Bifidobacterium breve attenuates murine dextran sodium sulfate-induced colitis and increases regulatory T cell responses.

PubMed

Zheng, Bin; van Bergenhenegouwen, Jeroen; Overbeek, Saskia; van de Kant, Hendrik J G; Garssen, Johan; Folkerts, Gert; Vos, Paul; Morgan, Mary E; Kraneveld, Aletta D

2014-01-01

While some probiotics have shown beneficial effects on preventing or treating colitis development, others have shown no effects. In this study, we have assessed the immunomodulating effects of two probiotic strains, Lactobacillus rhamnosus (L. rhamnosus) and Bifidobacterium breve (B. breve) on T cell polarization in vitro, using human peripheral blood mononuclear cells (PBMC), and in vivo, using murine dextran sodium sulfate (DSS) colitis model. With respect to the latter, the mRNA expression of T cell subset-associated transcription factors and cytokines in the colon was measured and the T helper type (Th) 17 and regulatory T cell (Treg) subsets were determined in the Peyer's patches. Both L. rhamnosus and B. breve incubations in vitro reduced Th17 and increased Th2 cell subsets in human PBMCs. In addition, B. breve incubation was also able to reduce Th1 and increase Treg cell subsets in contrast to L. rhamnosus. In vivo intervention with B. breve, but not L. rhamnosus, significantly attenuated the severity of DSS-induced colitis. In DSS-treated C57BL/6 mice, intervention with B. breve increased the expression of mRNA encoding for Th2- and Treg-associated cytokines in the distal colon. In addition, intervention with B. breve led to increases of Treg and decreases of Th17 cell subsets in Peyer's patches of DSS-treated mice. B. breve modulates T cell polarization towards Th2 and Treg cell-associated responses in vitro and in vivo. In vivo B. breve intervention ameliorates DSS-induced colitis symptoms and this protective effect may mediated by its effects on the T-cell composition.

Bifidobacterium breve Attenuates Murine Dextran Sodium Sulfate-Induced Colitis and Increases Regulatory T Cell Responses

PubMed Central

Zheng, Bin; van Bergenhenegouwen, Jeroen; Overbeek, Saskia; van de Kant, Hendrik J. G.; Garssen, Johan; Folkerts, Gert; Vos, Paul; Morgan, Mary E.; Kraneveld, Aletta D.

2014-01-01

While some probiotics have shown beneficial effects on preventing or treating colitis development, others have shown no effects. In this study, we have assessed the immunomodulating effects of two probiotic strains, Lactobacillus rhamnosus (L. rhamnosus) and Bifidobacterium breve (B. breve) on T cell polarization in vitro, using human peripheral blood mononuclear cells (PBMC), and in vivo, using murine dextran sodium sulfate (DSS) colitis model. With respect to the latter, the mRNA expression of T cell subset-associated transcription factors and cytokines in the colon was measured and the T helper type (Th) 17 and regulatory T cell (Treg) subsets were determined in the Peyer's patches. Both L. rhamnosus and B. breve incubations in vitro reduced Th17 and increased Th2 cell subsets in human PBMCs. In addition, B. breve incubation was also able to reduce Th1 and increase Treg cell subsets in contrast to L. rhamnosus. In vivo intervention with B. breve, but not L. rhamnosus, significantly attenuated the severity of DSS-induced colitis. In DSS-treated C57BL/6 mice, intervention with B. breve increased the expression of mRNA encoding for Th2- and Treg-associated cytokines in the distal colon. In addition, intervention with B. breve led to increases of Treg and decreases of Th17 cell subsets in Peyer's patches of DSS-treated mice. B. breve modulates T cell polarization towards Th2 and Treg cell-associated responses in vitro and in vivo. In vivo B. breve intervention ameliorates DSS-induced colitis symptoms and this protective effect may mediated by its effects on the T-cell composition. PMID:24787575

L1-mediated retrotransposition of murine B1 and B2 SINEs recapitulated in cultured cells.

PubMed

Dewannieux, Marie; Heidmann, Thierry

2005-06-03

SINEs are short interspersed nucleotide elements with transpositional activity, present at a high copy number (up to a million) in mammalian genomes. They are 80-400 bp long, non-coding sequences which derive either from the 7SL RNA (e.g. human Alus, murine B1s) or tRNA (e.g. murine B2s) polymerase III-driven genes. We have previously demonstrated that Alus very efficiently divert the enzymatic machinery of the autonomous L1 LINE (long interspersed nucleotide element) retrotransposons to transpose at a high rate. Here we show, using an ex vivo assay for transposition, that both B1 and B2 SINEs can be mobilized by murine LINEs, with the hallmarks of a bona fide retrotransposition process, including target site duplications of varying lengths and integrations into A-rich sequences. Despite different phylogenetic origins, transposition of the tRNA-derived B2 sequences is as efficient as that of the human Alus, whereas that of B1s is 20-100-fold lower despite a similar high copy number of these elements in the mouse genome. We provide evidence, via an appropriate nucleotide substitution within the B1 sequence in a domain essential for its intracellular targeting, that the current B1 SINEs are not optimal for transposition, a feature most probably selected for the host sake in the course of evolution.

Markers of murine embryonic and neural stem cells, neurons and astrocytes: reference points for developmental neurotoxicity testing

EPA Science Inventory

Developmental neurotoxicity (DNT) is a significant concern for environmental chemicals, as well as for food and drug constituents. The sensitivity of animal-based DNT models is unclear, and they are expensive and time consuming. Murine embryonic stem cells (mESC) recapitulate sev...

B cell activating factor is central to bleomycin- and IL-17-mediated experimental pulmonary fibrosis.

PubMed

François, Antoine; Gombault, Aurélie; Villeret, Bérengère; Alsaleh, Ghada; Fanny, Manoussa; Gasse, Paméla; Adam, Sylvain Marchand; Crestani, Bruno; Sibilia, Jean; Schneider, Pascal; Bahram, Seiamak; Quesniaux, Valérie; Ryffel, Bernhard; Wachsmann, Dominique; Gottenberg, Jacques-Eric; Couillin, Isabelle

2015-01-01

Idiopathic pulmonary fibrosis (IPF) is a progressive devastating, yet untreatable fibrotic disease of unknown origin. We investigated the contribution of the B-cell activating factor (BAFF), a TNF family member recently implicated in the regulation of pathogenic IL-17-producing cells in autoimmune diseases. The contribution of BAFF was assessed in a murine model of lung fibrosis induced by airway administered bleomycin. We show that murine BAFF levels were strongly increased in the bronchoalveolar space and lungs after bleomycin exposure. We identified Gr1(+) neutrophils as an important source of BAFF upon BLM-induced lung inflammation and fibrosis. Genetic ablation of BAFF or BAFF neutralization by a soluble receptor significantly attenuated pulmonary fibrosis and IL-1β levels. We further demonstrate that bleomycin-induced BAFF expression and lung fibrosis were IL-1β and IL-17A dependent. BAFF was required for rIL-17A-induced lung fibrosis and augmented IL-17A production by CD3(+) T cells from murine fibrotic lungs ex vivo. Finally we report elevated levels of BAFF in bronchoalveolar lavages from IPF patients. Our data therefore support a role for BAFF in the establishment of pulmonary fibrosis and a crosstalk between IL-1β, BAFF and IL-17A. Copyright © 2014 Elsevier Ltd. All rights reserved.

The Murine Factor H-Related Protein FHR-B Promotes Complement Activation.

PubMed

Cserhalmi, Marcell; Csincsi, Ádám I; Mezei, Zoltán; Kopp, Anne; Hebecker, Mario; Uzonyi, Barbara; Józsi, Mihály

2017-01-01

Factor H-related (FHR) proteins consist of varying number of complement control protein domains that display various degrees of sequence identity to respective domains of the alternative pathway complement inhibitor factor H (FH). While such FHR proteins are described in several species, only human FHRs were functionally investigated. Their biological role is still poorly understood and in part controversial. Recent studies on some of the human FHRs strongly suggest a role for FHRs in enhancing complement activation via competing with FH for binding to certain ligands and surfaces. The aim of the current study was the functional characterization of a murine FHR, FHR-B. To this end, FHR-B was expressed in recombinant form. Recombinant FHR-B bound to human C3b and was able to compete with human FH for C3b binding. FHR-B supported the assembly of functionally active C3bBb alternative pathway C3 convertase via its interaction with C3b. This activity was confirmed by demonstrating C3 activation in murine serum. In addition, FHR-B bound to murine pentraxin 3 (PTX3), and this interaction resulted in murine C3 fragment deposition due to enhanced complement activation in mouse serum. FHR-B also induced C3 deposition on C-reactive protein, the extracellular matrix (ECM) extract Matrigel, and endothelial cell-derived ECM when exposed to mouse serum. Moreover, mouse C3 deposition was strongly enhanced on necrotic Jurkat T cells and the mouse B cell line A20 by FHR-B. FHR-B also induced lysis of sheep erythrocytes when incubated in mouse serum with FHR-B added in excess. Altogether, these data demonstrate that, similar to human FHR-1 and FHR-5, mouse FHR-B modulates complement activity by promoting complement activation via interaction with C3b and via competition with murine FH.

Strategies of NF-κB signaling modulation by ectromelia virus in BALB/3T3 murine fibroblasts.

PubMed

Struzik, Justyna; Szulc-Dąbrowska, Lidia; Winnicka, Anna; Niemiałtowski, Marek

2015-10-01

Nuclear factor κB (NF-κB) is a pleiotropic transcription factor that regulates the expression of immune response genes. NF-κB signaling can be disrupted by pathogens that prevent host immune response. In this work, we examined the influence of ectromelia (mousepox) virus (ECTV) on NF-κB signaling in murine BALB/3T3 fibroblasts. Activation of NF-κB via tumor necrosis factor (TNF) receptor 1 (TNFR1) in these cells induces proinflammatory cytokine secretion. We show that ECTV does not recruit NF-κB to viral factories or induce NF-κB nuclear translocation in BALB/3T3 cells. Additionally, ECTV counteracts TNF-α-induced p65 NF-κB nuclear translocation during the course of infection. Inhibition of TNF-α-induced p65 nuclear translocation was also observed in neighboring cells that underwent fusion with ECTV-infected cells. ECTV inhibits the key step of NF-κB activation, i.e. Ser32 phosphorylation and degradation of inhibitor κBα (IκBα) induced by TNF-α. We also observed that ECTV prevents TNF-α-induced Ser536 of p65 phosphorylation in BALB/3T3 cells. Studying TNFR1 signaling provides information about regulation of inflammatory response and cell survival. Unraveling poxviral immunomodulatory strategies may be helpful in drug target identification as well as in vaccine development. Copyright © 2015 Elsevier Ltd. All rights reserved.

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 3 2012-04-01 2012-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 3 2013-04-01 2013-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 4 2014-04-01 2014-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

17 CFR 240.15b7-3T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Operational capability in a Year 2000 environment. 240.15b7-3T Section 240.15b7-3T Commodity and Securities Exchanges SECURITIES... § 240.15b7-3T Operational capability in a Year 2000 environment. (a) This section applies to every...

Concentration dependent survival and neural differentiation of murine embryonic stem cells cultured on polyethylene glycol dimethacrylate hydrogels possessing a continuous concentration gradient of n-cadherin derived peptide His-Ala-Val-Asp-Lle.

PubMed

Lim, Hyun Ju; Mosley, Matthew C; Kurosu, Yuki; Smith Callahan, Laura A

2017-07-01

N-cadherin cell-cell signaling plays a key role in the structure and function of the nervous system. However, few studies have incorporated bioactive signaling from n-cadherin into tissue engineering matrices. The present study uses a continuous gradient approach in polyethylene glycol dimethacrylate hydrogels to identify concentration dependent effects of n-cadherin peptide, His-Ala-Val-Asp-Lle (HAVDI), on murine embryonic stem cell survival and neural differentiation. The n-cadherin peptide was found to affect the expression of pluripotency marker, alkaline phosphatase, in murine embryonic stem cells cultured on n-cadherin peptide containing hydrogels in a concentration dependent manner. Increasing n-cadherin peptide concentrations in the hydrogels elicited a biphasic response in neurite extension length and mRNA expression of neural differentiation marker, neuron-specific class III β-tubulin, in murine embryonic stem cells cultured on the hydrogels. High concentrations of n-cadherin peptide in the hydrogels were found to increase the expression of apoptotic marker, caspase 3/7, in murine embryonic stem cells compared to that of murine embryonic stem cell cultures on hydrogels containing lower concentrations of n-cadherin peptide. Increasing the n-cadherin peptide concentration in the hydrogels facilitated greater survival of murine embryonic stem cells exposed to increasing oxidative stress caused by hydrogen peroxide exposure. The combinatorial approach presented in this work demonstrates concentration dependent effects of n-cadherin signaling on mouse embryonic stem cell behavior, underscoring the need for the greater use of systematic approaches in tissue engineering matrix design in order to understand and optimize bioactive signaling in the matrix for tissue formation. Single cell encapsulation is common in tissue engineering matrices. This eliminates cellular access to cell-cell signaling. N-cadherin, a cell-cell signaling molecule, plays a vital role in

Baicalin Inhibits IL-17-Mediated Joint Inflammation in Murine Adjuvant-Induced Arthritis

PubMed Central

Yang, Xue; Yang, Ji; Zou, Hejian

2013-01-01

T-helper-17 (Th17) cells are implicated in a number of inflammatory disorders including rheumatoid arthritis. Antagonism of Th17 cells is a treatment option for arthritis. Here, we report that Baicalin, a compound isolated from the Chinese herb Huangqin (Scutellaria baicalensis Georgi), relieved ankle swelling and protected the joint against inflammatory destruction in a murine adjuvant-induced arthritis model. Baicalin inhibited splenic Th17 cell population expansion in vivo. Baicalin prevented interleukin- (IL-) 17-mediated lymphocyte adhesion to cultured synoviocytes. Baicalin also blocked IL-17-induced intercellular adhesion molecule 1, vascular cell adhesion molecule 1, IL-6, and tumor necrosis factor-alpha mRNA expression in cultured synoviocytes. Collectively, these findings suggest that Baicalin downregulates the joint inflammation caused by IL-17, which is likely produced by an expanded population of splenic Th17 cells in experimental arthritis. Baicalin might be a promising novel therapeutic agent for treating rheumatoid arthritis in humans. PMID:23840239

Induction of murine embryonic stem cell differentiation by medicinal plant extracts

PubMed Central

Reynertson, Kurt A.; Charlson, Mary E.; Gudas, Lorraine J.

2010-01-01

Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly-cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from twelve species of ethnomedically utilized plants, we found fractions from three species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. PMID:20955699

Epigallocatechin gallate (EGCG), influences a murine WEHI-3 leukemia model in vivo through enhancing phagocytosis of macrophages and populations of T- and B-cells.

PubMed

Huang, An-Cheng; Cheng, Hsiu-Yueh; Lin, Tsu-Shun; Chen, Wen-Hsein; Lin, Ju-Hwa; Lin, Jen-Jyh; Lu, Chi-Cheng; Chiang, Jo-Hua; Hsu, Shu-Chun; Wu, Ping-Ping; Huang, Yi-Ping; Chung, Jing-Gung

2013-01-01

Epigallocatechin gallate (EGCG) is the major polyphenol in green tea, and has been reported to have anticancer effects on many types of cancer cells. However, there is no report to show its effects on the immune response in a murine leukemia mouse model. Thus, in the present study, we investigated the effects of EGCG on the immune responses of murine WEHI-3 leukemia cells in vivo. WEHI-3 cells were intraperitoneally injected into normal BALB/c mice to establish leukemic BALB/c mice, which were then oral-treated with or without EGCG at 5, 20 and 40 mg/kg for two weeks. The results indicated that EGCG did not change the weight of the animals, nor the liver or spleen when compared to vehicle (olive oil) -treated groups. Furthermore, EGCG increased the percentage of cluster of differentiation 3 (CD3) (T-cell), cluster of differentiation 19 (CD19) (B-cell) and Macrophage-3 antigen (Mac-3) (macrophage) but reduced the percentage of CD11b (monocyte) cell surface markers in EGCG-treated groups as compared with the untreated leukemia group. EGCG promoted the phagocytosis of macrophages from 5 mg/kg treatment and promoted natural killer cell activity at 40 mg/kg, increased T-cell proliferation at 40 mg/kg but promoted B-cell proliferation at all three doses. Based on these observations, it appears that EGCG might exhibit an immune response in the murine WEHI-3 cell line-induced leukemia in vivo.

Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.

PubMed

Winkler, Monica E; Mauritz, Christina; Groos, Stephanie; Kispert, Andreas; Menke, Sandra; Hoffmann, Anika; Gruh, Ina; Schwanke, Kristin; Haverich, Axel; Martin, Ulrich

2008-03-01

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.

Developmental staging of male murine embryonic gonad by SAGE analysis

PubMed Central

Lee, Tin-Lap; Li, Yunmin; Alba, Diana; Vong, Queenie P.; Wu, Shao-Ming; Baxendale, Vanessa; Rennert, Owen M.; Lau, Yun-Fai Chris; Chan, Wai-Yee

2012-01-01

Despite the identification of key genes such as Sry integral to embryonic gonadal development, the genomic classification and identification of chromosomal activation of this process is still poorly understood. To better understand the genetic regulation of gonadal development, we performed Serial Analysis of Gene Expression (SAGE) to profile the genes and novel transcripts, and an average of 152,000 tags from male embryonic gonads at E10.5 (embryonic day 10.5), E11.5, E12.5, E13.5, E15.5 and E17.5 were analyzed. A total of 275,583 non-singleton tags that do not map to any annotated sequence were identified in the six gonad libraries, and 47,255 tags were mapped to 24,975 annotated sequences, among which 987 sequences were uncharacterized. Utilizing an unsupervised pattern identification technique, we established molecular staging of male gonadal development. Rather than providing a static descriptive analysis, we developed algorithms to cluster the SAGE data and assign SAGE tags to a corresponding chromosomal position; these data are displayed in chromosome graphic format. A prominent increase in global genomic activity from E10.5 to E17.5 was observed. Important chromosomal regions related to the developmental processes were identified and validated based on established mouse models with developmental disorders. These regions may represent markers for early diagnosis for disorders of male gonad development as well as potential treatment targets. PMID:19376482

Applicability, usability, and limitations of murine embryonic imaging with optical coherence tomography and optical projection tomography

PubMed Central

Singh, Manmohan; Raghunathan, Raksha; Piazza, Victor; Davis-Loiacono, Anjul M.; Cable, Alex; Vedakkan, Tegy J.; Janecek, Trevor; Frazier, Michael V.; Nair, Achuth; Wu, Chen; Larina, Irina V.; Dickinson, Mary E.; Larin, Kirill V.

2016-01-01

We present an analysis of imaging murine embryos at various embryonic developmental stages (embryonic day 9.5, 11.5, and 13.5) by optical coherence tomography (OCT) and optical projection tomography (OPT). We demonstrate that while OCT was capable of rapid high-resolution live 3D imaging, its limited penetration depth prevented visualization of deeper structures, particularly in later stage embryos. In contrast, OPT was able to image the whole embryos, but could not be used in vivo because the embryos must be fixed and cleared. Moreover, the fixation process significantly altered the embryo morphology, which was quantified by the volume of the eye-globes before and after fixation. All of these factors should be weighed when determining which imaging modality one should use to achieve particular goals of a study. PMID:27375945

A step-wise approach for analysis of the mouse embryonic heart using 17.6 Tesla MRI

PubMed Central

Gabbay-Benziv, Rinat; Reece, E. Albert; Wang, Fang; Bar-Shir, Amnon; Harman, Chris; Turan, Ozhan M.; Yang, Peixin; Turan, Sifa

2018-01-01

Background The mouse embryo is ideal for studying human cardiac development. However, laboratory discoveries do not easily translate into clinical findings partially because of histological diagnostic techniques that induce artifacts and lack standardization. Aim To present a step-wise approach using 17.6 T MRI, for evaluation of mice embryonic heart and accurate identification of congenital heart defects. Subjects 17.5-embryonic days embryos from low-risk (non-diabetic) and high-risk (diabetic) model dams. Study design Embryos were imaged using 17.6 Tesla MRI. Three-dimensional volumes were analyzed using ImageJ software. Outcome measures Embryonic hearts were evaluated utilizing anatomic landmarks to locate the four-chamber view, the left- and right-outflow tracts, and the arrangement of the great arteries. Inter- and intra-observer agreement were calculated using kappa scores by comparing two researchers’ evaluations independently analyzing all hearts, blinded to the model, on three different, timed occasions. Each evaluated 16 imaging volumes of 16 embryos: 4 embryos from normal dams, and 12 embryos from diabetic dams. Results Inter-observer agreement and reproducibility were 0.779 (95% CI 0.653–0.905) and 0.763 (95% CI 0.605–0.921), respectively. Embryonic hearts were structurally normal in 4/4 and 7/12 embryos from normal and diabetic dams, respectively. Five embryos from diabetic dams had defects: ventricular septal defects (n = 2), transposition of great arteries (n = 2) and Tetralogy of Fallot (n = 1). Both researchers identified all cardiac lesions. Conclusion A step-wise approach for analysis of MRI-derived 3D imaging provides reproducible detailed cardiac evaluation of normal and abnormal mice embryonic hearts. This approach can accurately reveal cardiac structure and, thus, increases the yield of animal model in congenital heart defect research. PMID:27569369

Suppression of Murine Retrovirus Polypeptide Termination: Effect of Amber Suppressor tRNA on the Cell-Free Translation of Rauscher Murine Leukemia Virus, Moloney Murine Leukemia Virus, and Moloney Murine Sarcoma Virus 124 RNA

PubMed Central

Murphy, Edwin C.; Wills, Norma; Arlinghaus, Ralph B.

1980-01-01

The effect of suppressor tRNA's on the cell-free translation of several leukemia and sarcoma virus RNAs was examined. Yeast amber suppressor tRNA (amber tRNA) enhanced the synthesis of the Rauscher murine leukemia virus and clone 1 Moloney murine leukemia virus Pr200gag-pol polypeptides by 10- to 45-fold, but at the same time depressed the synthesis of Rauscher murine leukemia virus Pr65gag and Moloney murine leukemia virus Pr63gag. Under suppressor-minus conditions, Moloney murine leukemia virus Pr70gag was present as a closely spaced doublet. Amber tRNA stimulated the synthesis of the “upper” Moloney murine leukemia virus Pr70gag polypeptide. Yeast ochre suppressor tRNA appeared to be ineffective. Quantitative analyses of the kinetics of viral precursor polypeptide accumulation in the presence of amber tRNA showed that during linear protein synthesis, the increase in accumulated Moloney murine leukemia virus Pr200gag-pol coincided closely with the molar loss of Pr63gag. Enhancement of Pr200gag-pol and Pr70gag by amber tRNA persisted in the presence of pactamycin, a drug which blocks the initiation of protein synthesis, thus arguing for the addition of amino acids to the C terminus of Pr63gag as the mechanism behind the amber tRNA effect. Moloney murine sarcoma virus 124 30S RNA was translated into four major polypeptides, Pr63gag, P42, P38, and P23. In the presence of amber tRNA, a new polypeptide, Pr67gag, appeared, whereas Pr63gag synthesis was decreased. Quantitative estimates indicated that for every 1 mol of Pr67gag which appeared, 1 mol of Pr63gag was lost. Images PMID:7373716

Induction of murine embryonic stem cell differentiation by medicinal plant extracts

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

Reynertson, Kurt A.; Department of Pharmacology, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065; Charlson, Mary E.

Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extractsmore » for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells.« less

Induction of murine embryonic stem cell differentiation by medicinal plant extracts.

PubMed

Reynertson, Kurt A; Charlson, Mary E; Gudas, Lorraine J

2011-01-01

Epidemiological evidence indicates that diets high in fruits and vegetables provide a measure of cancer chemoprevention due to phytochemical constituents. Natural products are a rich source of cancer chemotherapy drugs, and primarily target rapidly cycling tumor cells. Increasing evidence indicates that many cancers contain small populations of resistant, stem-like cells that have the capacity to regenerate tumors following chemotherapy and radiation, and have been linked to the initiation of metastases. Our goal is to discover natural product-based clinical or dietary interventions that selectively target cancer stem cells, inducing differentiation. We adapted an alkaline phosphatase (AP) stain to assay plant extracts for the capacity to induce differentiation in embryonic stem (ES) cells. AP is a characteristic marker of undifferentiated ES cells, and this represents a novel approach to screening medicinal plant extracts. Following a survey of approximately 100 fractions obtained from 12 species of ethnomedically utilized plants, we found fractions from 3 species that induced differentiation, decreasing AP and transcript levels of pluripotency markers (Nanog, Oct-4, Rex-1). These fractions affected proliferation of murine ES, and human embryonal, prostate, and breast carcinoma cells in a dose-dependent manner. Several phytochemical constituents were isolated; the antioxidant phytochemicals ellagic acid and gallic acid were shown to affect viability of cultured breast carcinoma cells. Copyright © 2010 Elsevier Inc. All rights reserved.

Telomere sister chromatid exchange in telomerase deficient murine cells.

PubMed

Wang, Yisong; Giannone, Richard J; Liu, Yie

2005-10-01

We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape "end crisis". However, the possibility that ES cells were more permissive to genomic rearrangements than other cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.

Intrauterine air impairs embryonic postimplantation development in mice.

PubMed

Liu, Ruonan; Li, Yimeng; Miao, Yanping; Wei, Yanhui; Guan, Mo; Zhou, Rongyan; Li, Xiangyun

2017-12-01

Although most embryologists load air bubbles into the catheter along with embryos during embryo transfer, the effects of these air bubbles on embryo transfer success rate are not clear. Air bubbles were nonsurgically injected into unilateral uterine horns of mice to demonstrate the negative effects of intrauterine air bubbles on embryonic development. Our data showed that when air bubbles are nonsurgically injected into unilateral uterine horns of pregnant 4days mice the litter size is significantly decreased. Four days after the introduction of air, abnormal decidua and dead conceptuses were detected in the uterine horns receiving the air bubbles. In addition, intrauterine air also significantly impaired murine embryo transfer success rates, and induced an increase in endometrial capillary permeability and decidualization in mice on day 4 of pseudopregnancy. These results strongly indicated that the air bubbles loaded into embryo transfer catheters to bracket the embryo-containing medium may have negative effect on embryonic implantation and development. Intrauterine air impaired murine embryonic postimplantation development, and this provided some clues for improving embryo transfer techniques in human. Copyright © 2017 Elsevier B.V. All rights reserved.

A Critical Role for IL-17RB Signaling in HTLV-1 Tax-Induced NF-κB Activation and T-Cell Transformation

PubMed Central

Lavorgna, Alfonso; Matsuoka, Masao; Harhaj, Edward William

2014-01-01

Human T-cell leukemia virus type 1 (HTLV-1) infection is linked to the development of adult T-cell leukemia (ATL) and the neuroinflammatory disease HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 Tax protein functions as a potent viral oncogene that constitutively activates the NF-κB transcription factor to transform T cells; however, the underlying mechanisms remain obscure. Here, using next-generation RNA sequencing we identified the IL-25 receptor subunit IL-17RB as an aberrantly overexpressed gene in HTLV-1 immortalized T cells. Tax induced the expression of IL-17RB in an IκB kinase (IKK) and NF-κB-dependent manner. Remarkably, Tax activation of the canonical NF-κB pathway in T cells was critically dependent on IL-17RB expression. IL-17RB and IL-25 were required for HTLV-1-induced immortalization of primary T cells, and the constitutive NF-κB activation and survival of HTLV-1 transformed T cells. IL-9 was identified as an important downstream target gene of the IL-17RB pathway that drives the proliferation of HTLV-1 transformed cells. Furthermore, IL-17RB was overexpressed in leukemic cells from a subset of ATL patients and also regulated NF-κB activation in some, but not all, Tax-negative ATL cell lines. Together, our results support a model whereby Tax instigates an IL-17RB-NF-κB feed-forward autocrine loop that is obligatory for HTLV-1 leukemogenesis. PMID:25340344

Epidemiological investigation of the UGT2B17 polymorphism in doping control urine samples and its correlation to T/E ratios.

PubMed

Anielski, Patricia; Simmchen, Juliane; Wassill, Lars; Ganghofner, Dirk; Thieme, Detlef

2011-10-01

The deletion polymorphism of the enzyme UGT2B17 is known to correlate with the level of the testosterone to epitestosterone (T/E) ratio in urine specimen. Due to the importance of the T/E ratio to detect testosterone abuse in doping analysis, a PCR-ELISA system (Genotype® UGT test, AmplexDiagnostics) was established to identify the UGT2B17 phenotype in urine samples. Epidemiological investigations in a set of 674 routine doping controls (in- and out-of-competition) resulted in 22.8% homozygote gene-deleted and 74.5% UGT2B17-positive athletes. The validated test system has shown to be robust and sensitive: in only 18 cases (2.7%) isolation of cell material from urine failed. Following hydrolysis of glucuronidated conjugates, steroids were analyzed as bis-TMS derivatives by gas chromatography-mass spectrometry (GC-MS), for example, testosterone (T) and epitestosterone (E). Additionally, isotope ration mass spectrometry (IRMS) analysis and luteinizing hormone (LH) measurement were applied. Mean T/E ratios significantly correlated with the UGT2B17 phenotype (del: T/E 0.9; pos: 1.7), however the values did not differ as distinctive as reported in previous studies. Additionally, the T/E ratios in the gene-deleted group did not show a normal curve of distribution (median of T/E 0.5). Obviously, beside the UGT2B17 deletion further influences have to be taken into account, for example, polymorphisms or induction of other metabolizing enzymes. Our results indicate that the UGT2B17 polymorphism might be insufficient when utilized solely as a crucial parameter for individual interpretation of T/E in urine. Nevertheless, the detection of the UGT2B17-gene deletion in urine samples would provide additional information important for gathering evidence in analysis of steroids in doping control. Copyright © 2011 John Wiley & Sons, Ltd.

Embryonic cholecystitis and defective gallbladder contraction in the Sox17-haploinsufficient mouse model of biliary atresia

PubMed Central

Fujino, Ko; Igarashi, Hitomi; Imaimatsu, Kenya; Tsunekawa, Naoki; Hirate, Yoshikazu; Kurohmaru, Masamichi; Saijoh, Yukio; Kanai-Azuma, Masami

2017-01-01

The gallbladder excretes cytotoxic bile acids into the duodenum through the cystic duct and common bile duct system. Sox17 haploinsufficiency causes biliary atresia-like phenotypes and hepatitis in late organogenesis mouse embryos, but the molecular and cellular mechanisms underlying this remain unclear. In this study, transcriptomic analyses revealed the early onset of cholecystitis in Sox17+/− embryos, together with the appearance of ectopic cystic duct-like epithelia in their gallbladders. The embryonic hepatitis showed positive correlations with the severity of cholecystitis in individual Sox17+/− embryos. Embryonic hepatitis could be induced by conditional deletion of Sox17 in the primordial gallbladder epithelia but not in fetal liver hepatoblasts. The Sox17+/− gallbladder also showed a drastic reduction in sonic hedgehog expression, leading to aberrant smooth muscle formation and defective contraction of the fetal gallbladder. The defective gallbladder contraction positively correlated with the severity of embryonic hepatitis in Sox17+/− embryos, suggesting a potential contribution of embryonic cholecystitis and fetal gallbladder contraction in the early pathogenesis of congenital biliary atresia. PMID:28432216

Telomere sister chromatid exchange in telomerase deficient murine cells

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

Wang, Yisong; Giannone, Richard J; Liu, Yie

2005-01-01

We have recently demonstrated that several types of genomic rearrangements (i.e., telomere sister chromatid exchange (T-SCE), genomic-SCE, or end-to-end fusions) were more often detected in long-term cultured murine telomerase deficient embryonic stem (ES) cells than in freshly prepared murine splenocytes, even through they possessed similar frequencies of critically short telomeres. The high rate of genomic rearrangements in telomerase deficient ES cells, when compared to murine splenocytes, may reflect the cultured cells' gained ability to protect chromosome ends with eroded telomeres allowing them to escape 'end crisis'. However, the possibility that ES cells were more permissive to genomic rearrangements than othermore » cell types or that differences in the microenvironment or genetic background of the animals might consequentially determine the rate of T-SCEs or other genomic rearrangements at critically short telomeres could not be ruled out.« less

Heme oxygenase-1 ameliorates dextran sulfate sodium-induced acute murine colitis by regulating Th17/Treg cell balance.

PubMed

Zhang, Liya; Zhang, Yanjie; Zhong, Wenwei; Di, Caixia; Lin, Xiaoliang; Xia, Zhenwei

2014-09-26

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn's disease, is a group of autoimmune diseases characterized by nonspecific inflammation in the gastrointestinal tract. Recent investigations suggest that activation of Th17 cells and/or deficiency of regulatory T cells (Treg) is involved in the pathogenesis of IBD. Heme oxygenase (HO)-1 is a protein with a wide range of anti-inflammatory and immune regulatory function, which exerts significantly protective roles in various T cell-mediated diseases. In this study, we aim to explore the immunological regulation of HO-1 in the dextran sulfate sodium-induced model of experimental murine colitis. BALB/c mice were administered 4% dextran sulfate sodium orally; some mice were intraperitoneally pretreated with HO-1 inducer hemin or HO-1 inhibitor stannum protoporphyrin IX. The results show that hemin enhances the colonic expression of HO-1 and significantly ameliorates the symptoms of colitis with improved histological changes, accompanied by a decreased proportion of Th17 cells and increased number of Tregs in mesenteric lymph node and spleen. Moreover, induction of HO-1 down-regulates retinoic acid-related orphan receptor γt expression and IL-17A levels, while promoting Treg-related forkhead box p3 (Foxp3) expression and IL-10 levels in colon. Further study in vitro revealed that up-regulated HO-1 switched the naive T cells to Tregs when cultured under a Th17-inducing environment, which involved in IL-6R blockade. Therefore, HO-1 may exhibit anti-inflammatory activity in the murine model of acute experimental colitis via regulating the balance between Th17 and Treg cells, thus providing a possible novel therapeutic target in IBD. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

The excretory-secretory products of Echinococcus granulosus protoscoleces directly regulate the differentiation of B10, B17 and Th17 cells.

PubMed

Pan, Wei; Hao, Wen-Ting; Shen, Yu-Juan; Li, Xiang-Yang; Wang, Yan-Juan; Sun, Fen-Fen; Yin, Jian-Hai; Zhang, Jing; Tang, Ren-Xian; Cao, Jian-Ping; Zheng, Kui-Yang

2017-07-21

Excretory-secretory products (ESPs) released by helminths are well-known to regulate T cell responses in the host. However, their direct influence in the differentiation of naïve T cells, and especially B cells, remains largely unknown. This study investigated the effects of Echinococcus granulosus protoscoleces ESPs (EgPSC-ESPs) on the differentiation of IL-10-producing B cells (B10), IL-17A-producing B cells (B17) and Th17 cells. BALB/c mice injected with EgPSC were used to evaluate the in vivo profiles of B10, B17 and Th17 cells. In vitro purified CD19 + B and naïve CD4 + T cells were cultured in the presence of native, heat-inactivated or periodate-treated EgPSC-ESPs, and the differentiation of these cell subsets were compared. In contrast to the control group, infected mice showed higher frequencies of B10, B17 and Th17 cells, and higher levels of IL-10 and IL-17A in the sera. Interestingly, B17 cells were first identified to express CD19 + CD1d high . In vitro, B cells cultured with native ESPs exhibited a higher percentage of B10 cells but lower percentage of B17 and Th17 cells compared to the PBS group. Moreover, the relative expression of IL-10 and IL-17A mRNA were consistent with the altered frequencies. However, ESPs subjected to heat-inactivation or periodate treatment exhibited an inverse effect on the induction of these cell subsets. Our findings indicate that ESPs released by EgPSC can directly regulate the differentiation of B10, B17 and Th17 cells, which appear to be heat-labile and carbohydrate-dependent.

Baicalin maintains late-stage functional cardiomyocytes in embryoid bodies derived from murine embryonic stem cells.

PubMed

Tang, Meilin; Yin, Mengmeng; Tang, Ming; Liang, Huamin; Yu, Chong; Hu, Xinwu; Luo, Hongyan; Baudis, Birte; Haustein, Moritz; Khalil, Markus; Sarić, Tomo; Hescheler, Jürgen; Xi, Jiaoya

2013-01-01

Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to β-adrenergic and muscarinic stimulations. Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells. Copyright © 2013 S. Karger AG, Basel.

A Chitin-Like Component on Sclerotic Cells of Fonsecaea pedrosoi Inhibits Dectin-1-Mediated Murine Th17 Development by Masking β-Glucans

PubMed Central

Li, Ruoyu; Chen, Sharon C.-A.; Liu, Weihuang; Liu, Wei; Chen, Liuqing; Chen, Yao; Zhang, Xu; Tong, Zhongsheng; Xia, Yun; Xia, Ping; Wang, Yan; Duan, Yiqun

2014-01-01

Fonsecaea pedrosoi (F. pedrosoi), a major agent of chromoblastomycosis, has been shown to be recognized primarily by C-type lectin receptors (CLRs) in a murine model of chromoblastomycosis. Specifically, the β-glucan receptor, Dectin-1, mediates Th17 development and consequent recruitment of neutrophils, and is evidenced to have the capacity to bind to saprophytic hyphae of F. pedrosoi in vitro. However, when embedded in tissue, most etiological agents of chromoblastomycosis including F. pedrosoi will transform into the sclerotic cells, which are linked to the greatest survival of melanized fungi in tissue. In this study, using immunocompetent and athymic (nu/nu) murine models infected subcutaneously or intraperitoneally with F. pedrosoi, we demonstrated that T lymphocytes play an active role in the resolution of localized footpad infection, and there existed a significantly decreased expression of Th17-defining transcription factor Rorγt and inefficient recruitment of neutrophils in chronically infected spleen where the inoculated mycelium of F. pedrosoi transformed into the sclerotic cells. We also found that Dectin-1-expressing histocytes and neutrophils participated in the enclosure of transformed sclerotic cells in the infectious foci. Furthermore, we induced the formation of sclerotic cells in vitro, and evidenced a significantly decreased binding capacity of human or murine-derived Dectin-1 to the induced sclerotic cells in comparison with the saprophytic mycelial forms. Our analysis of β-glucans-masking components revealed that it is a chitin-like component, but not the mannose moiety on the sclerotic cells, that interferes with the binding of β-glucans by human or murine Dectin-1. Notably, we demonstrated that although Dectin-1 contributed to the development of IL-17A-producing CD3+CD4+ murine splenocytes upon in vitro-stimulation by saprophytic F. pedrosoi, the masking effect of chitin components partly inhibited Dectin-1-mediated Th17 development

A model system for testing gene vectors using murine tumor cells on the chorioallantoic membrane of the chick embryo.

PubMed

Dani, Sergio U; Espindola, Rachel

2002-06-30

We developed a model system for testing gene vectors, based on the growth of murine tumors on the chorioallantoic membrane (CAM) of embryonic chickens. The ability of selected murine cells to grow on the CAM was rated according to the following criteria: i) formation of tumor masses; ii) metastasis formation; iii) reproducibility; iv) yield, indicated as the number of embryos surviving to assessment time with visible tumors on the CAM; v) maintainability of the cell, both in the original host and the embryonic chick, or 'shuttle maintainability'; vi) detection by the naked eye, and vii) cost/benefit relation. The murine melanoma cell lineage, B16F10, which efficiently forms distinct, pigmented tumor masses and metastases on the CAM, performed better in this model than the murine B61 cell line. In vitro transduction of B16F10 cells with a recombinant adenovirus carrying a construct of the E. coli LacZ gene followed by inoculation onto the CAM resulted in beta-galactosidase expression in the tumor mass growing on the CAM. This model is potentially applicable to preclinical evaluation of gene vectors, especially for gene therapy of cancer.

Myeloid-derived suppressor cells in murine retrovirus-induced AIDS inhibit T- and B-cell responses in vitro that are used to define the immunodeficiency.

PubMed

Green, Kathy A; Cook, W James; Green, William R

2013-02-01

Myeloid-derived suppressor cells (MDSCs) have been characterized in several disease settings, especially in many tumor systems. Compared to their involvement in tumor microenvironments, however, MDSCs have been less well studied in their responses to infectious disease processes, in particular to retroviruses that induce immunodeficiency. Here, we demonstrate for the first time the development of a highly immunosuppressive MDSC population that is dependent on infection by the LP-BM5 retrovirus, which causes murine acquired immunodeficiency. These MDSCs express a cell surface marker signature (CD11b(+) Gr-1(+) Ly6C(+)) characteristic of monocyte-type MDSCs. Such MDSCs profoundly inhibit immune responsiveness by a cell dose- and substantially inducible nitric oxide synthase (iNOS)-dependent mechanism that is independent of arginase activity, PD-1-PD-L1 expression, and interleukin 10 (IL-10) production. These MDSCs display levels of immunosuppressive function in parallel with the extent of disease in LP-BM5-infected wild-type (w.t.) versus knockout mouse strains that are differentially susceptible to pathogenesis. These MDSCs suppressed not only T-cell but also B-cell responses, which are an understudied target for MDSC inhibition. The MDSC immunosuppression of B-cell responses was confirmed by the use of purified B responder cells, multiple B-cell stimuli, and independent assays measuring B-cell expansion. Retroviral load measurements indicated that the suppressive Ly6G(low/±) Ly6C(+) CD11b(+)-enriched MDSC subset was positive for LP-BM5, albeit at a significantly lower level than that of nonfractionated splenocytes from LP-BM5-infected mice. These results, including the strong direct MDSC inhibition of B-cell responsiveness, are novel for murine retrovirus-induced immunosuppression and, as this broadly suppressive function mirrors that of the LP-BM5-induced disease syndrome, support a possible pathogenic effector role for these retrovirus-induced MDSCs.

UV-B susceptibility and photoreactivation in embryonic development of the two-spotted spider mite, Tetranychus urticae.

PubMed

Yoshioka, Yoshio; Gotoh, Tetsuo; Suzuki, Takeshi

2018-05-14

Developmental errors are often induced in the embryos of many organisms by environmental stress. Ultraviolet-B radiation (UV-B) is one of the most serious environmental stressors in embryonic development. Here, we investigated susceptibility to UV-B (0.5 kJ m -2 ) in embryos of the two-spotted spider mite, Tetranychus urticae, to examine the potential use of UV-B in control of this important agricultural pest worldwide. Peak susceptibility to UV-B (0% hatchability) was found in T. urticae eggs 36-48 h after oviposition at 25 °C, which coincides with the stages of morphogenesis forming the germ band and initial limb primordia. However, hatchability recovered to ~ 80% when eggs irradiated with UV-B were subsequently exposed to visible radiation (VIS) at 10.2 kJ m -2 , driving photoreactivation (the photoenzymatic repair of DNA damage). The recovery effect decreased to 40-70% hatchability, depending on the embryonic developmental stage, when VIS irradiation was delayed for 4 h after the end of exposure to UV-B. Thus UV-B damage to T. urticae embryos is critical, particularly in the early stages of morphogenesis, and photoreactivation functions to mitigate UV-B damage, even in the susceptible stages, but immediate VIS irradiation is needed after exposure to UV-B. These findings suggest that nighttime irradiation with UV-B can effectively kill T. urticae eggs without subsequent photoreactivation and may be useful in the physical control of this species.

Elastin receptor (S-gal) occupancy by elastin peptides modulates T-cell response during murine emphysema.

PubMed

Meghraoui-Kheddar, Aïda; Pierre, Alexandre; Sellami, Mehdi; Audonnet, Sandra; Lemaire, Flora; Le Naour, Richard

2017-09-01

Chronic obstructive pulmonary disease and emphysema are associated with increased elastin peptides (EP) production because of excessive breakdown of lung connective tissue. We recently reported that exposure of mice to EP elicited hallmark features of emphysema. EP effects are largely mediated through a receptor complex that includes the elastin-binding protein spliced-galactosidase (S-gal). In previous studies, we established a correlation between cytokine production and S-gal protein expression in EP-treated immune cells. In this study, we investigated the S-gal-dependent EP effects on T-helper (Th) and T-cytotoxic (Tc) responses during murine EP-triggered pulmonary inflammation. C57BL/6J mice were endotracheally instilled with the valine-glycine-valine-alanine-proline-glycine (VGVAPG) elastin peptide, and, 21 days after treatment, local and systemic T-lymphocyte phenotypes were analyzed at cytokine and transcription factor expression levels by multicolor flow cytometry. Exposure of mice to the VGVAPG peptide resulted in a significant increase in the proportion of the CD4 + and CD8 + T cells expressing the cytokines IFN-γ or IL-17a and the transcription factors T-box expressed in T cells or retinoic acid-related orphan receptor-γt (RORγt) without effects on IL-4 and Gata-binding protein 3 to DNA sequence [A/T]GATA[A/G] expression. These effects were maximized when each T-cell subpopulation was challenged ex vivo with EP, and they were inhibited in vivo when an analogous peptide antagonizing the EP/S-gal interactions was instilled together with the VGVAPG peptide. This study demonstrates that, during murine emphysema, EP-S-gal interactions contribute to a Th-1 and Th-17 proinflammatory T-cell response combined with a Tc-1 response. Our study also highlights the S-gal receptor as a putative pharmacological target to modulate such an immune response. Copyright © 2017 the American Physiological Society.

Development of Mouse Embryonic Stem (ES) Cells: IV Differentiation to Mature T and B Lymphocytes after Implantation of Embryoid Bodies Into Nude Mice

PubMed Central

Mok, Hoyan

1995-01-01

Mouse embryonic stem (ES) cells in culture can differentiate into late stages of many lineage-committed precursor cells. Under appropriate organ-culture conditions, ES cels differentiate into lymphoidlike cells at a stage equivalent to lymphoid cells found in fetal liver. These hematopoietic precursors are located in cup-shaped structures found in some embryoid bodies; we called such embryoid bodies “ES fetuses.” In this study, we have followed the maturation of hematopoietic cells after implantation of ES fetuses into nude mice for 3 weeks. ES-cell-derived lymphoid cells-pre-B cells, mature B cells, and mature T cells were found in all lymphoid organs. Interestingly, there was also an increase of T cells of host origin. Because native nude mouse lack thymus, these T cells might be educated by thymuslike epithelium generated from ES fetuses. Practical applications of this combined in vitro and in vivo system are discussed. PMID:9700357

Comparison of frailty of primary neurons, embryonic, and aging mouse cortical layers.

PubMed

Fugistier, Patrick; Vallet, Philippe G; Leuba, Geneviève; Piotton, Françoise; Marin, Pascale; Bouras, Constantin; Savioz, Armand

2014-02-01

Superficial layers I to III of the human cerebral cortex are more vulnerable toward Aβ peptides than deep layers V to VI in aging. Three models of layers were used to investigate this pattern of frailty. First, primary neurons from E14 and E17 embryonic murine cortices, corresponding respectively to future deep and superficial layers, were treated either with Aβ(1-42), okadaic acid, or kainic acid. Second, whole E14 and E17 embryonic cortices, and third, in vitro separated deep and superficial layers of young and old C57BL/6J mice, were treated identically. We observed that E14 and E17 neurons in culture were prone to death after the Aβ and particularly the kainic acid treatment. This was also the case for the superficial layers of the aged cortex, but not for the embryonic, the young cortex, and the deep layers of the aged cortex. Thus, the aged superficial layers appeared to be preferentially vulnerable against Aβ and kainic acid. This pattern of vulnerability corresponds to enhanced accumulation of senile plaques in the superficial cortical layers with aging and Alzheimer's disease. Copyright © 2014 Elsevier Inc. All rights reserved.

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion

PubMed Central

Zhao, Guoyan; Penna, Vinay R.; Park, Eugene; Lauron, Elvin J.; Harvey, Ian B.; Beatty, Wandy L.; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H.; Wang, David

2017-01-01

ABSTRACT The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4+ T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological

A Murine Herpesvirus Closely Related to Ubiquitous Human Herpesviruses Causes T-Cell Depletion.

PubMed

Patel, Swapneel J; Zhao, Guoyan; Penna, Vinay R; Park, Eugene; Lauron, Elvin J; Harvey, Ian B; Beatty, Wandy L; Plougastel-Douglas, Beatrice; Poursine-Laurent, Jennifer; Fremont, Daved H; Wang, David; Yokoyama, Wayne M

2017-05-01

The human roseoloviruses human herpesvirus 6A (HHV-6A), HHV-6B, and HHV-7 comprise the Roseolovirus genus of the human Betaherpesvirinae subfamily. Infections with these viruses have been implicated in many diseases; however, it has been challenging to establish infections with roseoloviruses as direct drivers of pathology, because they are nearly ubiquitous and display species-specific tropism. Furthermore, controlled study of infection has been hampered by the lack of experimental models, and until now, a mouse roseolovirus has not been identified. Herein we describe a virus that causes severe thymic necrosis in neonatal mice, characterized by a loss of CD4 + T cells. These phenotypes resemble those caused by the previously described mouse thymic virus (MTV), a putative herpesvirus that has not been molecularly characterized. By next-generation sequencing of infected tissue homogenates, we assembled a contiguous 174-kb genome sequence containing 128 unique predicted open reading frames (ORFs), many of which were most closely related to herpesvirus genes. Moreover, the structure of the virus genome and phylogenetic analysis of multiple genes strongly suggested that this virus is a betaherpesvirus more closely related to the roseoloviruses, HHV-6A, HHV-6B, and HHV-7, than to another murine betaherpesvirus, mouse cytomegalovirus (MCMV). As such, we have named this virus murine roseolovirus (MRV) because these data strongly suggest that MRV is a mouse homolog of HHV-6A, HHV-6B, and HHV-7. IMPORTANCE Herein we describe the complete genome sequence of a novel murine herpesvirus. By sequence and phylogenetic analyses, we show that it is a betaherpesvirus most closely related to the roseoloviruses, human herpesviruses 6A, 6B, and 7. These data combined with physiological similarities with human roseoloviruses collectively suggest that this virus is a murine roseolovirus (MRV), the first definitively described rodent roseolovirus, to our knowledge. Many biological and

Iron oxide nanoparticles attenuate T helper 17 cell responses in vitro and in vivo.

PubMed

Hsiao, Yai-Ping; Shen, Chien-Chang; Huang, Chung-Hsiung; Lin, Yu-Chin; Jan, Tong-Rong

2018-05-01

Iron oxide nanoparticles (IONPs) have been shown to attenuate T helper (Th)1 and Th2 cell-mediated immunity in ovalbumin (OVA)-sensitized mice. The objective of this study is to investigate the effects of IONPs on the immune responses of Th17 cells, a subset of T cells involved in various inflammatory pathologies. For in vivo study, a murine model of delayed-type hypersensitivity (DTH) was employed. BALB/c mice received a single dose of IONPs (0.2-10 mg iron/kg) via the tail vein 1 h prior to ovalbumin (OVA) sensitization. Their footpads were subcutaneously challenged with OVA to induce DTH reactions. The expression of Th17 cell-related molecules in inflamed footpads were examined by immunohistochemistry. For in vitro study, OVA-primed splenocytes were directly exposed to IONPs (1-100 μg iron/mL), and then re-stimulated with OVA in culture. The expression of Th17 cell-related molecules were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. IONP administration attenuated the number of interleukin (IL)-6, IL-17, the transcription factor ROR-γ, and chemokine receptor 6 positive cells in OVA-challenged footpads, whereas the number of transforming growth factor-β, IL-23 and chemokine (C-C motif) ligand 20 positive cells was not altered. Direct exposure of OVA-primed splenocytes to IONPs suppressed the production of IL-6 and IL-17, and the mRNA expression of IL-17 and ROR-γt. These data indicate that exposure to IONPs attenuates Th17 cell responses in vivo and in vitro. Copyright © 2018. Published by Elsevier B.V.

