Small amounts of tissue preserve pancreatic function

PubMed Central

Lu, Zipeng; Yin, Jie; Wei, Jishu; Dai, Cuncai; Wu, Junli; Gao, Wentao; Xu, Qing; Dai, Hao; Li, Qiang; Guo, Feng; Chen, Jianmin; Xi, Chunhua; Wu, Pengfei; Zhang, Kai; Jiang, Kuirong; Miao, Yi

2016-01-01

Abstract Middle-segment preserving pancreatectomy (MPP) is a novel procedure for treating multifocal lesions of the pancreas while preserving pancreatic function. However, long-term pancreatic function after this procedure remains unclear. The aims of this current study are to investigate short- and long-term outcomes, especially long-term pancreatic endocrine function, after MPP. From September 2011 to December 2015, 7 patients underwent MPP in our institution, and 5 cases with long-term outcomes were further analyzed in a retrospective manner. Percentage of tissue preservation was calculated using computed tomography volumetry. Serum insulin and C-peptide levels after oral glucose challenge were evaluated in 5 patients. Beta-cell secreting function including modified homeostasis model assessment of beta-cell function (HOMA2-beta), area under the curve (AUC) for C-peptide, and C-peptide index were evaluated and compared with those after pancreaticoduodenectomy (PD) and total pancreatectomy. Exocrine function was assessed based on questionnaires. Our case series included 3 women and 2 men, with median age of 50 (37–81) years. Four patients underwent pylorus-preserving PD together with distal pancreatectomy (DP), including 1 with spleen preserved. The remaining patient underwent Beger procedure and spleen-preserving DP. Median operation time and estimated intraoperative blood loss were 330 (250–615) min and 800 (400–5500) mL, respectively. Histological examination revealed 3 cases of metastatic lesion to the pancreas, 1 case of chronic pancreatitis, and 1 neuroendocrine tumor. Major postoperative complications included 3 cases of delayed gastric emptying and 2 cases of postoperative pancreatic fistula. Imaging studies showed that segments representing 18.2% to 39.5% of the pancreas with good blood supply had been preserved. With a median 35.0 months of follow-ups on pancreatic functions, only 1 patient developed new-onset diabetes mellitus of the 4 preoperatively euglycemic patients. Beta-cell function parameters in this group of patients were quite comparable to those after Whipple procedure, and seemed better than those after total pancreatectomy. No symptoms of hypoglycemia were identified in any patient, although half of the patients reported symptoms of exocrine insufficiency. In conclusion, MPP is a feasible and effective procedure for middle-segment sparing multicentric lesions in the pancreas, and patients exhibit satisfied endocrine function after surgery. PMID:27861351

Preserved MHC-II antigen processing and presentation function in chronic HCV infection

PubMed Central

DH, Canaday; CJ, Burant; L, Jones; H, Aung; L, Woc-Colburn; DD, Anthony

2010-01-01

Individuals with chronic HCV infection have impaired response to vaccine, though the etiology remains to be elucidated. Dendritic cells (DC) and monocytes (MN) provide antigen uptake, processing, presentation, and costimulatory functions necessary to achieve optimal immune responses. The integrity of antigen processing and presentation function within these antigen presenting cells (APC) in the setting of HCV infection has been unclear. We used a novel T cell hybridoma system that specifically measures MHC-II antigen processing and presentation function of human APC. Results demonstrate MHC-II antigen processing and presentation function is preserved in both myeloid DC (mDC) and MN in the peripheral blood of chronically HCV-infected individuals, and indicates that an alteration in this function does not likely underlie the defective HCV-infected host response to vaccination. PMID:21055734

Donor Satellite Cell Engraftment is Significantly Augmented When the Host Niche is Preserved and Endogenous Satellite Cells are Incapacitated

PubMed Central

Boldrin, Luisa; Neal, Alice; Zammit, Peter S; Muntoni, Francesco; Morgan, Jennifer E

2012-01-01

Stem cell transplantation is already in clinical practice for certain genetic diseases and is a promising therapy for dystrophic muscle. We used the mdx mouse model of Duchenne muscular dystrophy to investigate the effect of the host satellite cell niche on the contribution of donor muscle stem cells (satellite cells) to muscle regeneration. We found that incapacitation of the host satellite cells and preservation of the muscle niche promote donor satellite cell contribution to muscle regeneration and functional reconstitution of the satellite cell compartment. But, if the host niche is not promptly refilled, or is filled by competent host satellite cells, it becomes nonfunctional and donor engraftment is negligible. Application of this regimen to aged host muscles also promotes efficient regeneration from aged donor satellite cells. In contrast, if the niche is destroyed, yet host satellite cells remain proliferation-competent, donor-derived engraftment is trivial. Thus preservation of the satellite cell niche, concomitant with functional impairment of the majority of satellite cells within dystrophic human muscles, may improve the efficiency of stem cell therapy. Stem Cells2012;30:1971–1984 PMID:22730231

Therapeutic microparticles functionalized with biomimetic cardiac stem cell membranes and secretome

PubMed Central

Tang, Junnan; Shen, Deliang; Caranasos, Thomas George; Wang, Zegen; Vandergriff, Adam C.; Allen, Tyler A.; Hensley, Michael Taylor; Dinh, Phuong-Uyen; Cores, Jhon; Li, Tao-Sheng; Zhang, Jinying; Kan, Quancheng; Cheng, Ke

2017-01-01

Stem cell therapy represents a promising strategy in regenerative medicine. However, cells need to be carefully preserved and processed before usage. In addition, cell transplantation carries immunogenicity and/or tumourigenicity risks. Mounting lines of evidence indicate that stem cells exert their beneficial effects mainly through secretion (of regenerative factors) and membrane-based cell–cell interaction with the injured cells. Here, we fabricate a synthetic cell-mimicking microparticle (CMMP) that recapitulates stem cell functions in tissue repair. CMMPs carry similar secreted proteins and membranes as genuine cardiac stem cells do. In a mouse model of myocardial infarction, injection of CMMPs leads to the preservation of viable myocardium and augmentation of cardiac functions similar to cardiac stem cell therapy. CMMPs (derived from human cells) do not stimulate T-cell infiltration in immuno-competent mice. In conclusion, CMMPs act as ‘synthetic stem cells’ which mimic the paracrine and biointerfacing activities of natural stem cells in therapeutic cardiac regeneration. PMID:28045024

Basic Techniques in Mammalian Cell Tissue Culture.

PubMed

Phelan, Katy; May, Kristin M

2016-11-01

Cultured mammalian cells are used extensively in cell biology studies. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Elastin overexpression by cell-based gene therapy preserves matrix and prevents cardiac dilation

PubMed Central

Li, Shu-Hong; Sun, Zhuo; Guo, Lily; Han, Mihan; Wood, Michael F G; Ghosh, Nirmalya; Alex Vitkin, I; Weisel, Richard D; Li, Ren-Ke

2012-01-01

After a myocardial infarction, thinning and expansion of the fibrotic scar contribute to progressive heart failure. The loss of elastin is a major contributor to adverse extracellular matrix remodelling of the infarcted heart, and restoration of the elastic properties of the infarct region can prevent ventricular dysfunction. We implanted cells genetically modified to overexpress elastin to re-establish the elastic properties of the infarcted myocardium and prevent cardiac failure. A full-length human elastin cDNA was cloned, subcloned into an adenoviral vector and then transduced into rat bone marrow stromal cells (BMSCs). In vitro studies showed that BMSCs expressed the elastin protein, which was deposited into the extracellular matrix. Transduced BMSCs were injected into the infarcted myocardium of adult rats. Control groups received either BMSCs transduced with the green fluorescent protein gene or medium alone. Elastin deposition in the infarcted myocardium was associated with preservation of myocardial tissue structural integrity (by birefringence of polarized light; P < 0.05 versus controls). As a result, infarct scar thickness and diastolic compliance were maintained and infarct expansion was prevented (P < 0.05 versus controls). Over a 9-week period, rats implanted with BMSCs demonstrated better cardiac function than medium controls; however, rats receiving BMSCs overexpressing elastin showed the greatest functional improvement (P < 0.01). Overexpression of elastin in the infarcted heart preserved the elastic structure of the extracellular matrix, which, in turn, preserved diastolic function, prevented ventricular dilation and preserved cardiac function. This cell-based gene therapy provides a new approach to cardiac regeneration. PMID:22435995

Preservation of Cell Structure, Metabolism, and Biotransformation Activity of Liver-On-Chip Organ Models by Hypothermic Storage.

PubMed

Gröger, Marko; Dinger, Julia; Kiehntopf, Michael; Peters, Frank T; Rauen, Ursula; Mosig, Alexander S

2018-01-01

The liver is a central organ in the metabolization of nutrition, endogenous and exogenous substances, and xenobiotic drugs. The emerging organ-on-chip technology has paved the way to model essential liver functions as well as certain aspects of liver disease in vitro in liver-on-chip models. However, a broader use of this technology in biomedical research is limited by a lack of protocols that enable the short-term preservation of preassembled liver-on-chip models for stocking or delivery to researchers outside the bioengineering community. For the first time, this study tested the ability of hypothermic storage of liver-on-chip models to preserve cell viability, tissue morphology, metabolism and biotransformation activity. In a systematic study with different preservation solutions, liver-on-chip function can be preserved for up to 2 d using a derivative of the tissue preservation solution TiProtec, containing high chloride ion concentrations and the iron chelators LK614 and deferoxamine, supplemented with polyethylene glycol (PEG). Hypothermic storage in this solution represents a promising method to preserve liver-on-chip function for at least 2 d and allows an easier access to liver-on-chip technology and its versatile and flexible use in biomedical research. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cold preservation with hyperbranched polyglycerol-based solution improves kidney functional recovery with less injury at reperfusion in rats

PubMed Central

Li, Shadan; Liu, Bin; Guan, Qiunong; Chafeeva, Irina; Brooks, Donald E; Nguan, Christopher YC; Kizhakkedathu, Jayachandran N; Du, Caigan

2017-01-01

Minimizing donor organ injury during cold preservation (including cold perfusion and storage) is the first step to prevent transplant failure. We recently reported the advantages of hyperbranched polyglycerol (HPG) as a novel substitute for hydroxyethyl starch in UW solution for both cold heart preservation and cold kidney perfusion. This study evaluated the functional recovery of the kidney at reperfusion after cold preservation with HPG solution. The impact of HPG solution compared to conventional UW and HTK solutions on tissue weight and cell survival at 4°C was examined using rat kidney tissues and cultured human umbilical vein endothelial cells (HUVECs), respectively. The kidney protection by HPG solution was tested in a rat model of cold kidney ischemia-reperfusion injury, and was evaluated by histology and kidney function. Here, we showed that preservation with HPG solution prevented cell death in cultured HUVECs and edema formation in kidney tissues at 4°C similar to UW solution, whereas HTK solution was less effective. In rat model of cold ischemia-reperfusion injury, the kidneys perfused and subsequently stored 1-hour with cold HPG solution showed less leukocyte infiltration, less tubular damage and better kidney function (lower levels of serum creatinine and blood urea nitrogen) at 48 h of reperfusion than those treated with UW or HTK solution. In conclusion, our data show the superiority of HPG solution to UW or HTK solution in the cold perfusion and storage of rat kidneys, suggesting that the HPG solution may be a promising candidate for improved donor kidney preservation prior to transplantation. PMID:28337272

Platform technology for scalable assembly of instantaneously functional mosaic tissues

PubMed Central

Zhang, Boyang; Montgomery, Miles; Davenport-Huyer, Locke; Korolj, Anastasia; Radisic, Milica

2015-01-01

Engineering mature tissues requires a guided assembly of cells into organized three-dimensional (3D) structures with multiple cell types. Guidance is usually achieved by microtopographical scaffold cues or by cell-gel compaction. The assembly of individual units into functional 3D tissues is often time-consuming, relying on cell ingrowth and matrix remodeling, whereas disassembly requires an invasive method that includes either matrix dissolution or mechanical cutting. We invented Tissue-Velcro, a bio-scaffold with a microfabricated hook and loop system. The assembly of Tissue-Velcro preserved the guided cell alignment realized by the topographical features in the 2D scaffold mesh and allowed for the instant establishment of coculture conditions by spatially defined stacking of cardiac cell layers or through endothelial cell coating. The assembled cardiac 3D tissue constructs were immediately functional as measured by their ability to contract in response to electrical field stimulation. Facile, on-demand tissue disassembly was demonstrated while preserving the structure, physical integrity, and beating function of individual layers. PMID:26601234

In vitro effects of preserved and unpreserved anti-allergic drugs on human corneal epithelial cells.

PubMed

Guzman-Aranguez, Ana; Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-11-01

Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures.

Mammalian Cell Tissue Culture.

PubMed

Phelan, Katy; May, Kristin M

2017-07-11

Cultured mammalian cells are used extensively in the field of human genetics. It requires a number of special skills in order to be able to preserve the structure, function, behavior, and biology of the cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Pluripotent Stem Cells for Retinal Tissue Engineering: Current Status and Future Prospects.

PubMed

Singh, Ratnesh; Cuzzani, Oscar; Binette, François; Sternberg, Hal; West, Michael D; Nasonkin, Igor O

2018-04-19

The retina is a very fine and layered neural tissue, which vitally depends on the preservation of cells, structure, connectivity and vasculature to maintain vision. There is an urgent need to find technical and biological solutions to major challenges associated with functional replacement of retinal cells. The major unmet challenges include generating sufficient numbers of specific cell types, achieving functional integration of transplanted cells, especially photoreceptors, and surgical delivery of retinal cells or tissue without triggering immune responses, inflammation and/or remodeling. The advances of regenerative medicine enabled generation of three-dimensional tissues (organoids), partially recreating the anatomical structure, biological complexity and physiology of several tissues, which are important targets for stem cell replacement therapies. Derivation of retinal tissue in a dish creates new opportunities for cell replacement therapies of blindness and addresses the need to preserve retinal architecture to restore vision. Retinal cell therapies aimed at preserving and improving vision have achieved many improvements in the past ten years. Retinal organoid technologies provide a number of solutions to technical and biological challenges associated with functional replacement of retinal cells to achieve long-term vision restoration. Our review summarizes the progress in cell therapies of retina, with focus on human pluripotent stem cell-derived retinal tissue, and critically evaluates the potential of retinal organoid approaches to solve a major unmet clinical need-retinal repair and vision restoration in conditions caused by retinal degeneration and traumatic ocular injuries. We also analyze obstacles in commercialization of retinal organoid technology for clinical application.

In Vitro Effects of Preserved and Unpreserved Anti-Allergic Drugs on Human Corneal Epithelial Cells

PubMed Central

Calvo, Patricia; Ropero, Inés; Pintor, Jesús

2014-01-01

Abstract Purpose: Treatment with topical eye drops for long-standing ocular diseases like allergy can induce detrimental side effects. The purpose of this study was to investigate in vitro cytotoxicity of commercially preserved and unpreserved anti-allergic eye drops on the viability and barrier function of monolayer and stratified human corneal-limbal epithelial cells. Methods: Cells were treated with unpreserved ketotifen solution, benzalkonium chloride (BAC)-containing anti-allergic drugs (ketotifen, olopatadine, levocabastine) as well as BAC alone. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine cell viability. Effects of compounds on barrier function were analyzed measuring transepithelial electrical resistance (TEER) to determine paracellular permeability and rose bengal assays to evaluate transcellular barrier formation. Results: The BAC-preserved anti-allergic formulations and BAC alone significantly reduced cell viability, monolayer cultures being more sensitive to damage by these solutions. Unpreserved ketotifen induced the least diminution in cell viability. The extent of decrease of cell viability was clearly dependent of BAC presence, but it was also affected by the different types of drugs when the concentration of BAC was low and the short time of exposure. Treatment with BAC-containing anti-allergic drugs and BAC alone resulted in increased paracellular permeability and loss of transcellular barrier function as indicated by TEER measurement and rose bengal assays. Conclusions: The presence of the preservative BAC in anti-allergic eye drop formulations contributes importantly to the cytotoxic effects induced by these compounds. Stratified cell cultures seem to be a more relevant model for toxicity evaluation induced on the ocular surface epithelia than monolayer cultures. PMID:25100331

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells

PubMed Central

Swioklo, Stephen; Constantinescu, Andrei

2016-01-01

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C–23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 106 cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. Significance Despite considerable advancement in the clinical application of cell-based therapies, major logistical challenges exist throughout the cell therapy supply chain associated with the storage and distribution of cells between the sites of manufacture and the clinic. A simple, low-cost system capable of preserving the viability and functionality of human adipose-derived stem cells (a cell with substantial clinical interest) at hypothermic temperatures (0°C–32°C) is presented. Such a system has considerable potential for extending the shelf life of cell therapy products at multiple stages throughout the cell therapy supply chain. PMID:26826163

Mammalian Cell Tissue Culture Techniques.

PubMed

Phelan, Katy; May, Kristin M

2016-06-01

Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and storage, recovering frozen stocks, and counting viable cells. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

Spermatogonial stem cells as a therapeutic alternative for fertility preservation of prepubertal boys

PubMed Central

Galuppo, Andrea Giannotti

2015-01-01

ABSTRACT Spermatogonial stem cells, which exist in the testicles since birth, are progenitors cells of male gametes. These cells are critical for the process of spermatogenesis, and not able to produce mature sperm cells before puberty due to their dependency of hormonal stimuli. This characteristic of the reproductive system limits the preservation of fertility only to males who are able to produce an ejaculate. This fact puts some light on the increase in survival rates of childhood cancer over the past decades because of improvements in the diagnosis and effective treatment in pediatric cancer patients. Therefore, we highlight one of the most important challenges concerning male fertility preservation that is the toxic effect of cancer therapy on reproductive function, especially the spermatogenesis. Currently, the experimental alternative for fertility preservation of prepubertal boys is the testicular tissue cryopreservationfor, for future isolation and spermatogonial stem cells transplantation, in order to restore the spermatogenesis. We present a brief review on isolation, characterization and culture conditions for the in vitro proliferation of spermatogonial stem cells, as well as the future perspectives as an alternative for fertility preservation in prepubertal boys. The possibility of restoring male fertility constitutes a research tool with an huge potential in basic and applied science. The development of these techniques may be a hope for the future of fertility preservation in cases that no other options exist, e.g, pediatric cancer patients. PMID:26761559

The E-domain region of mechano-growth factor inhibits cellular apoptosis and preserves cardiac function during myocardial infarction.

PubMed

Mavrommatis, Evangelos; Shioura, Krystyna M; Los, Tamara; Goldspink, Paul H

2013-09-01

Insulin-like growth factor-1 (IGF-1) isoforms are expressed via alternative splicing. Expression of the minor isoform IGF-1Eb [also known as mechano-growth factor (MGF)] is responsive to cell stress. Since IGF-1 isoforms differ in their E-domain regions, we are interested in determining the biological function of the MGF E-domain. To do so, a synthetic peptide analog was used to gain mechanistic insight into the actions of the E-domain. Treatment of H9c2 cells indicated a rapid cellular uptake mechanism that did not involve IGF-1 receptor activation but resulted in a nuclear localization. Peptide treatment inhibited the intrinsic apoptotic pathway in H9c2 cells subjected to cell stress with sorbitol by preventing the collapse of the mitochondrial membrane potential and inhibition of caspase-3 activation. Therefore, we administered the peptide at the time of myocardial infarction (MI) in mice. At 2 weeks post-MI cardiac function, gene expression and cell death were assayed. A significant decline in both systolic and diastolic function was evident in untreated mice based on PV loop analysis. Delivery of the E-peptide ameliorated the decline in function and resulted in significant preservation of cardiac contractility. Associated with these changes were an inhibition of pathologic hypertrophy and significantly fewer apoptotic nuclei in the viable myocardium of E-peptide-treated mice post-MI. We conclude that administration of the MGF E-domain peptide may provide a means of modulating local tissue IGF-1 autocrine/paracrine actions to preserve cardiac function, prevent cell death, and pathologic remodeling in the heart.

A novel procedure to improve functional preservation of hematopoietic stem and progenitor cells in cord blood stored at +4°c before cryopreservation.

PubMed

Chevaleyre, Jean; Rodriguez, Laura; Duchez, Pascale; Plainfossé, Marie; Dazey, Bernard; Lapostolle, Véronique; Vlaski, Marija; Brunet de la Grange, Philippe; Delorme, Bruno; Ivanovic, Zoran

2014-08-01

During storage and transportation of collected cord blood units (CBUs) to the bank prior to their processing and cryopreservation, it is imperative to preserve the functional capacities of a relatively small amount of cells of interest (stem and progenitor cells) which are critical for graft potency. To improve CBU storage efficiency, we conceived an approach based on the following two principles: (1) to provide a better nutritive and biochemical environment to stem and progenitor cells in CB and (2) to prevent the hyperoxygenation of these cells transferred from a low- (1.1%-4% O2 in the CB) to a high-oxygen (20%-21% O2 in atmosphere) concentration. Our hypothesis is confirmed by the functional assessment of stem cell (hematopoietic reconstitution capacity in immunodeficient mice-scid repopulating cell assay) and committed progenitor activities (capacity of in vitro colony formation and of ex vivo expansion) after the storage period with our medium (HP02) in gas-impermeable bags. This storage procedure maintains the full functional capacity of a CBU graft for 3 days with respect to day 0. Further, using this procedure, a graft stored 3 days at +4°C exhibits better functional capacities than one currently used in routine storage (CBUs stored at +4°C for 1 day in gas-permeable bags and without medium). We provided the proof of principle of our approach, developed a clinical-scale kit and performed a preclinical assay demonstrating the feasibility and efficiency of our CBU preservation protocol through all steps of preparation (volume reduction, freezing, and thawing).

Treatment of prediabetes

PubMed Central

Kanat, Mustafa; DeFronzo, Ralph A; Abdul-Ghani, Muhammad A

2015-01-01

Progression of normal glucose tolerance (NGT) to overt diabetes is mediated by a transition state called impaired glucose tolerance (IGT). Beta cell dysfunction and insulin resistance are the main defects in type 2 diabetes mellitus (type 2 DM) and even normoglycemic IGT patients manifest these defects. Beta cell dysfunction and insulin resistance also contribute to the progression of IGT to type 2 DM. Improving insulin sensitivity and/or preserving functions of beta-cells can be a rational way to normalize the GT and to control transition of IGT to type 2 DM. Loosing weight, for example, improves whole body insulin sensitivity and preserves beta-cell function and its inhibitory effect on progression of IGT to type 2 DM had been proven. But interventions aiming weight loss usually not applicable in real life. Pharmacotherapy is another option to gain better insulin sensitivity and to maintain beta-cell function. In this review, two potential treatment options (lifestyle modification and pharmacologic agents) that limits the IGT-type 2 DM conversion in prediabetic subjects are discussed. PMID:26464759

Histone Deacetylase Inhibitors Enhance Cytotoxicity Towards Breast Tumors While Preserving the Wound-Healing Function of Adipose-Derived Stem Cells.

PubMed

Koko, Kiavash R; Chang, Shaohua; Hagaman, Ashleigh L; Fromer, Marc W; Nolan, Ryan S; Gaughan, John P; Zhang, Ping; Carpenter, Jeffrey P; Brown, Spencer A; Matthews, Martha; Bird, Dorothy

2017-06-01

Paclitaxel improves the oncologic response of breast cancer resections; however, it may negatively affect the wound-healing potential of human adipose-derived stem cells (hASCs) for fat grafting and reconstructive surgery. Histone deacetylase inhibitors (HDACis) modify the epigenetic regulation of gene expression and stabilize microtubules similarly to paclitaxel, thus, creating a synergistic mechanism of cell cycle arrest. We aim to combine these drugs to enhance cytotoxicity towards breast cancer cells, while preserving the wound-healing function of hASCs for downstream reconstructive applications. Triple negative breast cancer cells (MBA-MB-231) and hASCs (institutional review board-approved clinical isolates) were treated with a standard therapeutic dose of paclitaxel (1.0 μM) or with low-dose paclitaxel (0.1 μM) combined with the HDACi suberoylanilide hydroxamic acid or trichostatin A. Cell viability, gene expression, apoptosis, and wound-healing/migration were measured via methylthiazol tetrazolium assay, quantitative real-time polymerase chain reaction, annexin V assay, and fibroblast scratch assay, respectively. Combined HDACi and low-dose paclitaxel therapy maintained cytotoxicity towards breast cancer cells and preserved adipose-derived stem cell viability. Histone deacetylase inhibitor demonstrated selective anti-inflammatory effects on adipose-derived stem cell gene expression and decreased expression of the proapoptotic gene FAS. Furthermore, HDACi therapy did not increase relative apoptosis within hASCs. A scratch assay demonstrated enhanced wound healing among injured fibroblasts indirectly co-cultured with HDACi-treated hASCs. Combining HDACi with low-dose paclitaxel improved cytotoxicity towards breast cancer cells and preserved hASC viability. Furthermore, enhanced wound healing was observed by improved migration in a fibroblast scratch assay. These results suggest that the addition of HDACi to taxane chemotherapy regimens may improve oncologic results and wound-healing outcomes after reconstructive surgery.

Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation.

PubMed

Hai, Bo; Zhao, Qingguo; Qin, Lizheng; Rangaraj, Dharanipathy; Gutti, Veera R; Liu, Fei

2016-05-01

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

The gastrin-releasing peptide analog bombesin preserves exocrine and endocrine pancreas morphology and function during parenteral nutrition

PubMed Central

Pierre, Joseph F.; Neuman, Joshua C.; Brill, Allison L.; Brar, Harpreet K.; Thompson, Mary F.; Cadena, Mark T.; Connors, Kelsey M.; Busch, Rebecca A.; Heneghan, Aaron F.; Cham, Candace M.; Jones, Elaina K.; Kibbe, Carly R.; Davis, Dawn B.; Groblewski, Guy E.; Kudsk, Kenneth A.

2015-01-01

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis. PMID:26185331

Hardwiring stem cell communication through tissue structure

PubMed Central

Xin, Tianchi; Greco, Valentina; Myung, Peggy

2016-01-01

Adult stem cells across diverse organs self-renew and differentiate to maintain tissue homeostasis. How stem cells receive input to preserve tissue structure and function largely relies on their communication with surrounding cellular and non-cellular elements. As such, how tissues are organized and patterned not only reflects organ function but also inherently hardwires networks of communication between stem cells and their environment to direct tissue homeostasis and injury repair. This review highlights how different methods of stem cell communication reflect the unique organization and function of diverse tissues. PMID:26967287

Insulin-Like Growth Factor-1 Preserves Salivary Gland Function After Fractionated Radiation

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

Limesand, Kirsten H., E-mail: limesank@u.arizona.ed; Department of Nutritional Sciences, University of Arizona, Tucson, AZ; Avila, Jennifer L.

Purpose: Radiotherapy for head-and-neck cancer consists of fractionated radiation treatments that cause significant damage to salivary glands leading to chronic salivary gland dysfunction with only limited prevention and treatment options currently available. This study examines the feasibility of IGF-1 in preserving salivary gland function following a fractionated radiation treatment regimen in a pre-clinical model. Methods and Materials: Mice were exposed to fractionated radiation, and salivary gland function and histological analyses of structure, apoptosis, and proliferation were evaluated. Results: In this study, we report that treatment with fractionated doses of radiation results in a significant level of apoptotic cells in FVBmore » mice after each fraction, which is significantly decreased in transgenic mice expressing a constitutively active mutant of Akt1 (myr-Akt1). Salivary gland function is significantly reduced in FVB mice exposed to fractionated radiation; however, myr-Akt1 transgenic mice maintain salivary function under the same treatment conditions. Injection into FVB mice of recombinant insulin-like growth factor-1 (IGF-1), which activates endogenous Akt, suppressed acute apoptosis and preserved salivary gland function after fractionated doses of radiation 30 to 90 days after treatment. FVB mice exposed to fractionated radiation had significantly lower levels of proliferating cell nuclear antigen-positive salivary acinar cells 90 days after treatment, which correlated with a chronic loss of function. In contrast, FVB mice injected with IGF-1 before each radiation treatment exhibited acinar cell proliferation rates similar to those of untreated controls. Conclusion: These studies suggest that activation of IGF-1-mediated pathways before head-and-neck radiation could modulate radiation-induced salivary gland dysfunction and maintain glandular homeostasis.« less

The Mitochondria-Targeted Antioxidant Mitoquinone Protects against Cold Storage Injury of Renal Tubular Cells and Rat Kidneys

PubMed Central

Mitchell, Tanecia; Rotaru, Dumitru; Saba, Hamida; Smith, Robin A. J.; Murphy, Michael P.

2011-01-01

The majority of kidneys used for transplantation are obtained from deceased donors. These kidneys must undergo cold preservation/storage before transplantation to preserve tissue quality and allow time for recipient selection and transport. However, cold storage (CS) can result in tissue injury, kidney discardment, or long-term renal dysfunction after transplantation. We have previously determined mitochondrial superoxide and other downstream oxidants to be important signaling molecules that contribute to CS plus rewarming (RW) injury of rat renal proximal tubular cells. Thus, this study's purpose was to determine whether adding mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to University of Wisconsin (UW) preservation solution could offer protection against CS injury. CS was initiated by placing renal cells or isolated rat kidneys in UW solution alone (4 h at 4°C) or UW solution containing MitoQ or its control compound, decyltriphenylphosphonium bromide (DecylTPP) (1 μM in vitro; 100 μM ex vivo). Oxidant production, mitochondrial function, cell viability, and alterations in renal morphology were assessed after CS exposure. CS induced a 2- to 3-fold increase in mitochondrial superoxide generation and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or renal damage. MitoQ treatment decreased oxidant production ∼2-fold, completely prevented mitochondrial dysfunction, and significantly improved cell viability and/or renal morphology, whereas DecylTPP treatment did not offer any protection. These findings implicate that MitoQ could potentially be of therapeutic use for reducing organ preservation damage and kidney discardment and/or possibly improving renal function after transplantation. PMID:21159749

The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renal tubular cells and rat kidneys.

PubMed

Mitchell, Tanecia; Rotaru, Dumitru; Saba, Hamida; Smith, Robin A J; Murphy, Michael P; MacMillan-Crow, Lee Ann

2011-03-01

The majority of kidneys used for transplantation are obtained from deceased donors. These kidneys must undergo cold preservation/storage before transplantation to preserve tissue quality and allow time for recipient selection and transport. However, cold storage (CS) can result in tissue injury, kidney discardment, or long-term renal dysfunction after transplantation. We have previously determined mitochondrial superoxide and other downstream oxidants to be important signaling molecules that contribute to CS plus rewarming (RW) injury of rat renal proximal tubular cells. Thus, this study's purpose was to determine whether adding mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to University of Wisconsin (UW) preservation solution could offer protection against CS injury. CS was initiated by placing renal cells or isolated rat kidneys in UW solution alone (4 h at 4°C) or UW solution containing MitoQ or its control compound, decyltriphenylphosphonium bromide (DecylTPP) (1 μM in vitro; 100 μM ex vivo). Oxidant production, mitochondrial function, cell viability, and alterations in renal morphology were assessed after CS exposure. CS induced a 2- to 3-fold increase in mitochondrial superoxide generation and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or renal damage. MitoQ treatment decreased oxidant production ~2-fold, completely prevented mitochondrial dysfunction, and significantly improved cell viability and/or renal morphology, whereas DecylTPP treatment did not offer any protection. These findings implicate that MitoQ could potentially be of therapeutic use for reducing organ preservation damage and kidney discardment and/or possibly improving renal function after transplantation.

Alginate-Encapsulation for the Improved Hypothermic Preservation of Human Adipose-Derived Stem Cells.

PubMed

Swioklo, Stephen; Constantinescu, Andrei; Connon, Che J

2016-03-01

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C-23°C) preservation of human adipose-derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature-dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 10(6) cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno-free, serum-free,current Good Manufacturing Practice-grade medium. We present a simple, low-cost system capable of enhancing the preservation of human adipose-derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. ©AlphaMed Press.

The Cell's Sophisticated Army to Defend Against Assaults on DNAThe Cell's Sophisticated Army to Defend Against Assaults on DNA | Center for Cancer Research

Cancer.gov

The maintenance of genome integrity and function is essen-tial for the survival of cells and organisms. Any damage to our genetic material must be immediately sensed and repaired to preserve a cell’s func-tional integrity. Cells are constantly faced with the challenge of protecting their DNA from assaults by damaging chemicals and ultraviolet light. DNA damage that escapes

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

PubMed

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

2016-04-01

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

Injection of Peptide nanogels preserves postinfarct diastolic function and prolongs efficacy of cell therapy in pigs.

PubMed

Lin, Yi-Dong; Chang, Ming-Yao; Cheng, Bill; Liu, Yen-Wen; Lin, Lung-Chun; Chen, Jyh-Hong; Hsieh, Patrick C H

2015-05-01

Accumulating evidence suggests that the benefits of cell therapy for cardiac repair are modest and transient due to progressive harmful cardiac remodeling as well as loss of transplanted cells. We previously demonstrated that injection of peptide nanofibers (NFs) reduces ventricular remodeling and facilitates cell retention at 1 month after acute myocardial infarction (MI) in pigs. However, it remains unclear whether these benefits still persist as the material is being degraded. In this study, 2 mL of placebo or NFs, with or without 1×10(8) mononuclear cells (MNCs), was injected into the pig myocardium after MI (n≥5 in each group), and cardiac function was assessed by echocardiography, including myocardial deformation analyses and catheterization at 3 months post-MI. Our results reveal that MNC-only injection slightly improved cardiac systolic function at 1 month post-MI, but this benefit was lost at later time points (ejection fraction: 42.0±2.3 in MI+normal saline [NS] and 43.5±1.1 in MI+MNCs). In contrast, NF-only injection resulted in improved cardiac diastolic function and reduced pathological remodeling at 3 months post-MI. Furthermore, combined injection of MNCs/NFs provided a greater and longer term cardiac performance (52.1±1.2 in MI+MNCs/NFs, p<0.001 versus MI+NS and MI+MNCs) and 11.3-fold transplanted cell retention. We also found that about 30% NFs remained at 3 months after injection; however, endogenous myofibroblasts were recruited to the NF-injected microenvironment to replace the degraded NFs and preserved cardiac dimensions and mechanics. In conclusion, we demonstrated that injection of NFs contributes to preservation of ventricular mechanical integrity and sustains MNC efficacy at 3 months postinjection.

The stress polarity pathway: AMPK ‘GIV’-es protection against metabolic insults

PubMed Central

Ghosh, Pradipta

2017-01-01

Loss of cell polarity impairs organ development and function; it can also serve as one of the first triggers for oncogenesis. In 2006-2007 two groups simultaneously reported the existence of a special pathway for maintaining epithelial polarity in the face of environmental stressors. In this pathway, AMPK, a key sensor of metabolic stress stabilizes tight junctions, preserves cell polarity, and thereby, maintains epithelial barrier functions. Accumulating evidence since has shown that pharmacologic activation of AMPK by Metformin protects the epithelial barrier against multiple environmental and pathological stressful states and suppresses tumorigenesis. How AMPK protects the epithelium remained unknown until recently Aznar et al. identified GIV/Girdin as a novel effector of AMPK at the cell-cell junctions; phosphorylation of GIV at a single site by AMPK appears to be both necessary and sufficient for strengthening tight junctions and preserving cell polarity and epithelial barrier function in the face of energetic stress. Here we review the fundamentals of this specialized signaling pathway that buttresses cell-cell junctions against stress-induced collapse and discuss its pathophysiologic relevance in the context of a variety of diseases, including cancers, diabetes, aging, and the growing list of beneficial effects of the AMPK-activator, Metformin. PMID:28209925

NMDAR antagonists for the treatment of diabetes mellitus-Current status and future directions.

PubMed

Welters, Alena; Klüppel, Carina; Mrugala, Jessica; Wörmeyer, Laura; Meissner, Thomas; Mayatepek, Ertan; Heiss, Christian; Eberhard, Daniel; Lammert, Eckhard

2017-09-01

Diabetes mellitus is characterized by chronically elevated blood glucose levels accelerated by a progressive decline of insulin-producing β-cells in the pancreatic islets. Although medications are available to transiently adjust blood glucose to normal levels, the effects of current drugs are limited when it comes to preservation of a critical mass of functional β-cells to sustainably maintain normoglycemia. In this review, we recapitulate recent evidence on the role of pancreatic N-methyl-D-aspartate receptors (NMDARs) in β-cell physiology, and summarize effects of morphinan-based NMDAR antagonists that are beneficial for insulin secretion, glucose tolerance and islet cell survival. We further discuss NMDAR-mediated molecular pathways relevant for neuronal cell survival, which may also be important for the preservation of β-cell function and mass. Finally, we summarize the literature for evidence on the role of NMDARs in the development of diabetic long-term complications, and highlight beneficial pharmacologic aspects of NMDAR antagonists in diabetic nephropathy, retinopathy as well as neuropathy. © 2017 John Wiley & Sons Ltd.

Cellular complexity captured in durable silica biocomposites

PubMed Central

Kaehr, Bryan; Townson, Jason L.; Kalinich, Robin M.; Awad, Yasmine H.; Swartzentruber, B. S.; Dunphy, Darren R.; Brinker, C. Jeffrey

2012-01-01

Tissue-derived cultured cells exhibit a remarkable range of morphological features in vitro, depending on phenotypic expression and environmental interactions. Translation of these cellular architectures into inorganic materials would provide routes to generate hierarchical nanomaterials with stabilized structures and functions. Here, we describe the fabrication of cell/silica composites (CSCs) and their conversion to silica replicas using mammalian cells as scaffolds to direct complex structure formation. Under mildly acidic solution conditions, silica deposition is restricted to the molecularly crowded cellular template. Inter- and intracellular heterogeneity from the nano- to macroscale is captured and dimensionally preserved in CSCs following drying and subjection to extreme temperatures allowing, for instance, size and shape preserving pyrolysis of cellular architectures to form conductive carbon replicas. The structural and behavioral malleability of the starting material (cultured cells) provides opportunities to develop robust and economical biocomposites with programmed structures and functions. PMID:23045634

Cryopreservation of Human Mesenchymal Stem Cells for Clinical Applications: Current Methods and Challenges.

PubMed

Yong, Kar Wey; Wan Safwani, Wan Kamarul Zaman; Xu, Feng; Wan Abas, Wan Abu Bakar; Choi, Jane Ru; Pingguan-Murphy, Belinda

2015-08-01

Mesenchymal stem cells (MSCs) hold many advantages over embryonic stem cells (ESCs) and other somatic cells in clinical applications. MSCs are multipotent cells with strong immunosuppressive properties. They can be harvested from various locations in the human body (e.g., bone marrow and adipose tissues). Cryopreservation represents an efficient method for the preservation and pooling of MSCs, to obtain the cell counts required for clinical applications, such as cell-based therapies and regenerative medicine. Upon cryopreservation, it is important to preserve MSCs functional properties including immunomodulatory properties and multilineage differentiation ability. Further, a biosafety evaluation of cryopreserved MSCs is essential prior to their clinical applications. However, the existing cryopreservation methods for MSCs are associated with notable limitations, leading to a need for new or improved methods to be established for a more efficient application of cryopreserved MSCs in stem cell-based therapies. We review the important parameters for cryopreservation of MSCs and the existing cryopreservation methods for MSCs. Further, we also discuss the challenges to be addressed in order to preserve MSCs effectively for clinical applications.

Hyperbranched Polyglycerol as a Colloid in Cold Organ Preservation Solutions

PubMed Central

Gao, Sihai; Guan, Qiunong; Chafeeva, Irina; Brooks, Donald E.; Nguan, Christopher Y. C.; Kizhakkedathu, Jayachandran N.; Du, Caigan

2015-01-01

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation. PMID:25706864

Hyperbranched polyglycerol as a colloid in cold organ preservation solutions.

PubMed

Gao, Sihai; Guan, Qiunong; Chafeeva, Irina; Brooks, Donald E; Nguan, Christopher Y C; Kizhakkedathu, Jayachandran N; Du, Caigan

2015-01-01

Hydroxyethyl starch (HES) is a common colloid in organ preservation solutions, such as in University of Wisconsin (UW) solution, for preventing graft interstitial edema and cell swelling during cold preservation of donor organs. However, HES has undesirable characteristics, such as high viscosity, causing kidney injury and aggregation of erythrocytes. Hyperbranched polyglycerol (HPG) is a branched compact polymer that has low intrinsic viscosity. This study investigated HPG (MW-0.5 to 119 kDa) as a potential alternative to HES for cold organ preservation. HPG was synthesized by ring-opening multibranching polymerization of glycidol. Both rat myocardiocytes and human endothelial cells were used as an in vitro model, and heart transplantation in mice as an in vivo model. Tissue damage or cell death was determined by both biochemical and histological analysis. HPG polymers were more compact with relatively low polydispersity index than HES in UW solution. Cold preservation of mouse hearts ex vivo in HPG solutions reduced organ damage in comparison to those in HES-based UW solution. Both size and concentration of HPGs contributed to the protection of the donor organs; 1 kDa HPG at 3 wt% solution was superior to HES-based UW solution and other HPGs. Heart transplants preserved with HPG solution (1 kDa, 3%) as compared with those with UW solution had a better functional recovery, less tissue injury and neutrophil infiltration in syngeneic recipients, and survived longer in allogeneic recipients. In cultured myocardiocytes or endothelial cells, significantly more cells survived after cold preservation with the HPG solution than those with the UW solution, which was positively correlated with the maintenance of intracellular adenosine triphosphate and cell membrane fluidity. In conclusion, HPG solution significantly enhanced the protection of hearts or cells during cold storage, suggesting that HPG is a promising colloid for the cold storage of donor organs and cells in transplantation.

Preserved seminiferous tubule integrity with spermatogonial survival and induction of Sertoli and Leydig cell maturation after long-term organotypic culture of prepubertal human testicular tissue.

PubMed

de Michele, F; Poels, J; Weerens, L; Petit, C; Evrard, Z; Ambroise, J; Gruson, D; Wyns, C

2017-01-01

Is an organotypic culture system able to provide the appropriate testicular microenvironment for in-vitro maturation of human immature testicular tissue (ITT)? Our organotypic culture system provided a microenvironment capable of preserving seminiferous tubule (ST) integrity and Leydig cell (LC) functionality and inducing Sertoli cell (SC) maturation. Cryopreservation of human ITT is a well-established strategy to preserve fertility in prepubertal boys affected by cancer, with a view for obtaining sperm. While spermatogenesis in mice has been replicated in organotypic culture, yielding reproductively efficient spermatozoa, this process has not yet been achieved in humans. The aim of this study was to in vitro mature frozen-thawed ITT. To this end, 1 mm 3 tissue fragments from three prepubertal patients aged 2 (P1), 11 (P2) and 12 (P3) years were placed in organotypic culture for 139 days. Culture media, supplemented with either testosterone or hCG, were compared. ST integrity and tissue viability were assessed by histological score and lactate dehydrogenase (LDH) levels in supernatants. Spermatogonia (SG), proliferating cells and proliferating SG were identified by the use of MAGE-A4 and Ki67 immunohistochemical markers. Glial cell line-derived neurotrophic factor (GDNF) was used as a marker of SC functionality, while SC maturation was evaluated by androgen receptor (AR), anti-Müllerian hormone (AMH) immunohistochemistry (IHC) and AMH immunoenzymatic assay. LC functionality was determined by testosterone levels in supernatants and by 3β-hydroxysteroid dehydrogenase (3β-HSD) IHC. Apoptosis was studied by IHC with active caspases 3 and 8 and by TUNEL (terminal deoxynubocleotidyl transferase-mediated dUTP nick end labeling) analysis. Tissue viability was preserved, as demonstrated by the decrease in and stabilization of LDH release, and evolution of ST scoring, with the percentage of well-preserved STs showing no statistical differences during culture in either medium. GDNF was expressed until Day 139, demonstrating SC functionality. Moreover, a significant reduction in AMH expression and release indicated SC maturation. Testosterone concentrations in supernatants increased in both culture media, demonstrating LC functionality with paracrine interactions. SG were present up to Day 139, although the ratio between MAGE-A4-positive cells and well-preserved tubules was significantly reduced over the course of culture (P ≤ 0.001). SCs exhibited a decreased proliferation rate over time (P ≤ 0.05). The proliferation rate of SG remained stable until Day 64, but over the total culture period (139 days), it was found to have decreased (P ≤ 0.05). The number of apoptotic cells did not vary during culture, nor was any statistical difference observed between the two culture media for any of the studied parameters. N/A LIMITATIONS, REASONS FOR CAUTION: Loss of SG constitutes a limitation for evaluating full functionality of spermatogonial stem cells and warrants further investigation. The scarcity of human immature material is the reason for the limited amount of tissue available for experiments, precluding more comprehensive analysis. Our culture system, mimicking the peripubertal testicular microenvironment with SC maturation, LC functionality and preserved paracrine interactions, and the first to use human ITT, opens the door to a deeper understanding of niche and culture conditions to obtain sperm from cryostored ITT, with the ultimate goal of restoring fertility after gonadotoxic treatments. This project was supported by a grant from the Fond National de la Recherche Scientifique de Belgique (grant Télevie N° 7.4554.14F and N° 7.4512.15F) and the Fondation Salus Sanguinis. No conflict of interest is declared. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Hemoglobin Function in Stored Blood.

DTIC Science & Technology

1977-12-31

reverse aide if neceseary and Identify by block number) Blood preservation, Red Cell Function, 2,3- Diphosphoglycerate , Adenine, Inosine, Methylene Blue...2,3-DPG, pH, and glucose levels of whole blood and packed cells studied in CPD-adenine with the following variables: pH, glucose concentrations...aimed directly at maintaining red cell 2,3-DPG levels during blood storage in order for transfused blood to deliver oxygen to the tissues immediately

Ebselen treatment prevents islet apoptosis, maintains intranuclear Pdx-1 and MafA levels, and preserves β-cell mass and function in ZDF rats.

PubMed

Mahadevan, Jana; Parazzoli, Susan; Oseid, Elizabeth; Hertzel, Ann V; Bernlohr, David A; Vallerie, Sara N; Liu, Chang-qin; Lopez, Melissa; Harmon, Jamie S; Robertson, R Paul

2013-10-01

We reported earlier that β-cell-specific overexpression of glutathione peroxidase (GPx)-1 significantly ameliorated hyperglycemia in diabetic db/db mice and prevented glucotoxicity-induced deterioration of β-cell mass and function. We have now ascertained whether early treatment of Zucker diabetic fatty (ZDF) rats with ebselen, an oral GPx mimetic, will prevent β-cell deterioration. No other antihyperglycemic treatment was given. Ebselen ameliorated fasting hyperglycemia, sustained nonfasting insulin levels, lowered nonfasting glucose levels, and lowered HbA1c levels with no effects on body weight. Ebselen doubled β-cell mass, prevented apoptosis, prevented expression of oxidative stress markers, and enhanced intranuclear localization of pancreatic and duodenal homeobox (Pdx)-1 and v-maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA), two critical insulin transcription factors. Minimal β-cell replication was observed in both groups. These findings indicate that prevention of oxidative stress is the mechanism whereby ebselen prevents apoptosis and preserves intranuclear Pdx-1 and MafA, which, in turn, is a likely explanation for the beneficial effects of ebselen on β-cell mass and function. Since ebselen is an oral antioxidant currently used in clinical trials, it is a novel therapeutic candidate to ameliorate fasting hyperglycemia and further deterioration of β-cell mass and function in humans undergoing the onset of type 2 diabetes.

A comparison of cryopreservation methods: Slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and CD34+ enumeration of human umbilical cord blood mononucleated cells

PubMed Central

2011-01-01

Background The finding of human umbilical cord blood as one of the most likely sources of hematopoietic stem cells offers a less invasive alternative for the need of hematopoietic stem cell transplantation. Due to the once-in-a-life time chance of collecting it, an optimum cryopreservation method that can preserve the life and function of the cells contained is critically needed. Methods Until now, slow-cooling has been the routine method of cryopreservation; however, rapid-cooling offers a simple, efficient, and harmless method for preserving the life and function of the desired cells. Therefore, this study was conducted to compare the effectiveness of slow- and rapid-cooling to preserve umbilical cord blood of mononucleated cells suspected of containing hematopoietic stem cells. The parameters used in this study were differences in cell viability, malondialdehyde content, and apoptosis level. The identification of hematopoietic stem cells themselves was carried out by enumerating CD34+ in a flow cytometer. Results Our results showed that mononucleated cell viability after rapid-cooling (91.9%) was significantly higher than that after slow-cooling (75.5%), with a p value = 0.003. Interestingly, the malondialdehyde level in the mononucleated cell population after rapid-cooling (56.45 μM) was also significantly higher than that after slow-cooling (33.25 μM), with a p value < 0.001. The apoptosis level in rapid-cooling population (5.18%) was not significantly different from that of the mononucleated cell population that underwent slow-cooling (3.81%), with a p value = 0.138. However, CD34+ enumeration was much higher in the population that underwent slow-cooling (23.32 cell/μl) than in the one that underwent rapid-cooling (2.47 cell/μl), with a p value = 0.001. Conclusions Rapid-cooling is a potential cryopreservation method to be used to preserve the umbilical cord blood of mononucleated cells, although further optimization of the number of CD34+ cells after rapid-cooling is critically needed. PMID:21943045

Phagocytosis of photoreceptor outer segments by transplanted human neural stem cells as a neuroprotective mechanism in retinal degeneration.

PubMed

Cuenca, Nicolás; Fernández-Sánchez, Laura; McGill, Trevor J; Lu, Bin; Wang, Shaomei; Lund, Raymond; Huhn, Stephen; Capela, Alexandra

2013-10-15

Transplantation of human central nervous system stem cells (HuCNS-SC) into the subretinal space of Royal College of Surgeons (RCS) rats preserves photoreceptors and visual function. To explore possible mechanism(s) of action underlying this neuroprotective effect, we performed a detailed morphologic and ultrastructure analysis of HuCNS-SC transplanted retinas. The HuCNS-SC were transplanted into the subretinal space of RCS rats. Histologic examination of the transplanted retinas was performed by light and electron microscopy. Areas of the retina adjacent to HuCNS-SC graft (treated regions) were analyzed and compared to control sections obtained from the same retina, but distant from the transplant site (untreated regions). The HuCNS-SC were detected as a layer of STEM 121 immunopositive cells in the subretinal space. In treated regions, preserved photoreceptor nuclei, as well as inner and outer segments were identified readily. In contrast, classic signs of degeneration were observed in the untreated regions. Interestingly, detailed ultrastructure analysis revealed a striking preservation of the photoreceptor-bipolar-horizontal cell synaptic contacts in the outer plexiform layer (OPL) of treated areas, in stark contrast with untreated areas. Finally, the presence of phagosomes and vesicles exhibiting the lamellar structure of outer segments also was detected within the cytosol of HuCNS-SC, indicating that these cells have phagocytic capacity in vivo. This study reveals the novel finding that preservation of specialized synaptic contacts between photoreceptors and second order neurons, as well as phagocytosis of photoreceptor outer segments, are potential mechanism(s) of HuCNS-SC transplantation, mediating functional rescue in retinal degeneration.

Importance of oestrogen receptors to preserve functional β-cell mass in diabetes.

PubMed

Tiano, Joseph P; Mauvais-Jarvis, Franck

2012-02-14

Protecting the functional mass of insulin-producing β cells of the pancreas is a major therapeutic challenge in patients with type 1 (T1DM) or type 2 diabetes mellitus (T2DM). The gonadal hormone 17β-oestradiol (E2) is involved in reproductive, bone, cardiovascular and neuronal physiology. In rodent models of T1DM and T2DM, treatment with E2 protects pancreatic β cells against oxidative stress, amyloid polypeptide toxicity, lipotoxicity and apoptosis. Three oestrogen receptors (ERs)--ERα, ERβ and the G protein-coupled ER (GPER)--have been identified in rodent and human β cells. Whereas activation of ERα enhances glucose-stimulated insulin biosynthesis, reduces islet toxic lipid accumulation and promotes β-cell survival from proapoptotic stimuli, activation of ERβ increases glucose-stimulated insulin secretion. However, activation of GPER protects β cells from apoptosis, raises glucose-stimulated insulin secretion and lipid homeostasis without affecting insulin biosynthesis. Oestrogens are also improving islet engraftment in rodent models of pancreatic islet transplantation. This Review describes developments in the role of ERs in islet insulin biosynthesis and secretion, lipid homeostasis and survival. Moreover, we discuss why and how enhancing ER action in β cells without the undesirable effect of general oestrogen therapy is a therapeutic avenue to preserve functional β-cell mass in patients with diabetes mellitus.

Initiation of ART during Early Acute HIV Infection Preserves Mucosal Th17 Function and Reverses HIV-Related Immune Activation

PubMed Central

Schuetz, Alexandra; Deleage, Claire; Sereti, Irini; Rerknimitr, Rungsun; Phanuphak, Nittaya; Phuang-Ngern, Yuwadee; Estes, Jacob D.; Sandler, Netanya G.; Sukhumvittaya, Suchada; Marovich, Mary; Jongrakthaitae, Surat; Akapirat, Siriwat; Fletscher, James L. K.; Kroon, Eugene; Dewar, Robin; Trichavaroj, Rapee; Chomchey, Nitiya; Douek, Daniel C.; O′Connell, Robert J.; Ngauy, Viseth; Robb, Merlin L.; Phanuphak, Praphan; Michael, Nelson L.; Excler, Jean-Louis; Kim, Jerome H.; de Souza, Mark S.; Ananworanich, Jintanat

2014-01-01

Mucosal Th17 cells play an important role in maintaining gut epithelium integrity and thus prevent microbial translocation. Chronic HIV infection is characterized by mucosal Th17 cell depletion, microbial translocation and subsequent immune-activation, which remain elevated despite antiretroviral therapy (ART) correlating with increased mortality. However, when Th17 depletion occurs following HIV infection is unknown. We analyzed mucosal Th17 cells in 42 acute HIV infection (AHI) subjects (Fiebig (F) stage I-V) with a median duration of infection of 16 days and the short-term impact of early initiation of ART. Th17 cells were defined as IL-17+ CD4+ T cells and their function was assessed by the co-expression of IL-22, IL-2 and IFNγ. While intact during FI/II, depletion of mucosal Th17 cell numbers and function was observed during FIII correlating with local and systemic markers of immune-activation. ART initiated at FI/II prevented loss of Th17 cell numbers and function, while initiation at FIII restored Th17 cell numbers but not their polyfunctionality. Furthermore, early initiation of ART in FI/II fully reversed the initially observed mucosal and systemic immune-activation. In contrast, patients treated later during AHI maintained elevated mucosal and systemic CD8+ T-cell activation post initiation of ART. These data support a loss of Th17 cells at early stages of acute HIV infection, and highlight that studies of ART initiation during early AHI should be further explored to assess the underlying mechanism of mucosal Th17 function preservation. PMID:25503054

Preserved immune functionality and high CMV-specific T-cell responses in HIV-infected individuals with poor CD4+ T-cell immune recovery.

PubMed

Gómez-Mora, Elisabet; García, Elisabet; Urrea, Victor; Massanella, Marta; Puig, Jordi; Negredo, Eugenia; Clotet, Bonaventura; Blanco, Julià; Cabrera, Cecilia

2017-09-15

Poor CD4 + T-cell recovery after cART has been associated with skewed T-cell maturation, inflammation and immunosenescence; however, T-cell functionality in those individuals has not been fully characterized. In the present study, we assessed T-cell function by assessing cytokine production after polyclonal, CMV and HIV stimulations of T-cells from ART-suppressed HIV-infected individuals with CD4 + T-cell counts >350 cells/μL (immunoconcordants) or <350 cells/μL (immunodiscordants). A group of HIV-uninfected individuals were also included as controls. Since CMV co-infection significantly affected T-cell maturation and polyfunctionality, only CMV + individuals were analyzed. Despite their reduced and skewed CD4 + T-cell compartment, immunodiscordant individuals showed preserved polyclonal and HIV-specific responses. However, CMV response in immunodiscordant participants was significantly different from immunoconcordant or HIV-seronegative individuals. In immunodiscordant subjects, the magnitude of IFN-γ + CD8 + and IL-2 + CD4 + T-cells in response to CMV was higher and differently associated with the CD4 + T-cell maturation profile., showing an increased frequency of naïve, central memory and EMRA CMV-specific CD4 + T-cells. In conclusion, CD4 + and CD8 + T-cell polyfunctionality was not reduced in immunodiscordant individuals, although heightened CMV-specific immune responses, likely related to subclinical CMV reactivations, may be contributing to the skewed T-cell maturation and the higher risk of clinical progression observed in those individuals.

Immune Interventions to Preserve Beta Cell Function in Type 1 Diabetes

PubMed Central

Ehlers, Mario R.

2015-01-01

Type 1 diabetes (T1D) is a chronic autoimmune disease that leads to destruction of pancreatic beta cells, lifelong dependence on insulin, and increased morbidity and mortality from diabetes-related complications. Preservation of residual beta cells at diagnosis is a major goal because higher levels of endogenous insulin secretion are associated with better short- and long-term outcomes. Over the past 3 decades, a variety of immune interventions have been evaluated in the setting of new-onset T1D, including nonspecific immunosuppression, pathway-specific immune modulation, antigen-specific therapies, and cellular therapies. To date, no single intervention has produced durable remission off-therapy in the majority of treated patients, but the field has gained valuable insights into disease mechanisms and potential immunologic correlates of success. In particular, T cell-directed therapies, including therapies that lead to partial depletion or modulation of effector T (Teff) cells and preservation or augmentation of regulatory T (Treg) cells, have shown the most success and will likely form the backbone of future approaches. The next phase will see evaluation of rational combinations, comprising one or more of the following: a Teff-depleting or modulating drug, a cytokine-based tolerogenic (Treg-promoting) agent, and an antigen-specific component. The long-term goal is to reestablish immunologic tolerance to beta cells, thereby preserving residual beta cells early after diagnosis or enabling restoration of beta cell mass from autologous stem cells or induced neogenesis in patients with established T1D. PMID:26225763

Cryopreservation of putative pre-pubertal bovine spermatogonial stem cells by slow freezing.

PubMed

Kim, Ki-Jung; Lee, Yong-An; Kim, Bang-Jin; Kim, Yong-Hee; Kim, Byung-Gak; Kang, Hyun-Gu; Jung, Sang-Eun; Choi, Sun-Ho; Schmidt, Jonathan A; Ryu, Buom-Yong

2015-04-01

Development of techniques for the preservation of mammalian spermatogonial stem cells (SSCs) is a critical step in commercial application of SSC based technologies, including species preservation, amplification of agriculturally valuable germ lines, and human fertility preservations. The objective of this study was to develop an efficient cryopreservation protocol for preservation of bovine SSCs using a slow freezing technique. To maximize the efficiency of SSC cryopreservation, the effects of various methods (tissue vs. cell freezing) and cryoprotective agents (trehalose, sucrose, and polyethylene glycol [PEG]) were tested. Following thawing, cells were enriched for undifferentiated spermatogonia by differential plating and evaluated for recovery rate, proliferation capacity, and apoptosis. Additionally, putative stem cell activity was assessed using SSC xenotransplantation. The recovery rate, and proliferation capacity of undifferentiated spermatogonia were significantly greater for germ cells frozen using tissue freezing methods compared to cell freezing methods. Cryopreservation in the presence of 200 mM trehalose resulted in significantly greater recovery rate, proliferation capacity, and apoptosis of germ cells compared to control. Furthermore, cryopreservation using the tissue freezing method in the presence of 200 mM trehalose resulted in the production of colonies of donor-derived germ cells after xenotransplantation into recipient mouse testes, indicating putative stem cell function. Collectively, these data indicate that cryopreservation using tissue freezing methods in the presence of 200 mM trehalose is an efficient cryopreservation protocol for bovine SSCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Nucleostemin rejuvenates cardiac progenitor cells and antagonizes myocardial aging.

PubMed

Hariharan, Nirmala; Quijada, Pearl; Mohsin, Sadia; Joyo, Anya; Samse, Kaitlen; Monsanto, Megan; De La Torre, Andrea; Avitabile, Daniele; Ormachea, Lucia; McGregor, Michael J; Tsai, Emily J; Sussman, Mark A

2015-01-20

Functional decline in stem cell-mediated regeneration contributes to aging associated with cellular senescence in c-kit+ cardiac progenitor cells (CPCs). Clinical implementation of CPC-based therapy in elderly patients would benefit tremendously from understanding molecular characteristics of senescence to antagonize aging. Nucleostemin (NS) is a nucleolar protein regulating stem cell proliferation and pluripotency. This study sought to demonstrate that NS preserves characteristics associated with "stemness" in CPCs and antagonizes myocardial senescence and aging. CPCs isolated from human fetal (fetal human cardiac progenitor cell [FhCPC]) and adult failing (adult human cardiac progenitor cell [AhCPC]) hearts, as well as young (young cardiac progenitor cell [YCPC]) and old mice (old cardiac progenitor cell [OCPC]), were studied for senescence characteristics and NS expression. Heterozygous knockout mice with 1 functional allele of NS (NS+/-) were used to demonstrate that NS preserves myocardial structure and function and slows characteristics of aging. NS expression is decreased in AhCPCs relative to FhCPCs, correlating with lowered proliferation potential and shortened telomere length. AhCPC characteristics resemble those of OCPCs, which have a phenotype induced by NS silencing, resulting in cell flattening, senescence, multinucleated cells, decreased S-phase progression, diminished expression of stemness markers, and up-regulation of p53 and p16. CPC senescence resulting from NS loss is partially p53 dependent and is rescued by concurrent silencing of p53. Mechanistically, NS induction correlates with Pim-1 kinase-mediated stabilization of c-Myc. Engineering OCPCs and AhCPCs to overexpress NS decreases senescent and multinucleated cells, restores morphology, and antagonizes senescence, thereby preserving phenotypic properties of "stemness." Early cardiac aging with a decline in cardiac function, an increase in senescence markers p53 and p16, telomere attrition, and accompanied CPC exhaustion is evident in NS+/- mice. Youthful properties and antagonism of senescence in CPCs and the myocardium are consistent with a role for NS downstream from Pim-1 signaling that enhances cardiac regeneration. Copyright © 2015 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Preservation of Antigen-Specific Functions of αβ T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy.

PubMed

Li Pira, Giuseppina; Di Cecca, Stefano; Biagini, Simone; Girolami, Elia; Cicchetti, Elisabetta; Bertaina, Valentina; Quintarelli, Concetta; Caruana, Ignazio; Lucarelli, Barbarella; Merli, Pietro; Pagliara, Daria; Brescia, Letizia Pomponia; Bertaina, Alice; Montanari, Mauro; Locatelli, Franco

2017-01-01

Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification.

Preservation of Antigen-Specific Functions of αβ T Cells and B Cells Removed from Hematopoietic Stem Cell Transplants Suggests Their Use As an Alternative Cell Source for Advanced Manipulation and Adoptive Immunotherapy

PubMed Central

Li Pira, Giuseppina; Di Cecca, Stefano; Biagini, Simone; Girolami, Elia; Cicchetti, Elisabetta; Bertaina, Valentina; Quintarelli, Concetta; Caruana, Ignazio; Lucarelli, Barbarella; Merli, Pietro; Pagliara, Daria; Brescia, Letizia Pomponia; Bertaina, Alice; Montanari, Mauro; Locatelli, Franco

2017-01-01

Hematopoietic stem cell transplantation is standard therapy for numerous hematological diseases. The use of haploidentical donors, sharing half of the HLA alleles with the recipient, has facilitated the use of this procedure as patients can rely on availability of a haploidentical donor within their family. Since HLA disparity increases the risk of graft-versus-host disease, T-cell depletion has been used to remove alloreactive lymphocytes from the graft. Selective removal of αβ T cells, which encompass the alloreactive repertoire, combined with removal of B cells to prevent EBV-related lymphoproliferative disease, proved safe and effective in clinical studies. Depleted αβ T cells and B cells are generally discarded as by-products. Considering the possible use of donor T cells for donor lymphocyte infusions or for generation of pathogen-specific T cells as mediators of graft-versus-infection effect, we tested whether cells in the discarded fractions were functionally intact. Response to alloantigens and to viral antigens comparable to that of unmanipulated cells indicated a functional integrity of αβ T cells, in spite of the manipulation used for their depletion. Furthermore, B cells proved to be efficient antigen-presenting cells, indicating that antigen uptake, processing, and presentation were fully preserved. Therefore, we propose that separated αβ T lymphocytes could be employed for obtaining pathogen-specific T cells, applying available methods for positive selection, which eventually leads to indirect allodepletion. In addition, these functional T cells could undergo additional manipulation, such as direct allodepletion or genetic modification. PMID:28386262

Nucleostemin Rejuvenates Cardiac Progenitor Cells and Antagonizes Myocardial Aging

PubMed Central

Hariharan, Nirmala; Quijada, Pearl; Mohsin, Sadia; Joyo, Anya; Samse, Kaitlen; Monsanto, Megan; De La Torre, Andrea; Avitabile, Daniele; Ormachea, Lucia; McGregor, Michael J.; Tsai, Emily J; Sussman, Mark A.

2015-01-01

BACKGROUND Functional decline in stem cell-mediated regeneration contributes to aging associated with cellular senescence in c-kit+ cardiac progenitor cells (CPCs). Clinical implementation of CPC-based therapy with elderly patients would benefit tremendously from understanding molecular characteristics of senescence to antagonize aging. Nucleostemin (NS) is a nucleolar protein regulating stem cell proliferation and pluripotency. OBJECTIVES The goal is to demonstrate that NS preserves characteristics associated with “stemness” in CPCs and antagonizes myocardial senescence and aging. METHODS CPCs isolated from human fetal (FhCPC) and adult failing (AhCPC) hearts, as well as young (YCPC) and old mice (OCPC), were studied for senescence characteristics and NS expression. Heterozygous knockout mice with one functional allele of NS (NS+/−) were used to demonstrate that NS preserves myocardial structure and function and slows characteristics of aging. RESULTS NS expression is decreased in AhCPCs relative to FhCPC, correlating with lowered proliferation potential and shortened telomere length. AhCPC characteristics resemble OCPCs, which have a phenotype induced by NS silencing, resulting in cell flattening, senescence, multinucleated cells, decreased S phase progression, diminished expression of stemness markers and up-regulation of p53 and p16. CPC senescence resulting from NS loss is partially p53 dependent and is rescued by concurrent silencing of p53. Mechanistically, NS induction correlates with Pim-1 kinase-mediated stabilization of c-Myc. Engineering OCPCs and AhCPCs to overexpress NS decreases senescent and multinucleated cells, restores morphology, and antagonizes senescence, thereby preserving phenotypic properties of “stemness.” Early cardiac aging with decline in cardiac function, increase in senescence markers p53 and p16, telomere attrition, and accompanied CPC exhaustion is evident in NS+/− mice. CONCLUSIONS Youthful properties and antagonism of senescence in CPCs and the myocardium is consistent with a role for NS downstream from Pim-1 signaling that enhances cardiac regeneration. PMID:25593054

Osteocalcin protects pancreatic beta cell function and survival under high glucose conditions

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

Kover, Karen, E-mail: kkover@cmh.edu; University of Missouri-Kansas City School of Medicine, Kansas City, MO 64108; Yan, Yun

Diabetes is characterized by progressive beta cell dysfunction and loss due in part to oxidative stress that occurs from gluco/lipotoxicity. Treatments that directly protect beta cell function and survival in the diabetic milieu are of particular interest. A growing body of evidence suggests that osteocalcin, an abundant non-collagenous protein of bone, supports beta cell function and proliferation. Based on previous gene expression data by microarray, we hypothesized that osteocalcin protects beta cells from glucose-induced oxidative stress. To test our hypothesis we cultured isolated rat islets and INS-1E cells in the presence of normal, high, or high glucose ± osteocalcin for up tomore » 72 h. Oxidative stress and viability/mitochondrial function were measured by H{sub 2}O{sub 2} assay and Alamar Blue assay, respectively. Caspase 3/7 activity was also measured as a marker of apoptosis. A functional test, glucose stimulated insulin release, was conducted and expression of genes/protein was measured by qRT-PCR/western blot/ELISA. Osteocalcin treatment significantly reduced high glucose-induced H{sub 2}O{sub 2} levels while maintaining viability/mitochondrial function. Osteocalcin also significantly improved glucose stimulated insulin secretion and insulin content in rat islets after 48 h of high glucose exposure compared to untreated islets. As expected sustained high glucose down-regulated gene/protein expression of INS1 and BCL2 while increasing TXNIP expression. Interestingly, osteocalcin treatment reversed the effects of high glucose on gene/protein expression. We conclude that osteocalcin can protect beta cells from the negative effects of glucose-induced oxidative stress, in part, by reducing TXNIP expression, thereby preserving beta cell function and survival. - Highlights: • Osteocalcin reduces glucose-induced oxidative stress in beta cells. • Osteocalcin preserves beta cell function and survival under stress conditions. • Osteocalcin reduces glucose-induced TXNIP expression in beta cells.« less

[Fertility preservation in patients with hematological malignancies].

PubMed

Kanda, Yoshinobu

2015-03-01

Antineoplastic chemotherapy and irradiation affect gonadal function and may lead to infertility. Recovery of gonadal function is frequently observed after conventional chemotherapy in young patients with hematological malignancies, but conditioning regimens before hematopoietic stem cell transplantation result in permanent gonadal failure. Cryopreservation of sperm is effective for male patients, but it becomes difficult even after a single cycle of chemotherapy and therefore should be accomplished before starting chemotherapy. Embryo freezing after in vitro fertilization of harvested oocytes is an established method to preserve fertility in female patients. In addition, harvesting and freezing of unfertilized oocytes is also being evaluated in a clinical study. However, collection of good oocytes after chemotherapy is difficult. In addition, oocyte harvesting is an invasive procedure and may be associated with hemorrhage or infectious complications. Ovarian shielding during total body irradiation allows ovary preservation in most female patients, but this cannot be performed in patients with active malignancies. Strategies for gonadal function preservation should be planned before starting treatment for hematological malignancies.

CD4-veCD8-ve CD30+ve T cells are detectable in human lung transplant patients and their proportion of the lymphocyte population after in vitro stimulation with donor spleen cells correlates with preservation of lung physiology.

PubMed

Polster, K; Walker, A; Fildes, J; Entwistle, G; Yonan, N; Hutchinson, I V; Leonard, C T

2005-06-01

Survival following lung transplantation is less than 50% at 5 years, mainly due to immune-mediated chronic rejection. Recently a novel subset of T cells, CD4-veCD8-ve CD30+ve, so-called double negative (DN) CD30+ve T cells, has been described and shown to be responsible for tolerance in an animal model of skin transplantation. We investigated 18 lung transplant recipients for the presence of DN CD30+ve T cells in resting peripheral blood and also following in vitro stimulation of recipient peripheral blood mononuclear cells (PBMCs) with donor spleen cells. Small percentages (0.2% to 6%) of DN T cells are detectable in resting PBMCs of human transplant patients (n = 18), but these did not correlate with allograft function, acute rejection episodes, HLA mismatch, or CMV status. On repeated stimulation of recipient PBMCs (two exposures) in vitro by donor spleen cells (2:1 ratio stimulators to responders) the percentage of DN CD30+ve T cells within the lymphocyte pool correlated with preservation of allograft lung function (both for FEV(1), P = .009, and FEF(25-75), P = .036) and was inversely correlated with grade of chronic rejection. On repeated exposure of recipient PBMCs to donor spleen cells with a 1:1 ratio the percentage of DN CD30+ve T cells correlated with the number of acute rejection episodes of grade 2 or greater. The total number of HLA mismatches correlated with the percentage DN CD30+ve T cells present after primary stimulation of recipient PBMCs with donor spleen cells (1:1 ratio). The number of mismatches at the B locus inversely correlated with the percentage of DN CD30+ve T cells after primary stimulation of recipient PBMCs with donor spleen cells (1:1 ratio; P = .031, n = 18). Percentages of DN CD30+ve T cells present following repeated stimulation of recipient PBMCs by donor spleen cells correlated with preservation of graft function following lung transplantation.

Previous Ingestion of Lactococcus lactis by Ethanol-Treated Mice Preserves Antigen Presentation Hierarchy in the Gut and Oral Tolerance Susceptibility.

PubMed

Alvarenga, Débora M; Perez, Denise A; Gomes-Santos, Ana C; Miyoshi, Anderson; Azevedo, Vasco; Coelho-Dos-Reis, Jordana G A; Martins-Filho, Olindo A; Faria, Ana Maria C; Cara, Denise C; Andrade, Marileia C

2015-08-01

Ethanol (EtOH) consumption is able to disturb the ovalbumin (OVA)-oral tolerance induction by interfering on the function of antigen presenting cells (APC), down-regulating dendritic cells (DCs) and macrophages and up-regulating B-lymphocytes and their function, which results in an overall allergic-type immune status. In this study, the potential of a priori administration of Lactococcus lactis (LL) in avoiding loss of oral tolerance in EtOH-treated mice was investigated. Female C57BL/6 mice received, by oral route, ad libitum wild-type (WT) LL or heat-shock protein producer (Hsp65) LL for 4 consecutive days. Seven days later, mice were submitted to short-term high-dose EtOH treatment. After 24 hours, stomach, intestine, spleen, mesenteric lymph nodes (mLN) specimens were collected for biomarkers analysis. Following EtOH-treatment protocol, a group of animals underwent single-gavage OVA-tolerance protocol and sera samples collected for antibody analysis. The ingestion of WT LL or Hsp65 LL is able to restore oral tolerance to OVA in EtOH-treated mice, by reducing local and systemic allergic outcomes such as gastric mast cells and gut-interleukin-4, as well as serum IgE. WT LL treatment prevents the decrease of mLN regulatory T cells induced by the EtOH treatment. Moreover, LL treatment preserves APC hierarchy and antigen presentation commitment in EtOH-treated mice, with conserved DC and macrophage activity over B lymphocytes in mLN and preserved macrophage activity over DC and B-cell subsets in the spleen. The present findings suggest that a priori ingestion of LL preserves essential mechanisms associated with oral tolerance induction that are disturbed by EtOH ingestion. Maintenance of mucosal homeostasis by preserving APC hierarchy and antigen presentation commitment could be associated with T-regulatory subset activities in the gastrointestinal tract. Copyright © 2015 by the Research Society on Alcoholism.

Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head

PubMed Central

2012-01-01

Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN. PMID:22356811

MicroRNAs in islet immunobiology and transplantation.

PubMed

Pileggi, Antonello; Klein, Dagmar; Fotino, Carmen; Bravo-Egaña, Valia; Rosero, Samuel; Doni, Marco; Podetta, Michele; Ricordi, Camillo; Molano, R Damaris; Pastori, Ricardo L

2013-12-01

The ultimate goal of diabetes therapy is the restoration of physiologic metabolic control. For type 1 diabetes, research efforts are focused on the prevention or early intervention to halt the autoimmune process and preserve β cell function. Replacement of pancreatic β cells via islet transplantation reestablishes physiologic β cell function in patients with diabetes. Emerging research shows that microRNAs (miRNAs), noncoding small RNA molecules produced by a newly discovered class of genes, negatively regulate gene expression. MiRNAs recognize and bind to partially complementary sequences of target messenger RNA (mRNA), regulating mRNA translation and affecting gene expression. Correlation between miRNA signatures and genome-wide RNA expression allows identification of multiple miRNA-mRNA pairs in biological processes. Because miRNAs target functionally related genes, they represent an exciting and indispensable approach for biomarkers and drug discovery. We are studying the role of miRNA in the context of islet immunobiology. Our research aims at understanding the mechanisms underlying pancreatic β cell loss and developing clinically relevant approaches for preservation and restoration of β cell function to treat insulin-dependent diabetes. Herein, we discuss some of our recent efforts related to the study of miRNA in islet inflammation and islet engraftment. Our working hypothesis is that modulation of the expression of specific microRNAs in the transplant microenvironment will be of assistance in enhancing islet engraftment and promoting long-term function.

Autophagy and self-preservation: a step ahead from cell plasticity?

PubMed

Galliot, Brigitte

2006-01-01

Silencing the SPINK-related gene Kazal1 in hydra gland cells induces an excessive autophagy of both gland and digestive cells, leading to animal death. Moreover, during regeneration, autophagosomes are immediately detected in regenerating tips, where Kazal1 expression is lowered. When Kazal1 is completely silenced, hydra no longer survive the amputation stress (Chera S, de Rosa R, Miljkovic-Licina M, Dobretz K, Ghila L, Kaloulis K, Galliot B. Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human Spink1 pancreatic phenotype. J Cell Sci 2006; 119:846-57). These results highlight the essential digestive and cytoprotective functions played by Kazal1 in hydra. In mammals, autophagy of exocrine pancreatic cells is also induced upon SPINK1/Spink3 inactivation, whereas Spink3 is activated in injured pancreatic cells. Hence SPINKs, by preventing an excessive autophagy, appear to act as key players of the stress-induced self-preservation program. In hydra, this program is a prerequisite to the early cellular transition, whereby digestive cells of the regenerating tips transform into a head-organizer center. Enhancing the self-preservation program in injured tissues might therefore be the condition for unmasking their potential cell and/or developmental plasticity.

Estrogen receptor α protects pancreatic β-cells from apoptosis by preserving mitochondrial function and suppressing endoplasmic reticulum stress.

PubMed

Zhou, Zhenqi; Ribas, Vicent; Rajbhandari, Prashant; Drew, Brian G; Moore, Timothy M; Fluitt, Amy H; Reddish, Britany R; Whitney, Kate A; Georgia, Senta; Vergnes, Laurent; Reue, Karen; Liesa, Marc; Shirihai, Orian; van der Bliek, Alexander M; Chi, Nai-Wen; Mahata, Sushil K; Tiano, Joseph P; Hewitt, Sylvia C; Tontonoz, Peter; Korach, Kenneth S; Mauvais-Jarvis, Franck; Hevener, Andrea L

2018-03-30

Estrogen receptor α (ERα) action plays an important role in pancreatic β-cell function and survival; thus, it is considered a potential therapeutic target for the treatment of type 2 diabetes in women. However, the mechanisms underlying the protective effects of ERα remain unclear. Because ERα regulates mitochondrial metabolism in other cell types, we hypothesized that ERα may act to preserve insulin secretion and promote β-cell survival by regulating mitochondrial-endoplasmic reticulum (EndoRetic) function. We tested this hypothesis using pancreatic islet-specific ERα knockout (PERαKO) mice and Min6 β-cells in culture with Esr1 knockdown (KD). We found that Esr1-KD promoted reactive oxygen species production that associated with reduced fission/fusion dynamics and impaired mitophagy. Electron microscopy showed mitochondrial enlargement and a pro-fusion phenotype. Mitochondrial cristae and endoplasmic reticulum were dilated in Esr1-KD compared with ERα replete Min6 β-cells. Increased expression of Oma1 and Chop was paralleled by increased oxygen consumption and apoptosis susceptibility in ERα-KD cells. In contrast, ERα overexpression and ligand activation reduced both Chop and Oma1 expression, likely by ERα binding to consensus estrogen-response element sites in the Oma1 and Chop promoters. Together, our findings suggest that ERα promotes β-cell survival and insulin secretion through maintenance of mitochondrial fission/fusion-mitophagy dynamics and EndoRetic function, in part by Oma1 and Chop repression.

A multicellular view of cytokinesis in epithelial tissue.

PubMed

Herszterg, Sophie; Pinheiro, Diana; Bellaïche, Yohanns

2014-05-01

The study of cytokinesis in single-cell systems provided a wealth of knowledge on the molecular and biophysical mechanisms controlling daughter cell separation. In this review, we outline recent advances in the understanding of cytokinesis in epithelial tissues. These findings provide evidence for how the cytokinetic machinery adapts to a multicellular context and how the cytokinetic machinery is itself exploited by the tissue for the preservation of tissue function and architecture during proliferation. We propose that cytokinesis in epithelia should be viewed as a multicellular process, whereby the biochemical and mechanical interactions between the dividing cell and its neighbors are essential for successful daughter cell separation while defining epithelial tissue organization and preserving tissue integrity. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sulforaphane Protects against High Cholesterol-Induced Mitochondrial Bioenergetics Impairments, Inflammation, and Oxidative Stress and Preserves Pancreatic β-Cells Function.

