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Background: The p53 isoform Δ133p53β is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53β is driving aggressive disease in RA., Methods: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53β. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software., Results: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53β mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53β., Conclusions: Induction of Δ133p53β in CD90 + synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53β could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53β could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6., (© 2023. The Author(s).)

Huge progress has been made in understanding the biology of innate lymphoid cells (ILC) by adopting several well-known concepts in T cell biology. As such, flow cytometry gating strategies and markers, such as CD90, have been applied to indentify ILC. Here, we report that most non-NK intestinal ILC have a high expression of CD90 as expected, but surprisingly a sub-population of cells exhibit low or even no expression of this marker. CD90-negative and CD90-low CD127 + ILC were present amongst all ILC subsets in the gut. The frequency of CD90-negative and CD90-low CD127 + ILC was dependent on stimulatory cues in vitro and enhanced by dysbiosis in vivo . CD90-negative and CD90-low CD127 + ILC were a potential source of IL-13, IFNγ and IL-17A at steady state and upon dysbiosis- and dextran sulphate sodium-elicited colitis. Hence, this study reveals that, contrary to expectations, CD90 is not constitutively expressed by functional ILC in the gut., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schroeder, Beattie, Lo, Zabinski, Powell, Neves, Jenner and Lord.)

Context: Modified BuShenYiQi formula (M-BYF) is derived from BuShenYiQi formula, used for the treatment of allergic asthma. The exact effect and mechanism of M-BYF on the improvement of asthma remain unclear., Objective: We investigated the mechanism underlying the therapeutic effect of M-BYF on allergic asthma., Materials and Methods: The asthma model was established in female BALB/c mice that were sensitized and challenged with ovalbumin (OVA). Mice in the treated groups were orally treated once a day with M-BYF (7, 14 and 28 g/kg/d) or dexamethasone before OVA challenge. Control and Model group received saline. Pathophysiological abnormalities and percentages of lung type 2 innate lymphoid cells (ILC2s) and Th9 cells were measured. Expression levels of type 2 cytokines and transcription factors required for these cells function and differentiation were analysed. Expression of vasoactive intestinal polypeptide (VIP)-VPAC2 signalling pathway-related proteins, and percentages of VIP expressing (VIP + ) cells and VPAC2, CD90 co-expressing (VPAC2 + CD90 + ) cells were detected., Results: M-BYF alleviated airway hyperresponsiveness, inflammation, mucus hypersecretion and collagen deposition in asthmatic mice. M-BYF down-regulated percentages of ILC2s and Th9 cells with lower expression of GATA3, PU.1 and IRF4, reduced IL-5, IL-13, IL-9 and VIP production. The decrease in the expression of VIP-VPAC2 signalling pathway and percentages of VIP + cells, VPAC2 + CD90 + cells were observed after M-BYF treatment. The LD 50 value of M-BYF was higher than 90 g/kg., Discussion and Conclusions: M-BYF alleviated experimental asthma by negatively regulating ILC2s and Th9 cells and the VIP-VPAC2 signalling pathway. These findings provide the theoretical basis for future research of M-BYF in asthma patient population.

Crohn's disease is an inflammatory disease of the gastrointestinal tract characterized by an aberrant response to microbial and environmental triggers. This includes an altered microbiome dominated by Enterobacteriaceae and in particular adherent-invasive E. coli (AIEC). Clinical evidence implicates periods of psychological stress in Crohn's disease exacerbation, and disturbances in the gut microbiome might contribute to the pathogenic mechanism. Here we show that stress-exposed mice develop ileal dysbiosis, dominated by the expansion of Enterobacteriaceae. In an AIEC colonisation model, stress-induced glucocorticoids promote apoptosis of CD45 + CD90 + cells that normally produce IL-22, a cytokine that is essential for the maintenance of ileal mucosal barrier integrity. Blockade of glucocorticoid signaling or administration of recombinant IL-22 restores mucosal immunity, prevents ileal dysbiosis, and blocks AIEC expansion. We conclude that psychological stress impairs IL-22-driven protective immunity in the gut, which creates a favorable niche for the expansion of pathobionts that have been implicated in Crohn's disease. Importantly, this work also shows that immunomodulation can counteract the negative effects of psychological stress on gut immunity and hence disease-associated dysbiosis., (© 2021. The Author(s).)

Background: Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease mediated by T helper type 2 (Th2) cells in acute phase. Group 2 innate lymphoid cells (ILCs) play a role in the initiation of the Th2 response. Although mold exposure is associated with the development of AD, studies on the underlying mechanisms are lacking. This study investigated whether group 2 ILCs are involved in inflammation in AD-like skin induced by Aspergillus fumigatus ( Af )., Methods: We investigated changes of group 2 ILCs population in Af -induced AD-like skin lesions. To induce AD-like skin lesions, Af extracts were applied to the dorsal skin of BALB/c and Rag1 -/- mice five times per week, with repeat exposures at 2-week intervals., Results: The clinical parameters were higher in the Af -treated group than in the control group. Histologic findings revealed epiderrmal and dermal thickening as well as eosinophil and mast cell infiltration into the skin of Af -treated mice. Populations of group 2 ILCs in the skin were also significantly higher in the Af -treated group. In addition, interleukin-33 mRNA expression was significantly higher in the skin lesions of the Af -treated mice. In the Rag1 -/- mice lacking mature lymphocytes, AD-like skin lesions were still induced by Af and ILCs depletion using an anti-CD90.2 mAb lowered the Af -induced inflammatory response., Conclusions: Group 2 ILCs may play a role in a murine model of Af -induced AD-like skin lesions., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2021 The Korean Academy of Medical Sciences.)

We investigated the role of mesothelin (Msln) and thymocyte differentiation antigen 1 (Thy1) in the activation of fibroblasts across multiple organs and demonstrated that Msln -/- mice are protected from cholestatic fibrosis caused by Mdr2 (multidrug resistance gene 2) deficiency, bleomycin-induced lung fibrosis, and UUO (unilateral urinary obstruction)-induced kidney fibrosis. On the contrary, Thy1 -/- mice are more susceptible to fibrosis, suggesting that a Msln-Thy1 signaling complex is critical for tissue fibroblast activation. A similar mechanism was observed in human activated portal fibroblasts (aPFs). Targeting of human MSLN + aPFs with two anti-MSLN immunotoxins killed fibroblasts engineered to express human mesothelin and reduced collagen deposition in livers of bile duct ligation (BDL)-injured mice. We provide evidence that antimesothelin-based therapy may be a strategy for treatment of parenchymal organ fibrosis., Competing Interests: The authors declare no competing interest.

Available therapies aimed at treating age-related osteoporosis are still insufficient. Therefore, designing reliable in vitro model for the analysis of molecular mechanisms underlying senile osteoporosis is highly required. We have isolated and characterized progenitor cells isolated from bone marrow (BMSCs) of osteoporotic mice strain SAM/P6 (BMSC SAM/P6 ). The cytophysiology of BMSC SAM/P6 was for the first time compared with BMSCs isolated from healthy BALB/c mice (BMSC BALB/c ). Characterization of the cells included evaluation of their multipotency, morphology and determination of specific phenotype. Viability of BMSCs cultures was determined in reference to apoptosis profile, metabolic activity, oxidative stress, mitochondrial membrane potential and caspase activation. Additionally, expression of relevant biomarkers was determined with RT-qPCR. Obtained results indicated that BMSC SAM/P6 and BMSC BALB/c show the typical phenotype of mesenchymal stromal cells (CD44+, CD73+, CD90+) and do not express CD45. Further, BMSC SAM/P6 were characterized by deteriorated multipotency, decreased metabolic activity and increased apoptosis occurrence, accompanied by elevated oxidative stress and mitochondria depolarisation. The transcriptome analyses showed that BMSC SAM/P6 are distinguished by lowered expression of molecules crucial for proper osteogenesis, including Coll-1, Opg and Opn. However, the expression of Trap, DANCR1 and miR-124-3p was significantly up-regulated. Obtained results show that BMSC SAM/P6 present features of progenitor cells with disturbed metabolism and could serve as appropriate model for in vitro investigation of age-dependent osteoporosis., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Intercellular communication through the secretion of soluble factors plays a vital role in a wide range of biological processes (e.g., homeostasis, immune response), yet identification and quantification of many of these factors can be challenging due to their degradation or sequestration in cell culture media prior to analysis. Here, we present a customizable bead-based system capable of simultaneously binding to live cells (through antibody-mediated cell tethering) and capturing cell-secreted molecules. Our functionalized beads capture secreted molecules (e.g., hepatocyte growth factor secreted by fibroblasts) that are diminished when sampled via traditional supernatant analysis techniques ( p < 0.05), effectively rescuing a reduced signal in the presence of neutralizing components in the cell culture media. Our system enables capture and analysis of molecules integral to chemical communication that would otherwise be markedly decreased prior to analysis.

