HuR Plays a Positive Role to Strengthen the Signaling Pathways of CD4+ T Cell Activation and Th17 Cell Differentiation.

After antigen and/or different cytokine stimulation, CD4+ T cells activated and differentiated into distinct T helper (Th) cells via differential T cell signaling pathways. Transcriptional regulation of the activation and differentiation of naïve CD4+ T cells into distinct lineage Th cells such as Th17 cells has been fully studied. However, the role of RNA-binding protein HuR in the signaling pathways of their activation and differentiation has not been well characterized. Here, we used HuR conditional knockout (HuR KO) CD4+ T cells to study mechanisms underlying HuR regulation of T cell activation and differentiation through distinct signaling pathways. Our work showed that, mechanistically, HuR positively promoted CD3g expression by binding its mRNA and enhanced the expression of downstream adaptor Zap70 and Malt1 in activated CD4+ T cells. Compared to WT Th0 cells, HuR KO Th0 cells with reduced Bcl-2 expression are much more susceptible to apoptosis than WT Th0 cells. We also found that HuR stabilized IL-6Rα mRNA and promoted IL-6Rα protein expression, thereby upregulating its downstream phosphorylation of Jak1 and Stat3 and increased level of phosphorylation of IκBα to facilitate Th17 cell differentiation. However, knockout of HuR increased IL-22 production in Th17 cells, which was due to HuR deficiency in reducing IL-22 transcription repressor c-Maf expression. These results highlight the importance of HuR in TCR signaling and IL-6/IL-6R axis driving naïve CD4+ T cell activation and differentiation into Th17 cells.

Rapid improvement in vitamin D status with dietary 25-hydroxycholecalciferol in vitamin D insufficient dogs.

Vitamin D insufficiency is associated with various disease processes. We determined whether consumption of a diet supplemented with HyD®, a 25-hydroxycholecalciferol (25(OH)D3) source, would safely increase plasma 25(OH)D3 concentrations in Golden Retrievers with low vitamin D status. We hypothesised that dietary supplementation with HyD® would rapidly increase and sustain plasma 25(OH)D3 levels in healthy Golden Retrievers with low vitamin D status compared with supplementation with vitamin D3. Of fifty-seven privately owned dogs recruited with written owner consent, eighteen dogs with low vitamin D status were identified and sorted between two groups to have similar initial plasma 25(OH)D3 concentrations, sex distributions, ages and body weights. Dogs of each group were fed a dry dog food supplemented with either 16 µg/kg of 25(OH)D3 as HyD® (n 10) or 81 µg/kg of cholecalciferol (D3) (n 8) for 4 months. Plasma 25(OH)D3 concentrations were determined monthly. A significant time effect (P < 0⋅001) and time by group interaction (P = 0⋅0045) were found for monthly determined plasma 25(OH)D3 concentrations. Dogs fed the HyD®-supplemented diet experienced a 40⋅5 % rise in plasma 25(OH)D3 values after 1 month (P < 0⋅001) and no change thereafter. Plasma 25(OH)D3 values of dogs supplemented with vitamin D3 did not increase (P > 0⋅05) and were less than values of dogs supplemented with HyD® (P = 0⋅044). With few exceptions, average haematologic, biochemical and urinalyses results remained within the reference range for both groups. Dietary supplementation with HyD® is sufficient to safely increase and sustain plasma 25(OH)D3 levels in healthy dogs.

Correction: Safety of a novel feed ingredient, Algal Oil containing EPA and DHA, in a gestation-lactation-growth feeding study in Beagle dogs.

[This corrects the article DOI: 10.1371/journal.pone.0217794.].

RNA-Binding Protein HuR Promotes Th17 Cell Differentiation and Can Be Targeted to Reduce Autoimmune Neuroinflammation.