NFκB signaling regulates embryonic and adult neurogenesis

PubMed Central

ZHANG, Yonggang; HU, Wenhui

2013-01-01

Both embryonic and adult neurogenesis involves the self-renewal/proliferation, survival, migration and lineage differentiation of neural stem/progenitor cells. Such dynamic process is tightly regulated by intrinsic and extrinsic factors and complex signaling pathways. Misregulated neurogenesis contributes much to a large range of neurodevelopmental defects and neurodegenerative diseases. The signaling of NFκB regulates many genes important in inflammation, immunity, cell survival and neural plasticity. During neurogenesis, NFκB signaling mediates the effect of numerous niche factors such as cytokines, chemokines, growth factors, extracellular matrix molecules, but also crosstalks with other signaling pathways such as Notch, Shh, Wnt/β-catenin. This review summarizes current progress on the NFκB signaling in all aspects of neurogenesis, focusing on the novel role of NFκB signaling in initiating early neural differentiation of neural stem cells and embryonic stem cells. PMID:24324484

Highly efficient gene transfer using a retroviral vector into murine T cells for preclinical chimeric antigen receptor-expressing T cell therapy

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

Kusabuka, Hotaka; Fujiwara, Kento; Tokunaga, Yusuke

Adoptive immunotherapy using chimeric antigen receptor-expressing T (CAR-T) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CAR-T cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CAR-T cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CAR-T cells by optimizing the period ofmore » pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen-specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. - Highlights: • We established highly efficient gene transduction protocols for murine T cells. • CD8{sup +} CAR-T cells had antigen-specific cytotoxic activity. • CD4{sup +} CAR-T cells secreted multiple cytokines by antigen stimulation. • This finding can contribute to the development of T-cell biology and immunotherapy.« less

Efficient femtosecond driven SOX 17 delivery into mouse embryonic stem cells: differentiation studies

NASA Astrophysics Data System (ADS)

Thobakgale, Lebogang; Manoto, Sello Lebohang; Lemboumba, Satuurnin Ombinda; Maaza, Malik; Mthunzi-Kufa, Patience

2017-02-01

Embryonic stem cells have great promise in regenerative medicine because of their ability to self-renew and differentiate into various cell types. Delivery of therapeutic genes into cells has already been achieved using of chemical agents and viral vectors with high transfection efficiencies. However, these methods have also been documented as toxic and in the latter case they can cause latent cell infections. In this study we use femtosecond laser pulses to optically deliver genetic material in mouse embryonic stem cells. Femtosecond laser pulses in contrast to the conventional approach, minimises the risk of unwanted side effects because photons are used to create transient pores on the membrane which allow free entry of molecules with no need for delivery agents. Using an Olympus microscope, fluorescence imaging of the samples post irradiation was performed and decreased expression of stage specific embryonic antigen one (SSEA-1) consistent with on-going cellular differentiation was observed. Our results also show that femtosecond laser pulses were effective in delivering SOX 17 plasmid DNA (pSOX17) which resulted in the differentiation of mouse embryonic stem cells into endoderm cells. We thus concluded that laser transfection of stem cells for the purpose of differentiation, holds potential for applications in tissue engineering as a method of generating new cell lines.

IL-25 promotes the function of CD4+CD25+ T regulatory cells and prolongs skin-graft survival in murine models.

PubMed

Tang, Jiayou; Zhou, Xiaohui; Liu, Jie; Meng, Qingshu; Han, Yang; Wang, Zhulin; Fan, Huimin; Liu, Zhongmin

2015-10-01

Interleukin (IL)-25, also known as IL-17E, belongs to the IL-17 family of cytokines. Unlike other IL-17 family members, IL-25 promotes Th2-type immune responses, stimulating IL-4, IL-5, and IL-13 production. Here, we employed murine models of skin graft to explore the role of IL-25 in suppression of graft rejection. We found that IL-25 expression is increased during allograft rejection, and allograft rejection was enhanced in IL-25 KO mice. IL-25 KO was associated with down-regulation of Foxp3 expression in CD4+ T cells. Further, while adoptive transfer of WT regulatory T cells (Tregs) protected against allograft rejection, adoptive transfer of IL-25 deficient Tregs failed to protect against allograft rejection. Exogenous IL-25 restored Foxp3 expression and Treg function in vitro. Moreover, IL-25 promoted phosphorylation of NFAT2. Thus, IL-25 may enhance Treg function by up-regulating NFAT2 phosphorylation. Our findings suggest that IL-25 can sustain Foxp3 expression, enhance the suppressive function of Tregs, and prolong skin-graft survival. Copyright © 2015 Elsevier B.V. All rights reserved.

Enhanced Cultivation Of Stimulated Murine B Cells

NASA Technical Reports Server (NTRS)

Sammons, David W.

1994-01-01

Method of in vitro cultivation of large numbers of stimulated murine B lymphocytes. Cells electrofused with other cells to produce hybridomas and monoclonal antibodies. Offers several advantages: polyclonally stimulated B-cell blasts cultivated for as long as 14 days, hybridomas created throughout culture period, yield of hybridomas increases during cultivation, and possible to expand polyclonally in vitro number of B cells specific for antigenic determinants first recognized in vivo.

PIF1 disruption or NBS1 hypomorphism does not affect chromosome healing or fusion resulting from double-strand breaks near telomeres in murine embryonic stem cells.

PubMed

Reynolds, Gloria E; Gao, Qing; Miller, Douglas; Snow, Bryan E; Harrington, Lea A; Murnane, John P

2011-11-10

Telomerase serves to maintain telomeric repeat sequences at the ends of chromosomes. However, telomerase can also add telomeric repeat sequences at DNA double-strand breaks (DSBs), a process called chromosome healing. Here, we employed a method of inducing DSBs near telomeres to query the role of two proteins, PIF1 and NBS1, in chromosome healing in mammalian cells. PIF1 was investigated because the PIF1 homolog in Saccharomyces cerevisiae inhibits chromosome healing, as shown by a 1000-fold increase in chromosome in PIF1-deficient cells. NBS1 was investigated because the functional homolog of NBS1 in S. cerevisiae, Xrs2, is part of the Mre11/Rad50/Xrs2 complex that is required for chromosome healing due to its role in the processing of DSBs and recruitment of telomerase. We found that disruption of mPif1 had no detectable effect on the frequency of chromosome healing at DSBs near telomeres in murine embryonic stem cells. Moreover, the Nbs1(ΔB) hypomorph, which is defective in the processing of DSBs, also had no detectable effect on the frequency of chromosome healing, DNA degradation, or gross chromosome rearrangements (GCRs) that result from telomeric DSBs. Although we cannot rule out small changes in chromosome healing using this system, it is clear from our results that knockout of PIF1 or the Nbs1(ΔB) hypomorph does not result in large differences in chromosome healing in murine cells. These results represent the first genetic assessment of the role of these proteins in chromosome healing in mammals, and suggest that murine cells have evolved mechanisms to ensure the functional redundancy of Pif1 or Nbs1 in the regulation of chromosome healing. Copyright © 2011 Elsevier B.V. All rights reserved.

CrxOS maintains the self-renewal capacity of murine embryonic stem cells

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

Saito, Ryota; Yamasaki, Tokiwa; Nagai, Yoko

2009-12-25

Embryonic stem (ES) cells maintain pluripotency by self-renewal. Several homeoproteins, including Oct3/4 and Nanog, are known to be key factors in maintaining the self-renewal capacity of ES cells. However, other genes required for the mechanisms underlying this process are still unclear. Here we report the identification by in silico analysis of a homeobox-containing gene, CrxOS, that is specifically expressed in murine ES cells and is essential for their self-renewal. ES cells mainly express the short isoform of endogenous CrxOS. Using a polyoma-based episomal expression system, we demonstrate that overexpression of the CrxOS short isoform is sufficient for maintaining the undifferentiatedmore » morphology of ES cells and stimulating their proliferation. Finally, using RNA interference, we show that CrxOS is essential for the self-renewal of ES cells, and provisionally identify foxD3 as a downstream target gene of CrxOS. To our knowledge, ours is the first delineation of the physiological role of CrxOS in ES cells.« less

AMPKα2 Suppresses Murine Embryonic Fibroblast Transformation and Tumorigenesis

PubMed Central

Phoenix, Kathryn N.; Devarakonda, Charan V.; Fox, Melissa M.; Stevens, Laura E.

2012-01-01

AMP-activated kinase (AMPK) is a key metabolic sensor and stress signaling kinase. AMPK activity is known to suppress anabolic processes such as protein and lipid biosynthesis and promote energy-producing pathways including fatty acid oxidation, resulting in increased cellular energy. In addition, AMPK localizes to centrosomes during cell division, plays a role in cellular polarization, and directly targets p53, affecting apoptosis. Two distinct catalytic AMPKα isoforms exist: α1 and α2. Multiple reports indicate that both common and distinct functions exist for each of the 2 α isoforms. AMPK activation has been shown to repress tumor growth, and it has been suggested that AMPK may function as a metabolic tumor suppressor. To evaluate the potential role of each of the AMPKα isoforms in modulating cellular transformation, susceptibility to Ras-induced transformation was evaluated in normal murine embryonic fibroblasts (MEFs) obtained from genetically deleted AMPKα1- or AMPKα2-null mice. This study demonstrated that while AMPKα1 is the dominant AMPK isoform expressed in MEFs, only the AMPKα2-null MEFs displayed increased susceptibility to H-RasV12 transformation in vitro and tumorigenesis in vivo. Conversely, AMPKα1-null MEFs, which demonstrated compensation with increased expression of AMPKα2, displayed minimal transformation susceptibility, decreased cell survival, decreased cell proliferation, and increased apoptosis. Finally, this study demonstrates that AMPKα2 was selectively responsible for targeting p53, thus contributing to the suppression of transformation and tumorigenic mechanisms. PMID:22893790

Bone marrow CD11b(+)F4/80(+) dendritic cells ameliorate collagen-induced arthritis through modulating the balance between Treg and Th17.

PubMed

Zhang, Lingling; Fu, Jingjing; Sheng, Kangliang; Li, Ying; Song, Shanshan; Li, Peipei; Song, Shasha; Wang, Qingtong; Chen, Jingyu; Yu, Jianhua; Wei, Wei

2015-03-01

Tolerogenic dendritic cells (DCs) are well-known to show an immunosuppressive function. In this study we determine the therapeutic effects and potential mechanisms of transferred bone marrow (BM) CD11b(+)F4/80(+) DCs on collagen-induced arthritis (CIA) in mice. Murine BM CD11b(+)F4/80(+) DCs were generated under the stimulation of GM-CSF and IL-4, and the function of BM CD11b(+) F4/80(+) DCs was identified by measuring the levels of IL-10, TGF-beta and indoleamine 2,3-dioxygenase (IDO). BM CD11b(+)F4/80(+) DCs were transferred to CIA mice by intravenous injections. The histopathology of joint and spleen were evaluated. T lymphocyte proliferation, Treg and Th17 subsets were analyzed. The expressions of Foxp3, Helios and RORγt in T lymphocytes co-cultured with BM CD11b(+)F4/80(+) DCs were measured in vitro. We found that BM CD11b(+)F4/80(+) DCs induced by GM-CSF and IL-4 could express high levels of IL-10, TGF-beta and IDO. BM CD11b(+)F4/80(+) DCs significantly reduced the pathologic scores in joints and spleens, which correlated significantly with the reduced T lymphocyte proliferation and Th17 cell number, and with the increased Tregs number. In vitro, OVA-pulsed BM CD11b(+)F4/80(+) DCs promoted Treg cell expansion, enhanced IL-10 and CTLA-4 protein expression, augmented Foxp3 and Helios mRNA expression, and inhibited RORγt and IL-17 mRNA expression. Taken together, BM CD11b(+)F4/80(+) DCs are able to ameliorate the development and severity of CIA, at least partly by inducing Foxp3(+) Treg cell expansion and suppressing Th17 function. The BM CD11b(+)F4/80(+) DCs might have a promising immunotherapeutic potential for autoimmune arthritis. Copyright © 2015 Elsevier B.V. All rights reserved.

Coxiella burnetii Avirulent Nine Mile Phase II Induces Caspase-1-Dependent Pyroptosis in Murine Peritoneal B1a B Cells.

PubMed

Schoenlaub, Laura; Cherla, Rama; Zhang, Yan; Zhang, Guoquan

2016-12-01

Our recent study demonstrated that virulent Coxiella burnetii Nine Mile phase I (NMI) is capable of infecting and replicating within peritoneal B1a cells and that B1a cells play an important role in host defense against C. burnetii infection in mice. However, it remains unknown if avirulent Nine Mile phase II (NMII) can infect and replicate in B1a cells and whether NMI and NMII can differentially interact with B1a cells. In this study, we examined if NMI and NMII can differentially modulate host cell apoptotic signaling in B1a cells. The results showed that NMII induced dose-dependent cell death in murine peritoneal B1a cells but NMI did not, suggesting that NMI and NMII may differentially activate host cell apoptotic signaling in B1a cells. Western blotting indicated that NMII-induced B1a cell death was not dependent on either caspase-3 or PARP-1 cleavage, but cleavage of caspase-1 was detected in NMII-infected B1a cells. In addition, inhibition or deficiency of caspase-1 activity blocked NMII-induced B1a cell death. These results suggest that NMII induces a caspase-1-dependent pyroptosis in murine peritoneal B1a cells. We also found that heat-killed NMII and type 4 secretion system (T4SS) mutant NMII were unable to induce B1a cell death and that NMII infection did not induce cell death in peritoneal B1a cells from Toll-like receptor 2 (TLR-2)- or NLRP3 inflammasome-deficient mice. These data suggest that NMII-induced caspase-1-dependent pyroptosis may require its T4SS and activation of the TLR-2 and NLRP3 signaling pathways. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

LPS induces direct death of IFN-gamma primed murine embryonic hepatocyte, BNL CL2 cells in a TNF-alpha independent manner.

PubMed

So, H S; Jung, B H; Yeum, S S; Park, J S; Kim, M S; Lee, J H; Chung, S Y; Choi, S; Chae, H J; Kim, H R; Ko, C B; Chung, H T; Park, R

2000-11-01

Although it has been well known that the role of LPS on liver damage is mediated through TNF-alpha, the mechanism by which LPS modulates the cytotoxicity of IFN-gamma on hepatocytes has not yet been clearly demonstrated. Here, we demonstrate that IFN-gamma mediated apoptosis in murine embryonic hepatocyte BNL CL2 cells is potentiated by the addition of LPS (0.5 microg/ml). Consistently, LPS markedly increases the catalytic activity of caspase 3-like protease but not caspase 1-like protease in IFN-gamma treated cells. In addition, TNF-alpha alone does not affect cell viability but rather it potentiates the cytotoxic effect of IFN-gamma on BNL CL2 cells. However, the cell viability of IFN-gamma/LPS treated cells is affected by the addition of polymyxin B but not by TNF binding protein I (TNF-BPI). These data suggest that the lipid moiety of LPS may mediate direct cytotoxicity of BNL CL2 cells in a TNF-alpha independent manner.

Human and murine very small embryonic-like cells represent multipotent tissue progenitors, in vitro and in vivo.

PubMed

Havens, Aaron M; Sun, Hongli; Shiozawa, Yusuke; Jung, Younghun; Wang, Jingcheng; Mishra, Anjali; Jiang, Yajuan; O'Neill, David W; Krebsbach, Paul H; Rodgerson, Denis O; Taichman, Russell S

2014-04-01

The purpose of this study was to determine the lineage progression of human and murine very small embryonic-like (HuVSEL or MuVSEL) cells in vitro and in vivo. In vitro, HuVSEL and MuVSEL cells differentiated into cells of all three embryonic germ layers. HuVSEL cells produced robust mineralized tissue of human origin compared with controls in calvarial defects. Immunohistochemistry demonstrated that the HuVSEL cells gave rise to neurons, adipocytes, chondrocytes, and osteoblasts within the calvarial defects. MuVSEL cells were also able to differentiate into similar lineages. First round serial transplants of MuVSEL cells into irradiated osseous sites demonstrated that ∼60% of the cells maintained their VSEL cell phenotype while other cells differentiated into multiple tissues at 3 months. Secondary transplants did not identify donor VSEL cells, suggesting limited self renewal but did demonstrate VSEL cell derivatives in situ for up to 1 year. At no point were teratomas identified. These studies show that VSEL cells produce multiple cellular structures in vivo and in vitro and lay the foundation for future cell-based regenerative therapies for osseous, neural, and connective tissue disorders.

Interleukin-17A-Deficient Mice Are Highly Susceptible to Toxoplasma gondii Infection Due to Excessively Induced T. gondii HSP70 and Interferon Gamma Production.

PubMed

Moroda, Masataka; Takamoto, Masaya; Iwakura, Yoichiro; Nakayama, Jun; Aosai, Fumie

2017-12-01

Interleukin17A (IL-17A) is known to be involved in the host defense against pathogens and the pathogenesis of autoimmune diseases. Previously, we showed that excessive amounts of interferon gamma (IFN-γ) play an important role in the pathogenesis of the lethal effects of Toxoplasma gondii by inducing anaphylactic responses. In the study described in this report, we examined the effects of IL-17A deficiency on murine host defense against oral T. gondii infection. IL-17A-deficient C57BL/6 (B6) mice exhibited higher rates of mortality than wild-type (WT) mice during the acute phase of T. gondii infection. CD4 + T cells in the mesenteric lymph nodes (mLNs) and ileum of T. gondii -infected IL-17A-deficient mice produced higher levels of IFN-γ than did those of WT mice. In addition, the level of T. gondii HSP70 ( T.g HSP70) expression was also significantly increased in the ileum, mLNs, liver, and spleen of infected IL-17A-deficient mice compared with that in WT mice. These elevated levels of expression of T.g HSP70 and IFN-γ in infected IL-17A-deficient mice were presumably linked to the IL-17A defect since they decreased to WT levels after treatment with recombinant IL-17A. Furthermore, IL-17A-deficient mice were highly susceptible to the anaphylactic effect of T.g HSP70, and the survival of IL-17A-deficient mice during the acute phase was improved by treatment with an anti- T.g HSP70 monoclonal antibody. These results suggest that IL-17A plays an important role in host survival against T. gondii infection by protecting the host from an anaphylactic reaction via the downregulation of T.g HSP70 and IFN-γ production. Copyright © 2017 American Society for Microbiology.

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers

PubMed Central

Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A.; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

2016-01-01

B-cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL+ MHC class-IHi CD86Hi B cells of unknown origin. Here we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. The 4BL cells induce expression of 4-1BBL and IFNγR1 on B1a cells resulting in subsequent up regulation of membrane TNFα (mTNFα) and CD86. As a result, B1a cells induce expression of granzyme B in CD8+T cells by targeting TNFR2 via mTNFα while providing co-stimulation with CD86. Thus, for the first time, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8+T cells. PMID:26983789

Tumor Regression and Delayed Onset Toxicity Following B7-H4 CAR T Cell Therapy

PubMed Central

Smith, Jenessa B; Lanitis, Evripidis; Dangaj, Denarda; Buza, Elizabeth; Poussin, Mathilde; Stashwick, Caitlin; Scholler, Nathalie; Powell, Daniel J

2016-01-01

B7-H4 protein is frequently overexpressed in ovarian cancer. Here, we engineered T cells with novel B7-H4-specific chimeric antigen receptors (CARs) that recognized both human and murine B7-H4 to test the hypothesis that B7-H4 CAR T cell therapy can be applied safely in preclinical models. B7-H4 CAR T cells specifically secreted IFN-γ and lysed B7-H4(+) targets. In vivo, B7-H4 CAR T cells displayed antitumor reactivity against B7-H4(+) human ovarian tumor xenografts. Unexpectedly, B7-H4 CAR T cell treatment reproducibly showed delayed, lethal toxicity 6–8 weeks after therapy. Comprehensive assessment of murine B7-H4 protein distribution uncovered expression in ductal and mucosal epithelial cells in normal tissues. Postmortem analysis revealed the presence of widespread histologic lesions that correlated with B7-H4(+) expression, and were inconsistent with graft versus host disease. Lastly, expression patterns of B7-H4 protein in normal human tissue were comparable to distribution in mice, advancing our understanding of B7-H4. We conclude that B7-H4 CAR therapy mediates control of cancer outgrowth. However, long-term engraftment of B7-H4 CAR T cells mediates lethal, off-tumor toxicity that is likely due to wide expression of B7-H4 in healthy mouse organs. This model system provides a unique opportunity for preclinical evaluation of safety approaches that limit CAR-mediated toxicity after tumor destruction in vivo. PMID:27439899

CXCR6 regulates the recruitment of pro-inflammatory IL-17A-producing T cells into atherosclerotic aortas

PubMed Central

Butcher, Matthew J.; Wu, Chih-I; Waseem, Tayab

2016-01-01

The adaptive immune response is involved in the development and progression of atherosclerosis and IL-17A+ cells play a role in this disease. Although elevated number of CD4+ IL-17A+ (Th17) and IL-17A+TCRγδ+ T cells are found within murine atherosclerotic aortas and human plaques, the mechanisms governing IL-17A+ T-cell migration to atherosclerotic lesions are unclear. The chemokine receptor CXCR6 is expressed on several T-cell subsets and plays a pro-atherogenic role in atherosclerosis. Here, we used CXCR6-deficient (Cxcr6 GFP/GFP) apolipoprotein E-deficient (Apoe −/−) mice to investigate the involvement of CXCR6 in the recruitment IL-17A+ T cells to atherosclerotic aortas. Flow cytometric analyses revealed reductions in Th17 and IL-17A+TCRγδ+ T cells within aged Cxcr6 GFP/GFP Apoe −/− aortas, in comparison with age-matched Cxcr6 GFP/+ Apoe −/− aortas. Although CXCR6-sufficient IL-17A+ T cells efficiently migrated toward CXCL16, the migration of CXCR6-deficient IL-17A+ T cells was abolished in transwell assays. Importantly, the recruitment of Cxcr6 GFP/GFP Apoe −/− IL-17A+ T cells into the aortas of Apoe −/− recipients was markedly reduced in short-term adoptive transfer experiments. Altogether these results demonstrate an important role of CXCR6 in the regulation of pathological Th17 and IL-17A+TCRγδ+ T-cell recruitment into atherosclerotic lesions. PMID:26614640

CXCR6 regulates the recruitment of pro-inflammatory IL-17A-producing T cells into atherosclerotic aortas.

PubMed

Butcher, Matthew J; Wu, Chih-I; Waseem, Tayab; Galkina, Elena V

2016-05-01

The adaptive immune response is involved in the development and progression of atherosclerosis and IL-17A(+) cells play a role in this disease. Although elevated number of CD4(+) IL-17A(+) (Th17) and IL-17A(+)TCRγδ(+) T cells are found within murine atherosclerotic aortas and human plaques, the mechanisms governing IL-17A(+) T-cell migration to atherosclerotic lesions are unclear. The chemokine receptor CXCR6 is expressed on several T-cell subsets and plays a pro-atherogenic role in atherosclerosis. Here, we used CXCR6-deficient (Cxcr6 (GFP/GFP) ) apolipoprotein E-deficient (Apoe (-/-) ) mice to investigate the involvement of CXCR6 in the recruitment IL-17A(+) T cells to atherosclerotic aortas. Flow cytometric analyses revealed reductions in Th17 and IL-17A(+)TCRγδ(+) T cells within aged Cxcr6 (GFP/GFP) Apoe (-/-) aortas, in comparison with age-matched Cxcr6 (GFP/+) Apoe (-/-) aortas. Although CXCR6-sufficient IL-17A(+) T cells efficiently migrated toward CXCL16, the migration of CXCR6-deficient IL-17A(+) T cells was abolished in transwell assays. Importantly, the recruitment of Cxcr6 (GFP/GFP) Apoe (-/-) IL-17A(+) T cells into the aortas of Apoe (-/-) recipients was markedly reduced in short-term adoptive transfer experiments. Altogether these results demonstrate an important role of CXCR6 in the regulation of pathological Th17 and IL-17A(+)TCRγδ(+) T-cell recruitment into atherosclerotic lesions. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

B cells expressing the transcription factor T-bet drive lupus-like autoimmunity

PubMed Central

Rubtsov, Anatoly V.; Thurman, Joshua M.; Mennona, Johanna M.; Kappler, John W.; Marrack, Philippa

2017-01-01

B cells contribute to multiple aspects of autoimmune disorders and may play a role in triggering disease. Thus, targeting B cells may be a promising strategy for treating autoimmune disorders. Better understanding of the B cell subsets that are responsible for the development of autoimmunity will be critical for developing efficient therapies. Here we have reported that B cells expressing the transcription factor T-bet promote the rapid appearance of autoantibodies and germinal centers in spontaneous murine models of systemic lupus erythematosus (SLE). Conditional deletion of T-bet from B cells impaired the formation of germinal centers and mitigated the development of kidney damage and rapid mortality in SLE mice. B cell–specific deletion of T-bet was also associated with lower activation of both B cells and T cells. Taken together, our results suggest that targeting T-bet–expressing B cells may be a potential target for therapy for autoimmune diseases. PMID:28240602

PHOX2B Is A Reliable Immunomarker in Distinguishing Peripheral Neuroblastic Tumors From CNS Embryonal Tumors.

PubMed

Alexandrescu, Sanda; Paulson, Vera; Dubuc, Adrian; Ligon, Azra; Lidov, Hart G

2018-05-14

The PHOX2B gene regulates neuronal maturation in the brain stem nuclei associated with cardiorespiratory function, and in the autonomic sympathetic and enteric nervous system. PHOX2B expression is a reliable immunomarker for peripheral neuroblastic tumors, however no systematic evaluation of CNS embryonal tumors was included in the studies. We encountered two cases in which the differential diagnosis included neuroblastoma and CNS embryonal tumor, and we hypothesized that PHOX2B immunostain would be helpful establishing the diagnosis. PHOX2B immunostain was performed on 29 pediatric cases, with adequate controls: 1 retroperitoneal embryonal tumor in a child with retinoblastoma (index1), 1 posterior fossa embryonal tumor in a child with a neuroblastoma (index2), 7 medulloblastomas, 4 atypical teratoid/rhabdoid tumors (ATRT), 4 retinoblastomas, 6 pineoblastomas, 4 embryonal tumors with multilayered rosettes (ETMR), and 2 CNS embryonal tumors, NEC. Cell lineage immunomarkers (GFAP, OLIG2, Synaptophysin, NeuN, CRX, PGP9.5), immunosurrogates for molecular alterations (beta-catenin, INI1, Lin28), array CGH and OncoPanel were performed as needed. Medulloblastomas, ATRTs, ETMRs, retinoblastomas and CNS embryonal tumors NOS were essentially negative for PHOX2B. Two (2) of 6 pineoblastomas had significant PHOX2B expression, while the rest were negative. Index1 was negative for PHOX2B and PGP 9.5, and positive for CRX, consistent with retinoblastoma. Index2 had diffuse PHOX2B expression, MYCN amplification and no copy number changes of medulloblastoma, in keeping with neuroblastoma. PHOX2B antibody is helpful in distinguishing between peripheral neuroblastic and CNS embryonal tumors, which are immunonegative, with the caveat that a subset of pineoblastomas has significant expression. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors.

PubMed

Gareau, Tia; Lara, Giovanna G; Shepherd, Robert D; Krawetz, Roman; Rancourt, Derrick E; Rinker, Kristina D; Kallos, Michael S

2014-04-01

Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue-engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm(2) ), 60 rpm (3 dyne/cm(2) )] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct-4, Nanog, Sox-2 and Rex-1 were assessed using gene expression profiles and flow-cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs. Copyright © 2012 John Wiley & Sons, Ltd.

A Minimal Chimera of Human Cyclin T1 and Tat Binds TAR and Activates Human Immunodeficiency Virus Transcription in Murine Cells

PubMed Central

Fujinaga, Koh; Irwin, Dan; Taube, Ran; Zhang, Fan; Geyer, Matthias; Peterlin, B. Matija

2002-01-01

The transcriptional elongation of human immunodeficiency virus type 1 (HIV-1) is mediated by the virally encoded transactivator Tat and its cellular cofactor, positive transcription elongation factor b (P-TEFb). The human cyclin T1 (hCycT1) subunit of P-TEFb forms a stable complex with Tat and the transactivation response element (TAR) RNA located at the 5′ end of all viral transcripts. Previous studies have demonstrated that hCycT1 binds Tat in a Zn2+-dependent manner via the cysteine at position 261, which is a tyrosine in murine cyclin T1. In the present study, we mutated all other cysteines and histidines that could be involved in this Zn2+-dependent interaction. Because all of these mutant proteins except hCycT1(C261Y) activated viral transcription in murine cells, no other cysteine or histidine in hCycT1 is responsible for this interaction. Next, we fused the N-terminal 280 residues in hCycT1 with Tat. Not only the full-length chimera but also the mutant hCycT1 with an N-terminal deletion to position 249, which retained the Tat-TAR recognition motif, activated HIV-1 transcription in murine cells. This minimal hybrid mutant hCycT1-Tat protein bound TAR RNA as well as human and murine P-TEFb in vitro. We conclude that this minimal chimera not only reproduces the high-affinity binding among P-TEFb, Tat, and TAR but also will be invaluable for determining the three-dimensional structure of this RNA-protein complex. PMID:12438619

Microfluidic-based patterning of embryonic stem cells for in vitro development studies.

PubMed

Suri, Shalu; Singh, Ankur; Nguyen, Anh H; Bratt-Leal, Andres M; McDevitt, Todd C; Lu, Hang

2013-12-07

In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments.

Microfluidic-based patterning of embryonic stem cells for in vitro development studies

PubMed Central

Suri, Shalu; Singh, Ankur; Nguyen, Anh H.; Bratt-Leal, Andres M.; McDevitt, Todd C.

2013-01-01

In vitro recapitulation of mammalian embryogenesis and examination of the emerging behaviours of embryonic structures require both the means to engineer complexity and accurately assess phenotypes of multicellular aggregates. Current approaches to study multicellular populations in 3D configurations are limited by the inability to create complex (i.e. spatially heterogeneous) environments in a reproducible manner with high fidelity thus impeding the ability to engineer microenvironments and combinations of cells with similar complexity to that found during morphogenic processes such as development, remodelling and wound healing. Here, we develop a multicellular embryoid body (EB) fusion technique as a higher-throughput in vitro tool, compared to a manual assembly, to generate developmentally relevant embryonic patterns. We describe the physical principles of the EB fusion microfluidic device design; we demonstrate that >60 conjoined EBs can be generated overnight and emulate a development process analogous to mouse gastrulation during early embryogenesis. Using temporal delivery of bone morphogenic protein 4 (BMP4) to embryoid bodies, we recapitulate embryonic day 6.5 (E6.5) during mouse embryo development with induced mesoderm differentiation in murine embryonic stem cells leading to expression of Brachyury-T-green fluorescent protein (T-GFP), an indicator of primitive streak development and mesoderm differentiation during gastrulation. The proposed microfluidic approach could be used to manipulate hundreds or more of individual embryonic cell aggregates in a rapid fashion, thereby allowing controlled differentiation patterns in fused multicellular assemblies to generate complex yet spatially controlled microenvironments. PMID:24113509

Aging Converts Innate B1a Cells into Potent CD8+ T Cell Inducers.

PubMed

Lee-Chang, Catalina; Bodogai, Monica; Moritoh, Kanako; Chen, Xin; Wersto, Robert; Sen, Ranjan; Young, Howard A; Croft, Michael; Ferrucci, Luigi; Biragyn, Arya

2016-04-15

B cell dysregulation in aging is thought to mostly occur in conventional B2 cells without affecting innate B1 cells. Elderly humans and mice also accumulate 4-1BBL(+)MHC class-I(Hi)CD86(Hi)B cells of unknown origin. In this article, we report that these cells, termed 4BL cells, are activated murine and possibly human B1a cells. The activation is mediated by aging human monocytes and murine peritoneal macrophages. They induce expression and activation of 4-1BBL and IFN-γR1 on B1a cells to subsequently upregulate membrane TNF-α and CD86. As a result, activated B1a/4BL cells induce expression of granzyme B in CD8(+)T cells by targeting TNFR2 via membrane TNF-α and providing costimulation with CD86. Thus, for the first time, to our knowledge, these results indicate that aging affects the function of B1a cells. Upon aging, these cells lose their tumor-supporting activity and become inducers of potentially antitumor and autoimmune CD8(+)T cells. Copyright © 2016 by The American Association of Immunologists, Inc.

mCLCA3 Modulates IL-17 and CXCL-1 Induction and Leukocyte Recruitment in Murine Staphylococcus aureus Pneumonia

PubMed Central

Dietert, Kristina; Reppe, Katrin; Mundhenk, Lars; Witzenrath, Martin; Gruber, Achim D.

2014-01-01

The human hCLCA1 and its murine ortholog mCLCA3 (calcium-activated chloride channel regulators) are exclusively expressed in mucus cells and linked to inflammatory airway diseases with increased mucus production, such as asthma, cystic fibrosis and chronic obstructive pulmonary disease. Both proteins have a known impact on the mucus cell metaplasia trait in these diseases. However, growing evidence points towards an additional role in innate immune responses. In the current study, we analyzed Staphylococcus aureus pneumonia, an established model to study pulmonary innate immunity, in mCLCA3-deficient and wild-type mice, focusing on the cellular and cytokine-driven innate inflammatory response. We compared clinical signs, bacterial clearance, leukocyte immigration and cytokine responses in the bronchoalveolar compartment, as well as pulmonary vascular permeability, histopathology, mucus cell number and mRNA expression levels of selected genes (mClca1 to 7, Muc5ac, Muc5b, Muc2, Cxcl-1, Cxcl-2, Il-17). Deficiency of mCLCA3 resulted in decreased neutrophilic infiltration into the bronchoalveolar space during bacterial infection. Only the cytokines IL-17 and the murine CXCL-8 homolog CXCL-1 were decreased on mRNA and protein levels during bacterial infection in mCLCA3-deficient mice compared to wild-type controls. However, no differences in clinical outcome, histopathology or mucus cell metaplasia were observed. We did not find evidence for regulation of any other CLCA homolog that would putatively compensate for the lack of mCLCA3. In conclusion, mCLCA3 appears to modulate leukocyte response via IL-17 and murine CXCL-8 homologs in acute Staphylococcus aureus pneumonia which is well in line with the proposed function of hCLCA1 as a signaling molecule acting on alveolar macrophages. PMID:25033194

Systemic Inflammation in Progressive Multiple Sclerosis Involves Follicular T-Helper, Th17- and Activated B-Cells and Correlates with Progression

PubMed Central

Christensen, Jeppe Romme; Börnsen, Lars; Ratzer, Rikke; Piehl, Fredrik; Khademi, Mohsen; Olsson, Tomas; Sørensen, Per Soelberg; Sellebjerg, Finn

2013-01-01

Pathology studies of progressive multiple sclerosis (MS) indicate a major role of inflammation including Th17-cells and meningeal inflammation with ectopic lymphoid follicles, B-cells and plasma cells, the latter indicating a possible role of the newly identified subset of follicular T-helper (TFH) cells. Although previous studies reported increased systemic inflammation in progressive MS it remains unclear whether systemic inflammation contributes to disease progression and intrathecal inflammation. This study aimed to investigate systemic inflammation in progressive MS and its relationship with disease progression, using flow cytometry and gene expression analysis of CD4+ and CD8+T-cells, B-cells, monocytes and dendritic cells. Furthermore, gene expression of cerebrospinal fluid cells was studied. Flow cytometry studies revealed increased frequencies of ICOS+TFH-cells in peripheral blood from relapsing-remitting (RRMS) and secondary progressive (SPMS) MS patients. All MS subtypes had decreased frequencies of Th1 TFH-cells, while primary progressive (PPMS) MS patients had increased frequency of Th17 TFH-cells. The Th17-subset, interleukin-23-receptor+CD4+T-cells, was significantly increased in PPMS and SPMS. In the analysis of B-cells, we found a significant increase of plasmablasts and DC-SIGN+ and CD83+B-cells in SPMS. ICOS+TFH-cells and DC-SIGN+B-cells correlated with disease progression in SPMS patients. Gene expression analysis of peripheral blood cell subsets substantiated the flow cytometry findings by demonstrating increased expression of IL21, IL21R and ICOS in CD4+T-cells in progressive MS. Cerebrospinal fluid cells from RRMS and progressive MS (pooled SPMS and PPMS patients) had increased expression of TFH-cell and plasmablast markers. In conclusion, this study is the first to demonstrate the potential involvement of activated TFH-cells in MS. The increased frequencies of Th17-cells, activated TFH- and B-cells parallel findings from pathology studies

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression.

PubMed

Klochendler-Yeivin, A; Fiette, L; Barra, J; Muchardt, C; Babinet, C; Yaniv, M

2000-12-01

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types.

The murine SNF5/INI1 chromatin remodeling factor is essential for embryonic development and tumor suppression

PubMed Central

Klochendler-Yeivin, Agnes; Fiette, Laurence; Barra, Jaqueline; Muchardt, Christian; Babinet, Charles; Yaniv, Moshe

2000-01-01

The assembly of eukaryotic DNA into nucleosomes and derived higher order structures constitutes a barrier for transcription, replication and repair. A number of chromatin remodeling complexes, as well as histone acetylation, were shown to facilitate gene activation. To investigate the function of two closely related mammalian SWI/SNF complexes in vivo, we inactivated the murine SNF5/INI1 gene, a common subunit of these two complexes. Mice lacking SNF5 protein stop developing at the peri-implantation stage, showing that the SWI/SNF complex is essential for early development and viability of early embryonic cells. Furthermore, heterozygous mice develop nervous system and soft tissue sarcomas. In these tumors the wild-type allele was lost, providing further evidence that SNF5 functions as a tumor suppressor gene in certain cell types. PMID:11263494

Maintenance and induction of murine embryonic stem cell differentiation using E-cadherin-Fc substrata without colony formation

NASA Astrophysics Data System (ADS)

Meng, Qing-Yuan; Akaike, Toshihiro

2013-03-01

Induced embryonic stem (ES) cells are expected to be promising cell resources for the observation of the cell behaviors in developmental biology as well as the implantation in cell treatments in human diseases. A recombinant E-cadherin substratum was developed as a cell recognizable substratum to maintain the ES cells' self-renewal and pluripotency at single cell level. Furthermore, the generation of various cell lineages in different germ layers, including hepatic or neural cells, was achieved on the chimeric protein layer precisely and effectively. The induction and isolation of specific cell population was carried out with the enhancing effect of other artificial extracellular matrices (ECMs) in enzyme-free process. The murine ES cell-derived cells showed highly morphological similarities and functional expressions to matured hepatocytes or neural progenitor cells.

PLGA nano/micro particles encapsulated with pertussis toxoid (PTd) enhances Th1/Th17 immune response in a murine model.

PubMed

Li, Pan; Asokanathan, Catpagavalli; Liu, Fang; Khaing, Kyi Kyi; Kmiec, Dorota; Wei, Xiaoqing; Song, Bing; Xing, Dorothy; Kong, Deling

2016-11-20

Poly(lactic-co-glycolic acid) (PLGA) based nano/micro particles were investigated as a potential vaccine platform for pertussis antigen. Presentation of pertussis toxoid as nano/micro particles (NP/MP) gave similar antigen-specific IgG responses in mice compared to soluble antigen. Notably, in cell line based assays, it was found that PLGA based nano/micro particles enhanced the phagocytosis of fluorescent antigen-nano/micro particles by J774.2 murine monocyte/macrophage cells compared to soluble antigen. More importantly, when mice were immunised with the antigen-nano/micro particles they significantly increased antigen-specific Th1 cytokines INF-γ and IL-17 secretion in splenocytes after in vitro re-stimulation with heat killed Bordetalla pertussis, indicating the induction of a Th1/Th17 response. Also, presentation of pertussis antigen in a NP/MP formulation is able to provide protection against respiratory infection in a murine model. Thus, the NP/MP formulation may provide an alternative to conventional acellular vaccines to achieve a more balanced Th1/Th2 immune response. Copyright © 2016 Elsevier B.V. All rights reserved.

Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production

PubMed Central

Byun, J-K; Moon, S-J; Jhun, J-Y; Kim, E-K; Park, J-S; Youn, J; Min, J-K; Park, S-H; Kim, H-Y; Cho, M-L

2014-01-01

Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti-arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co-stimulator (ICOS)+ follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg), CD19+ CD1dhigh CD5high, CD19+ CD25high forkhead box protein 3 (FoxP3)+ regulatory B (Breg) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS+GL-7+ germinal centre B cells and B220− CD138+ plasma cells in the spleens of rebamipide-treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose-dependent manner in vitro. Rebamipide-induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4+ T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA. PMID:24749771

Regulation of type 17 helper T-cell function by nitric oxide during inflammation

PubMed Central

Niedbala, Wanda; Alves-Filho, Jose C.; Fukada, Sandra Y.; Vieira, Silvio Manfredo; Mitani, Akio; Sonego, Fabiane; Mirchandani, Ananda; Nascimento, Daniele C.; Cunha, Fernando Q.; Liew, Foo Y.

2011-01-01

Type 17 helper T (Th17) cells are implicated in the pathogenesis many of human autoimmune diseases. Development of Th17 can be enhanced by the activation of aryl hydrocarbon receptor (AHR) whose ligands include the environmental pollutant dioxin, potentially linking environmental factors to the increased prevalence of autoimmune disease. We report here that nitric oxide (NO) can suppress the proliferation and function of polarized murine and human Th17 cells. NO also inhibits AHR expression in Th17 cells and the downstream events of AHR activation, including IL-22, IL-23 receptor, and Cyp1a1. Conversely, NO did not affect the polarization of Th17 cells from mice deficient in AHR. Furthermore, mice lacking inducible nitric oxide synthase (Nos2−/−) developed more severe experimental autoimmune encephalomyelitis than WT mice, with elevated AHR expression, increased IL-17A, and IL-22 synthesis. NO may therefore represent an important endogenous regulator to prevent overexpansion of Th17 cells and control of autoimmune diseases caused by environmental pollutants. PMID:21576463

Tumor necrosis factor alpha blockade exacerbates murine psoriasis-like disease by enhancing Th17 function and decreasing expansion of Treg cells.

PubMed

Ma, Hak-Ling; Napierata, Lee; Stedman, Nancy; Benoit, Stephen; Collins, Mary; Nickerson-Nutter, Cheryl; Young, Deborah A

2010-02-01

Patients with psoriasis and psoriatic arthritis respond well to tumor necrosis factor alpha (TNFalpha) blockers in general; however, there is now mounting evidence that a small cohort of patients with rheumatoid arthritis who receive TNFalpha blockers develop psoriasis. This study was undertaken to explore the mechanisms underlying TNFalpha blockade-induced exacerbation of skin inflammation in murine psoriasis-like skin disease. Skin inflammation was induced in BALB/c scid/scid mice after they received CD4+CD45RB(high)CD25- (naive CD4) T cells from donor mice. These mice were treated with either anti-interleukin-12 (anti-IL-12)/23p40 antibody or murine TNFRII-Fc fusion protein and were examined for signs of disease, including histologic features, various cytokine levels in the serum, and cytokine or FoxP3 transcripts in the affected skin and draining lymph node (LN) cells. In a separate study, naive CD4+ T cells were differentiated into Th1 or Th17 lineages with anti-CD3/28 magnetic beads and appropriate cytokines in the presence or absence of TNFalpha. Cytokine gene expression from these differentiated cells was also determined. Neutralization of TNFalpha exacerbated skin inflammation and markedly enhanced the expression of the proinflammatory cytokines IL-1beta, IL-6, IL-17, IL-21, and IL-22 but suppressed FoxP3 expression in the skin and reduced the number of FoxP3-positive Treg cells in the draining LNs. TNFalpha also demonstrated a divergent role during priming and reactivation of naive T cells. These results reveal a novel immunoregulatory role of TNFalpha on Th17 and Treg cells in some individuals, which may account for the exacerbation of skin inflammation in some patients who receive anti-TNF treatments.

Psoriatic inflammation causes hepatic inflammation with concomitant dysregulation in hepatic metabolism via IL-17A/IL-17 receptor signaling in a murine model.

PubMed

Al-Harbi, Naif O; Nadeem, Ahmed; Al-Harbi, Mohammed M; Zoheir, Khairy M A; Ansari, Mushtaq A; El-Sherbeeny, Ahmed M; Alanazi, Khalid M; Alotaibi, Moureq R; Ahmad, Sheikh F

2017-02-01

Psoriatic inflammation has been shown to be associated with cardiovascular dysfunction and systemic inflammation. Recently, psoriasis has also been linked to hepatic disorders, however underlying mechanism connecting the two are unknown. IL-17A being a central pro-inflammatory cytokine in the pathogenesis of psoriasis may be involved in hepatic inflammation through its receptor and downward signaling; however so far no study has investigated IL-17A related signaling in the liver during psoriasis in a murine model. Therefore, this study explored psoriasis-induced hepatic inflammation and concurrent metabolic changes. Mice were applied topically imiquimod (IMQ) to develop psoriatic inflammation. Additionally mice were also treated either with IL-17A or anti-IL17A antibody to explore the role of IL-17 related signaling in liver. Mice were then assessed for hepatic inflammation through assessment of inflammatory/oxidative stress markers (IL-17RC, NFκB, IL-6, MCP-1, IL-1β, GM-CSF, ICAM-1, iNOS, lipid peroxides and myeloperoxidase activity) as well as hepatic injury (alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase) and protein/lipid metabolic biomarkers (total proteins, albumin, total bilirubin, triglycerides, HDL cholesterol, and total cholesterol). IMQ treatment led to hepatic inflammation as evidenced by increased pro-inflammatory cytokines and oxidative stress with concomitant dysregulation in hepatic protein/lipid metabolism. Treatment with IL-17A further aggravated, whereas treatment with anti-IL17A antibody ameliorated IMQ-induced changes in hepatic injury/inflammation and protein/lipid metabolism. Our study shows for the first time that psoriatic inflammation leads to hepatic inflammation which results in dysregulated protein/lipid metabolism through IL-17RC/NFκB signaling. This could result in increased risk of cardiovascular dysfunction in patients with psoriasis. Copyright © 2016 Elsevier GmbH. All rights reserved.

Immunosuppressive Effect of B7-H4 Pathway in a Murine Systemic Lupus Erythematosus Model.

PubMed

Xiao, Ze Xiu; Zheng, Xu; Hu, Li; Wang, Julie; Olsen, Nancy; Zheng, Song Guo

2017-01-01

B7-H4, one of the co-stimulatory molecules of the B7 family, has been shown to play an important role in negatively regulating the adaptive immune response by inhibiting the proliferation, activation, and cytokine production of T cells. In this study, we investigate the role of B7-H4 in development of systemic lupus erythematosus (SLE). We investigated a murine model of SLE using transfer of bone marrow-derived dendritic cells (BMDCs) that were incubated with activated syngeneic lymphocyte-derived DNA. The recipient mouse produced anti-ds-DNA antibodies as well as displayed splenomegaly and lymphadenopathy as shown by significantly increased weights, and the kidneys showed lupus-like pathological changes include urine protein and glomerulonephritis with hyperplasia in glomeruli and increased mesangial cells and vasculitis with perivascular cell infiltration, glomerular deposition of IgG and complement C3. We showed that B7-H4 deficiency in BMDCs could cause greater production of anti-ds-DNA antibodies in transferred mice, and the lymph tissue swelling and the kidney lesions were also exacerbated with B7-H4 deficiency. Treatment with a B7-H4 antagonist antibody also aggravated the lupus model. Conversely, B7-H4 Ig alleviated the lupus manifestations. Therefore, we conclude that B7-H4 is a negative check point for the development of SLE in this murine model. These results suggest that this approach may have a clinical potential in treating human SLE.

Immunosuppressive Effect of B7-H4 Pathway in a Murine Systemic Lupus Erythematosus Model

PubMed Central

Xiao, Ze Xiu; Zheng, Xu; Hu, Li; Wang, Julie; Olsen, Nancy; Zheng, Song Guo

2017-01-01

B7-H4, one of the co-stimulatory molecules of the B7 family, has been shown to play an important role in negatively regulating the adaptive immune response by inhibiting the proliferation, activation, and cytokine production of T cells. In this study, we investigate the role of B7-H4 in development of systemic lupus erythematosus (SLE). We investigated a murine model of SLE using transfer of bone marrow-derived dendritic cells (BMDCs) that were incubated with activated syngeneic lymphocyte-derived DNA. The recipient mouse produced anti-ds-DNA antibodies as well as displayed splenomegaly and lymphadenopathy as shown by significantly increased weights, and the kidneys showed lupus-like pathological changes include urine protein and glomerulonephritis with hyperplasia in glomeruli and increased mesangial cells and vasculitis with perivascular cell infiltration, glomerular deposition of IgG and complement C3. We showed that B7-H4 deficiency in BMDCs could cause greater production of anti-ds-DNA antibodies in transferred mice, and the lymph tissue swelling and the kidney lesions were also exacerbated with B7-H4 deficiency. Treatment with a B7-H4 antagonist antibody also aggravated the lupus model. Conversely, B7-H4 Ig alleviated the lupus manifestations. Therefore, we conclude that B7-H4 is a negative check point for the development of SLE in this murine model. These results suggest that this approach may have a clinical potential in treating human SLE. PMID:29321778

Cited2 Gene Controls Pluripotency and Cardiomyocyte Differentiation of Murine Embryonic Stem Cells through Oct4 Gene*

PubMed Central

Li, Qiang; Ramírez-Bergeron, Diana L.; Dunwoodie, Sally L.; Yang, Yu-Chung

2012-01-01

Cited2 (CBP/p300-interacting transactivator with glutamic acid (E)/aspartic acid (D)-rich tail 2) is a transcriptional modulator critical for the development of multiple organs. Although many Cited2-mediated phenotypes and molecular events have been well characterized using in vivo genetic murine models, Cited2-directed cell fate decision in embryonic stem cells (ESCs) remains elusive. In this study, we examined the role of Cited2 in the maintenance of stemness and pluripotency of murine ESCs by a gene-targeting approach. Cited2 knock-out (Cited2Δ/−, KO) ESCs display defective differentiation. Loss of Cited2 in differentiating ESCs results in delayed silencing of the genes involved in the maintenance of pluripotency and self-renewal of stem cells (Oct4, Klf4, Sox2, and c-Myc) and the disturbance in cardiomyocyte, hematopoietic, and neuronal differentiation. In addition, Cited2 KO ESCs experience a delayed induction of cardiomyocyte differentiation-associated proteins, NFAT3 (along with the reduced expression of NFAT3 target genes, Nkx2.5 and β-MHC), N-cadherin, and smooth muscle actin. CITED2 is recruited to the Oct4 promoter to regulate its expression during early ESC differentiation. This is the first demonstration that Cited2 controls ESC pluripotency and differentiation via direct regulation of Oct4 gene expression. PMID:22761414

Icariin promotes expression of junctophilin 2 and Ca2+ related function during cardiomyocyte differentiation of murine embryonic stem cells.