Our understanding of mesenchymal cell subsets and their function in human lung affected by aging and in certain disease settings remains poorly described. We use a combination of flow cytometry, prospective cell-sorting strategies, confocal imaging, and modeling of microvessel formation using advanced microfluidic chip technology to characterize mesenchymal cell subtypes in human postnatal and adult lung. Tissue was obtained from patients undergoing elective surgery for congenital pulmonary airway malformations (CPAM) and other airway abnormalities including chronic obstructive pulmonary disease (COPD). In microscopically normal postnatal human lung, there was a fivefold higher mesenchymal compared with epithelial (EpCAM + ) fraction, which diminished with age. The mesenchymal fraction composed of CD90 + and CD90 + CD73 + cells was enriched in CXCL12 and platelet-derived growth factor receptor-α (PDGFRα) and located in close proximity to EpCAM + cells in the alveolar region. Surprisingly, alveolar organoids generated from EpCAM + cells supported by CD90 + subset were immature and displayed dysplastic features. In congenital lung lesions, cystic air spaces and dysplastic alveolar regions were marked with an underlying thick interstitium composed of CD90 + and CD90 + PDGFRα + cells. In postnatal lung, a subset of CD90 + cells coexpresses the pericyte marker CD146 and supports self-assembly of perfusable microvessels. CD90 + CD146 + cells from COPD patients fail to support microvessel formation due to fibrinolysis. Targeting the plasmin-plasminogen system during microvessel self-assembly prevented fibrin gel degradation, but microvessels were narrower and excessive contraction blocked perfusion. These data provide important new information regarding the immunophenotypic identity of key mesenchymal lineages and their change in a diverse setting of congenital lung lesions and COPD.

Increased T helper (Th)1/Th17 immune responses are a hallmark of Crohn's disease (CD) immunopathogenesis. CD90+ (myo-)fibroblasts (MFs) are abundant cells in the normal (N) intestinal mucosa contributing to mucosal tolerance via suppression of Th1 cell activity through cell surface membrane-bound PD-L1 (mPD-L1). CD-MFs have a decreased level of mPD-L1. Consequently, mPD-L1-mediated suppression of Th1 cells by CD-MFs is decreased, yet the mechanism responsible for the reduction in mPDL-1 is unknown. Increased expression of matrix metalloproteinases (MMPs) has been reported in CD. Herein we observed that when compared to N- and ulcerative colitis (UC)-MFs, CD-MFs increase in LPS-inducible levels of MMP-7 and -9 with a significant increase in both basal and inducible MMP-10. A similar pattern of MMP expression was observed in the CD-inflamed mucosa. Treatment of N-MFs with a combination of recombinant human MMP-7, -9 and -10 significantly decreased mPD-L1. In contrast, inhibition of MMP activity with MMP inhibitors or anti-MMP-10 neutralizing antibodies restores mPD-L1 on CD-MFs. CD-MFs demonstrated reduced capacity to suppress Th1 and Th17 responses from activated CD4+ T cells. By contrast, supplementation of the CD-MF:T-cell co-cultures with MMP inhibitors or anti-MMP neutralizing antibodies restored the CD-MF-mediated suppression. Our data suggest that (i) increased MMP-10 expression by CD-MFs and concomitant cleavage of PD-L1 from the surface of CD-MFs are likely to be one of the factors contributing to the decrease of mPD-L1-mediated suppression of Th1/Th17 cells in CD; and (ii) MMPs are likely to have a significant role in the intestinal mucosal immune responses., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mesangial proliferative glomerulonephritis (MsPGN) is an inflammatory disease, but both the nature of disease progression and its regulation remain unclear. In the present study, we monitored the course of anti-Thy1 nephritis from days 1 to 5 and established gene expression profiles at each time point using microarrays to explore the development of inflammation. According to the gene expression profiles, macrophage infiltration (triggered by CCL2 activation) was evident on day 1 and enhanced inflammation over the next few days. We screened for genes with expression levels similar to CCL2 and found that the upregulation of the circadian gene albumin D-site-binding protein (DBP) was involved in CCL2 activation in mesangial cells. More importantly, CCL2 expression showed oscillatory changes similar to DBP, and DBP induced peak CCL2 expression at 16:00 a clock on day 1 in the anti-Thy1 nephritis model. We knocked down DBP through transfection with a small interfering RNA (siRNA) and used RNA sequencing to identify the DBP-regulated TNF-α-CCL2 pathway. We performed chromatin immunoprecipitation sequencing (ChIP-Seq) and the dual luciferase assay to show that DBP bound to the TRIM55 promoter, regulating gene expression and in turn controlling the TNF-α-CCL2 pathway. In conclusion, DBP-regulated circadian CCL2 expression by the TRIM55-TNF pathway in injured mesangial cells at an early stage, which promoted macrophage recruitment and in turn triggered infiltration and inflammation in a model of anti-Thy1 nephritis.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with no effective treatment. Cancer cells, especially cancer stem cells (CSCs), redirect immune cells to evade immune surveillance and even coopt these immune cells to support their growth and metastasis. However, the identification of CSCs and how CSCs interact with immune cells in PDAC remain uncharacterized. Here, we report that CD90 is expressed on both stromal and tumor cells and that high expression of CD90 is related to a poor prognosis in patients with PDAC. The CD90 highly expressed (CD90 hi ) population in PDAC cells harbors high stemness features and tumorigenicity. Notably, CD90 acts as an anchor for monocyte/macrophage adhesion, providing a physical interaction between CD90 hi cells and monocytes/macrophages. In response, the crosstalk between CD90 hi cells and monocytes/macrophages promotes immunosuppressive features of immune cells, which enhance the stemness and epithelial-mesenchymal transition (EMT) of PDAC cells. Moreover, PD-L1 is dominantly expressed in the CD90 hi population, providing another strategy for these cells to evade immune surveillance. These findings provide an understanding of the biological significance of CD90 expression in PDAC cells and uncover a novel mechanism for how "stem-like" PDAC cells evade immune surveillance., (Copyright © 2019. Published by Elsevier B.V.)

Objective: The aim of this study was to investigate whether bone marrow stem cells (MSCs) derived exosomes in rats could promote osteoblast proliferation and improve osteoporosis via inhibiting cell apoptosis., Materials and Methods: MSCs in rats were isolated and cultured, followed by the identification of surface antigens via flow cytometry. The differentiation of MSCs was detected by alizarin red staining and oil red staining. After extraction from MSCs by ultracentrifugation, the size distribution of exosomes was detected by tunable resistive pulse sensing (TRPS). Specific antigens in MSCs-derived exosomes were determined by flow cytometry. Furthermore, the proliferation and viability of hFOB1.19 cells treated with MSCs-derived exosomes were detected by cell count kit-8 (CCK-8) assay. The effect of MSCs-derived exosomes on cell apoptosis was evaluated by flow cytometry. Protein expression levels of apoptosis-related genes in hFOB1.19 cells were detected by Western blot., Results: MSCs differentiated into osteoblasts and lipoblasts under different treatments. Meanwhile, MSCs-derived exosomes exhibited typical elongated morphology after isolation and culture for 1 and 3 days, respectively. Functionally, MSCs-derived exosomes could promote the viability of hFOB1.19 cells, and significantly increase the expression level of GLUT3. In addition, MSCs-derived exosomes remarkably downregulated apoptosis-related genes and decreased apoptosis in hFOB1.19 cells., Conclusions: MSCs-derived exosomes could promote osteoblast proliferation via inhibiting cell apoptosis, eventually improving osteoporosis.

