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Astrogliosis is a process by which astrocytes, when exposed to inflammation, exhibit hypertrophy, motility, and elevated expression of reactivity markers such as Glial Fibrillar Acidic Protein, Vimentin, and Connexin43. Since 1999, our laboratory in Chile has been studying molecular signaling pathways associated with "gliosis" and has reported that reactive astrocytes upregulate Syndecan 4 and αVβ3 Integrin, which are receptors for the neuronal glycoprotein Thy-1. Thy-1 engagement stimulates adhesion and migration of reactive astrocytes and induces neurons to retract neurites, thus hindering neuronal network repair. Reportedly, we have used DITNC1 astrocytes and neuron-like CAD cells to study signaling mechanisms activated by the Syndecan 4–αVβ3 Integrin/Thy-1 interaction. Importantly, the sole overexpression of β3 Integrin in non-reactive astrocytes turns them into reactive cells. In vitro, extensive passaging is a simile for "aging", and aged fibroblasts have shown β3 Integrin upregulation. However, it is not known if astrocytes upregulate β3 Integrin after successive cell passages. Here, we hypothesized that astrocytes undergoing long-term passaging increase β3 Integrin expression levels and behave as reactive astrocytes without needing pro-inflammatory stimuli. We used DITNC1 cells with different passage numbers to study reactivity markers using immunoblots, immunofluorescence, and astrocyte adhesion/migration assays. We also evaluated β3 Integrin levels by immunoblot and flow cytometry, as well as the neurotoxic effects of reactive astrocytes. Serial cell passaging mimicked the effects of inflammatory stimuli, inducing astrocyte reactivity. Indeed, in response to Thy-1, β3 Integrin levels, as well as cell adhesion and migration, gradually increased with multiple passages. Importantly, these long-lived astrocytes expressed and secreted factors that inhibited neurite outgrowth and caused neuronal death, just like reactive astrocytes in culture. Therefore, we describe two DITNC1 cell types: a non-reactive type that can be activated with Tumor Necrosis Factor (TNF) and another one that exhibits reactive astrocyte features even in the absence of TNF treatment. Our results emphasize the importance of passage numbers in cell behavior. Likewise, we compare the pro-inflammatory stimulus versus long-term in-plate passaging of cell cultures and introduce them as astrocyte models to study the reactivity process. [ABSTRACT FROM AUTHOR]

Background and Aims: Steatotic liver disease (SLD) is generally considered to represent a hepatic manifestation of metabolic syndrome and includes a disease spectrum comprising isolated steatosis, metabolic dysfunction‐associated steatohepatitis, liver fibrosis and ultimately cirrhosis. A better understanding of the detailed underlying pathogenic mechanisms of this transition is crucial for the design of new and efficient therapeutic interventions. Thymocyte differentiation antigen (Thy‐1, also known as CD90) expression on fibroblasts controls central functions relevant to fibrogenesis, including proliferation, apoptosis, cytokine responsiveness, and myofibroblast differentiation. Methods: The impact of Thy‐1 on the development of SLD and progression to fibrosis was investigated in high‐fat diet (HFD)‐induced SLD wild‐type and Thy‐1‐deficient mice. In addition, the serum soluble Thy‐1 (sThy‐1) concentration was analysed in patients with metabolic dysfunction‐associated SLD stratified according to steatosis, inflammation, or liver fibrosis using noninvasive markers. Results: We demonstrated that Thy‐1 attenuates the development of fatty liver and the expression of profibrogenic genes in the livers of HFD‐induced SLD mice. Mechanistically, Thy‐1 directly inhibits the profibrotic activation of nonparenchymal liver cells. In addition, Thy‐1 prevents palmitic acid‐mediated amplification of the inflammatory response of myeloid cells, which might indirectly contribute to the pronounced development of liver fibrosis in Thy‐1‐deficient mice. Serum analysis of patients with metabolically associated steatotic liver disease syndrome revealed that sThy‐1 expression is correlated with liver fibrosis status, as assessed by liver stiffness, the Fib4 score, and the NAFLD fibrosis score. Conclusion: Our data strongly suggest that Thy‐1 may function as a fibrosis‐protective factor in mouse and human SLD. [ABSTRACT FROM AUTHOR]

Objectives: Thy‐1 (CD90) is a glycosylphosphatidyl‐anchored protein belonging to the immunoglobulin family and is known to control mesenchymal stromal cells differentiation into osteoblasts or adipocytes. The study aimed to investigate the salivary levels of Thy‐1 in health, periodontitis, obesity, and any potential association. Materials and Methods: Seventy‐one participants were divided into four groups: healthy (H), subjects with periodontitis (P), obese individuals (O), and obese individuals having periodontitis (PO). Unstimulated whole saliva was collected from participants who were evaluated for periodontal parameters. The levels of Thy‐1 were measured with a commercially available ELISA kit. The data were statistically analyzed. Results: A significant difference in salivary Thy‐1 levels among different groups was observed. Periodontitis patients had the maximum, and obese individuals had the minimum Thy‐1 levels. Significant differences between H and P, H and PO, P and O, and O and PO were observed. Overall correlations between Thy‐1 and periodontal parameters and a positive correlation with pocket depth in group PO were noted. Conclusion: Thy‐1 could be detected in the saliva of all study participants. It is implied that a local inflammatory condition like periodontitis elevates the salivary levels of Thy‐1 with, and without obesity. [ABSTRACT FROM AUTHOR]

Micro/nano topological modification is critical for improving the in vivo behaviors of bone implants, regulating multiple cellular functions. Titania (TiO2) nanotubes show the capacity of promoting osteoblast-related cell differentiation and induce effective osseointegration, serving as a model material for studying the effects of micro/nano-topological modifications on cells. However, the intracellular signaling pathways by which TiO2 nanotubes regulate the osteogenic differentiation of stem cells are not fully defined. Thy-1 (CD90), a cell surface glycoprotein anchored by glycosylphosphatidylinositol, has been considered a key molecule in osteoblast differentiation in recent years. Nevertheless, whether the micro/nano topology of the implant surface leads to changes in Thy-1 is unknown, as well as whether these changes promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Here, TiO2 nanotubes of various diameters were prepared by adjusting the anodizing voltage. qPCR and immunoblot were carried out to assess the mechanism by which TiO2 nanotubes regulate Thy-1. The results revealed Ti plates harboring TiO2 nanotubes ∼100-nm diameter (TNT-100) markedly upregulated Thy-1. Subsequently, upregulated Thy-1 promoted the activation of Fyn/RhoA/MLC Ⅱ/F-actin axis, which enhanced the nuclear translocation of YAP. After Thy-1 knockdown by siRNA, the Fyn/RhoA/MLC Ⅱ/F-actin axis was significantly inhibited and TiO2 nanotubes showed decreased effects on osteogenic differentiation. Therefore, Thy-1 upregulation might be a major mechanism by which micro/nano-topological modification of TiO2 nanotubes promotes osteogenic differentiation in BMSCs. This study provides novel insights into the molecular mechanism of TiO2 nanotubes, which may help design improved bone implants for clinical application.