PubMed

Liang, Xingguang; Hong, Dongsheng; Huang, Yujie; Rao, Yuefeng; Ma, Kuifen; Huang, Mingzhu; Zhang, Xingguo; Lou, Yijia; Zhao, Qingwei

2015-12-01

Junctophilin2 (JP2) is a critical protein associated with cardiogenesis. Icariin (ICA) facilitated the directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes. However, little is known about the effects of ICA on JP2 during cardiac differentiation. Here, we explored whether ICA has effects on the expression and Ca2+ related function of JP2 during cardiomyocyte differentiation of ES cells in vitro. Embryonid bodies (EBs) formed by hanging drop were treated with 10(-7) mol/L ICA from day 5 to promote the cardiac differentiation. Percentage of beating EBs and number of beating area within EBs were monitored. Cardiomyocytes were purified by discontinuous percoll gradient centrifugation from EBs. The expression of JP2, α-actinin and troponin-T within EBs or isolated cardiomyocytes were analyzed by immunocytochemistry, western blot and flow cytometry. The transient Ca2+ release was characterized in cardiomyocytes treated with/without 10 mmol/L caffeine and 8 mmol/L Ca2+. Our results showed that ES cell-derived cardiomyocytes were well characterized with JP2 proteins. ICA promoted cardiomyocyte differentiation as indicated by an increased percentage of beating EBs and number of beating area within EBs. The expression of JP2, α-actinin and troponin-T were up-regulated both in EBs and isolated cardiomyocytes from EBs. Furthermore, ICA-induced JP2 expression was accompanied by a remarkable increase of the amplitude of Ca2+ transients in cardiomyocytes before/after caffeine and Ca2+ stimulating. In conclusion, ICA promotes in cardiac differentiation partly through regulating JP2 and improved the Ca2+ modulatory function of cardiomyocytes.

Mucosal tolerance disruption favors disease progression in an extraorbital lacrimal gland excision model of murine dry eye.

PubMed

Guzmán, Mauricio; Keitelman, Irene; Sabbione, Florencia; Trevani, Analía S; Giordano, Mirta N; Galletti, Jeremías G

2016-10-01

Dry eye is a highly prevalent immune disorder characterized by a dysfunctional tear film and a Th1/Th17 T cell response at the ocular surface. The specificity of these pathogenic effector T cells remains to be determined, but auto-reactivity is considered likely. However, we have previously shown that ocular mucosal tolerance to an exogenous antigen is disrupted in a scopolamine-induced murine dry eye model and that it is actually responsible for disease progression. Here we report comparable findings in an entirely different murine model of dry eye that involves resection of the extraorbital lacrimal glands but no systemic muscarinic receptor blockade. Upon ocular instillation of ovalbumin, a delayed breakdown in mucosal tolerance to this antigen was observed in excised but not in sham-operated mice, which was mediated by interferon γ- and interleukin 17-producing antigen-specific T cells. Consistently, antigen-specific regulatory T cells were detectable in sham-operated but not in excised mice. As for other models of ocular surface disorders, epithelial activation of the NF-κB pathway by desiccating stress was determinant in the mucosal immune outcome. Underscoring the role of mucosal tolerance disruption in dry eye pathogenesis, its prevention by a topical NF-κB inhibitor led to reduced corneal damage in excised mice. Altogether these results show that surgically originated desiccating stress also initiates an abnormal Th1/Th17 T cell response to harmless exogenous antigens that reach the ocular surface. This event might actually contribute to corneal damage and challenges the conception of dry eye as a strictly autoimmune disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom's syndrome gene.

PubMed

Chester, N; Kuo, F; Kozak, C; O'Hara, C D; Leder, P

1998-11-01

Bloom's syndrome is a human autosomal genetic disorder characterized at the cellular level by genome instability and increased sister chomatid exchanges (SCEs). Clinical features of the disease include proportional dwarfism and a predisposition to develop a wide variety of malignancies. The human BLM gene has been cloned recently and encodes a DNA helicase. Mouse embryos homozygous for a targeted mutation in the murine Bloom's syndrome gene (Blm) are developmentally delayed and die by embryonic day 13.5. The fact that the interrupted gene is the homolog of the human BLM gene was confirmed by its homologous sequence, its chromosomal location, and by demonstrating high numbers of SCEs in cultured murine Blm-/- fibroblasts. The proportional dwarfism seen in the human is consistent with the small size and developmental delay (12-24 hr) seen during mid-gestation in murine Blm-/- embryos. Interestingly, the growth retardation in mutant embryos can be accounted for by a wave of increased apoptosis in the epiblast restricted to early post-implantation embryogenesis. Mutant embryos do not survive past day 13.5, and at this time exhibit severe anemia. Red blood cells and their precursors from Blm-/- embryos are heterogeneous in appearance and have increased numbers of macrocytes and micronuclei. Both the apoptotic wave and the appearance of micronuclei in red blood cells are likely cellular consequences of damaged DNA caused by effects on replicating or segregating chromosomes.

Decreased Vδ2 γδ T cells associated with liver damage by regulation of Th17 response in patients with chronic hepatitis B.

PubMed

Wu, Xiaoli; Zhang, Ji-Yuan; Huang, Ang; Li, Yuan-Yuan; Zhang, Song; Wei, Jun; Xia, Siyuan; Wan, Yajuan; Chen, Weiwei; Zhang, Zheng; Li, Yangguang; Wen, Ti; Chen, Yan; Tanaka, Yoshimasa; Cao, Youjia; Wang, Puyue; Zhao, Liqing; Wu, Zhenzhou; Wang, Fu-Sheng; Yin, Zhinan

2013-10-15

 γδ T cells comprise a small subset of T cells and play a protective role against cancer and viral infections; however, their precise role in patients with chronic hepatitis B remains unclear.  Flow cytometry and immunofunctional assays were performed to analyze the impact of Vδ2 γδ (Vδ2) T cells in 64 immune-activated patients, 22 immune-tolerant carriers, and 30 healthy controls.  The frequencies of peripheral and hepatic Vδ2 T cells decreased with disease progression from immune tolerant to immune activated. In the latter group of patients, the decreases in peripheral and intrahepatic frequencies of Vδ2 T cells reversely correlated with alanine aminotransferase levels and histological activity index. These activated terminally differentiated memory phenotypic Vδ2 T cells exhibited impaired abilities in proliferation and chemotaxis, while maintained a relative intact interferon (IFN) γ production. Importantly, Vδ2 T cells, in vitro, significantly suppressed the production of cytokines associated with interleukin 17-producing CD4+ T (Th17) cells through both cell contact-dependent and IFN-γ-dependent mechanisms.  Inflammatory microenvironment in IA patients result in decreased numbers of Vδ2 T cells, which play a novel role by regulating the pathogenic Th17 response to protect the liver in patients with chronic hepatitis B.

Rebamipide attenuates autoimmune arthritis severity in SKG mice via regulation of B cell and antibody production.

PubMed

Byun, J-K; Moon, S-J; Jhun, J-Y; Kim, E-K; Park, J-S; Youn, J; Min, J-K; Park, S-H; Kim, H-Y; Cho, M-L

2014-10-01

Oxidative stress is involved in the pathophysiology of rheumatoid arthritis (RA). We investigated the therapeutic potential of rebamipide, a gastroprotective agent with a property of reactive oxygen species scavenger, on the development of inflammatory polyarthritis and the pathophysiological mechanisms by which rebamipide might confer anti-arthritic effects in SKG mice, an animal model of RA. Intraperitoneal (i.p.) injection of rebamipide attenuated the severity of clinical and histological arthritis. Rebampide treatment reduced the number of T helper type 1 (Th1), Th2, Th17, inducible T cell co-stimulator (ICOS)(+) follicular helper T (Tfh) transitional type (T2) and mature B cells in the spleen, but increased the number of regulatory T (Treg ), CD19(+) CD1d(high) CD5(high) , CD19(+) CD25(high) forkhead box protein 3 (FoxP3)(+) regulatory B (Breg ) cells, memory B cells, and transitional type 1 (T1) B cells. In addition, flow cytometric analysis revealed significantly decreased populations of FAS(+) GL-7(+) germinal centre B cells and B220(-) CD138(+) plasma cells in the spleens of rebamipide-treated SKG mice compared to controls. Rebamipide decreased germinal centre B cells and reciprocally induced Breg cells in a dose-dependent manner in vitro. Rebamipide-induced Breg cells had more suppressive capacity in relation to T cell proliferation and also inhibited Th17 differentiation from murine CD4(+) T cells. Together, these data show that i.p. administration of rebamipide suppresses arthritis severity by inducing Breg and Treg cells and suppressing Tfh and Th17 cells in a murine model of RA. © 2014 British Society for Immunology.

The Role of B Cells for in Vivo T Cell Responses to a Friend Virus-Induced Leukemia

NASA Astrophysics Data System (ADS)

Schultz, Kirk R.; Klarnet, Jay P.; Gieni, Randall S.; Hayglass, Kent T.; Greenberg, Philip D.

1990-08-01

B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4^+ helper and CD8^+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.

Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells.

PubMed

Bunea, Andrea; Rugină, Dumitriţa; Sconţa, Zoriţa; Pop, Raluca M; Pintea, Adela; Socaciu, Carmen; Tăbăran, Flaviu; Grootaert, Charlotte; Struijs, Karin; VanCamp, John

2013-11-01

Blueberry consumption is associated with health benefits contributing to a reduced risk for cardiovascular disease, diabetes and cancer. The aim of this study was to determine the anthocyanin profile of blueberry extracts and to evaluate their effects on B16-F10 metastatic melanoma murine cells. Seven blueberry cultivars cultivated in Romania were used. The blueberry extracts were purified over an Amberlite XAD-7 resin and a Sephadex LH-20 column, in order to obtain the anthocyanin rich fractions (ARF). The antioxidant activity of the ARF of all cultivars was evaluated by ABTS, CUPRAC and ORAC assays. High performance liquid chromatography followed by electrospray ionization mass spectrometry (HPLC-ESI-MS) was used to identify and quantify individual anthocyanins. The anthocyanin content of tested cultivars ranged from 101.88 to 195.01 mg malvidin-3-glucoside/100g fresh weight. The anthocyanin rich-fraction obtained from cultivar Torro (ARF-T) was shown to have the highest anthocyanin content and antioxidant activity, and inhibited B16-F10 melanoma murine cells proliferation at concentrations higher than 500 μg/ml. In addition, ARF-T stimulated apoptosis and increased total LDH activity in metastatic B16-F10 melanoma murine cells. These results indicate that the anthocyanins from blueberry cultivar could be used as a chemopreventive or adjuvant treatment for metastasis control. Copyright © 2013 Elsevier Ltd. All rights reserved.

B cell depletion reduces T cell activation in pancreatic islets in a murine autoimmune diabetes model.

PubMed

Da Rosa, Larissa C; Boldison, Joanne; De Leenheer, Evy; Davies, Joanne; Wen, Li; Wong, F Susan

2018-06-01

Type 1 diabetes is a T cell-mediated autoimmune disease characterised by the destruction of beta cells in the islets of Langerhans, resulting in deficient insulin production. B cell depletion therapy has proved successful in preventing diabetes and restoring euglycaemia in animal models of diabetes, as well as in preserving beta cell function in clinical trials in the short term. We aimed to report a full characterisation of B cell kinetics post B cell depletion, with a focus on pancreatic islets. Transgenic NOD mice with a human CD20 transgene expressed on B cells were injected with an anti-CD20 depleting antibody. B cells were analysed using multivariable flow cytometry. There was a 10 week delay in the onset of diabetes when comparing control and experimental groups, although the final difference in the diabetes incidence, following prolonged observation, was not statistically significant (p = 0.07). The co-stimulatory molecules CD80 and CD86 were reduced on stimulation of B cells during B cell depletion and repopulation. IL-10-producing regulatory B cells were not induced in repopulated B cells in the periphery, post anti-CD20 depletion. However, the early depletion of B cells had a marked effect on T cells in the local islet infiltrate. We demonstrated a lack of T cell activation, specifically with reduced CD44 expression and effector function, including IFN-γ production from both CD4 + and CD8 + T cells. These CD8 + T cells remained altered in the pancreatic islets long after B cell depletion and repopulation. Our findings suggest that B cell depletion can have an impact on T cell regulation, inducing a durable effect that is present long after repopulation. We suggest that this local effect of reducing autoimmune T cell activity contributes to delay in the onset of autoimmune diabetes.

A novel monoclonal antibody, C41, reveals IL-13Ralpha1 expression by murine germinal center B cells and follicular dendritic cells.

PubMed

Poudrier, J; Graber, P; Herren, S; Berney, C; Gretener, D; Kosco-Vilbois, M H; Gauchat, J F

2000-11-01

Responsiveness to IL-13 involves at least two chains, IL-4Ralpha and IL-13Ralpha1. Although mouse B cells express IL-4Ralpha, little is known about their expression of IL-13Ralpha chains. To investigate this topic further, we have generated a monoclonal antibody (C41) specific for murine IL-13Ralpha1. Using C41, IL-13Ralpha1 expression was detected on germinal center (GC) B cells by flow cytometry and immunohistochemistry. In addition, IL-13Ralpha1 was observed on follicular dendritic cells, but not interdigitating dendritic cells in the T cell areas. Furthermore, resting B cells also expressed IL-13Ralpha1, and in the presence of IL-13 produced increased amounts of IgM in response to in vitro CD40 stimulation. However, C41 was unable to neutralize this bioactivity. The distribution of IL-13Ralpha1 on murine B cells and during GC reactions suggests a role for IL-13 during B cell differentiation.

Aborted germinal center reactions and B cell memory by follicular T cells specific for a B cell receptor V region peptide.

PubMed

Heiser, Ryan A; Snyder, Christopher M; St Clair, James; Wysocki, Lawrence J

2011-07-01

A fundamental problem in immunoregulation is how CD4(+) T cells react to immunogenic peptides derived from the V region of the BCR that are created by somatic mechanisms, presented in MHC II, and amplified to abundance by B cell clonal expansion during immunity. BCR neo Ags open a potentially dangerous avenue of T cell help in violation of the principle of linked Ag recognition. To analyze this issue, we developed a murine adoptive transfer model using paired donor B cells and CD4 T cells specific for a BCR-derived peptide. BCR peptide-specific T cells aborted ongoing germinal center reactions and impeded the secondary immune response. Instead, they induced the B cells to differentiate into short-lived extrafollicular plasmablasts that secreted modest quantities of Ig. These results uncover an immunoregulatory process that restricts the memory pathway to B cells that communicate with CD4 T cells via exogenous foreign Ag.

Murine Cytomegalovirus Downregulates Interleukin-17 in Mice with Retrovirus-induced Immunosuppression that are Susceptible to Experimental Cytomegalovirus Retinitis

PubMed Central

Blalock, Emily L.; Chien, Hsin; Dix, Richard D.

2013-01-01

Interleukin-17 (IL-17), a proinflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by whole splenic CD4+ T cells. Based on additional studies using IL-10 −/− mice infected systemically with MCMV and IL-10 −/− mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10. PMID:23415673

Curcumin Attenuates Acute Graft-versus-Host Disease Severity via In Vivo Regulations on Th1, Th17 and Regulatory T Cells

PubMed Central

Lee, Sung-Hee; Yang, Eun-Ji; Min, Jun-Ki; Cho, Seok-Goo; Yang, Chul-Woo; Park, Sung-Hwan; Kim, Ho-Youn; Cho, Mi-La

2013-01-01

Background In this study we examined the in vivo and in vitro effects and mechanisms of action of curcumin on the development of acute graft-versus-host disease (GVHD) using a murine model. Methodology/Principal Findings Mixed lymphocyte reactions were used to determine the in vitro effects of curcumin. Treatment with curcumin attenuated alloreactive T cell proliferation and inhibited the production of interferon (IFN)-γ and interleukin (IL)-17. In a murine acute GVHD model, transplantation of curcumin-treated allogeneic splenocytes into irradiated recipient mice significantly reduced the clinical severity scores of acute GVHD manifested in the liver, skin, colon and lung as compared with animals receiving vehicle-treated splenocytes. c-Fos and c-Jun expression levels in the skin and intestine, which are major target organs, were analyzed using immunohistochemical staining. Expression of both proteins was reduced in epithelial tissues of skin and intestine from curcumin-treated GVHD animals. The IFN-γ-expressing CD4+ splenocytes and IFN-γ-expressing lymph node cells were dramatically decreased in curcumin-treated mice. In contrast, CD4+Foxp3+ splenocytes were increased in the curcumin-treated acute GVHD animals. Flow cytometric analysis revealed that animals transplanted with curcumin-treated allogeneic splenocytes showed increased populations of CD4+ regulatory T cells (Tregs) as well as CD8+ Treg cells, compared to animals administered vehicle-treated splenocytes. Curcumin-treated acute GVHD animals could have a change in B cell subpopulations. Conclusion/Significance In the present study, we investigated the efficacy and mechanism of action of curcumin treatment against acute GVHD. The acute GVHD mice administered with curcumin-treated splenocytes showed significantly reduced severity of acute GVHD. Curcumin exerted in vivo preventive effects on acute GVHD by reciprocal regulation of T helper 1 (Th1) and Treg (both CD4+ and CD8+ Treg) cell lineages as well as

MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation

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

Gong, Xi; Zhang, Kunshan; Wang, Yanlu

2013-10-04

Highlights: •We found that the 3′ UTR of the Fmr1 mRNA is a target of miR-130b. •MiR-130b suppresses the expression of Fmr1 in mouse embryonic stem cell. •MiR-130b alters the proliferation of mouse embryonic stem cell. •MiR-130b alters fate specification of mouse embryonic stem cell. -- Abstract: Fragile X syndrome, one of the most common forms of inherited mental retardation, is caused by expansion of the CGG repeat in the 5′-untranslated region of the X-linked Fmr1 gene, which results in transcriptional silencing and loss of expression of its encoded protein FMRP. The loss of FMRP increases proliferation and alters fatemore » specification in adult neural progenitor cells (aNPCs). However, little is known about Fmr1 mRNA regulation at the transcriptional and post-transcriptional levels. In the present study, we report that miR-130b regulated Fmr1 expression by directly targeting its 3′-untranslated region (3′ UTR). Up-regulation of miR-130b in mouse embryonic neural progenitor cells (eNPCs) decreased Fmr1 expression, markedly increased eNPC proliferation and altered the differentiation tendency of eNPCs, suggesting that antagonizing miR-130b may be a new therapeutic entry point for treating Fragile X syndrome.« less

Identification of distinct topographical surface microstructures favoring either undifferentiated expansion or differentiation of murine embryonic stem cells.

PubMed

Markert, Lotte D'Andrea; Lovmand, Jette; Foss, Morten; Lauridsen, Rune Hoff; Lovmand, Michael; Füchtbauer, Ernst-Martin; Füchtbauer, Annette; Wertz, Karin; Besenbacher, Flemming; Pedersen, Finn Skou; Duch, Mogens

2009-11-01

The potential of embryonic stem (ES) cells for both self-renewal and differentiation into cells of all three germ layers has generated immense interest in utilizing these cells for tissue engineering or cell-based therapies. However, the ability to culture undifferentiated ES cells without the use of feeder cells as well as means to obtain homogeneous, differentiated cell populations devoid of residual pluripotent ES cells still remain major challenges. Here we have applied murine ES cells to topographically microstructured surface libraries, BioSurface Structure Arrays (BSSA), and investigated whether these could be used to (i) identify topographically microstructured growth supports alleviating the need for feeder cells for expansion of undifferentiated ES cells and (ii) identify specific types of microstructures enforcing differentiation of ES cells. The BSSA surfaces arrays consisted of 504 different topographical microstructures each located in a tester field of 3 x 3 mm. The murine ES cell lines CJ7 and KH2 were seeded upon the BSSA libraries and specific topographical structures facilitating either undifferentiated ES cell growth or enhancing spreading indicative of differentiation of the ES cells were identified. Secondly serial passage of undifferentiated CJ7 ES cells on selected microstructures, identified in the screening of these BSSA libraries, showed that these cells had retained germ-line potential. These results indicate that one specific type of topographical surface microstructures, identified by the BSSA technology, can substitute for feeder cells and that another subset may be used to eliminate undifferentiated ES cells from a population of differentiated ES cells.

46 CFR 35.40-17 - Foam hose/monitor stations-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Foam hose/monitor stations-T/ALL. 35.40-17 Section 35.40... Requirements-TB/ALL. § 35.40-17 Foam hose/monitor stations—T/ALL. (a) At each required foam hose/monitor valve there shall be marked in not less than 2-inch red letters and figures: “FOAM STATION 1,” 2, 3, etc. (b...

46 CFR 35.40-17 - Foam hose/monitor stations-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Foam hose/monitor stations-T/ALL. 35.40-17 Section 35.40... Requirements-TB/ALL. § 35.40-17 Foam hose/monitor stations—T/ALL. (a) At each required foam hose/monitor valve there shall be marked in not less than 2-inch red letters and figures: “FOAM STATION 1,” 2, 3, etc. (b...

Granzyme B mediated function of Parvovirus B19-specific CD4+ T cells

PubMed Central

Kumar, Arun; Perdomo, Maria F; Kantele, Anu; Hedman, Lea; Hedman, Klaus; Franssila, Rauli

2015-01-01

A novel conception of CD4+ T cells with cytolytic potential (CD4+ CTL) is emerging. These cells appear to have a part in controlling malignancies and chronic infections. Human parvovirus B19 can cause a persistent infection, yet no data exist on the presence of B19-specific CD4+ CTLs. Such cells could have a role in the pathogenesis of some autoimmune disorders reported to be associated with B19. We explored the cytolytic potential of human parvovirus B19-specific T cells by stimulating peripheral blood mononuclear cell (PBMC) with recombinant B19-VP2 virus-like particles. The cytolytic potential was determined by enzyme immunoassay-based quantitation of granzyme B (GrB) and perforin from the tissue culture supernatants, by intracellular cytokine staining (ICS) and by detecting direct cytotoxicity. GrB and perforin responses with the B19 antigen were readily detectable in B19-seropositive individuals. T-cell depletion, HLA blocking and ICS experiments showed GrB and perforin to be secreted by CD4+ T cells. CD4+ T cells with strong GrB responses were found to exhibit direct cytotoxicity. As anticipated, ICS of B19-specific CD4+ T cells showed expected co-expression of GrB, perforin and interferon gamma (IFN-γ). Unexpectedly, also a strong co-expression of GrB and interleukin 17 (IL-17) was detected. These cells expressed natural killer (NK) cell surface marker CD56, together with the CD4 surface marker. To our knowledge, this is the first report on virus-specific CD4+ CTLs co-expressing CD56 antigen. Our results suggest a role for CD4+ CTL in B19 immunity. Such cells could function within both immune regulation and triggering of autoimmune phenomena such as systemic lupus erythematosus (SLE) or rheumatoid arthritis. PMID:26246896

ArtinM Mediates Murine T Cell Activation and Induces Cell Death in Jurkat Human Leukemic T Cells

PubMed Central

Oliveira-Brito, Patrícia Kellen Martins; Gonçalves, Thiago Eleutério; Vendruscolo, Patrícia Edivânia; Roque-Barreira, Maria Cristina

2017-01-01

The recognition of cell surface glycans by lectins may be critical for the innate and adaptive immune responses. ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus, activates antigen-presenting cells by recognizing TLR2 N-glycans and induces Th1 immunity. We recently demonstrated that ArtinM stimulated CD4+ T cells to produce proinflammatory cytokines. Here, we further studied the effects of ArtinM on adaptive immune cells. We showed that ArtinM activates murine CD4+ and CD8+ T cells, augmenting their positivity for CD25, CD69, and CD95 and showed higher interleukin (IL)-2 and interferon (IFN)-γ production. The CD4+ T cells exhibited increased T-bet expression in response to ArtinM, and IL-2 production by CD4+ and CD8+ T cells depended on the recognition of CD3εγ-chain glycans by ArtinM. The ArtinM effect on aberrantly-glycosylated neoplastic lymphocytes was studied in Jurkat T cells, in which ArtinM induced IL-2, IFN-γ, and IL-1β production, but decreased cell viability and growth. A higher frequency of AnnexinV- and propidium iodide-stained cells demonstrated the induction of Jurkat T cells apoptosis by ArtinM, and this apoptotic response was reduced by caspases and protein tyrosine kinase inhibitors. The ArtinM effects on murine T cells corroborated with the immunomodulatory property of lectin, whereas the promotion of Jurkat T cells apoptosis may reflect a potential applicability of ArtinM in novel strategies for treating lymphocytic leukemia. PMID:28665310

Corrective recombination of mouse immunoglobulin kappa alleles in Abelson murine leukemia virus-transformed pre-B cells.

PubMed Central

Feddersen, R M; Van Ness, B G

1990-01-01

Previous characterization of mouse immunoglobulin kappa gene rearrangement products cloned from murine plasmacytomas has indicated that two recombination events can take place on a single kappa allele (R. M. Feddersen and B. G. Van Ness, Proc. Natl. Acad. Sci. USA 82:4792-4797, 1985; M. A. Shapiro and M. Weigert, J. Immunol. 139:3834-3839, 1987). To determine whether multiple recombinations on a single kappa allele can contribute to the formation of productive V-J genes through corrective recombinations, we have examined several Abelson murine leukemia virus-transformed pre-B-cell clones which rearrange the kappa locus during cell culture. Clonal cell lines which had rearranged one kappa allele nonproductively while maintaining the other allele in the germ line configuration were grown, and secondary subclones, which subsequently expressed kappa protein, were isolated and examined for further kappa rearrangement. A full spectrum of rearrangement patterns was observed in this sequential cloning, including productive and nonproductive recombinations of the germ line allele and secondary recombinations of the nonproductive allele. The results show that corrective V-J recombinations, with displacement of the nonproductive kappa gene, occur with a significant frequency (6 of 17 kappa-producing subclones). Both deletion and maintenance of the primary (nonfunctional) V-J join, as a reciprocal product, were observed. Images PMID:2153918

Functional Heterogeneity in the CD4+ T Cell Response to Murine γ-Herpesvirus 68

PubMed Central

Hu, Zhuting; Blackman, Marcia A.; Kaye, Kenneth M.; Usherwood, Edward J.

2015-01-01

CD4+ T cells are critical for the control of virus infections, T cell memory and immune surveillance. Here we studied the differentiation and function of murine γ-herpesvirus 68 (MHV-68)-specific CD4+ T cells using gp150-specific TCR transgenic mice. This allowed a more detailed study of the characteristics of the CD4+ T cell response than previously available approaches for this virus. Most gp150-specific CD4+ T cells expressed T-bet and produced IFN-γ, indicating MHV-68 infection triggered differentiation of CD4+ T cells largely into the Th1 subset, whereas some became TFH and Foxp3+ regulatory T cells. These CD4+ T cells were protective against MHV-68 infection, in the absence of CD8+ T cells and B cells, and protection depended on IFN-γ secretion. Marked heterogeneity was observed in the CD4+ T cells, based on Ly6C expression. Ly6C expression positively correlated with IFN-γ, TNF-α and granzyme B production, T-bet and KLRG1 expression, proliferation and CD4+ T cell-mediated cytotoxicity. Ly6C expression inversely correlated with survival, CCR7 expression and secondary expansion potential. Ly6C+ and Ly6C− gp150-specific CD4+ T cells were able to interconvert in a bidirectional manner upon secondary antigen exposure in vivo. These results indicate that Ly6C expression is closely associated with antiviral activity in effector CD4+ T cells, but inversely correlated with memory potential. Interconversion between Ly6C+ and Ly6C− cells may maintain a balance between the two antigen-specific CD4+ T cell populations during MHV-68 infection. These findings have significant implications for Ly6C as a surface marker to distinguish functionally distinct CD4+ T cells during persistent virus infection. PMID:25662997

46 CFR 34.17-10 - Controls-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-10 Controls—T/ALL. (a) The foam agent, its container, measuring devices, and other items peculiar to the system shall be of an approved type. (b) The foam-producing material...

46 CFR 34.17-10 - Controls-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-10 Controls—T/ALL. (a) The foam agent, its container, measuring devices, and other items peculiar to the system shall be of an approved type. (b) The foam-producing material...

Na{sub 3}[B{sub 20}H{sub 17}NH{sub 3}]: Synthesis and liposomal delivery to murine tumors

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

Feakes, D.A.; Shelly, K.; Knobler, C.B.

1994-04-12

The polyhedral borane ion [n-B{sub 20}H{sub 18}]{sup 2{minus}} reacts with liquid ammonia in the presence of a suitable base to produce an apical-equatorial (ae) isomer of the [B{sub 20}H{sub 17}NH{sub 3}]{sup 3{minus}} ion, [1-(2{prime}-B{sub 10}H{sub 9})-2-NH{sub 3}B{sub 10}H{sub 8}]{sup 3{minus}}. The structure of this product has been confirmed by {sup 11}B NMR spectroscopy and x-ray crystallography. This species undergoes acid-catalyzed rearrangement to an apical-apical (a{sup 2}) isomer, [1-(1{prime}-B{sub 10}H{sub 9})-2-NH{sub 3}B{sub 10}H{sub 8}]{sup 3{minus}}, whose structure has been determined by {sup 11}B NMR spectroscopy. The sodium salts of both the ae and the a{sup 2} isomers of [B{sub 20}H{sub 17}NH{submore » 3}]{sup 3{minus}} have been encapsulated within small unilamellar liposomes, composed of distearoyl phosphatidyl-choline/cholesterol (1:1), and investigated as boron-delivery agents for boron neutron capture therapy (BNCT) of cancer. The biodistribution of boron was determined after the injection of liposomal suspensions into BALB/c mice bearing EMT6 tumors. Both [B{sub 20}H{sub 17}NH{sub 3}]{sup 3{minus}} isomers exhibited excellent tumor uptake and selectivity at very low injected doses, achieving peak tumor boron concentrations of 30-40 {mu}g of B/g of tissue and tumor/blood boron ratios of {approximately}5. The enhanced retention of the [B{sub 20}H{sub 17}NH{sub 3}]{sup 3{minus}} isomers by EMT6 tumors may be attributed to their facile intracellular oxidation. In another experiment, [ae-B{sub 20}H{sub 17}NH{sub 3}]{sup 3{minus}} was encapsulated in liposomes prepared with 5% PEG-2000-distearoyl phosphatidylethanolamine in the liposome membrane. As expected, these liposomes exhibited a longer circulation lifetime in the biodistribution experiment, resulting in the continued accumulation of boron in the tumor over the entire 48-hr experiment and reaching a maximum of 47 {mu}g of B/g of tumor.« less

One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system.

PubMed

Waese, Elaine Y L; Stanford, William L

2011-01-01

Cartilage defects have limited capacity for repair and are often replaced by fibrocartilage with inferior mechanical properties. To overcome the limitations of artificial joint replacement, high-throughput screens (HTS) could be developed to identify molecules that stimulate differentiation and/or proliferation of articular cartilage for drug therapy or tissue engineering. Currently embryonic stem cells (ESCs) can differentiate into articular cartilage by forming aggregates (embryoid body (EB), pellet, micromass), which are difficult to image. We present a novel, single-step method of generating murine ESC-derived chondrocytes in monolayer cultures under chemically defined conditions. Mesoderm induction was achieved in cultures supplemented with BMP4, activin A, or Wnt3a. Prolonged culture with sustained activin A, TGFβ3, or BMP4 supplementation led to robust chondrogenic induction. A short pulse of activin A or BMP4 also induced chondrogenesis efficiently while Wnt3a acted as a later inducer. Long-term supplementation with activin A or with activin A followed by TGFβ3 promoted articular cartilage formation. Thus, we devised a serum-free (SF) culture system to generate ESC-derived chondrocytes without the establishment of 3D cultures or the aid of cell sorting. Cultures were governed by the same signaling pathways as 3D ESC differentiation systems and limb bud mesenchyme or articular cartilage explant cultures. Copyright © 2010 Elsevier B.V. All rights reserved.

Expression of the ephrin receptor B2 in the embryonic chicken bursa of Fabricius

USDA-ARS?s Scientific Manuscript database

Chicken B-cells develop in a specific organ, the bursa of Fabricius. To understand the bursal microenvironment guiding B-cell development, previous studies identified ephrin (Eph) receptor B2 (EphB2) gene transcripts in the embryonic bursa. We hypothesize that the EphB2 receptors and their ligands r...

Glucocorticoid Signaling Enhances Expression of Glucose-Sensing Molecules in Immature Pancreatic Beta-Like Cells Derived from Murine Embryonic Stem Cells In Vitro.

PubMed

Ghazalli, Nadiah; Wu, Xiaoxing; Walker, Stephanie; Trieu, Nancy; Hsin, Li-Yu; Choe, Justin; Chen, Chialin; Hsu, Jasper; LeBon, Jeanne; Kozlowski, Mark T; Rawson, Jeffrey; Tirrell, David A; Yip, M L Richard; Ku, Hsun Teresa

2018-06-06

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. In this study, we aim to identify small molecules that affect immature beta cells. A cell-based assay, using pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative reverse transcription-polymerase chain reaction analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GR flox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM d-glucose and stimulated by 17 mM d-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in

Transiting exoplanets from the CoRoT space mission. XVII. The hot Jupiter CoRoT-17b: a very old planet

NASA Astrophysics Data System (ADS)

Csizmadia, Sz.; Moutou, C.; Deleuil, M.; Cabrera, J.; Fridlund, M.; Gandolfi, D.; Aigrain, S.; Alonso, R.; Almenara, J.-M.; Auvergne, M.; Baglin, A.; Barge, P.; Bonomo, A. S.; Bordé, P.; Bouchy, F.; Bruntt, H.; Carone, L.; Carpano, S.; Cavarroc, C.; Cochran, W.; Deeg, H. J.; Díaz, R. F.; Dvorak, R.; Endl, M.; Erikson, A.; Ferraz-Mello, S.; Fruth, Th.; Gazzano, J.-C.; Gillon, M.; Guenther, E. W.; Guillot, T.; Hatzes, A.; Havel, M.; Hébrard, G.; Jehin, E.; Jorda, L.; Léger, A.; Llebaria, A.; Lammer, H.; Lovis, C.; MacQueen, P. J.; Mazeh, T.; Ollivier, M.; Pätzold, M.; Queloz, D.; Rauer, H.; Rouan, D.; Santerne, A.; Schneider, J.; Tingley, B.; Titz-Weider, R.; Wuchterl, G.

2011-07-01

We report on the discovery of a hot Jupiter-type exoplanet, CoRoT-17b, detected by the CoRoT satellite. It has a mass of 2.43 ± 0.30 MJup and a radius of 1.02 ± 0.07 RJup, while its mean density is 2.82 ± 0.38 g/cm3. CoRoT-17b is in a circular orbit with a period of 3.7681 ± 0.0003 days. The host star is an old (10.7 ± 1.0 Gyr) main-sequence star, which makes it an intriguing object for planetary evolution studies. The planet's internal composition is not well constrained and can range from pure H/He to one that can contain ~380 earth masses of heavier elements. The CoRoT space mission, launched on December 27th 2006, has been developed and is operated by CNES, with the contribution of Austria, Belgium, Brazil, ESA (RSSD and Science Programme), Germany and Spain. Part of the observations were obtained at the Canada-France-Hawaii Telescope (CFHT) which is operated by the National Research Council of Canada, the Institut National des Sciences de l'Univers of the Centre National de la Recherche Scientifique of France, and the University of Hawaii. Based on observations made with HARPS spectrograph on the 3.6-m European Organisation for Astronomical Research in the Southern Hemisphere telescope at La Silla Observatory, Chile (ESO program 184.C-0639). Based on observations made with the IAC80 telescope operated on the island of Tenerife by the Instituto de Astrofísica de Canarias in the Spanish Observatorio del Teide. Part of the data presented herein were obtained at the W.M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W.M. Keck Foundation.

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia

PubMed Central

Paszkiewicz, Paulina J.; Fräßle, Simon P.; Srivastava, Shivani; Sommermeyer, Daniel; Hudecek, Michael; Sadelain, Michel; Liu, Lingfeng; Jensen, Michael C.; Riddell, Stanley R.; Busch, Dirk H.

2016-01-01

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies. PMID:27760047

Targeted antibody-mediated depletion of murine CD19 CAR T cells permanently reverses B cell aplasia.

PubMed

Paszkiewicz, Paulina J; Fräßle, Simon P; Srivastava, Shivani; Sommermeyer, Daniel; Hudecek, Michael; Drexler, Ingo; Sadelain, Michel; Liu, Lingfeng; Jensen, Michael C; Riddell, Stanley R; Busch, Dirk H

2016-11-01

The adoptive transfer of T cells that have been genetically modified to express a CD19-specific chimeric antigen receptor (CAR) is effective for treating human B cell malignancies. However, the persistence of functional CD19 CAR T cells causes sustained depletion of endogenous CD19+ B cells and hypogammaglobulinemia. Thus, there is a need for a mechanism to ablate transferred T cells after tumor eradication is complete to allow recovery of normal B cells. Previously, we developed a truncated version of the epidermal growth factor receptor (EGFRt) that is coexpressed with the CAR on the T cell surface. Here, we show that targeting EGFRt with the IgG1 monoclonal antibody cetuximab eliminates CD19 CAR T cells both early and late after adoptive transfer in mice, resulting in complete and permanent recovery of normal functional B cells, without tumor relapse. EGFRt can be incorporated into many clinical applications to regulate the survival of gene-engineered cells. These results support the concept that EGFRt represents a promising approach to improve safety of cell-based therapies.

Protein tyrosine phosphatase 1B (PTP1B) is required for cardiac lineage differentiation of mouse embryonic stem cells.

PubMed

Eshkiki, Zahra Shokati; Ghahremani, Mohammad Hossein; Shabani, Parisa; Firuzjaee, Sattar Gorgani; Sadeghi, Asie; Ghanbarian, Hossein; Meshkani, Reza

2017-01-01

Protein tyrosine phosphatase 1B (PTP1B) has been shown to regulate multiple cellular events such as differentiation, cell growth, and proliferation; however, the role of PTP1B in differentiation of embryonic stem (ES) cells into cardiomyocytes remains unexplored. In the present study, we investigated the effects of PTP1B inhibition on differentiation of ES cells into cardiomyocytes. PTP1B mRNA and protein levels were increased during the differentiation of ES cells into cardiomyocytes. Accordingly, a stable ES cell line expressing PTP1B shRNA was established. In vitro, the number and size of spontaneously beating embryoid bodies were significantly decreased in PTP1B-knockdown cells, compared with the control cells. Decreased expression of cardiac-specific markers Nkx2-5, MHC-α, cTnT, and CX43, as assessed by real-time PCR analysis, was further confirmed by immunocytochemistry of the markers. The results also showed that PTP1B inhibition induced apoptosis in both differentiated and undifferentiated ES cells, as presented by increasing the level of cleaved caspase-3, cytochrome C, and cleaved PARP. Further analyses revealed that PTP1B inhibition did not change proliferation and pluripotency of undifferentiated ES cells. Taken together, the data presented here suggest that PTP1B is essential for proper differentiation of ES cells into cardiomyocytes.

Dexamethasone facilitates erythropoiesis in murine embryonic stem cells differentiating into hematopoietic cells in vitro

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

Srivastava, Anand S.; Kaushal, Sharmeela; Mishra, Rangnath

2006-07-28

Differentiating embryonic stem (ES) cells are increasingly emerging as an important source of hematopoietic progenitors with a potential to be useful for both basic and clinical research applications. It has been suggested that dexamethasone facilitates differentiation of ES cells towards erythrocytes but the mechanism responsible for sequential expression of genes regulating this process are not well-understood. Therefore, we in vitro induced differentiation of murine ES cells towards erythropoiesis and studied the sequential expression of a set of genes during the process. We hypothesized that dexamethasone-activates its cognate nuclear receptors inducing up-regulation of erythropoietic genes such as GATA-1, Flk-1, Epo-R, andmore » direct ES cells towards erythropoietic differentiation. ES cells were cultured in primary hematopoietic differentiation media containing methyl-cellulose, IMDM, IL-3, IL-6, and SCF to promote embryoid body (EB) formation. Total RNA of day 3, 5, and 9-old EBs was isolated for gene expression studies using RT-PCR. Cells from day 9 EBs were subjected to secondary differentiation using three different cytokines and growth factors combinations: (1) SCF, EPO, dexamethasone, and IGF; (2) SCF, IL-3, IL-6, and TPO; and (3) SCF IL-3, IL-6, TPO, and EPO. Total RNA from day 12 of secondary differentiated ES cells was isolated to study the gene expression pattern during this process. Our results demonstrate an up-regulation of GATA-1, Flk-1, HoxB-4, Epo-R, and globin genes ({alpha}-globin, {beta}H-1 globin, {beta}-major globin, {epsilon} -globin, and {zeta}-globin) in the 9-day-old EBs, whereas, RNA from 5-day-old EBs showed expression of HoxB-4, {epsilon}-globin, {gamma}-globin, {beta}H1-globin, and Flk-1. Three-day-old EBs showed only HoxB-4 and Flk-1 gene expression and lacked expression of all globin genes. These findings indicate that erythropoiesis-specific genes are activated later in the course of differentiation. Gene expression studies

The Aryl Hydrocarbon Receptor: Differential Contribution to T Helper 17 and T Cytotoxic 17 Cell Development

PubMed Central

Hayes, Mark D.; Ovcinnikovs, Vitalijs; Smith, Andrew G.; Kimber, Ian; Dearman, Rebecca J.

2014-01-01

The aryl hydrocarbon receptor (AhR) has been shown to be required for optimal Thelper (Th) 17 cell activation. Th17 cells provide immunity against extracellular pathogens and are implicated in autoimmune diseases. Herein, the role of the AhR in cytokine production by Th17, and by the analogous population of T cytotoxic (Tc)17 cells, has been examined. Lymph node Tc (CD8+) and Th (CD4+) cells were isolated by negative selection from naive AhR+/− and AhR−/− mice and polarised to Tc1/Th1 or Tc17/Th17 phenotypes with appropriate cytokines. Cell differentiation was assessed as a function of mRNA and protein (ELISA and flow cytometry) expression for interferon (IFN)-γ and for key Th17 cytokines. In AhR+/− mice, Th17 cells displayed an exclusive IL-17 profile, which was markedly inhibited by a selective AhR antagonist to levels observed in AhR knockout mice. Addition of the natural AhR agonist 6-formylindolo[3,2-b]carbazole (FICZ) markedly enhanced Th17 cell activity in the heterozygotes. In contrast, Tc17 cells polarised into 3 distinct subsets: producing either IL-17 or IFN-γ alone, or both cytokines. Blocking AhR was also detrimental to Tc17 development, with reduced responses recorded in AhR−/− mice and antagonist-mediated reduction of IL-17 expression in the heterozygotes. However, Tc17 cells were largely refractory to exogenous FICZ, presumably because Tc17 cells express baseline AhR mRNA, but unlike Th17 cells, there is no marked up-regulation during polarisation. Thus, Th17 cell development is more dependent upon AhR activation than is Tc17 cell development, suggesting that endogenous AhR ligands play a much greater role in driving Th17 cell responses. PMID:25203682

CD4+ T-Cell- and Gamma Interferon-Dependent Protection against Murine Malaria by Immunization with Linear Synthetic Peptides from a Plasmodium yoelii 17-Kilodalton Hepatocyte Erythrocyte Protein

PubMed Central

Charoenvit, Yupin; Majam, Victoria Fallarme; Corradin, Giampietro; Sacci, John B.; Wang, Ruobing; Doolan, Denise L.; Jones, Trevor R.; Abot, Esteban; Patarroyo, Manuel E.; Guzman, Fanny; Hoffman, Stephen L.

1999-01-01

Most work on protective immunity against the pre-erythrocytic stages of malaria has focused on induction of antibodies that prevent sporozoite invasion of hepatocytes, and CD8+ T-cell responses that eliminate infected hepatocytes. We recently reported that immunization of A/J mice with an 18-amino-acid synthetic linear peptide from Plasmodium yoelii sporozoite surface protein 2 (SSP2) in TiterMax adjuvant induces sterile protection that is dependent on CD4+ T cells and gamma interferon (IFN-γ). We now report that immunization of inbred A/J mice and outbred CD1 mice with each of two linear synthetic peptides from the 17-kDa P. yoelii hepatocyte erythrocyte protein (HEP17) in the same adjuvant also induces protection against sporozoite challenge that is dependent on CD4+ T cells and IFN-γ. The SSP2 peptide and the two HEP17 peptides are recognized by B cells as well as T cells, and the protection induced by these peptides appears to be directed against the infected hepatocytes. In contrast to the peptide-induced protection, immunization of eight different strains of mice with radiation-attenuated sporozoites induces protection that is absolutely dependent on CD8+ T cells. Data represented here demonstrate that CD4+ T-cell-dependent protection can be induced by immunization with linear synthetic peptides. These studies therefore provide the foundation for an approach to pre-erythrocytic-stage malaria vaccine development, based on the induction of protective CD4+ T-cell responses, which will complement efforts to induce protective antibody and CD8+ T-cell responses. PMID:10531206

Trametes versicolor protein YZP activates regulatory B lymphocytes - gene identification through de novo assembly and function analysis in a murine acute colitis model.

PubMed

Kuan, Yen-Chou; Wu, Ying-Jou; Hung, Chih-Liang; Sheu, Fuu

2013-01-01

Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized. We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d(+) B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway. We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d(+) Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models.

Oral exposure to Listeria monocytogenes in aged IL-17RKO mice: A possible murine model to study listeriosis in susceptible populations.

PubMed

Alam, Mohammad S; Costales, Matthew; Cavanaugh, Christopher; Pereira, Marion; Gaines, Dennis; Williams, Kristina

2016-10-01

Foodborne Listeria monocytogenes (LM) is a cause of serious illness and death in the US. The case-fatality rate of invasive LM infection in the elderly population is >50%. The goal of this study is to establish a murine model of oral LM infection that can be used as a surrogate for human foodborne listeriosis in the geriatric population. Adult C57BL/6 (wild-type, WT) and adult or old IL17R-KO (knock-out) mice were gavaged with a murinized LM strain (Lmo-InlA m ) and monitored for body-weight loss and survivability. Tissues were collected and assayed for bacterial burden, histology, and cytokine responses. When compared to WT mice, adult IL17R-KO mice are more susceptible to LM infection and showed increased LM burden and tissue pathology and a higher mortality rate. Older LM-infected KO-mice lost significantly (p < 0.02, ANOVA) more body-weight and had a higher bacterial burden in the liver (p = 0.03) and spleen as compared to adult mice. Uninfected, aged KO-mice showed a higher baseline pro-inflammatory response when compared to uninfected adult-KO mice. After infection, the pro-inflammatory cytokine, IFN-γ, mRNA in the liver was higher in the adult mice as compared to the old mice. The anti-inflammatory cytokine, IL-10, mRNA and regulatory T-cells (CD4 + CD25 +h or CD4 + Foxp3 + ) cells in the aged mice increased significantly after infection as compared to adult mice. Expression of the T-cell activation marker, CD25 (IL-2Rα) in the aged mice did not increase significantly over baseline. These data suggest that aged IL17R-KO mice can be used as an in vivo model to study oral listeriosis and that aged mice are more susceptible to LM infection due to dysregulation of pro- and anti-inflammatory responses compared to adult mice, resulting in a protracted clearance of the infection. Published by Elsevier Ltd.

T-bet promotes the accumulation of encephalitogenic Th17 cells in the CNS.

PubMed

Grifka-Walk, Heather M; Segal, Benjamin M

2017-03-15

T-bet enhances the encephalitogenicity of myelin-reactive CD4 + T cells, however its mechanism of action is unknown. In this study we show that T-bet confers a competitive advantage for the accumulation of IL-23 conditioned Th17 effector cells in the central nervous system (CNS). Impaired migration of T-bet deficient Th17 cells to the CNS is associated with altered expression of adhesion molecules and chemokine receptors on their cell surface. Our data suggest that therapeutic targeting of T-bet in individuals with Th17-mediated autoimmune demyelinating disease may inhibit inflammatory infiltration of the CNS and, hence, clinical exacerbations. Copyright © 2016 Elsevier B.V. All rights reserved.

Dermal γδ T Cells Do Not Freely Re-Circulate Out of Skin and Produce IL-17 to Promote Neutrophil Infiltration during Primary Contact Hypersensitivity

PubMed Central

Jiang, Xiaodong; Park, Chang Ook; Geddes Sweeney, Jenna; Yoo, Min Jae; Gaide, Olivier; Kupper, Thomas Seth

2017-01-01

The role of mouse dermal γδ T cells in inflammatory skin disorders and host defense has been studied extensively. It is known that dendritic epidermal T cells (DETC) have a monomorphic γδ T cell receptor (TCR) and reside in murine epidermis from birth. We asked if dermal γδ cells freely re-circulated out of skin, or behaved more like dermal resident memory T cells (TRM) in mice. We found that, unlike epidermal γδ T cells (DETC), dermal γδ cells are not homogeneous with regard to TCR, express the tissue resident T cell markers CD69 and CD103, bear skin homing receptors, and produce IL-17 and IL-22. We created GFP+: GFP− parabiotic mice and found that dermal γδ T cells re-circulate very slowly—more rapidly than authentic αβ TCR TRM, but more slowly than the recently described dermal αβ TCR T migratory memory cells (TMM). Mice lacking the TCR δ gene (δ-/-) had a significant reduction of 2,4-dinitrofluorobenzene (DNFB)-induced contact hypersensitivity (CHS). We created mice deficient in dermal γδ T cells but not DETC, and these mice also showed a markedly reduced CHS response after DNFB challenge. The infiltration of effector T cells during CHS was not reduced in dermal γδ T cell-deficient mice; however, infiltration of Gr-1+CD11b+ neutrophils, as well as ear swelling, was reduced significantly. We next depleted Gr-1+ neutrophils in vivo, and demonstrated that neutrophils are required for ear swelling, the accepted metric for a CHS response. Depletion of IL-17-producing dermal Vγ4+ cells and neutralization of IL-17 in vivo, respectively, also led to a significantly reduced CHS response and diminished neutrophil infiltration. Our findings here suggest that dermal γδ T cells have an intermediate phenotype of T cell residence, and play an important role in primary CHS through producing IL-17 to promote neutrophil infiltration. PMID:28081153

Idiotypic properties of the murine anti-arsonate antibody response: B- and T-cell influences.