The opportunistic fungal pathogen Candida albicans frequently causes diseases such as oropharyngeal candidiasis (OPC) in immunocompromised individuals. Although it is well appreciated that the cytokine IL-17 is crucial for protective immunity against OPC, the cellular source and the regulation of this cytokine during infection are still a matter of debate. Here, we directly visualized IL-17 production in the tongue of experimentally infected mice, thereby demonstrating that this key cytokine is expressed by three complementary subsets of CD90+ leukocytes: RAG-dependent αβ and γδ T cells, as well as RAG-independent ILCs. To determine the regulation of IL-17 production at the onset of OPC, we investigated in detail the myeloid compartment of the tongue and found a heterogeneous and dynamic mononuclear phagocyte (MNP) network in the infected tongue that consists of Zbtb46-Langerin- macrophages, Zbtb46+Langerin+ dendritic cells (DCs) and Ly6C+ inflammatory monocytes. Of those, the Langerin+ DC population stands out by its unique capacity to co-produce the cytokines IL-1β, IL-6 and IL-23, all of which promote IL-17 induction in response to C. albicans in the oral mucosa. The critical role of Langerin+ DCs for the innate IL-17 response was confirmed by depletion of this cellular subset in vivo, which compromised IL-17 induction during OPC. In conclusion, our work revealed key regulatory factors and their cellular sources of innate IL-17-dependent antifungal immunity in the oral mucosa., Competing Interests: The authors have declared that no competing interests exist.

A key feature of glomerular diseases such as crescentic glomerulonephritis and focal segmental glomerulosclerosis is the activation, migration and proliferation of parietal epithelial cells. CD44-positive activated parietal epithelial cells have been identified in proliferative cellular lesions in glomerular disease. However, it remains unknown whether CD44-positive parietal epithelial cells contribute to the pathogenesis of scarring glomerular diseases. Here, we evaluated this in experimental crescentic glomerulonephritis and the transgenic anti-Thy1.1 model for collapsing focal segmental glomerulosclerosis in CD44-deficient (cd44-/-) and wild type mice. For both models albuminuria was significantly lower in cd44-/- compared to wild type mice. The number of glomerular Ki67-positive proliferating cells was significantly reduced in cd44-/- compared to wild type mice, which was associated with a reduced number of glomerular lesions in crescentic glomerulonephritis. In collapsing focal segmental glomerulosclerosis, the extracapillary proliferative cellular lesions were smaller in cd44-/- mice, but the number of glomerular lesions was not different compared to wild type mice. For crescentic glomerulonephritis the influx of granulocytes and macrophages into the glomerulus was similar. In vitro, the growth of CD44-deficient murine parietal epithelial cells was reduced compared to wild type parietal epithelial cells, and human parietal epithelial cell migration could be inhibited using antibodies directed against CD44. Thus, CD44-positive proliferating glomerular cells, most likely parietal epithelial cells, are essential in the pathogenesis of scarring glomerular disease., (Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Purpose of Review: Stroma is a broad term referring to the connective tissue matrix in which other cells reside. It is composed of diverse cell types with functions such as extracellular matrix maintenance, blood and lymph vessel development, and effector cell recruitment. The tissue microenvironment is determined by the molecular characteristics and relative abundances of different stromal cells such as fibroblasts, endothelial cells, pericytes, and mesenchymal precursor cells. Stromal cell heterogeneity is explained by embryonic developmental lineage, stages of differentiation to other cell types, and activation states. Interaction between immune and stromal cell types is critical to wound healing, cancer, and a wide range of inflammatory diseases. Here, we review recent studies of inflammatory diseases that use functional genomics and single-cell technologies to identify and characterize stromal cell types associated with pathogenesis., Recent Findings: High dimensional strategies using mRNA sequencing, mass cytometry, and fluorescence activated cell-sorting with fresh primary tissue samples are producing detailed views of what is happening in diseased tissue in rheumatoid arthritis, inflammatory bowel disease, and cancer. Fibroblasts positive for CD90 (Thy-1) are enriched in the synovium of rheumatoid arthritis patients. Single-cell RNA-seq studies will lead to more discoveries about the stroma in the near future., Summary: Stromal cells form the microenvironment of inflamed and diseased tissues. Functional genomics is producing an increasingly detailed view of subsets of stromal cells with pathogenic functions in rheumatic diseases and cancer. Future genomics studies will discover disease mechanisms by perturbing molecular pathways with chemokines and therapies known to affect patient outcomes. Functional genomics studies with large sample sizes of patient tissues will identify patient subsets with different disease phenotypes or treatment responses.

There is a lack of effective targeted therapies for the treatment of complement dependent diseases. We developed two recombinant Fc multimers, G207 and G211, with limited ability to interact with low/moderate affinity FcγRs, but with high avidity for C1q. These drugs effectively inhibited complement dependent cytotoxicity (CDC) in vitro, and prevented the deposition of C1q, C3b and MAC, on the surface of Ab-opsonized cells. Importantly, these inhibitory effects were both C1q dependent and independent. In order to determine the biologic relevance of our findings, we evaluated the clinical efficacy of these drugs in three different animal models, acute RBC hemolysis, anti-Thy-1 nephritis and passive Heymann's nephropathy (PHN), in which disease pathophysiology relies preferentially on complement activation. While G207 was protective in the anti-Thy-1 nephritis and PHN models, G211 was protective in all of the models tested and could effectively treat PHN. In the anti-Thy-1 nephritis model, G211 prevented the characteristic histologic changes associated with the disease and limited glomerular deposition of C3. Collectively, these data suggest that "complement preferential" Fc multimers offer a novel approach to the treatment of complement mediated diseases., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Antibody-mediated crosslinking of Thy-1 [also known as cluster of differentiation (CD)90], results in a T cell receptor (TcR)‑like signal; however, the impact of Thy‑1 stimulation in comparison to TcR stimulation on T cell activation and effector function has yet to be fully elucidated. In the present study, the outcome of Thy‑1‑ and TcR‑induced stimulation of T cells was investigated in mice, using fragment crystalizable (Fc) receptor‑bound antibodies and costimulatory signals provided by syngeneic lipopolysaccharide‑matured bone marrow‑derived dendritic cells. Compared with TcR signaling, Thy‑1 signaling initiated a less robust proliferative response in T cells, as determined by tritiated‑thymidine incorporation. In addition, enzyme‑linked immunosorbent assays revealed that interleukin‑2 production was reduced, and the expression of CD25 and cyclin D3 was weaker in Thy‑1‑stimulated cells, as determined by western blotting; however, the expression of cyclin‑dependent kinase 6 was similar to that in TcR‑induced T cells. Furthermore, western blotting demonstrated that the phosphorylation of ζ-chain‑associated protein kinase 70 and extracellular signal‑regulated kinase 1/2 was delayed following Thy‑1 stimulation. DNA fragmentation assays revealed that cytotoxic effector function was also slower to develop in Thy‑1‑stimulated T cells, required more time to be effective and was largely Ca2+‑independent; these findings suggested that Fas ligand rather than granule‑associated perforin was involved in T cell effector function. In conclusion, the present results suggested that Thy‑1 signaling may contribute to the regulation of T cell homeostasis and the development of non‑specific T cell‑mediated cytotoxicity. However, further studies are required to elucidate the exact physiological roles of TcR‑like signals that result from Thy‑1 crosslinking and to investigate the molecular mechanisms that are involved.