This document is an editorial titled "In celebration of women in cell adhesion and migration" that highlights women's contributions to the field of cell adhesion and migration. The editorial discusses a research topic consisting of six articles covering various areas such as wound healing, integrin adhesome, mechanosensing and mechanotransduction, and Leishmania infection. The authors emphasize the importance of understanding the physical principles underlying these cellular processes and the role of the microenvironment. Two specific articles are mentioned, one focusing on matricellular proteins and tissue healing, and the other exploring the role of the integrin receptor Thy-1. The text provides insights into the functions and interactions of these proteins and their impact on cellular processes. The editorial also discusses other research articles on topics such as mechanosensing, bone mineralization, breast cancer cells, and Leishmania infection. The importance of integrating mechanobiology into cell biology and molecular biology research is highlighted, as it can enhance our understanding of physiological processes and pathological developments. The author acknowledges the contributors to the research topic and declares no conflicts of interest. [Extracted from the article]

Gingival mesenchymal stem cells (GMSCs) are newly developed seed cells for tissue engineering owing to their easy isolation, abundance and high growth rates. Thy‐1 is an important regulatory molecule in the differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the function of Thy‐1 in the osteogenic differentiation of GMSCs by reducing the expression of Thy‐1 using a lentivirus. The results demonstrated that Thy‐1 knockdown promoted the osteogenic differentiation of GMSCs in vitro. Validation by RNA‐seq revealed an obvious decrease in Vcam1 and Sox9 gene expression with Thy‐1 knockdown. Kyoto Encyclopedia of Genes and Genomes pathway analysis suggested that the differentially expressed genes were enriched in the Wnt signalling pathway. We further demonstrated that Thy‐1 knockdown promoted osteogenic differentiation of GMSCs by activating the Wnt/β‐catenin signalling pathway. Therefore, Thy‐1 has a key regulatory role in the differentiation of GMSCs and maybe a core molecule connecting transcription factors related to the differentiation of MSCs. Our study also highlighted the potential of Thy‐1 to modify MSCs, which may help improve their use in tissue engineering. [ABSTRACT FROM AUTHOR]

Abstract Background Rapidly expanding clones (RECs) are one of the single-cell-derived mesenchymal stem cell clones sorted from human bone marrow mononuclear cells (BMMCs), which possess advantageous features. The RECs exhibit long-lasting proliferation potency that allows more than 10 repeated serial passages in vitro, considerably benefiting the manufacturing process of allogenic MSC-based therapeutic products. Although RECs aid the preparation of large-variation clone libraries for a greedy selection of better-quality clones, such a selection is only possible by establishing multiple-candidate cell banks for quality comparisons. Thus, there is a high demand for a novel method that can predict “low-risk and high-potency clones” early and in a feasible manner given the excessive cost and effort required to maintain such an establishment. Methods LNGFR and Thy-1 co-positive cells from BMMCs were single-cell-sorted into 96-well plates, and only fast-growing clones that reached confluency in 2 weeks were picked up and passaged as RECs. Fifteen RECs were prepared as passage 3 (P3) cryostock as the primary cell bank. From this cryostock, RECs were passaged until their proliferation limitation; their serial-passage limitation numbers were labeled as serial-passage potencies. At the P1 stage, phase-contrast microscopic images were obtained over 6–90 h to identify time-course changes of 24 morphological descriptors describing cell population information. Machine learning models were constructed using the morphological descriptors for predicting serial-passage potencies. The time window and field-of-view-number effects were evaluated to identify the most efficient image data usage condition for realizing high-performance serial-passage potency models. Results Serial-passage test results indicated variations of 7–13-repeated serial-passage potencies within RECs. Such potency values were predicted quantitatively with high performance (RMSE

We demonstrated previously that the hypothalamic supraoptic nucleus (SON) undergoes an axonal sprouting response following a unilateral lesion of the hypothalamo-neurohypophysial tract in a 35-day-old rat to repopulate the partially denervated neural lobe (NL). However, no sprouting occurs following the same injury in a 125-day-old rat. We previously reported a significant increase in Thy-1 protein in the SON of a 125-day-old rat compared to a 35-day-old rat in the absence of injury. Thy-1 is a cell surface glycoprotein shown to inhibit axonal outgrowth following injury; however, we did not look at axotomy's effect on Thy-1 in the SON. Therefore, we sought to determine the integrin ligands that bind Thy-1 in the SON and how axotomy impacts Thy-1. Like what others have shown, the co-immunoprecipitation analysis demonstrated that Thy-1 interacts with αvß3 and αvß5 integrin dimers in the SON. We used western blot analysis to examine protein levels of Thy-1 and integrin subunits following injury in the 35- and 125-day-old rat SON and NL. Our results demonstrated that Thy-1 protein levels increase in the lesion SON in a 35-day-old rat. The quantitative dual-fluorescent analysis showed that the increase in Thy-1 in the lesion SON occurred in astrocytes. There was no change in Thy-1 or integrin protein levels following injury in the 125-day-old following injury. Furthermore, the axotomy significantly decreased Thy-1 protein levels in the NL of both 35- and 125-day-old rats. These results provide evidence that Thy-1 protein levels are injury dependent in the magnocellular neurosecretory system. [ABSTRACT FROM AUTHOR]

Kidney fibrosis is a major culprit in the development and progression of chronic kidney disease (CKD), ultimately leading to the irreversible loss of organ function. Thymocyte differentiation antigen-1 (Thy-1) controls many core functions of fibroblasts relevant to fibrogenesis but is also found in a soluble form (sThy-1) in serum and urine. We investigated the association of sThy-1 with clinical parameters in patients with CKD receiving hemodialysis treatment compared to individuals with a preserved renal function. Furthermore, Thy-1 tissue expression was detected in a mouse model of diabetic CKD (eNOS−/−; db/db) and non-diabetic control mice (eNOS−/−). Serum and urinary sThy-1 concentrations significantly increased with deteriorating renal function, independent of the presence of diabetes. Serum creatinine is the major, independent, and inverse predictor of serum sThy-1 levels. Moreover, sThy-1 is not only predicted by markers of renal function but is also itself an independent and strong predictor of markers of renal function, i.e., serum creatinine. Mice with severe diabetic CKD show increased Thy-1 mRNA and protein expression in the kidney compared to control animals, as well as elevated urinary sThy-1 levels. Pro-fibrotic mediators, such as interleukin (IL)-4, IL-13, IL-6 and transforming growth factor β, increase Thy-1 gene expression and release of sThy-1 from fibroblasts. Our data underline the role of Thy-1 in the control of kidney fibrosis in CKD and raise the opportunity that Thy-1 may function as a renal antifibrotic factor. [ABSTRACT FROM AUTHOR]