PubMed

Conger, J D; Lewis, G K; Goodman, J W

1985-10-01

In a previous report characterizing the arsonate (ABA)-specific plaque-forming cell (PFC) responses of A/J mice induced by ABA-KLH, two interesting characteristics of the idiotypic (Id) profile were noted: (1) an apparent Id selectivity in the isotype switch since the earliest appearing IgG PFC in the primary response were significantly more "cross-reactive Id" (CRI)-dominant than the IgM PFC population, and, (2) a temporal waning of CRI dominance with time among IgG PFC, from 75-100% CRI+ PFC to about 25-45% CRI+ PFC in secondary responses. Experiments were performed to determine whether these effects are largely attributable to T or to B cells. Mice were immunized with a T-independent (TI) form of ABA (ABA-Brucella abortus) and apparent Id selectivity was observed; the earliest IgG PFC averaged 75% CRI+ while IgM PFC were only 39% CRI+. Due to the TI nature of the Ag, this provides suggestive, but not conclusive, evidence that the Id asymmetry in the isotype switch may be attributable to the direct interaction of Ag with B cells. Other studies addressed the temporal shift in CRI dominance. First, it was found that preexposure of mice to either KLH or to ABA (on an irrelevant carrier) resulted in diminished CRI dominance in subsequent "primary" responses to ABA-KLH. Secondly, adoptive transfer experiments with B and T cells from virgin mice (Bv, Tv) or ABA-KLH-primed mice (Bp, Tp) showed that recipients of Bv + Tp or Bp + Tv generated anti-ABA PFC responses with intermediate CRI levels. The Tv cells had some preferential tendency to activate CRI+ clones in the Bp population. The results demonstrate that CRI levels are jointly determined by the immune status of both B and T cells. A simple model is offered which accounts for early Id dominance and its gradual decline and has as its central postulate the assumption that CRI+ B cells in the virgin ABA-specific repertoire have an affinity advantage over CRI- clones.

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction.

PubMed

Oida, Takatoku; Weiner, Howard L

2010-11-24

It has been reported that human FOXP3(+) CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3(+) Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs. We generated anti-mouse LAP mAbs by immunizing TGF-β(-/-) animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3(+) CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4(+)CD25(-) T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4(+)CD25(-) T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3(+) but also on T cells that remained Foxp3(-) after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells. Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.

Development and characterization of Histoplasma capsulatum-reactive murine T-cell lines and clones

NASA Technical Reports Server (NTRS)

Deepe, George S., Jr.; Smith, James G.; Denman, David; Bullock, Ward E.; Sonnenfeld, Gerald

1986-01-01

Several Histoplasma capsulatum-reactive murine cloned T-cell lines (TCLs) were isolated from spleens of C57BL/6 mice immunized with viable H. capsulatum yeast cells, using the methodology of Kimoto and Fathman (1980). These T-cells were characterized phenotypically as Thy-1.2(+) Lyt-1(+) L3T4(+) Lyt-2(-), that is, as the helper/inducer phenotype. The cloned T cells proliferate in response to histoplasmin and, in some cases, to heterologous fungal anigens. Upon injection of mice with the antigen, the T-cells mediate local delayed-type hypersensitivity responses and, after stimulation, release regulatory lymphokines.

High dietary folate in pregnant mice leads to pseudo-MTHFR deficiency and altered methyl metabolism, with embryonic growth delay and short-term memory impairment in offspring.

PubMed

Bahous, Renata H; Jadavji, Nafisa M; Deng, Liyuan; Cosín-Tomás, Marta; Lu, Jessica; Malysheva, Olga; Leung, Kit-Yi; Ho, Ming-Kai; Pallàs, Mercè; Kaliman, Perla; Greene, Nicholas D E; Bedell, Barry J; Caudill, Marie A; Rozen, Rima

2017-03-01

Methylenetetrahydrofolate reductase (MTHFR) generates methyltetrahydrofolate for methylation reactions. Severe MTHFR deficiency results in homocystinuria and neurologic impairment. Mild MTHFR deficiency (677C > T polymorphism) increases risk for complex traits, including neuropsychiatric disorders. Although low dietary folate impacts brain development, recent concerns have focused on high folate intake following food fortification and increased vitamin use. Our goal was to determine whether high dietary folate during pregnancy affects brain development in murine offspring. Female mice were placed on control diet (CD) or folic acid-supplemented diet (FASD) throughout mating, pregnancy and lactation. Three-week-old male pups were evaluated for motor and cognitive function. Tissues from E17.5 embryos, pups and dams were collected for choline/methyl metabolite measurements, immunoblotting or gene expression of relevant enzymes. Brains were examined for morphology of hippocampus and cortex. Pups of FASD mothers displayed short-term memory impairment, decreased hippocampal size and decreased thickness of the dentate gyrus. MTHFR protein levels were reduced in FASD pup livers, with lower concentrations of phosphocholine and glycerophosphocholine in liver and hippocampus, respectively. FASD pup brains showed evidence of altered acetylcholine availability and Dnmt3a mRNA was reduced in cortex and hippocampus. E17.5 embryos and placentas from FASD dams were smaller. MTHFR protein and mRNA were reduced in embryonic liver, with lower concentrations of choline, betaine and phosphocholine. Embryonic brain displayed altered development of cortical layers. In summary, high folate intake during pregnancy leads to pseudo-MTHFR deficiency, disturbed choline/methyl metabolism, embryonic growth delay and memory impairment in offspring. These findings highlight the unintended negative consequences of supplemental folic acid. © The Author 2017. Published by Oxford University Press.

Factors Expressed by Murine Embryonic Pancreatic Mesenchyme Enhance Generation of Insulin-Producing Cells From hESCs

PubMed Central

Guo, Tingxia; Landsman, Limor; Li, Na; Hebrok, Matthias

2013-01-01

Islet transplantation has proven to be a successful strategy to restore normoglycemia in patients with type 1 diabetes (T1D). However, the dearth of cadaveric islets available for transplantation hampers the widespread application of this treatment option. Although human embryonic stem cells and induced pluripotent stem cells are capable of generating insulin-producing cells in vitro when provided with the appropriate inductive cues, the insulin-expressing cells that develop behave more like immature β-cells with minimal sensitivity to glucose stimulation. Here, we identify a set of signaling factors expressed in mouse embryonic mesenchyme during the time when foregut and pancreatic progenitors are specified and test their activities during in vitro differentiation of human embryonic stem cells. Several of the identified factors work in concert to expand the pancreatic progenitor pool. Interestingly, transforming growth factor (TGF)-β ligands, most potent in inducing pancreatic progenitors, display strong inhibitory effects on subsequent endocrine cell differentiation. Treatment with TGF-β ligands, followed by the addition of a TGF-β receptor antagonist, dramatically increased the number of insulin-producing cells in vitro, demonstrating the need for dynamic temporal regulation of TGF-β signaling during in vitro differentiation. These studies illustrate the need to precisely mimic the in vivo conditions to fully recapitulate pancreatic lineage specification in vitro. PMID:23305648

Trametes versicolor Protein YZP Activates Regulatory B Lymphocytes – Gene Identification through De Novo Assembly and Function Analysis in a Murine Acute Colitis Model

PubMed Central

Kuan, Yen-Chou; Wu, Ying-Jou; Hung, Chih-Liang; Sheu, Fuu

2013-01-01

Background Trametes versicolor (Yun-Zhi) is a medicinal fungus used as a chemotherapy co-treatment to enhance anti-tumor immunity. Although the efficacies of T. versicolor extracts have been documented, the active ingredients and mechanisms underlying the actions of these extracts remain uncharacterized. Results We purified a new protein, YZP, from the fruiting bodies of T. versicolor and identified the gene encoding YZP using RNA-seq and de novo assembly technologies. YZP is a 12-kDa non-glycosylated protein comprising 139 amino acids, including an 18-amino acids signal peptide. YZP induced a greater than 60-fold increase in IL-10 secretion in mice B lymphocytes; moreover, YZP specifically triggered the differentiation of CD1d+ B cells into IL-10-producing regulatory B cells (Bregs) and enhanced the expression of CD1d. YZP-induced B cells suppressed approximately 40% of the LPS-activated macrophage production of inflammatory cytokines in a mixed leukocyte reaction and significantly alleviated the disease activity and colonic inflammation in a DSS-induced acute colitis murine model. Furthermore, YZP activated Breg function via interaction with TLR2 and TLR4 and up-regulation of the TLR-mediated signaling pathway. Conclusions We purified a novel Breg-stimulating protein, YZP, from T. versicolor and developed an advanced approach combining RNA-seq and de novo assembly technologies.to clone its gene. We demonstrated that YZP activated CD1d+ Breg differentiation through TLR2/4-mediated signaling pathway, and the YZP-stimulated B cells exhibited anti-inflammatory efficacies in vitro and in murine acute colitis models. PMID:24019869

Abnormal differentiation, hyperplasia and embryonic/perinatal lethality in BK5-T/t transgenic mice

PubMed Central

Chen, Xin; Schneider-Broussard, Robin; Hollowell, Debra; McArthur, Mark; Jeter, Collene R.; Benavides, Fernando; DiGiovanni, John; Tang, Dean G.

2009-01-01

The cell-of-origin has a great impact on the types of tumors that develop and the stem/progenitor cells have long been considered main targets of malignant transformation. The SV40 large T and small t antigens (T/t), have been targeted to multiple differentiated cellular compartments in transgenic mice. In most of these studies, transgenic animals develop tumors without apparent defects in animal development. In this study, we used the bovine keratin 5 (BK5) promoter to target the T/t antigens to stem/progenitor cell-containing cytokeratin 5 (CK5) cellular compartment. A transgene construct, BK5-T/t, was made and microinjected into the male pronucleus of FVB/N mouse oocytes. After implanting ∼1700 embryos, only 7 transgenics were obtained, including 4 embryos (E9.5, E13, E15, and E20) and 3 postnatal animals, which died at P1, P2, and P18, respectively. Immunohistological analysis revealed aberrant differentiation and prominent hyperplasia in several transgenic CK5 tissues, especially the upper digestive organs (tongue, oral mucosa, esophagus, and forestomach) and epidermis, the latter of which also showed focal dysplasia. Altogether, these results indicate that constitutive expression of the T/t antigens in CK5 cellular compartment results in abnormal epithelial differentiation and leads to embryonic/perinatal animal lethality. PMID:19272531

Inotuzumab Ozogamicin Murine Analog–Mediated B-Cell Depletion Reduces Anti-islet Allo- and Autoimmune Responses

PubMed Central

Carvello, Michele; Petrelli, Alessandra; Vergani, Andrea; Lee, Kang Mi; Tezza, Sara; Chin, Melissa; Orsenigo, Elena; Staudacher, Carlo; Secchi, Antonio; Dunussi-Joannopoulos, Kyri; Sayegh, Mohamed H.; Markmann, James F.; Fiorina, Paolo

2012-01-01

B cells participate in the priming of the allo- and autoimmune responses, and their depletion can thus be advantageous for islet transplantation. Herein, we provide an extensive study of the effect of B-cell depletion in murine models of islet transplantation. Islet transplantation was performed in hyperglycemic B-cell–deficient(μMT) mice, in a purely alloimmune setting (BALB/c into hyperglycemic C57BL/6), in a purely autoimmune setting (NOD.SCID into hyperglycemic NOD), and in a mixed allo-/autoimmune setting (BALB/c into hyperglycemic NOD). Inotuzumab ozogamicin murine analog (anti-CD22 monoclonal antibody conjugated with calicheamicin [anti-CD22/cal]) efficiently depleted B cells in all three models of islet transplantation examined. Islet graft survival was significantly prolonged in B-cell–depleted mice compared with control groups in transplants of islets from BALB/c into C57BL/6 (mean survival time [MST]: 16.5 vs. 12.0 days; P = 0.004), from NOD.SCID into NOD (MST: 23.5 vs. 14.0 days; P = 0.03), and from BALB/c into NOD (MST: 12.0 vs. 5.5 days; P = 0.003). In the BALB/c into B-cell–deficient mice model, islet survival was prolonged as well (MST: μMT = 32.5 vs. WT = 14 days; P = 0.002). Pathology revealed reduced CD3+ cell islet infiltration and confirmed the absence of B cells in treated mice. Mechanistically, effector T cells were reduced in number, concomitant with a peripheral Th2 profile skewing and ex vivo recipient hyporesponsiveness toward donor-derived antigen as well as islet autoantigens. Finally, an anti-CD22/cal and CTLA4-Ig–based combination therapy displayed remarkable prolongation of graft survival in the stringent model of islet transplantation (BALB/c into NOD). Anti-CD22/cal–mediated B-cell depletion promotes the reduction of the anti-islet immune response in various models of islet transplantation. PMID:22076927

Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells.

PubMed

Forrai, Ariel; Boyle, Kristy; Hart, Adam H; Hartley, Lynne; Rakar, Steven; Willson, Tracy A; Simpson, Ken M; Roberts, Andrew W; Alexander, Warren S; Voss, Anne K; Robb, Lorraine

2006-03-01

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

Imaging of murine embryonic cardiovascular development using optical coherence tomography (Conference Presentation)

NASA Astrophysics Data System (ADS)

Huang, Yongyang; Degenhardt, Karl R.; Astrof, Sophie; Zhou, Chao

2016-03-01

We have demonstrated the capability of spectral domain optical coherence tomography (SDOCT) system to image full development of mouse embryonic cardiovascular system. Monitoring morphological changes of mouse embryonic heart occurred in different embryonic stages helps identify structural or functional cardiac anomalies and understand how these anomalies lead to congenital heart diseases (CHD) present at birth. In this study, mouse embryo hearts ranging from E9.5 to E15.5 were prepared and imaged in vitro. A customized spectral domain OCT system was used for imaging, with a central wavelength of 1310nm, spectral bandwidth of ~100nm and imaging speed of 47kHz A-scans/s. Axial resolution of this system was 8.3µm in air, and transverse resolution was 6.2 µm with 5X objective. Key features of mouse embryonic cardiovascular development such as vasculature remodeling into circulatory system, separation of atria and ventricles and emergence of valves could be clearly seen in three-dimensional OCT images. Optical clearing was applied to overcome the penetration limit of OCT system. With high resolution, fast imaging speed, 3D imaging capability, OCT proves to be a promising biomedical imaging modality for developmental biology studies, rivaling histology and micro-CT.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells*

PubMed Central

Ali, Ramadan A.; Camick, Christina; Wiles, Katherine; Walseth, Timothy F.; Slama, James T.; Bhattacharya, Sumit; Giovannucci, David R.; Wall, Katherine A.

2016-01-01

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca2+ mobilizing second messenger discovered to date, has been implicated in Ca2+ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca2+ signaling or the identity of the Ca2+ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca2+ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca2+ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca2+ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca2+ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca2+ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. PMID:26728458

Antibody-Independent Control of γ-Herpesvirus Latency via B Cell Induction of Anti-Viral T Cell Responses

PubMed Central

McClellan, Kelly B; Gangappa, Shivaprakash; Speck, Samuel H; Virgin, Herbert W.

2006-01-01

B cells can use antibody-dependent mechanisms to control latent viral infections. It is unknown whether this represents the sole function of B cells during chronic viral infection. We report here that hen egg lysozyme (HEL)-specific B cells can contribute to the control of murine γ-herpesvirus 68 (γHV68) latency without producing anti-viral antibody. HEL-specific B cells normalized defects in T cell numbers and proliferation observed in B cell−/− mice during the early phase of γHV68 latency. HEL-specific B cells also reversed defects in CD8 and CD4 T cell cytokine production observed in B cell−/− mice, generating CD8 and CD4 T cells necessary for control of latency. Furthermore, HEL-specific B cells were able to present virally encoded antigen to CD8 T cells. Therefore, B cells have antibody independent functions, including antigen presentation, that are important for control of γ-herpesvirus latency. Exploitation of this property of B cells may allow enhanced vaccine responses to chronic virus infection. PMID:16789842

Circulating FoxP3+ Regulatory T and Interleukin17-Producing Th17 Cells Actively Influence HBV Clearance in De Novo Hepatitis B Virus Infected Patients after Orthotopic Liver Transplantation

PubMed Central

Li, Jin; Yang, Mei; Liu, Yuan; Guo, Xiaodong; Li, Hanwei; Liu, Zhenwen; Zhao, Jingmin

2015-01-01

Objective To longitudinally investigate the role of FoxP3+ Regulatory T cells (Treg) and interleukin17-producing T helper 17 cells (Th17) in De Novo Hepatitis B Virus infection after orthotopic Liver Transplantation (DNHB-OLT), and analyze the possible correlation between these cells and HBV clearance of the disease. Methods We enrolled 12 control cases after orthotopic Liver Transplantation (OLT) and 24 patients, including 12 diagnosed with DNHB-OLT and 12 diagnosed with Acute Hepatitis B Virus infection (AHB), into the study from the liver transplantation and research center at Beijing 302 Hospital. Flow cytometry was used to detect the frequencies of Treg and Th17, and ELISA was applied to detect the concentration of IL6, IL22, TGF-β and IL2 in peripheral blood. We also measured the gene expression level by real time-quantitative PCR and protein expression using immunohistochemistry and western-blot. Furthermore, we divided DNHB-OLT patients into the clearance and non-clearance groups and examined longitudinally Th17, Treg cells at different times. Results The percentage of Treg cells, expression of FoxP3 mRNA and related anti-inflammatory cytokines such as IL2 and TGF-β1 in the DNHB-OLT group were significantly higher than that in the AHB and OLT groups. The percentage of Th17 cells, expression of RORγt mRNA and related pro-inflammatory cytokines such as IL17 and IL22 in the DNHB-OLT group were significantly lower than that in the AHB group, but the levels of these cytokines are very similar to the OLT group. The ratios of Treg to Th17 in the DNHB-OLT group were significantly higher than that in the OLT and AHB groups. Treg frequencies significantly correlated with HBV DNA, whereas IL17 frequencies didn’t significantly correlate with ALT. In DNHB-OLT patients, the clearance group was accompanied by a rapid increase in the Th17 cells during the first 4th week and afterwards continuously decrease to the control group, together with a continuously decrease in

Murine neural crest stem cells and embryonic stem cell-derived neuron precursors survive and differentiate after transplantation in a model of dorsal root avulsion.

PubMed

Konig, Niclas; Trolle, Carl; Kapuralin, Katarina; Adameyko, Igor; Mitrecic, Dinko; Aldskogius, Hakan; Shortland, Peter J; Kozlova, Elena N

2017-01-01

Spinal root avulsion results in paralysis and sensory loss, and is commonly associated with chronic pain. In addition to the failure of avulsed dorsal root axons to regenerate into the spinal cord, avulsion injury leads to extensive neuroinflammation and degeneration of second-order neurons in the dorsal horn. The ultimate objective in the treatment of this condition is to counteract degeneration of spinal cord neurons and to achieve functionally useful regeneration/reconnection of sensory neurons with spinal cord neurons. Here we compare survival and migration of murine boundary cap neural crest stem cells (bNCSCs) and embryonic stem cells (ESCs)-derived, predifferentiated neuron precursors after their implantation acutely at the junction between avulsed dorsal roots L3-L6 and the spinal cord. Both types of cells survived transplantation, but showed distinctly different modes of migration. Thus, bNCSCs migrated into the spinal cord, expressed glial markers and formed elongated tubes in the peripheral nervous system (PNS) compartment of the avulsed dorsal root transitional zone (DRTZ) area. In contrast, the ESC transplants remained at the site of implantation and differentiated to motor neurons and interneurons. These data show that both stem cell types successfully survived implantation to the acutely injured spinal cord and maintained their differentiation and migration potential. These data suggest that, depending on the source of neural stem cells, they can play different beneficial roles for recovery after dorsal root avulsion. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Ionizing radiation potentiates the induction of nitric oxide synthase by interferon-gamma (Ifn-gamma) or Ifn-gamma and lipopolysaccharide in bnl cl.2 murine embryonic liver cells: role of hydrogen peroxide.

PubMed

Yoo, J C; Pae, H O; Choi, B M; Kim, W I; Kim, J D; Kim, Y M; Chung, H T

2000-02-01

The effects of ionizing irradiation on the nitric oxide (NO) production in murine embryonic liver cell line, BNL CL.2 cells, were investigated. Various doses (5-40 Gy) of radiation made BNL CL.2 cells responsive to interferon-gamma alone for the production of NO in a dose-dependent manner. Small amounts of lipopolysaccharide (LPS) or tumor necrosis factor-alpha (TNF-alpha) synergized with IFN-gamma in the production of NO from irradiated BNL CL.2 cells, even though LPS or TNF-alpha alone did not induce NO production from the same cells. Immunoblots showed parallel induction of inducible nitric oxide synthase (iNOS). NO production in irradiated BNL CL.2 cells by IFN-gamma or IFN-gamma plus LPS was decreased by the addition of catalase, suggesting that H(2)O(2) produced by ionizing irradiation primed the cells to trigger NO production in response to IFN-gamma or IFN-gamma plus LPS. Furthermore, the treatment of nongamma-irradiated BNL CL.2 cells with H(2)O(2) made the cells responsive to IFN-gamma or IFN-gamma plus LPS for the production of NO. This study shows that ionizing irradiation has the ability to induce iNOS gene expression in responsive to IFN-gamma via the formation of H(2)O(2) in BNL CL.2 murine embryonic liver cells.

Hedgehog signaling in the murine melanoma microenvironment.

PubMed

Geng, Ling; Cuneo, Kyle C; Cooper, Michael K; Wang, Hong; Sekhar, Konjeti; Fu, Allie; Hallahan, Dennis E

2007-01-01

The Hedgehog intercellular signaling pathway regulates cell proliferation and differentiation. This pathway has been implicated to play a role in the pathogenesis of cancer and in embryonic blood vessel development. In the current study, Hedgehog signaling in tumor related vasculature and microenvironment was examined using human umbilical vein endothelial cells and B16F0 (murine melanoma) tumors models. Use of exogenous Sonic hedgehog (Shh) peptide significantly increased BrdU incorporation in endothelial cells in vitro by a factor of 2 (P < 0.001). The Hedgehog pathway antagonist cyclopamine effectively reduced Shh-induced proliferation to control levels. To study Hedgehog signaling in vivo a hind limb tumor model with the B16F0 cell line was used. Treatment with 25 mg/kg cyclopamine significantly attenuated BrdU incorporation in tumor cells threefold (P < 0.001), in tumor related endothelial cells threefold (P = 0.004), and delayed tumor growth by 4 days. Immunohistochemistry revealed that the Hedgehog receptor Patched was localized to the tumor stroma and that B16F0 cells expressed Shh peptide. Furthermore, mouse embryonic fibroblasts required the presence of B16F0 cells to express Patched in a co-culture assay system. These studies indicate that Shh peptide produced by melanoma cells induces Patched expression in fibroblasts. To study tumor related angiogenesis a vascular window model was used to monitor tumor vascularity. Treatment with cyclopamine significantly attenuated vascular formation by a factor of 2.5 (P < 0.001) and altered vascular morphology. Furthermore, cyclopamine reduced tumor blood vessel permeability to FITC labeled dextran while having no effect on normal blood vessels. These studies suggest that Hedgehog signaling regulates melanoma related vascular formation and function.

Baicalein induces cell death in murine T cell lymphoma via inhibition of thioredoxin system.

PubMed

Patwardhan, Raghavendra S; Pal, Debojyoti; Checker, Rahul; Sharma, Deepak; Sandur, Santosh K

2017-10-01

We have earlier demonstrated the radioprotective potential of baicalein using murine splenic lymphocytes. Here, we have studied the effect of baicalein on murine T cell lymphoma EL4 cells and investigated the underlying mechanism of action. We observed that baicalein induced a dose dependent cell death in EL4 cells in vitro and significantly reduced the frequency of cancer stem cells. Previously, we have reported that murine and human T cell lymphoma cells have increased oxidative stress tolerance capacity due to active thioredoxin system. Hence, we monitored the effect of baicalein on thioredoxin system in EL4 cells. Docking studies revealed that baicalein could bind to the active site of thioredoxin reductase. Baicalein treatment led to significant reduction in the activity of thioredoxin reductase and nuclear levels of thioredoxin-1 thereby increasing ASK1 levels and caspase-3 activity. Interestingly, CRISPR-Cas9 based knock-out of ASK1 or over-expression of thioredoxin-1 abolished anti-tumor effects of baicalein in EL4 cells. Further, baicalein administration significantly reduced intra-peritoneal tumor burden of EL4 cells in C57BL/6 mice. Thus, our study describes anti-tumor effects of baicalein in EL4 cells via inhibition of thioredoxin system. Copyright © 2017 Elsevier Ltd. All rights reserved.

Merkel Cell Polyomavirus Small T Antigen Induces Cancer and Embryonic Merkel Cell Proliferation in a Transgenic Mouse Model.

PubMed

Shuda, Masahiro; Guastafierro, Anna; Geng, Xuehui; Shuda, Yoko; Ostrowski, Stephen M; Lukianov, Stefan; Jenkins, Frank J; Honda, Kord; Maricich, Stephen M; Moore, Patrick S; Chang, Yuan

2015-01-01

Merkel cell polyomavirus (MCV) causes the majority of human Merkel cell carcinomas (MCC) and encodes a small T (sT) antigen that transforms immortalized rodent fibroblasts in vitro. To develop a mouse model for MCV sT-induced carcinogenesis, we generated transgenic mice with a flox-stop-flox MCV sT sequence homologously recombined at the ROSA locus (ROSAsT), allowing Cre-mediated, conditional MCV sT expression. Standard tamoxifen (TMX) administration to adult UbcCreERT2; ROSAsT mice, in which Cre is ubiquitously expressed, resulted in MCV sT expression in multiple organs that was uniformly lethal within 5 days. Conversely, most adult UbcCreERT2; ROSAsT mice survived low-dose tamoxifen administration but developed ear lobe dermal hyperkeratosis and hypergranulosis. Simultaneous MCV sT expression and conditional homozygous p53 deletion generated multi-focal, poorly-differentiated, highly anaplastic tumors in the spleens and livers of mice after 60 days of TMX treatment. Mouse embryonic fibroblasts from these mice induced to express MCV sT exhibited anchorage-independent cell growth. To examine Merkel cell pathology, MCV sT expression was also induced during mid-embryogenesis in Merkel cells of Atoh1CreERT2/+; ROSAsT mice, which lead to significantly increased Merkel cell numbers in touch domes at late embryonic ages that normalized postnatally. Tamoxifen administration to adult Atoh1CreERT2/+; ROSAsT and Atoh1CreERT2/+; ROSAsT; p53flox/flox mice had no effects on Merkel cell numbers and did not induce tumor formation. Taken together, these results show that MCV sT stimulates progenitor Merkel cell proliferation in embryonic mice and is a bona fide viral oncoprotein that induces full cancer cell transformation in the p53-null setting.

Cutting Edge: 2B4-Mediated Coinhibition of CD4+ T Cells Underlies Mortality in Experimental Sepsis.

PubMed

Chen, Ching-Wen; Mittal, Rohit; Klingensmith, Nathan J; Burd, Eileen M; Terhorst, Cox; Martin, Greg S; Coopersmith, Craig M; Ford, Mandy L

2017-09-15

Sepsis is a leading cause of death in the United States, but the mechanisms underlying sepsis-induced immune dysregulation remain poorly understood. 2B4 (CD244, SLAM4) is a cosignaling molecule expressed predominantly on NK cells and memory CD8 + T cells that has been shown to regulate T cell function in models of viral infection and autoimmunity. In this article, we show that 2B4 signaling mediates sepsis lymphocyte dysfunction and mortality. 2B4 expression is increased on CD4 + T cells in septic animals and human patients at early time points. Importantly, genetic loss or pharmacologic inhibition of 2B4 significantly increased survival in a murine cecal ligation and puncture model. Further, CD4-specific conditional knockouts showed that 2B4 functions on CD4 + T cell populations in a cell-intrinsic manner and modulates adaptive and innate immune responses during sepsis. Our results illuminate a novel role for 2B4 coinhibitory signaling on CD4 + T cells in mediating immune dysregulation. Copyright © 2017 by The American Association of Immunologists, Inc.

BLTK1 murine Leydig cells: a novel steroidogenic model for evaluating the effects of reproductive and developmental toxicants.

PubMed

Forgacs, Agnes L; Ding, Qi; Jaremba, Rosemary G; Huhtaniemi, Ilpo T; Rahman, Nafis A; Zacharewski, Timothy R

2012-06-01

Leydig cells are the primary site of androgen biosynthesis in males. Several environmental toxicants target steroidogenesis resulting in both developmental and reproductive effects including testicular dysgenesis syndrome. The aim of this study was to evaluate the effect of several structurally diverse endocrine disrupting compounds (EDCs) on steroidogenesis in a novel BLTK1 murine Leydig cell model. We demonstrate that BLTK1 cells possess a fully functional steroidogenic pathway that produces low basal levels of testosterone (T) and express all the necessary steroidogenic enzymes including Star, Cyp11a1, Cyp17a1, Hsd3b1, Hsd17b3, and Srd5a1. Recombinant human chorionic gonadotropin (rhCG) and forskolin (FSK) elicited concentration- and time-dependent induction of 3',5'-cyclic adenosine monophosphate, progesterone (P), and T, as well as the differential expression of Star, Hsd3b6, Hsd17b3, and Srd5a1 messenger RNA levels. The evaluation of several structurally diverse male reproductive toxicants including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), atrazine, prochloraz, triclosan, monoethylhexyl phthalate (MEHP), glyphosate, and RDX in BLTK1 cells suggests different modes of action perturb steroidogenesis. For example, prochloraz and triclosan antifungals reduced rhCG induction of T, consistent with published in vivo data but did not alter basal T levels. In contrast, atrazine and MEHP elicited modest induction of basal T but antagonized rhCG-mediated induction of T levels, whereas TCDD, glyphosate, and RDX had no effect on basal or rhCG induction of T in BLTK1 cells. These results suggest that BLTK1 cells maintain rhCG-inducible steroidogenesis and are a viable in vitro Leydig cell model to evaluate the effects of EDCs on steroidogenesis. This model can also be used to elucidate the different mechanisms underlying toxicant-mediated disruption of steroidogenesis.

Neutralization of the IL-17 axis diminishes neutrophil invasion and protects from ischemic stroke.

PubMed

Gelderblom, Mathias; Weymar, Anna; Bernreuther, Christian; Velden, Joachim; Arunachalam, Priyadharshini; Steinbach, Karin; Orthey, Ellen; Arumugam, Thiruma V; Leypoldt, Frank; Simova, Olga; Thom, Vivien; Friese, Manuel A; Prinz, Immo; Hölscher, Christoph; Glatzel, Markus; Korn, Thomas; Gerloff, Christian; Tolosa, Eva; Magnus, Tim

2012-11-01

The devastating effect of ischemic stroke is attenuated in mice lacking conventional and unconventional T cells, suggesting that inflammation enhances tissue damage in cerebral ischemia. We explored the functional role of αβ and γδ T cells in a murine model of stroke and distinguished 2 different T cell-dependent proinflammatory pathways in ischemia-reperfusion injury. IFN-γ produced by CD4(+) T cells induced TNF-α production in macrophages, whereas IL-17A secreted by γδ T cells led to neutrophil recruitment. The synergistic effect of TNF-α and IL-17A on astrocytes resulted in enhanced secretion of CXCL-1, a neutrophil chemoattractant. Application of an IL-17A-blocking antibody within 3 hours after stroke induction decreased infarct size and improved neurologic outcome in the murine model. In autoptic brain tissue of patients who had a stroke, we detected IL-17A-positive lymphocytes, suggesting that this aspect of the inflammatory cascade is also relevant in the human brain. We propose that selective targeting of IL-17A signaling might provide a new therapeutic option for the treatment of stroke.

Metabolism of murine TH 17 cells: Impact on cell fate and function.

PubMed

Wang, Ran; Solt, Laura A

2016-04-01

An effective adaptive immune response relies on the ability of lymphocytes to rapidly act upon a variety of insults. In T lymphocytes, this response includes cell growth, clonal expansion, differentiation, and cytokine production, all of which place a significant energy burden on the cell. Recent evidence shows that T-cell metabolic reprogramming is an essential component of the adaptive immune response and specific metabolic pathways dictate T-cell fate decisions, including the development of TH 17 versus T regulatory (Treg) cells. TH 17 cells have garnered significant attention due to their roles in the pathology of immune-mediated inflammatory diseases. Attempts to characterize TH 17 cells have demonstrated that they are highly dynamic, adjusting their function to environmental cues, which dictate their metabolic program. In this review, we highlight recent data demonstrating the impact of cellular metabolism on the TH 17/Treg balance and present factors that mediate TH 17-cell metabolism. Some examples of these include the differential impact of the mTOR signaling complexes on T-helper-cell differentiation, hypoxia inducible factor 1 alpha (HIF1α) promotion of glycolysis to favor TH 17-cell development, and ACC1-dependent de novo fatty acid synthesis favoring TH 17-cell development over Treg cells. Finally, we discuss the potential therapeutic options and the implications of modulating TH 17-cell metabolism for the treatment of TH 17-mediated diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of HSD17B1 and SRD5A1 in lymphocytes.

PubMed

Zhou, Z; Speiser, P W

1999-11-01

We previously reported lymphocyte expression of genes encoding enzymes required for steroid metabolism; however, only 17beta-HSD and 5alpha-reductase showed significant enzyme activity. We now investigate regulation of lymphocyte expression for genes encoding 17beta-HSD and 5alpha-reductase. Cultured human T and B lymphoid cell lines and peripheral blood mononuclear cells were treated with known regulators of steroidogenic gene expression including forskolin, PMA, ionomycin, various steroids, interleukin (IL)-4, and IL-6. Treatment with 10 or 50 microM forskolin resulted in a 20-60% reduction of expression for HSD17B1 (encoding 17beta-HSD I) in T and B lymphoid cell lines and peripheral blood mononuclear cells, although such a change was not observed in the expression of SRD5A1 (encoding 5alpha-reductase I). No significant changes were found when cells were treated for 24 h with various concentrations of PMA or ionomycin. Incubation with 10(-9) to 10(-7) M androstenedione or estradiol increased expression of HSD17B1, while testosterone decreased the expression of this gene. SRD5A1 expression was increased in the presence of 5alpha-DHT although no consistent changes were observed when the cells were treated with testosterone. Other steroids, including dexamethasone, progesterone, and 6-hydroxypregnanolone, produced no effects on expression of either HSD17B1 or SRD5A1. Treatment with 0.1-10 ng/ml of IL-4 or IL-6 also did not effect significant changes in gene expression. These data implicate the involvement of the cAMP-protein kinase signal transduction pathway in regulating lymphocyte expression of HSD17B1. Furthermore, it appears that lymphocyte HSD17B1 and SRD5A1 are regulated to some extent by specific steroids. Copyright 1999 Academic Press.

17 CFR 230.702(T)-230.703(T) - [Reserved

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 2 2011-04-01 2011-04-01 false [Reserved] 230.702(T)-230.703(T) Section 230.702(T)-230.703(T) Commodity and Securities Exchanges SECURITIES AND EXCHANGE... Small Business Investment Companies §§ 230.702(T)-230.703(T) [Reserved] Exemptions for Cross-Border...

The Murine Nck SH2/SH3 Adaptors Are Important for the Development of Mesoderm-Derived Embryonic Structures and for Regulating the Cellular Actin Network

PubMed Central

Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A.; Nash, Piers; Tafuri, Anna; Gertler, Frank B.; Pawson, Tony

2003-01-01

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated β-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1−/− Nck2−/− embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization. PMID:12808099

The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network.

PubMed

Bladt, Friedhelm; Aippersbach, Elke; Gelkop, Sigal; Strasser, Geraldine A; Nash, Piers; Tafuri, Anna; Gertler, Frank B; Pawson, Tony

2003-07-01

Mammalian Nck1 and Nck2 are closely related adaptor proteins that possess three SH3 domains, followed by an SH2 domain, and are implicated in coupling phosphotyrosine signals to polypeptides that regulate the actin cytoskeleton. However, the in vivo functions of Nck1 and Nck2 have not been defined. We have mutated the murine Nck1 and Nck2 genes and incorporated beta-galactosidase reporters into the mutant loci. In mouse embryos, the two Nck genes have broad and overlapping expression patterns. They are functionally redundant in the sense that mice deficient for either Nck1 or Nck2 are viable, whereas inactivation of both Nck1 and Nck2 results in profound defects in mesoderm-derived notochord and embryonic lethality at embryonic day 9.5. Fibroblast cell lines derived from Nck1(-/-) Nck2(-/-) embryos have defects in cell motility and in the organization of the lamellipodial actin network. These data suggest that the Nck SH2/SH3 adaptors have important functions in the development of mesodermal structures during embryogenesis, potentially linked to a role in cell movement and cytoskeletal organization.

CRISPR/Cas9-Mediated Genomic Deletion of the Beta-1, 4 N-acetylgalactosaminyltransferase 1 Gene in Murine P19 Embryonal Carcinoma Cells Results in Low Sensitivity to Botulinum Neurotoxin Type C.

PubMed

Tsukamoto, Kentaro; Ozeki, Chikako; Kohda, Tomoko; Tsuji, Takao

2015-01-01

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

MHC class II molecules control murine B cell responsiveness to lipopolysaccharide stimulation.

PubMed

Rodo, Joana; Gonçalves, Lígia A; Demengeot, Jocelyne; Coutinho, António; Penha-Gonçalves, Carlos

2006-10-01

LPS is a strong stimulator of the innate immune system and inducer of B lymphocyte activation. Two TLRs, TLR4 and RP105 (CD180), have been identified as mediators of LPS signaling in murine B cells, but little is known about genetic factors that are able to control LPS-induced cell activation. We performed a mouse genome-wide screen that aside from identifying a controlling locus mapping in the TLR4 region (logarithm of odds score, 2.77), also revealed that a locus closely linked to the MHC region (logarithm of odds score, 3.4) governed B cell responsiveness to LPS stimulation. Using purified B cells obtained from MHC congenic strains, we demonstrated that the MHC(b) haplotype is accountable for higher cell activation, cell proliferation, and IgM secretion, after LPS stimulation, when compared with the MHC(d) haplotype. Furthermore, B cells from MHC class II(-/-) mice displayed enhanced activation and proliferation in response to LPS. In addition, we showed that the MHC haplotype partially controls expression of RP105 (a LPS receptor molecule), following a pattern that resembles the LPS responsiveness phenotype. Together, our results strongly suggest that murine MHC class II molecules play a role in constraining the B cell response to LPS and that genetic variation at the MHC locus is an important component in controlling B cell responsiveness to LPS stimulation. This work raises the possibility that constraining of B cell responsiveness by MHC class II molecules may represent a functional interaction between adaptive and innate immune systems.

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells.

PubMed

Ali, Ramadan A; Camick, Christina; Wiles, Katherine; Walseth, Timothy F; Slama, James T; Bhattacharya, Sumit; Giovannucci, David R; Wall, Katherine A

2016-02-26

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca(2+) mobilizing second messenger discovered to date, has been implicated in Ca(2+) signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca(2+) signaling or the identity of the Ca(2+) stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca(2+) signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca(2+) signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca(2+) stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca(2+) signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca(2+) release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Electrophysiological properties of prion-positive cardiac progenitors derived from murine embryonic stem cells.

PubMed

Fujii, Hiroshi; Ikeuchi, Yu; Kurata, Yasutaka; Ikeda, Nobuhito; Bahrudin, Udin; Li, Peili; Nakayama, Yuji; Endo, Ryo; Hasegawa, Akira; Morikawa, Kumi; Miake, Junichiro; Yoshida, Akio; Hidaka, Kyoko; Morisaki, Takayuki; Ninomiya, Haruaki; Shirayoshi, Yasuaki; Yamamoto, Kazuhiro; Hisatome, Ichiro

2012-01-01

The prion protein (PrP) has been reported to serve as a surface maker for isolation of cardiomyogenic progenitors from murine embryonic stem (ES) cells. Although PrP-positive cells exhibited automaticity, their electrophysiological characteristics remain unresolved. The aim of the present study was therefore to investigate the electrophysiological properties of PrP-positive cells in comparison with those of HCN4p-or Nkx2.5-positive cells. Differentiation of AB1, HCN5p-EGFP and hcgp7 ES cells into cardiac progenitors was induced by embryoid body (EB) formation. EBs were dissociated and cells expressing PrP, HCN4-EGFP and/or Nkx2.5-GFP were collected via flow cytometry. Sorted cells were subjected to reverse transcriptase-polymerase chain reaction, immunostaining and patch-clamp experiments. PrP-positive cells expressed mRNA of undifferentiation markers, first and second heart field markers, and cardiac-specific genes and ion channels, indicating their commitment to cardiomyogenic progenitors. PrP-positive cells with automaticity showed positive and negative chronotropic responses to isoproterenol and carbamylcholine, respectively. Hyperpolarization-activated cation current (I(f)) was barely detectable, whereas Na(+) and L-type Ca(2+) channel currents were frequently observed. Their spontaneous activity was slowed by inhibition of sarcoplasmic reticulum Ca(2+) uptake and release but not by blocking I(f). The maximum diastolic potential of their spontaneous firings was more depolarized than that of Nkx2.5-GFP-positive cells. PrP-positive cells contained cardiac progenitors that separated from the lineage of sinoatrial node cells. PrP can be used as a marker to enrich nascent cardiac progenitors.

Collaboration between tumor-specific CD4+ T cells and B cells in anti-cancer immunity.

PubMed

Guy, Thomas V; Terry, Alexandra M; Bolton, Holly A; Hancock, David G; Zhu, Erhua; Brink, Robert; McGuire, Helen M; Shklovskaya, Elena; Fazekas de St. Groth, Barbara

2016-05-24

The role of B cells and antibodies in anti-tumor immunity is controversial, with both positive and negative effects reported in animal models and clinical studies. We developed a murine B16.F10 melanoma model to study the effects of collaboration between tumor-specific CD4+ T cells and B cells on tumor control. By incorporating T cell receptor transgenic T cells and B cell receptor isotype switching B cells, we were able to track the responses of tumor-reactive T and B cells and the development of anti-tumor antibodies in vivo. In the presence of tumor-specific B cells, the number of tumor-reactive CD4+ T cells was reduced in lymphoid tissues and the tumor itself, and this correlated with poor tumor control. B cells had little effect on the Th1 bias of the CD4+ T cell response, and the number of induced FoxP3+ regulatory cells (iTregs) generated from within the original naive CD4+ T cell inoculum was unrelated to the degree of B cell expansion. In response to CD4+ T cell help, B cells produced a range of isotype-switched anti-tumor antibodies, principally IgG1, IgG2a/c and IgG2b. In the absence of CD4+ T cells, B cells responded to agonistic anti-CD40 administration by switching to production of IgG2a/c and, to a lesser extent, IgG1, IgG3, IgA and IgE, which reduced the number of lung metastases after i.v. tumor inoculation but had no effect on the growth of subcutaneous tumors.

STAT3 signaling in CD4+ T cells is critical for the pathogenesis of chronic sclerodermatous graft-versus-host disease in a murine model.

PubMed

Radojcic, Vedran; Pletneva, Maria A; Yen, Hung-Rong; Ivcevic, Sanja; Panoskaltsis-Mortari, Angela; Gilliam, Anita C; Drake, Charles G; Blazar, Bruce R; Luznik, Leo

2010-01-15

Donor CD4+ T cells are thought to be essential for inducing delayed host tissue injury in chronic graft-versus-host disease (GVHD). However, the relative contributions of distinct effector CD4+ T cell subpopulations and the molecular pathways influencing their generation are not known. We investigated the role of the STAT3 pathway in a murine model of chronic sclerodermatous GVHD. This pathway integrates multiple signaling events during the differentiation of naive CD4+ T cells and impacts their homeostasis. We report that chimeras receiving an allograft containing STAT3-ablated donor CD4+ T cells do not develop classic clinical and pathological manifestations of alloimmune tissue injury. Analysis of chimeras showed that abrogation of STAT3 signaling reduced the in vivo expansion of donor-derived CD4+ T cells and their accumulation in GVHD target tissues without abolishing antihost alloreactivity. STAT3 ablation did not significantly affect Th1 differentiation while enhancing CD4+CD25+Foxp3+ T cell reconstitution through thymus-dependent and -independent pathways. Transient depletion of CD25+ T cells in chimeras receiving STAT3-deficient T cells resulted in delayed development of alloimmune gut and liver injury. This delayed de novo GVHD was associated with the emergence of donor hematopoietic stem cell-derived Th1 and Th17 cells. These results suggest that STAT3 signaling in graft CD4+ T cells links the alloimmune tissue injury of donor graft T cells and the emergence of donor hematopoietic stem cell-derived pathogenic effector cells and that both populations contribute, albeit in different ways, to the genesis of chronic GVHD after allogenic bone marrow transplantation in a murine model.

IL-17 and VEGF are necessary for efficient corneal nerve regeneration

USDA-ARS?s Scientific Manuscript database

The contribution of acute inflammation to sensory nerve regeneration was investigated in the murine cornea using a model of corneal abrasion that removes the stratified epithelium and subbasal nerve plexus. Abrasion induced accumulation of IL-17(+) CCR6(+) yo T cells, neutrophils, and platelets in t...

Embryonic GABA(B) receptor blockade alters cell migration, adult hypothalamic structure, and anxiety- and depression-like behaviors sex specifically in mice.

PubMed

Stratton, Matthew S; Staros, Michelle; Budefeld, Tomaz; Searcy, Brian T; Nash, Connor; Eitel, Chad; Carbone, David; Handa, Robert J; Majdic, Gregor; Tobet, Stuart A

2014-01-01

Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA(B) receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA(B) receptor to a 7-day critical period (E11-E17) during embryonic development. Experiments tested the role of GABA(B) receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA(B) receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA(B) receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA(B) receptor antagonist. Embryonic exposure to GABA(B) receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA(B) receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.

17 CFR 240.17Ad-21T - Operational capability in a Year 2000 environment.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Operational capability in a Year 2000 environment. 240.17Ad-21T Section 240.17Ad-21T Commodity and Securities Exchanges SECURITIES... Company Rules § 240.17Ad-21T Operational capability in a Year 2000 environment. (a) This section applies...

17 CFR 230.702(T)-230.703(T) - [Reserved

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false [Reserved] 230.702(T)-230.703(T) Section 230.702(T)-230.703(T) Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION GENERAL RULES AND REGULATIONS, SECURITIES ACT OF 1933 Regulation E-Exemption for Securities of...

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells.

PubMed

Wang, Dachun; Quan, Yuan; Yan, Qing; Morales, John E; Wetsel, Rick A

2015-10-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. This study reports the generation of a novel β2-microglobulin (B2M)-/- human embryonic stem cell (hESC) line. Differentiated mature cells from this line

Targeted Disruption of the β2-Microglobulin Gene Minimizes the Immunogenicity of Human Embryonic Stem Cells

PubMed Central

Quan, Yuan; Yan, Qing; Morales, John E.

2015-01-01

Human embryonic stem cells (hESCs) are a promising source of cells for tissue regeneration, yet histoincompatibility remains a major challenge to their clinical application. Because the human leukocyte antigen class I (HLA-I) molecules are the primary mediators of immune rejection, we hypothesized that cells derived from a hESC line lacking HLA-I expression could be transplanted without evoking a robust immune response from allogeneic recipients. In the present study, we used the replacement targeting strategy to delete exons 2 and 3 of β2-microglobulin on both gene alleles in hESCs. Because β2-microglobulin serves as the HLA-I light chain, disruption of the β2-microglobulin gene led to complete HLA-I deficiency on the cell surface of hESCs and their derivatives. Therefore, these cells were resistant to CD8+ T-cell-mediated destruction. Although interferon-γ (IFN-γ) treatment significantly induced β2-microglobulin expression, promoting CD8+ T cell-mediated killing of control hESCs and their derivatives, CD8+ T-cell-mediated cytotoxicity was barely observed with β2-microglobulin-null hESCs and their derivatives treated with IFN-γ. This genetic manipulation to disrupt HLA-I expression did not affect the self-renewal capacity, genomic stability, or pluripotency of hESCs. Despite being relatively sensitive to natural killer (NK) cell-mediated killing due to the lack of HLA-I expression, when transplanted into NK cell-depleted immunocompetent mice, β2-microglobulin-null hESCs developed into tumors resembling those derived from control hESCs in severe combined immunodeficiency mice. These results demonstrate that β2-microglobulin-null hESCs significantly reduce immunogenicity to CD8+ T cells and might provide a renewable source of cells for tissue regeneration without the need for HLA matching in the future. Significance This study reports the generation of a novel β2-microglobulin (B2M)−/− human embryonic stem cell (hESC) line. Differentiated mature cells

Decoy receptor 3 attenuates collagen-induced arthritis by modulating T cell activation and B cell expansion.

PubMed

Cheng, Chia-Pi; Sytwu, Huey-Kang; Chang, Deh-Ming

2011-12-01

To investigate the immune-modulated effects of decoy receptor 3 (DCR3) in an experimental model of rheumatoid arthritis (RA). We delivered DCR3 plasmid into collagen-induced arthritis (CIA) mice using the hydrodynamic method and evaluated the serum level of DCR3 protein by ELISA. After immunization, we assessed disease severity of arthritis incidence, arthritis scores, paw thickness, and means of arthritic limbs, and used hematoxylin and eosin staining to observe synovial hyperplasia. We analyzed numbers of murine splenocytes and inguinal lymphocyte cells, cell populations, and serum proinflammatory cytokines by flow cytometry. We investigated B cell proliferation by carboxyfluorescein succinimidyl ester assay. We evaluated serum levels of total IgG2a and type II collagen-specific IgG and IgG2a using ELISA. DCR3 expression in sera significantly attenuated disease severity in CIA mice. We found that DCR3 inhibited the volume of inguinal lymph nodes, numbers of CD19+ B cells, and populations of interferon-γ, interleukin 4 (IL-4), IL-17A, and Foxp3-producing CD4+ T cell in vivo. We found that DCR3 inhibited Pam3CSK4 (Toll-like receptor 1/2 ligand)-induced B220+ B cell proliferation in vitro. DCR3 treatment reduced the serum level of IL-6, total IgG2a, and CII-specific IgG2a antibody. We postulated that the protective effects of DCR3 in CIA resulted from modulation of the immune system by maintaining the B/T cell balance and decreasing lymphocyte expansion. We suggest DCR3 as a prophylactic and potential therapeutic agent in the treatment of RA.