IFN-γ is known to be predominantly produced by lymphoid cells such as certain subsets of T cells, NK cells, and other group 1 innate lymphoid cells. In this study, we used IFN-γ reporter mouse models to search for additional cells capable of secreting this cytokine. We identified a novel and rare population of nonconventional IFN-γ-producing cells of hematopoietic origin that were characterized by the expression of Thy1.2 and the lack of lymphoid, myeloid, and NK lineage markers. The expression of IFN-γ by this population was higher in the liver and lower in the spleen. Furthermore, these cells were present in mice lacking both the Rag2 and the common γ-chain (γc) genes (Rag2 -/- γc -/- ), indicating their innate nature and their γc cytokine independence. Rag2 -/- γc -/- mice are as resistant to Mycobacterium avium as Rag2 -/- mice, whereas Rag2 -/- mice lacking IFN-γ are more susceptible than either Rag2 -/- or Rag2 -/- γc -/- These lineage-negative CD45 + /Thy1.2 + cells are found within the mycobacterially induced granulomatous structure in the livers of infected Rag2 -/- γc -/- animals and are adjacent to macrophages that expressed inducible NO synthase, suggesting a potential protective role for these IFN-γ-producing cells. Accordingly, Thy1.2-specific mAb administration to infected Rag2 -/- γc -/- animals increased M. avium growth in the liver. Overall, our results demonstrate that a population of Thy1.2 + non-NK innate-like cells present in the liver expresses IFN-γ and can confer protection against M. avium infection in immunocompromised mice., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Cardiosphere-derived cells (CDCs) have been shown to reduce scar size and increase viable myocardium in human patients with mild/moderate myocardial infarction. Studies in rodent models suggest that CDC therapy may confer therapeutic benefits in patients with non-ischaemic dilated cardiomyopathy (DCM). We sought to determine the safety and efficacy of allogeneic CDC in a large animal (canine) model of spontaneous DCM. Canine CDCs (cCDCs) were grown from a donor dog heart. Similar to human CDCs, cCDCs express CD105 and are slightly positive for c-kit and CD90. Thirty million of allogeneic cCDCs was infused into the coronary vessels of Doberman pinscher dogs with spontaneous DCM. Adverse events were closely monitored, and cardiac functions were measured by echocardiography. No adverse events occurred during and after cell infusion. Histology on dog hearts (after natural death) revealed no sign of immune rejection from the transplanted cells., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Aim: To investigate the use of thermosensitive magnetoliposomes (TMs) loaded with magnetic iron oxide (Fe3O4) and the anti-cancer stem cell marker CD90 (CD90@TMs) to target and kill CD90+ liver cancer stem cells (LCSCs)., Methods: The hepatocellular carcinoma cell line Huh7 was used to separate CD90+ LCSCs by magnetic-activated cell sorting. CD90@TMs was characterized and their ability to target CD90+ LCSCs was determined. Experiments were used to investigate whether CD90@TMs combined with magnetic hyperthermia could effectively eliminate CD90+ LCSCs., Results: The present study demonstrated that CD90+ LCSCs with stem cells properties were successfully isolated. We also successfully prepared CD90@TMs that was almost spherical and uniform with an average diameter of 130±4.6 nm and determined that magnetic iron oxide could be incorporated and retained a superparamagnetic response. CD90@TMs showed good targeting and increased inhibition of CD90+ LCSCs in vitro and in vivo compared to TMs., Conclusions: CD90@TMs can be used for controlled and targeted delivery of anticancer drugs, which may offer a promising alternative for HCC therapy., Competing Interests: The author reports no conflicts of interest in this work.

Pancreatic neuroendocrine tumors (PanNETs) are a type of pancreatic cancer with limited therapeutic options. Consequently, most patients with advanced disease die from tumor progression. Current evidence indicates that a subset of cancer cells is responsible for tumor development, metastasis, and recurrence, and targeting these tumor-initiating cells is necessary to eradicate tumors. However, tumor-initiating cells and the biological processes that promote pathogenesis remain largely uncharacterized in PanNETs. Here we profile primary and metastatic tumors from an index patient and demonstrate that MET proto-oncogene activation is important for tumor growth in PanNET xenograft models. We identify a highly tumorigenic cell population within several independent surgically acquired PanNETs characterized by increased cell-surface protein CD90 expression and aldehyde dehydrogenase A1 (ALDHA1) activity, and provide in vitro and in vivo evidence for their stem-like properties. We performed proteomic profiling of 332 antigens in two cell lines and four primary tumors, and showed that CD47, a cell-surface protein that acts as a "don't eat me" signal co-opted by cancers to evade innate immune surveillance, is ubiquitously expressed. Moreover, CD47 coexpresses with MET and is enriched in CD90(hi)cells. Furthermore, blocking CD47 signaling promotes engulfment of tumor cells by macrophages in vitro and inhibits xenograft tumor growth, prevents metastases, and prolongs survival in vivo.

IL-6 is a pleiotropic cytokine increased in CRC and known to directly promote tumor growth. Colonic myofibroblasts/fibroblasts (CMFs or stromal cells) are CD90(+) innate immune cells representing up to 30% of normal colonic mucosal lamina propria cells. They are expanded in CRC tumor stroma, where they also known as a cancer associated fibroblasts (CAFs). Cells of mesenchymal origin, such as normal myofibroblasts/fibroblasts, are known to secrete IL-6; however, their contribution to the increase in IL-6 in CRC and to tumor-promoting inflammation is not well defined. Using in situ, ex vivo and coculture analyses we have demonstrated that the number of IL-6 producing CMFs is increased in CRC (C-CMFs) and they represent the major source of IL-6 in T2-T3 CRC tumors. Activity/expression of stem cell markers-aldehyde dehydrogenase and LGR5- was significantly up-regulated in colon cancer cells (SW480, Caco-2 or HT29) cultured in the presence of conditioned medium from tumor isolated C-CMFs in an IL-6 dependent manner. C-CMF and its derived condition medium, but not normal CMF isolated from syngeneic normal colons, induced differentiation of tumor promoting inflammatory T helper 17 cells (Th17) cell responses in an IL-6 dependent manner. Our study suggests that CD90(+) fibroblasts/myofibroblasts may be the major source of IL-6 in T2-T3 CRC tumors, which supports the stemness of tumor cells and induces an immune adaptive inflammatory response (a.k.a. Th17) favoring tumor growth. Taken together our data supports the notion that IL-6 producing CAFs (a.k.a. C-CMFs) may provide a useful target for treating or preventing CRCs., (© 2015 UICC.)

Recent studies show that spermatogonial stem cells (SSCs) are able to colonize and form mature spermatozoa following transplantation into germ cell depleted testes of recipient males. Therefore, efficient ways for enrichment and gene transfer into SSCs provides a powerful tool for production of transgenic animals. In order to adapt the technique to goats, three issues were addressed: (i) enrichment of the undifferentiated spermatogonia including SSCs using magnetic activated cell sorting (MACS), (ii) lentiviral vector-mediated transduction of an enhanced green fluorescent protein (EGFP) transgene into enriched cells, and (iii) transplantation of transduced undifferentiated spermatogonia into the germ cell depleted testes of immune-suppressed mice to assess for migration and colony formation ability. Enriched cells were transduced by lentiviral vectors and subsequently analyzed for expression of THY1, PLZF, VASA, UCHL1 and BCL6B genes. Cells were also analyzed for GFP and PLZF by flow cytometry. Enriched transduced cells were transplanted into germ cell depleted mice testis. Quantitative analysis of transcripts revealed that MACS-enrichment significantly increased the expression of SSC-characteristic genes THY1, PLZF, VASA, UCHL1 and BCL6B compared to non-enriched population (P≤0.05). EGFP transduction did not affect the expression levels of SSC-characteristic genes. Flow cytometry revealed that 72% of transduced-enriched cells were positive for EGFP. Finally, transduced-enriched goat SSCs could colonize within the cells into the seminiferous tubules of germ cell depleted recipient mice at higher frequency than non-enriched cells. The results indicated that enrichment of goat undifferentiated spermatogonia by magnetic-activated cell sorting for THY1 antibody combined with lentiviral vector-mediated transduction has the potential to be used for production of transgenic goats., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cluster of Differentiation 90 (CD90, Thy-1) has been proposed as one of the most important biomarkers in several cancer cells including cancer stem cells (CSCs). CD90 is considered as a potential normal stem cell and CSCs biomarker and also has been identified in lung cancer stem cells, hepatocellular carcinoma cells and high-grade gliomas. Using eukaryotic host systems involves complex procedures and frequently results in low protein yields. The expression of recombinant proteins in Escherichia coli is comparatively easier than eukaryotic host cells. The potential of large scale production of recombinant protein has made this system an economic production platform. In this study we expressed the extra-membrane domain of human CD90 (exCD90) antigen (Gln15-Cys130) in E. coli expression host cells. The epitope integrity of purified recombinant antigen was confirmed by antibody-antigen interaction using 5E10 anti-CD90 monoclonal antibody and binding study through ELISA and florescent staining of CD90(+) cells in a flow cytometry experiment., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Physiologically low O(2) tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0-5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O(2) tensions (3-10%). We hypothesized that O(2) concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34(+) -enriched cells was studied in co-culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O(2). 2% O(2) resulted in a significantly lower CD34(+) cell expansion (25-fold vs 60-, 64- and 92-fold at day 10 for 5%, 21%, 10% O(2), respectively). In turn, 10% O(2) promoted the highest CD34(+) CD90(+) cell expansion, reaching 22 ± 5.4- vs 5.6 ± 2.4- and 5.7 ± 2.0-fold for 2%, 5% and 21% O(2), respectively, after 14 days. Similar differentiation patterns were observed under different O(2) tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O(2). Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O(2) when compared to higher O(2) concentrations (5-21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O(2), can maximize the ex vivo expansion of UCB stem/progenitor cells in co-culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications., (Copyright © 2013 John Wiley & Sons, Ltd.)