Long-term survival of lung transplants lags behind other solid organs due to early onset of a fibrotic form of chronic rejection known as chronic lung allograft dysfunction (CLAD). Preventing CLAD is difficult as multiple immunologic and physiologic insults contribute to its development. Targeting fibroblast activation, which is the final common pathway leading to CLAD, offers the opportunity to ameliorate fibrosis irrespective of the initiating insult. Thy-1 is a surface glycoprotein that controls fibroblast differentiation and activation. To study the role of Thy-1 in CLAD, we utilized the minor antigen mismatched C57BL/6 (B6wild-type) or B6Thy-1-/−→C57BL/10 (B10) model of murine orthotopic lung transplantation with postoperative bacterial infection modeled by intratracheal lipopolysaccharide (LPS) administration. The effects of LPS on Thy-1 expression, proliferation, and gene expression were assessed in fibroblasts in vitro and the therapeutic potential of Thy-1 replacement was assessed in vivo. More severe CLAD was evident in B6Thy-1-/- →B10 grafts compared to B6wild-type →B10 grafts. LPS further accentuated fibrosis in B6wild-type →B10 grafts with some, but limited, effects on B6Thy-1-/- →B10 grafts. LPS contributed to Thy-1 loss from Thy-1(+) fibroblasts in vitro due to a decrease in mRNA expression. In addition, LPS promoted proliferation and upregulation of multiple inflammatory pathways in Thy-1(–) fibroblasts by gene expression analysis. Most importantly, replacement of Thy-1 through exogenous administration ameliorated the fibrotic phenotype post-LPS mediated modeling of infection. Our findings suggest that the loss of Thy-1 on fibroblasts is a previously unrecognized cause of CLAD and its replacement may offer therapeutic applications for amelioration of this disease post-transplantation in the setting of infectious stress responses. [ABSTRACT FROM AUTHOR]

Objective: Iatrogenic laryngotracheal stenosis (iLTS) is the pathologic narrowing of the glottis, subglottis, and/or trachea secondary to intubation or tracheostomy related injury. Patients with type 2 diabetes mellitus (T2DM) are more likely to develop iLTS. To date, the metabolomics and phenotypic expression of cell markers in fibroblasts derived from patients with T2DM and iLTS are largely unknown. Study Design: Controlled in vitro cohort study. Setting: Tertiary referral center (2017-2020). Methods: This in vitro study assessed samples from 6 patients with iLTS who underwent surgery at a single institution. Fibroblasts were isolated from biopsy specimens of laryngotracheal scar and normal-appearing trachea and compared with controls obtained from the trachea of rapid autopsy specimens. Patients with iLTS were subcategorized into those with and without T2DM. Metabolic substrates were identified by mass spectrometry, and cell protein expression was measured by flow cytometry. Results: T2DM iLTS-scar fibroblasts had a metabolically distinct profile and clustered tightly on a Pearson correlation heat map as compared with non-T2DM iLTS-scar fibroblasts. Levels of itaconate were elevated in T2DM iLTS-scar fibroblasts. Flow cytometry demonstrated that T2DM iLTS-scar fibroblasts were associated with higher CD90 expression (Thy-1; mean, 95%) when compared with non-T2DM iLTS-scar (mean, 83.6%; P =.0109) or normal tracheal fibroblasts (mean, 81.1%; P =.0042). Conclusions: Scar-derived fibroblasts from patients with T2DM and iLTS have a metabolically distinct profile. These fibroblasts are characterized by an increase in itaconate, a metabolite related to immune-induced scar remodeling, and can be identified by elevated expression of CD90 (Thy-1) in vitro. [ABSTRACT FROM AUTHOR]

Acute skin wound healing is a multistage process consisting of a plethora of tightly regulated signaling events in specialized cells. The Thy-1 (CD90) glycoprotein interacts with integrins and the heparan sulfate proteoglycan syndecan 4, generating a trimolecular complex that triggers bi-directional signaling to regulate diverse aspects of the wound healing process. These proteins can act either as ligands or receptors, and they are critical for the successful progression of wound healing. The expression of Thy-1, integrins, and syndecan 4 is controlled during the healing process, and the lack of expression of any of these proteins results in delayed wound healing. Here, we review and discuss the roles and regulatory events along the stages of wound healing that support the relevance of Thy-1, integrins, and syndecan 4 as crucial regulators of skin wound healing.

Abstract Background Ovarian cancer is the leading cause of gynecologic cancer death in the United States despite effective first-line systemic chemotherapy. Cancer stem cells (CSCs) retain the ability to self-renew and proliferate and may be a means of harboring disease that evades standard treatment strategies. We previously performed a high-throughput screen to assess differential protein expression in ovarian CSCs compared to non-CSCs and observed that Thy-1 was more highly expressed in CSCs. Our primary aim was to validate Thy-1 (CD90) as a cancer stem cell (CSC) marker in epithelial ovarian cancer (EOC), correlate with clinical outcomes, and assess as a potential therapeutic target. Results Kaplan Meier (KM) Plotter data were correlated with survival outcomes. Quantitative real-time PCR, flow cytometry, and immunoblots assessed RNA and protein expression. Limiting dilution assays assessed self-renewal capacity and proliferation assays assessed proliferative capacity. RNA in-situ hybridization was performed on patient specimens to assess feasibility. Thy-1 (CD90) is more highly expressed in ovarian CSCs than non-CSCs, in EOC compared to benign ovarian epithelium (P

The pathogenesis of Parkinson's disease involves fibrillization and deposition of alpha-synuclein (α-syn) into Lewy bodies. Accumulating evidence suggests that α-syn oligomers are particularly neurotoxic. Transgenic (tg) mice overexpressing wild-type human α-syn under the Thy-1 promoter (L61) reproduce many Parkinson's disease features, but the pathogenetic relevance of α-syn oligomers in this mouse model has not been studied in detail. Here, we report an age progressive increase of α-syn oligomers in the brain of L61 tg mice. Interestingly, more profound motor symptoms were observed in animals with higher levels of membrane-bound oligomers. As this tg model is X-linked, we also performed subset analyses, indicating that both sexes display a similar age-related increase in α-syn oligomers. However, compared with females, males featured increased brain levels of oligomers from an earlier age, in addition to a more severe behavioral phenotype with hyperactivity and thigmotaxis in the open field test. Taken together, our data indicate that α-syn oligomers are central to the development of brain pathology and behavioral deficits in the L61 tg α-syn mouse model. [Display omitted] • Age-related increase of oligomeric α-syn-levels in L61 mice was observed. • Males displayed increased levels of oligomeric α-syn earlier than females. • Oligomeric α-syn levels were higher in animals with severe motor impairments. [ABSTRACT FROM AUTHOR]