Site preference, magnetism and lattice vibrations of intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru)

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

Li, Jin-Chun; Qian, Ping, E-mail: qianpinghu@sohu.com; Zhang, Zhen-Feng

We present an atomistic study on the phase stability, site preference and lattice constants of the rare earth intermetallics Lu₂Fe 17–xT x (T=Cr, Mn, Ru). The calculated preferential occupation site of ternary element T is found to be the 4f site. The order of site preference is given as 4f, 12k, 12j and 6g for Lu₂Fe 17–xT x. The calculated lattice parameters are corresponding to the experimental results. We have calculated the magnetic moments of Lu₂Fe 17–xT x compounds. Results show that the calculated total magnetic moment of Lu₂Fe₁₇ compound is M=37.34 μ B/f.u. In addition, the total and partialmore » phonon densities of states are evaluated first for these complicated structures. - Graphical abstract: The vibrational modes are mostly excited by Fe atoms, Lu contributes to the lower frequencies modes, and the contribution of Ru atoms is the same as Fe atoms. Highlights: • There are no reports on lattice vibrations of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The phase stability and site preference are evaluated first for the complex structures of Lu₂(Fe, T) 17–x (T=Cr, Mn, Ru) compounds. • The lattice inversion method to obtain the interatomic pair potential is the unique one.« less

STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS

EPA Science Inventory

STRAIN-DEPENDENT SUSCEPTIBILITY TO TRANSPLACENTALLY-INDUCED MURINE LUNG TUMORS
M S Miller, J E Moore, M Xu, G B Nelson, S T Dance, N D Kock, J A Ross Wake Forest University, Winston-Salem, NC and USEPA, Research Triangle Park, NC

Previously, our laboratory demonstrated...

I-309/T cell activation gene-3 chemokine protects murine T cell lymphomas against dexamethasone-induced apoptosis.

PubMed

Van Snick, J; Houssiau, F; Proost, P; Van Damme, J; Renauld, J C

1996-09-15

We have previously reported that cytokines such as IL-9, IL-4, and IL-6 protect murine thymic lymphoma cell lines against dexamethasone-induced apoptosis. A similar activity, which could not be ascribed to any of these factors, was found in a number of human T cell supernatants that enabled mouse BW5147 thymic lymphoma not only to escape apoptosis but also to maintain proliferation. The protein responsible for this activity was purified to homogeneity from the culture medium of activated leukemic T cells and was found to be identical with the I-309 chemokine. Half-maximal anti-apoptotic activity was obtained with approximately 1 ng/ml, a concentration considerably lower than that required for the monocyte chemotactic activity of this molecule, as measured on THP-1 cells. The purified I-309 also improved the survival of two other mouse thymic lymphoma cell lines. This activity was as potent as that of IL-9, which was the strongest anti-apoptotic factor found to date for these cells. Similar results were obtained for BW5147 cells with recombinant I-309 and with T cell activation gene-3, the murine homologue of I-309, but not with other members of the chemokine family, including IL-8, neutrophil-activating peptide-2, granulocyte chemotactic protein-2, macrophage inflammatory protein-1a, RANTES (regulated upon activation, normal T cell expressed and secreted), monocyte chemotactic protein-1 (MCP-1), and MCP-2. MCP-3, however, showed a minor, but significant effect in this model. Unlike that of IL-9, the activity of I-309 was completely inhibited in the presence of pertussis toxin, indicating the involvement of a G protein in this process.

Secretory expression, purification and functional characterization of 17β-hydroxysteroid dehydrogenase type 1 from mammalian HEK293T cells.

PubMed

Chen, Jiong; Feng, Wei; Zhao, Yue

2017-09-01

17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) mainly catalyzes the reduction of estrone into estradiol. The enzymatic conversion is a critical step in estradiol accumulation in breast tissue, which is a valuable prognosis index of breast cancer disease. However, the source of 17β-HSD1 for inhibitor design is limited. In this study, the fragment encoding human 17β-HSD1 was successfully cloned and expressed in human embryonic kidney (HEK) 293T mammalian cells. The recombinant protein was purified by immobilized metal ion affinity chromatography yielding above 17 mg of purified 17β-HSD1 protein per liter of cell culture, with a specific activity of 8.54 μmoL/min/mg of protein for conversion of estradiol into estrone, with NAD + as cofactor at pH 9.2. Enzyme characterization studies revealed that the protein has estrogenic activity and the K m value for estrone is about 20 nM. The recombinant protein purified from transfected HEK293T cells had higher specific activity compared to that of the enzyme purified directly from placenta. The present data show that the mammalian cell expression system can provide active 17β-HSD1 which is functionally identical to its natural counterpart and easy to purify in qualities suitable for its structure-function study. Copyright © 2017 Elsevier Inc. All rights reserved.

Lin28b is sufficient to drive liver cancer and necessary for its maintenance in murine models

PubMed Central

Nguyen, Liem H.; Robinton, Daisy A.; Seligson, Marc; Wu, Linwei; Li, Lin; Rakheja, Dinesh; Comerford, Sarah; Ramezani, Saleh; Sun, Xiankai; Parikh, Monisha; Yang, Erin; Powers, John T.; Shinoda, Gen; Shah, Samar; Hammer, Robert; Daley, George Q.; Zhu, Hao

2014-01-01

SUMMARY Lin28a/b are RNA-binding proteins that influence stem cell maintenance, metabolism, and oncogenesis. Poorly differentiated, aggressive cancers often overexpress Lin28, but its role in tumor initiation or maintenance has not been definitively addressed. We report that LIN28B overexpression is sufficient to initiate hepatoblastoma and hepatocellular carcinoma in murine models. We also detected Lin28b overexpression in MYC-driven hepatoblastomas, and liver-specific deletion of Lin28a/b reduced tumor burden, extended latency, and prolonged survival. Both intravenous siRNA against Lin28b and conditional Lin28b deletion reduced tumor burden and prolonged survival. Igf2bp proteins are upregulated and Igf2bp3 is required in the context of LIN28B overexpression to promote growth. Thus, multiple murine models demonstrate that Lin28b is both sufficient to initiate liver cancer and necessary for its maintenance. PMID:25117712

Induction of IgA B cell differentiation of bone marrow-derived B cells by Peyer's patch autoreactive helper T cells.

PubMed

Kihira, T; Kawanishi, H

1995-08-01

The objective of this study was to demonstrate in vitro that bone marrow-derived pro/pre-B cells bearing mu mRNA can switch their Ig heavy-chain isotype to that of alpha mRNA-expressing B cells after contact with Peyer's patches-derived activated autoreactive CD4+ T cells. Bone marrow-derived pro/pre-B cells and activated autoreactive Peyer's patch, mesenteric lymph node, or spleen CD4+ T cells were co-cultured in the presence of recombinant (r) IL-2, rIL-7, and Con A for 3 days. The mixed cultured cells were isolated for preparation of total RNA. Dot/slot hybridization, using murine C mu (pu3741) and C alpha (P alpha J558) Ig heavy-chain cDNA probes, detected C mu and C alpha Ig heavy-chain mRNA transcripts. The magnitude of each mRNA expression was measured demsitometrically. In addition, the secreted class-specific Ig contents from the co-cultured supernatants were measured. The results indicate that activated autoreactive Peyer's patch and mesenteric lymph node CD4+ T cells provide a specific Ig heavy-chain switch from mu to alpha (Peyer's patch CD4+ T cells > mesenteric lymph node CD4+ T cells) in bone marrow-derived pro/pre-B cells and also assist to develop IgA-secreting plasma cells. The alpha heavy-chain switch and IgA production do not occur in the presence of activated autoreactive spleen CD4+ T cells. These results support the view that autoreactive gut Peyer's patch CD4+ T cells, at least, regulate IgA B cell heavy-chain switching and terminal differentiation during gut mucosal B cell development.

Mouse Cytotoxic T Cell-derived Granzyme B Activates the Mitochondrial Cell Death Pathway in a Bim-dependent Fashion*

PubMed Central

Catalán, Elena; Jaime-Sánchez, Paula; Aguiló, Nacho; Simon, Markus M.; Froelich, Christopher J.; Pardo, Julián

2015-01-01

Cytotoxic T cells (Tc) use perforin and granzyme B (gzmB) to kill virus-infected cells and cancer cells. Recent evidence suggests that human gzmB primarily induces apoptosis via the intrinsic mitochondrial pathway by either cleaving Bid or activating Bim leading to the activation of Bak/Bax and subsequent generation of active caspase-3. In contrast, mouse gzmB is thought to predominantly induce apoptosis by directly processing pro-caspase-3. However, in certain mouse cell types gzmB-mediated apoptosis mainly occurs via the mitochondrial pathway. To investigate whether Bim is involved under the latter conditions, we have now employed ex vivo virus-immune mouse Tc that selectively kill by using perforin and gzmB (gzmB+Tc) as effector cells and wild type as well as Bim- or Bak/Bax-deficient spontaneously (3T9) or virus-(SV40) transformed mouse embryonic fibroblast cells as targets. We show that gzmB+Tc-mediated apoptosis (phosphatidylserine translocation, mitochondrial depolarization, cytochrome c release, and caspase-3 activation) was severely reduced in 3T9 cells lacking either Bim or both Bak and Bax. This outcome was related to the ability of Tc cells to induce the degradation of Mcl-1 and Bcl-XL, the anti-apoptotic counterparts of Bim. In contrast, gzmB+Tc-mediated apoptosis was not affected in SV40-transformed mouse embryonic fibroblast cells lacking Bak/Bax. The data provide evidence that Bim participates in mouse gzmB+Tc-mediated apoptosis of certain targets by activating the mitochondrial pathway and suggest that the mode of cell death depends on the target cell. Our results suggest that the various molecular events leading to transformation and/or immortalization of cells have an impact on their relative resistance to the multiple gzmB+Tc-induced death pathways. PMID:25605735

Using Antigen-Specific B Cells to Combine Antibody and T Cell-Based Cancer Immunotherapy.

PubMed

Wennhold, Kerstin; Thelen, Martin; Schlößer, Hans Anton; Haustein, Natalie; Reuter, Sabrina; Garcia-Marquez, Maria; Lechner, Axel; Kobold, Sebastian; Rataj, Felicitas; Utermöhlen, Olaf; Chakupurakal, Geothy; Theurich, Sebastian; Hallek, Michael; Abken, Hinrich; Shimabukuro-Vornhagen, Alexander; von Bergwelt-Baildon, Michael

2017-09-01

Cancer immunotherapy by therapeutic activation of T cells has demonstrated clinical potential. Approaches include checkpoint inhibitors and chimeric antigen receptor T cells. Here, we report the development of an alternative strategy for cellular immunotherapy that combines induction of a tumor-directed T-cell response and antibody secretion without the need for genetic engineering. CD40 ligand stimulation of murine tumor antigen-specific B cells, isolated by antigen-biotin tetramers, resulted in the development of an antigen-presenting phenotype and the induction of a tumor antigen-specific T-cell response. Differentiation of antigen-specific B cells into antibody-secreting plasma cells was achieved by stimulation with IL21, IL4, anti-CD40, and the specific antigen. Combined treatment of tumor-bearing mice with antigen-specific CD40-activated B cells and antigen-specific plasma cells induced a therapeutic antitumor immune response resulting in remission of established tumors. Human CEA or NY-ESO-1-specific B cells were detected in tumor-draining lymph nodes and were able to induce antigen-specific T-cell responses in vitro, indicating that this approach could be translated into clinical applications. Our results describe a technique for the exploitation of B-cell effector functions and provide the rationale for their use in combinatorial cancer immunotherapy. Cancer Immunol Res; 5(9); 730-43. ©2017 AACR . ©2017 American Association for Cancer Research.

An antigen receptor-driven, interleukin 2-independent pathway for proliferation of murine cytolytic T lymphocyte clones

PubMed Central

1986-01-01

Proliferation of T lymphocytes can be induced by IL-2, either through an autocrine pathway in which the responding cell produces its own IL-2 or through an exocrine pathway in which IL-2 secreted by Th stimulates proliferation of IL-2-dependent CTL. However, proliferation of at least some CTL clones, such as CTL L3 and CTL dB45, also can be induced by stimulation of the antigen receptor in the absence of IL-2. Stimulation of these cloned CTL with T cell-depleted allogeneic spleen cells, allogeneic tumor cells, or immobilized mAb reactive with the T cell antigen receptor (TCR) induced thymidine incorporation, entry into cell cycle, and secretion of macrophage activating factor, but these stimuli did not induce the secretion of IL-2. Several observations indicated that such proliferation of cloned CTL induced by stimulation of the TCR was independent of IL-2; IL-2 could not be detected in supernatants from stimulated CTL cells. mAbs reactive with the murine IL-2-R efficiently blocked IL-2-mediated thymidine incorporation in cloned CTL and Th, but had no inhibitory effect on TCR-driven thymidine incorporation in the CTL clones. TCR-driven thymidine incorporation in cloned Th L2 cells was profoundly inhibited by these antibodies, indicating the operation of an IL-2-mediated autocrine pathway for proliferation in this cloned Th. When antibodies to the TCR were used to stimulate cloned CTL and Th, IFN-gamma mRNA was easily shown in the cloned CTL and Th. Although IL-2 mRNA could be detected in the cloned Th, it was never observed in the cloned CTL. These findings provide evidence for the existence of a TCR-mediated, IL-2-independent pathway for induction of cellular proliferation in cloned murine CTL. PMID:3486939

Evaluation of biological effects of intermediate frequency magnetic field on differentiation of embryonic stem cell.

PubMed

Yoshie, Sachiko; Ogasawara, Yuki; Ikehata, Masateru; Ishii, Kazuyuki; Suzuki, Yukihisa; Wada, Keiji; Wake, Kanako; Nakasono, Satoshi; Taki, Masao; Ohkubo, Chiyoji

2016-01-01

The embryotoxic effect of intermediate frequency (IF) magnetic field (MF) was evaluated using murine embryonic stem (ES) cells and fibroblast cells based on the embryonic stem cell test (EST). The cells were exposed to 21 kHz IF-MF up to magnetic flux density of 3.9 mT during the cell proliferation process (7 days) or the cell differentiation process (10 days) during which an embryonic body differentiated into myocardial cells. As a result, there was no significant difference in the cell proliferation between sham- and IF-MF-exposed cells for both ES and fibroblast cells. Similarly, the ratio of the number of ES-derived cell aggregates differentiated to myocardial cells to total number of cell aggregates was not changed by IF-MF exposure. In addition, the expressions of a cardiomyocytes-specific gene, Myl2 , and an early developmental gene, Hba-x , in the exposed cell aggregate were not altered. Since the magnetic flux density adopted in this study is much higher than that generated by an inverter of the electrical railway, an induction heating (IH) cooktop, etc . in our daily lives, these results suggested that IF-MF in which the public is exposed to in general living environment would not have embryotoxic effect.

Mediator Med23 deficiency enhances neural differentiation of murine embryonic stem cells through modulating BMP signaling.

PubMed

Zhu, Wanqu; Yao, Xiao; Liang, Yan; Liang, Dan; Song, Lu; Jing, Naihe; Li, Jinsong; Wang, Gang

2015-02-01

Unraveling the mechanisms underlying early neural differentiation of embryonic stem cells (ESCs) is crucial to developing cell-based therapies of neurodegenerative diseases. Neural fate acquisition is proposed to be controlled by a 'default' mechanism, for which the molecular regulation is not well understood. In this study, we investigated the functional roles of Mediator Med23 in pluripotency and lineage commitment of murine ESCs. Unexpectedly, we found that, despite the largely unchanged pluripotency and self-renewal of ESCs, Med23 depletion rendered the cells prone to neural differentiation in different differentiation assays. Knockdown of two other Mediator subunits, Med1 and Med15, did not alter the neural differentiation of ESCs. Med15 knockdown selectively inhibited endoderm differentiation, suggesting the specificity of cell fate control by distinctive Mediator subunits. Gene profiling revealed that Med23 depletion attenuated BMP signaling in ESCs. Mechanistically, MED23 modulated Bmp4 expression by controlling the activity of ETS1, which is involved in Bmp4 promoter-enhancer communication. Interestingly, med23 knockdown in zebrafish embryos also enhanced neural development at early embryogenesis, which could be reversed by co-injection of bmp4 mRNA. Taken together, our study reveals an intrinsic, restrictive role of MED23 in early neural development, thus providing new molecular insights for neural fate determination. © 2015. Published by The Company of Biologists Ltd.

Shifted T Helper Cell Polarization in a Murine Staphylococcus aureus Mastitis Model.

PubMed

Zhao, Yanqing; Zhou, Ming; Gao, Yang; Liu, Heyuan; Yang, Wenyu; Yue, Jinhua; Chen, Dekun

2015-01-01

Mastitis, one of the most costly diseases in dairy ruminants, is an inflammation of the mammary gland caused by pathogenic infection. The mechanisms of adaptive immunity against pathogens in mastitis have not been fully elucidated. To investigate T helper cell-mediated adaptive immune responses, we established a mastitis model by challenge with an inoculum of 4 × 106 colony-forming units of Staphylococcus aureus in the mammary gland of lactating mice, followed by quantification of bacterial burden and histological analysis. The development of mastitis was accompanied by a significant increase in both Th17 and Th1 cells in the mammary gland. Moreover, the relative expression of genes encoding cytokines and transcription factors involved in the differentiation and function of these T helper cells, including Il17, Rorc, Tgfb, Il1b, Il23, Ifng, Tbx21, and Il12, was greatly elevated in the infected mammary gland. IL-17 is essential for neutrophil recruitment to infected mammary gland via CXC chemokines, whereas the excessive IL-17 production contributes to tissue damage in mastitis. In addition, a shift in T helper cell polarization toward Th2 and Treg cells was observed 5 days post-infection, and the mRNA expression of the anti-inflammatory cytokine Il10 was markedly increased at day 7 post-infection. These results indicate that immune clearance of Staphylococcus aureus in mastitis is facilitated by the enrichment of Th17, Th1 and Th2 cells in the mammary gland mediated by pro-inflammatory cytokine production, which is tightly regulated by Treg cells and the anti-inflammatory cytokine IL-10.

Shifted T Helper Cell Polarization in a Murine Staphylococcus aureus Mastitis Model

PubMed Central

Zhao, Yanqing; Zhou, Ming; Gao, Yang; Liu, Heyuan; Yang, Wenyu; Yue, Jinhua; Chen, Dekun

2015-01-01

Mastitis, one of the most costly diseases in dairy ruminants, is an inflammation of the mammary gland caused by pathogenic infection. The mechanisms of adaptive immunity against pathogens in mastitis have not been fully elucidated. To investigate T helper cell-mediated adaptive immune responses, we established a mastitis model by challenge with an inoculum of 4 × 106 colony-forming units of Staphylococcus aureus in the mammary gland of lactating mice, followed by quantification of bacterial burden and histological analysis. The development of mastitis was accompanied by a significant increase in both Th17 and Th1 cells in the mammary gland. Moreover, the relative expression of genes encoding cytokines and transcription factors involved in the differentiation and function of these T helper cells, including Il17, Rorc, Tgfb, Il1b, Il23, Ifng, Tbx21, and Il12, was greatly elevated in the infected mammary gland. IL-17 is essential for neutrophil recruitment to infected mammary gland via CXC chemokines, whereas the excessive IL-17 production contributes to tissue damage in mastitis. In addition, a shift in T helper cell polarization toward Th2 and Treg cells was observed 5 days post-infection, and the mRNA expression of the anti-inflammatory cytokine Il10 was markedly increased at day 7 post-infection. These results indicate that immune clearance of Staphylococcus aureus in mastitis is facilitated by the enrichment of Th17, Th1 and Th2 cells in the mammary gland mediated by pro-inflammatory cytokine production, which is tightly regulated by Treg cells and the anti-inflammatory cytokine IL-10. PMID:26230498

MTORC1 EXPANDS TH17 AND IL-4+ DN T CELLS AND CONTRACTS TREGS IN SLE

PubMed Central

Kato, Hiroshi; Perl, Andras

2014-01-01

The mechanistic target of rapamycin (mTOR) is activated in CD4−CD8− double-negative (DN) T cells and its blockade is therapeutic in systemic lupus erythematosus (SLE) patients. Murine studies showed the involvement of mTOR complex 1 (mTORC1) and 2 (mTORC2) in the differentiation of Th1/Th17 cells and Th2 cells, respectively. Here, we investigated the roles of mTORC1 and mTORC2 in T-cell lineage development in SLE and matched healthy control (HC) subjects. mTORC1 activity was increased while mTORC2 was reduced as assessed by phosphorylation of their substrates pS6K or pS6RP and pAkt, respectively. Rapamycin inhibited mTORC1 and enhanced mTORC2. IL-4 expression was increased in freshly isolated CD8+ lupus T cells (SLE: 8.09±1.93%, HC: 3.61±0.49%; p=0.01). DN T cells had greater IL-4 expression than CD4+ or CD8+ T cells of SLE patients after 3 day in vitro stimulation, which was suppressed by rapamycin (control: 9.26±1.48%, rapamycin: 5.03±0.66%; p<0.001). GATA-3 expression was increased in CD8+ lupus T cells (p<0.01) and insensitive to rapamycin treatment. IFN-γ expression was reduced in all lupus T cell subsets (p=1.0×10−5) and also resisted rapamycin. IL-17 expression was increased in CD4+ lupus T cells (SLE: 3.62±0.66%, HC: 2.29±0.27%; p=0.019), which was suppressed by rapamycin (control: 3.91±0.79%, rapamycin: 2.22±0.60%; p<0.001). Frequency of Tregs was reduced in SLE (SLE: 1.83±0.25%, HC: 2.97±0.27%; p=0.0012). Rapamycin inhibited mTORC1 in Tregs and promoted their expansion. Neutralization of IL-17 but not IL-4 also expanded Tregs in SLE and HC subjects. These results indicate that mTORC1 expands IL-4+ DN T and Th17 cells and contracts Tregs in SLE. PMID:24683191

Role of interleukin (IL)-17 and T-helper (Th)17 cells in cancer.

PubMed

Song, Yang; Yang, Jian Ming

2017-11-04

Interleukin-17 (IL-17), a pleiotropic proinflammatory cytokine, is reported to be significantly generated by a distinct subset of CD4 + T-cells, upgrading cancer-elicited inflammation and preventing cancer cells from immune surveillance. T-helper (Th)17 cells produced from naive CD4 + T cells have recently been renowned and generally accepted, gaining eminence in cancer studies and playing the effective role in context of cancer. Th17 cells are the main source of IL-17-secreting cells, It was found that other cell types produced this cytokine as well, including Group 3 innate lymphoid cells (ILC3), δγT cells, invariant natural killer T (iNKT) cells, lymphoid-tissue inducer (LTi)-like cells and Natural killer (NK) cells. Th17-associated cytokines give impetus to tumor progression, or inducing angiogenesis and metastasis. This review demonstrates an understanding on how the pro- or antitumor function of Th17 cells and IL-17 may change cancer progression, leading to the appearance of complex and pivotal biologic activities in tumor. Copyright © 2017 Elsevier Inc. All rights reserved.

IL-17 suppresses immune effector functions in human papillomavirus-associated epithelial hyperplasia.

PubMed

Gosmann, Christina; Mattarollo, Stephen R; Bridge, Jennifer A; Frazer, Ian H; Blumenthal, Antje

2014-09-01

Persistent infection with high-risk human papillomaviruses (HPV) causes epithelial hyperplasia that can progress to cancer and is thought to depend on immunosuppressive mechanisms that prevent viral clearance by the host. IL-17 is a cytokine with diverse functions in host defense and in the pathology of autoimmune disorders, chronic inflammatory diseases, and cancer. We analyzed biopsies from patients with HPV-associated cervical intraepithelial neoplasia grade 2/3 and murine skin displaying HPV16 E7 protein-induced epithelial hyperplasia, which closely models hyperplasia in chronic HPV lesions. Expression of IL-17 and IL-23, a major inducer of IL-17, was elevated in both human HPV-infected and murine E7-expressing lesions. Using a skin-grafting model, we demonstrated that IL-17 in HPV16 E7 transgenic skin grafts inhibited effective host immune responses against the graft. IL-17 was produced by CD3(+) T cells, predominantly CD4(+) T cells in human, and CD4(+) and γδ T cells in mouse hyperplastic lesions. IL-23 and IL-1β, but not IL-18, induced IL-17 production in E7 transgenic skin. Together, these findings demonstrate an immunosuppressive role for IL-17 in HPV-associated epithelial hyperplasia and suggest that blocking IL-17 in persistent viral infection may promote antiviral immunity and prevent progression to cancer. Copyright © 2014 by The American Association of Immunologists, Inc.

Induction of regulatory T cells in Th1-/Th17-driven experimental autoimmune encephalomyelitis by zinc administration.

PubMed

Rosenkranz, Eva; Maywald, Martina; Hilgers, Ralf-Dieter; Brieger, Anne; Clarner, Tim; Kipp, Markus; Plümäkers, Birgit; Meyer, Sören; Schwerdtle, Tanja; Rink, Lothar

2016-03-01

The essential trace element zinc is indispensable for proper immune function as zinc deficiency accompanies immune defects and dysregulations like allergies, autoimmunity and an increased presence of transplant rejection. This point to the importance of the physiological and dietary control of zinc levels for a functioning immune system. This study investigates the capacity of zinc to induce immune tolerance. The beneficial impact of physiological zinc supplementation of 6 μg/day (0.3mg/kg body weight) or 30 μg/day (1.5mg/kg body weight) on murine experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis with a Th1/Th17 (Th, T helper) cell-dominated immunopathogenesis, was analyzed. Zinc administration diminished EAE scores in C57BL/6 mice in vivo (P<.05), reduced Th17 RORγT(+) cells (P<.05) and significantly increased inducible iTreg cells (P<.05). While Th17 cells decreased systemically, iTreg cells accumulated in the central nervous system. Cumulatively, zinc supplementation seems to be capable to induce tolerance in unwanted immune reactions by increasing iTreg cells. This makes zinc a promising future tool for treating autoimmune diseases without suppressing the immune system. Copyright © 2015 Elsevier Inc. All rights reserved.

Interferon-gamma alone triggers the production of nitric oxide from serum-starved BNL CL.2, murine embryonic liver cells.

PubMed

Pae, H O; Yoo, J C; Choi, B M; Paik, S G; Kim, Y H; Jin, H S; Chung, H T

1999-01-01

A previous study has demonstrated that both interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were needed to induce the production of nitric oxide (NO) in BNL CL.2 cells, murine embryonic liver cells. We here demonstrate that when BNL CL.2 cells were cultured with serum-free medium, they were induced to produce NO by the stimulation of IFN-gamma alone. BNL CL.2 cells were cultured with serum-free or serum-containing medium for 1-3 days and then stimulated to synthesize NO by IFN-gamma. Surprisingly, only serum-starved cells showed significant amount of nitrite accumulation and iNOS protein expression in response to IFN-gamma in dose- and time-dependent manners, but serum-supplied cells did not. When the cells were stimulated with IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), or LPS in combinations, only the combination of IFN-gamma and LPS produced more NO than that produced by IFN-gamma alone. The production of NO by the cells stimulated with IFN-gamma or IFN-gamma plus LPS was blocked by the addition of N(G)-monomethyl-L-arginine (N(G)MMA), a NO synthesis inhibitor. To address the intracellular signal pathway responsible for the production of NO by the cells stimulated with IFN-gamma aloneor IFN-gamma plus LPS, we examined the effects of several protein kinase inhibitors on the production of NO from the cells. The production of NO was significantly inhibited by protein tyrosine kinase (PTK) inhibitors, genistein and herbimycin A, but not by protein kinase A or C inhibitors. These results suggest that the deprivation of serum from BNL CL.2 cell culture medium might prime the cells to induce NO synthesis when the cells are triggered by IFN-gamma and the involvement of PTK signal transduction pathway in the expression of inducible NO synthase gene in murine hepatoma cells.

Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells.

PubMed

Park, Mi-Hee; Jung, Yu-Jin; Kim, Pyeung-Hyeun

2012-02-01

Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching.

γδ T Cells Regulate the Early Inflammatory Response to Bordetella pertussis Infection in the Murine Respiratory Tract

PubMed Central

Zachariadis, O.; Cassidy, J. P.; Brady, J.; Mahon, B. P.

2006-01-01

The role of γδ T cells in the regulation of pulmonary inflammation following Bordetella pertussis infection was investigated. Using a well-characterized murine aerosol challenge model, inflammatory events in mice with targeted disruption of the T-cell receptor δ-chain gene (γδ TCR−/− mice) were compared with those in wild-type animals. Early following challenge with B. pertussis, γδ TCR−/− mice exhibited greater pulmonary inflammation, as measured by intra-alveolar albumin leakage and lesion histomorphometry, yet had lower contemporaneous bacterial lung loads. The larger numbers of neutrophils and macrophages and the greater concentration of the neutrophil marker myeloperoxidase in bronchoalveolar lavage fluid from γδ TCR−/− mice at this time suggested that differences in lung injury were mediated through increased leukocyte trafficking into infected alveoli. Furthermore, flow cytometric analysis found the pattern of recruitment of natural killer (NK) and NK receptor+ T cells into airspaces differed between the two mouse types over the same time period. Taken together, these findings suggest a regulatory influence for γδ T cells over the early pulmonary inflammatory response to bacterial infection. The absence of γδ T cells also influenced the subsequent adaptive immune response to specific bacterial components, as evidenced by a shift from a Th1 to a Th2 type response against the B. pertussis virulence factor filamentous hemagglutinin in γδ TCR−/− mice. The findings are relevant to the study of conditions such as neonatal B. pertussis infection and acute respiratory distress syndrome where γδ T cell dysfunction has been implicated in the inflammatory process. PMID:16495558

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade.

PubMed

Li, Wen; Green, William R

2011-12-01

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.

Effects of overexpression of IL-1 receptor-associated kinase on NFkappaB activation, IL-2 production and stress-activated protein kinases in the murine T cell line EL4.

PubMed

Knop, J; Wesche, H; Lang, D; Martin, M U

1998-10-01

The association and activation of the IL-1 receptor-associated protein kinase (IRAK) to the IL-1 receptor complex is one of the earliest events detectable in IL-1 signal transduction. We generated permanent clones of the murine T cell line EL4 6.1 overexpressing human (h)IRAK to evaluate the role of this kinase in IL-1 signaling. Overexpression of hIRAK enhanced IL-1-stimulated activation of the transcription factor NFkappaB, whereas a truncated form (N-IRAK) specifically inhibited IL-1-dependent NFkappaB activity. In clones stably overexpressing hIRAK a weak constitutive activation of NFkappaB correlated with a low basal IL-2 production which was enhanced in an IL-1-dependent manner. Compared to the parental cell line the dose-response curve of IL-1-induced IL-2 production was shifted in both potency and efficacy. These results demonstrate that IRAK directly triggers NFkappaB-mediated gene expression in EL4 cells. Qualitatively different effects were observed for the IL-1-induced activation of stress-activated protein (SAP) kinases: permanent overexpression of IRAK did not affect the dose dependence but prolonged the kinetics of IL-1-induced activation of SAP kinases, suggesting that this signaling branch may be regulated by distinct mechanisms.

Ultrafast transient photocarrier dynamics of the bulk-insulating topological insulator B i1.5S b0.5T e1.7S e1.3

NASA Astrophysics Data System (ADS)

Choi, Young Gwan; Zhung, Chan June; Park, Sun-Hee; Park, Joonbum; Kim, Jun Sung; Kim, Seongheun; Park, Jaehun; Lee, J. S.

2018-02-01

Using optical-pump terahertz-probe spectroscopy, we investigated an ultrafast photocarrier relaxation behavior in a B i1.5S b0.5T e1.7S e1.3 (BSTS) single crystal, which is one of the most bulk-insulating topological insulators. Compared to n -type bulk-metallic B i2S e3 , we found that BSTS endows distinct behaviors in its photocarrier dynamics; the relaxation time turns out to be an order of magnitude longer, and the transient conductance spectrum exhibits a nonlinear increase as a function of the pumping power. Also, we observed an abrupt reduction of the photocarrier scattering rate in several picoseconds after the initial photoexcitation. We discuss these intriguing experimental observations based on a bulk-to-surface carrier injection assisted by the built-in electric field near the surface and electron-phonon scattering.

A Bone Anabolic Effect of RANKL in a Murine Model of Osteoporosis mediated through FoxP3+ CD8 T-cells

PubMed Central

Buchwald, Zachary S.; Yang, Chang; Nellore, Suman; Shashkova, Elena V.; Davis, Jennifer L.; Cline, Anna; Ko, Je; Novack, Deborah V.; DiPaolo, Richard; Aurora, Rajeev

2015-01-01

TNFα and IL-17 secreted by proinflammatory T-cells (TEFF) promote bone erosion by activating osteoclasts. We previously demonstrated that in addition to bone resorption, osteoclasts act as antigen presenting cells to induce FoxP3 in CD8 T-cells (TcREG). The osteoclast-induced regulatory CD8 T-cells limit bone resorption in ovariectomized mice (a murine model of postmenopausal osteoporosis). Here we show that while low-dose RANKL maximally induces TcREG via Notch signaling pathway to limit bone resorption, high-dose RANKL promotes bone resorption. In vitro, both TNFα and IL-17, cytokines that are abundant in ovariectomized animals, suppress TcREG induction by osteoclasts by repressing Notch ligand expression in osteoclasts but this effect can be counteracted by addition of RANKL. Ovariectomized mice treated with low-dose RANKL induced TcREG that suppressed bone resorption, decreased TEFF levels and increased bone formation. High dose RANKL had the expected osteolytic effect. Low dose RANKL administration in ovariectomized mice lacking CD8 T-cells was also osteolytic, confirming that TcREG mediate this bone anabolic effect. Our results show that while RANKL directly stimulates osteoclasts to resorb bone, it also controls the osteoclasts’ ability to induce regulatory T-cells, engaging an important negative feedback loop. In addition to the conceivable clinical relevance to treatment of osteoporosis, these observations have potential relevance to induction of tolerance and autoimmune diseases. PMID:25656537

Characterization of CD31 expression on murine and human neonatal T lymphocytes during development and activation

PubMed Central

Fike, Adam J.; Nguyen, Linda T.; Kumova, Ogan K.; Carey, Alison J.

2017-01-01

Background CD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell Receptor signalling by increasing the threshold for T cell activation. Therefore, CD31 could modulate neonatal tolerance and adaptive immune responses. Methods Lymphocytes were harvested from murine neonates at different ages, human late preterm and term cord blood, and adult peripheral blood. Human samples were activated over a five-day period to simulate acute inflammation. Mice were infected with influenza; lungs and spleens were harvested at days 6 and 9 post-infection and analyzed by flow cytometry. Results CD31 expressing neonatal murine CD4+ and CD8a+ T cells increase over the first week of life. Upon in vitro stimulation, human infants’ CD4+ and CD8a+ T cells shed CD31 faster in comparison to adults. In the context of acute infection, mice infected at 3-days old have an increased number of naive and activated CD31+ T lymphocytes at the site of infection at day 6 and 9 post-infection, as compared to 7-days old; however, the opposite is true in the periphery. Conclusion Differences in trafficking of CD31+ Cytotoxic T Lymphocytes (CTLs) during acute influenza infection could modulate tolerance and contribute to a dampened adaptive immune response in neonates. PMID:28355204

17 CFR 240.15b12-1T - Brokers or dealers engaged in a retail forex business.

Code of Federal Regulations, 2012 CFR

2012-04-01

... a retail forex business. 240.15b12-1T Section 240.15b12-1T Commodity and Securities Exchanges... § 240.15b12-1T Brokers or dealers engaged in a retail forex business. (a) Definitions. In addition to... et seq.). (2) Retail forex business means engaging in one or more retail forex transactions with the...

17 CFR 240.15b12-1T - Brokers or dealers engaged in a retail forex business.

Code of Federal Regulations, 2013 CFR

2013-04-01

... a retail forex business. 240.15b12-1T Section 240.15b12-1T Commodity and Securities Exchanges... § 240.15b12-1T Brokers or dealers engaged in a retail forex business. (a) Definitions. In addition to... et seq.). (2) Retail forex business means engaging in one or more retail forex transactions with the...

Temporal distribution of endogenous retinoids in the embryonic mouse mandible.

PubMed

Beeman, C S; Kronmiller, J E

1994-09-01

Retinoids play an important part in embryonic pattern formation. They are necessary for normal differentiation of odontogenic tissues and, in excess, disrupt the pattern of tooth formation. Excess retinoids produce supernumerary buds of the dental lamina in the diastema region of the mouse embryonic mandible where teeth do not normally form. This effect is coincident with an increase in epithelial proliferation and an alteration in epidermal growth factor mRNA expression (a gene product necessary for tooth formation). It was found by high-performance liquid chromatography that endogenous retinoids are present in the developing murine mandible and that concentrations of some retinoids reach a peak at the time of the initiation of odontogenesis (dental lamina formation).

Sensitivity to methylmercury toxicity is enhanced in oxoguanine glycosylase 1 knockout murine embryonic fibroblasts and is dependent on cellular proliferation capacity

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

Ondovcik, Stephanie L.; Tamblyn, Laura; McPherson, John Peter

2013-07-01

Methylmercury (MeHg) is a persistent environmental contaminant with potent neurotoxic action for which the underlying molecular mechanisms remain to be conclusively delineated. Our objectives herein were twofold: first, to corroborate our previous findings of an increased sensitivity of spontaneously-immortalized oxoguanine glycosylase 1-null (Ogg1{sup −/−}) murine embryonic fibroblasts (MEFs) to MeHg through generation of Simian virus 40 (SV40) large T antigen-immortalized wild-type and Ogg1{sup −/−} MEFs; and second, to determine whether MeHg toxicity is proliferation-dependent. As with the spontaneously-immortalized cells used previously, the SV40 large T antigen-immortalized cells exhibited similar tendencies to undergo MeHg-initiated cell cycle arrest, with increased sensitivity inmore » the Ogg1{sup −/−} MEFs as measured by clonogenic survival and DNA damage. Compared to exponentially growing cells, those seeded at a higher density exhibited compromised proliferation, which proved protective against MeHg-mediated cell cycle arrest and induction of DNA double strand breaks (DSBs), measured by phosphorylation of the core histone H2A variant (H2AX) on serine 139 (γH2AX), and by its functional confirmation by micronucleus assessment. This enhanced sensitivity of Ogg1{sup −/−} MEFs to MeHg toxicity using discrete SV40 immortalization corroborates our previous studies, and suggests a novel role for OGG1 in minimizing MeHg-initiated DNA lesions that trigger replication-associated DSBs. Furthermore, proliferative capacity may determine MeHg toxicity in vivo and in utero. Accordingly, variations in cellular proliferative capacity and interindividual variability in repair activity may modulate the risk of toxicological consequences following MeHg exposure. - Highlights: • SV40 large T antigen-immortalized Ogg1{sup −/−} cells are more sensitive to MeHg. • Sensitivity to MeHg is dependent on cellular proliferation capacity. • OGG1 maintains

7 CFR 15b.17 - Discrimination prohibited.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 1 2011-01-01 2011-01-01 false Discrimination prohibited. 15b.17 Section 15b.17... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.17 Discrimination prohibited. No... to discrimination under any program or activity receiving assistance from this Department. ...

7 CFR 15b.17 - Discrimination prohibited.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 1 2010-01-01 2010-01-01 false Discrimination prohibited. 15b.17 Section 15b.17... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.17 Discrimination prohibited. No... to discrimination under any program or activity receiving assistance from this Department. ...

7 CFR 15b.17 - Discrimination prohibited.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 1 2013-01-01 2013-01-01 false Discrimination prohibited. 15b.17 Section 15b.17... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.17 Discrimination prohibited. No... to discrimination under any program or activity receiving assistance from this Department. ...

7 CFR 15b.17 - Discrimination prohibited.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 1 2012-01-01 2012-01-01 false Discrimination prohibited. 15b.17 Section 15b.17... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.17 Discrimination prohibited. No... to discrimination under any program or activity receiving assistance from this Department. ...

7 CFR 15b.17 - Discrimination prohibited.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 1 2014-01-01 2014-01-01 false Discrimination prohibited. 15b.17 Section 15b.17... ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Accessibility § 15b.17 Discrimination prohibited. No... to discrimination under any program or activity receiving assistance from this Department. ...

Bupleurum chinense extract ameliorates an OVA-induced murine allergic asthma through the reduction of the Th2 and Th17 cytokines production by inactivation of NFκB pathway.

PubMed

Bui, Thi Tho; Piao, Chun Hua; Song, Chang Ho; Shin, Hee Soon; Chai, Ok Hee

2017-07-01

Bupleurum chinense belongs to the Bupleurum spp. family that has been used in traditional herbal medicine for over thousand years. It has been reported to have anti-inflammatory, anti-oxidant, hepato-protective, antipyretic, analgesic, anti-fibrotic and immunomodulatory effect. However, the effect of B. Chinense on allergic asthma remains unclear. This study investigated the immunomodulatory effects of B. Chinense extracts (BCE) on airway inflammation in asthmatic mice model. In the ovalbumin (OVA)-induced allergic asthma model, we evaluated the number of total cells, differential inflammatory cells and the production of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) and lung homogenate as well as histological structure. The levels of NFκB p65, IκBα, p-NFκB p65, p-IκBα and the total immunoglobulin (Ig) E, anti-OVA IgE, anti-OVA IgG were also examined. The oral administration of 200mg/kg BCE inhibited the accumulation of inflammatory cells especially eosinophils in BALF. Also, BCE regulated the imbalance of Th1, Th2 and Th17-related production, with attenuated the expression of GATA3, IL-1β, IL-4, IL-5, IL-6, TNF-α and RORγt, IL-17A in BALF and lung homogenate, meanwhile, up-regulated the secretion of INF-γ in lung homogenate. The levels of IgE, anti-OVA IgE, anti-OVA IgG1 and anti-OVA IgG2a were also suppressed by BCE treatment in serum. Futhermore, BCE inhibited the proinflammatory cytokines via inactivation of NFκB p65 phosphorylation and IκBα degradation in cytoplasm. The histological analysis showed that the infiltration of inflammatory cells, mucus hypersecretion and collagen fiber deposits were ameliorated in BCE treated mice. In addition, BCE induced the functional differentiation of naive CD4+ T cells forward to Th1 and Tr1 through producing INF-γ and IL-10. These results suggest that BCE may have therapeutic potential for treating allergic asthma through inhibiting Th2/Th17 cytokines production by inactivation of NFκB

17 CFR 240.10b-17 - Untimely announcements of record dates.

Code of Federal Regulations, 2010 CFR

2010-04-01

... distribution and if that security is a right or a warrant, the subscription price, (e) In any other property... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Untimely announcements of record dates. 240.10b-17 Section 240.10b-17 Commodity and Securities Exchanges SECURITIES AND EXCHANGE...

Diverse functions of miR-17-92 cluster microRNAs in T helper cells.

PubMed

Baumjohann, Dirk

2018-06-01

T helper (Th) cells are critically involved in adaptive immune responses against various pathogens. In contrast, dysregulated T helper cell responses are associated with a variety of diseases, including autoimmunity, allergies, and cancer. Differentiation of naïve CD4 + T cells into effector T helper cell subsets, including Th1, Th2, Th17, Treg, and T follicular helper (Tfh), requires precise dosing of signaling molecules and transcription factors. MicroRNAs (miRNAs), which are small endogenously expressed RNAs that regulate gene expression, play important roles in these processes. The miR-17-92 cluster, a miRNA polycistron also known as oncomiR-1, has emerged as a central integrator of gene expression events that govern T helper cell differentiation pathways. The complexity of miR-17-92-mediated gene regulation lies in the nature of this miRNA cluster, which consists of six different miRNAs. Individual miR-17-92 miRNAs, albeit initially transcribed as one transcript, can have cooperative or opposing effects on biological processes. Therefore, a better understanding of the molecular regulation of miR-17-92 and its downstream networks will provide important insights into T helper cell differentiation and diversity that may be harnessed for the design of advanced T cell-targeting therapies. Copyright © 2018 Elsevier B.V. All rights reserved.

CD1b-autoreactive T cells contribute to hyperlipidemia-induced skin inflammation in mice

PubMed Central

Bagchi, Sreya; He, Ying; Zhang, Hong; Cao, Liang; Van Rhijn, Ildiko; Moody, D. Branch; Gudjonsson, Johann E.

2017-01-01

A large proportion of human T cells are autoreactive to group 1 CD1 proteins, which include CD1a, CD1b, and CD1c. However, the physiological role of the CD1 proteins remains poorly defined. Here, we have generated a double-transgenic mouse model that expresses human CD1b and CD1c molecules (hCD1Tg) as well as a CD1b-autoreactive TCR (HJ1Tg) in the ApoE-deficient background (hCD1Tg HJ1Tg Apoe–/– mice) to determine the role of CD1-autoreactive T cells in hyperlipidemia-associated inflammatory diseases. We found that hCD1Tg HJ1Tg Apoe–/– mice spontaneously developed psoriasiform skin inflammation characterized by T cell and neutrophil infiltration and a Th17-biased cytokine response. Anti–IL-17A treatment ameliorated skin inflammation in vivo. Additionally, phospholipids and cholesterol preferentially accumulated in diseased skin and these autoantigens directly activated CD1b-autoreactive HJ1 T cells. Furthermore, hyperlipidemic serum enhanced IL-6 secretion by CD1b+ DCs and increased IL-17A production by HJ1 T cells. In psoriatic patients, the frequency of CD1b-autoreactive T cells was increased compared with that in healthy controls. Thus, this study has demonstrated the pathogenic role of CD1b-autoreactive T cells under hyperlipidemic conditions in a mouse model of spontaneous skin inflammation. As a large proportion of psoriatic patients are dyslipidemic, this finding is of clinical significance and indicates that self-lipid–reactive T cells might serve as a possible link between hyperlipidemia and psoriasis. PMID:28463230

Newly Identified TLR9 Stimulant, M6-395 Is a Potent Polyclonal Activator for Murine B Cells

PubMed Central

Park, Mi-Hee; Jung, Yu-Jin

2012-01-01

Background Toll-like receptors (TLRs) have been extensively studied in recent years. However, functions of these molecules in murine B cell biology are largely unknown. A TLR4 stimulant, LPS is well known as a powerful polyclonal activator for murine B cells. Methods In this study, we explored the effect of a murine TLR9 stimulant, M6-395 (a synthetic CpG ODNs) on B cell proliferation and Ig production. Results First, M6-395 was much more potent than LPS in augmenting B cell proliferation. As for Ig expression, M6-395 facilitated the expression of both TGF-β1-induced germ line transcript α (GLTα) and IL-4-induced GLTγ1 as levels as those by LPS and Pam3CSK4 (TLR1/2 agonist) : a certain Ig GLT expression is regarded as an indicative of the corresponding isotype switching recombination. However, IgA and IgG1 secretion patterns were quite different--these Ig isotype secretions by M6-395 were much less than those by LPS and Pam3CSK4. Moreover, the increase of IgA and IgG1 production by LPS and Pam3CSK4 was virtually abrogated by M6-395. The same was true for the secretion of IgG3. We found that this unexpected phenomena provoked by M6-395 is attributed, at least in part, to its excessive mitogenic nature. Conclusion Taken together, these results suggest that M6-395 can act as a murine polyclonal activator but its strong mitogenic activity is unfavorable to Ig isotype switching. PMID:22536167

Differentiation, phenotype, and function of interleukin-17-producing human Vγ9Vδ2 T cells.

PubMed

Caccamo, Nadia; La Mendola, Carmela; Orlando, Valentina; Meraviglia, Serena; Todaro, Matilde; Stassi, Giorgio; Sireci, Guido; Fournié, Jean Jacques; Dieli, Francesco

2011-07-07

In healthy adults, the major peripheral blood γδ T-cell subset expresses the Vγ9Vδ2 TCR and displays pleiotropic features. Here we report that coculture of naive Vγ9Vδ2 T cells with phosphoantigens and a cocktail of cytokines (IL-1-β, TGF-β, IL-6, and IL-23), leads to selective expression of the transcription factor RORγt and polarization toward IL-17 production. IL-17(+) Vγ9Vδ2 T cells express the chemokine receptor CCR6 and produce IL-17 but neither IL-22 nor IFN-γ; they have a predominant terminally differentiated (CD27(-)CD45RA(+)) phenotype and express granzyme B, TRAIL, FasL, and CD161. On antigen activation, IL-17(+) Vγ9Vδ2 T cells rapidly induce CXCL8-mediated migration and phagocytosis of neutrophils and IL-17-dependent production of β-defensin by epithelial cells, indicating that they may be involved in host immune responses against infectious microorganisms. Accordingly, an increased percentage of IL-17(+) Vγ9Vδ2 lymphocytes is detected in the peripheral blood and at the site of disease in children with bacterial meningitis, and this pattern was reversed after successful antibacterial therapy. Most notably, the phenotype of IL-17(+) Vγ9Vδ2 T cells in children with meningitis matches that of in vitro differentiated IL-17(+) Vγ9Vδ2 T cells. Our findings delineate a previously unknown subset of human IL-17(+) Vγ9Vδ2 T lymphocytes implicated in the pathophysiology of inflammatory responses during bacterial infections.

CD8+ T Cells Contribute to the Development of Coronary Arteritis in the Lactobacillus casei Cell Wall Extract-Induced Murine Model of Kawasaki Disease.

PubMed

Noval Rivas, Magali; Lee, Youngho; Wakita, Daiko; Chiba, Norika; Dagvadorj, Jargalsaikhan; Shimada, Kenichi; Chen, Shuang; Fishbein, Michael C; Lehman, Thomas J A; Crother, Timothy R; Arditi, Moshe

2017-02-01

Kawasaki disease (KD) is the leading cause of acquired heart disease among children in developed countries. Coronary lesions in KD in humans are characterized by an increased presence of infiltrating CD3+ T cells; however, the specific contributions of the different T cell subpopulations in coronary arteritis development remain unknown. Therefore, we sought to investigate the function of CD4+ and CD8+ T cells, Treg cells, and natural killer (NK) T cells in the pathogenesis of KD. We addressed the function of T cell subsets in KD development by using a well-established murine model of Lactobacillus casei cell wall extract (LCWE)-induced KD vasculitis. We determined which T cell subsets were required for development of KD vasculitis by using several knockout murine strains and depleting monoclonal antibodies. LCWE-injected mice developed coronary lesions characterized by the presence of inflammatory cell infiltrates. Frequently, this chronic inflammation resulted in complete occlusion of the coronary arteries due to luminal myofibroblast proliferation (LMP) as well as the development of coronary arteritis and aortitis. We found that CD8+ T cells, but not CD4+ T cells, NK T cells, or Treg cells, were required for development of KD vasculitis. The LCWE-induced murine model of KD vasculitis mimics many histologic features of the disease in humans, such as the presence of CD8+ T cells and LMP in coronary artery lesions as well as epicardial coronary arteritis. Moreover, CD8+ T cells functionally contribute to the development of KD vasculitis in this murine model. Therapeutic strategies targeting infiltrating CD8+ T cells might be useful in the management of KD in humans. © 2016, American College of Rheumatology.

Dendritic and tumor cell fusions transduced with adenovirus encoding CD40L eradicate B-cell lymphoma and induce a Th17-type response.