PubMed

Carrasco-Pozo, Catalina; Tan, Kah Ni; Gotteland, Martin; Borges, Karin

2017-01-01

Cholesterol plays an important role in inducing pancreatic β -cell dysfunction, leading to an impaired insulin secretory response to glucose. This study aimed to determine the protective effects of sulforaphane, a natural isothiocyanate Nrf2-inducer, against cholesterol-induced pancreatic β -cells dysfunction, through molecular and cellular mechanisms involving mitochondrial bioenergetics. Sulforaphane prevented cholesterol-induced alterations in the coupling efficiency of mitochondrial respiration, improving ATP turnover and spare capacity, and averted the impairment of the electron flow at complexes I, II, and IV. Sulforaphane also attenuated the cholesterol-induced activation of the NF κ B pathway, normalizing the expression of pro- and anti-inflammatory cytokines. In addition, it also inhibited the decrease in sirtuin 1 expression and greatly increased Pgc-1α expression in Min6 cells. Sulforaphane increased the expression of antioxidant enzymes downstream of the Nrf2 pathway and prevented lipid peroxidation induced by cholesterol. The antioxidant and anti-inflammatory properties of sulforaphane and its ability to protect and improve mitochondrial bioenergetic function contribute to its protective action against cholesterol-induced pancreatic β -cell dysfunction. Our data provide a scientifically tested foundation upon which sulforaphane can be developed as nutraceutical to preserve β -cell function and eventually control hyperglycemia.

Sulforaphane Protects against High Cholesterol-Induced Mitochondrial Bioenergetics Impairments, Inflammation, and Oxidative Stress and Preserves Pancreatic β-Cells Function

PubMed Central

Tan, Kah Ni; Gotteland, Martin

2017-01-01

Cholesterol plays an important role in inducing pancreatic β-cell dysfunction, leading to an impaired insulin secretory response to glucose. This study aimed to determine the protective effects of sulforaphane, a natural isothiocyanate Nrf2-inducer, against cholesterol-induced pancreatic β-cells dysfunction, through molecular and cellular mechanisms involving mitochondrial bioenergetics. Sulforaphane prevented cholesterol-induced alterations in the coupling efficiency of mitochondrial respiration, improving ATP turnover and spare capacity, and averted the impairment of the electron flow at complexes I, II, and IV. Sulforaphane also attenuated the cholesterol-induced activation of the NFκB pathway, normalizing the expression of pro- and anti-inflammatory cytokines. In addition, it also inhibited the decrease in sirtuin 1 expression and greatly increased Pgc-1α expression in Min6 cells. Sulforaphane increased the expression of antioxidant enzymes downstream of the Nrf2 pathway and prevented lipid peroxidation induced by cholesterol. The antioxidant and anti-inflammatory properties of sulforaphane and its ability to protect and improve mitochondrial bioenergetic function contribute to its protective action against cholesterol-induced pancreatic β-cell dysfunction. Our data provide a scientifically tested foundation upon which sulforaphane can be developed as nutraceutical to preserve β-cell function and eventually control hyperglycemia. PMID:28386307

Prevention of radiation-induced salivary gland dysfunction utilizing a CDK inhibitor in a mouse model.

PubMed

Martin, Katie L; Hill, Grace A; Klein, Rob R; Arnett, Deborah G; Burd, Randy; Limesand, Kirsten H

2012-01-01

Treatment of head and neck cancer with radiation often results in damage to surrounding normal tissues such as salivary glands. Permanent loss of function in the salivary glands often leads patients to discontinue treatment due to incapacitating side effects. It has previously been shown that IGF-1 suppresses radiation-induced apoptosis and enhances G2/M arrest leading to preservation of salivary gland function. In an effort to recapitulate the effects of IGF-1, as well as increase the likelihood of translating these findings to the clinic, the small molecule therapeutic Roscovitine, is being tested. Roscovitine is a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues. Treatment with Roscovitine prior to irradiation induced a significant increase in the percentage of cells in the G(2)/M phase, as demonstrated by flow cytometry. In contrast, mice treated with radiation exhibit no differences in the percentage of cells in G(2)/M when compared to unirradiated controls. Similar to previous studies utilizing IGF-1, pretreatment with Roscovitine leads to a significant up-regulation of p21 expression and a significant decrease in the number of PCNA positive cells. Radiation treatment leads to a significant increase in activated caspase-3 positive salivary acinar cells, which is suppressed by pretreatment with Roscovitine. Administration of Roscovitine prior to targeted head and neck irradiation preserves normal tissue function in mouse parotid salivary glands, both acutely and chronically, as measured by salivary output. These studies suggest that induction of transient G(2)/M cell cycle arrest by Roscovitine allows for suppression of apoptosis, thus preserving normal salivary function following targeted head and neck irradiation. This could have an important clinical impact by preventing the negative side effects of radiation therapy in surrounding normal tissues.

[Influence of raising oxygen content on function of platelet concentrate during preservation].

PubMed

Zhan, Tong; Xiao, Jian-Yu; Tao, Jing; Miao, Xi-Feng; Liu, Yan-Cun; Tang, Rong-Cai

2006-08-01

To explore the influence of raising oxygen (dissolved oxygen) content on function of platelet concentrate, the platelet concentrate was prepared by a CS-3000 plus blood cell separator. Experiments were divided into 2 groups: test group and control group. After raising oxygen content in platelet plasma under sterile operation, the platelet samples of two groups were preserved in oscillator with horizontal oscillation at 22 +/- 2 degrees C. The platelet count, platelet aggregation rate, lactic acid content and CD62p expression level of platelet were detected on 0, 1, 2, 3, 4, 5 days of platelet preservation. The results showed that the platelet count and platelet aggregation rate decreased with prolongation of preserved time, while the lactic acid content and CD62p expression level of platelet increased gradually. Compared with control group, there were significant differences in aggregation rate of platelet preserved for 2-3 days, and in CD62p expression level of platelet preserved for 1-3 days, while significant difference was found in lactic acid content of platelet preserved for 1-3 days. It is concluded that raising content of oxygen in platelet plasma can provide more oxygen to compensate oxygen supply deficiency for platelet metabolism and improve the efficiency of platelet oxygenic metabolism and the quality of platelet during preservation.

Maximising the use of freshly isolated human hepatocytes.

PubMed

Evans, Peter J

2016-01-01

Freshly isolated human hepatocytes are the best model for predicting adverse drug reactions. However, their preparation and use present the investigator with many variables that are beyond their control. These include operation continuity and timing, size and number of cut surfaces on liver tissue and the prior history of the patient. To exploit the potential of freshly isolated human hepatocytes a method is required to preserve the cells in their initial in vivo like state. This experimental pausing allows experiments to be prioritised at convenient times of the day. A novel approach for selecting viable human hepatocytes by functional attachment to a gelatin gel is described rather than relying on their physical characteristics. The cells are preserved as a monolayer on the semi-solid support at 10°C as single spherical entities. The hepatocytes can be released into suspension, when required, by a temperature transition to 37°C for 20min. The cells can be used in suspension or as a monolayer. The length of preservation depends upon the source tissue. Hepatocytes from normal liver can be maintained for at least 4days and demonstrated to have the same level of CYP3A4 and the enzymes involved in glucuronidation and sulphation as freshly isolated cells. Cells from fatty liver, attached to gelatin, vary in their preservation time but it is at least 24h and so confluent monolayers, that survive at 37°C can be generated the following day. The technique enables freshly isolated human hepatocytes to be used more effectively. They can be preserved in times of plenty so more experimentation is possible. Alternatively, with poorer fatty cells the initial attachment on gelatin enables confluent monolayers of lipid rich cells to be studied. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial respiration controls lysosomal function during inflammatory T cell responses

PubMed Central

Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Dolores Ledesma, Maria; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

2016-01-01

Summary The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4+ T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration-deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward pro-inflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD+ levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify novel strategies for intervention in mitochondrial-related diseases. PMID:26299452

Transplantation of BDNF-Secreting Mesenchymal Stem Cells Provides Neuroprotection in Chronically Hypertensive Rat Eyes

PubMed Central

Harper, Matthew M.; Grozdanic, Sinisa D.; Blits, Bas; Kuehn, Markus H.; Zamzow, Daniel; Buss, Janice E.; Kardon, Randy H.; Sakaguchi, Donald S.

2011-01-01

Purpose. To evaluate the ability of mesenchymal stem cells (MSCs) engineered to produce and secrete brain-derived neurotrophic factor (BDNF) to protect retinal function and structure after intravitreal transplantation in a rat model of chronic ocular hypertension (COH). Methods. COH was induced by laser cauterization of trabecular meshwork and episcleral veins in rat eyes. COH eyes received an intravitreal transplant of MSCs engineered to express BDNF and green fluorescent protein (BDNF-MSCs) or just GFP (GFP-MSCs). Computerized pupillometry and electroretinography (ERG) were performed to assess optic nerve and retinal function. Quantification of optic nerve damage was performed by counting retinal ganglion cells (RGCs) and evaluating optic nerve cross-sections. Results. After transplantation into COH eyes, BDNF-MSCs preserved significantly more retina and optic nerve function than GFP-MSC–treated eyes when pupil light reflex (PLR) and ERG function were evaluated. PLR analysis showed significantly better function (P = 0.03) in BDNF-MSC–treated eyes (operated/control ratio = 63.00% ± 11.39%) than GFP-MSC–treated eyes (operated/control ratio = 31.81% ± 9.63%) at 42 days after surgery. The BDNF-MSC–transplanted eyes also displayed a greater level of RGC preservation than eyes that received the GFP-MSCs only (RGC cell counts: BDNF-MSC–treated COH eyes, 112.2 ± 19.39 cells/section; GFP-MSC–treated COH eyes, 52.21 ± 11.54 cells/section; P = 0.01). Conclusions. The authors have demonstrated that lentiviral-transduced BDNF-producing MSCs can survive in eyes with chronic hypertension and can provide retina and optic nerve functional and structural protection. Transplantation of BDNF-producing stem cells may be a viable treatment strategy for glaucoma. PMID:21498611

Dietary antioxidants preserve endothelium-dependent vessel relaxation in cholesterol-fed rabbits.

PubMed Central

Keaney, J F; Gaziano, J M; Xu, A; Frei, B; Curran-Celentano, J; Shwaery, G T; Loscalzo, J; Vita, J A

1993-01-01

Recent evidence suggests that dietary therapy with lipid-soluble antioxidants may be beneficial for patients with atherosclerotic vascular disease but the potential mechanism(s) for these observations remain obscure. Abnormalities in endothelium-dependent control of vascular tone develop early in the course of atherosclerosis and may result from oxidative modification of low density lipoproteins. We examined the role of dietary antioxidants in preserving normal endothelial cell vasodilator function in cholesterol-fed rabbits with particular attention to possible effects on serum lipoproteins, low density lipoprotein oxidation, and atherogenesis. Male New Zealand White rabbits were fed diets containing no additive (controls), 1% cholesterol (cholesterol group), or 1% cholesterol chow supplemented with either beta-carotene (0.6 g/kg of chow) or alpha-tocopherol (1000 international units/kg of chow) for a 28-day period. After dietary therapy, thoracic aortae were harvested for assay of vascular function and for pathologic examination and tissue antioxidant levels. Compared to controls, acetylcholine- and A23187-mediated endothelium-dependent relaxations were significantly impaired in vessels from the cholesterol group (P < 0.001), whereas vessels from animals treated with beta-carotene or alpha-tocopherol demonstrated normal endothelium-dependent arterial relaxation. Preservation of endothelial function was associated with vascular incorporation of alpha-tocopherol and beta-carotene but was unrelated to plasma lipoprotein levels, smooth muscle cell function, or the extent of atherosclerosis. Increased low density lipoprotein resistance to ex vivo copper-mediated oxidation was observed only in the alpha-tocopherol group. Our results suggest that dietary antioxidants may benefit patients with atherosclerosis by preserving endothelial vasodilator function through a mechanism related to vascular tissue antioxidant content and not reflected by assay of low density lipoprotein resistance to ex vivo oxidation. PMID:8265642

Dinosaur peptides suggest mechanisms of protein survival.

PubMed

San Antonio, James D; Schweitzer, Mary H; Jensen, Shane T; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P R O

2011-01-01

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.

Renal Tubular Cell Mitochondrial Dysfunction Occurs Despite Preserved Renal Oxygen Delivery in Experimental Septic Acute Kidney Injury

PubMed Central

Pollen, Sean; Greco, Elisabetta; Courtneidge, Holly; Hall, Andrew M.; Duchen, Michael R.; Tam, Frederick W. K.; Unwin, Robert J.; Singer, Mervyn

2018-01-01

Objective: To explain the paradigm of significant renal functional impairment despite preserved hemodynamics and histology in sepsis-induced acute kidney injury. Design: Prospective observational animal study. Setting: University research laboratory. Subjects: Male Wistar rats. Intervention: Using a fluid-resuscitated sublethal rat model of fecal peritonitis, changes in renal function were characterized in relation to global and renal hemodynamics, and histology at 6 and 24 hours (n = 6–10). Sham-operated animals were used as comparison (n = 8). Tubular cell mitochondrial function was assessed using multiphoton confocal imaging of live kidney slices incubated in septic serum. Measurements and Main Results: By 24 hours, serum creatinine was significantly elevated with a concurrent decrease in renal lactate clearance in septic animals compared with sham-operated and 6-hour septic animals. Renal uncoupling protein-2 was elevated in septic animals at 24 hours although tubular cell injury was minimal and mitochondrial ultrastructure in renal proximal tubular cells preserved. There was no significant change in global or renal hemodynamics and oxygen delivery/consumption between sham-operated and septic animals at both 6- and 24-hour timepoints. In the live kidney slice model, mitochondrial dysfunction was seen in proximal tubular epithelial cells incubated with septic serum with increased production of reactive oxygen species, and decreases in nicotinamide adenine dinucleotide and mitochondrial membrane potential. These effects were prevented by coincubation with the reactive oxygen species scavenger, 4-hydroxy-2,2,6,6-tetramethyl-piperidin-1-oxyl. Conclusions: Renal dysfunction in sepsis occurs independently of hemodynamic instability or structural damage. Mitochondrial dysfunction mediated by circulating mediators that induce local oxidative stress may represent an important pathophysiologic mechanism. PMID:29293148

Atorvastatin helps preserve pancreatic β cell function in obese C57BL/6 J mice and the effect is related to increased pancreas proliferation and amelioration of endoplasmic-reticulum stress.

PubMed

Chen, Zhi-Yu; Liu, Shuai-Nan; Li, Cai-Na; Sun, Su-Juan; Liu, Quan; Lei, Lei; Gao, Li-Hui; Shen, Zhu-Fang

2014-06-21

3-Hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors or statins are competitive inhibitors of the rate-limiting enzyme in cholesterol biosynthesis. Currently, statins are used as first-line therapy in the treatment of diabetic dyslipidemia. However, effects of statins on β cell function remains unclear. This study aims to examine effects of atorvastatin treatment on pancreatic β cell function in obese C57BL/6 J mice and the possible mechanisms. Diet-induced obesity (DIO) C57BL/6 J mice were treated with atorvastatin (30 mg/kg/day) for 58 days. β cell function was assessed by hyperglycemic clamp and the area of insulin-positive β cells was examined by immunofluorescence. Gene expression was assessed by RT-PCR, and endoplasmic reticulum (ER) stress related proteins were examined by Western blot. Additionally, cell viability and apoptosis of the cholesterol-loaded NIT-1 cells were investigated after atorvastatin treatment. Hyperglycemic clamp study revealed that glucose infusion rate (GIR) and insulin stimulation ratio in atorvastatin-treated DIO mice were markedly higher than control mice (P < 0.05, P < 0.01 vs. con), indicating preserved β-cell sensitivity to glucose. Lipid profiles of plasma triglyceride (TG), pancreas TG and plasma cholesterol (CHO) were improved. Pancreas weight and weight index were improved significantly after atorvastatin treatment (P < 0.05 vs. con). Immunofluorescence results showed that atorvastatin-treated mice had significantly larger insulin-positive β cell area (P < 0.05 vs. con). Furthermore, RT-PCR and western blot showed that the mRNA and protein expression of pancreatic and duodenal homeobox 1 (Pdx1) in the pancreas were upregulated (P < 0.001, P < 0.01 vs. con). Moreover, the expression level of ER stress markers of activating transcription factor 4 (ATF4), CCAAT-enhancer-binding protein homologous protein (CHOP) and phosphorylated eukaryotic initiation factor 2α (eIF2α) were downregulated in the pancreas of atorvastatin-treated mice (P < 0.001, P < 0.01, P < 0.01 vs. con). Besides, atorvastatin protected the pancreatic β cell line of NIT-1 from cholesterol-induced apoptosis. Western blot showed increased expression of anti-apoptotic protein of B-cell lymphoma 2 (Bcl-2). Pancreatic β cell function of obese C57BL/6 J mice was preserved after atorvastatin treatment, and this improvement may be attributed to enhanced pancreas proliferation and amelioration of pancreatic ER stress.

Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration.

PubMed

Wang, Shaomei; Lu, Bin; Girman, Sergej; Holmes, Toby; Bischoff, Nicolas; Lund, Raymond D

2008-01-01

It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19. Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression. Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors. RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Comparing renal function preservation after laparoscopic radio frequency ablation assisted tumor enucleation and laparoscopic partial nephrectomy for clinical T1a renal tumor: using a 3D parenchyma measurement system.

PubMed

Zhu, Liangsong; Wu, Guangyu; Huang, Jiwei; Wang, Jianfeng; Zhang, Ruiyun; Kong, Wen; Xue, Wei; Huang, Yiran; Chen, Yonghui; Zhang, Jin

2017-05-01

To compare the renal function preservation between laparoscopic radio frequency ablation assisted tumor enucleation and laparoscopic partial nephrectomy. Data were analyzed from 246 patients who underwent laparoscopic radio frequency ablation assisted tumor enucleation and laparoscopic partial nephrectomy for solitary cT1a renal cell carcinoma from January 2013 to July 2015. To reduce the intergroup difference, we used a 1:1 propensity matching analysis. The functional renal parenchyma volume preservation were measured preoperative and 12 months after surgery. The total renal function recovery and spilt GFR was compared. Multivariable logistic analysis was used for predictive factors for renal function decline. After 1:1 propensity matching, each group including 100 patients. Patients in the laparoscopic radio frequency ablation assisted tumor enucleation had a smaller decrease in estimate glomerular filtration rate at 1 day (-7.88 vs -20.01%, p < 0.001), 3 months (-2.31 vs -10.39%, p < 0.001), 6 months (-2.16 vs -7.99%, p = 0.015), 12 months (-3.26 vs -8.03%, p = 0.012) and latest test (-3.24 vs -8.02%, p = 0.040), also had better functional renal parenchyma volume preservation (89.19 vs 84.27%, p < 0.001), lower decrease of the spilt glomerular filtration rate (-9.41 vs -17.13%, p < 0.001) at 12 months. The functional renal parenchyma volume preservation, warm ischemia time and baseline renal function were the important independent factors in determining long-term functional recovery. The laparoscopic radio frequency ablation assisted tumor enucleation technology has unique advantage and potential in preserving renal parenchyma without ischemia damage compared to conventional laparoscopic partial nephrectomy, and had a better outcome, thus we recommend this technique in selected T1a patients.

Transplantation of Embryonic Spinal Cord Derived Cells Helps to Prevent Muscle Atrophy after Peripheral Nerve Injury

PubMed Central

Ruven, Carolin; Li, Wen; Li, Heng; Wong, Wai-Man; Wu, Wutian

2017-01-01

Injuries to peripheral nerves are frequent in serious traumas and spinal cord injuries. In addition to surgical approaches, other interventions, such as cell transplantation, should be considered to keep the muscles in good condition until the axons regenerate. In this study, E14.5 rat embryonic spinal cord fetal cells and cultured neural progenitor cells from different spinal cord segments were injected into transected musculocutaneous nerve of 200–300 g female Sprague Dawley (SD) rats, and atrophy in biceps brachii was assessed. Both kinds of cells were able to survive, extend their axons towards the muscle and form neuromuscular junctions that were functional in electromyographic studies. As a result, muscle endplates were preserved and atrophy was reduced. Furthermore, we observed that the fetal cells had a better effect in reducing the muscle atrophy compared to the pure neural progenitor cells, whereas lumbar cells were more beneficial compared to thoracic and cervical cells. In addition, fetal lumbar cells were used to supplement six weeks delayed surgical repair after the nerve transection. Cell transplantation helped to preserve the muscle endplates, which in turn lead to earlier functional recovery seen in behavioral test and electromyography. In conclusion, we were able to show that embryonic spinal cord derived cells, especially the lumbar fetal cells, are beneficial in the treatment of peripheral nerve injuries due to their ability to prevent the muscle atrophy. PMID:28264437

Impact of aging on antigen presentation cell function of dendritic cells.

PubMed

Wong, Christine; Goldstein, Daniel R

2013-08-01

Older people exhibit increased mortality to infections and cancer as compared to younger people, indicating that aging impairs immunity. Dendritic cells (DCs) are key for bridging the innate and adaptive arms of the immune system by priming antigen specific T cells. Discerning how aging impacts DC function to initiate adaptive immune responses is of great biomedical importance as this could lead to the development of novel therapeutics to enhance immunity with aging. This review details reports indicating that aging impairs the antigen presenting function of DCs but highlights other studies indicating preserved DC function with aging. How aging impacts antigen presentation by DCs is complex and without a clear unifying biological underpinning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Transoral laser microsurgery for locally advanced (T3-T4a) supraglottic squamous cell carcinoma: Sixteen years of experience.

PubMed

Vilaseca, Isabel; Blanch, José Luis; Berenguer, Joan; Grau, Juan José; Verger, Eugenia; Muxí, África; Bernal-Sprekelsen, Manuel

2016-07-01

Controversy exists regarding treatment of advanced laryngeal cancer. The purpose of this study was to evaluate the oncologic and functional outcomes of T3 to T4a supraglottic squamous carcinomas treated with transoral laser microsurgery (TLM). We conducted a retrospective analysis from an SPSS database. Primary outcomes were: locoregional control, overall survival (OS), disease-specific survival (DSS), laryngectomy-free survival, and function-preservation rates. Secondary objectives were: rate of tracheostomies and gastrostomies according to age. Risk factors for local control and larynx preservation were also evaluated. One hundred fifty-four consecutive patients were chosen for this study. Median follow-up was 40.7 + /- 32.8 months. Five and 10-year OS, DSS, and laryngectomy-free survival were 55.6% and 47%, 67.6% and 58.6%, and 75.2% and 59.5%, respectively. Paraglottic involvement was an independent factor for larynx preservation. Six patients (3.9%) needed a definitive tracheostomy, a gastrostomy, or both. The gastrostomy rate was higher in the group of patients above 65 years of age (p = .03). Five-year laryngectomy-free survival with preserved function was 74.5%. TLM constitutes a true alternative for organ preservation in locally advanced supraglottic carcinomas with good oncologic and functional outcomes. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1050-1057, 2016. © 2016 Wiley Periodicals, Inc.

Speciation and preservation of CrVI and CrIII in finished drinking water matrices using collision cell ion chromatography-inductively coupled plasma-mass spectrometry

EPA Science Inventory

The polyatomic background at the major isotope of Cr was evaluated as a function of collision cell gas flow rate using three different mobile phases. The stability of CrVI was evaluated as a function of solution pH using an enriched 53CrVI. The recovery was ≥ 95% at pH 7.8 but...

Acute administration of ucf-101 ameliorates the locomotor impairments induced by a traumatic spinal cord injury.

PubMed

Reigada, D; Nieto-Díaz, M; Navarro-Ruiz, R; Caballero-López, M J; Del Águila, A; Muñoz-Galdeano, T; Maza, R M

2015-08-06

Secondary death of neural cells plays a key role in the physiopathology and the functional consequences of traumatic spinal cord injury (SCI). Pharmacological manipulation of cell death pathways leading to the preservation of neural cells is acknowledged as a main therapeutic goal in SCI. In the present work, we hypothesize that administration of the neuroprotective cell-permeable compound ucf-101 will reduce neural cell death during the secondary damage of SCI, increasing tissue preservation and reducing the functional deficits. To test this hypothesis, we treated mice with ucf-101 during the first week after a moderate contusive SCI. Our results reveal that ucf-101 administration protects neural cells from the deleterious secondary mechanisms triggered by the trauma, reducing the extension of tissue damage and improving motor function recovery. Our studies also suggest that the effects of ucf-101 may be mediated through the inhibition of HtrA2/OMI and the concomitant increase of inhibitor of apoptosis protein XIAP, as well as the induction of ERK1/2 activation and/or expression. In vitro assays confirm the effects of ucf-101 on both pathways as well as on the reduction of caspase cascade activation and apoptotic cell death in a neuroblastoma cell line. These results suggest that ucf-101 can be a promising therapeutic tool for SCI that deserves more detailed analyses. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

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

Li, Zhao; Jin, Zhu-Qiu, E-mail: zhu-qiu.jin@sdstate.edu

2012-08-31

Highlights: Black-Right-Pointing-Pointer Cardiac tight junctions are present between coronary endothelial cells. Black-Right-Pointing-Pointer Ischemic preconditioning preserves the structural and functional integrity of tight junctions. Black-Right-Pointing-Pointer Myocardial edema is prevented in hearts subjected to ischemic preconditioning. Black-Right-Pointing-Pointer Ischemic preconditioning enhances translocation of ZO-2 from cytosol to cytoskeleton. -- Abstract: Ischemic preconditioning (IPC) is one of the most effective procedures known to protect hearts against ischemia/reperfusion (IR) injury. Tight junction (TJ) barriers occur between coronary endothelial cells. TJs provide barrier function to maintain the homeostasis of the inner environment of tissues. However, the effect of IPC on the structure and function of cardiacmore » TJs remains unknown. We tested the hypothesis that myocardial IR injury ruptures the structure of TJs and impairs endothelial permeability whereas IPC preserves the structural and functional integrity of TJs in the blood-heart barrier. Langendorff hearts from C57BL/6J mice were prepared and perfused with Krebs-Henseleit buffer. Cardiac function, creatine kinase release, and myocardial edema were measured. Cardiac TJ function was evaluated by measuring Evans blue-conjugated albumin (EBA) content in the extravascular compartment of hearts. Expression and translocation of zonula occludens (ZO)-2 in IR and IPC hearts were detected with Western blot. A subset of hearts was processed for the observation of ultra-structure of cardiac TJs with transmission electron microscopy. There were clear TJs between coronary endothelial cells of mouse hearts. IR caused the collapse of TJs whereas IPC sustained the structure of TJs. IR increased extravascular EBA content in the heart and myocardial edema but decreased the expression of ZO-2 in the cytoskeleton. IPC maintained the structure of TJs. Cardiac EBA content and edema were reduced in IPC hearts. IPC enhanced the translocation of ZO-2 from cytosol to cytoskeleton. In conclusion, TJs occur in normal mouse heart. IPC preserves the integrity of TJ structure and function that are vulnerable to IR injury.« less

Biologically Complex Planar Cell Plasma Membranes Supported on Polyelectrolyte Cushions Enhance Transmembrane Protein Mobility and Retain Native Orientation.

PubMed

Liu, Han-Yuan; Chen, Wei-Liang; Ober, Christopher K; Daniel, Susan

2018-01-23

Reconstituted supported lipid bilayers (SLB) are widely used as in vitro cell-surface models because they are compatible with a variety of surface-based analytical techniques. However, one of the challenges of using SLBs as a model of the cell surface is the limited complexity in membrane composition, including the incorporation of transmembrane proteins and lipid diversity that may impact the activity of those proteins. Additionally, it is challenging to preserve the transmembrane protein native orientation, function, and mobility in SLBs. Here, we leverage the interaction between cell plasma membrane vesicles and polyelectrolyte brushes to create planar bilayers from cell plasma membrane vesicles that have budded from the cell surface. This approach promotes the direct incorporation of membrane proteins and other species into the planar bilayer without using detergent or reconstitution and preserves membrane constituents. Furthermore, the structure of the polyelectrolyte brush serves as a cushion between the planar bilayer and rigid supporting surface, limiting the interaction of the cytosolic domains of membrane proteins with this surface. Single particle tracking was used to analyze the motion of GPI-linked yellow fluorescent proteins (GPI-YFP) and neon-green fused transmembrane P2X2 receptors (P2X2-neon) and shows that this platform retains over 75% mobility of multipass transmembrane proteins in its native membrane environment. An enzyme accessibility assay confirmed that the protein orientation is preserved and results in the extracellular domain facing toward the bulk phase and the cytosolic side facing the support. Because the platform presented here retains the complexity of the cell plasma membrane and preserves protein orientation and mobility, it is a better representative mimic of native cell surfaces, which may find many applications in biological assays aimed at understanding cell membrane phenomena.

Dissecting the roles of ROCK isoforms in stress-induced cell detachment.

PubMed

Shi, Jianjian; Surma, Michelle; Zhang, Lumin; Wei, Lei

2013-05-15

The homologous Rho kinases, ROCK1 and ROCK2, are involved in stress fiber assembly and cell adhesion and are assumed to be functionally redundant. Using mouse embryonic fibroblasts (MEFs) derived from ROCK1(-/-) and ROCK2(-/-) mice, we have recently reported that they play different roles in regulating doxorubicin-induced stress fiber disassembly and cell detachment: ROCK1 is involved in destabilizing the actin cytoskeleton and cell detachment, whereas ROCK2 is required for stabilizing the actin cytoskeleton and cell adhesion. Here, we present additional insights into the roles of ROCK1 and ROCK2 in regulating stress-induced impairment of cell-matrix and cell-cell adhesion. In response to doxorubicin, ROCK1(-/-) MEFs showed significant preservation of both focal adhesions and adherens junctions, while ROCK2(-/-) MEFs exhibited impaired focal adhesions but preserved adherens junctions compared with the wild-type MEFs. Additionally, inhibition of focal adhesion or adherens junction formations by chemical inhibitors abolished the anti-detachment effects of ROCK1 deletion. Finally, ROCK1(-/-) MEFs, but not ROCK2(-/-) MEFs, also exhibited preserved central stress fibers and reduced cell detachment in response to serum starvation. These results add new insights into a novel mechanism underlying the anti-detachment effects of ROCK1 deletion mediated by reduced peripheral actomyosin contraction and increased actin stabilization to promote cell-cell and cell-matrix adhesion. Our studies further support the differential roles of ROCK isoforms in regulating stress-induced loss of central stress fibers and focal adhesions as well as cell detachment.

Non-Invasive Cell-Based Therapy for Traumatic Optic Neuropathy

DTIC Science & Technology

2015-06-01

Morphological and Functional Changes in an Animal Model of Retinitis Pigmentosa . Vis Neurosci, 2013: 1-13. Bin Lu, Catherine W. Morgans, Sergey Girman...of human retinal progenitor cells for treatment of retinitis pigmentosa 2013, ARVO, A0106. Benjamin Bakondi; YuChun Tsai; Bin Lu; Sergey...Systemic administration of MSCs significantly preserved retinal ganglion cell survival after TON. (d) Systemic administration of MSCs also promote limited

An injectable spheroid system with genetic modification for cell transplantation therapy.

PubMed

Uchida, Satoshi; Itaka, Keiji; Nomoto, Takahiro; Endo, Taisuke; Matsumoto, Yu; Ishii, Takehiko; Kataoka, Kazunori

2014-03-01

The new methodology to increase a therapeutic potential of cell transplantation was developed here by the use of three-dimensional spheroids of transplanting cells subsequent to the genetic modification with non-viral DNA vectors, polyplex nanomicelles. Particularly, spheroids in regulated size of 100-μm of primary hepatocytes transfected with luciferase gene were formed on the micropatterned culture plates coated with thermosensitive polymer, and were recovered in the form of injectable liquid suspension simply by cooling the plates. After subcutaneously transplanting these hepatocyte spheroids, efficient transgene expression was observed in host tissue for more than a month, whereas transplantation of a single-cell suspension from a monolayer culture resulted in an only transient expression. The spheroid system contributed to the preservation of innate functions of transplanted hepatocytes in the host tissue, such as albumin expression, thereby possessing high potential for expressing transgene. Intravital observation of transplanted cells showed that those from spheroid cultures had a tendency to localize in the vicinity of blood vessels, making a favorable microenvironment for preserving cell functionality. Furthermore, spheroids transfected with erythropoietin-expressing DNA showed a significantly higher hematopoietic effect than that of cell suspensions from monolayer cultures, demonstrating high potential of this genetically-modified spheroid transplantation system for therapeutic applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

Lovastatin inhibits T cell proliferation while preserving the cytolytic function of EBV-, CMV- and MART-1-specific CTLs

PubMed Central

Li, Dan; Li, Yufeng; Hernandez, Jessica A.; Patenia, Rebecca; Kim, Tae Kon; Khalili, Jahan; Dougherty, Mark C.; Hanley, Patrick J.; Bollard, Catherine M.; Komanduri, Krishna V.; Hwu, Patrick; Champlin, Richard E.; Radvanyi, Laszlo G.; Molldrem, Jeffrey J.; Ma, Qing

2016-01-01

Statin treatment has been shown to reduce graft-versus-host disease (GVHD) while preserving graft-versus-tumor (GVT) effect in allogeneic stem cell transplantation (allo-HCT). Herein, we investigated whether lovastatin treatment affects the function of human cytolytic T lymphocytes (CTLs). Upon TCR stimulation, lovastatin significantly inhibited the proliferation of both CD4+ and CD8+ T cells from healthy donors while their intracellular cytokine production including IFN-γ and TNF-α remained the same with a slight decrease of IL-2. Moreover, the specific lysis of target cells by CTL lines derived from patients and normal donors specific for EBV-encoded antigen LMP2 or CMV-encoded antigen pp65 was uncompromised in the presence of lovastatin. In addition, we evaluated the effect of lovastatin on the proliferation and effector function of the CD8+ tumor–infiltrating lymphocytes (TILs) derived from melanoma patients specific for MART-1 antigen. Lovastatin significantly reduced the expansion of antigen-specific TILs upon MART-1 stimulation. However, the effector function of TILs, including the specific lysis of target cells and secretion of cytokine IFN-γ, remained intact with lovastatin treatment. Taken together, these data demonstrated that lovastatin inhibits the proliferation of EBV-, CMV- and MART-1-specific CTLs without affecting cytolytic capacity. The differential effect of lovastatin on the proliferation versus cytoxicity of CTLs might shed some light on elucidating the possible mechanisms of GVHD and GVT effect elicited by alloimmune responses. PMID:20948439

The functional relevance of polyploidization in the skin.

PubMed

Trakala, Marianna; Malumbres, Marcos

2014-02-01

Cell proliferation and differentiation are tightly coupled through the regulation of the cell division cycle. To preserve specific functional properties in differentiated cells, distinct variants of the basic mitotic cell cycle are used in various mammalian tissues, leading to the formation of polyploid cells. In this issue of Experimental Dermatology, Gandarillas and Freije discuss the evidences for polyploidization in keratinocytes, a process whose physiological relevance is now becoming evident. A better evaluation of these unconventional cell cycles is required not only to improve our understanding of the development and structure of the epidermis but also for future therapies against skin diseases. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

IFATS Collection: Human Adipose Tissue-Derived Stem Cells Induce Angiogenesis and Nerve Sprouting Following Myocardial Infarction, in Conjunction with Potent Preservation of Cardiac Function

PubMed Central

Cai, Liying; Johnstone, Brian H.; Cook, Todd G.; Tan, Jian; Fishbein, Michael C.; Chen, Peng-Sheng; March, Keith L.

2010-01-01

The administration of therapeutic cell types, such as stem and progenitor cells, has gained much interest for the limitation or repair of tissue damage caused by a variety of insults. However, it is still uncertain whether the morphological and functional benefits are mediated predominantly via cell differentiation or paracrine mechanisms. Here, we assessed the extent and mechanisms of adipose-derived stromal/stem cells (ASC)-dependent tissue repair in the context of acute myocardial infarction. Human ASCs in saline or saline alone was injected into the peri-infarct region in athymic rats following left anterior descending (LAD) coronary artery ligation. Cardiac function and structure were evaluated by serial echocardiography and histology. ASC-treated rats consistently exhibited better cardiac function, by all measures, than control rats 1 month following LAD occlusion. Left ventricular (LV) ejection fraction and fractional shortening were improved in the ASC group, whereas LV remodeling and dilation were limited in the ASC group compared with the saline control group. Anterior wall thinning was also attenuated by ASC treatment, and post-mortem histological analysis demonstrated reduced fibrosis in ASC-treated hearts, as well as increased peri-infarct density of both arterioles and nerve sprouts. Human ASCs were persistent at 1 month in the peri-infarct region, but they were not observed to exhibit significant cardiomyocyte differentiation. Human ASCs preserve heart function and augment local angiogenesis and cardiac nerve sprouting following myocardial infarction predominantly by the provision of beneficial trophic factors. PMID:18772313

Acidosis overrides oxygen deprivation to maintain mitochondrial function and cell survival

PubMed Central

Khacho, Mireille; Tarabay, Michelle; Patten, David; Khacho, Pamela; MacLaurin, Jason G.; Guadagno, Jennifer; Bergeron, Richard; Cregan, Sean P.; Harper, Mary-Ellen; Park, David S.; Slack, Ruth S.

2014-01-01

Sustained cellular function and viability of high-energy demanding post-mitotic cells rely on the continuous supply of ATP. The utilization of mitochondrial oxidative phosphorylation for efficient ATP generation is a function of oxygen levels. As such, oxygen deprivation, in physiological or pathological settings, has profound effects on cell metabolism and survival. Here we show that mild extracellular acidosis, a physiological consequence of anaerobic metabolism, can reprogramme the mitochondrial metabolic pathway to preserve efficient ATP production regardless of oxygen levels. Acidosis initiates a rapid and reversible homeostatic programme that restructures mitochondria, by regulating mitochondrial dynamics and cristae architecture, to reconfigure mitochondrial efficiency, maintain mitochondrial function and cell survival. Preventing mitochondrial remodelling results in mitochondrial dysfunction, fragmentation and cell death. Our findings challenge the notion that oxygen availability is a key limiting factor in oxidative metabolism and brings forth the concept that mitochondrial morphology can dictate the bioenergetic status of post-mitotic cells. PMID:24686499

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation.

PubMed

Onofre, J; Baert, Y; Faes, K; Goossens, E

2016-11-01

Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS. Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Cryopreservation of testicular tissue or testicular cell suspensions: a pivotal step in fertility preservation

PubMed Central

Onofre, J.; Baert, Y.; Faes, K.; Goossens, E.

2016-01-01

BACKGROUND Germ cell depletion caused by chemical or physical toxicity, disease or genetic predisposition can occur at any age. Although semen cryopreservation is the first reflex for preserving male fertility, this cannot help out prepubertal boys. Yet, these boys do have spermatogonial stem cells (SSCs) that able to produce sperm at the start of puberty, which allows them to safeguard their fertility through testicular tissue (TT) cryopreservation. SSC transplantation (SSCT), TT grafting and recent advances in in vitro spermatogenesis have opened new possibilities to restore fertility in humans. However, these techniques are still at a research stage and their efficiency depends on the amount of SSCs available for fertility restoration. Therefore, maintaining the number of SSCs is a critical step in human fertility preservation. Standardizing a successful cryopreservation method for TT and testicular cell suspensions (TCSs) is most important before any clinical application of fertility restoration could be successful. OBJECTIVE AND RATIONALE This review gives an overview of existing cryopreservation protocols used in different animal models and humans. Cell recovery, cell viability, tissue integrity and functional assays are taken into account. Additionally, biosafety and current perspectives in male fertility preservation are discussed. SEARCH METHODS An extensive PubMED and MEDline database search was conducted. Relevant studies linked to the topic were identified by the search terms: cryopreservation, male fertility preservation, (immature)testicular tissue, testicular cell suspension, spermatogonial stem cell, gonadotoxicity, radiotherapy and chemotherapy. OUTCOMES The feasibility of fertility restoration techniques using frozen-thawed TT and TCS has been proven in animal models. Efficient protocols for cryopreserving human TT exist and are currently applied in the clinic. For TCSs, the highest post-thaw viability reported after vitrification is 55.6 ± 23.8%. Yet, functional proof of fertility restoration in the human is lacking. In addition, few to no data are available on the safety aspects inherent to offspring generation with gametes derived from frozen-thawed TT or TCSs. Moreover, clarification is needed on whether it is better to cryopreserve TT or TCS. WIDER IMPLICATIONS Fertility restoration techniques are very promising and expected to be implemented in the clinic in the near future. However, inter-center variability needs to be overcome and the gametes produced for reproduction purposes need to be subjected to safety studies. With the perspective of a future clinical application, there is a dire need to optimize and standardize cryopreservation and safety testing before using frozen-thawed TT of TCSs for fertility restoration. PMID:27566839

Normothermic machine perfusion reduces bile duct injury and improves biliary epithelial function in rat donor livers.

PubMed

Op den Dries, Sanna; Karimian, Negin; Westerkamp, Andrie C; Sutton, Michael E; Kuipers, Michiel; Wiersema-Buist, Janneke; Ottens, Petra J; Kuipers, Jeroen; Giepmans, Ben N; Leuvenink, Henri G D; Lisman, Ton; Porte, Robert J

2016-07-01

Bile duct injury may occur during liver procurement and transplantation, especially in livers from donation after circulatory death (DCD) donors. Normothermic machine perfusion (NMP) has been shown to reduce hepatic injury compared to static cold storage (SCS). However, it is unknown whether NMP provides better preservation of bile ducts. The aim of this study was to determine the impact of NMP on bile duct preservation in both DCD and non-DCD livers. DCD and non-DCD livers obtained from Lewis rats were preserved for 3 hours using either SCS or NMP, followed by 2 hours ex vivo reperfusion. Biomarkers of bile duct injury (gamma-glutamyltransferase and lactate dehydrogenase in bile) were lower in NMP-preserved livers compared to SCS-preserved livers. Biliary bicarbonate concentration, reflecting biliary epithelial function, was 2-fold higher in NMP-preserved livers (P < 0.01). In parallel with this, the pH of the bile was significantly higher in NMP-preserved livers (7.63 ± 0.02 and 7.74 ± 0.05 for non-DCD and DCD livers, respectively) compared with SCS-preserved livers (7.46 ± 0.02 and 7.49 ± 0.04 for non-DCD and DCD livers, respectively). Scanning and transmission electron microscopy of donor extrahepatic bile ducts demonstrated significantly decreased injury of the biliary epithelium of NMP-preserved donor livers (including the loss of lateral interdigitations and mitochondrial injury). Differences between NMP and SCS were most prominent in DCD livers. Compared to conventional SCS, NMP provides superior preservation of bile duct epithelial cell function and morphology, especially in DCD donor livers. By reducing biliary injury, NMP could have an important impact on the utilization of DCD livers and outcome after transplantation. Liver Transplantation 22 994-1005 2016 AASLD. © 2016 American Association for the Study of Liver Diseases.

Partial preservation of rod and cone ERG function following subretinal injection of ARPE-19 cells in RCS rats.

PubMed

Sauvé, Y; Pinilla, I; Lund, R D

2006-04-01

We quantified rod- and cone-related electroretinogram (ERG) responses following subretinal injections of the human-derived retinal pigment epithelial (hRPE) cell line ARPE-19 at age P23 to prevent progressive photoreceptor loss in the Royal College of Surgeons (RCS) rat. Culture medium-injected eyes served as sham controls. At P60, in comparison with sham-injected eyes, all recordings from hRPE-injected eyes showed preserved scotopic a- and b-waves, oscillatory potentials, double-flash-derived rod b-waves and photopic cone b-waves, and flicker critical fusion frequencies and amplitudes. Although the actual preservation did not exceed 10% of a-wave and 20% of b-wave amplitude values in non-dystrophic RCS and deteriorated rapidly by P90, rod- and cone-related ERG parameters were still recordable up to P120 unlike the virtually unresponsive sham-injected eyes.

Dinosaur Peptides Suggest Mechanisms of Protein Survival

PubMed Central

San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.; Kalluri, Raghu; Buckley, Michael; Orgel, Joseph P. R. O.

2011-01-01

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results show empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a ‘preservation motif’, and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival. PMID:21687667

Dinosaur Peptides Suggest Mechanisms of Protein Survival

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

San Antonio, James D.; Schweitzer, Mary H.; Jensen, Shane T.

Eleven collagen peptide sequences recovered from chemical extracts of dinosaur bones were mapped onto molecular models of the vertebrate collagen fibril derived from extant taxa. The dinosaur peptides localized to fibril regions protected by the close packing of collagen molecules, and contained few acidic amino acids. Four peptides mapped to collagen regions crucial for cell-collagen interactions and tissue development. Dinosaur peptides were not represented in more exposed parts of the collagen fibril or regions mediating intermolecular cross-linking. Thus functionally significant regions of collagen fibrils that are physically shielded within the fibril may be preferentially preserved in fossils. These results showmore » empirically that structure-function relationships at the molecular level could contribute to selective preservation in fossilized vertebrate remains across geological time, suggest a 'preservation motif', and bolster current concepts linking collagen structure to biological function. This non-random distribution supports the hypothesis that the peptides are produced by the extinct organisms and suggests a chemical mechanism for survival.« less

A role for Mfb1p in region-specific anchorage of high-functioning mitochondria and lifespan in Saccharomyces cerevisiae

PubMed Central

Pernice, Wolfgang M.; Vevea, Jason D.; Pon, Liza A.

2016-01-01

Previous studies indicate that replicative lifespan in daughter cells of Sacchraromyces cerevisiae depends on the preferential inheritance of young, high-functioning mitochondria. We report here that mitochondria are functionally segregated even within single mother cells in S. cerevisiae. A high-functioning population of mitochondria accumulates at the tip of the mother cell distal to the bud. We find that the mitochondrial F-box protein (Mfb1p) localizes to mitochondria in the mother tip and is required for mitochondrial anchorage at that site, independent of the previously identified anchorage protein Num1p. Deletion of MFB1 results in loss of the mother-tip-localized mitochondrial population, defects in mitochondrial function and premature replicative ageing. Inhibiting mitochondrial inheritance to buds, by deletion of MMR1, in mfb1Δ cells restores mitochondrial distribution, promotes mitochondrial function and extends replicative lifespan. Our results identify a mechanism that retains a reservoir of high-functioning mitochondria in mother cells and thereby preserves maternal reproductive capacity. PMID:26839174

A novel solution configuration on liquid-based endometrial cytology

PubMed Central

Wang, Qi; Han, Lu; Tuo, Xiaoqian; Hou, Huilian; Liu, Yu; Shi, Zan; Wang, Qing; Li, Yan; Sun, Chao; Xue, Xue

2018-01-01

Objective Early detection and diagnosis of endometrial carcinoma and precancerous change would undoubtedly become the most alluring part for researchers. With the emergence of endometrial brush samplers, a new upsurge in endometrial cytology is in the making. But endometrial specimens obtained by the endometrial brush samplers require special preservation solution. The objective of this study is to develop a new kind of endometrial-cell preservation solution and to test the availability compared with a patented liquid-based cell preservation solution. Methods In this controlled study, we had 5 endometrial cases collected with Li Brush from the First Affiliated Hospital of Xi'an Jiaotong University (09/2016 to 12/2016). The samples of each case were collected 2 times separately and perserved in different perservation solutions. One was a kind of novel endometrial cell preservation solution and the other was a kind of patented liquid-based cell (LBC) preservation solution. The endometrial cells were smeared on slides by using the ZP-C automated slide preparation system and stained with Papanicolaou stain. A semi-quantitative scoring system was used to analyze the quality of slides. Statistical analysis was performed using the Wilcoxon signed rank test on the SPSS program (SPSS 18.0). In all LBC preparations, endometrial cells from the novel endometrial cells preservation solution had more cell quantity, less red blood cell fragments, and the background was cleaner compared with control group. Although the novel endometrial-cell preservation solution showed cellularity and absence of blood and debris expressed by no statistically significant differences (p = 0.063 and 0.102 respectively). The preservation period of the two kinds of liquids was equivalent. Conclusions The novel endometrial-cell preservation solution is superior to the liquid-base cell preservation solution for cervical cells, with clear background, diagnostic cells and low cost. PMID:29401497

Treatment of giant cell tumor of bone: Current concepts.

PubMed

Puri, Ajay; Agarwal, Manish

2007-04-01

Giant cell tumor (GCT) of bone though one of the commonest bone tumors encountered by an orthopedic surgeon continues to intrigue treating surgeons. Usually benign, they are locally aggressive and may occasionally undergo malignant transformation. The surgeon needs to strike a balance during treatment between reducing the incidence of local recurrence while preserving maximal function.Differing opinions pertaining to the use of adjuvants for extension of curettage, the relative role of bone graft or cement to pack the defect and the management of recurrent lesions are some of the issues that offer topics for eternal debate.Current literature suggests that intralesional curettage strikes the best balance between controlling disease and preserving optimum function in the majority of the cases though there may be occasions where the extent of the disease mandates resection to ensure adequate disease clearance.An accompanying treatment algorithm helps outline the management strategy in GCT.

Kynurenic Acid Prevents Cytoskeletal Disorganization Induced by Quinolinic Acid in Mixed Cultures of Rat Striatum.

PubMed

Pierozan, Paula; Biasibetti-Brendler, Helena; Schmitz, Felipe; Ferreira, Fernanda; Pessoa-Pureur, Regina; Wyse, Angela T S

2018-06-01

Kynurenic acid (KYNA) is a neuroactive metabolite of tryptophan known to modulate a number of mechanisms involved in neural dysfunction. Although its activity in the brain has been widely studied, the effect of KYNA counteracting the actions of quinolinic acid (QUIN) remains unknown. The present study aims at describing the ability of 100 μM KYNA preventing cytoskeletal disruption provoked by QUIN in astrocyte/neuron/microglia mixed culture. KYNA totally preserved cytoskeletal organization, cell morphology, and redox imbalance in mixed cultures exposed to QUIN. However, KYNA partially prevented morphological alteration in isolated primary astrocytes and failed to protect the morphological alterations of neurons caused by QUIN exposure. Moreover, KYNA prevented QUIN-induced microglial activation and upregulation of ionized calcium-binding adapter molecule 1 (Iba-1) and partially preserved tumor necrosis factor-α (TNF-α) level in mixed cultures. TNF-α level was also partially preserved in astrocytes. In addition to the mechanisms dependent on redox imbalance and microglial activation, KYNA prevented downregulation of connexin-43 and the loss of functionality of gap junctions (GJs), preserving cell-cell contact, cytoskeletal organization, and cell morphology in QUIN-treated cells. Furthermore, the toxicity of QUIN targeting the cytoskeleton of mixed cultures was not prevented by the N-methyl-D-aspartate (NMDA) antagonist MK-801. We suggest that KYNA protects the integrity of the cytoskeleton of mixed cultures by complex mechanisms including modulating microglial activation preventing oxidative imbalance and misregulated GJs leading to disrupted cytoskeleton in QUIN-treated cells. This study contributed to elucidate the molecular basis of KYNA protection against QUIN toxicity.

FlowCam: Quantification and Classification of Phytoplankton by Imaging Flow Cytometry.

PubMed

Poulton, Nicole J

2016-01-01

The ability to enumerate, classify, and determine biomass of phytoplankton from environmental samples is essential for determining ecosystem function and their role in the aquatic community and microbial food web. Traditional micro-phytoplankton quantification methods using microscopic techniques require preservation and are slow, tedious and very laborious. The availability of more automated imaging microscopy platforms has revolutionized the way particles and cells are detected within their natural environment. The ability to examine cells unaltered and without preservation is key to providing more accurate cell concentration estimates and overall phytoplankton biomass. The FlowCam(®) is an imaging cytometry tool that was originally developed for use in aquatic sciences and provides a more rapid and unbiased method for enumerating and classifying phytoplankton within diverse aquatic environments.

Atorvastatin helps preserve pancreatic β cell function in obese C57BL/6 J mice and the effect is related to increased pancreas proliferation and amelioration of endoplasmic-reticulum stress

PubMed Central

2014-01-01

Background 3-Hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors or statins are competitive inhibitors of the rate-limiting enzyme in cholesterol biosynthesis. Currently, statins are used as first-line therapy in the treatment of diabetic dyslipidemia. However, effects of statins on β cell function remains unclear. This study aims to examine effects of atorvastatin treatment on pancreatic β cell function in obese C57BL/6 J mice and the possible mechanisms. Methods Diet-induced obesity (DIO) C57BL/6 J mice were treated with atorvastatin (30 mg/kg/day) for 58 days. β cell function was assessed by hyperglycemic clamp and the area of insulin-positive β cells was examined by immunofluorescence. Gene expression was assessed by RT-PCR, and endoplasmic reticulum (ER) stress related proteins were examined by Western blot. Additionally, cell viability and apoptosis of the cholesterol-loaded NIT-1 cells were investigated after atorvastatin treatment. Results Hyperglycemic clamp study revealed that glucose infusion rate (GIR) and insulin stimulation ratio in atorvastatin-treated DIO mice were markedly higher than control mice (P < 0.05, P < 0.01 vs. con), indicating preserved β-cell sensitivity to glucose. Lipid profiles of plasma triglyceride (TG), pancreas TG and plasma cholesterol (CHO) were improved. Pancreas weight and weight index were improved significantly after atorvastatin treatment (P < 0.05 vs. con). Immunofluorescence results showed that atorvastatin-treated mice had significantly larger insulin-positive β cell area (P < 0.05 vs. con). Furthermore, RT-PCR and western blot showed that the mRNA and protein expression of pancreatic and duodenal homeobox 1 (Pdx1) in the pancreas were upregulated (P < 0.001, P < 0.01 vs. con). Moreover, the expression level of ER stress markers of activating transcription factor 4 (ATF4), CCAAT-enhancer-binding protein homologous protein (CHOP) and phosphorylated eukaryotic initiation factor 2α (eIF2α) were downregulated in the pancreas of atorvastatin-treated mice (P < 0.001, P < 0.01, P < 0.01 vs. con). Besides, atorvastatin protected the pancreatic β cell line of NIT-1 from cholesterol-induced apoptosis. Western blot showed increased expression of anti-apoptotic protein of B-cell lymphoma 2 (Bcl-2). Conclusion Pancreatic β cell function of obese C57BL/6 J mice was preserved after atorvastatin treatment, and this improvement may be attributed to enhanced pancreas proliferation and amelioration of pancreatic ER stress. PMID:24950764

Bcl-2 prevents loss of mitochondria in CCCP-induced apoptosis.

PubMed

de Graaf, Aniek O; van den Heuvel, Lambert P; Dijkman, Henry B P M; de Abreu, Ronney A; Birkenkamp, Kim U; de Witte, Theo; van der Reijden, Bert A; Smeitink, Jan A M; Jansen, Joop H

2004-10-01

Bcl-2 family proteins regulate apoptosis at the level of mitochondria. To examine the mechanism of Bcl-2 function, we investigated the effects of the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) on two hematopoietic cell lines and Bcl-2 overexpressing transfectants. CCCP directly interferes with mitochondrial function and induces apoptosis. We show that Bcl-2 inhibits apoptosis and that the antiapoptotic effect of Bcl-2 takes place upstream of caspase activation and nuclear changes associated with apoptosis, since these were markedly inhibited in cells overexpressing Bcl-2. Bcl-2 does not prevent the decrease in mitochondrial membrane potential nor the alterations in cellular ATP content induced by CCCP in FL5.12 and Jurkat cells. A higher number of mitochondria was observed in untreated Bcl-2 transfected cells compared to parental cells, as shown by electron microscopy. Exposure to CCCP induced a dramatic decrease in the number of mitochondria and severely disrupted mitochondrial ultrastructure, with apparent swelling and loss of cristae in parental cells. Bcl-2 clearly diminished the disruption of mitochondrial structure and preserved a higher number of mitochondria. These data suggest that CCCP induces apoptosis by structural disruption of mitochondria and that Bcl-2 prevents apoptosis and mitochondrial degeneration by preserving mitochondrial integrity.

Evaluation of drug-metabolizing and functional competence of human hepatocytes incubated under hypothermia in different media for clinical infusion.

PubMed

Gómez-Lechón, María José; Lahoz, Agustín; Jiménez, Nuria; Bonora, Ana; Castell, José V; Donato, María Teresa

2008-01-01

Hepatocyte transplantation has been proposed as a method to support patients with liver insufficiency. Key factors for clinical cell transplantation to progress is to prevent hepatocyte damage, loss of viability and cell functionality, factors that depend on the nature of the tissue used for isolation to a large extent. The main sources of tissue for hepatocyte isolation are marginal livers that are unsuitable for transplantation, and segments from reduced cadaveric grafts. Hepatocellular transplantation requires infusing human hepatocytes in suspension over a period of minutes to hours. The beneficial effect of hypothermic preservation of hepatocytes in infusion medium has been reported, but how critical issues towards the success of cell transplantation, such as the composition of infusion medium and duration of hepatocyte storage will affect hepatocyte quality for clinical cell infusion has not been systematically investigated. Infusion media composition is phosphate-buffered saline containing anticoagulants and human serum albumin. The supplementation of infusion media with glucose or N-acetyl-cystein, or with both components at the same time, has been investigated. After isolation, hepatocytes were suspended in each infusion medium and a sample at the 0 time point was harvested for cell viability and functional assessment. Thereafter, cells were incubated in different infusion media agitated on a rocker platform to simulate the clinical infusion technique. The time course of hepatocyte viability, funtionality (drug-metabolizing enzymes, ureogenic capability, ATP, glycogen, and GSH levels), apoptosis (caspase-3 activation), and attachment and monolayer formation were analyzed. The optimal preservation of cell viability, attaching capacity, and functionality, particularly GSH and glycogen levels, as well as drug-metabolizing cytochrome P450 enzymes, was found in infusion media supplemented with 2 mM N-acetyl-cystein and 15 mM glucose.

Adenoviral Gene Transfer of Hepatic Stimulator Substance Confers Resistance Against Hepatic Ischemia–Reperfusion Injury by Improving Mitochondrial Function

PubMed Central

Jiang, Shu-Jun; Li, Wen

2013-01-01

Abstract Hepatic stimulator substance (HSS) has been suggested to protect liver cells from various toxins. However, the precise role of HSS in hepatic ischemia–reperfusion (I/R) injury remains unknown. This study aims to elucidate whether overexpression of HSS could attenuate hepatic ischemia–reperfusion injury and its possible mechanisms. Both in vivo hepatic I/R injury in mice and in vitro hypoxia–reoxygenation (H/R) in a cell model were used to evaluate the effect of HSS protection after adenoviral gene transfer. Moreover, a possible mitochondrial mechanism of HSS protection was investigated. Efficient transfer of the HSS gene into liver inhibited hepatic I/R injury in mice, as evidenced by improvement in liver function tests, the preservation of hepatic morphology, and a reduction in hepatocyte apoptosis. HSS overexpression also inhibited H/R-induced cell death, as detected by cell viability and cell apoptosis assays. The underlying mechanism of this hepatic protection might involve the attenuation of mitochondrial dysfunction and mitochondrial-dependent cell apoptosis, as shown by the good preservation of mitochondrial ultrastructure, mitochondrial membrane potential, and the inhibition of cytochrome c leakage and caspase activity. Moreover, the suppression of H/R-induced mitochondrial ROS production and the maintenance of mitochondrial respiratory chain complex activities may participate in this mechanism. This new function of HSS expands the possibility of its application for the prevention of I/R injury, such as hepatic resection and liver transplantation in clinical practice. PMID:23461564

Treatment of new-onset type 1 diabetes with peptide DiaPep277 is safe and associated with preserved beta-cell function: extension of a randomized, double-blind, phase II trial.

PubMed

Raz, I; Avron, A; Tamir, M; Metzger, M; Symer, L; Eldor, R; Cohen, I R; Elias, D

2007-05-01

Treatment with DiaPep277, a peptide derived from HSP60, has been shown to preserve beta-cell function in non-obese diabetic mouse (NOD) mice and in a trial with newly diagnosed human patients with type 1 diabetes treated over a 10-month period. This article extends the clinical trial observations to a total of 20 months of treatment to determine the safety and the effects of repeated doses of DiaPep277 on endogenous insulin secretion, metabolic control, and exogenous insulin requirements. Thirty-five male patients (aged 16-58) with a basal C-peptide greater than 0.1 nmol/L were assigned to periodic treatment with DiaPep277 (1 mg) or placebo for a 12-month treatment and 18-month observation protocol, later extended to an additional year of treatment. Stimulated C-peptide, HbA1c, and an exogenous insulin dose were the clinical endpoints. At 18 months, stimulated C-peptide concentrations had fallen in the placebo group (p = 0.0005) but were maintained in the DiaPep277 group. The need for exogenous insulin was higher in the placebo group than in the DiaPep277 group. Mean HbA1c concentrations were similar in both groups. After extension of the study, patients continuing treatment with DiaPep277 and those switched from placebo to DiaPep277 manifested a trend towards a greater preservation of beta-cell function compared to patients maintained on or switched to placebo. The safety profile of DiaPep277 was similar between the treatment and placebo groups, and no drug-related adverse events occurred. Periodic treatment of subjects with DiaPep277 over 2 years was safe and associated preservation of endogenous insulin secretion up to 18 months was observed. Copyright 2007 John Wiley & Sons, Ltd.

Intermittent Hypoxia and Stem Cell Implants Preserve Breathing Capacity in a Rodent Model of Amyotrophic Lateral Sclerosis

PubMed Central

Nichols, Nicole L.; Gowing, Genevieve; Satriotomo, Irawan; Nashold, Lisa J.; Dale, Erica A.; Suzuki, Masatoshi; Avalos, Pablo; Mulcrone, Patrick L.; McHugh, Jacalyn

2013-01-01

Rationale: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease causing paralysis and death from respiratory failure. Strategies to preserve and/or restore respiratory function are critical for successful treatment. Although breathing capacity is maintained until late in disease progression in rodent models of familial ALS (SOD1G93A rats and mice), reduced numbers of phrenic motor neurons and decreased phrenic nerve activity are observed. Decreased phrenic motor output suggests imminent respiratory failure. Objectives: To preserve or restore phrenic nerve activity in SOD1G93A rats at disease end stage. Methods: SOD1G93A rats were injected with human neural progenitor cells (hNPCs) bracketing the phrenic motor nucleus before disease onset, or exposed to acute intermittent hypoxia (AIH) at disease end stage. Measurements and Main Results: The capacity to generate phrenic motor output in anesthetized rats at disease end stage was: (1) transiently restored by a single presentation of AIH; and (2) preserved ipsilateral to hNPC transplants made before disease onset. hNPC transplants improved ipsilateral phrenic motor neuron survival. Conclusions: AIH-induced respiratory plasticity and stem cell therapy have complementary translational potential to treat breathing deficits in patients with ALS. PMID:23220913

Recommendations for the Definition of Clinical Responder in Insulin Preservation Studies

PubMed Central

Gitelman, Stephen E.; Palmer, Jerry P.

2014-01-01

Clinical responder studies should contribute to the translation of effective treatments and interventions to the clinic. Since ultimately this translation will involve regulatory approval, we recommend that clinical trials prespecify a responder definition that can be assessed against the requirements and suggestions of regulatory agencies. In this article, we propose a clinical responder definition to specifically assist researchers and regulatory agencies in interpreting the clinical importance of statistically significant findings for studies of interventions intended to preserve β-cell function in newly diagnosed type 1 diabetes. We focus on studies of 6-month β-cell preservation in type 1 diabetes as measured by 2-h–stimulated C-peptide. We introduce criteria (bias, reliability, and external validity) for the assessment of responder definitions to ensure they meet U.S. Food and Drug Administration and European Medicines Agency guidelines. Using data from several published TrialNet studies, we evaluate our definition (no decrease in C-peptide) against published alternatives and determine that our definition has minimum bias with external validity. We observe that reliability could be improved by using changes in C-peptide later than 6 months beyond baseline. In sum, to support efficacy claims of β-cell preservation therapies in type 1 diabetes submitted to U.S. and European regulatory agencies, we recommend use of our definition. PMID:24722251

Cold storage of rat hepatocyte suspensions for one week in a customized cold storage solution--preservation of cell attachment and metabolism.

PubMed

Pless-Petig, Gesine; Singer, Bernhard B; Rauen, Ursula

2012-01-01

Primary hepatocytes are of great importance for basic research as well as cell transplantation. However, their stability, especially in suspension, is very low. This feature severely compromises storage and shipment. Based on previous studies with adherent cells, we here assessed cold storage injury in rat hepatocyte suspensions and aimed to find a cold storage solution that preserves viability, attachment ability and functionality of these cells. Rat hepatocyte suspensions were stored in cell culture medium, organ preservation solutions and modified TiProtec solutions at 4°C for one week. Viability and cell volume were determined by flow cytometry. Thereafter, cells were seeded and density and metabolic capacity (reductive metabolism, forskolin-induced glucose release, urea production) of adherent cells were assessed. Cold storage injury in hepatocyte suspensions became evident as cell death occurring during cold storage or rewarming or as loss of attachment ability. Cell death during cold storage was not dependent on cell swelling and was almost completely inhibited in the presence of glycine and L-alanine. Cell attachment could be greatly improved by use of chloride-poor solutions and addition of iron chelators. Using a chloride-poor, potassium-rich storage solution containing glycine, alanine and iron chelators, cultures with 75% of the density of control cultures and with practically normal cell metabolism could be obtained after one week of cold storage. In the solution presented here, cold storage injury of hepatocyte suspensions, differing from that of adherent hepatocytes, was effectively inhibited. The components which acted on the different injurious processes were identified.

Different effects of amino acid-based and glucose-based dialysate from peritoneal dialysis patients on mesothelial cell ultrastructure and function.

PubMed

Chan, Tak-Mao; Leung, Jack Kok-Hung; Sun, Yuling; Lai, Kar-Neng; Tsang, Ryan Chi-Wai; Yung, Susan

2003-06-01

Peritoneal dialysis fluid (PDF) containing amino acids has been introduced recently aiming to improve the nutritional status of PD patients. Dextrose-based PDFs have been implicated in progressive functional and structural deterioration of the peritoneal membrane. Limited data are currently available regarding the effect of amino acid-based PDF on the function and ultrastructure of human peritoneal mesothelial cells (HPMCs), which play a critical role in peritoneal membrane pathophysiology. We investigated the effects of two commercially available PDFs, which utilized dextrose (1.5% Dianeal) or amino acids (1.1% Nutrineal) as the osmotic agent, obtained from patients after a 4 h dwell, on HPMC proliferation (MTT assay and cell counting) and viability [lactate dehydrogenase (LDH)release], interleukin-6 (IL-6) secretion (commercial enzyme-linked immunosorbent assay) and ultrastructure (scanning and transmission electron microscopy). Exposure of HPMCs to 1.5% Dianeal reduced cell proliferation, total cellular protein synthesis, IL-6 secretion and cell attachment, but prolonged the cell doubling time on recovery, and increased LDH release (P<0.001, P<0.001, P<0.0001, P<0.0001, P<0.001 and P<0.001, respectively). The 1.1% Nutrineal reduced HPMC proliferation (P<0.001) and increased IL-6 secretion (P<0.0001), but did not affect cell attachment, LDH release, protein synthesis or cell doubling time. Ultrastructural studies of HPMCs exposed to Dianeal showed cell flattening, increased cell surface area, reduced microvilli, and intracellular organelles compatible with dysfunctional mitochondria. In contrast, the ultrastructural morphology of HPMCs was relatively preserved after incubation with Nutrineal. Our results showed that HPMC ultrastructure, viability and protein synthesis were better preserved with amino acid-based PDF, compared with conventional dextrose-based PDF. The significance of IL-6 induction by Nutrineal remains to be elucidated.

The Late Devonian Gogo Formation Lägerstatte of Western Australia: Exceptional Early Vertebrate Preservation and Diversity

NASA Astrophysics Data System (ADS)

Long, John A.; Trinajstic, Kate

2010-05-01

The Gogo Formation of Western Australia preserves a unique Late Devonian (Frasnian) reef fauna. The exceptional three-dimensional preservation of macrofossils combined with unprecedented soft-tissue preservation (including muscle bundles, nerve cells, and umbilical structures) has yielded a particularly rich assemblage with almost 50 species of fishes described. The most significant discoveries have contributed to resolving placoderm phylogeny and elucidating their reproductive physiology. Specifically, these discoveries have produced data on the oldest known vertebrate embryos; the anatomy of the primitive actinopterygian neurocranium and phylogeny of the earliest actinopterygians; the histology, radiation, and plasticity of dipnoan (lungfish) dental and cranial structures; the anatomy and functional morphology of the extinct onychodonts; and the anatomy of the primitive tetrapodomorph head and pectoral fin.

Renal cell carcinoma: new insights and challenges for a clinician scientist.

PubMed

Shingarev, Roman; Jaimes, Edgar A

2017-08-01

There is a growing recognition of the complex interplay between renal cell cancer (RCC), kidney function, mechanical reduction of nephron mass, and systemic agents targeting the cancer. Earlier detection of RCC and rising life expectancy of cancer survivors places a greater emphasis on preservation of renal function after cancer resection and during systemic therapy. Unique adverse effects associated with RCC drugs not only help reveal cancer pathophysiology but also expand our knowledge of normal cell signaling and metabolism. In this review, we outline our current understanding of RCC biology and treatment, their bidirectional relationship with kidney function, and unmet research needs in this field. Copyright © 2017 the American Physiological Society.

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

DOE PAGES

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry; ...

2016-08-31

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

Preserving elemental content in adherent mammalian cells for analysis by synchrotron-based x-ray fluorescence microscopy

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

Jin, Qiaoling; Paunesku, Tatjana; Lai, Barry

Trace metals play important roles in biological function, and x-ray fluorescence microscopy (XFM) provides a way to quantitatively image their distribution within cells. The faithfulness of these measurements is dependent on proper sample preparation. Using mouse embryonic fibroblast NIH/3T3 cells as an example, we compare various approaches to the preparation of adherent mammalian cells for XFM imaging under ambient temperature. Direct side-by-side comparison shows that plunge-freezing-based cryoimmobilization provides more faithful preservation than conventional chemical fixation for most biologically important elements including P, S, Cl, K, Fe, Cu, Zn and possibly Ca in adherent mammalian cells. Although cells rinsed with freshmore » media had a great deal of extracellular background signal for Cl and Ca, this approach maintained cells at the best possible physiological status before rapid freezing and it does not interfere with XFM analysis of other elements. If chemical fixation has to be chosen, the combination of 3% paraformaldehyde and 1.5 % glutaraldehyde preserves S, Fe, Cu and Zn better than either fixative alone. Lastly, when chemically fixed cells were subjected to a variety of dehydration processes, air drying was proved to be more suitable than other drying methods such as graded ethanol dehydration and freeze drying. This first detailed comparison for x-ray fluorescence microscopy shows how detailed quantitative conclusions can be affected by the choice of cell preparation method.« less

P7C3 neuroprotective chemicals block axonal degeneration and preserve function after traumatic brain injury.

PubMed

Yin, Terry C; Britt, Jeremiah K; De Jesús-Cortés, Héctor; Lu, Yuan; Genova, Rachel M; Khan, Michael Z; Voorhees, Jaymie R; Shao, Jianqiang; Katzman, Aaron C; Huntington, Paula J; Wassink, Cassie; McDaniel, Latisha; Newell, Elizabeth A; Dutca, Laura M; Naidoo, Jacinth; Cui, Huxing; Bassuk, Alexander G; Harper, Matthew M; McKnight, Steven L; Ready, Joseph M; Pieper, Andrew A

2014-09-25

The P7C3 class of neuroprotective aminopropyl carbazoles has been shown to block neuronal cell death in models of neurodegeneration. We now show that P7C3 molecules additionally preserve axonal integrity after injury, before neuronal cell death occurs, in a rodent model of blast-mediated traumatic brain injury (TBI). This protective quality may be linked to the ability of P7C3 molecules to activate nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in nicotinamide adenine dinucleotide salvage. Initiation of daily treatment with our recently reported lead agent, P7C3-S243, 1 day after blast-mediated TBI blocks axonal degeneration and preserves normal synaptic activity, learning and memory, and motor coordination in mice. We additionally report persistent neurologic deficits and acquisition of an anxiety-like phenotype in untreated animals 8 months after blast exposure. Optimized variants of P7C3 thus offer hope for identifying neuroprotective agents for conditions involving axonal damage, neuronal cell death, or both, such as occurs in TBI. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

How preservation time changes the linear viscoelastic properties of porcine liver.

PubMed

Wex, C; Stoll, A; Fröhlich, M; Arndt, S; Lippert, H

2013-01-01

The preservation time of a liver graft is one of the crucial factors for the success of a liver transplantation. Grafts are kept in a preservation solution to delay cell destruction and cellular edema and to maximize organ function after transplantation. However, longer preservation times are not always avoidable. In this paper we focus on the mechanical changes of porcine liver with increasing preservation time, in order to establish an indicator for the quality of a liver graft dependent on preservation time. A time interval of 26 h was covered and the rheological properties of liver tissue studied using a stress-controlled rheometer. For samples of 1 h preservation time 0.8% strain was found as the limit of linear viscoelasticity. With increasing preservation time a decrease in the complex shear modulus as an indicator for stiffness was observed for the frequency range from 0.1 to 10 Hz. A simple fractional derivative representation of the Kelvin Voigt model was applied to gain further information about the changes of the mechanical properties of liver with increasing preservation time. Within the small shear rate interval of 0.0001-0.01 s⁻¹ the liver showed Newtonian-like flow behavior.

Molecular biological features of male germ cell differentiation

PubMed Central

HIROSE, MIKA; TOKUHIRO, KEIZO; TAINAKA, HITOSHI; MIYAGAWA, YASUSHI; TSUJIMURA, AKIRA; OKUYAMA, AKIHIKO; NISHIMUNE, YOSHITAKE

2007-01-01

Somatic cell differentiation is required throughout the life of a multicellular organism to maintain homeostasis. In contrast, germ cells have only one specific function; to preserve the species by conveying the parental genes to the next generation. Recent studies of the development and molecular biology of the male germ cell have identified many genes, or isoforms, that are specifically expressed in the male germ cell. In the present review, we consider the unique features of male germ cell differentiation. (Reprod Med Biol 2007; 6: 1–9) PMID:29699260

Intracerebroventricular gene therapy that delays neurological disease progression is associated with selective preservation of retinal ganglion cells in a canine model of CLN2 disease.

PubMed

Whiting, Rebecca E H; Jensen, Cheryl A; Pearce, Jacqueline W; Gillespie, Lauren E; Bristow, Daniel E; Katz, Martin L

2016-05-01

CLN2 disease is one of a group of lysosomal storage disorders called the neuronal ceroid lipofuscinoses (NCLs). The disease results from mutations in the TPP1 gene that cause an insufficiency or complete lack of the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1). TPP1 is involved in lysosomal protein degradation, and lack of this enzyme results in the accumulation of protein-rich autofluorescent lysosomal storage bodies in numerous cell types including neurons throughout the central nervous system and the retina. CLN2 disease is characterized primarily by progressive loss of neurological functions and vision as well as generalized neurodegeneration and retinal degeneration. In children the progressive loss of neurological functions typically results in death by the early teenage years. A Dachshund model of CLN2 disease with a null mutation in TPP1 closely recapitulates the human disorder with a progression from disease onset at approximately 4 months of age to end-stage at 10-11 months. Delivery of functional TPP1 to the cerebrospinal fluid (CSF), either by periodic infusion of the recombinant protein or by a single administration of a TPP1 gene therapy vector to the CSF, significantly delays the onset and progression of neurological signs and prolongs life span but does not prevent the loss of vision or modest retinal degeneration that occurs by 11 months of age. In this study we found that in dogs that received the CSF gene therapy treatment, the degeneration of the retina and loss of retinal function continued to progress during the prolonged life spans of the treated dogs. Eventually the normal cell layers of the retina almost completely disappeared. An exception was the ganglion cell layer. In affected dogs that received TPP1 gene therapy to the CSF and survived an average of 80 weeks, ganglion cell axons were present in numbers comparable to those of normal Dachshunds of similar age. The selective preservation of the retinal ganglion cells suggests that while TPP1 protein delivered via the CSF may protect these cells, preservation of the remainder of the retina will require delivery of normal TPP1 more directly to the retina, probably via the vitreous body. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

A lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor

USDA-ARS?s Scientific Manuscript database

Protein p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis and preserves barrier function by activation of EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study was to determine the mechanisms by which p40...

Implications of Differential Stress Response Activation Following Non-Frozen Hepatocellular Storage

PubMed Central

Corwin, William L.; Baust, John G.; Van Buskirk, Robert G.

2013-01-01

Hepatocytes are critical for numerous cell therapies and in vitro investigations. A limiting factor for their use in these applications is the ability to process and preserve them without loss of viability or functionality. Normal rat hepatocytes (NHEPs) and human hepatoma (C3A) cells were stored at either 4°C or 37°C to examine post-processing stress responses. Resveratrol and salubrinal were used during storage to determine how targeted molecular stress pathway modulation would affect cell survival. This study revealed that storage outcome is dependent upon numerous factors including: cell type, storage media, storage length, storage temperature, and chemical modulator. These data implicate a molecular-based stress response that is not universal but is specific to the set of conditions under which cells are stored. Further, these findings allude to the potential for targeted protection or destruction of particular cell types for numerous applications, from diagnostic cell selection to cell-based therapy. Ultimately, this study demonstrates the need for further in-depth molecular investigations into the cellular stress response to bioprocessing and preservation. PMID:24845253

Vildagliptin preserves the mass and function of pancreatic β cells via the developmental regulation and suppression of oxidative and endoplasmic reticulum stress in a mouse model of diabetes

PubMed Central

Hamamoto, S; Kanda, Y; Shimoda, M; Tatsumi, F; Kohara, K; Tawaramoto, K; Hashiramoto, M; Kaku, K

2013-01-01

Aim We investigated the molecular mechanisms by which vildagliptin preserved pancreatic β cell mass and function. Methods Morphological, biochemical and gene expression profiles of the pancreatic islets were investigated in male KK-Ay-TaJcl(KK-Ay) and C57BL/6JJcl (B6) mice aged 8 weeks which received either vildagliptin or a vehicle for 4 weeks. Results Body weight, food intake, fasting blood glucose, plasma insulin and active glucagon-like peptide-1 were unchanged with vildagliptin treatment in both mice. In KK-Ay mice treated with vildagliptin, increased plasma triglyceride (TG) level and islet TG content were decreased, insulin sensitivity significantly improved, and the glucose tolerance ameliorated with increases in plasma insulin levels. Furthermore, vildagliptin increased glucose-stimulated insulin secretion, islet insulin content and pancreatic β cell mass in both strains. By vildagliptin, the expression of genes involved in cell differentiation/proliferation was upregulated in both strains, those related to apoptosis, endoplasmic reticulum stress and lipid synthesis was decreased and those related to anti-apoptosis and anti-oxidative stress was upregulated, in KK-Ay mice. The morphological results were consistent with the gene expression profiles. Conclusion Vildagliptin increases β cell mass by not only directly affecting cell kinetics but also by indirectly reducing cell apoptosis, oxidative stress and endoplasmic reticulum stress in diabetic mice. PMID:22950702

Type 1 Diabetes TrialNet--an international collaborative clinical trials network.

PubMed

Skyler, Jay S; Greenbaum, Carla J; Lachin, John M; Leschek, Ellen; Rafkin-Mervis, Lisa; Savage, Peter; Spain, Lisa

2008-12-01

Type 1 Diabetes TrialNet is an international consortium of clinical research centers aimed at the prevention or delay of type 1 diabetes (T1D). The fundamental goal of TrialNet is to counter the T1D disease process by immune modulation and/or enhancement of beta cell proliferation and regeneration. To achieve this goal, TrialNet researchers are working to better understand the natural history of the disease, to identify persons at risk, and to clinically evaluate novel therapies that balance potential risks and benefits. The particular focus is on studies of preventive measures. In addition, TrialNet evaluates therapies in individuals with newly diagnosed T1D with preserved beta cell function to help determine the risk/benefit profile and gain an initial assessment of potential efficacy in preservation of beta cell function, so that promising agents can be studied in prevention trials. In addition, TrialNet evaluates methodologies that enhance the conduct of its clinical trials, which includes tests of outcome assessment methodology, the evaluation of surrogate markers, and mechanistic studies laying the foundation for future clinical trials.