Thy-1 is a cell surface protein that is expressed during the differentiation of retinal ganglion cells (RGCs). Optic nerve injury induces progressive loss in the number of RGCs expressing Thy-1. The rate of this loss is fastest during the first week after optic nerve injury and slower in subsequent weeks. This study was undertaken to determine whether oral treatment with a water-soluble N-hydroxy-2,2,6,6-tetramethylpiperidine derivative (OT-440) protects against loss of Thy-1 promoter activation following optic nerve crush and whether this effect targets the earlier quick phase or the later slow phase. The retina of mice expressing cyan fluorescent protein under control of the Thy-1 promoter (Thy1-CFP mice) was imaged using a blue-light confocal scanning laser ophthalmoscope (bCSLO). These mice then received oral OT-440 prepared in cream cheese or dissolved in water, or plain vehicle, for two weeks and were imaged again prior to unilateral optic nerve crush. Treatments and weekly imaging continued for four more weeks. Fluorescent neurons were counted in the same defined retinal areas imaged at each time point in a masked fashion. When the counts at each time point were directly compared, the numbers of fluorescent cells at each time point were greater in the animals that received OT-440 in cream cheese by 8%, 27%, 52% and 60% than in corresponding control animals at 1, 2, 3 and 4 weeks after optic nerve crush. Similar results were obtained when the vehicle was water. Rate analysis indicated the protective effect of OT-440 was greatest during the first two weeks and was maintained in the second two weeks after crush for both the cream cheese vehicle study and water vehicle study. Because most of the fluorescent cells detected by bCSLO are RGCs, these findings suggest that oral OT-440 can either protect against or delay early degenerative responses occurring in RGCs following optic nerve injury.

PurposeThis study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells.MethodsDifferentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimensional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method.ResultsMitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35+/-14% on day 2, but reduced expression by 36+/-4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death.ConclusionsElevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria.

Nerve transfer is the most common treatment for total brachial plexus avulsion injury. After nerve transfer, the movement of the injured limb may be activated by certain movements of the healthy limb at the early stage of recovery, i.e., trans-hemispheric reorganization. Previous studies have focused on functional magnetic resonance imaging and changes in brain-derived neurotrophic factor and growth associated protein 43, but there have been no proteomics studies. In this study, we designed a rat model of total brachial plexus avulsion injury involving contralateral C7 nerve transfer. Isobaric tags for relative and absolute quantitation and western blot assay were then used to screen differentially expressed proteins in bilateral motor cortices. We found that most differentially expressed proteins in both cortices of upper limb were associated with nervous system development and function (including neuron differentiation and development, axonogenesis, and guidance), microtubule and cytoskeleton organization, synapse plasticity, and transmission of nerve impulses. Two key differentially expressed proteins, neurofilament light (NFL) and Thy-1, were identified. In contralateral cortex, the NFL level was upregulated 2 weeks after transfer and downregulated at 1 and 5 months. The Thy-1 level was upregulated from 1 to 5 months. In the affected cortex, the NFL level increased gradually from 1 to 5 months. Western blot results of key differentially expressed proteins were consistent with the proteomic findings. These results indicate that NFL and Thy-1 play an important role in trans-hemispheric organization following total brachial plexus root avulsion and contralateral C7 nerve transfer.

CD90 is a membrane GPI-anchored protein with one Ig V-type superfamily domain that was initially described in mouse T cells. Besides the specific expression pattern and functions of CD90 that were described in normal tissues, i.e., neurons, fibroblasts and T cells, increasing evidences are currently highlighting the possible involvement of CD90 in cancer. This review first provides a brief overview on CD90 gene, mRNA and protein features and then describes the established links between CD90 and cancer. Finally, we report newly uncovered functional connections between CD90 and endoplasmic reticulum (ER) stress signaling and discuss their potential impact on cancer development.

Objective To investigate the expression of CD90 in human intrahepatic cholangiocellular carcinoma and its association with lymph node metastasis and poor prognosis. Methods A total of 49 samples of intrahepatic cholangiocellular carcinoma tissue and 49 samples of adjacent tissue were collected after surgical resection in The Affiliated Hospital of Chengde Medical College from March 2013 to March 2016, and 40 normal liver tissue samples were selected as control. Immunohistochemistry was used to measure the expression of CD90 in each sample, and its association with clinical indices, survival, and prognosis of patients with intrahepatic cholangiocellular carcinoma was analyzed. The t-test was used for comparison of continuous data between two groups; the chi-square test was used for comparison of categorical data between two groups, the Kruskal-Wallis H test was used for comparison between three groups and further comparison between two groups. The Kaplan-Meier curve was used to analyze postoperative recurrence and survival, and the log-rank test was used for comparison. Results The moderately positive expression rate of CD90 was 65.31% in intrahepatic cholangiocellular carcinoma tissue, 30.61% in adjacent normal tissue, and 0% in normal liver tissue, suggesting that the intrahepatic cholangiocellular carcinoma tissue had a significantly higher moderately positive expression rate of CD90 than adjacent normal tissue and normal liver tissue (P＜0.05). The expression of CD90 was associated with TNM stage and hilar lymph node metastasis in the patients with intrahepatic cholangiocellular carcinoma (χ2=12.837 and 17.824, both P＜0.001). The patients with negative/weakly positive expression of CD90 had significantly higher relapse-free survival rate and overall survival rate after surgery than those with moderately/strongly positive expression (χ2=3.845 and 4.152, P=0.025 and 0.021). Conclusion CD90 is highly expressed in human intrahepatic cholangiocellular carcinoma tissue, which is associated with hilar lymph node metastasis and poor prognosis and plays an important role in tumor development, progression, and metastasis. [ABSTRACT FROM AUTHOR]