PubMed

Alvarez, E; Moga, E; Barquinero, J; Sierra, J; Briones, J

2010-04-01

Fusion of dendritic cells and tumor cells (FCs) constitutes a promising tool for generating an antitumor response because of their capacity to present tumor antigens and provide appropriate costimulatory signals. CD40-CD40L interaction has an important role in the maturation and survival of dendritic cells and provides critical help for T-cell priming. In this study, we sought to improve the effectiveness of FC vaccines in a murine model of B-cell lymphoma by engineering FCs to express CD40L by means of an adenovirus encoding CD40L (Adv-CD40L). Before transduction with Adv-CD40L, no CD40L expression was detected in FCs, DCs or tumor cells. The surface expression of CD40L in FC transduced with Adv-CD40L (FC-CD40L) ranged between 50 and 60%. FC-CD40L showed an enhanced expression of CD80, CD86, CD54 and MHC class II molecules and elicited a strong in vitro immune response in a syngeneic mixed lymphocyte reaction. Furthermore, FC-CD40L showed enhanced migration to secondary lymphoid organs. Splenocytes from mice treated with FC-CD40L had a dramatic increase in the production of IL-17, IL-6 and IFN-gamma, compared with controls. Treatment with the FC-CD40L vaccine induced regression of established tumors and increased survival. Our data demonstrate that FC transduced with Adv-CD40L enhances the antitumor effect of FC vaccines in a murine lymphoma model and this is associated with an increased Th17-type immune response.

In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model.

PubMed

Tamai, Miho; Aoki, Mami; Nishimura, Akihito; Morishita, Koji; Tagawa, Yoh-ichi

2013-12-01

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL(mES), which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL(mES) culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. L-Ornithine is an intermediate of the urea cycle, but supplemental L-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL(mES), supplemental L-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL(mES) displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL(mES) will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.

The role of indoleamine 2,3-dioxygenase in LP-BPM5 murine retroviral disease progression.

PubMed

O'Connor, Megan A; Green, William R

2013-05-17

Indoleamine 2,3-dioxygenase (IDO) is an immunomodulatory intracellular enzyme involved in tryptophan degradation. IDO is induced during cancer and microbial infections by cytokines, ligation of co-stimulatory molecules and/or activation of pattern recognition receptors, ultimately leading to modulation of the immune response. LP-BM5 murine retroviral infection induces murine AIDS (MAIDS), which is characterized by profound and broad immunosuppression of T- and B-cell responses. Our lab has previously described multiple mechanisms regulating the development of immunodeficiency of LP-BM5-induced disease, including Programmed Death 1 (PD-1), IL-10, and T-regulatory (Treg) cells. Immunosuppressive roles of IDO have been demonstrated in other retroviral models, suggesting a possible role for IDO during LP-BM5-induced retroviral disease progression and/or development of viral load. Mice deficient in IDO (B6.IDO-/-) and wildtype C57BL/6 (B6) mice were infected with LP-BM5 murine retrovirus. MAIDS and LP-BM5 viral load were assessed at termination. As expected, IDO was un-inducible in B6.IDO-/- during LP-BM5 infection. B6.IDO-/- mice infected with LP-BM5 retrovirus succumbed to MAIDS as indicated by splenomegaly, serum hyper IgG2a and IgM, decreased responsiveness to B- and T-cell mitogens, conversion of a proportion of CD4+ T cells from Thy1.2+ to Thy1.2-, and increased percentages of CD11b+Gr-1+ cells. LP-BM5 infected B6.IDO-/- mice also demonstrated the development of roughly equivalent disease kinetics as compared to infected B6 mice. Splenic viral loads of B6 and B6.IDO-/- mice were also equivalent after infection as measured by LP-BM5-specific Def Gag and Eco Gag viral mRNA, determined by qRT-PCR. Collectively, these results demonstrate IDO neither plays an essential role, nor is required, in LP-BM5-induced disease progression or LP-BM5 viral load.

The role of indoleamine 2,3-dioxygenase in LP-BPM5 murine retroviral disease progression

PubMed Central

2013-01-01

Background Indoleamine 2,3-dioxygenase (IDO) is an immunomodulatory intracellular enzyme involved in tryptophan degradation. IDO is induced during cancer and microbial infections by cytokines, ligation of co-stimulatory molecules and/or activation of pattern recognition receptors, ultimately leading to modulation of the immune response. LP-BM5 murine retroviral infection induces murine AIDS (MAIDS), which is characterized by profound and broad immunosuppression of T- and B-cell responses. Our lab has previously described multiple mechanisms regulating the development of immunodeficiency of LP-BM5-induced disease, including Programmed Death 1 (PD-1), IL-10, and T-regulatory (Treg) cells. Immunosuppressive roles of IDO have been demonstrated in other retroviral models, suggesting a possible role for IDO during LP-BM5-induced retroviral disease progression and/or development of viral load. Methods Mice deficient in IDO (B6.IDO−/−) and wildtype C57BL/6 (B6) mice were infected with LP-BM5 murine retrovirus. MAIDS and LP-BM5 viral load were assessed at termination. Results As expected, IDO was un-inducible in B6.IDO−/− during LP-BM5 infection. B6.IDO−/− mice infected with LP-BM5 retrovirus succumbed to MAIDS as indicated by splenomegaly, serum hyper IgG2a and IgM, decreased responsiveness to B- and T-cell mitogens, conversion of a proportion of CD4+ T cells from Thy1.2+ to Thy1.2-, and increased percentages of CD11b+Gr-1+ cells. LP-BM5 infected B6.IDO−/− mice also demonstrated the development of roughly equivalent disease kinetics as compared to infected B6 mice. Splenic viral loads of B6 and B6.IDO−/− mice were also equivalent after infection as measured by LP-BM5-specific Def Gag and Eco Gag viral mRNA, determined by qRT-PCR. Conclusions Collectively, these results demonstrate IDO neither plays an essential role, nor is required, in LP-BM5-induced disease progression or LP-BM5 viral load. PMID:23680027

46 CFR 34.17-1 - Application-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Application-T/ALL. 34.17-1 Section 34.17-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-1 Application—T/ALL. (a) Where a fixed foam extinguishing system is...

46 CFR 34.17-1 - Application-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Application-T/ALL. 34.17-1 Section 34.17-1 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-1 Application—T/ALL. (a) Where a fixed foam extinguishing system is...

A Rapid Embryonic Stem Cell-Based Mouse Model for B-cell Lymphomas Driven by Epstein-Barr Virus Protein LMP1.

PubMed

Ba, Zhaoqing; Meng, Fei-Long; Gostissa, Monica; Huang, Pei-Yi; Ke, Qiang; Wang, Zhe; Dao, Mai N; Fujiwara, Yuko; Rajewsky, Klaus; Zhang, Baochun; Alt, Frederick W

2015-06-01

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) contributes to oncogenic human B-cell transformation. Mouse B cells conditionally expressing LMP1 are not predisposed to B-cell malignancies, as LMP1-expressing B cells are eliminated by T cells. However, mice with conditional B-cell LMP1 expression and genetic elimination of α/β and γ/δ T cells ("CLT" mice) die early in association with B-cell lymphoproliferation and lymphomagenesis. Generation of CLT mice involves in-breeding multiple independently segregating alleles. Thus, although introduction of additional activating or knockout mutations into the CLT model is desirable for further B-cell expansion and immunosurveillance studies, doing such experiments by germline breeding is time-consuming, expensive, and sometimes unfeasible. To generate a more tractable model, we generated clonal CLT embryonic stem (ES) cells from CLT embryos and injected them into RAG2-deficient blastocysts to generate chimeric mice, which, like germline CLT mice, harbor splenic CLT B cells and lack T cells. CLT chimeric mice generated by this RAG2-deficient blastocyst complementation ("RDBC") approach die rapidly in association with B-cell lymphoproliferation and lymphoma. Because CLT lymphomas routinely express the activation-induced cytidine deaminase (AID) antibody diversifier, we tested potential AID roles by eliminating the AID gene in CLT ES cells and testing them via RDBC. We found that CLT and AID-deficient CLT ES chimeras had indistinguishable phenotypes, showing that AID is not essential for LMP1-induced lymphomagenesis. Beyond expanding accessibility and utility of CLT mice as a cancer immunotherapy model, our studies provide a new approach for facilitating generation of genetically complex mouse cancer models. ©2015 American Association for Cancer Research.

46 CFR 34.17-15 - Piping-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Piping-T/ALL. 34.17-15 Section 34.17-15 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-15 Piping—T/ALL. (a) All piping, valves, and fittings shall meet the applicable...

Eicosapentaenoic and docosahexaenoic acid ethyl esters differentially enhance B-cell activity in murine obesity[S

PubMed Central

Teague, Heather; Harris, Mitchel; Fenton, Jenifer; Lallemand, Perrine; Shewchuk, Brian M.; Shaikh, Saame Raza

2014-01-01

EPA and DHA are not biologically equivalent; however, their individual activity on B cells is unknown. We previously reported fish oil enhanced murine B-cell activity in obesity. To distinguish between the effects of EPA and DHA, we studied the ethyl esters of EPA and DHA on murine B-cell function as a function of time. We first demonstrate that EPA and DHA maintained the obese phenotype, with no improvements in fat mass, adipose inflammatory cytokines, fasting insulin, or glucose clearance. We then tested the hypothesis that EPA and DHA would increase the frequency of splenic B cells. EPA and DHA differentially enhanced the frequency and/or percentage of select B-cell subsets, correlating with increased natural serum IgM and cecal IgA. We next determined the activities of EPA and DHA on ex vivo production of cytokines upon lipopolysaccharide stimulation of B cells. EPA and DHA, in a time-dependent manner, enhanced B-cell cytokines with DHA notably increasing IL-10. At the molecular level, EPA and DHA differentially enhanced the formation of ordered microdomains but had no effect on Toll-like receptor 4 mobility. Overall, the results establish differential effects of EPA and DHA in a time-dependent manner on B-cell activity in obesity, which has implications for future clinical studies. PMID:24837990

Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis

PubMed Central

Whibley, Natasha; Tritto, Elaine; Traggiai, Elisabetta; Kolbinger, Frank; Moulin, Pierre; Brees, Dominique; Coleman, Bianca M.; Mamo, Anna J.; Garg, Abhishek V.; Jaycox, Jillian R.; Siebenlist, Ulrich; Kammüller, Michael; Gaffen, Sarah L.

2016-01-01

Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency. PMID:26729813

Influence of sire and sire breed (Gyr versus Holstein) on establishment of pregnancy and embryonic loss in lactating Holstein cows during summer heat stress.

PubMed

Pegorer, Marcelo F; Vasconcelos, José L M; Trinca, Luzia A; Hansen, Peter J; Barros, Ciro M

2007-03-01

Heat stress has negative effects on pregnancy rates of lactating dairy cattle. There are genetic differences in tolerance to heat stress; Bos taurus indicus (B. t. indicus) cattle and embryos are more thermotolerant than Bos taurus taurus (B. t. taurus). In the present study, the effects of sire and sire breed on conception and embryonic/fetal loss rates of lactating Holstein cows during the Brazilian summer were determined. In Experiment 1, cows (n=302) were AI after estrus detection or at a fixed-time with semen from one Gyr (B. t. indicus) or one Holstein sire (B. t. taurus). Pregnancy was diagnosed 80 days after AI. In Experiment 2, cows (n=811) were AI with semen from three Gyr and two Holstein sires. Pregnancy was diagnosed at 30-40 and at 60-80 days after AI. Cows diagnosed pregnant at the first examination but non-pregnant at the second were considered as having lost their embryo or fetus. Data were analyzed by logistic regression. The model considered the effect of sire within breed, sire breed, days postpartum, period of lactation, and AI type (AI after estrus versus fixed-time). There was no effect of the AI type, days postpartum or milk production on conception or embryonic loss rates. The use of Gyr bulls increased pregnancy rate when compared to Holstein bulls [9.1% (60/657) versus 5.0% (23/456), respectively, P=0.008; data from Experiments 1 and 2 combined]. Additionally, in Experiment 2, cows inseminated using semen from sire #4 (Gyr) had lower embryonic loss (10%) when compared with other B. t. indicus (35.3% and 40%) or B. t. taurus sires (18.2% and 38.5%, P=0.03). In conclusion, the use of B. t. indicus sires may result in higher conception rates in lactating Holstein cows during summer heat stress. Moreover, sire can affect embryonic loss and selection of bulls according to this criterion may result in higher parturition rates in lactating Holstein cows.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients.

PubMed

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients.

Engagement of Cytotoxic T Lymphocyte–associated Antigen 4 (CTLA-4) Induces Transforming Growth Factor β (TGF-β) Production by Murine CD4+ T Cells

PubMed Central

Chen, Wanjun; Jin, Wenwen; Wahl, Sharon M.

1998-01-01

Evidence indicates that cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) may negatively regulate T cell activation, but the basis for the inhibitory effect remains unknown. We report here that cross-linking of CTLA-4 induces transforming growth factor β (TGF-β) production by murine CD4+ T cells. CD4+ T helper type 1 (Th1), Th2, and Th0 clones all secrete TGF-β after antibody cross-linking of CTLA-4, indicating that induction of TGF-β by CTLA-4 signaling represents a ubiquitous feature of murine CD4+ T cells. Stimulation of the CD3–T cell antigen receptor complex does not independently induce TGF-β, but is required for optimal CTLA-4–mediated TGF-β production. The consequences of cross-linking of CTLA-4, together with CD3 and CD28, include inhibition of T cell proliferation and interleukin (IL)-2 secretion, as well as suppression of both interferon γ (Th1) and IL-4 (Th2). Moreover, addition of anti–TGF-β partially reverses this T cell suppression. When CTLA-4 was cross-linked in T cell populations from TGF-β1 gene–deleted (TGF-β1−/−) mice, the T cell responses were only suppressed 38% compared with 95% in wild-type mice. Our data demonstrate that engagement of CTLA-4 leads to CD4+ T cell production of TGF-β, which, in part, contributes to the downregulation of T cell activation. CTLA-4, through TGF-β, may serve as a counterbalance for CD28 costimulation of IL-2 and CD4+ T cell activation. PMID:9815262

Negative regulation of retrovirus expression in embryonal carcinoma cells mediated by an intragenic domain.

PubMed

Loh, T P; Sievert, L L; Scott, R W

1988-11-01

An intragenic region spanning the tRNA primer binding site of a Moloney murine leukemia virus recombinant retrovirus was found to restrict expression specifically in embryonal carcinoma (EC) cells. When the inhibitory domain was present, the levels of steady-state RNA synthesized from integrated recombinant templates in stable cotransformation assays were reduced 20-fold in EC cells but not in C2 myoblast cells. Transient-cotransfection assays showed that repression of a template containing the EC-specific inhibitory component was relieved by an excess of specific competitor DNA. In addition, repression mediated by the inhibitory component was orientation independent. This evidence demonstrates the presence of a saturable, trans-acting negative regulatory factor(s) in EC cells and suggests that the interaction of the factor(s) with the intragenic inhibitory component occurs at the DNA level.

IL-17+ γδ T cells as kick-starters of inflammation.

PubMed

Papotto, Pedro H; Ribot, Julie C; Silva-Santos, Bruno

2017-05-18

Shortly after the discovery of interleukin 17 (IL-17)-producing CD4 + helper T cells (T H 17 cells), it was found that γδ T cells can also secrete large amounts of this pro-inflammatory cytokine. A decade later, it is now known that IL-17 + γδ T cells (γδ17 T cells) are often the main providers of IL-17A in various models of inflammatory diseases, while they also contribute to protective immune responses to infectious organisms. Due to an intricate thymic program of differentiation, γδ17 T cells are able to respond faster than T H 17 cells do and thus predominate in the early stages of inflammatory responses. Here we review the current knowledge of the development, activation and pathophysiological functions of γδ17 T cells, aiming to increase the awareness in the community of the therapeutic potential of this 'other side' of IL-17-mediated immune responses.

Nanobiotechnological Nanocapsules Containing Polyhemoglobin-Tyrosinase: Effects on Murine B16F10 Melanoma Cell Proliferation and Attachment

PubMed Central

Wang, Yun; Chang, Thomas M. S.

2012-01-01

We have reported previously that daily intravenous infusions of a soluble nanobiotechnological complex, polyhemoglobin-tyrosinase [polyHb-Tyr], can suppress the growth of murine B16F10 melanoma in a mouse model. In order to avoid the need for daily intravenous injections, we have now extended this further as follows. We have prepared two types of biodegradable nanocapsules containing [polyHb-Tyr]. One type is to increase the circulation time and decrease the frequency of injection and is based on polyethyleneglycol-polylactic acid (PEG-PLA) nanocapsules containing [polyHb-Tyr]. The other type is to allow for intratumoural or local injection and is based on polylactic acid (PLA) nanocapsules containing [polyHb-Tyr]. Cell culture studies show that it can inhibit the proliferation of murine B16F10 melanoma cells in the “proliferation model”. It can also inhibit the attachment of murine B16F10 melanoma cells in the “attachment model.” This could be due to the action of tyrosinase on the depletion of tyrosine or the toxic effect of tyrosine metabolites. The other component, polyhemoglobin (polyHb), plays a smaller role in nanocapsules containing [polyHb-Tyr], and this is most likely by its depletion of nitric oxide needed for melanoma cell growth. PMID:23209910

The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells.

PubMed

Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

2017-03-01

Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immunomodulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17‑34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17‑34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17‑34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17‑34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17‑34-mediated pigmentation. Taken together, these results suggest that LfB17‑34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17‑34 could be further developed for the treatment of hypopigmentation disorders.

The lactoferricin B-derived peptide, LfB17-34, induces melanogenesis in B16F10 cells

PubMed Central

Huang, Hsiu-Chin; Lin, Hsuan; Huang, Min-Chuan

2017-01-01

Lactoferricin B (LfcinB), a peptide of bovine lactoferrin (LfB), exhibits multiple biological functions, including antimicrobial, antiviral, antioxidant and immuno-modulatory activities. However, the role of LfcinB-related peptides in melanogenesis remains unclear. In this study, a set of five LfcinB-related peptides was examined. We found that LfB17-34, an 18-mer LfcinB-derived peptide, increased melanogenesis in B16F10 melanoma cells without significantly affecting cell viability. LfB17-34 increased in vitro tyrosinase activity and melanin content in a dose-dependent manner. The results of RT-qPCR and western blot analyses showed that LfB17-34 increased the mRNA and protein expression of tyrosinase and tyrosinase-related protein 1 (Trp1). Moreover, LfB17-34 inhibited the phosphorylation of MAPK/Erk, but not p38 and Akt, and constitutively active MEK was able to reverse the LfB17-34-enhanced pigmentation, melanin content, and tyrosinase activity, suggesting a role of Erk signaling in the process of LfB17-34-mediated pigmentation. Taken together, these results suggest that LfB17-34 induces melanogenesis in B16F10 cells primarily through increased tyrosinase expression and activity and that LfB17-34 could be further developed for the treatment of hypopigmentation disorders. PMID:28204812

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

PubMed

Xiao, Sheng; Yosef, Nir; Yang, Jianfei; Wang, Yonghui; Zhou, Ling; Zhu, Chen; Wu, Chuan; Baloglu, Erkan; Schmidt, Darby; Ramesh, Radha; Lobera, Mercedes; Sundrud, Mark S; Tsai, Pei-Yun; Xiang, Zhijun; Wang, Jinsong; Xu, Yan; Lin, Xichen; Kretschmer, Karsten; Rahl, Peter B; Young, Richard A; Zhong, Zhong; Hafler, David A; Regev, Aviv; Ghosh, Shomir; Marson, Alexander; Kuchroo, Vijay K

2014-04-17

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity. Copyright © 2014 Elsevier Inc. All rights reserved.

Human CD22 Inhibits Murine B Cell Receptor Activation in a Human CD22 Transgenic Mouse Model.

PubMed

Bednar, Kyle J; Shanina, Elena; Ballet, Romain; Connors, Edward P; Duan, Shiteng; Juan, Joana; Arlian, Britni M; Kulis, Michael D; Butcher, Eugene C; Fung-Leung, Wai-Ping; Rao, Tadimeti S; Paulson, James C; Macauley, Matthew S

2017-11-01

CD22, a sialic acid-binding Ig-type lectin (Siglec) family member, is an inhibitory coreceptor of the BCR with established roles in health and disease. The restricted expression pattern of CD22 on B cells and most B cell lymphomas has made CD22 a therapeutic target for B cell-mediated diseases. Models to better understand how in vivo targeting of CD22 translates to human disease are needed. In this article, we report the development of a transgenic mouse expressing human CD22 (hCD22) in B cells and assess its ability to functionally substitute for murine CD22 (mCD22) for regulation of BCR signaling, Ab responses, homing, and tolerance. Expression of hCD22 on transgenic murine B cells is comparable to expression on human primary B cells, and it colocalizes with mCD22 on the cell surface. Murine B cells expressing only hCD22 have identical calcium (Ca 2+ ) flux responses to anti-IgM as mCD22-expressing wild-type B cells. Furthermore, hCD22 transgenic mice on an mCD22 -/- background have restored levels of marginal zone B cells and Ab responses compared with deficiencies observed in CD22 -/- mice. Consistent with these observations, hCD22 transgenic mice develop normal humoral responses in a peanut allergy oral sensitization model. Homing of B cells to Peyer's patches was partially rescued by expression of hCD22 compared with CD22 -/- B cells, although not to wild-type levels. Notably, Siglec-engaging antigenic liposomes formulated with an hCD22 ligand were shown to prevent B cell activation, increase cell death, and induce tolerance in vivo. This hCD22 transgenic mouse will be a valuable model for investigating the function of hCD22 and preclinical studies targeting hCD22. Copyright © 2017 by The American Association of Immunologists, Inc.

HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR expression in infertile women with endometriosis.

PubMed

Osiński, Maciej; Wirstlein, Przemysław; Wender-Ożegowska, Ewa; Mikołajczyk, Mateusz; Jagodziński, Paweł Piotr; Szczepańska, Małgorzata

2018-01-01

The development of endometriosis is associated with changes in the expression of genes encoding the 3β-hydroxysteroid dehydrogenase type II (HSD3B2) and 17β-hydroxysteroid dehydrogenase type II (HSD17B2), estrogen receptors 1 (ESR1) and 2 (ESR2) and the androgen receptor (AR). However, little is known about the expression of HSD3B2, HSD17B1, HSD17B2, ESR1 ESR2 and AR during the endometrial phases in eutopic endometrium from infertile women with endometriosis. Using RT-qPCR analysis, we assessed the expression of the studied genes in the follicular and luteal phases in eutopic endometrium from fertile women (n = 17) and infertile women (n = 35) with endometriosis. In the mid-follicular eutopic endometrium, we observed a significant increase in HSD3B2 transcript levels in all infertile women with endometriosis (p = 0.003), in infertile women with stage I/II endometriosis (p = 0.008) and in infertile women with stage III/IV endometriosis (p = 0.009) compared to all fertile women. There was a significant increase in ESR1 tran-scripts in all infertile women with endometriosis (p = 0.008) and in infertile women with stage I/II endometriosis (p = 0.019) and in infertile women with stage III/IV endometriosis (p = 0.023) compared to all fertile women. In the mid-luteal eutopic endometrium, we did not observe significant differences in HSD3B2, HSD17B1, HSD17B2, ESR1, ESR2 and AR transcripts between infertile women with endometriosis and fertile women. Observed significant increase in HSD3B2 and ESR1 transcripts in follicular eutopic endometrium from infer-tile women with endometriosis may be related to abnormal biological effect of E2 in endometrium, further affecting the development of human embryos.

PKCλ/ι regulates Th17 differentiation and house dust mite-induced allergic airway inflammation.

PubMed

Yang, Yingying; Dong, Panpan; Zhao, Jing; Zhou, Wei; Zhou, Yonghua; Xu, Yongliang; Mei, Congjin; Guo, Fukun; Zheng, Yi; Yang, Jun-Qi

2018-03-01

Asthma is a chronic airway inflammation in which Th2 and Th17 cells play critical roles in its pathogenesis. We have reported that atypical protein kinase (PKC) λ/ι is a new regulator for Th2 differentiation and function. However, the role of PKCλ/ι for Th17 cells remains elusive. In this study, we explored the effect of PKCλ/ι on Th17 cells in the context of ex vivo cell culture systems and an in vivo murine model of allergic airway inflammation with the use of activated T cell-specific conditional PKCλ/ι-deficient mice. Our findings indicate that PKCλ/ι regulates Th17 cells. The secretion of Th17 effector cytokines, including IL-17, IL-21 and IL-22, were inhibited from PKCλ/ι-deficient T cells under non-skewing or Th17-skewing culture conditions. Moreover, the impaired Th17 differentiation and function by the PKCλ/ι-deficiency was associated with the downregulation of Stat3 and Rorγt, key Th17 transcription factors. We developed a model of Th17 and neutrophil-involved allergic airway inflammation by intratracheal inoculation of house dust mites. PKCλ/ι-deficiency significantly inhibited airway inflammations. The infiltrating cells in the lungs and bronchoalveolar lavage fluids were significantly reduced in conditional PKCλ/ι-deficient mice. Th17 effector cytokines were reduced in the bronchoalveolar lavage fluids and lungs at protein and mRNA levels. Thus, PKCλ/ι emerges as a critical regulator of Th17 differentiation and allergic airway hyperresponsiveness. Copyright © 2018 Elsevier B.V. All rights reserved.

Nitric oxide protects murine embryonic liver cells (BNL CL.2) from cytotoxicity induced by glucose deprivation.

PubMed

Pae, H O; Kim, H G; Paik, Y S; Paik, S G; Kim, Y M; Oh, G S; Chung, H T

2000-03-01

We investigated the protective effects of nitric oxide on cell death of murine embryonic liver cells (BNL CL.2) after glucose deprivation. Endogenous nitric oxide production by BNL CL.2 cells was induced by 6 hr pretreatment with interferon-gamma and lipopolysaccharide. We used sodium nitroprusside and S-nitroso-L-glutathione as exogenous nitric oxide-generating compounds. All agents were used at doses that did not show direct cytotoxicity as measured by crystal violet staining assay. In the BNL CL.2 cells, the viability dropped very steeply after 24 hr incubation with glucose-free media. Endogenous nitric oxide produced by treatment of the cells with interferon-gamma and lipopolysaccharide protected the cells from glucose deprivation-induced cytotoxicity, but did not protect them in the presence of the nitric oxide synthesis inhibitor, N(G)-monomethyl-L-arginine. Exogenous nitric oxide protected the cells from glucose deprivation-induced cytotoxicity in a concentration-dependent manner. Cytoprotection by nitric oxide donors was abolished by the use of nitric oxide scavenger, 2-phenyl-4,4,5,5,-tetramethylimidazole, but not by the soluble guanosine cyclase inhibitor, 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. In addition, cytoprotective effects comparable to endogenous or exogenous nitric oxide were not observed when the cells were incubated with dibutyl guanosine 3',5'-cyclic monophosphate. Based upon these results, we suggest that nitric oxide may enhance the cell survival of BNL CL.2 cells after glucose deprivation via a guanosine 3',5'-cyclic monophosphate-independent pathway.

Generation of a novel artificial TrkB agonist, BM17d99, using T7 phage-displayed random peptide libraries.

PubMed

Ohnishi, Toshiyuki; Sakamoto, Kotaro; Asami-Odaka, Asano; Nakamura, Kimie; Shimizu, Ayako; Ito, Takashi; Asami, Taiji; Ohtaki, Tetsuya; Inooka, Hiroshi

2017-01-29

Tropomyosin receptor kinase B (TrkB) is a known receptor of brain-derived neurotrophic factor (BDNF). Because it plays a critical role in the regulation of neuronal development, maturation, survival, etc., TrkB is a good target for drugs against central nervous system diseases. In this study, we aimed to generate peptidic TrkB agonists by applying random peptide phage display technology. After the phage panning against recombinant Fc-fused TrkB (TrkB-Fc), agonistic phages were directly screened against TrkB-expressing HEK293 cells. Through subsequent screening of the first-hit BM17 peptide-derived focus library, we successfully obtained the BM17d99 peptide, which had no sequence similarity with BDNF but had TrkB-binding capacity. We then synthesized a dimeric BM17d99 analog peptide that could phosphorylate or activate TrkB by facilitating receptor homodimerization. Treatment of TrkB-expressing HEK293 cells with the dimeric BM17d99 analog peptide significantly induced the phosphorylation of TrkB, suggesting that homodimerization of TrkB was enhanced by the dimeric peptide. This report demonstrates that our approach is useful for the generation of artificial peptidic agonists of cell surface receptors. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of IL-17B and IL-17F mRNA expression in peripheral blood mononuclear cells and association with clinical outcome of IBD patients

PubMed Central

Safari, Mohammad Taghi; Chaleshi, Vahid; Tarban, Peyman; Nourian, Mahyar; Balaii, Hedieh; Shahrokh, Shabnam; Asadzadeh Aghdaei, Hamid

2017-01-01

Aim: In this study, we determined the gene expression analysis of IL-17 gene family for early detection of subclinical inflammation among IBD patients. Background: Cytokines have a vital role in the pathogenesis of inflammatory bowel disease (IBD). Interleukin-17 is the signature cytokine of the recently identified T helper 17 (Th17) cell subset. IL-17F is mainly involved in mucosal host defense mechanisms whereas the functions of IL-17B remain largely elusive. Methods: In this cross-sectional study, IBD patients divided into two active and inactive groups. Peripheral blood mononuclear cells (PBMCs) from 38 IBD patients which 20 inactive samples and 18 active individuals were collected. Changes of IL-17 F and IL-17B mRNA expression level evaluated by quantitative-real time-PCR. Results: mRNA expression level of IL-17B and IL-17F in CD, UC, active and inactive groups have been assessed and there were no significant differences (P>0.05). Patients were classified into five different categories as follows: i) 5ASA; ii) 5ASA + Pred; iii) 5ASA + AZA; iv) 5ASA + Pred + AZA; v) 5ASA + Pred + AZA + IFX according to medication usage, expression of IL-17F and IL-17B had no differences (p>0.05). Conclusion: Evaluation of IL-17B and IL-17F mRNA expression level illustrate no difference among active and inactive patients. Therefore, IL-17B and IL-17F are not biomarkers in an Iranian IBD patients. PMID:29511476

Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

PubMed Central

Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

2008-01-01

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes

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

Cui, Ju, E-mail: juzi.cui@gmail.com; Pang, Jing; Lin, Ya-Jun

2016-08-05

Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with themore » induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. - Highlights: • The KIF5B level was up regulated during 3T3-L1 adipogenesis. • Endogenous KIF5B and adiponectin were partially colicalized. • Adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. • The secretion of adiponectin, but not leptin, is dependent on functional KIF5B.« less

Negative regulation of early polyomavirus expression in mouse embryonal carcinoma cells.

PubMed Central

Cremisi, C; Babinet, C

1986-01-01

Embryonal carcinoma cells are resistant to infection by polyomavirus (Py). We showed that this block was partially removed by inhibiting protein synthesis temporarily. The block was also partially removed when Py was coinfected with simian virus 40. Cycloheximide treatment of cells infected with Py mutants able to grow on PCC4 embryonal carcinoma cells led to 3- to 10-fold increases in the production of T-antigen-positive cells. At 31 degrees C, Py T-antigen expression was enhanced when the cells were treated with cycloheximide. We suggest that a negative labile regulatory protein(s) is synthesized in PCC4 cells, preventing the initiation of early Py transcription by binding to the noncoding sequence, especially the enhancer element B and perhaps also element A, and that the Py mutants retained a binding site(s). PMID:3016339

Restoring homeostasis of CD4+ T cells in hepatitis-B-virus-related liver fibrosis

PubMed Central

Cheng, Li-Sha; Liu, Yun; Jiang, Wei

2015-01-01

Immune-mediated liver injury is widely seen during hepatitis B virus (HBV) infection. Unsuccessful immune clearance of HBV results in chronic hepatitis and increases the risk of liver cirrhosis and hepatocellular carcinoma. HBV-related liver fibrosis (HBVLF), occurring as a result of HBV-induced chronic hepatitis, is a reversible, intermediate stage of chronic hepatitis B (CHB) and liver cirrhosis. Therefore, defining the pathogenesis of HBVLF is of practical significance for achieving better clinical outcomes. Recently, the homeostasis of CD4+ T cells was considered to be pivotal in the process of HBVLF. To better uncover the underlying mechanisms, in this review, we systematically retrospect the impacts of different CD4+ T-cell subsets on CHB and HBVLF. We emphasize CD4+ T-cell homeostasis and the important balance between regulatory T (Treg) and T helper 17 (Th17) cells. We discuss some cytokines associated with Treg and Th17 cells such as interleukin (IL)-17, IL-22, IL-21, IL-23, IL-10, IL-35 and IL-33, as well as surface molecules such as programmed cell death protein 1, cytotoxic T lymphocyte-associated antigen 4, T cell immunoglobulin domain and mucin domain-containing molecule 3 and cannabinoid receptor 2 that have potential therapeutic implications for the homeostasis of CD4+ T cells in CHB and HBVLF. PMID:26478664

Blockade of Syk ameliorates the development of murine sclerodermatous chronic graft-versus-host disease.

PubMed

Le Huu, Doanh; Kimura, Hiroshi; Date, Mutsumi; Hamaguchi, Yasuhito; Hasegawa, Minoru; Hau, Khang Tran; Fujimoto, Manabu; Takehara, Kazuhiko; Matsushita, Takashi

2014-06-01

Murine sclerodermatous chronic graft-versus-host disease (Scl-cGVHD) is a model for human Scl-cGVHD and systemic sclerosis (SSc). Syk is expressed in most of hematopoietic cells, fibroblasts, and endothelial cells. Syk is a protein tyrosine kinase that has an important role in transmitting signals from a variety of cell surface receptors. This study aims to investigate the effect of R788 (fostamatinib sodium), an oral prodrug that is rapidly converted to a potent inhibitor of Syk, R406, on Scl-cGVHD. R788 was orally administered twice a day to allogeneic recipients from day 14 to day 42 after bone marrow transplantation (BMT). In vitro, proliferation of GVHD-derived CD4(+) T cells and CD11b(+) cells was analyzed by R406. Allogeneic BMT increased Syk phosphorylation in T, B, and CD11b(+) cells. The administration of R788 attenuated severity and fibrosis of Scl-cGVHD. The elevated expressions of CXCR4 on T cells, B cells, and CD11b(+) cells were significantly down-regulated by R788 treatment. R788 reduced memory CD4(+) T cells (CD44(hi)CD62L(-)CD4(+)). R406 inhibited proliferation of GVHD CD4(+) T cells and CD11b(+) cells in vitro. In addition, R788 treatment, inhibited proliferation of CD11b(+) cells in Scl-cGVHD mice. R788 treatment also reduced skin mRNA expressions of MCP-1, MIP-1α, IFN-γ, IL-13, IL-17A, and TGF-β1, but not influenced RANTES, CXCL12, and TFN-α. Blockade of Syk suppressed migration factor of immune cells and antigen-specific memory CD4(+) T cells and proliferation and activation of GVHD CD4(+) T cells and CD11b(+) cells. The current studies suggested that Syk inhibitor is a potential candidate for use in treating patients with Scl-cGVHD and SSc. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Interleukin-17B Antagonizes Interleukin-25-Mediated Mucosal Inflammation.

PubMed

Reynolds, Joseph M; Lee, Young-Hee; Shi, Yun; Wang, Xiaohu; Angkasekwinai, Pornpimon; Nallaparaju, Kalyan C; Flaherty, Stephanie; Chang, Seon Hee; Watarai, Hiroshi; Dong, Chen

2015-04-21

The interleukin-17 (IL-17) family of cytokines has emerged as a critical player in inflammatory diseases. Among them, IL-25 has been shown to be important in allergic inflammation and protection against parasitic infection. Here we have demonstrated that IL-17B, a poorly understood cytokine, functions to inhibit IL-25-driven inflammation. IL-17B and IL-25, both binding to the interleukin-17 receptor B (IL-17RB), were upregulated in their expression after acute colonic inflammation. Individual inhibition of these cytokines revealed opposing functions in colon inflammation: IL-25 was pathogenic but IL-17B was protective. Similarly opposing phenotypes were observed in Citrobacter rodentium infection and allergic asthma. Moreover, IL-25 was found to promote IL-6 production from colon epithelial cells, which was inhibited by IL-17B. Therefore, our data demonstrate that IL-17B is an anti-inflammatory cytokine in the IL-17 family. Copyright © 2015 Elsevier Inc. All rights reserved.

Heparanase confers a growth advantage to differentiating murine embryonic stem cells, and enhances oligodendrocyte formation.

PubMed

Xiong, Anqi; Kundu, Soumi; Forsberg, Maud; Xiong, Yuyuan; Bergström, Tobias; Paavilainen, Tanja; Kjellén, Lena; Li, Jin-Ping; Forsberg-Nilsson, Karin

2017-10-01

Heparan sulfate proteoglycans (HSPGs), ubiquitous components of mammalian cells, play important roles in development and homeostasis. These molecules are located primarily on the cell surface and in the pericellular matrix, where they interact with a multitude of macromolecules, including many growth factors. Manipulation of the enzymes involved in biosynthesis and modification of HSPG structures alters the properties of stem cells. Here, we focus on the involvement of heparanase (HPSE), the sole endo-glucuronidase capable of cleaving of HS, in differentiation of embryonic stem cells into the cells of the neural lineage. Embryonic stem (ES) cells overexpressing HPSE (Hpse-Tg) proliferated more rapidly than WT ES cells in culture and formed larger teratomas in vivo. In addition, differentiating Hpse-Tg ES cells also had a higher growth rate, and overexpression of HPSE in NSPCs enhanced Erk and Akt phosphorylation. Employing a two-step, monolayer differentiation, we observed an increase in HPSE as wild-type (WT) ES cells differentiated into neural stem and progenitor cells followed by down-regulation of HPSE as these NSPCs differentiated into mature cells of the neural lineage. Furthermore, NSPCs overexpressing HPSE gave rise to more oligodendrocytes than WT cultures, with a concomitant reduction in the number of neurons. Our present findings emphasize the importance of HS, in neural differentiation and suggest that by regulating the availability of growth factors and, or other macromolecules, HPSE promotes differentiation into oligodendrocytes. Copyright © 2016 Elsevier B.V. All rights reserved.

Antibody blockade of IL-17 family cytokines in immunity to acute murine oral mucosal candidiasis.

PubMed

Whibley, Natasha; Tritto, Elaine; Traggiai, Elisabetta; Kolbinger, Frank; Moulin, Pierre; Brees, Dominique; Coleman, Bianca M; Mamo, Anna J; Garg, Abhishek V; Jaycox, Jillian R; Siebenlist, Ulrich; Kammüller, Michael; Gaffen, Sarah L

2016-06-01

Antibodies targeting IL-17A or its receptor, IL-17RA, are approved to treat psoriasis and are being evaluated for other autoimmune conditions. Conversely, IL-17 signaling is critical for immunity to opportunistic mucosal infections caused by the commensal fungus Candida albicans, as mice and humans lacking the IL-17R experience chronic mucosal candidiasis. IL-17A, IL-17F, and IL-17AF bind the IL-17RA-IL-17RC heterodimeric complex and deliver qualitatively similar signals through the adaptor Act1. Here, we used a mouse model of acute oropharyngeal candidiasis to assess the impact of blocking IL-17 family cytokines compared with specific IL-17 cytokine gene knockout mice. Anti-IL-17A antibodies, which neutralize IL-17A and IL-17AF, caused elevated oral fungal loads, whereas anti-IL-17AF and anti-IL-17F antibodies did not. Notably, there was a cooperative effect of blocking IL-17A, IL-17AF, and IL-17F together. Termination of anti-IL-17A treatment was associated with rapid C. albicans clearance. IL-17F-deficient mice were fully resistant to oropharyngeal candidiasis, consistent with antibody blockade. However, IL-17A-deficient mice had lower fungal burdens than anti-IL-17A-treated mice. Act1-deficient mice were much more susceptible to oropharyngeal candidiasis than anti-IL-17A antibody-treated mice, yet anti-IL-17A and anti-IL-17RA treatment caused equivalent susceptibilities. Based on microarray analyses of the oral mucosa during infection, only a limited number of genes were associated with oropharyngeal candidiasis susceptibility. In sum, we conclude that IL-17A is the main cytokine mediator of immunity in murine oropharyngeal candidiasis, but a cooperative relationship among IL-17A, IL-17AF, and IL-17F exists in vivo. Susceptibility displays the following hierarchy: IL-17RA- or Act1-deficiency > anti-IL-17A + anti-IL-17F antibodies > anti-IL-17A or anti-IL-17RA antibodies > IL-17A deficiency. © Society for Leukocyte Biology.

Infection and cellular defense dynamics in a novel 17β-estradiol murine model of chronic human group B streptococcus genital tract colonization reveal a role for hemolysin in persistence and neutrophil accumulation.

PubMed

Carey, Alison J; Tan, Chee Keong; Mirza, Shaper; Irving-Rodgers, Helen; Webb, Richard I; Lam, Alfred; Ulett, Glen C

2014-02-15

Genital tract carriage of group B streptococcus (GBS) is prevalent among adult women; however, the dynamics of chronic GBS genital tract carriage, including how GBS persists in this immunologically active host niche long term, are not well defined. To our knowledge, in this study, we report the first animal model of chronic GBS genital tract colonization using female mice synchronized into estrus by delivery of 17β-estradiol prior to intravaginal challenge with wild-type GBS 874391. Cervicovaginal swabs, which were used to measure bacterial persistence, showed that GBS colonized the vaginal mucosa of mice at high numbers (10(6)-10(7) CFU/swab) for at least 90 d. Cellular and histological analyses showed that chronic GBS colonization of the murine genital tract caused significant lymphocyte and PMN cell infiltrates, which were localized to the vaginal mucosal surface. Long-term colonization was independent of regular hormone cycling. Immunological analyses of 23 soluble proteins related to chemotaxis and inflammation showed that the host response to GBS in the genital tract comprised markers of innate immune activation including cytokines such as GM-CSF and TNF-α. A nonhemolytic isogenic mutant of GBS 874391, Δcyle9, was impaired for colonization and was associated with amplified local PMN responses. Induction of DNA neutrophil extracellular traps, which was observed in GBS-infected human PMNs in vitro in a hemolysin-dependent manner, appeared to be part of this response. Overall, this study defines key infection dynamics in a novel murine model of chronic GBS genital tract colonization and establishes previously unknown cellular and soluble defense responses to GBS in the female genital tract.

The Roles of T Helper 1, T Helper 17 and Regulatory T Cells in the Pathogenesis of Sarcoidosis.

PubMed

Mortaz, Esmaeil; Rezayat, Fatemeh; Amani, Davar; Kiani, Arda; Garssen, Johan; Adcock, Ian M; Velayati, Aliakbar

2016-08-01

Sarcoidosis is a systemic granulomatous disorder of unidentified etiology, with a heterogeneous clinical presentation. It is characterized by a reduced delayed-type hypersensitivity to tuberculin and common antigens. The balance between Th1, Th17 and Regulatory T(Treg) cells controls T-cell proliferation and activation.The Th17/Treg ratio in the peripheral blood and bronchoalveolar lavage fluidis increased in patients with active sarcoidosis. Amplified IL-17A expression in granulomas and the presence of IL-17A+, IL-17A+IL-4+ and IL-17A+IFN-γ+ memory T helper cells in the circulation and BAL indicate Th17 cell involvement in granuloma induction and/or maintenance in sarcoidosis. Sarcoidosis should therefore be considered as a Th1/Th17 multisystem disorder and anti-IL-17/Th17 approaches that control and reduce IL-17Amay be an option, therefore, for the treatment of sarcoidosis.Here we provide a short overview as to the role of Th17 cells as critical cells in the pathogenesis of sarcoidosis.

17 CFR 240.17h-2T - Risk assessment reporting requirements for brokers and dealers.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Risk assessment reporting... Organizations § 240.17h-2T Risk assessment reporting requirements for brokers and dealers. (a) Reporting requirements of risk assessment information required to be maintained by section 240.17h-1T. (1) Every broker...

The Sox17CreERT2 knock-in mouse line displays spatiotemporal activation of Cre recombinase in distinct Sox17 lineage progenitors.

PubMed

Engert, Silvia; Burtscher, Ingo; Kalali, Behnam; Gerhard, Markus; Lickert, Heiko

2013-11-01

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis. Copyright © 2013 Wiley Periodicals, Inc.

Periconceptional maternal one-carbon biomarkers are associated with embryonic development according to the Carnegie stages.

PubMed

Parisi, F; Rousian, M; Koning, A H J; Willemsen, S P; Cetin, I; Steegers-Theunissen, R P M

2017-03-01

Is periconceptional maternal one-carbon (I-C) metabolism associated with embryonic morphological development in non-malformed ongoing pregnancies? Serum vitamin B12, red blood cell (RBC) folate and plasma total homocysteine (tHcy) are associated with embryonic development according to the Carnegie stages. Derangements in maternal I-C metabolism affect reproductive and pregnancy outcomes, as well as future health of the offspring. Between 2010 and 2014, women with singleton ongoing pregnancies were enrolled in a prospective periconceptional cohort study. A total of 234 pregnancies, including 138 spontaneous or IUI pregnancies with strict pregnancy dating and 96 pregnancies derived from IVF, ICSI or cryopreserved embryo transfer (IVF/ICSI pregnancies), underwent longitudinal transvaginal three-dimensional ultrasound (3D US) scans from 6+0 up to 10+2 weeks of gestation. Carnegie stages were defined using internal and external morphologic criteria in a virtual reality system. Maternal venous blood samples were collected at enrollment for serum vitamin B12, RBC folate and plasma tHcy assessment. Associations between biomarker concentrations and longitudinal Carnegie stages were investigated using linear mixed models. We performed a median of three 3D US scans per pregnancy (range 1-5) resulting in 600 good quality data sets for the Carnegie stage annotation (80.5%). Vitamin B12 was positively associated with embryonic development in the total study population (β = 0.001 (95% CI: 0.000; 0.002), P < 0.05) and in the subgroup of strictly dated spontaneous pregnancies (β = 0.002 (95% CI: 0.001; 0.003), P < 0.05). Low vitamin B12 concentrations (-2SD, 73.4 pmol/l) were associated with delayed embryonic development by 1.4 days (95% CI: 1.3-1.4) compared with high concentrations (+2SD, 563.1 pmol/l). RBC folate was positively associated with Carnegie stages only in IVF/ICSI pregnancies (β = 0.001 (95% CI: 0.0005; 0.0015), P < 0.05). In this group, low RBC folate

8-CPT-cAMP/all-trans retinoic acid targets t(11;17) acute promyelocytic leukemia through enhanced cell differentiation and PLZF/RARα degradation

PubMed Central

Jiao, Bo; Ren, Zhi-Hong; Liu, Ping; Chen, Li-Juan; Shi, Jing-Yi; Dong, Ying; Ablain, Julien; Shi, Lin; Gao, Li; Hu, Jun-Pei; Ren, Rui-Bao; de Thé, Hugues; Chen, Zhu; Chen, Sai-Juan

2013-01-01

The refractoriness of acute promyelocytic leukemia (APL) with t(11;17)(q23;q21) to all-trans retinoic acid (ATRA)-based therapy concerns clinicians and intrigues basic researchers. By using a murine leukemic model carrying both promyelocytic leukemia zinc finger/retinoic acid receptor-α (PLZF/RARα) and RARα/PLZF fusion genes, we discovered that 8-chlorophenylthio adenosine-3′, 5′-cyclic monophosphate (8-CPT-cAMP) enhances cellular differentiation and improves gene trans-activation by ATRA in leukemic blasts. Mechanistically, in combination with ATRA, 8-CPT-cAMP activates PKA, causing phosphorylation of PLZF/RARα at Ser765 and resulting in increased dissociation of the silencing mediator for retinoic acid and thyroid hormone receptors/nuclear receptor corepressor from PLZF/RARα. This process results in changes of local chromatin and transcriptional reactivation of the retinoic acid pathway in leukemic cells. Meanwhile, 8-CPT-cAMP also potentiated ATRA-induced degradation of PLZF/RARα through its Ser765 phosphorylation. In vivo treatment of the t(11;17) APL mouse model demonstrated that 8-CPT-cAMP could significantly improve the therapeutic effect of ATRA by targeting a leukemia-initiating cell activity. This combined therapy, which induces enhanced differentiation and oncoprotein degradation, may benefit t(11;17) APL patients. PMID:23382200

A Dominantly Acting Murine Allele of Mcm4 Causes Chromosomal Abnormalities and Promotes Tumorigenesis

PubMed Central

Bagley, Bruce N.; Keane, Thomas M.; Maklakova, Vilena I.; Marshall, Jonathon G.; Lester, Rachael A.; Cancel, Michelle M.; Paulsen, Alex R.; Bendzick, Laura E.; Been, Raha A.; Kogan, Scott C.; Cormier, Robert T.; Kendziorski, Christina; Adams, David J.; Collier, Lara S.

2012-01-01

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4D573H). MCM4 is part of the heterohexameric complex of MCM2–7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4D573H to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities. PMID:23133403

A dominantly acting murine allele of Mcm4 causes chromosomal abnormalities and promotes tumorigenesis.

PubMed

Bagley, Bruce N; Keane, Thomas M; Maklakova, Vilena I; Marshall, Jonathon G; Lester, Rachael A; Cancel, Michelle M; Paulsen, Alex R; Bendzick, Laura E; Been, Raha A; Kogan, Scott C; Cormier, Robert T; Kendziorski, Christina; Adams, David J; Collier, Lara S

2012-01-01

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4(D573H)). MCM4 is part of the heterohexameric complex of MCM2-7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4(D573H) to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.

46 CFR 34.17-20 - Discharge outlets-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Discharge outlets-T/ALL. 34.17-20 Section 34.17-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-20 Discharge outlets—T/ALL. (a) Discharge outlets shall be of an approved...

46 CFR 34.17-20 - Discharge outlets-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Discharge outlets-T/ALL. 34.17-20 Section 34.17-20 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-20 Discharge outlets—T/ALL. (a) Discharge outlets shall be of an approved...

Inhibition of IKK/NF-κB Signaling Enhances Differentiation of Mesenchymal Stromal Cells from Human Embryonic Stem Cells.

PubMed

Deng, Peng; Zhou, Chenchen; Alvarez, Ruth; Hong, Christine; Wang, Cun-Yu

2016-04-12

Embryonic stem cell-derived mesenchymal stromal cells (MSCs; also known as mesenchymal stem cells) represent a promising source for bone regenerative medicine. Despite remarkable advances in stem cell biology, the molecular mechanism regulating differentiation of human embryonic stem cells (hESCs) into MSCs remains poorly understood. Here, we report that inhibition of IκB kinase (IKK)/nuclear factor kappa B (NF-κB) signaling enhances differentiation of hESCs into MSCs by expediting the loss of pluripotent markers and increasing the expression of MSC surface markers. In addition, a significantly higher quantity of MSCs was produced from hESCs with IKK/NF-κB suppression. These isolated MSCs displayed evident multipotency with capacity to terminally differentiate into osteoblasts, chondrocytes, and adipocytes in vitro and to form bone in vivo. Collectively, our data provide important insights into the role of NF-κB in mesenchymal lineage specification during hESC differentiation, suggesting that IKK inhibitors could be utilized as an adjuvant in generating MSCs for cell-mediated therapies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Cellular mechanisms of IL-17-induced blood-brain barrier disruption.

