated compared to the numbers in nonvaccinated animals. Our data suggest that day 40 postinfection may be a pivotal time point in determining vaccine efficacy and prolonged survival and that BCG promotes the capacity of T cells in the lungs to respond to infection.

Prenatal Alcohol Exposure Affects Progenitor Cell Numbers in Olfactory Bulbs and Dentate Gyrus of Vervet Monkeys.

PubMed

Burke, Mark W; Inyatkin, Alexey; Ptito, Maurice; Ervin, Frank R; Palmour, Roberta M

2016-10-27

Fetal alcohol exposure (FAE) alters hippocampal cell numbers in rodents and primates, and this may be due, in part, to a reduction in the number or migration of neuronal progenitor cells. The olfactory bulb exhibits substantial postnatal cellular proliferation and a rapid turnover of newly formed cells in the rostral migratory pathway, while production and migration of postnatal neurons into the dentate gyrus may be more complex. The relatively small size of the olfactory bulb, compared to the hippocampus, potentially makes this structure ideal for a rapid analysis. This study used the St. Kitts vervet monkey ( Chlorocebus sabeus ) to (1) investigate the normal developmental sequence of post-natal proliferation in the olfactory bulb and dentate gyrus and (2) determine the effects of naturalistic prenatal ethanol exposure on proliferation at three different ages (neonate, five months and two years). Using design-based stereology, we found an age-related decrease of actively proliferating cells in the olfactory bulb and dentate gyrus for both control and FAE groups. Furthermore, at the neonatal time point, the FAE group had fewer actively proliferating cells as compared to the control group. These data are unique with respect to fetal ethanol effects on progenitor proliferation in the primate brain and suggest that the olfactory bulb may be a useful structure for studies of cellular proliferation.

Th17 cells and CD4(+) multifunctional T cells in patients with systemic lupus erythematosus.

PubMed

Araújo, Júlio Antônio Pereira; Mesquita, Danilo; de Melo Cruvinel, Wilson; Salmazi, Karina Inácio; Kallás, Esper Georges; Andrade, Luis Eduardo Coelho

2016-01-01

Recent evidence suggests that abnormalities involving Th17 lymphocytes are associated with the pathophysiology of systemic lupus erythematosus (SLE). In addition, multifunctional T cells (MFT), i.e., those producing multiple cytokines simultaneously, are present in the inflammatory milieu and may be implicated in the autoimmune process observed in SLE. In the present study, we aimed to characterize the functional status of CD4(+) T cells in SLE by simultaneously determining the concentration of IL-2, IFN-γ and IL-17 in lymphocyte cultures under exogenous and self-antigenic stimuli. Eighteen patients with active disease, 18 with inactive disease, and 14 healthy controls had functional status of CD4(+) T cells analyzed. We found that SLE patients presented a decreased number of total CD4(+) cells, an increased number of activated T cells, and an increased frequency of Th17 cells compared to healthy controls (HC). MFT cells had increased frequency in SLE patients and there was an increased frequency of tri-functional MFT in patients with active SLE compared with those with inactive SLE. Interestingly, MTF cells produced larger amounts of IFNγ than mono-functional T cells in patients and controls. Taken together these data indicate the participation of recently activated Th17 cells and MTF cells in the SLE pathophysiology. Copyright © 2015 Elsevier Editora Ltda. All rights reserved.

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays.

PubMed

Raghavan, Shreya; Ward, Maria R; Rowley, Katelyn R; Wold, Rachel M; Takayama, Shuichi; Buckanovich, Ronald J; Mehta, Geeta

2015-07-01

Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant 3D in vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Spheroids had uniform geometry, with projected areas (42.60×10(3)μm-475.22×10(3)μm(2) for A2780 spheroids and 37.24×10(3)μm(2)-281.01×10(3)μm(2) for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell-cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70-80% viability) to cisplatin chemotherapy compared to 2D cultures (30-50% viability). Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. Copyright © 2015 Elsevier Inc. All rights reserved.

Gut-derived factors promote neurogenesis of CNS-neural stem cells and nudge their differentiation to an enteric-like neuronal phenotype.

PubMed

Kulkarni, Subhash; Zou, Bende; Hanson, Jesse; Micci, Maria-Adelaide; Tiwari, Gunjan; Becker, Laren; Kaiser, Martin; Xie, Xinmin Simon; Pasricha, Pankaj Jay

2011-10-01

Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.

Efficiency of donor cell preparation and recipient oocyte source for production of transgenic cloned dairy goats harboring human lactoferrin.

PubMed

Wan, Yong-Jie; Zhang, Yan-Li; Zhou, Zheng-Rong; Jia, Ruo-Xin; Li, Meng; Song, Hui; Wang, Zi-Yu; Wang, Li-Zhong; Zhang, Guo-Min; You, Ji-Hao; Wang, Feng

2012-08-01

The objective was to investigate the effects of the transgenic donor cell synchronization method, oocyte sources, and other factors, on production of hLF-gene nucleus transfer dairy goats. Three transfected cell lines from ear biopsies from three 3-mo-old Saanen dairy goats (designated Number 1, Number 2, and Number 3, respectively) were selected as karyoplast donors for somatic cell nuclear transfer (SCNT) after detailed identification (including PCR and sequencing of PCR products). In donor cell cycle synchronization studies, the apoptosis rate of hLF transgenic fibroblasts was not different (P > 0.05) after 3 days of serum starvation or 2 days of contact inhibition. Additionally, there was no effect (P > 0.05) on developmental capacity of reconstructed embryos; however, the kidding rate of recipients in the serum starvation group was higher than that in the contact inhibition group (18 vs. 0%, respectively). The production efficiency of the transgenic cloned goats using donor cells from the Number 1 dairy goat cell line was higher than those using the Number 2 and the Number 3 cell lines (kidding rates were 18, 2, and 0%, respectively, P < 0.05). The oocyte source did not significantly affect the pregnancy rate of hLF-transgenic cloned dairy goats, but more fetuses were aborted when using in vitro matured oocytes compared to in vivo matured oocytes. In summary, utilizing transfected 3-mo-old dairy goat fibroblasts as donor cells, seven live offspring were produced, and the hLF gene was successfully integrated. This study provided additional insights into preparation of donor cells and recipient oocytes for producing transgenic cloned goats through SCNT. Copyright © 2012 Elsevier Inc. All rights reserved.

Decreased natural killer cell activity in atopic eczema.

PubMed Central

Hall, T J; Rycroft, R; Brostoff, J

1985-01-01

We have studied NK cell activity in atopic and non-atopic subjects using a standard 51Cr-release assay and K562 target cells. In atopics (AT) with allergic rhinitis and/or asthma, NK cell activity was similar to that in non-atopic (N) subjects, whilst patients with severe atopic eczema (AE) had depressed NK cell activity compared to AT or N subjects. In addition, circulating T-cell numbers and Con A responsiveness was decreased in AE, although neither parameter was correlated with decreased NK cell activity. However, decreased NK cell activity in atopic eczema was positively correlated with decreased numbers of Fc gamma + lymphocytes (P = 0.01) and decreased effector: target cell binding (P = 0.05), and negatively correlated with increased monocytes in AE (P = 0.09). AE NK cell activity was equally or more sensitive to the inhibitory effects of drugs such as dibutyryl cyclic AMP, prostaglandins (PG) D2,E2 and histamine. The relative percentage increase in NK cell activity by the interferon inducer poly I:C was similar in AE patients and controls. The results suggest that reduced numbers of circulating NK cells and pre-NK cells account for the depressed level of NK cell activity in subjects with severe atopic eczema. PMID:3876984

Interactions between IGF-I, estrogen receptor-α (ERα), and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells.

PubMed

Mendoza, Rhone A; Enriquez, Marlene I; Mejia, Sylvia M; Moody, Emily E; Thordarson, Gudmundur

2011-01-01

Understanding of the interactions between estradiol (E₂) and IGF-I is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating noninterfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions, and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human GH plus epidermal growth factor, but E₂ did not cause an increase in the number of the IGF-IR.low cells compared to controls. The proliferation rate of IGF-IR.low cells was only reduced in response to E₂ compared to controls, whereas their basal and hormone-stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E₂, without affecting control cells. Furthermore, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. In conclusion, suppressing IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK, which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate.

Estrogen deficiency heterogeneously affects tissue specific stem cells in mice

PubMed Central

Kitajima, Yuriko; Doi, Hanako; Ono, Yusuke; Urata, Yoshishige; Goto, Shinji; Kitajima, Michio; Miura, Kiyonori; Li, Tao-Sheng; Masuzaki, Hideaki

2015-01-01

Postmenopausal disorders are frequently observed in various organs, but their relationship with estrogen deficiency and mechanisms remain unclear. As tissue-specific stem cells have been found to express estrogen receptors, we examined the hypothesis that estrogen deficiency impairs stem cells, which consequently contributes to postmenopausal disorders. Six-week-old C57BL/6 female mice were ovariectomized, following which they received 17β-estradiol replacement or vehicle (control). Sham-operated mice were used as healthy controls. All mice were killed for evaluation 2 months after treatments. Compared with the healthy control, ovariectomy significantly decreased uterine weight, which was partially recovered by 17β-estradiol replacement. Ovariectomy significantly increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, but impaired their capacity to grow mixed cell-type colonies in vitro. Estrogen replacement further increased the numbers of c-kit-positive hematopoietic stem/progenitor cells in bone marrow, without significantly affecting colony growth in vitro. The number of CD105-positive mesenchymal stem cells in bone marrow also significantly decreased after ovariectomy, but completely recovered following estrogen replacement. Otherwise, neither ovariectomy nor estrogen replacement changed the number of Pax7-positive satellite cells, which are a skeletal muscle-type stem cell. Estrogen deficiency heterogeneously affected tissue-specific stem cells, suggesting a likely and direct relationship with postmenopausal disorders. PMID:26245252

Mobilization of Peripheral Blood Stem Cells and Changes in the Concentration of Plasma Factors Influencing their Movement in Patients with Panic Disorder.

PubMed

Jabłoński, Marcin; Mazur, Jolanta Kucharska; Tarnowski, Maciej; Dołęgowska, Barbara; Pędziwiatr, Daniel; Kubiś, Ewa; Budkowska, Marta; Sałata, Daria; Wysiecka, Justyna Pełka; Kazimierczak, Arkadiusz; Reginia, Artur; Ratajczak, Mariusz Z; Samochowiec, Jerzy

2017-04-01

In this paper we examined whether stem cells and factors responsible for their movement may serve as new biological markers of anxiety disorders. The study was carried out on a group of 30 patients diagnosed with panic disorder (examined before and after treatment), compared to 30 healthy individuals forming the control group. We examined the number of circulating HSCs (hematopoetic stem cells) (Lin-/CD45 +/CD34 +) and HSCs (Lin-/CD45 +/AC133 +), the number of circulating VSELs (very small embryonic-like stem cells) (Lin-/CD45-/CD34 +) and VSELs (Lin-/CD45-/AC133 +), as well as the concentration of complement components: C3a, C5a and C5b-9, SDF-1 (stromal derived factor) and S1P (sphingosine-1-phosphate). Significantly lower levels of HSCs (Lin-/CD45 +/AC133 +) have been demonstrated in the patient group compared to the control group both before and after treatment. The level of VSELs (Lin-/CD45-/CD133 +) was significantly lower in the patient group before treatment as compared to the patient group after treatment.The levels of factors responsible for stem cell movement were significantly lower in the patient group compared to the control group before and after treatment. It was concluded that the study of stem cells and factors associated with their movement can be useful in the diagnostics of panic disorder, as well as differentiating between psychotic and anxiety disorders.

Plotting Lightning-Stroke Data

NASA Technical Reports Server (NTRS)

Tatom, F. B.; Garst, R. A.

1986-01-01

Data on lightning-stroke locations become easier to correlate with cloudcover maps with aid of new graphical treatment. Geographic region divided by grid into array of cells. Number of lightning strokes in each cell tabulated, and value representing density of lightning strokes assigned to each cell. With contour-plotting routine, computer draws contours of lightning-stroke density for region. Shapes of contours compared directly with shapes of storm cells.

VGF expression by T lymphocytes in patients with Alzheimer's disease

PubMed Central

Glorius, Sarah; Dobrowolny, Henrik; Greiner-Bohl, Sabrina; Mawrin, Christian; Bommhardt, Ursula; Hartig, Roland; Bogerts, Bernhard; Busse, Mandy

2015-01-01

Secretion of VGF is increased in cerebrospinal fluid and blood in neurodegenerative disorders like Alzheimer's disease (AD) and VGF is a potential biomarker for these disorders. We have shown that VGF is expressed in peripheral T cells and is correlated with T cell survival and cytokine secretion. The frequency of VGF+CD3+ T cells increases with normal aging. We found an increased number of VGF-expressing T cells in patients with AD compared to aged healthy controls, which was associated with enhanced HbA1c levels in blood. Upon treatment with rivastigmine, T cell proliferation and VGF expression in AD patients decreased to the level found in controls. Moreover, rapamycin treatment in vitro reduced the number of VGF+CD3+ cells in AD patients to control levels. PMID:26142708

Central Memory CD4+ T Cells Are Responsible for the Recombinant Bacillus Calmette-Guérin ΔureC::hly Vaccine's Superior Protection Against Tuberculosis

PubMed Central

Vogelzang, Alexis; Perdomo, Carolina; Zedler, Ulrike; Kuhlmann, Stefanie; Hurwitz, Robert; Gengenbacher, Martin; Kaufmann, Stefan H. E.

2014-01-01

Bacillus Calmette-Guérin (BCG) has been used for vaccination against tuberculosis for nearly a century. Here, we analyze immunity induced by a live tuberculosis vaccine candidate, recombinant BCG ΔureC::hly vaccine (rBCG), with proven preclinical and clinical safety and immunogenicity. We pursue in-depth analysis of the endogenous mycobacteria-specific CD4+ T-cell population, comparing the more efficacious rBCG with canonical BCG to determine which T-cell memory responses are prerequisites for superior protection against tuberculosis. rBCG induced higher numbers and proportions of antigen-specific memory CD4+ T cells than BCG, with a CXCR5+CCR7+ phenotype and low expression of the effector transcription factors T-bet and Bcl-6. We found that the superior protection of rBCG, compared with BCG, correlated with higher proportions and numbers of these central memory T cells and of T follicular helper cells associated with specific antibody responses. Adoptive transfer of mycobacteria-specific central memory T cells validated their critical role in protection against pulmonary tuberculosis. PMID:24943726

Severe pulmonary metastasis in obese and diabetic mice.

PubMed

Mori, Akinori; Sakurai, Hiroaki; Choo, Min-Kyung; Obi, Ryosuke; Koizumi, Keiichi; Yoshida, Chiho; Shimada, Yutaka; Saiki, Ikuo

2006-12-15

Although obesity is known as a risk factor for several human cancers, the association of obesity with cancer recurrence and metastasis remains to be characterized. Here, B16-BL6 melanoma and Lewis lung carcinoma cells were intravenously injected into diabetic (db/db) and obese (ob/ob) mice. The number of experimental lung colonies was markedly promoted in these mice when compared with C57BL/6 mice. In contrast, tumor growth at the implanted site was comparable when cells were inoculated orthotopically. The use of B16-BL6 cells stably transfected with the luciferase gene revealed that the increased metastasis reflected a difference mainly within 6 hr after the intravenous inoculation of tumor cells. Administration of recombinant leptin in ob/ob mice abolished the increase in metastasis early on as well as the decrease in the splenic NK cell number. In addition, depletion of NK cells by an anti-asialo-GM1 antibody abrogated the enhanced metastasis in db/db mice. These results demonstrate that metastasis is markedly promoted in diabetic and obese mice mainly because of decreased NK cell function during the early phase of metastasis. Copyright 2006 Wiley-Liss, Inc.

Abnormalities in human pluripotent cells due to reprogramming mechanisms

PubMed Central

Ma, Hong; Morey, Robert; O’Neil, Ryan C.; He, Yupeng; Daughtry, Brittany; Schultz, Matthew D.; Hariharan, Manoj; Nery, Joseph R.; Castanon, Rosa; Sabatini, Karen; Thiagarajan, Rathi D.; Tachibana, Masahito; Kang, Eunju; Tippner-Hedges, Rebecca; Ahmed, Riffat; Gutierrez, Nuria Marti; Van Dyken, Crystal; Polat, Alim; Sugawara, Atsushi; Sparman, Michelle; Gokhale, Sumita; Amato, Paula; Wolf, Don P.; Ecker, Joseph R.; Laurent, Louise C.; Mitalipov, Shoukhrat

2016-01-01

Human pluripotent stem cells hold potential for regenerative medicine, but available cell types have significant limitations. Although embryonic stem cells (ES cells) from in vitro fertilized embryos (IVF ES cells) represent the ‘gold standard’, they are allogeneic to patients. Autologous induced pluripotent stem cells (iPS cells) are prone to epigenetic and transcriptional aberrations. To determine whether such abnormalities are intrinsic to somatic cell reprogramming or secondary to the reprogramming method, genetically matched sets of human IVF ES cells, iPS cells and nuclear transfer ES cells (NT ES cells) derived by somatic cell nuclear transfer (SCNT) were subjected to genome-wide analyses. Both NT ES cells and iPS cells derived from the same somatic cells contained comparable numbers of de novo copy number variations. In contrast, DNA methylation and transcriptome profiles of NT ES cells corresponded closely to those of IVF ES cells, whereas iPS cells differed and retained residual DNA methylation patterns typical of parental somatic cells. Thus, human somatic cells can be faithfully reprogrammed to pluripotency by SCNT and are therefore ideal for cell replacement therapies. PMID:25008523

Formation of stable small cell number three-dimensional ovarian cancer spheroids using hanging drop arrays for preclinical drug sensitivity assays

PubMed Central

Raghavan, Shreya; Ward, Maria R.; Rowley, Katelyn R.; Wold, Rachel M.; Takayama, Shuichi; Buckanovich, Ronald J.; Mehta, Geeta

2015-01-01

Background Ovarian cancer grows and metastasizes from multicellular spheroidal aggregates within the ascites fluid. Multicellular tumor spheroids are therefore physiologically significant3Din vitro models for ovarian cancer research. Conventional hanging drop cultures require high starting cell numbers, and are tedious for long-term maintenance. In this study, we generate stable, uniform multicellular spheroids using very small number of ovarian cancer cells in a novel 384 well hanging drop array platform. Methods We used novel tumor spheroid platform and two ovarian cancer cell lines (A2780 and OVCAR3) to demonstrate the stable incorporation of as few as 10 cells into a single spheroid. Results Spheroids had uniform geometry, with projected areas (42.60 × 103 μm–475.22 × 103 μm2 for A2780 spheroids and 37.24 × 103 μm2–281.01 × 103 μm2 for OVCAR3 spheroids) that varied as a function of the initial cell seeding density. Phalloidin and nuclear stains indicated cells formed tightly packed spheroids with demarcated boundaries and cell–cell interaction within spheroids. Cells within spheroids demonstrated over 85% viability. 3D tumor spheroids demonstrated greater resistance (70–80% viability) to cisplatin chemotherapy compared to 2D cultures (30–50% viability). Conclusions Ovarian cancer spheroids can be generated from limited cell numbers in high throughput 384 well plates with high viability. Spheroids demonstrate therapeutic resistance relative to cells in traditional 2D culture. Stable incorporation of low cell numbers is advantageous when translating this research to rare patient-derived cells. This system can be used to understand ovarian cancer spheroid biology, as well as carry out preclinical drug sensitivity assays. PMID:25913133

ACEA (a highly selective cannabinoid CB1 receptor agonist) stimulates hippocampal neurogenesis in mice treated with antiepileptic drugs.

PubMed

Andres-Mach, Marta; Haratym-Maj, Agnieszka; Zagaja, Miroslaw; Rola, Radoslaw; Maj, Maciej; Chrościńska-Krawczyk, Magdalena; Luszczki, Jarogniew J

2015-10-22

Hippocampal neurogenesis plays a very important role in learning and memory functions. In a search for best neurological drugs that protect neuronal cells and stimulate neurogenesis with no side effects, cannabinoids proved to be a strong group of substances having many beneficial properties. The aim of this study was to evaluate the impact of ACEA (arachidonyl-2'-chloroethylamide--a highly selective cannabinoid CB1 receptor agonist) combined with a classical antiepileptic drug sodium valproate (VPA) on neural precursor cells' proliferation and differentiation in the mouse brain. All experiments were performed on adolescent CB57/BL male mice injected i.p. with VPA (10mg/kg), ACEA (10mg/kg) and PMSF (30 mg/kg) (phenylmethylsulfonyl fluoride--a substance protecting ACEA against degradation by the fatty-acid amidohydrolase) for 10 days. Next an acute response of proliferating neural precursor cells to ACEA and VPA administration was evaluated with Ki-67 staining (Time point 1). Next, in order to determine whether acute changes translated into long-term alterations in neurogenesis, proliferating cells were labeled with 5-bromo-2deoxyuridine (BrdU) followed by confocal microscopy used to determine the percentage of BrdU-labeled cells that showed mature cell phenotypes (Time point 2). Results indicate that ACEA with PMSF significantly increase the total number of Ki-67-positive cells when compared to the control group. Moreover, ACEA in combination with VPA increased the number of Ki-67-positive cells, whereas VPA administered alone had no impact on proliferating cells' population. Accordingly, neurogenesis study results indicate that the combination of ACEA+PMSF administered alone and in combination with VPA considerably increases the total number of BrdU-positive cells in comparison to the control group while ACEA+PMSF alone and in combination with VPA increased total numbers of BrdU-positive cells, newly born neurons and astrocytes as compared to VPA group but not to the control group. VPA administered alone decreased the number of newly born neurons with no significant impact on neurogenesis. These data provide substantial evidence that VPA administered chronically slightly decreases the proliferation and differentiation of newly born cells while combination of VPA+ACEA significantly increases the level of newborn neurons in the dentate subgranular zone. Copyright © 2015 Elsevier B.V. All rights reserved.

High Intensity Interval Training Increases Natural Killer Cell Number and Function in Obese Breast Cancer-challenged Mice and Obese Women.

PubMed

Barra, Nicole G; Fan, Isabella Y; Gillen, Jenna B; Chew, Marianne; Marcinko, Katarina; Steinberg, Gregory R; Gibala, Martin J; Ashkar, Ali A

2017-12-01

High intensity interval training (HIIT) boosts natural killer (NK) cell number and activity in normal weight breast cancer patients; however, whether this occurs in obese individuals is not well established. The goal of this study was to determine whether HIIT effectively boosts NK cells as a therapeutic strategy against breast cancer in an obese mouse model and in overweight/obese women. Diet induced female C57Bl/6 obese mice were assigned to undergo HIIT for four weeks or remain sedentary. Female participants were subjected to a six weeks HIIT protocol. HIIT mice acclimatized to treadmill running were subsequently injected with 5 × 10 5 polyoma middle T (MT) breast cancer cells intravenously. NK cell number and activation were monitored using flow cytometry, and tumor burden or lipid content evaluated from histological lung and liver tissues, respectively. In both mice and humans, circulating NK cell number and activation (CD3-NK1.1+CD27+ and CD3-CD56+, respectively) markedly increased immediately after HIIT. HIIT obese mice had reduced lung tumor burden compared to controls following MT challenge, and had diminished hepatic lipid deposition despite minimal body weight loss. Our findings demonstrate that HIIT can benefit obese individuals by enhancing NK cell number and activity, reducing tumor burden, and enhancing metabolic health.

High Intensity Interval Training Increases Natural Killer Cell Number and Function in Obese Breast Cancer-challenged Mice and Obese Women

PubMed Central

Barra, Nicole G.; Fan, Isabella Y.; Gillen, Jenna B.; Chew, Marianne; Marcinko, Katarina; Steinberg, Gregory R.; Gibala, Martin J.; Ashkar, Ali A.

2017-01-01

High intensity interval training (HIIT) boosts natural killer (NK) cell number and activity in normal weight breast cancer patients; however, whether this occurs in obese individuals is not well established. The goal of this study was to determine whether HIIT effectively boosts NK cells as a therapeutic strategy against breast cancer in an obese mouse model and in overweight/obese women. Diet induced female C57Bl/6 obese mice were assigned to undergo HIIT for four weeks or remain sedentary. Female participants were subjected to a six weeks HIIT protocol. HIIT mice acclimatized to treadmill running were subsequently injected with 5 × 105 polyoma middle T (MT) breast cancer cells intravenously. NK cell number and activation were monitored using flow cytometry, and tumor burden or lipid content evaluated from histological lung and liver tissues, respectively. In both mice and humans, circulating NK cell number and activation (CD3−NK1.1+CD27+ and CD3−CD56+, respectively) markedly increased immediately after HIIT. HIIT obese mice had reduced lung tumor burden compared to controls following MT challenge, and had diminished hepatic lipid deposition despite minimal body weight loss. Our findings demonstrate that HIIT can benefit obese individuals by enhancing NK cell number and activity, reducing tumor burden, and enhancing metabolic health. PMID:29302585

Experimental investigation of recirculating cells in laminar coaxial jets.

NASA Technical Reports Server (NTRS)

Warpinski, N. R.; Nagib, H. M.; Lavan, Z.

1972-01-01

Utilizing several unique means of introducing smoke into the flow field for careful visualization in addition to hot-wire techniques, experiments are performed in a specially designed facility producing laminar flows up to considerably high Reynolds numbers. Characteristics of the cells and the flow conditions that bring them about are documented by smoke photographs in the Reynolds number velocity ratio plane and the results are compared to previous analytical predictions. The cells are found to fall into three categories with different flow characteristics involving unsteadiness in position, and shear layer instabilities which result in higher mixing with the outer streams.-

MORPHOLOGICAL AND CULTURAL COMPARISON OF MICROORGANISMS IN SURFACE SOIL AND SUBSURFACE SEDIMENTS AT A PRISTINE STUDY SITE IN OKLAHOMA (JOURNAL VERSION)

EPA Science Inventory

Surface-soil and subsurface microfloras at the site of a shallow aquifer in Oklahoma were examined and compared with respect to (1) total and viable cell numbers, (2) colony and cell types that grew on various plating media, (3) cell morphologies seen in flotation films stripped ...

Immunohistowax processing, a new fixation and embedding method for light microscopy, which preserves antigen immunoreactivity and morphological structures: visualisation of dendritic cells in peripheral organs

PubMed Central

Pajak, B.; De Smedt, T.; Moulin, V.; De Trez, C.; Maldonado-Lopez, R.; Vansanten, G.; Briend, E.; Urbain, J.; Leo, O.; Moser, M.

2000-01-01

Aims—To describe a new fixation and embedding method for tissue samples, immunohistowax processing, which preserves both morphology and antigen immunoreactivity, and to use this technique to investigate the role of dendritic cells in the immune response in peripheral tissues. Methods—This technique was used to stain a population of specialised antigen presenting cells (dendritic cells) that have the unique capacity to sensitise naive T cells, and therefore to induce primary immune responses. The numbers of dendritic cells in peripheral organs of mice either untreated or injected with live Escherichia coli were compared. Results—Numbers of dendritic cells were greatly decreased in heart, kidney, and intestine after the inoculation of bacteria. The numbers of dendritic cells in the lung did not seem to be affected by the injection of E coli. However, staining of lung sections revealed that some monocyte like cells acquired morphological and phenotypic features of dendritic cells, and migrated into blood vessels. Conclusions—These observations suggest that the injection of bacteria induces the activation of dendritic cells in peripheral organs, where they play the role of sentinels, and/or their movement into lymphoid organs, where T cell priming is likely to occur. Key Words: dendritic cell • Escherichia coli • immunohistochemistry PMID:10961175

The correlation between NK cell and liver function in patients with primary hepatocellular carcinoma.

PubMed

Sha, Wei Hong; Zeng, Xiao Hui; Min, Lu

2014-05-01

This study aimed to detect the expression of natural killer (NK) cell receptor natural killer group 2D (NKG2D) in the peripheral blood of patients with primary hepatocellular carcinoma and to discuss the correlation between NK cell cytotoxicity and liver function. The number of NK cells and the expression of NK cell receptor NKG2D in peripheral blood were determined by flow cytometry in patients with primary hepatocellular carcinoma, hepatitis B cirrhosis, chronic hepatitis B, and healthy controls. When compared with patients in the healthy and the chronic hepatitis B groups, the primary hepatocellular carcinoma group showed significant decreases in all parameters, including the cytotoxicity of NK cells on K562 cells, expression rate of NKG2D in NK cells, number of NKG2D(+) NK cells, expression level of NKG2D, and number of NK cells (p<0.05). The activity of NK cells showed a positive correlation, whereas the Child-Pugh scores in the primary hepatocellular carcinoma and the hepatitis B cirrhosis groups showed a negative correlation with all parameters detected above. The decrease of NK cell activity in patients with primary hepatocellular carcinoma is closely related to their lower expression of NKG2D. Liver function affects the expression of NKG2D and the activity of NK cells.

Characterization of cell proliferation throughout the brain of the African cichlid fish Astatotilapia burtoni and its regulation by social status.

PubMed

Maruska, Karen P; Carpenter, Russ E; Fernald, Russell D

2012-10-15

New cells are added in the brains of all adult vertebrates, but fishes have some of the greatest potential for neurogenesis and gliogenesis among all taxa, partly due to their indeterminate growth. Little is known, however, about how social interactions influence cell proliferation in the brain of these fishes that comprise the largest group of vertebrates. We used 5-bromo-2'-deoxyuridine (BrdU) to identify and localize proliferation zones in the telencephalon, diencephalon, mesencephalon, and rhombencephalon that were primarily associated with ventricular surfaces in the brain of the African cichlid fish Astatotilapia burtoni. Cell migration was evident in some regions by 1 day post injection, and many newborn cells coexpressed the neuronal marker HuC/D at 30 days, suggesting they had differentiated into neurons. To test the hypothesis that social status and perception of an opportunity to rise in rank influenced cell proliferation, we compared numbers of BrdU-labeled cells in multiple brain nuclei among fish of different social status. Socially suppressed subordinate males had the lowest numbers of proliferating cells in all brain regions examined, but males that were given an opportunity to rise in status had higher cell proliferation rates within 1 day, suggesting rapid upregulation of brain mitotic activity associated with this social transition. Furthermore, socially isolated dominant males had similar numbers of BrdU-labeled cells compared with dominant males that were housed in a socially rich environment, suggesting that isolation has little effect on proliferation and that reduced proliferation in subordinates is a result of the social subordination. These results suggest that A. burtoni will be a useful model to analyze the mechanisms of socially induced neurogenesis in vertebrates. Copyright © 2012 Wiley Periodicals, Inc.

Effect of Early Neonatal Exposure to Deltamethrin on the Purkinje Cell Number in Rat Cerebellum

PubMed Central

Asari, Mohd Asnizam; Abdullah, Mohammad Shukri; Abdullah, Suryati

2008-01-01

Deltamethrin is a widely used insecticide belonging to the class of pyrethroid. Although the neurotoxicity of pyrethroids including deltamethrin is well established, it is still unclear whether exposure to deltamethrin during neonatal period has any deleterious effect on the survival of the Purkinje cells in the cerebellum. In the study, we investigated the total number of Purkinje cells in experimental rats exposed to deltamethrin using a stereological method, the fractionator. Deltamethrin in a dose of 1 mg/kg/day (corresponds to 20% of LD50 ) was administered through oral gavage to male pups from 2nd to 5th postnatal day (PND). At PND 21 the animals were sacrificed and their cerebelli were removed. The cerebelli were systematically sampled using the fractionator method and stained with cresyl fast violet. The number of the Purkinje cells was counted for each cerebellum. The results showed that there was no significant difference in the total number of Purkinje cells in the deltamethrin-treated group as compared to the control animals. This suggests that deltamethrin exposure at the current dosage during the neonatal period do not have any significant effect on the survival of the Purkinje cells in the cerebellum. PMID:22570585

The Effect of Taraxacum officinale Hydroalcoholic Extract on Blood Cells in Mice

PubMed Central

Modaresi, Mehrdad; Resalatpour, Narges

2012-01-01

Objectives. Dandelion (Taraxacum officinale) is a herbaceous perennial plant of the family Asteraceae and has medicinal and culinary uses. Dandelion has been used as a remedy for anemia, purifing the blood, and providing immune modulation. Therefore, the aim of this study was to investigate the effect of hydro alcoholic extract on blood cells in mice. Methods. Five groups each including ten adult female (Balb/C) mice weighing 30 ± 5 g were chosen. Normal saline was administered as placebo for group, and dandelion hydro alcoholic extract in doses of 50,100, and 200 mg/kg was injected intraperitoneally for 20 days to test groups and the last group was control group.WBC, RBC, HB, HCT, platelet, and other cells were measured with automated cell counter. Main Results. The number of RBC and the rate of HB in three doses of 100 and 200 mg/kg significantly increased (P < 0.05). As compared with control group, the number of WBC in three doses of 50, 100, and 200 mg/kg increased, but it was significantly in 200 mg/kg dandelion treated group as compared with control group(P < 0.05). The rate of platelet in three doses of 50, 100 and 200 mg/kg significantly decreased as compared with control group (P < 0.01). 3 doses of dandelion increased lymphocyte numbers significantly compared with controls. Conclusion. The study indicates efficacy of dandelion extract on RBC and HB in doses of 50, 100, and 200 mg/kg and in 200 mg/kg on WBC to achieve normal body balance. PMID:22844289

Regulatory T Cells in Peripheral Blood and Cerebrospinal Fluid of Syphilis Patients with and without Neurological Involvement

PubMed Central

Li, Kang; Wang, Cuini; Lu, Haikong; Gu, Xin; Guan, Zhifang; Zhou, Pingyu

2013-01-01

Background Syphilis, a sexually transmitted disease caused by spirochetal bacterium Treponema pallidum, can progress to affect the central nervous system, causing neurosyphilis. Accumulating evidence suggest that regulatory T cells (Tregs) may play an important role in the pathogenesis of syphilis. However, little is known about Treg response in neurosyphilis. Methodology/Principal Findings We analyzed Treg frequencies and Transforming Growth Factor-β (TGF-β) levels in the blood and CSF of 431 syphilis patients without neurological involvement, 100 neurosyphilis patients and 100 healthy donors. Suppressive function of Tregs in peripheral blood was also assessed. Among syphilis patients without neurological involvement, we found that secondary and serofast patients had increased Treg percentages, suppressive function and TGF-β levels in peripheral blood compared to healthy donors. Serum Rapid Plasma Reagin (RPR) titers were positively correlated with Treg numbers in these patients. Compared to these syphilis patients without neurological involvement, neurosyphilis patients had higher Treg frequency in peripheral blood. In the central nervous system, neurosyphilis patients had higher numbers of leukocytes in CSF compared to syphilis patients without neurological involvement. CD4+ T cells were the predominant cell type in the inflammatory infiltrates in CSF of neurosyphilis patients. Interestingly, among these neurosyphilis patients, a significant decrease in CSF CD4+ CD25high Treg percentage and number was observed in symptomatic neurosyphilis patients compared to those of asymptomatic neurosyphilis patients, which may be associated with low CSF TGF-β levels. Conclusions Our findings suggest that Tregs might play an important role in both bacterial persistence and neurologic compromise in the pathogenesis of syphilis. PMID:24244772

Warburg phenotype in renal cell carcinoma: high expression of glucose-transporter 1 (GLUT-1) correlates with low CD8(+) T-cell infiltration in the tumor.

PubMed

Singer, Katrin; Kastenberger, Michael; Gottfried, Eva; Hammerschmied, Christine G; Büttner, Maike; Aigner, Michael; Seliger, Barbara; Walter, Bernhard; Schlösser, Hans; Hartmann, Arndt; Andreesen, Reinhard; Mackensen, Andreas; Kreutz, Marina

2011-05-01

Many tumor cells are characterized by a dysregulated glucose metabolism associated with increased glycolysis in the presence of oxygen ("Warburg Effect"). Here, we analyzed for the first time a possible link between glucose metabolism and immune cell infiltration in renal cell carcinoma (RCC). RCC specimens revealed a highly significant increase in the expression of lactate dehydrogenase A (LDHA) and glucose-transporter 1 (GLUT-1) compared to the corresponding normal kidney tissue on mRNA level. Accordingly, tumor cell lines of different origin such as RCC, melanoma and hepatocellular carcinoma strongly expressed LDHA and GLUT-1 compared to their nonmalignant counterparts. In line with this finding, tumor cells secreted high amounts of lactate. High expression of GLUT-1 and LDH5, a tetramer of 4 LDHA subunits, was confirmed by tissue microarray analysis of 249 RCC specimens. Overall, 55/79 (69.6%) and 46/71 (64.7%) cases of clear cell carcinoma showed a constitutive, but heterogeneous expression of GLUT-1 and LDH5, respectively. The number of CD3(+), CD8(+) and FOXP3(+) T cells was significantly elevated in RCC lesions compared to normal kidney epithelium, but effector molecules such as granzyme B and perforin were decreased in tumor infiltrating T cells. Of interest, further analysis revealed an inverse correlation between GLUT-1 expression and the number of CD8(+) T cells in RCC lesions. Together, our data suggest that an accelerated glucose metabolism in RCC tissue is associated with a low infiltration of CD8(+) effector T cells. Targeting the glucose metabolism may represent an interesting tool to improve the efficacy of specific immunotherapeutic approaches in RCC. Copyright © 2010 UICC.

A Microfluidic Cell Concentrator

PubMed Central

Warrick, Jay; Casavant, Ben; Frisk, Megan; Beebe, David

2010-01-01

Cell concentration via centrifugation is a ubiquitous step in many cell culture procedures. At the macroscale, centrifugation suffers from a number of limitations particularly when dealing with small numbers of cells (e.g., less than 50,000). On the other hand, typical microscale methods for cell concentration can affect cell physiology and bias readouts of cell behavior and function. In this paper, we present a microfluidic concentrator device that utilizes the effects of gravity to allow cells to gently settle out of a suspension into a collection region without the use of specific adhesion ligands. Dimensional analysis was performed to compare different device designs and was verified with flow modeling to optimize operational parameters. We are able to concentrate low-density cell suspensions in a microfluidic chamber, achieving a cell loss of only 1.1 ± 0.6% (SD, n=7) with no observed loss during a subsequent cell staining protocol which incorporates ~36 complete device volume replacements. This method provides a much needed interface between rare cell samples and microfluidic culture assays. PMID:20843010

COUP-TF1 Modulates Thyroid Hormone Action in an Embryonic Stem-Cell Model of Cortical Pyramidal Neuronal Differentiation.

PubMed

Teng, Xiaochun; Liu, Yan-Yun; Teng, Weiping; Brent, Gregory A

2018-05-01

Thyroid hormone is critical for normal brain development and acts in a spatial and temporal specific pattern. Thyroid hormone excess, or deficiency, can lead to irreversible impairment of brain and sensory development. Chicken ovalbumin upstream-transcription factor 1 (COUP-TF1), expressed early in neuronal development, is essential to achieve normal brain structure. Thyroid hormone stimulation of gene expression is inversely correlated with the level of COUP-TF1 expression. An in vitro method of differentiating mouse embryonic stem (mES) cells into cortical neurons was utilized to study the influence of COUP-TF1 on thyroid hormone signaling in brain development. mES cells were cultured and differentiated in specific conditioned media, and a high percentage of nestin-positive progenitor neurons in the first stage, and cortical neurons in the second stage, was obtained with characteristic neuronal firing. The number of nestin-positive progenitors, as determined by fluorescence-activated cell sorting analysis, was significantly greater with triiodothyronine (T3) treatment compared to control (p < 0.05). T3 enhanced the expression of cortical neuron marker (Tbr1 and Rc3) mRNAs. After COUP-TF1 knockdown, the number of nestin-positive progenitors was reduced compared to control (p < 0.05), but the number increased with T3 treatment. The mRNA of cortical neuronal gene markers was measured after COUP-TF1 knockdown. In the presence of T3, the peak expression of neuron markers Emx1, Tbr1, Camkiv, and Rc3 mRNA was earlier, at day 18 of differentiation, compared to control cells, at day 22. Furthermore, after COUP-TF1 knockdown, T3 induction of Rc3 and Tbr1 mRNA was significantly enhanced compared to cells expressing COUP-TF1. These results indicate that COUP-TF1 plays an important role in modulating the timing and magnitude of T3-stimulated gene expression required for normal corticogenesis.

High-versus standard-dose filgrastim (rhG-CSF) for mobilization of peripheral-blood progenitor cells from allogeneic donors and CD34(+) immunoselection.

PubMed

Engelhardt, M; Bertz, H; Afting, M; Waller, C F; Finke, J

1999-07-01

The efficacy of a high- versus a standard-dose filgrastim (recombinant human granulocyte colony-stimulating factor, or rhG-CSF) regimen to mobilize peripheral-blood progenitor cells (PBPCs) for allogeneic transplantation was investigated in 75 healthy donors. From December 1994 to December 1997, 75 consecutive donors (median age, 38 years; range, 17 to 67 years) were assigned to two different schedules of rhG-CSF for PBPC mobilization. Fifty donors received 24 microg rhG-CSF/kg body weight (BW) divided into two daily subcutaneous injections (two doses of 12 microg, group A), whereas 25 were treated with 10 microg rhG-CSF once daily (group B). Apheresis was started on day 4 in group A and on day 5 in group B. Target CD34(+) cell numbers in apheresis products were >/= 4 x 10(6)/kg recipient BW. Cytokine priming and collection of PBPCs were equally well tolerated in both groups. Significantly higher CD34(+) cell numbers in group A with 3. 7 x 10(6)/kg recipient BW/apheresis (0.47 x 10(6)/L apheresis) compared with 2 x 10(6)/kg recipient BW/apheresis (0.25 x 10(6)/L apharesis) in group B were obtained (P <.05). Using standard aphereses (median, 9 L), two doses of 12 microg rhG-CSF/kg allowed collection of >/= 4 x 10(6)/kg CD34(+) cells with two aphereses (range, one to three) in group A versus three aphereses (range, one to six) in group B (P <.015). Donor age, sex, and BW influenced the collection of CD34(+) cell numbers: in particular, significantly higher apheresis results were obtained in donors younger than 40 years compared with donors older than 40 years of age (P <.05). In 65 CD34(+) selection procedures using avidin-biotin immunoabsorption columns (Ceprate SC System, CellPro, Bothell, WA), a median CD34(+) purity of 53%, CD34(+) recovery of 40%, and the collection of 2 x 10(6)/kg CD34(+) cells/selection were achieved. In group A with higher CD34(+) cells/kg/apheresis, CD34(+) purity, recovery, and cell yields were 60%, 45%, and 2.3 x 10(6)/kg/selection, respectively, as compared with 48%, 31%, and 0.7 x 10(6)/kg in group B (P <.05). Our results demonstrate that twice daily rhG-CSF (two doses of 12 microg/kg BM) compared with once daily rhG-CSF (10 microg/kg BW), in addition to being well tolerated, significantly improves PBPC mobilization, allows the collection of higher numbers of CD34(+) cells with one or two standard aphereses, and facilitates subsequent selection procedures in healthy allogeneic donors.

The kisspeptin/neurokinin B/dynorphin (KNDy) cell population of the arcuate nucleus: sex differences and effects of prenatal testosterone in sheep.

PubMed

Cheng, Guanliang; Coolen, Lique M; Padmanabhan, Vasantha; Goodman, Robert L; Lehman, Michael N

2010-01-01

Recent work in sheep has identified a neuronal subpopulation in the arcuate nucleus that coexpresses kisspeptin, neurokinin B, and dynorphin (referred to here as KNDy cells) and that mediate the negative feedback influence of progesterone on GnRH secretion. We hypothesized that sex differences in progesterone negative feedback are due to sexual dimorphism of KNDy cells and compared neuropeptide and progesterone receptor immunoreactivity in this subpopulation between male and female sheep. In addition, because sex differences in progesterone negative feedback and neurokinin B are due to the influence of testosterone (T) during fetal life, we determined whether prenatal T exposure would mimic sex differences in KNDy cells. Adult rams had nearly half the number of kisspeptin, neurokinin B, dynorphin, and progesterone receptor-positive cells in the arcuate nucleus as did females, but the percentage of KNDy cells colocalizing progesterone receptors remained high in both sexes. Prenatal T treatment also reduced the number of dynorphin, neurokinin B, and progesterone receptor-positive cells in the female arcuate nucleus; however, the number of kisspeptin cells remained high and at levels comparable to control females. Thus, sex differences in kisspeptin in the arcuate nucleus, unlike that of dynorphin and neurokinin B, are not due solely to exposure to prenatal T, suggesting the existence of different critical periods for multiple peptides coexpressed within the same neuron. In addition, the imbalance between inhibitory (dynorphin) and stimulatory (kisspeptin) neuropeptides in this subpopulation provides a potential explanation for the decreased ability of progesterone to inhibit GnRH neurons in prenatal T-treated ewes.

Chemoprevention studies of the flavonoids quercetin and rutin in normal and azoxymethane-treated mouse colon.

PubMed

Yang, K; Lamprecht, S A; Liu, Y; Shinozaki, H; Fan, K; Leung, D; Newmark, H; Steele, V E; Kelloff, G J; Lipkin, M

2000-09-01

In this study we investigated the chemopreventive effects of quercetin and rutin when added to standard AIN-76A diet and fed to normal and azoxymethane (AOM)-treated mice. Early changes in colonic mucosa were analyzed, including colonic cell proliferation, apoptotic cell death, cyclin D(1) expression and focal areas of dysplasia (FAD). The findings show that the number of colonic epithelial cells per crypt column increased (P: < 0.01) in each normal mouse group fed the flavonoids; AOM administration increased colonic crypt cell proliferation and resulted in a marked rise of bromodeoxyuridine-labeled cells in the lower proliferative zone of the crypt. Both supplementary dietary quercetin and rutin increased the apoptotic index and caused a redistribution of apoptotic cells along the crypt axis in normal mice fed a standard AIN-76A diet. The number of apoptotic cells/column and apoptotic indices markedly increased (P: < 0.01) in the AOM-treated group compared with untreated animals; apoptotic cells expanded throughout the colonic crypts after flavonoid supplementation and AOM administration. Positive cyclin D(1) expression was detected in mice on diets supplemented either with quercetin (P: < 0.01) or rutin (P: < 0.05). AOM administration resulted in the formation of FAD. Both the number of mice exhibiting FAD and the total numer of FAD observed were significantly reduced (P: < 0.01) in AOM-treated animals fed flavonoids compared with mice maintained on the standard AIN-76A diet. Surprisingly, however, quercetin alone was able to induce FAD in 22% of normal mice fed the standard AIN-76A diet.

Altered immune parameters correlate with infection-related hospitalizations in children with Down syndrome.

PubMed

Martínez, Elizabeth; Castañeda, Diana; Jaramillo, Sonia; Iregui, Alejandro; Quiñonez, Tatiana; Rodríguez, Jairo A; Herrera, Eddy; Gómez, Ana Milena; Rondón, Martin A; Prieto, Juan Carlos; Angel, Juana; Franco, Manuel A; Mesa, Martha C

2016-07-01

In addition to previously studied immunological variables, the relative expression of IFNGR2, IFNAR1, CD18, and CD275 (all encoded in chromosome 21) on circulating leucocytes and multifunctional T cells (evaluated by an intracellular cytokine/proliferation assay) were compared between children with Down syndrome (DS) and healthy controls (HC). As previously reported, numbers of lymphocytes, CD4(+) T cells, Treg cells, B cells, and levels of serum IgM were decreased, and levels of IgG and IgA were increased in children with DS. Moreover, the relative expression of CD18 on T and B cells (previously and not previously reported, respectively) were elevated in DS children (p⩽0.01). Age and numbers of B and Treg cells moderately correlated with retrospectively identified infection related hospitalizations (rho: 0.300-0.460, p⩽0.003). Age and the numbers of Treg cells also correlated with prospectively identified infection related hospitalizations. Future studies are necessary to clarify the role of these parameters in the immunity of DS patients. Copyright © 2016 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

Developmental sources of conservation and variation in the evolution of the primate eye.

PubMed

Dyer, Michael A; Martins, Rodrigo; da Silva Filho, Manoel; Muniz, José Augusto P C; Silveira, Luiz Carlos L; Cepko, Constance L; Finlay, Barbara L

2009-06-02

Conserved developmental programs, such as the order of neurogenesis in the mammalian eye, suggest the presence of useful features for evolutionary stability and variability. The owl monkey, Aotus azarae, has developed a fully nocturnal retina in recent evolution. Description and quantification of cell cycle kinetics show that embryonic cytogenesis is extended in Aotus compared with the diurnal New World monkey Cebus apella. Combined with the conserved mammalian pattern of retinal cell specification, this single change in retinal progenitor cell proliferation can produce the multiple alterations of the nocturnal retina, including coordinated reduction in cone and ganglion cell numbers, increase in rod and rod bipolar numbers, and potentially loss of the fovea.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells.

PubMed

Jung, YunJae

2015-12-01

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity.

VIPhyb, an antagonist of vasoactive intestinal peptide receptor, enhances cellular antiviral immunity in murine cytomegalovirus infected mice.

PubMed

Li, Jian-Ming; Darlak, Kasia A; Southerland, Lauren; Hossain, Mohammad S; Jaye, David L; Josephson, Cassandra D; Rosenthal, Hilary; Waller, Edmund K

2013-01-01

Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1-mediated cellular immunity. We previously reported that VIP-knockout (VIP-KO) mice have enhanced cellular immune responses and increased survival following murine cytomegalovirus (mCMV) infection in C57BL/6 mice. In this study, we tested whether treatment with a VIP receptor antagonistic peptide protects C57BL/6 and BALB/c mice from mCMV-infection. One week of daily subcutaneous injections of VIPhyb was non-toxic and did not alter frequencies of immune cell subsets in non-infected mice. VIPhyb administration to mCMV-infected C57BL/6 and BALB/c mice markedly enhanced survival, viral clearance, and reduced liver and lung pathology compared with saline-treated controls. The numbers of effector/memory CD8+ T-cells and mature NK cells were increased in VIPhyb-treated mice compared with PBS-treated groups. Pharmacological blockade of VIP-receptor binding or genetic blockade of VIP-signaling prevented the up-regulation of PD-L1 and PD-1 expression on DC and activated CD8+ T-cells, respectively, in mCMV-infected mice, and enhanced CD80, CD86, and MHC-II expression on conventional and plasmacytoid DC. VIPhyb-treatment increased type-I IFN synthesis, numbers of IFN-γ- and TNF-α-expressing NK cells and T-cells, and the numbers of mCMV-M45 epitope-peptide-MHC-I tetramer CD8+ T-cells following mCMV infection. VIP-treatment lowered the percentage of Treg cells in spleens compared with PBS-treated WT mice following mCMV infection, while significantly decreasing levels of serum VEGF induced by mCMV-infection. The mice in all treated groups exhibited similar levels of anti-mCMV antibody titers. Short-term administration of a VIP-receptor antagonist represents a novel approach to enhance innate and adaptive cellular immunity in a murine model of CMV infection.

Age-Related Changes in Mouse Taste Bud Morphology, Hormone Expression, and Taste Responsivity

PubMed Central

Shin, Yu-Kyong; Cong, Wei-na; Cai, Huan; Kim, Wook; Maudsley, Stuart; Martin, Bronwen

2012-01-01

Normal aging is a complex process that affects every organ system in the body, including the taste system. Thus, we investigated the effects of the normal aging process on taste bud morphology, function, and taste responsivity in male mice at 2, 10, and 18 months of age. The 18-month-old animals demonstrated a significant reduction in taste bud size and number of taste cells per bud compared with the 2- and 10-month-old animals. The 18-month-old animals exhibited a significant reduction of protein gene product 9.5 and sonic hedgehog immunoreactivity (taste cell markers). The number of taste cells expressing the sweet taste receptor subunit, T1R3, and the sweet taste modulating hormone, glucagon-like peptide-1, were reduced in the 18-month-old mice. Concordant with taste cell alterations, the 18-month-old animals demonstrated reduced sweet taste responsivity compared with the younger animals and the other major taste modalities (salty, sour, and bitter) remained intact. PMID:22056740

Age-related changes in mouse taste bud morphology, hormone expression, and taste responsivity.

PubMed

Shin, Yu-Kyong; Cong, Wei-na; Cai, Huan; Kim, Wook; Maudsley, Stuart; Egan, Josephine M; Martin, Bronwen

2012-04-01

Normal aging is a complex process that affects every organ system in the body, including the taste system. Thus, we investigated the effects of the normal aging process on taste bud morphology, function, and taste responsivity in male mice at 2, 10, and 18 months of age. The 18-month-old animals demonstrated a significant reduction in taste bud size and number of taste cells per bud compared with the 2- and 10-month-old animals. The 18-month-old animals exhibited a significant reduction of protein gene product 9.5 and sonic hedgehog immunoreactivity (taste cell markers). The number of taste cells expressing the sweet taste receptor subunit, T1R3, and the sweet taste modulating hormone, glucagon-like peptide-1, were reduced in the 18-month-old mice. Concordant with taste cell alterations, the 18-month-old animals demonstrated reduced sweet taste responsivity compared with the younger animals and the other major taste modalities (salty, sour, and bitter) remained intact.

Human Embryonic Stem Cell-Derived Neurons Are Highly Permissive for Varicella-Zoster Virus Lytic Infection.

PubMed

Sadaoka, Tomohiko; Schwartz, Cindi L; Rajbhandari, Labchan; Venkatesan, Arun; Cohen, Jeffrey I

2018-01-01

Varicella-zoster virus (VZV) is highly cell associated when grown in culture and has a much higher (4,000- to 20,000-fold increased) particle-to-PFU ratio in vitro than herpes simplex virus (HSV). In contrast, VZV is highly infectious in vivo by airborne transmission. Neurons are major targets for VZV in vivo ; in neurons, the virus can establish latency and reactivate to produce infectious virus. Using neurons derived from human embryonic stem cells (hESC) and cell-free wild-type (WT) VZV, we demonstrated that neurons are nearly 100 times more permissive for WT VZV infection than very-early-passage human embryonic lung cells or MRC-5 diploid human fibroblasts, the cells used for vaccine production or virus isolation. The peak titers achieved after infection were ∼10-fold higher in human neurons than in MRC-5 cells, and the viral genome copy number-to-PFU ratio for VZV in human neurons was 500, compared with 50,000 for MRC-5 cells. Thus, VZV may not necessarily have a higher particle-to-PFU ratio than other herpesviruses; instead, the cells previously used to propagate virus in vitro may have been suboptimal. Furthermore, based on electron microscopy, neurons infected with VZV produced fewer defective or incomplete viral particles than MRC-5 cells. Our data suggest that neurons derived from hESC may have advantages compared to other cells for studies of VZV pathogenesis, for obtaining stocks of virus with high titers, and for isolating VZV from clinical specimens. IMPORTANCE Varicella-zoster virus (VZV) causes chickenpox and shingles. Cell-free VZV has been difficult to obtain, both for in vitro studies and for vaccine production. While numerous cells lines have been tested for their ability to produce high titers of VZV, the number of total virus particles relative to the number of viral particles that can form plaques in culture has been reported to be extremely high relative to that in other viruses. We show that VZV grows to much higher titers in human neurons than in other cell types in vitro and that the number of total virus genomes relative to the number of viral particles that can form plaques in culture is much lower in human neurons than other cultured cells. These findings indicate that human neurons may be useful for studying VZV in vitro , for growing preparations of virus with high titers, and for isolating the virus from human samples. Copyright © 2017 American Society for Microbiology.

Number of negative lymph nodes as a prognostic factor in esophageal squamous cell carcinoma.