HIV virus-like particles (VLPs) present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC) resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV) + monophosphoryl lipid A (MPLA) at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively), which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively). In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8+ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4+ and CD8+ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses.

Human adult dental pulp tissue is a source of adult stem cells that have a potential to differentiate into various tissues, although the primary cell suspensions cultured from pulp tissue are mixtures of both stem cell and nonstem cell populations with heterogeneous phenotypes and various differentiation efficiencies. Therefore, cell surface protein markers on dental pulp stem cells are critical for detection and purification of stem cell populations. Yet, little is known about the cell surface molecules that are specifically associated with the undifferentiated and progenitor state of human adult dental pulp stem cells (hDPSCs). Presently, cell surface proteins expressed on hDPSCs were assessed by screening surface molecules specifically expressed on dentinogenic progenitors. Using a decoy immunization strategy, a set of monoclonal antibodies (MAbs) was generated against undifferentiated pulp progenitor cells. Forty-five hybridomas produced MAbs that interacted weakly, if at all, to differentiated pulp cells. Of these, 19 MAbs (18 IgG, 1 IgM) recognized surface molecules on undifferentiated hDPSCs. By multicolor flow cytometric analysis, 40%-60% of newly identified MAb-positive cells were demonstrated to be positive for the CD44 and CD90 mesenchymal markers. When MAb-positive cells were sorted from the heterogeneous pulp cell suspension, mineralization efficiency was increased three to five times compared with MAb-negative cells. The results suggest that the decoy immunization is an efficient method for isolation of MAbs against dentinogenic progenitors. These MAbs will be helpful for identification and enrichment of hDPSCs for efficient dentin regeneration.

Natural antibodies produced by CD5+ B1 B cells include anti-thymocyte autoantibody (ATA). Transgenic mice bearing the Ig-μ heavy chain of a prototypic ATA, V(H)3609Vκ21c, demonstrated a critical requirement for self-antigen in the accumulation of ATA B cells and production of high levels of serum ATA. Further work with ATA-μκ transgenic mice revealed that, while development of most B cells were blocked at an immature stage in spleen, some mature ATA B cells were present. ATA-μκ transgenic mice with varying levels of Thy-1 autoantigen showed a clear relationship between BCR crosslinking and B cell fate, with low levels generating marginal zone ATA B cells and complete antigen absence allowing maturation to follicular ATA B cells. Finally, different fates of developing ATA B cells encountering high levels self-antigen may be accounted for by variations in the response of newly formed B cells arising from foetal and adult development.

Anti-Thy1 glomerulonephritis is a rat nephritis model closely simulating human mesangial proliferative glomerulonephritis. It affects primarily the mesangium, yet displays substantial proteinuria during the course. This study investigated the molecular signals underlying proteinuria in this disease and the modulation of which by the known antiproteinuric agent, pentoxifylline. Male Wistar rats were randomly divided into a control group and nephritic groups with or without treatment with IMD-0354 (an IκB kinase inhibitor), SB431542 (an activin receptor-like kinase inhibitor) or pentoxifylline. Kidney sections were prepared for histological examinations. Glomeruli were isolated for mRNA and protein analysis. Urine samples were collected for protein and nephrin quantitation. One day after nephritis induction, proteinuria developed together with ultrastructural changes of the podocyte and downregulation of podocyte mRNA and protein expression. These were associated with upregulation of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β/activins mRNAs and activation of nuclear factor (NF)-κB p65 and Smad2/3. IMD-0354 attenuated proteinuria on d 1, whereas SB431542 decreased proteinuria on d 3 and 5, in association with partial restoration of downregulated podocyte mRNA and protein expression. Pentoxifylline attenuated proteinuria and nephrinuria through the course, plus inhibition of p-NF-κB p65 (d 1) and p-Smad2/3 (d 5) and partial reversal of downregulated podocyte mRNA and protein. Our data show that the pathogenesis of proteinuria in anti-Thy1 glomerulonephritis involves TNF-α and TGF-β/activin pathways, and the evolution of this process can be attenuated by pentoxifylline via downregulation of NF-κB and Smad signals and restoration of the podocyte component of the glomerular filtration barrier.

The skin has to effectively combat external attacks, while maintaining skin immune homeostasis under steady-state conditions. To fulfill these challenging tasks, the dermis harbors a variety of heterogeneous cell types that are able to suppress T-cell proliferation similar to bone marrow mesenchymal stromal cells. Here we show that plastic-adherent, human dermal cells induce FoxP3 expression in TCR-complex-stimulated CD25(-)CD4(+)CD45RA(+) T cells in the absence of CD28 co-ligation in a cell-contact-dependent manner. These FoxP3(+) T cells reveal an effective suppressive capacity in vitro. Moreover, we found that the vast majority of CD90(+) dermal cells are perivascularly located and generate a significantly higher percentage of regulatory T cells compared with cells expressing markers such as CD271 in vitro. Importantly, we further demonstrate that plastic-adherent dermal cells are also able to differentiate toward the endothelial lineage. Our data show that human skin harbors specific cell types with immunosuppressive potential, which are located in close vicinity to their likely operational area and provide evidence for a CD28-independent regulatory mechanism. Further, the differentiation potential into endothelial cells suggests the existence of a tissue-resident cell pool for vessel regeneration. These findings might have important implications for the clinical use of allogeneic dermal cells to rebuild an imbalanced human skin immune homeostasis.

Intraepithelial lymphocytes (IELs) play an important role in maintaining the physiology of the small intestine. The majority of mouse IELs express CD8αα and are either γδ or αβ T cells. Although the development and homing of CD8αα IELs have been studied in some detail, the factors controlling their homeostasis and positioning are incompletely understood. Here we demonstrate that G protein-coupled receptor 18 (GPR18) is abundantly expressed in CD8αα IELs and that mice lacking this orphan receptor have reduced numbers of γδT IELs. Mixed bone marrow chimera experiments reveal a markedly reduced contribution of GPR18-deficient cells to the CD8αα IEL compartment and a reduction in the CD8αβ T cell subset. These defects could be rescued by transduction with a GPR18-expressing retrovirus. The GPR18-deficient γδT IELs that remained in mixed chimeras had elevated Thy1, and there were less granzyme B(+) and Vγ7(+) cells, indicating a greater reduction in effector-type cells. Flow cytometric analysis indicated GPR18 deficiency more strongly affected the CD8αα cells in the intraepithelial compared with the adjacent lamina propria compartment. These findings establish a requirement for GPR18 in CD8αα and CD8αβ IELs, and we suggest the receptor has a role in augmenting the accumulation of CD8 T cells in the intraepithelial versus lamina propria compartment., (© 2014 Wang et al.)