Meiotic recombination generates rich diversity in NK cell receptor genes, alleles, and haplotypes

PubMed Central

Norman, Paul J.; Abi-Rached, Laurent; Gendzekhadze, Ketevan; Hammond, John A.; Moesta, Achim K.; Sharma, Deepti; Graef, Thorsten; McQueen, Karina L.; Guethlein, Lisbeth A.; Carrington, Christine V.F.; Chandanayingyong, Dasdayanee; Chang, Yih-Hsin; Crespí, Catalina; Saruhan-Direskeneli, Güher; Hameed, Kamran; Kamkamidze, Giorgi; Koram, Kwadwo A.; Layrisse, Zulay; Matamoros, Nuria; Milà, Joan; Park, Myoung Hee; Pitchappan, Ramasamy M.; Ramdath, D. Dan; Shiau, Ming-Yuh; Stephens, Henry A.F.; Struik, Siske; Tyan, Dolly; Verity, David H.; Vaughan, Robert W.; Davis, Ronald W.; Fraser, Patricia A.; Riley, Eleanor M.; Ronaghi, Mostafa; Parham, Peter

2009-01-01

Natural killer (NK) cells contribute to the essential functions of innate immunity and reproduction. Various genes encode NK cell receptors that recognize the major histocompatibility complex (MHC) Class I molecules expressed by other cells. For primate NK cells, the killer-cell immunoglobulin-like receptors (KIR) are a variable and rapidly evolving family of MHC Class I receptors. Studied here is KIR3DL1/S1, which encodes receptors for highly polymorphic human HLA-A and -B and comprises three ancient allelic lineages that have been preserved by balancing selection throughout human evolution. While the 3DS1 lineage of activating receptors has been conserved, the two 3DL1 lineages of inhibitory receptors were diversified through inter-lineage recombination with each other and with 3DS1. Prominent targets for recombination were D0-domain polymorphisms, which modulate enhancer function, and dimorphism at position 283 in the D2 domain, which influences inhibitory function. In African populations, unequal crossing over between the 3DL1 and 3DL2 genes produced a deleted KIR haplotype in which the telomeric “half” was reduced to a single fusion gene with functional properties distinct from its 3DL1 and 3DL2 parents. Conversely, in Eurasian populations, duplication of the KIR3DL1/S1 locus by unequal crossing over has enabled individuals to carry and express alleles of all three KIR3DL1/S1 lineages. These results demonstrate how meiotic recombination combines with an ancient, preserved diversity to create new KIR phenotypes upon which natural selection acts. A consequence of such recombination is to blur the distinction between alleles and loci in the rapidly evolving human KIR gene family. PMID:19411600

SIRT1 activation rescues doxorubicin-induced loss of functional competence of human cardiac progenitor cells.

PubMed

De Angelis, Antonella; Piegari, Elena; Cappetta, Donato; Russo, Rosa; Esposito, Grazia; Ciuffreda, Loreta Pia; Ferraiolo, Fiorella Angelica Valeria; Frati, Caterina; Fagnoni, Francesco; Berrino, Liberato; Quaini, Federico; Rossi, Francesco; Urbanek, Konrad

2015-01-01

The search for compounds able to counteract chemotherapy-induced heart failure is extremely important at the age of global cancer epidemic. The role of SIRT1 in the maintenance of progenitor cell homeostasis may contribute to its cardioprotective effects. SIRT1 activators, by preserving progenitor cells, could have a clinical relevance for the prevention of doxorubicin (DOXO)-cardiotoxicity. To determine whether SIRT1 activator, resveratrol (RES), interferes with adverse effects of DOXO on cardiac progenitor cells (CPCs): 1) human CPCs (hCPCs) were exposed in vitro to DOXO or DOXO+RES and their regenerative potential was tested in vivo in an animal model of DOXO-induced heart failure; 2) the in vivo effects of DOXO+RES co-treatment on CPCs were studied in a rat model. In contrast to healthy cells, DOXO-exposed hCPCs were ineffective in a model of anthracycline cardiomyopathy. The in vitro activation of SIRT1 decreased p53 acetylation, overcame suppression of the IGF-1/Akt pro-survival and anti-apoptotic signaling, enhanced oxidative stress defense and prevented senescence and growth arrest of hCPCs. Priming with RES counterbalanced the onset of dysfunctional phenotype in DOXO-exposed hCPCs, partly restoring their ability to repair the damage with improvement in cardiac function and animal survival. The in vivo co-treatment DOXO+RES prevented the anthracycline-induced alterations in CPCs, partly preserving cardiac function. SIRT1 activation protects DOXO-exposed CPCs and re-establishes their proper function. Pharmacological intervention at the level of tissue-specific progenitor cells may provide cardiac benefits for the growing population of long-term cancer survivors that are at risk of chemotherapy-induced cardiovascular toxicity. Copyright © 2015. Published by Elsevier Ireland Ltd.

Functional Stem Cell Integration into Neural Networks Assessed by Organotypic Slice Cultures.

PubMed

Forsberg, David; Thonabulsombat, Charoensri; Jäderstad, Johan; Jäderstad, Linda Maria; Olivius, Petri; Herlenius, Eric

2017-08-14

Re-formation or preservation of functional, electrically active neural networks has been proffered as one of the goals of stem cell-mediated neural therapeutics. A primary issue for a cell therapy approach is the formation of functional contacts between the implanted cells and the host tissue. Therefore, it is of fundamental interest to establish protocols that allow us to delineate a detailed time course of grafted stem cell survival, migration, differentiation, integration, and functional interaction with the host. One option for in vitro studies is to examine the integration of exogenous stem cells into an existing active neural network in ex vivo organotypic cultures. Organotypic cultures leave the structural integrity essentially intact while still allowing the microenvironment to be carefully controlled. This allows detailed studies over time of cellular responses and cell-cell interactions, which are not readily performed in vivo. This unit describes procedures for using organotypic slice cultures as ex vivo model systems for studying neural stem cell and embryonic stem cell engraftment and communication with CNS host tissue. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Intrinsic protective mechanisms of the neuron-glia network against glioma invasion.

PubMed

Iwadate, Yasuo; Fukuda, Kazumasa; Matsutani, Tomoo; Saeki, Naokatsu

2016-04-01

Gliomas arising in the brain parenchyma infiltrate into the surrounding brain and break down established complex neuron-glia networks. However, mounting evidence suggests that initially the network microenvironment of the adult central nervous system (CNS) is innately non-permissive to glioma cell invasion. The main players are inhibitory molecules in CNS myelin, as well as proteoglycans associated with astrocytes. Neural stem cells, and neurons themselves, possess inhibitory functions against neighboring tumor cells. These mechanisms have evolved to protect the established neuron-glia network, which is necessary for brain function. Greater insight into the interaction between glioma cells and the surrounding neuron-glia network is crucial for developing new therapies for treating these devastating tumors while preserving the important and complex neural functions of patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

Preservation of cardiac function by prolonged action potentials in mice deficient of KChIP2.

PubMed

Grubb, Søren; Aistrup, Gary L; Koivumäki, Jussi T; Speerschneider, Tobias; Gottlieb, Lisa A; Mutsaers, Nancy A M; Olesen, Søren-Peter; Calloe, Kirstine; Thomsen, Morten B

2015-08-01

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K(+) current (Ito,f) and with CaV1.2 to mediate the inward L-type Ca(2+) current (ICa,L). Anesthetized KChIP2(-/-) mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ₂ protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca(2+) entry into the cell, thereby augmenting Ca(2+)-induced Ca(2+) release. Ca(2+) transients in disaggregated KChIP2(-/-) cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca(2+)-induced Ca(2+) release event. We next functionally probed dyad structure, ryanodine receptor Ca(2+) sensitivity, and sarcoplasmic reticulum Ca(2+) load and found that increased temporal synchronicity of the Ca(2+) release in KChIP2(-/-) cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2(-/-) mice. Copyright © 2015 the American Physiological Society.

Intraovarian Transplantation of Female Germline Stem Cells Rescue Ovarian Function in Chemotherapy-Injured Ovaries.

PubMed

Xiong, Jiaqiang; Lu, Zhiyong; Wu, Meng; Zhang, Jinjin; Cheng, Jing; Luo, Aiyue; Shen, Wei; Fang, Li; Zhou, Su; Wang, Shixuan

2015-01-01

Early menopause and infertility often occur in female cancer patients after chemotherapy (CTx). For these patients, oocyte/embryo cryopreservation or ovarian tissue cryopreservation is the current modality for fertility preservation. However, the above methods are limited in the long-term protection of ovarian function, especially for fertility preservation (very few females with cancer have achieved pregnancy with cryopreserved ovarian tissue or eggs until now). In addition, the above methods are subject to their scope (females with no husband or prepubertal females with no mature oocytes). Thus, many females who suffer from cancers would not adopt the above methods pre- and post-CTx due to their uncertainty, safety and cost-effectiveness. Therefore, millions of women have achieved long-term survival after thorough CTx treatment and have desired to rescue their ovarian function and fertility with economic, durable and reliable methods. Recently, some studies showed that mice with infertility caused by CTx can produce normal offspring through intraovarian injection of exogenous female germline stem cells (FGSCs). Though exogenous FGSC can be derived from mice without immune rejection in the same strain, it is difficult to obtain human female germline stem cells (hFGSCs), and immune rejection could occur between different individuals. In this study, infertility in mice was caused by CTx, and the ability of FGSCs to restore ovarian function or even produce offspring was assessed. We had successfully isolated and purified the FGSCs from adult female mice two weeks after CTx. After infection with GFP-carrying virus, the FGSCs were transplanted into ovaries of mice with infertility caused by CTx. Finally, ovarian function was restored and the recipients produced offspring long-term. These findings showed that mice with CTx possessed FGSCs, restoring ovarian function and avoiding immune rejection from exogenous germline stem cells.

Antioxidants Complement the Requirement for Protein Chaperone Function to Maintain β-Cell Function and Glucose Homeostasis

PubMed Central

Han, Jaeseok; Song, Benbo; Kim, Jiun; Kodali, Vamsi K.; Pottekat, Anita; Wang, Miao; Hassler, Justin; Wang, Shiyu; Pennathur, Subramaniam; Back, Sung Hoon; Katze, Michael G.

2015-01-01

Proinsulin misfolding in the endoplasmic reticulum (ER) initiates a cell death response, although the mechanism(s) remains unknown. To provide insight into how protein misfolding may cause β-cell failure, we analyzed mice with the deletion of P58IPK/DnajC3, an ER luminal co-chaperone. P58IPK−/− mice become diabetic as a result of decreased β-cell function and mass accompanied by induction of oxidative stress and cell death. Treatment with a chemical chaperone, as well as deletion of Chop, improved β-cell function and ameliorated the diabetic phenotype in P58IPK−/− mice, suggesting P58IPK deletion causes β-cell death through ER stress. Significantly, a diet of chow supplemented with antioxidant dramatically and rapidly restored β-cell function in P58IPK−/− mice and corrected abnormal localization of MafA, a critical transcription factor for β-cell function. Antioxidant feeding also preserved β-cell function in Akita mice that express mutant misfolded proinsulin. Therefore defective protein folding in the β-cell causes oxidative stress as an essential proximal signal required for apoptosis in response to ER stress. Remarkably, these findings demonstrate that antioxidant feeding restores cell function upon deletion of an ER molecular chaperone. Therefore antioxidant or chemical chaperone treatment may be a promising therapeutic approach for type 2 diabetes. PMID:25795214

Etiopathogenesis of type 1 diabetes mellitus: prognostic factors for the evolution of residual β cell function

PubMed Central

2009-01-01

Type 1A diabetes mellitus (T1ADM) is a progressive autoimmune disease mediated by T lymphocytes with destruction of beta cells. Up to now, we do not have precise methods to assess the beta cell mass, "in vivo" or "ex-vivo". The studies about its genetic susceptibility show strong association with class II antigens of the HLA system (particularly DQ). Others genetics associations are weaker and depend on the population studied. A combination of precipitating events may occur at the beginning of the disease. There is a silent loss of immune-mediated beta cells mass which velocity has an inverse relation with the age, but it is influenced by genetic and metabolic factors. We can predict the development of the disease primarily through the determination of four biochemically islet auto antibodies against antigens like insulin, GAD65, IA2 and Znt8. Beta cell destruction is chronically progressive but at clinical diagnosis of the disease a reserve of these cells still functioning. The goal of secondary disease prevention is halt the autoimmune attack on beta cells by redirecting or dampening the immune system. It is remains one of the foremost therapeutic goals in the T1ADM. Glycemic intensive control and immunotherapeutic agents may preserve beta-cell function in newly diagnosed patients with T1ADM. It may be assessed through C-peptide values, which are important for glycemic stability and for the prevention of chronic complications of this disease. This article will summarize the etiopathogenesis mechanisms of this disease and the factors can influence on residual C-peptide and the strategies to it preservation. PMID:19961609

A STUDY OF THE EFFECTS OF STORAGE TEMPERATURE AND THAWING METHODS ON THE LONG-TERM PRESERVATION OF SELECTED STRAINS OF MICROORGANISMS AND MAMMALIAN CELLS.

DTIC Science & Technology

MICROORGANISMS, PRESERVATION), (*PRESERVATION, MICROORGANISMS), (*TISSUE CULTURE CELLS, PRESERVATION), MAMMALS, PENICILLIUM (PENICILLINS), VIBRIO, STAPHYLOCOCCUS AUREUS, FUNGI, STORAGE, FREEZING, VIABILITY

AKI after conditional and kidney-specific knockdown of Stanniocalcin-1

USDA-ARS?s Scientific Manuscript database

Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overex...

Non-Invasive Cell-Based Therapy for Traumatic Optic Neuropathy

DTIC Science & Technology

2013-10-01

Morgans, Sergey Girman, Raymond Lund and Shaomei Wang Retinal Morphological and Functional Changes in an Animal Model of Retinitis Pigmentosa . Vis...model was created. 2. Rat MSC and M-Sch were reliable produced for experiments. 3. Systemic administration of MSC significantly preserved retinal ...TON also promote retinal ganglion cell survival. From the first year study, we have shown that systemic administration of MSC can significantly

Nutritional Factors Affecting Adult Neurogenesis and Cognitive Function.

PubMed

Poulose, Shibu M; Miller, Marshall G; Scott, Tammy; Shukitt-Hale, Barbara

2017-11-01

Adult neurogenesis, a complex process by which stem cells in the hippocampal brain region differentiate and proliferate into new neurons and other resident brain cells, is known to be affected by many intrinsic and extrinsic factors, including diet. Neurogenesis plays a critical role in neural plasticity, brain homeostasis, and maintenance in the central nervous system and is a crucial factor in preserving the cognitive function and repair of damaged brain cells affected by aging and brain disorders. Intrinsic factors such as aging, neuroinflammation, oxidative stress, and brain injury, as well as lifestyle factors such as high-fat and high-sugar diets and alcohol and opioid addiction, negatively affect adult neurogenesis. Conversely, many dietary components such as curcumin, resveratrol, blueberry polyphenols, sulforaphane, salvionic acid, polyunsaturated fatty acids (PUFAs), and diets enriched with polyphenols and PUFAs, as well as caloric restriction, physical exercise, and learning, have been shown to induce neurogenesis in adult brains. Although many of the underlying mechanisms by which nutrients and dietary factors affect adult neurogenesis have yet to be determined, nutritional approaches provide promising prospects to stimulate adult neurogenesis and combat neurodegenerative diseases and cognitive decline. In this review, we summarize the evidence supporting the role of nutritional factors in modifying adult neurogenesis and their potential to preserve cognitive function during aging. © 2017 American Society for Nutrition.

Proregenerative Microenvironment Triggered by Donor Mesenchymal Stem Cells Preserves Renal Function and Structure in Mice with Severe Diabetes Mellitus.

PubMed

Ezquer, Fernando; Giraud-Billoud, Maximiliano; Carpio, Daniel; Cabezas, Fabián; Conget, Paulette; Ezquer, Marcelo

2015-01-01

The aim of our work was to evaluate, in an animal model of severe diabetes mellitus, the effect of mesenchymal stem cells (MSCs) administration on diabetic nephropathy (DN) progression. After diabetes induction, one group of mice received the vehicle (DM) and other group received a single dose of MSCs (DM + MSCs). DM + MSCs mice showed a significant improvement in functional parameters of the kidney compared with untreated mice. While DM mice presented marked histopathological changes characteristics of advanced stages of DN (fibrosis, glomerulosclerosis, glomerular basement membrane thickening, capillary occlusion, decreased podocyte density, and effacement of foot processes), DM + MSCs mice showed only slight tubular dilatation. The renoprotection was not associated with an improvement in diabetic condition and very low number of donor cells was found in the kidney of DM + MSCs mice, suggesting that renoprotection could be mediated by paracrine effects. Indeed, DM + MSC mice presented increased renal proliferation index, decreased renal apoptotic index and the restoration of proregenerative factors, and anti-inflammatory cytokines levels. Moreover, macrophage infiltration and oxidative stress damage were also reduced in DM + MSCs mice. Our data demonstrate that MSC administration triggers a proregenerative microenvironment in DN kidney, which allows the preservation of the renal function even if diabetes was uncorrected.

Proregenerative Microenvironment Triggered by Donor Mesenchymal Stem Cells Preserves Renal Function and Structure in Mice with Severe Diabetes Mellitus

PubMed Central

Ezquer, Fernando; Giraud-Billoud, Maximiliano; Carpio, Daniel; Cabezas, Fabián; Conget, Paulette

2015-01-01

The aim of our work was to evaluate, in an animal model of severe diabetes mellitus, the effect of mesenchymal stem cells (MSCs) administration on diabetic nephropathy (DN) progression. After diabetes induction, one group of mice received the vehicle (DM) and other group received a single dose of MSCs (DM + MSCs). DM + MSCs mice showed a significant improvement in functional parameters of the kidney compared with untreated mice. While DM mice presented marked histopathological changes characteristics of advanced stages of DN (fibrosis, glomerulosclerosis, glomerular basement membrane thickening, capillary occlusion, decreased podocyte density, and effacement of foot processes), DM + MSCs mice showed only slight tubular dilatation. The renoprotection was not associated with an improvement in diabetic condition and very low number of donor cells was found in the kidney of DM + MSCs mice, suggesting that renoprotection could be mediated by paracrine effects. Indeed, DM + MSC mice presented increased renal proliferation index, decreased renal apoptotic index and the restoration of proregenerative factors, and anti-inflammatory cytokines levels. Moreover, macrophage infiltration and oxidative stress damage were also reduced in DM + MSCs mice. Our data demonstrate that MSC administration triggers a proregenerative microenvironment in DN kidney, which allows the preservation of the renal function even if diabetes was uncorrected. PMID:26167475

DNA preservation in silk.

PubMed

Liu, Yawen; Zheng, Zhaozhu; Gong, He; Liu, Meng; Guo, Shaozhe; Li, Gang; Wang, Xiaoqin; Kaplan, David L

2017-06-27

The structure of DNA is susceptible to alterations at high temperature and on changing pH, irradiation and exposure to DNase. Options to protect and preserve DNA during storage are important for applications in genetic diagnosis, identity authentication, drug development and bioresearch. In the present study, the stability of total DNA purified from human dermal fibroblast cells, as well as that of plasmid DNA, was studied in silk protein materials. The DNA/silk mixtures were stabilized on filter paper (silk/DNA + filter) or filter paper pre-coated with silk and treated with methanol (silk/DNA + PT-filter) as a route to practical utility. After air-drying and water extraction, 50-70% of the DNA and silk could be retrieved and showed a single band on electrophoretic gels. 6% silk/DNA + PT-filter samples provided improved stability in comparison with 3% silk/DNA + filter samples and DNA + filter samples for DNA preservation, with ∼40% of the band intensity remaining at 37 °C after 40 days and ∼10% after exposure to UV light for 10 hours. Quantitative analysis using the PicoGreen assay confirmed the results. The use of Tris/borate/EDTA (TBE) buffer enhanced the preservation and/or extraction of the DNA. The DNA extracted after storage maintained integrity and function based on serving as a functional template for PCR amplification of the gene for zinc finger protein 750 (ZNF750) and for transgene expression of red fluorescence protein (dsRed) in HEK293 cells. The high molecular weight and high content of a crystalline beta-sheet structure formed on the coated surfaces likely accounted for the preservation effects observed for the silk/DNA + PT-filter samples. Although similar preservation effects were also obtained for lyophilized silk/DNA samples, the rapid and simple processing available with the silk-DNA-filter membrane system makes it appealing for future applications.

Sirtuin1 protects endothelial Caveolin-1 expression and preserves endothelial function via suppressing miR-204 and endoplasmic reticulum stress.

PubMed

Kassan, M; Vikram, A; Kim, Y R; Li, Q; Kassan, A; Patel, H H; Kumar, S; Gabani, M; Liu, J; Jacobs, J S; Irani, K

2017-02-09

Sirtuin1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including endothelial function. Caveolin1 (Cav1) is also an important determinant of endothelial function. We asked if Sirt1 governs endothelial Cav1 and endothelial function by regulating miR-204 expression and endoplasmic reticulum (ER) stress. Knockdown of Sirt1 in endothelial cells, and in vivo deletion of endothelial Sirt1, induced endothelial ER stress and miR-204 expression, reduced Cav1, and impaired endothelium-dependent vasorelaxation. All of these effects were reversed by a miR-204 inhibitor (miR-204 I) or with overexpression of Cav1. A miR-204 mimic (miR-204 M) decreased Cav1 in endothelial cells. In addition, high-fat diet (HFD) feeding induced vascular miR-204 and reduced endothelial Cav1. MiR-204-I protected against HFD-induced downregulation of endothelial Cav1. Moreover, pharmacologic induction of ER stress with tunicamycin downregulated endothelial Cav1 and impaired endothelium-dependent vasorelaxation that was rescued by overexpressing Cav1. In conclusion, Sirt1 preserves Cav1-dependent endothelial function by mitigating miR-204-mediated vascular ER stress.

Parenchymal preserving anatomic resections result in less pulmonary function loss in patients with Stage I non-small cell lung cancer.

PubMed

Macke, Ryan A; Schuchert, Matthew J; Odell, David D; Wilson, David O; Luketich, James D; Landreneau, Rodney J

2015-04-01

A suggested benefit of sublobar resection for stage I non-small cell lung cancer (NSCLC) compared to lobectomy is a relative preservation of pulmonary function. Very little objective data exist, however, supporting this supposition. We sought to evaluate the relative impact of both anatomic segmental and lobar resection on pulmonary function in patients with resected clinical stage I NSCLC. The records of 159 disease-free patients who underwent anatomic segmentectomy (n = 89) and lobectomy (n = 70) for the treatment of stage I NSCLC with pre- and postoperative pulmonary function tests performed between 6 to 36 months after resection were retrospectively reviewed. Changes in forced expiratory volume in one second (FEV1) and diffusion capacity of carbon monoxide (DLCO) were analyzed based upon the number of anatomic pulmonary segments removed: 1-2 segments (n = 77) or 3-5 segments (n = 82). Preoperative pulmonary function was worse in the lesser resection cohort (1-2 segments) compared to the greater resection group (3-5 segments) (FEV1(%predicted): 79% vs. 85%, p = 0.038; DLCO(%predicted): 63% vs. 73%, p = 0.010). A greater decline in FEV1 was noted in patients undergoing resection of 3-5 segments (FEV1 (observed): 0.1 L vs. 0.3 L, p = 0.003; and FEV1 (% predicted): 4.3% vs. 8.2%, p = 0.055). Changes in DLCO followed this same trend (DLCO(observed): 1.3 vs. 2.4 mL/min/mmHg, p = 0.015; and DLCO(% predicted): 3.6% vs. 5.9%, p = 0.280). Parenchymal-sparing resections resulted in better preservation of pulmonary function at a median of one year, suggesting a long-term functional benefit with small anatomic segmental resections (1-2 segments). Prospective studies to evaluate measurable functional changes, as well as quality of life, between segmentectomy and lobectomy with a larger patient cohort appear justified.

Male fertility preservation before gonadotoxic therapies

PubMed Central

Wyns, C.

2010-01-01

Background: Recent advances in cancer therapy have resulted in an increased number of long-term cancer survivors. Unfortunately, aggressive chemotherapy, radiotherapy and preparative regimens for bone marrow transplantation can severely affect male germ cells, including spermatogonial stem cells (SSCs), and lead to permanent loss of fertility. Different options for fertility preservation are dependent on the pubertal state of the patient. Methods: Relevant studies were identified by an extensive Medline search of English and French language articles. Results: Sperm cryopreservation prior to gonadotoxic treatment is a well established method after puberty. In case of ejaculation failure by masturbation, assisted ejaculation methods or testicular tissue sampling should be considered. Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell (SSC) protection in humans, current evidence supports the feasibility of immature testicular tissue (ITT) cryopreservation. The different cryopreservation protocols and available fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and in vitro maturation, are presented. Results obtained in humans are discussed in the light of lessons learned from animal studies. Conclusion: Advances in reproductive technology have made fertility preservation a real possibility in young patients whose gonadal function is threatened by gonadotoxic therapies. The putative indications for such techniques, as well as their limitations according to disease, are outlined. PMID:25302103

Male fertility preservation before gonadotoxic therapies.

PubMed

Wyns, C

2010-01-01

Recent advances in cancer therapy have resulted in an increased number of long-term cancer survivors. Unfortunately, aggressive chemotherapy, radiotherapy and preparative regimens for bone marrow transplantation can severely affect male germ cells, including spermatogonial stem cells (SSCs), and lead to permanent loss of fertility. Different options for fertility preservation are dependent on the pubertal state of the patient. Relevant studies were identified by an extensive Medline search of English and French language articles. Sperm cryopreservation prior to gonadotoxic treatment is a well established method after puberty. In case of ejaculation failure by masturbation, assisted ejaculation methods or testicular tissue sampling should be considered. Although no effective gonadoprotective drug is yet available for in vivo spermatogonial stem cell (SSC) protection in humans, current evidence supports the feasibility of immature testicular tissue (ITT) cryopreservation. The different cryopreservation protocols and available fertility restoration options from frozen tissue, i.e. cell suspension transplantation, tissue grafting and in vitro maturation, are presented. RESULTS obtained in humans are discussed in the light of lessons learned from animal studies. Advances in reproductive technology have made fertility preservation a real possibility in young patients whose gonadal function is threatened by gonadotoxic therapies. The putative indications for such techniques, as well as their limitations according to disease, are outlined.

Differential partitioning of triterpenes and triterpene esters in apple peel

USDA-ARS?s Scientific Manuscript database

Apple peel functions as a protective barrier against biotic and abiotic stresses, and preserving the integrity and appearance of peel critical for market acceptance. Peel epidermal cells and epicuticular wax are a rich source of secondary metabolites, including triterpenes. Several studies have ou...

Clinical indices of in vivo biocompatibility: the role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients.

PubMed

Mackenzie, Ruth; Holmes, Clifford J; Jones, Suzanne; Williams, John D; Topley, Nicholas

2003-12-01

Clinical indices of in vivo biocompatibility: The role of ex vivo cell function studies and effluent markers in peritoneal dialysis patients. Over the past 20 years, studies of the biocompatibility profile of peritoneal dialysis solutions (PDF) have evolved from initial in vitro studies assessing the impact of solutions on leukocyte function to evaluations of mesothelial cell behavior. More recent biocompatibility evaluations have involved assessments of the impact of PDF on membrane integrity and cell function in peritoneal dialysis (PD) patients. The development of ex vivo systems for the evaluation of in vivo cell function, and effluent markers of membrane integrity and inflammation in patients exposed both acutely and chronically to conventional and new PDF will be interpreted in the context of our current understanding of the biology of the dialyzed peritoneum. The available data indicate that exposure of the peritoneal environment to more biocompatible PDF is associated with improvements in peritoneal cell function, alterations in markers of membrane integrity, and reduced local inflammation. These data suggest that more biocompatible PDF will have a positive impact on host defense, peritoneal homeostasis, and the long-term preservation of peritoneal membrane function in PD patients.

Efficacy and Safety of Human Retinal Progenitor Cells

PubMed Central

Semo, Ma'ayan; Haamedi, Nasrin; Stevanato, Lara; Carter, David; Brooke, Gary; Young, Michael; Coffey, Peter; Sinden, John; Patel, Sara; Vugler, Anthony

2016-01-01

Purpose We assessed the long-term efficacy and safety of human retinal progenitor cells (hRPC) using established rodent models. Methods Efficacy of hRPC was tested initially in Royal College of Surgeons (RCS) dystrophic rats immunosuppressed with cyclosporine/dexamethasone. Due to adverse effects of dexamethasone, this drug was omitted from a subsequent dose-ranging study, where different hRPC doses were tested for their ability to preserve visual function (measured by optokinetic head tracking) and retinal structure in RCS rats at 3 to 6 months after grafting. Safety of hRPC was assessed by subretinal transplantation into wild type (WT) rats and NIH-III nude mice, with analysis at 3 to 6 and 9 months after grafting, respectively. Results The optimal dose of hRPC for preserving visual function/retinal structure in dystrophic rats was 50,000 to 100,000 cells. Human retinal progenitor cells integrated/survived in dystrophic and WT rat retina up to 6 months after grafting and expressed nestin, vimentin, GFAP, and βIII tubulin. Vision and retinal structure remained normal in WT rats injected with hRPC and there was no evidence of tumors. A comparison between dexamethasone-treated and untreated dystrophic rats at 3 months after grafting revealed an unexpected reduction in the baseline visual acuity of dexamethasone-treated animals. Conclusions Human retinal progenitor cells appear safe and efficacious in the preclinical models used here. Translational Relevance Human retinal progenitor cells could be deployed during early stages of retinal degeneration or in regions of intact retina, without adverse effects on visual function. The ability of dexamethasone to reduce baseline visual acuity in RCS dystrophic rats has important implications for the interpretation of preclinical and clinical cell transplant studies. PMID:27486556

In vitro effects of preservative-free and preserved prostaglandin analogs on primary cultured human conjunctival fibroblast cells.

PubMed

Kim, Eun Joo; Kim, Yeoun-Hee; Kang, Sun-Hee; Lee, Kyoo Won; Park, Young Jeung

2013-12-01

Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells. Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis. BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs. This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs formulations has not yet been firmly established. Alternatively preserved or preservative-free glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC.

In Vitro Effects of Preservative-free and Preserved Prostaglandin Analogs on Primary Cultured Human Conjunctival Fibroblast Cells

PubMed Central

Kim, Eun Joo; Kim, Yeoun-Hee; Kang, Sun-Hee; Lee, Kyoo Won

2013-01-01

Purpose Long-term use of topical medication is needed for glaucoma treatment. One of the most commonly prescribed classes of hypotensive agents are prostaglandin analogs (PGs) used as both first-line monotherapy; as well as in combination therapy with other hypotensive agents. Several side effects of eye drops can be caused by preservatives. The purpose of this study was to evaluate the effects of PGs with varying concentrations of benzalkonium chloride (BAC), alternative preservatives, or no preservatives on human conjunctival fibroblast cells. Methods Primary human conjunctival fibroblast cells were used in these experiments. Cells were exposed to the following drugs: BAC at different concentrations, bimatoprost 0.01% (with BAC 0.02%), latanoprost 0.005% (with BAC 0.02%), tafluprost 0.0015% with/without 0.001% BAC and travoprost 0.004% (with 0.001% Polyquad) for 15 and 30 minutes. Cell cytotoxicity was evaluated by phase-contrast microscopy to monitor morphological changes of cells, Counting Kit-8 (CCK-8) assay to cell viability, and fluorescent activated cell sorting (FACS) analysis to measure apoptosis. Results BAC caused cell shrinkage and detachment from the plate in a dose-dependent manner. Morphological changes were observed in cells treated with bimatoprost 0.01% and latanoprost 0.005%. However, mild cell shrinkage was noted in cells treated with tafluprost 0.0015%, while a non-toxic effect was noted with travoprost 0.004% and preservative-free tafluprost 0.0015%. CCK-8 assay and FACS analysis showed all groups had a significantly decreased cell viability and higher apoptosis rate compared with the control group. However, travoprost 0.004% and preservative-free tafluprost 0.0015% showed lower cytotoxicity and apoptosis rate than other drugs. Conclusions This in vitro study revealed that BAC-induced cytotoxicity is dose-dependent, although it is important to emphasize that the clinical significance of toxicity differences observed among the different PGs formulations has not yet been firmly established. Alternatively preserved or preservative-free glaucoma medications seem to be a reasonable and viable alternative to those preserved with BAC. PMID:24311931

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

PubMed

Mizuno, Mitsuru; Katano, Hisako; Otabe, Koji; Komori, Keiichiro; Kohno, Yuji; Fujii, Shizuka; Ozeki, Nobutake; Horie, Masafumi; Tsuji, Kunikazu; Koga, Hideyuki; Muneta, Takeshi; Sekiya, Ichiro

2017-06-13

In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 μL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide - and annexin V - cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. The viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.

Development of a living membrane comprising a functional human renal proximal tubule cell monolayer on polyethersulfone polymeric membrane.

PubMed

Schophuizen, Carolien M S; De Napoli, Ilaria E; Jansen, Jitske; Teixeira, Sandra; Wilmer, Martijn J; Hoenderop, Joost G J; Van den Heuvel, Lambert P W; Masereeuw, Rosalinde; Stamatialis, Dimitrios

2015-03-01

The need for improved renal replacement therapies has stimulated innovative research for the development of a cell-based renal assist device. A key requirement for such a device is the formation of a "living membrane", consisting of a tight kidney cell monolayer with preserved functional organic ion transporters on a suitable artificial membrane surface. In this work, we applied a unique conditionally immortalized proximal tubule epithelial cell (ciPTEC) line with an optimized coating strategy on polyethersulfone (PES) membranes to develop a living membrane with a functional proximal tubule epithelial cell layer. PES membranes were coated with combinations of 3,4-dihydroxy-l-phenylalanine and human collagen IV (Coll IV). The optimal coating time and concentrations were determined to achieve retention of vital blood components while preserving high water transport and optimal ciPTEC adhesion. The ciPTEC monolayers obtained were examined through immunocytochemistry to detect zona occludens 1 tight junction proteins. Reproducible monolayers were formed when using a combination of 2 mg ml(-1) 3,4-dihydroxy-l-phenylalanine (4 min coating, 1h dissolution) and 25 μg ml(-1) Coll IV (4 min coating). The successful transport of (14)C-creatinine through the developed living membrane system was used as an indication for organic cation transporter functionality. The addition of metformin or cimetidine significantly reduced the creatinine transepithelial flux, indicating active creatinine uptake in ciPTECs, most likely mediated by the organic cation transporter, OCT2 (SLC22A2). In conclusion, this study shows the successful development of a living membrane consisting of a reproducible ciPTEC monolayer on PES membranes, an important step towards the development of a bioartificial kidney. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Differences in the profile of protection afforded by TRO40303 and mild hypothermia in models of cardiac ischemia/reperfusion injury.

PubMed

Hansson, Magnus J; Llwyd, Osian; Morin, Didier; de Paulis, Damien; Arnoux, Thomas; Gouarné, Caroline; Koul, Sasha; Engblom, Henrik; Bordet, Thierry; Tissier, Renaud; Arheden, Haakan; Erlinge, David; Halestrap, Andrew P; Berdeaux, Alain; Pruss, Rebecca M; Schaller, Sophie

2015-08-05

The mode of protection against cardiac reperfusion injury by mild hypothermia and TRO40303 was investigated in various experimental models and compared to MitoQ in vitro. In isolated cardiomyocytes subjected to hypoxia/reoxygenation, TRO40303, MitoQ and mild hypothermia delayed mPTP opening, inhibited generation of mitochondrial superoxide anions at reoxygenation and improved cell survival. Mild hypothermia, but not MitoQ and TRO40303, provided protection in a metabolic starvation model in H9c2 cells and preserved respiratory function in isolated rat heart mitochondria submitted to anoxia/reoxygenation. In the Langendorff-perfused rat heart, only mild hypothermia provided protection of hemodynamic function and reduced infarct size following ischemia/reperfusion. In biopsies from the left ventricle of pigs subjected to in vivo occlusion/reperfusion, TRO40303 specifically preserved respiratory functions in the peri-infarct zone whereas mild hypothermia preserved both the ischemic core area and the peri-infarct zones. Additionally in this pig model, only hypothermia reduced infarct size. We conclude that mild hypothermia provided protection in all models by reducing the detrimental effects of ischemia, and when initiated before occlusion, reduced subsequent reperfusion damage leading to a smaller infarct. By contrast, although TRO40303 provided similar protection to MitoQ in vitro and offered specific protection against some aspects of reperfusion injury in vivo, this was insufficient to reduce infarct size. Copyright © 2015 Elsevier B.V. All rights reserved.

An extracatalytic function of CD45 in B cells is mediated by CD22

PubMed Central

Coughlin, Sarah; Noviski, Mark; Mueller, James L.; Chuwonpad, Ammarina; Raschke, William C.; Weiss, Arthur; Zikherman, Julie

2015-01-01

The receptor-like tyrosine phosphatase CD45 regulates antigen receptor signaling by dephosphorylating the C-terminal inhibitory tyrosine of the src family kinases. However, despite its abundance, the function of the large, alternatively spliced extracellular domain of CD45 has remained elusive. We used normally spliced CD45 transgenes either incorporating a phosphatase-inactivating point mutation or lacking the cytoplasmic domain to uncouple the enzymatic and noncatalytic functions of CD45 in lymphocytes. Although these transgenes did not alter T-cell signaling or development irrespective of endogenous CD45 expression, both partially rescued the phenotype of CD45-deficient B cells. We identify a noncatalytic role for CD45 in regulating tonic, but not antigen-mediated, B-cell antigen receptor (BCR) signaling through modulation of the function of the inhibitory coreceptor CD22. This finding has important implications for understanding how naïve B cells maintain tonic BCR signaling while restraining inappropriate antigen-dependent activation to preserve clonal “ignorance.” PMID:26561584

Neurotrophic Properties, Chemosensory Responses and Neurogenic Niche of the Human Carotid Body.

PubMed

Ortega-Sáenz, Patricia; Villadiego, Javier; Pardal, Ricardo; Toledo-Aral, Juan José; López-Barneo, José

2015-01-01

The carotid body (CB) is a polymodal chemoreceptor that triggers the hyperventilatory response to hypoxia necessary for the maintenance of O(2) homeostasis essential for the survival of organs such as the brain or heart. Glomus cells, the sensory elements in the CB, are also sensitive to hypercapnia, acidosis and, although less generally accepted, hypoglycemia. Current knowledge on CB function is mainly based on studies performed on lower mammals, but the information on the human CB is scant. Here we describe the structure, neurotrophic properties, and cellular responses to hypoxia and hypoglycemia of CBs dissected from human cadavers. The adult CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin-positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF). GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. As reported for other mammalian species, glomus cells responded to hypoxia by external Ca(2+)-dependent increase of cytosolic [Ca(2+)] and quantal catecholamine release. Human glomus cells are also responsive to hypoglycemia and together the two stimuli, hypoxia and hypoglycemia, can potentiate each other's effects. The chemo-sensory responses of glomus cells are also preserved at an advanced age. Interestingly, a neurogenic niche similar to that recently described in rodents is also preserved in the adult human CB. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.

Vascular endothelium summary statement II: Cardiovascular disease prevention and control.

PubMed

Mensah, George A; Ryan, Una S; Hooper, W Craig; Engelgau, Michael M; Callow, Allan D; Kapuku, Gaston K; Mantovani, Alberto

2007-05-01

The prevention and control of cardiovascular disease (CVD), principally ischemic heart disease and stroke, are a major clinical and public health challenge. Worldwide, CVD accounts for substantial morbidity and mortality. The major modifiable CVD risk factors are known and all of them cause endothelial activation and dysfunction. Preventing and controlling the established risk factors are associated with preserved endothelial function and reduced risk of CVD. Research advances that improve our understanding of strategies to preserve endothelial function or make the endothelial cells resilient to environmental insults may help improve our preventive interventions. This summary statement addresses the current state of the science with respect to endothelial dysfunction and CVD pathogenesis, diagnostic evaluation, and suggested strategies for public health practice and research.

THE PRESERVATION OF LIVING RED BLOOD CELLS IN VITRO

PubMed Central

Rous, Peyton; Turner, J. R.

1916-01-01

The erythrocytes of some species are much damaged when handled in salt solutions, as in washing with the centrifuge after the ordinary method. The injury is mechanical in character. It may express itself in hemolysis only after the cells have been kept for some days. It is greatest in the case of dog corpuscles, and well marked with sheep and rabbit cells. The fragility of the red cells, as indicated by washing or shaking them in salt solution is different, not only for different species, but for different individuals. It varies independently of the resistance to hypotonic solutions. The protection of fragile erythrocytes during washing is essential if they are to be preserved in vitro for any considerable time. The addition of a little gelatin (⅛ per cent) to the wash fluid suffices for this purpose, and by its use the period of survival in salt solutions of washed rabbit, sheep, and dog cells is greatly prolonged. Plasma, like gelatin, has marked protective properties. Though gelatin acts as a protective for red cells it is not preservative of them in the real sense. Cells do not last longer when it is added to the fluids in which they are kept. Locke's solution, though better probably than Ringer's solution, or a sodium chloride solution, as a medium in which to keep red cells, is ultimately harmful. The addition of innocuous colloids does not improve it. But the sugars, especially dextrose and saccharose, have a remarkable power to prevent its injurious action, and they possess, in addition, preservative qualities. Cells washed in gelatin-Locke's and placed in a mixture of Locke's solution with an isotonic, watery solution of a sugar remain intact for a long time,—nearly 2 months in the case of sheep cells. The kept cells go easily into suspension free of clumps, they pass readily through paper filters, take up and give off oxygen, and when used for the Wassermann reaction behave exactly as do fresh cells of the same individual. The best preservative solutions are approximately isotonic with the blood serum. If the cells are to be much handled gelatin should be present, for the sugars do not protect against mechanical injury. Different preservative mixtures are required for the cells of different species. Dog cells last longest in fluids containing dextrin as well as a sugar. The mixture best for red cells is not necessarily best for leukocytes. A simple and practical method of keeping rabbit and human erythrocytes is in citrated whole blood to which sugar solution is added. In citrated blood, as such, human red cells tend to break down rather rapidly, no matter what the proportion of citrate. Hemolysis is well marked after little more than a week. But in a mixture of 3 parts of human blood, 2 parts of isotonic citrate solution (3.8 per cent sodium citrate in water), and 5 parts of isotonic dextrose solution (5.4 per cent dextrose in water), the cells remain intact for about 4 weeks. Rabbit red cells can be kept for more than 3 weeks in citrated blood; and the addition of sugar lengthens the preservation only a little. The results differ strikingly with the amount of citrate employed. Hemolysis occurs relatively early when the smallest quantity is used that will prevent clotting. The optimum mixture has 3 parts of rabbit blood to 2 of isotonic citrate solution. In the second part of this paper experiments are detailed which prove that cells preserved by the methods here recorded function excellently when reintroduced into the body. PMID:19867981

Live Cell Imaging of a Fluorescent Gentamicin Conjugate

PubMed Central

Escobedo, Jorge O.; Chu, Yu-Hsuan; Wang, Qi; Steyger, Peter S.; Strongin, Robert M.

2012-01-01

Understanding cellular mechanisms of ototoxic and nephrotoxic drug uptake, intracellular distribution, and molecular trafficking across cellular barrier systems aids the study of potential uptake blockers that preserve sensory and renal function during critical life-saving therapy. Herein we report the design, synthesis characterization and evaluation of a fluorescent conjugate of the aminoglycoside antibiotic gentamicin. Live cell imaging results show the potential utility of this new material. Related gentamicin conjugates studied to date quench in live kindney cells, and have been largely restricted to use in fixed (delipidated) cells. PMID:22545403

Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples.

PubMed

Yu, Feiqiao Brian; Blainey, Paul C; Schulz, Frederik; Woyke, Tanja; Horowitz, Mark A; Quake, Stephen R

2017-07-05

Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.

RED BLOOD CELL STORAGE LESION

PubMed Central

Kor, Daryl J.; Van Buskirk, Camille M; Gajic, Ognjen

2009-01-01

The past two decades have witnessed increased scrutiny regarding efficacy and risk of the once unquestioned therapy of red blood cell (RBC) transfusion. Simultaneously, a variety of changes have been identified within the RBC and storage media during RBC preservation that are correlated with reduced tissue oxygenation and transfusion-associated adverse effects. These alterations are collectively termed the storage lesion and include extensive biochemical, biomechanical, and immunologic changes involving cells of diverse origin. Time-dependent falls is 2,3-diphosphoglycerate, intracellular RBC adenosine triphosphate, and nitric oxide have been shown to impact RBC deformability and delivery of oxygen to the end-organ. The accumulation of biologic response modifiers such as soluble CD40 ligand (sCD40L), lyso-phosphatidylcholine (lyso-PC), and Regulated on Activation, Normal T-cell Expressed and Secreted (RANTES) have been associated with altered recipient immune function as well. This review will address the alterations occurring within the RBC and storage media during RBC preservation and will address the potential clinical consequence thereof.

Reversible p53 inhibition prevents cisplatin ototoxicity without blocking chemotherapeutic efficacy.

PubMed

Benkafadar, Nesrine; Menardo, Julien; Bourien, Jérôme; Nouvian, Régis; François, Florence; Decaudin, Didier; Maiorano, Domenico; Puel, Jean-Luc; Wang, Jing

2017-01-01

Cisplatin is a widely used chemotherapy drug, despite its significant ototoxic side effects. To date, the mechanism of cisplatin-induced ototoxicity remains unclear, and hearing preservation during cisplatin-based chemotherapy in patients is lacking. We found activation of the ATM-Chk2-p53 pathway to be a major determinant of cisplatin ototoxicity. However, prevention of cisplatin-induced ototoxicity is hampered by opposite effects of ATM activation upon sensory hair cells: promoting both outer hair cell death and inner hair cell survival. Encouragingly, however, genetic or pharmacological ablation of p53 substantially attenuated cochlear cell apoptosis, thus preserving hearing. Importantly, systemic administration of a p53 inhibitor in mice bearing patient-derived triple-negative breast cancer protected auditory function, without compromising the anti-tumor efficacy of cisplatin. Altogether, these findings highlight a novel and effective strategy for hearing protection in cisplatin-based chemotherapy. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

The Effect of Disinfection on Viability and Function of Baboon Red Blood Cells and Platelets

DTIC Science & Technology

1997-07-11

blood cells was evaluated by their ability to transport oxygen as assessed by measurement of 2,3 diphosphoglycerate (DPG)14 and red blood cell p50,15...Blood collected from the bleeding time site (referred to as "shed blood") had a significantly reduced thromboxane A2 level . The ability of the...preserved or treated platelets to increase the shed blood thromboxane A2 level and reduce the 8; extended bleeding time is the measure of their

The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function

PubMed Central

Isern, Joan; García-García, Andrés; Martín, Ana M; Arranz, Lorena; Martín-Pérez, Daniel; Torroja, Carlos; Sánchez-Cabo, Fátima; Méndez-Ferrer, Simón

2014-01-01

Mesenchymal stem cells (MSCs) and osteolineage cells contribute to the hematopoietic stem cell (HSC) niche in the bone marrow of long bones. However, their developmental relationships remain unclear. In this study, we demonstrate that different MSC populations in the developing marrow of long bones have distinct functions. Proliferative mesoderm-derived nestin− MSCs participate in fetal skeletogenesis and lose MSC activity soon after birth. In contrast, quiescent neural crest-derived nestin+ cells preserve MSC activity, but do not generate fetal chondrocytes. Instead, they differentiate into HSC niche-forming MSCs, helping to establish the HSC niche by secreting Cxcl12. Perineural migration of these cells to the bone marrow requires the ErbB3 receptor. The neonatal Nestin-GFP+ Pdgfrα− cell population also contains Schwann cell precursors, but does not comprise mature Schwann cells. Thus, in the developing bone marrow HSC niche-forming MSCs share a common origin with sympathetic peripheral neurons and glial cells, and ontogenically distinct MSCs have non-overlapping functions in endochondrogenesis and HSC niche formation. DOI: http://dx.doi.org/10.7554/eLife.03696.001 PMID:25255216

The Cell's Sophisticated Army to Defend Against Assaults on DNAThe Cell's Sophisticated Army to Defend Against Assaults on DNA | Center for Cancer Research

Cancer.gov

The maintenance of genome integrity and function is essen-tial for the survival of cells and organisms. Any damage to our genetic material must be immediately sensed and repaired to preserve a cell’s func-tional integrity. Cells are constantly faced with the challenge of protecting their DNA from assaults by damaging chemicals and ultraviolet light. DNA damage that escapes repair can lead to a variety of genetic disorders and diseases, particularly cancer. To avoid this catastrophe, the cell employs an army of DNA repair factors that “rush to the scene” and initiate a cascade of events to repair the damage. Exactly how different repair factors sense DNA damage and orchestrate their concert-ed response is not well understood.

Patterns of conservation and change in honey bee developmental genes

PubMed Central

Dearden, Peter K.; Wilson, Megan J.; Sablan, Lisha; Osborne, Peter W.; Havler, Melanie; McNaughton, Euan; Kimura, Kiyoshi; Milshina, Natalia V.; Hasselmann, Martin; Gempe, Tanja; Schioett, Morten; Brown, Susan J.; Elsik, Christine G.; Holland, Peter W.H.; Kadowaki, Tatsuhiko; Beye, Martin

2006-01-01

The current insect genome sequencing projects provide an opportunity to extend studies of the evolution of developmental genes and pathways in insects. In this paper we examine the conservation and divergence of genes and developmental processes between Drosophila and the honey bee; two holometabolous insects whose lineages separated ∼300 million years ago, by comparing the presence or absence of 308 Drosophila developmental genes in the honey bee. Through examination of the presence or absence of genes involved in conserved pathways (cell signaling, axis formation, segmentation and homeobox transcription factors), we find that the vast majority of genes are conserved. Some genes involved in these processes are, however, missing in the honey bee. We have also examined the orthology of Drosophila genes involved in processes that differ between the honey bee and Drosophila. Many of these genes are preserved in the honey bee despite the process in which they act in Drosophila being different or absent in the honey bee. Many of the missing genes in both situations appear to have arisen recently in the Drosophila lineage, have single known functions in Drosophila, and act early in developmental pathways, while those that are preserved have pleiotropic functions. An evolutionary interpretation of these data is that either genes with multiple functions in a common ancestor are more likely to be preserved in both insect lineages, or genes that are preserved throughout evolution are more likely to co-opt additional functions. PMID:17065607

Nephrotic Syndrome and Idiopathic Membranous Nephropathy Associated with Autosomal-Dominant Polycystic Kidney Disease

PubMed Central

Peces, Ramón; Martínez-Ara, Jorge; Peces, Carlos; Picazo, Mariluz; Cuesta-López, Emilio; Vega, Cristina; Azorín, Sebastián; Selgas, Rafael

2011-01-01

We report the case of a 38-year-old male with autosomal-dominant polycystic kidney disease (ADPKD) and concomitant nephrotic syndrome secondary to membranous nephropathy (MN). A 3-month course of prednisone 60 mg daily and losartan 100 mg daily resulted in resistance. Treatment with chlorambucil 0.2 mg/kg daily, low-dose prednisone, plus an angiotensin-converting enzyme inhibitor (ACEI) and an angiotensin II receptor blocker (ARB) for 6 weeks resulted in partial remission of his nephrotic syndrome for a duration of 10 months. After relapse of the nephrotic syndrome, a 13-month course of mycophenolate mofetil (MFM) 2 g daily and low-dose prednisone produced complete remission for 44 months. After a new relapse, a second 24-month course of MFM and low-dose prednisone produced partial to complete remission of proteinuria with preservation of renal function. Thirty-six months after MFM withdrawal, complete remission of nephrotic-range proteinuria was maintained and renal function was preserved. This case supports the idea that renal biopsy is needed for ADPKD patients with nephrotic-range proteinuria in order to exclude coexisting glomerular disease and for appropriate treatment/prevention of renal function deterioration. To the best of our knowledge, this is the first reported case of nephrotic syndrome due to MN in a patient with ADPKD treated with MFM, with remission of proteinuria and preservation of renal function after more than 10 years. Findings in this patient also suggest that MFM might reduce cystic cell proliferation and fibrosis, preventing progressive renal scarring with preservation of renal function. PMID:21552769

Supportive therapies for prevention of hepatocellular carcinoma recurrence and preservation of liver function

PubMed Central

Takami, Taro; Yamasaki, Takahiro; Saeki, Issei; Matsumoto, Toshihiko; Suehiro, Yutaka; Sakaida, Isao

2016-01-01

Hepatocellular carcinoma (HCC) is one of the deadliest cancers in the world and is associated with a high risk of recurrence. The development of a wide range of new therapies is therefore essential. In this study, from the perspective of supportive therapy for the prevention of HCC recurrence and preservation of liver function in HCC patients, we surveyed a variety of different therapeutic agents. We show that branched chain amino acids (BCAA) supplementation and late evening snack with BCAA, strategies that address issues of protein-energy malnutrition, are important for liver cirrhotic patients with HCC. For chemoprevention of HCC recurrence, we show that viral control after radical treatment is important. We also reviewed the therapeutic potential of antiviral drugs, sorafenib, peretinoin, iron chelators. Sorafenib is a kinase inhibitor and a standard therapy in the treatment of advanced HCC. Peretinoin is a vitamin A-like molecule that targets the retinoid nuclear receptor to induce apoptosis and inhibit tumor growth in HCC cells. Iron chelators, such as deferoxamine and deferasirox, act to prevent cancer cell growth. These chelators may have potential as combination therapies in conjunction with peretinoin. Finally, we review the potential inhibitory effect of bone marrow cells on hepatocarcinogenesis. PMID:27621572

Intra-renal delivery of mesenchymal stem cells attenuates myocardial injury after reversal of hypertension in porcine renovascular disease.

PubMed

Eirin, Alfonso; Zhu, Xiang-Yang; Ferguson, Christopher M; Riester, Scott M; van Wijnen, Andre J; Lerman, Amir; Lerman, Lilach O

2015-01-19

Percutaneous transluminal renal angioplasty (PTRA) fails to fully improve cardiac injury and dysfunction in patients with renovascular hypertension (RVH). Mesenchymal stem cells (MSCs) restore renal function, but their potential for attenuating cardiac injury after reversal of RVH has not been explored. We hypothesized that replenishment of MSCs during PTRA would improve cardiac function and oxygenation, and decrease myocardial injury in porcine RVH. Pigs were studied after 16 weeks of RVH, RVH treated 4 weeks earlier with PTRA with or without adjunct intra-renal delivery of MSC (10^6 cells), and controls. Cardiac structure, function (fast-computed tomography (CT)), and myocardial oxygenation (Blood-Oxygen-Level-Dependent- magnetic resonance imaging) were assessed in-vivo. Myocardial microvascular density (micro-CT) and myocardial injury were evaluated ex-vivo. Kidney venous and systemic blood levels of inflammatory markers were measured and their renal release calculated. PTRA normalized blood pressure, yet stenotic-kidney glomerular filtration rate, similarly blunted in RVH and RVH + PTRA, normalized only in PTRA + MSC-treated pigs. PTRA attenuated left ventricular remodeling, whereas myocardial oxygenation, subendocardial microvascular density, and diastolic function remained decreased in RVH + PTRA, but normalized in RVH + PTRA-MSC. Circulating isoprostane levels and renal release of inflammatory cytokines increased in RVH and RVH + PTRA, but normalized in RVH + PTRA-MSC, as did myocardial oxidative stress, inflammation, collagen deposition, and fibrosis. Intra-renal MSC delivery during PTRA preserved stenotic-kidney function, reduced systemic oxidative stress and inflammation, and thereby improved cardiac function, oxygenation, and myocardial injury four weeks after revascularization, suggesting a therapeutic potential for adjunctive MSC delivery to preserve cardiac function and structure after reversal of experimental RVH.

Spaceflight of HUVEC: An Integrated eXperiment- SPHINX Onboard the ISS

NASA Astrophysics Data System (ADS)

Versari, S.; Maier, J. A. M.; Norfini, A.; Zolesi, V.; Bradamante, S.

2013-02-01

The spaceflight orthostatic challenge can promote in astronauts inadequate cardiovascular responses defined as cardiovascular deconditioning. In particular, disturbance of endothelial functions are known to lead to altered vascular performances, being the endothelial cells crucial in the maintenance of the functional integrity of the vascular wall. In order to evaluate whether weightlessness affects endothelial functions, we designed, developed, and performed the experiment SPHINX - SPaceflight of HUVEC: an INtegrated eXperiment - where HUVEC (Human Umbilical Vein Endothelial Cells) were selected as a macrovascular cell model system. SPHINX arrived at the International Space Station (ISS) onboard Progress 40P, and was processed inside Kubik 6 incubator for 7 days. At the end, all of the samples were suitably fixed and preserved at 6°C until return on Earth on Soyuz 23S.

Reconstruction of structure and function in tissue engineering of solid organs: Toward simulation of natural development based on decellularization.

PubMed

Zheng, Chen-Xi; Sui, Bing-Dong; Hu, Cheng-Hu; Qiu, Xin-Yu; Zhao, Pan; Jin, Yan

2018-04-27

Failure of solid organs, such as the heart, liver, and kidney, remains a major cause of the world's mortality due to critical shortage of donor organs. Tissue engineering, which uses elements including cells, scaffolds, and growth factors to fabricate functional organs in vitro, is a promising strategy to mitigate the scarcity of transplantable organs. Within recent years, different construction strategies that guide the combination of tissue engineering elements have been applied in solid organ tissue engineering and have achieved much progress. Most attractively, construction strategy based on whole-organ decellularization has become a popular and promising approach, because the overall structure of extracellular matrix can be well preserved. However, despite the preservation of whole structure, the current constructs derived from decellularization-based strategy still perform partial functions of solid organs, due to several challenges, including preservation of functional extracellular matrix structure, implementation of functional recellularization, formation of functional vascular network, and realization of long-term functional integration. This review overviews the status quo of solid organ tissue engineering, including both advances and challenges. We have also put forward a few techniques with potential to solve the challenges, mainly focusing on decellularization-based construction strategy. We propose that the primary concept for constructing tissue-engineered solid organs is fabricating functional organs based on intact structure via simulating the natural development and regeneration processes. Copyright © 2018 John Wiley & Sons, Ltd.

Toward Rare Blood Cell Preservation for RNA Sequencing.

PubMed

Vickovic, Sanja; Ahmadian, Afshin; Lewensohn, Rolf; Lundeberg, Joakim

2015-07-01

Cancer is driven by various events leading to cell differentiation and disease progression. Molecular tools are powerful approaches for describing how and why these events occur. With the growing field of next-generation DNA sequencing, there is an increasing need for high-quality nucleic acids derived from human cells and tissues-a prerequisite for successful cell profiling. Although advances in RNA preservation have been made, some of the largest biobanks still do not employ RNA blood preservation as standard because of limitations in low blood-input volume and RNA stability over the whole gene body. Therefore, we have developed a robust protocol for blood preservation and long-term storage while maintaining RNA integrity. Furthermore, we explored the possibility of using the protocol for preserving rare cell samples, such as circulating tumor cells. The results of our study confirmed that gene expression was not impacted by the preservation procedure (r(2) > 0.88) or by long-term storage (r(2) = 0.95), with RNA integrity number values averaging over 8. Similarly, cell surface antigens were still available for antibody selection (r(2) = 0.95). Lastly, data mining for fusion events showed that it was possible to detect rare tumor cells among a background of other cells present in blood irrespective of fixation. Thus, the developed protocol would be suitable for rare blood cell preservation followed by RNA sequencing analysis. Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Inner ear tissue preservation by rapid freezing: Improving fixation by high-pressure freezing and hybrid methods

PubMed Central

Bullen, A.; Taylor, R.R.; Kachar, B.; Moores, C.; Fleck, R.A.; Forge, A.

2014-01-01

In the preservation of tissues in as ‘close to life’ state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues. PMID:25016142

Transient protective effect of caspase inhibitors in RCS rat.

PubMed

Perche, O; Doly, M; Ranchon-Cole, I

2008-03-01

In most retinal degenerations in humans and in animal models, photoreceptor cells die by apoptosis. Although the biochemical features are similar in all apoptotic cells, different molecular events lead the cell to death. In the present study we used a rat model of inherited retinal degeneration, the RCS rats, to investigate the involvement of the proteases, caspases and/or calpains, in photoreceptor apoptosis. In the first experiments, rats were untreated or injected intravitreally at post natal day 27 (P27) with the large broad spectrum caspase inhibitor, ZVAD, the calpain inhibitor, MuhPhe, or with the vehicle, DMSO. Retinal status was evaluated at P35 and P42 by electroretinography, morphometry and apoptotic nuclei detection. DMSO and MuhPhe had no effect on RCS retinas as evidenced by equivalent loss of function and equivalent number of apoptotic cells than in untreated group. ZVAD transiently reduced apoptotic cells and preserved photoreceptor function at P35 but not at P42. These results suggest that caspases but not calpains are involved in retinal degeneration in the RCS. In the second experiments, RCS rats were injected twice at P27 and P35 with ZVAD or DMSO. Although ZVAD-treated retinas were preserved at P35 compared to the DMSO controls, the second injection of ZVAD did not extend the preserving effect to P42. Moreover, a single injection of ZVAD at P35 had no preserving effect at P42. All these data taken together suggest that caspases do not play a pivotal role after P35. In a fourth set of experiments, we used specific caspase inhibitors to elucidate which caspase was activated. The caspase-1/4 inhibitor (YVAD) or the caspase-3/7 inhibitor (DEVD) were injected intravitreally at P27 and retinal status was evaluated at P35 and P42. Electroretinograms and apoptotic nuclei detection demonstrated that YVAD and DEVD preserved photoreceptors at P35 but not at P42. These results suggest that both caspase-1/4 and caspase-3/7 play a major role in the apoptotic pathway between P27 and P35 in retinal degeneration of RCS rats. In this study, we show that 1/ the photoreceptor apoptotic process in the RCS rat involves caspases but not calpains, and 2/ the retinal degeneration seems to be composed of different phases involving different molecular players. Indeed, we have demonstrated that caspases are playing a major role at P35, but not at P42.

The Cryoprotectant Effect of Polysaccharides from Plants and Microalgae on Human White Blood Cells.

PubMed

Khudyakov, Andrey Nikolayevich; Polezhaeva, Tatyana Vitalyevna; Zaitseva, Oksana Olegovna; Gűnter, Elena Aleksandrovna; Solomina, Olga Nurzadinovna; Popeyko, Oksana Viktorovna; Shubakov, Anatolyi Aleksandrovich; Vetoshkin, Konstantin Aleksandrovich

2015-08-01

The use of carbohydrates as cryoprotectants is increasing. In this study the effects of incorporating polysaccharides extracted from plants and microalgae originating in northern Russia, into cryoprotectant solutions used to preserve human white blood cells were investigated. Cells in the presence of the polysaccharides were cooled to either -40°C or -80°C, using a two-step cooling process. The morphological and functional indicators of the cryopreserved leukocytes were assessed by light microscopy. When combined with glycerol, the pectin-polysaccharides Lemnan from common duckweed (Lemna minor L.) and Comaruman from marsh cinquefoil (Comarum palustre L), were capable of lowering the freezing point of the cryoprotectant solution and helped to preserve the integrity of the human white blood cell membranes at temperatures below zero. In addition, the increase in phagocytic activity of neutrophils was confirmed. In the context of the contemporary search for effective cell cryoprotectants, the results of this research demonstrate that the cryopreservation of biospecimens in a polysaccharide environment is a promising trend in applied medicine, which can be considered an alternative to traditional cryogenic nitrogen techniques.

Screening and Identification of Cryopreservative Agents for Human Cellular Biotechnology Experiments in Microgravity

NASA Technical Reports Server (NTRS)

Love,J.; Elliott, T.; Das, G. C.; Hammond, D. K.; Schwarzkopf, R. J.; Jones, L. B.; Baker, T. L.

2006-01-01

Dimethyl sulfoxide (DMSO) has been used as a standard cryopreservative agent for mammalian cell culture; however, prolonged exposure of thawed cells to DMSO can alter cell growth. While DMSO is easily eliminated in ground-based experiments, removal of DMSO in flight-based experiments is more difficult due to various on-orbit constraints. Failure of cryopreservation is due to a number of factors, including intracellular ice formation, solute effect, and apoptotic cell death following thawing. One objective of this study is to identify and characterize an alternative cryopreservative that could be used on the International Space Station (ISS). We systematically screened for potential permeating and non-permeating agents using a human colorectal carcinoma cell line, MIP-101. Cells were suspended in cryopreservation solution and frozen either following a two-step procedure involving initial cooling at -1 C/min overnight followed by storage in liquid nitrogen (LN2) vapor, or by freezing cells directly in the LN2 vapor phase at -10 C/min. Ability to preserve cellular function after one cycle of freeze-thawing was assessed by the recovery of viable cells in short and long-term cell culture experiments. Results showed that permeating preservatives glycerol (G) and ethylene glycol (EG) had an efficacy (80-110%) comparable to, if not better than, 7.5% DMSO; but, propylene glycol (PG) had a somewhat lesser efficacy. Among the non-permeating preservatives, trehalose, raffinose, and dextran exhibited significant protective effect (50-80%) relative to that offered by 7.5% DMSO, but at -10 C and not at -1 C/min cooling rate. Preliminary data thus suggest that a combination of permeating and non-permeating agents may have improved efficacy as a cryoprotectant and serve as an alternate to DMSO for experimentation on ISS.

Differences in mitochondrial function and morphology during cooling and rewarming between hibernator and non-hibernator derived kidney epithelial cells.

PubMed

Hendriks, Koen D W; Lupi, Eleonora; Hardenberg, Maarten C; Hoogstra-Berends, Femke; Deelman, Leo E; Henning, Robert H

2017-11-14

Hibernators show superior resistance to ischemia and hypothermia, also outside the hibernation season. Therefore, hibernation is a promising strategy to decrease cellular damage in a variety of fields, such as organ transplantation. Here, we explored the role of mitochondria herein, by comparing epithelial cell lines from a hibernator (hamster kidney cells, HaK) and a non-hibernator (human embryonic kidney cells, HEK293) during cold preservation at 4 °C and rewarming. Cell survival (Neutral Red), ATP and MDA levels, mitochondrial membrane potential (MMP), mitochondrial morphology (using fluorescent probes) and metabolism (seahorse XF) were assessed. Hypothermia induced dispersion of the tubular mitochondrial network, a loss of MMP, increased oxygen radical (MDA) and decreased ATP production in HEK293. In contrast, HaK maintained MMP and ATP production without an increase in oxygen radicals during cooling and rewarming, resulting in superior cell survival compared to HEK293. Further, normothermic HaK showed a dispersed mitochondrial network and higher respiratory and glycolysis capacity compared to HEK293. Disclosing the mechanisms that hibernators use to counteract cell death in hypothermic and ischemic circumstances may help to eventually improve organ preservation in a variety of fields, including organ transplantation.

Voluntary running exercise prevents β-cell failure in susceptible islets of the Zucker diabetic fatty rat.

PubMed

Delghingaro-Augusto, Viviane; Décary, Simon; Peyot, Marie-Line; Latour, Martin G; Lamontagne, Julien; Paradis-Isler, Nicolas; Lacharité-Lemieux, Marianne; Akakpo, Huguette; Birot, Olivier; Nolan, Christopher J; Prentki, Marc; Bergeron, Raynald

2012-01-15

Physical activity improves glycemic control in type 2 diabetes (T2D), but its contribution to preserving β-cell function is uncertain. We evaluated the role of physical activity on β-cell secretory function and glycerolipid/fatty acid (GL/FA) cycling in male Zucker diabetic fatty (ZDF) rats. Six-week-old ZDF rats engaged in voluntary running for 6 wk (ZDF-A). Inactive Zucker lean and ZDF (ZDF-I) rats served as controls. ZDF-I rats displayed progressive hyperglycemia with β-cell failure evidenced by falling insulinemia and reduced insulin secretion to oral glucose. Isolated ZDF-I rat islets showed reduced glucose-stimulated insulin secretion expressed per islet and per islet protein. They were also characterized by loss of the glucose regulation of fatty acid oxidation and GL/FA cycling, reduced mRNA expression of key β-cell genes, and severe reduction of insulin stores. Physical activity prevented diabetes in ZDF rats through sustaining β-cell compensation to insulin resistance shown in vivo and in vitro. Surprisingly, ZDF-A islets had persistent defects in fatty acid oxidation, GL/FA cycling, and β-cell gene expression. ZDF-A islets, however, had preserved islet insulin mRNA and insulin stores compared with ZDF-I rats. Physical activity did not prevent hyperphagia, dyslipidemia, or obesity in ZDF rats. In conclusion, islets of ZDF rats have a susceptibility to failure that is possibly due to altered β-cell fatty acid metabolism. Depletion of pancreatic islet insulin stores is a major contributor to islet failure in this T2D model, preventable by physical activity.

Complement inhibition prevents gut ischemia and endothelial cell dysfunction after hemorrhage/resuscitation.

PubMed

Fruchterman, T M; Spain, D A; Wilson, M A; Harris, P D; Garrison, R N

1998-10-01

Complement, a nonspecific immune response, is activated during hemorrhage/resuscitation (HEM/RES) and is involved in cellular damage. We hypothesized that activated complement injures endothelial cells (ETCs) and is responsible for intestinal microvascular hypoperfusion after HEM/RES. Four groups of rats were studied by in vivo videomicroscopy of the intestine: SHAM, HEM/RES, HEM/RES + sCR1 (complement inhibitor, 15 mg/kg intravenously given before resuscitation), and SHAM + sCR1. Hemorrhage was to 50% of mean arterial pressure for 60 minutes followed by resuscitation with shed blood plus an equal volume of saline. ETC function was assessed by response to acetylcholine. Resuscitation restored central hemodynamics to baseline after hemorrhage. After resuscitation, inflow A1 and premucosal A3 arterioles progressively constricted (-24% and -29% change from baseline, respectively), mucosal blood flow was reduced, and ETC function was impaired. Complement inhibition prevented postresuscitation vasoconstriction and gut ischemia. This protective effect appeared to involve preservation of ETC function in the A3 vessels (SHAM 76% of maximal dilation, HEM/RES 61%, HEM/RES + sCR1 74%, P < .05). Complement inhibition preserved ETC function after HEM/RES and maintained gut perfusion. Inhibition of complement activation before resuscitation may be a useful adjunct in patients experiencing major hemorrhage and might prevent the sequelae of gut ischemia.

Recommendations for the definition of clinical responder in insulin preservation studies.

PubMed

Beam, Craig A; Gitelman, Stephen E; Palmer, Jerry P

2014-09-01

Clinical responder studies should contribute to the translation of effective treatments and interventions to the clinic. Since ultimately this translation will involve regulatory approval, we recommend that clinical trials prespecify a responder definition that can be assessed against the requirements and suggestions of regulatory agencies. In this article, we propose a clinical responder definition to specifically assist researchers and regulatory agencies in interpreting the clinical importance of statistically significant findings for studies of interventions intended to preserve β-cell function in newly diagnosed type 1 diabetes. We focus on studies of 6-month β-cell preservation in type 1 diabetes as measured by 2-h-stimulated C-peptide. We introduce criteria (bias, reliability, and external validity) for the assessment of responder definitions to ensure they meet U.S. Food and Drug Administration and European Medicines Agency guidelines. Using data from several published TrialNet studies, we evaluate our definition (no decrease in C-peptide) against published alternatives and determine that our definition has minimum bias with external validity. We observe that reliability could be improved by using changes in C-peptide later than 6 months beyond baseline. In sum, to support efficacy claims of β-cell preservation therapies in type 1 diabetes submitted to U.S. and European regulatory agencies, we recommend use of our definition. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Plasmonic nanobubbles for target cell-specific gene and drug delivery and multifunctional processing of heterogeneous cell systems

NASA Astrophysics Data System (ADS)

Lukianova-Hleb, Ekaterina Y.; Huye, Leslie E.; Brenner, Malcolm K.; Lapotko, Dmitri O.

2014-03-01

Cell and gene cancer therapies require ex vivo cell processing of human grafts. Such processing requires at least three steps - cell enrichment, cell separation (destruction), and gene transfer - each of which requires the use of a separate technology. While these technologies may be satisfactory for research use, they are of limited usefulness in the clinical treatment setting because they have a low processing rate, as well as a low transfection and separation efficacy and specificity in heterogeneous human grafts. Most problematic, because current technologies are administered in multiple steps - rather than in a single, multifunctional, and simultaneous procedure - they lengthen treatment process and introduce an unnecessary level of complexity, labor, and resources into clinical treatment; all these limitations result in high losses of valuable cells. We report a universal, high-throughput, and multifunctional technology that simultaneously (1) inject free external cargo in target cells, (2) destroys unwanted cells, and (3) preserve valuable non-target cells in heterogeneous grafts. Each of these functions has single target cell specificity in heterogeneous cell system, processing rate > 45 mln cell/min, injection efficacy 90% under 96% viability of the injected cells, target cell destruction efficacy > 99%, viability of not-target cells >99% The developed technology employs novel cellular agents, called plasmonic nanobubbles (PNBs). PNBs are not particles, but transient, intracellular events, a vapor nanobubbles that expand and collapse in mere nanoseconds under optical excitation of gold nanoparticles with short picosecond laser pulses. PNBs of different, cell-specific, size (1) inject free external cargo with small PNBs, (2) Destroy other target cells mechanically with large PNBs and (3) Preserve non-target cells. The multi-functionality, precision, and high throughput of all-in-one PNB technology will tremendously impact cell and gene therapies and other clinical applications that depend on ex vivo processing of heterogeneous cell systems.

Ischemic preconditioning protects against gap junctional uncoupling in cardiac myofibroblasts.

PubMed

Sundset, Rune; Cooper, Marie; Mikalsen, Svein-Ole; Ytrehus, Kirsti

2004-01-01

Ischemic preconditioning increases the heart's tolerance to a subsequent longer ischemic period. The purpose of this study was to investigate the role of gap junction communication in simulated preconditioning in cultured neonatal rat cardiac myofibroblasts. Gap junctional intercellular communication was assessed by Lucifer yellow dye transfer. Preconditioning preserved intercellular coupling after prolonged ischemia. An initial reduction in coupling in response to the preconditioning stimulus was also observed. This may protect neighboring cells from damaging substances produced during subsequent regional ischemia in vivo, and may preserve gap junctional communication required for enhanced functional recovery during subsequent reperfusion.

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6β-deficient Dogs

PubMed Central

Pichard, Virginie; Provost, Nathalie; Mendes-Madeira, Alexandra; Libeau, Lyse; Hulin, Philippe; Tshilenge, Kizito-Tshitoko; Biget, Marine; Ameline, Baptiste; Deschamps, Jack-Yves; Weber, Michel; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

2016-01-01

We previously reported that subretinal injection of AAV2/5 RK.cpde6β allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials. PMID:26857842

AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6β-deficient Dogs.

PubMed

Pichard, Virginie; Provost, Nathalie; Mendes-Madeira, Alexandra; Libeau, Lyse; Hulin, Philippe; Tshilenge, Kizito-Tshitoko; Biget, Marine; Ameline, Baptiste; Deschamps, Jack-Yves; Weber, Michel; Le Meur, Guylène; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

2016-05-01

We previously reported that subretinal injection of AAV2/5 RK.cpde6β allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6β deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Identification of intestinal ion transport defects in microvillus inclusion disease.

PubMed

Kravtsov, Dmitri V; Ahsan, Md Kaimul; Kumari, Vandana; van Ijzendoorn, Sven C D; Reyes-Mugica, Miguel; Kumar, Anoop; Gujral, Tarunmeet; Dudeja, Pradeep K; Ameen, Nadia A

2016-07-01

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl(-)) and sodium (Na(+)) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na(+)), CFTR (Cl(-)), and SLC26A3 (DRA) (Cl(-)/HCO3 (-)) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl(-) and Na(+) stool loss in MVID diarrhea.

Identification of intestinal ion transport defects in microvillus inclusion disease

PubMed Central

Kravtsov, Dmitri V.; Ahsan, Md Kaimul; Kumari, Vandana; van Ijzendoorn, Sven C. D.; Reyes-Mugica, Miguel; Kumar, Anoop; Gujral, Tarunmeet; Dudeja, Pradeep K.

2016-01-01

Loss of function mutations in the actin motor myosin Vb (Myo5b) lead to microvillus inclusion disease (MVID) and death in newborns and children. MVID results in secretory diarrhea, brush border (BB) defects, villus atrophy, and microvillus inclusions (MVIs) in enterocytes. How loss of Myo5b results in increased stool loss of chloride (Cl−) and sodium (Na+) is unknown. The present study used Myo5b loss-of-function human MVID intestine, polarized intestinal cell models of secretory crypt (T84) and villus resembling (CaCo2BBe, C2BBe) enterocytes lacking Myo5b in conjunction with immunofluorescence confocal stimulated emission depletion (gSTED) imaging, immunohistochemical staining, transmission electron microscopy, shRNA silencing, immunoblots, and electrophysiological approaches to examine the distribution, expression, and function of the major BB ion transporters NHE3 (Na+), CFTR (Cl−), and SLC26A3 (DRA) (Cl−/HCO3−) that control intestinal fluid transport. We hypothesized that enterocyte maturation defects lead villus atrophy with immature secretory cryptlike enterocytes in the MVID epithelium. We investigated the role of Myo5b in enterocyte maturation. NHE3 and DRA localization and function were markedly reduced on the BB membrane of human MVID enterocytes and Myo5bKD C2BBe cells, while CFTR localization was preserved. Forskolin-stimulated CFTR ion transport in Myo5bKD T84 cells resembled that of control. Loss of Myo5b led to YAP1 nuclear retention, retarded enterocyte maturation, and a cryptlike phenotype. We conclude that preservation of functional CFTR in immature enterocytes, reduced functional expression of NHE3, and DRA contribute to Cl− and Na+ stool loss in MVID diarrhea. PMID:27229121

Reconstructing Carotenoid-Based and Structural Coloration in Fossil Skin.

PubMed

McNamara, Maria E; Orr, Patrick J; Kearns, Stuart L; Alcalá, Luis; Anadón, Pere; Peñalver, Enrique

2016-04-25

Evidence of original coloration in fossils provides insights into the visual communication strategies used by ancient animals and the functional evolution of coloration over time [1-7]. Hitherto, all reconstructions of the colors of reptile integument and the plumage of fossil birds and feathered dinosaurs have been of melanin-based coloration [1-6]. Extant animals also use other mechanisms for producing color [8], but these have not been identified in fossils. Here we report the first examples of carotenoid-based coloration in the fossil record, and of structural coloration in fossil integument. The fossil skin, from a 10 million-year-old colubrid snake from the Late Miocene Libros Lagerstätte (Teruel, Spain) [9, 10], preserves dermal pigment cells (chromatophores)-xanthophores, iridophores, and melanophores-in calcium phosphate. Comparison with chromatophore abundance and position in extant reptiles [11-15] indicates that the fossil snake was pale-colored in ventral regions; dorsal and lateral regions were green with brown-black and yellow-green transverse blotches. Such coloration most likely functioned in substrate matching and intraspecific signaling. Skin replicated in authigenic minerals is not uncommon in exceptionally preserved fossils [16, 17], and dermal pigment cells generate coloration in numerous reptile, amphibian, and fish taxa today [18]. Our discovery thus represents a new means by which to reconstruct the original coloration of exceptionally preserved fossil vertebrates. Copyright © 2016 Elsevier Ltd. All rights reserved.

Dosimetric feasibility of 4DCT-ventilation imaging guided proton therapy for locally advanced non-small-cell lung cancer.