PubMed

Ma, Mingquan; Tang, Peng; Jiang, Hongjing; Gong, Lei; Duan, Xiaofeng; Shang, Xiaobin; Yu, Zhentao

2017-10-01

The aim of this study is to investigate the number of negative lymph nodes (NLNs) as a prognostic factor for survival in patients with resected esophageal squamous cell carcinoma. A total of 381 esophageal squamous cell carcinoma patients who had underwent surgical resection as the primary treatment was enrolled into this retrospective study. The impact of number of NLNs on patient's overall survival was assessed and compared with the factors among the current tumor-nodes-metastasis (TNM) staging system. The number of NLNs was closely related to the overall survival, and the 5-year survival rate was 45.4% for number of NLNs of >20 (142 cases) and 26.4% for NLNs ≤ 20 (239 cases) (P = 0.001). In multivariate survival analysis, the number of NLNs remained an independent prognostic factor (P = 0.002) as did the other current TNM factors. For subgroup analysis, the predictive value of number of NLNs was significant in patients with T3 or T4 disease (P = 0.001) and patients with N1 and N2-3 disease (P = 0.025, 0.043), but not in patients with T1 or T2 disease or patients with N0 disease. The number of NLNs, which represents the extent of lymphadenectomy for esophageal squamous cell carcinoma, could impact the overall survival of patients with resected esophageal squamous cell carcinoma, especially among those with nodal-positive disease and advanced T-stage tumor. © 2016 John Wiley & Sons Australia, Ltd.

A Novel Assay for Easy and Rapid Quantification of Helicobacter pylori Adhesion.

PubMed

Skindersoe, Mette E; Rasmussen, Lone; Andersen, Leif P; Krogfelt, Karen A

2015-06-01

Reducing adhesion of Helicobacter pylori to gastric epithelial cells could be a new way to counteract infections with this organism. We here present a novel method for quantification of Helicobacter pylori adhesion to cells. Helicobacter pylori is allowed to adhere to AGS or MKN45g cells in a 96-well microtiter plate. Then wells are added saponin, which lyses the cells without affecting the bacteria. After addition of alamarBlue(®) (resazurin) and 1- to 2-hour incubation, fluorescence measurements can be used to quantify the number of adherent bacteria. By use of the method, we demonstrate that adhesion of both a sabA and babA deletion mutant of H. pylori is significantly reduced compared to the wild type. The method offers a number of applications and may be used to compare the adherence potential of different strains of H. pylori to either cells or different materials or to screen for potential anti-adhesive compounds. The results presented here suggest that this easy and reproducible assay is well suited for quantitative investigation of H. pylori adhesion. © 2015 John Wiley & Sons Ltd.

Changes in Hippocampal Volume are Correlated with Cell Loss but Not with Seizure Frequency in Two Chronic Models of Temporal Lobe Epilepsy

PubMed Central

Polli, Roberson S.; Malheiros, Jackeline M.; dos Santos, Renan; Hamani, Clement; Longo, Beatriz M.; Tannús, Alberto; Mello, Luiz E.; Covolan, Luciene

2014-01-01

Kainic acid (KA) or pilocarpine (PILO) have been used in rats to model human temporal lobe epilepsy (TLE) but the distribution and severity of structural lesions between these two models may differ. Magnetic resonance imaging (MRI) studies have used quantitative measurements of hippocampal T2 (T2HP) relaxation time and volume, but simultaneous comparative results have not been reported yet. The aim of this study was to compare the MRI T2HP and volume with histological data and frequency of seizures in both models. KA- and PILO-treated rats were imaged with a 2 T MRI scanner. T2HP and volume values were correlated with the number of cells, mossy fiber sprouting, and spontaneous recurrent seizures (SRS) frequency over the 9 months following status epilepticus (SE). Compared to controls, KA-treated rats had unaltered T2HP, pronounced reduction in hippocampal volume and concomitant cell reduction in granule cell layer, CA1 and CA3 at 3 months post SE. In contrast, hippocampal volume was unchanged in PILO-treated animals despite detectable increased T2HP and cell loss in granule cell layer, CA1 and CA3. In the following 6 months, MRI hippocampal volume remained stable with increase of T2HP signal in the KA-treated group. The number of CA1 and CA3 cells was smaller than age-matched CTL group. In contrast, PILO group had MRI volumetric reduction accompanied by reduction in the number of CA1 and CA3 cells. In this group, T2HP signal was unaltered at 6 or 9 months after status. Reductions in the number of cells were not progressive in both models. Notably, the SRS frequency was higher in PILO than in the KA model. The volumetry data correlated well with tissue damage in the epileptic brain, suggesting that MRI may be useful for tracking longitudinal hippocampal changes, allowing the assessment of individual variability and disease progression. Our results indicate that the temporal changes in hippocampal morphology are distinct for both models of TLE and that these are not significantly correlated to the frequency of SRS. PMID:25071699

Sex differences and the roles of sex steroids in apoptosis of sexually dimorphic nuclei of the preoptic area in postnatal rats.

PubMed

Tsukahara, S

2009-03-01

The brain contains several sexually dimorphic nuclei that exhibit sex differences with respect to cell number. It is likely that the control of cell number by apoptotic cell death in the developing brain contributes to creating sex differences in cell number in sexually dimorphic nuclei, although the mechanisms responsible for this have not been determined completely. The milieu of sex steroids in the developing brain affects sexual differentiation in the brain. The preoptic region of rats has two sexually dimorphic nuclei. The sexually dimorphic nucleus of the preoptic area (SDN-POA) has more neurones in males, whereas the anteroventral periventricular nucleus (AVPV) has a higher cell density in females. Sex differences in apoptotic cell number arise in the SDN-POA and AVPV of rats in the early postnatal period, and an inverse correlation exists between sex differences in apoptotic cell number and the number of living cells in the mature period. The SDN-POA of postnatal male rats exhibits a higher expression of anti-apoptotic Bcl-2 and lower expression of pro-apoptotic Bax compared to that in females and, as a potential result, apoptotic cell death via caspase-3 activation more frequently occurs in the SDN-POA of females. The patterns of expression of Bcl-2 and Bax in the SDN-POA of postnatal female rats are changed to male-typical ones by treatment with oestrogen, which is normally synthesised from testicular androgen and affects the developing brain in males. In the AVPV of postnatal rats, apoptotic regulation also differs between the sexes, although Bcl-2 expression is increased and Bax expression and caspase-3 activity are decreased in females. The mechanisms of apoptosis possibly contributing to the creation of sex differences in cell number and the roles of sex steroids in apoptosis are discussed.

Effects of perfluorinated chemicals on adipocyte development ...

EPA Pesticide Factsheets

Obesity is a growing concern in the US population. Current interest is high in the role played by environmental factors called obesogens that may contribute to obesity through developmental exposure. One class of potential obesogens is the family of perfluorinated chemicals used as surfactants in a variety of industrial applications. Given the importance of understanding the role these compounds play in lipid homeostasis we used pre-adipocyte 3T3-L1 mouse fibroblast cells (Zen-Bio, RTP NC) to study their effects on adipogenesis and lipid accumulation. These cells differentiate into adipocytes accumulating large lipid droplets. Cultures were treated with perfluorooctanoic acid (PFOA) (1-200uM), perfluorononanoic acid (PFNA) (5-lOOuM), perfluorooctane sulfonate (PFOS) (5O-300uM), and perfluorohexane sulfonate (PFHxS) (40- 250uM). Cell size number, and lipid content were assessed using morphomeiric analysis. All four compounds decreased cell size compared to control, and PFNA was most potent, in terms of lowest observed effect concentration (LOEC), whereas PFOA was least potent. Cell number increased for all perfluorinated chemicals tested, most potently for PFNA and least for PFOS. Interestingly, average lipid area per cell for all four chemicals decreased compared to control, but PFOS and PFHxS had increased total lipid area. Additionally, significant increases in total triglyceride were noted for all compounds compared to controls. PFOA and PFNA increased trigly

Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat

NASA Technical Reports Server (NTRS)

Bhat, G. K.; Yang, H.; Sridaran, R.

2001-01-01

The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.

Phenotypically non-suppressive cells predominate among FoxP3-positive cells in oral lichen planus.

PubMed

Schreurs, Olav; Karatsaidis, Andreas; Schenck, Karl

2016-11-01

Oral lichen planus (OLP) is a common T-cell-dominated oral chronic inflammatory disease occurring in periods of remission, quiescence, activity with pronounced inflammation, and acute ulceration. Cell infiltrates in OLP contain varying numbers of CD4 + T cells expressing the transcription factor FoxP3. FoxP3 + CD4 + T cells are, however, a heterogeneous cell population containing suppressive and non-suppressive cells, and their distribution in infiltrates from OLP is unknown. Biopsies were taken from normal oral mucosa (n = 8) and OLP lesions (n = 19), and a set of in situ methods for the determination of the functional phenotype of FoxP3 + CD4 + T cells was applied. Numbers of FoxP3 + CD4 + T cells were highest in the atrophic form of the disease, yet low in the ulcerative form. The main FoxP3 + CD4 + T-cell population observed was FoxP3 + CD45RA - CD25 + CD45RO + and CD15s - , a phenotype delineating a non-suppressive subset. Numbers of cells with an actively suppressing phenotype (FoxP3 + CD45RA - CD25 + CD45RO + and CD15s + ) were, however, about twice as high in reticular lesions as compared with the atrophic form. Many FoxP3 + CD4 + T cells expressed T-bet, the hallmark transcription factor for IFN-γ-producing T cells, indicating that they may enhance immune and inflammatory responses rather than suppress them. The absence of actively suppressing FoxP3 + CD4 + T cells may in part explain why OLP is a remarkably persisting condition, in spite of the presence of substantially high numbers of FoxP3 + CD4 + T cells. The findings emphasize that it is crucial to examine not only numbers but also functional phenotype of FoxP3 + CD4 + T cells in human tissues. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Increased numbers of pre-existing memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells1

PubMed Central

Joshi, Nikhil S.; Cui, Weiguo; Dominguez, Claudia; Chen, Jonathan H.; Hand, Timothy W.; Kaech, Susan M.

2011-01-01

Memory CD8 T cells acquire TEM properties following reinfection, and may reach terminally differentiated, senescent states (“Hayflick limit”) after multiple infections. The signals controlling this process are not well understood, but we found that the degree of 2o effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and pre-existing memory CD8 T cell number (i.e., 1o memory CD8 T cell precursor frequency) present during secondary infection. Compared to naïve cells, memory CD8 T cells were predisposed towards terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of antigen. TE cell formation following 2o or 3o infections was dependent on increased T-bet expression because T-bet+/− cells were resistant to these phenotypic changes. Larger numbers of pre-existing memory CD8 T cells limited the duration of 2o infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2o TE CD8 T cells that formed. Together, these data show that, over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with antigen or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by pre-existing memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies. PMID:21930973

Ghrelin receptor expression and colocalization with anterior pituitary hormones using a GHSR-GFP mouse line.

PubMed

Reichenbach, Alex; Steyn, Frederik J; Sleeman, Mark W; Andrews, Zane B

2012-11-01

Ghrelin is the endogenous ligand for the GH secretagogue receptor (GHSR) and robustly stimulates GH release from the anterior pituitary gland. Ghrelin also regulates the secretion of anterior pituitary hormones including TSH, LH, prolactin (PRL), and ACTH. However, the relative contribution of a direct action at the GHSR in the anterior pituitary gland vs. an indirect action at the GHSR in the hypothalamus remains undefined. We used a novel GHSR-enhanced green fluorescent protein (eGFP) reporter mouse to quantify GHSR coexpression with GH, TSH, LH, PRL, and ACTH anterior pituitary cells in males vs. females and in chow-fed or calorie-restricted (CR) mice. GHSR-eGFP-expressing cells were only observed in anterior pituitary. The number of GHSR-eGFP-expressing cells was higher in male compared with females, and CR did not affect the GHSR-eGFP cell number. Double staining revealed 77% of somatotrophs expressed GHSR-eGFP in both males and females. Nineteen percent and 12.6% of corticotrophs, 21% and 9% of lactotrophs, 18% and 19% of gonadotrophs, and 3% and 9% of males and females, respectively, expressed GHSR-eGFP. CR increased the number of TSH cells, but suppressed the number of lactotrophs and gonadotrophs, expressing GHSR-eGFP compared with controls. These studies support a robust stimulatory action of ghrelin via the GHSR on GH secretion and identify a previously unknown sexual dimorphism in the GHSR expression in the anterior pituitary. CR affects GHSR-eGFP expression on lactotrophs, gonadotrophs, and thyrotrophs, which may mediate reproductive function and energy metabolism during periods of negative energy balance. The low to moderate expression of GHSR-eGFP suggests that ghrelin plays a minor direct role on remaining anterior pituitary cells.

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses

PubMed Central

2014-01-01

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 μg/cm2 MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 μm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels. PMID:24479647

Mesenteric Th1 polarization and monocyte TNF-alpha production: first steps to systemic inflammation in rats with cirrhosis.

PubMed

Muñoz, Leticia; Albillos, Agustín; Nieto, Mónica; Reyes, Eduardo; Lledó, Lourdes; Monserrat, Jorge; Sanz, Eva; de la Hera, Antonio; Alvarez-Mon, Melchor

2005-08-01

A systemic inflammatory state with increased circulating tumor necrosis factor alpha (TNF-alpha) has been related to the bacterial infection susceptibility and hemodynamic derangement of patients with cirrhosis. We compared the activation status of immune cell subpopulations defined by 4-color cytometry in mesenteric and peripheral lymph nodes and blood of rats with CCl(4)-cirrhosis to define the immune response initiation site, the T-cell and monocyte contribution to pro-inflammatory cytokine production, as well as the pathogenic role of enteric bacteria in the cirrhosis immune response. Th1 cells and monocytes were expanded in the mesenteric nodes (P < .001) and blood (P < .001) of rats with cirrhosis, and activated to produce interferon gamma (P < .0001) and TNF-alpha (P < .0001), respectively. The greater numbers of recently activated CD134(+) Th cells in mesenteric nodes compared with blood, the correlation between their numbers in mesenteric nodes and blood (r = 0.66, P < .001), and the expansion of activated CD45RC(-) Th cells, which are unable to re-enter lymph nodes, in mesenteric nodes but not in blood or axillary nodes points to mesenteric nodes as the origin site of activated Th cells. Abrogation of bacterial translocation by bowel decontamination reduced the number of activated Th cells and monocytes, and normalized interferon gamma production by Th cells and TNF-alpha production by monocytes in mesenteric nodes and blood, respectively. In conclusion, in cirrhosis, enteric bacteria start off an orchestrated immune response cascade in mesenteric nodes involving Th1 polarization and monocyte activation to TNF-alpha production. Later, the recirculation of these activated effector immune cells into blood promotes systemic inflammation.

Analysis of renal cancer cell lines from two major resources enables genomics-guided cell line selection

NASA Astrophysics Data System (ADS)

Sinha, Rileen; Winer, Andrew G.; Chevinsky, Michael; Jakubowski, Christopher; Chen, Ying-Bei; Dong, Yiyu; Tickoo, Satish K.; Reuter, Victor E.; Russo, Paul; Coleman, Jonathan A.; Sander, Chris; Hsieh, James J.; Hakimi, A. Ari

2017-05-01

The utility of cancer cell lines is affected by the similarity to endogenous tumour cells. Here we compare genomic data from 65 kidney-derived cell lines from the Cancer Cell Line Encyclopedia and the COSMIC Cell Lines Project to three renal cancer subtypes from The Cancer Genome Atlas: clear cell renal cell carcinoma (ccRCC, also known as kidney renal clear cell carcinoma), papillary (pRCC, also known as kidney papillary) and chromophobe (chRCC, also known as kidney chromophobe) renal cell carcinoma. Clustering copy number alterations shows that most cell lines resemble ccRCC, a few (including some often used as models of ccRCC) resemble pRCC, and none resemble chRCC. Human ccRCC tumours clustering with cell lines display clinical and genomic features of more aggressive disease, suggesting that cell lines best represent aggressive tumours. We stratify mutations and copy number alterations for important kidney cancer genes by the consistency between databases, and classify cell lines into established gene expression-based indolent and aggressive subtypes. Our results could aid investigators in analysing appropriate renal cancer cell lines.

An essential role for the association of CD47 to SHPS-1 in skeletal remodeling.

PubMed

Maile, Laura A; DeMambro, Victoria E; Wai, Christine; Lotinun, Sutada; Aday, Ariel W; Capps, Byron E; Beamer, Wesley G; Rosen, Clifford J; Clemmons, David R

2011-09-01

Integrin-associated protein (IAP/CD47) has been implicated in macrophage-macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47(-/-) mice with Cd47(+/+) controls. Cd47(-/-) mice weighed less and had decreased areal bone mineral density compared with controls. Cd47(-/-) femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone-formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47(-/-) mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47(-/-) bone marrow cells was significantly decreased compared with wild-type cultures and was associated with a decrease in bone-resorption capacity. Furthermore, by disrupting the CD47-SHPS-1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS-1 phosphorylation, SHP-1 phosphatase recruitment, and subsequent dephosphorylation of non-muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47(-/-) mice. Our finding of cell-autonomous defects in Cd47(-/-) osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47(-/-) mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption. Copyright © 2011 American Society for Bone and Mineral Research.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Urushihara, Yusuke; Kobayashi, Junya; Matsumoto, Yoshihisa

Highlights: Black-Right-Pointing-Pointer We investigated the effect of DNA-PK inhibition on DSB repair using fish cells. Black-Right-Pointing-Pointer A radiation sensitive mutant RIC1 strain showed a low level of DNA-PK activity. Black-Right-Pointing-Pointer DNA-PK dysfunction leads defects in HR repair and DNA-PKcs autophosphorylation. Black-Right-Pointing-Pointer DNA-PK dysfunction leads a slight increase in the number of 53BP1 foci after DSBs. Black-Right-Pointing-Pointer DNA-PK dysfunction leads an alternative NHEJ that depends on 53BP1. -- Abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ andmore » aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after {gamma}-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of {gamma}H2AX foci after {gamma}-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of {gamma}H2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after {gamma}-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation in RIC1 cells. It is known that 53BP1 is involved in both DNA-PK dependent and independent NHEJ. Therefore we suggest that DNA-PK independent NHEJ repair DSBs under the condition of decreased DNA-PK activity, which causes reduction of HR efficiency.« less

CD20+ T cell numbers are decreased in untreated HIV-1 patients and recover after HAART.

PubMed

Förster, Friederike; Singla, Anuj; Arora, Sunil K; Schmidt, Reinhold E; Jacobs, Roland

2012-08-30

To elucidate if CD20(+) T cells are affected by HIV-1 infection and may have a prognostic value for the course of disease, numbers of CD20(+) T cells were determined in healthy controls, untreated and HAART-treated HIV-1 patients. Coexpression patterns of CD4, CD8, and CD38 were analysed on CD3(+)CD20(+) and CD3(+)CD20(-) T cells. We found a significant decrease of CD20(+) T cell numbers in untreated HIV-1 patients (1.4%) as compared to healthy controls (2.5%) which recovered under HAART (1.9%). Particularly, the CD8(+) T cell compartment was affected revealing significant differences between healthy controls (3.4%) and both treated (1.7%) and untreated (1.1%) patients. CD38 was expressed on a few CD20(+) T cells but preferentially on CD20(-) cells in all three groups. IFN-γ production was measured upon cell activation using PMA alone or in combination with ionomycin in order to assess functional capacities of the cells. PMA alone was much more effective in CD20(+) cells regardless of CD38 coexpression, indicating a supportive role of CD20 but not CD38 in T cell activation. Here we present data showing that CD3(+)CD20(+) T cells are decreased in untreated HIV-1 patients and normal numbers are restored under HAART. Expression of CD20 and CD38 is independently regulated on T cells. Contrary to CD38, CD20 can substitute ionophores for Ca(2+) flux in early T cell activation and also strongly amplify cell stimulation in the presence of Ca(2+) ionophores, indicating that CD20 contributes to T cell activation. Copyright © 2012 Elsevier B.V. All rights reserved.

New Developments in Mitigation of Microbiologically Influenced Corrosion

DTIC Science & Technology

2007-07-12

either bacterium. Higher R1, values for surfaces colonized by S . marcescens compared to those colonized by Pseudomonas sp. were attributed to higher...numbers of S . marcescens cells attached to the metal surface. Impedance spectra (see Figure 1) after immersion in sterile and inoculated VNSS demonstrated...NUMBER New Developments in Mitigation of Microbiologically Influenced Corrosion. 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 0601153N 6. AUTHOR( S ) 5d

Fluoxetine prevents the memory deficits and reduction in hippocampal cell proliferation caused by valproic acid.

PubMed

Welbat, Jariya Umka; Sangrich, Preeyanuch; Sirichoat, Apiwat; Chaisawang, Pornthip; Chaijaroonkhanarak, Wunnee; Prachaney, Parichat; Pannangrong, Wanassanun; Wigmore, Peter

2016-12-01

Valproic acid (VPA), a commonly used antiepileptic drug, has been reported to cause cognitive impairments in patients. In a previous study, using a rodent model, we showed that VPA treatment impaired cognition which was associated with a reduction in the cell proliferation required for hippocampal neurogenesis. The antidepressant fluoxetine has been shown to increase hippocampal neurogenesis and to reverse the memory deficits found in a number of pathological conditions. In the present study we investigated the protective effects of fluoxetine treatment against the impairments in memory and hippocampal cell proliferation produced by VPA. Male Sprague Dawley rats received daily treatment with fluoxetine (10mg/kg) by oral gavage for 21days. Some rats were co-administered with VPA (300mg/kg, twice daily i.p. injections) for 14days from day 8 to day 21 of the fluoxetine treatment. Spatial memory was tested using the novel object location (NOL) test. The number of proliferating cells present in the sub granular zone of the dentate gyrus was quantified using Ki67 immunohistochemistry at the end of the experiment. Levels of the receptor Notch1, the neurotrophic factor BDNF and the neural differentiation marker DCX were determined by Western blotting. VPA-treated rats showed memory deficits, a decrease in the number of proliferating cells in the sub granular zone and decreases in the levels of Notch1 and BDNF but not DCX compared to control animals. These changes in behavior, cell proliferation and Notch1 and BDNF were prevented in animals which had received both VPA and fluoxetine. Rats receiving fluoxetine alone did not show a significant difference in the number of proliferating cells or behavior compared to controls. These results demonstrated that the spatial memory deficits and reduction of cell proliferation produced by VPA can be ameliorated by the simultaneous administration of the antidepressant fluoxetine. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Physics constraints on double-pulse LIA engineering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ekdahl, Carl August Jr.

2015-05-20

The options for advanced-radiography double-pulse linear induction accelerators (LIA) under consideration naturally fall into three categories that differ by the number of cells required. Since the two major physics issues, beam breakup (BBU) and corkscrew, are also dependent on the number of cells, it may be useful for the decision process to review the engineering consequences of beam physics constraints for each class. The LIAs can be categorized three different ways, and this report compares the different categories based upon the physics of their beams.

Enhanced Expansion and Sustained Inductive Function of Skin‐Derived Precursor Cells in Computer‐Controlled Stirred Suspension Bioreactors

PubMed Central

Agabalyan, Natacha A.; Borys, Breanna S.; Sparks, Holly D.; Boon, Kathryn; Raharjo, Eko W.; Abbasi, Sepideh; Kallos, Michael S.

2016-01-01

Abstract Endogenous dermal stem cells (DSCs) reside in the adult hair follicle mesenchyme and can be isolated and grown in vitro as self‐renewing colonies called skin‐derived precursors (SKPs). Following transplantation into skin, SKPs can generate new dermis and reconstitute the dermal papilla and connective tissue sheath, suggesting they could have important therapeutic value for the treatment of skin disease (alopecia) or injury. Controlled cell culture processes must be developed to efficiently and safely generate sufficient stem cell numbers for clinical use. Compared with static culture, stirred‐suspension bioreactors generated fivefold greater expansion of viable SKPs. SKPs from each condition were able to repopulate the dermal stem cell niche within established hair follicles. Both conditions were also capable of inducing de novo hair follicle formation and exhibited bipotency, reconstituting the dermal papilla and connective tissue sheath, although the efficiency was significantly reduced in bioreactor‐expanded SKPs compared with static conditions. We conclude that automated bioreactor processing could be used to efficiently generate large numbers of autologous DSCs while maintaining their inherent regenerative function. Stem Cells Translational Medicine 2017;6:434–443 PMID:28191777

The influence of donor age on liver regeneration and hepatic progenitor cell populations.

PubMed

Ono, Yoshihiro; Kawachi, Shigeyuki; Hayashida, Tetsu; Wakui, Masatoshi; Tanabe, Minoru; Itano, Osamu; Obara, Hideaki; Shinoda, Masahiro; Hibi, Taizo; Oshima, Go; Tani, Noriyuki; Mihara, Kisyo; Kitagawa, Yuko

2011-08-01

Recent reports suggest that donor age might have a major impact on recipient outcome in adult living donor liver transplantation (LDLT), but the reasons underlying this effect remain unclear. The aims of this study were to compare liver regeneration between young and aged living donors and to evaluate the number of Thy-1+ cells, which have been reported to be human hepatic progenitor cells. LDLT donors were divided into 2 groups (Group O, donor age ≥ 50 years, n = 6 and Group Y, donor age ≤ 30 years, n = 9). The remnant liver regeneration rates were calculated on the basis of computed tomography volumetry on postoperative days 7 and 30. Liver tissue samples were obtained from donors undergoing routine liver biopsy or patients undergoing partial hepatectomy for metastatic liver tumors. Thy-1+ cells were isolated and counted using immunomagnetic activated cell sorting (MACS) technique. Donor liver regeneration rates were significantly higher in young donors compared to old donors (P = .042) on postoperative day 7. Regeneration rates were significantly higher after right lobe resection compared to rates after left lobe resection. The MACS findings showed that the number of Thy-1+ cells in the human liver consistently tended to decline with age. Our study revealed that liver regeneration is impaired with age after donor hepatectomy, especially after right lobe resection. The declining hepatic progenitor cell population might be one of the reasons for impaired liver regeneration in aged donors. Copyright © 2011 Mosby, Inc. All rights reserved.

Elevations in vascular markers and eosinophils in chronic spontaneous urticarial weals with low-level persistence in uninvolved skin

PubMed Central

Kay, AB; Ying, S; Ardelean, E; Mlynek, A; Kita, H; Clark, P; Maurer, M

2014-01-01

Background In chronic spontaneous urticaria (CSU) mast cell activation together with inflammatory changes in the skin are well documented and may play an important role in mechanisms of tissue oedema. Objectives To confirm and extend these observations by measuring microvascular markers, leucocytes and mast cell numbers in lesional and uninvolved skin and to compare findings with a control group. Methods Paired biopsies (one from 4–8-h spontaneous weals and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects and studied using immunohistochemistry and confocal microscopy using the lectin Ulex europaeus agglutinin 1 (UEA-1). Results Lesional skin in CSU contained significantly more CD31+ endothelial cells; CD31+ blood vessels, neutrophils, eosinophils, basophils and macrophages; and CD3+ T cells than nonlesional skin. Increased vascularity was confirmed by confocal imaging using the lectin UEA-1. Uninvolved skin from CSU contained significantly more CD31+ endothelial cells, CD31+ blood vessels and eosinophils compared with the control subjects. There was a threefold increase in mast cell numbers when CSU was compared with controls but no difference was observed between lesional and uninvolved skin. Conclusions Increased vascular markers together with eosinophil and neutrophil infiltration are features of lesional skin in CSU and might contribute to tissue oedema. Eosinophils and microvascular changes persist in uninvolved skin, which, together with increased mast cells, suggests that nonlesional skin is primed for further wealing. PMID:24665899

Effect of Thermodiffusion Nitriding on Cytocompatibility of Ti-6Al-4V Titanium Alloy

NASA Astrophysics Data System (ADS)

Pohrelyuk, I. M.; Tkachuk, O. V.; Proskurnyak, R. V.; Boiko, N. M.; Kluchivska, O. Yu.; Stoika, R. S.

2016-04-01

The nitrided layer was formed on the surface of Ti-6Al-4V titanium alloy by the thermodiffusion saturation in nitrogen at the atmospheric pressure. The study of the vitality of pseudonormal human embryo kidney cells of the HEK293T line showed that their cultivation in the presence of the untreated alloy sample is accompanied by a statistically significant reduction in the number of living cells compared with the control sample (untreated cells), whereas their cultivation in the presence of the nitrided alloy sample does not change the cell number considerably. In addition, it was shown that cell behavior in the presence of the nitrided sample differs only slightly from the control sample, whereas the growth of cells in the presence of the untreated alloy differed significantly from that in the control sample, demonstrating small groups of cells instead of their big clusters.

Secretory vesicles of immune cells contain only a limited number of interleukin 6 molecules.

PubMed

Verboogen, Daniëlle R J; Ter Beest, Martin; Honigmann, Alf; van den Bogaart, Geert

2018-05-01

Immune cells communicate by releasing large quantities of cytokines. Although the mechanisms of cytokine secretion are increasingly understood, quantitative knowledge of the number of cytokines per vesicle is still lacking. Here, we measured with quantitative microscopy the release rate of vesicles potentially carrying interleukin-6 (IL-6) in human dendritic cells. By comparing this to the total secreted IL-6, we estimate that secretory vesicles contain about 0.5-3 IL-6 molecules, but with a large spread among cells/donors. Moreover, IL-6 did not accumulate within most cells, indicating that synthesis and not trafficking is the bottleneck for IL-6 production. IL-6 accumulated in the Golgi apparatus only in ~ 10% of the cells. Understanding how immune cells produce cytokines is important for designing new immunomodulatory drugs. © 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Rebamipide protects against glaucoma eyedrop-induced ocular surface disorders in rabbits

PubMed Central

Kawaguchi, Ichiro; Higashide, Tomomi; Takeji, Yasuhiro; Sakurai, Kazushi; Kawaguchi, Chiaki; Sugiyama, Kazuhisa

2017-01-01

Purpose This study aimed to determine if rebamipide eyedrops can improve ocular surface damage caused by the use of glaucoma eyedrops. Methods Female Kbl:Dutch rabbits were used to evaluate glaucoma eyedrop-induced ocular surface damage; one eye of each rabbit was untreated and the other was administered glaucoma eyedrops for 30 days. To evaluate the effects of rebamipide on ocular surface damage, one eye of each rabbit was administered vehicle-treated glaucoma eyedrops and the other was administered rebamipide-treated glaucoma eyedrops for 30 days. Corneal and conjunctival epithelial damage was evaluated using fluorescein and rose bengal staining, respectively. Conjunctival inflammation was observed by light microscopy with hematoxylin-eosin staining. Dark cells (in which the corneal microvilli were damaged) were analyzed by scanning electron microscopy. Results There were no significant differences in fluorescein staining between the untreated and glaucoma eyedrop-treated groups; however, rose bengal staining and the number of inflammatory cells in the conjunctiva significantly increased after glaucoma eyedrop treatment. There was a four-fold increase in the number of dark cells in the glaucoma eyedrop-treated group compared to untreated. In contrast, in the conjunctiva of the rebamipide-treated glaucoma eyedrop group, rose bengal staining scores, the number of inflammatory cells, and the number of dark cells were decreased compared to the vehicle-treated glaucoma eyedrop group. Conclusions Results from our in vivo rabbit study demonstrated that short-term use of glaucoma eyedrops induces corneal epithelium disorders at the cellular level, but that simultaneous use of rebamipide has the potential to protect and repair the ocular surface. PMID:29049370

Rebamipide protects against glaucoma eyedrop-induced ocular surface disorders in rabbits.

PubMed

Kawaguchi, Ichiro; Kobayashi, Akira; Higashide, Tomomi; Takeji, Yasuhiro; Sakurai, Kazushi; Kawaguchi, Chiaki; Sugiyama, Kazuhisa

2017-01-01

This study aimed to determine if rebamipide eyedrops can improve ocular surface damage caused by the use of glaucoma eyedrops. Female Kbl:Dutch rabbits were used to evaluate glaucoma eyedrop-induced ocular surface damage; one eye of each rabbit was untreated and the other was administered glaucoma eyedrops for 30 days. To evaluate the effects of rebamipide on ocular surface damage, one eye of each rabbit was administered vehicle-treated glaucoma eyedrops and the other was administered rebamipide-treated glaucoma eyedrops for 30 days. Corneal and conjunctival epithelial damage was evaluated using fluorescein and rose bengal staining, respectively. Conjunctival inflammation was observed by light microscopy with hematoxylin-eosin staining. Dark cells (in which the corneal microvilli were damaged) were analyzed by scanning electron microscopy. There were no significant differences in fluorescein staining between the untreated and glaucoma eyedrop-treated groups; however, rose bengal staining and the number of inflammatory cells in the conjunctiva significantly increased after glaucoma eyedrop treatment. There was a four-fold increase in the number of dark cells in the glaucoma eyedrop-treated group compared to untreated. In contrast, in the conjunctiva of the rebamipide-treated glaucoma eyedrop group, rose bengal staining scores, the number of inflammatory cells, and the number of dark cells were decreased compared to the vehicle-treated glaucoma eyedrop group. Results from our in vivo rabbit study demonstrated that short-term use of glaucoma eyedrops induces corneal epithelium disorders at the cellular level, but that simultaneous use of rebamipide has the potential to protect and repair the ocular surface.

Vesicle Pool Size at the Salamander Cone Ribbon Synapse

PubMed Central

Bartoletti, Theodore M.; Babai, Norbert

2010-01-01

Cone light responses are transmitted to postsynaptic neurons by changes in the rate of synaptic vesicle release. Vesicle pool size at the cone synapse constrains the amount of release and can thus shape contrast detection. We measured the number of vesicles in the rapidly releasable and reserve pools at cone ribbon synapses by performing simultaneous whole cell recording from cones and horizontal or off bipolar cells in the salamander retinal slice preparation. We found that properties of spontaneously occurring miniature excitatory postsynaptic currents (mEPSCs) are representative of mEPSCs evoked by depolarizing presynaptic stimulation. Strong, brief depolarization of the cone stimulated release of the entire rapidly releasable pool (RRP) of vesicles. Comparing charge transfer of the EPSC with mEPSC charge transfer, we determined that the fast component of the EPSC reflects release of ∼40 vesicles. Comparing EPSCs with simultaneous presynaptic capacitance measurements, we found that horizontal cell EPSCs constitute 14% of the total number of vesicles released from a cone terminal. Using a fluorescent ribeye-binding peptide, we counted ∼13 ribbons per cone. Together, these results suggest each cone contacts a single horizontal cell at ∼2 ribbons. The size of discrete components in the EPSC amplitude histogram also suggested ∼2 ribbon contacts per cell pair. We therefore conclude there are ∼20 vesicles per ribbon in the RRP, similar to the number of vesicles contacting the plasma membrane at the ribbon base. EPSCs evoked by lengthy depolarization suggest a reserve pool of ∼90 vesicles per ribbon, similar to the number of additional docking sites further up the ribbon. PMID:19923246

The influence of magnetic fields exposure on neurite outgrowth in PC12 rat pheochromocytoma cells

NASA Astrophysics Data System (ADS)

Fan, W.; Ding, J.; Duan, W.; Zhu, Y. M.

2004-11-01

The aim of present work was to investigate the influence of magnetic fields exposure on neurite outgrowth in PC12 cells. The neurite number per cell, length of neurites and directions of neurite growth with respect to the direction of the magnetic field were analyzed after exposure to 50 Hz electromagnetic field for 96 h. A promotion was observed under a weak field (0.23 mT), as the average number of neurites per cell increased to 2.38±0.06 compared to 1.91±0.07 neurites/cell of the control dishes, while inhibition and directional outgrowth was evident under a relatively stronger field (1.32 mT). Our work shows that biological systems can be very sensitive to the strength of electromagnetic field.

Ultrasound-guided fine-needle aspiration biopsy of clinically suspicious thyroid nodules with an automatic aspirator: a novel technique.

PubMed

Nagarajah, James; Sheu-Grabellus, Sien-Yi; Farahati, Jamshid; Kamruddin, Kamer A; Bockisch, Andreas; Schmid, Kurt Werner; Görges, Rainer

2012-07-01

Fine-needle aspiration biopsy (FNAB) is a simple technique for the investigation of suspicious thyroid nodules. However, low success rates are reported in the literature. The aim of this prospective study was to compare the clinical performance and impact of an automatic aspirator, referred to here as Aspirator 3, to those of the manual technique for the FNAB of clinically suspicious thyroid nodules. One hundred nine consecutive patients with 121 clinically suspicious thyroid nodules underwent a biopsy twice of the same site with the clinically approved Aspirator 3 and with the manual technique. The number of follicular cell formations and the total number of follicular cells in the aspirate were counted using the ThinPrep® method. With the Aspirator 3, the total number and the mean number of extracted cell formations were significantly higher than the values achieved with the manual technique (total: 3222 vs. 1951, p=0.02; mean: 27 vs. 16). The total number of cells that were biopsied was also higher when the Aspirator 3 was utilized (47,480 vs. 23,080, p=0.005). Overall, the Aspirator 3 was superior in 65 biopsies, and the manual technique was superior in 39 biopsies. In terms of cell formations and the total number of cells aspirated, the Aspirator 3 was superior to the manual technique. Further, the Aspirator 3 was more convenient to use and had a greater precision in needle guidance.

Expression of interleukin-33 and its receptor ST2 in periapical granulomas and radicular cysts.

PubMed

Velickovic, Milena; Pejnovic, Nada; Petrovic, Renata; Mitrovic, Slobodanka; Jeftic, Ilija; Kanjevac, Tatjana; Lukic, Aleksandra

2016-01-01

Interleukin-33 (IL-33) is a recently identified cytokine belonging to the IL-1 family and ligand for the IL-1 receptor-related protein ST2. IL-33/ST2 signaling plays a critical role in allergy, autoimmunity, and chronic inflammatory disorders, but its role in the pathogenesis of periapical lesions is unknown. We aimed to investigate the expression patterns of IL-33 and ST2 in human periapical lesions. Periapical lesions (n = 36) and healthy periapical tissues (n = 10) were evaluated by immunohistochemistry using antibodies specific for human IL-33 and ST2. Lesion samples were further analyzed by double immunofluorescence to assess IL-33/ST2 co-expression. The numbers of IL-33- and ST2-positive fibroblasts were significantly higher in periapical lesions compared to healthy periapical tissues (both P < 0.05), while the numbers of IL-33- and ST2-positive endothelial cells were similar (both P > 0.05). There were no significant differences in the numbers of IL-33- and ST2-positive fibroblasts and endothelial cells between periapical granulomas and radicular cysts (all P > 0.05). Similarly, numbers of ST2-positive mononuclear cells did not differ between periapical granulomas and radicular cysts (P > 0.05). The majority of epithelial cells in radicular cysts were IL-33 positive, while the small proportion of epithelial cells was ST2 positive. Double immunofluorescence analysis revealed IL-33/ST2 co-expression in fibroblasts and endothelial cells. IL-33 and ST2 are expressed in periapical granulomas and radicular cysts. Increased numbers of IL-33- and ST2-positive fibroblasts in periapical lesions when compared to healthy periapical tissues suggest that IL-33/ST2 signaling may be involved in periapical inflammation and tissue fibrosis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transient Suppression of TGFβ Receptor Signaling Facilitates Human Islet Transplantation

PubMed Central

Fischbach, Shane; Song, Zewen; Gaffar, Iljana; Zimmerman, Ray; Wiersch, John; Prasadan, Krishna; Shiota, Chiyo; Guo, Ping; Ramachandran, Sabarinathan; Witkowski, Piotr

2016-01-01

Although islet transplantation is an effective treatment for severe diabetes, its broad application is greatly limited due to a shortage of donor islets. Suppression of TGFβ receptor signaling in β-cells has been shown to increase β-cell proliferation in mice, but has not been rigorously examined in humans. Here, treatment of human islets with a TGFβ receptor I inhibitor, SB-431542 (SB), significantly improved C-peptide secretion by β-cells, and significantly increased β-cell number by increasing β-cell proliferation. In addition, SB increased cell-cycle activators and decreased cell-cycle suppressors in human β-cells. Transplantation of SB-treated human islets into diabetic immune-deficient mice resulted in significant improvement in blood glucose control, significantly higher serum and graft insulin content, and significantly greater increases in β-cell proliferation in the graft, compared with controls. Thus, our data suggest that transient suppression of TGFβ receptor signaling may improve the outcome of human islet transplantation, seemingly through increasing β-cell number and function. PMID:26872091

Combined inhibition of autophagy and caspases fails to prevent developmental nurse cell death in the Drosophila melanogaster ovary.

PubMed

Peterson, Jeanne S; McCall, Kimberly

2013-01-01

During the final stages of Drosophila melanogaster oogenesis fifteen nurse cells, sister cells to the oocyte, degenerate as part of normal development. This process involves at least two cell death mechanisms, caspase-dependent cell death and autophagy, as indicated by apoptosis and autophagy markers. In addition, mutations affecting either caspases or autophagy partially reduce nurse cell removal, leaving behind end-stage egg chambers with persisting nurse cell nuclei. To determine whether apoptosis and autophagy work in parallel to degrade and remove these cells as is the case with salivary glands during pupariation, we generated mutants doubly affecting caspases and autophagy. We found no significant increase in either the number of late stage egg chambers containing persisting nuclei or in the number of persisting nuclei per egg chamber in the double mutants compared to single mutants. These findings suggest that there is another cell death mechanism functioning in the ovary to remove all nurse cell remnants from late stage egg chambers.

Ex-vivo expansion of red blood cells: how real for transfusion in humans?

PubMed

Migliaccio, Anna Rita; Masselli, Elena; Varricchio, Lilian; Whitsett, Carolyn

2012-03-01

Blood transfusion is indispensable for modern medicine. In developed countries, the blood supply is adequate and safe but blood for alloimmunized patients is often unavailable. Concerns are increasing that donations may become inadequate in the future as the population ages prompting a search for alternative transfusion products. Improvements in culture conditions and proof-of-principle studies in animal models have suggested that ex-vivo expanded red cells may represent such a product. Compared to other cell therapies transfusion poses the unique challenge of requiring great cell doses (2.5×10(12) cells vs 10(7) cells). Although production of such cell numbers is theoretically possible, current technologies generate red cells in numbers sufficient only for safety studies. It is conceived that by the time these studies will be completed, technical barriers to mass cell production will have been eliminated making transfusion with ex-vivo generated red cells a reality. Copyright © 2011 Elsevier Ltd. All rights reserved.

Tbo-Filgrastim versus Filgrastim during Mobilization and Neutrophil Engraftment for Autologous Stem Cell Transplantation.

PubMed

Elayan, Mohammed M; Horowitz, Justin G; Magraner, Jose M; Shaughnessy, Paul J; Bachier, Carlos

2015-11-01

There are limited data available supporting the use of the recombinant granulocyte colony-stimulating factor (G-CSF), tbo-filgrastim, rather than traditionally used filgrastim to mobilize peripheral blood stem cells (PBSC) or to accelerate engraftment after autologous stem cell transplantation (ASCT). We sought to compare the efficacy and cost of tbo-filgrastim to filgrastim in these settings. Patients diagnosed with lymphoma or plasma cell disorders undergoing G-CSF mobilization, with or without plerixafor, were included in this retrospective analysis. The primary outcome was total collected CD34(+) cells/kg. Secondary mobilization endpoints included peripheral CD34(+) cells/μL on days 4 and 5 of mobilization, adjunctive use of plerixafor, CD34(+) cells/kg collected on day 5, number of collection days and volumes processed, number of collections reaching 5 million CD34(+) cells/kg, and percent reaching target collection goal in 1 day. Secondary engraftment endpoints included time to neutrophil and platelet engraftment, number of blood product transfusions required before engraftment, events of febrile neutropenia, and length of stay. A total of 185 patients were included in the final analysis. Patients receiving filgrastim (n = 86) collected a median of 5.56 × 10(6) CD34(+) cells/kg, compared with a median of 5.85 × 10(6) CD34(+) cells/kg in the tbo-filgrastim group (n = 99; P = .58). There were no statistically significant differences in all secondary endpoints with the exception of apheresis volumes processed (tbo-filgrastim, 17.0 liters versus filgrastim, 19.7 liters; P < .01) and mean platelet transfusions (tbo-filgrastim, 1.7 units versus filgrastim, 1.4 units; P = .04). In conclusion, tbo-filgrastim demonstrated similar CD34(+) yield compared with filgrastim in mobilization and post-transplantation settings, with no clinically meaningful differences in secondary efficacy and safety endpoints. Furthermore, tbo-filgrastim utilization was associated with cost savings of approximately $1406 per patient utilizing average wholesale price. Copyright © 2015 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Effect of Hedera helix on lung histopathology in chronic asthma.

PubMed

Hocaoglu, Arzu Babayigit; Karaman, Ozkan; Erge, Duygu Olmez; Erbil, Guven; Yilmaz, Osman; Kivcak, Bijen; Bagriyanik, H Alper; Uzuner, Nevin

2012-12-01

Hedera helix is widely used to treat bronchial asthma for many years. However, effects of this herb on lung histopathology is still far from clear. We aimed to determine the effect of oral administration of Hedera helix on lung histopathology in a murine model of chronic asthma.BALB/c mice were divided into four groups; I (Placebo), II (Hedera helix), III (Dexamethasone) and IV (Control). All mice except controls were sensitized and challenged with ovalbumin. Then, mice in group I received saline, group II 100 mg/kg Hedera helix and group III 1 mg/kg dexamethasone via orogastic gavage once daily for one week. Airway histopathology was evaluated by using light and electron microscopy in all groups.Goblet cell numbers and thicknesses of basement membrane were found significantly lower in group II, but there was no statistically significant difference in terms of number of mast cells, thicknesses of epithelium and subepithelial smooth muscle layers between group I and II. When Hedera helix and dexamethasone groups were compared with each other, thickness of epithelium, subepithelial muscle layers, number of mast cells and goblet cells of group III were significantly ameliorated when compared with the group II. Although Hedera helix administration reduced only goblet cell counts and the thicknesses of basement membrane in the asthmatic airways, dexamethasone ameliorated all histopathologic parameters except thickness of basement membrane better than Hedera helix.

Allelic Variation of Ets1 Does Not Contribute to NK and NKT Cell Deficiencies in Type 1 Diabetes Susceptible NOD Mice

PubMed Central

Jordan, Margaret A.; Poulton, Lynn D.; Fletcher, Julie M.; Baxter, Alan G.

2009-01-01

The NOD mouse is a well characterized model of type 1 diabetes that shares several of the characteristics of Ets1-deficient targeted mutant mice, viz: defects in TCR allelic exclusion, susceptibility to a lupus like disease characterized by IgM and IgG autoantibodies and immune complex-mediated glomerulonephritis, and deficiencies of NK and NKT cells. Here, we sought evidence for allelic variation of Ets1 in mice contributing to the NK and NKT cell phenotypes of the NOD strain. ETS1 expression in NK and NKT cells was reduced in NOD mice, compared to C57BL/6 mice. Although NKT cells numbers were significantly correlated with ETS1 expression in both strains, NKT cell numbers were not linked to the Ets1 gene in a first backcross from NOD to C57BL/6 mice. These results indicate that allelic variation of Ets1 did not contribute to variation in NKT cell numbers in these mice. It remains possible that a third factor not linked to the Ets1 locus controls both ETS1 expression and subsequently NK and NKT cell phenotypes. PMID:19806240

Good manufacturing practice-compliant expansion of marrow-derived stem and progenitor cells for cell therapy.

PubMed

Gastens, Martin H; Goltry, Kristin; Prohaska, Wolfgang; Tschöpe, Diethelm; Stratmann, Bernd; Lammers, Dirk; Kirana, Stanley; Götting, Christian; Kleesiek, Knut

2007-01-01

Ex vivo expansion is being used to increase the number of stem and progenitor cells for autologous cell therapy. Initiation of pivotal clinical trials testing the efficacy of these cells for tissue repair has been hampered by the challenge of assuring safe and high-quality cell production. A strategy is described here for clinical-scale expansion of bone marrow (BM)-derived stem cells within a mixed cell population in a completely closed process from cell collection through postculture processing using sterile connectable devices. Human BM mononuclear cells (BMMNC) were isolated, cultured for 12 days, and washed postharvest using either standard open procedures in laminar flow hoods or using automated closed systems. Conditions for these studies were similar to long-term BM cultures in which hematopoietic and stromal components are cultured together. Expansion of marrow-derived stem and progenitor cells was then assessed. Cell yield, number of colony forming units (CFU), phenotype, stability, and multilineage differentiation capacity were compared from the single pass perfusion bioreactor and standard flask cultures. Purification of BMMNC using a closed Ficoll gradient process led to depletion of 98% erythrocytes and 87% granulocytes, compared to 100% and 70%, respectively, for manual processing. After closed system culture, mesenchymal progenitors, measured as CD105+CD166+CD14-CD45- and fibroblastic CFU, expanded 317- and 364-fold, respectively, while CD34+ hematopoietic progenitors were depleted 10-fold compared to starting BMMNC. Cultured cells exhibited multilineage differentiation by displaying adipogenic, osteogenic, and endothelial characteristics in vitro. No significant difference was observed between manual and bioreactor cultures. Automated culture and washing of the cell product resulted in 181 x 10(6) total cells that were viable and contained fibroblastic CFU for at least 24 h of storage. A combination of closed, automated technologies enabled production of good manufacturing practice (GMP)-compliant cell therapeutics, ready for use within a clinical setting, with minimal risk of microbial contamination.

Colonic stem cell data are consistent with the immortal model of stem cell division under non-random strand segregation.

PubMed

Walters, K

2009-06-01

Colonic stem cells are thought to reside towards the base of crypts of the colon, but their numbers and proliferation mechanisms are not well characterized. A defining property of stem cells is that they are able to divide asymmetrically, but it is not known whether they always divide asymmetrically (immortal model) or whether there are occasional symmetrical divisions (stochastic model). By measuring diversity of methylation patterns in colon crypt samples, a recent study found evidence in favour of the stochastic model, assuming random segregation of stem cell DNA strands during cell division. Here, the effect of preferential segregation of the template strand is considered to be consistent with the 'immortal strand hypothesis', and explore the effect on conclusions of previously published results. For a sample of crypts, it is shown how, under the immortal model, to calculate mean and variance of the number of unique methylation patterns allowing for non-random strand segregation and compare them with those observed. The calculated mean and variance are consistent with an immortal model that incorporates non-random strand segregation for a range of stem cell numbers and levels of preferential strand segregation. Allowing for preferential strand segregation considerably alters previously published conclusions relating to stem cell numbers and turnover mechanisms. Evidence in favour of the stochastic model may not be as strong as previously thought.

Inter-operator variation in ELISPOT analysis of measles virus-specific IFN-gamma-secreting T cells.

PubMed

Ryan, J E; Ovsyannikova, I G; Dhiman, N; Pinsky, N A; Vierkant, R A; Jacobson, R M; Poland, G A

2005-01-01

The ELISPOT assay is a highly sensitive technique used for the detection of individual cytokine releasing cells. We have developed an IFN-gamma ELISPOT assay utilizing unfractionated frozen peripheral blood mononuclear cells (PBMC) to quantify the frequency of measles virus (MV)-specific IFN-gamma-secreting T cells in 117 healthy children who had been previously immunized with two doses of the measles-mumps-rubella vaccine. We have also estimated the variability associated with the quantification of ELISPOT plates and compared the number of MV-specific IFN-gamma-secreting T cells for each subject as determined by two different operators of an ELISPOT reader. The median frequency of MV-specific IFN-gamma-producing memory T cells detected by this assay was 0.005 % and 0.01 % as determined by an in-house and commercial operator, respectively. Although we found a significant correlation (r = 0.83, p<0.0001) between the number of spots counted by the commercial and in-house operators of an ELISPOT reader, the median number of spots counted by the commercial operator was twice the number of spots counted by an in-house operator (p<0.001). This demonstrates the importance of using a common ELISPOT reader and operator, among other parameters, to quantify the number of spots when a large volume of plates are being scanned and analyzed.

A retrospective analysis of RET translocation, gene copy number gain and expression in NSCLC patients treated with vandetanib in four randomized Phase III studies.

PubMed

Platt, Adam; Morten, John; Ji, Qunsheng; Elvin, Paul; Womack, Chris; Su, Xinying; Donald, Emma; Gray, Neil; Read, Jessica; Bigley, Graham; Blockley, Laura; Cresswell, Carl; Dale, Angela; Davies, Amanda; Zhang, Tianwei; Fan, Shuqiong; Fu, Haihua; Gladwin, Amanda; Harrod, Grace; Stevens, James; Williams, Victoria; Ye, Qingqing; Zheng, Li; de Boer, Richard; Herbst, Roy S; Lee, Jin-Soo; Vasselli, James

2015-03-23

To determine the prevalence of RET rearrangement genes, RET copy number gains and expression in tumor samples from four Phase III non-small-cell lung cancer (NSCLC) trials of vandetanib, a selective inhibitor of VEGFR, RET and EGFR signaling, and to determine any association with outcome to vandetanib treatment. Archival tumor samples from the ZODIAC ( NCT00312377 , vandetanib ± docetaxel), ZEAL ( NCT00418886 , vandetanib ± pemetrexed), ZEPHYR ( NCT00404924 , vandetanib vs placebo) and ZEST ( NCT00364351 , vandetanib vs erlotinib) studies were evaluated by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) in 944 and 1102 patients. The prevalence of RET rearrangements by FISH was 0.7% (95% CI 0.3-1.5%) among patients with a known result. Seven tumor samples were positive for RET rearrangements (vandetanib, n = 3; comparator, n = 4). 2.8% (n = 26) of samples had RET amplification (innumerable RET clusters, or ≥7 copies in > 10% of tumor cells), 8.1% (n = 76) had low RET gene copy number gain (4-6 copies in ≥40% of tumor cells) and 8.3% (n = 92) were RET expression positive (signal intensity ++ or +++ in >10% of tumor cells). Of RET-rearrangement-positive patients, none had an objective response in the vandetanib arm and one patient responded in the comparator arm. Radiologic evidence of tumor shrinkage was observed in two patients treated with vandetanib and one treated with comparator drug. The objective response rate was similar in the vandetanib and comparator arms for patients positive for RET copy number gains or RET protein expression. We have identified prevalence for three RET biomarkers in a population predominated by non-Asians and smokers. RET rearrangement prevalence was lower than previously reported. We found no evidence of a differential benefit for efficacy by IHC and RET gene copy number gains. The low prevalence of RET rearrangements (0.7%) prevents firm conclusions regarding association of vandetanib treatment with efficacy in the RET rearrangement NSCLC subpopulation. Randomized Phase III clinical trials ( NCT00312377 , ZODIAC; NCT00418886 , ZEAL; NCT00364351 , ZEST; NCT00404924 , ZEPHYR).

Differential adipokine receptor expression on circulating leukocyte subsets in lean and obese children.

PubMed

Keustermans, Genoveva; van der Heijden, Laila B; Boer, Berlinda; Scholman, Rianne; Nuboer, Roos; Pasterkamp, Gerard; Prakken, Berent; de Jager, Wilco; Kalkhoven, Eric; Janse, Arieke J; Schipper, Henk S

2017-01-01

Childhood obesity prevalence has increased worldwide and is an important risk factor for type 2 diabetes (T2D) and cardiovascular disease (CVD). The production of inflammatory adipokines by obese adipose tissue contributes to the development of T2D and CVD. While levels of circulating adipokines such as adiponectin and leptin have been established in obese children and adults, the expression of adiponectin and leptin receptors on circulating immune cells can modulate adipokine signalling, but has not been studied so far. Here, we aim to establish the expression of adiponectin and leptin receptors on circulating immune cells in obese children pre and post-lifestyle intervention compared to normal weight control children. 13 obese children before and after a 1-year lifestyle intervention were compared with an age and sex-matched normal weight control group of 15 children. Next to routine clinical and biochemical parameters, circulating adipokines were measured, and flow cytometric analysis of adiponectin receptor 1 and 2 (AdipoR1, AdipoR2) and leptin receptor expression on peripheral blood mononuclear cell subsets was performed. Obese children exhibited typical clinical and biochemical characteristics compared to controls, including a higher BMI-SD, blood pressure and circulating leptin levels, combined with a lower insulin sensitivity index (QUICKI). The 1-year lifestyle intervention resulted in stabilization of their BMI-SD. Overall, circulating leukocyte subsets showed distinct adipokine receptor expression profiles. While monocytes expressed high levels of all adipokine receptors, NK and iNKT cells predominantly expressed AdipoR2, and B-lymphocytes and CD4+ and CD8+ T-lymphocyte subsets expressed AdipoR2 as well as leptin receptor. Strikingly though, leukocyte subset numbers and adipokine receptor expression profiles were largely similar in obese children and controls. Obese children showed higher naïve B-cell numbers, and pre-intervention also higher numbers of immature transition B-cells and intermediate CD14++CD16+ monocytes combined with lower total monocyte numbers, compared to controls. Furthermore, adiponectin receptor 1 expression on nonclassical CD14+CD16++ monocytes was consistently upregulated in obese children pre-intervention, compared to controls. However, none of the differences in leukocyte subset numbers and adipokine receptor expression profiles between obese children and controls remained significant after multiple testing correction. First, the distinct adipokine receptor profiles of circulating leukocyte subsets may partly explain the differential impact of adipokines on leukocyte subsets. Second, the similarities in adipokine receptor expression profiles between obese children and normal weight controls suggest that adipokine signaling in childhood obesity is primarily modulated by circulating adipokine levels, instead of adipokine receptor expression.