Dysregulated Th17 cell differentiation is associated with autoimmune diseases such as multiple sclerosis, which has no curative treatment. Understanding the molecular mechanisms of regulating Th17 cell differentiation will help find a novel therapeutic target for treating Th17 cell-mediated diseases. In this study, we investigated the cell-intrinsic processes by which RNA-binding protein HuR orchestrates Th17 cell fate decisions by posttranscriptionally regulating transcription factors Irf4 and Runx1 and receptor Il12rb1 expression, in turn promoting Th17 cell and Th1-like Th17 cell differentiation in C57BL/6J mice. Knockout of HuR altered the transcriptome of Th17 cells characterized by reducing the levels of RORγt, IRF4, RUNX1, and T-bet, thereby reducing the number of pathogenic IL-17+IFN-γ+CD4+ T cells in the spleen during experimental autoimmune encephalomyelitis. In keeping with the fact that HuR increased the abundance of adhesion molecule VLA-4 on Th17 cells, knockout of HuR impaired splenic Th17 cell migration to the CNS and abolished the disease. Accordingly, targeting HuR by its inhibitor DHTS inhibited splenic Th17 cell differentiation and reduced experimental autoimmune encephalomyelitis severity. In sum, we uncovered the molecular mechanism of HuR regulating Th17 cell functions, underscoring the therapeutic value of HuR for treatment of autoimmune neuroinflammation.

Safety of Algal Oil Containing EPA and DHA in cats during gestation, lactation and growth.

Algal Oil Containing EPA and DHA (AOCED) at approximately 50% was developed as a sustainable n-3 fatty acid source. AOCED was incorporated in diets at dose levels of 0%, 0.75%, 1.5% and 3.0% (w/w) and administered to healthy domestic shorthair female cats starting two weeks before mating, then during mating, gestation, lactation and to their kittens until they reached 32 weeks of age. The diets were made isocaloric and met the Association of American Feed Control Officials (AAFCO) nutrient requirements of cats for growth and reproduction. Dietary AOCED treatment did not affect the overall health, physiological parameters, food consumption and body weights of the queens and their kittens. No AOCED-related changes in haematology, coagulation or clinical chemistry parameters were observed in either generation when compared to control cats. Plasma levels of EPA and DHA were dose-dependently increased in both generations, demonstrating bioavailability of the fatty acids. In this study, safety of AOCED at levels up to 3.0% in the diet was demonstrated in cats with administration starting in utero and until kittens reached 32 weeks of age. Bioavailability of EPA and DHA in cats supports use of AOCED as a source of EPA and DHA for feline growth and reproduction.

Safety of a novel feed ingredient, Algal Oil containing EPA and DHA, in a gestation-lactation-growth feeding study in Beagle dogs.

An Algal Oil Containing EPA and DHA (AOCED) at ~50% was developed as a sustainable source of omega-3 fatty acids. AOCED was incorporated into extruded dry foods for dogs at 0, 0.75%, 1.5% and 3.0% levels (equivalent to 0, 7.5, 15 and 30 g/kg diet) on dry matter basis at the expense of chicken fat and fed to healthy female Beagle dogs starting at mating and throughout gestation and lactation. The offspring were fed their maternal corresponding diets for 26 weeks after weaning. AOCED-enriched diets were well tolerated by dogs in both generations and did not affect their overall health, physiological parameters, food consumption, body weights and body weight gains. There were no changes in hematology, clinical chemistry, and coagulation parameters in both generations of dogs fed the AOCED diets when compared to those in the control group. Plasma levels of DHA and EPA increased significantly and generally dose-dependently in both generations. The study demonstrated the safety of AOCED in dogs during gestation, lactation, and growth periods at dietary levels up to 3.0wt%, equivalent to 30 g/kg diet. AOCED's bioavailability as a source of DHA and EPA in dogs was demonstrated by the increased plasma concentrations of these nutritional lipids.

Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3' untranslated region (3'UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3'UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.

The RNA-binding protein HuR contributes to neuroinflammation by promoting C-C chemokine receptor 6 (CCR6) expression on Th17 cells.