Prevention of sexual acquisition of HIV in women requires a substantial increase in our knowledge about HIV-target cell availability and regulation in the female reproductive tract (FRT). In this study, we analyzed the phenotype and susceptibility to HIV infection of CD4(+) T cell in the endometrium (EM), endocervix (END), and ectocervix (ECT) of the FRT. We found that T helper type 17 (Th17) cells represent a major subset in FRT tissues analyzed and that Th17 cells were the main CD4(+) T-cell population expressing C-C motif chemokine receptor 5 (CCR5) and CD90. In premenopausal women, CD4(+) T cells and Th17 cells, in particular, were significantly lower in EM relative to END and ECT. Th17 cells were elevated in EM from postmenopausal women relative to premenopausal tissues but not changed in END and ECT. Susceptibility of CD4(+) T cells to HIV infection measured as intracellular p24 was lowest in the EM and highest in the ECT. Additionally, we found that Th17 cells co-expressing CCR5 and CD90 were the most susceptible to HIV infection. Our results provide valuable information for designing preventive strategies directed at targeting highly susceptible target cells in the FRT.

Signaling via programmed death ligand-1 (PD-L1) and PD-L2 is crucial for maintaining peripheral tolerance. CD90(+) myofibroblasts/fibroblasts (CMFs) are major programmed cell death-1 (PD-1) ligand-expressing cells in normal human colonic mucosa. CMFs suppress activated CD4(+) T cell proliferation via PD-1 ligands. It is not known whether signaling through TLRs contribute to the regulation PD-1 ligands on CMFs upon colonic mucosal tolerance. In this study, we demonstrated that stimulation of TLR4 on human CMFs upregulates PD-L1, but not PD-L2, and reinforces CMF-mediated suppression of CD4(+) T cell proliferation and IFN-γ production. TLR4-mediated upregulation of PD-L1 on CMFs involved NF-κB pathways and was JAK2 and MyD88 dependent. MyD88-dependent stimulation of TLR1/2 and TLR5 also upregulated PD-L1 expression on CMFs in culture. PD-L1 expression was drastically decreased in vivo in the colonic mucosa of mice devoid of MyD88. Induction of MyD88 deficiency in CMFs in fibroblast-specific MyD88 conditional knockout mice resulted in a strong increase in a mucosal IFN-γ expression concomitantly with the abrogation of PD-L1 expression in CMFs under homeostasis and epithelial injury induced by dextran sodium sulfate. Together, these data suggest that MyD88-dependent TLR stimulation of CMFs in the normal colonic mucosa may reinforce these cells' anti-inflammatory capacity and thus contribute to the maintenance of mucosal tolerance., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Natural killer (NK) cell development relies on signals provided from the bone marrow (BM) microenvironment. It is thought that lymphotoxin (LT) α1β2 expressed by the NK cell lineage interacts with BM stromal cells to promote NK cell development. However, we now report that a small number of RORγt(+) innate lymphoid cells (ILCs), and not CD3(-)NK1.1(+) cells, express LT to drive NK development. Similar to LT(-/-) or RORγt(-/-) mice, the mice conditionally lacking LTα1β2 on RORγt(+) ILCs experience a developmental arrest at the immature NK stages, between stages of NK development to the mature NK cell stage. This developmental block results in a functional deficiency in the clearance of NK-sensitive tumor cells. Reconstitution of Thy1(+) ILCs from BM or purified RORγt(+) ILCs from lamina propria lymphocytes into LT-deficient RORγt(+) BM cultures rescues NK cell development. These data highlight a previously undiscovered role of RORγt(+) ILCs for NK cell development and define LT from ILCs as an essential molecule for the stromal microenvironment supporting NK cell development., (© 2014 Kim et al.)

Epigenetic therapies, including DNA methyltransferase and histone deacetylase (HDAC) inhibitors, are increasingly being considered to treat hematological malignancies, but their effects on normal hematopoietic stem cells (HSCs) remain largely unexplored. We compared the effects of several HDAC inhibitors, including valproic acid (VPA) and trichostatin A (TSA), alone or in combination with 5-aza-2'-deoxycytidine (5azaD) on the expansion of HSCs. VPA induced the highest expansion of CD34+CD90+ cells and progenitor cells compared with other HDAC inhibitors or the sequential addition of 5azaD/TSA in culture. Xenotransplantation studies demonstrated that VPA prevents HSC loss, whereas 5azaD/TSA treatment leads to a net expansion of HSCs that retain serial transplantation ability. 5azaD/TSA-mediated HSC expansion was associated with increased histone acetylation and transient DNA demethylation, which corresponded with higher gene transcript levels. However, some genes with increased transcript levels lacked changes in methylation. Importantly, a global microarray analysis revealed a set of differentially expressed genes in 5azaD/TSA- and VPA-expanded CD34+ cells that might be involved in the expansion and maintenance of transplantable HSCs, respectively. In summary, our data indicate that treatment of HSCs with different chromatin-modifying agents results in either the expansion or maintenance of HSCs, an observation of potential therapeutic importance., (Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Mesenchymal stem cell (MSC) transplantation has the potential for organ repair. Nevertheless, some factors might lessen the regenerative potential of MSCs, e.g. donor age or systemic disease. It is thus important to carefully assess the patient's suitability for autologous MSC transplantation. Here we investigated the effects of chronic kidney disease (CKD) on MSC function. We isolated bone marrow MSCs from remnant kidney rats (RK) with CKD (CKD-RK-MSC) and found signs of premature senescence: spontaneous adipogenesis, reduced proliferation capacity, active senescence-associated-β-galactosidase, accumulation of actin and a modulated secretion profile. The functionality of CKD-RK-MSCs in vivo was tested in rats with acute anti-Thy1.1-nephritis, where healthy MSCs have been shown to be beneficial. Rats received healthy MSCs, CKD-RK-MSC or medium by injection into the left renal artery. Kidneys receiving healthy MSCs exhibited accelerated healing of glomerular lesions, whereas CKD-RK-MSC or medium exerted no benefit. The negative influence of advanced CKD/uremia on MSCs was confirmed in a second model of CKD, adenine nephropathy (AD). MSCs from rats with adenine nephropathy (CKD-AD-MSC) also exhibited cellular modifications and functional deficits in vivo. We conclude that CKD leads to a sustained loss of in vitro and in vivo functionality in MSCs, possibly due to premature cellular senescence. Considering autologous MSC therapy in human renal disease, studies identifying uremia-associated mechanisms that account for altered MSC function are urgently needed.

Cytoglobin (CYGB) is ubiquitously expressed in the cytoplasm of fibroblastic cells in many organs, including hepatic stellate cells. As yet, there is no specific marker with which to distinguish stellate cells from myofibroblasts in the human liver. To investigate whether CYGB can be utilized to distinguish hepatic stellate cells from myofibroblasts in normal and fibrotic human liver, human liver tissues damaged by infection with hepatitis C virus (HCV) and at different stages of fibrosis were obtained by liver biopsy. Immunohistochemistry was performed on histological sections of liver tissues using antibodies against CYGB, cellular retinol-binding protein-1 (CRBP-1), α-smooth muscle actin (α-SMA), thymocyte differentiation antigen 1 (Thy-1), and fibulin-2 (FBLN2). CYGB- and CRBP-1-positive cells were counted around fibrotic portal tracts in histological sections of the samples. The expression of several of the proteins listed above was examined in cultured mouse stellate cells. Quiescent stellate cells, but not portal myofibroblasts, expressed both CYGB and CRBP-1 in normal livers. In fibrotic and cirrhotic livers, stellate cells expressed both CYGB and α-SMA, whereas myofibroblasts around the portal vein expressed α-SMA, Thy-1, and FBLN2, but not CYGB. Development of the fibrotic stage was positively correlated with increases in Sirius red-stained, α-SMA-positive, and Thy-1-positive areas, whereas the number of CYGB- and CRBP-1-positive cells decreased with fibrosis development. Primary cultured mouse stellate cells expressed cytoplasmic CYGB at day 1, whereas they began to express α-SMA at the cellular margins at day 4. Thy-1 was undetectable throughout the culture period. In human liver tissues, quiescent stellate cells are CYGB positive. When activated, they also become α-SMA positive; however, they are negative for Thy-1 and FBLN2. Thus, CYGB is a useful marker with which to distinguish stellate cells from portal myofibroblasts in the damaged human liver.