Photoperiod affects the expression of sex and species differences in leukocyte number and leukocyte trafficking in congeneric hamsters.

PubMed

Bilbo, S D; Dhabhar, F S; Viswanathan, K; Saul, A; Nelson, R J

2003-11-01

Sex differences in immune function are well documented. These sex differences may be modulated by social and environmental factors. Individuals of polygynous species generally exhibit more pronounced sex differences in immune parameters than individuals of monogamous species, often displaying an energetic trade-off between enhanced immunity and high mating success. During winter, animals contend with environmental conditions (e.g. low temperatures and decreased food availability) that evoke energetic-stress responses; many mammals restrict reproduction in response to photoperiod as part of an annual winter coping strategy. To test the hypothesis that extant sex and species differences in immune surveillance may be modulated by photoperiod, we examined leukocyte numbers in males and females of two closely related hamster species (Phodopus). As predicted, uniparental P. sungorus exhibited a robust sex difference, with total white blood cells, total lymphocytes, T cells, and B cells higher in females than males, during long days when reproduction occurs, but not during short days when reproduction usually stops. In contrast, biparental male and female P. campbelli exhibited comparable leukocyte numbers during both long and short days. To study sex differences in stress responses, we also examined immune cell trafficking in response to an acute (2 h) restraint stressor. During stressful challenges, it appears beneficial for immune cells to exit the blood and move to primary immune defense areas such as the skin, in preparation for potential injury or infection. Acute stress moved lymphocytes and monocytes out of the blood in all animals. Blood cortisol concentrations were increased in P. sungorus females compared to males at baseline (52%) and in response to restraint stress (38%), but only in long days. P. campbelli males and females exhibited comparable blood cortisol and stress responses during both long and short days. Our results suggest that interactions among social factors and the environment play a significant role in modulating sex and seasonal alterations in leukocyte numbers and stress responses.

Precision oncology using a limited number of cells: optimization of whole genome amplification products for sequencing applications.

PubMed

Sho, Shonan; Court, Colin M; Winograd, Paul; Lee, Sangjun; Hou, Shuang; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2017-07-01

Sequencing analysis of circulating tumor cells (CTCs) enables "liquid biopsy" to guide precision oncology strategies. However, this requires low-template whole genome amplification (WGA) that is prone to errors and biases from uneven amplifications. Currently, quality control (QC) methods for WGA products, as well as the number of CTCs needed for reliable downstream sequencing, remain poorly defined. We sought to define strategies for selecting and generating optimal WGA products from low-template input as it relates to their potential applications in precision oncology strategies. Single pancreatic cancer cells (HPAF-II) were isolated using laser microdissection. WGA was performed using multiple displacement amplification (MDA), multiple annealing and looping based amplification (MALBAC) and PicoPLEX. Quality of amplified DNA products were assessed using a multiplex/RT-qPCR based method that evaluates for 8-cancer related genes and QC-scores were assigned. We utilized this scoring system to assess the impact of de novo modifications to the WGA protocol. WGA products were subjected to Sanger sequencing, array comparative genomic hybridization (aCGH) and next generation sequencing (NGS) to evaluate their performances in respective downstream analyses providing validation of the QC-score. Single-cell WGA products exhibited a significant sample-to-sample variability in amplified DNA quality as assessed by our 8-gene QC assay. Single-cell WGA products that passed the pre-analysis QC had lower amplification bias and improved aCGH/NGS performance metrics when compared to single-cell WGA products that failed the QC. Increasing the number of cellular input resulted in improved QC-scores overall, but a resultant WGA product that consistently passed the QC step required a starting cellular input of at least 20-cells. Our modified-WGA protocol effectively reduced this number, achieving reproducible high-quality WGA products from ≥5-cells as a starting template. A starting cellular input of 5 to 10-cells amplified using the modified-WGA achieved aCGH and NGS results that closely matched that of unamplified, batch genomic DNA. The modified-WGA protocol coupled with the 8-gene QC serve as an effective strategy to enhance the quality of low-template WGA reactions. Furthermore, a threshold number of 5-10 cells are likely needed for a reliable WGA reaction and product with high fidelity to the original starting template.

Morphologically Distinct Escherichia coli Bacteriophages Differ in Their Efficacy and Ability to Stimulate Cytokine Release In Vitro.

PubMed

Khan Mirzaei, Mohammadali; Haileselassie, Yeneneh; Navis, Marit; Cooper, Callum; Sverremark-Ekström, Eva; Nilsson, Anders S

2016-01-01

Due to a global increase in the range and number of infections caused by multi-resistant bacteria, phage therapy is currently experiencing a resurgence of interest. However, there are a number of well-known concerns over the use of phages to treat bacterial infections. In order to address concerns over safety and the poorly understood pharmacokinetics of phages and their associated cocktails, immunological characterization is required. In the current investigation, the immunogenicity of four distinct phages (taken from the main families that comprise the Caudovirales order) and their interaction with donor derived peripheral blood mononuclear cells and immortalized cell lines (HT-29 and Caco-2 intestinal epithelial cells) were investigated using standard immunological techniques. When exposed to high phage concentrations (10(9) PFU/well), cytokine driven inflammatory responses were induced from all cell types. Although phages appeared to inhibit the growth of intestinal epithelial cell lines, they also appear to be non-cytotoxic. Despite co-incubation with different cell types, phages maintained a high killing efficiency, reducing extended-spectrum beta-lactamase-producing Escherichia coli numbers by 1-4 log10 compared to untreated controls. When provided with a suitable bacterial host, phages were also able to actively reproduce in the presence of human cells resulting in an approximately 2 log10 increase in phage titer compared to the initial inoculum. Through an increased understanding of the complex pharmacokinetics of phages, it may be possible to address some of the safety concerns surrounding phage preparations prior to creating new therapeutic strategies.

Increased numbers of Foxp3-positive regulatory T cells in gastritis, peptic ulcer and gastric adenocarcinoma

PubMed Central

Cheng, Hsin-Hung; Tseng, Guan-Ying; Yang, Hsiao-Bai; Wang, Hung-Jung; Lin, Hwai-Jeng; Wang, Wen-Ching

2012-01-01

AIM: To determine the number of regulatory T cells (Tregs) in gastric mucosa of patients with gastritis, peptic ulcers and gastric cancer. METHODS: This study was a retrospective analysis of gastric antrum biopsy specimens from healthy controls (n = 22) and patients with gastritis (n = 30), peptic ulcer (n = 83), or gastric cancer (n = 32). Expression of CD4, CD25 and Foxp3 was determined by immunohistochemistry in three consecutive sections per sample. RESULTS: Compared with healthy controls, there was an increased number of CD25+ and Foxp3+ cells in patients with gastritis (P = 0.004 and P = 0.008), peptic ulcer (P < 0.001 and P < 0.001), and gastric cancer (P < 0.001 and P < 0.001). The ratio of CD25+/CD4+ or Foxp3+/CD4+ cells was also significantly higher in all disease groups (P < 0.001, respectively). The number of CD4+, CD25+, and Foxp3+ cells, and the ratio of CD25+/CD4+ and Foxp3+/CD4+ cells, were associated with the histological grade of the specimens, including acute inflammation, chronic inflammation, lymphoid follicle number, and Helicobacter pylori infection. The number of CD4+, CD25+ and Foxp3+ cells, and the ratio of CD25+/CD4+ and Foxp3+/CD4+ cells, were negatively associated with intestinal metaplasia among gastritis (P < 0.001, P < 0.001, P < 0.001, P = 0.002 and P = 0.002) and peptic ulcer groups (P = 0.013, P = 0.004, P < 0.001, P = 0.040 and P = 0.003). CONCLUSION: Tregs are positively associated with endoscopic findings of gastroduodenal diseases and histological grade but negatively associated with intestinal metaplasia in gastritis and peptic ulcer groups. PMID:22228968

Gender differences and lateralization in the distribution pattern of insulin-like growth factor-1 receptor in developing rat hippocampus: an immunohistochemical study.

PubMed

Hami, Javad; Kheradmand, Hamed; Haghir, Hossein

2014-03-01

Numerous investigators have provided data supporting essential roles for insulin-like growth factor-I (IGF-I) in development of the brain. The aim of this study was to immunohistochemically determine the distinct regional distribution pattern of IGF-1 receptor (IGF-IR) expression in various portions of newborn rat hippocampus on postnatal days 0 (P0), 7 (P7), and 14 (P14), with comparison between male/female and right/left hippocampi. We found an overall significant increase in distribution of IGF-IR-positive (IGF-IR+) cells in CA1 from P0 until P14. Although, no marked changes in distribution of IGF-IR+ cells in areas CA2 and CA3 were observed; IGF-IR+ cells in DG decreased until P14. The smallest number of immunoreactive cells was present in CA2 and the highest number in DG at P0. Moreover, in CA1, CA3, and DG, the number of IGF-IR+ cells was markedly higher in both sides of the hippocampus in females. Our data also showed a higher mean number of IGF-IR+ cells in the left hippocampus of female at P7. By contrast, male pups showed a significantly higher number of IGF-IR+ cells in the DG of the right hippocampus. At P14, the mean number of immunoreactive cells in CA1, CA3, and DG areas found to be significantly increased in left side of hippocampus of males, compared to females. These results indicate the existence of a differential distribution pattern of IGF-IR between left-right and male-female hippocampi. Together with other mechanisms, these differences may underlie sexual dimorphism and left-right asymmetry in the hippocampus.

Acid Sphingomyelinase (ASM) is a Negative Regulator of Regulatory T Cell (Treg) Development.

PubMed

Zhou, Yuetao; Salker, Madhuri S; Walker, Britta; Münzer, Patrick; Borst, Oliver; Gawaz, Meinrad; Gulbins, Erich; Singh, Yogesh; Lang, Florian

2016-01-01

Regulatory T cell (Treg) is required for the maintenance of tolerance to various tissue antigens and to protect the host from autoimmune disorders. However, Treg may, indirectly, support cancer progression and bacterial infections. Therefore, a balance of Treg function is pivotal for adequate immune responses. Acid sphingomyelinase (ASM) is a rate limiting enzyme involved in the production of ceramide by breaking down sphingomyelin. Previous studies in T-cells have suggested that ASM is involved in CD28 signalling, T lymphocyte granule secretion, degranulation, and vesicle shedding similar to the formation of phosphatidylserine-exposing microparticles from glial cells. However, whether ASM affects the development of Treg has not yet been described. Splenocytes, isolated Naive T lymphocytes and cultured T cells were characterized for various immune T cell markers by flow cytometery. Cell proliferation was measured by Carboxyfluorescein succinimidyl ester (CFSE) dye, cell cycle analysis by Propidium Iodide (PI), mRNA transcripts by q-RT PCR and protein expression by Western Blotting respectively. ASM deficient mice have higher number of Treg compared with littermate control mice. In vitro induction of ASM deficient T cells in the presence of TGF-β and IL-2 lead to a significantly higher number of Foxp3+ induced Treg (iTreg) compared with control T-cells. Further, ASM deficient iTreg has less AKT (serine 473) phosphorylation and Rictor levels compared with control iTreg. Ceramide C6 led to significant reduction of iTreg in both ASM deficient and WT mice. The reduction in iTreg leads to induction of IL-1β, IL-6 and IL-17 but not IFN-γ mRNA levels. ASM is a negative regulator of natural and iTreg. © 2016 The Author(s) Published by S. Karger AG, Basel.

Defibrotide in combination with granulocyte colony-stimulating factor significantly enhances the mobilization of primitive and committed peripheral blood progenitor cells in mice.

PubMed

Carlo-Stella, Carmelo; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Stucchi, Claudio; Cleris, Loredana; Formelli, Franca; Gianni, Massimo A

2002-11-01

Defibrotide is a polydeoxyribonucleotide, which significantly reduces the expression of adhesion molecules on endothelial cells. We investigated the activity of Defibrotide alone or in combination with recombinant human granulocyte colony-stimulating factor (rhG-CSF) to mobilize peripheral blood progenitor cells (PBPCs) in BALB/c mice. A 5-day treatment with Defibrotide alone (1-15 mg/mouse/day) had no effect on WBC counts, frequencies and absolute numbers of total circulating colony-forming cells (CFCs), i.e., granulocyte-macrophage colony-forming units, erythroid burst-forming units, and multilineage colony-forming units. As compared with mock-injected mice, administration of rhG-CSF alone (5 micro g/mouse/day) for 5 days significantly (P < or = 0.0001) increased WBC counts, CFC frequencies, and CFC absolute numbers by 2-, 13-, and 27-fold, respectively. As compared with control mice, the combined administration of Defibrotide (15 mg/mouse/day) and rhG-CSF significantly (P < or = 0.0001) increased WBC counts, frequencies and absolute numbers of CFCs by 4-, 38-, and 119-fold, respectively. As compared with rhG-CSF alone, administration of Defibrotide plus rhG-CSF resulted in a significant increase (P < or = 0.001) of the frequency of circulating long-term culture-initiating cells. In addition, transplantation of 2 x 10(5) rhG-CSF- or Defibrotide/rhG-CSF-mobilized mononuclear cells rescued 43% and 71% of recipient mice, respectively. Experiments of CFC homing performed in lethally irradiated or nonirradiated recipients showed that marrow homing of transplanted PBPCs was reduced by 3-fold in Defibrotide-treated animals as compared with mock-injected mice (P < or = 0.001), suggesting that the mobilizing effect of Defibrotide might be because of an effect on PBPC trafficking. In conclusion, our data demonstrate that Defibrotide synergizes with rhG-CSF and significantly increases the mobilization of a broad spectrum of PBPCs, including primitive and committed progenitor cells. These data might have relevant implications for autologous and allogeneic anticancer therapy in humans.

Intracellular protein determination using droplet-based immunoassays.

PubMed

Martino, Chiara; Zagnoni, Michele; Sandison, Mairi E; Chanasakulniyom, Mayuree; Pitt, Andrew R; Cooper, Jonathan M

2011-07-01

This paper describes the implementation of a sensitive, on-chip immunoassay for the analysis of intracellular proteins, developed using microdroplet technology. The system offers a number of analytical functionalities, enabling the lysis of low cell numbers, as well as protein detection and quantification, integrated within a single process flow. Cells were introduced into the device in suspension and were electrically lysed in situ. The cell lysate was subsequently encapsulated together with antibody-functionalized beads into stable, water-in-oil droplets, which were stored on-chip. The binding of intracellular proteins to the beads was monitored fluorescently. By analyzing many individual droplets and quantifying the data obtained against standard additions, we measured the level of two intracellular proteins, namely, HRas-mCitrine, expressed within HEK-293 cells, and actin-EGFP, expressed within MCF-7 cells. We determined the concentrations of these proteins over 5 orders of magnitude, from ~50 pM to 1 μM. The results from this semiautomated method were compared to those for determinations made using Western blots, and were found not only to be faster, but required a smaller number of cells.

Low Copy Numbers of DC-SIGN in Cell Membrane Microdomains: Implications for Structure and Function

PubMed Central

Liu, Ping; Wang, Xiang; Itano, Michelle S.; Neumann, Aaron K.; de Silva, Aravinda M.; Jacobson, Ken; Thompson, Nancy L.

2014-01-01

Presently, there are few estimates of the number of molecules occupying membrane domains. Using a total internal reflection fluorescence microscopy (TIRFM) imaging approach, based on comparing the intensities of fluorescently labeled microdomains with those of single fluorophores, we measured the occupancy of DC-SIGN, a C-type lectin, in membrane microdomains. DC-SIGN or its mutants were labeled with primary monoclonal antibodies (mAbs) in either dendritic cells (DCs) or NIH3T3 cells, or expressed as GFP fusions in NIH3T3 cells. The number of DC-SIGN molecules per microdomain ranges from only a few to over 20, while microdomain dimensions range from the diffraction limit to > 1μm. The largest fraction of microdomains, appearing at the diffraction limit, in either immature DCs or 3T3 cells contains only 4-8 molecules of DC-SIGN, consistent with our preliminary super-resolution Blink microscopy estimates. We further show that these small assemblies are sufficient to bind and efficiently internalize a small (~50nm) pathogen, dengue virus, leading to infection of host cells. PMID:24313910

Comparison of twin-cell centrifugal partition chromatographic columns with different cell volume.

PubMed

Goll, Johannes; Audo, Gregoire; Minceva, Mirjana

2015-08-07

Two twin-cell centrifugal partition chromatographic columns (SCPC 250 and SCPE-250-BIO, Armen Instrument, France) with the same column volume but different cell size and number were compared in terms of stationary phase retention and column efficiency. The columns were tested with two types of solvent systems: a commonly used organic solvent based biphasic system from the ARIZONA solvent system family and a polymer/salt based aqueous two phase system (ATPS). The efficiency of the columns was evaluated by pulse injection experiments of two benzenediols (pyrocatechol and hydroquinone) in the case of the ARIZONA system and a protein mixture (myoglobin and lysozyme) in the case of the ATPS. As result of high stationary phase retention, the column with the lower number of larger twin-cells (SCPE-250-BIO) is suitable for protein separations using ATPS. On the other hand, due to higher column efficiency, the column with the greater number of smaller cells (SCPC 250) is superior for batch elution separations performed with standard liquid-liquid chromatography organic solvent based biphasic systems. Copyright © 2015 Elsevier B.V. All rights reserved.

Modulation of cell adhesion and viability of cultured murine bone marrow cells by arsenobetaine, a major organic arsenic compound in marine animals.

PubMed

Sakurai, T; Fujiwara, K

2001-01-01

1. In this study, we investigated the biological effects of trimethyl (carboxymethyl) arsonium zwitterion, namely arsenobetaine (AsBe), which is a major organic arsenic compound in marine animals using murine bone marrow (BM) cells and compared them with those of an inorganic arsenical, sodium arsenite, in vitro. 2. Sodium arsenite showed strong cytotoxicity in BM cells, and its IC(50) was 6 microM. In contrast, AsBe significantly enhanced the viability of BM cells in a dose-dependent manner during a 72-h incubation; about a twofold increase in the viability of cells compared with that of control cells cultured with the medium alone was observed with a microM level of AsBe. 3. In morphological investigations, AsBe enhanced the numbers of large mature adherent cells, especially granulocytes, during a 72-h BM culture. When BM cells were cultured together with AsBe and a low dose (1 u ml(-1)) of recombinant murine granulocyte/macrophage colony-stimulating factor (rMu GM-CSF), significant additive-like increasing effects were observed on the numbers of both granulocytes and macrophages originated from BM cells. However, AsBe did not cause proliferation of BM cells at all as determined by colony-forming assay using a gelatinous medium. 4. These findings demonstrate the unique and potent biological effects in mammalian cells of AsBe, a major organic arsenic compound in various marine animals which are ingested daily as seafood in many countries.

Hypericin-mediated photocytotoxic effect on HT-29 adenocarcinoma cells is reduced by light fractionation with longer dark pause between two unequal light doses.

PubMed

Sacková, Veronika; Kuliková, Lucia; Mikes, Jaromír; Kleban, Ján; Fedorocko, Peter

2005-01-01

The present study demonstrates the in vitro effect of hypericin-mediated PDT with fractionated light delivery. Cells were photosensitized with unequal light fractions separated by dark intervals (1 or 6 h). We compared the changes in viability, cell number, survival, apoptosis and cell cycle on HT-29 cells irradiated with a single light dose (12 J/cm(2)) to the fractionated light delivery (1 + 11 J/cm(2)) 24 and 48 h after photodynamic treatment. We found that a fractionated light regime with a longer dark period resulted in a decrease of hypericin cytotoxicity. Both cell number and survival were higher after light sensitization with a 6-h dark interval. DNA fragmentation occurred after a single light-dose application, but in contrast no apoptotic DNA formation was detected with a 6-h dark pause. After fractionation the percentage of cells in the G1 phase of the cell cycle was increased, while the proportion of cells in the G2 phase decreased as compared to a single light-dose application, i.e. both percentage of cells in the G1 and G2 phase of the cell cycle were near control levels. We presume that the longer dark interval after the irradiation of cells by first light dose makes them resistant to the effect of the second illumination. These findings confirm that the light application scheme together with other photodynamic protocol components is crucial for the photocytotoxicity of hypericin.

Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application.

PubMed

Priglinger, E; Maier, J; Chaudary, S; Lindner, C; Wurzer, C; Rieger, S; Redl, H; Wolbank, S; Dungel, P

2018-06-01

A highly interesting source for adult stem cells is adipose tissue, from which the stromal vascular fraction (SVF)-a heterogeneous cell population including the adipose-derived stromal/stem cells-can be obtained. To enhance the regenerative potential of freshly isolated SVF cells, low-level light therapy (LLLT) was used. The effects of pulsed blue (475 nm), green (516 nm), and red (635 nm) light from light-emitting diodes applied on freshly isolated SVF were analysed regarding cell phenotype, cell number, viability, adenosine triphosphate content, cytotoxicity, and proliferation but also osteogenic, adipogenic, and proangiogenic differentiation potential. The colony-forming unit fibroblast assay revealed a significantly increased colony size after LLLT with red light compared with untreated cells, whereas the frequency of colony-forming cells was not affected. LLLT with green and red light resulted in a stronger capacity to form vascular tubes by SVF when cultured within 3D fibrin matrices compared with untreated cells, which was corroborated by increased number and length of the single tubes and a significantly higher concentration of vascular endothelial growth factor. Our study showed beneficial effects after LLLT on the vascularization potential and proliferation capacity of SVF cells. Therefore, LLLT using pulsed light-emitting diode light might represent a new approach for activation of freshly isolated SVF cells for direct clinical application. Copyright © 2018 John Wiley & Sons, Ltd.

Distinct abnormalities in the innate immune system of children with Down syndrome.

PubMed

Bloemers, Beatrijs L P; van Bleek, Grada M; Kimpen, Jan L L; Bont, Louis

2010-05-01

To analyze the frequency and phenotype of cells of the innate immune system in the peripheral blood of children with Down syndrome (DS). Flow cytometric analysis of expression of cell surface markers was performed in children with DS (n = 41) and healthy age-matched controls (n = 41). Compared with controls, children with DS had significantly lower absolute total leukocyte counts, lymphocytes, monocytes, and granulocytes, but 1.5-times higher absolute numbers of CD14(dim)CD16(+) monocytes (147 x 10(6)/L vs 93 x 10(6)/L; P = .02). This difference is fully explained by a higher percentage of CD14(dim)CD16(+) monocytes within the monocyte compartment (28.7% vs 13.4%; P <.001). The absolute numbers of myeloid dendritic cells were lower in DS (13.8 x 10(6)/L vs 22.7 x 10(6)/L; P <.001). The numbers of plasmacytoid dendritic cells and natural killer cells were normal. Absolute numbers of invariant natural killer T cells were very low overall, but significantly lower in children with DS than in controls (1.2 x 10(6)/L vs 3.7 x 10(6)/L; P = .01). Children with DS exhibited distinct abnormalities in cells of the innate immune system. Most strikingly, they had a high number of proinflammatory CD14(dim)CD16(+) monocytes. This elevated level of CD14(dim)CD16(+) monocytes may play an important role in the onset and maintenance of chronic inflammatory disease in DS.

Response of iNOS and its relationship with IL-22 and STAT3 in macrophage activity in the polar forms of leprosy.

PubMed

de Sousa, Jorge Rodrigues; de Sousa, Raphael Primo Martins; de Souza Aarão, Tinara Leila; Dias, Leonidas Braga; Oliveira Carneiro, Francisca Regina; Simões Quaresma, Juarez Antonio

2017-07-01

Leprosy is a chronic granulomatous infection that manifests as different clinical forms related to the immunological response. The aim of the study was to evaluated the response of IL-22, STAT3, CD68 and iNOS in leprosy skin lesions. The mean number IL-22 positive cells was 12.12±1.90cells/field in the TT form and 31.31±2.91cells/field in the LL form. STAT3 positive cells was 5.29±1.96 cells/field in the TT form, while this number was 11.13±3.48cells/field in the LL form. The mean number of CD68 positive cells was 25.18±6.21cells/field in the TT form and 62.81±8.13cells/field in the LL form. Quantitative analysis of iNOS revealed a significant difference, with the mean number of cells expressing the enzyme being 30.24±2.88cells/field in the TT form compared to 35.44±4.69cells/field in the LL form. Linear correlations in lesions of TT patients showed a moderate positive correlations between CD68 and iNOS, STAT3 and Inos, IL-22 and STAT3, and IL-22 and iNOS. Our results demonstrate that these factors can act synergistically to induce a microbicidal activity in the population of macrophages in the leprosy lesions. Copyright © 2017 Elsevier B.V. All rights reserved.

Altered levels of memory T cell subsets and common γc cytokines in Strongyloides stercoralis infection and partial reversal following anthelmintic treatment.

PubMed

Rajamanickam, Anuradha; Munisankar, Saravanan; Bhootra, Yukti; Dolla, Chandra Kumar; Thiruvengadam, Kannan; Nutman, Thomas B; Babu, Subash

2018-05-01

CD4+ and CD8+ T cells are central players in immunity to helminth infections. However, the role of T cell subsets in human helminth infections is not well understood. In addition, the common γc cytokines, IL-2, IL-4, IL-7, IL-9 and IL-15 play an important role in the maintenance of these CD4+ and CD8+ T cell subsets. To examine the major T cell subsets and their association with the common γc cytokines, the absolute numbers of CD4+ and CD8+ naïve, central memory, effector memory and effector cells and the plasma levels of IL-2, IL-4, IL-7, IL-9 and IL-15 were measured in Strongyloides stercoralis (Ss) infected (INF, n = 60), helminth-uninfected (UN, n = 58) and in post treatment INF individuals. Ss infection is characterized by significantly increased absolute numbers of naïve and decreased absolute numbers of central and effector memory CD4+ T cells in comparison to UN individuals. No significant difference in the numbers of CD8+ T cell subsets was observed between the groups. The numbers of naïve cells and central memory CD4+ T cells were significantly reversed after anthelmintic treatment. Circulating levels of IL-2, IL-7 and IL-15 were significantly diminished, whereas the levels of IL-4 and IL-9 were significantly increased in INF compared to UN individuals. Following anthelminthic treatment, IL-2, IL-7 and IL-15 levels were significantly increased, while IL-4 and IL-9 levels were significantly decreased. Our data also showed a significant positive correlation between the levels of IL-7 and the numbers of central and effector memory CD4+ T cells. Ss infection is characterized by alterations in the absolute numbers of CD4+ T cell subsets and altered levels of common γc cytokines IL-2, IL-4, IL-7, IL-9 and IL-15; alterations which are partially reversed after anthelmintic treatment.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

PubMed Central

Kusuma, Gina D.; Brennecke, Shaun P.; O’Connor, Andrea J.; Kalionis, Bill

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies. PMID:28152107

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion.

PubMed

Kusuma, Gina D; Brennecke, Shaun P; O'Connor, Andrea J; Kalionis, Bill; Heath, Daniel E

2017-01-01

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Understanding the antimicrobial activity behind thin- and thick-rolled copper plates.

PubMed

Yousuf, Basit; Ahire, Jayesh J; Dicks, Leon M T

2016-06-01

The aim of this study was to compare the antibacterial properties of the surfaces of copper plates that were rolled to a thickness of 25 and 100 μm. Differences in topology of 25- and 100-μm-thick copper plates were studied using scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray diffraction (XRD). Antibacterial activity of the copper surfaces was tested against strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, Streptococcus sp. BY1, Enterococcus sp. BY2, and Bacillus cereus BY3. Changes in viable cell numbers were determined by plating onto optimal growth media and staining with LIVE/DEAD BacLight™. Changes in metabolic activity were recorded by expression of the luciferase (lux) gene. Cell morphology was studied using SEM. Accumulation and diffusion of copper from cells were recorded using inductively coupled plasma mass spectroscopy (ICP-MS). Lipid and protein oxidation were recorded spectrophotometrically. Surfaces of 25-μm-thick copper plates were rough compared to that of 100-μm-thick copper plates. For most species, a five-log reduction in cell numbers, cell membrane instability, and a decline in metabolic activity were recorded after 15 min of exposure to 25-μm-thick copper plates. Copper accumulated in the cells, and lipids and proteins were oxidized. The rough surface of thinner copper plates (25 μm thick) released more copper and was more antimicrobial compared to thicker (100 μm) copper plates. Cell death was attributed to destabilization of the cell membrane, lipid peroxidation, and protein oxidation.

An Analysis of microRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-13-1-0082 TITLE: An Analysis of microRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells ...syndromes (MDS) to identify microRNAs (miRNAs) dysregulated in MDS hematopoietic stem cells (MDS HSCs) as compared with normal HSCs. MiRNAs differentially...the age-related predisposition for the development of MDS. 15. SUBJECT TERMS MicroRNAs, the myelodysplastic syndromes, hematopoietic stem cells

An Analysis of MicroRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells

DTIC Science & Technology

2015-10-01

AWARD NUMBER: W81XWH-13-1-0082 TITLE: An Analysis of microRNA Expression in the Myelodysplastic Syndromes Using Hematopoietic Stem Cells ...syndromes (MDS) to identify microRNAs (miRNAs) dysregulated in MDS hematopoietic stem cells (MDS HSCs) as compared with normal HSCs. MiRNAs differentially...the age-related predisposition for the development of MDS. 15. SUBJECT TERMS MicroRNAs, the myelodysplastic syndromes, hematopoietic stem cells

Aging-dependent decline of IL-10 producing B cells coincides with production of antinuclear antibodies but not rheumatoid factors.

PubMed

van der Geest, Kornelis S M; Lorencetti, Pedro G; Abdulahad, Wayel H; Horst, Gerda; Huitema, Minke; Roozendaal, Caroline; Kroesen, Bart-Jan; Brouwer, Elisabeth; Boots, Annemieke M H

2016-03-01

Aging is associated with development of autoimmunity. Loss of B cell tolerance in the elderly is suggested by an increased prevalence of anti-nuclear antibodies (ANAs) and rheumatoid factors (RFs). Accumulating evidence indicates that B cells also impact autoimmunity via secretion of cytokines. So far, few studies have directly assessed the effect of aging on the latter B cell function. Here, we determined if and how human aging influences the production of cytokines by B cells. In a cross-sectional study, we found that absolute numbers of circulating B cells were similar in 31 young (ages 19-39) and 73 old (age ≥ 60) individuals. Numbers of transitional B cells (CD19(+)CD27(-)CD38(High)CD24(High)) were decreased in old individuals, whereas numbers of naive and memory B cell subsets were comparable in young and old individuals. Short-term in vitro stimulation of whole blood samples revealed that numbers of B cells capable of producing TNF-α were similar in young and old individuals. In contrast, B cells capable of IL-10 production were decreased in old subjects. This decline of IL-10(+) B cells was observed in old individuals that were ANA positive, and in those that were negative for both ANAs and RFs. However, IL-10(+) B cells were remarkably well retained in the circulation of old subjects that were RF positive. Thus, pro-inflammatory TNF-α(+) B cells are retained in the elderly, whereas IL-10(+) B cells generally decline. In addition, our findings indicate that IL-10(+) B cells may differentially impact the development of ANAs and RFs in the elderly. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer.

PubMed

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-10-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer

PubMed Central

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-01-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer. PMID:28943918

Polyamine spermidine is an upstream negator of ethylene-regulated pathogenesis of botrytis cinerea in tomato leaf

USDA-ARS?s Scientific Manuscript database

Polyamines are biogenic polycationic compounds implicated in a number of processes including plant cell division, cell elongation, flowering, fruit set and development, fruit ripening, senescence and responses to abiotic stresses. Comparatively, little is known about their role in plant-microbe int...

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

PubMed Central

Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

2018-01-01

Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

Morphologic and Functional Effects of Gamma Secretase Inhibition on Splenic Marginal Zone B Cells

PubMed Central

de Vera Mudry, Maria Cristina; Regenass-Lechner, Franziska; Ozmen, Laurence; Altmann, Bernd; Festag, Matthias; Singer, Thomas; Müller, Lutz; Jacobsen, Helmut; Flohr, Alexander

2012-01-01

The γ-secretase complex is a promising target in Alzheimer's disease because of its role in the amyloidogenic processing of β-amyloid precursor protein. This enzyme also catalyzes the cleavage of Notch receptor, resulting in the nuclear translocation of intracellular Notch where it modulates gene transcription. Notch signaling is essential in cell fate decisions during embryogenesis, neuronal differentiation, hematopoiesis, and development of T and B cells, including splenic marginal zone (MZ) B cells. This B cell compartment participates in the early phases of the immune response to blood-borne bacteria and viruses. Chronic treatment with the oral γ-secretase inhibitor RO4929097 resulted in dose-dependent decreased cellularity (atrophy) of the MZ of rats and mice. Significant decreases in relative MZ B-cell numbers of RO4929097-treated animals were confirmed by flow cytometry. Numbers of MZ B cells reverted to normal after a sufficient RO4929097-free recovery period. Functional characterization of the immune response in relation to RO4929097-related MZ B cell decrease was assessed in mice vaccinated with inactivated vesicular stomatitis virus (VSV). Compared with the immunosuppressant cyclosporin A, RO4929097 caused only mild and reversible delayed early neutralizing IgM and IgG responses to VSV. Thus, the functional consequence of MZ B cell decrease on host defense is comparatively mild. PMID:23316412

Paneth and intestinal stem cells preserve their functional integrity during worsening of acute cellular rejection in small bowel transplantation.

PubMed

Pucci Molineris, M; Gonzalez Polo, V; Perez, F; Ramisch, D; Rumbo, M; Gondolesi, G E; Meier, D

2018-04-01

Graft survival after small bowel transplantation remains impaired due to acute cellular rejection (ACR), the leading cause of graft loss. Although it was shown that the number of enteroendocrine progenitor cells in intestinal crypts was reduced during mild ACR, no results of Paneth and intestinal stem cells localized at the crypt bottom have been shown so far. Therefore, we wanted to elucidate integrity and functionality of the Paneth and stem cells during different degrees of ACR, and to assess whether these cells are the primary targets of the rejection process. We compared biopsies from ITx patients with no, mild, or moderate ACR by immunohistochemistry and quantitative PCR. Our results show that numbers of Paneth and stem cells remain constant in all study groups, whereas the transit-amplifying zone is the most impaired zone during ACR. We detected an unchanged level of antimicrobial peptides in Paneth cells and similar numbers of Ki-67 + IL-22R + stem cells revealing cell functionality in moderate ACR samples. We conclude that Paneth and stem cells are not primary target cells during ACR. IL-22R + Ki-67 + stem cells might be an interesting target cell population for protection and regeneration of the epithelial monolayer during/after a severe ACR in ITx patients. © 2017 The American Society of Transplantation and the American Society of Transplant Surgeons.

Co-regulation of pluripotency and genetic integrity at the genomic level.

PubMed

Cooper, Daniel J; Walter, Christi A; McCarrey, John R

2014-11-01

The Disposable Soma Theory holds that genetic integrity will be maintained at more pristine levels in germ cells than in somatic cells because of the unique role germ cells play in perpetuating the species. We tested the hypothesis that the same concept applies to pluripotent cells compared to differentiated cells. Analyses of transcriptome and cistrome databases, along with canonical pathway analysis and chromatin immunoprecipitation confirmed differential expression of DNA repair and cell death genes in embryonic stem cells and induced pluripotent stem cells relative to fibroblasts, and predicted extensive direct and indirect interactions between the pluripotency and genetic integrity gene networks in pluripotent cells. These data suggest that enhanced maintenance of genetic integrity is fundamentally linked to the epigenetic state of pluripotency at the genomic level. In addition, these findings demonstrate how a small number of key pluripotency factors can regulate large numbers of downstream genes in a pathway-specific manner. Copyright © 2014. Published by Elsevier B.V.

Antral G-cell in gastrin and gastrin-cholecystokinin knockout animals.

PubMed

Friis-Hansen, Lennart; Wierup, Nils; Rehfeld, Jens F; Sundler, Frank

2005-07-01

The antral hormone gastrin is the key regulator of gastric acid secretion, mucosal growth and differentiation. Gastrin is synthesized in the endocrine G-cells in the antroduodenal mucosa. We have now examined the way in which the loss of gastrin alone or gastrin plus cholecystokinin (CCK) affects the antral G-cell. Immunohistochemistry, radioimmunoassay and quantitative real-time polymerase chain reaction techniques were employed to examine the expression of genes belonging to the G-cell secretory pathway in gastrin and gastrin-CCK knockout mice. Transmission electron microscopy was used to examine the ultrastructure of the G-cells. The number of G-cells increased but the secretory granules were few and abnormally small in the G-cells of both mouse models compared with wildtypes. Thus, gastrin is not necessary for the formation of G-cells as such but the lack of gastrin reduces the number and size of their secretory granules suggesting that gastrin is vital for the formation and/or maintenance of secretory granules in G-cells.

Potential over request in anemia laboratory tests in primary care in Spain.

PubMed

Salinas, María; López-Garrigós, Maite; Flores, Emilio; Uris, Joaquín; Leiva-Salinas, Carlos

2015-07-01

The aim was to study the inter-practice variability in anemia laboratory tests requested by general practitioners in Spain, to evaluate for a potential requesting inappropriateness. Laboratories from diverse Spanish regions filled out the number of cell blood count, ferritin, folate, iron, transferrin, and vitamin B12 requested by general practitioners during 2012. The number of test requests per 1000 inhabitants and ratios of related tests requests were calculated. The results obtained in hospitals from different areas (urban, rural, or urban-rural), type of management (public or private), and geographic regions were compared. There was a high variability in the number of test requests and ratios of related tests. Cell blood count was over requested in rural areas and in hospitals with private management. Andalucía was the community with the lowest number of iron requests and the lowest folate/vitamin B12 indicator value. Iron and transferrin seemed over requested in some areas; as were folate and ferritin when compared to vitamin B12 and cell blood count, respectively. The differences observed between areas indicate that other factors besides clinical reasons could be behind that variability and emphasize the need to accomplish interventions to improve the appropriate use of anemia laboratory tests.

Canonical Wnt Signaling as a Specific Marker of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2013-02-01

get enough sorted mammary cells for the transplantation experiments. We are currently working with our Flow Cytometry Core to sort Lin-/CD24+/CD49...activity our flow cytometry data suggests t here is a 2-fold increase in the number of FOG+ MEC’s in BATgal animals compared to contro ls which...this populat ion of cells is enriched for stem cell activity. Flow cytometry will determine the percentage of FOG+ cells within pre-neoplastic BATgai

Persistent low thymic activity and non-cardiac mortality in children with chromosome 22q11·2 microdeletion and partial DiGeorge syndrome

PubMed Central

Eberle, P; Berger, C; Junge, S; Dougoud, S; Büchel, E Valsangiacomo; Riegel, M; Schinzel, A; Seger, R; Güngör, T

2009-01-01

A subgroup of patients with 22q11·2 microdeletion and partial DiGeorge syndrome (pDGS) appears to be susceptible to non-cardiac mortality (NCM) despite sufficient overall CD4+ T cells. To detect these patients, 20 newborns with 22q11·2 microdeletion and congenital heart disease were followed prospectively for 6 years. Besides detailed clinical assessment, longitudinal monitoring of naive CD4+ and cytotoxic CD3+CD8+ T cells (CTL) was performed. To monitor thymic activity, we analysed naive platelet endothelial cell adhesion molecule-1 (CD31+) expressing CD45RA+RO−CD4+ cells containing high numbers of T cell receptor excision circle (TREC)-bearing lymphocytes and compared them with normal values of healthy children (n = 75). Comparing two age periods, low overall CD4+ and naive CD4+ T cell numbers were observed in 65%/75%, respectively, of patients in period A (< 1 year) declining to 22%/50%, respectively, of patients in period B (> 1/< 7 years). The percentage of patients with low CTLs (< P10) remained robust until school age (period A: 60%; period B: 50%). Low numbers of CTLs were associated with abnormally low naive CD45RA+RO−CD4+ T cells. A high-risk (HR) group (n = 11) and a standard-risk (SR) (n = 9) group were identified. HR patients were characterized by low numbers of both naive CD4+ and CTLs and were prone to lethal infectious and lymphoproliferative complications (NCM: four of 11; cardiac mortality: one of 11) while SR patients were not (NCM: none of nine; cardiac mortality: two of nine). Naive CD31+CD45RA+RO−CD4+, naive CD45RA+RO−CD4+ T cells as well as TRECs/106 mononuclear cells were abnormally low in HR and normal in SR patients. Longitudinal monitoring of naive CD4+ and cytotoxic T cells may help to discriminate pDGS patients at increased risk for NCM. PMID:19040613

[Effect of aurora kinase B inhibitor AZD1152 in the treatment of cisplatin-resistant ovarian carcinoma].

PubMed

Ma, Ya-xi; Li, Xiu-zhen

2013-01-01

To investigate whether AZD1152 (AZD), the selective inhibitor of aurora kinase B, may play a role in the treatment of cisplatin-resistant ovarian carcinoma when administrated alone or in combination with cisplatin. Hey (cisplatin-resistant ovarian cancer cell line) cells were analyzed. According to the treatment plan, Hey cells were divided into four groups (AZD group, cisplatin group, AZD + cisplatin group and control group). Methyl thiazolyl tetrazolium (MTT) assay was used to test the cells proliferation, caspase-3/7 activity analysis was used to analyze cells apoptosis, and fluorescence in-situ hybridization (FISH) assay was used to determine the copy the number of chromosome 7 and checked the copy numbers of hTERC gene and C-myc gene. MTT test showed that proliferation of AZD group was lower than that in control group (P < 0.01). The cells proliferation with the treatment with 10 and 20 nmol/L AZD for 24 hours was (81.4 ± 3.6)% and (81.4 ± 3.6)% respectively, and the cells proliferation for 48 hours was (43.1 ± 2.0)% and (38.5 ± 1.6)% respectively, which was significantly lower than control group (100%, P < 0.01); Treated with the same concentration of AZD, inhibition of proliferation was significantly enhanced as the time extended (P < 0.01). Proliferation in group AZD + cisplatin was lower than that in cisplatin group (P < 0.01) which suggest that there were additive effects after combined AZD with cisplatin. Compared with control group, caspase-3/7 activity in AZD group increased significantly (P = 0.000), and the same results was seen between AZD + cisplatin group and cisplatin group or AZD group (all P < 0.01). Compared with cisplatin group or control group, the copy numbers of hTERC, C-myc and the number of chromosome were significantly increased in AZD group and AZD + cisplat group (all P < 0.05). AZD could inhibit ovarian cancer cells proliferation and induce cells apoptosis significantly. AZD alone or in combination with cisplatin may result in the increased cells polyploidy.

Is the PentaBDE replacement, tris (1,3-dichloro-2-propyl) phosphate (TDCPP), a developmental neurotoxicant? Studies in PC12 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dishaw, Laura V.; Powers, Christina M.; Ryde, Ian T.

Organophosphate flame retardants (OPFRs) are used as replacements for the commercial PentaBDE mixture that was phased out in 2004. OPFRs are ubiquitous in the environment and detected at high concentrations in residential dust, suggesting widespread human exposure. OPFRs are structurally similar to neurotoxic organophosphate pesticides, raising concerns about exposure and toxicity to humans. This study evaluated the neurotoxicity of tris (1,3-dichloro-2-propyl) phosphate (TDCPP) compared to the organophosphate pesticide, chlorpyrifos (CPF), a known developmental neurotoxicant. We also tested the neurotoxicity of three structurally similar OPFRs, tris (2-chloroethyl) phosphate (TCEP), tris (1-chloropropyl) phosphate (TCPP), and tris (2,3-dibromopropyl) phosphate (TDBPP), and 2,2 Primemore » ,4,4 Prime -tetrabromodiphenyl ether (BDE-47), a major component of PentaBDE. Using undifferentiated and differentiating PC12 cells, changes in DNA synthesis, oxidative stress, differentiation into dopaminergic or cholinergic neurophenotypes, cell number, cell growth and neurite growth were assessed. TDCPP displayed concentration-dependent neurotoxicity, often with effects equivalent to or greater than equimolar concentrations of CPF. TDCPP inhibited DNA synthesis, and all OPFRs decreased cell number and altered neurodifferentiation. Although TDCPP elevated oxidative stress, there was no adverse effect on cell viability or growth. TDCPP and TDBPP promoted differentiation into both neuronal phenotypes, while TCEP and TCPP promoted only the cholinergic phenotype. BDE-47 had no effect on cell number, cell growth or neurite growth. Our results demonstrate that different OPFRs show divergent effects on neurodifferentiation, suggesting the participation of multiple mechanisms of toxicity. Additionally, these data suggest that OPFRs may affect neurodevelopment with similar or greater potency compared to known and suspected neurotoxicants.« less

Assessment and modeling of E-waste generation based on growth rate from different telecom companies in the State of Kuwait.

PubMed

Al-Anzi, Bader S; Al-Burait, Abdul Aziz; Thomas, Ashly; Ong, Chi Siang

2017-12-01

The present work assesses the production rate of cell phone e-waste in Kuwait by comparing the number of clients in three telecommunication service providers like Zain, Ooredoo, and Viva in the state of Kuwait over a period of 7 years from 2008 to 2015. An online survey was conducted to evaluate the growth in the number of clients in three cell phone companies, and the data analysis was carried out using statistical package for the social sciences (SPSS) software. The prediction of the growth percentage of the number of clients in each telecommunication company was analyzed using analysis of variance (ANOVA) test and followed by the regression model. The study shows that there is an increase in the number of clients in all three companies (Zain, Ooredoo, and Viva) between year 2008 and 2015, and it was estimated that approximately 7.9 million cell phone users would be achieved in the first quarter of 2015. Based on this predicted number of cell phone users, the production of e-waste would be 3 kt per year with an average growth of 12.7%.

Immunohistochemical analysis of FoxP3+ cells in periapical granulomas and radicular cysts.

PubMed

Peixoto, Raniel Fernandes; Pereira, Joabe dos Santos; Nonaka, Cassiano Francisco Weege; Silveira, Ericka Janine Dantas da; Miguel, Márcia Cristina da Costa

2012-09-01

To compare the number of FoxP3(+) cells between periapical granulomas (PGs) and radicular cysts (RCs), and to correlate this number with the intensity of the inflammatory infiltrate in these lesions and with epithelial thickness of RCs. Thirty PGs and 30 RCs were submitted to immunohistochemical analysis using an anti-FoxP3 polyclonal antibody. FoxP3(+) cells were counted under a light microscope (×400 magnification) in five fields and the mean value was calculated for each specimen. Statistical tests were used to evaluate differences in the number of FoxP3(+) cells according to type of lesion (PG vs. RC), intensity of the inflammatory infiltrate (grade I/II vs. grade III), and epithelial thickness of RCs (atrophic vs. hyperplastic). FoxP3(+) cells were detected in most PGs (93.3%) and RCs (93.3%). The median number of FoxP3(+) cells was 2.40 in PGs and 1.00 in RCs, with this difference being statistically significant (P=0.005). No significant differences in the number of FoxP3(+) cells were observed in terms of the intensity of the inflammatory infiltrate (P=0.465) or epithelial thickness of RCs (P=0.737). The present results suggest a greater participation of regulatory T cells in the modulation of the inflammatory response in PGs. In addition, the presence of a less effective regulatory environment in RCs, together with the high levels of inflammatory mediators as reported in the literature, may contribute to the greater growth potential of these lesions. Copyright © 2012 Elsevier Ltd. All rights reserved.

Droplet Microarray Based on Superhydrophobic-Superhydrophilic Patterns for Single Cell Analysis.

PubMed

Jogia, Gabriella E; Tronser, Tina; Popova, Anna A; Levkin, Pavel A

2016-12-09

Single-cell analysis provides fundamental information on individual cell response to different environmental cues and is a growing interest in cancer and stem cell research. However, current existing methods are still facing challenges in performing such analysis in a high-throughput manner whilst being cost-effective. Here we established the Droplet Microarray (DMA) as a miniaturized screening platform for high-throughput single-cell analysis. Using the method of limited dilution and varying cell density and seeding time, we optimized the distribution of single cells on the DMA. We established culturing conditions for single cells in individual droplets on DMA obtaining the survival of nearly 100% of single cells and doubling time of single cells comparable with that of cells cultured in bulk cell population using conventional methods. Our results demonstrate that the DMA is a suitable platform for single-cell analysis, which carries a number of advantages compared with existing technologies allowing for treatment, staining and spot-to-spot analysis of single cells over time using conventional analysis methods such as microscopy.

Multiple cells express interleukin 17 in oral squamous cell carcinoma.

PubMed

Avadhani, Avadhoot V; Parachuru, Venkata P B; Milne, Trudy; Seymour, Gregory J; Rich, Alison M

2017-01-01

Interleukin (IL)-17 is a pro-inflammatory cytokine with pro- and antitumour effects. The aim of this study was to investigate the presence and potential sources of IL-17 in oral squamous cell carcinoma (OSCC). Immunohistochemistry was used to label and compare IL-17 + cells in the tissue sections of OSCC and inflammatory controls (IC), n = 14 for both. In OSCC, the comparison was made between the number of IL-17 + cells in the tumoral islands (TI), tumour-stroma interface (TS) and more distant stroma (DS). Cells expressing IL-17 were identified using double-labelling immunofluorescence and examined using laser scanning microscopy. The production of IL-17 from tumour cells was determined in the culture supernatants of OSCC cell lines, SCC4, SCC15 and SCC25, using sandwich ELISA. Significantly more IL-17 + cells were observed in OSCC compared with IC (Mann-Whitney, P < 0.0001). In OSCC, the numbers of IL-17 + cells were not significantly different in three compartments, TI, TS and DS (one-way ANOVA, P > 0.05). However, the TI had significantly fewer IL-17 + cells than the combined stroma (both TS and DS together, Mann-Whitney, P < 0.01). Laser scanning microscopy revealed helper T cells, cytotoxic T cells, macrophages and mast cells co-expressed IL-17. ELISA experiments did not detect IL-17 in the supernatants of OSCC cell lines. Although the tumour cells themselves did not express IL-17, a range of cell types did, suggesting multiple cellular sources for IL-17 in OSCC. The spatial distribution of IL-17 + cells suggests specific interactions with cells within the tumour microenvironment, implying that IL-17 + cells are likely to play a role in the pathogenesis of OSCC. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Involvement of H-ras in erythroid differentiation of TF1 and human umbilical cord blood CD34 cells.

PubMed

Ge, Y; Li, Z H; Marshall, M S; Broxmeyer, H E; Lu, L

1998-06-01

To investigate the role of the ras gene in erythroid differentiation, a human erythroleukemic cell line, TF1, was transduced with a selectable retroviral vector carrying a mammalian wild type H-ras gene or a cytoplasmic dominant negative RAS1 gene. Transduction of TF1 cells with the wild type H-ras gene resulted in changes of cell types and up-regulation of erythroid-specific gene expression similar to that seen in differentiating erythroid cells. The number of red blood cell containing colonies derived from TF1 cells transduced with wild type H-ras cDNA was significantly increased and the cells in the colonies were more hemoglobinized as estimated by a deeper red color compared to those colony cells from mock or dominant negative RAS1 gene transduced TF1 cells, suggesting increased erythroid differentiation of TF1 cells after transduction of wild type H-ras in vitro. The mRNA levels of beta- and gamma-, but not alpha-, globin genes were significantly higher in H-ras transduced TF1 cells than those in TF1 cells transduced with mock or dominant negative RAS1 gene. Moreover, a 4kb pre-mRNA of the Erythropoietin receptor (EpoR) was highly expressed only in H-ras transduced TF1 cells. Additionally, human umbilical cord blood (CB) CD34 cells which are highly enriched for hematopoietic stem/progenitor cells were transduced with the same retroviral vectors to evaluate in normal primary cells the activities of H-ras in erythroid differentiation. Increased numbers of erythroid cell containing colonies (BFU-E and CFU-GEMM) were observed in CD34 cells transduced with the H-ras cDNA, compared to that from mock transduced cells. These data suggest a possible role for ras in erythroid differentiation.

Generating Chimeric Mice by Using Embryos from Nonsuperovulated BALB/c Mice Compared with Superovulated BALB/c and Albino C57BL/6 Mice.

PubMed

Esmail, Michael Y; Qi, Peimin; Connor, Aurora Burds; Fox, James G; García, Alexis

2016-01-01

The reliable generation of high-percentage chimeras from gene-targeted C57BL/6 embryonic stem cells has proven challenging, despite optimization of cell culture and microinjection techniques. To improve the efficiency of this procedure, we compared the generation of chimeras by using 3 different inbred, albino host, embryo-generating protocols: BALB/cAnNTac (BALB/c) donor mice superovulated at 4 wk of age, 12-wk-old BALB/c donor mice without superovulation, and C57BL/6NTac-Tyr(tm1Arte) (albino B6) mice superovulated at 4 wk of age. Key parameters measured included the average number of injectable embryos per donor, the percentage of live pups born from the total number of embryos transferred to recipients, and the number of chimeric pups with high embryonic-stem-cell contribution by coat color. Although albino B6 donors produced significantly more injectable embryos than did BALB/c donors, 12-wk-old BALB/c donor produced high-percentage (at least 70%) chimeras more than 2.5 times as often as did albino B6 mice and 20 times more efficiently than did 4-wk-old BALB/c donors. These findings clearly suggest that 12-wk-old BALB/c mice be used as blastocyst donors to reduce the number of mice used to generate each chimera, reduce the production of low-percentage chimeras, and maximize the generation of high-percentage chimeras from C57BL/6 embryonic stem cells.

Peripheral leukocyte subpopulations and catecholamine levels in astronauts as a function of mission duration

NASA Technical Reports Server (NTRS)

Mills, P. J.; Meck, J. V.; Waters, W. W.; D'Aunno, D.; Ziegler, M. G.

2001-01-01

OBJECTIVE: The objective of this study was to determine the effects of spaceflight duration on immune cells and their relationship to catecholamine levels. METHODS: Eleven astronauts who flew aboard five different US Space Shuttle flights ranging in duration from 4 to 16 days were studied before launch and after landing. RESULTS: Consistent with prior studies, spaceflight was associated with a significant increase in the number of circulating white blood cells (p <.01), including neutrophils (p <.01), monocytes (p <.05), CD3+CD4+ T-helper cells (p <.05), and CD19+ B cells (p <.01). In contrast, the number of CD3-CD16+56+ natural killer cells was decreased (p <.01). Plasma norepinephrine levels were increased at landing (p <.01) and were significantly correlated with the number of white blood cells (p <.01), neutrophils (p <.01), monocytes (p <.01), and B cells (p <.01). Astronauts who were in space for approximately 1 week showed a significantly larger increase on landing in plasma norepinephrine (p =.02) and epinephrine (p =.03) levels, as well as number of circulating CD3+CD4+ T-helper cells (p <.05) and CD3+CD8+ T-cytotoxic cells (p <.05) as compared with astronauts in space for approximately 2 weeks. CONCLUSIONS: The data suggest that the stress of spaceflight and landing may lead to a sympathetic nervous system-mediated redistribution of circulating leukocytes, an effect potentially attenuated after longer missions.