In both multiple sclerosis and experimental autoimmune encephalomyelitis (EAE), the C-C chemokine receptor 6 (CCR6) is critical for pathogenic T helper 17 (Th17) cell migration to the central nervous system (CNS). Whereas many cytokines and their receptors are potently regulated via post-transcriptional mechanisms in response to various stimuli, how CCR6 expression is post-transcriptionally regulated in Th17 cells is unknown. Here, using RNA-binding protein HuR conditional knock-out (KO) and wild-type (WT) mice, we present evidence that HuR post-transcriptionally regulates CCR6 expression by binding to and stabilizing Ccr6 mRNA and by promoting CCR6 translation. We also found that HuR down-regulates several microRNA expressions, which could target the 3'-UTR of Ccr6 mRNA for decay. Accordingly, knock-out of HuR reduced CCR6 expression on Th17 cells and impaired their migration to CNS compared with the response of WT Th17 cells and thereby ameliorated EAE. Together, these findings highlight how HuR contributes to Th17 cell-mediated autoimmune neuroinflammation and support the notion that targeting HuR might be a potential therapeutic intervention for managing autoimmune disorders of the CNS.

NOD.H-2h4 mice: an important and underutilized animal model of autoimmune thyroiditis and Sjogren's syndrome.

NOD.H-2h4 mice express the K haplotype on the NOD genetic background. They spontaneously develop thyroiditis and Sjogren's syndrome, but they do not develop diabetes. Although autoimmune thyroid diseases and Sjogren's syndrome are highly prevalent autoimmune diseases in humans, there has been relatively little emphasis on the use of animal models of these diseases for understanding basic mechanisms involved in development and therapy of chronic organ-specific autoimmune diseases. The goal of this review is to highlight some of the advantages of NOD.H-2h4 mice for studying basic mechanisms involved in development of autoimmunity. NOD.H-2h4 mice are one of relatively few animal models that develop organ-specific autoimmune diseases spontaneously, i.e., without a requirement for immunization with antigen and adjuvant, and in both sexes in a relatively short period of time. Thyroiditis and Sjogren's syndrome in NOD.H-2h4 mice are chronic autoimmune diseases that develop relatively early in life and persist for the life of the animal. Because the animals do not become clinically ill, the NOD.H-2h4 mouse provides an excellent model to test therapeutic protocols over a long period of time. The availability of several mutant mice on this background provides a means to address the impact of particular cells and molecules on the autoimmune diseases. Moreover, to our knowledge, this is the only animal model in which the presence or absence of a single cytokine, IFN-γ, is sufficient to completely inhibit one autoimmune thyroid disease, with a completely distinct autoimmune thyroid disease developing when it is absent.

The immunomodulator AS101 suppresses production of inflammatory cytokines and ameliorates the pathogenesis of experimental autoimmune encephalomyelitis.

We reported that AS101 (organotellurium compound, trichloro(dioxoethylene-O,O') tellurate) inhibited the differentiation of Th17 cells and reduced the production of IL-17 and GM-CSF. In addition, AS101 promoted the production of IL-2 in activated T cells. Flow cytometric analysis showed that AS101 inhibited Th17 cell proliferation. AS101 blocked the activation of transcriptional factor NFAT, Stat3, and RORγt, and increased activation of Erk1/2, suggesting a mechanism of action of AS101. We further demonstrated that AS101 was effective in amelioration of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Finally, by real-time PCR analysis we showed that AS101 reduces the IL-17, IFN-γ, GM-CSF, and IL-6 mRNA expression in inflammatory cells of spinal cords. Additionally, flow cytometry analysis also indicated that the CD4+ T cells and IL-17 and GM-CSF-producing cells were reduced in the spinal cords of AS101 treated mice compared to those treated with PBS.

Posttranscriptional gene regulation of IL-17 by the RNA-binding protein HuR is required for initiation of experimental autoimmune encephalomyelitis.

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17-producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3' untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4(+) Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17(+) cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3' untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4(+) T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

Agonistic anti-CD40 induces thyrocyte proliferation and promotes thyroid autoimmunity by increasing CD40 expression on thyroid epithelial cells.