Background: Alcohol exposure induces TGFβ1 and renders the lung susceptible to injury and disrepair. We determined that TGFβ1 regulates myofibroblast differentiation through the loss of Thy‐1 expression and consequent induction of α‐SMA. TGFβ1 is important for T helper 17 (Th17) differentiation and IL‐17 secretion, which in turn participates in tissue repair. We hypothesized that alcohol induces Th17 differentiation via TGFβ1 and that IL‐17 produced by these cells contributes to the development of profibrotic lung myofibroblasts. Methods: Primary lung fibroblasts (PLFs) were treated with alcohol, TGFβ1, and IL‐17 and then analyzed for Thy‐1 expression and cell morphology. Naïve and Th17‐polarized CD4+ T cells were exposed to alcohol and assessed for IL‐17 expression. CD4+ T cells from alcohol‐fed mice were analyzed for Th17 and IL‐17 expression. Lungs of control‐fed, bleomycin‐treated and alcohol‐fed, bleomycin‐treated mice were analyzed for IL‐17 protein expression. Results: Alcohol‐treated PLFs expressed lower levels of Thy‐1 than untreated cells. TGFβ1 or IL‐17 exposure suppressed PLF Thy‐1 expression. When administered together, TGFβ1 and IL‐17 additively down‐regulated Thy‐1 expression. Exposure of naïve and Th17‐polarized CD4+ T cells to alcohol induced the Th17 phenotype and augmented their production of IL‐17. CD4+ Th17+ levels are elevated in the peripheral compartment but not in the lungs of alcohol‐fed animals. Treatment of the PLFs with IL‐17 and alcohol induced α‐SMA expression. Induction of α‐SMA and myofibroblast morphology by IL‐17 occurred selectively in a Thy‐1− fibroblast subpopulation. Chronic alcohol ingestion augmented lung‐specific IL‐17 expression following bleomycin‐induced lung injury. Conclusions: Alcohol exposure skews T cells toward a Th17 immune response that in turn primes the lung for fibroproliferative disrepair through loss of Thy‐1 expression and induction of myofibroblast differentiation. These effects suggest that IL‐17 and TGFβ1 contribute to fibroproliferative disrepair in the lung and targeting these proteins could limit morbidity and mortality following lung injury in alcoholic individuals. [ABSTRACT FROM AUTHOR]

Thy-1 is a small membrane glycoprotein and member of the immunoglobulin superfamily of cell adhesion molecules. It is abundantly expressed in many cell types including neurons and is anchored to the outer membrane leaflet via a glycosyl phosphatidylinositol tail. Thy-1 displays a number of interesting properties such as fast lateral diffusion, which allows it to get in and out of membrane nanodomains with different lipid composition. Thy-1 displays a broad expression in different cell types and plays confirmed roles in cell development, adhesion and differentiation. Here, we explored the functions of Thy-1 in neuronal signaling, initiated by extracellular binding of αVβ3 integrin, may strongly dependent on the lipid content of the cell membrane. Also, we assort literature suggesting the association of Thy-1 with specific components of lipid rafts such as sialic acid containing glycosphingolipids, called gangliosides. Furthermore, we argue that Thy-1 positioning in nanodomains may be influenced by gangliosides. We propose that the traditional conception of Thy-1 localization in rafts should be reconsidered and evaluated in detail based on the potential diversity of neuronal nanodomains.

Thy-1 was discovered over 50 years ago, and in that time investigators from a broad variety of fields have described numerous and heterogeneous biological functions of Thy-1 in multiple contexts. As an outwardly facing cell surface molecule, it is well positioned to receive extracellular signals; previously reviewed studies have confirmed an important role in cell-cell and cell-matrix adhesion, cell migration, and regulation of outside-in signaling. More recent studies reviewed here expand the repertoire of Thy-1 effects on signaling pathways, and reveal novel roles in mechanotransduction, cellular differentiation, viral entry, and extracellular vesicle binding and internalization. All of these studies contribute to understanding Thy-1 as a context-dependent integrator of a diverse range of extracellular information, and provide impetus for further studies, some of which are suggested here.

The glycosylphosphatidylinositol (GPI) anchored glycoprotein Thy-1 has been prevalently expressed on the surface of various cell types. The biological function of Thy-1 ranges from T cell activation, cell adhesion, neurite growth, differentiation, metastasis and fibrogenesis and has been extensively reviewed elsewhere. However, current discoveries implicate Thy-1 also functions as a key mechanotransduction mediator. In this review, we will be focusing on the role of Thy-1 in translating extracellular mechanic cues into intracellular biological cascades. The mechanotransduction capability of Thy-1 relies on trans and cis interaction between Thy-1 and RGD-binding integrins; and will be discussed in depth in the review.

Thy-1 (CD90) is a glycosylphosphatidylinositol-anchored protein (GPI-AP) with signaling properties that is abundant on mouse T cells. Upon antibody-mediated crosslinking, Thy-1 provides a T cell receptor (TcR)-like signal that is sufficient to drive CD4+ T cell proliferation and differentiation into effector cells when costimulatory signals are provided by syngeneic lipopolysaccharide-matured bone marrow-derived dendritic cells. In this study, we investigated the impact of Thy-1 signaling on the production of the T helper (Th) cell subset-associated cytokines, interferon (IFN) γ, interleukin (IL)-4 and IL-17A, as well as the in vitro polarization of highly purified resting CD4+ T cells into Th1, Th2, and Th17 cells. Although CD8+ T cells expressed more Thy-1 than CD4+ T cells, both T cell populations were equally responsive to Thy-1 stimulation. In contrast to TcR stimulation of CD3+ T cells, which favored IFNγ and IL-4 production, Thy-1 signaling favored IL-17 synthesis, indicating a previously unidentified difference between the consequences of Thy-1 and TcR signal transduction. Moreover, Thy-1 signaling preferentially induced the Th17-associated transcription factor RORγt in CD4+ T cells. As with TcR signaling, Thy-1 stimulation of CD4+ T cells under the appropriate polarizing conditions resulted in Th1, Th2 or Th17 cell induction; however, Thy-1 stimulation induced nearly 7- and 2-fold more IL-4 and IL-17A, respectively, but only slightly more IFNγ. The ability to provide a TcR-like signal capable of promoting T helper cell differentiation and cytokine synthesis was not common to all GPI-APs since cross-linking of Ly6A/E with mitogenic mAb did not promote substantial production of IFNγ, IL-4 or IL-17, although there was a substantial proliferative response. The preferential induction of RORγt and Th17 cytokine synthesis as a consequence of Thy-1 signaling suggests a default T helper cell response that may enhance host defense against extracellular pathogens.