The Number of Point Mutations in Induced Pluripotent Stem Cells and Nuclear Transfer Embryonic Stem Cells Depends on the Method and Somatic Cell Type Used for Their Generation.

PubMed

Araki, Ryoko; Mizutani, Eiji; Hoki, Yuko; Sunayama, Misato; Wakayama, Sayaka; Nagatomo, Hiroaki; Kasama, Yasuji; Nakamura, Miki; Wakayama, Teruhiko; Abe, Masumi

2017-05-01

Induced pluripotent stem cells hold great promise for regenerative medicine but point mutations have been identified in these cells and have raised serious concerns about their safe use. We generated nuclear transfer embryonic stem cells (ntESCs) from both mouse embryonic fibroblasts (MEFs) and tail-tip fibroblasts (TTFs) and by whole genome sequencing found fewer mutations compared with iPSCs generated by retroviral gene transduction. Furthermore, TTF-derived ntESCs showed only a very small number of point mutations, approximately 80% less than the number observed in iPSCs generated using retrovirus. Base substitution profile analysis confirmed this greatly reduced number of point mutations. The point mutations in iPSCs are therefore not a Yamanaka factor-specific phenomenon but are intrinsic to genome reprogramming. Moreover, the dramatic reduction in point mutations in ntESCs suggests that most are not essential for genome reprogramming. Our results suggest that it is feasible to reduce the point mutation frequency in iPSCs by optimizing various genome reprogramming conditions. We conducted whole genome sequencing of ntES cells derived from MEFs or TTFs. We thereby succeeded in establishing TTF-derived ntES cell lines with far fewer point mutations. Base substitution profile analysis of these clones also indicated a reduced point mutation frequency, moving from a transversion-predominance to a transition-predominance. Stem Cells 2017;35:1189-1196. © 2017 AlphaMed Press.

Bacteria-to-Archaea ratio depending on soil depth and agrogenic impact

NASA Astrophysics Data System (ADS)

Semenov, Mikhail; Manucharova, Natalia; Kuzyakov, Yakov

2014-05-01

Archaeal communities and their potential roles in the soil ecosystem are affected by a number of soil proprerties and environmental factors. Competitive interactions between Archaea and Bacteria play a particular role in spread and abundance of these two domains. Therefore, the goal of the study was to evaluate the Bacteria-to-Archaea ratio in different soils. The research was carried out at field and natural ecosystems of European part of Russia. Samples were collected within the soil profiles (3-6 horizons) of chernozem and kastanozem with distinctly different agrogenic impact. In situ hybridization with fluorescently labeled rRNA-targeted oligonucleotide probes (FISH) was used to determine the abundance of metabolically active cells of Archaea and Bacteria. The Cmic, Corg, C/N, DNA content and growth characteristics have been analyzed as well. Determination of number of metabolically active cells in chernozem under arable land and forest revealed that abundance of Archaea in topsoil under forest was higher more than 2 times comparing with arable land, but leveled off in the deeper horizons. Plowing of Chernozem decreased amount of archaeal and bacterial active cells simultaneously, however, Bacteria were more resistant to agrogenic impact than Archaea. Determination of the taxonomic composition within Bacteria domain showed a significant decrease in the abundance of phylogenetic groups Firmicutes and Actinobacteria in the topsoil under arable land comparing to the forest, which is the main reason for the declining of the total amount of prokaryotic cells. In kastanozem significant change in the number of metabolically active cells due to plowing was detected only within 40 cm soil layer, and this effect disappeared in lower horizons. The number of Archaea was higher in the upper horizons of arable as compared to virgin soil. Conversely, the number of Bacteria in the upper layers of the soil after plowing kastanozem decreased. Relationship between soil organic carbon content and the amount of soil metabolically active Bacteria and Archaea cells revealed that distribution of both Bacteria and Archaea throughout the soil profile was governed by organic matter content. Thus, the organic matter content seemed to be the main factor of declining Bacteria-to- Archaea ratio down the profile (from 7.1 to 4.2 for virgin soil and from 5 to 3.9 for arable soil). In conclusion, Archaea out-compete Bacteria under conditions of reduced energy supply.

Upregulation of GRAIL is associated with impaired CD4 T cell proliferation in sepsis.

PubMed

Aziz, Monowar; Yang, Weng-Lang; Matsuo, Shingo; Sharma, Archna; Zhou, Mian; Wang, Ping

2014-03-01

The loss of numbers and functionality of CD4 T cells is observed in sepsis; however, the mechanism remains elusive. Gene related to anergy in lymphocytes (GRAIL) is critical for the impairment of CD4 T cell proliferation. We therefore sought to examine the role of GRAIL in CD4 T cell proliferation during sepsis. Sepsis was induced in 10-wk-old male C57BL/6 mice by cecal ligation and puncture. Splenocytes were isolated and subjected to flow cytometry to determine CD4 T cell contents. CD4 T cell proliferation was assessed by CFSE staining, and the expression of GRAIL in splenocytes was measured by immunohistochemistry, real-time PCR, and flow cytometry. The expressions of IL-2 and early growth response-2 were determined by real-time PCR. As compared with shams, the numbers of CD4 T cells were significantly reduced in spleens. Septic CD4 T cells were less efficient in proliferation than shams. The IL-2 expression was significantly reduced, whereas the GRAIL expression was significantly increased in septic mice splenocytes as compared with shams. The small interfering RNA-mediated knockdown of GRAIL expression re-established the CD4 T cell proliferation ability ex vivo. Similarly, the treatment with recombinant murine IL-2 to the septic CD4 T cells restored their proliferation ability by downregulating GRAIL expression. Our findings reveal a novel association of the increased GRAIL expression with impaired CD4 T cell proliferation, implicating an emerging therapeutic tool in sepsis.

Spontaneous cell sorting of fibroblasts and keratinocytes creates an organotypic human skin equivalent.

PubMed

Wang, C K; Nelson, C F; Brinkman, A M; Miller, A C; Hoeffler, W K

2000-04-01

We show that an inherent ability of two distinct cell types, keratinocytes and fibroblasts, can be relied upon to accurately reconstitute full-thickness human skin including the dermal-epidermal junction by a cell-sorting mechanism. A cell slurry containing both cell types added to silicone chambers implanted on the backs of severe combined immunodeficient mice sorts out to reconstitute a clearly defined dermis and stratified epidermis within 2 wk, forming a cell-sorted skin equivalent. Immunostaining of the cell-sorted skin equivalent with human cell markers showed patterns similar to those of normal full-thickness skin. We compared the cell-sorted skin equivalent model with a composite skin model also made on severe combined immunodeficient mice. The composite grafts were constructed from partially differentiated keratinocyte sheets placed on top of a dermal equivalent constructed of devitalized dermis. Electron microscopy revealed that both models formed ample numbers of normal appearing hemidesmosomes. The cell-sorted skin equivalent model, however, had greater numbers of keratin intermediate filaments within the basal keratinocytes that connected to hemidesmosomes, and on the dermal side both collagen filaments and anchoring fibril connections to the lamina densa were more numerous compared with the composite model. Our results may provide some insight into why, in clinical applications for treating burns and other wounds, composite grafts may exhibit surface instability and blistering for up to a year following grafting, and suggest the possible usefulness of the cell-sorted skin equivalent in future grafting applications.

Comparative Analysis of Dibutyric cAMP and Butyric Acid on the Differentiation of Human Eosinophilic Leukemia EoL-1 Cells

PubMed Central

2015-01-01

Purification of enough numbers of circulating eosinophils is difficult because eosinophils account for less than 5% peripheral blood leukocytes. Human eosinophilic leukemia EoL-1 cells have been considered an in vitro source of eosinophils as they can differentiate into mature eosinophil-like cells when incubated with dibutyryl cAMP (dbcAMP) or butyric acid. In this study, the viability and phenotypic maturation of EoL-1 cells stimulated by either dbcAMP or butyric acid were comparatively analyzed. After treatment with 100 µM dbcAMP or 0.5 µM butyric acid, EoL-1 cells showed morphological signs of differentiation, although the number of nonviable EoL-1 cells was significantly increased following butyric acid treatment. Stimulation of EoL-1 cells with 0.5 µM butyric acid more effectively induced the expression of mature eosinophil markers than stimulation with dbcAMP. These results suggest that treatment of EoL-1 cells with 0.5 µM butyric acid for limited duration could be an effective strategy for inducing their differentiation. Considering that expression of CCR3 was not sufficient in EoL-1 cells stimulated with 0.5 µM butyric acid, treatment of the chemically stimulated EoL-1 cells with cytokines, which primarily support eosinophil maturation, would help to obtain differentiated EoL-1 cells with greater functional maturity. PMID:26770185

Transitional boundary layer in low-Prandtl-number convection at high Rayleigh number

NASA Astrophysics Data System (ADS)

Schumacher, Joerg; Bandaru, Vinodh; Pandey, Ambrish; Scheel, Janet

2016-11-01

The boundary layer structure of the velocity and temperature fields in turbulent Rayleigh-Bénard flows in closed cylindrical cells of unit aspect ratio is revisited from a transitional and turbulent viscous boundary layer perspective. When the Rayleigh number is large enough the boundary layer dynamics at the bottom and top plates can be separated into an impact region of downwelling plumes, an ejection region of upwelling plumes and an interior region (away from side walls) that is dominated by a shear flow of varying orientation. This interior plate region is compared here to classical wall-bounded shear flows. The working fluid is liquid mercury or liquid gallium at a Prandtl number of Pr = 0 . 021 for a range of Rayleigh numbers of 3 ×105 <= Ra <= 4 ×108 . The momentum transfer response to these system parameters generates a fluid flow in the closed cell with a macroscopic flow Reynolds number that takes values in the range of 1 . 8 ×103 <= Re <= 4 . 6 ×104 . It is shown that particularly the viscous boundary layers for the largest Ra are highly transitional and obey some properties that are directly comparable to transitional channel flows at friction Reynolds numbers below 100. This work is supported by the Deutsche Forschungsgemeinschaft.

Immune cells in the normal ovary and spontaneous ovarian tumors in the laying hen (Gallus domesticus) model of human ovarian cancer.

PubMed

Bradaric, Michael J; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L; Yu, Yi; Abramowicz, Jacques S; Bahr, Janice M; Luborsky, Judith L

2013-01-01

Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials.

Immune Cells in the Normal Ovary and Spontaneous Ovarian Tumors in the Laying Hen (Gallus domesticus) Model of Human Ovarian Cancer

PubMed Central

Bradaric, Michael J.; Penumatsa, Krishna; Barua, Animesh; Edassery, Seby L.; Yu, Yi; Abramowicz, Jacques S.; Bahr, Janice M.; Luborsky, Judith L.

2013-01-01

Background Spontaneous ovarian cancer in chickens resembles human tumors both histologically and biochemically. The goal was to determine if there are differences in lymphocyte content between normal ovaries and ovarian tumors in chickens as a basis for further studies to understand the role of immunity in human ovarian cancer progression. Methods Hens were selected using grey scale and color Doppler ultrasound to determine if they had normal or tumor morphology. Cells were isolated from ovaries (n = 6 hens) and lymphocyte numbers were determined by flow cytometry using antibodies to avian CD4 and CD8 T and B (Bu1a) cells. Ovarian sections from another set of hens (n = 26) were assessed to verify tumor type and stage and to count CD4, CD8 and Bu1a immunostained cells by morphometric analysis. Results T and B cells were more numerous in ovarian tumors than in normal ovaries by flow cytometry and immunohistochemistry. There were less CD4+ cells than CD8+ and Bu1a+ cells in normal ovaries or ovarian tumors. CD8+ cells were the dominant T cell sub-type in both ovarian stroma and in ovarian follicles compared to CD4+ cells. Bu1a+ cells were consistently found in the stroma of normal ovaries and ovarian tumors but were not associated with follicles. The number of immune cells was highest in late stage serous tumors compared to endometrioid and mucinous tumors. Conclusions The results suggest that similar to human ovarian cancer there are comparatively more immune cells in chicken ovarian tumors than in normal ovaries, and the highest immune cell content occurs in serous tumors. Thus, this study establishes a foundation for further study of tumor immune responses in a spontaneous model of ovarian cancer which will facilitate studies of the role of immunity in early ovarian cancer progression and use of the hen in pre-clinical vaccine trials. PMID:24040191

Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro

PubMed Central

Zhou, Liping; Zhang, Xiaowei; Zhou, Panpan; Li, Xue; Xu, Xuejing; Shi, Qing; Li, Dong; Ju, Xiuli

2017-01-01

Successfully expanding hematopoietic stem cells (HSCs) is advantageous for clinical HSC transplantation. The present study investigated the influence of testosterone on the proliferation, antigen phenotype and expression of hematopoiesis-related genes in umbilical cord blood-derived cluster of differentiation (CD)34+ cells under normoxic or hypoxia conditions. Cord blood (CB) CD34+ cells were separated using magnetic activated cell sorting. A cytokine cocktail and feeder cells were used to stimulate the expansion of CD34+ cells under normoxic (20% O2) and hypoxic (1% O2) conditions for 7 days and testosterone was added accordingly. Cells were identified using flow cytometry and reconstruction capacity was determined using a colony-forming unit (CFU) assay. The effects of oxygen concentration and testosterone on the expression of hematopoietic-related genes, including homeobox (HOX)A9, HOXB2, HOXB4, HOXC4 and BMI-1, were measured using reverse transcription-quantitative polymerase chain reaction. The results indicated that the number of CFUs and total cells in the testosterone group increased under normoxic and hypoxic conditions compared with the corresponding control groups. Furthermore, the presence of testosterone increased the number of CFU-erythroid colonies. In liquid culture, the growth of CD34+ cells was rapid under normoxic conditions compared with under hypoxic conditions, however CD34+ cells were maintained in an undifferentiated state under hypoxic conditions. The addition of testosterone under hypoxia promoted the differentiation of CD34+ cells into CD34+CD38+CD71+ erythroid progenitor cells. Furthermore, it was determined that the expression of hematopoietic-related genes was significantly increased (P<0.05) in the hypoxia testosterone group compared with the other groups. Therefore, the results of the current study indicate that a combination of hypoxia and testosterone may be a promising cultivation condition for HSC/hemopoietic progenitor cell expansion ex vivo. PMID:29067121

Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro.

PubMed

Zhou, Liping; Zhang, Xiaowei; Zhou, Panpan; Li, Xue; Xu, Xuejing; Shi, Qing; Li, Dong; Ju, Xiuli

2017-11-01

Successfully expanding hematopoietic stem cells (HSCs) is advantageous for clinical HSC transplantation. The present study investigated the influence of testosterone on the proliferation, antigen phenotype and expression of hematopoiesis-related genes in umbilical cord blood-derived cluster of differentiation (CD)34 + cells under normoxic or hypoxia conditions. Cord blood (CB) CD34 + cells were separated using magnetic activated cell sorting. A cytokine cocktail and feeder cells were used to stimulate the expansion of CD34 + cells under normoxic (20% O 2 ) and hypoxic (1% O 2 ) conditions for 7 days and testosterone was added accordingly. Cells were identified using flow cytometry and reconstruction capacity was determined using a colony-forming unit (CFU) assay. The effects of oxygen concentration and testosterone on the expression of hematopoietic-related genes, including homeobox (HOX)A9, HOXB2, HOXB4, HOXC4 and BMI-1, were measured using reverse transcription-quantitative polymerase chain reaction. The results indicated that the number of CFUs and total cells in the testosterone group increased under normoxic and hypoxic conditions compared with the corresponding control groups. Furthermore, the presence of testosterone increased the number of CFU-erythroid colonies. In liquid culture, the growth of CD34 + cells was rapid under normoxic conditions compared with under hypoxic conditions, however CD34 + cells were maintained in an undifferentiated state under hypoxic conditions. The addition of testosterone under hypoxia promoted the differentiation of CD34 + cells into CD34 + CD38 + CD71 + erythroid progenitor cells. Furthermore, it was determined that the expression of hematopoietic-related genes was significantly increased (P<0.05) in the hypoxia testosterone group compared with the other groups. Therefore, the results of the current study indicate that a combination of hypoxia and testosterone may be a promising cultivation condition for HSC/hemopoietic progenitor cell expansion ex vivo .

Regulation of diet-induced adipose tissue and systemic inflammation by salicylates and pioglitazone.

PubMed

Kim, Myung-Sunny; Yamamoto, Yasuhiko; Kim, Kyungjin; Kamei, Nozomu; Shimada, Takeshi; Liu, Libin; Moore, Kristin; Woo, Ju Rang; Shoelson, Steven E; Lee, Jongsoon

2013-01-01

It is increasingly accepted that chronic inflammation participates in obesity-induced insulin resistance and type 2 diabetes (T2D). Salicylates and thiazolidinediones (TZDs) both have anti-inflammatory and anti-hyperglycemic properties. The present study compared the effects of these drugs on obesity-induced inflammation in adipose tissue (AT) and AT macrophages (ATMs), as well as the metabolic and immunological phenotypes of the animal models. Both drugs improved high fat diet (HFD)-induced insulin resistance. However, salicylates did not affect AT and ATM inflammation, whereas Pioglitazone improved these parameters. Interestingly, HFD and the drug treatments all modulated systemic inflammation as assessed by changes in circulating immune cell numbers and activation states. HFD increased the numbers of circulating white blood cells, neutrophils, and a pro-inflammatory monocyte subpopulation (Ly6C(hi)), whereas salicylates and Pioglitazone normalized these cell numbers. The drug treatments also decreased circulating lymphocyte numbers. These data suggest that obesity induces systemic inflammation by regulating circulating immune cell phenotypes and that anti-diabetic interventions suppress systemic inflammation by normalizing circulating immune phenotypes.

Is the appearance of macrophages in pulmonary tissue related to time of asphyxia?

PubMed

Vacchiano, G; D'Armiento, F; Torino, R

2001-01-01

In order to connect the appearance of macrophages and giant cells in pulmonary tissue with the time of asphyxia the authors analyzed 50 asphyxiated human lungs paying their attention on the number of alveolar and interstitial macrophages and giant cells. They compared histological specimens of 25 asphixiated humans lungs following a slow asphyxia (30 min or more) with 25 histological specimens of asphyxiated human lungs following a rapid asphyxia (10-15 min). Alveolar and interstitial macrophages and giant cells per section, were considered and numbered. Controls were done on histological examination of traumatized lungs. In the pulmonary alveoli following on acute asphyxia there were 27.7+/-4.4 macrophages per section. Subjects dead after a slow asphyxiation showed 68.2+/-7.1 alveolar macrophages per section (p<0.001). Interstitial macrophages were also frequently present. No differences are detectable in the number of polynuclear giant cells between rapidly and slowly asphyxiated human lungs. The number of alveolar and interstitial macrophages per section can be considered as a further histological evidence of a slow asphyxia and can differentiate a slow asphyxia from an acute one.

Effects of the oral administration of the exopolysaccharide produced by Lactobacillus kefiranofaciens on the gut mucosal immunity.

PubMed

Vinderola, Gabriel; Perdigón, Gabriela; Duarte, Jairo; Farnworth, Edward; Matar, Chantal

2006-12-01

The probiotic effects ascribed to lactic acid bacteria (LAB) and their fermented dairy products arise not only from whole microorganisms and cell wall components but also from peptides and extracellular polysaccharides (exopolysaccharides) produced during the fermentation of milk. There is a lack of knowledge concerning the immune mechanisms induced by exopolysaccharides produced by lactic acid bacteria, which would allow a better understanding of the functional effects described to them. The aim of this study was to investigate the in vivo immunomodulating capacity of the exopolysaccharide produced by Lactobacillus kefiranofaciens by analyzing the profile of cytokines and immunoglobulins induced at the intestinal mucosa level, in the intestinal fluid and blood serum. BALB/c mice received the exopolysaccharide produced by L. kefiranofaciens for 2, 5 or 7 consecutive days. At the end of each period of administration, control and treated mice were sacrificed and the numbers of IgA+ and IgG+ cells were determined on histological slices of the small and large intestine by immunofluorescence. Cytokines (IL-4, IL-6, IL-10, IL-12, IFNgamma and TNFalpha) were also determined in the gut lamina propria as well as in the intestinal fluid and blood serum. There was an increase of IgA+ cells in the small and large intestine lamina propria, without change in the number of IgG+ cells in the small intestine. This study reports the effects of the oral administration of the exopolysaccharide produced by L. kefiranofaciens in the number of IgA+ cells in the small and large intestine, comparing simultaneously the production of cytokines by cells of the lamina propria and in the intestinal fluid and blood serum. The increase in the number of IgA+ cells was not simultaneously accompanied by an enhance of the number of IL-4+ cells in the small intestine. This finding would be in accordance with the fact that, in general, polysaccharide antigens elicit a T-independent immune response. For IL-10+, IL-6+ and IL-12+ cells, the values found were slightly increased compared to control values, while IFNgamma+ and TNFalpha+ cells did not change compared to control values. The effects observed on immunoglobulins and in all the cytokines assayed in the large intestine after kefiran administration were of greater magnitude than the ones observed in the small intestine lamina propria, which may be due to the saccharolytic action of the colonic microflora. In the intestinal fluid, only IL-4 and IL-12 increased compared to control values. In blood serum, all the cytokines assayed followed a pattern of production quite similar to the one found for them in the small intestine lamina propria. We observed that the exopolysaccharide induced a gut mucosal response and it was able to up and down regulate it for protective immunity, maintaining intestinal homeostasis, enhancing the IgA production at both the small and large intestine level and influencing the systemic immunity through the cytokines released to the circulating blood.

A Study of the Efficiency of Spatial Indexing Methods Applied to Large Astronomical Databases

NASA Astrophysics Data System (ADS)

Donaldson, Tom; Berriman, G. Bruce; Good, John; Shiao, Bernie

2018-01-01

Spatial indexing of astronomical databases generally uses quadrature methods, which partition the sky into cells used to create an index (usually a B-tree) written as database column. We report the results of a study to compare the performance of two common indexing methods, HTM and HEALPix, on Solaris and Windows database servers installed with a PostgreSQL database, and a Windows Server installed with MS SQL Server. The indexing was applied to the 2MASS All-Sky Catalog and to the Hubble Source catalog. On each server, the study compared indexing performance by submitting 1 million queries at each index level with random sky positions and random cone search radius, which was computed on a logarithmic scale between 1 arcsec and 1 degree, and measuring the time to complete the query and write the output. These simulated queries, intended to model realistic use patterns, were run in a uniform way on many combinations of indexing method and indexing level. The query times in all simulations are strongly I/O-bound and are linear with number of records returned for large numbers of sources. There are, however, considerable differences between simulations, which reveal that hardware I/O throughput is a more important factor in managing the performance of a DBMS than the choice of indexing scheme. The choice of index itself is relatively unimportant: for comparable index levels, the performance is consistent within the scatter of the timings. At small index levels (large cells; e.g. level 4; cell size 3.7 deg), there is large scatter in the timings because of wide variations in the number of sources found in the cells. At larger index levels, performance improves and scatter decreases, but the improvement at level 8 (14 min) and higher is masked to some extent in the timing scatter caused by the range of query sizes. At very high levels (20; 0.0004 arsec), the granularity of the cells becomes so high that a large number of extraneous empty cells begin to degrade performance. Thus, for the use patterns studied here the database performance is not critically dependent on the exact choices of index or level.

Cellular basis of neuroepithelial bending during mouse spinal neural tube closure

PubMed Central

McShane, Suzanne G.; Molè, Matteo A.; Savery, Dawn; Greene, Nicholas D. E; Tam, Patrick P.L.; Copp, Andrew J.

2015-01-01

Summary Bending of the neural plate at paired dorsolateral hinge points (DLHPs) is required for neural tube closure in the spinal region of the mouse embryo. As a step towards understanding the morphogenetic mechanism of DLHP development, we examined variations in neural plate cellular architecture and proliferation during closure. Neuroepithelial cells within the median hinge point (MHP) contain nuclei that are mainly basally located and undergo relatively slow proliferation, with a 7 h cell cycle length. In contrast, cells in the dorsolateral neuroepithelium, including the DLHP, exhibit nuclei distributed throughout the apico-basal axis and undergo rapid proliferation, with a 4 h cell cycle length. As the neural folds elevate, cell numbers increase to a greater extent in the dorsolateral neural plate that contacts the surface ectoderm, compared with the more ventromedial neural plate where cells contact paraxial mesoderm and notochord. This marked increase in dorsolateral cell number cannot be accounted for solely on the basis of enhanced cell proliferation in this region. We hypothesised that neuroepithelial cells may translocate in a ventral-to-dorsal direction as DLHP formation occurs, and this was confirmed by vital cell labelling in cultured embryos. The translocation of cells into the neural fold, together with its more rapid cell proliferation, leads to an increase in cell density dorsolaterally compared with the more ventromedial neural plate. These findings suggest a model in which DLHP formation may proceed through ‘buckling’ of the neuroepithelium at a dorso-ventral boundary marked by a change in cell-packing density. PMID:26079577

Functional and quantitative alterations in T lymphocyte subpopulations in acute toxoplasmosis.

PubMed

Luft, B J; Kansas, G; Engleman, E G; Remington, J S

1984-11-01

The cellular immune response to Toxoplasma gondii has been studied in 23 patients with acute toxoplasma infection. Abnormalities of T cell subpopulations included a marked and significant elevation in suppressor (Leu 2) T cells in patients with prolonged symptoms due to acute infection and either a decrease in the number of T helper cells or an increase in the number of suppressor cells--or both--in patients with asymptomatic lymphadenopathy. There was no significant difference in lymphocyte proliferation to phytohemagglutinin or pokeweed mitogen among the various groups tested. The peak lymphocyte response to toxoplasma antigen, however, was significantly depressed in patients with acute infection compared with that in chronically infected control patients. The kinetics of the depression were consistent with the induction of a non-Leu 2 suppressor cell. These results demonstrate marked quantitative alterations in T lymphocyte subpopulations and functional alterations of T cells to toxoplasma antigen during infection with T. gondii.

Design and interpretation of cell trajectory assays

PubMed Central

Bowden, Lucie G.; Simpson, Matthew J.; Baker, Ruth E.

2013-01-01

Cell trajectory data are often reported in the experimental cell biology literature to distinguish between different types of cell migration. Unfortunately, there is no accepted protocol for designing or interpreting such experiments and this makes it difficult to quantitatively compare different published datasets and to understand how changes in experimental design influence our ability to interpret different experiments. Here, we use an individual-based mathematical model to simulate the key features of a cell trajectory experiment. This shows that our ability to correctly interpret trajectory data is extremely sensitive to the geometry and timing of the experiment, the degree of motility bias and the number of experimental replicates. We show that cell trajectory experiments produce data that are most reliable when the experiment is performed in a quasi-one-dimensional geometry with a large number of identically prepared experiments conducted over a relatively short time-interval rather than a few trajectories recorded over particularly long time-intervals. PMID:23985736

Proliferative responses to canine thyroglobulin of peripheral blood mononuclear cells from hypothyroid dogs.

PubMed

Tani, Hiroyuki; Nabetani, Tomoyo; Sasai, Kazumi; Baba, Eiichiroh

2005-04-01

The immune responses of hypothyroid dogs to canine thyroglobulin (cTg) were evaluated for the proliferative ability of peripheral blood mononuclear cells (PBMC). PBMC from three hypothyroid dogs with high titers of thyroglobulin autoantibody (TgAA) and 3 clinically normal dogs were cultured with 5, 10, or 20 microg/ml of cTg for 72 hr. The proliferative responses of the cells were determined by the level of incorporated BrdU. The numbers of cells expressing Thy-1, CD4, CD8 and IgG in the PBMC were counted by the immunofluorescence method. Proliferative responses to cTg were observed in the cells from hypothyroid dogs. The number of cells expressing IgG and CD8 in the hypothyroid dogs tended to be high compared with the clinically normal dogs. The CD4+ cells in cultures from hypothyroid dogs increased depending upon the amount of cTg. There was a significant (P<0.05) positive correlation between the number of CD4+ cells and the concentration of cTg in the cultures from hypothyroid dogs. These findings suggest a possible relationship between canine hypothyroidism and cellular immunity. Loss of self tolerance to thyroid antigens in CD4+ T cells may play an important role in the development of canine hypothyroidism.

Online Medicine for Pregnant Women

PubMed Central

Davidesko, Sharon; Segal, David

2014-01-01

Objective. To assess the use of cell phones and email as means of communication between pregnant women and their gynecologists and family physicians. Study Design. A cross-sectional study of pregnant women at routine followup. One hundred and twenty women participated in the study. Results. The mean age was 27.4 ± 3.4 years. One hundred nineteen women owned a cell phone and 114 (95%) had an email address. Seventy-two women (60%) had their gynecologist's cell phone number and 50 women (42%) had their family physician's cell phone number. More women contacted their gynecologist via cell phone or email during pregnancy compared to their family physician (P = 0.005 and 0.009, resp.). Most preferred to communicate with their physician via cell phone at predetermined times, but by email at any time during the day (P < 0.0001). They would use cell phones for emergencies or unusual problems but preferred email for other matters (P < 0.0001). Conclusions. Pregnant women in the Negev region do not have a preference between the use of cell phones or email for medical consultation with their gynecologist or family physician. The provision of the physician's cell phone numbers or email address together with the provision of guidelines and resources could improve healthcare services. PMID:25132848

Vaccine draining lymph nodes are a source of antigen-specific B cells.

PubMed

Pero, Stephanie C; Sun, Yu-Jing; Shukla, Girja S; Carman, Chelsea L; Krag, Christopher C; Teuscher, Cory; Krementsov, Dimitry N; Krag, David N

2017-03-01

Our research is focused on using vaccine draining lymph nodes as a source of immune cells to better understand the immune response and to attempt to generate new anti-cancer reagents. Following a vaccine, harvesting the lymph node can only be done once. We endeavored to determine the range of times that B cells secreting anti-KLH antibodies were present in the node of KLH-vaccinated mice. Following vaccination the total number of mononuclear cells (MNCs) increased in the vaccine-draining lymph node (VDN). The percentage of MNCs that were B cells nearly doubled. B cells recovered from the node that secreted anti-KLH antibodies were evident by day 7. The number continued to increase and then slowly decreased over the observed time range to 28days after vaccination. The VDN, compared to the spleen, the bone marrow and the nonVDN, contained a higher percentage of B cells that secreted anti-KLH antibodies. After a vaccine, there is a multi-week window of time when an increasing number of B cells are present in a VDN that secrete anti-KLH antibodies. These results support using the VDN as a source for B cells that secrete anti-vaccine antibodies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Online medicine for pregnant women.

PubMed

Davidesko, Sharon; Segal, David; Peleg, Roni

2014-01-01

Objective. To assess the use of cell phones and email as means of communication between pregnant women and their gynecologists and family physicians. Study Design. A cross-sectional study of pregnant women at routine followup. One hundred and twenty women participated in the study. Results. The mean age was 27.4 ± 3.4 years. One hundred nineteen women owned a cell phone and 114 (95%) had an email address. Seventy-two women (60%) had their gynecologist's cell phone number and 50 women (42%) had their family physician's cell phone number. More women contacted their gynecologist via cell phone or email during pregnancy compared to their family physician (P = 0.005 and 0.009, resp.). Most preferred to communicate with their physician via cell phone at predetermined times, but by email at any time during the day (P < 0.0001). They would use cell phones for emergencies or unusual problems but preferred email for other matters (P < 0.0001). Conclusions. Pregnant women in the Negev region do not have a preference between the use of cell phones or email for medical consultation with their gynecologist or family physician. The provision of the physician's cell phone numbers or email address together with the provision of guidelines and resources could improve healthcare services.

Circulating dendritic cell precursors in chronic kidney disease: a cross-sectional study.

PubMed

Paul, Katharina; Kretzschmar, Daniel; Yilmaz, Atilla; Bärthlein, Barbara; Titze, Stephanie; Wolf, Gunter; Busch, Martin

2013-12-10

Dendritic cells (DC) are professional antigen-presenting cells in the immune system. They patrol the blood as circulating dendritic cell precursors (DCP). Decreased blood DCP count has been shown to be related to atherosclerotic plaque burden. Since chronic kidney disease (CKD) is associated with chronic inflammation and increased cardiovascular risk, the aim of our study was to investigate a potential effect of CKD on circulating DCP numbers especially in patients with a history of cardiovascular disease. The number of circulating myeloid (mDCP), plasmacytoid (pDCP), and total DCP (tDCP) was analysed by flow cytometry in 245 patients with CKD stage 3 (with and without known cardiovascular events) and 85 coronary healthy controls. In addition, data were compared with a historical group of 130 patients with known coronary artery disease (CAD). Compared to controls, patients with CKD 3 revealed a significant decrease in circulating mDCP (-29%), pDCP (-43%), and tDCP (-38%) (P < 0.001, respectively). Compared with CAD-patients, the decrease in circulating DCP in CKD was comparable or even more pronounced indicating a potential role for DCP in cardiovascular risk potentiation due to CKD. Based on previous findings in CAD, the marked decrease of DCP in CKD implicates a potential role for DCP as a mediator of cardiovascular disease. Whether DCP in CKD may act as new cardiovascular biomarkers needs to be established in future prospective trials.

Metastatic Lung Lesions as a Preferred Resection Site for Immunotherapy With Tumor Infiltrating Lymphocytes.

PubMed

Ben-Avi, Ronny; Itzhaki, Orit; Simansky, David; Zippel, Dov; Markel, Gal; Ben Nun, Alon; Schachter, Jacob; Besser, Michal J

2016-06-01

Adoptive cell therapy with tumor infiltrating lymphocytes (TIL) yields 50% response rates in metastatic melanoma and shows promising clinical results in other solid tumors. Autologous TIL cultures are isolated from resected tumor tissue, expanded ex vivo to large numbers and reinfused to the preconditioned patient. In this prospective study, we validate the origin of the tumor biopsy and its effect on T-cell function and clinical response. One hundred forty-four patients underwent surgery and 79 patients were treated with TIL adoptive cell therapy. Cultures from lung tissue were compared with other origins. The success rate of establishing TIL culture from lung tissue was significantly higher compared with nonlung tissue (94% vs. 72%, respectively, P≤0.003). Lung-derived TIL cultures gave rise to higher cell numbers (P≤0.011) and exhibited increased in vitro antitumor reactivity. The average fold expansion for lung-derived TIL during a rapid expansion procedure was 1349±557 compared with 1061±473 for nonlung TIL (P≤0.038). Patients treated with TIL cultures of lung origin (compared with nonlung) had prolonged median overall survival (29 vs. 9.5 mo; P≤0.065). Given the remarkable advancement in minimally invasive thoracic surgery and the results of this study, we suggest efforts should be taken to resect lung metastasis rather than other sites to generate TIL cultures for clinical use.

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry.

PubMed

Wang, Meiyao; Misakian, Martin; He, Hua-Jun; Bajcsy, Peter; Abbasi, Fatima; Davis, Jeffrey M; Cole, Kenneth D; Turko, Illarion V; Wang, Lili

2014-01-01

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells. Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry. The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10(5) and (0.85 ± 0.11) × 10(5), respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC. Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

T-cell homeostasis in breast cancer survivors with persistent fatigue.

PubMed

Bower, Julienne E; Ganz, Patricia A; Aziz, Najib; Fahey, John L; Cole, Steve W

2003-08-06

Approximately 30% of women successfully treated for breast cancer suffer persistent fatigue of unknown origin. Recent studies linking inflammatory processes to central nervous system-mediated fatigue led us to examine cellular immune system status in 20 fatigued breast cancer survivors and 19 matched non-fatigued breast cancer survivors. Fatigued survivors, compared with non-fatigued survivors, had statistically significantly increased numbers of circulating T lymphocytes (mean 31% increase, 95% confidence interval [CI] = 6% to 56%; P =.015 by two-sided analysis of variance [ANOVA]), with pronounced elevation in the numbers of CD4+ T lymphocytes (mean 41% increase, 95% CI = 15% to 68%; P =.003 by two-sided ANOVA) and CD56+ effector T lymphocytes (mean 52% increase, 95% CI = 4% to 99%; P =.027 by two-sided ANOVA). These changes were independent of patient demographic and treatment characteristics. Absolute numbers of B cells, natural killer cells, granulocytes, and monocytes were not altered. The increased numbers of circulating T cells correlated with elevations in the level of serum interleukin 1 receptor antagonist (for CD3+ cells, r =.56 and P =.001; for CD3+/CD4+ cells, r =.68 and P<.001, by Spearman rank correlation). Results of this study suggest that persistent fatigue in breast cancer survivors might be associated with a chronic inflammatory process involving the T-cell compartment. These results require confirmation in a larger study that is specifically designed to address this hypothesis.

Analysis of Cell Turnover in the Bronchiolar Epithelium Through the Normal Aging Process.

PubMed

Ortega-Martínez, Marta; Rodríguez-Flores, Laura E; Ancer-Arellano, Adriana; Cerda-Flores, Ricardo M; de-la-Garza-González, Carlos; Ancer-Rodríguez, Jesús; Jaramillo-Rangel, Gilberto

2016-08-01

Aging is associated with changes in the lung that leads to a decrease in its function. Alterations in structure and function in the small airways are well recognized in chronic lung diseases. The aim of this study was the assessment of cell turnover in the bronchiolar epithelium of mouse through the normal aging process. Lungs from CD1 mice at the age of 2, 6, 12, 18, or 24 months were fixed in neutral-buffered formalin and paraffin-embedded. Proliferating cell nuclear antigen was examined by immunohistochemistry. Apoptosis was analyzed by in situ end-labeling of fragmented DNA. Epithelial dimensions were analyzed by morphometry. The 2-month-old mice showed significantly higher number of proliferating cells when compared with mice at all other age groups. The number of apoptotic cells in mice at 24 months of age was significantly greater than in mice at all other age groups. Thus, the number of epithelial cells decreased as the age of the subject increased. We also found reductions in both area and height of the bronchiolar epithelium in mice at 18 and 24 months of age. We found a decrease in the total number of epithelial cells in the aged mice, which was accompanied by a thinning of the epithelium. These changes reflect a dysregulated tissue regeneration process in the bronchiolar epithelium that might predispose to respiratory diseases in elderly subjects.

Chromosome Aberrations in Cells Infected with Bovine Papillomavirus: Comparing Cutaneous Papilloma, Esophagus Papilloma, and Urinary Bladder Lesion Cells

PubMed Central

Campos, S. R. C.; Melo, T. C.; Assaf, S.; Araldi, R. P.; Mazzuchelli-de-Souza, J.; Sircili, M. P.; Carvalho, R. F.; Roperto, F.; Beçak, W.; Stocco, R. C.

2013-01-01

The majority of malignant cells present genetic instability with chromosome number changes plus segmental defects: these changes involve intact chromosomes and breakage-induced alterations. Some pathways of chromosomal instability have been proposed as random breakage, telomere fusion, and centromere fission. Chromosome alterations in tumor cells have been described in animal models and in vitro experiments. One important question is about possible discrepancies between animal models, in vitro studies, and the real events in cancer cells in vivo. Papillomaviruses are relevant agents in oncogenic processes related to action on host genome. Recently, many reports have discussed the presence of virus DNA in peripheral blood, in humans and in animals infected by papillomaviruses. The meaning of this event is of controversy: possible product of apoptosis occurring in cancer cells, metastasized cancer cells, or active DNA sequences circulating in bloodstream. This study compares chromosome aberrations detected in bovine cells, in peripheral blood cells, and in BPV lesion cells: the literature is poor in this type of study. Comparing chromosome aberrations described in the different cells, a common mechanism in their origin, can be suggested. Furthermore blood cells can be evaluated as an effective way of virus transmission. PMID:24298391

Necrotic cell death by hydrogen peroxide in immortal DF-1 chicken embryo fibroblast cells expressing deregulated MnSOD and catalase.

PubMed

Kim, H; You, S; Kong, B W; Foster, L K; Farris, J; Foster, D N

2001-08-22

The reactive oxygen species are known as endogenous toxic oxidant damaging factors in a variety of cell types, and in response, the antioxidant genes have been implicated in cell proliferation, senescence, immortalization, and tumorigenesis. The expression of manganese superoxide dismutase mRNA was shown to increase in most of the immortal chicken embryo fibroblast (CEF) cells tested, while expression of catalase mRNA appeared to be dramatically decreased in all immortal CEF cells compared to their primary counterparts. The expression of copper-zinc superoxide dismutase mRNA was shown to increase slightly in some immortal CEF cells. The glutathione peroxidase expressed relatively similar levels in both primary and immortal CEF cells. As primary and immortal DF-1 CEF cells were treated with 10-100 microM of hydrogen peroxide (concentrations known to be sublethal in human diploid fibroblasts), immortal DF-1 CEF cells were shown to be more sensitive to hydrogen peroxide, and total cell numbers were dramatically reduced when compared with primary cell counterparts. This increased sensitivity to hydrogen peroxide in immortal DF-1 cells occurred without evident changes in either antioxidant gene expression, mitochondrial membrane potential, cell cycle distribution or chromatin condensation. However, the total number of dead cells without chromatin condensation was dramatically elevated in immortal DF-1 CEFs treated with hydrogen peroxide, indicating that the inhibition of immortal DF-1 cell growth by low concentrations of hydrogen peroxide is due to increased necrotic cell death, but not apoptosis. Taken together, our observation suggests that the balanced antioxidant function might be important for cell proliferation in response to toxic oxidative damage by hydrogen peroxide.

Evaluation of efficacy of 1% Crystal Violet & Nuclear Fast Red stain compared to Haematoxyline & Eosin stain for assessing mitotic figures in oral premalignant and malignant lesions.

PubMed

Motiwale, Gauri; Jaiswal, Shradha; Vikey, Ashok; Motiwale, Tejas; Bagulkar, Bhupesh; Bhat, Atul; Kapoor, Prakhar

2016-07-01

Various chromosomal arrangements in cells undergoing division are referred to as Mitotic figure (MF). The abnormal excess of mitotic figures is commonly seen in oral epithelial dysplasia (ED) and oral squamous cell carcinoma (OSCC). In present study, we compared the number of mitotic figures in normal oral mucosa, epithelial dysplasia & OSCC sections with haematoxyline & eosine (H&E) and 1%Crystal Violet & Nuclear Fast Red (CV&NFR) stain, also the efficacy of the CV&NFR stain as compared to H & E stain. We investigated the correlation between the number of mitotic figures & grades of OSCC. Study sample comprised of two serial sections of archival blocks of normal oral mucosa & diagnosed cases of epithelial dysplasia & OSCC. One slide stained with H& E & the other one with 1% CV & NFR. Mitotic figures were counted with the grid eyepiece. There was significant increase in number of MFs in oral ED and OSCC in comparison with normal oral mucosa. There was a highly significant increase in number of MFs in CV&NFR stained tissue sections when compared with H & E stain. Metaphase is the most commonly observed phase of mitosis. In summary, our study proposes the use of Crystal violet & Nuclear fast red stain as a selective stain for better contrast & easy identification MFs. © 2016 Old City Publishing, Inc.

Solar granulation and statistical crystallography: A modeling approach using size-shape relations

NASA Technical Reports Server (NTRS)

Noever, D. A.

1994-01-01

The irregular polygonal pattern of solar granulation is analyzed for size-shape relations using statistical crystallography. In contrast to previous work which has assumed perfectly hexagonal patterns for granulation, more realistic accounting of cell (granule) shapes reveals a broader basis for quantitative analysis. Several features emerge as noteworthy: (1) a linear correlation between number of cell-sides and neighboring shapes (called Aboav-Weaire's law); (2) a linear correlation between both average cell area and perimeter and the number of cell-sides (called Lewis's law and a perimeter law, respectively) and (3) a linear correlation between cell area and squared perimeter (called convolution index). This statistical picture of granulation is consistent with a finding of no correlation in cell shapes beyond nearest neighbors. A comparative calculation between existing model predictions taken from luminosity data and the present analysis shows substantial agreements for cell-size distributions. A model for understanding grain lifetimes is proposed which links convective times to cell shape using crystallographic results.

Interactions between insulin-like growth factor-I, estrogen receptor-α (ERα) and ERβ in regulating growth/apoptosis of MCF-7 human breast cancer cells

PubMed Central

Mendoza, Rhone A.; Enriquez, Marlene I; Mejia, Sylvia M; Moody, Emily E; Thordarson, Gudmundur

2011-01-01

Understanding of the interactions between estradiol (E2) and insulin-like growth factor-I (IGF-I) is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating non-interfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human growth hormone plus epidermal growth factor, but E2 did not cause increase in the number of the IGF-IR.low cells compared to controls. Proliferation rate of IGF-IR.low cells was only reduced in response to E2 compared to controls, whereas their basal and hormone stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E2, without affecting control cells. Further, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. Summary, suppressing the IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate. PMID:20974640

Blood T-cell receptor diversity decreases during the course of HIV infection, but the potential for a diverse repertoire persists

PubMed Central

Young, Jennifer J.; Schmidt, Diane; Zhang, Qianjun; Hoh, Rebecca; Busch, Michael; Martin, Jeffrey; Deeks, Steven; McCune, Joseph M.

2012-01-01

HIV infection results in a decrease in circulating CD4+ T-cell and naive T-cell numbers. If such losses were associated with an erosion of T-cell receptor (TCR) repertoire diversity in the peripheral T-cell pool, this might exacerbate the state of persistent immunodeficiency. Existing methods for the analysis of the TCR repertoire have demonstrated skewed distributions of TCR genes in HIV-infected subjects but cannot directly measure TCR diversity. Here we used AmpliCot, a quantitative assay based on DNA hybridization kinetics, to measure TCR diversity in a cross-sectional comparison of 19 HIV-infected persons to 18 HIV-uninfected controls. HIV-infected persons had a 10-fold decrease in total TCR repertoire diversity in 1.5 mL of blood compared with uninfected controls, with decreased diversity correlating most closely with a lower CD4+ T-cell percentage. Nonetheless, the TCR repertoire diversity of sort-purified T-cell subpopulations in HIV-infected and HIV-uninfected subjects was comparable. These observations suggest that the TCR repertoire diversity changes in whole blood during HIV disease progression are primarily the result of changes in the number and proportion of T-cell subpopulations and that most HIV-infected persons may retain a sufficiently diverse TCR repertoire to permit immune reconstitution with antiretroviral therapy alone, without thymopoiesis. PMID:22371879

Factors for C-Kit Expression in Cardiac Outgrowth Cells and Human Heart Tissue.

PubMed

Matsushita, Satoshi; Minematsu, Kazuo; Yamamoto, Taira; Inaba, Hirotaka; Kuwaki, Kenji; Shimada, Akie; Yokoyama, Yasutaka; Amano, Atsushi

2017-12-12

We determined the factors associated with the expression of c-kit in the heart and the proliferation of c-kit-positive (c-kit pos ) cardiac stem cells among the outgrowth cells cultured from human cardiac explants.Samples of the right atrium (RA), left atrium (LA), and left ventricle obtained from patients during open-heart surgery were processed for cell culture of outgrowth cells and tissue analysis. The total number of growing cells and the population of c-kit pos cells were measured and compared with c-kit expression in native tissues and characteristics of the patients according to the region of the heart.We analyzed 452 samples from 334 patients. Atrial fibrillation (AF) in the patients reduced the number of outgrowth cells from the RA and LA, and aging was a co-factor for the LA. The c-kit pos population from the RA was associated with serum brain natriuretic peptide (BNP). C-kit expression in native tissue was also associated with BNP expression. However, we observed no relationship in expression between outgrowth cells and native tissue. In addition, the RA tissue provided the highest number of c-kit pos cells, and the left ventricle provided the lowest.C-kit was weakly expressed in response to damage. In addition, no correlation between outgrowth cells and native tissue was found for c-kit expression.

The effect of diabetes on the wound healing potential of adipose-tissue derived stem cells.

PubMed

Kim, Sue Min; Kim, Yun Ho; Jun, Young Joon; Yoo, Gyeol; Rhie, Jong Won

2016-03-01

To investigate whether diabetes mellitus affects the wound-healing-promoting potential of adipose tissue-derived stem cells, we designed a wound-healing model using diabetic mice. We compared the degree of wound healing between wounds treated with normal adipose tissue-derived stem cells and wounds treated with diabetic adipose tissue-derived stem cells. We evaluated the wound-healing rate, the epithelial tongue distance, the area of granulation tissue, the number of capillary and the number of Ki-67-stained cells. The wound-healing rate was significantly higher in the normal adipose tissue-derived stem cells group than in the diabetic adipose tissue-derived stem cells group; it was also significantly higher in the normal adipose tissue-derived stem cells group than in the control group. Although the diabetic adipose tissue-derived stem cells group showed a better wound-healing rate than the control group, the difference was not statistically significant. Similar trends were observed for the other parameters examined: re-epithelisation and keratinocyte proliferation; granulation tissue formation; and dermal regeneration. However, with regard to the number of capillary, diabetic adipose tissue-derived stem cells retained their ability to promote neovasculisation and angiogenesis. These results reflect the general impairment of the therapeutic potential of diabetic adipose tissue-derived stem cells in vivo. © 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Independent replication of mitochondrial genes supports the transcriptional program in developing fiber cells of cotton (Gossypium hirsutum L.).

PubMed

Thyssen, Gregory N; Song, Xianliang; Naoumkina, Marina; Kim, Hee-Jin; Fang, David D

2014-07-01

The mitochondrial genomes of flowering plants exist both as a "master circle" chromosome and as numerous subgenomic sublimons that are generated by intramolecular recombination. Differential stability or replication of these sublimons allows individual mitochondrial gene copy numbers to vary independently between different cell types and developmental stages. Our objective was to determine the relationship between mitochondrial gene copy number and transcript abundance in the elongating fiber cells of Upland cotton (Gossypium hirsutum L.). We compared RNA and DNA from cotton fiber cells at five developmental time points from early elongation through secondary cell wall thickening from the Ligon-lintless 2 (Li2) short fiber mutant and its wild type near isogenic line (NIL) DP5690. Mitochondrial gene copy number decreased from 3 to 8-DPA in the developing cotton fiber cells while transcript levels remained low. As secondary cell wall biosynthesis began in developing fibers, the expression levels and copy numbers of mitochondrial genes involved in energy production and respiration were up-regulated in wild type cotton DP5690. However, the short fiber mutant Li2, failed to increase expression of these genes, which include three subunits of ATP synthase, atp1, atp8 and atp9 and two cytochrome genes cox1 and cob. At the same time, Li2 failed to increase the copy numbers of these highly expressed genes. Surprisingly, we found that when mitochondrial genes were highly transcribed, they also had very high copy numbers. This observation suggests that in developing cotton fibers, increased mitochondrial sublimon replication may support increases in gene transcription. Published by Elsevier B.V.

The number of preproghrelin mRNA expressing cells is increased in mice with activity-based anorexia.

PubMed

François, Marie; Barde, Swapnali; Achamrah, Najate; Breton, Jonathan; do Rego, Jean-Claude; Coëffier, Moïse; Hökfelt, Tomas; Déchelotte, Pierre; Fetissov, Sergueï O

2015-06-01

Plasma levels of ghrelin, an orexigenic peptide, are increased during conditions of chronic starvation, such as in patients with anorexia nervosa. However, it is not known whether such increase can be related to the number of preproghrelin mRNA-expressing cells in the stomach, and if chronic starvation may activate a tentative central ghrelin production. In this work, in situ hybridization technique was used to analyze the presence and number of preproghrelin mRNA-expressing cells in the stomach and the hypothalamus of mice with activity-based anorexia (ABA) induced by the combination of running wheel activity with progressive, during 10 days, feeding-time restriction (FTR) and compared with sedentary FTR, ABA pair-fed (PF) and ad libitum-fed control mice. All food-restricted mice lost more than 20% of body weight. Body weight loss was similar in ABA and PF mice, but it was more pronounced than in FTR mice. Food intake was also lower in ABA than in FTR mice. Preproghrelin mRNA-expressing cells in the stomach were increased proportionally to the body weight loss in all food-restricted groups with the highest number in ABA mice. No preproghrelin mRNA-producing cells were detectable in the hypothalamus of either control or food-restricted mice. Thus, the increased number of gastric preproghrelin mRNA-producing cells during chronic starvation proportionally to the body weight loss and reduced food intake may underlie increased plasma ghrelin. Hyperactivity-induced anorexia appears to further increase the number of preproghrelin mRNA-producing cells in the stomach. No evidence was found for ghrelin expression in the hypothalamus, not even in any of the present experimental models. Copyright © 2015 Elsevier Ltd. All rights reserved.

Obesity induced by a high-fat diet is associated with increased immune cell entry into the central nervous system.

PubMed

Buckman, Laura B; Hasty, Alyssa H; Flaherty, David K; Buckman, Christopher T; Thompson, Misty M; Matlock, Brittany K; Weller, Kevin; Ellacott, Kate L J

2014-01-01

Obesity is associated with chronic low-grade inflammation in peripheral tissues caused, in part, by the recruitment of inflammatory monocytes into adipose tissue. Studies in rodent models have also shown increased inflammation in the central nervous system (CNS) during obesity. The goal of this study was to determine whether obesity is associated with recruitment of peripheral immune cells into the CNS. To do this we used a bone marrow chimerism model to track the entry of green-fluorescent protein (GFP) labeled peripheral immune cells into the CNS. Flow cytometry was used to quantify the number of GFP(+) immune cells recruited into the CNS of mice fed a high-fat diet compared to standard chow fed controls. High-fat feeding resulted in obesity associated with a 30% increase in the number of GFP(+) cells in the CNS compared to control mice. Greater than 80% of the GFP(+) cells recruited to the CNS were also CD45(+) CD11b(+) indicating that the GFP(+) cells displayed characteristics of microglia/macrophages. Immunohistochemistry further confirmed the increase in GFP(+) cells in the CNS of the high-fat fed group and also indicated that 93% of the recruited cells were found in the parenchyma and had a stellate morphology. These findings indicate that peripheral immune cells can be recruited to the CNS in obesity and may contribute to the inflammatory response. Copyright © 2013 Elsevier Inc. All rights reserved.

Intestinal immune cells in Strongyloides stercoralis infection.

PubMed Central

Trajman, A; MacDonald, T T; Elia, C C

1997-01-01

BACKGROUND: Strongyloides stercoralis can cause a wide spectrum of disease in man, ranging from a chronic asymptomatic infection to a hyperinfective, often fatal syndrome. In rodents, spontaneous expulsion of Strongyloides spp occurs after experimental infection. Mast cells, goblet cells, and eosinophils have been identified as possible effectors of this expulsion. AIMS: To investigate intestinal histopathology and mucosal immunity in immunocompetent patients with chronic S stercoralis infection. METHODS: Jejunal biopsies were performed in 19 immunocompetent patients with a positive stool examination for S stercoralis and few or no symptoms, and in seven healthy controls. Specimens were processed for histopathological analysis and stained by the immunoperoxidase technique, using the following monoclonal antibodies: CD2, CD3, CD4, CD8, anti-T cell receptor (TcR) gamma/delta, RFD1 and RFD7 (two different macrophage markers), Ki67+ (proliferating) cells, antihuman leucocyte antigen (HLA)-DR, and anticollagen IV. In addition, CD25+ cells, mast cells, IgE expressing cells, calprotectin containing cells, and neutrophil elastase positive cells were stained by the alkaline phosphatase method. RESULTS: Jejunal morphology and the numbers of different T cell subsets, mast cells, IgE expressing cells, eosinophils, and goblet cells were unaffected by S stercoralis infection. Conversely, the numbers of mature macrophages and dividing enterocytes in the crypts were reduced significantly. Crypt enterocytes did not express HLA-DR in both groups. The expression of HLA-DR by villus enterocytes was also comparable in patients and controls. There were no activated (CD25+) cells in the mucosa of either patients or controls. CONCLUSIONS: Compared with seven healthy uninfected volunteers, a group of 19 Brazilians with clinically mild strongyloides infection showed no abnormality of mucosal structure and no increase in non-specific inflammatory cells. Likewise, there was no increase in mucosal T cells or macrophages. Images PMID:9516879

Stem Cells in Aggregate Form to Enhance Chondrogenesis in Hydrogels

PubMed Central

Sridharan, BanuPriya; Lin, Staphany M.; Hwu, Alexander T.; Laflin, Amy D.; Detamore, Michael S.