PubMed

Huppert, Jula; Closhen, Dorothea; Croxford, Andrew; White, Robin; Kulig, Paulina; Pietrowski, Eweline; Bechmann, Ingo; Becher, Burkhard; Luhmann, Heiko J; Waisman, Ari; Kuhlmann, Christoph R W

2010-04-01

Recently T-helper 17 (Th17) cells were demonstrated to disrupt the blood-brain barrier (BBB) by the action of IL-17A. The aim of the present study was to examine the mechanisms that underlie IL-17A-induced BBB breakdown. Barrier integrity was analyzed in the murine brain endothelial cell line bEnd.3 by measuring the electrical resistance values using electrical call impedance sensing technology. Furthermore, in-cell Western blots, fluorescence imaging, and monocyte adhesion and transendothelial migration assays were performed. Experimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 mice. IL-17A induced NADPH oxidase- or xanthine oxidase-dependent reactive oxygen species (ROS) production. The resulting oxidative stress activated the endothelial contractile machinery, which was accompanied by a down-regulation of the tight junction molecule occludin. Blocking either ROS formation or myosin light chain phosphorylation or applying IL-17A-neutralizing antibodies prevented IL-17A-induced BBB disruption. Treatment of mice with EAE using ML-7, an inhibitor of the myosin light chain kinase, resulted in less BBB disruption at the spinal cord and less infiltration of lymphocytes via the BBB and subsequently reduced the clinical characteristics of EAE. These observations indicate that IL-17A accounts for a crucial step in the development of EAE by impairing the integrity of the BBB, involving augmented production of ROS.-Huppert, J., Closhen, D., Croxford, A., White, R., Kulig, P., Pietrowski, E., Bechmann, I., Becher, B., Luhmann, H. J., Waisman, A., Kuhlmann, C. R. W. Cellular mechanisms of IL-17-induced blood-brain barrier disruption.

Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

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

Ouji, Yukiteru; Yoshikawa, Masahide; Shiroi, Akira

2006-06-30

We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/cmore » nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis.« less

Influenza viruses production: Evaluation of a novel avian cell line DuckCelt®-T17.

PubMed

Petiot, Emma; Proust, Anaïs; Traversier, Aurélien; Durous, Laurent; Dappozze, Frédéric; Gras, Marianne; Guillard, Chantal; Balloul, Jean-Marc; Rosa-Calatrava, Manuel

2018-05-24

The influenza vaccine manufacturing industry is looking for production cell lines that are easily scalable, highly permissive to multiple viruses, and more effective in term of viral productivity. One critical characteristic of such cell lines is their ability to grow in suspension, in serum free conditions and at high cell densities. Influenza virus causing severe epidemics both in human and animals is an important threat to world healthcare. The repetitive apparition of influenza pandemic outbreaks in the last 20years explains that manufacturing sector is still looking for more effective production processes to replace/supplement embryonated egg-based process. Cell-based production strategy, with a focus on avian cell lines, is one of the promising solutions. Three avian cell lines, namely duck EB66®cells (Valneva), duck AGE.CR® cells (Probiogen) and quail QOR/2E11 cells (Baxter), are now competing with traditional mammalian cell platforms (Vero and MDCK cells) used for influenza vaccine productions and are currently at advance stage of commercial development for the manufacture of influenza vaccines. The DuckCelt®-T17 cell line presented in this work is a novel avian cell line developed by Transgene. This cell line was generated from primary embryo duck cells with the constitutive expression of the duck telomerase reverse transcriptase (dTERT). The DuckCelt®-T17 cells were able to grow in batch suspension cultures and serum-free conditions up to 6.5×10 6 cell/ml and were easily scaled from 10ml up to 3l bioreactor. In the present study, DuckCelt®-T17 cell line was tested for its abilities to produce various human, avian and porcine influenza strains. Most of the viral strains were produced at significant infectious titers (>5.8 log TCID50/ml) with optimization of the infection conditions. Human strains H1N1 and H3N2, as well as all the avian strains tested (H5N2, H7N1, H3N8, H11N9, H12N5) were the most efficiently produced with highest titre reached of 9

Inorganic arsenic represses interleukin-17A expression in human activated Th17 lymphocytes

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

Morzadec, Claudie; Macoch, Mélinda; Robineau, Marc

2012-08-01

Trivalent inorganic arsenic [As(III)] is an efficient anticancer agent used to treat patients suffering from acute promyelocytic leukemia. Recently, experimental studies have clearly demonstrated that this metalloid can also cure lymphoproliferative and/or pro-inflammatory syndromes in different murine models of chronic immune-mediated diseases. T helper (Th) 1 and Th17 lymphocytes play a central role in development of these diseases, in mice and humans, especially by secreting the potent pro-inflammatory cytokine interferon-γ and IL-17A, respectively. As(III) impairs basic functions of human T cells but its ability to modulate secretion of pro-inflammatory cytokines by differentiated Th lymphocytes is unknown. In the present study,more » we demonstrate that As(III), used at concentrations clinically achievable in plasma of patients, has no effect on the secretion of interferon-γ from Th1 cells but almost totally blocks the expression and the release of IL-17A from human Th17 lymphocytes co-stimulated for five days with anti-CD3 and anti-CD28 antibodies, in the presence of differentiating cytokines. In addition, As(III) specifically reduces mRNA levels of the retinoic-related orphan receptor (ROR)C gene which encodes RORγt, a key transcription factor controlling optimal IL-17 expression in fully differentiated Th17 cells. The metalloid also blocks initial expression of IL-17 gene induced by the co-stimulation, probably in part by impairing activation of the JNK/c-Jun pathway. In conclusion, our results demonstrate that As(III) represses expression of the major pro-inflammatory cytokine IL-17A produced by human Th17 lymphocytes, thus strengthening the idea that As(III) may be useful to treat inflammatory immune-mediated diseases in humans. -- Highlights: ► Arsenic inhibits secretion of IL-17A from human naïve and memory Th17 lymphocytes. ► Arsenic represses early expression of IL-17A gene in human activated T lymphocytes. ► Arsenic interferes with

Essential roles for Cdx in murine primitive hematopoiesis.

PubMed

Brooke-Bisschop, Travis; Savory, Joanne G A; Foley, Tanya; Ringuette, Randy; Lohnes, David

2017-02-15

The Cdx transcription factors play essential roles in primitive hematopoiesis in the zebrafish where they exert their effects, in part, through regulation of hox genes. Defects in hematopoiesis have also been reported in Cdx mutant murine embryonic stem cell models, however, to date no mouse model reflecting the zebrafish Cdx mutant hematopoietic phenotype has been described. This is likely due, in part, to functional redundancy among Cdx members and the early lethality of Cdx2 null mutants. To circumvent these limitations, we used Cre-mediated conditional deletion to assess the impact of concomitant loss of Cdx1 and Cdx2 on murine primitive hematopoiesis. We found that Cdx1/Cdx2 double mutants exhibited defects in primitive hematopoiesis and yolk sac vasculature concomitant with reduced expression of several genes encoding hematopoietic transcription factors including Scl/Tal1. Chromatin immunoprecipitation analysis revealed that Scl was occupied by Cdx2 in vivo, and Cdx mutant hematopoietic yolk sac differentiation defects could be rescued by expression of exogenous Scl. These findings demonstrate critical roles for Cdx members in murine primitive hematopoiesis upstream of Scl. Copyright © 2017 Elsevier Inc. All rights reserved.

An increase in telomere sister chromatid exchange in murine embryonic stem cells possessing critically shortened telomeres

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

Wang, Yisong; Giannone, Richard J; Wu, Jun

Telomerase deficiency leads to a progressive loss of telomeric DNA that eventually triggers cell apoptosis in human primary cells during prolonged growth in culture. Rare survivors can maintain telomere length through either activation of telomerase or recombination-based telomere lengthening, and thus proliferate indefinitely. We have explored the possibility that telomeres may be maintained through telomere sister chromatid exchange (T-SCE) in murine telomere reverse transcriptase-deficient (mTert -/-) splenocytes and ES cells. Because telomerase deficiency leads to gradual loss of telomeric DNA in mTert -/- splenocytes and ES cells and eventually to chromosomes with telomere signal-free ends (SFEs), we examined these cellmore » types for evidence of sister chromatid exchange at telomeres, and observed an increase in T-SCEs only in a subset of mTert -/- splenocytes or ES cells that possessed multiple SFEs. Furthermore, T-SCEs were more often detected in ES cells than in splenocytes that harbored a similar frequency of SFEs. In mTert heterozygous (mTert +/-) ES cells or splenocytes, which are known to exhibit a decrease in average telomere length but no SFEs, no increase in T-SCE was observed. In addition to T-SCE, other genomic rearrangements (i.e., SCE) were also significantly increased in mTert -/- ES cells possessing critically short telomeres, but not in splenocytes. Our results suggest that animals and cell culture differ in their ability to carry out genomic rearrangements as a means of maintaining telomere integrity when telomeres become critically shortened.« less

Diminished potential for B-lymphoid differentiation after murine leukemia virus infection in vivo and in EML hematopoietic progenitor cells.

PubMed

Finstad, Samantha L; Rosenberg, Naomi; Levy, Laura S

2007-07-01

Infection with a recombinant murine-feline gammaretrovirus, MoFe2, or with the parent virus, Moloney murine leukemia virus, caused significant reduction in B-lymphoid differentiation of bone marrow at 2 to 8 weeks postinfection. The suppression was selective, in that myeloid potential was significantly increased by infection. Analysis of cell surface markers and immunoglobulin H gene rearrangements in an in vitro model demonstrated normal B-lymphoid differentiation after infection but significantly reduced viability of differentiating cells. This reduction in viability may confer a selective advantage on undifferentiated lymphoid progenitors in the bone marrow of gammaretrovirus-infected animals and thereby contribute to the establishment of a premalignant state.

Response to programmed cell death-1 blockade in a murine melanoma syngeneic model requires costimulation, CD4, and CD8 T cells

PubMed Central

Moreno, Blanca Homet; Zaretsky, Jesse M.; Garcia-Diaz, Angel; Tsoi, Jennifer; Parisi, Giulia; Robert, Lidia; Meeth, Katrina; Ndoye, Abibatou; Bosenberg, Marcus; Weeraratna, Ashani T.; Graeber, Thomas G.; Comin-Anduix, Begoña; Hu-Lieskovan, Siwen; Ribas, Antoni

2016-01-01

The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor-host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN-/-). Anti–PD-1 or anti–PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor associated macrophages in the tumors after PD-1 blockade. Compared to YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression from chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. PMID:27589875

Signed weighted gene co-expression network analysis of transcriptional regulation in murine embryonic stem cells

PubMed Central

Mason, Mike J; Fan, Guoping; Plath, Kathrin; Zhou, Qing; Horvath, Steve

2009-01-01

Background Recent work has revealed that a core group of transcription factors (TFs) regulates the key characteristics of embryonic stem (ES) cells: pluripotency and self-renewal. Current efforts focus on identifying genes that play important roles in maintaining pluripotency and self-renewal in ES cells and aim to understand the interactions among these genes. To that end, we investigated the use of unsigned and signed network analysis to identify pluripotency and differentiation related genes. Results We show that signed networks provide a better systems level understanding of the regulatory mechanisms of ES cells than unsigned networks, using two independent murine ES cell expression data sets. Specifically, using signed weighted gene co-expression network analysis (WGCNA), we found a pluripotency module and a differentiation module, which are not identified in unsigned networks. We confirmed the importance of these modules by incorporating genome-wide TF binding data for key ES cell regulators. Interestingly, we find that the pluripotency module is enriched with genes related to DNA damage repair and mitochondrial function in addition to transcriptional regulation. Using a connectivity measure of module membership, we not only identify known regulators of ES cells but also show that Mrpl15, Msh6, Nrf1, Nup133, Ppif, Rbpj, Sh3gl2, and Zfp39, among other genes, have important roles in maintaining ES cell pluripotency and self-renewal. We also report highly significant relationships between module membership and epigenetic modifications (histone modifications and promoter CpG methylation status), which are known to play a role in controlling gene expression during ES cell self-renewal and differentiation. Conclusion Our systems biologic re-analysis of gene expression, transcription factor binding, epigenetic and gene ontology data provides a novel integrative view of ES cell biology. PMID:19619308

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells

PubMed Central

West, Michael D.; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B.; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-01

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1, encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro-derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition. PMID:29487692

Use of deep neural network ensembles to identify embryonic-fetal transition markers: repression of COX7A1 in embryonic and cancer cells.

PubMed

West, Michael D; Labat, Ivan; Sternberg, Hal; Larocca, Dana; Nasonkin, Igor; Chapman, Karen B; Singh, Ratnesh; Makarev, Eugene; Aliper, Alex; Kazennov, Andrey; Alekseenko, Andrey; Shuvalov, Nikolai; Cheskidova, Evgenia; Alekseev, Aleksandr; Artemov, Artem; Putin, Evgeny; Mamoshina, Polina; Pryanichnikov, Nikita; Larocca, Jacob; Copeland, Karen; Izumchenko, Evgeny; Korzinkin, Mikhail; Zhavoronkov, Alex

2018-01-30

Here we present the application of deep neural network (DNN) ensembles trained on transcriptomic data to identify the novel markers associated with the mammalian embryonic-fetal transition (EFT). Molecular markers of this process could provide important insights into regulatory mechanisms of normal development, epimorphic tissue regeneration and cancer. Subsequent analysis of the most significant genes behind the DNNs classifier on an independent dataset of adult-derived and human embryonic stem cell (hESC)-derived progenitor cell lines led to the identification of COX7A1 gene as a potential EFT marker. COX7A1 , encoding a cytochrome C oxidase subunit, was up-regulated in post-EFT murine and human cells including adult stem cells, but was not expressed in pre-EFT pluripotent embryonic stem cells or their in vitro -derived progeny. COX7A1 expression level was observed to be undetectable or low in multiple sarcoma and carcinoma cell lines as compared to normal controls. The knockout of the gene in mice led to a marked glycolytic shift reminiscent of the Warburg effect that occurs in cancer cells. The DNN approach facilitated the elucidation of a potentially new biomarker of cancer and pre-EFT cells, the embryo-onco phenotype, which may potentially be used as a target for controlling the embryonic-fetal transition.

Survival and characteristics of murine leukaemic and normal stem cells after hyperthermia: a murine model for human bone marrow purging.

PubMed

Gidáli, J; Szamosvölgyi, S; Fehér, I; Kovács, P

1990-01-01

The effect of hyperthermia in vitro on the survival and leukaemogenic effectiveness of WEHI 3-B cells and on the survival and transplantation efficiency of bone marrow cells was compared in a murine model system. Normal murine clonogenic haemopoietic cells (day 9 CFU-S and CFU-GM) proved to be significantly less sensitive to 42.5 degrees C hyperthermia (Do values: 54.3 and 41.1 min, respectively) than leukaemic clonogenic cells (CFU-L) derived from suspension culture or from bone marrow of leukaemic mice (Do: 17.8 min). Exposure for 120 min to 42.5 degrees C reduced the surviving fraction of CFU-L to 0.002 and that of CFU-S to 0.2. If comparable graft sizes were transplanted from normal or heat exposed bone marrow, 60-day survival of supralethally irradiated mice was similar. Surviving WEHI 3-B cells were capable of inducing leukaemia in vivo. The two log difference in the surviving fraction of CFU-L and CFU-S after 120 min exposure to 42.5 degrees C suggests that hyperthermia ex vivo may be a suitable purging method for autologous bone marrow transplantation.

A panel of Trypanosoma brucei strains tagged with blue and red-shifted luciferases for bioluminescent imaging in murine infection models.

PubMed

Van Reet, Nick; Van de Vyver, Hélène; Pyana, Patient Pati; Van der Linden, Anne Marie; Büscher, Philippe

2014-08-01

Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better kinetics of their substrate D-luciferin.

A Panel of Trypanosoma brucei Strains Tagged with Blue and Red-Shifted Luciferases for Bioluminescent Imaging in Murine Infection Models

PubMed Central

Van Reet, Nick; Van de Vyver, Hélène; Pyana, Patient Pati; Van der Linden, Anne Marie; Büscher, Philippe

2014-01-01

Background Genetic engineering with luciferase reporter genes allows monitoring Trypanosoma brucei (T.b.) infections in mice by in vivo bioluminescence imaging (BLI). Until recently, luminescent T.b. models were based on Renilla luciferase (RLuc) activity. Our study aimed at evaluating red-shifted luciferases for in vivo BLI in a set of diverse T.b. strains of all three subspecies, including some recently isolated from human patients. Methodology/Principal findings We transfected T.b. brucei, T.b. rhodesiense and T.b. gambiense strains with either RLuc, click beetle red (CBR) or Photinus pyralis RE9 (PpyRE9) luciferase and characterised their in vitro luciferase activity, growth profile and drug sensitivity, and their potential for in vivo BLI. Compared to RLuc, the red-shifted luciferases, CBR and PpyRE9, allow tracking of T.b. brucei AnTaR 1 trypanosomes with higher details on tissue distribution, and PpyRE9 allows detection of the parasites with a sensitivity of at least one order of magnitude higher than CBR luciferase. With CBR-tagged T.b. gambiense LiTaR1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT in an acute, subacute and chronic infection model respectively, we observed differences in parasite tropism for murine tissues during in vivo BLI. Ex vivo BLI on the brain confirmed central nervous system infection by all luminescent strains of T.b. brucei AnTaR 1, T.b. rhodesiense RUMPHI and T.b. gambiense 348 BT. Conclusions/Significance We established a genetically and phenotypically diverse collection of bioluminescent T.b. brucei, T.b. gambiense and T.b. rhodesiense strains, including drug resistant strains. For in vivo BLI monitoring of murine infections, we recommend trypanosome strains transfected with red-shifted luciferase reporter genes, such as CBR and PpyRE9. Red-shifted luciferases can be detected with a higher sensitivity in vivo and at the same time they improve the spatial resolution of the parasites in the entire body due to the better

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 17 Commodity and Securities Exchanges 3 2014-04-01 2014-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 17 Commodity and Securities Exchanges 2 2013-04-01 2013-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 17 Commodity and Securities Exchanges 2 2011-04-01 2011-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

17 CFR 200.80b - Appendix B-SEC releases.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 17 Commodity and Securities Exchanges 2 2012-04-01 2012-04-01 false Appendix B-SEC releases. 200.80b Section 200.80b Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION ORGANIZATION... corporate reorganization releases, and the litigation releases are contained in the SEC Docket, which may be...

Effect of antioxidant of bamboo leaves on gene expression associated with mouse embryonic fibroblast reproduction and embryonic development.

PubMed

Yu, Feng; Qian, Xiaowei; Zeng, Zhanghui; Zhao, Xiaoli; Hou, Rong; Zhang, Zhihe; Bian, Hongwu; Han, Ning; Wang, Junhui; Zhu, Muyuan

2017-11-01

Antioxidant of bamboo leaves (AOB) was certified to be a natural antioxidant by the Chinese Ministry of Health in 2003. However, the effects of AOB on animal reproductive and developmental functions remain unclear. The present study aimed to investigate the effects of different concentrations of AOB on mouse embryonic fibroblast (MEF) cells, and to examine the underlying molecular mechanism through which AOB affects the proliferation and apoptosis of MEFs. MEFs prepared from individual embryos were treated with various dosages of AOB. Cell viability and apoptosis were detected by MTT and flow cytometry assays, respectively. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were used for the detection of mRNA and protein expression. Functional annotation of differentially‑expressed genes was performed according to the Gene Ontology database and Kyoto Encyclopedia of Genes and Genomes pathway analysis. Compared with the control group, ~50% of MEF cells were inhibited following treatment with a 400 µg/ml concentration of AOB. Treatment with 400 µg/ml AOB for 72 h significantly increased the apoptotic rate of MEF cells compared with the control group. Following treatment with AOB, dehydrogenase/reductase 9, phospholipase A2 group IVE and platelet derived growth factor B were downregulated, while 17 other genes were upregulated in MEF cells. Treatment with AOB markedly increased the expression of phosphorylated extracellular signal‑regulated kinase (ERK), β‑catenin, transcription factor SOX‑17, calcium‑binding tyrosine phosphorylation‑regulated protein, and cholesterol side chain cleavage enzyme mitochondrial (P<0.01). Additionally, the ERK pathway inhibitor U0126 and Wnt pathway inhibitor dickkopf‑related protein 1 markedly suppressed the expression of the above genes (P<0.01). AOB may impact the expression of proteins associated with embryonic fibroblast reproduction and embryonic development through

Inflammatory T helper 17 cells promote depression-like behavior in mice.

PubMed

Beurel, Eléonore; Harrington, Laurie E; Jope, Richard S

2013-04-01

Recognition of substantial immune-neural interactions is revising dogmas about their insular actions and revealing that immune-neural interactions can substantially impact central nervous system functions. The inflammatory cytokine interleukin-6 promotes susceptibility to depression and drives production of inflammatory T helper 17 (Th17) T cells, raising the hypothesis that in mouse models, Th17 cells promote susceptibility to depression-like behaviors. Behavioral characteristics were measured in male mice administered Th17 cells, CD4(+) cells, or vehicle and in retinoid-related orphan receptor-γT (RORγT)(+/GFP) mice or male mice treated with RORγT inhibitor or anti-interleukin-17A antibodies. Mouse brain Th17 cells were elevated by learned helplessness and chronic restraint stress, two common depression-like models. Th17 cell administration promoted learned helplessness in 89% of mice in a paradigm where no vehicle-treated mice developed learned helplessness, and impaired novelty suppressed feeding and social interaction behaviors. Mice deficient in the RORγT transcription factor necessary for Th17 cell production exhibited resistance to learned helplessness, identifying modulation of RORγT as a potential intervention. Treatment with the RORγT inhibitor SR1001, or anti-interleukin-17A antibodies to abrogate Th17 cell function, reduced Th17-dependent learned helplessness. These findings indicate that Th17 cells are increased in the brain during depression-like states, promote depression-like behaviors in mice, and specifically inhibiting the production or function of Th17 cells reduces vulnerability to depression-like behavior, suggesting antidepressant effects may be attained by targeting Th17 cells. Copyright © 2013 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Lipopolysaccharide-induced murine embryonic resorption involves changes in endocannabinoid profiling and alters progesterone secretion and inflammatory response by a CB1-mediated fashion.

PubMed

Wolfson, Manuel L; Correa, Fernando; Leishman, Emma; Vercelli, Claudia; Cymeryng, Cora; Blanco, Julieta; Bradshaw, Heather B; Franchi, Ana María

2015-08-15

Genital tract infections are a common complication of human pregnancy that can result in miscarriage. We have previously shown that a lipopolysaccharide (LPS) induces embryonic resorption in a murine model of inflammatory miscarriage. This is accompanied by a dramatic decrease in systemic progesterone levels associated with a robust pro-inflammatory response that results in embryo resorption. Here, we tested the hypothesis that the endogenous cannabinoid system (eCS), through cannabinoid receptor 1 (CB1), plays a role in regulating progesterone levels and, therefore, the pro-inflammatory response. We show that LPS treatment in pregnant mice causes significant changes in the eCS ligands, which are reversed by progesterone treatment. We further show the CB1-KO mice maintain higher plasma progesterone levels after LPS treatment, which is associated with a feebler uterine inflammatory response and a significant drop in embryo resorption. These data suggest that manipulation of CB1 receptors and/or ligands is a potential therapeutic avenue to decrease infection-induced miscarriage. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Embryonal rhabdomyosarcoma with a der(16)t(1;16) translocation.

PubMed

Kapels, Kayla M; Nishio, Jun; Zhou, Ming; Qualman, Stephen J; Bridge, Julia A

2007-04-01

Embryonal rhabdomyosarcoma (ERMS) is the most common subtype of RMS that predominantly involves the genitourinary tract and the head and neck regions in children younger than 10 years of age. Cytogenetically, ERMS is most frequently hyperdiploid, with extra copies of chromosomes 2, 7, 8, 11, 12, 13, and 20. No consistent structural chromosomal alteration has been identified in ERMS. In contrast, a t(2;13)(q35;q14) or t(1;13)(q36;q14) corresponding to PAX3-FOXO1A (previously FKHR) and PAX7-FOXO1A gene fusions are considered tumor-specific anomalies for alveolar RMS (ARMS). Occasionally, a recurrent secondary structural rearrangement involving chromosomes 1 and 16 is seen in translocation-positive ARMS, a der(16)t(1;16) resulting in an imbalance of 1q and 16q material. Conventional cytogenetic analysis of an ERMS arising in the urinary bladder of a 22-month-old male child revealed this nonrandom secondary chromosomal aberration, der(16)(1;16)(q22;q24), in a hyperdiploid complement with extra copies of chromosomes 2, 7, 8, 10, 12, 13, 19, and 20. Subsequent analyses showed tumor cells to be negative for FOXO1A, PAX3, or PAX7 gene locus rearrangements (by fluorescence in situ hybridization) and also negative for PAX3-FOXO1A and PAX7-FOXO1A fusion transcripts (by reverse transcriptase-polymerase chain reaction). These results suggest that the unbalanced t(1;16) translocation may be seen in RMSs lacking a primary genetic rearrangement.

Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

PubMed Central

Zwaenepoel, Karen; Louis, Justin V; Goris, Jozef; Janssens, Veerle

2008-01-01

Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice. PMID:18715506

4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

PubMed Central

1984-01-01

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts. PMID:6538202

Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice

PubMed Central

Liao, Chien-Wei; Fan, Chia-Kwung; Kao, Ting-Chang; Ji, Dar-Der; Su, Kua-Eyre; Lin, Yun-Ho; Cho, Wen-Long

2008-01-01

Background Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor β1 (TGF-β1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), β-amyloid precursor proteins (AβPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT. Methods BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR. Results Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-β1, S100B, NF-L, tTG, AβPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain. Conclusion Further studies are needed to

Brain injury-associated biomarkers of TGF-beta1, S100B, GFAP, NF-L, tTG, AbetaPP, and tau were concomitantly enhanced and the UPS was impaired during acute brain injury caused by Toxocara canis in mice.

PubMed

Liao, Chien-Wei; Fan, Chia-Kwung; Kao, Ting-Chang; Ji, Dar-Der; Su, Kua-Eyre; Lin, Yun-Ho; Cho, Wen-Long

2008-06-24

Because the outcomes and sequelae after different types of brain injury (BI) are variable and difficult to predict, investigations on whether enhanced expressions of BI-associated biomarkers (BIABs), including transforming growth factor beta1 (TGF-beta1), S100B, glial fibrillary acidic protein (GFAP), neurofilament light chain (NF-L), tissue transglutaminases (tTGs), beta-amyloid precursor proteins (AbetaPP), and tau are present as well as whether impairment of the ubiquitin-proteasome system (UPS) is present have been widely used to help delineate pathophysiological mechanisms in various BIs. Larvae of Toxocara canis can invade the brain and cause BI in humans and mice, leading to cerebral toxocariasis (CT). Because the parasitic burden is light in CT, it may be too cryptic to be detected in humans, making it difficult to clearly understand the pathogenesis of subtle BI in CT. Since the pathogenesis of murine toxocariasis is very similar to that in humans, it appears appropriate to use a murine model to investigate the pathogenesis of CT. BIAB expressions and UPS function in the brains of mice inoculated with a single dose of 250 T. canis embryonated eggs was investigated from 3 days (dpi) to 8 weeks post-infection (wpi) by Western blotting and RT-PCR. Results revealed that at 4 and 8 wpi, T. canis larvae were found to have invaded areas around the choroid plexus but without eliciting leukocyte infiltration in brains of infected mice; nevertheless, astrogliosis, an indicator of BI, with 78.9~142.0-fold increases in GFAP expression was present. Meanwhile, markedly increased levels of other BIAB proteins including TGF-beta1, S100B, NF-L, tTG, AbetaPP, and tau, with increases ranging 2.0~12.0-fold were found, although their corresponding mRNA expressions were not found to be present at 8 wpi. Concomitantly, UPS impairment was evidenced by the overexpression of conjugated ubiquitin and ubiquitin in the brain. Further studies are needed to determine whether there is an

Subpopulations of M-MDSCs from mice infected by an immunodeficiency-causing retrovirus and their differential suppression of T- vs B-cell responses.

PubMed

O'Connor, Megan A; Fu, Whitney W; Green, Kathy A; Green, William R

2015-11-01

Monocytic (CD11b(+)Ly6G(±/Lo)Ly6C(+)) myeloid derived suppressor cells (M-MDSCs) expand following murine retroviral LP-BM5 infection and suppress ex vivo polyclonal T-cell and B-cell responses. M-MDSCs 3 weeks post LP-BM5 infection have decreased suppression of T-cell, but not B-cell, responses and alterations in the degree of iNOS/NO dependence of suppression. M-MDSCs from LP-BM5 infected mice were sorted into four quadrant populations (Ly6C/CD11b density): all quadrants suppressed B-cell responses, but only M-MDSCs expressing the highest levels of Ly6C and CD11b (Q2) significantly suppressed T-cell responses. Further subdivision of this Q2 population revealed the Ly6C(+/Hi) M-MDSC subpopulation as the most suppressive, inhibiting T- and B-cell responses in a full, or partially, iNOS/NO-dependent manner, respectively. In contrast, the lower/moderate levels of suppression by the Ly6C(+/Lo) and Ly6C(+/Mid) M-MDSC Q2 subpopulations, whether versus T- and/or B-cells, displayed little/no iNOS dependency for suppression. These results highlight differential phenotypic and functional immunosuppressive M-MDSC subsets in a retroviral immunodeficiency model. Published by Elsevier Inc.

Adeno-associated virus type 2 enhances goose parvovirus replication in embryonated goose eggs

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

Malkinson, Mertyn; Winocour, Ernest

The autonomous goose parvovirus (GPV) and the human helper-dependent adeno-associated virus type 2 (AAV2) share a high degree of homology. To determine if this evolutionary relationship has a biological impact, we studied viral replication in human 293 cells and in embryonated goose eggs coinfected with both viruses. Similar experiments were performed with the minute virus of mice (MVM), an autonomous murine parvovirus with less homology to AAV2. In human 293 cells, both GPV and MVM augmented AAV2 replication. In contrast, AAV2 markedly enhanced GPV replication in embryonated goose eggs under conditions where a similar effect was not observed with MVM.more » AAV2 did not replicate in embryonated goose eggs and AAV2 inactivated by UV-irradiation also enhanced GPV replication. To our knowledge, this is the first report that a human helper-dependent member of the Parvoviridae can provide helper activity for an autonomous parvovirus in a natural host.« less

B and T lymphocyte attenuator restricts the protective immune response against experimental malaria.

PubMed

Adler, Guido; Steeg, Christiane; Pfeffer, Klaus; Murphy, Theresa L; Murphy, Kenneth M; Langhorne, Jean; Jacobs, Thomas

2011-11-15

The immune response against the blood stage of malaria has to be tightly regulated to allow for vigorous antiplasmodial activity while restraining potentially lethal immunopathologic damage to the host like cerebral malaria. Coinhibitory cell surface receptors are important modulators of immune activation. B and T lymphocyte attenuator (BTLA) (CD272) is a coinhibitory receptor expressed by most leukocytes, with the highest expression levels on T and B cells, and is involved in the maintenance of peripheral tolerance by dampening the activation of lymphocytes. The function of BTLA is described in several models of inflammatory disorders and autoimmunity, but its function in infectious diseases is less well characterized. Also, little is known about the influence of BTLA on non-T cells. In this study, we analyzed the function of BTLA during blood-stage malaria infection with the nonlethal Plasmodium yoelii strain 17NL. We show that BTLA knockout mice exhibit strongly reduced parasitemia and clear the infection earlier compared with wild-type mice. This increased resistance was seen before the onset of adaptive immune mechanisms and even in the absence of T and B cells but was more pronounced at later time points when activation of T and B cells was observed. We demonstrate that BTLA regulates production of proinflammatory cytokines in a T cell-intrinsic way and B cell intrinsically regulates the production of P. yoelii 17NL-specific Abs. These results indicate that the coinhibitory receptor BTLA plays a critical role during experimental malaria and attenuates the innate as well as the subsequent adaptive immune response.

Intratumoral Infection with Murine Cytomegalovirus Synergizes with PD-L1 Blockade to Clear Melanoma Lesions and Induce Long-term Immunity

PubMed Central

Erkes, Dan A; Xu, Guangwu; Daskalakis, Constantine; Zurbach, Katherine A; Wilski, Nicole A; Moghbeli, Toktam; Hill, Ann B; Snyder, Christopher M

2016-01-01

Cytomegalovirus is an attractive cancer vaccine platform because it induces strong, functional CD8+ T-cell responses that accumulate over time and migrate into most tissues. To explore this, we used murine cytomegalovirus expressing a modified gp100 melanoma antigen. Therapeutic vaccination by the intraperitoneal and intradermal routes induced tumor infiltrating gp100-specific CD8+ T-cells, but provided minimal benefit for subcutaneous lesions. In contrast, intratumoral infection of established tumor nodules greatly inhibited tumor growth and improved overall survival in a CD8+ T-cell-dependent manner, even in mice previously infected with murine cytomegalovirus. Although murine cytomegalovirus could infect and kill B16F0s in vitro, infection was restricted to tumor-associated macrophages in vivo. Surprisingly, the presence of a tumor antigen in the virus only slightly increased the efficacy of intratumoral infection and tumor-specific CD8+ T-cells in the tumor remained dysfunctional. Importantly, combining intratumoral murine cytomegalovirus infection with anti-PD-L1 therapy was synergistic, resulting in tumor clearance from over half of the mice and subsequent protection against tumor challenge. Thus, while a murine cytomegalovirus-based vaccine was poorly effective against established subcutaneous tumors, direct infection of tumor nodules unexpectedly delayed tumor growth and synergized with immune checkpoint blockade to promote tumor clearance and long-term protection. PMID:27434584

Huntingtin Protein is Essential for Mitochondrial Metabolism, Bioenergetics and Structure in Murine Embryonic Stem Cells

PubMed Central

Ismailoglu, Ismail; Chen, Qiuying; Popowski, Melissa; Yang, Lili; Gross, Steven S.; Brivanlou, Ali H.

2014-01-01

Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington's disease (HD), while htt-/- mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3,700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt-/-, extended poly-Q (Htt-Q140/7), and wildtype mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells, did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt-/- mESCs, including: (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt-/- early embryonic lethality. PMID:24780625

Huntingtin protein is essential for mitochondrial metabolism, bioenergetics and structure in murine embryonic stem cells.

PubMed

Ismailoglu, Ismail; Chen, Qiuying; Popowski, Melissa; Yang, Lili; Gross, Steven S; Brivanlou, Ali H

2014-07-15

Mutations in the Huntington locus (htt) have devastating consequences. Gain-of-poly-Q repeats in Htt protein causes Huntington's disease (HD), while htt(-/-) mutants display early embryonic lethality. Despite its importance, the function of Htt remains elusive. To address this, we compared more than 3700 compounds in three syngeneic mouse embryonic stem cell (mESC) lines: htt(-/-), extended poly-Q (Htt-Q140/7), and wild-type mESCs (Htt-Q7/7) using untargeted metabolite profiling. While Htt-Q140/7 cells did not show major differences in cellular bioenergetics, we find extensive metabolic aberrations in htt(-/-) mESCs, including (i) complete failure of ATP production despite preservation of the mitochondrial membrane potential; (ii) near-maximal glycolysis, with little or no glycolytic reserve; (iii) marked ketogenesis; (iv) depletion of intracellular NTPs; (v) accelerated purine biosynthesis and salvage; and (vi) loss of mitochondrial structural integrity. Together, our findings reveal that Htt is necessary for mitochondrial structure and function from the earliest stages of embryogenesis, providing a molecular explanation for htt(-/-) early embryonic lethality. Copyright © 2014 Elsevier Inc. All rights reserved.

CD8+ T cells complement antibodies in protecting against yellow fever virus.

PubMed

Bassi, Maria R; Kongsgaard, Michael; Steffensen, Maria A; Fenger, Christina; Rasmussen, Michael; Skjødt, Karsten; Finsen, Bente; Stryhn, Anette; Buus, Søren; Christensen, Jan P; Thomsen, Allan R

2015-02-01

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4(+) T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8(+) T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8(+) T cells from these animals revealed a pivotal role for CD8(+) T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8(+) T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8(+) T cells have been demonstrated to possess antiviral activity in vivo. Copyright © 2015 by The American Association of Immunologists, Inc.

Donor B cells in Transplants Augment Clonal Expansion and Survival of Pathogenic CD4+ T cells That Mediate Autoimmune-like Chronic GVHD

PubMed Central

Young, James S; Wu, Tao; Chen, Yuhong; Zhao, Dongchang; Liu, Hongjun; Yi, Tangsheng; Johnston, Heather; Racine, Jeremy; Li, Xiaofan; Wang, Audrey; Todorov, Ivan; Zeng, Defu

2013-01-01

We reported that both donor CD4+ T and B cells in transplants were required for induction of an autoimmune-like chronic graft versus host disease (cGVHD) in a murine model of DBA/2 donor to BALB/c recipient, but mechanisms whereby donor B cells augment cGVHD pathogenesis remain unknown. Here, we report that, although donor B cells have little impact on acute GVHD (aGVHD) severity, they play an important role in augmenting the persistence of tissue damage in the acute and chronic GVHD overlapping target organs (i.e. skin and lung); they also markedly augment damage in a prototypical cGVHD target organ- the salivary gland. During cGVHD pathogenesis, donor B cells are activated by donor CD4+ T cells to upregulate MHC II and co-stimulatory molecules. Acting as efficient APCs, donor B cells augment donor CD4+ T clonal expansion, autoreactivity, IL-7Rα expression, and survival. These qualitative changes markedly augment donor CD4+ T cells' capacity in mediating autoimmune-like cGVHD, so that they mediate disease in the absence of donor B cells in secondary recipients. Therefore, a major mechanism whereby donor B cells augment cGVHD is through augmenting the clonal expansion, differentiation and survival of pathogenic CD4+ T cells. PMID:22649197

Developmental plasticity of murine and human Foxp3(+) regulatory T cells.

PubMed

Liston, Adrian; Piccirillo, Ciriaco A

2013-01-01

Murine and human CD4(+) regulatory T (Treg) cells expressing the Forkhead box p3 (Foxp3) transcription factor represent a distinct, highly differentiated CD4(+) T cell lineage that is programmed for dominant self-tolerance and control of immune responses against a variety of foreign antigens. Sustained Foxp3 expression in these cells drives the differentiation of a regulatory phenotype and ensures the stability of their suppressive functions under a variety of inflammatory settings. Some recent studies have challenged this premise and advanced the notion that Foxp3(+) Treg cells manifest a high degree of functional plasticity that enables them to adapt and reprogram into effector-like T cells in response to various inflammatory stimuli. The concept of Treg cell plasticity remains highly contentious, with a high degree of variation in measured plasticity potential observed under different experimental conditions. In this chapter, we propose a unifying model of Treg cell plasticity, which hypothesizes that the stable fates of regulatory and effector T (Teff) cell lineages allow transient plasticity into the alternative lineage under a discrete set of microenvironmental influences associated with, respectively, the initiation and resolution phases of infection. This model utilizes a theoretical framework consistent with the requirements for effective immune regulation and accounts for both the extraordinary long-term stability of Treg cells and the observed fate plasticity. Copyright © 2013 Elsevier Inc. All rights reserved.

Signal Transduction in T Cell Activation and Tolerance

DTIC Science & Technology

1993-01-01

chains and ’ chains may transduce different signals in intact T cells. These studies demonstrate that while c- deficient and c-containing TCR complexes...three independently derived pairs of CD45- and CD45+ murine T cell lymphomas, the CD45- expressing cells were consistently deficient in...D.B., Larsen, A. and Wilson, C.B. (1986) Reduced interferon-gamma mRNA levels in human neonates: Evidence for an intrinsic T cell deficiency yi 114

CXCR4 blockade decreases CD4+ T cell exhaustion and improves survival in a murine model of polymicrobial sepsis.

PubMed

Ramonell, Kimberly M; Zhang, Wenxiao; Hadley, Annette; Chen, Ching-Wen; Fay, Katherine T; Lyons, John D; Klingensmith, Nathan J; McConnell, Kevin W; Coopersmith, Craig M; Ford, Mandy L

2017-01-01

Sepsis is a dysregulated systemic response to infection involving many inflammatory pathways and the induction of counter-regulatory anti-inflammatory processes that results in a state of immune incompetence and can lead to multi-organ failure. CXCR4 is a chemokine receptor that, following ligation by CXCL12, directs cells to bone marrow niches and also plays an important role in T cell cosignaling and formation of the immunological synapse. Here, we investigated the expression and function of CXCR4 in a murine model of polymicrobial sepsis. Results indicate that CXCR4 is selectively upregulated on naïve CD4+ and CD8+ T cells and CD4+ central memory T cells following the induction of sepsis, and that CXCR4 antagonism resulted in a significant decrease in sepsis-induced mortality. We probed the mechanistic basis for these findings and found that CXCR4 antagonism significantly increased the number of peripheral CD4+ and CD8+ T cells following sepsis. Moreover, mice treated with the CXCR4 antagonist contained fewer PD-1+ LAG-3+ 2B4+ cells, suggesting that blockade of CXCR4 mitigates CD4+ T cell exhaustion during sepsis. Taken together, these results characterize CXCR4 as an important pathway that modulates immune dysfunction and mortality following sepsis, which may hold promise as a target for future therapeutic intervention in septic patients.

Immunostaining to visualize murine enteric nervous system development.

PubMed

Barlow-Anacker, Amanda J; Erickson, Christopher S; Epstein, Miles L; Gosain, Ankush

2015-04-29

The enteric nervous system is formed by neural crest cells that proliferate, migrate and colonize the gut. Following colonization, neural crest cells must then differentiate into neurons with markers specific for their neurotransmitter phenotype. Cholinergic neurons, a major neurotransmitter phenotype in the enteric nervous system, are identified by staining for choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Historical efforts to visualize cholinergic neurons have been hampered by antibodies with differing specificities to central nervous system versus peripheral nervous system ChAT. We and others have overcome this limitation by using an antibody against placental ChAT, which recognizes both central and peripheral ChAT, to successfully visualize embryonic enteric cholinergic neurons. Additionally, we have compared this antibody to genetic reporters for ChAT and shown that the antibody is more reliable during embryogenesis. This protocol describes a technique for dissecting, fixing and immunostaining of the murine embryonic gastrointestinal tract to visualize enteric nervous system neurotransmitter expression.

Immunogenicity and protective efficacy of a recombinant yellow fever vaccine against the murine malarial parasite Plasmodium yoelii.

PubMed

Stoyanov, Cristina T; Boscardin, Silvia B; Deroubaix, Stephanie; Barba-Spaeth, Giovanna; Franco, David; Nussenzweig, Ruth S; Nussenzweig, Michel; Rice, Charles M

2010-06-23

The live-attenuated yellow fever vaccine (YF17D) is one of the safest and most effective vaccines available today. Here, YF17D was genetically altered to express the circumsporozoite protein (CSP) from the murine malarial parasite Plasmodium yoelii. Reconstituted recombinant virus was viable and exhibited robust CSP expression. Immunization of naïve mice resulted in extensive proliferation of adoptively transferred CSP-specific transgenic CD8(+) T-cells. A single immunization of naïve mice with recombinant YF17D resulted in robust production of IFN-gamma by CD8(+) T-cells and IFN-gamma and IL-2 by CD4(+) T-cells. A prime-boost regimen consisting of recombinant virus followed by a low-dose of irradiated sporozoites conferred protection against challenge with P. yoelii. Taken together, these results show that recombinant YF17D can efficiently express CSP in culture, and prime a protective immune response in vivo. (c) 2010 Elsevier Ltd. All rights reserved.

Murine AIDS Protects Mice Against Experimental Cerebral Malaria: Down-Regulation by Interleukin 10 a T-Helper Type 1 CD4^+ Cell-Mediated Pathology

NASA Astrophysics Data System (ADS)

Eckwalanga, Michel; Marussig, Myriam; Dias Tavares, Marisa; Bouanga, Jean Claude; Hulier, Elisabeth; Henriette Pavlovitch, Jana; Minoprio, Paola; Portnoi, Denis; Renia, Laurent; Mazier, Dominique

1994-08-01

The retrovirus LP-BM5 murine leukemia virus induces murine AIDS in C57BL/6 mice that has many similarities with human AIDS; Plasmodium berghei ANKA causes experimental cerebral malaria in the same strain of mice. The outcome of malaria infection was studied in mice concurrently infected with the two pathogens. The retrovirus significantly reduced the gravity of the neurological manifestations associated with Plasmodium berghei ANKA infection. The protection against experimental cerebral malaria induced by murine AIDS increased with duration of viral infection and, hence, with the severity of the immunodeficiency. Interleukin 10, principally from splenic T cells, was shown to play a crucial role in this protection.

Role of Melatonin in the Regulation of Differentiation of T Cells Producing Interleukin-17 (Th17).

PubMed

Kuklina, E M; Glebezdina, N S; Nekrasova, I V

2016-03-01

We studied the ability of melatonin in physiological and pharmacological concentrations to induce and/or regulate differentiation of T cells producing IL-17 (Th17). This hormone produced the opposite effect on CD4+T cells, which depended on their activation status. Melatonin induced the synthesis of IL-17A by intact T cells, but had little effect on activated cells. Melatonin in high (pharmacological) concentration decreased the intracellular expression of this cytokine under conditions of polyclonal activation. Melatonin had a dose-depended effect. Taking into the fact that Th17 cells play an important role in the immune defense, it can be suggested that the regulation of their activity by melatonin contributes to this process.

Vγ4 T Cells Inhibit the Pro-healing Functions of Dendritic Epidermal T Cells to Delay Skin Wound Closure Through IL-17A

PubMed Central

Li, Yashu; Wang, Yangping; Zhou, Lina; Liu, Meixi; Liang, Guangping; Yan, Rongshuai; Jiang, Yufeng; Hao, Jianlei; Zhang, Xiaorong; Hu, Xiaohong; Huang, Yong; Wang, Rupeng; Yin, Zhinan; Wu, Jun; Luo, Gaoxing; He, Weifeng

2018-01-01

Dendritic epidermal T cells (DETCs) and dermal Vγ4 T cells engage in wound re-epithelialization and skin inflammation. However, it remains unknown whether a functional link between Vγ4 T cell pro-inflammation and DETC pro-healing exists to affect the outcome of skin wound closure. Here, we revealed that Vγ4 T cell-derived IL-17A inhibited IGF-1 production by DETCs to delay skin wound healing. Epidermal IL-1β and IL-23 were required for Vγ4 T cells to suppress IGF-1 production by DETCs after skin injury. Moreover, we clarified that IL-1β rather than IL-23 played a more important role in inhibiting IGF-1 production by DETCs in an NF-κB-dependent manner. Together, these findings suggested a mechanistic link between Vγ4 T cell-derived IL-17A, epidermal IL-1β/IL-23, DETC-derived IGF-1, and wound-healing responses in the skin. PMID:29483920

Type17 T-cells in Central Nervous System Autoimmunity and Tumors

PubMed Central

Okada, Hideho; Khoury, Samia J.

2012-01-01

Interleukin-17 (IL-17) producing Type17 T-cells, specifically T-helper (Th)17 cells reactive to central nervous system (CNS) autoantigens, manifest a higher migratory capability to the CNS parenchyma compared with other T-cell subpopulations due to their ability to penetrate the blood brain barrier (BBB). In the field of cancer immunotherapy, there are now a number of cell therapy approaches including early studies using T-cells transduced with chimeric antigen receptors in hematologic malignancy, suggesting that the use of T-cells or genetically modified T-cells could have a significant role in effective cancer therapy. However, the successful application of this strategy in solid tumors, such as CNS tumors, requires careful consideration of critical factors to improve the tumor-homing of T-cells. The current review is dedicated to discuss recent findings on the role of Type17 T-cells in CNS autoimmunity and cancer. The insight gained from these findings may lead to the development of novel therapeutic and prophylactic strategies for CNS autoimmunity and tumors. PMID:22454247

Role of the UGT2B17 deletion in exemestane pharmacogenetics

PubMed Central

Luo, Shaman; Chen, Gang; Truica, Cristina; Baird, Cynthia C.; Leitzel, Kim; Lazarus, Philip

2017-01-01

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients’ urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs. subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs. subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs. subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo. PMID:28534527

Role of the UGT2B17 deletion in exemestane pharmacogenetics.

PubMed

Luo, S; Chen, G; Truica, C; Baird, C C; Leitzel, K; Lazarus, P

2018-04-01

Exemestane (EXE) is an aromatase inhibitor used for the prevention and treatment of breast cancer. The major metabolic pathway for EXE is reduction to form the active 17β-dihydro-EXE (17β-DHE) and subsequent glucuronidation to 17β-hydroxy-EXE-17-O-β-D-glucuronide (17β-DHE-Gluc) by UGT2B17. The aim of the present study was to determine the effects of UGT2B17 copy number variation on the levels of urinary and plasma 17β-DHE-Gluc and 17β-DHE in patients taking EXE. Ninety-six post-menopausal Caucasian breast cancer patients with ER+ breast tumors taking 25 mg EXE daily were recruited into this study. UGT2B17 copy number was determined by a real-time PCR copy number variant assay and the levels of EXE, 17β-DHE and 17β-DHE-Gluc were quantified by UPLC/MS in patients' urine and plasma. A 39-fold decrease (P<0.0001) in the levels of creatinine-adjusted urinary 17β-DHE-Gluc was observed among UGT2B17 (*2/*2) subjects vs subjects with the UGT2B17 (*1/*1) genotype. The plasma levels of 17β-DHE-Gluc was decreased 29-fold (P<0.0001) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with UGT2B17 (*1/*1) genotype. The levels of plasma EXE-adjusted 17β-DHE was 28% higher (P=0.04) in subjects with the UGT2B17 (*2/*2) genotype vs subjects with the UGT2B17 (*1/*1) genotype. These data indicate that UGT2B17 is the major enzyme responsible for 17β-DHE-Gluc formation in vivo and that the UGT2B17 copy number variant may play a role in inter-individual variability in 17β-DHE levels in vivo.

Impact of stirred suspension bioreactor culture on the differentiation of murine embryonic stem cells into cardiomyocytes.