CD40 is expressed on cells of the immune system and in some tissues that are targets for autoimmune-mediated damage. It is not known if CD40 expression in target tissues plays a role in the pathology of autoimmune diseases. This study shows that agonistic anti-CD40 induces strong and sustained proliferation of thyroid epithelial cells (TECs), or thyrocytes, in IFN-γ(-/-) autoimmune-prone NOD and NOD.H-2h4 mice. TEC proliferation is accompanied by greatly increased expression of CD40 on TECs, development of fibrosis and hypothyroidism, and increased expression of proinflammatory molecules in thyroids. Bone marrow chimera experiments indicate that TEC expression of CD40 is required for anti-CD40-induced TEC proliferation, but lymphoid cells do not have to express CD40. TEC proliferation is reduced in wild-type mice given anti-CD40, presumably because they produce IFN-γ, which inhibits TEC proliferation. CD40 also increases on TECs during development of an autoimmune thyroid disease characterized by TEC hyperproliferation that develops spontaneously in IFN-γ(-/-) NOD.H-2h4 mice. TEC hyperproliferation development is accelerated in mice given agonistic anti-CD40. These studies provide new information regarding the role of target tissue expression of CD40 in development of autoimmunity and suggest that use of agonistic anti-CD40 for tumor therapy could result in autoimmune disease.

Agonistic anti-CD40 promotes early development and increases the incidence of severe thyroid epithelial cell hyperplasia (TEC H/P) in CD4-/- mice.

IFN-γ-/-NOD.H-2h4 mice develop thyroid epithelial cell hyperplasia (TEC H/P) characterised by abnormal proliferation of thyrocytes and infiltration of thyroids by CD4+ and CD8+ T cells, macrophages and dendritic cells. CD8+ T cells from mice with severe TEC H/P transfer similar lesions to SCID recipients, whereas CD4+ T cells transfer mild TEC H/P. CD4- and CD8- deficient IFN-γ-/-NOD.H-2h4 mice were generated to determine if CD4+ T cells were required for initial activation of the CD8+ T cells that transfer TEC H/P. After 6-8 months on NaI water, only 2 of 60 CD8-/- mice developed severe TEC H/P, whereas 31 of 101 CD4-/- mice developed severe TEC H/P and fibrosis comparable in severity to that of IFN-γ-/- mice. However, splenocytes from CD4-/- mice with severe TEC H/P did not effectively transfer severe TEC H/P to SCID recipients. When CD4-/- donors were given agonistic anti-CD40 mAb, most developed severe TEC H/P and their cells transferred severe TEC H/P to SCID recipients. These results indicate that agonistic anti-CD40 can provide an important signal for activation of autoreactive CD8+ T cells that transfer severe TEC H/P. Therefore, targeting or blocking CD40 could provide effective therapy for diseases involving hyperplasia and fibrosis mediated by CD8+ T cells.

Transient depletion of B cells in young mice results in activation of regulatory T cells that inhibit development of autoimmune disease in adults.

B-cell depletion therapy can be effective for treating B-cell lymphomas as well as many human and murine autoimmune diseases. B-cell-deficient mice are normally resistant to spontaneous autoimmune thyroiditis (SAT), but they develop SAT if regulatory T cells are transiently depleted during the first 3-6 weeks after birth. This was also a critical time when B-cell depletion effectively inhibited development of SAT in adult mice. The current study was undertaken to test the hypothesis that transient depletion of B cells using anti-CD20 would be sufficient to suppress SAT if B cells were depleted early in life and that inhibition of SAT would be due to the activity of Treg that functioned most effectively when B cells were absent or low. The results presented here support this hypothesis and indicate that development of autoimmune disease in adults is effectively inhibited when anti-CD20 is administered 1-3 weeks after birth. After 3 weeks, transient B-cell depletion is no longer effective, and B-cell depletion must be maintained to effectively suppress autoimmune disease. B-cell depletion in 1- to 3-week-old mice depletes all B-cell subsets, whereas B-cell depletion initiated in adults spares many marginal zone B cells. Following early B-cell depletion, splenic Treg increase in number, and depletion of Treg reverses the inhibitory effect of anti-CD20 on disease development. Early transient depletion of B cells could be useful for preventing autoimmune disease in individuals at high risk for developing autoimmune diseases as adults.