2015-01-01

There are a variety of exciting hydrogel technologies being explored for cartilage regenerative medicine. Our overall goal is to explore whether using stem cells in an aggregate form may be advantageous in these applications. 3D stem cell aggregates hold great promise as they may recapitulate the in vivo skeletal tissue condensation, a property that is not typically observed in 2D culture. We considered two different stem cell sources, human umbilical cord Wharton’s jelly cells (hWJCs, currently being used in clinical trials) and rat bone marrow-derived mesenchymal stem cells (rBMSCs). The objective of the current study was to compare the influence of cell phenotype, aggregate size, and aggregate number on chondrogenic differentiation in a generic hydrogel (agarose) platform. Despite being differing cell sources, both rBMSC and hWJC aggregates were consistent in outperforming cell suspension control groups in biosynthesis and chondrogenesis. Higher cell density impacted biosynthesis favorably, and the number of aggregates positively influenced chondrogenesis. Therefore, we recommend that investigators employing hydrogels consider using cells in an aggregate form for enhanced chondrogenic performance. PMID:26719986

Celastrol supports survival of retinal ganglion cells injured by optic nerve crush.

PubMed

Kyung, Haksu; Kwong, Jacky M K; Bekerman, Vlad; Gu, Lei; Yadegari, Daniel; Caprioli, Joseph; Piri, Natik

2015-06-03

The present study evaluates the effect of celastrol on the survival of retinal ganglion cells (RGCs) injured by optic nerve crush (ONC). Celastrol, a quinine methide triterpene extracted from the perennial vine Tripterygium wilfordii (Celastraceae), has been identified as a potential neuroprotective candidate in a comprehensive drug screen against various neurodegenerative diseases. Two weeks after ONC, the average density of remaining RGCs in retinas of animals treated with daily intraperitoneal (i.p.) injections of celastrol (1mg/kg) was approximately 1332 cells/mm(2), or 40.8% of the Celastrol/Control group. In retinas of the Vehicle/ONC group about 381 RGCs/mm(2) were counted, which is 9.6% of the total number of RGCs in the DMSO/Control group. This corresponds to approximately a 250% increase in RGC survival mediated by celastrol treatment compared to Vehicle/ONC group. Furthermore, the average RGC number in retinas of ONC animals treated with a single intravitreal injection of 1mg/kg or 5mg/kg of celastrol was increased by approximately 80% (760 RGCs/mm(2)) and 78% (753 RGCs/mm(2)), respectively, compared to Vehicle/ONC controls (422 cells/mm(2)). Injection of 0.2mg/kg of celastrol had no significant effect on cell survival, with the average number of RGCs being 514 cells/mm(2) in celastrol-treated animals versus 422 cells/mm(2) in controls. The expression levels of Hsp70, Hsf1, Hsf2, HO-1 and TNF-alpha in the retina were analyzed to evaluate the roles of these proteins in the celastrol-mediated protection of injured RGCs. No statistically significant change in HO-1, Hsf1 and Hsp70 levels was seen in animals with ONC. An approximately 2 fold increase in Hsf2 level was observed in celastrol-treated animals with or without injury. Hsf2 level was also increased 1.8 fold in DMSO-treated animals with ONC injury compared to DMSO-treated animals with no injury suggesting that Hsf2 induction has an injury-induced component. Expression of TNF-alpha in retinas of celastrol-treated uninjured and ONC animals was reduced by approximately 2 and 1.5 fold compared to vehicle treated animals, respectively. The observed results suggest that mechanisms underlying celastrol׳s RGC protective effect are associated with inhibition of TNF-alpha-mediated cell death. Copyright © 2015 Elsevier B.V. All rights reserved.

Quantitative grading of a human blastocyst: optimal inner cell mass size and shape.

PubMed

Richter, K S; Harris, D C; Daneshmand, S T; Shapiro, B S

2001-12-01

To investigate the predictive value of quantitative measurements of blastocyst morphology on subsequent implantation rates after transfer. Prospective observational study. Private assisted reproductive technology center. One hundred seventy-four IVF patients receiving transfers of expanded blastocyst-stage embryos on day 5 (n = 112) or day 6 (n = 62) after oocyte retrieval. None. Blastocyst diameter, number of trophectoderm cells, inner cell mass (ICM) size, ICM shape, and implantation and pregnancy rates. Blastocyst diameter and trophectoderm cell numbers were unrelated to implantation rates. Day 5 expanded blastocysts with ICMs of >4,500 microm(2) implanted at a higher rate than did those with smaller ICMs (55% vs. 31%). Day 5 expanded blastocysts with slightly oval ICMs implanted at a higher rate (58%) compared with those with either rounder ICMs (7%) or more elongated ICMs (33%). Implantation rates were highest (71%) for embryos with both optimal ICM size and shape. Pregnancy rates were higher for day 5 transfers of optimally shaped ICMs compared with day 5 transfers of optimally sized ICMs. Quantitative measurements of the inner cell mass are highly indicative of blastocyst implantation potential. Blastocysts with relatively large and/or slightly oval ICMs are more likely to implant than other blastocysts.

Sparing of extraocular muscle in aging and muscular dystrophies: A myogenic precursor cell hypothesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kallestad, Kristen M.; Hebert, Sadie L.; McDonald, Abby A.

2011-04-01

The extraocular muscles (EOM) are spared from pathology in aging and many forms of muscular dystrophy. Despite many studies, this sparing remains an enigma. The EOM have a distinct embryonic lineage compared to somite-derived muscles, and we have shown that they continuously remodel throughout life, maintaining a population of activated satellite cells even in aging. These data suggested the hypothesis that there is a population of myogenic precursor cells (mpcs) in EOM that is different from those in limb, with either elevated numbers of stem cells and/or mpcs with superior proliferative capacity compared to mpcs in limb. Using flow cytometry,more » EOM and limb muscle mononuclear cells were compared, and a number of differences were seen. Using two different cell isolation methods, EOM have significantly more mpcs per mg muscle than limb skeletal muscle. One specific subpopulation significantly increased in EOM compared to limb was positive for CD34 and negative for Sca-1, M-cadherin, CD31, and CD45. We named these the EOMCD34 cells. Similar percentages of EOMCD34 cells were present in both newborn EOM and limb muscle. They were retained in aged EOM, whereas the population decreased significantly in adult limb muscle and were extremely scarce in aged limb muscle. Most importantly, the percentage of EOMCD34 cells was elevated in the EOM from both the mdx and the mdx/utrophin{sup -/-} (DKO) mouse models of DMD and extremely scarce in the limb muscles of these mice. In vitro, the EOMCD34 cells had myogenic potential, forming myotubes in differentiation media. After determining a media better able to induce proliferation in these cells, a fusion index was calculated. The cells isolated from EOM had a 40% higher fusion index compared to the same cells isolated from limb muscle. The EOMCD34 cells were resistant to both oxidative stress and mechanical injury. These data support our hypothesis that the EOM may be spared in aging and in muscular dystrophies due to a subpopulation of mpcs, the EOMCD34 cells, that are retained in significantly higher percentages in normal, mdx and DKO mice EOM, appear to be resistant to elevated levels of oxidative stress and toxins, and actively proliferate throughout life. Current studies are focused on further defining the EOMCD34 cell subtype molecularly, with the hopes that this may shed light on a cell type with potential therapeutic use in patients with sarcopenia, cachexia, or muscular dystrophy.« less

Elevations in vascular markers and eosinophils in chronic spontaneous urticarial weals with low-level persistence in uninvolved skin.

PubMed

Kay, A B; Ying, S; Ardelean, E; Mlynek, A; Kita, H; Clark, P; Maurer, M

2014-09-01

In chronic spontaneous urticaria (CSU) mast cell activation together with inflammatory changes in the skin are well documented and may play an important role in mechanisms of tissue oedema. To confirm and extend these observations by measuring microvascular markers, leucocytes and mast cell numbers in lesional and uninvolved skin and to compare findings with a control group. Paired biopsies (one from 4-8-h spontaneous weals and one from uninvolved skin) were taken from eight patients with CSU and nine control subjects and studied using immunohistochemistry and confocal microscopy using the lectin Ulex europaeus agglutinin 1 (UEA-1). Lesional skin in CSU contained significantly more CD31+ endothelial cells; CD31+ blood vessels, neutrophils, eosinophils, basophils and macrophages; and CD3+ T cells than nonlesional skin. Increased vascularity was confirmed by confocal imaging using the lectin UEA-1. Uninvolved skin from CSU contained significantly more CD31+ endothelial cells, CD31+ blood vessels and eosinophils compared with the control subjects. There was a threefold increase in mast cell numbers when CSU was compared with controls but no difference was observed between lesional and uninvolved skin. Increased vascular markers together with eosinophil and neutrophil infiltration are features of lesional skin in CSU and might contribute to tissue oedema. Eosinophils and microvascular changes persist in uninvolved skin, which, together with increased mast cells, suggests that nonlesional skin is primed for further wealing. © 2014 The Authors. British Journal of Dermatology published by John Wiley & Sons Ltd on behalf of British Association of Dermatologists.

Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design

PubMed Central

Zeidler-Erdely, Patti C.; Antonini, James M.; Meighan, Terence G.; Young, Shih-Houng; Eye, Tracy J.; Hammer, Mary Ann; Erdely, Aaron

2016-01-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable. PMID:27251196

Comparison of cell counting methods in rodent pulmonary toxicity studies: automated and manual protocols and considerations for experimental design.

PubMed

Zeidler-Erdely, Patti C; Antonini, James M; Meighan, Terence G; Young, Shih-Houng; Eye, Tracy J; Hammer, Mary Ann; Erdely, Aaron

2016-08-01

Pulmonary toxicity studies often use bronchoalveolar lavage (BAL) to investigate potential adverse lung responses to a particulate exposure. The BAL cellular fraction is counted, using automated (i.e. Coulter Counter®), flow cytometry or manual (i.e. hemocytometer) methods, to determine inflammatory cell influx. The goal of the study was to compare the different counting methods to determine which is optimal for examining BAL cell influx after exposure by inhalation or intratracheal instillation (ITI) to different particles with varying inherent pulmonary toxicities in both rat and mouse models. General findings indicate that total BAL cell counts using the automated and manual methods tended to agree after inhalation or ITI exposure to particle samples that are relatively nontoxic or at later time points after exposure to a pneumotoxic particle when the response resolves. However, when the initial lung inflammation and cytotoxicity was high after exposure to a pneumotoxic particle, significant differences were observed when comparing cell counts from the automated, flow cytometry and manual methods. When using total BAL cell count for differential calculations from the automated method, depending on the cell diameter size range cutoff, the data suggest that the number of lung polymorphonuclear leukocytes (PMN) varies. Importantly, the automated counts, regardless of the size cutoff, still indicated a greater number of total lung PMN when compared with the manual method, which agreed more closely with flow cytometry. The results suggest that either the manual method or flow cytometry would be better suited for BAL studies where cytotoxicity is an unknown variable.

The production of glial cell line-derived neurotrophic factor by human sertoli cells is substantially reduced in sertoli cell-only testes.

PubMed

Singh, D; Paduch, D A; Schlegel, P N; Orwig, K E; Mielnik, A; Bolyakov, A; Wright, W W

2017-05-01

Do human Sertoli cells in testes that exhibit the Sertoli cell-only (SCO) phenotype produce substantially less glial cell line-derived neurotrophic factor (GDNF) than Sertoli cells in normal testes? In human SCO testes, both the amounts of GDNF mRNA per testis and the concentration of GDNF protein per Sertoli cell are markedly reduced as compared to normal testes. In vivo, GDNF is required to sustain the numbers and function of mouse spermatogonial stem cells (SSCs) and their immediate progeny, transit-amplifying progenitor spermatogonia. GDNF is expressed in the human testis, and the ligand-binding domain of the GDNF receptor, GFRA1, has been detected on human SSCs. The numbers and/or function of these stem cells are markedly reduced in some infertile men, resulting in the SCO histological phenotype. We determined the numbers of human spermatogonia per mm2 of seminiferous tubule surface that express GFRA1 and/or UCHL1, another marker of human SSCs. We measured GFRA1 mRNA expression in order to document the reduced numbers and/or function of SSCs in SCO testes. We quantified GDNF mRNA in testes of humans and mice, a species with GDNF-dependent SSCs. We also compared GDNF mRNA expression in human testes with normal spermatogenesis to that in testes exhibiting the SCO phenotype. As controls, we also measured transcripts encoding two other Sertoli cell products, kit ligand (KITL) and clusterin (CLU). Finally, we compared the amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes were obtained from beating heart organ donors. Biopsies of testes from men who were infertile due to maturation arrest or the SCO phenotype were obtained as part of standard care during micro-testicular surgical sperm extraction. Cells expressing GFRA1, UCHL1 or both on whole mounts of normal human seminiferous tubules were identified by immunohistochemistry and confocal microscopy and their numbers were determined by image analysis. Human GDNF mRNA and GFRA1 mRNA were quantified by use of digital PCR and Taqman primers. Transcripts encoding mouse GDNF and human KITL, CLU and 18 S rRNA, used for normalization of data, were quantified by use of real-time PCR and Taqman primers. Finally, we used two independent methods, flow cytometric analysis of single cells and ELISA assays of homogenates of whole testis biopsies, to compare amounts of GDNF per Sertoli cell in normal and SCO testes. Normal human testes contain a large population of SSCs that express GFRA1, the ligand-binding domain of the GDNF receptor. In human SCO testes, GFRA1 mRNA was detected but at markedly reduced levels. Expression of GDNF mRNA and the amount of GDNF protein per Sertoli cell were also significantly reduced in SCO testes. These results were observed in multiple, independent samples, and the reduced amount of GDNF in Sertoli cells of SCO testes was demonstrated using two different analytical approaches. N/A. There currently are no approved protocols for the in vivo manipulation of human testis GDNF concentrations. Thus, while our data suggest that insufficient GDNF may be the proximal cause of some cases of human male infertility, our results are correlative in nature. We propose that insufficient GDNF expression may contribute to the infertility of some men with an SCO testicular phenotype. If their testes contain some SSCs, an approach that increases their testicular GDNF concentrations might expand stem cell numbers and possibly sperm production. This research was funded by the Eunice Kennedy Shriver National Institute for Child Health and Human Development, National Centers for Translational Research in Reproduction and Infertility Program (NCTRI) Grant 1R01HD074542-04, as well as grants R01 HD076412-02 and P01 HD075795-02 and the U.S.-Israel Binational Science Foundation. Support for this research was also provided by NIH P50 HD076210, the Robert Dow Foundation, the Frederick & Theresa Dow Wallace Fund of the New York Community Trust and the Brady Urological Foundation. There are no competing interests. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

[Effects on blood cell numbers and cytokines of dermal application rocket kerosene in mice].

PubMed

Xu, Bingxin; Wang, Jianying; Liu, Zhiguo; Li, Chenglin; Yang, Heming; Lou, Xiaotong; Li, Jianzhong; Cui, Yan

2015-09-01

To detect the number of cells and the level of IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-γ and IL-17 cytokines in the peripheral blood of mice exposed to rocket kerosene by skin. ICR mice were randomly divided into the normal control group and RK experimental group (400 µl×1 group). RK undiluted fuel were applied directly to the dorsal skin of the mice. In control groups were treated with sesame oil (SO). the number of blood cells were detected by automatic blood cell counter and the level of IL-2, IL-4, IL-6, IL-10, TNF-alpha, IFN-γ and IL-17 cytokines in serum were detected by using flow cytometry and BD CBA Flex set kit. Compared with the normal group, WBC and LYM had a decreasing tendency 2 h and decreased significantly 6 h, 12 h and 1 d after RK exposure (P<0.05). They increased significantly 7 d after RK exposure (P<0.05). Compared with the normal group, the level of IL-6 increased significantly 2 h, 6 h, 12 h,1 d and 3 d (P<0.05). The level of TNF-α increased significantly 2h, 3d, 5d and 7d (P<0.05). The level of IL-10 increased significantly 2 h, 6 h, 3 d, 5 d and 7 d (P<0.05). The level of IFN-γ increased significantly 6 h and 3 d (P< 0.05). The level of IL-17 significantly increased 3 d, 5 d and 7d (P<0.05). RK can change the number of immune cells, causing the immune cytokine changes in mice after RK cutaneous exposure.

Changes in mast cell number and stem cell factor expression in human skin after radiotherapy for breast cancer.

PubMed

Westbury, Charlotte B; Freeman, Alex; Rashid, Mohammed; Pearson, Ann; Yarnold, John R; Short, Susan C

2014-05-01

Mast cells are involved in the pathogenesis of radiation fibrosis and may be a therapeutic target. The mechanism of increased mast cell number in relation to acute and late tissue responses in human skin was investigated. Punch biopsies of skin 1 and 15-18 months after breast radiotherapy and a contralateral control biopsy were collected. Mast cells were quantified by immunohistochemistry using the markers c-Kit and tryptase. Stem cell factor (SCF) and collagen-1 expression was analysed by qRT-PCR. Clinical photographic scores were performed at post-surgical baseline and 18 months and 5 years post-radiotherapy. Primary human dermal microvascular endothelial cell (HDMEC) cultures were exposed to 2Gy ionising radiation and p53 and SCF expression was analysed by Western blotting and ELISA. Dermal mast cell numbers were increased at 1 (p=0.047) and 18 months (p=0.040) using c-Kit, and at 18 months (p=0.024) using tryptase immunostaining. Collagen-1 mRNA in skin was increased at 1 month (p=0.047) and 18 months (p=0.032) and SCF mRNA increased at 1 month (p=0.003). None of 16 cases scored had a change in photographic appearance at 5 years, compared to baseline. SCF expression was not increased in HDMECs irradiated in vitro. Increased mast cell number was associated with up-regulated collagen-1 expression in human skin at early and late time points. This increase could be secondary to elevated SCF expression at 1 month after radiotherapy. Although mast cells accumulate around blood vessels, no endothelial cell secretion of SCF was seen after in vitro irradiation. Modification of mast cell number and collagen-1 expression may be observed in skin at 1 and 18 months after radiotherapy in breast cancer patients with no change in photographic breast appearance at 5 years. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

FOXP3-expressing CD4(+) T-cell numbers increase in areas of duodenal gastric metaplasia and are associated to CD4(+) T-cell aggregates in the duodenum of Helicobacter pylori-infected duodenal ulcer patients.

PubMed

Kindlund, Bert; Sjöling, Asa; Hansson, Malin; Edebo, Anders; Hansson, Lars-Erik; Sjövall, Henrik; Svennerholm, Ann-Mari; Lundin, B Samuel

2009-06-01

We have previously demonstrated that Helicobacter pylori infection is associated with an increased number of CD4(+)CD25(high) regulatory T cells in the gastric and duodenal mucosa. In this study, we determined the number and localization of CD4(+) cells expressing the regulatory T-cell-specific transcription factor FOXP3 in the antrum and duodenum of duodenal ulcer patients, asymptomatic carriers, and uninfected individuals. We also determined gene expression levels of FOXP3 as well as anti- and proinflammatory cytokines before and after H. pylori eradication. Cellular FOXP3 expression was studied by immunofluorescence and flow cytometry, and transcription levels of FOXP3, interleukin (IL)-10, transforming growth factor-beta, CD4, and interferon-gamma were analyzed by real-time reverse transcription-polymerase chain reaction. We found an increased (6-fold) frequency of CD4(+)FOXP3(+) T cells in H. pylori-infected gastric mucosa; interestingly 26% of these cells did not co-express CD25. The increase of FOXP3-expressing T cells in the antrum of infected individuals was dependent on the presence of H. pylori, since eradication therapy resulted in 4-fold lower levels of FOXP3 and IL-10 mRNA in the antrum. Furthermore, higher numbers of CD4(+)FOXP3(+) T cells were found in areas of duodenal gastric metaplasia in the duodenum of duodenal ulcer patients compared to duodenal gastric metaplasia of asymptomatic individuals and healthy mucosa in both patient groups. In duodenal ulcer patients, the CD4(+)FOXP3(+) T cells were more highly associated to aggregates in the duodenal mucosa. The numbers of CD4(+)FOXP3(+) T cells are increased and localized in CD4(+) T-cell aggregates in areas of duodenal gastric metaplasia in duodenal ulcer patients.

Age-related decline in mitochondrial DNA copy number in isolated human pancreatic islets.

PubMed

Cree, L M; Patel, S K; Pyle, A; Lynn, S; Turnbull, D M; Chinnery, P F; Walker, M

2008-08-01

Pancreatic beta cell function has been shown to decline with age in man. Depletion of mitochondrial DNA (mtDNA) copy number is associated with impaired insulin secretion in pancreatic beta cell lines, and decreased mtDNA copy number has been observed with age in skeletal muscle in man. We investigated whether mtDNA copy number decreases with age in human pancreatic beta cells, which might in turn contribute to the age-related decline in insulin secretory capacity. We quantified mtDNA copy number in isolated human islet preparations from 15 pancreas donors aged between 17 and 75 years. Islets (n = 20) were individually hand-picked and pooled from each donor isolate for the quantification of mtDNA copy number and deleted mtDNA (%), which were determined using real-time PCR methods. There was a significant negative correlation between mtDNA copy number and islet donor age (r = -0.53, p = 0.044). mtDNA copy number was significantly decreased in islet preparations from donors aged > or =50 years (n = 8) compared with those aged <50 years (n = 7) (median [interquartile range]: 418 [236-503] vs 596 [554-729] mtDNA copy number/diploid genome; p = 0.032). None of the islet preparations harboured high levels of deleted mtDNA affecting the major arc. Given the correlation between mtDNA content and respiratory chain activity, the age-related decrease in mtDNA copy number that we observed in human pancreatic islet preparations may contribute to the age-dependent decline in pancreatic beta cell insulin secretory capacity.

Is There Any Reason to Prefer Cord Blood Instead of Adult Donors for Hematopoietic Stem Cell Transplants?

PubMed

Beksac, Meral

2015-01-01

As cord blood (CB) enables rapid access and tolerance to HLA mismatches, a number of unrelated CB transplants have reached 30,000. Such transplant activity has been the result of international accreditation programs maintaining highly qualified cord blood units (CBUs) reaching more than 600,000 CBUs stored worldwide. Efforts to increase stem cell content or engraftment rate of the graft by ex vivo expansion, modulation by molecules such as fucose, prostaglandin E2 derivative, complement CD26 inhibitors, or CXCR4/CXCL12 axis have been able to accelerate engraftment speed and rate. Furthermore, introduction of reduced intensity conditioning protocols, better HLA matching, and recognition of the importance of HLA-C have improved CB transplants success by decreasing transplant-related mortality. CB progenitor/stem cell content has been compared with adult stem cells revealing higher long-term repopulating capacity compared to bone marrow-mesenchymal stromal cells and lesser oncogenic potential than progenitor-induced stem cells. This chapter summarizes the advantages and disadvantages of CB compared to adult stem cells within the context of stem cell biology and transplantation.

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release.

PubMed

Balasundaram, B; Harrison, S T L

2006-06-05

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation. Copyright 2006 Wiley Periodicals, Inc.

Enniatin A1, enniatin B1 and beauvericin on HepG2: Evaluation of toxic effects.

PubMed

Juan-García, Ana; Ruiz, María-José; Font, Guillermina; Manyes, Lara

2015-10-01

Hepatotoxicity of three Fusarium mycotoxins, beauvericin (BEA) and two enniatins (ENNs) ENN A1 and ENN B1, in hepatocarcinoma cells (HepG2) were evaluated and compared. Concentrations used were 1.5 and 3 μM at 24, 48 and 72 h for each mycotoxin. Flow cytometry was used to examine enniatins effects on cell proliferation, to characterize the cell cycle phase where the cells blocked and to study the mitochondria role in ENNs-induced apoptosis. ENN B1 treated cells showed a time dependent G1 blockade at both concentrations used. ENN A1 and BEA decreased the apoptotic-necrotic percentage of cells comparing to control and disrupted the MMP as observed by TMRM and ToPro-3 fluorochromes signal. It is proposed a decreasing mycotoxin order by number of effects as follows: BEA > ENN B1 > ENN A1, with 47, 20 and 16%, respectively out of all situations compared. Copyright © 2015 Elsevier Ltd. All rights reserved.

Impaired removal of Vβ8(+) lymphocytes aggravates colitis in mice deficient for B cell lymphoma-2-interacting mediator of cell death (Bim).

PubMed

Leucht, K; Caj, M; Fried, M; Rogler, G; Hausmann, M

2013-09-01

We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vβ8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation. © 2013 British Society for Immunology.

Testosterone Attenuates Group 2 Innate Lymphoid Cell-Mediated Airway Inflammation.

PubMed

Cephus, Jacqueline-Yvonne; Stier, Matthew T; Fuseini, Hubaida; Yung, Jeffrey A; Toki, Shinji; Bloodworth, Melissa H; Zhou, Weisong; Goleniewska, Kasia; Zhang, Jian; Garon, Sarah L; Hamilton, Robert G; Poloshukin, Vasiliy V; Boyd, Kelli L; Peebles, R Stokes; Newcomb, Dawn C

2017-11-28

Sex hormones regulate many autoimmune and inflammatory diseases, including asthma. As adults, asthma prevalence is 2-fold greater in women compared to men. The number of group 2 innate lymphoid cells (ILC2) is increased in patients with asthma, and we investigate how testosterone attenuates ILC2 function. In patients with moderate to severe asthma, we determine that women have an increased number of circulating ILC2 compared to men. ILC2 from adult female mice have increased IL-2-mediated ILC2 proliferation versus ILC2 from adult male mice, as well as pre-pubescent females and males. Further, 5α-dihydrotestosterone, a hormone downstream of testosterone, decreases lung ILC2 numbers and IL-5 and IL-13 expression from ILC2. In vivo, testosterone attenuated Alternaria-extract-induced IL-5+ and IL-13+ ILC2 numbers and lung eosinophils by intrinsically decreasing lung ILC2 numbers, as well as by decreasing expression of IL-33 and thymic stromal lymphopoietin (TSLP), ILC2-stimulating cytokines. Collectively, these findings provide a foundational understanding of sexual dimorphism in ILC2 function. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Effects of Maternal Behavior Induction and Pup Exposure on Neurogenesis in Adult, Virgin Female Rats

PubMed Central

Furuta, Miyako; Bridges, Robert S.

2009-01-01

The states of pregnancy and lactation bring about a range of physiological and behavioral changes in the adult mammal that prepare the mother to care for her young. Cell proliferation increases in the subventricular zone (SVZ) of the female rodent brain during both pregnancy and lactation when compared to that in cycling, diestrous females. In the present study, the effects of maternal behavior induction and pup exposure on neurogenesis in nulliparous rats were examined in order to determine whether maternal behavior itself, independent of pregnancy and lactation, might affect neurogenesis. Adult, nulliparous, Sprague-Dawley, female rats were exposed daily to foster young in order to induce maternal behavior. Following the induction of maternal behavior each maternal subject plus females that were exposed to pups for a comparable number of test days, but did not display maternal behavior, and subjects that had received no pup exposure were injected with bromodeoxyuridine (BrdU, 90 mg/kg, i.v.). Brain sections were double-labeled for BrdU and the neural marker, NeuN, to examine the proliferating cell population. Increases in the number of double-labeled cells were found in the maternal virgin brain when compared with the number of double-labeled cells present in non-maternal, pup-exposed nulliparous rats and in females not exposed to young. No changes were evident in the dentate gyrus of the hippocampus as a function of maternal behavior. These data indicate that in nulliparous female rats maternal behavior itself is associated with the stimulation of neurogenesis in the SVZ. PMID:19712726

Kisspeptin expression features in the arcuate and anteroventral periventricular nuclei of hypothalamus of letrozole-induced polycystic ovarian syndrome in rats.

PubMed

Aliabadi, Elham; Namavar, Mohammad Reza; Mortezaee, Keywan; Toolee, Heidar; Keshtgar, Sara; Mirkhani, Hossein; Akbari, Mohammad; Rastegar, Tayebeh; Solhjoo, Somayeh

2017-11-01

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of the reproductive system characterized by polycystic ovaries and androgen excess. Letrozole is a nonsteroidal aromatase inhibitor that is used in experimental research to induce PCOS. Kisspeptin is an essential protein in regulation of cyclicity. Kisspeptin receptor is expressed in the hypothalamus and pituitary glands, and kisspeptin containing neurons are affected from sex steroid hormones. We aimed to investigate the number of kisspeptin-positive cells in the arcuate (Arc) and anteroventral periventricular nuclei (AVPV) of hypothalamus in the letrozole-induced PCOS. 40 female Wistar rats were divided into the proestrus control, diestrus control, proestrus vehicle, diestrus vehicle and letrozole. Animals were sacrificed after 3 weeks, and sera, ovary and brain samples were harvested for further evaluations. Letrozole group had high weight gain, high numbers of ovarian follicular cysts, high levels of luteinizing hormone and testosterone and increase number of kisspeptin-positive cells in the Arc nucleus, as compared with the control groups (P ≤ 0.05 vs. proestrus control and proestrus vehicle). Letrozole group showed a decrease in the number of kisspeptin-positive cells in the AVPV nucleus (P ≤ 0.05 vs. proestrus control and proestrus vehicle). Our findings show that the number of kisspeptin-positive cells may be affected from letrozole, and that the changes in the number of these cells may be in favor of the appearance of PCOS features in this group.

Evaluation of transport conditions for autologous bone marrow-derived mesenchymal stromal cells for therapeutic application in horses

PubMed Central

Espina, Miguel; Jülke, Henriette; Brehm, Walter; Ribitsch, Iris; Winter, Karsten

2016-01-01

Background. Mesenchymal stromal cells (MSCs) are increasingly used for clinical applications in equine patients. For MSC isolation and expansion, a laboratory step is mandatory, after which the cells are sent back to the attending veterinarian. Preserving the biological properties of MSCs during this transport is paramount. The goal of the study was to compare transport-related parameters (transport container, media, temperature, time, cell concentration) that potentially influence characteristics of culture expanded equine MSCs. Methods. The study was arranged in three parts comparing (I) five different transport containers (cryotube, two types of plastic syringes, glass syringe, CellSeal), (II) seven different transport media, four temperatures (4 °C vs. room temperature; −20 °C vs. −80 °C), four time frames (24 h vs. 48 h; 48 h vs. 72 h), and (III) three MSC concentrations (5 × 106, 10 × 106, 20 × 106 MSC/ml). Cell viability (Trypan Blue exclusion; percent and total number viable cell), proliferation and trilineage differentiation capacity were assessed for each test condition. Further, the recovered volume of the suspension was determined in part I. Each condition was evaluated using samples of six horses (n = 6) and differentiation protocols were performed in duplicates. Results. In part I of the study, no significant differences in any of the parameters were found when comparing transport containers at room temperature. The glass syringe was selected for all subsequent evaluations (highest recoverable volume of cell suspension and cell viability). In part II, media, temperatures, or time frames had also no significant influence on cell viability, likely due to the large number of comparisons and small sample size. Highest cell viability was observed using autologous bone marrow supernatant as transport medium, and “transport” at 4 °C for 24 h (70.6% vs. control group 75.3%); this was not significant. Contrary, viability was unacceptably low (<40%) for all freezing protocols at −20 °C or −80 °C, particularly with bone marrow supernatant or plasma and DMSO. In part III, various cell concentrations also had no significant influence on any of the evaluated parameters. Chondrogenic differentiation showed a trend towards being decreased for all transport conditions, compared to control cells. Discussion. In this study, transport conditions were not found to impact viability, proliferation or ability for trilineage differentiation of MSCs, most likely due to the small sample size and large number of comparisons. The unusual low viability after all freezing protocols is in contrast to previous equine studies. Potential causes are differences in the freezing, but also in thawing method. Also, the selected container (glass syringe) may have impacted viability. Future research may be warranted into the possibly negative effect of transport on chondrogenic differentiation. PMID:27019778

Manufacturing models permitting roll out/scale out of clinically led autologous cell therapies: regulatory and scientific challenges for comparability.

PubMed

Hourd, Paul; Ginty, Patrick; Chandra, Amit; Williams, David J

2014-08-01

Manufacturing of more-than-minimally manipulated autologous cell therapies presents a number of unique challenges driven by complex supply logistics and the need to scale out production to multiple manufacturing sites or near the patient within hospital settings. The existing regulatory structure in Europe and the United States imposes a requirement to establish and maintain comparability between sites. Under a single market authorization, this is likely to become an unsurmountable burden beyond two or three sites. Unless alternative manufacturing approaches can be found to bridge the regulatory challenge of comparability, realizing a sustainable and investable business model for affordable autologous cell therapy supply is likely to be extremely demanding. Without a proactive approach by the regulators to close this "translational gap," these products may not progress down the development pipeline, threatening patient accessibility to an increasing number of clinician-led autologous cellular therapies that are already demonstrating patient benefits. We propose three prospective manufacturing models for the scale out/roll out of more-than-minimally manipulated clinically led autologous cell therapy products and test their prospects for addressing the challenge of product comparability with a selected expert reference panel of US and UK thought leaders. This paper presents the perspectives and insights of the panel and identifies where operational, technological and scientific improvements should be prioritized. The main purpose of this report is to solicit feedback and seek input from key stakeholders active in the field of autologous cell therapy in establishing a consensus-based manufacturing approach that may permit the roll out of clinically led autologous cell therapies. Copyright © 2014 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Soliciting Strategies for Developing Cell-Based Reference Materials to Advance Mesenchymal Stromal Cell Research and Clinical Translation

PubMed Central

Viswanathan, Sowmya; Keating, Armand; Deans, Robert; Hematti, Peiman; Prockop, Darwin; Stroncek, David F.; Stacey, Glyn; Weiss, Dan J.; Mason, Christopher

2014-01-01

The mesenchymal stromal cell (MSC) field continues to rapidly progress with a number of clinical trials initiated and completed, with some reported successes in multiple clinical indications, and a growing number of companies established. The field, nevertheless, faces several challenges. Persistent issues include the definition of a MSC and comparability between MSC preparations. This is because of inherent cell heterogeneity, the absence of markers that are unique to MSCs, and the difficulty in precisely defining them by developmental origin. Differences in the properties of MSCs also depend on the site of tissue harvest, phenotypic and genotypic characteristics of the donor and the isolation, and storage and expansion methods used. These differences may be sufficient to ensure that attributes of the final MSC product could differ in potentially significant ways. Since there are currently no gold standards, we propose using a reference material to establish methods of comparability among MSC preparations. We suggest four possible “ruler scenarios” and a method for global distribution. We further suggest that critical to establishing a reference material is the need to define protocols for comparing cells. The main purpose of this article is to solicit input in establishing a consensus-based comparison. A comparative approach will be critical to all stages of translation to better clarify mechanisms of MSC actions, define an optimal cell manufacturing process, ensure best practice clinical investigations, extend the use of an MSC product for new indications, protect an MSC product from imitators, and develop uniform reimbursement policies. Importantly, a reference material may enable a consensus on a practical definition of MSCs. PMID:24422625

Effects of selective type I and II adrenal steroid agonists on immune cell distribution.

PubMed

Miller, A H; Spencer, R L; hassett, J; Kim, C; Rhee, R; Ciurea, D; Dhabhar, F; McEwen, B; Stein, M

1994-11-01

Adrenal steroids exert their effects through two distinct adrenal steroid receptor subtypes; the high affinity type I, or mineralocorticoid, receptor and the lower affinity type II, or glucocorticoid, receptor. Adrenal steroids have well known effects on immune cell distribution, and although both type I and II receptors are expressed in immune cells and tissues, few data exist on the relative effects mediated through these two receptor subtypes. Accordingly, we administered selective type I and II adrenal steroid receptor agonists to young adult male Sprague-Dawley rats for 7 days and then measured immune cell distribution in the peripheral blood and spleen. Results were compared with those of similar studies using the naturally occurring glucocorticoid of the rat, corticosterone, which binds both type I and II receptors. The majority of the well characterized effects of adrenal steroids on peripheral blood immune cells (increased neutrophils and decreased lymphocytes and monocytes) were reproduced by the type II receptor agonist, RU28362. RU28362 decreased the numbers of all lymphocyte subsets [T-cells, B-cells, and natural killer (NK) cells] to very low absolute levels. The largest relative decrease (i.e. in percentage) was seen in B-cells, whereas NK cells exhibited the least relative decrease and actually showed a 2-fold increase in relative percentage during RU28362 treatment. Similar to RU28362, the type I receptor agonist, aldosterone, significantly reduced the number of lymphocytes and monocytes. In contrast to RU28362, however, aldosterone significantly decreased the number of neutrophils. Moreover, aldosterone decreased the number of T-helper cells and NK cells, while having no effect on the number of B-cells or T-suppressor/cytotoxic cells. Corticosterone at physiologically relevant concentrations had potent effects on immune cell distribution, which were indistinguishable from those of the type II receptor agonist, RU28362. Taken together, these results indicate that effects of adrenal steroids on immune cell distribution are dependent on the receptor subtype involved as well as the specific cell type targeted. These factors allow for varied and complex effects of adrenal steroids on the immune system under physiological conditions.

Marrow stromal cells from patients affected by MPS I differentially support haematopoietic progenitor cell development.

PubMed

Baxter, M A; Wynn, R F; Schyma, L; Holmes, D K; Wraith, J E; Fairbairn, L J; Bellantuono, I

2005-01-01

Bone marrow transplantation is the therapy of choice in patients affected by MPS I (Hurler syndrome), but a high incidence of rejection limits the success of this treatment. The deficiency of alpha-L-iduronidase (EC 1.2.3.76), one of the enzymes responsible for the degradation of glycosaminoglycans, results in accumulation of heparan and dermatan sulphate in these patients. Heparan sulphate and dermatan sulphate are known to be important components of the bone marrow microenvironment and critical for haematopoietic cell development. In this study we compared the ability of marrow stromal cells from MPS I patients and healthy donors to support normal haematopoiesis in Dexter-type long term culture. We found an inverse stroma/supernatant ratio in the number of clonogenic progenitors, particularly the colony-forming unit granulocyte-machrophage in MPS I cultures when compared to normal controls. No alteration in the adhesion of haematopoietic cells to the stroma of MPS I patients was found, suggesting that the altered distribution in the number of clonogenic progenitors is probably the result of an accelerated process of differentiation and maturation. The use of alpha-L-iduronidase gene-corrected marrow stromal cells re-established normal haematopoiesis in culture, suggesting that correction of the bone marrow microenvironment with competent enzyme prior to transplantation might help establishment of donor haematopoiesis.

Failure of nitric oxide production by macrophages and decrease in CD4+ T cells in oral paracoccidioidomycosis: possible mechanisms that permit local fungal multiplication.

PubMed

Batista, Aline Carvalho; Soares, Cleverson Teixeira; Lara, Vanessa Soares

2005-01-01

Paracoccidioidomycosis is a chronic granulomatous disease that induces a specific inflammatory and immune response. The participation of nitric oxide (NO), a product of the inducible nitric oxide synthase enzyme (iNOS), as an important fungicidal molecule against Paracoccidioides brasiliensis has been demonstrated. In order to further characterize the Oral Paracoccidioidomycosis (OP), we undertook an immunohistochemical study of iNOS+, CD45RO+, CD3+, CD8+, CD20+, CD68+ cells and mast cells. The samples were distributed in groups according to the number of viable fungi per mm2. Our results showed weak immunolabeling for iNOS in the multinucleated giant cells (MNGC) and in most of the mononuclear (MN) cells, and the proportion of iNOS+ MN/MNGC cells in the OP were comparable to Control (clinically healthy oral tissues). Additionally, our analysis revealed a similarity in the number of CD4+ cells between the Control and the OP groups with higher numbers of fungi. These findings suggest that a low expression of iNOS and a decrease in the CD4+ T cells in OP may represent possible mechanisms that permit the local fungal multiplication and maintenance of active oral lesions.

Lower numbers of circulating natural killer T (NK T) cells in individuals with human T lymphotropic virus type 1 (HTLV-1) associated neurological disease

PubMed Central

Ndhlovu, L C; Snyder-Cappione, J E; Carvalho, K I; Leal, F E; Loo, C P; bruno, F R; Jha, A R; Devita, D; Hasenkrug, A M; Barbosa, H M R; Segurado, A C; Nixon, D F; Murphy, E L; Kallas, E G

2009-01-01

Human T lymphotropic virus type 1 (HTLV-1) infects 10–20 million people worldwide. The majority of infected individuals are asymptomatic; however, approximately 3% develop the debilitating neurological disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). There is also currently no cure, vaccine or effective therapy for HTLV-1 infection, and the mechanisms for progression to HAM/TSP remain unclear. NK T cells are an immunoregulatory T cell subset whose frequencies and effector functions are associated critically with immunity against infectious diseases. We hypothesized that NK T cells are associated with HAM/TSP progression. We measured NK T cell frequencies and absolute numbers in individuals with HAM/TSP infection from two cohorts on two continents: São Paulo, Brazil and San Francisco, CA, USA, and found significantly lower levels when compared with healthy subjects and/or asymptomatic carriers. Also, the circulating NK T cell compartment in HAM/TSP subjects is comprised of significantly more CD4+ and fewer CD8+ cells than healthy controls. These findings suggest that lower numbers of circulating NK T cells and enrichment of the CD4+ NK T subset are associated with HTLV-1 disease progression. PMID:19778295

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

PubMed Central

Higa, Futoshi; Kusano, Nobuchika; Tateyama, Masao; Shinzato, Takashi; Arakaki, Noriko; Kawakami, Kazuyoshi; Saito, Atsushi

1998-01-01

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. PMID:9574712

Induction of morphological changes in death-induced cancer cells monitored by holographic microscopy.

PubMed

El-Schich, Zahra; Mölder, Anna; Tassidis, Helena; Härkönen, Pirkko; Falck Miniotis, Maria; Gjörloff Wingren, Anette

2015-03-01

We are using the label-free technique of holographic microscopy to analyze cellular parameters including cell number, confluence, cellular volume and area directly in the cell culture environment. We show that death-induced cells can be distinguished from untreated counterparts by the use of holographic microscopy, and we demonstrate its capability for cell death assessment. Morphological analysis of two representative cell lines (L929 and DU145) was performed in the culture flasks without any prior cell detachment. The two cell lines were treated with the anti-tumour agent etoposide for 1-3days. Measurements by holographic microscopy showed significant differences in average cell number, confluence, volume and area when comparing etoposide-treated with untreated cells. The cell volume of the treated cell lines was initially increased at early time-points. By time, cells decreased in volume, especially when treated with high doses of etoposide. In conclusion, we have shown that holographic microscopy allows label-free and completely non-invasive morphological measurements of cell growth, viability and death. Future applications could include real-time monitoring of these holographic microscopy parameters in cells in response to clinically relevant compounds. Copyright © 2015 Elsevier Inc. All rights reserved.

Plasma levels of endothelial and B-cell-derived microparticles are restored by fingolimod treatment in multiple sclerosis patients.

PubMed

Zinger, Anna; Latham, Sharissa L; Combes, Valery; Byrne, Scott; Barnett, Michael H; Hawke, Simon; Grau, Georges E

2016-12-01

No molecular marker can monitor disease progression and treatment efficacy in multiple sclerosis (MS). Circulating microparticles represent a potential snapshot of disease activity at the blood brain barrier. To profile plasma microparticles by flow cytometry in MS and determine how fingolimod could impact endothelial microparticles production. In non-treated MS patients compared to healthy and fingolimod-treated patients, endothelial microparticles were higher, while B-cell-microparticle numbers were lower. Fingolimod dramatically reduced tumour necrosis factor (TNF)-induced endothelial microparticle release in vitro. Fingolimod restored dysregulated endothelial and B-cell-microparticle numbers, which could serve as a biomarker in MS. © The Author(s), 2016.

Changes in T-lymphocyte distribution associated with ingestion of aldicarb-contaminated drinking water: a follow-up study.

PubMed

Mirkin, I R; Anderson, H A; Hanrahan, L; Hong, R; Golubjatnikov, R; Belluck, D

1990-02-01

The carbamate pesticide, aldicarb, is the most commonly found man-made groundwater contaminant in Wisconsin. A 1985 study linked ingestion of aldicarb-contaminated drinking water with altered T-cell distributions, specifically an increase in the mean number of CD8+ (T8) T cells. To further evaluate this finding, a follow-up study was done in 1987. Of the 50 Portage County, Wisconsin, women who participated in the first study, 45 participated in the follow-up: 18 formerly exposed and 27 formerly unexposed. In our follow-up study, only 5 women were found to be currently exposed to aldicarb. This group of 5 women, compared to 39 unexposed women who had peripheral blood specimens taken, had an increased percentage of lymphocytes and an increased number of CD2+ T cells, due to an increased number of total CD8+ T cells. Although the number of exposed persons was small, the increases in percentage lymphocytes and absolute numbers of CD2+ and CD8+ T cells were consistent with a dose-response relationship. No identified drinking water contaminant other than aldicarb could explain these findings. These results support earlier evidence linking aldicarb exposure and lymphocyte distribution changes. Although adverse clinical effects have not been documented, the widespread use of this chemical and consequent potential for widespread exposure indicate a clear need for further research on this issue.

Graviceptor development in jellyfish ephyrae in space and on Earth

NASA Technical Reports Server (NTRS)

Spangenberg, D. B.; Jernigan, T.; Philput, C.; Lowe, B.

1994-01-01

Graviceptor (rhopalium) development in Aurelia aurita ephyrae which developed on Earth and in space during the nine-day NASA SLS-1 mission was compared. The space-developed ephyrae made graviceptors which were morphologically similar to those of their ground-based controls. Rhopalia of both groups developed statocysts with statoliths, ocelli, ciliated mechanoreceptor cells, and immature touch-plates with one type of hair cell. The number of rhopalia formed per arm of ephyrae of both groups revealed no significant differences. The number of statoliths formed per rhopalium was statistically higher in ephyrae which were induced to form in space with iodine than in L(Launch)+8h controls. Statolith numbers were not significantly different between Earth-formed control ephyrae and those formed from polyps induced on Earth and then sent into space 24h and 48h later. Statolith loss from rhopalia was significantly enhanced in the space-maintained ephyrae in artificial sea water (ASW) as compared to their controls. Ephyrae formed through thyroxine treatment and those maintained in thyroxine in space had statolith numbers comparable to thyroxine-treated controls. Pulsing abnormalitities seen in some space-developed ephyrae suggest that some space-formed ephyrae may have developed abnormal rhopalia because normal rhopalia development and function is necessary for normal pulsing.

In vivo effects of endotoxin on DNA synthesis in rat nasal epithelium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harkema, J.R.; Hotchkiss, J.A.

Airway inflammation in bacterial infections is characterized by the presence of neutrophils and often epithelial injury and repair. Release of endotoxin from bacteria may contribute to these processes. The purpose of this study was to determine the in vivo effects of repeated endotoxin exposure on DNA synthesis in rat nasal epithelium in the presence and absence of neutrophilic influx. Rats were intranasally instilled, once a day for 3 days, with endotoxin or saline (controls). Before the first and third instillations, half of the saline and endotoxin-instilled animals were depleted of circulating blood neutrophils by administering a rabbit anti-rat neutrophil antiserum.more » Rats were sacrificed 6 or 24 h after the last instillation. Two hours prior to sacrifice, rats were intraperitoneally injected with bromodeoxyuridine (BrdU), an analog of thymidine that is incorporated in the nucleus of cells in the S-phase of the cell cycle. Nasal tissues were processed for light microscopy and immunohistochemical detection of BrdU in nasal epithelial cells. The numbers of nasal epithelial cells, BrdU-labeled epithelial nuclei, and neutrophils per millimeter of basal lamina in the epithelium lining the nasal turbinates in the proximal nasal passages were determined by morphometric analysis. The authors did not observe a neutrophilic influx in the nasal tissues of neutrophil-depleted rats at 6 or 24 h after the last endotoxin instillation; however, the numbers of nasal epithelial cells and the BrdU-labeling index were significantly increased compared to saline-instilled controls. In contrast, non-neutrophil-depleted rats instilled with endotoxin had a marked neutrophilic influx, but no significant differences in the number of nasal epithelial cells at 6 or 24 h, compared to controls. In addition, the BrdU-labeling index in neutrophil-sufficient rats was increased only 6 h after the last instillation, compared to controls.« less

Dosimetry in radiobiological studies with the heavy ion beam of the Warsaw cyclotron

NASA Astrophysics Data System (ADS)

Kaźmierczak, U.; Banaś, D.; Braziewicz, J.; Czub, J.; Jaskóła, M.; Korman, A.; Kruszewski, M.; Lankoff, A.; Lisowska, H.; Malinowska, A.; Stępkowski, T.; Szefliński, Z.; Wojewódzka, M.

2015-12-01

The aim of this study was to verify various dosimetry methods in the irradiation of biological materials with a 12C ion beam at the Heavy Ion Laboratory of the University of Warsaw. To this end the number of ions hitting the cell nucleus, calculated on the basis of the Si-detector system used in the set-up, was compared with the number of ion tracks counted in irradiated Solid State Nuclear Track Detectors and with the number of ion tracks detected in irradiated Chinese Hamster Ovary cells processed for the γ-H2AX assay. Tests results were self-consistent and confirmed that the system serves its dosimetric purpose.

Single-cell analysis of transcription kinetics across the cell cycle

PubMed Central

Skinner, Samuel O; Xu, Heng; Nagarkar-Jaiswal, Sonal; Freire, Pablo R; Zwaka, Thomas P; Golding, Ido

2016-01-01

Transcription is a highly stochastic process. To infer transcription kinetics for a gene-of-interest, researchers commonly compare the distribution of mRNA copy-number to the prediction of a theoretical model. However, the reliability of this procedure is limited because the measured mRNA numbers represent integration over the mRNA lifetime, contribution from multiple gene copies, and mixing of cells from different cell-cycle phases. We address these limitations by simultaneously quantifying nascent and mature mRNA in individual cells, and incorporating cell-cycle effects in the analysis of mRNA statistics. We demonstrate our approach on Oct4 and Nanog in mouse embryonic stem cells. Both genes follow similar two-state kinetics. However, Nanog exhibits slower ON/OFF switching, resulting in increased cell-to-cell variability in mRNA levels. Early in the cell cycle, the two copies of each gene exhibit independent activity. After gene replication, the probability of each gene copy to be active diminishes, resulting in dosage compensation. DOI: http://dx.doi.org/10.7554/eLife.12175.001 PMID:26824388

A new hand-held microfluidic cytometer for evaluating irradiation damage by analysis of the damaged cells distribution.

PubMed

Wang, Junsheng; Fan, Zhiqiang; Zhao, Yile; Song, Younan; Chu, Hui; Song, Wendong; Song, Yongxin; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2016-03-17

Space radiation brings uneven damages to cells. The detection of the distribution of cell damage plays a very important role in radiation medicine and the related research. In this paper, a new hand-held microfluidic flow cytometer was developed to evaluate the degree of radiation damage of cells. The device we propose overcomes the shortcomings (e.g., large volume and high cost) of commercial flow cytometers and can evaluate the radiation damage of cells accurately and quickly with potential for onsite applications. The distribution of radiation-damaged cells is analyzed by a simultaneous detection of immunofluorescence intensity of γ-H2AX and resistance pulse sensor (RPS) signal. The γ-H2AX fluorescence intensity provides information of the degree of radiation damage in cells. The ratio of the number of cells with γ-H2AX fluorescence signals to the total numbers of cells detected by RPS indicates the percentage of the cells that are damaged by radiation. The comparison experiment between the developed hand-held microfluidic flow cytometer and a commercial confocal microscope indicates a consistent and comparable detection performance.

A new hand-held microfluidic cytometer for evaluating irradiation damage by analysis of the damaged cells distribution

NASA Astrophysics Data System (ADS)

Wang, Junsheng; Fan, Zhiqiang; Zhao, Yile; Song, Younan; Chu, Hui; Song, Wendong; Song, Yongxin; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

2016-03-01

Space radiation brings uneven damages to cells. The detection of the distribution of cell damage plays a very important role in radiation medicine and the related research. In this paper, a new hand-held microfluidic flow cytometer was developed to evaluate the degree of radiation damage of cells. The device we propose overcomes the shortcomings (e.g., large volume and high cost) of commercial flow cytometers and can evaluate the radiation damage of cells accurately and quickly with potential for onsite applications. The distribution of radiation-damaged cells is analyzed by a simultaneous detection of immunofluorescence intensity of γ-H2AX and resistance pulse sensor (RPS) signal. The γ-H2AX fluorescence intensity provides information of the degree of radiation damage in cells. The ratio of the number of cells with γ-H2AX fluorescence signals to the total numbers of cells detected by RPS indicates the percentage of the cells that are damaged by radiation. The comparison experiment between the developed hand-held microfluidic flow cytometer and a commercial confocal microscope indicates a consistent and comparable detection performance.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms.

PubMed

Chen, DaYang; Zhen, HeFu; Qiu, Yong; Liu, Ping; Zeng, Peng; Xia, Jun; Shi, QianYu; Xie, Lin; Zhu, Zhu; Gao, Ya; Huang, GuoDong; Wang, Jian; Yang, HuanMing; Chen, Fang

2018-03-21

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Trps1 deficiency inhibits the morphogenesis of secondary hair follicles via decreased Noggin expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sun, Yujing; Nakanishi, Masako; Sato, Fuyuki

Highlights: • The number of secondary hair follicles is reduced by half in Trps1 KO embryonic skin compared to wild-type skin. • Noggin expression is significantly decreased and BMP signaling is promoted in Trps1 KO embryonic skin. • Treatment with a Noggin or BMP inhibitor rescued the decreased number of hair follicles in Trps1 KO skin graft cultures. • Cell proliferation and apoptosis of the epidermis were normalized by Noggin treatment. - Abstract: A representative phenotype of patients with tricho-rhino-phalangeal syndrome (TRPS) is sparse hair. To understand the developmental defects of these patient’s hair follicles, we analyzed the development ofmore » hair follicles histologically and biochemically using Trps1 deficient (KO) mice. First, we compared the numbers of primary hair follicles in wild-type (WT) and KO embryos at different developmental stages. No differences were observed in the E14.5 skins of WT and KO mice. However, at later time points, KO fetal skin failed to properly develop secondary hair follicles, and the number of secondary hair follicles present in E18.5 KO skin was approximately half compared to that of WT skin. Sonic hedgehog expression was significantly decreased in E17.5 KO skin, whereas no changes were observed in Eda/Edar expression in E14.5 or E17.5 skins. In addition, Noggin expression was significantly decreased in E14.5 and E17.5 KO skin compared to WT skin. In parallel with the suppression of Noggin expression, BMP signaling was promoted in the epidermal cells of KO skins compared to WT skins as determined by immunohistochemistry for phosphorylated Smad1/5/8. The reduced number of secondary hair follicles was restored in skin graft cultures treated with a Noggin and BMP inhibitor. Furthermore, decreased cell proliferation, and increased apoptosis in KO skin was rescued by Noggin treatment. Taken together, we conclude that hair follicle development in Trps1 KO embryos is impaired directly or indirectly by decreased Noggin expression.« less

Comparative study of different procedures for the separation of peripheral blood mononuclear cells in cytokine-induced killer cell immunotherapy for hepatocarcinoma.