Healthy bone remodeling results from a balanced bone formation and bone resorption realized by bone-forming osteoblasts and bone-resorbing osteoclasts, respectively. Recently, Thy-1 (CD90) was identified as positive regulator of osteoblast differentiation and activation, thus, promoting bone formation while concurrently inhibiting adipogenesis and obesity in mice. Additionally, Thy-1 did not affect bone resorption. An obesity-related co-morbidity that is increasing in prevalence is a disturbed bone formation resulting in an increased fracture risk. The underlying mechanisms of obesity-induced bone alterations are not yet fully elucidated and therefore therapy options for efficient bone-anabolic treatments are limited. Therefore, we investigated the impact of Thy-1 on bone metabolism under obese conditions. Indeed, high fat diet (HFD) induced obese mice lacking Thy-1 (Thy-1−/−) showed increased body fat mass compared to wildtype (WT) mice while bone mass (−38%) and formation (−57%) were decreased as shown by micro-computed tomography (μCT) measurement, histological analysis, and fourier-transform infrared spectroscopy (FTIR). Interestingly, under obese conditions, lack of Thy-1 affected both osteoblast and osteoclast function. Number (−30%) and activity of osteoblasts were decreased in obese Thy-1−/− mice while osteoclast number (+39%) and activity were increased. Facilitated bone marrow fat accumulation (+56%) in obese Thy-1−/− mice compared to obese WT mice was associated with upregulated tumor necrosis factor α (Tnfα, +46%) and colony stimulating factor 1 receptor (Csf1r) expression, strong promoters of osteoclast differentiation. Moreover, lack of Thy-1 was accompanied by a reduction of osteoprotegerin (Tnfrsf11b) expression (−36%), an inhibitor of osteoclast differentiation. Altered Tnfα, Csf1r, and Tnfrsf11b expression might be responsible for elevated osteoclast activity in obese Thy-1-deficient mice. In summary, our findings show that lack of Thy-1 promotes obesity under HFD conditions while concurrently decreasing bone mass and formation. Mechanistic studies revealed that under obese conditions lack of Thy-1 impairs both bone formation and bone resorption.

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. [ABSTRACT FROM AUTHOR]

Our previous reports indicate that ligand-induced α V β 3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca 2 + to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether α V β 3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported α V β 3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca 2 + , hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an α V β 3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca 2 + entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release. [ABSTRACT FROM AUTHOR]

Acute respiratory distress syndrome (ARDS) is a common clinical disorder characterized by pulmonary edema leading to acute lung damage and arterial hypoxemia. Pulmonary fibrosis is a progressive, fibrotic lung disorder, whose pathogenesis in ARDS remains speculative. LincRNA-p21 was a novel regulator of cell proliferation, apoptosis and DNA damage response. This study aims to investigate the effects and mechanism of lincRNA-p21 on pulmonary fibrosis in ARDS. Purified 10 mg/kg LPS was dropped into airways of C57BL/6 mice. Expression levels of lincRNA-p21 and Thy-1 were measured by real-time PCR or western blotting. Proliferation of lung fibroblasts was analyzed by BrdU incorporation assay. Lung and BAL collagen contents were estimated using colorimetric Sircol assay. LincRNA-p21 expression was time-dependently increased and Thy-1 expression was time-dependently reduced in a mouse model of ARDS and in LPS-treated lung fibroblasts. Meanwhile, lung fibroblast proliferation was also time-dependently elevated in LPS-treated lung fibroblasts. In addition, lung fibroblast proliferation could be promoted by lincRNA-p21 overexpression and LPS treatment, however, the elevated lung fibroblast proliferation was further abrogated by Thy-1 overexpression or lincRNA-p21 interference. And Thy-1 interference could elevate cell viability of lung fibroblasts and rescue the reduction of lung fibroblast proliferation induced by lincRNA-p21 interference. Moreover, lincRNA-p21 overexpression dramatically inhibited acetylation of H3 and H4 at the Thy-1 promoter and Thy-1 expression levels in HLF1 cells. Finally, lincRNA-p21 interference rescued LPS-induced increase of lung and BAL collagen contents. LincRNA-p21 could lead to pulmonary fibrosis in ARDS by inhibition of the expression of Thy-1. [ABSTRACT FROM AUTHOR]

Background: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs. Methods: We investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors. Results: The present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression. Conclusion: Our study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro. [ABSTRACT FROM AUTHOR]

Use of multi-walled carbon nanotubes (MWCNT) is growing which increases occupational exposures to these materials. Their toxic potential makes it important to have an in-depth understanding of the inflammation and disease that develops due to exposure. Epigenetics is one area of interest that has been quickly developing to assess disease processes due to its ability to change gene expression and thus the lung environment after exposure. In this study, promoter methylation of inflammatory genes (IFN-γandTNF-α) was measured after MWCNT exposure using the pyrosequencing assay and found to correlate with initial cytokine production. In addition, methylation of a gene involved in tissue fibrosis (Thy-1) was also altered in a way that matched collagen deposition. In addition to using epigenetics to better understand disease processes, it has also been used as a biomarker of exposure and disease. In this study, global methylation was determined in the lung to ascertain whether MWCNT alter global methylation at the site of exposure and if those alterations coincide with disease development. Then, global methylation levels were determined in the blood to ascertain whether global methylation could be used as a biomarker of exposure in a more easily accessible tissue. Using the LuUminometric Methylation Assay (LUMA) and 5-Methylcytosine (5-mC) Quantification assay, we found that MWCNT lead to DNA hypomethylation in the lung and blood, which coincided with disease development. This study provides initial data showing that alterations in gene-specific methylation correspond with an inflammatory response to MWCNT exposure. In addition, global DNA methylation in the lung and blood coincides with MWCNT-induced disease development, suggesting its potential as a biomarker of both exposure and disease development. [ABSTRACT FROM AUTHOR]

Blood vessels in brain tumors, particularly glioblastomas, have been shown to express CD90. CD90+ cells in and around blood vessels in cancers including brain tumors have been identified as endothelial cells, cancer stem cells, fibroblasts or pericytes. In this study, we aimed to determine the nature or type(s) of cells that express CD90 in human brain tumors as well as an experimental rat glioma model by double immunofluorescence staining. The majority of CD90+ cells in human glioblastoma tissue expressed CD31, CD34 and von Willebrand factor, suggesting that they were endothelial cells. Vasculatures in a metastatic brain tumor and meningioma also expressed CD90. CD90+ cells often formed glomeruloid structures, typical of angiogenesis in malignant tumors, not only in glioblastoma but also in metastatic tumors. Some cells in the middle and outer layers of the vasculatures expressed CD90. Similar results were obtained in the rat glioma model. There were cells expressing both α-smooth muscle actin and CD90 in the middle layer of blood vessels, indicating that smooth muscle cells and/or pericytes may express CD90. CD90+ vasculatures were surrounded by tumor-associated macrophages (TAMs). Thus, in addition to endothelial cells, some other types of cells, such as smooth muscle cells, pericytes and fibroblasts constituting the vasculature walls in brain tumors expressed CD90. Because CD90 has been shown to interact with integrins expressed by circulating monocytes, CD90 might be involved in angiogenesis through recruitment and functional regulation of TAMs in tumors. CD90+ vasculatures may also interact with tumor cells through interactions with integrins. Because CD90 was not expressed by vasculatures in normal brain tissue, it might be a possible therapeutic target to suppress angiogenesis and tumor growth. [ABSTRACT FROM AUTHOR]