TGF-ß promotes proliferation of thyroid epithelial cells in IFN-γ(-/-) mice by down-regulation of p21 and p27 via AKT pathway.

IFN-γ(-/-) NOD.H-2h4 mice develop an autoimmune disease characterized by hyperplasia and proliferation of thyroid epithelial cells (TEC H/P). Proliferating TECs produce TGF-ß, and IFN-γ inhibits TEC H/P. In the present study, cultured TECs were used to directly determine the mechanisms by which these cytokines act on TECs to result in proliferation or inhibition of proliferation. With TECs from IFN-γ(-/-) NOD.H-2h4 mice or mice expressing the dominant negative TGF-ß type II receptor on TECs, TGF-ß was shown to promote TEC proliferation and IFN-γ was shown to inhibit TEC proliferation in vitro. TGF-ß may promote TEC proliferation by down-regulating antiproliferative molecules p21 and p27, whereas IFN-γ may inhibit proliferation by up-regulating antiproliferative molecules p18 and p21 and down-regulating the pro-proliferative molecule cyclin D. Inhibition of AKT abolished the effect of TGF-ß on p21 and p27, resulting in similar proliferation of TGF-ß-treated and control TECs. Increased expression of proliferating cell nuclear antigen (PCNA), TGF-ß, and p-AKT and decreased expression of p21 and p27 by proliferating TECs correlated with the proliferative state of TEC H/P. Taken together, the results suggest that TGF-ß promotes TEC proliferation by down-regulating p21 and p27 via the AKT pathway in IFN-γ(-/-) NOD.H-2h4 mice, which may have significant implications for development of effective therapeutic strategies targeting the TGF-ß and AKT pathways for treatment of hyperplasia and/or neoplasia.

CD8+ T cells induce thyroid epithelial cell hyperplasia and fibrosis.

CD8(+) T cells can be important effector cells in autoimmune inflammation, generally because they can damage target cells by cytotoxicity. This study shows that activated CD8(+) T cells induce thyroid epithelial cell hyperplasia and proliferation and fibrosis in IFN-γ(-/-) NOD.H-2h4 SCID mice in the absence of CD4(+) T cells. Because CD8(+) T cells induce proliferation rather than cytotoxicity of target cells, these results describe a novel function for CD8(+) T cells in autoimmune disease. In contrast to the ability of purified CD8(+) T cells to induce thyrocyte proliferation, CD4(+) T cells or CD8 T cell-depleted splenocytes induced only mild thyroid lesions in SCID recipients. T cells in both spleens and thyroids highly produce TNF-α. TNF-α promotes proliferation of thyrocytes in vitro, and anti-TNF-α inhibits development of thyroid epithelial cell hyperplasia and proliferation in SCID recipients of IFN-γ(-/-) splenocytes. This suggests that targeting CD8(+) T cells and/or TNF-α may be effective for treating epithelial cell hyperplasia and fibrosis.

Transgenic expression of TGF-beta on thyrocytes inhibits development of spontaneous autoimmune thyroiditis and increases regulatory T cells in thyroids of NOD.H-2h4 mice.

Transgenic NOD.H-2h4 mice expressing TGF-beta under control of the thyroglobulin promoter were generated to assess the role of TGF-beta in the development of thyrocyte hyperplasia. In contrast to nontransgenic littermates, which develop lymphocytic spontaneous autoimmune thyroiditis (L-SAT), all TGF-beta transgenic (Tg) mice given NaI water for 2-7 mo developed thyroid lesions characterized by severe thyroid epithelial cell hyperplasia and proliferation, with fibrosis and less lymphocyte infiltration than in nontransgenic mice. Most Tg mice produced less anti-mouse thyroglobulin autoantibody than did wild type (WT) mice. T cells from Tg and WT mice were equivalent in their ability to induce L-SAT after transfer to SCID or TCRalpha(-/-) mice. WT lymphocytes could transfer experimental autoimmune thyroiditis or L-SAT to Tg mice, indicating that the transgenic environment did not prevent migration of lymphocytes to the thyroid. Thyroids of Tg mice had higher frequencies of Foxp3(+) regulatory T cells (Tregs) compared with nontransgenic WT mice. Transient depletion of Tregs by anti-CD25 resulted in increased infiltration of inflammatory cells into thyroids of transgenic mice. Treg depletion also resulted in increased anti-mouse thyroglobulin autoantibody responses and increased expression of IFN-gamma and IFN-gamma-inducible chemokines in thyroids of Tg mice. The results suggest that spontaneous autoimmune thyroiditis is inhibited in mice expressing transgenic TGF-beta on thyrocytes, at least in part, because there is an increased frequency of Tregs in their thyroids.