PubMed

Huang, Qijie; Jabbour, Salma K; Xiao, Zhiyan; Yue, Ning; Wang, Xiao; Cao, Hongbin; Kuang, Yu; Zhang, Yin; Nie, Ke

2018-04-25

The principle aim of this study is to incorporate 4DCT ventilation imaging into functional treatment planning that preserves high-functioning lung with both double scattering and scanning beam techniques in proton therapy. Eight patients with locally advanced non-small-cell lung cancer were included in this study. Deformable image registration was performed for each patient on their planning 4DCTs and the resultant displacement vector field with Jacobian analysis was used to identify the high-, medium- and low-functional lung regions. Five plans were designed for each patient: a regular photon IMRT vs. anatomic proton plans without consideration of functional ventilation information using double scattering proton therapy (DSPT) and intensity modulated proton therapy (IMPT) vs. functional proton plans with avoidance of high-functional lung using both DSPT and IMPT. Dosimetric parameters were compared in terms of tumor coverage, plan heterogeneity, and avoidance of normal tissues. Our results showed that both DSPT and IMPT plans gave superior dose advantage to photon IMRTs in sparing low dose regions of the total lung in terms of V5 (volume receiving 5Gy). The functional DSPT only showed marginal benefit in sparing high-functioning lung in terms of V5 or V20 (volume receiving 20Gy) compared to anatomical plans. Yet, the functional planning in IMPT delivery, can further reduce the low dose in high-functioning lung without degrading the PTV dosimetric coverages, compared to anatomical proton planning. Although the doses to some critical organs might increase during functional planning, the necessary constraints were all met. Incorporating 4DCT ventilation imaging into functional proton therapy is feasible. The functional proton plans, in intensity modulated proton delivery, are effective to further preserve high-functioning lung regions without degrading the PTV coverage.

Preserving HIV-specific T cell responses: does timing of antiretroviral therapy help?

PubMed

Macatangay, Bernard J C; Rinaldo, Charles R

2015-01-01

HIV-specific T cell responses are likely to have an important role in HIV cure strategies that aim for long-lasting viral control without antiretroviral therapy (ART). An important issue in enhancing virus-specific T cell responses is whether timing of ART can influence their magnitude and breadth. Early ART is associated with lower T cell activation, preservation of T cell numbers, smaller DNA and RNA reservoir size, and, in a single study (VISCONTI), control of plasma viremia after treatment interruption. The prevention of T cell destruction by early ART is associated with relatively low anti-HIV CD8⁺ T cell responses but stronger CD4⁺ T helper function. The relatively lower CD8⁺T cell response, which is presumably due to rapid lowering of HIV antigen burden after early ART, appears sufficient to control residual viral replication as well as viral rebound upon treatment interruption. Available evidence of starting ART during acute or early HIV infection has shown benefit in both virologic and immunologic parameters despite the lower HIV-specific CD8⁺ T cell responses observed. Encouraging as this is, more extensive data are necessary to evaluate its role in combination with immunotherapeutic and latency activation strategies that are being assessed in various HIV cure-related studies.

Aggravation of cold-induced injury in Vero-B4 cells by RPMI 1640 medium - identification of the responsible medium components.

PubMed

Pless-Petig, Gesine; Metzenmacher, Martin; Türk, Tobias R; Rauen, Ursula

2012-10-10

In modern biotechnology, there is a need for pausing cell lines by cold storage to adapt large-scale cell cultures to the variable demand for their products. We compared various cell culture media/solutions for cold storage of Vero-B4 kidney cells, a cell line widely used in biotechnology. Cold storage in RPMI 1640 medium, a recommended cell culture medium for Vero-B4 cells, surprisingly, strongly enhanced cold-induced cell injury in these cells in comparison to cold storage in Krebs-Henseleit buffer or other cell culture media (DMEM, L-15 and M199). Manufacturer, batch, medium supplements and the most likely components with concentrations outside the range of the other media/solutions (vitamin B12, inositol, biotin, p-aminobenzoic acid) did not cause this aggravation of cold-induced injury in RPMI 1640. However, a modified Krebs-Henseleit buffer with a low calcium concentration (0.42 mM), a high concentration of inorganic phosphate (5.6 mM), and glucose (11.1 mM; i.e. concentrations as in RPMI 1640) evoked a cell injury and loss of metabolic function corresponding to that observed in RPMI 1640. Deferoxamine improved cell survival and preserved metabolic function in modified Krebs-Henseleit buffer as well as in RPMI 1640. Similar Ca2+ and phosphate concentrations did not increase cold-induced cell injury in the kidney cell line LLC-PK1, porcine aortic endothelial cells or rat hepatocytes. However, more extreme conditions (Ca2+ was nominally absent and phosphate concentration raised to 25 mM as in the organ preservation solution University of Wisconsin solution) also increased cold-induced injury in rat hepatocytes and porcine aortic endothelial cells. These data suggest that the combination of low calcium and high phosphate concentrations in the presence of glucose enhances cold-induced, iron-dependent injury drastically in Vero-B4 cells, and that a tendency for this pathomechanism also exists in other cell types.

Geobiological Comparisons of Preservation Potential within Hypersaline Mineral-Microbe Systems

NASA Astrophysics Data System (ADS)

Perl, S. M.; Celestian, A. J.; Vaishampayan, P.; Seuylemezian, A.; Mahseredjian, T.; Baxter, B.; Corsetti, F. A.

2017-12-01

The purpose of these investigations is to show comparative measurements between known biological sources of biomarkers and biosignatures and how they can be independently verified, within instrumentation limits, by laboratory investigations analogous to future surface missions to Mars and Europa. Precipitated hypersaline mineralogy can provide a biotic record of microbial activity and habitation within evaporating lake systems. The extent of microbial preservation is a direct relationship between the magnitudes of aqueous activity post-precipitation, original or in-situ biological habitats, dissolution events due to chemical weathering, and organic matter degradation due to UV exposure and desiccation. Chemical biomarkers and physical biosignatures to be quantified and correlated based from preserved DNA as the most sensitive biomarker to more recalcitrant biomarkers such as lipids and Total Organic Carbon (TOC). Moreover the timing of cell movement during nutrient cycling within specific evaporite minerals can be associated to the formation of physical biosignatures as a function of already active and abundant biomarkers allowing for relative timelines of biogenic actions (e.g., nutrient cycling, cell division) to be correlated together. Our investigation has compared hypersaline biotic activity within different photosynthetic and chemosynthetic settings to quantify preservation and detection profiles given measured DNA as the source validation standard and micron-scale Raman measurements for specific paleoenvironmental mineral sampling.

Human Langerhans Cells with Pro-inflammatory Features Relocate within Psoriasis Lesions

PubMed Central

Eidsmo, Liv; Martini, Elisa

2018-01-01

Psoriasis is a common skin disease that presents with well-demarcated patches of inflammation. Recurrent disease in fixed areas of the skin indicates a localized disease memory that is preserved in resolved lesions. In line with such concept, the involvement of tissue-resident immune cells in psoriasis pathology is increasingly appreciated. Langerhans cells (LCs) are perfectly placed to steer resident T cells and local tissue responses in psoriasis. Here, we present an overview of the current knowledge of LCs in human psoriasis, including findings that highlight pro-inflammatory features of LCs in psoriasis lesions. We also review the literature on conflicting data regarding LC localization and functionality in psoriasis. Our review highlights that further studies are needed to elucidate the molecular mechanisms that drive LCs functionality in inflammatory diseases. PMID:29520279

Proinsulin slows retinal degeneration and vision loss in the P23H rat model of retinitis pigmentosa.

PubMed

Fernández-Sánchez, Laura; Lax, Pedro; Isiegas, Carolina; Ayuso, Eduard; Ruiz, José M; de la Villa, Pedro; Bosch, Fatima; de la Rosa, Enrique J; Cuenca, Nicolás

2012-12-01

Proinsulin has been characterized as a neuroprotective molecule. In this work we assess the therapeutic potential of proinsulin on photoreceptor degeneration, synaptic connectivity, and functional activity of the retina in the transgenic P23H rat, an animal model of autosomal dominant retinitis pigmentosa (RP). P23H homozygous rats received an intramuscular injection of an adeno-associated viral vector serotype 1 (AAV1) expressing human proinsulin (hPi+) or AAV1-null vector (hPi-) at P20. Levels of hPi in serum were determined by enzyme-linked immunosorbent assay (ELISA), and visual function was evaluated by electroretinographic (ERG) recording at P30, P60, P90, and P120. Preservation of retinal structure was assessed by immunohistochemistry at P120. Human proinsulin was detected in serum from rats injected with hPi+ at all times tested, with average hPi levels ranging from 1.1 nM (P30) to 1.4 nM (P120). ERG recordings showed an amelioration of vision loss in hPi+ animals. The scotopic b-waves were significantly higher in hPi+ animals than in control rats at P90 and P120. This attenuation of visual deterioration correlated with a delay in photoreceptor degeneration and the preservation of retinal cytoarchitecture. hPi+ animals had 48.7% more photoreceptors than control animals. Presynaptic and postsynaptic elements, as well as the synaptic contacts between photoreceptors and bipolar or horizontal cells, were preserved in hPi+ P23H rats. Furthermore, in hPi+ rat retinas the number of rod bipolar cell bodies was greater than in control rats. Our data demonstrate that hPi expression preserves cone and rod structure and function, together with their contacts with postsynaptic neurons, in the P23H rat. These data strongly support the further development of proinsulin-based therapy to counteract retinitis pigmentosa.

Mesenchymal stem cells support hepatocyte function in engineered liver grafts.

PubMed

Kadota, Yoshie; Yagi, Hiroshi; Inomata, Kenta; Matsubara, Kentaro; Hibi, Taizo; Abe, Yuta; Kitago, Minoru; Shinoda, Masahiro; Obara, Hideaki; Itano, Osamu; Kitagawa, Yuko

2014-01-01

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.

Impact of Cardiac Progenitor Cells on Heart Failure and Survival in Single Ventricle Congenital Heart Disease.

PubMed

Sano, Toshikazu; Ousaka, Daiki; Goto, Takuya; Ishigami, Shuta; Hirai, Kenta; Kasahara, Shingo; Ohtsuki, Shinichi; Sano, Shunji; Oh, Hidemasa

2018-03-30

Intracoronary administration of cardiosphere-derived cells (CDCs) in patients with single ventricles resulted in a short-term improvement in cardiac function. To test the hypothesis that CDC infusion is associated with improved cardiac function and reduced mortality in patients with heart failure. We evaluated the effectiveness of CDCs using an integrated cohort study in 101 patients with single ventricles, including 41 patients who received CDC infusion and 60 controls treated with staged palliation alone. Heart failure with preserved ejection fraction (EF) or reduced EF was stratified by the cardiac function after surgical reconstruction. The main outcome measure was to evaluate the magnitude of improvement in cardiac function and all-cause mortality at 2 years. Animal studies were conducted to clarify the underlying mechanisms of heart failure with preserved EF and heart failure with reduced EF phenotypes. At 2 years, CDC infusion increased ventricular function (stage 2: +8.4±10.0% versus +1.6±6.4%, P =0.03; stage 3: +7.9±7.5% versus -1.1±5.5%, P <0.001) compared with controls. In all available follow-up data, survival did not differ between the 2 groups (log-rank P =0.225), whereas overall patients treated by CDCs had lower incidences of late failure ( P =0.022), adverse events ( P =0.013), and catheter intervention ( P =0.005) compared with controls. CDC infusion was associated with a lower risk of adverse events (hazard ratio, 0.411; 95% CI, 0.179-0.942; P =0.036). Notably, CDC infusion reduced mortality ( P =0.038) and late complications ( P <0.05) in patients with heart failure with reduced EF but not with heart failure with preserved EF. CDC-treated rats significantly reversed myocardial fibrosis with differential collagen deposition and inflammatory responses between the heart failure phenotypes. CDC administration in patients with single ventricles showed favorable effects on ventricular function and was associated with reduced late complications except for all-cause mortality after staged procedures. Patients with heart failure with reduced EF but not heart failure with preserved EF treated by CDCs resulted in significant improvement in clinical outcome. URL: http://www.clinicaltrials.gov. Unique identifiers: NCT01273857 and NCT01829750. © 2018 American Heart Association, Inc.

Premises for fowl sperm preservation based on applied bioenergetics.

PubMed

Froman, D P

2014-02-01

The primary goal of this work was to test whether the sperm mobility assay could be used to derive mathematical relationships from which predictions could be made about sperm cell function. A precondition was random sampling from a pool of sperm. This precondition was met by centrifuging mobile sperm through 12% (wt/vol) Accudenz containing the Ca(2+) chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and then holding washed sperm at 20°C within buffered potassium chloride. These 2 conditions rendered washed sperm immobile at 20°C. Resumption of sperm mobility was independent of time (P > 0.8558) when sperm were reactivated at body temperature with 2 mM Ca(2+) in isotonic sodium chloride at pH 7.4. Reactivated sperm mobility was 93% of the prewash control. Subsequent experiments served to define a dose response, predict optimal conditions for in vitro sperm mobility, and show how sperm can recover from an imposed non-physiological condition. Thus, functions were derived from which predictions were made. Whereas the utility of BAPTA treatment was confirmed in a new context, such utility did not address the question of whole-cell Ca(2+) flux during sperm cell manipulation. This issue is pivotal for the application of bioenergetics to fowl sperm preservation. Therefore, the secondary goal of this research was to investigate sperm cell Ca(2+) flux using a simulation of conditions encountered by sperm during centrifugation through 12% (wt/vol) Accudenz. These conditions included a temperature of 30°C, a Ca(2+) sink, and no exogenous substrate. Sperm motion was measured with a Hobson SpermTracker. Data points conformed to parabolic functions when motile concentration and velocity were plotted as functions of time. In each case, maximums were observed, e.g., 26 min for motile concentration. The upswing was attributed to a redistribution of intracellular Ca(2+) whereas the downswing was attributed to sperm cell Ca(2+) depletion. A pronounced isothermal increase was observed for each variable when the Ca(2+) sink was overcome with exogenous Ca(2+). Experimental outcomes supported four testable premises applicable to fowl sperm preservation research: 1) the importance of sperm mobility phenotype, 2) the relationship between mitochondrial Ca(2+) cycling and sperm mobility, 3) the utility of the sperm mobility assay for predicting experimental outcomes, and 4) understanding mitochondrial Ca(2+) cycling in terms of whole-cell Ca(2+) flux.

The hair follicle bulge: a niche for adult stem cells.

PubMed

Pasolli, Hilda Amalia

2011-08-01

Adult stem cells (SCs) are essential for tissue homeostasis and wound repair. They have the ability to both self-renew and differentiate into multiple cell types. They often reside in specialized microenvironments or niches that preserve their proliferative and tissue regenerative capacity. The murine hair follicle (HF) has a specialized and permanent compartment--the bulge, which safely lodges SCs and provides the necessary molecular cues to regulate their function. The HF undergoes cyclic periods of destruction, regeneration, and rest, making it an excellent system to study SC biology.

Sciatic (Popliteal Fossa) Catheter for Pediatric Pain Management of Sickle Cell Crisis: A Case Report.

PubMed

Weber, Garret; Liao, Sherry; Burns, Micah Alexander

2017-11-15

Sickle cell crisis, or vaso-occlusive crisis (VOC), is a major cause of hospitalizations for adults and children with sickle cell disease, and is associated with increased morbidity and mortality. Despite prompt pharmacological treatment and multimodal pain management, acute pain during a VOC is often not adequately controlled in the pediatric population. We placed a continuous popliteal sciatic nerve block under ultrasound guidance in a pediatric patient for localized refractory pain during a VOC, resulting in improved pain control with preserved sensorimotor function.

A droplet-merging platform for comparative functional analysis of m1 and m2 macrophages in response to e. coli-induced stimuli.

PubMed

Hondroulis, Evangelia; Movila, Alexandru; Sabhachandani, Pooja; Sarkar, Saheli; Cohen, Noa; Kawai, Toshihisa; Konry, Tania

2017-03-01

Microfluidic droplets are used to isolate cell pairs and prevent crosstalk with neighboring cells, while permitting free motility and interaction within the confined space. Dynamic analysis of cellular heterogeneity in droplets has provided insights in various biological processes. Droplet manipulation methods such as fusion and fission make it possible to precisely regulate the localized environment of a cell in a droplet and deliver reagents as required. Droplet fusion strategies achieved by passive mechanisms preserve cell viability and are easier to fabricate and operate. Here, we present a simple and effective method for the co-encapsulation of polarized M1 and M2 macrophages with Escherichia coli (E. coli) by passive merging in an integrated droplet generation, merging, and docking platform. This approach facilitated live cell profiling of effector immune functions in situ and quantitative functional analysis of macrophage heterogeneity. Biotechnol. Bioeng. 2017;114: 705-709. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Biomaterials and Culture Technologies for Regenerative Therapy of Liver Tissue.

PubMed

Perez, Roman A; Jung, Cho-Rok; Kim, Hae-Won

2017-01-01

Regenerative approach has emerged to substitute the current extracorporeal technologies for the treatment of diseased and damaged liver tissue. This is based on the use of biomaterials that modulate the responses of hepatic cells through the unique matrix properties tuned to recapitulate regenerative functions. Cells in liver preserve their phenotype or differentiate through the interactions with extracellular matrix molecules. Therefore, the intrinsic properties of the engineered biomaterials, such as stiffness and surface topography, need to be tailored to induce appropriate cellular functions. The matrix physical stimuli can be combined with biochemical cues, such as immobilized functional groups or the delivered actions of signaling molecules. Furthermore, the external modulation of cells, through cocultures with nonparenchymal cells (e.g., endothelial cells) that can signal bioactive molecules, is another promising avenue to regenerate liver tissue. This review disseminates the recent approaches of regenerating liver tissue, with a focus on the development of biomaterials and the related culture technologies. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Direct Actions of Kisspeptins on GnRH Neurons Permit Attainment of Fertility but are Insufficient to Fully Preserve Gonadotropic Axis Activity

PubMed Central

León, Silvia; Barroso, Alexia; Vázquez, María J.; García-Galiano, David; Manfredi-Lozano, María; Ruiz-Pino, Francisco; Heras, Violeta; Romero-Ruiz, Antonio; Roa, Juan; Schutz, Günther; Kirilov, Milen; Gaytan, Francisco; Pinilla, Leonor; Tena-Sempere, Manuel

2016-01-01

Kisspeptins, ligands of the receptor, Gpr54, are potent stimulators of puberty and fertility. Yet, whether direct kisspeptin actions on GnRH neurons are sufficient for the whole repertoire of their reproductive effects remains debatable. To dissect out direct vs. indirect effects of kisspeptins on GnRH neurons in vivo, we report herein the detailed reproductive/gonadotropic characterization of a Gpr54 null mouse line with selective re-introduction of Gpr54 expression only in GnRH cells (Gpr54−/−Tg; rescued). Despite preserved fertility, adult rescued mice displayed abnormalities in gonadal microstructure, with signs of precocious ageing in females and elevated LH levels with normal-to-low testosterone secretion in males. Gpr54−/−Tg rescued mice showed also altered gonadotropin responses to negative feedback withdrawal, while luteinizing hormone responses to various gonadotropic regulators were variably affected, with partially blunted relative (but not absolute) responses to kisspeptin-10, NMDA and the agonist of tachykinin receptors, NK2R. Our data confirm that direct effects of kisspeptins on GnRH cells are sufficient to attain fertility. Yet, such direct actions appear to be insufficient to completely preserve proper functionality of gonadotropic axis, suggesting a role of kisspeptin signaling outside GnRH cells. PMID:26755241

Central control of glucose homeostasis: the brain--endocrine pancreas axis.

PubMed

Thorens, B

2010-10-01

A large body of data gathered over the last decades has delineated the neuronal pathways that link the central nervous system with the autonomic innervation of the endocrine pancreas, which controls alpha- and beta-cell secretion activity and mass. These are important regulatory functions that are certainly keys for preserving the capacity of the endocrine pancreas to control glucose homeostasis over a lifetime. Identifying the cells involved in controlling the autonomic innervation of the endocrine pancreas, in response to nutrient, hormonal and environmental cues and how these cues are detected to activate neuronal activity are important goals of current research. Elucidation of these questions may possibly lead to new means for preserving or restoring defects in insulin and glucagon secretion associated with type 2 diabetes. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae.

PubMed

Jacq, Maxime; Arthaud, Christopher; Manuse, Sylvie; Mercy, Chryslène; Bellard, Laure; Peters, Katharina; Gallet, Benoit; Galindo, Jennifer; Doan, Thierry; Vollmer, Waldemar; Brun, Yves V; VanNieuwenhze, Michael S; Di Guilmi, Anne Marie; Vernet, Thierry; Grangeasse, Christophe; Morlot, Cecile

2018-05-15

Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.

Adenosine triphosphate postconditioning is associated with better preserved global and regional cardiac function during myocardial ischemia and reperfusion: a speckle tracking imaging-based echocardiologic study.

PubMed

Ren, Min; Liu, Yujie; Zhao, Huiya; Dong, Shixia; Jiang, Zhonghui; Li, Keting; Tian, Jiawei

2016-10-01

Effects of ischemic postconditioning (IPostC) and adenosine triphosphate (ATP)-mediated pharmacologic postconditioning (ATP-PPostC) on cardiac function were evaluated by speckle tracking imaging (STI)-based echocardiography. A myocardial I/R model was induced in rabbits by reversible ligation of the left ventricular branch of coronary artery. Rabbits were randomized into three groups: ischemia and reperfusion (IR) (no further intervention), IPostC, and ATP-PPostC groups. Cardiac function was evaluated by conventional and STI-based echocardiography. Myocardial necrosis, apoptosis, and myocardial mRNAs of apoptosis-related proteins (Bcl-2 and Bax) were evaluated. Speckle tracking imaging (STI)-based echocardiography revealed that IPostC and ATP-PPostC were associated with better preserved global and regional cardiac function, as indicated by significantly increased GLSrsys, GLSrd, GLSsys, SrLsys, SrLd, and SLsys in both groups (all P<.5). Subsequent pathologic studies indicate that the percentage of necrotic myocardium and permillage of apoptotic cells were significantly lower in the IPostC and ATP-PPostC groups than in the IR group (all P<.05). Moreover, both IPostC and ATP-PPostC were associated with increased Bcl-2 mRNA levels and reduced Bax mRNA levels. IPostC and ATP-PPostC may exert cardioprotective functions by better preservation of cardiac function during the I/R process and at least partly via attenuation of myocardial apoptosis. © 2016 John Wiley & Sons Ltd.

Glycine ameliorates lung reperfusion injury after cold preservation in an ex vivo rat lung model.

PubMed

Omasa, Mitsugu; Fukuse, Tatsuo; Toyokuni, Shinya; Mizutani, Yoichi; Yoshida, Hiroshi; Ikeyama, Kazuyuki; Hasegawa, Seiki; Wada, Hiromi

2003-03-15

The role of glycine has not been investigated in lung ischemia-reperfusion injury after cold preservation. Furthermore, the role of apoptosis after reperfusion following cold preservation has not been fully understood. Lewis rats were divided into three groups (n=6 each). In the GLY(-) and GLY(+) groups, isolated lungs were preserved for 15 hr at 4 degrees C after a pulmonary artery (PA) flush using our previously developed preservation solution (ET-K; extracellular-type trehalose containing Kyoto), with or without the addition of glycine (5 mM). In the Fresh group, isolated lungs were reperfused immediately after a PA flush with ET-K. They were reperfused for 60 min with an ex vivo perfusion model. Pulmonary function, oxidative stress, apoptosis, and tumor necrosis factor (TNF)-alpha expression were assessed after reperfusion. Shunt fraction and peak inspiratory pressure after reperfusion in the GLY(-) group were significantly higher than those in the GLY(+) and Fresh groups. Oxidative damage and apoptosis in the alveolar epithelial cells of the GLY(-) group, assessed by immunohistochemical staining and quantification of 8-hydroxy-2'-deoxyguanosine and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling method, were significantly higher than those of the GLY(+) and Fresh groups. There were correlations among shunt fraction, oxidative damage, and apoptosis. There was no expression of TNF-alpha messenger RNA in all groups evaluated by the reverse transcription-polymerase chain reaction. Glycine attenuates ischemia/reperfusion injury after cold preservation by reducing oxidative damage and suppressing apoptosis independent of TNF-alpha in this model. The suppression of apoptosis might ameliorate lung function after reperfusion.

Constituents of Bile, Bilirubin and TUDCA, Protect Against Oxidative Stress-Induced Retinal Degeneration

PubMed Central

Oveson, Brian C.; Iwase, Takeshi; Hackett, Sean F.; Lee, Sun Young; Usui, Shinichi; Sedlak, Thomas W.; Snyder, Solomon H.; Campochiaro, Peter A.; Sung, Jennifer U.

2014-01-01

Two constituents of bile, bilirubin and tauroursodeoxycholic acid (TUDCA), have antioxidant activity. However, bilirubin can also cause damage to some neurons and glial cells, particularly immature neurons. In this study, we tested the effects of bilirubin and TUDCA in two models in which oxidative stress contributes to photoreceptor cell death, prolonged light exposure and rd10+/+ mice. In albino BALB/c mice, intraperitoneal (IP) injection of 5 mg/kg of bilirubin or 500 mg/kg of TUDCA prior to exposure to 5,000 lux of white light for 8 hours significantly reduced loss of rod and cone function assessed by electroretinograms (ERGs). Both treatments also reduced light-induced accumulation of superoxide radicals in the outer retina, rod cell death assessed by outer nuclear layer (ONL) thickness, and disruption of cone inner and outer segments. In rd10+/+ mice, IP injections of 5 or 50 mg/kg of bilirubin or 500 mg/kg of TUDCA every 3 days starting at postnatal day (P) 6, caused significant preservation of cone cell number and cone function at P50. Rods were not protected at P50, but both bilirubin and TUDCA provided modest preservation of ONL thickness and rod function at P30. These data suggest that correlation of serum bilirubin levels with rate of vision loss in patients with retinitis pigmentosa (RP) could provide a useful strategy to test the hypothesis that cones die from oxidative damage in patients with RP. If proof-of-concept is established, manipulation of bilirubin levels and administration of TUDCA could be tested in interventional trials. PMID:21054389

Epithelial cell kinase-B61: an autocrine loop modulating intestinal epithelial migration and barrier function.

PubMed

Rosenberg, I M; Göke, M; Kanai, M; Reinecker, H C; Podolsky, D K

1997-10-01

Epithelial cell kinase (Eck) is a member of a large family of receptor tyrosine kinases whose functions remain largely unknown. Expression and regulation of Eck and its cognate ligand B61 were analyzed in the human colonic adenocarcinoma cell line Caco-2. Immunocytochemical staining demonstrated coexpression of Eck and B61 in the same cells, suggestive of an autocrine loop. Eck levels were maximal in preconfluent cells. In contrast, B61 levels were barely detectable in preconfluent cells and increased progressively after the cells reached confluence. Caco-2 cells cultured in the presence of added B61 showed a significant reduction in the levels of dipeptidyl peptidase and sucrase-isomaltase mRNA, markers of Caco-2 cell differentiation. Cytokines interleukin-1beta (IL-1beta), basic fibroblast growth factor, IL-2, epidermal growth factor, and transforming growth factor-beta modulated steady-state levels of Eck and B61 mRNA and regulated Eck activation as assessed by tyrosine phosphorylation. Functionally, stimulation of Eck by B61 resulted in increased proliferation, enhanced barrier function, and enhanced restitution of injured epithelial monolayers. These results suggest that the Eck-B61 interaction, a target of regulatory peptides, plays a role in intestinal epithelial cell development, migration, and barrier function, contributing to homeostasis and preservation of continuity of the epithelial barrier.

Investigations on the change of texture of plant cells due to preservative treatments by digital holographic microscopy

NASA Astrophysics Data System (ADS)

Vora, Priyanka; Anand, Arun

2014-10-01

Texture change is observed in preserved fruits and vegetables. Responsible factors for texture change during preservative treatments are cell morphology, cell wall structure, cell turger, water content and some biochemical components, and also the environmental conditions. Digital Holographic microscopy (DHM) is a quantitative phase contrast imaging technique, which provides three dimensional optical thickness profiles of transparent specimen. Using DHM the morphology of plant cells preserved by refrigeration or stored in vinegar or in sodium chloride can be obtained. This information about the spatio-temporal evolution of optical volume and thickness can be an important tool in area of food processing. Also from the three dimensional images, the texture of the cell can be retrieved and can be investigated under varying conditions.

Vγ9 and Vδ2 T cell antigen receptor genes and butyrophilin 3 (BTN3) emerged with placental mammals and are concomitantly preserved in selected species like alpaca (Vicugna pacos).

PubMed

Karunakaran, Mohindar M; Göbel, Thomas W; Starick, Lisa; Walter, Lutz; Herrmann, Thomas

2014-04-01

Human Vγ9Vδ2 T cells recognize phosphorylated products of isoprenoid metabolism (phosphoantigens) PAg with TCR comprising Vγ9JP γ-chains and Vδ2 δ-chains dependent on butyrophilin 3 (BTN3) expressed by antigen-presenting cells. They are massively activated in many infections and show anti-tumor activity and so far, they have been considered to exist only in higher primates. We performed a comprehensive analysis of databases and identified the three genes in species of both placental magnorders, but not in rodents. The common occurrence or loss of in silico translatable Vγ9, Vδ2, and BTN3 genes suggested their co-evolution based on a functional relationship. In the peripheral lymphocytes of alpaca (Vicugna pacos), characteristic Vγ9JP rearrangements and in-frame Vδ2 rearrangements were found and could be co-expressed in a TCR-negative mouse T cell hybridoma where they rescued CD3 expression and function. Finally, database sequence analysis of the extracellular domain of alpaca BTN3 revealed complete conservation of proposed PAg binding residues of human BTN3A1. In summary, we show emergence and preservation of Vγ9 and Vδ2 TCR genes with the gene of the putative antigen-presenting molecule BTN3 in placental mammals and lay the ground for analysis of alpaca as candidate for a first non-primate species to possess Vγ9Vδ2 T cells.

Inner ear tissue preservation by rapid freezing: improving fixation by high-pressure freezing and hybrid methods.

PubMed

Bullen, A; Taylor, R R; Kachar, B; Moores, C; Fleck, R A; Forge, A

2014-09-01

In the preservation of tissues in as 'close to life' state as possible, rapid freeze fixation has many benefits over conventional chemical fixation. One technique by which rapid freeze-fixation can be achieved, high pressure freezing (HPF), has been shown to enable ice crystal artefact-free freezing and tissue preservation to greater depths (more than 40 μm) than other quick-freezing methods. Despite increasingly becoming routine in electron microscopy, the use of HPF for the fixation of inner ear tissue has been limited. Assessment of the quality of preservation showed routine HPF techniques were suitable for preparation of inner ear tissues in a variety of species. Good preservation throughout the depth of sensory epithelia was achievable. Comparison to chemically fixed tissue indicated that fresh frozen preparations exhibited overall superior structural preservation of cells. However, HPF fixation caused characteristic artefacts in stereocilia that suggested poor quality freezing of the actin bundles. The hybrid technique of pre-fixation and high pressure freezing was shown to produce cellular preservation throughout the tissue, similar to that seen in HPF alone. Pre-fixation HPF produced consistent high quality preservation of stereociliary actin bundles. Optimising the preparation of samples with minimal artefact formation allows analysis of the links between ultrastructure and function in inner ear tissues. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Air-dried cells from the anhydrobiotic insect, Polypedilum vanderplanki, can survive long term preservation at room temperature and retain proliferation potential after rehydration.

PubMed

Watanabe, Kazuyo; Imanishi, Shigeo; Akiduki, Gaku; Cornette, Richard; Okuda, Takashi

2016-08-01

Pv11, a cell line derived from the anhydrobiotic insect, Polypedilum vanderplanki, was preserved in a dry form (only 6% residual moisture) at room temperature for up to 251 days and restarted proliferating after rehydration. A previous study already reported survival of Pv11 cells after desiccation, but without subsequent proliferation. Here, the protocol was improved to increase survival and achieve proliferation of Pv11 cells after dry storage. The method basically included preincubation, desiccation and rehydration processes and each step was investigated. So far, preincubation in a 600 mM trehalose solution for 48 h before dehydration was the most favourable preconditioning to achieve successful dry preservation of Pv11 cells, allowing about 16% of survival after rehydration and subsequent cell proliferation. Although the simple air-dry method established for Pv11 cells here was not applicable for successful dry-preservation of other insect cell lines, Pv11 is the first dry-preservable animal cell line and will surely contribute not only to basic but also applied sciences. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples

PubMed Central

Yu, Feiqiao Brian; Blainey, Paul C; Schulz, Frederik; Woyke, Tanja; Horowitz, Mark A; Quake, Stephen R

2017-01-01

Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell. DOI: http://dx.doi.org/10.7554/eLife.26580.001 PMID:28678007

Cardiac Stem Cell Hybrids Enhance Myocardial Repair

PubMed Central

Quijada, Pearl; Salunga, Hazel T.; Hariharan, Nirmala; Cubillo, Jonathan D.; El-Sayed, Farid G.; Moshref, Maryam; Bala, Kristin M.; Emathinger, Jacqueline M.; La Torre, Andrea De; Ormachea, Lucia; Alvarez, Roberto; Gude, Natalie A.; Sussman, Mark A.

2015-01-01

Rationale Dual cell transplantation of cardiac progenitor cells (CPCs) and mesenchymal stem cells (MSCs) after infarction improves myocardial repair and performance in large animal models relative to delivery of either cell population. Objective To demonstrate that CardioChimeras (CCs) formed by fusion between CPCs and MSCs have enhanced reparative potential in a mouse model of myocardial infarction relative to individual stem cells or combined cell delivery. Methods and Results Two distinct and clonally derived CCs, CC1 and CC2 were utilized for this study. CCs improved left ventricular anterior wall thickness (AWT) at 4 weeks post injury, but only CC1 treatment preserved AWT at 18 weeks. Ejection fraction was enhanced at 6 weeks in CCs, and functional improvements were maintained in CCs and CPC + MSC groups at 18 weeks. Infarct size was decreased in CCs, whereas CPC + MSC and CPC parent groups remained unchanged at 12 weeks. CCs exhibited increased persistence, engraftment, and expression of early commitment markers within the border zone relative to combinatorial and individual cell population-injected groups. CCs increased capillary density and preserved cardiomyocyte size in the infarcted regions suggesting CCs role in protective paracrine secretion. Conclusions CCs merge the application of distinct cells into a single entity for cellular therapeutic intervention in the progression of heart failure. CCs are a novel cell therapy that improves upon combinatorial cell approaches to support myocardial regeneration. PMID:26228030

Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells

PubMed Central

Samse, Kaitlen; Emathinger, Jacqueline; Hariharan, Nirmala; Quijada, Pearl; Ilves, Kelli; Völkers, Mirko; Ormachea, Lucia; De La Torre, Andrea; Orogo, Amabel M.; Alvarez, Roberto; Din, Shabana; Mohsin, Sadia; Monsanto, Megan; Fischer, Kimberlee M.; Dembitsky, Walter P.; Gustafsson, Åsa B.; Sussman, Mark A.

2015-01-01

Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated β-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-XL and decreasing cell death after H2O2 treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics. PMID:25882843

Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells.

PubMed

Samse, Kaitlen; Emathinger, Jacqueline; Hariharan, Nirmala; Quijada, Pearl; Ilves, Kelli; Völkers, Mirko; Ormachea, Lucia; De La Torre, Andrea; Orogo, Amabel M; Alvarez, Roberto; Din, Shabana; Mohsin, Sadia; Monsanto, Megan; Fischer, Kimberlee M; Dembitsky, Walter P; Gustafsson, Åsa B; Sussman, Mark A

2015-05-29

Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated β-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-XL and decreasing cell death after H2O2 treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Calcium glycerophosphate preserves transepithelial integrity in the Caco-2 model of intestinal transport.

PubMed

Datta, Palika; Weis, Margaret T

2015-08-14

To assess the direct effects of ischemia on intestinal epithelial integrity. Furthermore, clinical efforts at mitigating the effect of hypoperfusion on gut permeability have focused on restoring gut vascular function. We report that, in the Caco-2 cell model of transepithelial transport, calcium glycerophosphate (CGP), an inhibitor of intestinal alkaline phosphatase F3, has a significant effect to preserve transepithelial electrical resistance (TEER) and to attenuate increases in mannitol flux rates during hypoxia or cytokine stimulation. The effect was observable even at concentrations as low as 1 μmol/L. As celiac disease is also marked by a loss of gut epithelial integrity, the effect of CGP to attenuate the effect of the α-gliadin peptide 31-55 was also examined. In this instance, CGP exerted little effect of preservation of TEER, but significantly attenuated peptide induced increase in mannitol flux. It appears that CGP treatment might synergize with other therapies to preserve gut epithelial integrity.

Calcium glycerophosphate preserves transepithelial integrity in the Caco-2 model of intestinal transport

PubMed Central

Datta, Palika; Weis, Margaret T

2015-01-01

AIM: To assess the direct effects of ischemia on intestinal epithelial integrity. Furthermore, clinical efforts at mitigating the effect of hypoperfusion on gut permeability have focused on restoring gut vascular function. METHODS: We report that, in the Caco-2 cell model of transepithelial transport, calcium glycerophosphate (CGP), an inhibitor of intestinal alkaline phosphatase F3, has a significant effect to preserve transepithelial electrical resistance (TEER) and to attenuate increases in mannitol flux rates during hypoxia or cytokine stimulation. RESULTS: The effect was observable even at concentrations as low as 1 μmol/L. As celiac disease is also marked by a loss of gut epithelial integrity, the effect of CGP to attenuate the effect of the α-gliadin peptide 31-55 was also examined. In this instance, CGP exerted little effect of preservation of TEER, but significantly attenuated peptide induced increase in mannitol flux. CONCLUSION: It appears that CGP treatment might synergize with other therapies to preserve gut epithelial integrity. PMID:26290632

Suppression of class I and II histone deacetylases blunts pressure-overload cardiac hypertrophy.

PubMed

Kong, Yongli; Tannous, Paul; Lu, Guangrong; Berenji, Kambeez; Rothermel, Beverly A; Olson, Eric N; Hill, Joseph A

2006-06-06

Recent work has demonstrated the importance of chromatin remodeling, especially histone acetylation, in the control of gene expression in the heart. In cell culture models of cardiac hypertrophy, pharmacological suppression of histone deacetylases (HDACs) can either blunt or amplify cell growth. Thus, HDAC inhibitors hold promise as potential therapeutic agents in hypertrophic heart disease. In the present investigation, we studied 2 broad-spectrum HDAC inhibitors in a physiologically relevant banding model of hypertrophy, observing dose-responsive suppression of ventricular growth that was well tolerated in terms of both clinical outcome and cardiac performance measures. In both short-term (3-week) and long-term (9-week) trials, cardiomyocyte growth was blocked by HDAC inhibition, with no evidence of cell death or apoptosis. Fibrotic change was diminished in hearts treated with HDAC inhibitors, and collagen synthesis in isolated cardiac fibroblasts was blocked. Preservation of systolic function in the setting of blunted hypertrophic growth was documented by echocardiography and by invasive pressure measurements. The hypertrophy-associated switch of adult and fetal isoforms of myosin heavy chain expression was attenuated, which likely contributed to the observed preservation of systolic function in HDAC inhibitor-treated hearts. Together, these data suggest that HDAC inhibition is a viable therapeutic strategy that holds promise in the treatment of load-induced heart disease.

Calorie Restriction Attenuates Terminal Differentiation of Immune Cells.

PubMed

White, Matthew J; Beaver, Charlotte M; Goodier, Martin R; Bottomley, Christian; Nielsen, Carolyn M; Wolf, Asia-Sophia F M; Boldrin, Luisa; Whitmore, Charlotte; Morgan, Jennifer; Pearce, Daniel J; Riley, Eleanor M

2016-01-01

Immune senescence is a natural consequence of aging and may contribute to frailty and loss of homeostasis in later life. Calorie restriction increases healthy life-span in C57BL/6J (but not DBA/2J) mice, but whether this is related to preservation of immune function, and how it interacts with aging, is unclear. We compared phenotypic and functional characteristics of natural killer (NK) cells and T cells, across the lifespan, of calorie-restricted (CR) and control C57BL/6 and DBA/2 mice. Calorie restriction preserves a naïve T cell phenotype and an immature NK cell phenotype as mice age. The splenic T cell populations of CR mice had higher proportions of CD11a - CD44 lo cells, lower expression of TRAIL, KLRG1, and CXCR3, and higher expression of CD127, compared to control mice. Similarly, splenic NK cells from CR mice had higher proportions of less differentiated CD11b - CD27 + cells and correspondingly lower proportions of highly differentiated CD11b + CD27 - NK cells. Within each of these subsets, cells from CR mice had higher expression of CD127, CD25, TRAIL, NKG2A/C/E, and CXCR3 and lower expression of KLRG1 and Ly49 receptors compared to controls. The effects of calorie restriction on lymphoid cell populations in lung, liver, and lymph nodes were identical to those seen in the spleen, indicating that this is a system-wide effect. The impact of calorie restriction on NK cell and T cell maturation is much more profound than the effect of aging and, indeed, calorie restriction attenuates these age-associated changes. Importantly, the effects of calorie restriction on lymphocyte maturation were more marked in C57BL/6 than in DBA/2J mice indicating that delayed lymphocyte maturation correlates with extended lifespan. These findings have implications for understanding the interaction between nutritional status, immunity, and healthy lifespan in aging populations.

Azilsartan, an angiotensin II type 1 receptor blocker, attenuates tert-butyl hydroperoxide-induced endothelial cell injury through inhibition of mitochondrial dysfunction and anti-inflammatory activity.

PubMed

Liu, Hao; Mao, Ping; Wang, Jia; Wang, Tuo; Xie, Chang-Hou

2016-03-01

Angiotensin II type 1 receptor (AT1-R) blockers protect against brain ischemia by mechanisms dependent on and independent of arterial blood pressure. However, the effects of AT1-R blockers on brain endothelial cell injury and detailed mechanisms remain unclear. The goal of this study is to investigate whether azilsartan, an AT1-R blocker, could attenuate oxidative injury in endothelial cells via regulating mitochondrial function and inflammatory responses. We found that treatment with azilsartan suppressed tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in murine brain endothelial cells (mBECs) by increasing cell viability, decreasing lactate dehydrogenase (LDH) release and inhibiting cell apoptosis. Azilsartan significantly inhibited reactive oxygen species (ROS) generation and lipid peroxidation, but had no effect on antioxidant system. We also detected preserved mitochondrial function after azilsartan treatment, as evidenced by increased mitochondrial membrane potential (MMP), reduced cytochrome c release, preserved ATP synthesis and inhibited mitochondrial swelling. In addition, azilsartan differently regulated expression of inflammatory cytokines and increased the activation of endothelial nitric oxide synthase (eNOS). Pretreatment with eNOS inhibitor L-NIO partially prevented the azilsartan-induced regulation of cytokines and protection. Furthermore, azilsartan-induced protection in our in vitro model was shown to be associated with protein stability of peroxisome proliferator-activated receptor-γ (PPAR-γ). Overall, our data suggest that the AT1-R blocker azilsartan may have therapeutic values in treating endothelial dysfunction associated neurological disorders through anti-oxidative and anti-inflammatory properties. Copyright © 2016 Elsevier Ltd. All rights reserved.

Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene

PubMed Central

Seandel, Marco; Butler, Jason M.; Kobayashi, Hideki; Hooper, Andrea T.; White, Ian A.; Zhang, Fan; Vertes, Eva L.; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V.; Hackett, Neil R.; Rabbany, Sina; Boyer, Julie L.; Rafii, Shahin

2008-01-01

Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1+ ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1+ ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1+ ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1+ ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1+ ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-κB pathways. Therefore, E4ORF1+ ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis. PMID:19036927

Generation of a functional and durable vascular niche by the adenoviral E4ORF1 gene.

PubMed

Seandel, Marco; Butler, Jason M; Kobayashi, Hideki; Hooper, Andrea T; White, Ian A; Zhang, Fan; Vertes, Eva L; Kobayashi, Mariko; Zhang, Yan; Shmelkov, Sergey V; Hackett, Neil R; Rabbany, Sina; Boyer, Julie L; Rafii, Shahin

2008-12-09

Vascular cells contribute to organogenesis and tumorigenesis by producing unknown factors. Primary endothelial cells (PECs) provide an instructive platform for identifying factors that support stem cell and tumor homeostasis. However, long-term maintenance of PECs requires stimulation with cytokines and serum, resulting in loss of their angiogenic properties. To circumvent this hurdle, we have discovered that the adenoviral E4ORF1 gene product maintains long-term survival and facilitates organ-specific purification of PECs, while preserving their vascular repertoire for months, in serum/cytokine-free cultures. Lentiviral introduction of E4ORF1 into human PECs (E4ORF1(+) ECs) increased the long-term survival of these cells in serum/cytokine-free conditions, while preserving their in vivo angiogenic potential for tubulogenesis and sprouting. Although E4ORF1, in the absence of mitogenic signals, does not induce proliferation of ECs, stimulation with VEGF-A and/or FGF-2 induced expansion of E4ORF1(+) ECs in a contact-inhibited manner. Indeed, VEGF-A-induced phospho MAPK activation of E4ORF1(+) ECs is comparable with that of naive PECs, suggesting that the VEGF receptors remain functional upon E4ORF1 introduction. E4ORF1(+) ECs inoculated in implanted Matrigel plugs formed functional, patent, humanized microvessels that connected to the murine circulation. E4ORF1(+) ECs also incorporated into neo-vessels of human tumor xenotransplants and supported serum/cytokine-free expansion of leukemic and embryonal carcinoma cells. E4ORF1 augments survival of PECs in part by maintaining FGF-2/FGF-R1 signaling and through tonic Ser-473 phosphorylation of Akt, thereby activating the mTOR and NF-kappaB pathways. Therefore, E4ORF1(+) ECs establish an Akt-dependent durable vascular niche not only for expanding stem and tumor cells but also for interrogating the roles of vascular cells in regulating organ-specific vascularization and tumor neo-angiogenesis.

Human umbilical cord tissue-derived mesenchymal stromal cells attenuate remodeling after myocardial infarction by proangiogenic, antiapoptotic, and endogenous cell-activation mechanisms

PubMed Central

2014-01-01

Introduction Among the plethora of cells under investigation to restore a functional myocardium, mesenchymal stromal cells (MSCs) have been granted considerable interest. However, whereas the beneficial effects of bone marrow MSCs (BM-MSCs) in the context of the diseased heart are widely reported, data are still scarce on MSCs from the umbilical cord matrix (UCM-MSCs). Herein we report on the effect of UCM-MSC transplantation to the infarcted murine heart, seconded by the dissection of the molecular mechanisms at play. Methods Human umbilical cord tissue-derived MSCs (UCX®), obtained by using a proprietary technology developed by ECBio, were delivered via intramyocardial injection to C57BL/6 females subjected to permanent ligation of the left descending coronary artery. Moreover, medium produced by cultured UCX® preconditioned under normoxia (CM) or hypoxia (CMH) was collected for subsequent in vitro assays. Results Evaluation of the effects upon intramyocardial transplantation shows that UCX® preserved cardiac function and attenuated cardiac remodeling subsequent to myocardial infarction (MI). UCX® further led to increased capillary density and decreased apoptosis in the injured tissue. In vitro, UCX®-conditioned medium displayed (a) proangiogenic activity by promoting the formation of capillary-like structures by human umbilical vein endothelial cells (HUVECs), and (b) antiapoptotic activity in HL-1 cardiomyocytes subjected to hypoxia. Moreover, in adult murine cardiac Sca-1+ progenitor cells (CPCs), conditioned medium enhanced mitogenic activity while activating a gene program characteristic of cardiomyogenic differentiation. Conclusions UCX® preserve cardiac function after intramyocardial transplantation in a MI murine model. The cardioprotective effects of UCX® were attributed to paracrine mechanisms that appear to enhance angiogenesis, limit the extent of the apoptosis, augment proliferation, and activate a pool of resident CPCs. Overall, these results suggest that UCX® should be considered an alternative cell source when designing new therapeutic approaches to treat MI. PMID:24411922

Preservation media, durations and cell concentrations of short-term storage affect key features of human adipose-derived mesenchymal stem cells for therapeutic application.

PubMed

Zhang, Fengli; Ren, Huaijuan; Shao, Xiaohu; Zhuang, Chao; Chen, Yantian; Qi, Nianmin

2017-01-01

Adipose-derived mesenchymal stem cells (ADSCs) have shown great potential in the treatment of various diseases. However, the optimum short-term storage condition of ADSCs in 2∼8 °C is rarely reported. This study aimed at optimizing a short-term storage condition to ensure the viability and function of ADSCs before transplantation. Preservation media and durations of storage were evaluated by cell viability, apoptosis, adhesion ability and colony-forming unit (CFU) capacity of ADSCs. The abilities of cell proliferation and differentiation were used to optimize cell concentrations. Optimized preservation condition was evaluated by cell surface markers, cell cycle and immunosuppressive capacity. A total of 5% human serum albumin in multiple electrolytes (ME + HSA) was the optimized medium with high cell viability, low cluster rate, good adhesion ability and high CFU capacity of ADSCs. Duration of storage should be limited to 24 h to ensure the quality of ADSCs before transplantation. A concentration of 5 × 10 6 cells/ml was the most suitable cell concentration with low late stage apoptosis, rapid proliferation and good osteogenic and adipogenic differentiation ability. This selected condition did not change surface markers, cell cycle, indoleamine 2, 3-dioxygenase 1 (IDO1) gene expression and kynurenine (Kyn) concentration significantly. In this study, ME + HSA was found to be the best medium, most likely due to the supplement of HSA which could protect cells, the physiological pH (7.4) of ME and sodium gluconate ingredient in ME which could provide energy for cells. Duration should be limited to 24 h because of reduced nutrient supply and increased waste and lactic acid accumulation during prolonged storage. To keep cell proliferation and limit lactic acid accumulation, the proper cell concentration is 5× 10 6 cells/ml. Surface markers, cell cycle and immunosuppressive capacity did not change significantly after storage using the optimized condition, which confirmed our results that this optimized short-term storage condition of MSCs has a great potential for the application of cell therapy.

Developmental potential of murine germinal vesicle stage cumulus-oocyte complexes following exposure to dimethylsulphoxide or cryopreservation: loss of membrane integrity of cumulus cells after thawing.

PubMed

Ruppert-Lingham, C J; Paynter, S J; Godfrey, J; Fuller, B J; Shaw, R W

2003-02-01

Cumulus cells of the cumulus-oocyte complex (COC) are important in oocyte maturation. Thus, in preserving immature oocytes it is prudent to also preserve their associated cumulus cells. The survival and function of oocytes and their associated cumulus cells was assessed following cryopreservation or exposure to cryoprotectant without freezing. Immature COCs were collected from mice primed with pregnant mare's serum. COCs were either slow-cooled or exposed to 1.5 mol/l dimethylsulphoxide without freezing. Treated and fresh COCs were stained for membrane integrity or, after in-vitro maturation and IVF, were assessed for developmental capability. Development of cumulus-denuded fresh oocytes, as well as denuded and frozen-thawed oocytes co-cultured with fresh cumulus cells, was assessed. Slow-cooled oocytes had significantly reduced coverage by intact cumulus cells compared with fresh COCs. Cumulus cell association and developmental capability were not substantially affected by exposure to cryoprotectant without freezing. Denuded fresh oocytes and cryopreserved COCs had decreased developmental potential that was not overcome by co-culture with fresh cumulus cells. Loss of association between oocyte and cumulus cells was induced by cryopreservation, but not by treatment with cryoprotectant alone. The data indicate that direct physical contact between cumulus cells and the oocyte, throughout maturation, improves subsequent embryo development.

HEMOXCell, a New Oxygen Carrier Usable as an Additive for Mesenchymal Stem Cell Culture in Platelet Lysate-Supplemented Media.

PubMed

Le Pape, Fiona; Cosnuau-Kemmat, Lucie; Richard, Gaëlle; Dubrana, Frédéric; Férec, Claude; Zal, Franck; Leize, Elisabeth; Delépine, Pascal

2017-04-01

Human mesenchymal stem cells (MSCs) are promising candidates for therapeutic applications such as tissue engineering. However, one of the main challenges is to improve oxygen supply to hypoxic areas to reduce oxygen gradient formation while preserving MSC differentiation potential and viability. For this purpose, a marine hemoglobin, HEMOXCell, was evaluated as an oxygen carrier for culturing human bone marrow MSCs in vitro for future three-dimensional culture applications. Impact of HEMOXCell on cell growth and viability was assessed in human platelet lysate (hPL)-supplemented media. Maintenance of MSC features, such as multipotency and expression of MSC specific markers, was further investigated by biochemical assays and flow cytometry analysis. Our experimental results highlight its oxygenator potential and indicate that an optimal concentration of 0.025 g/L HEMOXCell induces a 25%-increase of the cell growth rate, preserves MSC phenotype, and maintains MSC differentiation properties; a two-fold higher concentration induces cell detachment without altering cell viability. Our data suggest the potential interest of HEMOXCell as a natural oxygen carrier for tissue engineering applications to oxygenate hypoxic areas and to maintain cell viability, functions and "stemness." These features will be further tested within three-dimensional scaffolds. © 2017 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Skeletal muscle regeneration and impact of aging and nutrition.

PubMed

Domingues-Faria, Carla; Vasson, Marie-Paule; Goncalves-Mendes, Nicolas; Boirie, Yves; Walrand, Stephane

2016-03-01

After skeletal muscle injury a regeneration process takes place to repair muscle. Skeletal muscle recovery is a highly coordinated process involving cross-talk between immune and muscle cells. It is well known that the physiological activities of both immune cells and muscle stem cells decline with advancing age, thereby blunting the capacity of skeletal muscle to regenerate. The age-related reduction in muscle repair efficiency contributes to the development of sarcopenia, one of the most important factors of disability in elderly people. Preserving muscle regeneration capacity may slow the development of this syndrome. In this context, nutrition has drawn much attention: studies have demonstrated that nutrients such as amino acids, n-3 polyunsaturated fatty acids, polyphenols and vitamin D can improve skeletal muscle regeneration by targeting key functions of immune cells, muscle cells or both. Here we review the process of skeletal muscle regeneration with a special focus on the cross-talk between immune and muscle cells. We address the effect of aging on immune and skeletal muscle cells involved in muscle regeneration. Finally, the mechanisms of nutrient action on muscle regeneration are described, showing that quality of nutrition may help to preserve the capacity for skeletal muscle regeneration with age. Copyright © 2015 Elsevier B.V. All rights reserved.

Higher Molecular Weight Polyethylene Glycol Increases Cell Proliferation While Improving Barrier Function in an In Vitro Colon Cancer Model

PubMed Central

Bharadwaj, Shruthi; Vishnubhotla, Ramana; Shan, Sun; Chauhan, Chinmay; Cho, Michael; Glover, Sarah C.

2011-01-01

Polyethylene glycol (PEG) has been previously shown to protect against enteric pathogens and prevent colon cancer invasion. To determine if PEG could indeed protect against previously observed pro-invasive effects of commensal E. coli and EPEC, Caco-2 cells grown in an in vitro model of colon cancer were infected with strains of human commensal E. coli or EPEC and treated with 10% PEG 3350, PEG 8000, and PEG 20,000, respectively. At 24 hours after infection, MMP-1 and MMP-13 activities, cell cluster thickness, depth of invasion, and proliferation were determined using standard molecular biology techniques and advanced imaging. We found that higher molecular weight PEG, especially PEG 8000 and 20,000, regardless of bacterial infection, increased proliferation and depth of invasion although a decrease in cellular density and MMP-1 activity was also noted. Maximum proliferation and depth of invasion of Caco-2 cells was observed in scaffolds treated with a combination of commensal E. coli strain, HS4 and PEG 8000. In conclusion, we found that PEG 8000 increased cell proliferation and led to the preservation of cell density in cells treated with commensal bacteria. This is important, because the preservation of a proliferative response in colon cancer results in a more chemo-responsive tumor. PMID:21976966

Higher molecular weight polyethylene glycol increases cell proliferation while improving barrier function in an in vitro colon cancer model.

PubMed

Bharadwaj, Shruthi; Vishnubhotla, Ramana; Shan, Sun; Chauhan, Chinmay; Cho, Michael; Glover, Sarah C

2011-01-01

Polyethylene glycol (PEG) has been previously shown to protect against enteric pathogens and prevent colon cancer invasion. To determine if PEG could indeed protect against previously observed pro-invasive effects of commensal E. coli and EPEC, Caco-2 cells grown in an in vitro model of colon cancer were infected with strains of human commensal E. coli or EPEC and treated with 10% PEG 3350, PEG 8000, and PEG 20,000, respectively. At 24 hours after infection, MMP-1 and MMP-13 activities, cell cluster thickness, depth of invasion, and proliferation were determined using standard molecular biology techniques and advanced imaging. We found that higher molecular weight PEG, especially PEG 8000 and 20,000, regardless of bacterial infection, increased proliferation and depth of invasion although a decrease in cellular density and MMP-1 activity was also noted. Maximum proliferation and depth of invasion of Caco-2 cells was observed in scaffolds treated with a combination of commensal E. coli strain, HS4 and PEG 8000. In conclusion, we found that PEG 8000 increased cell proliferation and led to the preservation of cell density in cells treated with commensal bacteria. This is important, because the preservation of a proliferative response in colon cancer results in a more chemo-responsive tumor.

Effectiveness of early intensive therapy on β-cell preservation in type 1 diabetes.

PubMed

Buckingham, Bruce; Beck, Roy W; Ruedy, Katrina J; Cheng, Peiyao; Kollman, Craig; Weinzimer, Stuart A; DiMeglio, Linda A; Bremer, Andrew A; Slover, Robert; Tamborlane, William V

2013-12-01

To assess effectiveness of inpatient hybrid closed-loop control (HCLC) followed by outpatient sensor-augmented pump (SAP) therapy initiated within 7 days of diagnosis of type 1 diabetes on the preservation of β-cell function at 1 year. Sixty-eight individuals (mean age 13.3 ± 5.7 years; 35% female, 92% Caucasian) were randomized to HCLC followed by SAP therapy (intensive group; N = 48) or to the usual-care group treated with multiple daily injections or insulin pump therapy (N = 20). Primary outcome was C-peptide concentrations during mixed-meal tolerance tests at 12 months. Intensive-group participants initiated HCLC a median of 6 days after diagnosis for a median duration of 71.3 h, during which median participant mean glucose concentration was 140 mg/dL (interquartile range 134-153 mg/dL). During outpatient SAP, continuous glucose monitor (CGM) use decreased over time, and at 12 months, only 33% of intensive participants averaged sensor use ≥6 days/week. In the usual-care group, insulin pump and CGM use were initiated prior to 12 months by 15 and 5 participants, respectively. Mean HbA1c levels were similar in both groups throughout the study. At 12 months, the geometric mean (95% CI) of C-peptide area under the curve was 0.43 (0.34-0.52) pmol/mL in the intensive group and 0.52 (0.32-0.75) pmol/mL in the usual-care group (P = 0.49). Thirty-seven (79%) intensive and 16 (80%) usual-care participants had a peak C-peptide concentration ≥0.2 pmol/mL (P = 0.30). In new-onset type 1 diabetes, HCLC followed by SAP therapy did not provide benefit in preserving β-cell function compared with current standards of care.

Intramyocardial injection of autologous cardiospheres or cardiosphere-derived cells preserves function and minimizes adverse ventricular remodeling in pigs with heart failure post-myocardial infarction.

PubMed

Lee, Shuo-Tsan; White, Anthony J; Matsushita, Satoshi; Malliaras, Konstantinos; Steenbergen, Charles; Zhang, Yiqiang; Li, Tao-Sheng; Terrovitis, John; Yee, Kristine; Simsir, Sinan; Makkar, Raj; Marbán, Eduardo

2011-01-25

The purpose of this study was to test the safety and efficacy of direct injection of cardiosphere-derived cells (CDCs) and their 3-dimensional precursors, cardiospheres, for cellular cardiomyoplasty in a mini-pig model of heart failure after myocardial infarction. Intracoronary administration of CDCs has been demonstrated to reduce infarct size and improve hemodynamic indexes in the mini-pig model, but intramyocardial injection of CDCs or cardiospheres has not been assessed in large animals. Autologous cardiospheres or CDCs grown from endomyocardial biopsies were injected through thoracotomy 4 weeks after anteroseptal myocardial infarction. Engraftment optimization with luciferase-labeled CDCs guided the choice of cell dose (0.5 million cells/site) and target tissue (20 peri-infarct sites). Pigs were randomly allocated to placebo (n = 11), cardiospheres (n = 8), or CDCs (n = 10). Functional data were acquired before injection and again 8 weeks later, after which organs were harvested for histopathology. Beyond the immediate perioperative period, all animals survived to protocol completion. Ejection fraction was equivalent at baseline, but at 8 weeks was higher than placebo in both of the cell-treated groups (placebo vs. CDC, p = 0.01; placebo vs. cardiospheres, p = 0.01). Echocardiographic and hemodynamic indexes of efficacy improved disproportionately with cardiospheres; likewise, adverse remodeling was more attenuated with cardiospheres than with CDCs. Provocative electrophysiologic testing showed no differences among groups, and no tumors were found. Dosage-optimized direct injection of cardiospheres or CDCs is safe and effective in preserving ventricular function in porcine ischemic cardiomyopathy. Although CDCs and cardiospheres have equivalent effects on left ventricular ejection fraction, cardiospheres are superior in improving hemodynamics and regional function, and in attenuating ventricular remodeling. Copyright © 2011 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

PubMed

Wang, Xiangmin; Pan, Bin; Honda, Goichi; Wang, Xintao; Hashimoto, Yuko; Ohkawara, Hiroshi; Xu, Kailin; Zeng, Lingyu; Ikezoe, Takayuki

2018-06-14

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Here, we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from GPR15 knock-out mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal ECs numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. Use of TME5C may be a promising treatment strategy to prevent or treat lethal complications such as sinusoidal obstruction syndrome whose pathogenesis is based on endothelial insults. Copyright © 2018, Ferrata Storti Foundation.

Suppression of lethal autoimmunity by regulatory T cells with a single TCR specificity

PubMed Central

Hemmers, Saskia; Schizas, Michail; Faire, Mehlika B.; Konopacki, Catherine; Schmidt-Supprian, Marc; Germain, Ronald N.

2017-01-01

The regulatory T cell (T reg cell) T cell receptor (TCR) repertoire is highly diverse and skewed toward recognition of self-antigens. TCR expression by T reg cells is continuously required for maintenance of immune tolerance and for a major part of their characteristic gene expression signature; however, it remains unknown to what degree diverse TCR-mediated interactions with cognate self-antigens are required for these processes. In this study, by experimentally switching the T reg cell TCR repertoire to a single T reg cell TCR, we demonstrate that T reg cell function and gene expression can be partially uncoupled from TCR diversity. An induced switch of the T reg cell TCR repertoire to a random repertoire also preserved, albeit to a limited degree, the ability to suppress lymphadenopathy and T helper cell type 2 activation. At the same time, these perturbations of the T reg cell TCR repertoire led to marked immune cell activation, tissue inflammation, and an ultimately severe autoimmunity, indicating the importance of diversity and specificity for optimal T reg cell function. PMID:28130403

Correlation between the mechanical and histological properties of liver tissue.

PubMed

Yarpuzlu, Berkay; Ayyildiz, Mehmet; Tok, Olgu Enis; Aktas, Ranan Gulhan; Basdogan, Cagatay

2014-01-01

In order to gain further insight into the mechanisms of tissue damage during the progression of liver diseases as well as the liver preservation for transplantation, an improved understanding of the relation between the mechanical and histological properties of liver is necessary. We suggest that this relation can only be established truly if the changes in the states of those properties are investigated dynamically as a function of post mortem time. In this regard, we first perform mechanical characterization experiments on three bovine livers to investigate the changes in gross mechanical properties (stiffness, viscosity, and fracture toughness) for the preservation periods of 5, 11, 17, 29, 41 and 53h after harvesting. Then, the histological examination is performed on the samples taken from the same livers to investigate the changes in apoptotic cell count, collagen accumulation, sinusoidal dilatation, and glycogen deposition as a function of the same preservation periods. Finally, the correlation between the mechanical and histological properties is investigated via the Spearman's Rank-Order Correlation method. The results of our study show that stiffness, viscosity, and fracture toughness of bovine liver increase as the preservation period is increased. These macroscopic changes are very strongly correlated with the increase in collagen accumulation and decrease in deposited glycogen level at the microscopic level. Also, we observe that the largest changes in mechanical and histological properties occur after the first 11-17h of preservation. © 2013 Elsevier Ltd. All rights reserved.

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

PubMed Central

2011-01-01

Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls. PMID:22088094

Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen.

PubMed

Givens, Robert M; Mesner, Larry D; Hamlin, Joyce L; Buck, Michael J; Huberman, Joel A

2011-11-16

Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls.

3D Cell Printing of Functional Skeletal Muscle Constructs Using Skeletal Muscle-Derived Bioink.

PubMed

Choi, Yeong-Jin; Kim, Taek Gyoung; Jeong, Jonghyeon; Yi, Hee-Gyeong; Park, Ji Won; Hwang, Woonbong; Cho, Dong-Woo

2016-10-01

Engineered skeletal muscle tissues that mimic the structure and function of native muscle have been considered as an alternative strategy for the treatment of various muscular diseases and injuries. Here, it is demonstrated that 3D cell-printing of decellularized skeletal muscle extracellular matrix (mdECM)-based bioink facilitates the fabrication of functional skeletal muscle constructs. The cellular alignment and the shape of the tissue constructs are controlled by 3D cell-printing technology. mdECM bioink provides the 3D cell-printed muscle constructs with a myogenic environment that supports high viability and contractility as well as myotube formation, differentiation, and maturation. More interestingly, the preservation of agrin is confirmed in the mdECM, and significant increases in the formation of acetylcholine receptor clusters are exhibited in the 3D cell-printed muscle constructs. In conclusion, mdECM bioink and 3D cell-printing technology facilitate the mimicking of both the structural and functional properties of native muscle and hold great promise for producing clinically relevant engineered muscle for the treatment of muscular injuries. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A hybrid substratum for primary hepatocyte culture that enhances hepatic functionality with low serum dependency

PubMed Central

Meng, Qingyuan; Tao, Chunsheng; Qiu, Zhiye; Akaike, Toshihiro; Cui, Fuzhai; Wang, Xiumei

2015-01-01

Cell culture systems have proven to be crucial for the in vitro maintenance of primary hepatocytes and the preservation of hepatic functional expression at a high level. A poly-(N-p-vinylbenzyl-4-O-β-D-galactopyranosyl-D-gluconamide) matrix can recognize cells and promote liver function in a spheroid structure because of a specific galactose–asialoglycoprotein receptor interaction. Meanwhile, a fusion protein, E-cadherin-Fc, when incubated with various cells, has shown an enhancing effect on cellular viability and metabolism. Therefore, a hybrid substratum was developed for biomedical applications by using both of these materials to combine their advantages for primary hepatocyte cultures. The isolated cells showed a monolayer aggregate morphology on the coimmobilized surface and displayed higher functional expression than cells on traditional matrices. Furthermore, the hybrid system, in which the highest levels of cell adhesion and hepatocellular metabolism were achieved with the addition of 1% fetal bovine serum, showed a lower serum dependency than the collagen/gelatin-coated surface. Accordingly, this substrate may attenuate the negative effects of serum and further contribute to establishing a defined culture system for primary hepatocytes. PMID:25848252

Effect of solution and post-mortem time on mechanical and histological properties of liver during cold preservation.

PubMed

Ayyildiz, Mehmet; Aktas, Ranan Gulhan; Basdogan, Cagatay

2014-01-01

In liver transplantation, the donor and recipient are in different locations most of the time, and longer preservation periods are inevitable. Hence, the choice of the preservation solution and the duration of the preservation period are critical for the success of the transplant surgery. In this study, we examine the mechanical and histological properties of the bovine liver tissue stored in Lactated Ringer's (control), HTK and UW solutions as a function of preservation period. The mechanical experiments are conducted with a shear rheometer on cylindrical tissue samples extracted from 3 bovine livers and the change in viscoelastic material properties of the bovine liver is characterized using the fractional derivative Kelvin-Voigt Model. Also, the histological examinations are performed on the same liver samples under a light microscope. The results show that the preservation solution and period have a significant effect on the mechanical and histological properties of the liver tissue. The storage and loss shear moduli, the number of the apoptotic cells, the collagen accumulation, and the sinusoidal dilatation increase, and the glycogen deposition decreases as the preservation period is longer. Based on the statistical analyses, we observe that the liver tissue is preserved well in all three solutions for up to 11 h. After then, UW solution provides a better preservation up to 29 h. However, for preservation periods longer than 29 h, HTK is a more effective preservation solution based on the least amount of change in mechanical properties. On the other hand, the highest correlation between the mechanical and histological properties is observed for the liver samples preserved in UW solution.

The 6-hydroxychromanol derivative SUL-109 ameliorates renal injury after deep hypothermia and rewarming in rats.

PubMed

Vogelaar, Pieter C; Roorda, Maurits; de Vrij, Edwin L; Houwertjes, Martin C; Goris, Maaike; Bouma, Hjalmar; van der Graaf, Adrianus C; Krenning, Guido; Henning, Robert H

2018-04-11

Mitochondrial dysfunction plays an important role in kidney damage in various pathologies, including acute and chronic kidney injury and diabetic nephropathy. In addition to the well-studied ischaemia/reperfusion (I/R) injury, hypothermia/rewarming (H/R) also inflicts acute kidney injury. Substituted 6-hydroxychromanols are a novel class of mitochondrial medicines that ameliorate mitochondrial oxidative stress and protect the mitochondrial network. To identify a novel 6-hydroxychromanol that protects mitochondrial structure and function in the kidney during H/R, we screened multiple compounds in vitro and subsequently assessed the efficacy of the 6-hydroxychromanol derivatives SUL-109 and SUL-121 in vivo to protect against kidney injury after H/R in rats. Human proximal tubule cell viability was assessed following exposure to H/R for 48/4 h in the presence of various 6-hydroxychromanols. Selected compounds (SUL-109, SUL-121) or vehicle were administered to ketamine-anaesthetized male Wistar rats (IV 135 µg/kg/h) undergoing H/R at 15°C for 3 h followed by rewarming and normothermia for 1 h. Metabolic parameters and body temperature were measured throughout. In addition, renal function, renal injury, histopathology and mitochondrial fitness were assessed. H/R injury in vitro lowered cell viability by 94 ± 1%, which was counteracted dose-dependently by multiple 6-hydroxychomanols derivatives. In vivo, H/R in rats showed kidney injury molecule 1 expression in the kidney and tubular dilation, accompanied by double-strand DNA breaks and protein nitrosylation. SUL-109 and SUL-121 ameliorated tubular kidney damage, preserved mitochondrial mass and maintained cortical adenosine 5'-triphosphate (ATP) levels, although SUL-121 did not reduce protein nitrosylation. The substituted 6-hydroxychromanols SUL-109 and SUL-121 ameliorate kidney injury during in vivo H/R by preserving mitochondrial mass, function and ATP levels. In addition, both 6-hydroxychromanols limit DNA damage, but only SUL-109 also prevented protein nitrosylation in tubular cells. Therefore SUL-109 offers a promising therapeutic strategy to preserve kidney mitochondrial function.

Effects of benzalkonium chloride- or polyquad-preserved fixed combination glaucoma medications on human trabecular meshwork cells

PubMed Central

Ammar, David A.

2011-01-01

Purpose We investigated the potential short and long-term effects in cultured human trabecular meshwork (TM) cells of various topical glaucoma formulations containing different preservatives. Methods We tested the fixed combination medications 0.004% travoprost plus 0.5% timolol preserved with either 0.015% benzalkonium chloride (BAK; DuoTrav®), or with 0.001% polyquad (PQ; DuoTrav® BAK-free); and 0.005% latanoprost plus 0.5% timolol preserved with 0.020% BAK (Xalacom®). Also tested was a range of BAK concentrations (0.001%–0.020%) in balanced salt solution (BSS). Cells were treated for 25 min at 37 °C with solutions diluted 1:10 and 1:100 to mimic the reduced penetration of topical preparations to the anterior chamber. The percentage of live cells was determined immediately after treatment through the uptake of the fluorescent vital dye calcein-AM. To determine any long-term effects, we assayed release of matrix metalloproteinase 9 (MMP-9) and apoptosis 24 h after treatments. Results BAK demonstrated a dose-dependent reduction in TM cell viability, ranging from 71±5% live cells at 0.001% BAK (diluted 1:10) to 33±3% live cells at 0.020% BAK (diluted 1:10). Travoprost (0.004%) plus 0.5% timolol preserved with 0.015% BAK had statistically fewer live TM cells (79±7%) than the same preparation preserved with 0.001% polyquad® (PQ; 93±1%; p<0.001). Latanoprost plus timolol preserved with 0.020% BAK (29±9% live cells) was similar to the 0.020% BAK (33±3%) treatment. However, travoprost plus timolol preserved in 0.015% BAK had significantly more live cells (83±12%) than the 1:10 dilution of 0.015% BAK (49±10%). We also found 0.020% BAK (diluted 1:100) resulted in elevated levels of extracellular MMP-9 at 24 h. Conclusions These results demonstrate that the substitution of the preservative BAK from topical ophthalmic drugs results in greater in vitro viability of TM cells. Travoprost with timolol, but not latanoprost with timolol, countered some of the toxic BAK effects. BAK treatment appeared to cause elevated levels of MMP-9, a matrix metalloproteinase implicated in the pathogenesis of glaucoma. PMID:21750606

Effects of benzalkonium chloride- or polyquad-preserved fixed combination glaucoma medications on human trabecular meshwork cells.

PubMed

Ammar, David A; Kahook, Malik Y

2011-01-01

We investigated the potential short and long-term effects in cultured human trabecular meshwork (TM) cells of various topical glaucoma formulations containing different preservatives. We tested the fixed combination medications 0.004% travoprost plus 0.5% timolol preserved with either 0.015% benzalkonium chloride (BAK; DuoTrav®), or with 0.001% polyquad (PQ; DuoTrav(®) BAK-free); and 0.005% latanoprost plus 0.5% timolol preserved with 0.020% BAK (Xalacom(®)). Also tested was a range of BAK concentrations (0.001%-0.020%) in balanced salt solution (BSS). Cells were treated for 25 min at 37 °C with solutions diluted 1:10 and 1:100 to mimic the reduced penetration of topical preparations to the anterior chamber. The percentage of live cells was determined immediately after treatment through the uptake of the fluorescent vital dye calcein-AM. To determine any long-term effects, we assayed release of matrix metalloproteinase 9 (MMP-9) and apoptosis 24 h after treatments. BAK demonstrated a dose-dependent reduction in TM cell viability, ranging from 71±5% live cells at 0.001% BAK (diluted 1:10) to 33±3% live cells at 0.020% BAK (diluted 1:10). Travoprost (0.004%) plus 0.5% timolol preserved with 0.015% BAK had statistically fewer live TM cells (79±7%) than the same preparation preserved with 0.001% polyquad® (PQ; 93±1%; p<0.001). Latanoprost plus timolol preserved with 0.020% BAK (29±9% live cells) was similar to the 0.020% BAK (33±3%) treatment. However, travoprost plus timolol preserved in 0.015% BAK had significantly more live cells (83±12%) than the 1:10 dilution of 0.015% BAK (49±10%). We also found 0.020% BAK (diluted 1:100) resulted in elevated levels of extracellular MMP-9 at 24 h. These results demonstrate that the substitution of the preservative BAK from topical ophthalmic drugs results in greater in vitro viability of TM cells. Travoprost with timolol, but not latanoprost with timolol, countered some of the toxic BAK effects. BAK treatment appeared to cause elevated levels of MMP-9, a matrix metalloproteinase implicated in the pathogenesis of glaucoma. © 2011 Molecular Vision

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy.

PubMed

Tschernutter, M; Schlichtenbrede, F C; Howe, S; Balaggan, K S; Munro, P M; Bainbridge, J W B; Thrasher, A J; Smith, A J; Ali, R R

2005-04-01

The Royal College of Surgeons (RCS) rat is a well-characterized model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding , a receptor tyrosine kinase found in RPE cells, that is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

REST and stress resistance in ageing and Alzheimer's disease

NASA Astrophysics Data System (ADS)

Lu, Tao; Aron, Liviu; Zullo, Joseph; Pan, Ying; Kim, Haeyoung; Chen, Yiwen; Yang, Tun-Hsiang; Kim, Hyun-Min; Drake, Derek; Liu, X. Shirley; Bennett, David A.; Colaiácovo, Monica P.; Yankner, Bruce A.

2014-03-01

Human neurons are functional over an entire lifetime, yet the mechanisms that preserve function and protect against neurodegeneration during ageing are unknown. Here we show that induction of the repressor element 1-silencing transcription factor (REST; also known as neuron-restrictive silencer factor, NRSF) is a universal feature of normal ageing in human cortical and hippocampal neurons. REST is lost, however, in mild cognitive impairment and Alzheimer's disease. Chromatin immunoprecipitation with deep sequencing and expression analysis show that REST represses genes that promote cell death and Alzheimer's disease pathology, and induces the expression of stress response genes. Moreover, REST potently protects neurons from oxidative stress and amyloid β-protein toxicity, and conditional deletion of REST in the mouse brain leads to age-related neurodegeneration. A functional orthologue of REST, Caenorhabditis elegans SPR-4, also protects against oxidative stress and amyloid β-protein toxicity. During normal ageing, REST is induced in part by cell non-autonomous Wnt signalling. However, in Alzheimer's disease, frontotemporal dementia and dementia with Lewy bodies, REST is lost from the nucleus and appears in autophagosomes together with pathological misfolded proteins. Finally, REST levels during ageing are closely correlated with cognitive preservation and longevity. Thus, the activation state of REST may distinguish neuroprotection from neurodegeneration in the ageing brain.

Survival or Revival: Long-Term Preservation Induces a Reversible Viable but Non-Culturable State in Methane-Oxidizing Bacteria

PubMed Central

Hoefman, Sven; Van Hoorde, Koenraad; Boon, Nico; Vandamme, Peter; De Vos, Paul; Heylen, Kim

2012-01-01

Knowledge on long-term preservation of micro-organisms is limited and research in the field is scarce despite its importance for microbial biodiversity and biotechnological innovation. Preservation of fastidious organisms such as methane-oxidizing bacteria (MOB) has proven difficult. Most MOB do not survive lyophilization and only some can be cryopreserved successfully for short periods. A large-scale study was designed for a diverse set of MOB applying fifteen cryopreservation or lyophilization conditions. After three, six and twelve months of preservation, the viability (via live-dead flow cytometry) and culturability (via most-probable number analysis and plating) of the cells were assessed. All strains could be cryopreserved without a significant loss in culturability using 1% trehalose in 10-fold diluted TSB (TT) as preservation medium and 5% DMSO as cryoprotectant. Several other cryopreservation and lyophilization conditions, all of which involved the use of TT medium, also allowed successful preservation but showed a considerable loss in culturability. We demonstrate here that most of these non-culturables survived preservation according to viability assessment indicating that preservation induces a viable but non-culturable (VBNC) state in a significant fraction of cells. Since this state is reversible, these findings have major implications shifting the emphasis from survival to revival of cells in a preservation protocol. We showed that MOB cells could be significantly resuscitated from the VBNC state using the TT preservation medium. PMID:22539945

Hematopoietic stem cells are acutely sensitive to Acd shelterin gene inactivation

PubMed Central

Jones, Morgan; Osawa, Gail; Regal, Joshua A.; Weinberg, Daniel N.; Taggart, James; Kocak, Hande; Friedman, Ann; Ferguson, David O.; Keegan, Catherine E.; Maillard, Ivan

2013-01-01

The shelterin complex plays dual functions in telomere homeostasis by recruiting telomerase and preventing the activation of a DNA damage response at telomeric ends. Somatic stem cells require telomerase activity, as evidenced by progressive stem cell loss leading to bone marrow failure in hereditary dyskeratosis congenita. Recent work demonstrates that dyskeratosis congenita can also arise from mutations in specific shelterin genes, although little is known about shelterin functions in somatic stem cells. We found that mouse hematopoietic stem cells (HSCs) are acutely sensitive to inactivation of the shelterin gene Acd, encoding TPP1. Homozygosity for a hypomorphic acd allele preserved the emergence and expansion of fetal HSCs but led to profoundly defective function in transplantation assays. Upon complete Acd inactivation, HSCs expressed p53 target genes, underwent cell cycle arrest, and were severely depleted within days, leading to hematopoietic failure. TPP1 loss induced increased telomeric fusion events in bone marrow progenitors. However, unlike in epidermal stem cells, p53 deficiency did not rescue TPP1-deficient HSCs, indicating that shelterin dysfunction has unique effects in different stem cell populations. Because the consequences of telomere shortening are progressive and unsynchronized, acute loss of shelterin function represents an attractive alternative for studying telomere crisis in hematopoietic progenitors. PMID:24316971

Deterministic Migration-Based Separation of White Blood Cells.