PubMed

Liu, Hui; Li, Jianyu; Wang, Fengmei; Gao, Yingtang; Luo, Ying; Wang, Peng; Li, Chenglong; Zhu, Zhengyan

2015-04-01

Cytokine-induced killer (CIK) cell immunotherapy exhibits significant advantages in the clinical treatment of tumors. This study was designed to compare the biological characteristics of autologous CIK cells from patients with hepatocarcinoma following different procedures for the separation of peripheral blood mononuclear cells (PBMCs). Forty-four hepatocarcinoma patients were enrolled and distributed into two groups. PBMCs were isolated either using a blood cell separator (apheresis method) or Ficoll lymphocyte separation medium (Ficoll method). The total amount, collection efficacy, and cell status of PBMCs in the two groups were determined. According to the number and status of collected PBMCs, different cultivation procedures were used for their amplification and activation and the proliferation ability, phenotype, and killing activity of CIK cells in the two groups were evaluated. Our results indicated that the number of collected PBMCs in the apheresis group was far more than that in the Ficoll group. However, the isolation rate was lower, and more cellular debris was observed in the apheresis group, which may be the cause of some untoward effects. Following in vitro culture, the enrichment time of CIK cells was longer in the Ficoll group, and the percentages of CD3(+)CD4(+) (Th) and CD4(+)CD25(+) (Treg) cells were higher. In the apheresis group, the percentages of CD3(-)CD56(+) (NK) and CD3(+)CD56(+) (NKT) cells were higher, and the CIK cells exhibited a higher cytolytic activity against HepG2 hepatoma cells. In conclusion, different procedures for PBMCs separation can influence the biological activities of CIK cells, and the apheresis method is more effective at enhancing the antitumor efficacy of CIK cells. However, significant attention should be paid to the possibility of adverse reactions in apheresis donors.

The effects of exercise and stress on the survival and maturation of adult-generated granule cells

PubMed Central

Snyder, Jason S.; Glover, Lucas R.; Sanzone, Kaitlin M.; Kamhi, J. Frances; Cameron, Heather A.

2009-01-01

Stress strongly inhibits proliferation of granule cell precursors in the dentate gyrus, while voluntary running has the opposite effect. Few studies, however, have examined the possible effects of these environmental manipulations on the maturation and survival of young granule cells. We examined number of surviving granule cells and the proportion of young neurons that were functionally mature, as defined by seizure-induced immediate-early gene expression, in 14 and 21 day-old granule cells in mice that were given access to a running wheel, restrained daily for 2 hours, or given no treatment during this period. Importantly, treatments began two days after BrdU injection, to isolate effects on survival from those on cell proliferation. We found a large increase in granule cell survival in running mice compared with controls at both time points. In addition, running increased the proportion of granule cells expressing the immediate-early gene Arc in response to seizures, suggesting that it speeds incorporation into circuits, i.e., functional maturation. Stressed mice showed no change in Arc expression, compared to control animals, but, surprisingly, showed a transient increase in survival of 14-day-old granule cells, which was gone 7 days later. Examination of cell proliferation, using the endogenous mitotic marker proliferating cell nuclear antigen (PCNA) showed an increase in cell proliferation after 12 days of running but not after 19 days of running. The number of proliferating cells was unchanged 24 hours after the 12th or 19th episode of daily restraint stress. These findings demonstrate that running has strong effects on survival and maturation of young granule cells as well as their birth and that stress can have positive but short-lived effects on granule cell survival. PMID:19156854

Generation of recombinant canine interleukin-15 and evaluation of its effects on the proliferation and function of canine NK cells.

PubMed

Lee, Soo-Hyeon; Shin, Dong-Jun; Kim, Sang-Ki

2015-05-15

Interleukin-15 (IL-15) is a pleiotropic cytokine that plays a pivotal role in both innate and adaptive immunity. IL-15 is also a promising cytokine for treating cancer. Despite the growing importance of the clinical use of IL-15 for immunotherapy, no attempts have been made to generate a recombinant canine IL-15 (rcIL-15) and to examine its effects on the antitumor activities of immune effector cells in dogs. Here, we generated an rcIL-15 protein consisting of Asn-49-Ser-162 with a C-terminal His tag and examined its functions ex vivo in terms of the proliferation and antitumor effects on canine non-B, non-T, large granular natural killer (NK) cells. Non-B, non-T, large granular NK cells rapidly expanded in response to stimulation with rcIL-15 in the presence of IL-2, and a majority of the cells that selectively expanded over 21 days exhibited a CD3(-)CD5(-)CD4(-)CD8(+/-)CD21(-) phenotype. Purified rcIL-15 significantly enhanced the expansion rate of canine NK cells derived from peripheral blood mononuclear cells compared to human IL-15, or culture in the absence of IL-15 for 21 days (p<0.05). Purified rcIL-15 was superior at enhancing the effector function of NK cells compared to human IL-15. The cytotoxic activity against canine thyroid adenocarcinoma (CTAC) cells, interferon-γ production, and the mRNA expression levels of perforin and granzyme B of expanded NK cells cultured with rcIL-15 were significantly elevated compared to those cultured with human IL-15 or without IL-15 (p<0.05). Intravenous administration of rcIL-15 significantly increased the numbers of lymphocytes in the peripheral blood of dogs on days 6, 8, and 11 after injection compared to numbers before administration (p<0.05). The results of this study suggest that the rcIL-15 protein, consisting of Asn-49-Ser-162, enhanced the proliferation and antitumor effects of canine NK cells and promoted the generation of lymphocytes in dogs. Copyright © 2015 Elsevier B.V. All rights reserved.

Increased expression of RANTES, CCR3 and CCR5 in the lesional skin of patients with atopic eczema.

PubMed

Kato, Yoko; Pawankar, Ruby; Kimura, Yoichi; Kawana, Seiji

2006-01-01

Atopic eczema (AE) is a relapsing inflammatory disease based on IgE sensitization and characterized by peripheral blood eosinophilia and eosinophil infiltration into the lesional skin. In the patch test reaction of AE by allergens, an increased infiltration of activated eosinophils has been demonstrated peaking at 24-48 h. Regulated on activation normal T cell expressed and secreted (RANTES/CCL5) is a chemokine that induces eosinophil migration, and CCR3 and CCR5 are the receptors of RANTES. In order to further clarify the pathomechanisms of eosinophil infiltration in ongoing chronic inflammation in the skin of patients with AE and its relation to disease severity, we examined the expression of RANTES and its receptors CCR3 and CCR5 in challenged and unchallenged lesional skin of AE. We examined the number of RANTES+ cells, CCR3+ cells, CCR5+cells, activated (EG2+) eosinophils and CD3+ T cells in normal skin of healthy volunteers, and in challenged lesional skin (24 h after mite patch test) as well as unchallenged lesional skin of AE patients by immunohistochemistry. The cellular source of RANTES, CCR3 and CCR5 was analyzed by double immunohistochemistry using specific antibodies to RANTES, CCR3 or CCR5, and antibodies to ECP (EG2) or CD3. The numbers of RANTES+ cells, CCR3+ cells, CCR5+ cells, EG2+ cells and CD3+ cells were all significantly increased in challenged (mite patch-tested) lesional skin of AE patients as compared to those in unchallenged lesional skin and normal skin. The numbers of these cells in unchallenged lesional skin were greater than those in normal skin. The number of EG2+ cells in the unchallenged lesional skin correlated with both the peripheral blood eosinophil count and the SCORAD index. The number of EG2+ cells in challenged lesional skin correlated with the number of CCR5+ cells. Activated eosinophils and T cells expressed RANTES and various proportions of these cells were CCR3+ and CCR5+ in both challenged and unchallenged lesional skin. Taken together, these results suggest that RANTES as well as its receptors CCR3 and CCR5 may play important roles in the orchestration of eosinophil infiltration in ongoing chronic inflammation in AE, and also reflect the severity of disease. Copyright (c) 2006 S. Karger AG, Basel.

Systemic immune cell response in rats after pulmonary exposure to manganese-containing particles collected from welding aerosols.

PubMed

Antonini, James M; Zeidler-Erdely, Patti C; Young, Shih-Houng; Roberts, Jenny R; Erdely, Aaron

2012-01-01

Welding fume inhalation affects the immune system of exposed workers. Manganese (Mn) in welding fume may induce immunosuppressive effects. The goal was to determine if Mn in welding fume alters immunity by reducing the number of circulating total leukocytes and specific leukocyte sub-populations. Sprague-Dawley rats were treated by intratracheal instillation (ITI) with either a single dose (2.00 mg/rat) or repeated doses (0.125 or 2.00 mg/rat for 7 weeks) with welding fumes that contained different levels of Mn. Additional rats were treated by ITI once a week for 7 weeks with the two doses of manganese chloride (MnCl₂). Bronchoalveolar lavage was performed to assess lung inflammation. Also, whole blood was recovered, and the number of circulating total leukocytes, as well as specific lymphocyte subsets, was determined by flow cytometry. The welding fume highest in Mn content significantly increased lung inflammation, injury, and production of inflammatory cytokines and chemokines compared to all other treatment groups. In addition, the same group expressed significant decreases in the number of circulating CD4⁺ and CD8⁺ T-lymphocytes after a single exposure, and significant reductions in the number of circulating total lymphocytes, primarily CD4⁺ and CD8⁺ T-lymphocytes, after repeated exposures (compared to control values). Repeated MnCl₂ exposure led to a trend of a reduction (but not statistically significant) in circulating total lymphocytes, attributable to the changes in the CD4⁺ T-lymphocyte population levels. The welding fume with the lower concentration of Mn had no significant effect on the numbers of blood lymphocytes and lymphocyte subsets compared to control values. Evidence from this study indicates that pulmonary exposure to certain welding fumes cause decrements in systemic immune cell populations, specifically circulating T-lymphocytes, and these alterations in immune cell number are not dependent exclusively on Mn, but likely a combination of other metals present in welding fume.

Human mast cell and airway smooth muscle cell interactions: implications for asthma.

PubMed

Page, S; Ammit, A J; Black, J L; Armour, C L

2001-12-01

Asthma is characterized by inflammation, hyperresponsiveness, and remodeling of the airway. Human mast cells (HMCs) play a central role in all of these changes by releasing mediators that cause exaggerated bronchoconstriction, induce human airway smooth muscle (HASM) cell proliferation, and recruit and activate inflammatory cells. Moreover, the number of HMCs present on asthmatic HASM is increased compared with that on nonasthmatic HASM. HASM cells also have the potential to actively participate in the inflammatory process by synthesizing cytokines and chemokines and expressing surface molecules, which have the capacity to perpetuate the inflammatory mechanisms present in asthma. This review specifically examines how the mediators of HMCs have the capacity to modulate many functions of HASM; how the synthetic function of HASM, particularly through the release and expression of stem cell factor, has the potential to influence HMC number and activation in an extraordinarily potent and proinflammatory manner; and how these interactions between HMCs and HASM have potential consequences for airway structure and inflammation relevant to the disease process of asthma.

Increased Th9 cells and IL-9 levels accelerate disease progression in experimental atherosclerosis.

PubMed

Li, Qing; Ming, Tingting; Wang, Yuanmin; Ding, Shaowei; Hu, Chaojie; Zhang, Cuiping; Cao, Qi; Wang, Yiping

2017-01-01

Atherosclerosis (AS) is the number one killer in developed countries, and currently considered a chronic inflammatory disease. The central role of T cells in the pathogenesis of atherosclerosis is well documented. However, little is known about the newly described T cell subset-Th9 cells and their role in AS pathogenesis. Here, the amounts of Th9 cells as well as their key transcription factors and relevant cytokines during atherosclerosis were assessed in ApoE -/- mice and age-matched C57BL/6J mice. Significantly increased Th9 cell number, Th9 related cytokine (IL-9), and key transcription factor (PU.1) were found in ApoE -/- mice compared with age-matched C57BL/6J mice. Additionally, treatment with rIL-9 accelerated atherosclerotic development, which was attenuated by anti-IL-9 antibodies. These data suggested that both Th9 cells and related IL-9 play key roles in the pathogenesis of atherosclerosis, and antibodies against these antigens offer a novel therapeutic approach in AS treatment.

Methylsulfonylmethane suppresses hepatic tumor development through activation of apoptosis

PubMed Central

Kim, Joo-Hyun; Shin, Hye-Jun; Ha, Hye-Lin; Park, Young-Ho; Kwon, Tae-Ho; Jung, Mi-Ra; Moon, Hyung-Bae; Cho, Eun-Sang; Son, Hwa-Young; Yu, Dae-Yeul

2014-01-01

AIM: To investigate the effect of methylsulfonylmethane (MSM), recently reported to have anti-cancer effects, in liver cancer cells and transgenic mice. METHODS: Three liver cancer cell lines, HepG2, Huh7-Mock and Huh7-H-rasG12V, were used. Cell growth was measured by Cell Counting Kit-8 and soft agar assay. Western blot analysis was used to detect caspases, poly (ADP-ribose) polymerase (PARP), and B-cell lymphoma 2 (Bcl-2) expressions. For in vivo study, we administered MSM to H-ras12V transgenic mice for 3 mo. RESULTS: MSM decreased the growth of HepG2, Huh7-Mock and Huh7-H-rasG12V cells in a dose-dependent manner. That was correlated with significantly increased apoptosis and reduced cell numbers in MSM treated cells. Cleaved caspase-8, cleaved caspase-3 and cleaved PARP were remarkably increased in the liver cancer cells treated with 500 mmol/L of MSM; however, Bcl-2 was slightly decreased in 500 mmol/L. Liver tumor development was greatly inhibited in the H-ras12V transgenic mice treated with MSM, compared to control, by showing reduced tumor size and number. Cleaved PARP was significantly increased in non-tumor treated with MSM compared to control. CONCLUSION: Liver injury was also significantly attenuated in the mice treated with MSM. Taken together, all the results suggest that MSM has anti-cancer effects through inducing apoptosis in liver cancer. PMID:24575169

Differential Regulation of Mouse B Cell Development by Transforming Growth Factor β1

PubMed Central

Kaminski, Denise A.; Letterio, John J.; Burrows, Peter D.

2002-01-01

Transforming growth factor β (TGFβ) can inhibit the in vitro proliferation, survival and differentiation of B cell progenitors, mature B lymphocytes and plasma cells. Here we demonstrate unexpected, age-dependent reductions in the bone marrow (BM) B cell progenitors and immature B cells in TGFβ1-/- mice. To evaluate TGFβ responsiveness during normal B lineage development, cells were cultured in interleukin 7 (IL7)±TGFβ. Picomolar doses of TGFβ1 reduced pro-B cell recoveries at every timepoint. By contrast, the pre-B cells were initially reduced in number, but subsequently increased compared to IL7 alone, resulting in a 4-fold increase in the growth rate for the pre-B cell population. Analysis of purified BM sub-populations indicated that pro-B cells and the earliest BP1- pre-B cells were sensitive to the inhibitory effects of TGFβ1. However, the large BP1+ pre-B cells, although initially reduced, were increased in number at days 5 and 7 of culture. These results indicate that TGFβ1 is important for normal B cell development in vivo, and that B cell progenitors are differentially affected by the cytokine according to their stage of differentiation. PMID:12739785

Software-aided automatic laser optoporation and transfection of cells

PubMed Central

Georg Breunig, Hans; Uchugonova, Aisada; Batista, Ana; König, Karsten

2015-01-01

Optoporation, the permeabilization of a cell membrane by laser pulses, has emerged as a powerful non-invasive and highly efficient technique to induce transfection of cells. However, the usual tedious manual targeting of individual cells significantly limits the addressable cell number. To overcome this limitation, we present an experimental setup with custom-made software control, for computer-automated cell optoporation. The software evaluates the image contrast of cell contours, automatically designates cell locations for laser illumination, centres those locations in the laser focus, and executes the illumination. By software-controlled meandering of the sample stage, in principle all cells in a typical cell culture dish can be targeted without further user interaction. The automation allows for a significant increase in the number of treatable cells compared to a manual approach. For a laser illumination duration of 100 ms, 7-8 positions on different cells can be targeted every second inside the area of the microscope field of view. The experimental capabilities of the setup are illustrated in experiments with Chinese hamster ovary cells. Furthermore, the influence of laser power is discussed, with mention on post-treatment cell survival and optoporation-efficiency rates. PMID:26053047

Expression of BAFF receptors in muscle tissue of myositis patients with anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies.

PubMed

Kryštůfková, Olga; Barbasso Helmers, Sevim; Venalis, Paulius; Malmström, Vivianne; Lindroos, Eva; Vencovský, Jiří; Lundberg, Ingrid E

2014-10-10

Anti-Jo-1 and anti-Ro52 autoantibodies are common in patients with myositis, but the mechanisms behind their production are not known. Survival of autoantibody-producing cells is dependent on B-cell-activating factor of the tumour necrosis factor family (BAFF). BAFF levels are elevated in serum of anti-Jo-1-positive myositis patients and are influenced by type-I interferon (IFN). IFN-producing cells and BAFF mRNA expression are present in myositis muscle. We investigated expression of the receptors for BAFF in muscle tissue in relation to anti-Jo-1 and anti-Ro52/anti-Ro60 autoantibodies and type-I IFN markers. Muscle biopsies from 23 patients with myositis selected based on autoantibody profile and 7 healthy controls were investigated for expression of BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). Nineteen samples were assessed for plasma (CD138) and B-cell (CD19) markers. The numbers of positive cells per area were compared with the expression of plasmacytoid dendritic cell (pDC) marker blood dendritic cell antigen-2 (BDCA-2) and IFNα/β-inducible myxovirus resistance-1 protein (MX-1). BAFF-R, BCMA and TACI were expressed in five, seven and seven patients, respectively, and more frequently in anti-Jo-1-positive and/or anti-Ro52/anti-Ro60-positive patients compared to controls and to patients without these autoantibodies (P = BAFF-R: 0.007, BCMA: 0.03 and TACI: 0.07). A local association of receptors with B and plasma cells was confirmed by confocal microscopy. The numbers of CD138-positive and BCMA-positive cells were correlated (r = 0.79; P = 0.001). Expression of BDCA-2 correlated with numbers of CD138-positive cells and marginally with BCMA-positive cells (r = 0.54 and 0.42, respectively; P = 0.04 and 0.06, respectively). There was a borderline correlation between the numbers of positively stained TACI cells and MX-1 areas (r = 0.38, P = 0.08). The expression pattern of receptors for BAFF on B and plasma cells in muscle suggests a local role for BAFF in autoantibody production in muscle tissues of patients with myositis who have anti-Jo-1 or anti-Ro52/anti-Ro60 autoantibodies. BAFF production could be influenced by type-I IFN produced by pDCs. Thus, B-cell-related molecular pathways may participate in the pathogenesis of myositis in this subset of patients.

Heat Transport Enhancement of Turbulent Thermal Convection by Inserted Channels

NASA Astrophysics Data System (ADS)

Xia, Ke-Qing; Zhang, Lu

2017-11-01

We report an experimental study on the heat transport properties of turbulent Rayleigh Benard Convection (RBC) in a rectangular cell with two types of 3D-printed structures inserted inside. The first one splits the original rectangular cell into 60 identical sub cells whose aspect ratio is 1:1:10 (length, width, height). The second one splits the cell into 30 sub cells, each with a 1:2:10 aspect ratio and a baffle in the center. We find that for large Rayleigh numbers (Ra), the Nusselt numbers (Nu) of both structures increase compared with that of the empty rectangular cell. An enhancement in Nu as much as 20% is found for the second type of insertion at Rayleigh number 2 ×109 . Moreover, the Nu-Ra scaling shows a transition with both geometries. The particle image velocimetry (PIV) measurement within a single sub unit indicates that the transition may be related to the laminar to turbulent transition in flow field. Direct numerical simulations (DNS) confirm the experimental results. Our results demonstrate the potential in using insertions to enhance passive heat transfer. This work was supported by the Research Grants Council (RGC) of HKSAR (Nos. CUHK404513 and CUHK14301115).

Ganglion Cell and Displaced Amacrine Cell Density Distribution in the Retina of the Howler Monkey (Alouatta caraya)

PubMed Central

Muniz, José Augusto Pereira Carneiro; de Athaide, Luana Modesto; Gomes, Bruno Duarte; Finlay, Barbara L.; Silveira, Luiz Carlos de Lima

2014-01-01

Unlike all other New World (platyrrine) monkeys, both male and female howler monkeys (Alouatta sp.) are obligatory trichromats. In all other platyrrines, only females can be trichromats, while males are always dichromats, as determined by multiple behavioral, electrophysiological, and genetic studies. In addition to obligatory trichromacy, Alouatta has an unusual fovea, with substantially higher peak cone density in the foveal pit than every other diurnal anthropoid monkey (both platyrrhines and catarrhines) and great ape yet examined, including humans. In addition to documenting the general organization of the retinal ganglion cell layer in Alouatta, the distribution of cones is compared to retinal ganglion cells, to explore possible relationships between their atypical trichromacy and foveal specialization. The number and distribution of retinal ganglion cells and displaced amacrine cells were determined in six flat-mounted retinas from five Alouatta caraya. Ganglion cell density peaked at 0.5 mm between the fovea and optic nerve head, reaching 40,700–45,200 cells/mm2. Displaced amacrine cell density distribution peaked between 0.5–1.75 mm from the fovea, reaching mean values between 2,050–3,100 cells/mm2. The mean number of ganglion cells was 1,133,000±79,000 cells and the mean number of displaced amacrine cells was 537,000±61,800 cells, in retinas of mean area 641±62 mm2. Ganglion cell and displaced amacrine cell density distribution in the Alouatta retina was consistent with that observed among several species of diurnal Anthropoidea, both platyrrhines and catarrhines. The principal alteration in the Alouatta retina appears not to be in the number of any retinal cell class, but rather a marked gradient in cone density within the fovea, which could potentially support high chromatic acuity in a restricted central region. PMID:25546077

Tracking the Invasion of Small Numbers of Cells in Paper-Based Assays with Quantitative PCR.

PubMed

Truong, Andrew S; Lochbaum, Christian A; Boyce, Matthew W; Lockett, Matthew R

2015-11-17

Paper-based scaffolds are an attractive material for culturing mammalian cells in a three-dimensional environment. There are a number of previously published studies, which utilize these scaffolds to generate models of aortic valves, cardiac ischemia and reperfusion, and solid tumors. These models have largely relied on fluorescence imaging and microscopy to quantify cells in the scaffolds. We present here a polymerase chain reaction (PCR)-based method, capable of quantifying multiple cell types in a single culture with the aid of DNA barcodes: unique sequences of DNA introduced to the genome of individual cells or cell types through lentiviral transduction. PCR-based methods are highly specific and are amenable to high-throughput and multiplexed analyses. To validate this method, we engineered two different breast cancer lines to constitutively express either a green or red fluorescent protein. These cells lines allowed us to directly compare the ability of fluorescence imaging (of the fluorescent proteins) and qPCR (of the unique DNA sequences of the fluorescent proteins) to quantify known numbers of cells in the paper based-scaffolds. We also used both methods to quantify the distribution of these breast cell lines in homotypic and heterotypic invasion assays. In the paper-based invasion assays, a single sheet of paper containing cells suspended in a hydrogel was sandwiched between sheets of paper containing only hydrogel. The stack was incubated, and the cells invaded the adjacent layers. The individual sheets of the invasion assay were then destacked and the number of cells in each layer quantified. Our results show both methods can accurately detect cell populations of greater than 500 cells. The qPCR method can repeatedly and accurately detect as few as 50 cells, allowing small populations of highly invasive cells to be detected and differentiated from other cell types.

Chronic Binge Alcohol Administration Increases Intestinal T-Cell Proliferation and Turnover in Rhesus Macaques.

PubMed

Veazey, Ronald S; Amedee, Angela; Wang, Xiaolei; Bernice Kaack, M; Porretta, Constance; Dufour, Jason; Welsh, David; Happel, Kyle; Pahar, Bapi; Molina, Patricia E; Nelson, Steve; Bagby, Gregory J

2015-08-01

Alcohol use results in changes in intestinal epithelial cell turnover and microbial translocation, yet less is known about the consequences on intestinal lymphocytes in the gut. Here, we compared T-cell subsets in the intestine of macaques before and after 3 months of chronic alcohol administration to examine the effects of alcohol on intestinal T-cell subsets. Rhesus macaques received either alcohol or isocaloric sucrose as a control treatment daily over a 3-month period via indwelling gastric catheters. Intestinal lymphocyte subsets were identified in biopsy samples by flow cytometry. Twenty-four hours prior to sampling, animals were inoculated with bromo-deoxyuridine (BrdU) to assess lymphocyte proliferation. Immunohistochemistry was performed on tissue samples to quantitate CD3+ cells. Animals receiving alcohol had increased rates of intestinal T-cell turnover of both CD4+ and CD8+ T cells as reflected by increased BrdU incorporation. However, absolute numbers of T cells were decreased in intestinal tissues as evidenced by immunohistochemistry for total CD3 expression per mm(2) intestinal lamina propria in tissue sections. Combining immunohistochemistry and flow cytometry data showed that the absolute numbers of CD8+ T cells were significantly decreased, whereas absolute numbers of total CD4+ T cells were minimally decreased. Collectively, these data indicate that alcohol exposure to the small intestine results in marked loss of CD3+ T cells, accompanied by marked increases in CD4+ and CD8+ T-cell proliferation and turnover, which we speculate is an attempt to maintain stable numbers of T cells in tissues. This suggests that alcohol results in accelerated T-cell turnover in the gut, which may contribute to premature T-cell senescence. Further, these data indicate that chronic alcohol administration results in increased levels of HIV target cells (proliferating CD4+ T cells) that may support higher levels of HIV replication in intestinal tissues. Copyright © 2015 by the Research Society on Alcoholism.

Tier-2 studies on monocrotaline immunotoxicity in C57BL/6 mice.

PubMed

Deyo, J A; Kerkvliet, N I

1991-01-01

Monocrotaline (MCT) is a member of a class of naturally occurring phytotoxins known as pyrrolizidine alkaloids, and is a toxicological concern to both man and his livestock. The purpose of these studies was to evaluate the effect of a 14-day oral MCT (0-100 mg/kg per day) exposure on the functional integrity of various immunocyte effector systems in C57BL/6 mice, as well as to investigate potential mechanisms for its immunotoxicity. Decreases in lymphoid organ weights and cellularity, and resident peritoneal exudate cell (PEC) number were only observed after exposure to the highest dose of 100 mg/kg MCT. This dose also inhibited NK cell cytotoxicity, while the total number of NK lytic units per spleen was decreased (-53%) after exposure to 50 mg/kg MCT. Following i.p. injection of SRBC, the percentage of PEC macrophages containing engulfed SRBC was significantly increased in MCT-exposed mice, while the percentage of large vacuolated (activated) macrophages was decreased in a dose-dependent manner. Exposure to MCT significantly decreased the total number of Ig+ cells without altering the number of CD4+ and CD8+ cells. The antibody responses (PFC/10(6) spleen cells) to two T cell-independent antigens, TNP-LPS and DNP-Ficoll, were significantly decreased at all MCT doses, and the degree of suppression of both responses was identical at coincident doses. MCT exposure (25 mg/kg) significantly suppressed the blastogenic response to the T cell mitogen concanavalin A (-38%), and to the B cell mitogen lipopolysaccharide (-58%). These results indicate that exposure to MCT can alter the functional integrity of various immune effector responses in a dose-dependent manner, and suggest that the B cell may be a relatively more sensitive target of MCT immunotoxicity compared to T cells, macrophages and NK cells.

Cryopreservation of cord blood CD34+ cells before or after thrombopoietin expansion differentially affects early platelet recovery in NOD SCID mice.

PubMed

van Hensbergen, Yvette; van der Garde, Mark; Brand, Anneke; Slot, Manon C; de Graaf-Dijkstra, Alice; Watt, Suzanne; Zwaginga, Jaap Jan

2015-07-01

Expansion of human cord blood (CB) CD34+ cells with thrombopoietin (TPO) can accelerate delayed platelet (PLT) recovery after transplantation into immunodeficient mice. Clinical implementation, however, will depend on practical and effective protocols. The best timing of TPO expansion in relation to cryopreservation in this respect is unknown. In this study, we evaluated whether the order of cryopreservation and TPO expansion affected the expansion rate and numbers of clonogenic hematopoietic progenitor cells in vitro or PLT and longer-term hematopoietic repopulation in NOD SCID mice in vivo. Our results demonstrate higher expansion rates and the generation of higher numbers of multilineage and megakaryocytic progenitors (granulocyte, erythrocyte, monocyte, megakaryocyte colony-forming units and megakaryocyte colony-forming units) in vitro when freshly isolated CB CD34+ cells are first cultured with TPO and then cryopreserved and thawed as compared to TPO expansion after CD34+ cell cryopreservation. In contrast, the cells produced with the latter strategy showed higher expression of CD62L and a superior stromal cell-derived factor-1α-mediated migration. This might play a role in an also observed superior early PLT recovery after transplantation of these cells into NOD SCID mice. The hematopoietic engraftment in the marrow 6 weeks after transplantation was not different between the two strategies. Although TPO expansion before cryopreservation would yield higher nucleated cell and clonogenic myeloid and megakaryocyte cell numbers and enable earlier availability, CB TPO expansion after cryopreservation is likely to be clinically more effective, despite the lower number of cells obtained after expansion. Moreover, the latter strategy is logistically more feasible. © 2015 AABB.

Vismodegib, an antagonist of hedgehog signaling, directly alters taste molecular signaling in taste buds.

PubMed

Yang, Hyekyung; Cong, Wei-Na; Yoon, Jeong Seon; Egan, Josephine M

2015-02-01

Vismodegib, a highly selective inhibitor of hedgehog (Hh) pathway, is an approved treatment for basal-cell carcinoma. Patients on treatment with vismodegib often report profound alterations in taste sensation. The cellular mechanisms underlying the alterations have not been studied. Sonic Hh (Shh) signaling is required for cell growth and differentiation. In taste buds, Shh is exclusively expressed in type IV taste cells, which are undifferentiated basal cells and the precursors of the three types of taste sensing cells. Thus, we investigated if vismodegib has an inhibitory effect on taste cell turnover because of its known effects on Hh signaling. We gavaged C57BL/6J male mice daily with either vehicle or 30 mg/kg vismodegib for 15 weeks. The gustatory behavior and immunohistochemical profile of taste cells were examined. Vismodegib-treated mice showed decreased growth rate and behavioral responsivity to sweet and bitter stimuli, compared to vehicle-treated mice. We found that vismodegib-treated mice had significant reductions in taste bud size and numbers of taste cells per taste bud. Additionally, vismodegib treatment resulted in decreased numbers of Ki67- and Shh-expressing cells in taste buds. The numbers of phospholipase Cβ2- and α-gustducin-expressing cells, which contain biochemical machinery for sweet and bitter sensing, were reduced in vismodegib-treated mice. Furthermore, vismodegib treatment resulted in reduction in numbers of T1R3, glucagon-like peptide-1, and glucagon-expressing cells, which are known to modulate sweet taste sensitivity. These results suggest that inhibition of Shh signaling by vismodegib treatment directly results in alteration of taste due to local effects in taste buds. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.

Vismodegib, an antagonist of hedgehog signaling, directly alters taste molecular signaling in taste buds

PubMed Central

Yang, Hyekyung; Cong, Wei-na; Yoon, Jeong Seon; Egan, Josephine M

2015-01-01

Vismodegib, a highly selective inhibitor of hedgehog (Hh) pathway, is an approved treatment for basal-cell carcinoma. Patients on treatment with vismodegib often report profound alterations in taste sensation. The cellular mechanisms underlying the alterations have not been studied. Sonic Hh (Shh) signaling is required for cell growth and differentiation. In taste buds, Shh is exclusively expressed in type IV taste cells, which are undifferentiated basal cells and the precursors of the three types of taste sensing cells. Thus, we investigated if vismodegib has an inhibitory effect on taste cell turnover because of its known effects on Hh signaling. We gavaged C57BL/6J male mice daily with either vehicle or 30 mg/kg vismodegib for 15 weeks. The gustatory behavior and immunohistochemical profile of taste cells were examined. Vismodegib-treated mice showed decreased growth rate and behavioral responsivity to sweet and bitter stimuli, compared to vehicle-treated mice. We found that vismodegib-treated mice had significant reductions in taste bud size and numbers of taste cells per taste bud. Additionally, vismodegib treatment resulted in decreased numbers of Ki67- and Shh-expressing cells in taste buds. The numbers of phospholipase Cβ2- and α-gustducin-expressing cells, which contain biochemical machinery for sweet and bitter sensing, were reduced in vismodegib-treated mice. Furthermore, vismodegib treatment resulted in reduction in numbers of T1R3, glucagon-like peptide-1, and glucagon-expressing cells, which are known to modulate sweet taste sensitivity. These results suggest that inhibition of Shh signaling by vismodegib treatment directly results in alteration of taste due to local effects in taste buds. PMID:25354792

Skeletal unloading causes resistance of osteoprogenitor cells to parathyroid hormone and to insulin-like growth factor-I

NASA Technical Reports Server (NTRS)

Kostenuik, P. J.; Harris, J.; Halloran, B. P.; Turner, R. T.; Morey-Holton, E. R.; Bikle, D. D.

1999-01-01

Skeletal unloading decreases bone formation and osteoblast number in vivo and decreases the number and proliferation of bone marrow osteoprogenitor (BMOp) cells in vitro. We tested the ability of parathyroid hormone (PTH) to stimulate BMOp cells in vivo by treating Sprague Dawley rats (n = 32) with intermittent PTH(1-34) (1 h/day at 8 microg/100 g of body weight), or with vehicle via osmotic minipumps during 7 days of normal weight bearing or hind limb unloading. Marrow cells were flushed from the femur and cultured at the same initial density for up to 21 days. PTH treatment of normally loaded rats caused a 2.5-fold increase in the number of BMOp cells, with similar increases in alkaline phosphatase (ALP) activity and mineralization, compared with cultures from vehicle-treated rats. PTH treatment of hind limb unloaded rats failed to stimulate BMOp cell number, ALP activity, or mineralization. Hind limb unloading had no significant effect on PTH receptor mRNA or protein levels in the tibia. Direct in vitro PTH challenge of BMOp cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on BMOp cells was mediated indirectly by a PTH-induced factor. We hypothesize that this factor is insulin-like growth factor-I (IGF-I), which stimulated the in vitro proliferation and differentiation of BMOp cells isolated from normally loaded bone, but not from unloaded bone. These results suggest that IGF-I mediates the ability of PTH to stimulate BMOp cell proliferation in normally loaded bone, and that BMOp cells in unloaded bone are resistant to the anabolic effect of intermittent PTH therapy due to their resistance to IGF-I.

Prenatal and early postnatal NOAEL-dose clothianidin exposure leads to a reduction of germ cells in juvenile male mice

PubMed Central

YANAI, Shogo; HIRANO, Tetsushi; OMOTEHARA, Takuya; TAKADA, Tadashi; YONEDA, Naoki; KUBOTA, Naoto; YAMAMOTO, Anzu; MANTANI, Youhei; YOKOYAMA, Toshifumi; KITAGAWA, Hiroshi; HOSHI, Nobuhiko

2017-01-01

Neonicotinoids are pesticides used worldwide. They bind to insect nicotinic acetylcholine receptors (nAChRs) with high affinity. We previously reported that clothianidin (CTD), one of the latest neonicotinoids, reduced antioxidant expression and induced germ cell death in the adult testis of vertebrates. Here, we investigated the male reproductive toxicity of prenatal and early postnatal exposure to CTD, because it is likely that developmental exposure more severely affects the testis compared to adults due to the absence of the blood-testis barrier. Pregnant C57BL/6 mice were given water gel blended with CTD (0, 10 or 50 mg/kg/day; no-observed-adverse-effect-level [NOAEL for mice]: 47.2 mg/kg/day) between gestational day 1 and 14 days post-partum. We then examined the testes of male offspring at postnatal day 14. The testis weights and the numbers of germ cells per seminiferous tubule were decreased in the CTD-50 group, and abnormal tubules containing no germ cells appeared. Nevertheless, the apoptotic cell number and proliferative activity were not significantly different between the control and CTD-exposed groups. There were no significant differences in the androgen-related parameters, such as the Leydig cell volume per testis, the Sertoli cell number and the tubule diameter. The present study is the first demonstration that in utero and lactational exposures to CTD at around the NOAEL for mice reduce the germ cell number, but our findings suggest that these exposures do not affect steroidogenesis in Leydig cells during prenatal or early postnatal life. PMID:28579575

Increased numbers of peripheral blood CD34+ cells in dogs with canine atopic dermatitis.

PubMed

Bruet, Vincent; Lieubeau, Blandine; Herve, Julie; Roussel, Anne; Imparato, Laëtitia; Desfontis, Jean-Claude; Bourdeau, Patrick

2015-06-01

The bone marrow may be involved in human atopic diseases, as shown by the release of CD34+ cells into the peripheral blood. The aim was to determine the numbers of CD34+ cells in atopic dogs. The following three groups of dogs were studied: 27 dogs with nonfood-induced atopic dermatitis (NFICAD); 16 dogs with nonallergic inflammatory diseases; and 13 healthy control dogs. Dogs with NFICAD were selected after fulfilment of Favrot's criteria and exclusion of other pruritic dermatoses, including flea infestation and adverse reaction to foods. The Canine Atopic Dermatitis Extent and Severity Index (CADESI)-03 and a Visual Analog Scale (VAS) score for pruritus were used to quantify clinical signs. A phycoerythrin-conjugated anticanine CD34 antibody was used to stain peripheral blood CD34+ cells, and these were enumerated using a flow cytometer. The CD34+ cell counts were compared between groups and tested (in the NFICAD group) for correlation with the severity of clinical signs. The numbers of peripheral CD34+ cells in dogs with NFICAD (median 1.7) were statistically higher than in dogs with other nonallergic inflammatory diseases (median 1.0; P = 0.01) and healthy control dogs (median 0.9; P = 0.009). In dogs with NFICAD, there was no correlation between CD34+ cell numbers and CADESI-03 scores or owner-assessed pruritus (VAS score). The results of this study suggest the possible involvement of CD34+ cells in dogs with NFICAD. The role of CD34+ cells in the aetiopathogenesis of canine atopic dermatitis remains to be determined. © 2014 ESVD and ACVD.

Enhanced expression of PD-1 and other activation markers by CD4+ T cells of young but not old patients with metastatic melanoma.

PubMed

van den Brom, Rob R H; van der Geest, Kornelis S M; Brouwer, Elisabeth; Hospers, Geke A P; Boots, Annemieke M H

2018-06-01

The biological behavior of melanoma is unfavorable in the elderly when compared to young subjects. We hypothesized that differences in T-cell responses might underlie the distinct behavior of melanoma in young and old melanoma patients. Therefore, we investigated the circulating T-cell compartment of 34 patients with metastatic melanoma and 42 controls, which were classified as either young or old. Absolute numbers of CD4+ T cells were decreased in young and old melanoma patients when compared to the age-matched control groups. Percentages of naive and memory CD4+ T cells were not different when comparing old melanoma patients to age-matched controls. Percentages of memory CD4+ T cells tended to be increased in young melanoma patients compared to young controls. Proportions of naive CD4+ T cells were lower in young patients than in age-matched controls, and actually comparable to those in old patients and controls. This was accompanied with increased percentages of memory CD4+ T cells expressing HLA-DR, Ki-67, and PD-1 in young melanoma patients in comparison to the age-matched controls, but not in old patients. Proportions of CD45RA-FOXP3 high memory regulatory T cells were increased in young and old melanoma patients when compared to their age-matched controls, whereas those of CD45RA+FOXP3 low naive regulatory T cells were similar. We observed no clear modulation of the circulating CD8+ T-cell repertoire in melanoma patients. In conclusion, we show that CD4+ T cells of young melanoma patients show signs of activation, whereas these signs are less clear in CD4+ T cells of old patients.

The role of exogenous neural stem cells transplantation in cerebral ischemic stroke.

PubMed

Chen, Lukui; Qiu, Rong; Li, Lushen; He, Dan; Lv, Haiqin; Wu, Xiaojing; Gu, Ning

2014-11-01

To observe the effects of neural stem cells (NSCs) transplantation in rats' striatum and subventricular zone (SVZ) in rat models of focal cerebral ischemia and reperfusion. Hippocampus was extracted from fetal rats with 14 days of gestation. Suspension culture was used to isolate and culture the rat's NSCs. A cerebral ischemia and reperfusion rat's model was made on the left side of the brain through occlusion of the left middle cerebral artery. Neurological signs were assessed by Zea Longa's five-grade scale, with scores 1, 2, and 3 used to determine the successful establishment of the rat's model. The NSCs were stereotaxically injected into the left striatum 24 hours after the successful rat's model was built. Rats were then randomly divided into 5 groups, namely, normal group, sham operation group, ischemia group, PBS transplantation group, and NSCs transplantation group, each of which was observed on day 3, day 7, and day 14. The ischemia-related neurological deficits were assessed by using a 7-point evaluation criterion. Forelimb injuries were evaluated in all rats using the foot-fault approach. Infarct size changes were observed through TTC staining and cell morphology and structure in the infarct region were investigated by Nissl staining. Apoptosis and apoptosis-positive cell counts were studied by Tunel assay. Expressions of double-labeling positive cells in the striatum and subventricular zone (SVZ) were observed by BrdU/NeuN and BrdU/GFAP fluorescent double-labeling method and the number of positive cells in the striatum and SVZ was counted. Results from the differently treated groups showed that right hemiplegia occurred in the ischemia group, PBS transplantation group, and NSCs transplantation group in varying degrees. Compared with the former two groups, there was least hemiplegia in the NSCs transplantation group. The TTC staining assay showed that rats in the NSCs transplantation group had smaller infarct volume than those from the PBS transplantation group. The Nissl dyeing showed that there was a large area of neuronal necrosis and apoptosis in the ischemia and PBS transplantation groups, and damage was mainly focused in the striatum. Degeneration and damage of nerve cells were significantly reduced in the NSCs transplantation group. The Tunel assay showed that the number of apoptosis-positive cells in the NSCs transplantation group was less than that in the PBS transplantation group at each time point. Double immunofluorescent labeling showed that the proliferation of endogenous neural stem cells began at the third day, reaching the peak at the 7th day, and was significantly reduced at the 14th day in the SVZ. The number of BrdU/NeuN increased significantly in the NSCs transplantation group compared to that in the PBS transplantation group (P < 0.05). The number of BrdU/GFAP decreased significantly in the NSCs transplantation group compared to that of PBS transplantation group (P < 0.05). The number of BrdU/GFAP-positive cells in the striatum was observed to be much more in the PBS transplantation group than in the NSCs transplantation group. Both neurological deficits and coordination capacity of rats with cerebral ischemia were significantly improved via transplantation of the neural stem cells. In conclusion, transplantation of neural stem cells can therefore possibly promote the differentiation of endogenous NSCs into neurons and reduce their differentiation towards glial cells. Transplantation of the neural stem cells may also change the ischemic microenvironment of striatum, possibly inhibiting the proliferation of glial cells.

The effect of partially stabilized zirconia on the biological properties of HA/HDPE composites in vitro.

PubMed

Sadi, A Yari; Shokrgozar, M A; Homaeigohar, S Sh; Hosseinalipour, M; Khavandi, A; Javadpour, J

2006-05-01

The effect of partially stabilized zirconia (PSZ) on the biological properties of the hyroxyapatite - high density polyethylene (HA/HDPE) composites was studied by investigating the simultaneous effect of hydroxyapatite and PSZ volume fractions on the in vitro response of human osteoblast cells. The biocompatibility of composite samples with different volume fraction of HA and PSZ powders was assessed by proliferation, alkaline phosphatase (ALP) and cell attachment assays on the osteoblast cell line (G-292) in different time periods. The effect of composites on the behavior of G-292 cells was compared with those of HDPE and TPS (Tissue Culture Poly Styrene as negative control) samples. Results showed a higher proliferation rate of G-292 cells in the presence of composite samples as compared to the HDPE sample after 7 and 14 days of incubation period. ALP production rate in all composite samples was higher than HDPE and TPS samples. The number of adhered cells on the composite samples was higher than the number adhered on the HDPE and TPS samples after the above mentioned incubation periods. These findings indicates that the addition of PSZ does not have any adverse affect on the biocompatibility of HA/HDPE composites. In fact in some experiments PSZ added HA/HDPE composites performed better in proliferation, differentiation and attachment of osteoblastic cells.

mTOR is necessary for proper satellite cell activity and skeletal muscle regeneration.

PubMed

Zhang, Pengpeng; Liang, Xinrong; Shan, Tizhong; Jiang, Qinyang; Deng, Changyan; Zheng, Rong; Kuang, Shihuan

The serine/threonine kinase mammalian target of rapamycin (mTOR) is a key regulator of protein synthesis, cell proliferation and energy metabolism. As constitutive deletion of Mtor gene results in embryonic lethality, the function of mTOR in muscle stem cells (satellite cells) and skeletal muscle regeneration remains to be determined. In this study, we established a satellite cell specific Mtor conditional knockout (cKO) mouse model by crossing Pax7(CreER) and Mtor(flox/flox) mice. Skeletal muscle regeneration after injury was severely compromised in the absence of Mtor, indicated by increased number of necrotic myofibers infiltrated by Evans blue dye, and reduced number and size of regenerated myofibers in the Mtor cKO mice compared to wild type (WT) littermates. To dissect the cellular mechanism, we analyzed satellite cell-derived primary myoblasts grown on single myofibers or adhered to culture plates. The Mtor cKO myoblasts exhibited defective proliferation and differentiation kinetics when compared to myoblasts derived from WT littermates. At the mRNA and protein levels, the Mtor cKO myoblasts expressed lower levels of key myogenic determinant genes Pax7, Myf5, Myod, Myog than did the WT myoblasts. These results suggest that mTOR is essential for satellite cell function and skeletal muscle regeneration through controlling the expression of myogenic genes. Copyright © 2015 Elsevier Inc. All rights reserved.

Chronic heart failure and aging - effects of exercise training on endothelial function and mechanisms of endothelial regeneration: Results from the Leipzig Exercise Intervention in Chronic heart failure and Aging (LEICA) study.

PubMed

Sandri, Marcus; Viehmann, Manuel; Adams, Volker; Rabald, Kristin; Mangner, Norman; Höllriegel, Robert; Lurz, Philipp; Erbs, Sandra; Linke, Axel; Kirsch, Katharina; Möbius-Winkler, Sven; Thiery, Joachim; Teupser, Daniel; Hambrecht, Rainer; Schuler, Gerhard; Gielen, Stephan

2016-03-01

A reduction in number and function of endothelial progenitor cells (EPCs) occurs in both physiologic aging and chronic heart failure (CHF). We assessed whether disease and aging have additive effects on EPCs or whether beneficial effects of exercise training are diminished in old age. We randomized 60 patients with stable CHF and 60 referent controls to a training or a control group. To detect possible aging effects we included subjects below 55 (young) and above 65 years (older). Subjects in the training group exercised four times daily at 60% to 70% of VO2max for four weeks under supervision. At baseline and after the intervention the number and function of EPCs were assessed. As compared with young referent controls, older referent controls showed at baseline a reduced EPC number (young: 190 ± 37 CD34/KDR positive cells/ml blood; older: 131 ± 26 CD34/KDR positive cells/ml blood; p < 0.05) and function (young: 230 ± 41 migrated cells/1000 plated cells; older: 185 ± 28 cells/1000 plated cells; p < 0.05). In young and older CHF patients EPC-number (young: 85 ± 21 CD34/KDR positive cells/ml blood; older: 78 ± 20 CD34/KDR positive cells/ml blood) and EPC-function (young: 113 ± 26 cells/1000 plated cells; older: 120 ± 27 cells/1000 plated cells) were impaired. As a result of exercise training, EPC function improved by 24% in older referent controls (p < 0.05), while it remained unchanged in young training referent controls and controls respectively. In young and older patients with CHF four weeks of exercise training resulted in a significant improvement in EPC numbers and EPC function (young: number +66% function +43%; p < 0.05; older: number +69% function +36%; p < 0.05). These results were accompanied by a significant increase in flow mediated dilatation in the training groups of young/older CHF patients and in older referent controls. Four weeks of exercise training are effective in improving EPC number and EPC function in CHF patients. These training effects were not impaired among older patients, emphasizing the potentials of rehabilitation interventions in a patient group where CHF has a high prevalence. © The European Society of Cardiology 2015.

A convergence algorithm for correlation of breech face images based on the congruent matching cells (CMC) method.

PubMed

Chen, Zhe; Song, John; Chu, Wei; Soons, Johannes A; Zhao, Xuezeng

2017-11-01

The Congruent Matching Cells (CMC) method was invented at the National Institute of Standards and Technology (NIST) for accurate firearm evidence identification and error rate estimation. The CMC method is based on the principle of discretization. The toolmark image of the reference sample is divided into correlation cells. Each cell is registered to the cell-sized area of the compared image that has maximum surface topography similarity. For each resulting cell pair, one parameter quantifies the similarity of the cell surface topography and three parameters quantify the pattern congruency of the registration position and orientation. An identification (declared match) requires a significant number of CMCs, that is, cell pairs that meet both similarity and pattern congruency requirements. The use of cell correlations reduces the effects of "invalid regions" in the compared image pairs and increases the correlation accuracy. The identification accuracy of the CMC method can be further improved by considering a feature named "convergence," that is, the tendency of the x-y registration positions of the correlated cell pairs to converge at the correct registration angle when comparing same-source samples at different relative orientations. In this paper, the difference of the convergence feature between known matching (KM) and known non-matching (KNM) image pairs is characterized, based on which an improved algorithm is developed for breech face image correlations using the CMC method. Its advantage is demonstrated by comparison with three existing CMC algorithms using four datasets. The datasets address three different brands of consecutively manufactured pistol slides, with significant differences in the distribution overlap of cell pair topography similarity for KM and KNM image pairs. For the same CMC threshold values, the convergence algorithm demonstrates noticeably improved results by reducing the number of false-positive or false-negative CMCs in a comparison. Published by Elsevier B.V.

The lateral line receptor array of cyprinids from different habitats.

PubMed

Schmitz, Anke; Bleckmann, Horst; Mogdans, Joachim

2014-04-01

The lateral line system of teleost fishes consists of an array of superficial and canal neuromasts (CN). Number and distribution of neuromasts and the morphology of the lateral line canals vary across species. We investigated the morphology of the lateral line system in four diurnal European cyprinids, the limnophilic bitterling (Rhodeus sericeus), the indifferent gudgeon (Gobio gobio), and ide (Leuciscus idus), and the rheophilic minnow (Phoxinus phoxinus). All fish had lateral line canals on head and trunk. The total number of both, CN and superficial neuromasts (SN), was comparable in minnow and ide but was greater than in gudgeon and bitterling. The ratio of SNs to CNs for the head was comparable in minnow and bitterling but was greater in gudgeon and ide. The SN-to-CN ratio for the trunk was greatest in bitterling. Polarization of hair cells in CNs was in the direction of the canal. Polarization of hair cells in SNs depended on body area. In cephalic SNs, hair cell polarization was dorso-ventral or rostro-caudal. In trunk SNs, it was rostro-caudal on lateral line scales and dorso-ventral on other trunk scales. On the caudal fin, hair cell polarization was rostro-caudal. The data show that, in the four species studied here, number, distribution, and orientation of CNs and SNs cannot be unequivocally related to habitat. Copyright © 2013 Wiley Periodicals, Inc.

Escherichia coli growth under modeled reduced gravity

NASA Technical Reports Server (NTRS)

Baker, Paul W.; Meyer, Michelle L.; Leff, Laura G.

2004-01-01

Bacteria exhibit varying responses to modeled reduced gravity that can be simulated by clino-rotation. When Escherichia coli was subjected to different rotation speeds during clino-rotation, significant differences between modeled reduced gravity and normal gravity controls were observed only at higher speeds (30-50 rpm). There was no apparent affect of removing samples on the results obtained. When E. coli was grown in minimal medium (at 40 rpm), cell size was not affected by modeled reduced gravity and there were few differences in cell numbers. However, in higher nutrient conditions (i.e., dilute nutrient broth), total cell numbers were higher and cells were smaller under reduced gravity compared to normal gravity controls. Overall, the responses to modeled reduced gravity varied with nutrient conditions; larger surface to volume ratios may help compensate for the zone of nutrient depletion around the cells under modeled reduced gravity.

Adoptive transfer of acute lung injury.

PubMed

Moxley, M A; Baird, T L; Corbett, J A

2000-11-01

In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.

Induction of tissue transglutaminase by dexamethasone: its correlation to receptor number and transglutaminase-mediated cell death in a series of malignant hamster fibrosarcomas.

PubMed Central

Johnson, T S; Scholfield, C I; Parry, J; Griffin, M

1998-01-01

Treatment of the hamster fibrosarcoma cell lines (Met B, D and E) and BHK-21 hamster fibroblast cells with the glucocorticoid dexamethasone led to a powerful dose-dependent mRNA-synthesis-dependent increase in transglutaminase activity, which can be correlated with dexamethasone-responsive receptor numbers in each cell line. Increasing the number of dexamethasone-responsive receptors by transfection of cells with the HG1 glucocorticoid receptor protein caused an increase in transglutaminase activity that was proportional to the level of transfected receptor. In all experiments the levels of the tissue transglutaminase-mediated detergent-insoluble bodies was found to be comparable with increases in transglutaminase activity. Despite an increase in detergent-insoluble body formation, an increase in apoptosis as measured by DNA fragmentation was not found. Incubation of cells with the non-toxic competitive transglutaminase substrate fluorescein cadaverine led to the incorporation of this fluorescent amine into cellular proteins when cells were damaged after exposure to trypsin during cell passage. These cross-linked proteins containing fluorescein cadaverine were shown to be present in the detergent-insoluble bodies, indicating that the origin of these bodies is via activation of tissue transglutaminase after cell damage by trypsinization rather than apoptosis per se, since Met B cells expressing the bcl-2 cDNA were not protected from detergent-insoluble body formation. We describe a novel mechanism of cell death related to tissue transglutaminase expression and cell damage. PMID:9512467

Mast cells mediate neutrophil recruitment during atherosclerotic plaque progression.

PubMed

Wezel, Anouk; Lagraauw, H Maxime; van der Velden, Daniël; de Jager, Saskia C A; Quax, Paul H A; Kuiper, Johan; Bot, Ilze

2015-08-01

Activated mast cells have been identified in the intima and perivascular tissue of human atherosclerotic plaques. As mast cells have been described to release a number of chemokines that mediate leukocyte fluxes, we propose that activated mast cells may play a pivotal role in leukocyte recruitment during atherosclerotic plaque progression. Systemic IgE-mediated mast cell activation in apoE(-/-)μMT mice resulted in an increase in atherosclerotic lesion size as compared to control mice, and interestingly, the number of neutrophils was highly increased in these lesions. In addition, peritoneal mast cell activation led to a massive neutrophil influx into the peritoneal cavity in C57Bl6 mice, whereas neutrophil numbers in mast cell deficient Kit(W(-sh)/W(-sh)) mice were not affected. Within the newly recruited neutrophil population, increased levels of CXCR2(+) and CXCR4(+) neutrophils were observed after mast cell activation. Indeed, mast cells were seen to contain and release CXCL1 and CXCL12, the ligands for CXCR2 and CXCR4. Intriguingly, peritoneal mast cell activation in combination with anti-CXCR2 receptor antagonist resulted in decreased neutrophil recruitment, thus establishing a prominent role for the CXCL1/CXCR2 axis in mast cell-mediated neutrophil recruitment. Our data suggest that chemokines, and in particular CXCL1, released from activated mast cells induce neutrophil recruitment to the site of inflammation, thereby aggravating the ongoing inflammatory response and thus affecting plaque progression and destabilization. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Two cell-counting factors regulate the aggregate size of the cellular slime mold Dictyostelium discoideum.