Autoimmune uveitis is characterized by crossing of blood-retinal barrier (BRB) by autoaggressive immune cells. Equine recurrent uveitis (ERU) is a valuable spontaneous model for autoimmune uveitis and analyses of differentially expressed proteins in ERU unraveled changed protein clusters in target tissues and immune system. Healthy eyes are devoid of leukocytes. In ERU, however, leukocytes enter the inner eye and subsequently destroy it. Molecular mechanisms enabling cell migration through BRB still remain elusive. Previously, we detected decreased talin 1 expression in blood-derived granulocytes of ERU cases, linking the innate immune system to ERU. Because changes in leukocyte protein expression pattern may play a role in pathological abnormalities leading to migration ability, we aimed at identifying interactors of talin 1 in leukocytes with immunoprecipitation, followed by LC-MS/MS for candidate identification. This enabled us to identify CD90 (Thy1) as novel interactor of talin 1 besides several other interactors. In blood-derived granulocytes from healthy individuals, CD90 was highly abundant and significantly reduced in ERU, especially in effector cells. Connection between talin 1 and CD90 and their expression differences in inflammation is an interesting novel finding allowing deeper insight into immune response of innate immune system and granulocyte migration ability in this organ-specific autoimmune disease.

Background: Chronic progressive mesangioproliferative nephropathy represents a major cause of end-stage renal disease worldwide. Until now, effective approaches to stop or even slow its progression are limited. We tested the effects of an inhibitor of PDGF receptor, abl and c-kit tyrosine kinases, Imatinib, in a chronic progressive model of mesangioproliferative glomerulosclerosis., Methods: Anti-thy1 glomerulosclerosis was induced by injection of anti-thy1 antibody into uninephrectomized Wistar rats. One week after disease induction, according to the degree of proteinuria, animals were stratified and assigned to chronic glomerulosclerosis (cGS) and cGS plus Imatinib (10 mg/kg body weight/day). In week 20, renoprotective actions of Imatinib were analyzed by a set of functional, histological and molecular biological parameters., Results: Untreated cGS rats showed elevation of systolic blood pressure and marked progression in proteinuria, renal fibrosis, cell infiltration, cell proliferation and function lost. Administration of Imatinib went along significantly with lower systolic blood pressure (-10 mmHg) and proteinuria (-33%). Imatinib administration was paralled by significant reductions in tubulointerstitial accumulation of matrix proteins (-44%), collagen I deposition (-86%), expression of TGF-beta1 (-30%), production of fibronectin (-23%), myofibroblast differentiation (-87%), macrophage infiltration (-36%) and cell proliferation (-45%), respectively. In comparison with untreated cGS animals, Imatinib therapy lowered also blood creatinine (-41%) and blood urea concentrations (-36%) and improved creatinine clearance (+25%). Glomerular fibrotic changes were lowered moderately by Imatinib., Conclusions: Therapy with Imatinib limits the progressive course of chronic anti-thy1 glomerulosclerosis towards tubulointerstitial fibrosis and renal insufficiency. This was paralleled by direct and indirect sign of TGF-β1 and PDGF inhibition. The findings suggest that the pharmacological principal of inhibition of tyrosine kinases with drugs such as Imatinib might serve as approach for limiting progression of human mesangioproliferative glomerulosclerosis.

CD8(+) T cells are responsible for killing cells of the body that have become infected or oncogenically transformed. In order to do so, effector CD8(+) T cells must recognize their cognate antigenic peptide bound to a MHC class I molecule that has been directly presented by the target cell. Due to the rapid nature of antigen presentation, it is believed that antigenic peptides are derived from a subset of newly synthesized proteins which are degraded almost immediately following synthesis and termed Defective Ribosomal Products or DRiPs. We have recently reported on a bioassay which can distinguish antigen presentation of DRiP substrates from other forms of rapidly degraded proteins and found that poly-ubiquitin chain disassembly may be necessary for efficient DRiP presentation. The AAA ATPase p97 protein is necessary for efficient cross-presentation of antigens on MHC class I molecules and plays an important role in extracting mis-folded proteins from the endoplasmic reticulum. Here, we find that genetic ablation or chemical inhibition of p97 does not diminish DRiP antigen presentation to any great extent nor does it alter the levels of MHC class I molecules on the cell surface, despite our observations that p97 inhibition increased the levels of poly-ubiquitinated proteins in the cell. These data demonstrate that inhibiting poly-ubiquitin chain disassembly alone is insufficient to abolish DRiP presentation.

Although a substantial number of T cells may escape depletion following in vivo mAb treatment in patients undergoing immunosuppression, their specific tissue location and phenotypic characteristics in different peripheral lymphoid tissues have not been analyzed in detail. Here we investigated the survival of CD4(+) T cells immediately following anti-Thy-1 mAb treatment in mice. We found a preferential survival of CD4(+) T cells expressing Thy-1 antigen in the Peyer's patches (PP) and also in mesenteric lymph nodes (MLN), where the relative majority of the surviving CD4(+) T cells displayed CD44(high)/CD62L(-) phenotype corresponding to effector memory T-cell features. These CD4(+) T cells also expressed CXCR5 and PD-1 (programmed cell death-1) markers characteristic for follicular Th cells (TFH). We also demonstrate that the immediate survival of these cells does not involve proliferation and is independent of IL-7. Induction of germinal center formation in spleen enhanced while the dissolution of follicular architecture by lymphotoxin-β receptor antagonist treatment slightly reduced TFH survival. Our results thus raise the possibility that the follicles within PP and MLN may create natural support niches for the preferential survival of TFH cells of the memory phenotype, thus allowing their escape during T-cell depletion.

Chondrocytes express a number of cell-surface molecules that mediate cell-cell or cell-matrix interactions. Identification and full characterization of new chondrocyte surface molecules will lead to a better understanding of the function of the chondrocyte. Researchers used primary human chondrocytes as an immunogen, and various monoclonal antibodies (MAbs) were generated using standard hybridoma technology. A monoclonal antibody named 5D2 was selected for further characterization. The antigen recognized by 5D2 MAb is expressed by primary human chondrocytes, primary synovial fibroblasts, synovial fibroblast cell lines (SW982), primary skin fibroblasts, and osteoblasts, but not expressed in blood cells. Biochemical analysis revealed that the 5D2 antigen is a protein with a molecular weight of approximately 25-35 kDa. Protein identification by mass spectrometry and molecular cloning revealed that 5D2 antigen is identical to the Thy-1 molecule. Furthermore we confirmed this specificity of the antibody by the isolated and cloned Thy-1 gene to the COS-7 and probed it with the 5D2 antibody using Western blot analysis. We examined the role of the Thy-1 molecule in arthritis models and tissue; one was papain-induced rat arthritis, the other was immunohistological staining of osteoarthritic (OA) human articular cartilage. OA cartilage showed a higher expression of Thy-1 as compared with normal tissue in all experimental approaches. The in vitro studies showed that the inflammatory cytokine interleukin-1β up-regulated Thy-1 molecule expression in the cartilage tissue. It can be concluded that the Thy-1 might be a potential biomarker for cartilage pathogenesis, degradation, and metabolic turnover.

Thymocyte differentiation antigen-1 (Thy-1) is a glycosylphosphatidylinositol (GPI)-linked cell surface glycoprotein expressed on numerous cell types, which regulates signals affecting cell adhesion, migration, differentiation, and survival. In addition, Thy-1 has been detected in the serum, cerebral spinal fluid, wound fluid from venous ulcers, synovial fluid from joints in rheumatoid arthritis, and, more recently, urine. We previously detected Thy-1 in the conditioned media of cytokine-stimulated lung fibroblasts, suggesting that Thy-1 shedding may be a response to cellular stress. Soluble and membrane-bound forms of Thy-1 from in vivo sources have been shown to be identical in size when deglycosylated, suggesting that soluble Thy-1 is separated from the diacyl glycerol portion of its GPI anchor by hydrolysis within the GPI moiety. For Thy-1- and other GPI-anchored proteins, delipidation induces a stable change in conformation that manifests itself in a change in antibody affinity for soluble forms. Using epitope-tagged recombinant soluble Thy-1, we report that widely available monoclonal antibodies to human Thy-1 are unable to detect soluble Thy-1 by immunoblotting. We re-evaluated the Thy-1 that we previously reported in the conditioned media of normal human lung fibroblasts and found it to be entirely insoluble. These findings suggest that most Thy-1 reported in body fluids retains its GPI anchor and may be associated with membrane fragments or vesicles. This phenomenon should be considered in the generation of antibodies and controls for Thy-1 bioassays. Furthermore, the changes in Thy-1 conformation with delipidation, beyond affecting antibody affinity, likely affect the ligand affinity and biological function of soluble vs released membrane-associated forms.