PubMed

Kim, Byeongyeon; Choi, Young Joon; Seo, Hyekyung; Shin, Eui-Cheol; Choi, Sungyoung

2016-10-01

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Intermittent fasting preserves beta-cell mass in obesity-induced diabetes via the autophagy-lysosome pathway.

PubMed

Liu, Haiyan; Javaheri, Ali; Godar, Rebecca J; Murphy, John; Ma, Xiucui; Rohatgi, Nidhi; Mahadevan, Jana; Hyrc, Krzysztof; Saftig, Paul; Marshall, Connie; McDaniel, Michael L; Remedi, Maria S; Razani, Babak; Urano, Fumihiko; Diwan, Abhinav

2017-01-01

Obesity-induced diabetes is characterized by hyperglycemia, insulin resistance, and progressive beta cell failure. In islets of mice with obesity-induced diabetes, we observe increased beta cell death and impaired autophagic flux. We hypothesized that intermittent fasting, a clinically sustainable therapeutic strategy, stimulates autophagic flux to ameliorate obesity-induced diabetes. Our data show that despite continued high-fat intake, intermittent fasting restores autophagic flux in islets and improves glucose tolerance by enhancing glucose-stimulated insulin secretion, beta cell survival, and nuclear expression of NEUROG3, a marker of pancreatic regeneration. In contrast, intermittent fasting does not rescue beta-cell death or induce NEUROG3 expression in obese mice with lysosomal dysfunction secondary to deficiency of the lysosomal membrane protein, LAMP2 or haplo-insufficiency of BECN1/Beclin 1, a protein critical for autophagosome formation. Moreover, intermittent fasting is sufficient to provoke beta cell death in nonobese lamp2 null mice, attesting to a critical role for lysosome function in beta cell homeostasis under fasting conditions. Beta cells in intermittently-fasted LAMP2- or BECN1-deficient mice exhibit markers of autophagic failure with accumulation of damaged mitochondria and upregulation of oxidative stress. Thus, intermittent fasting preserves organelle quality via the autophagy-lysosome pathway to enhance beta cell survival and stimulates markers of regeneration in obesity-induced diabetes.

Comparison of surgical outcomes after pneumonectomy and pulmonary function-preserving surgery for non-small cell lung cancer.

PubMed

Higuchi, Mitsunori; Takagi, Hironori; Ozaki, Yuki; Inoue, Takuya; Watanabe, Yuzuru; Yamaura, Takumi; Fukuhara, Mitsuro; Muto, Satoshi; Okabe, Naoyuki; Matsumura, Yuki; Hasegawa, Takeo; Osugi, Jun; Hoshino, Mika; Shio, Yutaka; Suzuki, Hiroyuki

2018-04-17

According to previous reports, lobectomy with bronchoplasty or angioplasty is a more feasible surgery than pneumonectomy for central-type non-small cell lung cancer. However, few studies have compared both the short- and long-term outcomes between pneumonectomy and pulmonary function-preserving surgery. From January 2004 to December 2015, 18 patients underwent pneumonectomy (Group PN) and 12 patients underwent pulmonary function-preserving surgery (group PS) at Fukushima Medical University Hospital. Clinicopathological factors were statistically compared between the two groups. The operation times in Group PN and Group PS were 285.9±27.9 and 271.3±99.2 min, respectively (p=0.613), while the amounts of intraoperative bleeding were 324.8±248.9 and 164.5±116.6 g, respectively (p=0.020). The duration of chest drainage and hospitalization after surgery in both groups were not significantly different but there was a tendency toward shorter periods of these durations in Group PS. The 5-year disease-free survival (DFS) rate in Group PN and PS was 51.4% and 74.1%, respectively, without a significant difference (p=0.298). The 5-year overall survival (OS) rate in Group PN and PS was 52.5% and 56.6%, respectively, also without a significant difference (p=0.748). The 5-year OS rate was inferior to the 5-year DFS rate in Group PS, and the 5-year OS rate was not better than the 5-year DFS rate in Group PN. The short-term results were better in Group PS than PN. However, the long-term results in both groups were similar. Other causes of death influenced OS in both groups; this result might have been affected by the surgical procedures.

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing

PubMed Central

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A.; Alves, Paula M.

2016-01-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. Significance The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. PMID:27025693

Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing.

PubMed

Correia, Cláudia; Koshkin, Alexey; Carido, Madalena; Espinha, Nuno; Šarić, Tomo; Lima, Pedro A; Serra, Margarida; Alves, Paula M

2016-05-01

To fully explore the potential of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), efficient methods for storage and shipment of these cells are required. Here, we evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage. We showed that two-dimensional (2D) monolayers of hPSC-CMs can be efficiently stored at 4°C for 3 days without compromising cell viability. However, cell viability decreased when the cold storage interval was extended to 7 days. We demonstrated that hPSC-CMs are more resistant to prolonged hypothermic storage-induced cell injury in three-dimensional aggregates than in 2D monolayers, showing high cell recoveries (>70%) after 7 days of storage. Importantly, hPSC-CMs maintained their typical (ultra)structure, gene and protein expression profile, electrophysiological profiles, and drug responsiveness. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic/industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. This study established effective clinically compatible strategies for cold (4°C) storage of hPSC-CMs cultured as two-dimensional (2D) monolayers and three-dimensional (3D) aggregates. Cell recovery of 2D monolayers of hPSC-CMs was found to be dependent on the time of storage, and 3D cell aggregates were more resistant to prolonged cold storage than 2D monolayers. Of note, it was demonstrated that 7 days of cold storage did not affect hPSC-CM ultrastructure, phenotype, or function. This study provides important insights into the cold preservation of PSC-CMs that could be valuable in improving global commercial distribution of hPSC-CMs. ©AlphaMed Press.

Comparison of Different Adult Stem Cell Types for Treatment of Myocardial Ischemia

PubMed Central

van der Bogt, Koen E.A.; Sheikh, Ahmad Y.; Schrepfer, Sonja; Hoyt, Grant; Cao, Feng; Ransohoff, Katie; Swijnenburg, Rutger-Jan; Pearl, Jeremy; Fischbein, Michael; Contag, Christopher H.; Robbins, Robert C.; Wu, Joseph C.

2013-01-01

Introduction A comparative analysis of the efficacy of different cell candidates for the treatment of heart disease remains to be described. This study is designed to evaluate the therapeutic efficacy of 4 cell types in a murine model of myocardial infarction. Methods Bone marrow mononuclear cells (MN), mesenchymal stem cells (MSC), skeletal myoblasts (SkMb) and fibroblasts (Fibro) were isolated from male L2G transgenic mice (FVB background) that constitutively express firefly luciferase (Fluc) and green fluorescence protein (GFP). Cells were characterized by flow cytometry, bioluminescence imaging (BLI), and luminometry. Female FVB mice (n=60) underwent LAD ligation and were randomized into 5 groups to intramyocardially receive one cell type (5 × 105) or PBS as control. Cell survival was measured in vivo by BLI and ex vivo by TaqMan PCR at week 6. Cardiac function was assessed by echocardiography and invasive hemodynamic measurements were made at week 6. Results Fluc expression correlated with the cell number in all groups (r2 >0.93). In vivo BLI revealed acute donor cell death of MSC, SkMb, and Fibro within 3 weeks after transplantation. By contrast, cardiac signals were still present after 6 weeks in the MN group, as confirmed by TaqMan PCR (P<0.01). Echocardiography showed significant preservation of fractional shortening in the MN group compared to controls (P<0.05). Measurements of left ventricular end-systolic/diastolic volumes revealed that the least amount of ventricular dilatation occurred in the MN group (P<0.05). Histology confirmed the presence of MN, although there was no evidence of transdifferentiation by donor MN into cardiomyocytes. Conclusion This is the first study to directly compare a variety of cell candidates for myocardial therapy. Compared to MSC, SkMB, and Fibro, our results suggest that MN cells exhibit a more favorable survival pattern, which translates into a more robust preservation of cardiac function. PMID:18824743

Mangiferin protects mitochondrial function by preserving mitochondrial hexokinase-II in vessel endothelial cells.

PubMed

Song, Junna; Li, Yi; Song, Junmei; Hou, Fangjie; Liu, Baolin; Li, Aiying

2017-07-01

Hexokinase-II (HK-II) confers protection against cell death and this study was designed to investigate the effect of mangiferin on the regulation of mitochondrial HK-II. In vessel endothelial cells, saturated fatty acid palmitate (PA) stimulation induced HK-II detachment from mitochondria due to cellular acidification. Mangiferin reduced lactate accumulation by improving pyruvate dehydrogenase activity, promoted Akt translocation to HK-II and prevented HK-II detachment from mitochondria. Knockdown of Akt2 diminished the protective effect of mangiferin on mitochondrial HK-II, confirming the role of Akt in the regulation of HK-II. Mangiferin prevented mitochondrial permeability transition pore opening, restored mitochondrial membrane potential and thereby protected cell from apoptosis. In high-fat diet fed mice, oral administration of mangiferin induced Akt phosphorylation, increased HK-II binding to mitochondria and resultantly protected vessel endothelial function, demonstrating its protective effect on endothelial integrity in vivo. This finding provided a novel strategy for the protection of mitochondrial function in the endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein and cell micropatterning and its integration with micro/nanoparticles assembly.

PubMed

Yap, F L; Zhang, Y

2007-01-15

Micropatterning of proteins and cells has become very popular over the past decade due to its importance in the development of biosensors, microarrays, tissue engineering and cellular studies. This article reviews the techniques developed for protein and cell micropatterning and its biomedical applications. The prospect of integrating micro and nanoparticles with protein and cell micropatterning is discussed. The micro/nanoparticles are assembled into patterns and form the substrate for proteins and cell attachment. The assembled particles create a micro or nanotopography, depending on the size of the particles employed. The nonplanar structure can increase the surface area for biomolecules attachment and therefore enhance the sensitivity for detection in biosensors. Furthermore, a nanostructured substrate can influence the conformation and functionality of protein attached to it, while cellular response in terms of morphology, adhesion, proliferation, differentiation, etc. can be affected by a surface expressing micro or nanoscale structures. Proteins and cells tend to lose their normal functions upon attachment to substrate. By recognizing the types of topography that are favourable for preserving proteins and cell behaviour, and integrating it with micropattering will lead to the development of functional protein and cell patterns.

The intestinal B-cell response in celiac disease

PubMed Central

Mesin, Luka; Sollid, Ludvig M.; Niro, Roberto Di

2012-01-01

The function of intestinal immunity is to provide protection toward pathogens while preserving the composition of the microflora and tolerance to orally fed nutrients. This is achieved via a number of tightly regulated mechanisms including production of IgA antibodies by intestinal plasma cells. Celiac disease is a common gut disorder caused by a dysfunctional immune regulation as signified, among other features, by a massive intestinal IgA autoantibody response. Here we review the current knowledge of this B-cell response and how it is induced, and we discuss key questions to be addressed in future research. PMID:23060888

Hydrogen Supplementation of Preservation Solution Improves Viability of Osteochondral Grafts

PubMed Central

Yamada, Takuya; Onuma, Kenji; Kuzuno, Jun; Ujihira, Masanobu; Kurokawa, Ryosuke; Sakai, Rina; Takaso, Masashi

2014-01-01

Allogenic osteochondral tissue (OCT) is used for the treatment of large cartilage defects. Typically, OCTs collected during the disease-screening period are preserved at 4°C; however, the gradual reduction in cell viability during cold preservation adversely affects transplantation outcomes. Therefore, improved storage methods that maintain the cell viability of OCTs are needed to increase the availability of high-quality OCTs and improve treatment outcomes. Here, we evaluated whether long-term hydrogen delivery to preservation solution improved the viability of rat OCTs during cold preservation. Hydrogen-supplemented Dulbecco's Modified Eagles Medium (DMEM) and University of Wisconsin (UW) solution both significantly improved the cell viability of OCTs during preservation at 4°C for 21 days compared to nonsupplemented media. However, the long-term cold preservation of OCTs in DMEM containing hydrogen was associated with the most optimal maintenance of chondrocytes with respect to viability and morphology. Our findings demonstrate that OCTs preserved in DMEM supplemented with hydrogen are a promising material for the repair of large cartilage defects in the clinical setting. PMID:25506061

Long-term topical application of preservative-free prostaglandin analogues evokes macrophage infiltration in the ocular adnexa.

PubMed

Trzeciecka, Anna; Paterno, J Jussi; Toropainen, Elisa; Koskela, Ali; Podracka, Lucia; Korhonen, Eveliina; Kauppinen, Anu; Kaarniranta, Kai; Smedowski, Adrian

2016-10-05

Success of the long-term glaucoma therapy and preservation of the visual function strongly depend on patients' compliance which may be affected by the inconvenience of treatment and its side effects. Recently, introduction of preservative-free anti-glaucoma agents has become an important step towards improved glaucoma care by eliminating the negative effects of preservatives on the eye surface. Although, newly developed eye drop formulations do not contain standard preservatives, they still can be harmful to ocular surface due to other excipients. In this study, we compared tolerability of commercial preservative-free (pf) prostaglandin analogues (pf tafluprost, pf latanoprost and pf bimatoprost) in long-term topical application in rabbits in vivo. We found that after eight weeks treatment, pf latanoprost was the worst tolerated among the tested drops. It expressed increased conjunctival redness and blinking frequency. Furthermore, it caused increased LDH release in the aqueous humour, infiltration of macrophages in the eyelids and visible defects in conjunctival goblet cells. However, we did not detect increased levels of inflammatory markers in the tear fluid or in the aqueous humour. Based on our study, we suspect that these negative effects are related to excipients included in pf latanoprost formulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Biomanufacturing of Therapeutic Cells: State of the Art, Current Challenges, and Future Perspectives.

PubMed

Roh, Kyung-Ho; Nerem, Robert M; Roy, Krishnendu

2016-06-07

Stem cells and other functionally defined therapeutic cells (e.g., T cells) are promising to bring hope of a permanent cure for diseases and disorders that currently cannot be cured by conventional drugs or biological molecules. This paradigm shift in modern medicine of using cells as novel therapeutics can be realized only if suitable manufacturing technologies for large-scale, cost-effective, reproducible production of high-quality cells can be developed. Here we review the state of the art in therapeutic cell manufacturing, including cell purification and isolation, activation and differentiation, genetic modification, expansion, packaging, and preservation. We identify current challenges and discuss opportunities to overcome them such that cell therapies become highly effective, safe, and predictively reproducible while at the same time becoming affordable and widely available.

Sirtuins, Bioageing, and Cancer

PubMed Central

McGuinness, D.; McGuinness, D. H.; McCaul, J. A.; Shiels, P. G.

2011-01-01

The Sirtuins are a family of orthologues of yeast Sir2 found in a wide range of organisms from bacteria to man. They display a high degree of conservation between species, in both sequence and function, indicative of their key biochemical roles. Sirtuins are heavily implicated in cell cycle, cell division, transcription regulation, and metabolism, which places the various family members at critical junctures in cellular metabolism. Typically, Sirtuins have been implicated in the preservation of genomic stability and in the prolongation of lifespan though many of their target interactions remain unknown. Sirtuins play key roles in tumourigenesis, as some have tumour-suppressor functions and others influence tumours through their control of the metabolic state of the cell. Their links to ageing have also highlighted involvement in various age-related and degenerative diseases. Here, we discuss the current understanding of the role of Sirtuins in age-related diseases while taking a closer look at their roles and functions in maintaining genomic stability and their influence on telomerase and telomere function. PMID:21766030

A Parvovirus B19 synthetic genome: sequence features and functional competence.

PubMed

Manaresi, Elisabetta; Conti, Ilaria; Bua, Gloria; Bonvicini, Francesca; Gallinella, Giorgio

2017-08-01

Central to genetic studies for Parvovirus B19 (B19V) is the availability of genomic clones that may possess functional competence and ability to generate infectious virus. In our study, we established a new model genetic system for Parvovirus B19. A synthetic approach was followed, by design of a reference genome sequence, by generation of a corresponding artificial construct and its molecular cloning in a complete and functional form, and by setup of an efficient strategy to generate infectious virus, via transfection in UT7/EpoS1 cells and amplification in erythroid progenitor cells. The synthetic genome was able to generate virus with biological properties paralleling those of native virus, its infectious activity being dependent on the preservation of self-complementarity and sequence heterogeneity within the terminal regions. A virus of defined genome sequence, obtained from controlled cell culture conditions, can constitute a reference tool for investigation of the structural and functional characteristics of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Preservation of high glycolytic phenotype by establishing new acute lymphoblastic leukemia cell lines at physiologic oxygen concentration

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

Sheard, Michael A., E-mail: msheard@chla.usc.edu; Ghent, Matthew V., E-mail: mattghent@gmail.com; Cabral, Daniel J., E-mail: dcabral14@gmail.com

2015-05-15

Cancer cells typically exhibit increased glycolysis and decreased mitochondrial oxidative phosphorylation, and they continue to exhibit some elevation in glycolysis even under aerobic conditions. However, it is unclear whether cancer cell lines employ a high level of glycolysis comparable to that of the original cancers from which they were derived, even if their culture conditions are changed to physiologically relevant oxygen concentrations. From three childhood acute lymphoblastic leukemia (ALL) patients we established three new pairs of cell lines in both atmospheric (20%) and physiologic (bone marrow level, 5%) oxygen concentrations. Cell lines established in 20% oxygen exhibited lower proliferation, survival,more » expression of glycolysis genes, glucose consumption, and lactate production. Interestingly, the effects of oxygen concentration used during cell line initiation were only partially reversible when established cell cultures were switched from one oxygen concentration to another for eight weeks. These observations indicate that ALL cell lines established at atmospheric oxygen concentration can exhibit relatively low levels of glycolysis and these levels are semi-permanent, suggesting that physiologic oxygen concentrations may be needed from the time of cell line initiation to preserve the high level of glycolysis commonly exhibited by leukemias in vivo. - Highlights: • Establishing new ALL cell lines in 5% oxygen resulted in higher glycolytic expression and function. • Establishing new ALL cell lines in 5% oxygen resulted in higher proliferation and lower cell death. • The divergent metabolic phenotypes selected in 5% and 20% oxygen are semi-permanent.« less

Cell-Permeable Parkin Proteins Suppress Parkinson Disease-Associated Phenotypes in Cultured Cells and Animals

PubMed Central

Duong, Tam; Kim, Jaetaek; Ruley, H. Earl; Jo, Daewoong

2014-01-01

Parkinson’s disease (PD) is a neurodegenerative disorder of complex etiology characterized by the selective loss of dopaminergic neurons, particularly in the substantia nigra. Parkin, a tightly regulated E3 ubiquitin ligase, promotes the survival of dopaminergic neurons in both PD and Parkinsonian syndromes induced by acute exposures to neurotoxic agents. The present study assessed the potential of cell-permeable parkin (CP-Parkin) as a neuroprotective agent. Cellular uptake and tissue penetration of recombinant, enzymatically active parkin was markedly enhanced by the addition of a hydrophobic macromolecule transduction domain (MTD). The resulting CP-Parkin proteins (HPM13 and PM10) suppressed dopaminergic neuronal toxicity in cells and mice exposed to 6-hydroxydopamine (6-OHDH) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). These included enhanced survival and dopamine expression in cultured CATH.a and SH-SY5Y neuronal cells; and protection against MPTP-induced damage in mice, notably preservation of tyrosine hydroxylase-positive cells with enhanced dopamine expression in the striatum and midbrain, and preservation of gross motor function. These results demonstrate that CP-Parkin proteins can compensate for intrinsic limitations in the parkin response and provide a therapeutic strategy to augment parkin activity in vivo. PMID:25019626

Physiological actions of preservative agents: prospective of use of modern microbiological techniques in assessing microbial behaviour in food preservation.

PubMed

Brul, Stanley; Coote, Peter; Oomes, Suus; Mensonides, Femke; Hellingwerf, Klaas; Klis, Frans

2002-11-15

In this mini-review, various aspects of homeostasis of microbial cells and its perturbation by antimicrobial agents will be discussed. First, outlining the position that the physiological studies on microbial behaviour using the modern molecular tools should have in food science sets the scene for the studies. Subsequently, the advent of functional genomics is discussed that allows full coverage of cellular reactions at unprecedented levels. Examples of weak organic acid resistance, the stress response against natural antimicrobial agents and responses against physicochemical factors show how we can now "open the black box" that microbes are, look inside and begin to understand how different cellular signalling cables are wired together. Using the analogy with machines, it will be indicated how the use of various signalling systems depends on the availability of substrates "fuel" to let the systems act in the context of the minimum energetic requirement cells have to let their housekeeping systems run. The outlook illustrates how new insights might be used to device knowledge-based rather than empirical combinations of preservation systems and how risk assessment models might be deviced that link the mechanistic insight to risk distributions of events in food manufacturing.

Decellularized Swine Dental Pulp as a Bioscaffold for Pulp Regeneration

PubMed Central

Hu, Lei; Gao, Zhenhua; Zhu, Zhao; Zhang, Chunmei; Wang, Jinsong

2017-01-01

Endodontic regeneration shows promise in treating dental pulp diseases; however, no suitable scaffolds exist for pulp regeneration. Acellular natural extracellular matrix (ECM) is a favorable scaffold for tissue regeneration since the anatomical structure and ECM of the natural tissues or organs are well-preserved. Xenogeneic ECM is superior to autologous or allogeneic ECM in tissue engineering for its unlimited resources. This study investigated the characteristics of decellularized dental pulp ECM from swine and evaluated whether it could mediate pulp regeneration. Dental pulps were acquired from the mandible anterior teeth of swine 12 months of age and decellularized with 10% sodium dodecyl sulfate (SDS) combined with Triton X-100. Pulp regeneration was conducted by seeding human dental pulp stem cells into decellularized pulp and transplanted subcutaneously into nude mice for 8 weeks. The decellularized pulp demonstrated preserved natural shape and structure without any cellular components. Histological analysis showed excellent ECM preservation and pulp-like tissue, and newly formed mineralized tissues were regenerated after being transplanted in vivo. In conclusion, decellularized swine dental pulp maintains ECM components favoring stem cell proliferation and differentiation, thus representing a suitable scaffold for improving clinical outcomes and functions of teeth with dental pulp diseases. PMID:29387727

Appropriate therapy for type 2 diabetes mellitus in view of pancreatic β-cell glucose toxicity: "the earlier, the better".

PubMed

Kaneto, Hideaki; Matsuoka, Taka-Aki; Kimura, Tomohiko; Obata, Atsushi; Shimoda, Masashi; Kamei, Shinji; Mune, Tomoatsu; Kaku, Kohei

2016-03-01

Pancreatic β-cells secrete insulin when blood glucose levels become high; however, when β-cells are chronically exposed to hyperglycemia, β-cell function gradually deteriorates, which is known as β-cell glucose toxicity. In the diabetic state, nuclear expression of the pancreatic transcription factors pancreatic and duodenal homeobox 1 (PDX-1) and v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein A (MafA) is decreased. In addition, incretin receptor expression in β-cells is decreased, which is likely involved in the impairment of incretin effects in diabetes. Clinically, it is important to select appropriate therapy for type 2 diabetes mellitus (T2DM) so that β-cell function can be preserved. In addition, when appropriate pharmacological interventions against β-cell glucose toxicity are started at the early stages of diabetes, β-cell function is substantially restored, which is not observed if treatment is started at advanced stages. These observations indicate that it is likely that downregulation of pancreatic transcription factors and/or incretin receptors is involved in β-cell dysfunction observed in T2DM and it is very important to start appropriate pharmacological intervention against β-cell glucose toxicity in the early stages of diabetes. © 2015 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

Predictors of splenic function preservation in children with sickle cell anemia treated with hydroxyurea.

PubMed

Nottage, Kerri A; Ware, Russell E; Winter, Bryan; Smeltzer, Matthew; Wang, Winfred C; Hankins, Jane S; Dertinger, Stephen D; Shulkin, Barry; Aygun, Banu

2014-11-01

More than 90% of children with sickle cell anemia (SCA) lose splenic function by the age of 2 yrs. Splenic function may improve with hydroxyurea, but previous studies are conflicting. We prospectively evaluated the effect of hydroxyurea on splenic filtrative function. Children with SCA enrolled in the Hydroxyurea Study of Long-Term Effects (HUSTLE-NCT00305175) underwent clinical evaluations including Tc(99) m liver-spleen (LS) scans before hydroxyurea initiation and after 3 yrs of treatment to maximum tolerated dose (MTD). LS scans were classified as follows: no uptake, <10% uptake, decreased but ≥10% uptake, and normal. Mean age (N = 40) was 9.1 yrs, range 2.3-17.0. After 3 yrs of treatment, 13 (33%) had uptake on LS scan. These 13 children were younger (median age 6.0 vs. 10.6 yrs, P = 0.008), had a higher HbF at baseline (mean 10.2% vs. 5.8%, P = 0.004) and after 3 yrs (22.9% vs. 13.9%, P < 0.001), achieved MTD more rapidly (median 288 vs. 358 d, P = 0.021), and were more likely to have baseline splenic uptake (P < 0.001). Hydroxyurea at MTD is associated with preserved or improved splenic filtrative function, with 33% demonstrating LS scan uptake after 3 yrs. Younger age, higher %HbF, and baseline splenic function are associated with a favorable outcome. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Glycogen metabolism protects against metabolic insult to preserve carotid body function during glucose deprivation

PubMed Central

Holmes, Andrew P; Turner, Philip J; Carter, Paul; Leadbeater, Wendy; Ray, Clare J; Hauton, David; Buckler, Keith J; Kumar, Prem

2014-01-01

The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ∼33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge. PMID:25063821

The functions and clinical applications of tumor-derived exosomes

PubMed Central

Shao, Yingkuan; Shen, Yanwei; Chen, Ting; Xu, Fei; Chen, Xuewen; Zheng, Shu

2016-01-01

Exosomes are extracellular vesicles with diameters ranging from 30 to 150 nm. They can be secreted by all cell types and transfer information in the form of their contents, which include proteins, lipids and nucleic acids, to other cells throughout the body. They have roles in normal physiological processes as well as in disease development. Here, we review recent findings regarding tumor-derived exosomes, including methods for their extraction and preservation. We also describe the actions of exosomes in tumorigenesis. The exosomal antigen-presenting effect during antitumor immune responses and its suppressive function in immune tolerance are discussed. Finally, we describe the potential application of exosomes to cancer therapy and liquid biopsy. PMID:27517627

Is antioxidant potential of the mitochondrial targeted ubiquinone derivative MitoQ conserved in cells lacking mtDNA?

PubMed

Lu, Chao; Zhang, Dawei; Whiteman, Matthew; Armstrong, Jeffrey S

2008-03-01

MitoQ has been developed as a mitochondrial targeted antioxidant for diseases associated with oxidative stress. Here we show that MitoQ blocks the generation of reactive oxygen species (ROS) and mitochondrial protein thiol oxidation, and preserves mitochondrial function and ultrastructure after glutathione (GSH) depletion. Furthermore, the antioxidant effect of MitoQ is conserved in cells lacking mitochondrial DNA, indicating that its antioxidant properties do not depend on a functional electron transport chain (ETC). Our results elucidate the antioxidant mechanism of MitoQ and suggest that it may be a useful therapeutic for disorders associated with a dysfunctional ETC and increased ROS production.

Scalable human ES culture for therapeutic use: propagation, differentiation, genetic modification and regulatory issues.

PubMed

Rao, M

2008-01-01

Embryonic stem cells unlike most adult stem cell populations can replicate indefinitely while preserving genetic, epigenetic, mitochondrial and functional profiles. ESCs are therefore an excellent candidate cell type for providing a bank of cells for allogenic therapy and for introducing targeted genetic modifications for therapeutic intervention. This ability of prolonged self-renewal of stem cells and the unique advantages that this offers for gene therapy, discovery efforts, cell replacement, personalized medicine and other more direct applications requires the resolution of several important manufacturing, gene targeting and regulatory issues. In this review, we assess some of the advance made in developing scalable culture systems, improvement in vector design and gene insertion technology and the changing regulatory landscape.

INPP5E Preserves Genomic Stability through Regulation of Mitosis.

PubMed

Sierra Potchanant, Elizabeth A; Cerabona, Donna; Sater, Zahi Abdul; He, Ying; Sun, Zejin; Gehlhausen, Jeff; Nalepa, Grzegorz

2017-03-15

The partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development. Copyright © 2017 Sierra Potchanant et al.

Inner retinal preservation in rat models of retinal degeneration implanted with subretinal photovoltaic arrays.

PubMed

Light, Jacob G; Fransen, James W; Adekunle, Adewumi N; Adkins, Alice; Pangeni, Gobinda; Loudin, James; Mathieson, Keith; Palanker, Daniel V; McCall, Maureen A; Pardue, Machelle T

2014-11-01

Photovoltaic arrays (PVA) implanted into the subretinal space of patients with retinitis pigmentosa (RP) are designed to electrically stimulate the remaining inner retinal circuitry in response to incident light, thereby recreating a visual signal when photoreceptor function declines or is lost. Preservation of inner retinal circuitry is critical to the fidelity of this transmitted signal to ganglion cells and beyond to higher visual targets. Post-implantation loss of retinal interneurons or excessive glial scarring could diminish and/or eliminate PVA-evoked signal transmission. As such, assessing the morphology of the inner retina in RP animal models with subretinal PVAs is an important step in defining biocompatibility and predicting success of signal transmission. In this study, we used immunohistochemical methods to qualitatively and quantitatively compare inner retinal morphology after the implantation of a PVA in two RP models: the Royal College of Surgeons (RCS) or transgenic S334ter-line 3 (S334ter-3) rhodopsin mutant rat. Two PVA designs were compared. In the RCS rat, we implanted devices in the subretinal space at 4 weeks of age and histologically examined them at 8 weeks of age and found inner retinal morphology preservation with both PVA devices. In the S334ter-3 rat, we implanted devices at 6-12 weeks of age and again, inner retinal morphology was generally preserved with either PVA design 16-26 weeks post-implantation. Specifically, the length of rod bipolar cells and numbers of cholinergic amacrine cells were maintained along with their characteristic inner plexiform lamination patterns. Throughout the implanted retinas we found nonspecific glial reaction, but none showed additional glial scarring at the implant site. Our results indicate that subretinally implanted PVAs are well-tolerated in rodent RP models and that the inner retinal circuitry is preserved, consistent with our published results showing implant-evoked signal transmission. Published by Elsevier Ltd.

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

PubMed

Orynbayeva, Zulfiya; Sensenig, Richard; Polyak, Boris

2015-05-01

To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

Cadaveric renal preservation with hyperosmolar, intracellular hypothermic washout solution and cold storage.

PubMed

Diethelm, A G; Blackstone, E H

1978-07-01

All 54 kidneys obtained from heart-beating cadavers functioned when preserved by a brief washout using a hypothermic, hyperosmolar, hyperkalemic perfusate, followed by cold storage. The duration of preservation ranged from two hours and 57 minutes to 39 hours and 47 minutes. Two other kidneys retrieved from a nonheart-beating cadaver and preserved by the same technique failed to function because of irreversible acute tubular necrosis. Fifty-six consecutive transplant patients were divided into four groups according to the period of preservation. There was no correlation between graft rejection, frequency of post-transplant dialysis, long term graft function and survival time, when the duration of preservation was less than 24 hours. The advantages of this technique included technical simplicity, low cost, minimal risk of graft infection and easy transportation. The two primary disadvantages were an apparent 24 to 30 hour limit of organ preservation with prompt function and the inability to determine intrarenal perfusion pressure during preservation, thereby missing an important parameter of graft viability.

Doxorubicin Action on Mitochondria: Relevance to Osteosarcoma Therapy?

PubMed

Armstrong, Jo; Dass, Crispin R

2018-01-01

The mitochondria may very well determine the final commitment of the cell to death, particularly in times of energy stress. Cancer chemotherapeutics such as the anthracycline doxorubicin perturb mitochondrial structure and function in tumour cells, as evidenced in osteosarcoma, for which doxorubicin is used clinically as frontline therapy. This same mechanism of cell inhibition is also pertinent to doxorubicin's primary cause of side-effects, that to the cardiac tissue, culminating in such dire events as congestive heart failure. Reactive oxygen species are partly to blame for this effect on the mitochondria, which impact the electron transport chain. As this review highlights that, there is much more to be learnt about the mitochondria and how it is affected by such effective but toxic drugs as doxorubicin. Such information will aid researchers who search for cancer treatment able to preserve mitochondrial number and function in normal cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

microRNA expression in the neural retina: Focus on Müller glia.

PubMed

Quintero, Heberto; Lamas, Mónica

2018-03-01

The neural retina hosts a unique specialized type of macroglial cell that not only preserves retinal homeostasis, function, and integrity but also may serve as a source of new neurons during regenerative processes: the Müller cell. Precise microRNA-driven mechanisms of gene regulation impel and direct the processes of Müller glia lineage acquisition from retinal progenitors during development, the triggering of their response to retinal degeneration and, in some cases, Müller cell reprogramming and regenerative events. In this review we survey the recent reports describing, through functional assays, the regulatory role of microRNAs in Müller cell physiology, differentiation potential, and retinal pathology. We discuss also the evidence based on expression analysis that points out the relevance of a Müller glia-specific microRNA signature that would orchestrate these processes. © 2017 Wiley Periodicals, Inc.

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

PubMed

Autour, Alexis; C Y Jeng, Sunny; D Cawte, Adam; Abdolahzadeh, Amir; Galli, Angela; Panchapakesan, Shanker S S; Rueda, David; Ryckelynck, Michael; Unrau, Peter J

2018-02-13

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Extraction of nucleic acids from yeast cells and plant tissues using ethanol as medium for sample preservation and cell disruption.

PubMed

Linke, Bettina; Schröder, Kersten; Arter, Juliane; Gasperazzo, Tatiana; Woehlecke, Holger; Ehwald, Rudolf

2010-09-01

Here we report that dehydrated ethanol is an excellent medium for both in situ preservation of nucleic acids and cell disruption of plant and yeast cells. Cell disruption was strongly facilitated by prior dehydration of the ethanol using dehydrated zeolite. Following removal of ethanol, nucleic acids were extracted from the homogenate pellet using denaturing buffers. The method provided DNA and RNA of high yield and integrity. Whereas cell wall disruption was essential for extraction of DNA and large RNA molecules, smaller molecules such as tRNAs could be selectively extracted from undisrupted, ethanol-treated yeast cells. Our results demonstrate the utility of absolute ethanol for sample fixation, cell membrane and cell wall disruption, as well as preservation of nucleic acids during sample storage.

Hydrogen Gas Ameliorates Hepatic Reperfusion Injury After Prolonged Cold Preservation in Isolated Perfused Rat Liver.

PubMed

Shimada, Shingo; Wakayama, Kenji; Fukai, Moto; Shimamura, Tsuyoshi; Ishikawa, Takahisa; Fukumori, Daisuke; Shibata, Maki; Yamashita, Kenichiro; Kimura, Taichi; Todo, Satoru; Ohsawa, Ikuroh; Taketomi, Akinobu

2016-12-01

Hydrogen gas reduces ischemia and reperfusion injury (IRI) in the liver and other organs. However, the precise mechanism remains elusive. We investigated whether hydrogen gas ameliorated hepatic I/R injury after cold preservation. Rat liver was subjected to 48-h cold storage in University of Wisconsin solution. The graft was reperfused with oxygenated buffer with or without hydrogen at 37° for 90 min on an isolated perfusion apparatus, comprising the H 2 (+) and H 2 (-) groups, respectively. In the control group (CT), grafts were reperfused immediately without preservation. Graft function, injury, and circulatory status were assessed throughout the perfusion. Tissue samples at the end of perfusion were collected to determine histopathology, oxidative stress, and apoptosis. In the H 2 (-) group, IRI was indicated by a higher aspartate aminotransferase (AST), alanine aminotransferase (ALT) leakage, portal resistance, 8-hydroxy-2-deoxyguanosine-positive cell rate, apoptotic index, and endothelial endothelin-1 expression, together with reduced bile production, oxygen consumption, and GSH/GSSG ratio (vs. CT). In the H 2 (+) group, these harmful changes were significantly suppressed [vs. H 2 (-)]. Hydrogen gas reduced hepatic reperfusion injury after prolonged cold preservation via the maintenance of portal flow, by protecting mitochondrial function during the early phase of reperfusion, and via the suppression of oxidative stress and inflammatory cascades thereafter. Copyright © 2016 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

PubMed Central

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

2018-01-01

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

Effect of three decellularisation protocols on the mechanical behaviour and structural properties of sheep aortic valve conduits.

PubMed

Khorramirouz, Reza; Sabetkish, Shabnam; Akbarzadeh, Aram; Muhammadnejad, Ahad; Heidari, Reza; Kajbafzadeh, Abdol-Mohammad

2014-09-01

To determine the best method for decellularisation of aortic valve conduits (AVCs) that efficiently removes the cells while preserving the extracellular matrix (ECM) by examining the valvular and conduit sections separately. Sheep AVCs were decellularised by using three different protocols: detergent-based (1% SDS+1% SDC), detergent and enzyme-based (Triton+EDTA+RNase and DNase), and enzyme-based (Trypsin+RNase and DNase) methods. The efficacy of the decellularisation methods to completely remove the cells while preserving the ECM was evaluated by histological evaluation, scanning electron microscopy (SEM), hydroxyproline analysis, tensile test, and DAPI staining. The detergent-based method completely removed the cells and left the ECM and collagen content in the valve and conduit sections relatively well preserved. The detergent and enzyme-based protocol did not completely remove the cells, but left the collagen content in both sections well preserved. ECM deterioration was observed in the aortic valves (AVs), but the ultrastructure of the conduits was well preserved, with no media distortion. The enzyme-based protocol removed the cells relatively well; however, mild structural distortion and poor collagen content was observed in the AVs. Incomplete cell removal (better than that observed with the detergent and enzyme-based protocol), poor collagen preservation, and mild structural distortion were observed in conduits treated with the enzyme-based method. The results suggested that the detergent-based methods are the most effective protocols for cell removal and ECM preservation of AVCs. The AVCs treated with this detergent-based method may be excellent scaffolds for recellularisation. Copyright © 2014 Medical University of Bialystok. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Changes in Ocular Surface Characteristics after Switching from Benzalkonium Chloride-Preserved Latanoprost to Preservative-Free Tafluprost or Benzalkonium Chloride-Preserved Tafluprost.

PubMed

Tokuda, Naoto; Kitaoka, Yasushi; Matsuzawa, Akiko; Tsukamoto, Ayaka; Sase, Kana; Sakae, Shinsuke; Takagi, Hitoshi

2017-01-01

The aim of the present study was to examine the effects of switching from Latanoprost ophthalmic solution containing a preservative to preservative-free Tafluprost ophthalmic solution or Tafluprost containing a preservative on ocular surfaces. Forty patients (40 eyes) with glaucoma (mean age: 62.0 ± 10.9 years) using Latanoprost with preservative for six months or longer were assigned either to a Tafluprost-containing-preservative group (20 eyes) or preservative-free-Tafluprost group (20 eyes). The intraocular pressure, corneal epithelial barrier function (fluorescein uptake concentration with fluorophotometer FL-500), superficial punctate keratopathy (AD classification), and tear film breakup time (TBUT) were assessed before switching and at 12 weeks after switching. No significant differences in intraocular pressure were noted after switching in either group. Corneal epithelial barrier function was improved significantly after switching in both the Tafluprost-containing-preservative and the preservative-free-Tafluprost groups. There were no significant differences in AD scores after switching in the Tafluprost-containing-preservative group, but significant improvements were noted in the preservative-free-Tafluprost group. No significant differences in TBUT were noted in the Tafluprost-containing-preservative or preservative-free-Tafluprost groups after switching. After switching from preservative Latanoprost to Tafluprost containing-preservative or preservative-free Tafluprost, corneal epithelial barrier function was improved while the intraocular pressure reduction was retained.

Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis.

PubMed

Abdelsamed, Hossam A; Moustaki, Ardiana; Fan, Yiping; Dogra, Pranay; Ghoneim, Hazem E; Zebley, Caitlin C; Triplett, Brandon M; Sekaly, Rafick-Pierre; Youngblood, Ben

2017-06-05

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell-mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (T EM ), and longer-lived central memory (T CM ) and stem cell memory (T SCM ) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7- and IL-15-mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of T CM and T SCM memory cells resulted in phenotypic conversion into T EM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired T EM -associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells. © 2017 Abdelsamed et al.

Early liraglutide treatment is better in glucose control, β-cell function improvement and mass preservation in db/db mice.

PubMed

Shao, Yimin; Yuan, Geheng; Feng, Yan; Zhang, Junqing; Guo, Xiaohui

2014-02-01

Glucagon-like peptide-1 (GLP-1) has been proved to have effects of anti-hyperglycemia and β-cell preservation. However, it is still unclear whether there are differences between early and late GLP-1 intervention in type 2 diabetes mellitus (T2DM). We divided the mice into 5 groups: early treated group (n=7, 8-week old, fasting glucose>10mmol/l), late treated group (n=7, 10-week old, fasting glucose>20mmol/l), early control group (n=7), late control group (n=7) and wild type group (n=7). Treated group was injected with liraglutide (a GLP-1 analog) 300μg/kg bid for 4 weeks, while control group was given saline at the same time. The results showed that compared with control group, food intake and body weight gain were reduced in both early and late treated group (p<0.05), and there was no significance between the two treated groups. Early liraglutide intervention showed better improvements in glucose control, acute insulin response to glucose (AIRg) and disposition index (before vs. after treatment, AIRg 1.01±0.53 vs. 2.98±0.63, disposition index 10.81±0.89 vs. 27.4±2.15) than late intervention (AIRg 0.99±0.02 vs. 1.41±0.32, disposition index 3.47±0.38 vs. 6.43±1.62, p=0.001). The histopathology of the pancreas showed the estimated β-cell mass (BCM) was increased more in early treated group than that in late one (0.03 vs. 0.01g). Expressions of the proliferation related genes PDX-1, MafA and GLP-1 receptor (GLP-1R) in early treated group were 1.81, 2.57 and 1.59 times as much as that in late treated group. In conclusion, early liraglutide intervention was better in glucose control, β-cell function improvement and β-cell mass preservation. Copyright © 2014 Elsevier Inc. All rights reserved.

Lipidomics Characterization of Biosynthetic and Remodeling Pathways of Cardiolipins in Genetically and Nutritionally Manipulated Yeast Cells.

PubMed

Tyurina, Yulia Y; Lou, Wenjia; Qu, Feng; Tyurin, Vladimir A; Mohammadyani, Dariush; Liu, Jenney; Hüttemann, Maik; Frasso, Michael A; Wipf, Peter; Bayir, Hülya; Greenberg, Miriam L; Kagan, Valerian E

2017-01-20

Cardioipins (CLs) are unique tetra-acylated phospholipids of mitochondria and define the bioenergetics and regulatory functions of these organelles. An unresolved paradox is the high uniformity of CL molecular species (tetra-linoleoyl-CL) in the heart, liver, and skeletal muscles-in contrast to their high diversification in the brain. Here, we combined liquid chromatography-mass-spectrometry-based phospholipidomics with genetic and nutritional manipulations to explore CLs' biosynthetic vs postsynthetic remodeling processes in S. cerevisiae yeast cells. By applying the differential phospholipidomics analysis, we evaluated the contribution of Cld1 (CL-specific phospholipase A) and Taz1 (acyl-transferase) as the major regulatory mechanisms of the remodeling process. We further established that nutritional "pressure" by high levels of free fatty acids triggered a massive synthesis of homoacylated molecular species in all classes of phospholipids, resulting in the preponderance of the respective homoacylated CLs. We found that changes in molecular speciation of CLs induced by exogenous C18-fatty acids (C18:1 and C18:2) in wild-type (wt) cells did not occur in any of the remodeling mutant cells, including cld1Δ, taz1Δ, and cld1Δtaz1Δ. Interestingly, molecular speciation of CLs in wt and double mutant cells cld1Δtaz1Δ was markedly different. Given that the bioenergetics functions are preserved in the double mutant, this suggests that the accumulated MLCL-rather than the changed CL speciation-are the likely major contributors to the mitochondrial dysfunction in taz1Δ mutant cells (also characteristic of Barth syndrome). Biochemical studies of Cld1 specificity and computer modeling confirmed the hydrolytic selectivity of the enzyme toward C16-CL substrates and the preservation of C18:1-containing CL species.

TAT-Mediated Delivery of Tousled Protein to Salivary Glands Protects Against Radiation-Induced Hypofunction

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

Sunavala-Dossabhoy, Gulshan, E-mail: gsunav@lsuhsc.edu; Palaniyandi, Senthilnathan; Richardson, Charles

2012-09-01

Purpose: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect againstmore » IR-induced salivary hypofunction. Methods: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. Results: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D{sub 0} = 4.13 {+-} 1.0 Gy; {alpha}/{beta} = 0 Gy) compared with cells transduced with the TAT peptide (D{sub 0} = 4.91 {+-} 1.0 Gy; {alpha}/{beta} = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). Conclusions: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.« less

Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

PubMed Central

Knies, Diana; Medenhoff, Sergej; Wabnitz, Guido; Luckner-Minden, Claudia; Feldmeyer, Nadja; Voss, Ralf-Holger; Kropf, Pascale; Müller, Ingrid; Conradi, Roland; Samstag, Yvonne; Theobald, Matthias; Ho, Anthony D.; Goldschmidt, Hartmut; Hundemer, Michael

2013-01-01

Tumor-growth is often associated with the expansion of myeloid derived suppressor cells that lead to local or systemic arginine depletion via the enzyme arginase. It is generally assumed that this arginine deficiency induces a global shut-down of T cell activation with ensuing tumor immune escape. While the impact of arginine depletion on polyclonal T cell proliferation and cytokine secretion is well documented, its influence on chemotaxis, cytotoxicity and antigen specific activation of human T cells has not been demonstrated so far. We show here that chemotaxis and early calcium signaling of human T cells are unimpaired in the absence of arginine. We then analyzed CD8+ T cell activation in a tumor peptide as well as a viral peptide antigen specific system: (i) CD8+ T cells with specificity against the MART-1aa26–35*A27L tumor antigen expanded with in vitro generated dendritic cells, and (ii) clonal CMV pp65aa495–503 specific T cells and T cells retrovirally transduced with a CMV pp65aa495–503 specific T cell receptor were analyzed. Our data demonstrate that human CD8+ T cell antigen specific cytotoxicity and perforin secretion are completely preserved in the absence of arginine, while antigen specific proliferation as well as IFN-γ and granzyme B secretion are severely compromised. These novel results highlight the complexity of antigen specific T cell activation and demonstrate that human T cells can preserve important activation-induced effector functions in the context of arginine deficiency. PMID:23717444

Sub-physiological oxygen levels optimal for growth and survival of human atrial cardiac stem cells.

PubMed

RajendranNair, Deepthi Sreerengam; Karunakaran, Jayakumar; Nair, Renuka R

2017-08-01

Cardiac stem cells reside in niches where the oxygen levels are close to 3%. For cytotherapy, cells are conventionally expanded in ambient oxygen (21% O 2 ) which represents hyperoxia compared to the oxygen tension of niches. Cardiosphere-derived cells (CDCs) are then transplanted to host tissue with lower-O 2 levels. The high-O 2 gradient can reduce the efficacy of cultured cells. Based on the assumption that minimizing injury due to O 2 gradients will enhance the yield of functionally efficient cells, CDCs were cultured in 3% O 2 and compared with cells maintained in ambient O 2 . CDCs were isolated from human right atrial explants and expanded in parallel in 21 and 3% oxygen and compared with regard to survival, proliferation, and retention of stemness. Increased cell viability even in the tenth passage and enhanced cardiosphere formation was observed in cells expanded in 3% O 2 . The cell yield from seven passages was fourfold higher for cells cultured in 3% O 2 . Preservation of stemness in hypoxic environment was evident from the proportion of c-kit-positive cells and reduced myogenic differentiation. Hypoxia promoted angiogenesis and reduced the tendency to differentiate to noncardiac lineages (adipocytes and osteocytes). Mimicking the microenvironment at transplantation, when shifted to 5% O 2 , viability and proliferation rate were significantly higher for CDCs expanded in 3% O 2 . Expansion of CDCs, from atria in sub-physiological oxygen, helps in obtaining a higher yield of healthy cells with better preservation of stem cell characteristics. The cells so cultured are expected to improve engraftment and facilitate myocardial regeneration.

Effects of glaucoma medications and preservatives on cultured human trabecular meshwork and non-pigmented ciliary epithelial cell lines.

PubMed

Ammar, David A; Kahook, Malik Y

2011-10-01

We investigated the potential cytotoxicity of various topical ophthalmic glaucoma formulations containing different preservatives in cultured human trabecular meshwork (TM) and non-pigmented ciliary epithelial (NPCE) cell lines. We tested 0.004% travoprost preserved with either 0.015% benzalkonium chloride (BAK), sofZia or 0.001% Polyquad (PQ); and 0.005% latanoprost preserved with 0.020% BAK. We also tested a range of BAK concentrations in balanced salt solution (BSS). TM cells were treated for 10 min at 37°C with solutions diluted 1:10 to mimic the reduced penetration of topical preparations to the anterior chamber. Viability was determined by the uptake of the fluorescent vital dye calcein-AM (n = 6). BAK solutions (diluted 1:10) demonstrated a dose-dependent reduction in cell viability in both cell types (TM and NPCE). With a 1:10 dilution of 0.020% BAK, there were significantly more living NPCE cells (89 ± 6%) than TM cells (57 ± 6%; p < 0.001). In TM cells, travoprost + BAK had statistically fewer live cells (83 ± 5%) than both travoprost + sofZia (97 ± 5%) and travoprost + PQ (97 ± 6%; p < 0.05). Compared with BSS-treated NPCE cells, travoprost had statistically fewer live cells (p < 0.05) when preserved with BAK (85 ± 16%), sofZia (91 ± 6%) or PQ (94 ± 2%). These results demonstrate that substitution of BAK from topical ophthalmic drugs results in greater viability of cultured TM cells, the cells involved in the conventional outflow pathway. Cultured NPCE, responsible for aqueous inflow, appear more resilient to BAK.

PolyI.polyC12U-mediated inhibition of loss of alloantigen responsiveness viral replication in human CD4+ T cell clones exposed to human immunodeficiency virus in vitro.

PubMed Central

Laurence, J; Kulkosky, J; Friedman, S M; Posnett, D N; Ts'o, P O

1987-01-01

Two alloreactive human CD4+ T cell clones, recognizing HLA-DR2 and HLA-DR1 determinants, lost their specific proliferative capacity after infection with HIV. This system was used to explore the effect of polyI.polyC12U on HIV replication and immune suppression. The mismatched double-stranded RNA blocked HIV-associated particulate reverse transcriptase activity and viral-mediated cytopathic effects. Also, polyI.polyC12U preserved the alloreactivity of T cell clones after exposure to HIV.PolyI.polyC12U appeared to act at a level subsequent to host cell infection and reverse transcription. It had no effect on the enhancement of gene expression by the HIV transcription unit tatIII. These findings indicate that early in the course of infection of CD4+ T lymphocytes, HIV can directly abrogate proliferation to specific allodeterminants, and that this function is preserved in the presence of polyI.polyC12U. They also provide insight into the mechanism of antiviral action of a class of agent with potential clinical utility in AIDS. Images PMID:2960696

Cell structure and function and response to chemotherapy in tumors heterotransplanted into the subrenal capsule of mice and rats.

PubMed

Stenbäck, F; Kangas, L; Wasenius, V M

1985-12-01

Specimens from 16 freshly biopsied human tumors, two mammary adenocarcinomas, ten ovarian adenocarcinomas, two squamous cell carcinomas, one malignant histiocytoma and one chondrosarcoma of the bone, two human ovarian adenocarcinomas established by transplantation into nude mice and two adenocarcinomas induced in rat mammary gland were transplanted under the renal capsule of 510 normal immunocompetent mice and 180 rats and the effects of chemotherapy were evaluated. The results showed successful transplantation of all types of tumors in both animal species. Morphological analysis revealed preserved glandular structures with surface microvilli, mucin and CEA production and partially preserved basement membranes. Treatment with cyclophosphamide, vinblastine, adriamycin and cisplatin caused cell shrinkage, degradation and partial or total disappearance of the tumor cells. Vascularization was distinct in all specimens. A cellular infiltrate was found frequently but not consistently. A common end stage was a fibrotic scar with no cellular activity, occasionally giving a misleading impression of a growing tumor on gross observation. The results were obtained rapidly and suggest that the subrenal capsule assay would be useful for evaluating the sensitivity of human tumors to therapeutic manipulation, but needs supplementary histological examination.

Clinical effects of phosphodiesterase 3A mutations in inherited hypertension with brachydactyly.

PubMed

Toka, Okan; Tank, Jens; Schächterle, Carolin; Aydin, Atakan; Maass, Philipp G; Elitok, Saban; Bartels-Klein, Eireen; Hollfinger, Irene; Lindschau, Carsten; Mai, Knut; Boschmann, Michael; Rahn, Gabriele; Movsesian, Matthew A; Müller, Thomas; Doescher, Andrea; Gnoth, Simone; Mühl, Astrid; Toka, Hakan R; Wefeld-Neuenfeld, Yvette; Utz, Wolfgang; Töpper, Agnieszka; Jordan, Jens; Schulz-Menger, Jeanette; Klussmann, Enno; Bähring, Sylvia; Luft, Friedrich C

2015-10-01

Autosomal-dominant hypertension with brachydactyly is a salt-independent Mendelian syndrome caused by activating mutations in the gene encoding phosphodiesterase 3A. These mutations increase the protein kinase A-mediated phosphorylation of phosphodiesterase 3A resulting in enhanced cAMP-hydrolytic affinity and accelerated cell proliferation. The phosphorylated vasodilator-stimulated phosphoprotein is diminished, and parathyroid hormone-related peptide is dysregulated, potentially accounting for all phenotypic features. Untreated patients die prematurely of stroke; however, hypertension-induced target-organ damage is otherwise hardly apparent. We conducted clinical studies of vascular function, cardiac functional imaging, platelet function in affected and nonaffected persons, and cell-based assays. Large-vessel and cardiac functions indeed seem to be preserved. The platelet studies showed normal platelet function. Cell-based studies demonstrated that available phosphodiesterase 3A inhibitors suppress the mutant isoforms. However, increasing cGMP to indirectly inhibit the enzyme seemed to have particular use. Our results shed more light on phosphodiesterase 3A activation and could be relevant to the treatment of severe hypertension in the general population. © 2015 American Heart Association, Inc.

Cochlear implants and ex vivo BDNF gene therapy protect spiral ganglion neurons.

PubMed

Rejali, Darius; Lee, Valerie A; Abrashkin, Karen A; Humayun, Nousheen; Swiderski, Donald L; Raphael, Yehoash

2007-06-01

Spiral ganglion neurons often degenerate in the deaf ear, compromising the function of cochlear implants. Cochlear implant function can be improved by good preservation of the spiral ganglion neurons, which are the target of electrical stimulation by the implant. Brain derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in experimentally deafened ears. Providing enhanced levels of BDNF in human ears may be accomplished by one of several different methods. The goal of these experiments was to test a modified design of the cochlear implant electrode that includes a coating of fibroblast cells transduced by a viral vector with a BDNF gene insert. To accomplish this type of ex vivo gene transfer, we transduced guinea pig fibroblasts with an adenovirus with a BDNF gene cassette insert, and determined that these cells secreted BDNF. We then attached BDNF-secreting cells to the cochlear implant electrode via an agarose gel, and implanted the electrode in the scala tympani. We determined that the BDNF expressing electrodes were able to preserve significantly more spiral ganglion neurons in the basal turns of the cochlea after 48 days of implantation when compared to control electrodes. This protective effect decreased in the higher cochlear turns. The data demonstrate the feasibility of combining cochlear implant therapy with ex vivo gene transfer for enhancing spiral ganglion neuron survival.

Suppression of Class I and II Histone Deacetylases Blunts Pressure-Overload Cardiac Hypertrophy

PubMed Central

Kong, Yongli; Tannous, Paul; Lu, Guangrong; Berenji, Kambeez; Rothermel, Beverly A.; Olson, Eric N.; Hill, Joseph A.

2014-01-01

Background Recent work has demonstrated the importance of chromatin remodeling, especially histone acetylation, in the control of gene expression in the heart. In cell culture models of cardiac hypertrophy, pharmacological suppression of histone deacetylases (HDACs) can either blunt or amplify cell growth. Thus, HDAC inhibitors hold promise as potential therapeutic agents in hypertrophic heart disease. Methods and Results In the present investigation, we studied 2 broad-spectrum HDAC inhibitors in a physiologically relevant banding model of hypertrophy, observing dose-responsive suppression of ventricular growth that was well tolerated in terms of both clinical outcome and cardiac performance measures. In both short-term (3-week) and long-term (9-week) trials, cardiomyocyte growth was blocked by HDAC inhibition, with no evidence of cell death or apoptosis. Fibrotic change was diminished in hearts treated with HDAC inhibitors, and collagen synthesis in isolated cardiac fibroblasts was blocked. Preservation of systolic function in the setting of blunted hypertrophic growth was documented by echocardiography and by invasive pressure measurements. The hypertrophy-associated switch of adult and fetal isoforms of myosin heavy chain expression was attenuated, which likely contributed to the observed preservation of systolic function in HDAC inhibitor–treated hearts. Conclusions Together, these data suggest that HDAC inhibition is a viable therapeutic strategy that holds promise in the treatment of load-induced heart disease. PMID:16735673

PAX4 preserves endoplasmic reticulum integrity preventing beta cell degeneration in a mouse model of type 1 diabetes mellitus.

PubMed

Mellado-Gil, José Manuel; Jiménez-Moreno, Carmen María; Martin-Montalvo, Alejandro; Alvarez-Mercado, Ana Isabel; Fuente-Martin, Esther; Cobo-Vuilleumier, Nadia; Lorenzo, Petra Isabel; Bru-Tari, Eva; Herrera-Gómez, Irene de Gracia; López-Noriega, Livia; Pérez-Florido, Javier; Santoyo-López, Javier; Spyrantis, Andreas; Meda, Paolo; Boehm, Bernhard O; Quesada, Ivan; Gauthier, Benoit R

2016-04-01

A strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM. Two groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements. PAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death. The coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.

Cell patterning by laser-assisted bioprinting.

PubMed

Devillard, Raphaël; Pagès, Emeline; Correa, Manuela Medina; Kériquel, Virginie; Rémy, Murielle; Kalisky, Jérôme; Ali, Muhammad; Guillotin, Bertrand; Guillemot, Fabien

2014-01-01

The aim of tissue engineering is to produce functional three-dimensional (3D) tissue substitutes. Regarding native organ and tissue complexity, cell density and cell spatial 3D organization, which influence cell behavior and fate, are key parameters in tissue engineering. Laser-Assisted Bioprinting (LAB) allows one to print cells and liquid materials with a cell- or picoliter-level resolution. Thus, LAB seems to be an emerging and promising technology to fabricate tissue-like structures that have the physiological functionality of their native counterparts. This technology has additional advantages such as automation, reproducibility, and high throughput. It makes LAB compatible with the (industrial) fabrication of 3D constructs of physiologically relevant sizes. Here we present exhaustively the numerous steps that allow printing of viable cells with a well-preserved micrometer pattern. To facilitate the understanding of the whole cell patterning experiment using LAB, it is discussed in two parts: (1) preprocessing: laser set-up, bio-ink cartridge and bio-paper preparation, and pattern design; and (2) processing: bio-ink printing on the bio-paper. Copyright © 2014 Elsevier Inc. All rights reserved.

Perfusion-decellularized matrix: using nature's platform to engineer a bioartificial heart.

PubMed

Ott, Harald C; Matthiesen, Thomas S; Goh, Saik-Kia; Black, Lauren D; Kren, Stefan M; Netoff, Theoden I; Taylor, Doris A

2008-02-01

About 3,000 individuals in the United States are awaiting a donor heart; worldwide, 22 million individuals are living with heart failure. A bioartificial heart is a theoretical alternative to transplantation or mechanical left ventricular support. Generating a bioartificial heart requires engineering of cardiac architecture, appropriate cellular constituents and pump function. We decellularized hearts by coronary perfusion with detergents, preserved the underlying extracellular matrix, and produced an acellular, perfusable vascular architecture, competent acellular valves and intact chamber geometry. To mimic cardiac cell composition, we reseeded these constructs with cardiac or endothelial cells. To establish function, we maintained eight constructs for up to 28 d by coronary perfusion in a bioreactor that simulated cardiac physiology. By day 4, we observed macroscopic contractions. By day 8, under physiological load and electrical stimulation, constructs could generate pump function (equivalent to about 2% of adult or 25% of 16-week fetal heart function) in a modified working heart preparation.

[The phylogenetic theory of pathology. The arterial hypertension--a test of metabolic disorder. The biological basis of damage of target organs (a lecture)].

PubMed

Titov, V N

2013-05-01

The increase of blood tension is a diagnostic test of disorders of homeostasis, trophology, endoecology and adaptation in paracrine regulated coenosis of cells. This conditions results in disorder of microcirculation in the distal section of arterial race and in compensatory increase of blood tension in its proximal section. The increase of blood tension disturbs the function of paracrine coenosis of cells which have one's own system of hemo- and hydrodynamics such as brain with system of spinal liquor and kidneys with local pool of primary urine. They counteract the rise of blood tension and activate local, humoral system of renin-angiotensin-II increasing peripheral resistance to blood flow. At that, the compensatory blood tension becomes even higher. The aldosterone and natriuretic peptides are functional synergists. So, they preserve and excrete ions of Na+ and support the stability of unified pool of intercellular medium ("Inner Ocean" of organism) where all cells live. The parameters of this pool are limited most strictly in vivo. If at the level of nephron the conditions are formed that can alter the parameters of unified pool of intercellular medium the vasomotor center rises blood tension from the level of organism "forcing" nephrons to re-establish the parameters of this pool and normalize the biological functions and biological reactions. The blood pressure increase under pathology of kidneys is caused because of pathological compensation at the level of organism mediated by vegetal nervous system and dictated by necessity to preserve the parameters of inner medium of organism.

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

PubMed

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

2018-04-01

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

Lipid Rafts in Mast Cell Biology

PubMed Central

Silveira e Souza, Adriana Maria Mariano; Mazucato, Vivian Marino; Jamur, Maria Célia; Oliver, Constance

2011-01-01

Mast cells have long been recognized to have a direct and critical role in allergic and inflammatory reactions. In allergic diseases, these cells exert both local and systemic responses, including allergic rhinitis and anaphylaxis. Mast cell mediators are also related to many chronic inflammatory conditions. Besides the roles in pathological conditions, the biological functions of mast cells include roles in innate immunity, involvement in host defense mechanisms against parasites, immunomodulation of the immune system, tissue repair, and angiogenesis. Despite their growing significance in physiological and pathological conditions, much still remains to be learned about mast cell biology. This paper presents evidence that lipid rafts or raft components modulate many of the biological processes in mast cells, such as degranulation and endocytosis, play a role in mast cell development and recruitment, and contribute to the overall preservation of mast cell structure and organization. PMID:21490812

Effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation depend on treatment dose, treatment duration and meal contents

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

Arakawa, Masayuki; Ebato, Chie; Mita, Tomoya

2009-12-18

Beta-cell proliferation is regulated by various metabolic demands including peripheral insulin resistance, obesity, and hyperglycemia. In addition to enhancement of glucose-induced insulin secretion, agonists for glucagon-like peptide-1 receptor (GLP-1R) stimulate proliferation and inhibit apoptosis of beta-cells, thereby probably preserve beta-cell mass. To evaluate the beta-cell preserving actions of GLP-1R agonists, we assessed the acute and chronic effects of exendin-4 on beta-cell proliferation, mass and glucose tolerance in C57BL/6J mice under various conditions. Short-term administration of high-dose exendin-4 transiently stimulated beta-cell proliferation. Comparative transcriptomic analysis showed upregulation of IGF-1 receptor and its downstream effectors in islets. Treatment of mice with exendin-4more » daily for 4 weeks (long-term administration) and feeding high-fat diet resulted in significant inhibition of weight gain and improvement of glucose tolerance with reduced insulin secretion and beta-cell mass. These findings suggest that long-term GLP-1 treatment results in insulin sensitization of peripheral organs, rather than enhancement of beta-cell proliferation and function, particularly when animals are fed high-fat diet. Thus, the effects of exendin-4 on glucose tolerance, insulin secretion, and beta-cell proliferation largely depend on treatment dose, duration of treatment and meal contents. While GLP-1 enhances proliferation of beta-cells in some diabetic mice models, our results suggest that GLP-1 stimulates beta-cell growth only when expansion of beta-cell mass is required to meet metabolic demands.« less

Decellularised skeletal muscles allow functional muscle regeneration by promoting host cell migration.

PubMed

Urciuolo, Anna; Urbani, Luca; Perin, Silvia; Maghsoudlou, Panagiotis; Scottoni, Federico; Gjinovci, Asllan; Collins-Hooper, Henry; Loukogeorgakis, Stavros; Tyraskis, Athanasios; Torelli, Silvia; Germinario, Elena; Fallas, Mario Enrique Alvarez; Julia-Vilella, Carla; Eaton, Simon; Blaauw, Bert; Patel, Ketan; De Coppi, Paolo

2018-05-30

Pathological conditions affecting skeletal muscle function may lead to irreversible volumetric muscle loss (VML). Therapeutic approaches involving acellular matrices represent an emerging and promising strategy to promote regeneration of skeletal muscle following injury. Here we investigated the ability of three different decellularised skeletal muscle scaffolds to support muscle regeneration in a xenogeneic immune-competent model of VML, in which the EDL muscle was surgically resected. All implanted acellular matrices, used to replace the resected muscles, were able to generate functional artificial muscles by promoting host myogenic cell migration and differentiation, as well as nervous fibres, vascular networks, and satellite cell (SC) homing. However, acellular tissue mainly composed of extracellular matrix (ECM) allowed better myofibre three-dimensional (3D) organization and the restoration of SC pool, when compared to scaffolds which also preserved muscular cytoskeletal structures. Finally, we showed that fibroblasts are indispensable to promote efficient migration and myogenesis by muscle stem cells across the scaffolds in vitro. This data strongly support the use of xenogeneic acellular muscles as device to treat VML conditions in absence of donor cell implementation, as well as in vitro model for studying cell interplay during myogenesis.

TanCAR: A Novel Bispecific Chimeric Antigen Receptor for Cancer Immunotherapy

PubMed Central

Grada, Zakaria; Hegde, Meenakshi; Byrd, Tiara; Shaffer, Donald R; Ghazi, Alexia; Brawley, Vita S; Corder, Amanda; Schönfeld, Kurt; Koch, Joachim; Dotti, Gianpietro; Heslop, Helen E; Gottschalk, Stephen; Wels, Winfried S; Baker, Matthew L; Ahmed, Nabil

2013-01-01

Targeted T cells are emerging as effective non-toxic therapies for cancer. Multiple elements, however, contribute to the overall pathogenesis of cancer through both distinct and redundant mechanisms. Hence, targeting multiple cancer-specific markers simultaneously could result in better therapeutic efficacy. We created a functional chimeric antigen receptor—the TanCAR, a novel artificial molecule that mediates bispecific activation and targeting of T cells. We demonstrate the feasibility of cumulative integration of structure and docking simulation data using computational tools to interrogate the design and predict the functionality of such a complex bispecific molecule. Our prototype TanCAR induced distinct T cell reactivity against each of two tumor restricted antigens, and produced synergistic enhancement of effector functions when both antigens were simultaneously encountered. Furthermore, the TanCAR preserved the cytolytic ability of T cells upon loss of one of the target molecules and better controlled established experimental tumors by recognition of both targets in an animal disease model. This proof-of-concept approach can be used to increase the specificity of effector cells for malignant versus normal target cells, to offset antigen escape or to allow for targeting the tumor and its microenvironment. PMID:23839099

Effects of early stress on adult affiliative behavior.

PubMed

Henry, J P; Wang, S

1998-11-01

The recently evolved mammalian species preservative behavior as opposed to the ancient self preservative behavior involves parental care, nursing, social interaction, pair bonding and mutual defense. Gonadal steroids together with oxytocin are critical for this affiliative, attachment behavior. When there is stressful loss of control, gonadotrophins are diminished, and the self preservative, fight-flight catecholamine coping response takes priority. It is suggested that self preservation is associated with left hemispheric brain function and that species preservation is associated with right hemispheric function. Stress during infancy that is severe enough to create insecure attachment has a dissociative effect, disrupting right hemispheric emotional functioning and species preservative behavior, and a permanent bias towards self preservation can become an adult trait. In such a person with impaired affiliation, corticoid responses may be deficient. The coronary type A behavior pattern common in our society exhibits some of this deficiency in species preservative activity.

Cell Viability and Functionality of Probiotic Bacteria in Dairy Products

PubMed Central

Vinderola, Gabriel; Binetti, Ana; Burns, Patricia; Reinheimer, Jorge

2011-01-01

Probiotic bacteria, according to the definition adopted by the World Health Organization in 2002, are live microorganisms, which when administered in adequate amounts confer a health benefit to the host. Recent studies show that the same probiotic strain produced and/or preserved under different storage conditions, may present different responses regarding their susceptibility to the adverse conditions of the gastrointestinal tract, its capacity to adhere to the intestinal epithelium, or its immunomodulating capacity, the functionality being affected without changes in cell viability. This could imply that the control of cell viability is not always enough to guarantee the functionality (probiotic capacity) of a strain. Therefore, a new challenge arises for food technologists and microbiologists when it comes to designing and monitoring probiotic food: to be able to monitor the functionality of a probiotic microorganism throughout all the stages the strain goes through from the moment it is produced and included in the food vehicle, until the moment of consumption. Conventional methodological tools or others still to be developed must be used. The application of cell membrane functionality markers, the use of tests of resistance to intestinal barriers, the study of surface properties and the application of in vivo models come together as complementary tools to assess the actual capacity of a probiotic organism in a specific food, to exert functional effects regardless of the number of viable cells present at the moment of consumption. PMID:21833320

Use of fibroblast growth factor 2 for expansion of chondrocytes and tissue engineering

NASA Technical Reports Server (NTRS)

Vunjak-Novakovic, Gordana (Inventor); Martin, Ivan (Inventor); Freed, Lisa E. (Inventor); Langer, Robert (Inventor)

2003-01-01

The present invention provides an improved method for expanding cells for use in tissue engineering. In particular the method provides specific biochemical factors to supplement cell culture medium during the expansion process in order to reproduce events occurring during embryonic development with the goal of regenerating tissue equivalents that resemble natural tissues both structurally and functionally. These specific biochemical factors improve proliferation of the cells and are capable of de-differentiation mature cells isolated from tissue so that the differentiation potential of the cells is preserved. The bioactive molecules also maintain the responsiveness of the cells to other bioactive molecules. Specifically, the invention provides methods for expanding chondrocytes in the presence of fibroblast growth factor 2 for use in regeneration of cartilage tissue.

Towards a biocompatible artificial lung: Covalent functionalization of poly(4-methylpent-1-ene) (TPX) with cRGD pentapeptide

PubMed Central

Möller, Lena; Hess, Christian; Paleček, Jiří; Su, Yi; Haverich, Axel

2013-01-01

Summary Covalent multistep coating of poly(methylpentene), the membrane material in lung ventilators, by using a copper-free “click” approach with a modified cyclic RGD peptide, leads to a highly biocompatible poly(methylpentene) surface. The resulting modified membrane preserves the required excellent gas-flow properties while being densely seeded with lung endothelial cells. PMID:23504394

Functional high-intensity training improves pancreatic β-cell function in adults with type 2 diabetes.

PubMed

Nieuwoudt, Stephan; Fealy, Ciarán E; Foucher, Julie A; Scelsi, Amanda R; Malin, Steven K; Pagadala, Mangesh; Rocco, Michael; Burguera, Bartolome; Kirwan, John P

2017-09-01

Type 2 diabetes (T2D) is characterized by reductions in β-cell function and insulin secretion on the background of elevated insulin resistance. Aerobic exercise has been shown to improve β-cell function, despite a subset of T2D patients displaying "exercise resistance." Further investigations into the effectiveness of alternate forms of exercise on β-cell function in the T2D patient population are needed. We examined the effect of a novel, 6-wk CrossFit functional high-intensity training (F-HIT) intervention on β-cell function in 12 sedentary adults with clinically diagnosed T2D (54 ± 2 yr, 166 ± 16 mg/dl fasting glucose). Supervised training was completed 3 days/wk, comprising functional movements performed at a high intensity in a variety of 10- to 20-min sessions. All subjects completed an oral glucose tolerance test and anthropometric measures at baseline and following the intervention. The mean disposition index, a validated measure of β-cell function, was significantly increased (PRE: 8.4 ± 3.1, POST: 11.5 ± 3.5, P = 0.02) after the intervention. Insulin processing inefficiency in the β-cell, expressed as the fasting proinsulin-to-insulin ratio, was also reduced (PRE: 2.40 ± 0.37, POST: 1.78 ± 0.30, P = 0.04). Increased β-cell function during the early-phase response to glucose correlated significantly with reductions in abdominal body fat ( R 2 = 0.56, P = 0.005) and fasting plasma alkaline phosphatase ( R 2 = 0.55, P = 0.006). Mean total body-fat percentage decreased significantly (Δ: -1.17 0.30%, P = 0.003), whereas lean body mass was preserved (Δ: +0.05 ± 0.68 kg, P = 0.94). We conclude that F-HIT is an effective exercise strategy for improving β-cell function in adults with T2D. Copyright © 2017 the American Physiological Society.

3D porous calcium-alginate scaffolds cell culture system improved human osteoblast cell clusters for cell therapy.

PubMed

Chen, Ching-Yun; Ke, Cherng-Jyh; Yen, Ko-Chung; Hsieh, Hui-Chen; Sun, Jui-Sheng; Lin, Feng-Huei

2015-01-01

Age-related orthopedic disorders and bone defects have become a critical public health issue, and cell-based therapy is potentially a novel solution for issues surrounding bone tissue engineering and regenerative medicine. Long-term cultures of primary bone cells exhibit phenotypic and functional degeneration; therefore, culturing cells or tissues suitable for clinical use remain a challenge. A platform consisting of human osteoblasts (hOBs), calcium-alginate (Ca-Alginate) scaffolds, and a self-made bioreactor system was established for autologous transplantation of human osteoblast cell clusters. The Ca-Alginate scaffold facilitated the growth and differentiation of human bone cell clusters, and the functionally-closed process bioreactor system supplied the soluble nutrients and osteogenic signals required to maintain the cell viability. This system preserved the proliferative ability of cells and cell viability and up-regulated bone-related gene expression and biological apatite crystals formation. The bone-like tissue generated could be extracted by removal of calcium ions via ethylenediaminetetraacetic acid (EDTA) chelation, and exhibited a size suitable for injection. The described strategy could be used in therapeutic application and opens new avenues for surgical interventions to correct skeletal defects.

In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes

PubMed Central

Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão‐Pires, Inês; van der Velden, Jolanda

2017-01-01

Key points This paper describes a novel model that allows exploration of matrix‐induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function.Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca2+ handling and myofilament function.Cell shortening and Ca2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix.Matrix stiffness‐impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness.Matrix stiffness‐induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Abstract Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte–matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix‐induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca2+ handling but does not alter myofilament‐generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness‐induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness‐induced changes in cardiomyocyte function could not be explained by adaptation in the microtubules. Additionally, cardiomyocytes isolated from stiff hearts of the obese ZSF1 rat model of heart failure with preserved ejection fraction show more pronounced reduction in unloaded shortening in response to matrix stiffening. Taken together, we introduce a method that allows evaluation of the influence of ECM properties on cardiomyocyte function separate from the passive inhibitory component of a stiff matrix. As such, it adds an important and physiologically relevant tool to investigate the functional consequences of cardiomyocyte–matrix interactions. PMID:28485491

Cytotoxic effect of commercially available methylprednisolone acetate with and without reduced preservatives on dorsal root ganglion sensory neurons in rats.

PubMed

Knezevic, Nebojsa Nick; Candido, Kenneth D; Cokic, Ivan; Krbanjevic, Aleksandar; Berth, Sarah L; Knezevic, Ivana

2014-01-01

Epidural and intrathecal injections of methylprednisolone acetate (MPA) have become the most commonly performed interventional procedures in the United States and worldwide in the last 2 decades. However neuraxial MPA injection has been dogged by controversy regarding the presence of different additives used in commercially prepared glucocorticoids. We previously showed that MPA could be rendered 85% free of polyethylene glycol (PEG) by a simple physical separation of elements in the suspension. The objective of the present study was to explore a possible cytotoxic effect of commercially available MPA (with intact or reduced preservatives) on rat sensory neurons. We exposed primary dissociated rat dorsal root ganglia (DRG) sensory neurons to commercially available MPA for 24 hours with either the standard (commercial) concentration of preservatives or to different fractions following separation (MPA suspension whose preservative concentration had been reduced, or fractions containing higher concentrations of preservatives). Cells were stained with the TUNEL assay kit to detect apoptotic cells and images were taken on the Bio-Rad Laser Sharp-2000 system. We also detected expression of caspase-3, as an indicator of apoptosis in cell lysates. We exposed sensory neurons from rat DRG to different concentrations of MPA from the original commercially prepared vial. TUNEL assay showed dose-related responses and increased percentages of apoptotic cells with increasing concentrations of MPA. Increased concentrations of MPA caused 1.5 - 2 times higher caspase-3 expression in DRG sensory neurons than in control cells (ANOVA, P = 0.001). Our results showed that MPA with reduced preservatives caused significantly less apoptosis observed with TUNEL assay labeling (P < 0.001) and caspase-3 immunoblotting (P = 0.001) than in neurons exposed to MPA from a commercially prepared vial or "clear phase" that contained higher concentrations of preservatives. Even though MPA with reduced preservatives caused 12.5% more apoptosis in DRG sensory neurons than in control cells, post hoc analysis showed no differences between these 2 groups. Our data was collected from in vitro isolated rat DRG neurons. There is a possibility that in vivo neurons have different extents of vulnerability compared to isolated neurons. Results of the present study identified a cytotoxic effect of commercially available MPA with preservatives or with a "clear phase" containing higher concentrations of preservatives on primary isolated rat DRG sensory neurons. This was shown by TUNEL positive assay and by increased caspase-3 expression as one of the final executing steps in apoptotic pathways in DRG neurons. However, our results showed no statistically significant difference between the control cells (saline-treated) and cells treated with MPA with reduced concentrations of preservatives, pointing out that either PEG or myristylgamma-picolinium chloride (MGPC) or their combination have harmful effects on these cells. Reduction of concentrations of preservatives from commercially available MPA suspensions by using the simple method of inverting vials for 2 hours could be considered useful in clinical practice to enhance the safety of this depot steroid when injected neuraxially.

Prediction of preservative sensitization potential using surface marker CD86 and/or CD54 expression on human cell line, THP-1.

PubMed

Sakaguchi, Hitoshi; Miyazawa, Masaaki; Yoshida, Yukiko; Ito, Yuichi; Suzuki, Hiroyuki

2007-02-01

Preservatives are important components in many products, but have a history of purported allergy. Several assays [e.g., guinea pig maximization test (GPMT), local lymph node assay (LLNA)] are used to evaluate allergy potential of preservatives. We recently developed the human Cell Line Activation Test (h-CLAT), an in vitro skin sensitization test using human THP-1 cells. This test evaluates the augmentation of CD86 and CD54 expression, which are key events in the sensitization process, as an indicator of allergy following treatment with test chemical. Earlier, we found that a sub-toxic concentration was needed for the up-regulation of surface marker expression. In this study, we further evaluate the capability of h-CLAT to predict allergy potential using eight preservatives. Cytotoxicity was determined using propidium iodide with flow cytometry analysis and five doses that produce a 95, 85, 75, 65, and 50% cell viability were selected. If a material did not have any cytotoxicity at the highest technical dose (HTD), five doses are set using serial 1.3 dilutions of the HTD. The test materials used were six known allergic preservatives (e.g., methylchloroisothiazolinone/methylisothiazolinone, formaldehyde), and two non-allergic preservatives (methylparaben and 4-hydroxybenzoic acid). All allergic preservatives augmented CD86 and/or CD54 expression, indicating h-CLAT correctly identified the allergens. No augmentation was observed with the non-allergic preservatives; also correctly identified by h-CLAT. In addition, we report two threshold concentrations that may be used to categorize skin sensitization potency like the LLNA estimated concentration that yield a three-fold stimulation (EC3) value. These corresponding values are the estimated concentration which gives a relative fluorescence intensity (RFI) = 150 for CD86 and an RFI = 200 for CD54. These data suggest that h-CLAT, using THP-1 cells, may be able to predict the allergy potential of preservatives and possibility classify the potency of an allergen.