PubMed

Shafa, Mehdi; Krawetz, Roman; Zhang, Yuan; Rattner, Jerome B; Godollei, Anna; Duff, Henry J; Rancourt, Derrick E

2011-12-14

Embryonic stem cells (ESCs) can proliferate endlessly and are able to differentiate into all cell lineages that make up the adult organism. Under particular in vitro culture conditions, ESCs can be expanded and induced to differentiate into cardiomyocytes in stirred suspension bioreactors (SSBs). However, in using these systems we must be cognizant of the mechanical forces acting upon the cells. The effect of mechanical forces and shear stress on ESC pluripotency and differentiation has yet to be clarified. The purpose of this study was to investigate the impact of the suspension culture environment on ESC pluripotency during cardiomyocyte differentiation. Murine D3-MHC-neo(r) ESCs formed embyroid bodies (EBs) and differentiated into cardiomyocytes over 25 days in static culture and suspension bioreactors. G418 (Geneticin) was used in both systems from day 10 to enrich for cardiomyocytes by eliminating non-resistant, undifferentiated cells. Treatment of EBs with 1 mM ascorbic acid and 0.5% dimethyl sulfoxide from day 3 markedly increased the number of beating EBs, which displayed spontaneous and cadenced contractile beating on day 11 in the bioreactor. Our results showed that the bioreactor differentiated cells displayed the characteristics of fully functional cardiomyocytes. Remarkably, however, our results demonstrated that the bioreactor differentiated ESCs retained their ability to express pluripotency markers, to form ESC-like colonies, and to generate teratomas upon transplantation, whereas the cells differentiated in adherent culture lost these characteristics. This study demonstrates that although cardiomyocyte differentiation can be achieved in stirred suspension bioreactors, the addition of medium enhancers is not adequate to force complete differentiation as fluid shear forces appear to maintain a subpopulation of cells in a transient pluripotent state. The development of successful ESC differentiation protocols within suspension bioreactors demands a

Rebamipide suppresses collagen-induced arthritis through reciprocal regulation of th17/treg cell differentiation and heme oxygenase 1 induction.

PubMed

Moon, Su-Jin; Park, Jin-Sil; Woo, Yun-Ju; Lim, Mi-Ae; Kim, Sung-Min; Lee, Seon-Yeong; Kim, Eun-Kyung; Lee, Hee Jin; Lee, Weon Sun; Park, Sang-Hi; Jeong, Jeong-Hee; Park, Sung-Hwan; Kim, Ho-Youn; Cho, Mi-La; Min, Jun-Ki

2014-04-01

Rebamipide, a gastroprotective agent, has the ability to scavenge reactive oxygen radicals. Increased oxidative stress is implicated in the pathogenesis of rheumatoid arthritis (RA). We undertook this study to investigate the impact of rebamipide on the development of arthritis and the pathophysiologic mechanisms by which rebamipide attenuates arthritis severity in a murine model of RA. Collagen-induced arthritis (CIA) was induced in DBA/1J mice. Anti-type II collagen antibody titers and interleukin-17 (IL-17) levels were determined using enzyme-linked immunosorbent assay. The expression of transcription factors was analyzed by immunostaining and Western blotting. Frequencies of IL-17-producing CD4+ T cells (Th17 cells) and CD4+CD25+FoxP3+ Treg cells were analyzed by flow cytometry. Rebamipide reduced the clinical arthritis score and severity of histologic inflammation and cartilage destruction in a dose-dependent manner. The joints isolated from rebamipide-treated mice with CIA showed decreased expression of nitrotyrosine, an oxidative stress marker. Rebamipide-treated mice showed lower circulating levels of type II collagen-specific IgG, IgG1, and IgG2a. Whereas the number of Th17 cells in spleens was decreased in rebamipide-treated mice with CIA, a significant increase in the number of Treg cells in spleens was observed. In vitro, rebamipide inhibited Th17 cell differentiation through STAT-3/retinoic acid receptor-related orphan nuclear receptor γt and reciprocally induced Treg cell differentiation through FoxP3. Rebamipide increased Nrf2 nuclear activities in murine CD4+ T cells and LBRM-33 murine T lymphoma cells. Heme oxygenase 1 (HO-1) expression in the spleens was markedly increased in rebamipide-treated mice. The inhibitory effects of rebamipide on joint inflammation are associated with recovery from an imbalance between Th17 cells and Treg cells and with activation of an Nrf2/HO-1 antioxidant pathway. Copyright © 2014 by the American College of

Rapamycin efficiently promotes cardiac differentiation of mouse embryonic stem cells.

PubMed

Lu, Qin; Liu, Yinan; Wang, Yang; Wang, Weiping; Yang, Zhe; Li, Tao; Tian, Yuyao; Chen, Ping; Ma, Kangtao; Jia, Zhuqing; Zhou, Chunyan

2017-06-30

To investigate the effects of rapamycin on cardiac differentiation, murine embryonic stem cells (ESCs) were induced into cardiomyocytes by 10 -4 M ascorbic acid (AA), 20 nM rapamycin alone or 0.01% solvent DMSO. We found that rapamycin alone was insufficient to initiate cardiomyogenesis. Then, the ESCs were treated with AA and rapamycin (20 nM) or AA and DMSO (0.01%) as a control. Compared with control, mouse ESCs (mESCs) treated with rapamycin (20 nM) and AA yielded a significantly higher percentage of cardiomyocytes, as confirmed by the percentage of beating embryonic bodies (EBs), the immunofluorescence and FACS analysis. Rapamycin significantly increased the expression of a panel of cardiac markers including Gata 4, α- Mhc , β- Mhc , and Tnnt 2. Additionally, rapamycin enhanced the expression of mesodermal and cardiac transcription factors such as Mesp 1, Brachyury T, Eomes, Isl 1 , Gata 4 , Nkx 2.5 , Tbx 5, and Mef2c. Mechanistic studies showed that rapamycin inhibits Wnt/β-catenin and Notch signaling but promotes the expression of fibroblast growth factor ( Fgf 8), Fgf 10, and Nodal at early stage, and bone morphogenetic protein 2 ( Bmp 2) at later stages. Sequential treatment of rapamycin showed that rapamycin promotes cardiac differentiation at the early and later stages. Interestingly, another mammalian target of rapamycin (mTOR) inhibitor Ku0063794 (1 µM) had similar effects on cardiomyogenesis. In conclusion, our results highlight a practical approach to generate cardiomyocytes from mESCs by rapamycin. © 2017 The Author(s).

Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus.

PubMed

Chesebro, John; Hrycaj, Steven; Mahfooz, Najmus; Popadić, Aleksandar

2009-05-01

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development.

Diverging functions of Scr between embryonic and post-embryonic development in a hemimetabolous insect, Oncopeltus fasciatus

PubMed Central

Chesebro, John; Hrycaj, Steven; Mahfooz, Najmus; Popadić, Aleksandar

2009-01-01

Hemimetabolous insects undergo an ancestral mode of development in which embryos hatch into first nymphs that resemble miniature adults. While recent studies have shown that homeotic (hox) genes establish segmental identity of first nymphs during embryogenesis, no information exists on the function of these genes during post-embryogenesis. To determine whether and to what degree hox genes influence the formation of adult morphologies, we performed a functional analysis of Sex combs reduced (Scr) during post-embryonic development in Oncopeltus fasciatus. The main effect was observed in prothorax of Scr-RNAi adults, and ranged from significant alterations in its size and shape to a near complete transformation of its posterior half toward a T2-like identity. Furthermore, while the consecutive application of Scr-RNAi at both of the final two post-embryonic stages (fourth and fifth) did result in formation of ectopic wings on T1, the individual applications at each of these stages did not. These experiments provide two new insights into evolution of wings. First, the role of Scr in wing repression appears to be conserved in both holo- and hemimetabolous insects. Second, the prolonged Scr-depletion (spanning at least two nymphal stages) is both necessary and sufficient to restart wing program. At the same time, other structures that were previously established during embryogenesis are either unaffected (T1 legs) or display only minor changes (labium) in adults. These observations reveal a temporal and spatial divergence of Scr roles during embryonic (main effect in labium) and post-embryonic (main effect in prothorax) development. PMID:19382295

Murine cell glycolipids customization by modular expression of glycosyltransferases.

PubMed

Cid, Emili; Yamamoto, Miyako; Buschbeck, Marcus; Yamamoto, Fumiichiro

2013-01-01

Functional analysis of glycolipids has been hampered by their complex nature and combinatorial expression in cells and tissues. We report an efficient and easy method to generate cells with specific glycolipids. In our proof of principle experiments we have demonstrated the customized expression of two relevant glycosphingolipids on murine fibroblasts, stage-specific embryonic antigen 3 (SSEA-3), a marker for stem cells, and Forssman glycolipid, a xenoantigen. Sets of genes encoding glycosyltansferases were transduced by viral infection followed by multi-color cell sorting based on coupled expression of fluorescent proteins.

Nck recruitment to Eph receptor, EphB1/ELK, couples ligand activation to c-Jun kinase.

PubMed

Stein, E; Huynh-Do, U; Lane, A A; Cerretti, D P; Daniel, T O

1998-01-16

Eph family receptor tyrosine kinases signal axonal guidance, neuronal bundling, and angiogenesis; yet the signaling systems that couple these receptors to targeting and cell-cell assembly responses are incompletely defined. Functional links to regulators of cytoskeletal structure are anticipated based on receptor mediated cell-cell aggregation and migratory responses. We used two-hybrid interaction cloning to identify EphB1-interactive proteins. Six independent cDNAs encoding the SH2 domain of the adapter protein, Nck, were recovered in a screen of a murine embryonic library. We mapped the EphB1 subdomain that binds Nck and its Drosophila homologue, DOCK, to the juxtamembrane region. Within this subdomain, Tyr594 was required for Nck binding. In P19 embryonal carcinoma cells, activation of EphB1 (ELK) by its ligand, ephrin-B1/Fc, recruited Nck to native receptor complexes and activated c-Jun kinase (JNK/SAPK). Transient overexpression of mutant EphB1 receptors (Y594F) blocked Nck recruitment to EphB1, attenuated downstream JNK activation, and blocked cell attachment responses. These findings identify Nck as an important intermediary linking EphB1 signaling to JNK.

CD4 T cells play important roles in maintaining IL-17-producing γδ T-cell subsets in naive animals.

PubMed

Do, Jeong-Su; Visperas, Anabelle; O'Brien, Rebecca L; Min, Booki

2012-04-01

A proportional balance between αβ and γδ T-cell subsets in the periphery is exceedingly well maintained by a homeostatic mechanism. However, a cellular mechanism underlying the regulation remains undefined. We recently reported that a subset of developing γδ T cells spontaneously acquires interleukin (IL)-17-producing capacity even within naive animals through a transforming growth factor (TGF)β1-dependent mechanism, thus considered 'innate' IL-17-producing cells. Here, we report that γδ T cells generated within αβ T cell (or CD4 T cell)-deficient environments displayed altered cytokine profiles; particularly, 'innate' IL-17 expression was significantly impaired compared with those in wild-type mice. Impaired IL-17 production in γδ T cells was directly related to CD4 T-cell deficiency, because depletion of CD4 T cells in wild-type mice diminished and adoptive CD4 T-cell transfer into T-cell receptor β-/- mice restored IL-17 expression in γδ T cells. CD4 T cell-mediated IL-17 expression required TGFβ1. Moreover, Th17 but not Th1 or Th2 effector CD4 T cells were highly efficient in enhancing γδ T-cell IL-17 expression. Taken together, our results highlight a novel CD4 T cell-dependent mechanism that shapes the generation of IL-17+ γδ T cells in naive settings.

The mutational landscape of MYCN, Lin28b and ALK F1174L driven murine neuroblastoma mimics human disease.

PubMed

De Wilde, Bram; Beckers, Anneleen; Lindner, Sven; Kristina, Althoff; De Preter, Katleen; Depuydt, Pauline; Mestdagh, Pieter; Sante, Tom; Lefever, Steve; Hertwig, Falk; Peng, Zhiyu; Shi, Le-Ming; Lee, Sangkyun; Vandermarliere, Elien; Martens, Lennart; Menten, Björn; Schramm, Alexander; Fischer, Matthias; Schulte, Johannes; Vandesompele, Jo; Speleman, Frank

2018-02-02

Genetically engineered mouse models have proven to be essential tools for unraveling fundamental aspects of cancer biology and for testing novel therapeutic strategies. To optimally serve these goals, it is essential that the mouse model faithfully recapitulates the human disease. Recently, novel mouse models for neuroblastoma have been developed. Here, we report on the further genomic characterization through exome sequencing and DNA copy number analysis of four of the currently available murine neuroblastoma model systems ( ALK, Th- MYCN, Dbh- MYCN and Lin28b ). The murine tumors revealed a low number of genomic alterations - in keeping with human neuroblastoma - and a positive correlation of the number of genetic lesions with the time to onset of tumor formation was observed. Gene copy number alterations are the hallmark of both murine and human disease and frequently affect syntenic genomic regions. Despite low mutational load, the genes mutated in murine disease were found to be enriched for genes mutated in human disease. Taken together, our study further supports the validity of the tested mouse models for mechanistic and preclinical studies of human neuroblastoma.

Ibrutinib treatment ameliorates murine chronic graft-versus-host disease

PubMed Central

Dubovsky, Jason A.; Flynn, Ryan; Du, Jing; Harrington, Bonnie K.; Zhong, Yiming; Kaffenberger, Benjamin; Yang, Carrie; Towns, William H.; Lehman, Amy; Johnson, Amy J.; Muthusamy, Natarajan; Devine, Steven M.; Jaglowski, Samantha; Serody, Jonathan S.; Murphy, William J.; Munn, David H.; Luznik, Leo; Hill, Geoffrey R.; Wong, Henry K.; MacDonald, Kelli K.P.; Maillard, Ivan; Koreth, John; Elias, Laurence; Cutler, Corey; Soiffer, Robert J.; Antin, Joseph H.; Ritz, Jerome; Panoskaltsis-Mortari, Angela; Byrd, John C.; Blazar, Bruce R.

2014-01-01

Chronic graft-versus-host disease (cGVHD) is a life-threatening impediment to allogeneic hematopoietic stem cell transplantation, and current therapies do not completely prevent and/or treat cGVHD. CD4+ T cells and B cells mediate cGVHD; therefore, targeting these populations may inhibit cGVHD pathogenesis. Ibrutinib is an FDA-approved irreversible inhibitor of Bruton’s tyrosine kinase (BTK) and IL-2 inducible T cell kinase (ITK) that targets Th2 cells and B cells and produces durable remissions in B cell malignancies with minimal toxicity. Here, we evaluated whether ibrutinib could reverse established cGVHD in 2 complementary murine models, a model interrogating T cell–driven sclerodermatous cGVHD and an alloantibody-driven multiorgan system cGVHD model that induces bronchiolar obliterans (BO). In the T cell–mediated sclerodermatous cGVHD model, ibrutinib treatment delayed progression, improved survival, and ameliorated clinical and pathological manifestations. In the alloantibody-driven cGVHD model, ibrutinib treatment restored pulmonary function and reduced germinal center reactions and tissue immunoglobulin deposition. Animals lacking BTK and ITK did not develop cGVHD, indicating that these molecules are critical to cGVHD development. Furthermore, ibrutinib treatment reduced activation of T and B cells from patients with active cGVHD. Our data demonstrate that B cells and T cells drive cGVHD and suggest that ibrutinib has potential as a therapeutic agent, warranting consideration for cGVHD clinical trials. PMID:25271622

The 17D-204 Vaccine Strain-Induced Protection against Virulent Yellow Fever Virus Is Mediated by Humoral Immunity and CD4+ but not CD8+ T Cells.

PubMed

Watson, Alan M; Lam, L K Metthew; Klimstra, William B; Ryman, Kate D

2016-07-01

A gold standard of antiviral vaccination has been the safe and effective live-attenuated 17D-based yellow fever virus (YFV) vaccines. Among more than 500 million vaccinees, only a handful of cases have been reported in which vaccinees developed a virulent wild type YFV infection. This efficacy is presumed to be the result of both neutralizing antibodies and a robust T cell response. However, the particular immune components required for protection against YFV have never been evaluated. An understanding of the immune mechanisms that underlie 17D-based vaccine efficacy is critical to the development of next-generation vaccines against flaviviruses and other pathogens. Here we have addressed this question for the first time using a murine model of disease. Similar to humans, vaccination elicited long-term protection against challenge, characterized by high neutralizing antibody titers and a robust T cell response that formed long-lived memory. Both CD4+ and CD8+ T cells were polyfunctional and cytolytic. Adoptive transfer of immune sera or CD4+ T cells provided partial protection against YFV, but complete protection was achieved by transfer of both immune sera and CD4+ T cells. Thus, robust CD4+ T cell activity may be a critical contributor to protective immunity elicited by highly effective live attenuated vaccines.

The 17D-204 Vaccine Strain-Induced Protection against Virulent Yellow Fever Virus Is Mediated by Humoral Immunity and CD4+ but not CD8+ T Cells

PubMed Central

Lam, L. K. Metthew; Klimstra, William B.

2016-01-01

A gold standard of antiviral vaccination has been the safe and effective live-attenuated 17D-based yellow fever virus (YFV) vaccines. Among more than 500 million vaccinees, only a handful of cases have been reported in which vaccinees developed a virulent wild type YFV infection. This efficacy is presumed to be the result of both neutralizing antibodies and a robust T cell response. However, the particular immune components required for protection against YFV have never been evaluated. An understanding of the immune mechanisms that underlie 17D-based vaccine efficacy is critical to the development of next-generation vaccines against flaviviruses and other pathogens. Here we have addressed this question for the first time using a murine model of disease. Similar to humans, vaccination elicited long-term protection against challenge, characterized by high neutralizing antibody titers and a robust T cell response that formed long-lived memory. Both CD4+ and CD8+ T cells were polyfunctional and cytolytic. Adoptive transfer of immune sera or CD4+ T cells provided partial protection against YFV, but complete protection was achieved by transfer of both immune sera and CD4+ T cells. Thus, robust CD4+ T cell activity may be a critical contributor to protective immunity elicited by highly effective live attenuated vaccines. PMID:27463517

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes

PubMed Central

Tarnawski, Laura; Xian, Xiaojie; Monnerat, Gustavo; Macaulay, Iain C.; Malan, Daniela; Borgman, Andrew; Wu, Sean M.; Fleischmann, Bernd K.; Jovinge, Stefan

2015-01-01

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes. PMID:26323090

Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

PubMed

Choi, Seung-Chul; Xu, Zhiwei; Li, Wei; Yang, Hong; Roopenian, Derry C; Morse, Herbert C; Morel, Laurence

2018-05-01

Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4 + T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4 + T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4 + T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4 + T cells were introduced into Rag1 -/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4 + T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4 + T cells in a nonredundant manner with myeloid/stromal cells. Copyright © 2018 by The American Association of Immunologists, Inc.

Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation.

PubMed

Stennard, Fiona A; Costa, Mauro W; Lai, Donna; Biben, Christine; Furtado, Milena B; Solloway, Mark J; McCulley, David J; Leimena, Christiana; Preis, Jost I; Dunwoodie, Sally L; Elliott, David E; Prall, Owen W J; Black, Brian L; Fatkin, Diane; Harvey, Richard P

2005-05-01

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program, defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical, repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20, underlie both congenital heart disease and adult cardiomyopathies.

T Helper 17 Cells Interplay with CD4+CD25highFoxp3+ Tregs in Regulation of Inflammations and Autoimmune Diseases

PubMed Central

Mai, Jietang; Wang, Hong; Yang#, Xiao-Feng

2010-01-01

Interleukin-17 (IL-17)-secreting T helper 17 cells (Th17) are a recently identified CD4+ T helper subset that has been implicated in various inflammatory and autoimmune diseases. Th17, along with CD4+CD25high Foxp3+ regulatory T cells (Tregs) and other newly emergent T helper subsets, Th9 and Tfh, have expanded the Th1-Th2 paradigm. Although this newly proposed six-subset paradigm significantly improved our understanding on the differentiation of CD4+ T helper cell subsets and the regulation of T helper cells in inflammation and autoimmunity, many questions remain to be answered. In this overview, we will briefly review the following issues: a) Old Th1-Th2 paradigm versus new multi-subset paradigm; b) Structural features of IL-17 family cytokines; c) Th17 cells; d) Effects of IL-17 on various cell types and tissues; e) IL-17 receptor and signaling pathways; f) Th17-mediated inflammations; and g) Protective mechanisms of IL-17 in infections. Lastly, we will look into the interaction of Th17 and Treg in autoimmune diseases and inflammation: Th17 cells interplay with Tregs. Regulation of autoimmunity and inflammation lies in the interplays of the different T helper subsets, therefore, better understanding of these subsets’ interactions with one another would greatly improve our approaches in developing therapy to combat inflammatory and autoimmune diseases. PMID:20515737

Th1 and Th17 Immunocompetence in Humanized NOD/SCID/γC-KO mice

PubMed Central

Rajesh, Deepika; Zhou, Ying; Jankowska-Gan, Ewa; Ronneburg, Drew Allan; Dart, Melanie M; Torrealba, Jose; Burlingham, William J

2010-01-01

We evaluated the immunocompetence of human T cells in humanized NOD-scid IL2r-γ-null (Hu—NSG) mice bearing a human thymic organoid, after multilinegage reconstitution with isogeneic human leukocytes. Delayed type hypersensitivity (DTH) response was assessed by a direct footpad challenge of the immunized hu-NSG host, or by transfer of splenocytes from immunized hu-NSG, along with antigen, into footpads of CB17 SCID mice [trans-vivo (tv) DTH]. Both methods revealed cellular immunity to tetanus toxoid (TT) or collagen type V (ColV). Immunohistochemical analysis of the swollen footpads revealed infiltration of human CD45+ cells, including CD3+ T cells, CD68+ macrophages and murine Ly6G+ neutrophils. We observed a significant correlation between % circulating human CD4+ cells and the direct DTH swelling response to TT. The tvDTH response to TT was inhibited by anti-IFNγ, while the tvDTH response to collagen V was inhibited by anti IL-17 antibody, mimicking the cytokine bias of adult human T cells to these antigens. Hu-NSG mice were also capable of mounting a B cell response (primarily IgM) to TT antigen. The activation of either Th1- or Th17 - dependent cellular immune response supports the utility of Hu-NSG mice as a surrogate model of allograft rejection and autoimmunity. PMID:20298731

In Vitro T-Cell Generation From Adult, Embryonic, and Induced Pluripotent Stem Cells: Many Roads to One Destination.

PubMed

Smith, Michelle J; Webber, Beau R; Mohtashami, Mahmood; Stefanski, Heather E; Zúñiga-Pflücker, Juan Carlos; Blazar, Bruce R

2015-11-01

T lymphocytes are critical mediators of the adaptive immune system and have the capacity to serve as therapeutic agents in the areas of transplant and cancer immunotherapy. While T cells can be isolated and expanded from patients, T cells derived in vitro from both hematopoietic stem/progenitor cells (HSPCs) and human pluripotent stem cells (hPSCs) offer great potential advantages in generating a self-renewing source of T cells that can be readily genetically modified. T-cell differentiation in vivo is a complex process requiring tightly regulated signals; providing the correct signals in vitro to induce T-cell lineage commitment followed by their development into mature, functional, single positive T cells, is similarly complex. In this review, we discuss current methods for the in vitro derivation of T cells from murine and human HSPCs and hPSCs that use feeder-cell and feeder-cell-free systems. Furthermore, we explore their potential for adoption for use in T-cell-based therapies. © 2015 AlphaMed Press.

Concurrent CCR7 Overexpression and RelB Knockdown in Immature Dendritic Cells Induces Immune Tolerance and Improves Skin-Graft Survival in a Murine Model.

PubMed

Dong, Zhiwei; Chen, Yajie; Peng, Yuan; Wang, Fan; Yang, Zichen; Huang, Guangtao; Chen, Yu; Yuan, Zhiqiang; Cao, Tongtong; Peng, Yizhi

2017-01-01

Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection. © 2017 The Author(s). Published by S. Karger AG, Basel.

Increased T follicular helper cells and germinal center B cells are required for cGVHD and bronchiolitis obliterans

PubMed Central

Flynn, Ryan; Du, Jing; Veenstra, Rachelle G.; Reichenbach, Dawn K.; Panoskaltsis-Mortari, Angela; Taylor, Patricia A.; Freeman, Gordon J.; Serody, Jonathan S.; Murphy, William J.; Munn, David H.; Sarantopoulos, Stefanie; Luznik, Leo; Maillard, Ivan; Koreth, John; Cutler, Corey; Soiffer, Robert J.; Antin, Joseph H.; Ritz, Jerome; Dubovsky, Jason A.; Byrd, John C.; MacDonald, Kelli P.; Hill, Geoff R.; Blazar, Bruce R.

2014-01-01

Chronic graft-versus-host disease (cGVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Having shown that germinal center (GC) formation and immunoglobulin deposition are required for multiorgan system cGVHD and associated bronchiolitis obliterans syndrome (BOS) in a murine model, we hypothesized that T follicular helper (Tfh) cells are necessary for cGVHD by supporting GC formation and maintenance. We show that increased frequency of Tfh cells correlated with increased GC B cells, cGVHD, and BOS. Although administering a highly depletionary anti-CD20 monoclonal antibody (mAb) to mice with established cGVHD resulted in peripheral B-cell depletion, B cells remained in the lung, and BOS was not reversed. BOS could be treated by eliminating production of interleukin-21 (IL-21) by donor T cells or IL-21 receptor (IL-21R) signaling of donor B cells. Development of BOS was dependent upon T cells expressing the chemokine receptor CXCR5 to facilitate T-cell trafficking to secondary lymphoid organ follicles. Blocking mAbs for IL-21/IL-21R, inducible T-cell costimulator (ICOS)/ICOS ligand, and CD40L/CD40 hindered GC formation and cGVHD. These data provide novel insights into cGVHD pathogenesis, indicate a role for Tfh cells in these processes, and suggest a new line of therapy using mAbs targeting Tfh cells to reverse cGVHD. PMID:24820310

Murine T cell activation is regulated by surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide)

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

Warford, Jordan, E-mail: jordan.warford@dal.ca; Doucette, Carolyn D., E-mail: carolyn.doucette@dal.ca; Hoskin, David W., E-mail: d.w.hoskin@dal.ca

2014-01-10

Highlights: •Surfen is the first inhibitor of glycosaminoglycan function to be studied in murine T cells. •Surfen reduces T cell proliferation stimulated in vitro and in vivo. •Surfen reduces CD25 expression in T cells activated in vivo but not in vitro. •Surfen increases T cell proliferation when T cell receptor activation is bypassed. •Surfen’s effects are blocked by co-administration of heparin sulfate. -- Abstract: Surfen (bis-2-methyl-4-amino-quinolyl-6-carbamide) binds to glycosaminoglycans (GAGs) and has been shown to influence their function, and the function of proteoglycans (complexes of GAGs linked to a core protein). T cells synthesize, secrete and express GAGs and proteoglycansmore » which are involved in several aspects of T cell function. However, there are as yet no studies on the effect of GAG-binding agents such as surfen on T cell function. In this study, surfen was found to influence murine T cell activation. Doses between 2.5 and 20 μM produced a graduated reduction in the proliferation of T cells activated with anti-CD3/CD28 antibody-coated T cell expander beads. Surfen (20 mg/kg) was also administered to mice treated with anti-CD3 antibody to activate T cells in vivo. Lymphocytes from surfen-treated mice also showed reduced proliferation and lymph node cell counts were reduced. Surfen reduced labeling with a cell viability marker (7-ADD) but to a much lower extent than its effect on proliferation. Surfen also reduced CD25 (the α-subunit of the interleukin (IL)-2 receptor) expression with no effect on CD69 expression in T cells treated in vivo but not in vitro. When receptor activation was bypassed by treating T cells in vitro with phorbyl myristate acetate (10 ng/ml) and ionomycin (100 ng/ml), surfen treatment either increased proliferation (10 μM) or had no effect (2.5, 5 and 20 μM). In vitro treatment of T cells with surfen had no effect on IL-2 or interferon-γ synthesis and did not alter proliferation of the IL-2

CD4+ cell-derived interleukin-17 in a model of dysregulated, Borrelia-induced arthritis.

PubMed

Hansen, Emily S; Johnson, Megan E; Schell, Ronald F; Nardelli, Dean T

2016-10-01

Lyme borreliosis, which is caused in the United States by the spirochete Borrelia burgdorferi, may manifest as different arrays of signs, symptoms and severities between infected individuals. Recent studies have indicated that particularly severe forms of Lyme borreliosis in humans are associated with an increased Th17 response. Here, we hypothesized that a murine model combining the dysregulated immune response of an environment lacking interleukin-10 (IL-10) with a robust T-cell-driven inflammatory response would reflect arthritis associated with the production of IL-17 by CD4+ cells. We demonstrate that IL-10 regulates the production of IL-17 by Borrelia-primed CD4+ cells early after interaction with Lyme spirochetes in vitro and that infection of Borrelia-primed mice with B. burgdorferi leads to significant production of IL-17 that contributes to the development of severe arthritis. These results extend our previous findings by demonstrating that a dysregulated adaptive immune response to Lyme spirochetes can contribute to severe, Th17-associated arthritis. These findings may lead to therapeutic measures for individuals with particularly severe symptoms of Lyme borreliosis. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

In utero imaging of mouse embryonic development with optical coherence tomography

NASA Astrophysics Data System (ADS)

Syed, Saba H.; Dickinson, Mary E.; Larin, Kirill V.; Larina, Irina V.

2011-03-01

Studying progression of congenital diseases in animal models can greatly benefit from live embryonic imaging Mouse have long served as a model of mammalian embryonic developmental processes, however, due to intra-uterine nature of mammalian development live imaging is challenging. In this report we present results on live mouse embryonic imaging in utero with Optical Coherence Tomography. Embryos from 12.5 through 17.5 days post-coitus (dpc) were studied through the uterine wall. In longitudinal studies, same embryos were imaged at developmental stages 13.5, 15.5 and 17.5 dpc. This study suggests that OCT can serve as a powerful tool for live mouse embryo imaging. Potentially this technique can contribute to our understanding developmental abnormalities associated with mutations, toxic drugs.

DNA methyltransferase inhibition increases efficacy of adoptive cellular immunotherapy of murine breast cancer.

PubMed

Terracina, Krista P; Graham, Laura J; Payne, Kyle K; Manjili, Masoud H; Baek, Annabel; Damle, Sheela R; Bear, Harry D

2016-09-01

Adoptive T cell immunotherapy is a promising approach to cancer treatment that currently has limited clinical applications. DNA methyltransferase inhibitors (DNAMTi) have known potential to affect the immune system through multiple mechanisms that could enhance the cytotoxic T cell responses, including: upregulation of tumor antigen expression, increased MHC class I expression, and blunting of myeloid derived suppressor cells (MDSCs) expansion. In this study, we have investigated the effect of combining the DNAMTi, decitabine, with adoptive T cell immunotherapy in the murine 4T1 mammary carcinoma model. We found that expression of neu, MHC class I molecules, and several murine cancer testis antigens (CTA) was increased by decitabine treatment of 4T1 cells in vitro. Decitabine also increased expression of multiple CTA in two human breast cancer cell lines. Decitabine-treated 4T1 cells stimulated greater IFN-gamma release from tumor-sensitized lymphocytes, implying increased immunogenicity. Expansion of CD11b + Gr1 + MDSC in 4T1 tumor-bearing mice was significantly diminished by decitabine treatment. Decitabine treatment improved the efficacy of adoptive T cell immunotherapy in mice with established 4T1 tumors, with greater inhibition of tumor growth and an increased cure rate. Decitabine may have a role in combination with existing and emerging immunotherapies for breast cancer.

IL-1R and MyD88 signalling in CD4+ T cells promote Th17 immunity and atherosclerosis.

PubMed

Engelbertsen, Daniel; Rattik, Sara; Wigren, Maria; Vallejo, Jenifer; Marinkovic, Goran; Schiopu, Alexandru; Björkbacka, Harry; Nilsson, Jan; Bengtsson, Eva

2018-01-01

The role of CD4+ T cells in atherosclerosis has been shown to be dependent on cytokine cues that regulate lineage commitment into mature T helper sub-sets. In this study, we tested the roles of IL-1R1 and MyD88 signalling in CD4+ T cells in atherosclerosis. We transferred apoe-/-myd88+/+ or apoe-/-myd88-/- CD4+ T cells to T- and B-cell-deficient rag1-/-apoe-/- mice fed high fat diet. Mice given apoe-/-myd88-/- CD4+ T cells exhibited reduced atherosclerosis compared with mice given apoe-/-myd88+/+ CD4+ T cells. CD4+ T cells from apoe-/-myd88-/- produced less IL-17 but similar levels of IFN-γ. Treatment of human CD4+ T cells with a MyD88 inhibitor inhibited IL-17 secretion in vitro. Transfer of il1r1-/- CD4+ T cells recapitulated the phenotype seen by transfer of myd88-/- CD4+ T cells with reduced lesion development and a reduction in Th17 and IL-17 production compared with wild type CD4+ T cell recipients. Relative collagen content of lesions was reduced in mice receiving il1r1-/- CD4+ T cells. We demonstrate that both IL1R and MyD88 signalling in CD4+ T cells promote Th17 immunity, plaque growth and may regulate plaque collagen levels. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions please email: journals.permissions@oup.com.

Osteogenic gene expression of murine osteoblastic (MC3T3-E1) cells under cyclic tension

NASA Astrophysics Data System (ADS)

Kao, C. T.; Chen, C. C.; Cheong, U.-I.; Liu, S. L.; Huang, T. H.

2014-08-01

Low-level laser therapy (LLLT) can promote cell proliferation. The remodeling ability of the tension side of orthodontic teeth affects post-orthodontic stability. The purpose of the present study was to investigate the osteogenic effects of LLLT on osteoblast-like cells treated with a simulated tension system that provides a mechanical tension regimen. Murine osteoblastic (MC3T3-E1) cells were cultured in a Flexcell strain unit with programmed loads of 12% elongation at a frequency of 0.5 Hz for 24 and 48 h. The cultured cells were treated with a low-level diode laser using powers of 5 J and 10 J. The proliferation of MC3T3-E1 cells was determined using the Alamar Blue assay. The expression of osteogenic genes (type I collagen (Col-1), osteopontin (OPN), osteocalcin (OC), osteoprotegerin (OPG), receptor activator of nuclear factor kappa B ligand (RANKL), bone morphologic protein (BMP-2), and bone morphologic protein (BMP-4)) in MC3T3-E1 cells was analyzed using reverse transcription polymerase chain reaction (RT-PCR). The data were analyzed using one-way analysis of variance. The proliferation rate of tension-cultured MC3T3-E1 cells under 5 J and 10 J LLLT increased compared with that of the control group (p < 0.05). Prominent mineralization of the MC3T3-E1 cells was visible using a von Kossa stain in the 5 J LLLT group. Osteogenic genes (Col-1, OC, OPG and BMP-2) were significantly expressed in the MC3T3-E1 cells treated with 5 J and 10 J LLLT (p < 0.05). LLLT in tension-cultured MC3T3-E1 cells showed synergistic osteogenic effects, including increases in cell proliferation and Col-1, OPN, OC, OPG and BMP-2 gene expression. LLLT might be beneficial for bone remodeling on the tension side of orthodontics.

Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment.

PubMed

Liu, Xiaoling; Xu, Qian; Liu, Weiwei; Yao, Guodong; Zhao, Yeli; Xu, Fanxing; Hayashi, Toshihiko; Fujisaki, Hitomi; Hattori, Shunji; Tashiro, Shin-Ichi; Onodera, Satoshi; Yamato, Masayuki; Ikejima, Takashi

2018-04-01

Migration of fibroblast-like preadipocytes is important for the development of adipose tissue, whereas excessive migration is often responsible for impaired adipose tissue related with obesity and fibrotic diseases. Type I collagen (collagen I) is the most abundant component of extracellular matrix and has been shown to regulate fibroblast migration in vitro, but its role in adipose tissue is not known. Silibinin is a bioactive natural flavonoid with antioxidant and antimetastasis activities. In this study, we found that type I collagen coating promoted the proliferation and migration of murine 3T3-L1 preadipocytes in a dose-dependent manner, implying that collagen I could be an extracellular signal. Regarding the mechanisms of collagen I-stimulated 3T3-L1 migration, we found that NF-κB p65 is activated, including the increased nuclear translocation of NF-κB p65 as well as the upregulation of NF-κB p65 phosphorylation and acetylation, accompanied by the increased expressions of proinflammatory factors and the generation of reactive oxygen species (ROS). Reduction of collagen I-enhanced migration of cells by treatment with silibinin was associated with suppression of NF-κB p65 activity and ROS generation, and negatively correlated with the increasing sirt1 expression. Taken together, the enhanced migration of 3T3-L1 cells induced on collagen I-coated dish is mediated by the activation of NF-κB p65 function and ROS generation that can be alleviated with silibinin by upregulation of sirt1, leading to the repression of NF-κB p65 function and ROS generation.

In vitro and in vivo activities of posaconazole and amphotericin B in a murine invasive infection by Mucor circinelloides: poor efficacy of posaconazole.

PubMed

Salas, Valentina; Pastor, F Javier; Calvo, Enrique; Alvarez, Eduardo; Sutton, Deanna A; Mayayo, Emilio; Fothergill, Anette W; Rinaldi, Michael G; Guarro, Josep

2012-05-01

The in vitro susceptibility of 17 strains of Mucor circinelloides to amphotericin B and posaconazole was ascertained by using broth microdilution and disk diffusion methods and by determining the minimal fungicidal concentration (MFC). We evaluated the efficacy of posaconazole at 40 mg/kg of body weight/day and amphotericin B at 0.8 mg/kg/day in a neutropenic murine model of disseminated infection by M. circinelloides by using 6 different strains tested previously in vitro. In general, most of the posaconazole MICs were within the range of susceptibility or intermediate susceptibility, while the small inhibition zone diameters (IZDs) were indicative of nonsusceptibility for all isolates tested. The MFCs were ≥ 3 dilutions higher than the corresponding MICs. In contrast, amphotericin B showed good activity against all of the strains tested regardless of the method used. The in vivo studies demonstrated that amphotericin B was effective in prolonging survival and reducing the fungal load. Posaconazole showed poor in vivo efficacy with no correlation with the MIC values. The results suggested that posaconazole should be used with caution in the treatment of infections caused by Mucor circinelloides or by strains of Mucor not identified to the species level.

EFFECT OF ACUTE STRESS ON PLASMA B-CORTICOSTERONE, ESTRADIOL-17B AND TESTOSTERONE CONCENTRATIONS IN JUVENILE AMERICAN ALLIGATORS COLLECTED FROM THREE SITES WITHIN THE KISSIMMEE-EVERGLADES DRAINAGE BASIN IN FLORIDA (USA)

EPA Science Inventory

The effect of acute stress on plasma b-corticosterone (B), testosterone (T) and estradiol-17b (E2), concentrations in juvenile alligators collected from sites with varying sediment contaminants was examined in this study. Dramatic increases in plasma B concentrations were observe...

46 CFR 34.17-5 - Quantity of foam required-T/ALL.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 46 Shipping 1 2011-10-01 2011-10-01 false Quantity of foam required-T/ALL. 34.17-5 Section 34.17-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-5 Quantity of foam required—T/ALL. (a) Area protected. (1) For machinery...

46 CFR 34.17-5 - Quantity of foam required-T/ALL.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 46 Shipping 1 2014-10-01 2014-10-01 false Quantity of foam required-T/ALL. 34.17-5 Section 34.17-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-5 Quantity of foam required—T/ALL. (a) Area protected. (1) For machinery...

46 CFR 34.17-5 - Quantity of foam required-T/ALL.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 46 Shipping 1 2012-10-01 2012-10-01 false Quantity of foam required-T/ALL. 34.17-5 Section 34.17-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-5 Quantity of foam required—T/ALL. (a) Area protected. (1) For machinery...

46 CFR 34.17-5 - Quantity of foam required-T/ALL.

Code of Federal Regulations, 2013 CFR

2013-10-01

... 46 Shipping 1 2013-10-01 2013-10-01 false Quantity of foam required-T/ALL. 34.17-5 Section 34.17-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-5 Quantity of foam required—T/ALL. (a) Area protected. (1) For machinery...

46 CFR 34.17-5 - Quantity of foam required-T/ALL.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 46 Shipping 1 2010-10-01 2010-10-01 false Quantity of foam required-T/ALL. 34.17-5 Section 34.17-5 Shipping COAST GUARD, DEPARTMENT OF HOMELAND SECURITY TANK VESSELS FIREFIGHTING EQUIPMENT Fixed Foam Extinguishing Systems, Details § 34.17-5 Quantity of foam required—T/ALL. (a) Area protected. (1) For machinery...

Histone deacetylase 1 and 2 are essential for murine neural crest proliferation, pharyngeal arch development, and craniofacial morphogenesis.

PubMed

Milstone, Zachary J; Lawson, Grace; Trivedi, Chinmay M

2017-12-01

Craniofacial anomalies involve defective pharyngeal arch development and neural crest function. Copy number variation at 1p35, containing histone deacetylase 1 (Hdac1), or 6q21-22, containing Hdac2, are implicated in patients with craniofacial defects, suggesting an important role in guiding neural crest development. However, the roles of Hdac1 and Hdac2 within neural crest cells remain unknown. The neural crest and its derivatives express both Hdac1 and Hdac2 during early murine development. Ablation of Hdac1 and Hdac2 within murine neural crest progenitor cells cause severe hemorrhage, atrophic pharyngeal arches, defective head morphogenesis, and complete embryonic lethality. Embryos lacking Hdac1 and Hdac2 in the neural crest exhibit decreased proliferation and increased apoptosis in both the neural tube and the first pharyngeal arch. Mechanistically, loss of Hdac1 and Hdac2 upregulates cyclin-dependent kinase inhibitors Cdkn1a, Cdkn1b, Cdkn1c, Cdkn2b, Cdkn2c, and Tp53 within the first pharyngeal arch. Our results show that Hdac1 and Hdac2 function redundantly within the neural crest to regulate proliferation and the development of the pharyngeal arches by means of repression of cyclin-dependent kinase inhibitors. Developmental Dynamics 246:1015-1026, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Evaluation of ACE, SP17, and FSHB as candidates for stallion fertility in Hanoverian warmblood horses.

PubMed

Giesecke, K; Hamann, H; Stock, K F; Klewitz, J; Martinsson, G; Distl, O; Sieme, H

2011-07-01

The research of fertility in humans and other mammals has strongly advanced in the recent years. The examination of molecular mechanisms influencing horse fertility is relatively recent. We chose the angiotensin converting enzyme (ACE), the sperm autoantigenic protein 17 (SP17) and the follicle stimulating hormone (FSHB) as candidates for determining stallion fertility and to analyze associations of intragenic single nucleotide polymorphisms (SNPs), flanking microsatellites and candidate-gene linked haplotypes with the pregnancy rate per oestrus (PRO) in 179 Hanoverian stallions. Fertility traits analyzed were the least square means of PRO for stallions (LSMs) and the paternal and embryonic component of breeding values for PRO (BVs). We detected nine SNPs and two flanking microsatellites in ACE, eight SNPs and two flanking microsatellites in SP17 and four SNPs and one flanking microsatellite in FSHB. Three SP17-associated SNPs and the two flanking microsatellites showed significant association with the embryonic component of BVs and one SP17-associated microsatellite was also significantly associated with the paternal component of BVs. Two ACE-associated SNPs were significantly associated with the embryonic component of BVs. Significantly associated haplotypes were shown for all three candidate genes and the tested fertility parameters. The final regression analysis model indicated that haplotypes of all three candidate genes significantly contributed to the paternal and embryonic fertility components of PRO. This is the first report of associations of ACE, SP17 and FSHB with fertility traits of stallions. Copyright © 2011 Elsevier B.V. All rights reserved.

Measurement of the ratio $$B(t \\to W b)/B(t \\to W q)$$ in $$t\\bar{t}$$ dilepton channel at CDF

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

Galloni, Camilla

2012-01-01

My analysis is based on the number of b-jets found in t¯t events using the dilepton sample with at least 2 jets in the final state. The charged leptons could be either electrons or muons. Tau leptons are not included. We use SecVtx algorithm, based on the reconstruction of a secondary vertex in the event, in order to identify a jet coming from b-quark fragmentation (b-tagging). Due to the high purity of the t¯t signal in dilepton events it is possible to perform a kinematic measurement of the t¯t cross section. Our strategy is to use this result to makemore » prediction on the number of t¯t events. We divide our sample in subsets according to dilepton type (combination of the lepton type), number of jets in the final states and events with zero, one or two tags. The comparison between events and the prediction, given by the sum of the expected t¯t estimate and the background yield, in each subsample is made using a Likelihood function. Our measured value for R is the one which maximizes the Likelihood, i.e. gives the best match between our expectation and the observed data. We measure: p¯p!t¯t = 7.05±0.53stat±0.42lumi , R= 0.86±0.06 (stat+syst) and, in the hypothesis of CKM matrix unitarity with three quark generations, | Vtb | = 0.93 ± 0.03. Our analysis on the p¯p!t¯t was performed independently of the official dilepton analisys on the t¯t production cross section. So it represents also a valuable crosscheck for the official analysis. In chapter 1, a brief introduction to the theoretical framework is given. The standard model of elementary particles and the Quantum Cromodynamic theories are introduced. Then the top quark is presented, with a short descpription of its properties, as its mass, its production mode and its cross section. Some previous results on R are listed as well. Later we present the experiment that collected our data, both the collider (chapter 2) and the detector (CDF)(chapter 3). In chapter 4 we describe the physics object

The role of transforming growth factor β in T helper 17 differentiation.

PubMed

Zhang, Song

2018-04-23

T helper 17 (Th17) cells play critical roles in inflammatory and autoimmune diseases. The lineage-specific transcription factor RORγt is the key regulator for Th17 cell fate commitment. A substantial number of studies have established the importance of transforming growth factor β (TGF-β) -dependent pathways in inducing RORγt expression and Th17 differentiation. TGF-β superfamily members TGF-β 1 , TGF-β 3 or activin A, in concert with interleukin-6 or interleukin-21, differentiate naive T cells into Th17 cells. Alternatively, Th17 differentiation can occur through TGF-β-independent pathways. However, the mechanism of how TGF-β-dependent and TGF-β-independent pathways control Th17 differentiation remains controversial. This review focuses on the perplexing role of TGF-β in Th17 differentiation, depicts the requirement of TGF-β for Th17 development, and underscores the multiple mechanisms underlying TGF-β-promoted Th17 generation, pathogenicity and plasticity. With new insights and comprehension from recent findings, this review specifically tackles the involvement of the canonical TGF-β signalling components, SMAD2, SMAD3 and SMAD4, summarizes diverse SMAD-independent mechanisms, and highlights the importance of TGF-β signalling in balancing the reciprocal conversion of Th17 and regulatory T cells. Finally, this review includes discussions and perspectives and raises important mechanistic questions about the role of TGF-β in Th17 generation and function. © 2018 John Wiley & Sons Ltd.

Genetic manipulation of murine embryonic stem cells with enhanced green fluorescence protein and sulfatase-modifying factor I genes.

PubMed

Zhao, Guoying; Karageorgos, Litsa; Hutchinson, Rhonda G; Hopwood, John J; Hemsley, Kim

2010-05-01

Mucopolysaccharidosis type IIIA (MPS IIIA) is a lysosomal storage disorder (LSD) in which an absence of sulfamidase results in incomplete degradation and subsequent accumulation of its substrate, heparan sulfate. Most neurodegenerative LSD remain untreatable. However, therapy options, such as gene, enzyme end cell therapy, are under investigation. Previously, we have constructed an embryonic stem (ES) cell line (NS21) that over-expresses human sulphamidase as a potential treatment for murine MPS IIIA. In the present study the sulfatase-modifying factor I (SUMF1) and enhanced green fluorescence protein (eGFP) genes were co-introduced under a cytomegalovirus (CMV) promoter into NS21 cells, to enhance further sulfamidase activity and provide a marker for in vivo cell tracking, respectively. eGFP was also introduced under the control of the human elongation factor-1alpha (hEF-1alpha) promoter to compare the stability of transgene expression. During differentiation of ES cells into glial precursors, SUMF1 was down-regulated and was hardly detectable by day 18 of differentiation. Likewise, eGFP expression was heterogeneous and highly unstable. Use of a human EF-1alpha promoter resulted in more homogeneous eGFP expression, with approximately 50% of cells eGFP positive following differentiation into glial precursors. Compared with NS21 cells, the outgrowth of eGFP-expressing cells was not as confluent when differentiated into glial precursors. Our data suggest that SUMF1 enhances sulfamidase activity in ES cells, hEF-1alpha is a stronger promoter than CMV for ES cells and over-expression of eGFP may affect cell growth and contribute to unstable gene expression.

Puerarin Suppresses the Self-Renewal of Murine Embryonic Stem Cells by Inhibition of REST-MiR-21 Regulatory Pathway.

PubMed

Yin, Mengmeng; Yuan, Yin; Cui, Yurong; Hong, Xian; Luo, Hongyan; Hu, Xinwu; Tang, Ming; Hescheler, Jurgen; Xi, Jiaoya

2015-01-01

Puerarin shows a wide range of biological activities, including affecting the cardiac differentiation from murine embryonic stem (mES) cells. However, little is known about its effect and mechanism of action on the self-renewal of mES cells. This study aimed to determine the effect of puerarin on the self-renewal and pluripotency of mES cells and its underlying mechanisms. RT-PCR and real-time PCR were used to detect the transcripts of core transcription factors, specific markers for multiple lineages, REST and microRNA-21 (miR-21). Colony-forming assay was performed to estimate the self-renewal capacity of mES cells. Western blotting and wortmannin were employed to explore the role of PI3K/Akt signaling pathway in the inhibitory action of puerarin on REST transcript. Transfected mES cells with antagomir21 were used to confirm the role of miR-21 in the action of puerarin on cell self-renewal. Puerarin significantly decreased the percentage of the self-renewal colonies, and suppressed the transcripts of Oct4, Nanog, Sox2, c-Myc and REST. Besides, PECAM, NCAM and miR-21 were up-regulated both under the self-renewal conditions and at day 4 of differentiation. The PI3K inhibitor wortmannin successfully reversed the mRNA expression changes of REST, Nanog and Sox2. Transfection of antagomir21 efficiently reversed the effects of puerarin on mES cells self-renewal. Inhibition of REST-miR-21 regulatory pathway may be the key mechanism of puerarin-induced suppression of mES cells self-renewal.

Antigen-specific IL-23/17 pathway activation by murine semi-mature DC-like cells

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

Nagasaka, Shinya; Iwasaki, Takumi; Okano, Tomoko

We analyzed the phenotype and function of bone marrow-derived dendritic cells (DCs) induced in vitro without using any serum during the late stage of cultivation. These 'serum-free' DCs (SF-DCs) possessed the ability to induce T cell proliferation as well as antibody responses, indicating that they were functional DCs. Surprisingly, the SF-DCs akin to semi-mature DCs in terms of both phenotypic and functional characteristics. The SF-DCs did not produce IL-12 but produced large amounts of IL-23 following lipopolysaccharide stimulation. The antigen-specific production of IL-17 by CD4{sup +} T cells co-cultured with OVA-loaded SF-DCs was significantly higher than that with OVA-loaded conventionalmore » DCs. These results suggest that SF-DCs tend to produce IL-23 and can consequently induce the IL-17 producing CD4{sup +} T cells. The semi-mature DC-like cells reported here will be useful vehicles for DC immunization and might contribute to studies on the possible involvement of semi-mature DCs in Th17 cell differentiation.« less

IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer.