Reversing multiple age-related pathologies by controlling the senescence-associated secretory phenotype of stem cells

Regenerative medicine by cell transplantation is a novel therapy for treating end-stage organ failure and tissue damage. Cell-based therapy based on the transplantation of neural stem/progenitor cells （NSPCs） represents an attractive strategy for the treatment of neurodegenerative diseases, but obtaining large numbers of these cells is difficult and their differentiation potential is strictly restricted in a spatiotemporally-regulated manner during central nervous system （CNS） development. Therefore, embryonic stem cells and induced pluripotent stem cells represent an attractive alternative for cell-transplantation therapy in regenerative medicine.

The role of Islet-1 in cell specification,differentiation,and maintenance of phenotypes in the vertebrate neural retina

Many blinding diseases,such as retinitis pigmentosa,age-related macular degeneration,and glaucoma involve the permanent loss of retinal neurons,especially photoreceptors or the centrally projecting retinal ganglion cells.Stem cells have been proposed as a potential source of cells for neuronal transplantation.

Memory CD8+ T cell differentiation in viral infection: A cell for all seasons

Chronic viral infections such as hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) are major global health problems affecting more than 500 million people worldwide. Virus-specific CD8+ T cells play an important role in the course and outcome of these viral infections and it is hypothesized that altered or impaired differentiation of virus- specific CD8+ T cells contributes to the development of persistence and/or disease progression. A deeper understanding of the mechanisms responsible for functional differentiation of CD8+ T cells is essential for the generation of successful therapies aiming to strengthen the adaptive component of the immune system.

Cell biomechanics and its applications in human disease diagnosis

Certain diseases are known to cause changes in the physical and biomechanical properties of cells.These include cancer,malaria,and sickle cell anemia among others.Typically,such physical property changes can result in several fold increases or decreases in cell stiffness,which are significant and can result in severe pathology and eventual catastrophic breakdown of the bodily functions.While there are developed biochemical and biological assays to detect the onset or presence of diseases,there is always a need to develop more rapid,precise,and sensitive methods to detect and diagnose diseases.Biomechanical property changes can play a significant role in this regard.As such,research into disease biomechanics can not only give us an in-depth knowledge of the mechanisms underlying disease progression,but can also serve as a powerful tool for detection and diagnosis.This article provides some insights into opportunities for how significant changes in cellular mechanical properties during onset or progression of a disease can be utilized as useful means for detection and diagnosis.We will also showcase several technologies that have already been developed to perform such detection and diagnosis.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

Phenotypic Modulation of Mesangial Cells in Diabetic Rats and Effect of Tujian Mixture (菟箭合剂) on It

Objective: To explore whether there is phenotypic modulation of mesangial cells in streptozotocin (STZ) induced diabetic rats and study the effect of Tujian Mixture (TJM) on it. Methods: SD rats were divided into the normal control group , the unilateral nephrectomized control group , the STZ induced diabetes mellitus with unilateral nephrectomy model group , the Valsartan treated group (VT group, n=8) and the TJM treated group , rats in the latter two groups were modeled as in the DM group and treated with Valsartan (20 mg/kg·d) and TJM (20g/kg·d) respectively for 12 weeks. The expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β 1 (TGF-β 1) in rats’ glomeruli were observed by immunohistochemistry assay, and the ratio of α-SMA and TGF-β 1 positive area/total glomerule tuft area (SMA/GT and TGF/GT) were analyzed using computer-assisted image analysis software. Results: In the NC and the QC groups, only trace of α-SMA positive staining was found. But there was prominant α-SMA positive staining in glomeruli of the DM group, with SMA/GT and TGF/GT increased significantly , and marked increase of 24 hrs proteinuria excretion ( P<0 01). As compared with the DM group, the three indexes were all significantly lower in the VT and ZY groups , and the lowering of proteinuria was more significant in the ZY group than that in the VT group (P<0 01). Conclusion: The expression of α-SMA in glomeruli in STZ induced diabetic rats with unilateral nephrectomy is pronounced, indicating that phenotypic modulation of mesangial cells involvement in the pathogenesis of diabetic nephropathy. TJM and Valsartan can reduce 24 hrs proteinuria excretion, inhibit the phenotypic modulation of mesangial cells and the expression of TGF-β 1 in glomeruli of diabetic rats, and the effect of TJM is more potent than that of Valsartan in lowering urinary protein excretion..

Conservation of T cell epitopes between seasonal influenza viruses and the novel influenza A H7N9 virus

A novel avian influenza A(H7N9) virus recently emerged in the Yangtze River delta and caused diseases, often severe, in over 130 people. This H7N9 virus appeared to infect humans with greater ease than previous avian influenza virus subtypes such as H5N1 and H9N2. While there are other potential explanations for this large number of human infections with an avian influenza virus, we investigated whether a lack of conserved T-cell epitopes between endemic H1N1 and H3N2 influenza viruses and the novel H7N9 virus contributes to this observation. Here we demonstrate that a number of T cell epitopes are conserved between endemic H1N1 and H3N2 viruses and H7N9 virus. Most of these conserved epitopes are from viral internal proteins. The extent of conservation between endemic human seasonal influenza and avian influenza H7N9 was comparable to that with the highly pathogenic avian influenza H5N1. Thus, the ease of inter-species transmission of H7N9 viruses(compared with avian H5N1 viruses) cannot be attributed to the lack of conservation of such T cell epitopes. On the contrary, our findings predict significant T-cell based cross-reactions in the human population to the novel H7N9 virus. Our findings also have implications for H7N9 virus vaccine design.

Characterization of recombinant humanized collagen type Ⅲ and its influence on cell behavior and phenotype

Collagen made a tremendous impact in the field of regenerative medicine as a bioactive material.For decades,collagen has been used not only as a scaffolding material but also as an active component in regulating cells'biological behavior and phenotype.However,animal-derived collagen as a major source suffered from problems of immunogenicity,risk of viral infection,and the unclear relationship between bioactive sequence and function.Recombinant humanized collagen(rhCol)provided alternatives for regenerative medicine with more controllable risks.However,the characterization of rhCol and the interaction between rhCol and cells still need further investigation,including cell behavior and phenotype.The current study preliminarily demonstrated that recombinant humanized collagen typeⅢ(rhColⅢ)conformed to the theoretical amino acid sequence and had an advanced structure resembling bovine collagen.Furthermore,rhColⅢcould facilitate basal biological behaviors of human skin fibroblasts,such as adhesion,proliferation and migration.rhColⅢwas beneficial for some extracellular matrix-expressing cell phenotypes.The study would shed light on the mechanism research of rhCol and cell interactions and further understanding of effectiveness in tissue regeneration.

Effect of retinoid X receptor alpha (RXRα) transfection on the proliferation and phenotype of rat hepatic stellate cells in vitro

Objective To study the effect of retinoid X receptor alpha (RXRα) transfection plus treatment with the RXRα ligand, 9-cis-RA, on the proliferation and phenotype of platelet-derived growth factor (PDGF)-activated hepatic stellate cells (HSCs). Methods PDGF activated rat hepatic stellate cells were transfected with eukaryotic expression vector pcDNA3.1- human RXRα, and confirmed by Western blot. Proliferation of transfected HSC was assayed by bromodeoxyuridine (BrdU) incorporation as well as MTT, and the phenotype (α-smooth muscle actin, desmin) was observed by immunocytochemistry with image analysis. Results Transfection of the RXRα gene and treatment with ligand 9-cis-RA of PDGF-activated HSCs extended the increased expression of RXRα protein for at least 168 hours. Cell proliferation and expressions of alpha-smooth muscle actin (α-SMA) and desmin were blocked, compared with groups of sham-transfected, PDGF-activated, no transfection, no ligand treatment, and irrelevant ligand treated HSCs. Conclusion Transfection with the RXRα gene followed by 9-cis-RA ligand treatment will inhibit the proliferation and reverse the phenotype of activated HSC.

Characterization of recombinant humanized collagen type III and its influence on cell behavior and phenotype

Collagen made a tremendous impact in the field of regenerative medicine as a bioactive material.For decades,collagen has been used not only as a scaffolding material but also as an active component in regulating cells’biological behavior and phenotype.However,animal-derived collagen as a major source suffered from problems of immunogenicity,risk of viral infection,and the unclear relationship between bioactive sequence and function.Recombinant humanized collagen(rhCol)provided alternatives for regenerative medicine with more controllable risks.However,the characterization of rhCol and the interaction between rhCol and cells still need further investigation,including cell behavior and phenotype.The current study preliminarily demonstrated that recombinant humanized collagen type III(rhCol III)conformed to the theoretical amino acid sequence and had an advanced structure resembling bovine collagen.Furthermore,rhCol III could facilitate basal biological behaviors of human skin fibroblasts,such as adhesion,proliferation and migration.rhCol III was beneficial for some extracellular matrix-expressing cell phenotypes.The study would shed light on the mechanism research of rhCol and cell interactions and further understanding of effectiveness in tissue regeneration.

Effect of interleukin-10 on the phenotype and function of cultured human dendritic cells

Dendritic cells （DCs） are potent antigen presenting cells （APCs）, and are able to induce tissue like kidney immune-inflammatory responses. DCs can either stimulate immune responses or induce immune tolerance, according to its mature states. DCs undergo a series of maturational steps that allow them-to up-regulate surface adhesion molecules and co-stimulation molecules, as well as increase secretion of interleukin （ IL ）-12,

Biological features of mesenchymal stem cells from human bone marrow

Objective To study the biological characteristics of mesenchymal stem cells (MSCs) from human bone marrow. Methods A culture of mesenchymal stem cells was initiated from bone marrow low-density mononuclear cells separated by Percoll Centrifugation and maintained in low-glucose Dulbecco's modified Eagle's medium (DMEM) with 10% selected fetal calf serum. Cell growth pattern and its responses to cytokines were evaluated by trypan blue exclusion and MTT test, respectively. Cell cycle and surface antigenic features were analyzed by flow cytometry technique. Cytochemistry characteristics of MSCs were determined. Results Easy-handling methods to isolate and culture expand MSCs were developed in this study. MSCs were unique in their phenotypes. They were positive for CD29, CD44, CD166, and negative for CD34, CD45, HLA-DR and Ulex europaeus. Cytochemistry evaluation showed that MSCs were homogeneously positive for acid α-naphthl acetate esterase (ANAE), glycogen (periodic acid Schiff reaction, PAS), and negative for acid phosphatase (ACP) and the Sudan black reaction (SB). Around 5% of them were positive for alkaline phosphatase (ALP). The cells had a population doubling time of 30 hours and cell cycle analysis showed that approximately 10% of them were in S phase. MSCs grew at significantly different rates when incubated in the presence of various recombinant human cytokines, of which interferon γ, tumor necrosis factor α, stem cell factor and insulin-like growth factor promoted the proliferation of MSCs dramatically, while others tested had no effects on cell growth. Conclusions MSCs are a homogenous population of cells that have unique growth, phenotypical and cytochemical characteristics. Furthermore, the diverse responses of MSCs to different cytokines provide a clue for the selection of optimal expansion and maintenance of MSCs.

Functional annotation map of natural compounds in traditional Chinese medicines library: TCMs with myocardial protection as a case

The chemical complexity of traditional Chinese medicines(TCMs) makes the active and functional annotation of natural compounds challenging. Herein, we developed the TCMs-Compounds Functional Annotation platform(TCMs-CFA) for large-scale predicting active compounds with potential mechanisms from TCM complex system, without isolating and activity testing every single compound one by one. The platform was established based on the integration of TCMs knowledge base, chemome profiling, and high-content imaging. It mainly included:(1) selection of herbal drugs of target based on TCMs knowledge base;(2) chemome profiling of TCMs extract library by LC-MS;(3) cytological profiling of TCMs extract library by high-content cell-based imaging;(4) active compounds discovery by combining each mass signal and multi-parametric cell phenotypes;(5) construction of functional annotation map for predicting the potential mechanisms of lead compounds. In this stud TCMs with myocardial protection were applied as a case study, and validated for the feasibility and utility of the platform. Seven frequently used herbal drugs(Ginseng, etc.) were screened from 100,000 TCMs formulas for myocardial protection and subsequently prepared as a library of 700 extracts. By using TCMs-CFA platform, 81 lead compounds, including 10 novel bioactive ones, were quickly identified by correlating 8089mass signals with 170,100 cytological parameters from an extract library. The TCMs-CFA platform described a new evidence-led tool for the rapid discovery process by data mining strategies, which is valuable for novel lead compounds from TCMs. All computations are done through Python and are publicly available on GitHub.

Depletion but Activation of CD56dimCD16+ NK Cells in Acute Infection with Severe Fever with Thrombocytopenia Syndrome Virus

Severe fever with thrombocytopenia syndrome(SFTS)is an emerging infectious disease with high mortality(12%–30%).The mechanism by which the SFTS bunyavirus(SFTSV)causes severe illness remains unclear.To evaluate the phenotypic and functional characteristics of the NK cell subsets in SFTS patients,twenty-nine SFTS patients were sequentially sampled from admission until recovery.Phenotypic and functional characteristics of NK cell subsets in circulating blood were analysed via flow cytometry.Then,correlations between NK cell subset frequencies and the SFTS index(SFTSI)were evaluated in all SFTS patients(15 mild,14 severe)upon admission.The frequencies of CD56dimCD16+NK cells were greatly decreased in early SFTSV infection and were negatively correlated with disease severity.Additionally,higher Ki-67 and granzyme B expression and relatively lower NKG2 A expression in CD56dimCD16+NK cells were observed in acute infection.Moreover,the effector function of CD56dimNK cells was increased in the acute phase compared with the recovery phase in nine severe SFTS patients.Additionally,interleukin(IL)-15,interferon(IFN)-a,IL-18 and IFN-c secretion was markedly increased during early infection.Collectively,despite depletion of CD56dimCD16+NK cells,activation and functional enhancement of CD56dimCD16+NK cells were still observed,suggesting their involvement in defence against early SFTSV infection.