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies. [ABSTRACT FROM AUTHOR]

Multicystic and unicystic ameloblastomas are benign odontogenic tumors that present distinct biological behavior. The investigation of stem cells has become an important branch of tumor biology, with several studies addressing the possible role of these cells in tumor growth, angiogenesis, progression, infiltration and invasiveness. This study evaluated the immunohistochemical expression of CD90(Thy-1) and P75NTR stem cell markers in multicystic and unicystic ameloblastomas. Seventeen (17) samples of ameloblastomas (multicystic, n = 10; unicystic, n = 7) were submitted to immunohistochemical reactions and graded semi-quantitatively. The Kolmogorov-Smirnov test was used to verify possible differences in CD90 and P75NTR expressions between multicystic and unicystic ameloblastomas (p < 0.05). CD90 immunostaining was observed in all multicystic ameloblastoma specimens (n = 10), in the cytoplasm of the fibroblasts and vascular endothelial cells of the tumor stroma, near the neoplastic odontogenic epithelia. The staining of stromal CD90 was significantly higher in multicystic than in unicystic ameloblastomas (p = 0.003). Nuclear P75NTR immunostaining was observed in all ameloblastoma specimens. A significant difference was seen in the epithelial staining of P75NTR between multicystic and unicystic types (p = 0.007). The increased expression of CD90 and P75NTR found in multicystic ameloblastomas suggests a behavioral biological difference between multicystic and unicystic ameloblastomas, as well as a difference in ameloblastoma development. [ABSTRACT FROM AUTHOR]

In this study, possible thyrotoxicosis-related histological changes in testicular tissues of rats with experimentally induced thyrotoxicosis model were evaluated on cellular connections and stem cell markers. Two experimental groups, thyrotoxicosis and control, each consisting of eight animals were used. Rats in the thyrotoxicosis group were injected intraperitoneally with 3,3′,5-triiodo-l-thyronine (50 µg/100 g body weight/day) for 10 days. At the end of the study, animals in both groups were anesthetized, and blood samples were collected for biochemical analyses. Their testes were dissected out and histological procedure was conducted to perform further histochemical, immunohistochemical analyses and tissue expression analysis by real-time polymerase chain reaction. Expression of the stem cell markers such as c-kit and Thy-1 significantly decreased in the testes of the thyrotoxicosis group compared with the control group; however, Nanog expression was not detected in any of the groups. Similarly, connexin 43 and occludin expressions were also found to be significantly lower in the thyrotoxicosis group. These results on cellular connections are supported with the tissue expression analysis. Our findings are indicative of supporting microenvironmental tissue decay rather than parenchyma damage, which has been actually ignored in the literature. In conclusion, experimental thyrotoxicosis model may have adverse effects on the cell junctional complexes, cell–cell interactions, and pluripotency capacity. [ABSTRACT FROM PUBLISHER]

Survival niches within GALT allow follicular Th cells to escape depletion by an anti-Thy-1 mAb.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 CD44high/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. [ABSTRACT FROM PUBLISHER]

Abstract: Interstitial fibrosis is regarded as the common final pathway in chronic renal failure. Myofibroblasts play an important role in the renal fibrosis through producing extracellular matrices. In addition to expressions of cytoskeletons such as vimentin, desmin and α-smooth muscle actin (α-SMA), Thy-1 expression was investigated in cisplatin-induced rat renal interstitial fibrosis, to clarify the characteristics of myofibroblasts. Immunohistochemically, myofibroblasts in the renal fibrotic lesions reacted to vimentin, desmin and α-SMA in varying degrees, and the expression degrees were increased with advancing fibrosis. Vimentin expression was the greatest and the increased expression retained even in scar at end stages, whereas desmin and α-SMA expressions were almost completely decreased in scar. In double immunofluorescence, there were myofibroblasts reacting to both vimentin/desmin, desmin/α-SMA or α-SMA/vimentin, indicating that renal myofibroblasts can simultaneously express different cytoskeletons. Thy-1 expression in renal myofibroblasts was increased according to progressing fibrosis; however, the increased expression was decreased in scar, similar to desmin and α-SMA expressions. Some myofibroblasts expressing Thy-1 reacted simultaneously to vimentin or desmin, but there were no cells reacting to both Thy-1 and α-SMA. Because well-differentiated myofibroblasts are characterized mainly by α-SMA expression and the pericytes (immature stromal stem cells) showed a positive reaction to Thy-1, renal myofibroblasts might be originated from immature mesenchymal cells through loosing Thy-1 expression. This study for the first time shows that renal myofibroblasts can variously exhibit such mesenchymal markers as vimentin, desmin, α-SMA and Thy-1; particularly, Thy-1 immunohistochemistry would be used to detect myofibroblasts at early stages in analyzing chemically induced renal lesions. [Copyright &y& Elsevier]

Abstract: Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron–astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell–cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell–cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement. [Copyright &y& Elsevier]

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75)-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell™) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75 expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75 at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types. [ABSTRACT FROM AUTHOR]

Abstract: Background: Thy-1 (CD90) is a glycophosphatidyl-inositol (GPI) linked, cell surface glycoprotein located in non-caveolar lipid raft microdomains. Objective: The biological functions of Thy-1 in many tissues are well known, however, its role in skin wound healing remains unclear. Methods and results: In fibroblasts cells, Thy-1 affects cell migration into the wound, cell proliferation and the cytoskeleton structure. Additionally, Thy-1 is mainly expressed in the wound dermis. Here, we compared the in vivo aspects of the repair process with and without Thy-1 siRNA treatment. Temporally blocking Thy-1 in skin wound regions worsens the quality of healing and retards the rate of wound healing. Specifically, the level of TGF-β1 at the wound continuously increased. Conclusion: These data suggest that blocking Thy-1 at wound areas using siRNA reduces repair and affects the re-epithelialization and over-expression of TGF-β1 of the wound during the skin healing process. [Copyright &y& Elsevier]