Utility of Cytospin and Cell block Technology in Evaluation of Body Fluids and Urine Samples: A Comparative Study.

PubMed

Qamar, Irmeen; Rehman, Suhailur; Mehdi, Ghazala; Maheshwari, Veena; Ansari, Hena A; Chauhan, Sunanda

2018-01-01

Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material.

Olfactory Ensheathing Cell Transplantation after a Complete Spinal Cord Transection Mediates Neuroprotective and Immunomodulatory Mechanisms to Facilitate Regeneration

PubMed Central

Khankan, Rana R.; Griffis, Khris G.; Haggerty-Skeans, James R.; Zhong, Hui; Roy, Roland R.; Edgerton, V. Reggie

2016-01-01

Multiple neural and peripheral cell types rapidly respond to tissue damage after spinal cord injury to form a structurally and chemically inhibitory scar that limits axon regeneration. Astrocytes form an astroglial scar and produce chondroitin sulfate proteoglycans (CSPGs), activate microglia, and recruit blood-derived immune cells to the lesion for debris removal. One beneficial therapy, olfactory ensheathing cell (OEC) transplantation, results in functional improvements and promotes axon regeneration after spinal cord injury. The lack of an OEC-specific marker, however, has limited the investigation of mechanisms underlying their proregenerative effects. We compared the effects of enhanced green fluorescent protein-labeled fibroblast (FB) and OEC transplants acutely after a complete low-thoracic spinal cord transection in adult rats. We assessed the preservation of neurons and serotonergic axons, the levels of inhibitory CSPGs and myelin debris, and the extent of immune cell activation between 1 and 8 weeks postinjury. Our findings indicate that OECs survive longer than FBs post-transplantation, preserve axons and neurons, and reduce inhibitory molecules in the lesion core. Additionally, we show that OECs limit immune-cell activation and infiltration, whereas FBs alter astroglial scar formation and increase immune-cell infiltration and concomitant secondary tissue damage. Administration of cyclosporine-A to enhance graft survival demonstrated that immune suppression can augment OEC contact-mediated protection of axons and neurons during the first 2 weeks postinjury. Collectively, these data suggest that OECs have neuroprotective and immunomodulatory mechanisms that create a supportive environment for neuronal survival and axon regeneration after spinal cord injury. SIGNIFICANCE STATEMENT Spinal cord injury creates physical and chemical barriers to axon regeneration. We used a complete spinal cord transection model and olfactory ensheathing cell (OEC) or fibroblast (FB; control) transplantation as a repair strategy. OECs, but not FBs, intermingled with astrocytes, facilitated astroglial scar border formation and sequestered invading peripheral cells. OECs attenuated immune cell infiltration, reduced secondary tissue damage, protected neurons and axons in the lesion core, and helped clear myelin debris. Immunosuppression enhanced survival of OECs and FBs, but only OEC transplantation promoted scaffold formation in the lesion site that facilitated axon regeneration and neuron preservation. PMID:27277804

The preservative polyquaternium-1 increases cytoxicity and NF-kappaB linked inflammation in human corneal epithelial cells

PubMed Central

Paimela, Tuomas; Ryhänen, Tuomas; Kauppinen, Anu; Marttila, Liisa; Salminen, Antero

2012-01-01

Purpose In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell culture. Methods HCE-2 cells were exposed to the commercial eye drops Travatan, Systane Ultra, Xalatan, and the preservative BAK. Cell viability was determined using colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by release of lactate dehydrogenase (LDH). Induction of apoptosis was measured with a using a colorimetric caspase-3 assay kit. DNA binding of the nuclear factor kappa B (NF-κB) transcription factor, and productions of the proinflammatory cytokines, interleukins IL-6 and IL-8, were determined using an enzyme-linked immunosorbent assay (ELISA) method. Results Cell viability, as measured by the MTT assay, declined by up to 50% after exposure to Travatan or Systane Ultra solutions which contain 0.001% PQ-1. BAK at 0.02% rather than at 0.001% concentration evoked total cell death signs on HCE-2 cells. In addition, cell membrane permeability, as measured by LDH release, was elevated by sixfold with Travatan and by a maximum threefold with Systane Ultra. Interestingly, Travatan and Systane Ultra activated NF-κB and elevated the secretion of inflammation markers IL-6 by 3 to eightfold and IL-8 by 1.5 to 3.5 fold, respectively, as analyzed with ELISA. Conclusions Eye drops containing PQ-1 evoke cytotoxicity and enhance the NF-κB driven inflammation reaction in cultured HCE-2 cells. Our results indicate that these harmful effects of ocular solutions preserved with PQ-1 should be further evaluated in vitro and in vivo. PMID:22605930

Acute Slices of Mice Testis Seminiferous Tubules Unveil Spontaneous and Synchronous Ca2+ Oscillations in Germ Cell Clusters1

PubMed Central

Sánchez-Cárdenas, Claudia; Guerrero, Adán; Treviño, Claudia Lydia; Hernández-Cruz, Arturo; Darszon, Alberto

2012-01-01

ABSTRACT Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca2+ ([Ca2+]i); however, very few studies exist on [Ca2+]i dynamics in these cells. Other tissues display Ca2+ oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca2+ imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca2+]i changes of living germ cells in situ within the SST preparation. Ca2+ imaging revealed that a subpopulation of male germ cells display spontaneous [Ca2+]i fluctuations resulting from Ca2+ entry possibly throughout CaV3 channels. These [Ca2+]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca2+ fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca2+ oscillations for at least 10 min. Synchronous Ca2+ oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca2+]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca2+ signaling phenomena, functional cell-cell communication, and multicellular functional arrangements. PMID:22914313

Long-term Culture and Cloning of Primary Human Bronchial Basal Cells that Maintain Multipotent Differentiation Capacity and CFTR Channel Function.

PubMed

Peters-Hall, Jennifer Ruth; Coquelin, Melissa L; Torres, Michael J; LaRanger, Ryan; Alabi, Busola Ruth; Sho, Sei; Calva-Moreno, Jose Francisco; Thomas, Philip J; Shay, Jerry William

2018-05-03

While primary cystic fibrosis (CF) and non-CF human bronchial epithelial basal cells (HBECs) accurately represent in vivo phenotypes, one barrier to their wider use has been a limited ability to clone and expand cells in sufficient numbers to produce rare genotypes using genome editing tools. Recently, conditional reprogramming of cells (CRC) with a ROCK inhibitor and culture on an irradiated fibroblast feeder layer resulted in extension of the lifespan of HBECs, but differentiation capacity and CF transmembrane conductance regulator (CFTR) function decreased as a function of passage. This report details modifications to the standard HBEC CRC protocol (Mod CRC), including the use of bronchial epithelial growth medium instead of F-medium and 2% oxygen instead of 21% oxygen, that extend HBEC lifespan while preserving multipotent differentiation capacity and CFTR function. Critically, Mod CRC conditions support clonal growth of primary HBECs from a single cell and the resulting clonal HBEC population maintains multipotent differentiation capacity, including CFTR function, permitting gene editing of these cells. As a proof of concept, CRISPR/Cas9 genome editing and cloning was used to introduce insertions/deletions in CFTR exon 11. Mod CRC conditions overcome many barriers to the expanded use of HBECs for basic research and drug screens. Importantly, Mod CRC conditions support the creation of isogenic cell lines in which CFTR is mutant or wild-type in the same genetic background with no history of CF to enable determination of the primary defects of mutant CFTR.

Curcumin-functionalized silk biomaterials for anti-aging utility.

PubMed

Yang, Lei; Zheng, Zhaozhu; Qian, Cheng; Wu, Jianbing; Liu, Yawen; Guo, Shaozhe; Li, Gang; Liu, Meng; Wang, Xiaoqin; Kaplan, David L

2017-06-15

Curcumin is a natural antioxidant that is isolated from turmeric (Curcuma longa) and exhibits strong free radical scavenging activity, thus functional for anti-aging. However, poor stability and low solubility of curcumin in aqueous conditions limit its biomedical applications. Previous studies have shown that the anti-oxidation activity of curcumin embedded in silk fibroin films could be well preserved, resulting in the promoted adipogenesis from human mesenchymal stem cells (hMSCs) cultured on the surface of the films. In the present study, curcumin was encapsulated in both silk fibroin films (silk/cur films) and nanoparticles (silk/cur NPs), and their anti-aging effects were compared with free curcumin in solution, with an aim to elucidate the mechanism of anti-aging of silk-associated curcumin and to better serve biomedical applications in the future. The morphology and structure of silk/cur film and silk/cur NP were characterized using SEM, FTIR and DSC, indicating characteristic stable beta-sheet structure formation in the materials. Strong binding of curcumin molecules to the beta-sheet domains of silk fibroin resulted in the slow release of curcumin with well-preserved activity from the materials. For cell aging studies, rat bone marrow mesenchymal stem cells (rBMSCs) were cultured in the presence of free curcumin (FC), silk/cur film and silk/cur NP, and cell proliferation and markers of aging (P53, P16, HSP70 gene expression and β-Galactosidase activity) were examined. The results indicated that cell aging was retarded in all FC, silk/cur NP and silk/cur film samples, with the silk-associated curcumin superior to the FC. Copyright © 2017 Elsevier Inc. All rights reserved.

Protection by sulforaphane from type 1 diabetes-induced testicular apoptosis is associated with the up-regulation of Nrf2 expression and function

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

Jiang, Xin; Bai, Yang; Zhang, Zhiguo

Diabetes-induced testicular apoptosis is predominantly due to increased oxidative stress. The nuclear factor-erythroid 2-related factor 2 (Nrf2), as a master transcription factor in controlling anti-oxidative systems, is able to be induced by sulforaphane (SFN). To examine whether SFN prevents testicular apoptosis, type 1 diabetic mouse model was induced with multiple low-dose streptozotocin. Diabetic and age-matched control mice were treated with and without SFN at 0.5 mg/kg daily in five days of each week for 3 months and then kept until 6 months. Diabetes significantly increased testicular apoptosis that was associated with endoplasmic reticulum stress and mitochondrial cell death pathways, shownmore » by the increased expression of C/EBP homologous protein (CHOP), cleaved caspase-12, Bax to Bcl2 expression ratio, and cleaved caspase-3. Diabetes also significantly increased testicular oxidative damage, inflammation and fibrosis, and decreased germ cell proliferation. All these diabetic effects were significantly prevented by SFN treatment for the first 3 months, and the protective effect could be sustained at 3 months after SFN treatment. SFN was able to up-regulate Nrf2 expression and function. The latter was reflected by the increased phosphorylation of Nrf2 at Ser40 and expression of Nrf2 downstream antioxidants at mRNA and protein levels. These results suggest that type 1 diabetes significantly induced testicular apoptosis and damage along with increasing oxidative stress and cell death and suppressing Nrf2 expression and function. SFN is able to prevent testicular oxidative damage and apoptosis in type 1 diabetes mice, which may be associated with the preservation of testicular Nrf2 expression and function under diabetic condition. - Highlights: • Sulforaphane (SFN) could attenuate diabetes-induced germ cell apoptosis. • SFN could preserve germ cell proliferation under diabetic conditions. • SFN testicular protection was sustained until 3 months after administration. • SFN prevents testicular oxidative damage and inflammation in diabetic mice. • SFN testicular protection from diabetic damage is associated with Nrf2 activation.« less

Bioengineered intestinal muscularis complexes with long-term spontaneous and periodic contractions

PubMed Central

Wang, Qianqian; Wang, Ke; Solorzano-Vargas, R. Sergio; Lin, Po-Yu; Walthers, Christopher M.; Thomas, Anne-Laure; Martín, Martín G.

2018-01-01

Although critical for studies of gut motility and intestinal regeneration, the in vitro culture of intestinal muscularis with peristaltic function remains a significant challenge. Periodic contractions of intestinal muscularis result from the coordinated activity of smooth muscle cells (SMC), the enteric nervous system (ENS), and interstitial cells of Cajal (ICC). Reproducing this activity requires the preservation of all these cells in one system. Here we report the first serum-free culture methodology that consistently maintains spontaneous and periodic contractions of murine and human intestinal muscularis cells for months. In this system, SMC expressed the mature marker myosin heavy chain, and multipolar/dipolar ICC, uniaxonal/multipolar neurons and glial cells were present. Furthermore, drugs affecting neural signals, ICC or SMC altered the contractions. Combining this method with scaffolds, contracting cell sheets were formed with organized architecture. With the addition of intestinal epithelial cells, this platform enabled up to 11 types of cells from mucosa, muscularis and serosa to coexist and epithelial cells were stretched by the contracting muscularis cells. The method constitutes a powerful tool for mechanistic studies of gut motility disorders and the functional regeneration of the engineered intestine. PMID:29718926

Hyperbranched polyglycerol is superior to glucose for long-term preservation of peritoneal membrane in a rat model of chronic peritoneal dialysis.

PubMed

Du, Caigan; Mendelson, Asher A; Guan, Qiunong; Dairi, Ghida; Chafeeva, Irina; da Roza, Gerald; Kizhakkedathu, Jayachandran N

2016-12-13

Replacing glucose with a better biocompatible osmotic agent in peritoneal dialysis (PD) solutions is needed in PD clinic. We previously demonstrated the potential of hyperbranched polyglycerol (HPG) as a replacement for glucose. This study further investigated the long-term effects of chronic exposure to HPG as compared to a glucose-based conventional PD solution on peritoneal membrane (PM) structure and function in rats. Adult male Wistar rats received once-daily intraperitoneal injection of 10 mL of HPG solution (1 kDa, HPG 6%) compared to Physioneal™ 40 (PYS, glucose 2.27%) or electrolyte solution (Control) for 3 months. The overall health conditions were determined by blood chemistry analysis. The PM function was determined by ultrafiltration, and its injury by histological and transcriptome-based pathway analyses. Here, we showed that there was no difference in the blood chemistry between rats receiving the HPG and the Control, while PYS increased serum alkaline phosphatase, globulin and creatinine and decreased serum albumin. Unlike PYS, HPG did not significantly attenuate PM function, which was associated with smaller change in both the structure and the angiogenesis of the PM and less cells expressing vascular endothelial growth factor, α-smooth muscle actin and MAC387 (macrophage marker). The pathway analysis revealed that there were more inflammatory signaling pathways functioning in the PM of PYS group than those of HPG or Control, which included the signaling for cytokine production in both macrophages and T cells, interleukin (IL)-6, IL-10, Toll-like receptors, triggering receptor expressed on myeloid cells 1 and high mobility group box 1. The results from this experimental study indicate the superiority of HPG to glucose in the preservation of the peritoneum function and structure during the long-term PD treatment, suggesting the potential of HPG as a novel osmotic agent for PD.

Bone Marrow Mononuclear Cell Transplantation Restores Inflammatory Balance of Cytokines after ST Segment Elevation Myocardial Infarction

PubMed Central

Alestalo, Kirsi; Miettinen, Johanna A.; Vuolteenaho, Olli; Huikuri, Heikki; Lehenkari, Petri

2015-01-01

Background Acute myocardial infarction (AMI) launches an inflammatory response and a repair process to compensate cardiac function. During this process, the balance between proinflammatory and anti-inflammatory cytokines is important for optimal cardiac repair. Stem cell transplantation after AMI improves tissue repair and increases the ventricular ejection fraction. Here, we studied in detail the acute effect of bone marrow mononuclear cell (BMMNC) transplantation on proinflammatory and anti-inflammatory cytokines in patients with ST segment elevation myocardial infarction (STEMI). Methods Patients with STEMI treated with thrombolysis followed by percutaneous coronary intervention (PCI) were randomly assigned to receive either BMMNC or saline as an intracoronary injection. Cardiac function was evaluated by left ventricle angiogram during the PCI and again after 6 months. The concentrations of 27 cytokines were measured from plasma samples up to 4 days after the PCI and the intracoronary injection. Results Twenty-six patients (control group, n = 12; BMMNC group, n = 14) from the previously reported FINCELL study (n = 80) were included to this study. At day 2, the change in the proinflammatory cytokines correlated with the change in the anti-inflammatory cytokines in both groups (Kendall’s tau, control 0.6; BMMNC 0.7). At day 4, the correlation had completely disappeared in the control group but was preserved in the BMMNC group (Kendall’s tau, control 0.3; BMMNC 0.7). Conclusions BMMNC transplantation is associated with preserved balance between pro- and anti-inflammatory cytokines after STEMI in PCI-treated patients. This may partly explain the favorable effect of stem cell transplantation after AMI. PMID:26690350

MICOS and phospholipid transfer by Ups2-Mdm35 organize membrane lipid synthesis in mitochondria.

PubMed

Aaltonen, Mari J; Friedman, Jonathan R; Osman, Christof; Salin, Bénédicte; di Rago, Jean-Paul; Nunnari, Jodi; Langer, Thomas; Tatsuta, Takashi

2016-06-06

Mitochondria exert critical functions in cellular lipid metabolism and promote the synthesis of major constituents of cellular membranes, such as phosphatidylethanolamine (PE) and phosphatidylcholine. Here, we demonstrate that the phosphatidylserine decarboxylase Psd1, located in the inner mitochondrial membrane, promotes mitochondrial PE synthesis via two pathways. First, Ups2-Mdm35 complexes (SLMO2-TRIAP1 in humans) serve as phosphatidylserine (PS)-specific lipid transfer proteins in the mitochondrial intermembrane space, allowing formation of PE by Psd1 in the inner membrane. Second, Psd1 decarboxylates PS in the outer membrane in trans, independently of PS transfer by Ups2-Mdm35. This latter pathway requires close apposition between both mitochondrial membranes and the mitochondrial contact site and cristae organizing system (MICOS). In MICOS-deficient cells, limiting PS transfer by Ups2-Mdm35 and reducing mitochondrial PE accumulation preserves mitochondrial respiration and cristae formation. These results link mitochondrial PE metabolism to MICOS, combining functions in protein and lipid homeostasis to preserve mitochondrial structure and function. © 2016 Aaltonen et al.

Preserving and enhancing the functionality of highways in Texas : workshop.

DOT National Transportation Integrated Search

2010-01-01

Workshop Objectives: : To promote the importance of Highway Functionality : To review functionality in highway lifecycle : To provide how to materials to preserve, maintain, and enhance functionality : To promote coordination be...

Preserving and enhancing the functionality of highways in Texas : workshop.

DOT National Transportation Integrated Search

2010-01-01

Workshop Objectives: To promote the importance of Highway Functionality To review functionality in highway lifecycle To provide how to materials to preserve, maintain, and enhance functionality To promote coordination between Tx...

Monomeric cocoa catechins enhance β-cell function by increasing mitochondrial respiration.

PubMed

Rowley, Thomas J; Bitner, Benjamin F; Ray, Jason D; Lathen, Daniel R; Smithson, Andrew T; Dallon, Blake W; Plowman, Chase J; Bikman, Benjamin T; Hansen, Jason M; Dorenkott, Melanie R; Goodrich, Katheryn M; Ye, Liyun; O'Keefe, Sean F; Neilson, Andrew P; Tessem, Jeffery S

2017-11-01

A hallmark of type 2 diabetes (T2D) is β-cell dysfunction and the eventual loss of functional β-cell mass. Therefore, mechanisms that improve or preserve β-cell function could be used to improve the quality of life of individuals with T2D. Studies have shown that monomeric, oligomeric and polymeric cocoa flavanols have different effects on obesity, insulin resistance and glucose tolerance. We hypothesized that these cocoa flavanols may have beneficial effects on β-cell function. INS-1 832/13-derived β-cells and primary rat islets cultured with a monomeric catechin-rich cocoa flavanol fraction demonstrated enhanced glucose-stimulated insulin secretion, while cells cultured with total cocoa extract and with oligomeric or polymeric procyanidin-rich fraction demonstrated no improvement. The increased glucose-stimulated insulin secretion in the presence of the monomeric catechin-rich fraction corresponded with enhanced mitochondrial respiration, suggesting improvements in β-cell fuel utilization. Mitochondrial complex III, IV and V components are up-regulated after culture with the monomer-rich fraction, corresponding with increased cellular ATP production. The monomer-rich fraction improved cellular redox state and increased glutathione concentration, which corresponds with nuclear factor, erythroid 2 like 2 (Nrf2) nuclear localization and expression of Nrf2 target genes including nuclear respiratory factor 1 (Nrf1) and GA binding protein transcription factor alpha subunit (GABPA), essential genes for increasing mitochondrial function. We propose a model by which monomeric cocoa catechins improve the cellular redox state, resulting in Nrf2 nuclear migration and up-regulation of genes critical for mitochondrial respiration, glucose-stimulated insulin secretion and ultimately improved β-cell function. These results suggest a mechanism by which monomeric cocoa catechins exert their effects as an effective complementary strategy to benefit T2D patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Degranulation and shrinkage of dark cells in eccrine glands and elevated serum carcinoembryonic antigen in patients with acquired idiopathic generalized anhidrosis.

PubMed

Sano, K; Asahina, M; Uehara, T; Matsumoto, K; Araki, N; Okuyama, R

2017-12-01

Acquired idiopathic generalized anhidrosis (AIGA) is characterized by anhidrosis/hypohidrosis without other autonomic and neurological dysfunctions. Pathologically, AIGA is considered to usually present no significant morphological alterations in eccrine glands, the secretory portion which consists of clear cells, dark cells, and myoepithelial cells. AIGA patients recently have been reported to show high serum concentrations of carcinoembryonic antigen (CEA). Our aim is to reveal morphological abnormalities of dark cells and investigate their relationship with serum CEA. We performed comparative analysis of eccrine glands between sweat-preserved and non-sweating skin in four AIGA patients. Serum CEA concentrations in 22 cases with AIGA were measured with healthy volunteers. Furthermore, we semiquantitatively investigated dermcidin, FoxA1 and CEA expression in eccrine glands of 12 cases with AIGA and 5 cases with non-AIGA. Marked degranulation and shrinkage of dark cells consistently occurred in AIGA. Furthermore, high serum CEA concentrations were found in 14 of 22 AIGA patients (over 60%), but serum CEA levels were not correlated with CEA expression in eccrine glands. Dermcidin expression in dark cells apparently decreased in AIGA patients, severely in those with high serum CEA and moderately in those with low serum CEA, while well-preserved expression was found in non-AIGA subjects. Our study suggests morphological damage and molecular dysregulation of dark cells, leading to impairment of their functions in AIGA patients. Severely damaged dark cells correspond to high serum CEA. Accordingly, these pathological changes in eccrine dark cells may be involved in anhidrosis/hypohidrosis of AIGA. © 2017 European Academy of Dermatology and Venereology.

Poised Regeneration of Zebrafish Melanocytes Involves Direct Differentiation and Concurrent Replenishment of Tissue-Resident Progenitor Cells.

PubMed

Iyengar, Sharanya; Kasheta, Melissa; Ceol, Craig J

2015-06-22

Efficient regeneration following injury is critical for maintaining tissue function and enabling organismal survival. Cells reconstituting damaged tissue are often generated from resident stem or progenitor cells or from cells that have dedifferentiated and become proliferative. While lineage-tracing studies have defined cellular sources of regeneration in many tissues, the process by which these cells execute the regenerative process is largely obscure. Here, we have identified tissue-resident progenitor cells that mediate regeneration of zebrafish stripe melanocytes and defined how these cells reconstitute pigmentation. Nearly all regeneration melanocytes arise through direct differentiation of progenitor cells. Wnt signaling is activated prior to differentiation, and inhibition of Wnt signaling impairs regeneration. Additional progenitors divide symmetrically to sustain the pool of progenitor cells. Combining direct differentiation with symmetric progenitor divisions may serve as a means to rapidly repair injured tissue while preserving the capacity to regenerate. Copyright © 2015 Elsevier Inc. All rights reserved.

Optical Magnetometry using Multipass Cells with overlapping beams

NASA Astrophysics Data System (ADS)

McDonough, Nathaniel David; Lucivero, Vito Giovanni; Dural, Nezih; Romalis, Michael

2017-04-01

In recent years, multipass cells with cylindrical mirrors have proven to be a successful way of making highly sensitive atomic magnetometers. In such cells a small laser beam makes 40 to 100 passes within the cell without significant overlap with itself. Here we describe a new multi-pass geometry which uses spherical mirrors to reflect the probe beam multiple times over the same cell region. Such geometry reduces the effects of atomic diffusion while preserving the advantages of multi-pass cells over standing-wave cavities, namely a deterministic number of passes and absence of interference. We have fabricated several cells with this geometry and obtained good agreement between the measured and calculated levels of quantum spin noise. We will report on our effort to characterize the diffusion spin-correlation function in these cells and operation of the cell as a magnetometer. This work is supported by DARPA.

Will stem cell therapies be safe and effective for treating spinal cord injuries?

PubMed Central

Thomas, Katharine E.; Moon, Lawrence D. F.

2017-01-01

Introduction A large number of different cells including embryonic and adult stem cells have been transplanted into animal models of spinal cord injury, and in many cases these procedures have resulted in modest sensorimotor benefits. In October 2010 the world’s first clinical trial using human embryonic stem cells began, using stem cells converted into oligodendrocyte precursor cells. Sources of data In this review we examine some of the publically-available pre-clinical evidence that some of these cell types improve outcome in animal models of spinal cord injury. Much evidence is not available for public scrutiny, however, being private commercial property of various stem cell companies. Areas of agreement Transplantation of many different types of stem and progenitor cell enhances spontaneous recovery of function when transplanted acutely after spinal cord injury in animal models. Areas of disagreement The common mechanism(s) whereby the generic procedure of cellular transplantation enhances recovery of function are not well understood, although a range of possibilities are usually cited (including preservation of tissue, remyelination, axon sprouting, glial cell replacement). Only in exceptional cases has it been shown that functional recovery depends causally on the survival and differentiation of the transplanted cells. There is no agreement about the optimal cell type for transplantation: candidate stem cells have not yet been compared with each other or with other cell types (e.g., autologous Schwann cells) in a single study. Areas timely for developing research Transplantation of cells into animals with a long lifespan is important to determine whether or not tumours will eventually form. It will also be important to determine whether long-term survival of cells is required for functional recovery, and if so, how many are optimal. PMID:21586446

The preservation of living cells with biocompatible microparticles

NASA Astrophysics Data System (ADS)

Yang, Jing; Zhu, Yingnan; Xu, Tong; Pan, Chao; Cai, Nana; Huang, He; Zhang, Lei

2016-07-01

Biomedical applications of living cells have rapidly expanded in many fields such as toxic detection, drug screening, and regenerative medicine, etc. Efficient methods to support cell survival and maintain activity in vitro have become increasingly important. However, traditional cryopreservation for living cell-based applications is limited by several problems. Here, we report that magnetic hydrogel microparticles can physically assemble into a 3D environment for efficient cell preservation in physiological conditions, avoiding any chemical reactions that would damage the cells. Two representative cell lines (loosely and firmly adherent) were tested to evaluate the versatility of this method. The results showed that cell longevity was significantly extended to at least 15 days, while the control cell samples without microparticles quickly died within 3 days. Moreover, after preservation, cells can be easily retrieved by applying a magnet to separate the magnetic particles. This strategy can also inhibit cell over-proliferation while avoiding the use of temperature extremes or toxic cryoprotectants that are essential in cryopreservation.

Molecular analysis of neutrophil spontaneous apoptosis reveals a strong role for the pro-apoptotic BH3-only protein Noxa.

PubMed

Kirschnek, S; Vier, J; Gautam, S; Frankenberg, T; Rangelova, S; Eitz-Ferrer, P; Grespi, F; Ottina, E; Villunger, A; Häcker, H; Häcker, G

2011-11-01

Neutrophils enter the peripheral blood from the bone marrow and die after a short time. Molecular analysis of spontaneous neutrophil apoptosis is difficult as these cells die rapidly and cannot be easily manipulated. We use conditional Hoxb8 expression to generate mouse neutrophils and test the regulation of apoptosis by extensive manipulation of B-cell lymphoma protein 2 (Bcl-2)-family proteins. Spontaneous apoptosis was preceded by downregulation of anti-apoptotic Bcl-2 proteins. Loss of the pro-apoptotic Bcl-2 homology domain (BH3)-only protein Bcl-2-interacting mediator of cell death (Bim) gave some protection, but only neutrophils deficient in both BH3-only proteins, Bim and Noxa, were strongly protected against apoptosis. Function of Noxa was at least in part neutralization of induced myeloid leukemia cell differentiation protein (Mcl-1) in neutrophils and progenitors. Loss of Bim and Noxa preserved neutrophil function in culture, and apoptosis-resistant cells remained in circulation in mice. Apoptosis regulated by Bim- and Noxa-driven loss of Mcl-1 is thus the final step in neutrophil differentiation, required for the termination of neutrophil function and neutrophil-dependent inflammation.

Pathophysiological Changes to the Peritoneal Membrane during PD-Related Peritonitis: The Role of Mesothelial Cells

PubMed Central

Yung, Susan; Chan, Tak Mao

2012-01-01

The success of peritoneal dialysis (PD) is dependent on the structural and functional integrity of the peritoneal membrane. The mesothelium lines the peritoneal membrane and is the first line of defense against chemical and/or bacterial insult. Peritonitis remains a major complication of PD and is a predominant cause of technique failure, morbidity and mortality amongst PD patients. With appropriate antibiotic treatment, peritonitis resolves without further complications, but in some PD patients excessive peritoneal inflammatory responses lead to mesothelial cell exfoliation and thickening of the submesothelium, resulting in peritoneal fibrosis and sclerosis. The detrimental changes in the peritoneal membrane structure and function correlate with the number and severity of peritonitis episodes and the need for catheter removal. There is evidence that despite clinical resolution of peritonitis, increased levels of inflammatory and fibrotic mediators may persist in the peritoneal cavity, signifying persistent injury to the mesothelial cells. This review will describe the structural and functional changes that occur in the peritoneal membrane during peritonitis and how mesothelial cells contribute to these changes and respond to infection. The latter part of the review discusses the potential of mesothelial cell transplantation and genetic manipulation in the preservation of the peritoneal membrane. PMID:22577250

Fatal autoimmunity results from the conditional deletion of Snai2 and Snai3

PubMed Central

Pioli, Peter D.; Chen, Xinjian; Weis, Janis J.; Weis, John H.

2015-01-01

Transcriptional regulation of gene expression is a key component of orchestrating proper immune cell development and function. One strategy for maintaining these transcriptional programs has been the evolution of transcription factor families with members possessing overlapping functions. Using the germ line deletion of Snai2 combined with the hematopoietic specific deletion of Snai3, we report that these factors function redundantly to preserve the development of B and T cells. Such animals display severe lymphopenia, alopecia and dermatitis as well as profound autoimmunity manifested by the production of high levels of autoantibodies as early as 3 weeks of age and die by 30 days after birth. Autoantibodies included both IgM and IgG isotypes and were reactive against cytoplasmic and membranous components. A regulatory T cell defect contributed to the autoimmune response in that adoptive transfer of wild type regulatory T cells alleviated symptoms of autoimmunity. Additionally, transplantation of Snai2/Snai3 double deficient bone marrow into Snai2 sufficient Rag2−/− recipients resulted in autoantibody generation. The results demonstrated that appropriate expression of Snai2 and Snai3 in cells of hematopoietic derivation plays an important role in development and maintenance of immune tolerance. PMID:25732600

Phagocyte-Myocyte Interactions and Consequences during Hypoxic Wound Healing

PubMed Central

Zhang, Shuang; Dehn, Shirley; DeBerge, Matthew; Rhee, KJ; Hudson, Barry; Thorp, Edward

2014-01-01

Myocardial infarction (MI), secondary to atherosclerotic plaque rupture and occlusive thrombi, triggers acute margination of inflammatory neutrophils and monocyte phagocyte subsets to the damaged heart, the latter of which may give rise briefly to differentiated macrophage-like or dendritic-like cells. Within the injured myocardium, a primary function of these phagocytic cells is to remove damaged extracellular matrix, necrotic and apoptotic cardiac cells, as well as immune cells that turn over. Recognition of dying cellular targets by phagocytes triggers intracellular signaling, particularly in macrophages, wherein cytokines and lipid mediators are generated to promote inflammation resolution, fibrotic scarring, angiogenesis, and compensatory organ remodeling. These actions cooperate in an effort to preserve myocardial contractility and prevent heart failure. Immune cell function is modulated by local tissue factors that include secreted protease activity, oxidative stress during clinical reperfusion, and hypoxia. Importantly, experimental evidence suggests that monocyte function and phagocytosis efficiency is compromised in the setting of MI risk factors, including hyperlipidemia and ageing, however underlying mechanisms remain unclear. Herein we review seminal phagocyte and cardiac molecular factors that lead to, and culminate in, the recognition and removal of dying injured myocardium, the effects of hypoxia, and their relationship to cardiac infarct size and heart healing. PMID:24862542

Relevance of Endoplasmic Reticulum Stress Cell Signaling in Liver Cold Ischemia Reperfusion Injury

PubMed Central

Folch-Puy, Emma; Panisello, Arnau; Oliva, Joan; Lopez, Alexandre; Castro Benítez, Carlos; Adam, René; Roselló-Catafau, Joan

2016-01-01

The endoplasmic reticulum (ER) is involved in calcium homeostasis, protein folding and lipid biosynthesis. Perturbations in its normal functions lead to a condition called endoplasmic reticulum stress (ERS). This can be triggered by many physiopathological conditions such as alcoholic steatohepatitis, insulin resistance or ischemia-reperfusion injury. The cell reacts to ERS by initiating a defensive process known as the unfolded protein response (UPR), which comprises cellular mechanisms for adaptation and the safeguarding of cell survival or, in cases of excessively severe stress, for the initiation of the cell death program. Recent experimental data suggest the involvement of ERS in ischemia/reperfusion injury (IRI) of the liver graft, which has been considered as one of major problems influencing outcome after liver transplantation. The purpose of this review is to summarize updated data on the molecular mechanisms of ERS/UPR and the consequences of this pathology, focusing specifically on solid organ preservation and liver transplantation models. We will also discuss the potential role of ERS, beyond the simple adaptive response and the regulation of cell death, in the modification of cell functional properties and phenotypic changes. PMID:27231901

Mesenchymal stem-cell potential in cartilage repair: an update

PubMed Central

Mazor, M; Lespessailles, E; Coursier, R; Daniellou, R; Best, T M; Toumi, H

2014-01-01

Articular cartilage damage and subsequent degeneration are a frequent occurrence in synovial joints. Treatment of these lesions is a challenge because this tissue is incapable of quality repair and/or regeneration to its native state. Non-operative treatments endeavour to control symptoms and include anti-inflammatory medications, viscosupplementation, bracing, orthotics and activity modification. Classical surgical techniques for articular cartilage lesions are frequently insufficient in restoring normal anatomy and function and in many cases, it has not been possible to achieve the desired results. Consequently, researchers and clinicians are focusing on alternative methods for cartilage preservation and repair. Recently, cell-based therapy has become a key focus of tissue engineering research to achieve functional replacement of articular cartilage. The present manuscript is a brief review of stem cells and their potential in the treatment of early OA (i.e. articular cartilage pathology) and recent progress in the field. PMID:25353372

Altered Memory Circulating T Follicular Helper-B Cell Interaction in Early Acute HIV Infection

PubMed Central

Muir, Roshell; Metcalf, Talibah; Tardif, Virginie; Takata, Hiroshi; Phanuphak, Nittaya; Kroon, Eugene; Colby, Donn J.; Trichavaroj, Rapee; Valcour, Victor; Robb, Merlin L.; Michael, Nelson L.; Ananworanich, Jintanat; Trautmann, Lydie; Haddad, Elias K.

2016-01-01

The RV254 cohort of HIV-infected very early acute (4thG stage 1 and 2) (stage 1/2) and late acute (4thG stage 3) (stage 3) individuals was used to study T helper- B cell responses in acute HIV infection and the impact of early antiretroviral treatment (ART) on T and B cell function. To investigate this, the function of circulating T follicular helper cells (cTfh) from this cohort was examined, and cTfh and memory B cell populations were phenotyped. Impaired cTfh cell function was observed in individuals treated in stage 3 when compared to stage 1/2. The cTfh/B cell cocultures showed lower B cell survival and IgG secretion at stage 3 compared to stage 1/2. This coincided with lower IL-10 and increased RANTES and TNF-α suggesting a role for inflammation in altering cTfh and B cell responses. Elevated plasma viral load in stage 3 was found to correlate with decreased cTfh-mediated B cell IgG production indicating a role for increased viremia in cTfh impairment and dysfunctional humoral response. Phenotypic perturbations were also evident in the mature B cell compartment, most notably a decrease in resting memory B cells in stage 3 compared to stage 1/2, coinciding with higher viremia. Our coculture assay also suggested that intrinsic memory B cell defects could contribute to the impaired response despite at a lower level. Overall, cTfh-mediated B cell responses are significantly altered in stage 3 compared to stage 1/2, coinciding with increased inflammation and a reduction in memory B cells. These data suggest that early ART for acutely HIV infected individuals could prevent immune dysregulation while preserving cTfh function and B cell memory. PMID:27463374

Dolomitized cells within chert of the Permian Assistência Formation, Paraná Basin, Brazil

NASA Astrophysics Data System (ADS)

Calça, Cléber P.; Fairchild, Thomas R.; Cavalazzi, Barbara; Hachiro, Jorge; Petri, Setembrino; Huila, Manuel Fernando Gonzalez; Toma, Henrique E.; Araki, Koiti

2016-04-01

Dolomitic microscopic structures in the form of microspheres, "horseshoe- shaped" objects, and thin botryoidal crusts found within microfossiliferous chert within stromatolites of the Evaporite Bed (EB) of the Permian Assistência Formation, Irati Subgroup, Paraná Basin, Brazil, have been investigated by means of optical microscopy, X-ray fluorescence, scanning electron microscopy, Raman spectrometry and energy-dispersive X-ray spectrometry. The microspheres were identified as dolomitized coccoidal cyanobacteria based on similarity in size, spheroidal and paired hemispheroidal morphologies and colonial habit to co-occurring silicified organic-walled cyanobacteria embedded within the same microfabric and rock samples. The co-occurrence of dolomite, pyrite framboids, and abundant dispersed carbonaceous material and silicified cells is consistent with a hypersaline depositional environment with abundant cyanobacterial mats and elevated Mg2 +/Ca2 + ratios and reducing conditions with active anoxic microbial processes near the water-(bio)sediment interface. The abundance of extracellular polymeric substances facilitated anoxic microbial processes (sulfate reduction), providing essential conditions for possible primary microbially induced dolomitization. In most of the dolomitized cells dolomite occurs only as an external layer; in fully dolomitized cells magnesium is richest in the outermost layer. Presumably, the dolomitization process was favored by the presence of anoxic microbial degraders and negatively charged functional groups at the surface of the cyanobacterial cells. Botryoidal dolomite rims of silica-filled fenestrae formed by a similar process and inherited the botryoidal morphology of the cell as originally lining the fenestrae. Silicification interrupted the dolomitization of the largely organic biosediment, mostly by permineralization, but locally by substitution, thereby preserving not only dolomitic microspheres, but also huge numbers of structurally well-preserved organic-walled cyanobacteria and portions of microbial mat. Clearly, dolomitization began very early in the microbial mats, prior to compaction of the sediment or full obliteration of cellular remains, followed very closely by silicification thereby impeding continued degradation and providing a window onto very well-preserved Permian microbial mats.

Freezing Responses in DMSO-Based Cryopreservation of Human iPS Cells: Aggregates Versus Single Cells.

PubMed

Li, Rui; Yu, Guanglin; Azarin, Samira M; Hubel, Allison

2018-05-01

Inadequate preservation methods of human induced pluripotent stem cells (hiPSCs) have impeded efficient reestablishment of cell culture after the freeze-thaw process. In this study, we examined roles of the cooling rate, seeding temperature, and difference between cell aggregates (3-50 cells) and single cells in controlled rate freezing of hiPSCs. Intracellular ice formation (IIF), post-thaw membrane integrity, cell attachment, apoptosis, and cytoskeleton organization were evaluated to understand the different freezing responses between hiPSC single cells and aggregates, among cooling rates of 1, 3, and 10°C/min, and between seeding temperatures of -4°C and -8°C. Raman spectroscopy images of ice showed that a lower seeding temperature (-8°C) did not affect IIF in single cells, but significantly increased IIF in aggregates, suggesting higher sensitivity of aggregates to supercooling. In the absence of IIF, Raman images showed greater variation of dimethyl sulfoxide concentration across aggregates than single cells, suggesting cryoprotectant transport limitations in aggregates. The ability of cryopreserved aggregates to attach to culture substrates did not correlate with membrane integrity for the wide range of freezing parameters, indicating inadequacy of using only membrane integrity-based optimization metrics. Lower cooling rates (1 and 3°C/min) combined with higher seeding temperature (-4°C) were better at preventing IIF and preserving cell function than a higher cooling rate (10°C/min) or lower seeding temperature (-8°C), proving the seeding temperature range of -7°C to -12°C from literature to be suboptimal. Unique f-actin cytoskeletal organization into a honeycomb-like pattern was observed in postpassage and post-thaw colonies and correlated with successful reestablishment of cell culture.

Gap junction-mediated calcium waves define communication networks among murine postnatal neural progenitor cells.

PubMed

Lacar, Benjamin; Young, Stephanie Z; Platel, Jean-Claude; Bordey, Angélique

2011-12-01

In the postnatal neurogenic niche, two populations of astrocyte-like cells (B cells) persist, one acting as neural progenitor cells (NPCs, B1 cells) and one forming a structural boundary between the neurogenic niche and the striatum (B2 cells, niche astrocytes). Despite being viewed as two distinct entities, we found that B1 and B2 cells express the gap junction protein connexin 43 and display functional coupling involving 50-60 cells. Using neonatal electroporation to label slowly cycling radial glia-derived B1 cells, which send a basal process onto blood vessels, we further confirmed dye coupling between NPCs. To assess the functionality of the coupling, we used calcium imaging in a preparation preserving the three-dimensional architecture of the subventricular zone. Intercellular calcium waves were observed among B cells. These waves travelled bidirectionally between B1 and B2 cells and propagated on blood vessels. Inter-B-cell calcium waves were absent in the presence of a gap junction blocker but persisted with purinergic receptor blockers. These findings show that privileged microdomains of communication networks exist among NPCs and niche astrocytes. Such functional coupling between these two cell types suggests that niche astrocytes do not merely have a structural role, but may play an active role in shaping the behavior of NPCs. © 2011 The Authors. European Journal of Neuroscience © 2011 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

The Transplantation of hBM-MSCs Increases Bone Neo-Formation and Preserves Hearing Function in the Treatment of Temporal Bone Defects - on the Experience of Two Month Follow Up.

PubMed

Školoudík, Lukáš; Chrobok, Viktor; Kočí, Zuzana; Popelář, Jiří; Syka, Josef; Laco, Jan; Filipová, Alžběta; Syková, Eva; Filip, Stanislav

2018-06-03

Temporal bone reconstruction is a persisting problem following middle ear cholesteatoma surgery. Seeking to advance the clinical transfer of stem cell therapy we attempted the reconstruction of temporal bone using a composite bioartificial graft based on a hydroxyapatite bone scaffold combined with human bone marrow-derived mesenchymal stromal cells (hBM-MSCs). The aim of this study was to evaluate the effect of the combined biomaterial on the healing of postoperative temporal bone defects and the preservation of physiological hearing functions in a guinea pig model. The treatment's effect could be observed at 1 and 2 months after implantation of the biomaterial, as opposed to the control group. The clinical evaluation of our results included animal survival, clinical signs of an inflammatory response, and exploration of the tympanic bulla. Osteogenesis, angiogenesis, and inflammation were evaluated by histopathological analyses, whereas hBM-MSCs survival was evaluated by immunofluorescence assays. Hearing capacity was evaluated by objective audiometric methods, i.e. auditory brainstem responses and otoacoustic emission. Our study shows that hBM-MSCs, in combination with hydroxyapatite scaffolds, improves the repair of bone defects providing a safe and effective alternative in their treatment following middle ear surgery due to cholesteatoma.

Isotropic Versus Bipolar Functionalized Biomimetic Artificial Basement Membranes and Their Evaluation in Long-Term Human Cell Co-Culture.

PubMed

Rossi, Angela; Wistlich, Laura; Heffels, Karl-Heinz; Walles, Heike; Groll, Jürgen

2016-08-01

In addition to dividing tissues into compartments, basement membranes are crucial as cell substrates and to regulate cellular behavior. The development of artificial basement membranes is indispensable for the ultimate formation of functional engineered tissues; however, pose a challenge due to their complex structure. Herein, biodegradable electrospun polyester meshes are presented, exhibiting isotropic or bipolar bioactivation as a biomimetic and biofunctional model of the natural basement membrane. In a one-step preparation process, reactive star-shaped prepolymer additives, which generate a hydrophilic fiber surface, are electrospun with cell-adhesion-mediating peptides, derived from major components of the basement membrane. Human skin cells adhere to the functionalized meshes, and long-term co-culture experiments confirm that the artificial basement membranes recapitulate and preserve tissue specific functions. Several layers of immortalized human keratinocytes grow on the membranes, differentiating toward the surface and expressing typical epithelial markers. Fibroblasts migrate into the reticular lamina mimicking part of the mesh. Both cells types begin to produce extracellular matrix proteins and to remodel the initial membrane. It is shown at the example of skin that the artificial basement membrane design provokes biomimetic responses of different cell types and can thus be used as basis for the future development of basement membrane containing tissues. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Utility of Cytospin and Cell block Technology in Evaluation of Body Fluids and Urine Samples: A Comparative Study

PubMed Central

Qamar, Irmeen; Rehman, Suhailur; Mehdi, Ghazala; Maheshwari, Veena; Ansari, Hena A.; Chauhan, Sunanda

2018-01-01

Background: Cytologic examination of body fluids commonly involves the use of direct or sediment smears, cytocentrifuge preparations, membrane filter preparations, or cell block sections. Cytospin and cell block techniques are extremely useful in improving cell yield of thin serous effusions and urine samples, and ensure high diagnostic efficacy. Materials and Methods: We studied cytospin preparations and cell block sections prepared from 180 samples of body fluids and urine samples to compare the relative efficiency of cell retrieval, preservation of cell morphology, ease of application of special stains, and diagnostic efficacy. Samples were collected and processed to prepare cytospin smears and cell block sections. Results: We observed that overall, cell yield and preservation of individual cell morphology were better in cytospin preparations as compared to cell blocks, while preservation of architectural pattern was better in cell block sections. The number of suspicious cases also decreased on cell block sections, with increased detection of malignancy. It was difficult to prepare cell blocks from urine samples due to low cellularity. Conclusions: Cytospin technology is a quick, efficient, and cost-effective method of increasing cell yield in hypocellular samples, with better preservation of cell morphology. Cell blocks are better prepared from high cellularity fluids; however, tissue architecture is better studied, with improved rate of diagnosis and decrease in ambiguous results. Numerous sections can be prepared from a small amount of material. Special stains and immunochemical stains can be easily applied to cell blocks. It also provides a source of archival material. PMID:29643653

Morphological variability, lectin binding and Na+,K+-activated adenosine triphosphatase activity of isolated Müller (glial) cells from the rabbit retina.

PubMed

Reichenbach, A; Dettmer, D; Brückner, G; Neumann, M; Birkenmeyer, G

1985-03-22

Rabbit retinal Müller cells were isolated by means of papaine and mechanical dissociation. These cells were shown to have a well preserved morphology and to preserve viability for many hours. Intense wheat germ agglutinin binding occurs on the photoreceptor side of Müller cells, especially in the microvillous region. Rabbit retinal Müller cells have a Na+,K+-activated adenosine triphosphatase activity in the same order of magnitude as brain astroglial cells.

Antibody Response to a T-Cell-Independent Antigen Is Preserved after Splenic Artery Embolization for Trauma

PubMed Central

Olthof, D. C.; Lammers, A. J. J.; van Leeuwen, E. M. M.; Hoekstra, J. B. L.; ten Berge, I. J. M.

2014-01-01

Splenic artery embolization (SAE) is increasingly being used as a nonoperative management strategy for patients with blunt splenic injury following trauma. The aim of this study was to assess the splenic function of patients who were embolized. A clinical study was performed, with splenic function assessed by examining the antibody response to polysaccharide antigens (pneumococcal 23-valent polysaccharide vaccine), B-cell subsets, and the presence of Howell-Jolly bodies (HJB). The data were compared to those obtained from splenectomized patients and healthy controls (HC) who had been included in a previously conducted study. A total of 30 patients were studied: 5 who had proximal SAE, 7 who had distal SAE, 8 who had a splenectomy, and 10 HC. The median vaccine-specific antibody response of the SAE patients (fold increase, 3.97) did not differ significantly from that of the HC (5.29; P = 0.90); however, the median response of the splenectomized patients (2.30) did differ (P = 0.003). In 2 of the proximally embolized patients and none of the distally embolized patients, the ratio of the IgG antibody level postvaccination compared to that prevaccination was <2. There were no significant differences in the absolute numbers of lymphocytes or B-cell subsets between the SAE patients and the HC. HJB were not observed in the SAE patients. The splenic immune function of embolized patients was preserved, and therefore routine vaccination appears not to be indicated. Although the median antibody responses did not differ between the patients who underwent proximal SAE and those who underwent distal SAE, 2 of the 5 proximally embolized patients had insufficient responses to vaccination, whereas none of the distally embolized patients exhibited an insufficient response. Further research should be done to confirm this finding. PMID:25185578

CD11c(hi) Dendritic Cells Regulate Ly-6C(hi) Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation.

PubMed

Kim, Jin Hyoung; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Patil, Ajit Mahadev; Han, Young Woo; Park, Sang-Youel; Lee, John Hwa; Kim, Koanhoi; Eo, Seong Kug

2015-12-02

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b(+)Ly-6C(hi) monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11c(hi)PDCA-1(int/lo) DCs without alteration in CD11c(int)PDCA-1(hi) plasmacytoid DC number, we found that CD11c(hi) DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b(+)Ly-6C(hi) monocytes and higher expression of CC chemokines. More interestingly, selective CD11c(hi) DC ablation provided altered differentiation and function of infiltrated CD11b(+)Ly-6C(hi) monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b(+)Ly-6C(hi) monocytes generated in CD11c(hi) DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11c(hi) DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b(+)Ly-6C(hi) monocytes.

Three-dimensional hepatocyte culture system for the study of Echinococcus multilocularis larval development

PubMed Central

Chen, Bing; Yan, Hongbin; Zhao, Yannan; Lou, Zhongzi; Li, Jianqiu; Fu, Baoquan; Zhu, Xingquan; McManus, Donald P.; Dai, Jianwu; Jia, Wanzhong

2018-01-01

Background Hepatocyte-based metacestode culture is an attractive method to study alveolar echinococcosis (AE), but it is limited by the relatively short lifespan of cultured hepatocytes in maintaining their normal function. Methodology/principal findings We describe a three-dimensional (3D) hepatic culture system developed from co-cultured hepatocytes and mesenchymal stem cells using a collagen scaffold to study the development of Echinococcus multilocularis larvae. This 3D culture system preserved the function of hepatocytes for a longer period of time than their monolayer counterparts, with albumin secretion, 7-ethoxyresorufin O-deethylation activity, urea synthesis, CYP3A4 and CYP2D6 activity being highly preserved for 21–28 days. The expression levels of hepatocyte-specific genes including CLDN-3, Bsep, AFP, G6P, A1AT, CYP3A4 and NR1I3 were significantly higher in the 3D cultured system compared with their monolayer counterparts after 14-days in culture. Additionally, in the presence of 3D cultured hepatocytes, 81.2% of E. multilocularis protoscoleces rapidly de-differentiated into infective vesicles within eight weeks. Transcriptomic analyses revealed 807 differentially expressed genes between cultured vesicles and protoscoleces, including 119 genes uniquely expressed in protoscoleces, and 242 genes uniquely expressed in vesicles. These differentially expressed genes were mainly involved in parasite growth relating to the G-protein coupled receptor activity pathway, substrate-specific transmembrane transporter activity, cell-cell adhesion process, and potentially with neuroactive ligand-receptor interaction. Conclusions/significance This culture system provides a valuable advance in prolonging hepatocyte functionality, a foundation for future in-depth analysis of the host-parasite interaction in AE, and a useful model to evaluate potential therapeutic strategies to treat AE. PMID:29538424

Vitrification of mouse embryo-derived ICM cells: a tool for preserving embryonic stem cell potential?

PubMed

Desai, Nina; Xu, Jing; Tsulaia, Tamara; Szeptycki-Lawson, Julia; AbdelHafez, Faten; Goldfarb, James; Falcone, Tommaso

2011-02-01

Vitrification technology presents new opportunities for preservation of embryo derived stem cells without first establishing a viable ESC line. This study tests the feasibility of cryopreserving ICM cells using vitrification. ICMs from mouse embryos were isolated and vitrified in HSV straws or on cryoloops. Upon warming, the vitrified ICMs were cultured and observed for attachment and morphology. Colonies were passaged every 3-6 days. ICMs and ICM-derived ESC colonies were tested for expression of stem cell specific markers. ICMs vitrified on both the cryoloop and the HSV straw had high survival rates. ICM derived ESCs remained undifferentiated for several passages and demonstrated expression of typical stem cell markers; SSEA-1, Sox-2, Oct 4 and alkaline phosphatase. This is the first report on successful vitrification of isolated ICMs and the subsequent derivation of ESC colonies. Vitrification of isolated ICMs is a novel approach for preservation of the "stem cell source" material.

Neuron-glia signaling and the protection of axon function by Schwann cells.

PubMed

Quintes, Susanne; Goebbels, Sandra; Saher, Gesine; Schwab, Markus H; Nave, Klaus-Armin

2010-03-01

The interaction between neurons and glial cells is a feature of all higher nervous systems. In the vertebrate peripheral nervous system, Schwann cells ensheath and myelinate axons thereby allowing rapid saltatory conduction and ensuring axonal integrity. Recently, some of the key molecules in neuron-Schwann cell signaling have been identified. Neuregulin-1 (NRG1) type III presented on the axonal surface determines the myelination fate of axons and controls myelin sheath thickness. Recent observations suggest that NRG1 regulates myelination via the control of Schwann cell cholesterol biosynthesis. This concept is supported by the finding that high cholesterol levels in Schwann cells are a rate-limiting factor for myelin protein production and transport of the major myelin protein P0 from the endoplasmic reticulum into the growing myelin sheath. NRG1 type III activates ErbB receptors on the Schwann cell, which leads to an increase in intracellular PIP3 levels via the PI3-kinase pathway. Surprisingly, enforced elevation of PIP3 levels by inactivation of the phosphatase PTEN in developing and mature Schwann cells does not entirely mimic NRG1 type III stimulated myelin growth, but predominantly causes focal hypermyelination starting at Schmidt-Lanterman incisures and nodes of Ranvier. This indicates that the glial transduction of pro-myelinating signals has to be under tight and life-long control to preserve integrity of the myelinated axon. Understanding the cross talk between neurons and Schwann cells will help to further define the role of glia in preserving axonal integrity and to develop therapeutic strategies for peripheral neuropathies such as CMT1A.

Percutaneous CT-Guided Cryoablation as an Alternative Treatment for an Extensive Pelvic Bone Giant Cell Tumor.

PubMed

Panizza, Pedro Sergio Brito; de Albuquerque Cavalcanti, Conrado Furtado; Yamaguchi, Nise Hitomi; Leite, Claudia Costa; Cerri, Giovanni Guido; de Menezes, Marcos Roberto

2016-02-01

A giant cell tumor (GCT) is an intermediate grade, locally aggressive neoplasia. Despite advances in surgical and clinical treatments, cases located on the spine and pelvic bones remain a significant challenge. Failure of clinical treatment with denosumab and patient refusal of surgical procedures (hemipelvectomy) led to the use of cryoablation. We report the use of percutaneous CT-guided cryoablation as an alternative treatment, shown to be a minimally invasive, safe, and effective option for a GCT with extensive involvement of the pelvic bones and allowed structural and functional preservation of the involved bones.

Percutaneous CT-Guided Cryoablation as an Alternative Treatment for an Extensive Pelvic Bone Giant Cell Tumor

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

Panizza, Pedro Sergio Brito; Albuquerque Cavalcanti, Conrado Furtado de; Yamaguchi, Nise Hitomi

2016-02-15

A giant cell tumor (GCT) is an intermediate grade, locally aggressive neoplasia. Despite advances in surgical and clinical treatments, cases located on the spine and pelvic bones remain a significant challenge. Failure of clinical treatment with denosumab and patient refusal of surgical procedures (hemipelvectomy) led to the use of cryoablation. We report the use of percutaneous CT-guided cryoablation as an alternative treatment, shown to be a minimally invasive, safe, and effective option for a GCT with extensive involvement of the pelvic bones and allowed structural and functional preservation of the involved bones.

Facile and one-pot synthesis of uniform PtRu nanoparticles on polydopamine-modified multiwalled carbon nanotubes for direct methanol fuel cell application.

PubMed

Chen, Fengxia; Ren, Junkai; He, Qian; Liu, Jun; Song, Rui

2017-07-01

A facile, environment-friendly and one-pot synthesis method for the preparation of high performance PtRu electrocatalysts on the multiwalled carbon nanotubes (MWCNTs) is reported. Herein, bimetallic PtRu electrocatalysts are deposited onto polydopamine (Pdop) - functionalized MWCNTs by mildly stirring at room temperature. Without the use of expensive chemicals or corrosive acids, this noncovalent functionalization of MWCNTs by Pdop is simple, facile and eco-friendly, and thus preserving the integrity and electronic structure of MWCNTs. Due to the well improved dispersion and the decreased size of alloy nanoparticles, the PtRu electrocatalysts on Pdop-functionalized MWCNTs show much better dispersion, higher electrochemically active surface area, and higher electrocatalytic activity for the electrooxidation of methanol in direct methanol fuel cells, compared with the conventional acid-treated MWCNTs. Copyright © 2017 Elsevier Inc. All rights reserved.

Methylene Blue Protects Astrocytes against Glucose Oxygen Deprivation by Improving Cellular Respiration

PubMed Central

Roy Choudhury, Gourav; Winters, Ali; Rich, Ryan M.; Ryou, Myoung-Gwi; Gryczynski, Zygmunt; Yuan, Fang; Yang, Shao-Hua; Liu, Ran

2015-01-01

Astrocytes outnumber neurons and serve many metabolic and trophic functions in the mammalian brain. Preserving astrocytes is critical for normal brain function as well as for protecting the brain against various insults. Our previous studies have indicated that methylene blue (MB) functions as an alternative electron carrier and enhances brain metabolism. In addition, MB has been shown to be protective against neurodegeneration and brain injury. In the current study, we investigated the protective role of MB in astrocytes. Cell viability assays showed that MB treatment significantly protected primary astrocytes from oxygen-glucose deprivation (OGD) & reoxygenation induced cell death. We also studied the effect of MB on cellular oxygen and glucose metabolism in primary astrocytes following OGD-reoxygenation injury. MB treatment significantly increased cellular oxygen consumption, glucose uptake and ATP production in primary astrocytes. In conclusion our study demonstrated that MB protects astrocytes against OGD-reoxygenation injury by improving astrocyte cellular respiration. PMID:25848957

Evolution and tinkering: what do a protein kinase, a transcriptional regulator and chromosome segregation/cell division proteins have in common?

PubMed

Derouiche, Abderahmane; Shi, Lei; Kalantari, Aida; Mijakovic, Ivan

2016-02-01

In this study, we focus on functional interactions among multi-domain proteins which share a common evolutionary origin. The examples we develop are four Bacillus subtilis proteins, which all possess an ATP-binding Walker motif: the bacterial tyrosine kinase (BY-kinase) PtkA, the chromosome segregation protein Soj (ParA), the cell division protein MinD and a transcription regulator SalA. These proteins have arisen via duplication of the ancestral ATP-binding domain, which has undergone fusions with other functional domains in the process of divergent evolution. We point out that these four proteins, despite having very different physiological roles, engage in an unusually high number of binary functional interactions. Namely, MinD attracts Soj and PtkA to the cell pole, and in addition, activates the kinase function of PtkA. SalA also activates the kinase function of PtkA, and it gets phosphorylated by PtkA as well. The consequence of this phosphorylation is the activation of SalA as a transcriptional repressor. We hypothesize that these functional interactions remain preserved during divergent evolution and represent a constraint on the process of evolutionary "tinkering", brought about by fusions of different functional domains.

Functional Heart Valve Scaffolds Obtained by Complete Decellularization of Porcine Aortic Roots in a Novel Differential Pressure Gradient Perfusion System

PubMed Central

Sierad, Leslie Neil; Shaw, Eliza Laine; Bina, Alexander; Brazile, Bryn; Rierson, Nicholas; Patnaik, Sourav S.; Kennamer, Allison; Odum, Rebekah; Cotoi, Ovidiu; Terezia, Preda; Branzaniuc, Klara; Smallwood, Harrison; Deac, Radu; Egyed, Imre; Pavai, Zoltan; Szanto, Annamaria; Harceaga, Lucian; Suciu, Horatiu; Raicea, Victor; Olah, Peter; Simionescu, Agneta; Liao, Jun; Movileanu, Ionela

2015-01-01

There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for whole-root decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open–close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality. PMID:26467108

Impact of β-hydroxy β-methylbutyrate (HMB) on age-related functional deficits in mice.

PubMed

Munroe, Michael; Pincu, Yair; Merritt, Jennifer; Cobert, Adam; Brander, Ryan; Jensen, Tor; Rhodes, Justin; Boppart, Marni D

2017-01-01

β-Hydroxy β-methylbutyrate (HMB) is a metabolite of the essential amino acid leucine. Recent studies demonstrate a decline in plasma HMB concentrations in humans across the lifespan, and HMB supplementation may be able to preserve muscle mass and strength in older adults. However, the impact of HMB supplementation on hippocampal neurogenesis and cognition remains largely unexplored. The purpose of this study was to simultaneously evaluate the impact of HMB on muscle strength, neurogenesis and cognition in young and aged mice. In addition, we evaluated the influence of HMB on muscle-resident mesenchymal stem/stromal cell (Sca-1 + CD45 - ; mMSC) function to address these cells potential to regulate physiological outcomes. Three month-old (n=20) and 24 month-old (n=18) female C57BL/6 mice were provided with either Ca-HMB or Ca-Lactate in a sucrose solution twice per day for 5.5weeks at a dose of 450mg/kg body weight. Significant decreases in relative peak and mean force, balance, and neurogenesis were observed in aged mice compared to young (age main effects, p≤0.05). Short-term HMB supplementation did not alter activity, balance, neurogenesis, or cognitive function in young or aged mice, yet HMB preserved relative peak force in aged mice. mMSC gene expression was significantly reduced with age, but HMB supplementation was able to recover expression of select growth factors known to stimulate muscle repair (HGF, LIF). Overall, our findings demonstrate that while short-term HMB supplementation does not appear to affect neurogenesis or cognitive function in young or aged mice, HMB may maintain muscle strength in aged mice in a manner dependent on mMSC function. Copyright © 2016 Elsevier Inc. All rights reserved.

High hydrostatic pressure as a method to preserve fresh-cut Hachiya persimmons: A structural approach.

PubMed

Vázquez-Gutiérrez, José Luis; Quiles, Amparo; Vonasek, Erica; Jernstedt, Judith A; Hernando, Isabel; Nitin, Nitin; Barrett, Diane M

2016-12-01

The "Hachiya" persimmon is the most common astringent cultivar grown in California and it is rich in tannins and carotenoids. Changes in the microstructure and some physicochemical properties during high hydrostatic pressure processing (200-400 MPa, 3 min, 25 ℃) and subsequent refrigerated storage were analyzed in this study in order to evaluate the suitability of this non-thermal technology for preservation of fresh-cut Hachiya persimmons. The effects of high-hydrostatic pressure treatment on the integrity and location of carotenoids and tannins during storage were also analyzed. Significant changes, in particular diffusion of soluble compounds which were released as a result of cell wall and membrane damage, were followed using confocal microscopy. The high-hydrostatic pressure process also induced changes in physicochemical properties, e.g. electrolyte leakage, texture, total soluble solids, pH and color, which were a function of the amount of applied hydrostatic pressure and may affect the consumer acceptance of the product. Nevertheless, the results indicate that the application of 200 MPa could be a suitable preservation treatment for Hachiya persimmon. This treatment seems to improve carotenoid extractability and tannin polymerization, which could improve functionality and remove astringency of the fruit, respectively. © The Author(s) 2016.

Maintenance of CCL5 mRNA stores by post-effector and memory CD8 T cells is dependent on transcription and is coupled to increased mRNA stability.

PubMed

Marçais, Antoine; Tomkowiak, Martine; Walzer, Thierry; Coupet, Charles-Antoine; Ravel-Chapuis, Aymeric; Marvel, Jacqueline

2006-10-01

Immunological memory is associated with the display of improved effector functions by cells of the adaptive immune system. The storage of untranslated mRNA coding for the CCL5 chemokine by CD8 memory cells is a new process supporting the immediate display of an effector function. Here, we show that, after induction during the primary response, high CCL5 mRNA levels are specifically preserved in CD8 T cells. We have investigated the mechanisms involved in the long-term maintenance of CCL5 mRNA levels by memory CD8 T cells. We demonstrate that the CCL5 mRNA half-life is increased in memory CD8 T cells and that these cells constitutively transcribe ccl5 gene. By inhibiting ccl5 transcription using IL-4, we demonstrate the essential role of transcription in the maintenance of CCL5 mRNA stores. Finally, we show that these stores are spontaneously reconstituted when the inhibitory signal is removed, indicating that the transcription of ccl5 is a default feature of memory CD8 T cells imprinted in their genetic program.

Clear cell carcinoma of the uterine cervix: clinical characteristics and feasibility of fertility-preserving treatment.

PubMed

Jiang, Xiang; Jin, Ying; Li, Yan; Huang, Hui-Fang; Wu, Ming; Shen, Keng; Pan, Ling-Ya

2014-01-01

The objective of this retrospective study was to analyze the clinical characteristics and prognosis of clear cell adenocarcinoma (CCA) in the post-diethylstilbestrol (DES) era and to evaluate the feasibility of fertility-preserving treatment. The records of 32 patients with CCAs who were treated at Peking Union Medical College Hospital from August 1986 to June 2012 were retrospectively reviewed. Three of the patients had undergone fertility-preserving treatment. The incidence of CCA among cervical adenocarcinomas was 15.2%. The median age was 38 years: 11 patients (34.4%) were diagnosed before 30 years of age and two (6.3%) after 70 years of age. Ten patients (31.2%) were nulliparous. No patient had been exposed to DES. Twenty-nine patients (90.6%) presented with obvious symptoms, and the cervix appeared abnormal in 26 patients (81.3%). Cervical Papanicolaou (Pap) tests were abnormal in all four patients in whom they were performed (three had high-grade squamous intraepithelial lesions and one had atypical squamous cells of undetermined significance). The distribution by stage was 56.3% stage I, 34.4% stage II, 6.3% stage III, and 3.1% stage IV. Treatments mainly included surgery for patients with stage I to IIA CCA and radiochemotherapy for patients with advanced CCA. The overall 5-year progression-free survival was 72.2%. Patients with stage I to IIA CCA had better 5-year progression-free survival than did patients with stage IIB to IV CCA (81.5% versus 40.0%, P=0.003). The three patients who had undergone fertility-preserving treatment had no recurrences. CCA may also affect adolescents and children without prior DES exposure, who are often misdiagnosed as having functional uterine bleeding. Radiotherapy appears to be effective for local control but to have no effect on distant recurrences. In our study, the prognosis of patients with early-stage CCA, including those who had undergone fertility-preserving treatment, was not inferior to that of patients with other types of cervical adenocarcinoma.

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

PubMed Central

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

2017-01-01

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

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

PubMed

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

2017-01-01

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

The Living Eye “Disarms” Uncommitted Autoreactive T Cells by Converting Them to FoxP3+ Regulatory Cells Following Local Antigen Recognition

PubMed Central

Zhou, Ru; Horai, Reiko; Silver, Phyllis B; Mattapallil, Mary J; Zárate-Bladés, Carlos R; Chong, Wai Po; Chen, Jun; Rigden, Rachael C; Villasmil, Rafael; Caspi, Rachel R

2011-01-01

Immune privilege is used by the eye, brain, reproductive organs and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable, and therefore also the most “privileged” of tissues, but paradoxically, remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using FoxP3-GFP reporter mice expressing a retina-specific T cell receptor, we now show that uncommitted T cells rapidly convert in the living eye to FoxP3+ Tregs in a process involving retinal antigen recognition, de novo FoxP3 induction and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye due to its function in the chemistry of vision. Non-converted T cells showed evidence of priming, but appeared restricted from expressing effector function in the eye. Preexisting ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment, and instead caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue. PMID:22238462

Ipsilateral renal function preservation after robot-assisted partial nephrectomy (RAPN): an objective analysis using mercapto-acetyltriglycine (MAG3) renal scan data and volumetric assessment.

PubMed

Zargar, Homayoun; Akca, Oktay; Autorino, Riccardo; Brandao, Luis Felipe; Laydner, Humberto; Krishnan, Jayram; Samarasekera, Dinesh; Stein, Robert J; Kaouk, Jihad H

2015-05-01

To objectively assess ipsilateral renal function (IRF) preservation and factors influencing it after robot-assisted partial nephrectomy (RAPN). Our database was queried to identify patients who had undergone RAPN from 2007 to 2013 and had complete pre- and postoperative mercapto-acetyltriglycine (MAG3) renal scan assessment. The estimated glomerular filtration rate (eGFR) for the operated kidney was calculated by multiplying the percentage of contribution from the renal scan by the total eGFR. IRF preservation was defined as a ratio of the postoperative eGFR for the operated kidney to the preoperative eGFR for the operated kidney. The percentage of total eGFR preservation was calculated in the same manner (postoperative eGFR/preoperative eGFR × 100). The amount of healthy rim of renal parenchyma removed was assessed by deducting the volume of tumour from the volume of the PN specimen assessed on pathology. Multivariable linear regression was used for analysis. In all, 99 patients were included in the analysis. The overall median (interquartile range) total eGFR preservation and IRF preservation for the operated kidney was 83.83 (75.2-94.1)% and 72 (60.3-81)%, respectively (P < 0.01). On multivariable analysis, volume of healthy rim of renal parenchyma removed, warm ischaemia time (WIT) > 30 min, body mass index (BMI) and operated kidney preoperative eGFR were predictive of IRF preservation. Using total eGFR tends to overestimate the degree of renal function preservation after RAPN. This is particularly relevant when studying factors affecting functional outcomes after nephron-sparing surgery. IRF may be a more precise assessment method in this setting. Operated kidney baseline renal function, BMI, WIT >30 min, and amount of resected healthy renal parenchyma represent the factors with a significant impact on the IRF preservation. RAPN provides significant preservation of renal function as shown by objective assessment criteria. © 2014 The Authors. BJU International © 2014 BJU International.

Nutritional Factors and Preservation of C-Peptide in Youth With Recently Diagnosed Type 1 Diabetes

PubMed Central

Mayer-Davis, Elizabeth J.; Dabelea, Dana; Crandell, Jamie L.; Crume, Tessa; D’Agostino, Ralph B.; Dolan, Lawrence; King, Irena B.; Lawrence, Jean M.; Norris, Jill M.; Pihoker, Catherine; The, Natalie

2013-01-01

OBJECTIVE To test the novel hypothesis that nutritional factors previously associated with type 1 diabetes etiology or with insulin secretion are prospectively associated with fasting C-peptide (FCP) concentration among youth recently diagnosed with type 1 diabetes. RESEARCH DESIGN AND METHODS Included were 1,316 youth with autoantibody-positive type 1 diabetes who participated in the SEARCH for Diabetes in Youth study (baseline disease duration, 9.9 months; SD, 6.3). Nutritional exposures included breastfeeding and age at introduction of complementary foods, baseline plasma long-chain omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), vitamin D, vitamin E, and, from a baseline food frequency questionnaire, estimated intake of the branched-chain amino acid leucine and total carbohydrate. Multiple linear regression models were conducted to relate each nutritional factor to baseline FCP adjusted for demographics, disease-related factors, and other confounders. Prospective analyses included the subset of participants with preserved β-cell function at baseline (baseline FCP ≥0.23 ng/mL) with additional adjustment for baseline FCP and time (mean follow-up, 24.3 months; SD, 8.2; n = 656). FCP concentration was analyzed as log(FCP). RESULTS In adjusted prospective analyses, baseline EPA (P = 0.02), EPA plus DHA (P = 0.03), and leucine (P = 0.03) were each associated positively and significantly with FCP at follow-up. Vitamin D was unexpectedly inversely associated with FCP (P = 0.002). CONCLUSIONS Increased intake of branched-chain amino acids and long-chain omega-3 fatty acids may support preservation of β-cell function. This represents a new direction for research to improve prognosis for type 1 diabetes. PMID:23801797

Acellular vascular matrix grafts from human placenta chorion: Impact of ECM preservation on graft characteristics, protein composition and in vivo performance.

PubMed

Schneider, Karl H; Enayati, Marjan; Grasl, Christian; Walter, Ingrid; Budinsky, Lubos; Zebic, Gabriel; Kaun, Christoph; Wagner, Anja; Kratochwill, Klaus; Redl, Heinz; Teuschl, Andreas H; Podesser, Bruno K; Bergmeister, Helga

2018-05-29

Small diameter vascular grafts from human placenta, decellularized with either Triton X-100 (Triton) or SDS and crosslinked with heparin were constructed and characterized. Graft biochemical properties, residual DNA, and protein composition were evaluated to compare the effect of the two detergents on graft matrix composition and structural alterations. Biocompatibility was tested in vitro by culturing the grafts with primary human macrophages and in vivo by subcutaneous implantation of graft conduits (n = 7 per group) into the flanks of nude rats. Subsequently, graft performance was evaluated using an aortic implantation model in Sprague Dawley rats (one month, n = 14). In situ graft imaging was performed using MRI angiography. Retrieved specimens were analyzed by electromyography, scanning electron microscopy, histology and immunohistochemistry to evaluate cell migration and the degree of functional tissue remodeling. Both decellularization methods resulted in grafts of excellent biocompatibility in vitro and in vivo, with low immunogenic potential. Proteomic data revealed removal of cytoplasmic proteins with relative enrichment of ECM proteins in decelluarized specimens of both groups. Noteworthy, LC-Mass Spectrometry analysis revealed that 16 proteins were exclusively preserved in Triton decellularized specimens in comparison to SDS-treated specimens. Aortic grafts showed high patency rates, no signs of thrombus formation, aneurysms or rupture. Conduits of both groups revealed tissue-specific cell migration indicative of functional remodeling. This study strongly suggests that decellularized allogenic grafts from the human placenta have the potential to be used as vascular replacement materials. Both detergents produced grafts with low residual immunogenicity and appropriate mechanical properties. Observed differences in graft characteristics due to preservation method had no impact on successful in vivo performance in the rodent model. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nutritional factors and preservation of C-peptide in youth with recently diagnosed type 1 diabetes: SEARCH Nutrition Ancillary Study.

PubMed

Mayer-Davis, Elizabeth J; Dabelea, Dana; Crandell, Jamie L; Crume, Tessa; D'Agostino, Ralph B; Dolan, Lawrence; King, Irena B; Lawrence, Jean M; Norris, Jill M; Pihoker, Catherine; The, Natalie

2013-07-01

To test the novel hypothesis that nutritional factors previously associated with type 1 diabetes etiology or with insulin secretion are prospectively associated with fasting C-peptide (FCP) concentration among youth recently diagnosed with type 1 diabetes. Included were 1,316 youth with autoantibody-positive type 1 diabetes who participated in the SEARCH for Diabetes in Youth study (baseline disease duration, 9.9 months; SD, 6.3). Nutritional exposures included breastfeeding and age at introduction of complementary foods, baseline plasma long-chain omega-3 fatty acids including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), vitamin D, vitamin E, and, from a baseline food frequency questionnaire, estimated intake of the branched-chain amino acid leucine and total carbohydrate. Multiple linear regression models were conducted to relate each nutritional factor to baseline FCP adjusted for demographics, disease-related factors, and other confounders. Prospective analyses included the subset of participants with preserved β-cell function at baseline (baseline FCP ≥0.23 ng/mL) with additional adjustment for baseline FCP and time (mean follow-up, 24.3 months; SD, 8.2; n = 656). FCP concentration was analyzed as log(FCP). In adjusted prospective analyses, baseline EPA (P = 0.02), EPA plus DHA (P = 0.03), and leucine (P = 0.03) were each associated positively and significantly with FCP at follow-up. Vitamin D was unexpectedly inversely associated with FCP (P = 0.002). Increased intake of branched-chain amino acids and long-chain omega-3 fatty acids may support preservation of β-cell function. This represents a new direction for research to improve prognosis for type 1 diabetes.

[Clinical characteristics and medium-term prognosis of patients with heart failure and preserved systolic function. Do they differ in systolic dysfunction?].

PubMed

Ojeda, Soledad; Anguita, Manuel; Muñoz, Juan F; Rodríguez, Marcos T; Mesa, Dolores; Franco, Manuel; Ureña, Isabel; Vallés, Federico

2003-11-01

To assess the prevalence, clinical profile and medium-term prognosis in patients with heart failure and preserved systolic ventricular function compared to those with systolic dysfunction. 153 patients were included, 62 with preserved systolic ventricular function (left ventricular ejection fraction > or = 45%) and 91 with impaired systolic ventricular function (left ventricular ejection fraction < 45%). The mean follow-up period was 25 10 months. Mean age was similar (66 10 vs. 65 10; p = 0.54). There was a higher proportion of women among patients with preserved systolic function (53% vs. 28%; p < 0.01). Ischemic and idiopathic cardiomyopathy were the most common causes of heart failure in patients with systolic dysfunction, whereas valvular disease and hypertensive cardiopathy were the most common in patients with preserved systolic function. Angiotensin-converting enzyme inhibitors and beta-blockers were more often prescribed in patients with impaired systolic ventricular function (86% vs. 52%; p < 0.01 and 33% vs. 11%; p < 0.01, respectively). There were no differences between the groups in terms of mortality rate (37% vs. 29%), readmission rate for other causes (29% vs. 23%), readmission rate for heart failure (45% vs. 45%), cumulative survival (51% vs. 62%) and the likelihood of not being readmitted for heart failure (50% vs. 52%). In the multivariate analysis, left ventricular ejection fraction was not a predictor of death or readmission because of heart failure. In a large proportion of patients with heart failure, systolic ventricular function is preserved. Despite the clinical differences between patients with preserved and impaired systolic ventricular function, the medium-term prognosis was similar in both groups.

Modulation of GSK-3 provides cellular and functional neuroprotection in the rd10 mouse model of retinitis pigmentosa.

PubMed

Sánchez-Cruz, Alonso; Villarejo-Zori, Beatriz; Marchena, Miguel; Zaldivar-Díez, Josefa; Palomo, Valle; Gil, Carmen; Lizasoain, Ignacio; de la Villa, Pedro; Martínez, Ana; de la Rosa, Enrique J; Hernández-Sánchez, Catalina

2018-04-16

Retinitis pigmentosa (RP) is a group of hereditary retinal neurodegenerative conditions characterized by primary dysfunction and death of photoreceptor cells, resulting in visual loss and, eventually, blindness. To date, no effective therapies have been transferred to clinic. Given the diverse genetic etiology of RP, targeting common cellular and molecular retinal alterations has emerged as a potential therapeutic strategy. Using the Pde6b rd10/rd10 mouse model of RP, we investigated the effects of daily intraperitoneal administration of VP3.15, a small-molecule heterocyclic GSK-3 inhibitor. Gene expression was analyzed by quantitative PCR and protein expression and phosphorylation by Western blot. Photoreceptor preservation was evaluated by histological analysis and visual function was assessed by electroretinography. In rd10 retinas, increased expression of pro-inflammatory markers and reactive gliosis coincided with the early stages of retinal degeneration. Compared with wild-type controls, GSK-3β expression (mRNA and protein) remained unchanged during the retinal degeneration period. However, levels of GSK-3β Ser9 and its regulator Akt Ser473 were increased in rd10 versus wild-type retinas. In vivo administration of VP3.15 reduced photoreceptor cell loss and preserved visual function. This neuroprotective effect was accompanied by a decrease in the expression of neuroinflammatory markers. These results provide proof of concept of the therapeutic potential of VP3.15 for the treatment of retinal neurodegenerative conditions in general, and RP in particular.