PubMed

Okuwa, T; Katayama, T; Takano, A; Kodaira, K; Yasukawa, H

2001-12-01

Countin, a cell-counting factor in Dictyostelium discoideum, is considered to limit the maximum size of the multicellular structure, because a countin null strain forms a huge fruiting body compared to that of the wild-type. A novel gene, countin2, that is highly homologous to countin (40% identity in amino acid sequence) was identified in the D. discoideum genome. The countin2 null strain formed a 1.7-fold higher number of the aggregates, resulting in smaller fruiting bodies compared with those of wild-type cells. Thus, the Countin2 protein is thought to limit the minimum size of the multicellular structure. The size and number of aggregates formed by a mixture of countin null and countin2 null strains were the same as those of the wild-type. These findings demonstrate that a combination of Countin and Countin2 proteins determines the appropriate size of the multicellular structure of D. discoideum.

Characterization of stem and progenitor cells in the dental pulp of erupted and unerupted murine molars

PubMed Central

Balic, Anamaria; Aguila, H. Leonardo; Caimano, Melissa J.; Francone, Victor P.; Mina, Mina

2010-01-01

In the past few years there have been significant advances in the identification of putative stem cells also referred to as “mesenchymal stem cells” (MSC) in dental tissues including the dental pulp. It is thought that MSC in dental pulp share certain similarities with MSC isolated from other tissues. However, cells in dental pulp are still poorly characterized. This study focused on the characterization of progenitor and stem cells in dental pulps of erupted and unerupted mice molars. Our study showed that dental pulps from unerupted molars contain a significant number of cells expressing CD90+/CD45-, CD117+/CD45-, Sca-1+/CD45- and little if any CD45+ cells. Our in vitro functional studies showed that dental pulp cells from unerupted molars displayed extensive osteo-dentinogenic potential but were unable to differentiate into chondrocytes and adipocytes. Dental pulp from erupted molars displayed a reduced number of cells, contained higher percentage of CD45+ and lower percentage of cells expressing CD90+/CD45-, CD117+/CD45- as compared to unerupted molars. In vitro functional assays demonstrated the ability of a small fraction of cells to differentiate into odontoblasts, osteoblasts, adipocytes and chondrocytes. There was a significant reduction in the osteo-dentinogenic potential of the pulp cells derived from erupted molars compared to unerupted molars. Furthermore, the adipogenic and chondrogenic differentiation of pulp cells from erupted molars was dependent on a long induction period and infrequent. Based on these findings we propose that the dental pulp of the erupted molars contain a small population of multipotent cells, whereas the dental pulp of the unerupted molars does not contain multipotent cells but is enriched in osteo-dentinogenic progenitors engaged in the formation of coronal and radicular odontoblasts. PMID:20193787

Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients

PubMed Central

Li, Chunyan; Bai, Jingchao; Hao, Xiaomeng; Zhang, Sheng; Hu, Yunhui; Zhang, Xiaobei; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lee, Mong-Hong; Zhang, J

2014-01-01

Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients. PMID:24621502

Effect of postmortem time interval on in vitro culture potential of goat skin tissues stored at room temperature.

PubMed

Singh, Mahipal; Ma, Xiaoling; Sharma, Anil

2012-09-01

Animal cloning using somatic cell nuclear transfer technology has renewed the interest in postmortem tissue storage, since these tissues can be used to reintroduce the lost genes back into the breeding pool in animal agriculture, preserve the genetic diversity, and revive the endangered species. However, for successful cloning of animals, integrity of nuclear DNA is essential. Cell viability and their potential to in vitro culture ensure nuclear integrity. The aim of this study was to determine the limits of postmortem time interval within which live cells can be recovered from goat skin tissues. To test the postmortem tissue storage limits, we cultured 2-3 mm(2) skin pieces (n = 70) from the ears of three breeds of goats (n = 7) after 0, 2, 4, and 6 days of postmortem storage at 24°C. After 10 days of culture, outgrowth of fibroblast-like cells (>50 cells) around the explants was scored. All the explants irrespective of breed displayed outgrowth of cells on the dish containing fresh tissues (i.e., day 0 of storage). However, the number of explants exhibiting outgrowth reduced with increasing time interval. Only 53.85 % explants displayed outgrowth after 2 days of tissue storage. The number of explants displaying outgrowth was much smaller after 4 (16.67 %) and 6 days (13.3 %) of storage. In general, the number of outgrowing cells per explant, on a given day, also decreased with increasing postmortem storage time interval. To test the differences between cell cultures, we established secondary cultures from one of the goats exhibiting outgrowth of cells after 6 days of tissue storage and compared them to similar cells from fresh tissues. Comparison of both the cell lines revealed similar cell morphology and growth curves and had doubling times of 23.04 and 22.56 h, respectively. These results suggest that live cells can be recovered from goat (and perhaps other animal) tissues stored at room temperature even after 6 days of their death with comparable growth profiles and, thus, can be used for tissue banking for preservation of superior genetics, genetic diversity, and cloning of animals.

Amelioration of cerebellar dysfunction in rats following postnatal ethanol exposure using low-intensity pulsed ultrasound.

PubMed

Bolbanabad, Hiva Mohammadi; Anvari, Enayat; Rezai, Mohammad Jafar; Moayeri, Ardashir; Kaffashian, Mohammad Reza

2017-04-01

The neonatal development stage of the cerebellum in rats is equivalent to a human foetus in the third trimester of pregnancy. In this stage, cell proliferation, migration, differentiation, and synaptogenesis occur. Clinical and experimental findings have shown that ethanol exposure during brain development causes a variety of disruptions to the brain, including neurogenesis depression, delayed neuronal migration, changes in neurotransmitter synthesis, and neuronal depletion.During postnatal cerebellar development, neurons are more vulnerable to the destructive effects of ethanol. The effects of low-intensity pulsed ultrasound (LIPUS) on the number of cells and thickness of the cell layers within the cerebellar cortex were examined during the first two postnatal weeks in rats following postnatal ethanol exposure. Postpartum rats were distributed randomly into six groups. Normal saline was injected intraperitoneally into control animals and ethanol (20%) was injected into the intervention groups for three consecutive days. Intervention groups received LIPUS at different frequencies (3 or 5MHz), after administration of ethanol. After transcardial perfusion, the rat's brain was removed, and a complete series of sagittal cerebellum sections were obtained by systematic random manner. Photomicrographs were made with Motic digital cameras and analysed using Nikon digital software. The numbers of granular cells decreased in ethanol-treated rats compared to the control group. LIPUS, administered at (3 or 5MHz), combined with ethanol administration resulted in a reduction of ethanol's effects. Using 5MHz LIPUS resulted in significantly higher numbers of granular cells in the internal layer compared to the control rats. Using 3 or 5MHz LIPUS alone resulted in a significant enhancement in the granular cells of the molecular layer. A significant reduction was seen in the thickness of the external granular layer in ethanol-treated rats. This study showed that exposure to LIPUS can affect the number of granular cells and thickness of the cell layer within the cerebellar cortex in neonatal rats. LIPUS also could attenuate ethanol toxicity effects on the cerebellum. Copyright © 2017 Elsevier B.V. All rights reserved.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis.

PubMed

Dores, C; Rancourt, D; Dobrinski, I

2015-05-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density, or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here, we report the use of stirred suspension bioreactors (SSB) to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: SSB followed by differential plating. After 66 h of culture, germ cell enrichment in SSBs provided 7.3 ± 1.0-fold (n = 9), differential plating 9.8 ± 2.4-fold (n = 6) and combination of both methods resulted in 9.1 ± 0.3-fold enrichment of germ cells from the initial germ cell population (n = 3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the SSB allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability owing to handling. © 2015 American Society of Andrology and European Academy of Andrology.

Stirred suspension bioreactors as a novel method to enrich germ cells from pre-pubertal pig testis

PubMed Central

Dores, Camila; Rancourt, Derrick; Dobrinski, Ina

2015-01-01

To study spermatogonial stem cells the heterogeneous testicular cell population first needs to be enriched for undifferentiated spermatogonia, which contain the stem cell population. When working with non-rodent models, this step requires working with large numbers of cells. Available cell separation methods rely on differential properties of testicular cell types such as expression of specific cell surface proteins, size, density or differential adhesion to substrates to separate germ cells from somatic cells. The objective of this study was to develop an approach that allowed germ cell enrichment while providing efficiency of handling large cell numbers. Here we report the use of stirred suspension bioreactors to exploit the adhesion properties of Sertoli cells to enrich cells obtained from pre-pubertal porcine testes for undifferentiated spermatogonia. We also compared the bioreactor approach with an established differential plating method and the combination of both: stirred suspension bioreactor followed by differential plating. After 66 hours of culture, germ cell enrichment in stirred suspension bioreactors provided 7.3±1.0 fold (n=9), differential plating 9.8±2.4 fold (n=6) and combination of both methods resulted in 9.1±0.3 fold enrichment of germ cells from the initial germ cell population (n=3). To document functionality of cells recovered from the bioreactor, we demonstrated that cells retained their functional ability to reassemble seminiferous tubules de novo after grafting to mouse hosts and to support spermatogenesis. These results demonstrate that the stirred suspension bioreactor allows enrichment of germ cells in a controlled and scalable environment providing an efficient method when handling large cell numbers while reducing variability due to handling. PMID:25877677

Activated Pancreatic Stellate Cells Sequester CD8+ T-Cells to Reduce Their Infiltration of the Juxtatumoral Compartment of Pancreatic Ductal Adenocarcinoma

PubMed Central

Ene-Obong, Abasi; Clear, Andrew J.; Watt, Jennifer; Wang, Jun; Fatah, Rewas; Riches, John C.; Marshall, John F.; Chin-Aleong, Joanne; Chelala, Claude; Gribben, John G.; Ramsay, Alan G.; Kocher, Hemant M.

2013-01-01

Background & Aims Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent desmoplastic microenvironment that contains many different immune cells. Activated pancreatic stellate cells (PSCs) contribute to the desmoplasia. We investigated whether distinct stromal compartments are differentially infiltrated by different types of immune cells. Method We used tissue microarray analysis to compare immune cell infiltration of different pancreatico-biliary diseased tissues (PDAC, ampullary carcinoma, cholangiocarcinoma, mucinous cystic neoplasm, chronic inflammation, and chronic pancreatitis), and juxtatumoral stromal (<100 μm from tumor) and panstromal compartments. We investigated the association between immune infiltrate and patient survival times. We analyzed T-cell migration and tumor infiltration in LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mice, and the effects of all-trans retinoic acid (ATRA) on these processes. Results Juxtatumoral compartments in PDAC samples from 2 independent groups of patients contained increased numbers of myeloperoxidase+ and CD68+ cells, compared with panstromal compartments. However, juxtatumoral compartments of PDACs contained fewer CD8+, FoxP3+, CD56+, or CD20+ cells than panstromal compartments, a distinction absent in ampullary carcinomas and cholangiocarcinomas. Patients with PDACs that had high densities of CD8+ T-cells in the juxtatumoral compartment had longer survival times than patients with lower densities. In KPC mice, administration of ATRA, which renders PSCs quiescent, increased numbers of CD8+ T-cells in juxtatumoral compartments. We found that activated PSCs express cytokines, chemokines, and adhesion molecules that regulate T-cell migration. In vitro migration assays showed that CD8+ T-cells from PDAC patients had increased chemotaxis towards activated PSCs, which secrete CXCL12, compared with quiescent PSC or tumor cells. These effects could be reversed by knockdown of CXCL12 or treatment of PSCs with ATRA. Conclusion Based on studies of human PDAC samples and KPC mice, activated PSCs appear to reduce migration of CD8+ T-cells to juxtatumoral stromal compartments, preventing their access to cancer cells. Deregulated signaling by activated PSCs could prevent an effective anti-tumor immune response. PMID:23891972

Growth and activation of natural killer cells ex vivo from children with neuroblastoma for adoptive cell therapy.

PubMed

Liu, Yin; Wu, Hong-Wei; Sheard, Michael A; Sposto, Richard; Somanchi, Srinivas S; Cooper, Laurence J N; Lee, Dean A; Seeger, Robert C

2013-04-15

Adoptive transfer of natural killer (NK) cells combined with tumor-specific monoclonal antibodies (mAb) has therapeutic potential for malignancies. We determined if large numbers of activated NK (aNK) cells can be grown ex vivo from peripheral blood mononuclear cells (PBMC) of children with high-risk neuroblastoma using artificial antigen-presenting cells (aAPC). Irradiated K562-derived Clone 9.mbIL21 aAPC were cocultured with PBMC, and propagated NK cells were characterized with flow cytometry, cytotoxicity assays, Luminex multicytokine assays, and a nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model of disseminated neuroblastoma. Coculturing patient PBMC with aAPC for 14 days induced 2,363- ± 443-fold expansion of CD56(+)CD3(-)CD14(-) NK cells with 83% ± 3% purity (n = 10). Results were similar to PBMC from normal donors (n = 5). Expression of DNAM-1, NKG2D, FcγRIII/CD16, and CD56 increased 6- ± 3-, 10- ± 2-, 21- ± 20-, and 18- ± 3-fold, respectively, on day 14 compared with day 0, showing activation of NK cells. In vitro, aNK cells were highly cytotoxic against neuroblastoma cell lines and killing was enhanced with GD2-specific mAb ch14.18. When mediating cytotoxicity with ch14.18, release of TNF-α, granulocyte macrophage colony-stimulating factor, IFN-γ, sCD40L, CCL2/MCP-1, CXCL9/MIG, and CXCL11/I-TAC by aNK cells increased 4-, 5-, 6-, 15-, 265-, 917-, and 363-fold (151-9,121 pg/mL), respectively, compared with aNK cells alone. Survival of NOD/SCID mice bearing disseminated neuroblastoma improved when treated with thawed and immediately intravenously infused cryopreserved aNK cells compared with untreated mice and was further improved when ch14.18 was added. Propagation of large numbers of aNK cells that maintain potent antineuroblastoma activities when cryopreserved supports clinical testing of adoptive cell therapy with ch14.18.

Significantly higher number of fetal cells in the maternal circulation of women with pre-eclampsia.

PubMed

Jansen, M W; Korver-Hakkennes, K; van Leenen, D; Visser, W; in 't Veld, P A; de Groot, C J; Wladimiroff, J W

2001-12-01

Although the pathophysiology of pre-eclampsia is unknown, several studies have indicated that abnormal placentation early in pregnancy might play a key role. It has recently been suggested that this abnormal placentation may result in transfusion of fetal cells (feto-maternal transfusion) in women with pre-eclampsia. In the present study, fetal nucleated red blood cells were isolated from 20 women with pre-eclampsia and 20 controls using a very efficient magnetic activated cell sorting (MACS) protocol. The number of male cells was determined using two-color fluorescence in situ hybridization (FISH) for X and Y chromosomes. Significantly more XY cells could be detected in women with pre-eclampsia (0.61+/-1.2 XY cells/ml blood) compared to women with uncomplicated pregnancies (0.02+/-0.04 XY cells/ml blood) (Mann-Whitney U-test, p<0.001). These results suggest that fetal cell trafficking is enhanced in women with pre-eclampsia, and this finding may contribute to the understanding of the pathophysiology of the disease. Copyright 2001 John Wiley & Sons, Ltd.

Effects of Nano-MnO2 on Dopaminergic Neurons and the Spatial Learning Capability of Rats

PubMed Central

Li, Tao; Shi, Tingting; Li, Xiaobo; Zeng, Shuilin; Yin, Lihong; Pu, Yuepu

2014-01-01

This study aimed to observe the effect of intracerebrally injected nano-MnO2 on neurobehavior and the functions of dopaminergic neurons and astrocytes. Nano-MnO2, 6-OHDA, and saline (control) were injected in the substantia nigra and the ventral tegmental area of Sprague-Dawley rat brains. The neurobehavior of rats was evaluated by Morris water maze test. Tyrosine hydroxylase (TH), inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP) expressions in rat brain were detected by immunohistochemistry. Results showed that the escape latencies of nano-MnO2 treated rat increased significantly compared with control. The number of TH-positive cells decreased, GFAP- and iNOS-positive cells increased significantly in the lesion side of the rat brains compared with the contralateral area in nano-MnO2 group. The same tendencies were observed in nano-MnO2-injected rat brains compared with control. However, in the the positive control, 6-OHDA group, escape latencies increased, TH-positive cell number decreased significantly compared with nano-MnO2 group. The alteration of spatial learning abilities of rats induced by nano-MnO2 may be associated with dopaminergic neuronal dysfunction and astrocyte activation. PMID:25101772

Simple and efficient generation of virus-specific T cells for adoptive therapy using anti-4-1BB antibody.

PubMed

Imahashi, Nobuhiko; Nishida, Tetsuya; Goto, Tatsunori; Terakura, Seitaro; Watanabe, Keisuke; Hanajiri, Ryo; Sakemura, Reona; Imai, Misa; Kiyoi, Hitoshi; Naoe, Tomoki; Murata, Makoto

2015-01-01

Although recent studies of virus-specific T-cell (VST) therapy for viral infections after allogeneic hematopoietic stem cell transplantation have shown promising results, simple and less time-intensive and labor-intensive methods are required to generate VSTs for the wider application of VST therapy. We investigated the efficacy of anti-CD28 and anti-4-1BB antibodies, which can provide T cells with costimulatory signals similar in strength to those of antigen-presenting cells, in generating VSTs. When peripheral blood mononuclear cells were stimulated with viral peptides together with isotype control, anti-CD28, or anti-4-1BB antibodies, anti-4-1BB antibodies yielded the highest numbers of VSTs, which were on an average 7.9 times higher than those generated with isotype control antibody. The combination of anti-CD28 and anti-4-1BB antibodies did not result in increased numbers of VSTs compared with anti-4-1BB antibody alone. Importantly, the positive effect of anti-4-1BB antibody was observed regardless of the epitopes of the VSTs. In contrast, the capacity of dendritic cells (DCs) to generate VSTs differed considerably depending on the epitopes of the VSTs. Furthermore, the numbers of VSTs generated with DCs were at most similar to those generated with the anti-4-1BB antibody. Generation of VSTs with anti-4-1BB antibody did not result in excessive differentiation or deteriorated function of the generated VSTs compared with those generated with control antibody or DCs. In conclusion, VSTs can be generated rapidly and efficiently by simply stimulating peripheral blood mononuclear cells with viral peptide and anti-4-1BB antibody without using antigen-presenting cells. We propose using anti-4-1BB antibody as a novel strategy to generate VSTs for adoptive therapy.

nbCNV: a multi-constrained optimization model for discovering copy number variants in single-cell sequencing data.

PubMed

Zhang, Changsheng; Cai, Hongmin; Huang, Jingying; Song, Yan

2016-09-17

Variations in DNA copy number have an important contribution to the development of several diseases, including autism, schizophrenia and cancer. Single-cell sequencing technology allows the dissection of genomic heterogeneity at the single-cell level, thereby providing important evolutionary information about cancer cells. In contrast to traditional bulk sequencing, single-cell sequencing requires the amplification of the whole genome of a single cell to accumulate enough samples for sequencing. However, the amplification process inevitably introduces amplification bias, resulting in an over-dispersing portion of the sequencing data. Recent study has manifested that the over-dispersed portion of the single-cell sequencing data could be well modelled by negative binomial distributions. We developed a read-depth based method, nbCNV to detect the copy number variants (CNVs). The nbCNV method uses two constraints-sparsity and smoothness to fit the CNV patterns under the assumption that the read signals are negatively binomially distributed. The problem of CNV detection was formulated as a quadratic optimization problem, and was solved by an efficient numerical solution based on the classical alternating direction minimization method. Extensive experiments to compare nbCNV with existing benchmark models were conducted on both simulated data and empirical single-cell sequencing data. The results of those experiments demonstrate that nbCNV achieves superior performance and high robustness for the detection of CNVs in single-cell sequencing data.

Exposure cell number during feeder cell growth-arrest by Mitomycin C is a critical pharmacological aspect in stem cell culture system.

PubMed

Chugh, Rishi Man; Chaturvedi, Madhusudan; Yerneni, Lakshmana Kumar

2016-01-01

Growth-arrested feeder cells following Mitomycin C treatment are instrumental in stem cell culture allowing development of regenerative strategies and alternatives to animal testing in drug discovery. The concentration of Mitomycin C and feeder cell type was described to affect feeder performance but the criticality of feeder cell exposure density was not addressed. We hypothesize that the exposure cell density influences the effectiveness of Mitomycin C in an arithmetic manner. Three different exposure cell densities of Swiss 3T3 fibroblasts were treated with a range of Mitomycin C concentrations for 2h. The cells were replaced and the viable cells counted on 3, 6, 9, 12 and 20days. The cell extinctions were compared with doses per cell which were derived by dividing the product of concentration and volume of Mitomycin C solution with exposure cell number. The periodic post-treatment feeder cell extinctions were not just dependent on Mitomycin C concentration but also on dose per cell. Analysis of linearity between viable cell number and Mitomycin C dose per cell derived from the concentrations of 3 to 10μg/ml revealed four distinct categories of growth-arrest. Confluent cultures exposed to low concentration showed growth-arrest failure. The in vitro cell density titration can facilitate prediction of a compound's operational in vivo dosing. For containing the growth arrest failure, an arithmetic volume derivation strategy is proposed by fixing the exposure density to a safe limit. The feeder extinction characteristics are critical for streamlining the stem cell based pharmacological and toxicological assays. Copyright © 2016 Elsevier Inc. All rights reserved.

Label-free high-throughput detection and quantification of circulating melanoma tumor cell clusters by linear-array-based photoacoustic tomography

NASA Astrophysics Data System (ADS)

Hai, Pengfei; Zhou, Yong; Zhang, Ruiying; Ma, Jun; Li, Yang; Shao, Jin-Yu; Wang, Lihong V.

2017-04-01

Circulating tumor cell (CTC) clusters, arising from multicellular groupings in a primary tumor, greatly elevate the metastatic potential of cancer compared with single CTCs. High-throughput detection and quantification of CTC clusters are important for understanding the tumor metastatic process and improving cancer therapy. Here, we applied a linear-array-based photoacoustic tomography (LA-PAT) system and improved the image reconstruction for label-free high-throughput CTC cluster detection and quantification in vivo. The feasibility was first demonstrated by imaging CTC cluster ex vivo. The relationship between the contrast-to-noise ratios (CNRs) and the number of cells in melanoma tumor cell clusters was investigated and verified. Melanoma CTC clusters with a minimum of four cells could be detected, and the number of cells could be computed from the CNR. Finally, we demonstrated imaging of injected melanoma CTC clusters in rats in vivo. Similarly, the number of cells in the melanoma CTC clusters could be quantified. The data showed that larger CTC clusters had faster clearance rates in the bloodstream, which agreed with the literature. The results demonstrated the capability of LA-PAT to detect and quantify melanoma CTC clusters in vivo and showed its potential for tumor metastasis study and cancer therapy.

Inner ear changes in mucopolysaccharidosis type I/Hurler syndrome.

PubMed

Kariya, Shin; Schachern, Patricia A; Nishizaki, Kazunori; Paparella, Michael M; Cureoglu, Sebahattin

2012-10-01

Mucopolysaccharidosis type I/Hurler syndrome is an autosomal recessive disease caused by a deficiency of α-L-iduronidase activity. Recurrent middle ear infections and hearing loss are common complications in Hurler syndrome. Although sensorineural and conductive components occur, the mechanism of sensorineural hearing loss has not been determined. The purpose of this study is to evaluate the quantitative inner ear histopathology of the temporal bones of patients with Hurler syndrome. Eleven temporal bones from 6 patients with Hurler syndrome were examined. Age-matched healthy control samples consisted of 14 temporal bones from 7 cases. Temporal bones were serially sectioned in the horizontal plane and stained with hematoxylin and eosin. The number of spiral ganglion cells, loss of cochlear hair cells, area of stria vascularis, and cell density of spiral ligament were evaluated using light microscopy. There was no significant difference between Hurler syndrome and healthy controls in the number of spiral ganglion cells, area of stria vascularis, or cell density of spiral ligament. The number of cochlear hair cells in Hurler syndrome was significantly decreased compared with healthy controls. Auditory pathophysiology in the central nerve system in Hurler syndrome remains unknown; however, decreased cochlear hair cells may be one of the important factors for the sensorineural component of hearing loss.

Facilitated Engraftment of Isolated Islets Coated With Expanded Vascular Endothelial Cells for Islet Transplantation.

PubMed

Barba-Gutierrez, D Alonso; Daneri-Navarro, A; Villagomez-Mendez, J Jesus Alejandro; Kanamune, J; Robles-Murillo, A Karina; Sanchez-Enriquez, S; Villafan-Bernal, J Rafael; Rivas-Carrillo, J D

2016-03-01

Diabetes is complex disease, which involves primary metabolic changes followed by immunological and vascular pathophysiological adjustments. However, it is mostly characterized by an unbalanced decreased number of the β-cells unable to maintain the metabolic requirements and failure to further regenerate newly functional pancreatic islets. The objective of this study was to analyze the properties of the endothelial cells to facilitate the islet cells engraftment after islet transplantation. We devised a co-cultured engineer system to coat isolated islets with vascular endothelial cells. To assess the cell integration of cell-engineered islets, we stained them for endothelial marker CD31 and nuclei counterstained with DAPI dye. We comparatively performed islet transplantations into streptozotocin-induced diabetic mice and recovered the islet grafts for morphometric analyses on days 3, 7, 10, and 30. Blood glucose levels were measured continuously after islet transplantation to monitor the functional engraftment and capacity to achieve metabolic control. Cell-engineered islets showed a well-defined rounded shape after co-culture when compared with native isolated islets. Furthermore, the number of CD31-positive cells layered on the islet surface showed a direct proportion with engraftment capacities and less TUNEL-positive cells on days 3 and 7 after transplantation. We observed that vascular endothelial cells could be functional integrated into isolated islets. We also found that islets that are coated with vascular endothelial cells increased their capacity to engraft. These findings indicate that islets coated with endothelial cells have a greater capacity of engraftment and thus establish a definitely vascular network to support the metabolic requirements. Copyright © 2016 Elsevier Inc. All rights reserved.

Cigarette smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell metaplasia.

PubMed

Mebratu, Yohannes A; Schwalm, Kurt; Smith, Kevin R; Schuyler, Mark; Tesfaigzi, Yohannes

2011-06-01

Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. We screened for dysregulated expression of the Bcl-2 family members. We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis.

Cigarette Smoke Suppresses Bik To Cause Epithelial Cell Hyperplasia and Mucous Cell Metaplasia

PubMed Central

Mebratu, Yohannes A.; Schwalm, Kurt; Smith, Kevin R.; Schuyler, Mark; Tesfaigzi, Yohannes

2011-01-01

Rationale: Aberrant regulation of airway epithelial cell numbers in airways leads to increased mucous secretions in chronic lung diseases such as chronic bronchitis. Because the Bcl-2 family of proteins is crucial for airway epithelial homeostasis, identifying the players that reduce cigarette smoke (CS)-induced mucous cell metaplasia can help to develop effective therapies. Objectives: To identify the Bcl-2 family of proteins that play a role in reducing CS-induced mucous cell metaplasia. Methods: We screened for dysregulated expression of the Bcl-2 family members. Measurements and Main Results: We identified Bik to be significantly reduced in bronchial brushings of patients with chronic epithelial cell hyperplasia compared with nondiseased control subjects. Reduced Bik but increased MUC5AC mRNA levels were also detected when normal human airway epithelial cells (HAECs) were exposed to CS or when autopsy tissues from former smokers with and without chronic bronchitis were compared. Similarly, exposure of C57Bl/6 mice to CS resulted in increased numbers of epithelial and mucous cells per millimeter of basal lamina, along with reduced Bik but increased Muc5ac expression, and this change was sustained even when mice were allowed to recover in filtered air for 8 weeks. Restoring Bik expression significantly suppressed CS-induced mucous cell metaplasia in differentiated primary HAEC cultures and in airways of mice in vivo. Bik blocked nuclear translocation of phospho-ERK1/2 to induce apoptosis of HAECs. The conserved Leu61 within Bik and ERK1/2 activation were essential to induce cell death in hyperplastic mucous cells. Conclusions: These studies show that CS suppresses Bik expression to block airway epithelia cell death and thereby increases epithelial cell hyperplasia in chronic bronchitis. PMID:21317312

Multi-nucleate retinal pigment epithelium cells of the human macula exhibit a characteristic and highly specific distribution.

PubMed

Starnes, Austin C; Huisingh, Carrie; McGwin, Gerald; Sloan, Kenneth R; Ablonczy, Zsolt; Smith, R Theodore; Curcio, Christine A; Ach, Thomas

2016-01-01

The human retinal pigment epithelium (RPE) is reportedly 3% bi-nucleated. The importance to human vision of multi-nucleated (MN)-RPE cells could be clarified with more data about their distribution in central retina. Nineteen human RPE-flatmounts (9 ≤ 51 years, 10 > 80 years) were imaged at 12 locations: 3 eccentricities (fovea, perifovea, near periphery) in 4 quadrants (superior, inferior, temporal, nasal). Image stacks of lipofuscin-attributable autofluorescence and phalloidin labeled F-actin cytoskeleton were obtained using a confocal fluorescence microscope. Nuclei were devoid of autofluorescence and were marked using morphometric software. Cell areas were approximated by Voronoi regions. Mean number of nuclei per cell among eccentricity/quadrant groups and by age were compared using Poisson and binominal regression models. A total of 11,403 RPE cells at 200 locations were analyzed: 94.66% mono-, 5.31% bi-, 0.02% tri-nucleate, and 0.01% with 5 nuclei. Age had no effect on number of nuclei. There were significant regional differences: highest frequencies of MN-cells were found at the perifovea (9.9%) and near periphery (6.8%). The fovea lacked MN-cells almost entirely. The nasal quadrant had significantly more MN-cells compared to other quadrants, at all eccentricities. This study demonstrates MN-RPE cells in human macula. MN-cells may arise due to endoreplication, cell fusion, or incomplete cell division. The topography of MN-RPE cells follows the topography of photoreceptors; with near-absence at the fovea (cones only) and high frequency at perifovea (highest rod density). This distribution might reflect specific requirements of retinal metabolism or other mechanisms addressable in further studies.

Dimensionless numbers and correlating equations for the analysis of the membrane-gas diffusion electrode assembly in polymer electrolyte fuel cells

NASA Astrophysics Data System (ADS)

Gyenge, E. L.

The Quraishi-Fahidy method [Can. J. Chem. Eng. 59 (1981) 563] was employed to derive characteristic dimensionless numbers for the membrane-electrolyte, cathode catalyst layer and gas diffuser, respectively, based on the model presented by Bernardi and Verbrugge for polymer electrolyte fuel cells [AIChE J. 37 (1991) 1151]. Monomial correlations among dimensionless numbers were developed and tested against experimental and mathematical modeling results. Dimensionless numbers comparing the bulk and surface-convective ionic conductivities, the electric and viscous forces and the current density and the fixed surface charges, were employed to describe the membrane ohmic drop and its non-linear dependence on current density due to membrane dehydration. The analysis of the catalyst layer yielded electrode kinetic equivalents of the second Damköhler number and Thiele modulus, influencing the penetration depth of the oxygen reduction front based on the pseudohomogeneous film model. The correlating equations for the catalyst layer could describe in a general analytical form, all the possible electrode polarization scenarios such as electrode kinetic control coupled or not with ionic and/or oxygen mass transport limitation. For the gas diffusion-backing layer correlations are presented in terms of the Nusselt number for mass transfer in electrochemical systems. The dimensionless number-based correlating equations for the membrane electrode assembly (MEA) could provide a practical approach to quantify single-cell polarization results obtained under a variety of experimental conditions and to implement them in models of the fuel cell stack.

Characterization of stem cells in Dupuytren's disease.

PubMed

Hindocha, S; Iqbal, S A; Farhatullah, S; Paus, R; Bayat, A

2011-02-01

Dupuytren's disease (DD) is a common fibroproliferative disease of unknown origin. The source of abnormal cells leading to DD formation remains underexplored. In addition to fascia, palmar skin and fat-derived cells may be a potential source of cells causing DD. This study aimed to profile haematopoietic and mesenchymal stem cells in different DD tissue components compared with tissue removed at carpal tunnel surgery (control). Biopsies were taken from the diseased cord, nodule, perinodular fat and skin overlying the nodule of ten patients with DD and compared with control tissue from seven patients having surgery for carpal tunnel syndrome. Fluorescence-activated cell sorting (FACS), immunohistochemistry and quantitative real-time polymerase chain reaction (QRT-PCR) were used to identify expression of selected stem cell markers. FACS and QRT-PCR analysis identified the highest RNA expression and number of cells positive for adipocyte stem cell markers (CD13 and CD29) in the DD nodule in comparison with carpal tunnel control tissue (P = 0·053). CD34 RNA was overexpressed, and a higher percentage of these cells was present in DD skin compared with carpal tunnel skin (P = 0·001). Each structural component of DD (cord, nodule, perinodular fat and skin) had distinct stem cell populations. These findings support the hypothesis that DD may result from mesenchymal progenitor cell expansion.

Functional Analysis of Dendritic Cells Generated from T-iPSCs from CD4+ T Cell Clones of Sjögren's Syndrome.

PubMed

Iizuka-Koga, Mana; Asashima, Hiromitsu; Ando, Miki; Lai, Chen-Yi; Mochizuki, Shinji; Nakanishi, Mahito; Nishimura, Toshinobu; Tsuboi, Hiroto; Hirota, Tomoya; Takahashi, Hiroyuki; Matsumoto, Isao; Otsu, Makoto; Sumida, Takayuki

2017-05-09

Although it is important to clarify the pathogenic functions of T cells in human samples, their examination is often limited due to difficulty in obtaining sufficient numbers of dendritic cells (DCs), used as antigen-presenting cells, especially in autoimmune diseases. We describe the generation of DCs from induced pluripotent stem cells derived from T cells (T-iPSCs). We reprogrammed CD4+ T cell clones from a patient with Sjögren's syndrome (SS) into iPSCs, which were differentiated into DCs (T-iPS-DCs). T-iPS-DCs had dendritic cell-like morphology, and expressed CD11c, HLA-DR, CD80, CD86, and also BDCA-3. Compared with monocyte-derived DCs, the capacity for antigen processing was similar, and T-iPS-DCs induced the proliferative response of autoreactive CD4+ T cells. Moreover, we could evaluate T cell functions of the patient with SS. In conclusion, we obtained adequate numbers of DCs from T-iPSCs, which could be used to characterize pathogenic T cells in autoimmune diseases such as SS. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

ERK-GluR1 phosphorylation in trigeminal spinal subnucleus caudalis neurons is involved in pain associated with dry tongue.

PubMed

Nakaya, Yuka; Tsuboi, Yoshiyuki; Okada-Ogawa, Akiko; Shinoda, Masamichi; Kubo, Asako; Chen, Jui Yen; Noma, Noboru; Batbold, Dulguun; Imamura, Yoshiki; Sessle, Barry J; Iwata, Koichi

2016-01-01

Dry mouth is known to cause severe pain in the intraoral structures, and many dry mouth patients have been suffering from intraoral pain. In development of an appropriate treatment, it is crucial to study the mechanisms underlying intraoral pain associated with dry mouth, yet the detailed mechanisms are not fully understood. To evaluate the mechanisms underlying pain related to dry mouth, the dry-tongue rat model was developed. Hence, the mechanical or heat nocifensive reflex, the phosphorylated extracellular signal-regulated kinase and phosphorylated GluR1-IR immunohistochemistries, and the single neuronal activity were examined in the trigeminal spinal subnucleus caudalis of dry-tongue rats. The head-withdrawal reflex threshold to mechanical, but not heat, stimulation of the tongue was significantly decreased on day 7 after tongue drying. The mechanical, but not heat, responses of trigeminal spinal subnucleus caudalis nociceptive neurons were significantly enhanced in dry-tongue rats compared to sham rats on day 7. The number of phosphorylated extracellular signal-regulated kinase-immunoreactive cells was also significantly increased in the trigeminal spinal subnucleus caudalis following noxious stimulation of the tongue in dry-tongue rats compared to sham rats on day 7. The decrement of the mechanical head-withdrawal reflex threshold (HWT) was reversed during intracisternal administration of the mitogen-activated protein kinase kinase 1 inhibitor, PD98059. The trigeminal spinal subnucleus caudalis neuronal activities and the number of phosphorylated extracellular signal-regulated kinase-immunoreactive cells following noxious mechanical stimulation of dried tongue were also significantly decreased following intracisternal administration of PD98059 compared to vehicle-administrated rats. Increased number of the phosphorylated GluR1-IR cells was observed in the trigeminal spinal subnucleus caudalis of dry-tongue rats, and the number of phosphorylated GluR1-IR cells was significantly reduced in PD98059-administrated rats compared to the vehicle-administrated tongue-dry rats. These findings suggest that the pERK-pGluR1 cascade is involved in central sensitization of trigeminal spinal subnucleus caudalis nociceptive neurons, thus resulting in tongue mechanical hyperalgesia associated with tongue drying. © The Author(s) 2016.

A redefinition of the representation of mammary cells and enzyme activities in a lactating dairy cow model.

PubMed

Hanigan, M D; Rius, A G; Kolver, E S; Palliser, C C

2007-08-01

The Molly model predicts various aspects of digestion and metabolism in the cow, including nutrient partitioning between milk and body stores. It has been observed previously that the model underpredicts milk component yield responses to nutrition and consequently overpredicts body energy store responses. In Molly, mammary enzyme activity is represented as an aggregate of mammary cell numbers and activity per cell with minimal endocrine regulation. Work by others suggests that mammary cells can cycle between active and quiescent states in response to various stimuli. Simple models of milk production have demonstrated the utility of this representation when using the model to simulate variable milking and nutrient restriction. It was hypothesized that replacing the current representation of mammary cells and enzyme activity in Molly with a representation of active and quiescent cells and improving the representation of endocrine control of cell activity would improve predictions of milk component yield. The static representation of cell numbers was replaced with a representation of cell growth during gestation and early lactation periods and first-order cell death. Enzyme capacity for fat and protein synthesis was assumed to be proportional to cell numbers. Enzyme capacity for lactose synthesis was represented with the same equation form as for cell numbers. Data used for parameter estimation were collected as part of an extended lactation trial. Cows with North American or New Zealand genotypes were fed 0, 3, or 6 kg of concentrate dry matter daily during a 600-d lactation. The original model had root mean square prediction errors of 17.7, 22.3, and 19.8% for lactose, protein, and fat yield, respectively, as compared with values of 8.3, 9.4, and 11.7% for the revised model, respectively. The original model predicted body weight with an error of 19.7% vs. 5.7% for the revised model. Based on these observations, it was concluded that representing mammary synthetic capacity as a function of active cell numbers and revisions to endocrine control of cell activity was meritorious.

Effects of donor fibroblast cell type and transferred cloned embryo number on the efficiency of pig cloning.

PubMed

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan; Wu, Zhenfang

2013-02-01

Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150-199, 200-249, 250-299, 300-349, or 350-450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53 ± 0.34) was similar with that associated with P,D,L,Y-FFBs (2.72 ± 0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47 ± 0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150-199 to 200-450 per recipient. However, increase of the number of transferred embryos from 200-249 to 250-450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250-450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200-249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs.

Effects of Donor Fibroblast Cell Type and Transferred Cloned Embryo Number on the Efficiency of Pig Cloning

PubMed Central

Li, Zicong; Shi, Junsong; Liu, Dewu; Zhou, Rong; Zeng, Haiyu; Zhou, Xiu; Mai, Ranbiao; Zeng, Shaofen; Luo, Lvhua; Yu, Wanxian; Zhang, Shouquan

2013-01-01

Abstract Currently, cloning efficiency in pigs is very low. Donor cell type and number of cloned embryos transferred to an individual surrogate are two major factors that affect the successful rate of somatic cell nuclear transfer (SCNT) in pigs. This study aimed to compare the influence of different donor fibroblast cell types and different transferred embryo numbers on recipients' pregnancy rate and delivery rate, the average number of total clones born, clones born alive and clones born healthy per litter, and the birth rate of healthy clones (=total number of healthy cloned piglets born /total number of transferred cloned embryos). Three types of donor fibroblasts were tested in large-scale production of cloned pigs, including fetal fibroblasts (FFBs) from four genetically similar Western swine breeds of Pietrain (P), Duroc (D), Landrace (L), and Yorkshire (Y), which are referred to as P,D,LY-FFBs, adult fibroblasts (AFBs) from the same four breeds, which are designated P,D,L,Y-AFBs, and AFBs from a Chinese pig breed of Laiwu (LW), which is referred to as LW-AFBs. Within each donor fibroblast cell type group, five transferred cloned embryo number groups were tested. In each embryo number group, 150–199, 200–249, 250–299, 300–349, or 350–450 cloned embryos were transferred to each individual recipient sow. For the entire experiment, 92,005 cloned embryos were generated from nearly 115,000 matured oocytes and transferred to 328 recipients; in total, 488 cloned piglets were produced. The results showed that the mean clones born healthy per litter resulted from transfer of embryos cloned from LW-AFBs (2.53±0.34) was similar with that associated with P,D,L,Y-FFBs (2.72±0.29), but was significantly higher than that resulted from P,D,L,Y-AFBs (1.47±0.18). Use of LW-AFBs as donor cells for SCNT resulted in a significantly higher pregnancy rate (72.00% vs. 59.30% and 48.11%) and delivery rate (60.00% vs. 45.93% and 35.85%) for cloned embryo recipients, and a significantly higher birth rate of healthy clones (0.5009% vs. 0.3362% and 0.2433%) than that resulting from P,D,L,Y-AFBs and P,D,L,Y-FFBs. This suggests that using LW-AFBs as donor cells results in a higher cloning efficiency in pigs, compared with the other two donor fibroblast cell types. The birth rate of healthy clones was significantly improved when the number of transferred cloned embryos was increased from 150–199 to 200–450 per recipient. However, increase of the number of transferred embryos from 200–249 to 250–450 per surrogate did not change the birth rate of healthy clones. This suggests that transfer of excessive (250–450) cloned embryos to an individual surrogate is not necessary for increasing the cloning efficiency in pigs, and the relatively optimal number of reconstructed embryos transferred to individual recipient is 200–249. Furthermore, our results indicated that the numbers of total born clones, clones born alive, and clones born healthy per litter have a significantly high positive correlation with each other. The present study provides useful information for improving SCNT efficiency in pigs. PMID:23256540

Cold thyroid nodules show a marked increase in proliferation markers.

PubMed

Krohn, Knut; Stricker, Ingo; Emmrich, Peter; Paschke, Ralf

2003-06-01

Thyroid follicular adenomas and adenomatous thyroid nodules are a frequent finding in geographical areas with iodine deficiency. They occur as hypofunctioning (scintigraphically cold) or hyperfunctioning (scintigraphically hot) nodules. Their predominant clonal origin suggests that they result from clonal expansion of a single cell, which is very likely the result of a prolonged increase in proliferation compared with non-affected surrounding cells. To test whether increased cell proliferation is detectable in cold thyroid nodules, we studied paraffin-embedded tissue from 40 cold thyroid nodules and their surrounding normal thyroid tissue for the occurrence of the proliferating cell nuclear antigen (PCNA) and Ki-67 (MIB-1 antibody) epitopes as markers for cell proliferation. All 40 thyroid nodules were histologically well characterized and have been studied for molecular characteristics before. The labeling index (number of labeled cells versus total cell number) for nodular and surrounding tissue was calculated. In 33 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for PCNA was detectable. In 19 cold thyroid nodules a significant (p < or = 0.05) increase in the labeling index for Ki-67 was detectable. Moreover, surrounding tissues with lymphocyte infiltration showed a significantly higher labeling index for both PCNA and Ki-67 compared with normal surrounding tissue. These findings are first evidence that an increased thyroid epithelial cell proliferation is a uniform feature common to most cold nodules. However, the increase of proliferation markers shows a heterogeneity that is not correlated with histopathologic, molecular, or clinical characteristics.

Interstitial cells of Cajal in chagasic megaesophagus.

PubMed

de Lima, Marcus Aurelho; Cabrine-Santos, Marlene; Tavares, Marcelo Garcia; Gerolin, Gustavo Pacheco; Lages-Silva, Eliane; Ramirez, Luis Eduardo

2008-08-01

Chagasic visceromegalies are the most important digestive manifestations of Chagas disease and are characterized by motor disorders and dilation of organs such as esophagus and colon. One of the theories raised to explain the physiopathogenesis of chagasic megas is the plexus theory. Recent studies have shown a reduction of interstitial cells of Cajal (ICCs) in the colon of chagasic patients. These cells are present throughout the gastrointestinal tract and are considered to be pacemaker cells, that is, they are responsible for coordinating peristalsis and for mediating nerve impulses. In view of the lack of studies on these cells in megaesophagus and the previous observation of a reduction of ICCs in chagasic megacolons, we compared the distribution of ICCs in the esophagus of chagasic and nonchagasic patients to contribute to a better understanding of the physiopathogenesis of this esophageal disease. Esophageal biopsy samples from 10 chagasic and 5 nonchagasic patients were used. Cells were identified with the anti-CD117 antibody. The number of ICCs was quantified in longitudinal and circular muscle layers and myenteric plexus. The results were analyzed statistically by comparison of means. An intense reduction in the number of ICCs was observed in muscle layers and in the myenteric plexus of patients with megaesophagus. We conclude that there is an intense reduction of ICCs in the esophagus of chagasic patients when compared to nonchagasic patients, a finding supporting the important role of these cells in gastrointestinal tract motility. A deficiency in these cells might be implied in the genesis of megaesophagus.

First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels.

PubMed

Wani, Nisar Ahmad; Vettical, Binoy S; Hong, Seung B

2017-01-01

Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5μM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term.

First cloned Bactrian camel (Camelus bactrianus) calf produced by interspecies somatic cell nuclear transfer: A step towards preserving the critically endangered wild Bactrian camels

PubMed Central

Vettical, Binoy S.; Hong, Seung B.

2017-01-01

Studies were conducted to explore the possibility of employing dromedary camel (Camelus dromedarius) oocytes as recipient cytoplasts for the development of interspecies somatic cell nuclear transfer (iSCNT) embryos using skin fibroblast cells of an adult Bactrian camel (Camelus bactrianus) and Llama (Llama glama) as donor nuclei. Also, the embryos reconstructed with Bactrian cells were transferred into the uterus of synchronized dromedary camel recipients to explore the possibility of using them as surrogate mothers. Serum-starved skin fibroblast cells were injected into the perivitelline space of enucleated mature oocytes, collected from super-stimulated dromedary camels, and fused using an Eppendorf electroporator. After activation with 5μM ionomycin and 6-dimethylaminopurine, they were cultured at 38.5°C in an atmosphere of 5% CO2, 5% O2, and 90% N2 in air. In experiment 1, Day 7 blastocysts were stained with Hoechst to count their cell numbers, while in experiment 2, they were transferred to synchronized dromedary recipients. A lower number (P < 0.05) of blastocysts were obtained from reconstructs utilizing fibroblast cells from Llama when compared with those reconstructed with dromedary and Bactrian fibroblast cells. However, no difference was observed in their cell numbers. In experiment 2, a higher (P < 0.05) proportion of blastocysts were obtained from the cleaved embryos reconstructed with Bactrian fibroblast cells when compared to those reconstructed with dromedary cells. Twenty-six Day 7 blastocysts reconstructed with Bactrian cells were transferred to 23 synchronized dromedary recipients with 5 pregnancies established on Day 30, however, only one of the pregnancies developed to term and a healthy calf weighing 33 kgs was born after completing 392 days of gestation. Unfortunately, the calf died on day 7 due to acute septicemia. In conclusion, the present study reports, for the first time, birth of a cloned Bactrian calf by iSCNT using dromedary camel as a source for oocytes as well as a surrogate for carrying the pregnancy to term. PMID:28545049

Evaluation of the osteogenic differentiation of gingiva-derived stem cells grown on culture plates or in stem cell spheroids: Comparison of two- and three-dimensional cultures.

PubMed

Lee, Sung-Il; Ko, Youngkyung; Park, Jun-Beom

2017-09-01

Three-dimensional cell culture systems provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates. The current study aimed to evaluate the osteogenic differentiation potential of gingiva-derived stem cells cultured in two-dimensional or three-dimensional systems. To the best of our knowledge, the present study is the first to compare the growth of gingiva-derived stem cells in monolayer culture to a three-dimensional culture system with microwells. For three-dimensional culture, gingiva-derived stem cells were isolated and seeded into polydimethylsiloxane-based concave micromolds. Alkaline phosphatase activity and alizarin red S staining assays were then performed to evaluate osteogenesis and the degree of mineralization, respectively. Stem cell spheroids had a significantly increased level of alkaline phosphatase activity and mineralization compared with cells from the two-dimensional culture. In addition, an increase in mineralized deposits was observed with an increase in the loading cell number. The results of present study indicate that gingiva-derived stem cell spheroids exhibit an increased osteogenic potential compared with stem cells from two-dimensional culture. This highlights the potential of three-dimensional culture systems using gingiva-derived stem cells for regenerative medicine applications requiring stem cells with osteogenic potential.

Differences in AMY1 Gene Copy Numbers Derived from Blood, Buccal Cells and Saliva Using Quantitative and Droplet Digital PCR Methods: Flagging the Pitfall.

PubMed

Ooi, Delicia Shu Qin; Tan, Verena Ming Hui; Ong, Siong Gim; Chan, Yiong Huak; Heng, Chew Kiat; Lee, Yung Seng

2017-01-01

The human salivary (AMY1) gene, encoding salivary α-amylase, has variable copy number variants (CNVs) in the human genome. We aimed to determine if real-time quantitative polymerase chain reaction (qPCR) and the more recently available Droplet Digital PCR (ddPCR) can provide a precise quantification of the AMY1 gene copy number in blood, buccal cells and saliva samples derived from the same individual. Seven participants were recruited and DNA was extracted from the blood, buccal cells and saliva samples provided by each participant. Taqman assay real-time qPCR and ddPCR were conducted to quantify AMY1 gene copy numbers. Statistical analysis was carried out to determine the difference in AMY1 gene copy number between the different biological specimens and different assay methods. We found significant within-individual difference (p<0.01) in AMY1 gene copy number between different biological samples as determined by qPCR. However, there was no significant within-individual difference in AMY1 gene copy number between different biological samples as determined by ddPCR. We also found that AMY1 gene copy number of blood samples were comparable between qPCR and ddPCR, while there is a significant difference (p<0.01) between AMY1 gene copy numbers measured by qPCR and ddPCR for both buccal swab and saliva samples. Despite buccal cells and saliva samples being possible sources of DNA, it is pertinent that ddPCR or a single biological sample, preferably blood sample, be used for determining highly polymorphic gene copy numbers like AMY1, due to the large within-individual variability between different biological samples if real time qPCR is employed.

Model of in vitro healing to test the influence of dedifferentiated Crithmum maritimum cells on dermal repair and epidermal regeneration.

PubMed

Lequeux, C; Lhoste, A; Rovere, M R; Montastier, C; Damour, O

2011-01-01

The aim was to test the influence of dedifferentiated Crithmum maritimum cells (dCMC), totipotent vegetal stem cells, on epidermal regeneration in perfect homeostasis using a skin equivalent (SE) model. SE are prepared by seeding fibroblasts on a collagen-glycosaminoglycan-chitosan dermal substrate (DS) epidermalized by keratinocytes 3 weeks later. The originality of this present study lies in the systemic administration of dCMC from the moment when fibroblasts are seeded in the DS right through to the reconstruction of the SE. The thickness of the epidermis as well as the number of proliferating cells expressing Ki-67 and layers expressing terminal differentiation marker (filaggrin) were compared in the dCMC-treated SE versus an untreated control group. dCMC accelerated the complete regeneration and differentiation of the epidermis compared to the negative control (35 days instead of 42 days). Histology showed a multilayered, thick and differentiated epithelium after 35 days of culture. The basal and suprabasal layers had increased 4.88 ± 0.41 times versus the negative control (Mann-Whitney U test: p < 0.001). This result was attributed to the greater proliferation of basal cells because the cell numbers expressing the Ki-67 proliferation marker had increased significantly compared to the negative control (Mann-Whitney U test: p < 0.001). Moreover, dCMC allowed the differentiated epithelium to recover because only treated SE expressed the terminal differentiation marker filaggrin. Our data show that dCMC enhance epidermal cell grafts by stimulating their regeneration and differentiation in perfect homeostasis. They allow the epidermis to recover its structure for protective functions faster than the negative control. Copyright © 2010 S. Karger AG, Basel.

Robust detection of chromosomal interactions from small numbers of cells using low-input Capture-C

PubMed Central

Oudelaar, A. Marieke; Davies, James O.J.; Downes, Damien J.; Higgs, Douglas R.

2017-01-01

Abstract Chromosome conformation capture (3C) techniques are crucial to understanding tissue-specific regulation of gene expression, but current methods generally require large numbers of cells. This hampers the investigation of chromatin architecture in rare cell populations. We present a new low-input Capture-C approach that can generate high-quality 3C interaction profiles from 10 000–20 000 cells, depending on the resolution used for analysis. We also present a PCR-free, sequencing-free 3C technique based on NanoString technology called C-String. By comparing C-String and Capture-C interaction profiles we show that the latter are not skewed by PCR amplification. Furthermore, we demonstrate that chromatin interactions detected by Capture-C do not depend on the degree of cross-linking by performing experiments with varying formaldehyde concentrations. PMID:29186505

Infant feeding with soy formula milk: effects on puberty progression, reproductive function and testicular cell numbers in marmoset monkeys in adulthood.

PubMed

Tan, Karen A L; Walker, Marion; Morris, Keith; Greig, Irene; Mason, J Ian; Sharpe, Richard M

2006-04-01

This marmoset study addresses concerns about feeding human male infants with soy formula milk (SFM). From age 4 to 5 days, seven male co-twin sets were fed standard formula milk (SMA) or SFM for 5-6 weeks; blood samples were subsequently collected at 10-week intervals. Testes from co-twins killed at 120-138 weeks were fixed for cell counts. SFM- and SMA-fed twins showed normal weight gain; puberty started and progressed normally, based on blood testosterone measurements. Body weight, organ weights (prostate, seminal vesicles, pituitary, thymus and spleen) and penis length were comparable in co-twins. All SMA- and 6/7 SFM-fed males were fertile. Unexpectedly, testis weight (P = 0.041), Sertoli (P = 0.025) and Leydig cell (P = 0.026) numbers per testis were consistently increased in SFM-fed co-twins; the increase in Leydig cell numbers was most marked in males with consistently low-normal testosterone levels. Seminiferous epithelium volume per tubule showed a less consistent, non-significant increase in SFM-fed males; raised germ cell numbers per testis, probably due to increased Sertoli cells, conceivably resulted in larger testes. Average lumen size, although greater in SFM-fed group, was inconsistent between co-twins and the difference was not significant. Infant feeding with SFM has no gross adverse reproductive effects in male marmosets, though it alters testis size and cell composition, and there is consistent, if indirect, evidence for possible 'compensated Leydig cell failure'. Similar and perhaps larger changes likely occur in adult men who were fed SFM as infants.

Effects of alpha-lipoic acid on retinal ganglion cells, retinal thicknesses, and VEGF production in an experimental model of diabetes.

PubMed

Kan, Emrah; Alici, Ömer; Kan, Elif Kılıç; Ayar, Ahmet

2017-12-01

The purpose of the present study was to investigate the effect of alpha-lipoic acid (ALA) on the thicknesses of various retinal layers and on the numbers of retinal ganglion cells and vascular endothelial growth factor levels in experimental diabetic mouse retinas. Twenty-one male BALB/C mice were made diabetic by the intraperitoneal administration of streptozotocin (200 mg/kg). One week after the induction of diabetes, the mice were divided randomly into three groups: control group (non-diabetic mice treated with alpha-lipoic acid, n = 7), diabetic group (diabetic mice without treatment, n = 7), and alpha-lipoic acid treatment group (diabetic mice with alpha-lipoic acid treatment, n = 7). At the end of the 8th week, the thicknesses of the inner nuclear layer (INL), outer nuclear layer (ONL), and full-length retina were measured; also retinal ganglion cells and VEGF expressions were counted on the histological sections of the mouse retinas and compared with each other. The thicknesses of the full-length retina, ONL, and INL were significantly reduced in the diabetic group compared to the control and ALA treatment groups (p = 0.001), whereas the thicknesses of these layers did not show a significant difference between ALA treatment and control groups. The number of ganglion cells in the diabetic group was significantly lower than those in the control and ALA treatment groups (p = 0.001). The VEGF expression was significantly higher in the diabetic group and mostly observed in the ganglion cell and inner nuclear layers compared to the control and ALA treatment groups (p = 0.001). Therefore, the number of ganglion cells and VEGF levels did not show significant differences between the ALA treatment and control groups (p = 0.7). Our results show that alpha-lipoic acid treatment may have an impact on reducing VEGF levels, protecting ganglion cells, and preserving the thicknesses of the inner and outer layers in diabetic mouse retinas.

Flexible method for monitoring fuel cell voltage

DOEpatents

Mowery, Kenneth D.; Ripley, Eugene V.

2002-01-01

A method for equalizing the measured voltage of each cluster in a fuel cell stack wherein at least one of the clusters has a different number of cells than the identical number of cells in the remaining clusters by creating a pseudo voltage for the different cell numbered cluster. The average cell voltage of the all of the cells in the fuel cell stack is calculated and multiplied by a constant equal to the difference in the number of cells in the identical cell clusters and the number of cells in the different numbered cell cluster. The resultant product is added to the actual voltage measured across the different numbered cell cluster to create a pseudo voltage which is equivalent in cell number to the number of cells in the other identical numbered cell clusters.

Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

PubMed Central

González, Sheyla; Ibáñez, Elena

2010-01-01

Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536

Altered status of CD4(+)CD25(+) regulatory T cells in patients with acute coronary syndromes.

PubMed

Mor, Adi; Luboshits, Galia; Planer, David; Keren, Gad; George, Jacob

2006-11-01

Considerable evidence supports the role of innate and adaptive immunity in the progression and destabilization of the atheromatous plaque. Naturally occurring CD4(+)CD25(+) regulatory T cells (Tregs) are a subpopulation of lymphocytes that are capable of suppressing the progression of experimental autoimmune disorders. We have hypothesized that peripheral numbers and function of Tregs would be deranged in patients with acute coronary syndromes (ACS). Peripheral numbers of Tregs were evaluated by FACS employing labelled antibodies to CD4 and CD25. Functional suppressive properties of Tregs were assayed by establishing a triple-cell culture in which purified Tregs were incubated with irradiated antigen-presenting cells and anti-CD3-activated responder T cells. Proliferation in the presence or absence of oxidized LDL (oxLDL) was evaluated by thymidine incorporation. mRNA and protein content of foxp3, a master transcriptional regulator of Tregs, were determined for all subjects. Patients with ACS exhibited significantly reduced numbers of peripheral Tregs as compared with patients with stable angina and normal coronary artery subjects. Moreover, oxLDL induced a more profound reduction in Treg numbers in patients with ACS. Tregs in ACS patients were significantly compromised as their ability to suppress responder CD4(+)CD25(-) T-cell proliferation was attenuated. mRNA and protein content of foxp3 were significantly reduced in purified Tregs obtained from patients with ACS. In patients with ACS, naturally occurring CD4(+)CD25(+) Treg numbers are reduced and their functional properties compromised. These findings may aid in understanding the mechanisms leading to culprit plaque associated T-cell activation in patients with ACS.

Electromagnetic and Electrohydraulic Shock Wave Lithotripsy-Induced Urothelial Damage: Is There a Difference?

PubMed

Mustafa, Mahmoud; Aburas, Honood; Helo, Fatima M; Qarawi, Lailah

2017-02-01

To evaluate and compare the acute effect of electromagnetic and electrohydraulic extracorporeal shockwave lithotripsy (SWL) on the urothelial layers of kidney and ureter. Fifty patients, 29 males (58%) and 21 females (42%), with an average age of 51.68 years (range: 37-70) who underwent SWL application in two different centers were included. Twenty-eight patients (56%) were treated with electrohydraulic and 22 (44%) were treated with electromagnetic lithotripsy. Urinary cytologic examinations were done immediately before and after SWL therapy and 10 days later. The average numbers of epithelial cells, red blood cells (RBC), and myocytes were counted under 40 × magnification. There were significant differences in the number of epithelial cells and RBC before and after immediate application of SWL: 1.66 and 14.9 cells/field, (p = 0.001), 5.44 and 113.45 cells/field, respectively (p = 0.001). The number of RBC was significantly higher in patients treated with electromagnetic lithotripsy than those treated with electrohydraulic: 141.9 and 93.4 cells/field, respectively (p = 0.02). No myocyte or basement membrane elements were detected in any of the cytologic examinations. Cytologic examinations done after 10 days of SWL therapy revealed recovery of all abnormal cytologic findings. The acute increments in the number of epithelial cells and RBC after SWL were statistically significant but it was not permanent. SWL-induced urinary urothelial lesion is limited to the mucosal layer and there was no evidence of damage to the basal membrane or muscle layer. Electromagnetic lithotripsy caused high numbers of RBC than the electrohydraulic device on the postimmediate urine cytologic examination.

Increased mast cell numbers in a calcaneal tendon overuse model.

PubMed

Pingel, J; Wienecke, J; Kongsgaard, M; Behzad, H; Abraham, T; Langberg, H; Scott, A

2013-12-01

Tendinopathy is often discovered late because the initial development of tendon pathology is asymptomatic. The aim of this study was to examine the potential role of mast cell involvement in early tendinopathy using a high-intensity uphill running (HIUR) exercise model. Twenty-four male Wistar rats were divided in two groups: running group (n = 12); sedentary control group (n = 12). The running-group was exposed to the HIUR exercise protocol for 7 weeks. The calcaneal tendons of both hind limbs were dissected. The right tendon was used for histologic analysis using Bonar score, immunohistochemistry, and second harmonic generation microscopy (SHGM). The left tendon was used for quantitative polymerase chain reaction (qPCR) analysis. An increased tendon cell density in the runners were observed compared to the controls (P = 0.05). Further, the intensity of immunostaining of protein kinase B, P = 0.03; 2.75 ± 0.54 vs 1.17 ± 0.53, was increased in the runners. The Bonar score (P = 0.05), and the number of mast cells (P = 0.02) were significantly higher in the runners compared to the controls. Furthermore, SHGM showed focal collagen disorganization in the runners, and reduced collagen density (P = 0.03). IL-3 mRNA levels were correlated with mast cell number in sedentary animals. The qPCR analysis showed no significant differences between the groups in the other analyzed targets. The current study demonstrates that 7-week HIUR causes structural changes in the calcaneal tendon, and further that these changes are associated with an increased mast cell density. © 2013 The Authors. Scand J Med Sci Sports published by John Wiley & Sons Ltd.

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media

PubMed Central

Hennings, Justin M.; Zimmer, Randall L.; Nabli, Henda; Davis, J. Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L.

2015-01-01

Objective: Validate single versus sequential culture media for murine embryo development. Design: Prospective laboratory experiment. Setting: Assisted Reproduction Laboratory. Animals: Murine embryos. Interventions: Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. Main Outcome Measures: On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4’,6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Results: Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Conclusions: Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. PMID:26668049

Improved Murine Blastocyst Quality and Development in a Single Culture Medium Compared to Sequential Culture Media.

PubMed

Hennings, Justin M; Zimmer, Randall L; Nabli, Henda; Davis, J Wade; Sutovsky, Peter; Sutovsky, Miriam; Sharpe-Timms, Kathy L

2016-03-01

Validate single versus sequential culture media for murine embryo development. Prospective laboratory experiment. Assisted Reproduction Laboratory. Murine embryos. Thawed murine zygotes cultured for 3 or 5 days (d3 or d5) in single or sequential embryo culture media developed for human in vitro fertilization. On d3, zygotes developing to the 8 cell (8C) stage or greater were quantified using 4',6-diamidino-2-phenylindole (DAPI), and quality was assessed by morphological analysis. On d5, the number of embryos reaching the blastocyst stage was counted. DAPI was used to quantify total nuclei and inner cell mass nuclei. Localization of ubiquitin C-terminal hydrolase L1 (UCHL1) and ubiquitin C-terminal hydrolase L3 (UCHL3) was reference points for evaluating cell quality. Comparing outcomes in single versus to sequential media, the odds of embryos developing to the 8C stage on d3 were 2.34 time greater (P = .06). On d5, more embryos reached the blastocyst stage (P = <.0001), hatched, and had significantly more trophoblast cells (P = .005) contributing to the increased total cell number. Also at d5, localization of distinct cytoplasmic UCHL1 and nuclear UCHL3 was found in high-quality hatching blastocysts. Localization of UCHL1 and UCHL3 was diffuse and inappropriately dispersed throughout the cytoplasm in low-quality nonhatching blastocysts. Single medium yields greater cell numbers, an increased growth rate, and more hatching of murine embryos. Cytoplasmic UCHL1 and nuclear UHCL3 localization patterns were indicative of embryo quality. Our conclusions are limited to murine embryos but one might speculate that single medium may also be more beneficial for human embryo culture. Human embryo studies are needed. © The Author(s) 2015.

Testing for nonrandom shape similarity between sister cells using automated shape comparison

NASA Astrophysics Data System (ADS)

Guo, Monica; Marshall, Wallace F.

2009-02-01

Several reports in the biological literature have indicated that when a living cell divides, the two daughter cells have a tendency to be mirror images of each other in terms of their overall cell shape. This phenomenon would be consistent with inheritance of spatial organization from mother cell to daughters. However the published data rely on a small number of examples that were visually chosen, raising potential concerns about inadvertent selection bias. We propose to revisit this issue using automated quantitative shape comparison methods which would have no contribution from the observer and which would allow statistical testing of similarity in large numbers of cells. In this report we describe a first order approach to the problem using rigid curve matching. Using test images, we compare a pointwise correspondence based distance metric with a chamfer matching strategy and find that the latter provides better correspondence and smaller distances between aligned curves, especially when we allow nonrigid deformation of the outlines in addition to rotation.

A sensitive ELISA for measuring the adhesion of leukocytic cells to human endothelial cells.

PubMed

Krakauer, T

1994-12-28

A new, sensitive ELISA using monoclonal antibodies reactive with surface molecules specific for various leukocytes was devised to measure the attachment of these cells to cultured monolayers of human umbilical vein endothelial cells. Preparations of peripheral blood mononuclear cells, a human monocytic cell line (THP-1) and a human lymphoblastic T cell line (MOLT-4) were used to test the sensitivity of this method and compare it with the conventional 51Cr-radiolabeled cell assay. The extent of adhesion to endothelial cells was assayed by measuring the optical density produced by a complex of peroxidase-labeled streptavidin, biotin-conjugated F(ab')2 anti-mouse Ig and monoclonal antibody on fixed leukocytic cells that had adhered to endothelial cells. This method is fast and sensitive, eliminates the use of radioisotopes, and, because the detection uses a specific marker on the cell of interest, can be used in preparations of unseparated mixtures of cells. As this is a microassay, using relatively small number of cells and reagents, the methodology can be applied to screen a large number of therapeutic agents that may regulate adhesion. Using this method, the anti-inflammatory corticosteroid, dexamethasone, was found to inhibit the adhesion of THP-1 and MOLT-4 cells to cytokine-activated endothelial cells.

Investigating the Effect of Carbon Nanotube Diameter and Wall Number in Carbon Nanotube/Silicon Heterojunction Solar Cells

PubMed Central

Grace, Tom; Yu, LePing; Gibson, Christopher; Tune, Daniel; Alturaif, Huda; Al Othman, Zeid; Shapter, Joseph

2016-01-01

Suspensions of single-walled, double-walled and multi-walled carbon nanotubes (CNTs) were generated in the same solvent at similar concentrations. Films were fabricated from these suspensions and used in carbon nanotube/silicon heterojunction solar cells and their properties were compared with reference to the number of walls in the nanotube samples. It was found that single-walled nanotubes generally produced more favorable results; however, the double and multi-walled nanotube films used in this study yielded cells with higher open circuit voltages. It was also determined that post fabrication treatments applied to the nanotube films have a lesser effect on multi-walled nanotubes than on the other two types. PMID:28344309

[The influence of immobilized fibronectin on karyotypic variability of two rat kangaroo kidney cell lines].

PubMed

Polianskaia, G G; Goriachaia, T S; Pinaev, G P

2007-01-01

The numerical and structural karyotypic variability has been investigated in "markerless" Rat kangaroo kidney cell lines NBL-3-17 and NBL-3-11 when cultivating on a fibronectin-coated surface. In cell line NBL-3-17, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of cell distribution for the chromosome number has changed. These changes involve a significant decrease in frequency of cells with modal number of chromosomes, and an increase in frequency of cells with lower chromosomal number. Many new additional structural variants of the karyotype (SVK) appear. The observed alterations seem to be due preference adhesion of cells with lower chromosome number, disturbances of mitotic apparatus and selection of SVK, which are more adopted to changes in culture conditions. Detachment of cells from the fibronectin-coated surface, followed by 5 days cultivation on a hydrophilic surface restored control distribution. In cell line NBL-3-11, cultivated on the fibronectin-coated surface for 1, 2, 4 and 8 days, the character of numerical karyotypic variability did not change compared to control variants. In cell line NBL-3-17 the frequency of chromosomal aberrations under cultivation on the fibronectin-coated surface for 1, 2, 4 and 8 days did not change relative to control variants. In cell line NBL-3-11 the frequency of chromosomal aberrations under the same conditions significantly increases, mainly at the expence of chromosomal, chromatid breaks and dicentrics (telomeric association) relative to control variants. We discuss possible reasons of differences in the character of numerical and structural karyotypic variability between cell lines NBL-3-17 (hypotriploid) and NBL-3-11 (hypodiploid) under cultivation on fibronectin. The reasons of the observed interline karyotypic differences possibly consist in peculiarity of karyotypic structure of cell line NBL-3-11 and in the change of gene expression, namely in a dose of certain functioning genes in the hypotryploid cell line NBL-3-17.

Developmental changes of mast cell populations in the cerebral meninges of the rat

PubMed Central

Michaloudi, Helen; Batzios, Christos; Chiotelli, Maria; Papadopoulos, Georgios C

2007-01-01

It is known that both the dura and the pia mater attract and support the differentiation of mast cells. The present study shows that unevenly distributed mast cells in the cerebral meninges of the rat can be found in perivascular sites and vessel ramification points, but can also be unrelated to the meningeal vasculature. It also documents changes in the number, localization and staining preferences of the mast cells in the two meninges of the developing and mature rat brain. Quantitative examination of all types of histochemically differentiated meningeal mast cells reveals no major (although some exist) differences between right and left side subpopulations, but strongly suggests a different origin and fate of the dural and the pial mast cells. The number of dural mast cells, already high from postnatal day 0, although declining from postnatal day 21 onwards, remains conspicuous up to postnatal day 180. In contrast, pial mast cells are comparatively very few in the first day of the postnatal life, and despite a transient significant increase in the following two weeks, they reach almost zero levels from postnatal day 21. PMID:17822416

Developmental changes of mast cell populations in the cerebral meninges of the rat.

PubMed

Michaloudi, Helen; Batzios, Christos; Chiotelli, Maria; Papadopoulos, Georgios C

2007-10-01

It is known that both the dura and the pia mater attract and support the differentiation of mast cells. The present study shows that unevenly distributed mast cells in the cerebral meninges of the rat can be found in perivascular sites and vessel ramification points, but can also be unrelated to the meningeal vasculature. It also documents changes in the number, localization and staining preferences of the mast cells in the two meninges of the developing and mature rat brain. Quantitative examination of all types of histochemically differentiated meningeal mast cells reveals no major (although some exist) differences between right and left side subpopulations, but strongly suggests a different origin and fate of the dural and the pial mast cells. The number of dural mast cells, already high from postnatal day 0, although declining from postnatal day 21 onwards, remains conspicuous up to postnatal day 180. In contrast, pial mast cells are comparatively very few in the first day of the postnatal life, and despite a transient significant increase in the following two weeks, they reach almost zero levels from postnatal day 21.

UV-laser microdissection and mRNA expression analysis of individual neurons from postmortem Parkinson's disease brains.

PubMed

Gründemann, Jan; Schlaudraff, Falk; Liss, Birgit

2011-01-01

Cell specificity of gene expression analysis is essential to avoid tissue sample related artifacts, in particular when the relative number of target cells present in the compared tissues varies dramatically, e.g., when comparing dopamine neurons in midbrain tissues from control subjects with those from Parkinson's disease (PD) cases. Here, we describe a detailed protocol that combines contact-free UV-laser microdissection and quantitative PCR of reverse-transcribed RNA of individual neurons from postmortem human midbrain tissue from PD patients and unaffected controls. Among expression changes in a variety of dopamine neuron marker, maintenance, and cell-metabolism genes, we found that α-synuclein mRNA levels were significantly elevated in individual neuromelanin-positive dopamine midbrain neurons from PD brains when compared to those from matched controls.

Effects of cell-attachment and extracellular matrix on bone formation in vivo in collagen-hydroxyapatite scaffolds.

PubMed

Villa, Max M; Wang, Liping; Rowe, David W; Wei, Mei

2014-01-01

Cell-based tissue engineering can be used to replace missing or damaged bone, but the optimal methods for delivering therapeutic cells to a bony defect have not yet been established. Using transgenic reporter cells as a donor source, two different collagen-hydroxyapatite (HA) scaffolds, and a critical-size calvarial defect model, we investigated the effect of a cell-attachment period prior to implantation, with or without an extracellular matrix-based seeding suspension, on cell engraftment and osteogenesis. When quantitatively compared, the in-house scaffold implanted immediately had a higher mean radiopacity than in-house scaffolds incubated overnight. Both scaffold types implanted immediately had significantly higher area fractions of donor cells, while the in-house collagen-HA scaffolds implanted immediately had higher area fractions of the mineralization label compared with groups incubated overnight. When the cell loading was compared in vitro for each delivery method using the in-house scaffold, immediate loading led to higher numbers of delivered cells. Immediate loading may be preferable in order to ensure robust bone formation in vivo. The use of a secondary ECM carrier improved the distribution of donor cells only when a pre-attachment period was applied. These results have improved our understanding of cell delivery to bony defects in the context of in vivo outcomes.

Quality of preimplantation embryos recovered in vivo from dairy cows in relation to their body condition.

PubMed

Makarevich, A V; Stádník, L; Kubovičová, E; Hegedüšová, Z; Holásek, R; Louda, F; Beran, J; Nejdlová, M

2016-06-01

This study examined the impact of cow body condition on the quality of bovine preimplantation embryos. The embryos (n = 107) were flushed from dairy cows and classified according to a five-point scale body condition score (BCS2 n = 17; BCS3 n = 31; BCS4 n = 11) on the 7th day after insemination and then analyzed for development, dead cell index (DCI), cell number and actin cytoskeleton quality. The highest embryo recovery rate (P < 0.05) was recorded in the BCS3 group and the lowest in the BCS4 group. More transferable (morula, blastocyst) embryos were obtained from the BCS4 cows (79%), compared with the BCS2 (64%) or BCS3 (63%) animals. However, cell numbers were higher in the BCS2 and BCS3 groups (P < 0.05) compared with the BCS4 embryos. Conversely, the DCI was lowest in the BCS2 (3.88%; P < 0.05) and highest in the BCS4 (6.56%) embryos. The proportion of embryos with the best actin quality (grade I) was higher in the BCS2 and BCS3 cows compared with the BCS4 group. Almost 25% of all embryos showed fragmented morphology and a higher DCI (5.65%) than normal morulas (1.76%). More fragmented embryos were revealed in the BCS2 (28.6%) and BCS4 (31.25%) groups, and less (19.15%) in the BCS3 group. The cell numbers in such embryos were lower in the BCS4 (22.57) than in the BCS2 (46.25) or BCS3 (42.4) groups. In conclusion, the body condition of dairy cows affects the quality of preimplantation embryos. A BCS over 3.0 resulted in a higher incidence of poor (fragmented) embryos.

Non-small cell lung carcinoma therapy using mTOR-siRNA.

PubMed

Matsubara, Hirochika; Sakakibara, Kenji; Kunimitsu, Tamo; Matsuoka, Hiroyasu; Kato, Kaori; Oyachi, Noboru; Dobashi, Yoh; Matsumoto, Masahiko

2012-01-01

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition.

Non-small cell lung carcinoma therapy using mTOR-siRNA

PubMed Central

Matsubara, Hirochika; Sakakibara, Kenji; Kunimitsu, Tamo; Matsuoka, Hiroyasu; Kato, Kaori; Oyachi, Noboru; Dobashi, Yoh; Matsumoto, Masahiko

2012-01-01

Molecular targeting agents play important roles in non-small-cell lung cancer (NSCLC) therapy. Published studies have investigated new drugs categorized as molecular targeting agents that inhibit the mammalian target of rapamycin (mTOR). We focused on a small interfering RNA (siRNA) that specifically inhibits mTOR and has fewer side effects. To evaluate the antitumor effects of the siRNA, cell proliferation, apoptosis, and migration were assessed. In the study group, the siRNA was transfected into NSCLC cells. The number of cells present after 6 days of culture was counted to determine changes in cell proliferation. The level of apoptosis was evaluated by the detection of DNA-histone complexes in the cytoplasmic fraction using an absorption spectrometer. Changes in migration were evaluated by calculating the number of cells that passed through a specific filter using a commercial chemotaxis assay kit. mTOR-siRNA transfection inhibited cell proliferation as indicated by 37.3% (p = 0.034) decrease in the number of cells compared with the control cells. Analysis of the level of apoptosis in NSCLC cells revealed 16.7% (p = 0.016) increase following mTOR-siRNA transfection, and mTOR-siRNA transfection significantly inhibited cell migration by 39.2% (p = 0.0001). We confirmed that mTOR-siRNA induces apoptosis and inhibits the proliferation and migration of NSCLC cells in vitro. Further studies using mTOR-siRNA may aid in the development of an alternative therapy that maximizes the antineoplastic effect of mTOR inhibition. PMID:22400071

Tunneling Nanotubes are Novel Cellular Structures That Communicate Signals Between Trabecular Meshwork Cells.

PubMed

Keller, Kate E; Bradley, John M; Sun, Ying Ying; Yang, Yong-Feng; Acott, Ted S

2017-10-01

The actin cytoskeleton of trabecular meshwork (TM) cells plays a role in regulating aqueous humor outflow. Many studies have investigated stress fibers, but F-actin also assembles into other supramolecular structures including filopodia. Recently, specialized filopodia called tunneling nanotubes (TNTs) have been described, which communicate molecular signals and organelles directly between cells. Here, we investigate TNT formation by TM cells. Human TM cells were labeled separately with the fluorescent dyes, DiO and DiD, or with mitochondrial dye. Fixed or live TM cells were imaged using confocal microscopy. Image analysis software was used to track fluorescent vesicles and count the number and length of filopodia. The number of fluorescently labeled vesicles transferred between cells was counted in response to specific inhibitors of the actin cytoskeleton. Human TM tissue was stained with phalloidin. Live-cell confocal imaging of cultured TM cells showed transfer of fluorescently labeled vesicles and mitochondria via TNTs. In TM tissue, a long (160 μm) actin-rich cell process bridged an intertrabecular space and did not adhere to the substratum. Treatment of TM cells with CK-666, an Arp2/3 inhibitor, significantly decreased the number and length of filopodia, decreased transfer of fluorescently labeled vesicles and induced thick stress fibers compared to vehicle control. Conversely, inhibiting stress fibers using Y27632 increased transfer of vesicles and induced long cell processes. Identification of TNTs provides a means by which TM cells can directly communicate with each other over long distances. This may be particularly important to overcome limitations of diffusion-based signaling in the aqueous humor fluid environment.

Cryopreservation of Circulating Tumor Cells for Enumeration and Characterization.

PubMed

Nejlund, Sarah; Smith, Julie; Kraan, Jaco; Stender, Henrik; Van, Mai N; Langkjer, Sven T; Nielsen, Mikkel T; Sölétormos, György; Hillig, Thore

2016-08-01

A blood sample containing circulating tumor cells (CTCs) may serve as a surrogate for metastasis in invasive cancer. Cryopreservation will provide new opportunities in management of clinical samples in the laboratory and allow collection of samples over time for future analysis of existing and upcoming cancer biomarkers. Blood samples from healthy volunteers were spiked with high (∼500) and low (∼50) number of tumor cells from culture. The samples were stored at -80C with cryopreservative dimethyl sulfoxide mixed with Roswell Park Memorial Institute 1640 medium. Flow cytometry tested if cryopreservation affected specific biomarkers regularly used to detect CTCs, i.e. cytokeratin (CK) and epithelial cell adhesion molecule (EpCAM) and white blood cell specific lymphocyte common antigen (CD45). After various time intervals (up to 6 months), samples were thawed and tumor cell recovery (enumeration) was examined. Clinical samples may differ from cell line studies, so the cryopreservation protocol was tested on 17 patients with invasive breast cancer and tumor cell recovery was examined. Two blood samples were drawn from each patient. Biomarkers, CK, CD45, and EpCAM, were not affected by the freezing and thawing procedures. Cryopreserved samples (n = 2) spiked with a high number of tumor cells (∼500) had a ∼90% recovery compared with the spiked fresh samples. In samples spiked with lower numbers of tumor cells (median = 43 in n = 5 samples), the recovery was 63% after cryopreservation (median 27 tumor cells), p = 0.03. With an even lower number of spiked tumor cells (median = 3 in n = 8 samples), the recovery rate of tumor cells after cryopreservation did not seem to be affected (median = 8), p = 0.09. Time of cryopreservation did not affect recovery. When testing the effect of cryopreservation on enumeration in clinical samples, no difference was observed in the number of CTCs between the fresh and the cryopreserved samples based on n = 17 pairs, p = 0.83; however, the variation was large. This large variation was confirmed by clinically paired fresh samples (n = 64 pairs), where 95% of the samples (<30 CTCs) vary in number up to ±15 CTCs, p = 0.18. A small loss of CTCs after cryopreservation may be expected; however, cryopreservation of CTCs for biomarker characterization for clinical applications seems promising.

Changes in the inner ear structures in cystic fibrosis patients.

PubMed

Pauna, Henrique F; Monsanto, Rafael C; Kurata, Natsuko; Paparella, Michael M; Cureoglu, Sebahattin

2017-01-01

Although prolonged use of antibiotics is very common in cystic fibrosis (CF) patients, no studies have assessed the changes in both cochlear and peripheral vestibular systems in this population. We used human temporal bones to analyze the density of vestibular dark, transitional, and hair cells in specimens from CF patients who were exposed to several types of antibiotics, as compared with specimens from an age-matched control group with no history of ear disease or antibiotic use. Additionally, we analyzed the changes in the elements of the cochlea (hair cells, spiral ganglion neurons, and the area of the stria vascularis). Data was gathered using differential interference contrast microscopy and light microscopy. In the CF group, 83% of patients were exposed to some ototoxic drugs, such as aminoglycosides. As compared with the control group, the density of both type I and type II vestibular hair cells was significantly lower in all structures analyzed; the number of dark cells was significantly lower in the lateral and posterior semicircular canals. We noted a trend toward a lower number of both inner and outer cochlear hair cells at all turns of the cochlea. The number of spiral ganglion neurons in Rosenthal's canal at the apical turn of the cochlea was significantly lower; furthermore, the area of the stria vascularis at the apical turn of the cochlea was significantly smaller. Deterioration of cochlear and vestibular structures in CF patients might be related to their exposure to ototoxic antibiotics. Well-designed case-control studies are necessary to rule out the effect of CF itself. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Changes in the Inner Ear Structures in Cystic Fibrosis Patients

PubMed Central

Pauna, Henrique F.; Monsanto, Rafael C.; Kurata, Natsuko; Paparella, Michael M.; Cureoglu, Sebahattin

2016-01-01

Objective Although prolonged use of antibiotics is very common in cystic fibrosis (CF) patients, no studies have assessed the changes in both cochlear and peripheral vestibular systems in this population. Methods We used human temporal bones to analyze the density of vestibular dark, transitional, and hair cells in specimens from CF patients who were exposed to several types of antibiotics, as compared with specimens from an age-matched control group with no history of ear disease or antibiotic use. Additionally, we analyzed the changes in the elements of the cochlea (hair cells, spiral ganglion neurons, and the area of the stria vascularis). Data was gathered using differential interference contrast microscopy and light microscopy. Results In the CF group, 83% of patients were exposed to some ototoxic drugs, such as aminoglycosides. As compared with the control group, the density of both type I and type II vestibular hair cells was significantly lower in all structures analyzed; the number of dark cells was significantly lower in the lateral and posterior semicircular canals. We noted a trend toward a lower number of both inner and outer cochlear hair cells at all turns of the cochlea. The number of spiral ganglion neurons in Rosenthal’s canal at the apical turn of the cochlea was significantly lower; furthermore, the area of the stria vascularis at the apical turn of the cochlea was significantly smaller. Conclusions Deterioration of cochlear and vestibular structures in CF patients might be related to their exposure to ototoxic antibiotics. Well-designed case-control studies are necessary to rule out the effect of CF itself. PMID:28012509

Programmed death ligand 1 expression and CD8+ tumor-infiltrating lymphocyte density differences between paired primary and brain metastatic lesions in non-small cell lung cancer.

PubMed

Zhou, Jie; Gong, Zhihua; Jia, Qingzhu; Wu, Yan; Yang, Zhen-Zhou; Zhu, Bo

2018-04-15

Immunotherapy targeting the programmed cell death-1/programmed death ligand 1(PD-L1) pathway has shown promising antitumor activity in brain metastases (BMs) of non-small cell lung cancer (NSCLC) patients with an acceptable safety profile; however, the response rates often differ between primary lesions and intracranial lesions. Studies are necessary to identify detailed characterizations of the response biomarkers. In this study, we aimed to compare the differences of PD-L1 expression and CD8 + tumor-infiltrating lymphocyte (TIL) density, two major response biomarkers of PD-1/PD-L1 blockade, between paired primary and brain metastatic lesions in advanced NSCLC. We observed that among primary lesions or BMs, only a small number of patients harbored common PD-L1 expression on both tumor cells and tumor-infiltrating immune cells. Additionally, we found that the numbers of CD8 + TILs were significantly fewer in BMs than in primary lung cancers. Low stromal CD8 + TIL numbers in BMs were associated with significantly shorter overall survival compared to high stromal CD8 + TIL counts. Notably, we demonstrated a discrepancy in PD-L1 expression and CD8 + TIL density between primary lung cancers and their corresponding BMs. Such heterogeneities are significantly associated with the time at which BMs occurred. Our study emphasizes the spatial and temporal heterogeneity of biomarkers for anti-PD-1/PD-L1 therapy, which should be concerned in clinical practice. Copyright © 2018 Elsevier Inc. All rights reserved.

How to count cells: the advantages and disadvantages of the isotropic fractionator compared with stereology.

PubMed

Herculano-Houzel, Suzana; von Bartheld, Christopher S; Miller, Daniel J; Kaas, Jon H

2015-04-01

The number of cells comprising biological structures represents fundamental information in basic anatomy, development, aging, drug tests, pathology and genetic manipulations. Obtaining unbiased estimates of cell numbers, however, was until recently possible only through stereological techniques, which require specific training, equipment, histological processing and appropriate sampling strategies applied to structures with a homogeneous distribution of cell bodies. An alternative, the isotropic fractionator (IF), became available in 2005 as a fast and inexpensive method that requires little training, no specific software and only a few materials before it can be used to quantify total numbers of neuronal and non-neuronal cells in a whole organ such as the brain or any dissectible regions thereof. This method entails transforming a highly anisotropic tissue into a homogeneous suspension of free-floating nuclei that can then be counted under the microscope or by flow cytometry and identified morphologically and immunocytochemically as neuronal or non-neuronal. We compare the advantages and disadvantages of each method and provide researchers with guidelines for choosing the best method for their particular needs. IF is as accurate as unbiased stereology and faster than stereological techniques, as it requires no elaborate histological processing or sampling paradigms, providing reliable estimates in a few days rather than many weeks. Tissue shrinkage is also not an issue, since the estimates provided are independent of tissue volume. The main disadvantage of IF, however, is that it necessarily destroys the tissue analyzed and thus provides no spatial information on the cellular composition of biological regions of interest.

Chlamydia trahomatis in cell culture. I. Comparison of efficiencies of infection in several chemically defined media, at various pH and temperature values, and after exposure to diethylaminoethyl-dextran

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rota, T.R.; Nichols, R.L.

1973-10-01

Three chemically defined cell culture media, Eagle minimum essential medium (MEM) with Earle basal salt solution, Eagle MEM with Hanks basal salt solution, and a modified Eagle MEM, were tested and found capable of supporting the development of Chlamydia trachomatis in /sup 60/Co-treated McCoy cells. The enhancement of trachoma infection by diethylaminoethyl-dextran (DEAE-D) was greater at pH values closer to neutrality than at and other pH values measured at the start of the experiments. Centrifugation of the trachoma inoculum onto cell monolayers at 33 C increased the number of inclusions when compared to centrifugation at 20 C. When the inoculummore » was centrifuged onto cell monolayers and subsequent incubation was at temperatures ranging from 34 to 39 C, the greatest number of inclusions was observed after incubation from 35 through 37 C. Enhancement of the trachoma infection by DEAE-D was tested at temperatures ranging from 35 to 37 C. These cultures had three- to fivefold increases in inclusions when compared to previously reported experiments in which DEAE-D- treated cultures were incubated at 34 C. (auth)« less

Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice.

PubMed

Li, D; Yee, J A; Thompson, L U; Yan, L

1999-07-19

We investigated the effect of dietary supplementation with secoisolariciresinol diglycoside (SDG), a lignan precursor isolated from flaxseed, on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147 or 293 micromol/kg (equivalent to SDG provided in the 2.5, 5 or 10% flaxseed diet). Mice were fed the diet for 2 weeks before and after an intravenous injection of 0.6 x 10(5) tumor cells. At necropsy, the number and size of tumors that formed in the lungs were determined. The median number of tumors in the control group was 62, and those in the SDG-supplemented groups were 38, 36 and 29, respectively. The last was significantly different from the control (P < 0.01). Dietary supplementation with SDG at 73, 147 and 293 micromol/kg also decreased tumor size (tumor cross-sectional area and volume) in a dose-dependent manner compared with the control values. These results show that SDG reduced pulmonary metastasis of melanoma cells and inhibited the growth of metastatic tumors that formed in the lungs. It is concluded that dietary supplementation with SDG reduces experimental metastasis of melanoma cells in mice.

Target sequencing and CRISPR/Cas editing reveal simultaneous loss of UTX and UTY in urothelial bladder cancer.

PubMed

Ahn, Jinwoo; Kim, Kwang Hyun; Park, Sanghui; Ahn, Young-Ho; Kim, Ha Young; Yoon, Hana; Lee, Ji Hyun; Bang, Duhee; Lee, Dong Hyeon

2016-09-27

UTX is a histone demethylase gene located on the X chromosome and is a frequently mutated gene in urothelial bladder cancer (UBC). UTY is a paralog of UTX located on the Y chromosome. We performed target capture sequencing on 128 genes in 40 non-metastatic UBC patients. UTX was the most frequently mutated gene (30%, 12/40). Of the genetic alterations identified, 75% were truncating mutations. UTY copy number loss was detected in 8 male patients (22.8%, 8/35). Of the 9 male patients with UTX mutations, 6 also had copy number loss (66.7%). To evaluate the functional roles of UTX and UTY in tumor progression, we designed UTX and UTY single knockout and UTX-UTY double knockout experiments using a CRISPR/Cas9 lentiviral system, and compared the proliferative capacities of two UBC cell lines in vitro. Single UTX or UTY knockout increased cell proliferation as compared to UTX-UTY wild-type cells. UTX-UTY double knockout cells exhibited greater proliferation than single knockout cells. These findings suggest both UTX and UTY function as dose-dependent suppressors of UBC development. While UTX escapes X chromosome inactivation in females, UTY may function as a male homologue of UTX, which could compensate for dosage imbalances.

I 5683 you: dialing phone numbers on cell phones activates key-concordant concepts.

PubMed

Topolinski, Sascha

2011-03-01

When people perform actions, effects associated with the actions are activated mentally, even if those effects are not apparent. This study tested whether sequences of simulations of virtual action effects can be integrated into a meaning of their own. Cell phones were used to test this hypothesis because pressing a key on a phone is habitually associated with both digits (dialing numbers) and letters (typing text messages). In Experiment 1, dialing digit sequences induced the meaning of words that share the same key sequence (e.g., 5683, LOVE). This occurred even though the letters were not labeled on the keypad, and participants were not aware of the digit-letter correspondences. In Experiment 2, subjects preferred dialing numbers implying positive words (e.g., 37326, DREAM) over dialing numbers implying negative words (e.g., 75463, SLIME). In Experiment 3, subjects preferred companies with phone numbers implying a company-related word (e.g., LOVE for a dating agency, CORPSE for a mortician) compared with companies with phone numbers implying a company-unrelated word.

A Comparative Morphometric Analysis of Three Cranial Nerves in Two Phocids: The Hooded Seal (Cystophora cristata) and the Harbor Seal (Phoca vitulina).

PubMed

Wohlert, Dennis; Kröger, Jürgen; Witt, Martin; Schmitt, Oliver; Wree, Andreas; Czech-Damal, Nicole; Siebert, Ursula; Folkow, Lars; Hanke, Frederike D

2016-03-01

While our knowledge about the senses of pinnipeds has increased over the last decades almost nothing is known about the organization of the neuroanatomical pathways. In a first approach to this field of research, we assessed the total number of myelinated axons of three cranial nerves (CNs) in the harbor (Phoca vitulina, Pv) and hooded seal (Cystophora cristata, Cc). Axons were counted in semithin sections of the nerves embedded in Epon and stained with toluidine blue. In both species, the highest axon number was found within the optic nerve (Pv 187,000 ± 8,000 axons, Cc 481,600 ± 1,300 axons). Generally, considering absolute axon numbers, far more axons were counted within the optic and trigmenial nerve (Pv 136,700 ± 2,500 axons, Cc 179,300 ± 6,900 axons) in hooded in comparison to harbor seals. The axon counts of the vestibulocochlear nerve are nearly identical for both species (Pv 87,100 ± 8,100 axons, Cc 86,600 ± 2,700 axons). However, when comparing cell density, the cell density is almost equal for all nerves for both species except for the optic nerve in which cell density was particularly higher than in the other nerves and higher in hooded in comparison to harbor seals. We here present the first comparative analysis of three CNs in two phocid seals. While the CNs of these closely related species share some general characteristics, pronounced differences in axon numbers/densities are apparent. These differences seem to reflect differences in e.g. size, habitat, and/or functional significance of the innervated sensory systems. © 2015 Wiley Periodicals, Inc.

Development of an Ammonium Sulfate DNA Extraction Method for Obtaining Amplifiable DNA in a Small Number of Cells and Its Application to Clinical Specimens

PubMed Central

Oh, Seo Young; Kim, Wook Youn; Hwang, Tae Sook; Han, Hye Seung; Lim, So Dug; Kim, Wan Seop

2013-01-01

DNA extraction from microdissected cells has become essential for handling clinical specimens with advances in molecular pathology. Conventional methods have limitations for extracting amplifiable DNA from specimens containing a small number of cells. We developed an ammonium sulfate DNA extraction method (A) and compared it with two other methods (B and C). DNA quality and quantity, β-globin amplification, and detectability of two cancer associated gene mutations were evaluated. Method A showed the best DNA yield, particularly when the cell number was very low. Amplification of the β-globin gene using DNA from the SNU 790 cell line and papillary thyroid carcinoma (PTC) cells extracted with Method A demonstrated the strongest band. BRAF V600E mutation analysis using ethanol-fixed PTC cells from a patient demonstrated both a “T” peak increase and an adjacent “A” peak decrease when 25 and 50 cells were extracted, whereas mutant peaks were too low to be analyzed using the other two methods. EGFR mutation analysis using formalin-fixed paraffin-embedded lung cancer tissues demonstrated a mutant peak with Method A, whereas the mutant peak was undetectable with Methods B or C. Method A yielded the best DNA quantity and quality with outstanding efficiency, particularly when paucicellular specimens were used. PMID:23691506

Cryopreservation of Human Mucosal Leukocytes.

PubMed

Hughes, Sean M; Shu, Zhiquan; Levy, Claire N; Ferre, April L; Hartig, Heather; Fang, Cifeng; Lentz, Gretchen; Fialkow, Michael; Kirby, Anna C; Adams Waldorf, Kristina M; Veazey, Ronald S; Germann, Anja; von Briesen, Hagen; McElrath, M Juliana; Dezzutti, Charlene S; Sinclair, Elizabeth; Baker, Chris A R; Shacklett, Barbara L; Gao, Dayong; Hladik, Florian

2016-01-01

Understanding how leukocytes in the cervicovaginal and colorectal mucosae respond to pathogens, and how medical interventions affect these responses, is important for developing better tools to prevent HIV and other sexually transmitted infections. An effective cryopreservation protocol for these cells following their isolation will make studying them more feasible. To find an optimal cryopreservation protocol for mucosal mononuclear leukocytes, we compared cryopreservation media and procedures using human vaginal leukocytes and confirmed our results with endocervical and colorectal leukocytes. Specifically, we measured the recovery of viable vaginal T cells and macrophages after cryopreservation with different cryopreservation media and handling procedures. We found several cryopreservation media that led to recoveries above 75%. Limiting the number and volume of washes increased the fraction of cells recovered by 10-15%, possibly due to the small cell numbers in mucosal samples. We confirmed that our cryopreservation protocol also works well for both endocervical and colorectal leukocytes. Cryopreserved leukocytes had slightly increased cytokine responses to antigenic stimulation relative to the same cells tested fresh. Additionally, we tested whether it is better to cryopreserve endocervical cells on the cytobrush or in suspension. Leukocytes from cervicovaginal and colorectal tissues can be cryopreserved with good recovery of functional, viable cells using several different cryopreservation media. The number and volume of washes has an experimentally meaningful effect on the percentage of cells recovered. We provide a detailed, step-by-step protocol with best practices for cryopreservation of mucosal leukocytes.

The sow endosalpinx at different stages of the oestrous cycle and at anoestrus: studies on morphological changes and infiltration by cells of the immune system.

PubMed

Jiwakanon, J; Persson, E; Kaeoket, K; Dalin, A-M

2005-02-01

The aim of this study was to investigate the morphological changes of the sow endosalpinx and the distribution of leukocytes throughout the oestrous cycle and at anoestrus. Nineteen crossbred sows (Swedish Landrace x Swedish Yorkshire) at late dioestrus (three), prooestrus (three), oestrus (three), early dioestrus (three), dioestrus (three) and anoestrus (four) were used. Oviductal samples from three different parts (isthmus, ampulla and infundibulum), taken immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue or stored in a freezer at -70 degrees C until analysed by immunohistochemistry (prooestrus and anoestrus) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. During all stages, a lower degree of morphological changes (pseudostratification, mitosis and secretory granules) was found in the isthmus compared with ampulla and infundibulum. In ampulla and infundibulum, pseudostratification, mitotic activity and secretory granules of the epithelium were high at prooestrus/oestrus. Cytoplasmic protrusions of epithelial cells with some extruded nuclei were prominent in ampulla and infundibulum at all stages except for oestrus and early dioestrus. Lymphocytes as well as CD2- and CD3-positive cells were the predominant immune cells in the epithelial layer. The numbers of lymphocytes and CD3-positive cells did not differ among segments and stages. Numbers of CD2-positive cells did not differ between prooestrus and anoestrus while the numbers were significantly higher in the infundibulum than in ampulla and isthmus. Neutrophils were only occasionally found and mainly in the infundibulum. In the subepithelial connective tissue layer, the two most commonly observed immune cell types were lymphocytes and plasma cells. The numbers of lymphocytes as well as CD2- and CD3-positive cells was lower in isthmus than in the other segments (p < or = 0.001). Higher numbers of plasma cells (p < or = 0.001) were found in infundibulum than in ampulla and isthmus. The numbers of lymphocytes and plasma cells were not significantly different between stages of the oestrous cycle. However, the number of neutrophils differed and were highest at prooestrus in ampulla and infundibulum. The numbers of CD2-, CD3- and CD79-positive cells did not differ between prooestrus and anoestrus whereas for CD14- and SWC3-positive cells, the numbers were higher at prooestrus (p < or = 0.05) than at anoestrus. In the oviduct, the morphology differed in ampulla and infundibulum with oestrous cycle stages, which indicates an effect by ovarian steroid hormones. The immune cell infiltration was less influenced by cyclic changes. However, the immune cell infiltration (in the connective tissue) in the upper part, especially infundibulum, differed significantly from the one in the lower part, isthmus, indicating different immune functions within various parts of the oviduct.

Engineering cord blood to improve engraftment after cord blood transplant

PubMed Central

Dave, Hema; Bollard, Catherine M.; Shpall, Elizabeth J.

2017-01-01

Umbilical cord blood transplant (CBT) has traditionally been associated with slower engraftment of neutrophils, delayed immune reconstitution and consequently higher risk of infections as compared with peripheral blood progenitor cell (PBPC) or bone marrow (BM) transplants. This is primarily due to low numbers of total nucleated cells (TNCs) and the naive nature of CB immune cells. The use of double unit CB transplant (DCBT) increases the total cell dose in the graft, but it still does not produce as rapid engraftment as seen with PBPC or even BM transplants. Herein, we discuss strategies to improve engraftment after CBT. We describe methods of (I) expansion of CB graft ex vivo to increase the total cell dose; and (II) enhancement of BM homing capability of CB progenitor cells; (III) ex vivo expansion of CB derived T cells for improving T cell function against viruses, tumors and protection from graft versus host disease (GVHD). With these novel approaches, engraftment after CBT is now reaching levels comparable to that of other graft types. PMID:28607915

Comparison of autogeneic and allogeneic natural killer cells immunotherapy on the clinical outcome of recurrent breast cancer

PubMed Central

Liang, Shuzhen; Xu, Kecheng; Niu, Lizhi; Wang, Xiaohua; Liang, Yingqing; Zhang, Mingjie; Chen, Jibing; Lin, Mao

2017-01-01

In the present study, we aimed to compare the clinical outcome of autogeneic and allogeneic natural killer (NK) cells immunotherapy for the treatment of recurrent breast cancer. Between July 2016 and February 2017, 36 patients who met the enrollment criteria were randomly assigned to two groups: autogeneic NK cells immunotherapy group (group I, n=18) and allogeneic NK cells immunotherapy group (group II, n=18). The clinical efficacy, quality of life, immune function, circulating tumor cell (CTC) level, and other related indicators were evaluated. We found that allogeneic NK cells immunotherapy has better clinical efficacy than autogeneic therapy. Moreover, allogeneic NK cells therapy improves the quality of life, reduces the number of CTCs, reduces carcinoembryonic antigen and cancer antigen 15-3 (CA15-3) expression, and significantly enhances immune function. To our knowledge, this is the first clinical trial to compare the clinical outcome of autogeneic and allogeneic NK cells immunotherapy for recurrent breast cancer. PMID:28894383

Effect of PDGF-BB combined with EDTA gel on adhesion and proliferation to the root surface.

PubMed

Belal, Mahmoud Helmy; Watanabe, Hisashi; Ichinose, Shizuko; Ishikawa, Isao

2012-07-01

Periodontal regeneration using EDTA or PDGF showed promising results, but the effect of combined application was still unclear. This study aimed to verify the effect of EDTA and/or PDGF application on root adhesion and proliferation of PDL fibroblast cells. Eighty specimens were prepared from forty periodontitis teeth and made five groups: (1) diseased (untreated), (2) SRP (scaling root planing), (3) EDTA (24%), (4) PDGF (25 ng/ml) and (5) Combined application of EDTA and PDGF. Periodontal ligament cells were cultured on the above conditioned dentin plate, and SEM examination was preformed and cells were counted within a representative standard area for both cell morphology and density. All groups including untreated showed significantly increase of adhered cells from baseline to 7 days. Among them, rate of increase was much higher in EDTA, PDGF, and combined groups. ANOVA test indicated that the number of cells in PDGF and combined groups was significantly higher than diseased group at 1 day. On day 7, PDGF and combined groups showed significantly higher number of adhesion cells than that found in the diseased, SRP and EDTA groups. Thus, root conditioning with EDTA enhanced cell adhesion more than SRP alone. There was no significant difference of cell number between PDGF and combined group. Combined application of EDTA and PDGF increased significantly PDL cell adhesion than EDTA alone. PDGF alone, however, also showed comparable effect to combined application at all periods. Thus, synergistic effect between PDGF and EDTA was not observed.

High levels of FOXP3⁺ regulatory T cells in gastric MALT lymphoma predict responsiveness to Helicobacter pylori eradication.

PubMed

Iwaya, Yugo; Kobayashi, Motohiro; Momose, Masanobu; Hiraoka, Nobuyoshi; Sakai, Yasuhiro; Akamatsu, Taiji; Tanaka, Eiji; Ohtani, Haruo; Fukuda, Minoru; Nakayama, Jun

2013-10-01

Although Helicobacter pylori eradication is a first-line treatment of gastric MALT lymphoma, roughly 25% of patients do not respond to treatment. CD4⁺ FOXP3⁺ regulatory T (Treg) cells regulate immune responses in physiological conditions and various inflammatory conditions, including H. pylori-associated diseases. Our goal was to determine how Treg cells affect responsiveness to H. pylori eradication therapy. We performed dual immunohistochemistry for CD4 and FOXP3 to evaluate the prevalence of FOXP3⁺ Treg cells in the stomach of 63 patients with MALT lymphoma and 55 patients with chronic active gastritis. Receiver operating characteristic analysis was carried out to determine the best cut-off point in differentiating H. pylori eradication responders from nonresponders. Both the FOXP3⁺/CD4⁺ cell ratio and the absolute number of FOXP3⁺ cells per high-power field in MALT lymphoma were significantly greater in H. pylori eradication responders compared with nonresponders, suggesting that Treg cells function in regression mechanisms of MALT lymphomas. Cut-off points with good sensitivities and specificities were obtained to predict eradication outcome. A high number of Treg cells or a high ratio of Treg cells to the total number of CD4⁺ T cells in gastric MALT lymphoma could predict responsiveness to eradication therapy. © 2013 John Wiley & Sons Ltd.

Buccal Micronucleus Cytome Assay in Sickle Cell Disease

PubMed Central

Naga, Mallika Bokka Sri Satya; Gour, Shreya; Nallagutta, Nalini; Velidandla, Surekha; Manikya, Sangameshwar

2016-01-01

Introduction Sickle Cell Anaemia (SCA) is a commonly inherited blood disorder preceded by episodes of pain, chronic haemolytic anaemia and severe infections. The underlying phenomenon which causes this disease is the point mutation in the haemoglobin beta gene (Hbβ) found on chromosome 11 p. Increased oxidative stress leads to DNA damage. DNA damage occurring in such conditions can be studied by the buccal micronucleus cytome assay, which is a minimally invasive method for studying chromosomal instability, cell death and regenerative potential of human buccal tissue. Aim To evaluate genomic instability in patients with sickle cell disease by buccal micronucleus cytome assay. Materials and Methods The study included 40 sickle cell anemia patients (Group A) and 40 age and sex matched controls (Group B). Buccal swabs were collected and stained with Papanicolaou (PAP). Number of cells with micronucleus, binuclei, nuclear bud, pyknosis and karyolysis were counted in two groups as parameters for the evaluation of genome stability. Results All the analysis was done using t-test. A p-value of <0.001 was considered statistically significant. There was a statistically significant increase in micronuclei number in SCA patients when compared with controls. Karyolytic (un-nucleated) cell number in Group A was more than to those of the controls. Conclusion The results might suggest that patients with sickle cell anaemia have genome instability which is represented by the presence of micronuclei in the somatic cells. Presence of apoptotic cells might only indicate the bodily damage to the tissue as a result of the disease. PMID:27504413

Buccal Micronucleus Cytome Assay in Sickle Cell Disease.

PubMed

Naga, Mallika Bokka Sri Satya; Gour, Shreya; Nallagutta, Nalini; Ealla, Kranti Kiran Reddy; Velidandla, Surekha; Manikya, Sangameshwar

2016-06-01

Sickle Cell Anaemia (SCA) is a commonly inherited blood disorder preceded by episodes of pain, chronic haemolytic anaemia and severe infections. The underlying phenomenon which causes this disease is the point mutation in the haemoglobin beta gene (Hbβ) found on chromosome 11 p. Increased oxidative stress leads to DNA damage. DNA damage occurring in such conditions can be studied by the buccal micronucleus cytome assay, which is a minimally invasive method for studying chromosomal instability, cell death and regenerative potential of human buccal tissue. To evaluate genomic instability in patients with sickle cell disease by buccal micronucleus cytome assay. The study included 40 sickle cell anemia patients (Group A) and 40 age and sex matched controls (Group B). Buccal swabs were collected and stained with Papanicolaou (PAP). Number of cells with micronucleus, binuclei, nuclear bud, pyknosis and karyolysis were counted in two groups as parameters for the evaluation of genome stability. All the analysis was done using t-test. A p-value of <0.001 was considered statistically significant. There was a statistically significant increase in micronuclei number in SCA patients when compared with controls. Karyolytic (un-nucleated) cell number in Group A was more than to those of the controls. The results might suggest that patients with sickle cell anaemia have genome instability which is represented by the presence of micronuclei in the somatic cells. Presence of apoptotic cells might only indicate the bodily damage to the tissue as a result of the disease.

Histiocyte-like cells expressing factor XIIIa do not belong to the neoplastic cell population in malignant fibrous histiocytoma.

PubMed

Szollosi, Zoltan; Nemeth, Tamas; Egervari, Kristof; Nemes, Zoltan

2005-01-01

The term malignant fibrous histiocytoma (MFH) is widely used for pleomorphic soft tissue sarcomas without a specific line of differentiation. MFH is included in the category of fibrohistiocytic soft tissue tumors. MFH has a broad range of histological appearances, and it has several subtypes. All of these subtypes are composed of spindled fibroblast-like cells, undifferentiated cells, and histiocytic or histiocyte-like cells. A large number of fibroblast-like and pleomorphic cells express factor XIIIa in MFH. The cytological pleomorphism of factor XIIIa cells suggests that these cells may belong to the neoplastic population. It is equally possible that the factor XIIIa-positive cells are only activated stromal cells. The relation of factor XIIIa-positive cells to the neoplastic cell population in MFH is addressed in the present study. A morphometric approach compares the measure of nuclear pleomorphism of the factor XIIIa-positive cells with that of the factor XIIIa-negative tumor cells in high-grade MFH. The immunohistochemical approach compares the factor XIIIa-positive and -negative cell populations with regard to mutations of p53 tumor suppressor gene in p53-positive MFH cases. We selected 58 cases of soft tissue pleomorphic or storiform-pleomorphic MFH on the basis of histopathological examinations. A combination of incident light immunofluorescence for factor XIIIa and transmitted light examination for nuclear staining was used for morphometrical analysis. We found cytoplasmic factor XIIIa positivity in at least 2% of cells in 39 cases; the number of factor XIIIa-positive cells was under 0.5% in two cases, and the number of factor-positive cells ranged between 0.5% and 2% in 13 cases. Eighteen cases were analyzed with nuclear morphometry. We found that mean nuclear area and mean nuclear Ferret diameter in factor XIIIa-positive cells differed significantly from those of the tumor cells in all cases. The mean nuclear roundness factor differed significantly only in four cases. The latter finding showed that the microscopic polymorphism of factor XIIIa cells is measurable and is not merely a suspicion. The immunohistochemical positivity for p53 positivity can be accepted as the manifestation of a missense mutation of TP53 gene and as a marker of neoplastic cells. The simultaneous immunohistochemical detection of factor XIIIa and p53 in the same section revealed that factor XIIIa-positive cells were invariably p53 negative in MFH. This finding implies that the factor XIIIa cell population is non-neoplastic and belongs to the stromal component of MFH.