Comparison of sensitivity of Th1, Th2, and Th17 cells to Fas-mediated apoptosis.

Following activation through the TCR, CD4+ T cells can differentiate into three major subsets: Th1, Th2, and Th17 cells. IL-17-secreting Th17 cells play an important role in the pathogenesis of several autoimmune diseases and in immune responses to pathogens, but little is known about the regulation of apoptosis in Th17 cells. In this study, the sensitivity of in vitro-polarized Th1, Th2, and Th17 cells to Fas-mediated apoptosis was compared directly by different methods. The order of sensitivity of T cell subsets to Fas-mediated apoptosis is: Th1 > Th17 > Th2. The greater sensitivity of Th17 cells to Fas-mediated apoptosis compared with Th2 cells correlated with their higher expression of FasL and comparable expression of the antiapoptotic molecule FLIP. The decreased sensitivity of Th17 compared with Th1 cells correlated with the higher expression of FLIP by Th17 cells. Transgenic overexpression of FLIP in T cells protected all three subsets from Fas-mediated apoptosis. These findings provide new knowledge for understanding how survival of different subsets of T cells is regulated.

TGF-beta promotes thyroid epithelial cell hyperplasia and fibrosis in IFN-gamma-deficient NOD.H-2h4 mice.

IFN-gamma(-/-)NOD.H-2h4 mice given 0.05% NaI in their water develop severe thyroid epithelial cell (thyrocyte) hyperplasia and proliferation (TEC H/P) and fibrosis. Proliferating thyrocytes of IFN-gamma(-/-) mice with TEC H/P produce TGF-beta as demonstrated by immunohistochemical staining and in situ hybridization. Strong expression of activating phosphorylated Smad-2/3 and weak expression of inhibitory Smad-7 by proliferating thyrocytes correlate with the severity of TEC H/P. Splenocytes from IFN-gamma(-/-) mice with severe TEC H/P transfer severe TEC H/P to IFN-gamma(-/-)NOD.H-2h4.SCID mice. Mice given anti-TGF-beta had markedly reduced thyrocyte proliferation and decreased fibrosis compared with mouse Ig-treated controls, suggesting that TGF-beta plays an important role in development of TEC H/P induced by activated splenocytes. Moreover, transgenic IFN-gamma(-/-)NOD.H-2h4 mice expressing TGF-beta on thyrocytes all develop fibrosis and moderate to severe TEC H/P with accelerated kinetics, directly demonstrating a role for TGF-beta in severe TEC H/P and fibrosis.

Cells captured from spatium intermusculare by porous material exhibit the characteristics of stem cells.

Most of the current methods to capture stem cells are very complicated. Our new discovery of acquiring adult stem cells by implanting three dimension (3-D) porous material into the spatium intermusculare of mice hind limbs would bring hope to achieve autologous stem cells transplantation. We discovered that a great number of cells migrated into the 3-D porous material implanted in vivo. Furthermore, the migrating cells exhibited stem cell properties (CD34(+), Sca-1(+), GFAP(+), alphafetoprotein(+)) and were hematogeous (CD45(+)) and CD105(+). The ability of migrating cells to undergo differentiation into hematopoietic lineages was tested with methylcellulose medium. These findings demonstrate that the cells captured from spatium intermusculare by implanting 3-D porous material exhibit the characteristics of stem cells.