Nicotinamide Forestalls Pathology and Cognitive Decline in Alzheimer Mice: Evidence for Improved Neuronal Bioenergetics and Autophagy Procession

PubMed Central

Liu, Dong; Pitta, Michael; Jiang, Haiyang; Lee, Jong-Hwan; Zhang, Guofeng; Chen, Xinzhi; Kawamoto, Elisa M.; Mattson, Mark P.

2012-01-01

Impaired brain energy metabolism and oxidative stress are implicated in cognitive decline and the pathological accumulations of amyloid β-peptide (Aβ) and hyperphosphorylated Tau (p-Tau) in Alzheimer's disease (AD). To determine whether improving brain energy metabolism will forestall disease progress in AD, the impact of the NAD+ precursor nicotinamide on brain cell mitochondrial function and macroautophagy, bioenergetics-related signaling and cognitive performance were studied in cultured neurons and in a mouse model of AD. Oxidative stress resulted in decreased mitochondrial mass, mitochondrial degeneration and autophagosome accumulation in neurons. Nicotinamide preserved mitochondrial integrity and autophagy function, and reduced neuronal vulnerability to oxidative/metabolic insults and Aβ toxicity. NAD+ biosynthesis, autophagy and PI3K signaling were required for the neuroprotective action of nicotinamide. Treatment of 3xTgAD mice with nicotinamide for 8 months resulted in improved cognitive performance, and reduced Aβ and p-Tau pathologies in hippocampus and cerebral cortex. Nicotinamide treatment preserved mitochondrial integrity, and improved autophagy-lysosome procession by enhancing lysosome/autolysosome acidification to reduce autophagosome accumulation. Treatment of 3xTgAD mice with nicotinamide resulted in elevated levels of activated neuroplasticity-related kinases (Akt and ERKs) and the transcription factor cyclic AMP response element-binding protein in the hippocampus and cerebral cortex. Thus, nicotinamide suppresses AD pathology and cognitive decline in a mouse model of AD by a mechanism involving improved brain bioenergetics with preserved functionality of mitochondria and the autophagy system. PMID:23273573

Functionally-fitted energy-preserving integrators for Poisson systems

NASA Astrophysics Data System (ADS)

Wang, Bin; Wu, Xinyuan

2018-07-01

In this paper, a new class of energy-preserving integrators is proposed and analysed for Poisson systems by using functionally-fitted technology. The integrators exactly preserve energy and have arbitrarily high order. It is shown that the proposed approach allows us to obtain the energy-preserving methods derived in [12] by Cohen and Hairer (2011) and in [1] by Brugnano et al. (2012) for Poisson systems. Furthermore, we study the sufficient conditions that ensure the existence of a unique solution and discuss the order of the new energy-preserving integrators.

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca²⁺-sensitive K⁺ current in miniature swine with LV hypertrophy.

PubMed

Emter, Craig A; Tharp, Darla L; Ivey, Jan R; Ganjam, Venkataseshu K; Bowles, Douglas K

2011-10-01

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ET(A)) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K(+) currents (I(K(+))) in coronary smooth muscle cells. Raising internal Ca(2+) from 200 to 500 nM increased Ca(2+)-sensitive K(+) current in HF-TR and control, but not HF animals. In conclusion, an ET(A)-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca(2+)-sensitive I(K(+)) was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca(2+)-sensitive I(K(+)), illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy.

Low-intensity interval exercise training attenuates coronary vascular dysfunction and preserves Ca2+-sensitive K+ current in miniature swine with LV hypertrophy

PubMed Central

Tharp, Darla L.; Ivey, Jan R.; Ganjam, Venkataseshu K.; Bowles, Douglas K.

2011-01-01

Coronary vascular dysfunction has been observed in several models of heart failure (HF). Recent evidence indicates that exercise training is beneficial for patients with HF, but the precise intensity and underlying mechanisms are unknown. Left ventricular (LV) hypertrophy can play a significant role in the development of HF; therefore, the purpose of this study was to assess the effects of low-intensity interval exercise training on coronary vascular function in sedentary (HF) and exercise trained (HF-TR) aortic-banded miniature swine displaying LV hypertrophy. Six months postsurgery, in vivo coronary vascular responses to endothelin-1 (ET-1) and adenosine were measured in the left anterior descending coronary artery. Baseline and maximal coronary vascular conductance were similar between all groups. ET-1-induced reductions in coronary vascular conductance (P < 0.05) were greater in HF vs. sedentary control and HF-TR groups. Pretreatment with the ET type A (ETA) receptor blocker BQ-123 prevented ET-1 hypersensitivity in HF animals. Whole cell voltage clamp was used to characterize composite K+ currents (IK+) in coronary smooth muscle cells. Raising internal Ca2+ from 200 to 500 nM increased Ca2+-sensitive K+ current in HF-TR and control, but not HF animals. In conclusion, an ETA-receptor-mediated hypersensitivity to ET-1, elevated resting LV wall tension, and decreased coronary smooth muscle cell Ca2+-sensitive IK+ was found in sedentary animals with LV hypertrophy. Low-intensity interval exercise training preserved normal coronary vascular function and smooth muscle cell Ca2+-sensitive IK+, illustrating a potential mechanism underlying coronary vascular dysfunction in a large-animal model of LV hypertrophy. Our results demonstrate the potential clinical impact of exercise on coronary vascular function in HF patients displaying pathological LV hypertrophy. PMID:21841018

Glycogen metabolism protects against metabolic insult to preserve carotid body function during glucose deprivation.

PubMed

Holmes, Andrew P; Turner, Philip J; Carter, Paul; Leadbeater, Wendy; Ray, Clare J; Hauton, David; Buckler, Keith J; Kumar, Prem

2014-10-15

The view that the carotid body (CB) type I cells are direct physiological sensors of hypoglycaemia is challenged by the finding that the basal sensory neuronal outflow from the whole organ is unchanged in response to low glucose. The reason for this difference in viewpoint and how the whole CB maintains its metabolic integrity when exposed to low glucose is unknown. Here we show that, in the intact superfused rat CB, basal sensory neuronal activity was sustained during glucose deprivation for 29.1 ± 1.2 min, before irreversible failure following a brief period of excitation. Graded increases in the basal discharge induced by reducing the superfusate PO2 led to proportional decreases in the time to the pre-failure excitation during glucose deprivation which was dependent on a complete run-down in glycolysis and a fall in cellular energy status. A similar ability to withstand prolonged glucose deprivation was observed in isolated type I cells. Electron micrographs and immunofluorescence staining of rat CB sections revealed the presence of glycogen granules and the glycogen conversion enzymes glycogen synthase I and glycogen phosphorylase BB, dispersed throughout the type I cell cytoplasm. Furthermore, pharmacological attenuation of glycogenolysis and functional depletion of glycogen both significantly reduced the time to glycolytic run-down by ∼33 and 65%, respectively. These findings suggest that type I cell glycogen metabolism allows for the continuation of glycolysis and the maintenance of CB sensory neuronal output in periods of restricted glucose delivery and this may act as a key protective mechanism for the organ during hypoglycaemia. The ability, or otherwise, to preserve energetic status may thus account for variation in the reported capacity of the CB to sense physiological glucose concentrations and may even underlie its function during pathological states associated with augmented CB discharge. © 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.

The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant Cells.

PubMed

Datta, Sandipan; Baudouin, Christophe; Brignole-Baudouin, Francoise; Denoyer, Alexandre; Cortopassi, Gino A

2017-04-01

Benzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as "dry eye" and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects. Mitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3). Benzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 μM) and O2 consumption (IC50, 10.9 μM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107-667 μM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 μM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 μM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect. Benzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients.

The Eye Drop Preservative Benzalkonium Chloride Potently Induces Mitochondrial Dysfunction and Preferentially Affects LHON Mutant Cells

PubMed Central

Datta, Sandipan; Baudouin, Christophe; Brignole-Baudouin, Francoise; Denoyer, Alexandre; Cortopassi, Gino A.

2017-01-01

Purpose Benzalkonium chloride (BAK) is the most commonly used eye drop preservative. Benzalkonium chloride has been associated with toxic effects such as “dry eye” and trabecular meshwork degeneration, but the underlying biochemical mechanism of ocular toxicity by BAK is unclear. In this study, we propose a mechanistic basis for BAK's adverse effects. Method Mitochondrial O2 consumption rates of human corneal epithelial primary cells (HCEP), osteosarcoma cybrid cells carrying healthy (control) or Leber hereditary optic neuropathy (LHON) mutant mtDNA [11778(G>A)], were measured before and after acute treatment with BAK. Mitochondrial adenosine triphosphate (ATP) synthesis and cell viability were also measured in the BAK-treated control: LHON mutant and human-derived trabecular meshwork cells (HTM3). Results Benzalkonium chloride inhibited mitochondrial ATP (IC50, 5.3 μM) and O2 consumption (IC50, 10.9 μM) in a concentration-dependent manner, by directly targeting mitochondrial complex I. At its pharmaceutical concentrations (107–667 μM), BAK inhibited mitochondrial function >90%. In addition, BAK elicited concentration-dependent cytotoxicity to cybrid cells (IC50, 22.8 μM) and induced apoptosis in HTM3 cells at similar concentrations. Furthermore, we show that BAK directly inhibits mitochondrial O2 consumption in HCEP cells (IC50, 3.8 μM) at 50-fold lower concentrations than used in eye drops, and that cells bearing mitochondrial blindness (LHON) mutations are further sensitized to BAK's mitotoxic effect. Conclusions Benzalkonium chloride inhibits mitochondria of human corneal epithelial cells and cells bearing LHON mutations at pharmacologically relevant concentrations, and we suggest this is the basis of BAK's ocular toxicity. Prescribing BAK-containing eye drops should be avoided in patients with mitochondrial deficiency, including LHON patients, LHON carriers, and possibly primary open-angle glaucoma patients. PMID:28444329

REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

EPA Science Inventory

This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium parvum as a function of preservation method and oocyst age. Simila...

REMOVAL OF CRYPTOSPORIDIUM BY IN-LINE FILTRATION AS A FUNCTION OF OOCYST AGE AND PRESERVATION METHOD

EPA Science Inventory

This study examined the impacts of oocyst preservation method and age on the removal of seeded Cryptosporidium oocysts by in-line filtration. An existing study has investigated the infectivity of Cryptosporidium Parvum as a function of preservation method and oocyst age. Simila...

Prostate Cancer Patient Characteristics Associated With a Strong Preference to Preserve Sexual Function and Receipt of Active Surveillance.

PubMed

Broughman, James R; Basak, Ramsankar; Nielsen, Matthew E; Reeve, Bryce B; Usinger, Deborah S; Spearman, Kiayni C; Godley, Paul A; Chen, Ronald C

2018-04-01

Men with early-stage prostate cancer have multiple options that have similar oncologic efficacy but vary in terms of their impact on quality of life. In low-risk cancer, active surveillance is the option that best preserves patients' sexual function, but it is unknown if patient preference affects treatment selection. Our objectives were to identify patient characteristics associated with a strong preference to preserve sexual function and to determine whether patient preference and baseline sexual function level are associated with receipt of active surveillance in low-risk cancer. In this population-based cohort of men with localized prostate cancer, baseline patient-reported sexual function was assessed using a validated instrument. Patients were also asked whether preservation of sexual function was very, somewhat, or not important. Prostate cancer disease characteristics and treatments received were abstracted from medical records. A modified Poisson regression model with robust standard errors was used to compute adjusted risk ratio (aRR) estimates. All statistical tests were two-sided. Among 1194 men, 52.6% indicated a strong preference for preserving sexual function. Older men were less likely to have a strong preference (aRR = 0.98 per year, 95% confidence interval [CI] = 0.97 to 0.99), while men with normal sexual function were more likely (vs poor function, aRR = 1.59, 95% CI = 1.39 to 1.82). Among 568 men with low-risk cancer, there was no clear association between baseline sexual function or strong preference to preserve function with receipt of active surveillance. However, strong preference may differnetially impact those with intermediate baseline function vs poor function (Pinteraction = .02). Treatment choice may not always align with patients' preferences. These findings demonstrate opportunities to improve delivery of patient-centered care in early prostate cancer.

The consequence of delayed fixation on subsequent preservation of urine cells.

PubMed

Ahmed, Hussain G; Tom, Murtada Am

2011-01-01

Degenerative changes caused by delays in urine preservation contribute to false-negative and false-positive interpretation of urothelial disease in cytology. The aim of this study is to assess whether the delay of fixation of urine samples makes any significant difference to urine cytology and morphology, and the limit of acceptability of delay for routine use in the hospital laboratory. Three cell collection fluids were evaluated by analyzing the preservation and degeneration of cells in urine samples. In this study, 50 voided urine specimens were taken at random from females complaining of vaginal discharge. Each specimen was divided into three sterile containers. The first was immediately centrifugated and the deposit was smeared onto a cleaned micro slide and immediately fixed into 95% ethyl alcohol for 15 minutes. The remaining two were prepared in the same manner, however, the second after two hours of collection and the third after four hours of collection. The degree of degeneration and thus the preservation were assessed by a table of chosen criteria, then ranked and analyzed using Friedman's nonparametric test, at p=0.05. The results showed a significant difference between the preservation and the delay in urine fixation, p<0.0001. Any delay in fixation of urine specimen for cytology affects the preservation of cells, which may result in miss diagnosis. It is recommended that urine samples for cytology should be fixed immediately after collection.

Toxicity of cosmetic preservatives on human ocular surface and adnexal cells.

PubMed

Chen, Xiaomin; Sullivan, David A; Sullivan, Amy Gallant; Kam, Wendy R; Liu, Yang

2018-05-01

Cosmetic products, such as mascara, eye shadow, eyeliner and eye makeup remover are used extensively to highlight the eyes or clean the eyelids, and typically contain preservatives to prevent microbial growth. These preservatives include benzalkonium chloride (BAK) and formaldehyde (FA)-releasing preservatives. We hypothesize that these preservatives, at concentrations (BAK = 1 mg/ml; FA = 0.74 mg/ml) approved for consumer use, are toxic to human ocular surface and adnexal cells. Accordingly, we tested the influence of BAK and FA on the morphology, survival, and proliferation and signaling ability of immortalized human meibomian gland (iHMGECs), corneal (iHCECs) and conjunctival (iHConjECs) epithelial cells. iHMGECs, iHCECs and iHConjECs were cultured with different concentrations of BAK (5 μg/ml to 0.005 μg/ml) or FA (1 mg/ml to 1 μg/ml) under basal, proliferating or differentiating conditions up to 7 days. We used low BAK levels, because we found that 0.5 mg/ml and 50 μg/ml BAK killed iHMGECs within 1 day after a 15 min exposure. Experimental procedures included analyses of cell appearance, cell number, and neutral lipid content (LipidTox), lysosome accumulation (LysoTracker) and AKT signaling in all 3 cell types. Our results demonstrate that BAK and FA cause dose-dependent changes in the morphology, survival, proliferation and AKT signaling of iHMGECs, iHCECs and iHConjECs. Many of the concentrations tested induced cell atrophy, poor adherence, decreased proliferation and death, after 5 days of exposure. Cellular signaling, as indicated by AKT phosphorylation after 15 (FA) or 30 (BAK) minutes of treatment, was also reduced in a dose-dependent fashion in all 3 cell types, irrespective of whether cells had been cultured under proliferating or differentiating conditions. Our results support our hypothesis and demonstrate that the cosmetic preservatives, BAK and FA, exert many toxic effects on cells of the ocular surface and adnexa. Copyright © 2018 Elsevier Ltd. All rights reserved.

Pulsed electromagnetic field improves cardiac function in response to myocardial infarction.

PubMed

Hao, Chang-Ning; Huang, Jing-Juan; Shi, Yi-Qin; Cheng, Xian-Wu; Li, Hao-Yun; Zhou, Lin; Guo, Xin-Gui; Li, Rui-Lin; Lu, Wei; Zhu, Yi-Zhun; Duan, Jun-Li

2014-01-01

Extracorporeal pulsed electromagnetic field (PEMF) has been shown the ability to improve regeneration in various ischemic episodes. Here, we examined whether PEMF therapy facilitate cardiac recovery in rat myocardial infarction (MI), and the cellular/molecular mechanisms underlying PEMF-related therapy was further investigated. The MI rats were exposed to active PEMF for 4 cycles per day (8 minutes/cycle, 30 ± 3 Hz, 5 mT) after MI induction. The data demonstrated that PEMF treatment significantly inhibited cardiac apoptosis and improved cardiac systolic function. Moreover, PEMF treatment increased capillary density, the levels of vascular endothelial growth factor (VEGF) and hypoxic inducible factor-1α in infarct border zone. Furthermore, the number and function of circulating endothelial progenitor cells were advanced in PEMF treating rats. In vitro, PEMF induced the degree of human umbilical venous endothelial cells tubulization and increased soluble pro-angiogenic factor secretion (VEGF and nitric oxide). In conclusion, PEMF therapy preserves cardiac systolic function, inhibits apoptosis and trigger postnatal neovascularization in ischemic myocardium.

Preserved speech abilities and compensation following prefrontal damage.

PubMed

Buckner, R L; Corbetta, M; Schatz, J; Raichle, M E; Petersen, S E

1996-02-06

Lesions to left frontal cortex in humans produce speech production impairments (nonfluent aphasia). These impairments vary from subject to subject and performance on certain speech production tasks can be relatively preserved in some patients. A possible explanation for preservation of function under these circumstances is that areas outside left prefrontal cortex are used to compensate for the injured brain area. We report here a direct demonstration of preserved language function in a stroke patient (LF1) apparently due to the activation of a compensatory brain pathway. We used functional brain imaging with positron emission tomography (PET) as a basis for this study.

Placental Mesenchymal Stromal Cells Rescue Ambulation in Ovine Myelomeningocele

PubMed Central

Brown, Erin G.; Lankford, Lee; Keller, Benjamin A.; Pivetti, Christopher D.; Sitkin, Nicole A.; Beattie, Michael S.; Bresnahan, Jacqueline C.; Farmer, Diana L.

2015-01-01

Myelomeningocele (MMC)—commonly known as spina bifida—is a congenital birth defect that causes lifelong paralysis, incontinence, musculoskeletal deformities, and severe cognitive disabilities. The recent landmark Management of Myelomeningocele Study (MOMS) demonstrated for the first time in humans that in utero surgical repair of the MMC defect improves lower limb motor function, suggesting a capacity for improved neurologic outcomes in this disorder. However, functional recovery was incomplete, and 58% of the treated children were unable to walk independently at 30 months of age. In the present study, we demonstrate that using early gestation human placenta-derived mesenchymal stromal cells (PMSCs) to augment in utero repair of MMC results in significant and consistent improvement in neurologic function at birth in the rigorous fetal ovine model of MMC. In vitro, human PMSCs express characteristic MSC markers and trilineage differentiation potential. Protein array assays and enzyme-linked immunosorbent assay show that PMSCs secrete a variety of immunomodulatory and angiogenic cytokines. Compared with adult bone marrow MSCs, PMSCs secrete significantly higher levels of brain-derived neurotrophic factor and hepatocyte growth factor, both of which have known neuroprotective capabilities. In vivo, functional and histopathologic analysis demonstrated that human PMSCs mediate a significant, clinically relevant improvement in motor function in MMC lambs and increase the preservation of large neurons within the spinal cord. These preclinical results in the well-established fetal ovine model of MMC provide promising early support for translating in utero stem cell therapy for MMC into clinical application for patients. Significance This study presents placenta-derived mesenchymal stromal cell (PMSC) treatment as a potential therapy for myelomeningocele (MMC). Application of PMSCs can augment current in utero surgical repair in the well-established and rigorously applied fetal lamb model of MMC. Treatment with human PMSCs significantly and dramatically improved neurologic function and preserved spinal cord neuron density in experimental animals. Sixty-seven percent of the PMSC-treated lambs were able to ambulate independently, with two exhibiting no motor deficits whatsoever. In contrast, none of the lambs treated with the vehicle alone were capable of ambulation. The locomotor rescue demonstrated in PMSC-treated lambs indicates great promise for future clinical trials to improve paralysis in children afflicted with MMC. PMID:25911465

Antifibrotic Therapy in Simian Immunodeficiency Virus Infection Preserves CD4+ T-Cell Populations and Improves Immune Reconstitution With Antiretroviral Therapy

PubMed Central

Estes, Jacob D.; Reilly, Cavan; Trubey, Charles M.; Fletcher, Courtney V.; Cory, Theodore J.; Piatak, Michael; Russ, Samuel; Anderson, Jodi; Reimann, Thomas G.; Star, Robert; Smith, Anthony; Tracy, Russell P.; Berglund, Anna; Schmidt, Thomas; Coalter, Vicky; Chertova, Elena; Smedley, Jeremy; Haase, Ashley T.; Lifson, Jeffrey D.; Schacker, Timothy W.

2015-01-01

Even with prolonged antiretroviral therapy (ART), many human immunodeficiency virus-infected individuals have <500 CD4+ T cells/µL, and CD4+ T cells in lymphoid tissues remain severely depleted, due in part to fibrosis of the paracortical T-cell zone (TZ) that impairs homeostatic mechanisms required for T-cell survival. We therefore used antifibrotic therapy in simian immunodeficiency virus-infected rhesus macaques to determine whether decreased TZ fibrosis would improve reconstitution of peripheral and lymphoid CD4+ T cells. Treatment with the antifibrotic drug pirfenidone preserved TZ architecture and was associated with significantly larger populations of CD4+ T cells in peripheral blood and lymphoid tissues. Combining pirfenidone with an ART regimen was associated with greater preservation of CD4+ T cells than ART alone and was also associated with higher pirfenidone concentrations. These data support a potential role for antifibrotic drug treatment as adjunctive therapy with ART to improve immune reconstitution. PMID:25246534

Bacterial Species and Biochemical Characteristic Investigations of Nostoc flagelliforme Concentrates during its Storage.

PubMed

Yue, Lifang; Lv, Hexin; Zhen, Jing; Jiang, Shengping; Jia, Shiru; Shen, Shigang; Gao, Lu; Dai, Yujie

2016-04-28

Preservation of fresh algae plays an important role in algae seed subculture and aquaculture. The determination and examination of the changes of cell viability, composition, and bacterial species during storage would help to take suitable preservation methods to prolong the preservation time of fresh algae. Nostoc flagelliforme is a kind of edible cyanobacterium with important herbal and dietary values. This article investigated the changes of bacterial species and biochemical characteristics of fresh N. flagelliforme concentrate during natural storage. It was found that the viability of cells decreased along with the storage time. Fourteen bacteria strains in the algae concentrate were identified by PCR-DGGE and were grouped into four phyla, including Cyanobacteria, Firmicutes, Proteobacteria, and Bacteroidetes. Among them, Enterococcus viikkiensis may be a concern in the preservation. Eleven volatile organic compounds were identified from N. flagelliforme cells, in which geosmin could be treated as an indicator of the freshness of N. flagelliforme. The occurrence of indole compound may be an indicator of the degradation of cells.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting

PubMed Central

Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

2016-01-01

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling. PMID:26858399

The living eye "disarms" uncommitted autoreactive T cells by converting them to Foxp3(+) regulatory cells following local antigen recognition.

PubMed

Zhou, Ru; Horai, Reiko; Silver, Phyllis B; Mattapallil, Mary J; Zárate-Bladés, Carlos R; Chong, Wai Po; Chen, Jun; Rigden, Rachael C; Villasmil, Rafael; Caspi, Rachel R

2012-02-15

Immune privilege is used by the eye, brain, reproductive organs, and gut to preserve structural and functional integrity in the face of inflammation. The eye is arguably the most vulnerable and, therefore, also the most "privileged" of tissues; paradoxically, it remains subject to destructive autoimmunity. It has been proposed, although never proven in vivo, that the eye can induce T regulatory cells (Tregs) locally. Using Foxp3-GFP reporter mice expressing a retina-specific TCR, we now show that uncommitted T cells rapidly convert in the living eye to Foxp3(+) Tregs in a process involving retinal Ag recognition, de novo Foxp3 induction, and proliferation. This takes place within the ocular tissue and is supported by retinoic acid, which is normally present in the eye because of its function in the chemistry of vision. Nonconverted T cells showed evidence of priming but appeared restricted from expressing effector function in the eye. Pre-existing ocular inflammation impeded conversion of uncommitted T cells into Tregs. Importantly, retina-specific T cells primed in vivo before introduction into the eye were resistant to Treg conversion in the ocular environment and, instead, caused severe uveitis. Thus, uncommitted T cells can be disarmed, but immune privilege is unable to protect from uveitogenic T cells that have acquired effector function prior to entering the eye. These findings shed new light on the phenomenon of immune privilege and on its role, as well as its limitations, in actively controlling immune responses in the tissue.

Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting.

PubMed

Ma, Xuanyi; Qu, Xin; Zhu, Wei; Li, Yi-Shuan; Yuan, Suli; Zhang, Hong; Liu, Justin; Wang, Pengrui; Lai, Cheuk Sun Edwin; Zanella, Fabian; Feng, Gen-Sheng; Sheikh, Farah; Chien, Shu; Chen, Shaochen

2016-02-23

The functional maturation and preservation of hepatic cells derived from human induced pluripotent stem cells (hiPSCs) are essential to personalized in vitro drug screening and disease study. Major liver functions are tightly linked to the 3D assembly of hepatocytes, with the supporting cell types from both endodermal and mesodermal origins in a hexagonal lobule unit. Although there are many reports on functional 2D cell differentiation, few studies have demonstrated the in vitro maturation of hiPSC-derived hepatic progenitor cells (hiPSC-HPCs) in a 3D environment that depicts the physiologically relevant cell combination and microarchitecture. The application of rapid, digital 3D bioprinting to tissue engineering has allowed 3D patterning of multiple cell types in a predefined biomimetic manner. Here we present a 3D hydrogel-based triculture model that embeds hiPSC-HPCs with human umbilical vein endothelial cells and adipose-derived stem cells in a microscale hexagonal architecture. In comparison with 2D monolayer culture and a 3D HPC-only model, our 3D triculture model shows both phenotypic and functional enhancements in the hiPSC-HPCs over weeks of in vitro culture. Specifically, we find improved morphological organization, higher liver-specific gene expression levels, increased metabolic product secretion, and enhanced cytochrome P450 induction. The application of bioprinting technology in tissue engineering enables the development of a 3D biomimetic liver model that recapitulates the native liver module architecture and could be used for various applications such as early drug screening and disease modeling.

Timing of Bone Marrow Cell Delivery Has Minimal Effects on Cell Viability and Cardiac Recovery Following Myocardial Infarction

PubMed Central

Swijnenburg, Rutger-Jan; Govaert, Johannes A.; van der Bogt, Koen E.A.; Pearl, Jeremy I.; Huang, Mei; Stein, William; Hoyt, Grant; Vogel, Hannes; Contag, Christopher H.; Robbins, Robert C.; Wu, Joseph C.

2011-01-01

Background Despite ongoing clinical trials, the optimal time for delivery of bone marrow mononuclear cells (BMCs) following myocardial infarction (MI) is unclear. We compared the viability and effects of transplanted BMCs on cardiac function in the acute and sub-acute inflammatory phases of MI. Methods and Results The time-course of acute inflammatory cell infiltration was quantified by FACS analysis of enzymatically digested hearts of FVB mice (n=12) following LAD ligation. Mac-1+Gr-1high neutrophil infiltration peaked at day 4. BMCs were harvested from transgenic FVB mice expressing firefly luciferase (Fluc) and green fluorescent protein (GFP). Afterwards, 2.5×106 BMCs were injected into the left ventricle of wild-type FVB mice either immediately (Acute BMC) or 7 days (Sub-acute BMC) after MI, or after a sham procedure (n=8 per group). In vivo bioluminescence imaging (BLI) showed an early signal increase in both BMC groups at day 7, followed by a non-significant trend (P=0.203) towards improved BMC survival in the Sub-acute BMC group that persisted until the BLI signal reached background levels after 42 days. Compared to controls (MI + saline injection), echocardiography showed a significant preservation of fractional shortening at 4 weeks (Acute BMC vs saline; P<0.01) and 6 weeks (both BMC groups vs saline; P<0.05), but no significant differences between the two BMC groups. FACS analysis of BMC injected hearts at day 7 revealed that GFP+ BMCs expressed hematopoietic (CD45, Mac-1, Gr-1), minimal progenitor (Sca-1, c-kit), and no endothelial (CD133, Flk-1) or cardiac (Trop-T) cell markers. Conclusion Timing of BMC delivery has minimal effects on intramyocardial retention and preservation of cardiac function. In general, there is poor long-term engraftment and BMCs tend to adopt inflammatory cell phenotypes. PMID:19920031

Developing a preservation policy and procedure statement for a health sciences library.

PubMed Central

Paulson, B A

1989-01-01

The preconditions for creating a preservation policy document in a health sciences library are an existing preservation policy for the institution of which it is a part and administrative support for preservation. The assumption underlying preservation activity, from the formulation of general guidelines to the detail of operating procedure, is that collection development and preservation are complementary functions. Documentation of operational procedures in some detail should be a part of the statement. Since preservation activity cuts across functional library structures, all management staff should be involved in the planning process and be made aware of their responsibilities. The creation of a preservation policy statement will highlight unaddressed issues, procedural inadequacies, and differences in staff perceptions of priorities, but a written statement provides a framework for setting priorities and making decisions. PMID:2758183

Basal exon skipping and nonsense-associated altered splicing allows bypassing complete CEP290 loss-of-function in individuals with unusually mild retinal disease.

PubMed

Barny, Iris; Perrault, Isabelle; Michel, Christel; Soussan, Mickael; Goudin, Nicolas; Rio, Marlène; Thomas, Sophie; Attié-Bitach, Tania; Hamel, Christian; Dollfus, Hélène; Kaplan, Josseline; Rozet, Jean-Michel; Gerard, Xavier

2018-05-16

CEP290 mutations cause a spectrum of ciliopathies from Leber congenital amaurosis type 10 (LCA10) to embryo-lethal Meckel syndrome (MKS). Using panel-based molecular diagnosis testing for inherited retinal diseases, we identified two individuals with some preserved vision despite biallelism for presumably truncating CEP290 mutations. The first one carried a homozygous 1 base-pair deletion in exon 17, introducing a premature termination codon (PTC) in exon 18 (c.1666del; p.Ile556Phefs*17). mRNA analysis revealed a basal exon skipping (BES) of exon 18, providing mutant cells with the ability to escape protein truncation, while disrupting the reading frame in controls. The second individual harbored compound heterozygous nonsense mutations in exon 8 (c.508A>T, p.Lys170*) and exon 32 (c.4090G>T, p.Glu1364*), respectively. Some CEP290 lacking exon 8 were detected in mutant fibroblasts but not in controls whereas some skipping of exon 32 occurred in both lines, but with higher amplitude in the mutant. Considering that the deletion of either exon maintains the reading frame in either line, skipping in mutant cells likely involves nonsense-associated altered splicing (NAS) alone (exon 8), or with BES (exon 32). Skipping of PTC-containing exons in mutant cells allowed production of CEP290 isoforms with preserved ability to assemble into a high molecular weight complex and to interact efficiently with proteins important for cilia formation and intraflagellar trafficking. In contrast, studying LCA10 and MKS fibroblasts we show moderate to severe cilia alterations, providing support for a correlation between disease severity and the ability of cells to express shortened, yet functional, CEP290 isoforms.

Cryopreservation of Fish Spermatogonial Cells: The Future of Natural History Collections.

PubMed

Hagedorn, Mary M; Daly, Jonathan P; Carter, Virginia L; Cole, Kathleen S; Jaafar, Zeehan; Lager, Claire V A; Parenti, Lynne R

2018-04-18

As global biodiversity declines, the value of biological collections increases. Cryopreserved diploid spermatogonial cells meet two goals: to yield high-quality molecular sequence data; and to regenerate new individuals, hence potentially countering species extinction. Cryopreserved spermatogonial cells that allow for such mitigative measures are not currently in natural history museum collections because there are no standard protocols to collect them. Vertebrate specimens, especially fishes, are traditionally formalin-fixed and alcohol-preserved which makes them ideal for morphological studies and as museum vouchers, but inadequate for molecular sequence data. Molecular studies of fishes routinely use tissues preserved in ethanol; yet tissues preserved in this way may yield degraded sequences over time. As an alternative to tissue fixation methods, we assessed and compared previously published cryopreservation methods by gating and counting fish testicular cells with flow cytometry to identify presumptive spermatogonia A-type cells. Here we describe a protocol to cryopreserve tissues that yields a high percentage of viable spermatogonial cells from the testes of Asterropteryx semipunctata, a marine goby. Material cryopreserved using this protocol represents the first frozen and post-thaw viable spermatogonial cells of fishes archived in a natural history museum to provide better quality material for re-derivation of species and DNA preservation and analysis.

The preservation and degradation of filamentous bacteria and biomolecules within iron oxide deposits at Rio Tinto, Spain.

PubMed

Preston, L J; Shuster, J; Fernández-Remolar, D; Banerjee, N R; Osinski, G R; Southam, G

2011-05-01

One of the keys to understanding and identifying life on other planets is to study the preservation of organic compounds and their precursor micro-organisms on Earth. Rio Tinto in southwestern Spain is a well documented site of microbial preservation within iron sulphates and iron oxides over a period of 2.1 Ma. This study has investigated the preservation of filamentous iron oxidising bacteria and organics through optical microscopy, scanning electron microscopy (SEM) and Fourier transform infra-red (FTIR) spectroscopy, from laboratory cultures of natural samples to contemporary natural materials to million-year old river terraces. Up to 40% elemental carbon and >7% nitrogen has been identified within microbial filaments and cell clusters in all samples through SEM EDS analyses. FTIR spectroscopy identified C-H(x) absorption bands between 2960 and 2800 cm(-1), Amide I and II absorption bands at 1656 and 1535 cm(-1), respectively and functional group vibrations from within nucleic acids at 917, 1016 and 1124 cm(-1). Absorption bands tracing the diagenetic transformation of jarosite to goethite to hematite through the samples are also identified. This combination of mineralogy, microbial morphology and biomolecular evidence allows us to further understand how organic fossils are created and preserved in iron-rich environments, and ultimately will aid in the search for the earliest life on Earth and potential organics on Mars. © 2011 Blackwell Publishing Ltd.

Effects of different fixation and freeze substitution methods on the ultrastructural preservation of ZYMV-infected Cucurbita pepo (L.) leaves.

PubMed

Zechmann, Bernd; Müller, Maria; Zellnig, Günther

2005-08-01

Different fixation protocols [chemical fixation, plunge and high pressure freezing (HPF)] were used to study the effects of Zucchini yellow mosaic virus (ZYMV) disease on the ultrastructure of adult leaves of Styrian oil pumpkin plants (Cucurbita pepo L. subsp. pepo var. styriaca Greb.) with the transmission electron microscope. Additionally, different media were tested for freeze substitution (FS) to evaluate differences in the ultrastructural preservation of cryofixed plant leaf cells. FS was either performed in (i) 2% osmium tetroxide in anhydrous acetone containing 0.2% uranyl acetate, (ii) 0.01% safranin in anhydrous acetone, (iii) 0.5% glutaraldehyde in anhydrous acetone or (iv) anhydrous acetone. No ultrastructural differences were found in well-preserved cells of plunge and high pressure frozen samples. Cryofixed cells showed a finer granulated cytosol and smoother membranes, than what was found in chemically fixed samples. HPF led in comparison to plunge frozen plant material to an excellent preservation of vascular bundle cells. The use of FS-media such as anhydrous acetone, 0.01% safranin and 0.5% glutaraldehyde led to low membrane contrast and did not preserve the inner fine structures of mitochondria. Additionally, the use of 0.5% glutaraldehyde caused the cytosol to be fuzzy and partly loosened. ZYMV-induced ultrastructural alterations like cylindrical inclusions and dilated ER-cisternae did not differ between chemically fixed and cryofixed cells and were found within the cytosol of infected leaf cells and within sieve tube elements. The results demonstrate specific structural differences depending on the FS-medium used, which has to be considered for investigations of selected cell structures.

Xenon treatment attenuates early renal allograft injury associated with prolonged hypothermic storage in rats.

PubMed

Zhao, Hailin; Yoshida, Akira; Xiao, Wei; Ologunde, Rele; O'Dea, Kieran P; Takata, Masao; Tralau-Stewart, Catherine; George, Andrew J T; Ma, Daqing

2013-10-01

Prolonged hypothermic storage elicits severe ischemia-reperfusion injury (IRI) to renal grafts, contributing to delayed graft function (DGF) and episodes of acute immune rejection and shortened graft survival. Organoprotective strategies are therefore needed for improving long-term transplant outcome. The aim of this study is to investigate the renoprotective effect of xenon on early allograft injury associated with prolonged hypothermic storage. Xenon exposure enhanced the expression of heat-shock protein 70 (HSP-70) and heme oxygenase 1 (HO-1) and promoted cell survival after hypothermia-hypoxia insult in human proximal tubular (HK-2) cells, which was abolished by HSP-70 or HO-1 siRNA. In the brown Norway to Lewis rat renal transplantation, xenon administered to donor or recipient decreased the renal tubular cell death, inflammation, and MHC II expression, while delayed graft function (DGF) was therefore reduced. Pathological changes associated with acute rejection, including T-cell, macrophage, and fibroblast infiltration, were also decreased with xenon treatment. Donors or recipients treated with xenon in combination with cyclosporin A had prolonged renal allograft survival. Xenon protects allografts against delayed graft function, attenuates acute immune rejection, and enhances graft survival after prolonged hypothermic storage. Furthermore, xenon works additively with cyclosporin A to preserve post-transplant renal function.

Targeting Cellular Calcium Homeostasis to Prevent Cytokine-Mediated Beta Cell Death.

PubMed

Clark, Amy L; Kanekura, Kohsuke; Lavagnino, Zeno; Spears, Larry D; Abreu, Damien; Mahadevan, Jana; Yagi, Takuya; Semenkovich, Clay F; Piston, David W; Urano, Fumihiko

2017-07-17

Pro-inflammatory cytokines are important mediators of islet inflammation, leading to beta cell death in type 1 diabetes. Although alterations in both endoplasmic reticulum (ER) and cytosolic free calcium levels are known to play a role in cytokine-mediated beta cell death, there are currently no treatments targeting cellular calcium homeostasis to combat type 1 diabetes. Here we show that modulation of cellular calcium homeostasis can mitigate cytokine- and ER stress-mediated beta cell death. The calcium modulating compounds, dantrolene and sitagliptin, both prevent cytokine and ER stress-induced activation of the pro-apoptotic calcium-dependent enzyme, calpain, and partly suppress beta cell death in INS1E cells and human primary islets. These agents are also able to restore cytokine-mediated suppression of functional ER calcium release. In addition, sitagliptin preserves function of the ER calcium pump, sarco-endoplasmic reticulum Ca 2+ -ATPase (SERCA), and decreases levels of the pro-apoptotic protein thioredoxin-interacting protein (TXNIP). Supporting the role of TXNIP in cytokine-mediated cell death, knock down of TXNIP in INS1-E cells prevents cytokine-mediated beta cell death. Our findings demonstrate that modulation of dynamic cellular calcium homeostasis and TXNIP suppression present viable pharmacologic targets to prevent cytokine-mediated beta cell loss in diabetes.

Clusterin Reduces Cold Ischemia-Reperfusion Injury in Heart Transplantation Through Regulation of NF-kB Signaling and Bax/Bcl-xL Expression.

PubMed

Liu, Guodong; Zhang, Hongmei; Hao, Fengyun; Hao, Jing; Pan, Lixiao; Zhao, Qing; Wo, Jinshan

2018-01-01

Ischemia-reperfusion (I/R) injury is an unavoidable event occurring during heart transplantation and is a key factor in graft failure and the long-term survival rate of recipients. Therefore, there is an urgent need for the development of new therapies to prevent I/R injury. Clusterin is a hetero-dimeric glycoprotein with an antiapoptotic function. In this study, we investigated whether clusterin was cardioprotective in heart transplantation against I/R injury using an in vivo rat model and an in vitro cell culture system, and examined the underlying mechanisms of I/R injury. Heart grafts from wild-type C57BL/6 mice were preserved in UW solution (control) or UW solution containing recombinant human apolipoprotein-J (hr clusterin) for 24 h. The preserved hearts were implanted into recipient mice of the same strain as the donors for 72 h, and the heart grafts were then taken for histopathological and gene expression analyses. An in vitro ischemia reperfusion model using H9C2 cells or H9C2/clusterin cDNA cells was constructed. The expression of clusterin, p65, Bax, Bcl-xL, IL-1β, and TNF-α protein and mRNA in heart tissue and H9C2 cells was detected by western blot, reverse transcription-polymerase chain reaction (RT-PCR), and quantitative RT-PCR assays; IL-1β and TNF-α protein was detected by enzyme-linked immunosorbent assays; NF-kB activity was detected by an electrophoretic mobility shift assay; cell apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometric analyses. Cold I/R caused severe morphologic myocardial injury to heart grafts from wild-type C57BL/6 mice, whereas grafts from hr clusterin preservation showed less damage, as demonstrated by decreased cell apoptosis/death, decreased neutrophil infiltration, and the preservation of the normal structure of the heart. Clusterin reduced the expression of p65, pre-inflammatory IL-1β, and TNF-α, and the pro-apoptotic gene Bax, while it enhanced the expression of the anti-apoptotic gene Bcl-xL in vitro and in vivo. Clusterin inhibited cell apoptosis/death and reduced pre-inflammatory. Clusterin is a promising target for preventing cold I/R injury in heart transplantation. This study also shows that the resultant protective effects of clusterin are mediated by NF-κB signaling and Bax/Bcl-xL expression. © 2018 The Author(s). Published by S. Karger AG, Basel.

Assessment of organ culture for the conservation of human skin allografts.

PubMed

Hautier, A; Sabatier, F; Stellmann, P; Andrac, L; Nouaille De Gorce, Y; Dignat-George, F; Magalon, G

2008-03-01

Human skin allografts are used in the treatment of severe burns and their preservation is therefore critical for optimal clinical benefit. Current preservation methods, such as 4 degrees C storage or cryopreservation, cannot prevent the decrease of tissue viability. The aim of this study was to assess viability and function of skin allografts in a new skin organ culture model, allowing conservation parameters as close as possible to physiological conditions: 32 degrees C, air-liquid interface and physiological skin tension. Twelve skin samples, harvested from 6 living surgical donors, were conserved 35 days in two conditions: conservation at 4 degrees C and organ culture. Viability and function of skin samples were investigated at Day 0, 7, 14, 21, 28 and 35 using cell culture methods (trypan blue exclusion, Colony Forming Efficiency and Growth Rate), histopathological and histoenzymological studies (Ki67 immunostaining). In the two conditions, fibroblast and keratinocyte viability was progressively affected by storage, with a significant decrease observed after 35 days. No statistical difference could be observed between the two conditions. The two methods were also comparable regarding alterations of fibroblast and keratinocyte culture parameters, which were respectively significantly reduced at Day 7 and 21, compared to fresh skin. By contrast, histopathological and histoenzymological studies revealed a better preservation of skin architecture and proliferative potential at 4 degrees C, as compared to organ culture. These results indicate that skin organ culture does not provide significant advantages for skin allograft preservation. However, its potential use as an experimental model to study skin physiology and wound healing should be further evaluated.

High-pressure freezing and freeze substitution of Arabidopsis for electron microscopy.

PubMed

Austin, Jotham R

2014-01-01

The objectives of electron microscopy ultrastructural studies are to examine cellular architecture and relate the cell's structural machinery to dynamic functional roles. This aspiration is difficult to achieve if specimens have not been adequately preserved in a "living state"; hence specimen preparation is of the utmost importance for the success of any electron micrographic study. High-pressure freezing (HPF)/freeze substitution (FS) has long been recognized as the primer technique for the preservation of ultrastructure in biological samples. In most cases a basic HPF/freeze substitution protocol is sufficient to obtain superior ultrastructural preservation and structural contrast, which allows one to use more advanced microscopy techniques such as 3D electron tomography. However, for plant tissues, which have a thick cell wall, large water-filled vacuoles, and air spaces (all of which are detrimental to cryopreservation), these basic HPF/FS protocols often yield undesirable results. In particular, ice crystal artifacts and the staining of membrane systems are often poorly or negatively stained, which make 3D segmentation of a tomogram difficult. To overcome these problems, various aspects of the HPF/FS protocol can be altered, including the cryo-filler(s) used, freeze substitution cocktail, and the resin infiltration process. This chapter will describe these modifications for the preparation of plant tissues for routine electron microscopic studies, immunocytochemistry, and 3D tomographic electron imaging.

In vitro model to study the effects of matrix stiffening on Ca2+ handling and myofilament function in isolated adult rat cardiomyocytes.

PubMed

van Deel, Elza D; Najafi, Aref; Fontoura, Dulce; Valent, Erik; Goebel, Max; Kardux, Kim; Falcão-Pires, Inês; van der Velden, Jolanda

2017-07-15

This paper describes a novel model that allows exploration of matrix-induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function. Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca 2+ handling and myofilament function. Cell shortening and Ca 2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix. Matrix stiffness-impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness. Matrix stiffness-induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. Extracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte-matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix-induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca 2+ handling but does not alter myofilament-generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness-induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness-induced changes in cardiomyocyte function could not be explained by adaptation in the microtubules. Additionally, cardiomyocytes isolated from stiff hearts of the obese ZSF1 rat model of heart failure with preserved ejection fraction show more pronounced reduction in unloaded shortening in response to matrix stiffening. Taken together, we introduce a method that allows evaluation of the influence of ECM properties on cardiomyocyte function separate from the passive inhibitory component of a stiff matrix. As such, it adds an important and physiologically relevant tool to investigate the functional consequences of cardiomyocyte-matrix interactions. © 2017 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.

Consensus for nonmelanoma skin cancer treatment: basal cell carcinoma, including a cost analysis of treatment methods.

PubMed

Kauvar, Arielle N B; Cronin, Terrence; Roenigk, Randall; Hruza, George; Bennett, Richard

2015-05-01

Basal cell carcinoma (BCC) is the most common cancer in the US population affecting approximately 2.8 million people per year. Basal cell carcinomas are usually slow-growing and rarely metastasize, but they do cause localized tissue destruction, compromised function, and cosmetic disfigurement. To provide clinicians with guidelines for the management of BCC based on evidence from a comprehensive literature review, and consensus among the authors. An extensive review of the medical literature was conducted to evaluate the optimal treatment methods for cutaneous BCC, taking into consideration cure rates, recurrence rates, aesthetic and functional outcomes, and cost-effectiveness of the procedures. Surgical approaches provide the best outcomes for BCCs. Mohs micrographic surgery provides the highest cure rates while maximizing tissue preservation, maintenance of function, and cosmesis. Mohs micrographic surgery is an efficient and cost-effective procedure and remains the treatment of choice for high-risk BCCs and for those in cosmetically sensitive locations. Nonsurgical modalities may be used for low-risk BCCs when surgery is contraindicated or impractical, but the cure rates are lower.

Cell-free synthetic biology for environmental sensing and remediation.

PubMed

Karig, David K

2017-06-01

The fields of biosensing and bioremediation leverage the phenomenal array of sensing and metabolic capabilities offered by natural microbes. Synthetic biology provides tools for transforming these fields through complex integration of natural and novel biological components to achieve sophisticated sensing, regulation, and metabolic function. However, the majority of synthetic biology efforts are conducted in living cells, and concerns over releasing genetically modified organisms constitute a key barrier to environmental applications. Cell-free protein expression systems offer a path towards leveraging synthetic biology, while preventing the spread of engineered organisms in nature. Recent efforts in the areas of cell-free approaches for sensing, regulation, and metabolic pathway implementation, as well as for preserving and deploying cell-free expression components, embody key steps towards realizing the potential of cell-free systems for environmental sensing and remediation. Copyright © 2017 The Author. Published by Elsevier Ltd.. All rights reserved.

Printing of Three-Dimensional Tissue Analogs for Regenerative Medicine

PubMed Central

Lee, Vivian K.; Dai, Guohao

2016-01-01

3-D cell printing, which can accurately deposit cells, biomaterial scaffolds and growth factors in precisely defined spatial patterns to form biomimetic tissue structures, has emerged as a powerful enabling technology to create live tissue and organ structures for drug discovery and tissue engineering applications. Unlike traditional 3-D printing that uses metals, plastics and polymers as the printing materials, cell printing has to be compatible with living cells and biological matrix. It is also required that the printing process preserves the biological functions of the cells and extracellular matrix, and to mimic the cell-matrix architectures and mechanical properties of the native tissues. Therefore, there are significant challenges in order to translate the technologies of traditional 3-D printing to cell printing, and ultimately achieve functional outcomes in the printed tissues. So it is essential to develop new technologies specially designed for cell printing and in-depth basic research in the bioprinted tissues, such as developing novel biomaterials specifically for cell printing applications, understanding the complex cell-matrix remodeling for the desired mechanical properties and functional outcomes, establishing proper vascular perfusion in bioprinted tissues, etc. In recent years, many exciting research progresses have been made in the 3-D cell printing technology and its application in engineering live tissue constructs. This review paper summarized the current development in 3-D cell printing technologies; focus on the outcomes of the live printed tissues and their potential applications in drug discovery and regenerative medicine. Current challenges and limitations are highlighted, and future directions of 3-D cell printing technology are also discussed. PMID:27066784

Multiphoton-generated localized electron plasma for membrane permeability modification in single cells

NASA Astrophysics Data System (ADS)

Merritt, T.; Leblanc, M.; McMillan, J.; Westwood, J.; Khodaparast, G. A.

2014-03-01

Successful incorporation of a specific macromolecule into a single cell would be ideal for characterizing trafficking dynamics through plasmodesmata or for studying intracellular localizations. Here, we demonstrate NIR femtosecond laser-mediated infiltration of a membrane impermeable dextran-conjugated dye into living cells of Arabidopsis thaliana seedling stems. Based on the reactions of fluorescing vacuoles of transgenic cells and artificial cell walls comprised of nanocellulose, laser intensity and exposure time were adjusted to avoid deleterious effects. Using these plant-tailored laser parameters, cells were injected with the fluorophores and long-term dye retention was observed, all while preserving vital cell functions. This method is ideal for studies concerning cell-to-cell interactions and potentially paves the way for introducing transgenes to specific cells. This work was supported by NSF award IOS-0843372 to JHW, with additional support from and U.S. Department of Agriculture Hatch Project no. 135997, and by the Institute of Critical Technology and Applied Sciences (ICTAS) at Virginia Tech.

Niche players

PubMed Central

Seandel, Marco; Falciatori, Ilaria; Shmelkov, Sergey V.; Kim, Jiyeon; James, Daylon; Rafii, Shahin

2010-01-01

The undifferentiated spermatogonia of adult mouse testes are composed of both true stem cells and committed progenitors. It is unclear what normally prevents these adult germ cells from manifesting multipotency. The critical elements of the spermatogonial stem cell niche, while poorly understood, are thought to be composed of Sertoli cells with several other somatic cell types in close proximity. We recently discovered a novel orphan G-protein coupled receptor (GPR125) that is restricted to undifferentiated spermatogonia within the testis. GPR125 expression was maintained when the progenitor cells were extracted from the in vivo niche and propagated under growth conditions that recapitulate key elements of the niche. Such conditions preserved the ability of the cells to generate multipotent derivatives, known as multipotent adult spermatogonial derived progenitor cells (MASCs). Upon differentiation, the latter produced a variety tissues including functional endothelium, illustrating the potential applications of such cells. Thus, GPR125 represents a novel target for purifying adult stem and progenitors from tissues, with the goal of developing autologous multipotent cell lines. PMID:18256534

Three dimensional culture of the murine osteoblastic cell line OCT-1 on collagen coated microcarriers

NASA Astrophysics Data System (ADS)

Lau, P.; Hellweg, C. E.; Kirchner, S.; Baumstark-Khan, C.

2005-08-01

During long-term space missions, astronauts suffer from the loss of minerals especially from weightbearing bones due to prolonged sojourn under microgravity. Bone loss during space flight is about 1-2% per month. Bone is continually being remodelled under the influence of three types of highly specialized cells. Osteoblasts, the bone forming cells, osteoclasts, the bone resorbing cells and finally osteocytes preserve the homeostasis of bone formation and resorption. In vitro 3- dimensional cell culture of osteoblastic cell lines on microcarrier beads might be a better model to evaluate changes in bone cell morphology, function and differentiation under influence of spaceflight related factors than the conventional 2-D monolayer culture technique. Furthermore, it allows production of a greater amount of cells compared to the monolayer culture. Aim of this study is to examine the effects of culturing the immortalized murine osteoblastic cell line OCT-1 in a 3- dimensional environment on cell morphology and proliferation rate.

Effects of pressure and temperature on the survival rate of adherent A-172 cells

NASA Astrophysics Data System (ADS)

Yasuhara, Ryo; Kushida, Ryo; Ishii, Shiwori; Yamanoha, Banri; Shimizu, Akio

2013-06-01

Preservation of cells under high pressure is an important alternative to cryopreservation. We studied the effect of temperature (4, 25, 37°C) and pressure (0.1-350 MPa) on the survival rate of A-172 glioblastoma cells. The survival rate was not changed by brief (10 min) pressurization of up to 150 MPa, but the survival rate began to decrease from 150 MPa, and most of the A-172 cells died when treated with over 200 MPa. Lengthy pressurization (4 days) at lower pressure (upto 20.1 MPa) without medium exchange showed complex results. The survival rate of cells preserved at 25°C showed two maxima at 1.6 and 20.1 MPa. After preservation, cells adhered and proliferated in the same way as normal cells when cultured at 37°C in a CO2 incubator. The other two temperatures, 4° and 37°C, showed no maximum survival rate. Therefore, a high survival rate can be maintained with high pressure treatment.

[Fertility preservation in boys: spermatogonial stem cell transplantation and testicular grafting].

PubMed

Goossens, E; Tournaye, H

2013-09-01

Spermatogonial stem cells (SSC) are the founder cells of spermatogenesis and are responsible for the lifelong production of spermatozoa. The cryopreservation and transplantation of these cells has been proposed as a fertility preservation strategy for young boys at risk for stem cell loss, i.e. patients undergoing chemotherapy for cancer or as a conditioning treatment for bone marrow transplantation. To prevent lifelong sterility in boys, two fertility restoration strategies are being developed: the injection of SSC and the grafting of testicular tissue containing SSC. Depending on the disease of the patient one of these two approaches will be applicable. Grafting has the advantage that SSC can reside within their natural niche, preserving the interactions between germ cells and their supporting cells and may therefore be regarded as the first choice strategy. However, in cases where the risk for malignant contamination of the testicular tissue is real, e.g. leukemia, transplantation of SSC by injection is preferable over grafting. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Honey bees preferentially consume freshly-stored pollen

USDA-ARS?s Scientific Manuscript database

Honey bees collect and store pollen in cells in a preserved form known as stored pollen, or beebread. To preserve pollen, bees add nectar and honey to collected pollen to form stored pollen. Bees eat stored pollen from a wide selection of pollen cells that have been stored for different lengths of...

Clinical methods of cryopreservation for donor lymphocyte infusions vary in their ability to preserve functional T-cell subpopulations.

PubMed

Worsham, D Nicole; Reems, Jo-Anna; Szczepiorkowski, Zbigniew M; McKenna, David H; Leemhuis, Thomas; Mathew, Aby J; Cancelas, Jose A

2017-06-01

Cryopreserved donor lymphocyte infusion (DLI) products are manufactured and administered to treat relapse after allogeneic hematopoietic stem cell transplantation. Reported clinical responses to DLIs vary broadly, even within the same group of patients. While there is an implicit recognition of the fact that different manufacturing protocols may have specific effects on different cell types, cryopreservation protocols are frequently derived from our experience in the cryopreservation of stem cell products and do not account for the heterogeneous functional nature of DLI T-cell populations. Here, we report the results of a prospective, multicenter trial on the effect of four different cryopreservation solutions that were used to freeze DLIs compared to control DLIs that were refrigerated overnight. Cryopreserved postthawed and refrigerated specimens were analyzed side by side for their T-cell subpopulation content and viability, as well as T-cell proliferation, cytokine secretion, and cytotoxic activities. This study indicates that "homemade" 10% dimethyl sulfoxide (DMSO) results in reduced viability of different CD4+ T-cell populations, including T-helper, T-cytotoxic, and T-regulatory populations, and a decrease in their proliferative and cytotoxic response to immunologically relevant stimuli, while the use of solutions containing 5% DMSO with intracellular-like cryoprotectant stabilizers maintains T-cell function at levels similar to refrigerated control samples. This study has important implications in determining the best cryoprotectant solution for specific clinical applications in allogeneic immunotherapy. © 2017 AABB.

Double-hydrophobic elastin-like polypeptides with added functional motifs: Self-assembly and cytocompatibility.

PubMed

Le, Duc H T; Tsutsui, Yoko; Sugawara-Narutaki, Ayae; Yukawa, Hiroshi; Baba, Yoshinobu; Ohtsuki, Chikara

2017-09-01

We have recently developed a novel double-hydrophobic elastin-like triblock polypeptide called GPG, designed after the uneven distribution of two different hydrophobic domains found in elastin, an extracellular matrix protein providing elasticity and resilience to tissues. Upon temperature trigger, GPG undergoes a sequential self-assembling process to form flexible beaded nanofibers with high homogeneity and excellent dispersibility in water. Given that GPG might be a potential elastin-mimetic material, we sought to explore the biological activities of this block polypeptide. Besides GPG, several functionalized derivatives were also constructed by fusing functional motifs such as KAAK or KAAKGRGDS at the C-terminal of GPG. Although the added motifs affected the kinetics of fiber formation and β-sheet contents, all three GPGs assembled into beaded nanofibers at the physiological temperature. The resulting GPG nanofibers preserved their beaded structures in cell culture medium; therefore, they were coated on polystyrene substrates to study their cytocompatibility toward mouse embryonic fibroblasts, NIH-3T3. Among the three polypeptides, GPG having the cell-binding motif GRGDS derived from fibronectin showed excellent cell adhesion and cell proliferation properties compared to other conventional materials, suggesting its promising applications as extracellular matrices for mammalian cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2475-2484, 2017. © 2017 Wiley Periodicals, Inc.

Scaling down of a clinical three-dimensional perfusion multicompartment hollow fiber liver bioreactor developed for extracorporeal liver support to an analytical scale device useful for hepatic pharmacological in vitro studies.

PubMed

Zeilinger, Katrin; Schreiter, Thomas; Darnell, Malin; Söderdahl, Therese; Lübberstedt, Marc; Dillner, Birgitta; Knobeloch, Daniel; Nüssler, Andreas K; Gerlach, Jörg C; Andersson, Tommy B

2011-05-01

Within the scope of developing an in vitro culture model for pharmacological research on human liver functions, a three-dimensional multicompartment hollow fiber bioreactor proven to function as a clinical extracorporeal liver support system was scaled down in two steps from 800 mL to 8 mL and 2 mL bioreactors. Primary human liver cells cultured over 14 days in 800, 8, or 2 mL bioreactors exhibited comparable time-course profiles for most of the metabolic parameters in the different bioreactor size variants. Major drug-metabolizing cytochrome P450 activities analyzed in the 2 mL bioreactor were preserved over up to 23 days. Immunohistochemical studies revealed tissue-like structures of parenchymal and nonparenchymal cells in the miniaturized bioreactor, indicating physiological reorganization of the cells. Moreover, the canalicular transporters multidrug-resistance-associated protein 2, multidrug-resistance protein 1 (P-glycoprotein), and breast cancer resistance protein showed a similar distribution pattern to that found in human liver tissue. In conclusion, the down-scaled multicompartment hollow fiber technology allows stable maintenance of primary human liver cells and provides an innovative tool for pharmacological and kinetic studies of hepatic functions with small cell numbers.

Phagocyte-myocyte interactions and consequences during hypoxic wound healing.

PubMed

Zhang, Shuang; Dehn, Shirley; DeBerge, Matthew; Rhee, Ki-Jong; Hudson, Barry; Thorp, Edward B

2014-01-01

Myocardial infarction (MI), secondary to atherosclerotic plaque rupture and occlusive thrombi, triggers acute margination of inflammatory neutrophils and monocyte phagocyte subsets to the damaged heart, the latter of which may give rise briefly to differentiated macrophage-like or dendritic-like cells. Within the injured myocardium, a primary function of these phagocytic cells is to remove damaged extracellular matrix, necrotic and apoptotic cardiac cells, as well as immune cells that turn over. Recognition of dying cellular targets by phagocytes triggers intracellular signaling, particularly in macrophages, wherein cytokines and lipid mediators are generated to promote inflammation resolution, fibrotic scarring, angiogenesis, and compensatory organ remodeling. These actions cooperate in an effort to preserve myocardial contractility and prevent heart failure. Immune cell function is modulated by local tissue factors that include secreted protease activity, oxidative stress during clinical reperfusion, and hypoxia. Importantly, experimental evidence suggests that monocyte function and phagocytosis efficiency is compromised in the setting of MI risk factors, including hyperlipidemia and ageing, however underlying mechanisms remain unclear. Herein we review seminal phagocyte and cardiac molecular factors that lead to, and culminate in, the recognition and removal of dying injured myocardium, the effects of hypoxia, and their relationship to cardiac infarct size and heart healing. Copyright © 2014 Elsevier Inc. All rights reserved.

Regulation of pancreatic islet beta-cell mass by growth factor and hormone signaling.

PubMed

Huang, Yao; Chang, Yongchang

2014-01-01

Dysfunction and destruction of pancreatic islet beta cells is a hallmark of diabetes. Better understanding of cellular signals in beta cells will allow development of therapeutic strategies for diabetes, such as preservation and expansion of beta-cell mass and improvement of beta-cell function. During the past several decades, the number of studies analyzing the molecular mechanisms, including growth factor/hormone signaling pathways that impact islet beta-cell mass and function, has increased exponentially. Notably, somatolactogenic hormones including growth hormone (GH), prolactin (PRL), and insulin-like growth factor-1 (IGF-1) and their receptors (GHR, PRLR, and IGF-1R) are critically involved in beta-cell growth, survival, differentiation, and insulin secretion. In this chapter, we focus more narrowly on GH, PRL, and IGF-1 signaling, and GH-IGF-1 cross talk. We also discuss how these signaling aspects contribute to the regulation of beta-cell proliferation and apoptosis. In particular, our novel findings of GH-induced formation of GHR-JAK2-IGF-1R protein complex and synergistic effects of GH and IGF-1 on beta-cell signaling, proliferation, and antiapoptosis lead to a new concept that IGF-1R may serve as a proximal component of GH/GHR signaling. © 2014 Elsevier Inc. All rights reserved.

Preserved function of the plasma membrane calcium pump of red blood cells from diabetic subjects with high levels of glycated haemoglobin.

PubMed

Bookchin, Robert M; Etzion, Zipora; Lew, Virgilio L; Tiffert, Teresa

2009-03-01

The activity of the plasma membrane Ca(2+)-pump decreases steeply throughout the 120 days lifespan of normal human red blood cells. Experiments with isolated membrane preparations showed that glycation of a lysine residue near the catalytic site of the pump ATPase had a powerful inhibitory effect. This prompted the question of whether glycation is the mechanism of age-related decline in pump activity in vivo. It is important to investigate this mechanism because the Ca(2+) pump is a major regulator of Ca(2+) homeostasis in all cells. Its impaired activity in diabetic patients, continuously exposed to high glycation rates, may thus contribute to varied tissue pathology in this disease. We measured Ca(2+)-pump activity as a function of red cell age in red cells from diabetics continuously exposed to high glucose concentrations, as documented by their high mean levels of glycated haemoglobin. The distribution of Ca(2+)-pump activities was indistinguishable from that in non-diabetics, and the pattern of activity decline with cell age in the diabetics' red cells was identical to that observed in red cells from non-diabetics. These results indicate that in intact cells the Ca(2+) pump is protected from glycation-induced inactivation.

Aging-associated inflammation promotes selection for adaptive oncogenic events in B cell progenitors.

PubMed

Henry, Curtis J; Casás-Selves, Matias; Kim, Jihye; Zaberezhnyy, Vadym; Aghili, Leila; Daniel, Ashley E; Jimenez, Linda; Azam, Tania; McNamee, Eoin N; Clambey, Eric T; Klawitter, Jelena; Serkova, Natalie J; Tan, Aik Choon; Dinarello, Charles A; DeGregori, James

2015-12-01

The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRAS(V12), or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRAS(V12)-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation--a common feature of aging--has the potential to limit aging-associated oncogenesis.

Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone-rod dystrophy.

PubMed

Lhériteau, Elsa; Petit, Lolita; Weber, Michel; Le Meur, Guylène; Deschamps, Jack-Yves; Libeau, Lyse; Mendes-Madeira, Alexandra; Guihal, Caroline; François, Achille; Guyon, Richard; Provost, Nathalie; Lemoine, Françoise; Papal, Samantha; El-Amraoui, Aziz; Colle, Marie-Anne; Moullier, Philippe; Rolling, Fabienne

2014-02-01

For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

Treatment of multiple synchronous misdiagnosed renal cell cancers in a young patient affected by a "de novo" Von Hippel-Lindau syndrome.

PubMed

Allasia, Marco; Battaglia, Antonino; Pasini, Barbara; Gazzera, Carlo; Calandri, Marco; Bosio, Andrea; Gontero, Paolo; Destefanis, Paolo

2017-02-28

Von Hippel-Lindau (VHL) disease is an autosomal dominant inherited syndrome occurring in one out of 36,000 live births. Diagnosis could be a challenge in patients with no familial VHL history. Renal cancer (RCC) represents one of the most important manifestations. RCC is usually recurrent and multifocal. Actually treating RCC in VHL patients represent a clinical dilemma: the oncological outcomes must be balanced against renal function preservation. A young man with a negative familial history was referred to our department with seven misdiagnosed renal masses. VHL disease was determined through genetic test. The multiple RCCs were treated by surgery and percutaneous thermal ablation by radiofrequency ablation (RFA) with complete control of RCC and no impairment of renal function. This case history confirms that VHL disease has to be suspected in young patients with evidence of synchronous multiple renal masses and in presence of specific clinical criteria.RFA appears to be safe in terms of oncological radicalism and in renal function preservation.In hereditary RCC, we should purpose, whenever it is possible, minimally invasive treatment in terms of low hospital stay and a minimal loss of renal tissue.

Preservation in the Age of Google: Digitization, Digital Preservation, and Dilemmas

ERIC Educational Resources Information Center

Conway, Paul

2010-01-01

The cultural heritage preservation community now functions largely within the environment of digital technologies. This article begins by juxtaposing definitions of the terms "digitization for preservation" and "digital preservation" within a sociotechnical environment for which Google serves as a relevant metaphor. It then reviews two reports…

Slow Cooling Cryopreservation Optimized to Human Pluripotent Stem Cells.

PubMed

Miyazaki, Takamichi; Suemori, Hirofumi

2016-01-01

Human pluripotent stem cells (hPSCs) have the potential for unlimited expansion and differentiation into cells that form all three germ layers. Cryopreservation is one of the key processes for successful applications of hPSCs, because it allows semi-permanent preservation of cells and their easy transportation. Most animal cell lines, including mouse embryonic stem cells, are standardly cryopreserved by slow cooling; however, hPSCs have been difficult to preserve and their cell viability has been extremely low whenever cryopreservation has been attempted.Here, we investigate the reasons for failure of slow cooling in hPSC cryopreservation. Cryopreservation involves a series of steps and is not a straightforward process. Cells may die due to various reasons during cryopreservation. Indeed, hPSCs preserved by traditional methods often suffer necrosis during the freeze-thawing stages, and the colony state of hPSCs prior to cryopreservation is a major factor contributing to cell death.It has now become possible to cryopreserve hPSCs using conventional cryopreservation methods without any specific equipment. This review summarizes the advances in this area and discusses the optimization of slow cooling cryopreservation for hPSC storage.

Central pancreatectomy for benign pancreatic pathology/trauma: is it a reasonable pancreas-preserving conservative surgical strategy alternative to standard major pancreatic resection?

PubMed

Johnson, Maria A; Rajendran, Shanmugasundaram; Balachandar, Tirupporur G; Kannan, Devy G; Jeswanth, Satyanesan; Ravichandran, Palaniappan; Surendran, Rajagopal

2006-11-01

The aim of this study was to assess the technical feasibility, safety and outcome of central pancreatectomy (CP) with pancreaticogastrostomy or pancreaticojejunostomy in appropriately selected patients with benign central pancreatic pathology/trauma. Benign lesions/trauma of the pancreatic neck and proximal body pose an interesting surgical challenge. CP is an operation that allows resection of benign tumours located in the pancreatic isthmus that are not suitable for enucleation. Between January 2000 and December 2005, eight central pancreatectomies were carried out. There were six women and two men with a mean age of 35.7 years. The cephalic pancreatic stump is oversewn and the distal stump is anastomosed end-to-end with a Roux-en-Y jejunal loop in two and with the stomach in six patients. The indications for CP were: non-functional islet cell tumours in two patients, traumatic pancreatic neck transection in two and one each for insulinoma, solid pseudopapillary tumour, splenic artery pseudoaneurysm and pseudocyst. Pancreatic exocrine function was evaluated by a questionnaire method. Endocrine function was evaluated by blood glucose level. Morbidity rate was 37.5% with no operative mortality. Mean postoperative hospital stay was 10.5 days. Neither of the patients developed pancreatic fistula nor required reoperations or interventional radiological procedures. At a mean follow up of 26.4 months, no patient had evidence of endocrine or exocrine pancreatic insufficiency, all the patients were alive and well without clinical and imaging evidence of disease recurrence. When technically feasible, CP is a safe, pancreas-preserving pancreatectomy for non-enucleable benign pancreatic pathology/trauma confined to pancreatic isthmus that allows for cure of the disease without loss of substantial amount of normal pancreatic parenchyma with preservation of exocrine/endocrine function and without interruption of enteric continuity.

IDO decreases glycolysis and glutaminolysis by activating GCN2K, while it increases fatty acid oxidation by activating AhR, thus preserving CD4+ T‑cell survival and proliferation.

PubMed

Eleftheriadis, Theodoros; Pissas, Georgios; Liakopoulos, Vassilios; Stefanidis, Ioannis

2018-07-01

It is generally hypothesized in the literature that indoleamine 2,3‑dioxygenase (IDO), by degrading L‑tryptophan along the kynurenine pathway, suppresses CD4+ T‑cell function by inducing apoptosis, inhibiting proliferation and promoting differentiation towards a regulatory phenotype. These effects are either accompanied or directly lead to alterations in cell metabolism. The present study evaluated the pathways that govern the effect of IDO on the utilization of the three main energy sources in CD4+ T‑cells. Two‑way mixed lymphocyte reactions were performed with or without oleate and/or the IDO inhibitor 1‑methyl‑DL‑tryptophan. In addition, isolated CD4+ T‑cells cultured in an oleate‑containing medium were activated in the presence or not of the general control nonderepressible 2 kinase (GCN2K) activator tryptophanol. L‑tryptophan, glucose and free fatty acid consumption, cell proliferation, apoptosis and the levels of key proteins involved in IDO‑mediated signal transduction, and glucose, glutamine and free fatty acid utilization were assessed. The results indicate that IDO decreased glycolysis and glutaminolysis by activating GCN2K, resulting in activation of AMP‑activated protein kinase (AMPK). In parallel with AMPK activation, IDO‑induced activation of aryl hydrocarbon receptor increased the expression of all carnitine palmitoyltransferase I isoenzymes, leading ultimately to increased free fatty acid oxidation and preservation of CD4+ T‑cell survival and proliferation. Thus, contrary to what is generally hypothesized, in a normal environment containing fatty acids, the immunosuppressive effect of IDO may not be due to a decrease in CD4+ T‑cell survival and proliferation, since IDO supplies the required energy for cell survival and proliferation by increasing free fatty acid oxidation.

Magnetic stem cell targeting to the inner ear

NASA Astrophysics Data System (ADS)

Le, T. N.; Straatman, L.; Yanai, A.; Rahmanian, R.; Garnis, C.; Häfeli, U. O.; Poblete, T.; Westerberg, B. D.; Gregory-Evans, K.

2017-12-01

Severe sensorineural deafness is often accompanied by a loss of auditory neurons in addition to injury of the cochlear epithelium and hair cell loss. Cochlear implant function however depends on a healthy complement of neurons and their preservation is vital in achieving optimal results. We have developed a technique to target mesenchymal stem cells (MSCs) to a deafened rat cochlea. We then assessed the neuroprotective effect of systematically delivered MSCs on the survival and function of spiral ganglion neurons (SGNs). MSCs were labeled with superparamagnetic nanoparticles, injected via the systemic circulation, and targeted using a magnetized cochlea implant and external magnet. Neurotrophic factor concentrations, survival of SGNs, and auditory function were assessed at 1 week and 4 weeks after treatments and compared against multiple control groups. Significant numbers of magnetically targeted MSCs (>30 MSCs/section) were present in the cochlea with accompanied elevation of brain-derived neurotrophic factor and glial cell-derived neurotrophic factor levels (p < 0.001). In addition we saw improved survival of SGNs (approximately 80% survival at 4 weeks). Hearing threshold levels in magnetically targeted rats were found to be significantly better than those of control rats (p < 0.05). These results indicate that magnetic targeting of MSCs to the cochlea can be accomplished with a magnetized cochlear permalloy implant and an external magnet. The targeted stem cells release neurotrophic factors which results in improved SGN survival and hearing recovery. Combining magnetic cell-based therapy and cochlear implantation may improve cochlear implant function in treating deafness.

Optically Transparent Wood from a Nanoporous Cellulosic Template: Combining Functional and Structural Performance.

PubMed

Li, Yuanyuan; Fu, Qiliang; Yu, Shun; Yan, Min; Berglund, Lars

2016-04-11

Optically transparent wood (TW) with transmittance as high as 85% and haze of 71% was obtained using a delignified nanoporous wood template. The template was prepared by removing the light-absorbing lignin component, creating nanoporosity in the wood cell wall. Transparent wood was prepared by successful impregnation of lumen and the nanoscale cellulose fiber network in the cell wall with refractive-index-matched prepolymerized methyl methacrylate (MMA). During the process, the hierarchical wood structure was preserved. Optical properties of TW are tunable by changing the cellulose volume fraction. The synergy between wood and PMMA was observed for mechanical properties. Lightweight and strong transparent wood is a potential candidate for lightweight low-cost, light-transmitting buildings and transparent solar cell windows.

Nanometer-Sized Diamond Particle as a Probe for Biolabeling

PubMed Central

Chao, Jui-I.; Perevedentseva, Elena; Chung, Pei-Hua; Liu, Kuang-Kai; Cheng, Chih-Yuan; Chang, Chia-Ching; Cheng, Chia-Liang

2007-01-01

A novel method is proposed using nanometer-sized diamond particles as detection probes for biolabeling. The advantages of nanodiamond's unique properties were demonstrated in its biocompatibility, nontoxicity, easily detected Raman signal, and intrinsic fluorescence from its natural defects without complicated pretreatments. Carboxylated nanodiamond's (cND's) penetration ability, noncytotoxicity, and visualization of cND-cell interactions are demonstrated on A549 human lung epithelial cells. Protein-targeted cell interaction visualization was demonstrated with cND-lysozyme complex interaction with bacteria Escherichia coli. It is shown that the developed biomolecule-cND complex preserves the original functions of the test protein. The easily detected natural fluorescent and Raman intrinsic signals, penetration ability, and low cytotoxicity of cNDs render them promising agents in multiple medical applications. PMID:17513352

Preserving residual renal function in dialysis patients: an update on evidence to assist clinical decision making

PubMed Central

Jensen, Jens Dam; Peters, Christian Daugaard; Jespersen, Bente

2011-01-01

It has been documented that preservation of residual renal function in dialysis patients improves quality of life as well as survival. Clinical trials on strategies to preserve residual renal function are clearly lacking. While waiting for more results from clinical trials, patients will benefit from clinicians being aware of available knowledge. The aim of this review was to offer an update on current evidence assisting doctors in clinical practice. PMID:25949486

Cognitive and fine motor deficits in a pediatric sickle cell disease cohort of mixed ethnic origin.

PubMed

Burkhardt, Luise; Lobitz, Stephan; Koustenis, Elisabeth; Rueckriegel, Stefan Mark; Hernáiz Driever, Pablo

2017-02-01

Cerebrovascular disease is an important feature of pediatric sickle cell disease (SCD) and may lead to cognitive and motor impairment. Our cross-sectional study examined the incidence and severity of these impairments in a pediatric cohort without clinical cerebrovascular events from Berlin of mixed ethnic origin. Thirty-two SCD patients (mean age 11.14 years, range 7.0-17.25 years; males 14) were evaluated for full-scale intelligence (IQ) (German version WISC-III), fine motor function (digital writing tablet), and executive function (planning, attention, working memory, and visual-spatial abilities) with the Amsterdam Neuropsychological Tasks (ANT) program and the Tower of London (ToL). Data on clinical risk factors were retrieved from medical records. Full-scale IQ of patients was preserved, whereas performance IQ was significantly reduced (91.19 (SD 12.17) d = 0.7, p = 0.007). SCD patients scored significantly lower than healthy peers when tested for executive and fine motor functions, e.g., planning time in the ToL (6.73 s (SD 3.21) vs. 5.9 s in healthy peers (SD 2.33), d = 0.5, p = <0.001) and frequency on the writing tablet (mean z score -0.79, d = 0.7, p < 0.001). No clinical risk factors were significantly associated with incidence and severity of cognitive and motor deficits. Despite the preservation of full-scale IQ, our SCD cohort of mixed origin exhibited inferior executive abilities and reduced fine motor skills. Our study is limited by the small size of our cohort as well as the lack for control of sociodemographic and socioeconomic factors modulating higher functions but highlights the need for early screening, prevention, and specific interventions for these deficits.

Mms1 is an assistant for regulating G-quadruplex DNA structures.

PubMed

Schwindt, Eike; Paeschke, Katrin

2018-06-01

The preservation of genome stability is fundamental for every cell. Genomic integrity is constantly challenged. Among those challenges are also non-canonical nucleic acid structures. In recent years, scientists became aware of the impact of G-quadruplex (G4) structures on genome stability. It has been shown that folded G4-DNA structures cause changes in the cell, such as transcriptional up/down-regulation, replication stalling, or enhanced genome instability. Multiple helicases have been identified to regulate G4 structures and by this preserve genome stability. Interestingly, although these helicases are mostly ubiquitous expressed, they show specificity for G4 regulation in certain cellular processes (e.g., DNA replication). To this date, it is not clear how this process and target specificity of helicases are achieved. Recently, Mms1, an ubiquitin ligase complex protein, was identified as a novel G4-DNA-binding protein that supports genome stability by aiding Pif1 helicase binding to these regions. In this perspective review, we discuss the question if G4-DNA interacting proteins are fundamental for helicase function and specificity at G4-DNA structures.

Parameters of the Immune System and Vitamin D Levels in Old Individuals.