Human hematopoietic stem cells reside in the CD34+CD38-CD90+ population in cord blood and bone marrow. However, this cell fraction is heterogeneous, and the phenotype of the rare primitive stem cells remains poorly defined. We here report that primitive cord blood CD34+CD38-CD90+ stem cells, with the ability to reconstitute NOD/SCID-IL2Rγ(c) null (NSG) mice long-term, at 24 weeks after transplantation, can be prospectively isolated at an increased purity by using integrin α2 receptor as an additional stem cell marker. Using a limiting dilution transplantation assay, we found a highly significant enrichment of multilineage reconstituting stem cells in the CD34+CD38-CD90+ cell fraction expressing the integrin α2 receptor, with a frequency of 1/29 cells, as compared to a frequency of 1/157 in the corresponding integrin α2- cells. In line with this, long-term reconstituting stem cells within the cord blood CD34+CD38- cell population were significantly enriched in the integrin α2+ fraction, while stem cells and progenitors reconstituting short-term, at 8-12 weeks, were heterogeneous in integrin α2 expression. Global gene expression profiling revealed that the lineage-marker negative (Lin-) CD34+CD38-CD90+CD45RA- integrin α2+ cell population was molecularly distinct from the integrin α2- cell population and the more mature Lin-CD34+CD38-CD90-CD45RA- cell population. Our findings identify integrin α2 as a novel stem cell marker, which improves prospective isolation of the primitive human hematopoietic stem cells within the CD34+CD38-CD90+ cell population for experimental and therapeutic stem cell applications., (Copyright © 2012 AlphaMed Press.)

We have shown that the decrease in oxygen tension in the culture medium of multipotent mesenchymal stromal cells (MMSCs) results in a short-term reduction in the proportion of CD73(+)-cells in the population, without effecting the number of cells expressing other constitutive surface markers (CD90 and CD105). In this case, the heterogeneity of the cell population declined: large spread cells disappeared. The proliferative activity of MMSCs significantly increased and remained stable in conditions in which the oxygen content was close to the tissue oxygen levels (5% O2). At lower oxygen concentration, proliferative activity of the cells gradually reduced from passages 3-4. The increase in proliferative activity was not accompanied by increased expression of telomerase gene indicateding the alsance of cell transformation. However, genome-wide analysis of MMSC gene expression level revealed changes in expression of cyclins (CCND2 and PCNA), regulatory subunit cyclin-dependent kinase (CKS2) and an inhibitor of cyclin-dependent kinase (CDKN2C), regulating the cell cycle, which is obviously facilitated the increase in the proliferative capacity of cells at lower oxygen tension.

Gastric cancer is associated with chronic inflammation and Helicobacter pylori infection. Th17 cells are CD4(+) T cells associated with infections and inflammation; but their role and mechanism of induction during carcinogenesis is not understood. Gastric myofibroblasts/fibroblasts (GMF) are abundant class II MHC expressing cells that act as novel antigen presenting cells. Here we have demonstrated the accumulation of Th17 in H. pylori-infected human tissues and in the gastric tumor microenvironment. GMF isolated from human gastric cancer and H. pylori infected tissues co-cultured with CD4(+) T cells induced substantially higher levels of Th17 than GMF from normal tissues in an IL-6, TGF-β, and IL-21 dependent manner. Th17 required interaction with class II MHC on GMF for activation and proliferation. These studies suggest that Th17 are induced during both H. pylori infection and gastric cancer in the inflammatory milieu of gastric stroma and may be an important link between inflammation and carcinogenesis.

Glycosylphosphatidylinositol-anchored protein Thy-1 is abundantly expressed on the cell surface of neurons and T lymphocytes in rodents. Although Thy-1 is known to bind integrins as a ligand and to mediate neurite outgrowth and immune responses, its precise function is not fully understood. Previously we produced several anti rat Thy-1 monoclonal antibodies and identified one, 2E11, which induces PC12 cell neurite outgrowth. Here we screened antibodies that inhibit 2E11-induced neurite outgrowth and stimulate or inhibit rat thymocyte aggregation. Since Thy-1 lacks an intracellular region, it requires other membrane-bound molecules for the signal transduction. Hence these antibodies are hypothesized to play key roles in the interaction between Thy-1 and signaling molecules. To elucidate the mechanisms of antibody-induced Thy-1 functions, antibody characterization and epitope determination were carried out. Thy-1 cleavage and mutation revealed that the antibodies recognize not only amino acid sequences, but also the three-dimensional structures consisting of immunoglobulin-like domains. Two antibodies were suggested to bind spatially close to the integrin binding site and crosslink Thy-1 molecules, while a third antibody is believed to inhibit Thy-1 crosslinking and subsequent Thy-1 signaling. The antibodies reported here may therefore function as crosslinkers, agonists, or antagonists that modify Thy-1 signaling.

Background: Previous studies have demonstrated that angiotensin Type I receptor blockade (ARB) reduces proteinuria, reverses glomerular injury and glomerulosclerosis in rat models of diabetic nephropathy and glomerulonephritis. However, the cellular and molecular mechanisms are unclear. To investigate the role of cells of the bone marrow (BM) in glomerular repair seen during ARB administration, we induced progressive glomerulosclerosis in enhanced green fluorescent protein BM chimeric rats by a single injection of anti-Thy 1.1 monoclonal antibody, followed by unilateral nephrectomy., Methods: Cohorts of rats received valsartan or no treatment from Week 2 to Week 8 after induction of disease. Renal function, urinary protein excretion and histological changes were examined 8 weeks after anti-Thy-1.1 monoclonal antibody injection., Results: Valsartan administration improved renal function, reduced severity of glomerulosclrosis and markedly reduced mortality. Valsartan administration promoted regeneration of the glomerular tuft, lowered proteinuria and resulted in enhanced vascular endothelial growth factor (VEGF) expression in the cortex and glomerular tuft. In addition, valsartan promoted increased recruitment of BM-derived cells (BMDCs) many of which expressed VEGF and likely contributed directly to glomerular repair. Nearly all BMDCs recruited to the glomerulus expressed the monocyte/macrophage marker CD68., Conclusions: In conclusion, the data shows that ARB by valsartan prevents glomerulosclerosis progression by enhancing glomerular capillary repair which is associated with the recruitment of VEGF producing 'reparative' monocytes and macrophages from the BM.

Eph receptor tyrosine kinases and their ligands (ephrins) have a pivotal role in the homeostasis of many adult organs and are widely expressed in the kidney. Glomerular diseases beginning with mesangiolysis can recover, with podocytes having a critical role in this healing process. We studied here the role of Eph signaling in glomerular disease recovery following mesangiolytic Thy1.1 nephritis in rats. EphB4 and ephrinBs were expressed in healthy glomerular podocytes and were upregulated during Thy1.1 nephritis, with EphB4 strongly phosphorylated around day 9. Treatment with NPV-BHG712, an inhibitor of EphB4 phosphorylation, did not cause glomerular changes in control animals. Nephritic animals treated with vehicle did not have morphological evidence of podocyte injury or loss; however, application of this inhibitor to nephritic rats induced glomerular microaneurysms, podocyte damage, and loss. Prolonged NPV-BHG712 treatment resulted in increased albuminuria and dysregulated mesangial recovery. Additionally, NPV-BHG712 inhibited capillary repair by intussusceptive angiogenesis (an alternative to sprouting angiogenesis), indicating a previously unrecognized role of podocytes in regulating intussusceptive vessel splitting. Thus, our results identify EphB4 signaling as a pathway allowing podocytes to survive transient capillary collapse during glomerular disease.