The recently emerged concept of cancer stem cell (CSC) has led to a new hypothesis on the basis for tumor progression. Basically, the CSC theory hypothesizes the presence of a hierarchically organized and relatively rare cell population, which is responsible for tumor initiation, self-renewal, and maintenance, in addition to accumulation of mutation and resistance to chemotherapy. CSCs have recently been described in breast cancer. Different genetic markers have been used to isolate breast CSCs, none of which have been correlated with the tumorigenicity or metastatic potential of the cells, limiting their precise characterization and clinical application in the development of therapeutic protocols. Here, we sought for subpopulations of CSCs by analyzing 10 judiciously chosen stem cell markers in a normal breast cell line (MCF10-A) and in four human breast cancer cell lines (MCF-7, MDA-MB-231, MDA-MB-435, and Hs578-T) displaying different degrees of metastatic and invasiveness potential. We were able to identify two markers, which are differentially expressed in nontumorigenic versus tumor cells. The CD90 marker was highly expressed in the malignant cell lines. Interestingly, the CD14 molecule displayed higher expression levels in the nontumorigenic cell line. Therefore, we demonstrated that these two markers, which are more commonly used to isolate and characterize stem cells, are differentially expressed in breast tumor cells, when compared with nontumorigenic breast cells. © 2012 International Society for Advancement of Cytometry [ABSTRACT FROM AUTHOR]

Background: Pulmonary fibrosis is a debilitating and lethal disease with no effective treatment options. Understanding the pathological processes at play will direct the application of novel therapeutic avenues. Hypoxia has been implicated in the pathogenesis of pulmonary fibrosis yet the precise mechanism by which it contributes to disease progression remains to be fully elucidated. It has been shown that chronic hypoxia can alter DNA methylation patterns in tumour-derived cell lines. This epigenetic alteration can induce changes in cellular phenotype with promoter methylation being associated with gene silencing. Of particular relevance to idiopathic pulmonary fibrosis (IPF) is the observation that Thy-1 promoter methylation is associated with a myofibroblast phenotype where loss of Thy-1 occurs alongside increased alpha smooth muscle actin (α-SMA) expression. The initial aim of this study was to determine whether hypoxia regulates DNA methylation in normal human lung fibroblasts (CCD19Lu). As it has been reported that hypoxia suppresses Thy-1 expression during lung development we also studied the effect of hypoxia on Thy-1 promoter methylation and gene expression. Methods: CCD19Lu were grown for up to 8 days in hypoxia and assessed for global changes in DNA methylation using flow cytometry. Real-time PCR was used to quantify expression of Thy-1, α-SMA, collagen I and III. Genomic DNA was bisulphite treated and methylation specific PCR (MSPCR) was used to examine the methylation status of the Thy-1 promoter. Results: Significant global hypermethylation was detected in hypoxic fibroblasts relative to normoxic controls and was accompanied by increased expression of myofibroblast markers. Thy-1 mRNA expression was suppressed in hypoxic cells, which was restored with the demethylating agent 5-aza-20-deoxycytidine. MSPCR revealed that Thy-1 became methylated following fibroblast exposure to 1% O2. Conclusion: These data suggest that global and gene-specific changes in DNA methylation may play an important role in fibroblast function in hypoxia. [ABSTRACT FROM AUTHOR]

The article discusses a research study which found that a novel vitamin K/1, 2,3-epoxide reductase (VKOR) inhibitor, 3-acetyl-5-methyltetronic acid (AMT) reduced experimental glomerulonephritis (GN). The results of in vitro VKOR inhibitory activity assay and tissue concentrations of AMT are presented. Observations and findings from histopathological examination, blood chemistry and urinalysis after administration of AMT in Thy-1 GN rats are presented.

Abstract: Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, β1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells. [Copyright &y& Elsevier]

Abstract: Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell–cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel’s law, whereas the number of female mice followed Mendel’s law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR. [Copyright &y& Elsevier]

Introduction: Human adipose derived stem cells (hADSCs), with their impressive differentiation potential, may be used in autologous cell therapy or grafting to replace damaged tissues. Low intensity laser irradiation (LILI) has been shown to influence the behaviour of various cells, including stem cells. Aims: This study aimed to investigate the effect of LILI on hADSCs 24, 48 or 72 h post-irradiation and their differentiation potential into smooth muscle cells (SMCs). Methodology: hADSCs were exposed to a 636 nm diode laser at a fluence of 5 J/cm. hADSCs were differentiated into SMCs using retinoic acid (RA). Morphology was assessed by inverted light and differential interference contrast (DIC) microscopy. Proliferation and viability of hADSCs was assessed by optical density (OD), Trypan blue staining and adenosine triphosphate (ATP) luminescence. Expression of stem cell markers, β1-integrin and Thy-1, and SMC markers, smooth muscle alpha actin (SM-αa), desmin, smooth muscle myosin heavy chain (SM-MHC) and smoothelin, was assessed by immunofluorescent staining and real-time reverse transcriptase polymerase chain reaction (RT-PCR). Results: Morphologically, hADSCs did not show any differences and there was an increase in viability and proliferation post-irradiation. Immunofluorescent staining showed expression of β1-integrin and Thy-1 72 h post-irradiation. RT-PCR results showed a down regulation of Thy-1 48 h post-irradiation. Differentiated SMCs were confirmed by morphology and expression of SMC markers. Conclusion: LILI at a wavelength of 636 nm and a fluence of 5 J/cm does not induce differentiation of isolated hADSCs over a 72 h period, and increases cellular viability and proliferation. hADSCs can be differentiated into SMCs within 14 days using RA. [ABSTRACT FROM AUTHOR]

Abstract: Fish oil containing n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is well known to prevent the progression of IgA nephropathy. However, the mechanism through which fish oil prevents the progression of renal injury remains uncertain. We tried to clarify the effects of EPA on tubulointerstitial injury in the kidney both in vivo and in vitro. We examined the effects of EPA, especially to focus on nuclear factor kappa B (NF-κB), using Thy-1 nephritis models. Also the mechanism of EPA was investigated using small-interfering RNA (siRNA) in lipopolysaccharide (LPS)-stimulated proximal tubular epithelial cells (PTECs). In Thy-1 nephritis models, EPA significantly inhibited tubulointerstitial injury and the infiltration of macrophages into tubulointerstitial lesions except severe glomerular injury at early stage. Compared with control animals, NF-κB activation was significantly augmented in the Thy-1 nephritic kidney. However, treatment with EPA significantly reduced NF-κB activation, down-regulated the expressions of NF-κB-dependent molecules. Also in LPS-stimulated PTECs, LPS augmented NF-κB activation and the expression of NF-κB-dependent molecules. As in the case with the Thy-1 nephritis models, treatment with EPA inhibited them, prevented the degradation of IκBα in LPS-stimulated PTECs. Pre-treatment with siRNA for IκBα abolished the inhibitory effect of EPA on LPS-induced NF-κB activation, suggesting that EPA inhibited NF-κB activation by regulating IκBα. Our results indicate that EPA prevents the early progression of tubulointerstitial injury in Thy-1 nephritis models, and the inhibitory effect of EPA on the expression of inflammatory molecules via the regulation of IκBα in cultured cells may explain this mechanism. [Copyright &y& Elsevier]