PubMed

Alves, Amanda Soares; Ishimura, Mayari Eika; Duarte, Yeda Aparecida de Oliveira; Bueno, Valquiria

2018-01-01

The increased number of individuals older than 80 years, centenarians, and supercentenarians is not a synonym for healthy aging, since severe infections, hospitalization, and disability are frequently observed. In this context, a possible strategy is to preserve the main characteristics/functions of the immune system with the aim to cause less damage to the organism during the aging process. Vitamin D acts on bone marrow, brain, breast, malignant cells, and immune system and has been recommended as a supplement. We aimed to evaluate whether immune parameters and vitamin D serum levels are correlated. We evaluated some features of the immune system using the peripheral blood of individuals older than 80 years ( n  = 12) compared to young subjects ( n  = 10). In addition, we correlated these findings with vitamin D serum levels. Old individuals presented metabolic parameters of healthy aging and maintained preserved some features of immunity such as CD4/CD8 ratio, and low production of pro-inflammatory cytokines after stimulus. On the other hand, we observed increase in the frequency of myeloid-derived suppressor cells, reduction in circulating leukocytes, in the percentage of total CD8+, and in CD8+ Naïve T cells, in addition to increase in the percentage of CD8+ effector memory re-expressing CD45RA (EMRA) T cells. We found seropositivity for CMV in 97.7%, which was correlated with the decrease of CD8+ Naïve T cells and increase in CD8+ EMRA T cells. Vitamin D levels were insufficient in 50% of old individuals and correlated positively with total CD8+ T cells and negatively with CD8+ EMRA T cells. In the studied population, longevity was correlated to maintenance of some immune parameters. Considering the limitations of the study as size of the sample and lack of functional assays, it was found that vitamin D in old individuals was correlated to some features of the immune system, mainly in the CD8 compartment.

Guidebook on preserving the functionality of state highways in Texas.

DOT National Transportation Integrated Search

2010-05-01

The purpose of this project was to identify the sources of deterioration of state highway : functionality that occur over time and what actions can be taken to preserve, recover, and : enhance functionality. Congestion and operational problems slow t...

A supporting role of Chinese National Immortalized Cell Bank in life science research.

PubMed

Xu, Chong-feng; Duan, Zi-yuan

2017-01-20

A biorepository of human samples is essential to support the research of life science. Lymphoblastoid B cell line (LCL), which is easy to be prepared and can reproduce indefinitely, is a convenient form of sample preservation. LCLs are established from human B cells transformed by Epstein-Barr virus (EBV). Chinese National Immortalized Cell Bank has preserved human LCLs from different ethnic groups in China. As there are many studies on the nature of LCLs and public available resources with genome-wide data for LCLs, they have been widely applied in genetics, immunology, pharmacogenetics/genomics, regenerative medicine, cancer pathogenesis and immunotherapy, screening and generation of fully human neutralizing monoclonal antibodies and study on EBV pathogenesis. Here, we review the characteristics of LCLs and their contributions to scientific research, and introduce preserved samples in Chinese National Immortalized Cell Bank to the scientific community. We hope this bank can support more areas in the scientific research.

Interpretable dimensionality reduction of single cell transcriptome data with deep generative models.

PubMed

Ding, Jiarui; Condon, Anne; Shah, Sohrab P

2018-05-21

Single-cell RNA-sequencing has great potential to discover cell types, identify cell states, trace development lineages, and reconstruct the spatial organization of cells. However, dimension reduction to interpret structure in single-cell sequencing data remains a challenge. Existing algorithms are either not able to uncover the clustering structures in the data or lose global information such as groups of clusters that are close to each other. We present a robust statistical model, scvis, to capture and visualize the low-dimensional structures in single-cell gene expression data. Simulation results demonstrate that low-dimensional representations learned by scvis preserve both the local and global neighbor structures in the data. In addition, scvis is robust to the number of data points and learns a probabilistic parametric mapping function to add new data points to an existing embedding. We then use scvis to analyze four single-cell RNA-sequencing datasets, exemplifying interpretable two-dimensional representations of the high-dimensional single-cell RNA-sequencing data.

Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels.

PubMed

Bertassoni, Luiz E; Cardoso, Juliana C; Manoharan, Vijayan; Cristino, Ana L; Bhise, Nupura S; Araujo, Wesleyan A; Zorlutuna, Pinar; Vrana, Nihal E; Ghaemmaghami, Amir M; Dokmeci, Mehmet R; Khademhosseini, Ali

2014-06-01

Fabrication of three dimensional (3D) organoids with controlled microarchitectures has been shown to enhance tissue functionality. Bioprinting can be used to precisely position cells and cell-laden materials to generate controlled tissue architecture. Therefore, it represents an exciting alternative for organ fabrication. Despite the rapid progress in the field, the development of printing processes that can be used to fabricate macroscale tissue constructs from ECM-derived hydrogels has remained a challenge. Here we report a strategy for bioprinting of photolabile cell-laden methacrylated gelatin (GelMA) hydrogels. We bioprinted cell-laden GelMA at concentrations ranging from 7 to 15% with varying cell densities and found a direct correlation between printability and the hydrogel mechanical properties. Furthermore, encapsulated HepG2 cells preserved cell viability for at least eight days following the bioprinting process. In summary, this work presents a strategy for direct-write bioprinting of a cell-laden photolabile ECM-derived hydrogel, which may find widespread application for tissue engineering, organ printing and the development of 3D drug discovery platforms.

Memory T cells: A helpful guard for allogeneic hematopoietic stem cell transplantation without causing graft-versus-host disease.

PubMed

Huang, Wei; Chao, Nelson J

2017-12-01

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (AHSCT) and the major cause of nonrelapse morbidity and mortality of AHSCT. In AHSCT, donor T cells facilitate hematopoietic stem cell (HSC) engraftment, contribute to anti-infection immunity, and mediate graft-versus-leukemia (GVL) responses. However, activated alloreactive T cells also attack recipient cells in vital organs, leading to GVHD. Different T-cell subsets, including naïve T (T N ) cells, memory T (T M ) cells, and regulatory T (T reg ) cells mediate different forms of GVHD and GVL; T N cells mediate severe GVHD, whereas T M cells do not cause GVHD, but preserve T-cell function including GVL. In addition, metabolic reprogramming controls T-cell differentiation and activation in these disease states. This minireview focuses on the role and the related mechanisms of T M cells in AHSCT, and the potential manipulation of T cells in AHSCT. Copyright © 2017 King Faisal Specialist Hospital & Research Centre. Published by Elsevier B.V. All rights reserved.

Effects of sample treatments on genome recovery via single-cell genomics

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

Clingenpeel, Scott; Schwientek, Patrick; Hugenholtz, Philip

2014-06-13

It is known that single-cell genomics is a powerful tool for accessing genetic information from uncultivated microorganisms. Methods of handling samples before single-cell genomic amplification may affect the quality of the genomes obtained. Using three bacterial strains we demonstrate that, compared to cryopreservation, lower-quality single-cell genomes are recovered when the sample is preserved in ethanol or if the sample undergoes fluorescence in situ hybridization, while sample preservation in paraformaldehyde renders it completely unsuitable for sequencing.

Syngeneic Schwann cell transplantation preserves vision in RCS rat without immunosuppression.

PubMed

McGill, Trevor J; Lund, Raymond D; Douglas, Robert M; Wang, Shaomei; Lu, Bin; Silver, Byron D; Secretan, Matt R; Arthur, Jennifer N; Prusky, Glen T

2007-04-01

To evaluate the efficacy of immunologically compatible Schwann cells transplanted without immunosuppression in the RCS rat retina to preserve vision. Syngeneic (dystrophic RCS) Schwann cells harvested from sciatic nerves were cultured and transplanted into one eye of dystrophic RCS rats at an early stage of retinal degeneration. Allogeneic (Long-Evans) Schwann cells and unoperated eyes served as controls. Vision through transplanted and unoperated eyes was then quantified using two visual behavior tasks, one measuring the spatial frequency and contrast sensitivity thresholds of the optokinetic response (OKR) and the other measuring grating acuity in a perception task. Spatial frequency thresholds measured through syngeneically transplanted eyes maintained near normal spatial frequency sensitivity for approximately 30 weeks, whereas thresholds through control eyes deteriorated to less than 20% of normal over the same period. Contrast sensitivity was preserved through syngeneically transplanted eyes better than through allogeneic and unoperated eyes, at all spatial frequencies. Grating acuity measured through syngeneically transplanted eyes was maintained at approximately 60% of normal, whereas acuity of allogeneically transplanted eyes was significantly lower at approximately 40% of normal. The ability of immunoprivileged Schwann cell transplants to preserve vision in RCS rats indicates that transplantation of syngeneic Schwann cells holds promise as a preventive treatment for retinal degenerative disease.

Invasive squamous cell carcinoma originating from a giant penile condyloma.

PubMed

Sir, Emin; Gungor, Melike; Ucer, Oktay; Kebat, Tulu

2017-05-01

In this case study, we present an unusual case with squamous cell carcinoma originating from a giant condyloma acuminata completely surrounding the penis. A 57-year-old circumcised heterosexual male patient presented with a penile lesion existing for 20 years. Incisional biopsy revealed acanthosis of the squamous epithelium. The patient was operated on under spinal anaesthesia. The lesion was resected circumferentially with macroscopic clearance, resulting in complete degloving of the penile shaft. Neurovascular bundles were preserved. The penile skin was constructed with a split thickness skin graft. Histopathological analysis of the lesion revealed an invasive and well-differentiated squamous cell carcinoma arising on a condyloma, and the surgical margins were free from tumour. The patient was staged as G2 T1 N0 M0 and was followed for one year. He did not have any erectile dysfunction and could engage in intercourse. Pelvic tomographic and physical examination findings did not reveal any episode of recurrence or metastasis. When encountering patients with giant condyloma acuminata, it should not be forgotten that it may be accompanied by squamous cell carcinoma. In addition, tissue excision should be as extensive as possible while keeping in mind the importance of the function. This is the first case of a penile-degloving surgery for giant penile condyloma, supporting conservative and preserving penile surgery for such tumours.

A High Frequency of HIV-Specific Circulating Follicular Helper T Cells Is Associated with Preserved Memory B Cell Responses in HIV Controllers.

PubMed

Claireaux, M; Galperin, M; Benati, D; Nouël, A; Mukhopadhyay, M; Klingler, J; de Truchis, P; Zucman, D; Hendou, S; Boufassa, F; Moog, C; Lambotte, O; Chakrabarti, L A

2018-05-08

Follicular helper T cells (Tfh) play an essential role in the affinity maturation of the antibody response by providing help to B cells. To determine whether this CD4 + T cell subset may contribute to the spontaneous control of HIV infection, we analyzed the phenotype and function of circulating Tfh (cTfh) in patients from the ANRS CO21 CODEX cohort who naturally controlled HIV-1 replication to undetectable levels and compared them to treated patients with similarly low viral loads. HIV-specific cTfh (Tet + ), detected by Gag-major histocompatibility complex class II (MHC-II) tetramer labeling in the CD45RA - CXCR5 + CD4 + T cell population, proved more frequent in the controller group ( P = 0.002). The frequency of PD-1 expression in Tet + cTfh was increased in both groups (median, >75%) compared to total cTfh (<30%), but the intensity of PD-1 expression per cell remained higher in the treated patient group ( P = 0.02), pointing to the persistence of abnormal immune activation in treated patients. The function of cTfh, analyzed by the capacity to promote IgG secretion in cocultures with autologous memory B cells, did not show major differences between groups in terms of total IgG production but proved significantly more efficient in the controller group when measuring HIV-specific IgG production. The frequency of Tet + cTfh correlated with HIV-specific IgG production ( R = 0.71 for Gag-specific and R = 0.79 for Env-specific IgG, respectively). Taken together, our findings indicate that key cTfh-B cell interactions are preserved in controlled HIV infection, resulting in potent memory B cell responses that may play an underappreciated role in HIV control. IMPORTANCE The rare patients who spontaneously control HIV replication in the absence of therapy provide a unique model to identify determinants of an effective anti-HIV immune response. HIV controllers show signs of particularly efficient antiviral T cell responses, while their humoral response was until recently considered to play only a minor role in viral control. However, emerging evidence suggests that HIV controllers maintain a significant but "silent" antiviral memory B cell population that can be reactivated upon antigenic stimulation. We report that cTfh help likely contributes to the persistence of controller memory B cell responses, as the frequency of HIV-specific cTfh correlated with the induction of HIV-specific antibodies in functional assays. These findings suggest that T follicular help may contribute to HIV control and highlight the need for inducing such help in HIV vaccine strategies that aim at eliciting persistent B cell responses. Copyright © 2018 Claireaux et al.

Contemporary approaches to modulating the nitric oxide-cGMP pathway in cardiovascular disease

PubMed Central

Kraehling, Jan R.; Sessa, William C.

2017-01-01

Endothelial cells lining the vessel wall control important aspects of vascular homeostasis. In particular, the production of endothelium-derived nitric oxide and activation of soluble guanylate cyclase promotes endothelial quiescence and governs vasomotor function and proportional remodeling of blood vessels. Here, we discuss novel approaches to improve endothelial nitric oxide generation and preserve its bioavailability. We also discuss therapeutic opportunities aimed at activation of soluble guanylate cyclase for multiple cardiovascular indications. PMID:28360348

An optimized protocol for handling and processing fragile acini cultured with the hanging drop technique.

PubMed

Snyman, Celia; Elliott, Edith

2011-12-15

The hanging drop three-dimensional culture technique allows cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix. The fragile acini are, however, difficult to preserve during processing steps for advanced microscopic investigation. We describe adaptations to the protocol for handling of hanging drop cultures to include investigation using confocal, scanning, and electron microscopy, with minimal loss of cell culture components. Copyright © 2011 Elsevier Inc. All rights reserved.

Wolfram Syndrome: A Case Report and Review of Clinical Manifestations, Genetics Pathophysiology, and Potential Therapies

PubMed Central

McMillan, J. M.; Au, P. Y. B.; Suchowersky, O.

2018-01-01

Background Classical Wolfram syndrome (WS) is a rare autosomal recessive disorder caused by mutations in WFS1, a gene implicated in endoplasmic reticulum (ER) and mitochondrial function. WS is characterized by insulin-requiring diabetes mellitus and optic atrophy. A constellation of other features contributes to the acronym DIDMOAD (Diabetes Insipidus, Diabetes Mellitus, Optic Atrophy, and Deafness). This review seeks to raise awareness of this rare form of diabetes so that individuals with WS are identified and provided with appropriate care. Case We describe a woman without risk factors for gestational or type 2 diabetes who presented with gestational diabetes (GDM) at the age of 39 years during her first and only pregnancy. Although she had optic atrophy since the age of 10 years, WS was not considered as her diagnosis until she presented with GDM. Biallelic mutations in WFS1 were identified, supporting a diagnosis of classical WS. Conclusions The distinct natural history, complications, and differences in management reinforce the importance of distinguishing WS from other forms of diabetes. Recent advances in the genetics and pathophysiology of WS have led to promising new therapeutic considerations that may preserve β-cell function and slow progressive neurological decline. Insight into the pathophysiology of WS may also inform strategies for β-cell preservation for individuals with type 1 and 2 diabetes. PMID:29850290

Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells.

PubMed

Sasaki, Natsuki; Nakamura, Masayuki; Kodama, Akiko; Urata, Yuka; Shiokawa, Nari; Hayashi, Takehiro; Sano, Akira

2016-11-01

The autophagy pathway has recently been implicated in several neurodegenerative diseases. Recently, it was reported that chorein-depleted cells showed accumulation of autophagic markers and impaired autophagic flux. Here, we demonstrate that chorein overexpression preserves cell viability from starvation-induced cell death in human embryonic kidney 293 (HEK293) cells. Subsequent coimmunoprecipitation and reverse coimmunoprecipitation assays using extracts from chorein that stably overexpressed HEK293 cells revealed that chorein interacts with α-tubulin and histone deacetylase 6, a known α-tubulin deacetylater and central component of basal autophagy. Indeed, acetylated α-tubulin immunoreactivity was significantly decreased in chorein that stably overexpressed HEK293 cells. These results suggest that chorein/histone deacetylase 6/α-tubulin interactions may play an important role in starvation-induced cell stress, and their disruption may be one of the molecular pathogenic mechanisms of chorea-acanthocytosis.-Sasaki, N., Nakamura, M., Kodama, A., Urata, Y., Shiokawa, N., Hayashi, T., Sano, A. Chorein interacts with α-tubulin and histone deacetylase 6, and overexpression preserves cell viability during nutrient deprivation in human embryonic kidney 293 cells. © FASEB.

Klinefelter syndrome and fertility: sperm preservation should not be offered to children with Klinefelter syndrome.

PubMed

Franik, S; Hoeijmakers, Y; D'Hauwers, K; Braat, D D M; Nelen, W L M; Smeets, D; Claahsen-van der Grinten, H L; Ramos, L; Fleischer, K

2016-09-01

Should fertility preservation be offered to children with Klinefelter syndrome (KS)? Current evidence shows that fertility preservation should not be offered to adolescents with KS younger than 16 years because of lower retrieval rates for germ cells by testicular sperm extraction (TESE) compared with retrieval rates for adolescents and adults between 16 and 30 years. KS, the most common chromosomal disorder in men leading to non-obstructive azoospermia, is caused by the presence of at least one additional X chromosome. The onset of puberty in adolescents with KS leads to progressive degeneration of the testicular environment. The impact of the subsequent tissue degeneration on fertility potential of patients with KS is unknown, but in previous literature it has been suggested that fertility preservation should be started in adolescents as early as possible. However spermatozoa can be found by TESE in about 50% of adults with KS despite severe testicular degeneration. This review discusses the current evidence for fertility preservation in children and adolescents and possible prognostic markers for fertility treatment in KS. An extensive literature search was conducted, searching Pubmed, Embase, Cinahl and Web of Science from origin until April 2016 for 'Klinefelter syndrome' and 'fertility' and various synonyms. Titles and abstracts have been scanned manually by the authors for eligibility. In total 76 studies were found to be eligible for inclusion in this review. Information from the papers was extracted separately by two authors. Various studies have shown that pre-pubertal children with KS already have a reduced number of germ cells despite a normal hormonal profile during childhood. The presence of spermatozoa in the ejaculate of adolescents with KS is extremely rare. Using TESE, the retrieval rates of spermatozoa for adolescents younger than 16 years old are much lower (0-20%) compared with those for adolescents and young adults between 16 and 30 years old (40-70%). Although spermatogonia can be found by TESE in about half of the peri-pubertal adolescents, there are currently no clinically functional techniques for their future use. Children and adolescents need to be informed that early fertility preservation before the age of 16 cannot guarantee fertility later in life and may even reduce the chances for offspring by removing functional immature germ cells which may possibly develop into spermatozoa after puberty. Furthermore, except for the age of patients with KS, there are no identified factors that can reliably be used as a predictive marker for fertility preservation. Most of the evidence presented in this review is based on studies including a small number of adolescents with KS. Therefore, the studies may have been underpowered to detect clinically significant differences for their various outcomes, especially for potential predictive factors for fertility preservation, such as hormone levels. Furthermore, the population of patients with KS diagnosed during childhood might be different from the adult population with KS where the diagnosis is based on infertility. Results based on comparisons between the two groups must be interpreted with caution. Despite the limitations, this review summarizes the current evidence for managing fertility preservation in patients with KS to provide optimal health care. There was no funding for this study. S.F., Y.H., K.D., W.L.M.N., D.S., H.L.C.-v.d.G. and L.R. declare to have no conflicts of interests. D.D.M.B. reports grants from Merck Serono, grants from Ferring and grants from MSD, outside the submitted work. K.F. reports personal fees from MSD (commercial sponsor), personal fees from Ferring (commercial sponsor), grants from Merck-Serono (commercial sponsor), grants from Ferring (commercial sponsor) and grants from MSD (commercial sponsor), outside the submitted work. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Retinal Remodeling in Human Retinitis Pigmentosa

PubMed Central

Jones, B.W.; Pfeiffer, R.L.; Ferrell, W. D.; Watt, C.B.; Marmor, M.; Marc, R.E.

2016-01-01

Retinitis Pigmentosa (RP) in the human is a progressive, currently irreversible neural degenerative disease usually caused by gene defects that disrupt the function or architecture of the photoreceptors. While RP can initially be a disease of photoreceptors, there is increasing evidence that the inner retina becomes progressively disorganized as the outer retina degenerates. These alterations have been extensively described in animal models, but remodeling in humans has not been as well characterized. This study, using computational molecular phenotyping (CMP) seeks to advance our understanding of the retinal remodeling process in humans. We describe cone mediated preservation of overall topology, retinal reprogramming in the earliest stages of the disease in retinal bipolar cells, and alterations in both small molecule and protein signatures of neurons and glia. Furthermore, while Müller glia appear to be some of the last cells left in the degenerate retina, they are also one of the first cell classes in the neural retina to respond to stress which may reveal mechanisms related to remodeling and cell death in other retinal cell classes. Also fundamentally important is the finding that retinal network topologies are altered. Our results suggest interventions that presume substantial preservation of the neural retina will likely fail in late stages of the disease. Even early intervention offers no guarantee that the interventions will be immune to progressive remodeling. Fundamental work in the biology and mechanisms of disease progression are needed to support vision rescue strategies. PMID:27020758

Effects of Four Formulations of Prostaglandin Analogs on Eye Surface Cells. A Comparative Study

PubMed Central

Pérez-Roca, Fernando; Rodrigo-Morales, Esther; Garzón, Ingrid; Oliveira, Ana-Celeste; Martín-Piedra, Miguel-Ángel; Carriel, Víctor; Ortiz-Pérez, Ana-Isabel; Sánchez-Montesinos, Indalecio; Campos, Antonio; Alaminos, Miguel

2015-01-01

We evaluated the cytotoxic effects of four prostaglandin analogs (PGAs) used to treat glaucoma. First we established primary cultures of conjunctival stromal cells from healthy donors. Then cell cultures were incubated with different concentrations (0, 0.1, 1, 5, 25, 50 and 100%) of commercial formulations of bimatoprost, tafluprost, travoprost and latanoprost for increasing periods (5 and 30 min, 1 h, 6 h and 24 h) and cell survival was assessed with three different methods: WST-1, MTT and calcein/AM-ethidium homodimer-1 assays. Our results showed that all PGAs were associated with a certain level of cell damage, which correlated significantly with the concentration of PGA used, and to a lesser extent with culture time. Tafluprost tended to be less toxic than bimatoprost, travoprost and latanoprost after all culture periods. The results for WST-1, MTT and calcein/AM-ethidium homodimer-1 correlated closely. When the average lethal dose 50 was calculated, we found that the most cytotoxic drug was latanoprost, whereas tafluprost was the most sparing of the ocular surface in vitro. These results indicate the need to design novel PGAs with high effectiveness but free from the cytotoxic effects that we found, or at least to obtain drugs that are functional at low dosages. The fact that the commercial formulation of tafluprost used in this work was preservative-free may support the current tendency to eliminate preservatives from eye drops for clinical use. PMID:26067827

Periodontitis aggravated pancreatic β-cell dysfunction in diabetic mice through interleukin-12 regulation on Klotho.

PubMed

Liu, Yihua; Zhang, Qiuli

2016-05-01

Recent studies have shown that periodontitis can contribute to adipose tissue inflammation and subsequent systemic insulin resistance in the obese rat model. However, the related inflammatory mechanism is not yet clear. The present study aims to investigate the effects of periodontitis on the function of pancreatic β-cells with pro-inflammatory cytokines-related immune mechanism in a mouse model. C57BL/6-db/db and inbred C57BL/6 mice were chosen here to establish a mouse model with periodontitis, which was induced by ligatures for 8 weeks. Glucose-stimulated insulin secretion was introduced to evaluate the function of pancreatic islets and β-cells. Serum levels of pro-inflammatory cytokines and Klotho were also measured, and the correlation between immunostimulation and Klotho level was deeply investigated in vitro. Pancreatic β-cell failure, with insulin resistance, was observed in db/db mice, while periodontitis could aggravate β-cell dysfunction-related features. Serum levels of interleukin (IL)-12 and Klotho showed a negatively synergistic change, whereas the expression of Klotho was also inhibited under IL-12 treatment in MIN6 β-cells or isolated islets. Furthermore, IL-12-induced immune stimulation and also decreased insulin secretion were proven to be reversed by Klotho overexpression. Periodontitis aggravated pancreatic β-cell failure in diabetic mice. Further in vitro studies showed IL-12 regulation on Klotho, while Klotho also acted as an inhibitor on IL-12, indicating the potential of Klotho for preserving pancreatic β-cell function in diabetes.

Nanomaterial Solutions for the Protection of Insulin Producing Beta Cells

NASA Astrophysics Data System (ADS)

Atchison, Nicole Ann

Islet transplantation is a promising treatment for type 1 diabetes. However, even with the many successes, islet transplantation has yet to reach its full potential. Limited islet sources, loss of cell viability during isolation and culture, and post-transplant graft loss are a few of the issues preventing extensive use of islet transplantation. The application of biomaterial systems to alleviate some of the stresses affecting islet viability has led to improvements in isolation and transplantation outcomes, but problems persist. In this work we approach two distinct issues affecting islet viability; ischemic conditions and immunological attack post-transplant. Ischemic conditions have been linked to a loss of islet graft function and occur during organ preservation, islet isolation and culture, and after islets are transplanted. We show that liposomal delivery of adenosine triphosphate (ATP) to beta cells can limit cell death and loss of function in ischemic conditions. We demonstrate that by functionalizing liposomes with the fibronectin-mimetic peptide PR_b, delivery of liposomes to porcine islets and rat beta cells is increased compared to nontargeted controls. Additionally, liposomes are shown to protect by providing both ATP and lipids to the ischemic cells. The delivery of ATP was investigated here but application of PR_b functionalized liposomes could be extended to other interesting cargos as well. The second area of investigation involves encapsulation of islets with silica nanoparticles to create a permselective barrier. Silica nanoparticles are an interesting material for encapsulation given their ability to be fine-tuned and further functionalized. We demonstrate that size-tunable, fluorescent silica nanoparticles can be assembled layer-by-layer on the surface of cells and that silica nanoparticle encapsulated islets are able to secrete insulin in response to a glucose challenge.

3D patterned substrates for bioartificial blood vessels - The effect of hydrogels on aligned cells on a biomaterial surface.

PubMed

Zhao, Xinxin; Irvine, Scott Alexander; Agrawal, Animesh; Cao, Ye; Lim, Pei Qi; Tan, Si Ying; Venkatraman, Subbu S

2015-10-01

The optimal bio-artificial blood vessel construct is one that has a compliant tubular core with circumferentially aligned smooth muscle cells (SMCs). Obtaining this well-aligned pattern of SMCs on a scaffold is highly beneficial as this cellular orientation preserves the SMC contractile phenotype. We used 3D patterning to create channels on a polycaprolactone (PCL) scaffold; SMCs were then found to be aligned within the microchannels. To preserve this alignment, and to provide a protective coating that could further incorporate cells, we evaluated the use of two hydrogels, one based on poly(ethylene glycol) diacrylate (PEGDA) and the other based on gelatin. Hydrogels were either physically coated on the PCL surfaces or covalently linked via suitable surface modification of PCL. For covalent immobilization of PEGDA hydrogel, alkene groups were introduced on PCL, while for gelatin covalent linkage, serum proteins were introduced. It is, however, crucial that the hydrogel coating does not disrupt the cellular patterning and distribution. We show in this work that both the process of coating as well as the nature of the coating are critical to preservation of the aligned SMCs. The covalent coating methods involving the crosslinking of hydrogels with the surface of PCL films promoted hydrogel retention time on the film as compared with physical deposition. Furthermore, subsequent hydrogel degradation is affected by the components of the cell culture medium, hinting at a possible route to in vivo biodegradation. Surface features control cellular orientation and subsequently influence their functionality, a useful effect for cellularized biomedical devices. Such devices also can benefit from protective and cell friendly hydrogel coatings. However, literature is lacking on the fate of cells that have endured hydrogel coating whilst orientated on a biomaterial surface. In particular, elucidation of the cells ability to remain adherent and orientated post hydrogel addition. Coating requires two procedures that may be deleterious to the orientated cells: the surface pretreatment for gel binding and the hydrogel crosslinking reaction. We compare transglutaminase gelatin crosslinking and UV initiated PEGDA crosslinking, coated onto smooth muscle cells orientated on patterned PCL surfaces. This original study will be of considerable use to the wider biomaterials community. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Reconceiving the hippocampal map as a topological template

PubMed Central

Dabaghian, Yuri; Brandt, Vicky L; Frank, Loren M

2014-01-01

The role of the hippocampus in spatial cognition is incontrovertible yet controversial. Place cells, initially thought to be location-specifiers, turn out to respond promiscuously to a wide range of stimuli. Here we test the idea, which we have recently demonstrated in a computational model, that the hippocampal place cells may ultimately be interested in a space's topological qualities (its connectivity) more than its geometry (distances and angles); such higher-order functioning would be more consistent with other known hippocampal functions. We recorded place cell activity in rats exploring morphing linear tracks that allowed us to dissociate the geometry of the track from its topology. The resulting place fields preserved the relative sequence of places visited along the track but did not vary with the metrical features of the track or the direction of the rat's movement. These results suggest a reinterpretation of previous studies and new directions for future experiments. DOI: http://dx.doi.org/10.7554/eLife.03476.001 PMID:25141375

Graft-versus-host disease in the ovary potentially causes female infertility after allogeneic hematopoietic stem cell transplantation.

PubMed

Shimoji, Sonoko; Hashimoto, Daigo; Teshima, Takanori

2017-01-01

Ovarian failure-associated infertility is a serious late complication for female patients who have undergone allogeneic hematopoietic stem cell transplantation (SCT). Although the role of a pretransplant conditioning regimen has been well appreciated, the increasing application of reduced-intensity conditioning has led us to reconsider other factors possibly affecting ovarian function after allogeneic SCT. We recently reported that graft-versus-host disease (GVHD) targets granulosa cells of the ovarian follicles, thereby significantly reducing ovarian reserves and fertility after SCT. We also found that ovarian GVHD impairs fertility independently of the toxicities of the conditioning regimens, and pharmacological GVHD prophylaxis preserves fertility after SCT. For the first time, these results demonstrated that GVHD targets the ovary and impairs ovarian functions and fertility, thereby having important clinical implications in young female transplant recipients with nonmalignant diseases, for whom minimally toxic regimens are used. Here we review recently published articles regarding clinical and basic researches on female infertility after SCT.

Nucleic Acid Encoding A Lectin-Derived Progenitor Cell Preservation Factor

DOEpatents

Colucci, M. Gabriella; Chrispeels, Maarten J.; Moore, Jeffrey G.

2001-10-30

The invention relates to an isolated nucleic acid molecule that encodes a protein that is effective to preserve progenitor cells, such as hematopoietic progenitor cells. The nucleic acid comprises a sequence defined by SEQ ID NO:1, a homolog thereof, or a fragment thereof. The encoded protein has an amino acid sequence that comprises a sequence defined by SEQ ID NO:2, a homolog thereof, or a fragment thereof that contains an amino acid sequence TNNVLQVT. Methods of using the encoded protein for preserving progenitor cells in vitro, ex vivo, and in vivo are also described. The invention, therefore, include methods such as myeloablation therapies for cancer treatment wherein myeloid reconstitution is facilitated by means of the specified protein. Other therapeutic utilities are also enabled through the invention, for example, expanding progenitor cell populations ex vivo to increase chances of engraftation, improving conditions for transporting and storing progenitor cells, and facilitating gene therapy to treat and cure a broad range of life-threatening hematologic diseases.

Mitochondrial Uncoupler Prodrug of 2,4-Dinitrophenol, MP201, Prevents Neuronal Damage and Preserves Vision in Experimental Optic Neuritis

PubMed Central

Khan, Reas S.; Geisler, John G.

2017-01-01

The ability of novel mitochondrial uncoupler prodrug of 2,4-dinitrophenol (DNP), MP201, to prevent neuronal damage and preserve visual function in an experimental autoimmune encephalomyelitis (EAE) model of optic neuritis was evaluated. Optic nerve inflammation, demyelination, and axonal loss are prominent features of optic neuritis, an inflammatory optic neuropathy often associated with the central nervous system demyelinating disease multiple sclerosis. Currently, optic neuritis is frequently treated with high-dose corticosteroids, but treatment fails to prevent permanent neuronal damage and associated vision changes that occur as optic neuritis resolves, thus suggesting that additional therapies are required. MP201 administered orally, once per day, attenuated visual dysfunction, preserved retinal ganglion cells (RGCs), and reduced RGC axonal loss and demyelination in the optic nerves of EAE mice, with limited effects on inflammation. The prominent mild mitochondrial uncoupling properties of MP201, with slow elimination of DNP, may contribute to the neuroprotective effect by modulating the entire mitochondria's physiology directly. Results suggest that MP201 is a potential novel treatment for optic neuritis. PMID:28680531

Suppression of HSP27 Restores Retinal Function and Protects Photoreceptors From Apoptosis in a Light-Induced Retinal Degeneration Animal Model.

PubMed

Chien, Chih-Cheng; Huang, Chi-Jung; Tien, Lu-Tai; Cheng, Yu-Che; Ke, Chia-Ying; Lee, Yih-Jing

2017-06-01

We used a light-induced retinal degeneration animal model to investigate possible roles of heat shock protein 27 (HSP27) in retinal/photoreceptor protection. Sprague-Dawley rats were used for the light-induced retinal degeneration animal model. The histology of eye sections was observed for morphologic changes in the retina. Cell apoptosis was examined in each group using the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay, and electroretinography was used to evaluate retinal function. Protein and mRNA expression levels of different retinal cell markers were also detected through immunofluorescence staining, Western blotting, and real-time PCR. The thickness of the outer nuclear layer significantly decreased after 7-day light exposure. Moreover, we injected a viral vector for silencing HSP27 expression into the eyes and observed that photoreceptors were better preserved in the HSP27-suppressed (sHSP27) retina 2 weeks after injection. HSP27 suppression also reduced retinal cell apoptosis caused by light exposure. In addition, the loss of retinal function caused by light exposure was reversed on suppressing HSP27 expression. We subsequently found that the expression of the Rho gene and immunofluorescence staining of rhodopsin and arrestin (cell markers for photoreceptors) increased in sHSP27-treated retinas. HSP27 suppression did not affect the survival of ganglion and amacrine cells. Retinal cell apoptosis and functional loss were observed after 7-day light exposure. However, in the following 2 weeks after light exposure, HSP27 suppression may initiate a protective effect for retinal cells, particularly photoreceptors, from light-induced retinal degeneration.

Effect of storage temperature on cultured epidermal cell sheets stored in xenobiotic-free medium.

PubMed

Jackson, Catherine; Aabel, Peder; Eidet, Jon R; Messelt, Edward B; Lyberg, Torstein; von Unge, Magnus; Utheim, Tor P

2014-01-01

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic-free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.

Risk factors for bowel dysfunction after sphincter-preserving rectal cancer surgery: a prospective study using the Memorial Sloan Kettering Cancer Center bowel function instrument.

PubMed

Ihn, Myong Hoon; Kang, Sung-Bum; Kim, Duck-Woo; Oh, Heung-Kwon; Lee, Soo Young; Hong, Sa Min

2014-08-01

Until recently, no studies have prospectively evaluated bowel function after sphincter-preserving surgery for rectal cancer with the use of a validated bowel function scoring system. The aim of this study was to investigate possible risk factors for altered bowel function after sphincter-preserving surgery. This was a prospective study. The study was conducted between January 2006 and May 2012 at the authors' institution. Patients who underwent sphincter-preserving rectal cancer surgery were recruited. Bowel function was assessed 1 day before (baseline) and at 1 year after sphincter-preserving surgery or temporary ileostomy takedown with the use of the Memorial Sloan Kettering Cancer Center questionnaire. Multivariable analysis was performed to identify the factors associated with altered bowel function after surgery. Overall, 266 patients were eligible for the analysis. The tumor was located in the upper, middle, and lower rectum in 68 (25.5%), 113 (42.5%), and 85 (32.0%) patients. Intersphincteric resection and temporary ileostomy were performed in 18 (6.8%) and 129 (48.5%) patients. The mean Memorial Sloan Kettering Cancer Center score was 64.5 ± 7.6 at 1 year after sphincter-preserving surgery or temporary ileostomy takedown. The Memorial Sloan Kettering Cancer Center score decreased in 163/266 patients (61.3%) between baseline and 1 year after surgery. Tumor location (p = 0.01), operative method (p = 0.03), anastomotic type (p = 0.01), and temporary ileostomy (p = 0.01) were associated with altered bowel function after sphincter-preserving surgery in univariate analyses. In multivariable analysis, only tumor location was independently associated with impaired bowel function after sphincter-preserving rectal cancer surgery. This study was limited by its nonrandomized design and the lack of measurement before preoperative chemoradiotherapy. We suggest that preoperative counseling should be implemented to inform patients of the risk of bowel dysfunction, especially in patients with lower rectal cancer, although this study cannot exclude the effect of chemoradiotherapy owing to the limitation of study.

Balancing self-renewal against genome preservation in stem cells: How do they manage to have the cake and eat it too?

PubMed

Tsai, Robert Y L

2016-05-01

Stem cells are endowed with the awesome power of self-renewal and multi-lineage differentiation that allows them to be major contributors to tissue homeostasis. Owing to their longevity and self-renewal capacity, they are also faced with a higher risk of genomic damage compared to differentiated cells. Damage on the genome, if not prevented or repaired properly, will threaten the survival of stem cells and culminate in organ failure, premature aging, or cancer formation. It is therefore of paramount importance that stem cells remain genomically stable throughout life. Given their unique biological and functional requirement, stem cells are thought to manage genotoxic stress somewhat differently from non-stem cells. The focus of this article is to review the current knowledge on how stem cells escape the barrage of oxidative and replicative DNA damage to stay in self-renewal. A clear statement on this subject should help us better understand tissue regeneration, aging, and cancer.

Homeostatic plasticity shapes cell-type-specific wiring in the retina

PubMed Central

Tien, Nai-Wen; Soto, Florentina; Kerschensteiner, Daniel

2017-01-01

SUMMARY Convergent input from different presynaptic partners shapes the responses of postsynaptic neurons. Whether developing postsynaptic neurons establish connections with each presynaptic partner independently, or balance inputs to attain specific responses is unclear. Retinal ganglion cells (RGCs) receive convergent input from bipolar cell types with different contrast responses and temporal tuning. Here, using optogenetic activation and pharmacogenetic silencing, we found that type 6 bipolar cells (B6) dominate excitatory input to ONα-RGCs. We generated mice in which B6 cells were selectively removed from developing circuits (B6-DTA). In B6-DTA mice, ONα-RGCs adjusted connectivity with other bipolar cells in a cell-type-specific manner. They recruited new partners, increased synapses with some existing partners, and maintained constant input from others. Patch clamp recordings revealed that anatomical rewiring precisely preserved contrast- and temporal frequency response functions of ONα-RGCs, indicating that homeostatic plasticity shapes cell-type-specific wiring in the developing retina to stabilize visual information sent to the brain. PMID:28457596

Pathway modulations and epigenetic alterations in ovarian tumorbiogenesis

PubMed Central

Saldanha, Sabita N.; Tollefsbol, Trygve O.

2013-01-01

Cellular pathways are numerous and are highly integrated in function in the control of cellular systems. They collectively regulate cell division, proliferation, survival and apoptosis of cells and mutagenesis of key genes that control these pathways can initiate neoplastic transformations. Understanding these pathways is crucial to future therapeutic and preventive strategies of the disease. Ovarian cancers are of three major types; epithelial, germ-cell and stromal. However, ovarian cancers of epithelial origin, arising from the mesothelium, are the predominant form. Of the subtypes of ovarian cancer, the high-grade serous tumors are fatal, with low survival rate due to late detection and poor response to treatments. Close examination of preserved ovarian tissues and in vitro studies have provided insights into the mechanistic changes occurring in cells mediated by a few key genes. This review will focus on pathways and key genes of the pathways that are mutated or have aberrant functions in the pathology of ovarian cancer. Non-genetic mechanisms that are gaining prominence in the pathology of ovarian cancer, miRNAs and epigenetics, will also be discussed in the review. PMID:24105793

Small-molecule MDM2 antagonists reveal aberrant p53 signaling in cancer: Implications for therapy

PubMed Central

Tovar, Christian; Rosinski, James; Filipovic, Zoran; Higgins, Brian; Kolinsky, Kenneth; Hilton, Holly; Zhao, Xiaolan; Vu, Binh T.; Qing, Weiguo; Packman, Kathryn; Myklebost, Ola; Heimbrook, David C.; Vassilev, Lyubomir T.

2006-01-01

The p53 tumor suppressor retains its wild-type conformation and transcriptional activity in half of all human tumors, and its activation may offer a therapeutic benefit. However, p53 function could be compromised by defective signaling in the p53 pathway. Using a small-molecule MDM2 antagonist, nutlin-3, to probe downstream p53 signaling we find that the cell-cycle arrest function of the p53 pathway is preserved in multiple tumor-derived cell lines expressing wild-type p53, but many have a reduced ability to undergo p53-dependent apoptosis. Gene array analysis revealed attenuated expression of multiple apoptosis-related genes. Cancer cells with mdm2 gene amplification were most sensitive to nutlin-3 in vitro and in vivo, suggesting that MDM2 overexpression may be the only abnormality in the p53 pathway of these cells. Nutlin-3 also showed good efficacy against tumors with normal MDM2 expression, suggesting that many of the patients with wild-type p53 tumors may benefit from antagonists of the p53–MDM2 interaction. PMID:16443686

Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival

PubMed Central

Rice, Dennis S.; Calandria, Jorgelina M.; Gordon, William C.; Jun, Bokkyoo; Zhou, Yongdong; Gelfman, Claire M.; Li, Songhua; Jin, Minghao; Knott, Eric J.; Chang, Bo; Abuin, Alex; Issa, Tawfik; Potter, David; Platt, Kenneth A.; Bazan, Nicolas G.

2015-01-01

The identification of pathways necessary for photoreceptor and retinal pigment epithelium (RPE) function is critical to uncover therapies for blindness. Here we report the discovery of adiponectin receptor 1 (AdipoR1) as a regulator of these cells’ functions. Docosahexaenoic acid (DHA) is avidly retained in photoreceptors, while mechanisms controlling DHA uptake and retention are unknown. Thus, we demonstrate that AdipoR1 ablation results in DHA reduction. In situ hybridization reveals photoreceptor and RPE cell AdipoR1 expression, blunted in AdipoR1−/− mice. We also find decreased photoreceptor-specific phosphatidylcholine containing very long-chain polyunsaturated fatty acids and severely attenuated electroretinograms. These changes precede progressive photoreceptor degeneration in AdipoR1−/− mice. RPE-rich eyecup cultures from AdipoR1−/− reveal impaired DHA uptake. AdipoR1 overexpression in RPE cells enhances DHA uptake, whereas AdipoR1 silencing has the opposite effect. These results establish AdipoR1 as a regulatory switch of DHA uptake, retention, conservation and elongation in photoreceptors and RPE, thus preserving photoreceptor cell integrity. PMID:25736573

Hydrogen Sulfide Attenuates the Recruitment of CD11b+Gr-1+ Myeloid Cells and Regulates Bax/Bcl-2 Signaling in Myocardial Ischemia Injury

PubMed Central

Zhang, Youen; Li, Hua; Zhao, Gang; Sun, Aijun; Zong, Nobel C.; Li, Zhaofeng; Zhu, Hongming; Zou, Yunzeng; Yang, Xiangdong; Ge, Junbo

2014-01-01

Hydrogen sulfide, an endogenous signaling molecule, plays an important role in the physiology and pathophysiology of the cardiovascular system. Using a mouse model of myocardial infarction, we investigated the anti-inflammatory and anti-apoptotic effects of the H2S donor sodium hydrosulfide (NaHS). The results demonstrated that the administration of NaHS improved survival, preserved left ventricular function, limited infarct size, and improved H2S levels in cardiac tissue to attenuate the recruitment of CD11b+Gr-1+ myeloid cells and to regulate the Bax/Bcl-2 pathway. Furthermore, the cardioprotective effects of NaHS were enhanced by inhibiting the migration of CD11b+Gr-1+ myeloid cells from the spleen into the blood and by attenuating post-infarction inflammation. These observations suggest that the novel mechanism underlying the cardioprotective function of H2S is secondary to a combination of attenuation the recruitment of CD11b+Gr-1+ myeloid cells and regulation of the Bax/Bcl-2 apoptotic signaling. PMID:24758901

Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis

PubMed Central

Zilberman, Yuliya; Abrams, Joshua; Anderson, Dorian C.

2017-01-01

During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc-42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARHGAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC-42 to control actin organization and AJ protein levels during epithelial morphogenesis. PMID:28903999

Calcineurin A beta deficiency ameliorates HFD-induced hypothalamic astrocytosis in mice.

PubMed

Pfuhlmann, Katrin; Schriever, Sonja C; Legutko, Beata; Baumann, Peter; Harrison, Luke; Kabra, Dhiraj G; Baumgart, Emily Violette; Tschöp, Matthias H; Garcia-Caceres, Cristina; Pfluger, Paul T

2018-02-08

ᅟ: Astrocytosis is a reactive process involving cellular, molecular, and functional changes to facilitate neuronal survival, myelin preservation, blood brain barrier function and protective glial scar formation upon brain insult. The overall pro- or anti-inflammatory impact of reactive astrocytes appears to be driven in a context- and disease-driven manner by modulation of astrocytic Ca 2+ homeostasis and activation of Ca 2+ /calmodulin-activated serine/threonine phosphatase calcineurin. Here, we aimed to assess whether calcineurin is dispensable for astrocytosis in the hypothalamus driven by prolonged high fat diet (HFD) feeding. Global deletion of calcineurin A beta (gene name: Ppp3cb) led to a decrease of glial fibrillary acidic protein (GFAP)-positive cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) of mice exposed chronically to HFD. The concomitant decrease in Iba1-positive microglia in the VMH further suggests a modest impact of Ppp3cb deletion on microgliosis. Pharmacological inhibition of calcineurin activity by Fk506 had no impact on IBA1-positive microglia in hypothalami of mice acutely exposed to HFD for 1 week. However, Fk506-treated mice displayed a decrease in GFAP levels in the ARC. In vivo effects could not be replicated in cell culture, where calcineurin inhibition by Fk506 had no effect on astrocytic morphology, astrocytic cell death, GFAP, and vimentin protein levels or microglia numbers in primary hypothalamic astrocytes and microglia co-cultures. Further, adenoviral overexpression of calcineurin subunit Ppp3r1 in primary glia culture did not lead to an increase in GFAP fluorescence intensity. Overall, our results point to a prominent role of calcineurin in mediating hypothalamic astrocytosis as response to acute and chronic HFD exposure. Moreover, discrepant findings in vivo and in cell culture indicate the necessity of studying astrocytes in their "natural" environment, i.e., preserving an intact hypothalamic microenvironment with neurons and non-neuronal cells in close proximity.

Regulation of mesenchymal stem cell 3D microenvironment: From macro to microfluidic bioreactors.

PubMed

Sart, Sébastien; Agathos, Spiros N; Li, Yan; Ma, Teng

2016-01-01

Human mesenchymal stem cells (hMSCs) have emerged as an important cell type in cell therapy and tissue engineering. In these applications, maintaining the therapeutic properties of hMSCs requires tight control of the culture environments and the structural cell organizations. Bioreactor systems are essential tools to achieve these goals in the clinical-scale expansion and tissue engineering applications. This review summarizes how different bioreactors provide cues to regulate the structure and the chemico-mechanical microenvironment of hMSCs with a focus on 3D organization. In addition to conventional bioreactors, recent advances in microfluidic bioreactors as a novel approach to better control the hMSC microenvironment are also discussed. These advancements highlight the key role of bioreactor systems in preserving hMSC's functional properties by providing dynamic and temporal regulation of in vitro cellular microenvironment. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The significance of cell-related challenges in the clinical application of tissue engineering.

PubMed

Almela, Thafar; Brook, Ian M; Moharamzadeh, Keyvan

2016-12-01

Tissue engineering is increasingly being recognized as a new approach that could alleviate the burden of tissue damage currently managed with transplants or synthetic devices. Making this novel approach available in the future for patients who would potentially benefit is largely dependent on understanding and addressing all those factors that impede the translation of this technology to the clinic. Cell-associated factors in particular raise many challenges, including those related to cell sources, up- and downstream techniques, preservation, and the creation of in vitro microenvironments that enable cells to grow and function as far as possible as they would in vivo. This article highlights the main confounding issues associated with cells in tissue engineering and how these issues may hinder the advancement of therapeutic tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3157-3163, 2016. © 2016 Wiley Periodicals, Inc.

Nitric oxide-donating statin improves multiple functions of circulating angiogenic cells

PubMed Central

Mangialardi, G; Monopoli, A; Ongini, E; Spinetti, G; Fortunato, O; Emanueli, C; Madeddu, P

2011-01-01

BACKGROUND AND PURPOSE Statins, a major component of the prevention of cardiovascular disease, aid progenitor cell functions in vivo and in vitro. Statins bearing a NO-releasing moiety were developed for their enhanced anti-inflammatory/anti-thrombotic properties. Here, we investigated if the NO-donating atorvastatin (NCX 547) improved the functions of circulating angiogenic cells (CACs). EXPERIMENTAL APPROACH Circulating angiogenic cells (CACs) were prepared from peripheral blood monocytes of healthy volunteers and type-2 diabetic patients and were cultured in low (LG) or high glucose (HG) conditions, in presence of atorvastatin or NCX 547 (both at 0.1 µM) or vehicle. Functional assays (outgrowth, proliferation, viability, senescence and apoptosis) were performed in presence of the endothelial NOS inhibitor L-NIO, the NO scavenger c-PTIO or vehicle. KEY RESULTS Culturing in HG conditions lowered NO in CACs, inhibited outgrowth, proliferation, viability and migration, and induced cell senescence and apoptosis. NCX 547 fully restored NO levels and functions of HG-cultured CACs, while atorvastatin prevented only apoptosis in CACs. The activity of Akt, a pro-survival kinase, was increased by atorvastatin in LG-cultured but not in HG-cultured CACs, whereas NCX 547 increased Akt activity in both conditions. L-NIO partially blunted and c-PTIO prevented NCX 547-induced improvements in CAC functions. Finally, NCX 547 improved outgrowth and migration of CACs prepared from patients with type 2 diabetes. CONCLUSIONS AND IMPLICATIONS NCX 547 was more effective than atorvastatin in preserving functions of CACs. This property adds to the spectrum of favourable actions that would make NO-releasing statins more effective agents for treating cardiovascular disease. PMID:21486281

CD4 T Cell Epitope Specificity and Cytokine Potential Are Preserved as Cells Transition from the Lung Vasculature to Lung Tissue following Influenza Virus Infection.

PubMed

DiPiazza, Anthony; Laniewski, Nathan; Rattan, Ajitanuj; Topham, David J; Miller, Jim; Sant, Andrea J

2018-07-01

Pulmonary CD4 T cells are critical in respiratory virus control, both by delivering direct effector function and through coordinating responses of other immune cells. Recent studies have shown that following influenza virus infection, virus-specific CD4 T cells are partitioned between pulmonary vasculature and lung tissue. However, very little is known about the peptide specificity or functional differences of CD4 T cells within these two compartments. Using a mouse model of influenza virus infection in conjunction with intravascular labeling in vivo , the cell surface phenotype, epitope specificity, and functional potential of the endogenous polyclonal CD4 T cell response was examined by tracking nine independent CD4 T cell epitope specificities. These studies revealed that tissue-localized CD4 cells were globally distinct from vascular cells in expression of markers associated with transendothelial migration, residency, and micropositioning. Despite these differences, there was little evidence for remodeling of the viral epitope specificity or cytokine potential as cells transition from vasculature to the highly inflamed lung tissue. Our studies also distinguished cells in the pulmonary vasculature from peripheral circulating CD4 T cells, providing support for the concept that the pulmonary vasculature does not simply reflect circulating cells that are trapped within the narrow confines of capillary vessels but rather is enriched in transitional cells primed in the draining lymph node that have specialized potential to enter the lung tissue. IMPORTANCE CD4 T cells convey a multitude of functions in immunity to influenza, including those delivered in the lymph node and others conveyed by CD4 T cells that leave the lymph node, enter the blood, and extravasate into the lung tissue. Here, we show that the transition of recently primed CD4 cells detected in the lung vasculature undergo profound changes in expression of markers associated with tissue localization as they establish residence in the lung. However, this transition does not edit CD4 T cell epitope specificity or the cytokine potential of the CD4 T cells. Thus, CD4 T cells that enter the infected lung can convey diverse functions and have a sufficiently broad viral antigen specificity to detect the complex array of infected cells within the infected tissue, offering the potential for more effective protective function. Copyright © 2018 American Society for Microbiology.

Cell and gene therapy for severe heart failure patients: The time and place for Pim-1 Kinase

PubMed Central

Siddiqi, Sailay; Sussman, Mark A

2014-01-01

Regenerative therapy in severe heart failure patients presents a challenging set of circumstances including a damaged myocardial environment that accelerates senescence in myocytes and cardiac progenitor cells. Failing myocardium suffers from deterioration of contractile function coupled with impaired regenerative potential that drives the heart toward decompensation. Efficacious regenerative cell therapy for severe heart failure requires disruption of this vicious circle that can be accomplished by alteration of the compromised myocyte phenotype and rejuvenation of progenitor cells. This review focuses upon potential for Pim-1 kinase to mitigate chronic heart failure by improving myocyte quality through preservation of mitochondrial integrity, prevention of hypertrophy and inhibition of apoptosis. In addition, cardiac progenitors engineered with Pim-1 possess enhanced regenerative potential, making Pim-1 an important player in future treatment of severe heart failure. PMID:23984924

Blood Preservation Study.

DTIC Science & Technology

1983-01-01

1977. 3. Wood LA and Beutler E: The effect of periodic mixing on the preservation of 2,3- diphosphoglycerate (2,3-DPG) levels in stored blood. Blood 42:17...ATP levels and the viability of red cells was investigated; and several procedures for protein extraction from red cells were performed. DO 1473... levels are a disappointing parameter with respect to predicting the viability of stored red cells (5-8). There is a great need to identify measurements

Differentiation-associated alteration in human monocyte-macrophage accessory cell function.

PubMed

Mayernik, D G; Ul-Haq, A; Rinehart, J J

1983-05-01

Human monocyte (Mo) to macrophage (Mx) differentiation is associated with marked and well studied changes in morphology, biochemical parameters, and effector cell function. Nevertheless, the comparative accessory cell (AC) function of blood Mo and differentiated Mx has not been carefully studied. We, therefore, examined the kinetics and mechanisms of change in AC function during in vitro Mo to Mx differentiation. The system utilized has two distinctive features: blood Mo and resultant cultured Mx represent a cohort of cells derived from the bone marrow within a 12-hr period. Moreover, the in vitro derived Mx utilized herein have been characterized extensively and are functionally and biochemically similar to pulmonary macrophages (PMx). In the experiments reported, AC functions of blood Mo, Mx derived from Mo after 1 to 6 days of culture, and PMx was compared. AC were cultured with nylon wool column-purified autologous T cells and were stimulated with concanavalin A (Con A) or streptokinase-streptodornase (SKSD). Blood T cell proliferation to Con A or SKSD was inhibited greater than 90% by the removal of Mo and was reconstituted by 20% Mo. Mx derived from Mo by culture for 1 to 3 days exhibited the same (or better) AC function as Mo when T cells were stimulated with either SKSD or Con A. In marked contrast, Mx derived from 6-day cultures exhibited less than or equal to 15% of Mo (i.e., control) capacity to support T cell proliferative response to SKSD. Six-day Mx support T cell proliferation to Con A was somewhat variable. Similar to 6-day cultured Mx, PMx failed to function as AC. The mechanism of loss of AC function was examined: a) cultured Mx maintained Ia antigen positivity for greater than 8 days; b) mixing experiments with Mo + 6-day cultured Mx or Mo + PMx demonstrated no T cell suppression; c) the normal capacity of most 6-day cultured Mx to support Con A but not SKSD induced T cell proliferation, apparently ruled out the loss of the ability to deliver a nonspecific "second signal" as the involved mechanism; d) inhibition of Mo to Mx differentiation by dexamethasone preserved AC activity. Thus, human culture-derived Mx and PMx exhibit deficit AC function through loss of an undefined mechanism. However, loss of AC antigen processing or presentation may occur.

Factors That Influence a Mother's Willingness to Preserve Umbilical Cord Blood: A Survey of 5120 Chinese Mothers.

PubMed

Lu, Haiyan; Chen, Yanwen; Lan, Qiaofen; Liao, Huanjin; Wu, Jing; Xiao, Haiyan; Dickerson, Carol A; Wu, Ping; Pan, Qingjun

2015-01-01

Umbilical Cord blood (UCB), which contains a substantive number of stem cells, could be widely used in transplants to treat a variety of oncologic, genetic, hematologic, and immunodeficiency disorders. However, only a small portion of mothers preserve or donate their UCB in China. The limited availability of UCB has hampered stem cell research and therapy nowadays. To date, no systemic investigations regarding factors that influence a mother's willingness to preserve UCB have been performed in China. In the current study, we are trying to determine those factors which will provide useful information for national health policy development and will raise awareness of the importance of UCB preservation. During 2011 to 2013, 5120 mothers with the average age of 26.1±8.4 years were included in this study. Those mothers participated in a standardized survey. The information gathered consisted of delivery time, occupation, level of education, knowledge of preservation of UCB, willingness to store UCB, and related concerns. The results have been analyzed with SPSS 16.0. The results showed that first-time mothers showed a greater willingness to preserve their UCB (73.3%) compared to those having their second (48.9%) or third child (40.3%). Mothers who were employed at Government Agencies and Organizations were more willing to preserve their UCB (87.3%) than those employed at factories (62.0%), and those who were unemployed (27.3%). Mothers holding master's or college degrees were more willing to preserve their UCB (72.5% and 71.1%, respectively) than mothers with high school diplomas (48.7%) or those who only went to preliminary school or middle school (40.7%). The two strongest factors that influenced an unwillingness to preserve UCB were the high cost and concerns regarding the safety of the preservation. The results showed that mothers with higher education or those having better occupations are more likely to preserve their UCB in China. These mothers have related knowledge and understand the importance of the preservation and they could more readily afford the relatively high cost. The government, clinicians and UCB banks should combine efforts to take measures, such as increasing public knowledge of the importance of UCB preservation and decreasing the high cost for its storage will most likely increase the frequency of UCB preservation which will further benefit stem cell research and therapy.

[The protective effect of dexpanthenol in nasal sprays. First results of cytotoxic and ciliary-toxic studies in vitro].

PubMed

Klöcker, N; Verse, T; Rudolph, P

2003-03-01

In Germany more than 60 million units of nasal decongestants are prescribed or sold over the counter. The cytotoxic and ciliary-toxic potential of alpha-sympathomimetic decongestants is well established. Furthermore, in many of the marketed products preservatives are added, predominantly benzalchonium-chloride, which can lead to a further alteration of cell- and ciliary function. Recently a protective effect of dexpanthenol was found for the human nasal mucosa. The objective of the present studies was to prove the hypothesis that dexpanthenol is able to neutralise the toxic effects of both alpha-sympathomimetic decongestants, in particular those of xylometazoline, and those of benzalconium-chloride. Therefore, systematic cytotoxic and ex vivo in vitro ciliary-toxic studies were performed. After exposition to xylometazoline in concentrations of 0.1 % and 0.05 %, the influence of dexpanthenol (5 %) and benzalconium-chloride (0,01 %) was assessed by determination of a) cell growth of FL-cells of human amnion origin, and b) ciliary beat frequency of human nasal mucosa. All tests were performed placebo-controlled. Both hypotheses were confirmed. Dexpanthenol (5 %) reduces statistically significantly the concentration-dependent toxic effects of xylometazoline, and benzalchonium-cloride regarding cell growth and ciliary beat frequency (p < 0.001). The combination of xylometazoline with dexpanthenol, while benzalconium-chloride is eliminated, resulted in a further significant increase of cell growth and ciliary beat frequency (p < 0.001), similar to control. The additive application of dexpanthenol (5 %) with nasal decongestants and/or with preserved nasal sprays seems to be able to reduce the cell- and ciliary-toxic effects of these substances.

NOX2 Deficiency Protects Against Streptozotocin-Induced β-Cell Destruction and Development of Diabetes in Mice

PubMed Central

Xiang, Fu-Li; Lu, Xiangru; Strutt, Brenda; Hill, David J.; Feng, Qingping

2010-01-01

OBJECTIVE The role of NOX2-containing NADPH oxidase in the development of diabetes is not fully understood. We hypothesized that NOX2 deficiency decreases reactive oxygen species (ROS) production and immune response and protects against streptozotocin (STZ)-induced β-cell destruction and development of diabetes in mice. RESEARCH DESIGN AND METHODS Five groups of mice—wild-type (WT), NOX2−/−, WT treated with apocynin, and WT adoptively transferred with NOX2−/− or WT splenocytes—were treated with multiple-low-dose STZ. Blood glucose and insulin levels were monitored, and an intraperitoneal glucose tolerance test was performed. Isolated WT and NOX2−/− pancreatic islets were treated with cytokines for 48 h. RESULTS Significantly lower blood glucose levels, higher insulin levels, and better glucose tolerance was observed in NOX2−/− mice and in WT mice adoptively transferred with NOX2−/− splenocytes compared with the respective control groups after STZ treatment. Compared with WT, β-cell apoptosis, as determined by TUNEL staining, and insulitis were significantly decreased, whereas β-cell mass was significantly increased in NOX2−/− mice. In response to cytokine stimulation, ROS production was significantly decreased, and insulin secretion was preserved in NOX2−/− compared with WT islets. Furthermore, proinflammatory cytokine release induced by concanavalin A was significantly decreased in NOX2−/− compared with WT splenocytes. CONCLUSIONS NOX2 deficiency decreases β-cell destruction and preserves islet function in STZ-induced diabetes by reducing ROS production, immune response, and β-cell apoptosis. PMID:20627937

Experiment K-6-23. Effect of spaceflight on levels and function of immune cells

NASA Technical Reports Server (NTRS)

Mandel, A. D.; Sonnenfeld, G.; Berry, W.; Taylor, G.; Wellhausen, S. R.; Konstantinova, I.; Lesnyak, A.; Fuchs, B.

1990-01-01

Two different immunology experiments were performed on samples received from rats flown on Cosmos 1887. In the first experiment, rat bone marrow cells were examined in Moscow for their response to colony stimulating factor-M. In the second experiment, rat spleen and bone marrow cells were stained in Moscow with a variety of antibodies directed against cell surface antigenic markers. These cells were preserved and shipped to the United States where they were subjected to analysis on a flow cytometer. The results of the studies indicate that bone marrow cells from flown rats showed a decreased response to colony stimulating factor than did bone marrow cells from control rats. There was a higher percentage of spleen cells from flown rats staining positively for pan-T-cell, suppressor-T-cell and innate interleukin-2 receptor antigens than from control animals. In addition, a higher percentage of cells that appeared to be part of the myelogenous population of bone marrow cells from flown rats stained positively for surface immunoglobulin than did equivalent cells from control rats.

PRMT7 Preserves Satellite Cell Regenerative Capacity.

PubMed

Blanc, Roméo Sébastien; Vogel, Gillian; Chen, Taiping; Crist, Colin; Richard, Stéphane

2016-02-16

Regeneration of skeletal muscle requires the continued presence of quiescent muscle stem cells (satellite cells), which become activated in response to injury. Here, we report that whole-body protein arginine methyltransferase PRMT7(-/-) adult mice and mice conditionally lacking PRMT7 in satellite cells using Pax7-CreERT2 both display a significant reduction in satellite cell function, leading to defects in regenerative capacity upon muscle injury. We show that PRMT7 is preferentially expressed in activated satellite cells and, interestingly, PRMT7-deficient satellite cells undergo cell-cycle arrest and premature cellular senescence. These defects underlie poor satellite cell stem cell capacity to regenerate muscle and self-renew after injury. PRMT7-deficient satellite cells express elevated levels of the CDK inhibitor p21CIP1 and low levels of its repressor, DNMT3b. Restoration of DNMT3b in PRMT7-deficient cells rescues PRMT7-mediated senescence. Our findings define PRMT7 as a regulator of the DNMT3b/p21 axis required to maintain muscle stem cell regenerative capacity. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Multifunctional Mitochondrial AAA Proteases

PubMed Central

Glynn, Steven E.

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle. PMID:28589125

Multifunctional Mitochondrial AAA Proteases.

PubMed

Glynn, Steven E

2017-01-01

Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

[Valuation and prospect of function preserving gastrectomy].

PubMed

Wang, Shuchang; Yu, Site; Xu, Jia; Zhao, Gang

2017-10-25

Preserving gastric function and improving quality of life (QOL) is the tendency of surgery for early gastric cancer. Function preserving gastrectomy (FPG) is applied to modify the extent of surgery and to achieve better quality of life at the premise of radical resection. Pylorus-preserving gastrectomy is the most favorable approach of FPG with oncological safety, which can improve nutritional status and QOL via preserving pylorus and vagal nerve. Proximal gastrectomy is widely accepted as FPG for early upper 1/3 gastric cancer. However, the most optimal way of anastomosis is not yet solved. Sentinel node navigation is currently the most accurate approach for intraoperative diagnosis of lymph node metastasis, which stimulates the development of many kinds of FPG procedures for individual treatment. Nevertheless, more efforts should be made to reduce false negative rate of sentinel node biopsy. Herein we discuss the valuation and prospect of FPG.

Preservation and compensation: The functional neuroanatomy of insight and working memory in schizophrenia☆

PubMed Central

Sapara, Adegboyega; ffytche, Dominic H.; Birchwood, Max; Cooke, Michael A.; Fannon, Dominic; Williams, Steven C.R.; Kuipers, Elizabeth; Kumari, Veena

2014-01-01

Background Poor insight in schizophrenia has been theorised to reflect a cognitive deficit that is secondary to brain abnormalities, localized in the brain regions that are implicated in higher order cognitive functions, including working memory (WM). This study investigated WM-related neural substrates of preserved and poor insight in schizophrenia. Method Forty stable schizophrenia outpatients, 20 with preserved and 20 with poor insight (usable data obtained from 18 preserved and 14 poor insight patients), and 20 healthy participants underwent functional magnetic resonance imaging (fMRI) during a parametric ‘n-back’ task. The three groups were preselected to match on age, education and predicted IQ, and the two patient groups to have distinct insight levels. Performance and fMRI data were analysed to determine how groups of patients with preserved and poor insight differed from each other, and from healthy participants. Results Poor insight patients showed lower performance accuracy, relative to healthy participants (p = 0.01) and preserved insight patients (p = 0.08); the two patient groups were comparable on symptoms and medication. Preserved insight patients, relative to poor insight patients, showed greater activity most consistently in the precuneus and cerebellum (both bilateral) during WM; they also showed greater activity than healthy participants in the inferior–superior frontal gyrus and cerebellum (bilateral). Group differences in brain activity did not co-vary significantly with performance accuracy. Conclusions The precuneus and cerebellum function contribute to preserved insight in schizophrenia. Preserved insight as well as normal-range WM capacity in schizophrenia sub-groups may be achieved via compensatory neural activity in the frontal cortex and cerebellum. PMID:24332795

Cryopreservation of Human Pluripotent Stem Cells in Defined Medium

PubMed Central

Liu, Weiwei; Chen, Guokai

2014-01-01

This protocol describes a cryopreservation procedure using an enzyme-free dissociation method to harvest cells and preserve cells in albumin-free chemically defined E8 medium for human pluripotent stem cells (hPSCs). The dissociation by EDTA/PBS produces small cell aggregates that allow high survival efficiency in passaging and cryopreservation. The preservation in E8 medium eliminates serum or other animal products, and is suitable for the increasing demand for high quality hPSCs in translational research. In combination with the special feature of EDTA/PBS dissociation, this protocol allows efficient cryopreservation in more time-saving manner. PMID:25366897

Follistatin-Like 3 Enhances the Function of Endothelial Cells Derived from Pluripotent Stem Cells by Facilitating β-Catenin Nuclear Translocation Through Inhibition of Glycogen Synthase Kinase-3β Activity.

PubMed

Kelaini, Sophia; Vilà-González, Marta; Caines, Rachel; Campbell, David; Eleftheriadou, Magdalini; Tsifaki, Marianna; Magee, Corey; Cochrane, Amy; O'neill, Karla; Yang, Chunbo; Stitt, Alan W; Zeng, Lingfang; Grieve, David J; Margariti, Andriana

2018-03-23

The fight against vascular disease requires functional endothelial cells (ECs) which could be provided by differentiation of induced Pluripotent Stem Cells (iPS Cells) in great numbers for use in the clinic. However, the great promise of the generated ECs (iPS-ECs) in therapy is often restricted due to the challenge in iPS-ECs preserving their phenotype and function. We identified that Follistatin-Like 3 (FSTL3) is highly expressed in iPS-ECs, and, as such, we sought to clarify its possible role in retaining and improving iPS-ECs function and phenotype, which are crucial in increasing the cells' potential as a therapeutic tool. We overexpressed FSTL3 in iPS-ECs and found that FSTL3 could induce and enhance endothelial features by facilitating β-catenin nuclear translocation through inhibition of glycogen synthase kinase-3β activity and induction of Endothelin-1. The angiogenic potential of FSTL3 was also confirmed both in vitro and in vivo. When iPS-ECs overexpressing FSTL3 were subcutaneously injected in in vivo angiogenic model or intramuscularly injected in a hind limb ischemia NOD.CB17-Prkdcscid/NcrCrl SCID mice model, FSTL3 significantly induced angiogenesis and blood flow recovery, respectively. This study, for the first time, demonstrates that FSTL3 can greatly enhance the function and maturity of iPS-ECs. It advances our understanding of iPS-ECs and identifies a novel pathway that can be applied in cell therapy. These findings could therefore help improve efficiency and generation of therapeutically relevant numbers of ECs for use in patient-specific cell-based therapies. In addition, it can be particularly useful toward the treatment of vascular diseases instigated by EC dysfunction. Stem Cells 2018. © 2018 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

Differentiation as symbiosis.

PubMed

Chigira, M; Watanabe, H

1994-07-01

Preservation of the identity of DNA is the ultimate goal of multicellular organisms. An abnormal DNA sequence in cells within an individual means its parasitic nature in cell society as shown in tumors. Somatic gene arrangement and gene mutation in development may be considered as de novo formation of parasites. It is likely that the developmental process with genetic alterations means symbiosis between altered cells and germ line cells preserving genetic information without alterations, when somatic alteration of DNA sequence is a major mechanism of differentiation. According to the selfish gene theory of Dawkins, germ line cells permit symbiosis when somatic cell society derives clear profit for the replication of original DNA copies.

High-Affinity Rb Binding, p53 Inhibition, Subcellular Localization, and Transformation by Wild-Type or Tumor-Derived Shortened Merkel Cell Polyomavirus Large T Antigens

PubMed Central

Borchert, Sophie; Czech-Sioli, Manja; Neumann, Friederike; Schmidt, Claudia; Wimmer, Peter; Dobner, Thomas

2014-01-01

ABSTRACT Interference with tumor suppressor pathways by polyomavirus-encoded tumor antigens (T-Ags) can result in transformation. Consequently, it is thought that T-Ags encoded by Merkel cell polyomavirus (MCPyV), a virus integrated in ∼90% of all Merkel cell carcinoma (MCC) cases, are major contributors to tumorigenesis. The MCPyV large T-Ag (LT-Ag) has preserved the key functional domains present in all family members but has also acquired unique regions that flank the LxCxE motif. As these regions may mediate unique functions, or may modulate those shared with T-Ags of other polyomaviruses, functional studies of MCPyV T-Ags are required. Here, we have performed a comparative study of full-length or MCC-derived truncated LT-Ags with regard to their biochemical characteristics, their ability to bind to retinoblastoma (Rb) and p53 proteins, and their transforming potential. We provide evidence that full-length MCPyV LT-Ag may not directly bind to p53 but nevertheless can significantly reduce p53-dependent transcription in reporter assays. Although early region expression constructs harboring either full-length or MCC-derived truncated LT-Ag genes can transform primary baby rat kidney cells, truncated LT-Ags do not bind to p53 or reduce p53-dependent transcription. Interestingly, shortened LT-Ags exhibit a very high binding affinity for Rb, as shown by coimmunoprecipitation and in vitro binding studies. Additionally, we show that truncated MCPyV LT-Ag proteins are expressed at higher levels than those for the wild-type protein and are able to partially relocalize Rb to the cytoplasm, indicating that truncated LT proteins may have gained additional features that distinguish them from the full-length protein. IMPORTANCE MCPyV is one of the 12 known polyomaviruses that naturally infect humans. Among these, it is of particular interest since it is the only human polyomavirus known to be involved in tumorigenesis. MCPyV is thought to be causally linked to MCC, a rare skin tumor. In these tumors, viral DNA is monoclonally integrated into the genome of the tumor cells in up to 90% of all MCC cases, and the integrated MCV genomes, furthermore, harbor signature mutations in the so-called early region that selectively abrogate viral replication while preserving cell cycle deregulating functions of the virus. This study describes comparative studies of early region T-Ag protein characteristics, their ability to bind to Rb and p53, and their transforming potential. PMID:24371076

Erythroleukemia cells acquire an alternative mitophagy capability.

PubMed

Wang, Jian; Fang, Yixuan; Yan, Lili; Yuan, Na; Zhang, Suping; Xu, Li; Nie, Meilan; Zhang, Xiaoying; Wang, Jianrong

2016-04-19

Leukemia cells are superior to hematopoietic cells with a normal differentiation potential in buffering cellular stresses, but the underlying mechanisms for this leukemic advantage are not fully understood. Using CRISPR/Cas9 deletion of the canonical autophagy-essential gene Atg7, we found that erythroleukemia K562 cells are armed with two sets of autophagic machinery. Alternative mitophagy is functional regardless of whether the canonical autophagic mechanism is intact or disrupted. Although canonical autophagy defects attenuated cell cycling, proliferation and differentiation potential, the leukemia cells retained their abilities for mitochondrial clearance and for maintaining low levels of reactive oxygen species (ROS) and apoptosis. Treatment with a specific inducer of mitophagy revealed that the canonical autophagy-defective erythroleukemia cells preserved a mitophagic response. Selective induction of mitophagy was associated with the upregulation and localization of RAB9A on the mitochondrial membrane in both wild-type and Atg7(-/-) leukemia cells. When the leukemia cells were treated with the alternative autophagy inhibitor brefeldin A or when the RAB9A was knocked down, this mitophagy was prohibited. This was accompanied by elevated ROS levels and apoptosis as well as reduced DNA damage repair. Therefore, the results suggest that erythroleukemia K562 cells possess an ATG7-independent alternative mitophagic mechanism that functions even when the canonical autophagic process is impaired, thereby maintaining the ability to respond to stresses such as excessive ROS and DNA damage.

In Vivo Functional Selection Identifies Cardiotrophin-1 as a Cardiac Engraftment Factor for Mesenchymal Stromal Cells.

PubMed

Bortolotti, Francesca; Ruozi, Giulia; Falcione, Antonella; Doimo, Sara; Dal Ferro, Matteo; Lesizza, Pierluigi; Zentilin, Lorena; Banks, Lawrence; Zacchigna, Serena; Giacca, Mauro

2017-10-17

Transplantation of cells into the infarcted heart has significant potential to improve myocardial recovery; however, low efficacy of cell engraftment still limits therapeutic benefit. Here, we describe a method for the unbiased, in vivo selection of cytokines that improve mesenchymal stromal cell engraftment into the heart both in normal conditions and after myocardial infarction. An arrayed library of 80 secreted factors, including most of the currently known interleukins and chemokines, were individually cloned into adeno-associated viral vectors. Pools from this library were then used for the batch transduction of bone marrow-derived mesenchymal stromal cells ex vivo, followed by intramyocardial cell administration in normal and infarcted mice. Three weeks after injection, vector genomes were recovered from the few persisting cells and identified by sequencing DNA barcodes uniquely labeling each of the tested cytokines. The most effective molecule identified by this competitive engraftment screening was cardiotrophin-1, a member of the interleukin-6 family. Intracardiac injection of mesenchymal stromal cells transiently preconditioned with cardiotrophin-1 preserved cardiac function and reduced infarct size, parallel to the persistence of the transplanted cells in the healing hearts for at least 2 months after injection. Engraftment of cardiotrophin-1-treated mesenchymal stromal cells was consequent to signal transducer and activator of transcription 3-mediated activation of the focal adhesion kinase and its associated focal adhesion complex and the consequent acquisition of adhesive properties by the cells. These results support the feasibility of selecting molecules in vivo for their functional properties with adeno-associated viral vector libraries and identify cardiotrophin-1 as a powerful cytokine promoting cell engraftment and thus improving cell therapy of the infarcted myocardium. © 2017 American Heart Association, Inc.

Different tissue phagocytes sample apoptotic cells to direct distinct homeostasis programs.

PubMed

Cummings, Ryan J; Barbet, Gaetan; Bongers, Gerold; Hartmann, Boris M; Gettler, Kyle; Muniz, Luciana; Furtado, Glaucia C; Cho, Judy; Lira, Sergio A; Blander, J Magarian

2016-11-24

Recognition and removal of apoptotic cells by professional phagocytes, including dendritic cells and macrophages, preserves immune self-tolerance and prevents chronic inflammation and autoimmune pathologies. The diverse array of phagocytes that reside within different tissues, combined with the necessarily prompt nature of apoptotic cell clearance, makes it difficult to study this process in situ. The full spectrum of functions executed by tissue-resident phagocytes in response to homeostatic apoptosis, therefore, remains unclear. Here we show that mouse apoptotic intestinal epithelial cells (IECs), which undergo continuous renewal to maintain optimal barrier and absorptive functions, are not merely extruded to maintain homeostatic cell numbers, but are also sampled by a single subset of dendritic cells and two macrophage subsets within a well-characterized network of phagocytes in the small intestinal lamina propria. Characterization of the transcriptome within each subset before and after in situ sampling of apoptotic IECs revealed gene expression signatures unique to each phagocyte, including macrophage-specific lipid metabolism and amino acid catabolism, and a dendritic-cell-specific program of regulatory CD4 + T-cell activation. A common 'suppression of inflammation' signature was noted, although the specific genes and pathways involved varied amongst dendritic cells and macrophages, reflecting specialized functions. Apoptotic IECs were trafficked to mesenteric lymph nodes exclusively by the dendritic cell subset and served as critical determinants for the induction of tolerogenic regulatory CD4 + T-cell differentiation. Several of the genes that were differentially expressed by phagocytes bearing apoptotic IECs overlapped with susceptibility genes for inflammatory bowel disease. Collectively, these findings provide new insights into the consequences of apoptotic cell sampling, advance our understanding of how homeostasis is maintained within the mucosa and set the stage for development of novel therapeutics to alleviate chronic inflammatory diseases such as inflammatory bowel disease.

Mouse pancreas tissue slice culture facilitates long-term studies of exocrine and endocrine cell physiology in situ.

PubMed

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ.

Mouse Pancreas Tissue Slice Culture Facilitates Long-Term Studies of Exocrine and Endocrine Cell Physiology in situ

PubMed Central

Marciniak, Anja; Selck, Claudia; Friedrich, Betty; Speier, Stephan

2013-01-01

Studies on pancreatic cell physiology rely on the investigation of exocrine and endocrine cells in vitro. Particularly, in the case of the exocrine tissue these studies have suffered from a reduced functional viability of acinar cells in culture. As a result not only investigations on dispersed acinar cells and isolated acini were limited in their potential, but also prolonged studies on pancreatic exocrine and endocrine cells in an intact pancreatic tissue environment were unfeasible. To overcome these limitations, we aimed to establish a pancreas tissue slice culture platform to allow long-term studies on exocrine and endocrine cells in the intact pancreatic environment. Mouse pancreas tissue slice morphology was assessed to determine optimal long-term culture settings for intact pancreatic tissue. Utilizing optimized culture conditions, cell specificity and function of exocrine acinar cells and endocrine beta cells were characterized over a culture period of 7 days. We found pancreas tissue slices cultured under optimized conditions to have intact tissue specific morphology for the entire culture period. Amylase positive intact acini were present at all time points of culture and acinar cells displayed a typical strong cell polarity. Amylase release from pancreas tissue slices decreased during culture, but maintained the characteristic bell-shaped dose-response curve to increasing caerulein concentrations and a ca. 4-fold maximal over basal release. Additionally, endocrine beta cell viability and function was well preserved until the end of the observation period. Our results show that the tissue slice culture platform provides unprecedented maintenance of pancreatic tissue specific morphology and function over a culture period for at least 4 days and in part even up to 1 week. This analytical advancement now allows mid -to long-term studies on the cell biology of pancreatic disorder pathogenesis and therapy in an intact surrounding in situ. PMID:24223842