The inflammation-associated protein TSG-6 cross-links hyaluronan via hyaluronan-induced TSG-6 oligomers

Tumor necrosis factor-stimulated gene-6 (TSG-6) is a hyaluronan (HA)-binding protein that plays important roles in inflammation and ovulation. TSG-6-mediated cross-linking of HA has been proposed as a functional mechanism (e.g. for regulating leukocyte adhesion), but direct evidence for cross-linking is lacking, and we know very little about its impact on HA ultrastructure. Here we used films of polymeric and oligomeric HA chains, end-grafted to a solid support, and a combination of surface-sensitive biophysical techniques to quantify the binding of TSG-6 into HA films and to correlate binding to morphological changes. We find that full-length TSG-6 binds with pronounced positive cooperativity and demonstrate that it can cross-link HA at physiologically relevant concentrations. Our data indicate that cooperative binding of full-length TSG-6 arises from HA-induced protein oligomerization and that the TSG-6 oligomers act as cross-linkers. In contrast, the HA-binding domain of TSG-6 (the Link module) alone binds without positive cooperativity and weaker than the full-length protein. Both the Link module and full-length TSG-6 condensed and rigidified HA films, and the degree of condensation scaled with the affinity between the TSG-6 constructs and HA. We propose that condensation is the result of protein-mediated HA cross-linking. Our findings firmly establish that TSG-6 is a potent HA cross-linking agent and might hence have important implications for the mechanistic understanding of the biological function of TSG-6 (e.g. in inflammation).     

Up-regulation of cyclooxygenase-2 expression by TSG-6 protein in macrophage cell line

TNF-stimulated gene 6 (TSG-6) encodes a 35 kDa inducible secreted glycoprotein important in inflammation and female fertility. Previous studies have shown that TSG-6 has anti-inflammatory activity in models of acute and chronic inflammation. In the present study, we show that treatment of the RAW 264.7 murine macrophage cell line with TSG-6 protein up-regulates the expression of inducible cyclooxygenase-2 (COX-2), a key enzyme in inflammation and immune responses. This action of TSG-6 protein was abolished by heat denaturation, trypsin digestion, or anti-TSG-6 antibodies. TSG-6 treatment also resulted in a rapid increase in COX-2 mRNA levels, suggesting that TSG-6 up-regulates COX-2 gene expression. Up-regulation of COX-2 was accompanied by an increase in the production of prostaglandins, especially PGD2. As the PGD2 metabolite, 15-deoxy-Delta12,14-PGJ2, can act as a negative regulator of inflammation, these TSG-6 actions may explain, at least in part, the anti-inflammatory effect of TSG-6 observed in the intact organism.     

Subcongenic analysis of genetic basis for impaired development of invariant NKT cells in NOD mice

Reduced numbers and function of invariant NKT (iNKT) cells partially contribute to type 1 diabetes (T1D) development in NOD mice. Previous linkage analysis identified a genetic locus on chromosome 2 controlling numbers of thymic iNKT cells. Interestingly, this locus resides within the Idd13 region that distinguishes NOD mice from the closely genetically related, but strongly T1D-resistant NOR strain. Thus, we tested if a genetic variant that confers T1D resistance in NOR mice may do so by enhancing iNKT cell numbers. iNKT cells were enumerated by an α-GalCer analog loaded CD1d tetramer in NOD and NOR mice as well as in NOD stocks carrying NOR-derived congenic regions on chromosome 1, 2, or 4. Significantly, more thymic and splenic iNKT cells were present in NOR than NOD mice. The NOR-derived Idd13 region on chromosome 2 contributed the most significant effect on increasing iNKT cell numbers. Subcongenic analyses indicated that at least two genes within the Idd13 region regulate iNKT cell numbers. These results further define the genetic basis for numerical iNKT cell defects contributing to T1D development in NOD mice.     

Alterations in oviductal cilia morphology and reduced expression of axonemal dynein in diabetic NOD mice

In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.     

The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice

The p38 mitogen-activated protein kinase (MAPK) pathway is important in Th1 immunity, macrophage activation, and apoptosis. Since they may be associated with beta-cell destruction during the development of type 1 diabetes, we investigated the role of the p38 MAPK pathway in female nonobese diabetic (NOD) mice. Phosphorylated p38 MAPK was observed immunohistochemically in CD4+ cells that had infiltrated into the islets and part of beta-cells, increasing in proportion to the severity of insulitis. Continuous oral administration of 0.08% FR167653, a specific p38 MAPK pathway inhibitor, significantly reduced the ex vivo production of interferon-gamma by splenic Th1 cells without affecting interleukin-4 production by Th2 cells. FR167653 administration from 4-30 weeks of age prevented NOD mice from developing diabetes without affecting the severity of insulitis. Treatment with FR167653 after insulitis had developed (i.e. from 10-30 weeks of age) also prevented diabetes, further suggesting that treatment with the p38 MAPK pathway inhibitor keeps insulitis benign in NOD mice, partly by inhibiting Th1 immunity. These findings suggest that p38 MAPK is a key mediator that switches insulitis from benign to destructive in the development of type 1 diabetes.     

TSG-6 protein, a negative regulator of inflammatory arthritis, forms a ternary complex with murine mast cell tryptases and heparin

TSG-6 (TNF-α-stimulated gene/protein 6), a hyaluronan (HA)-binding protein, has been implicated in the negative regulation of inflammatory tissue destruction. However, little is known about the tissue/cell-specific expression of TSG-6 in inflammatory processes, due to the lack of appropriate reagents for the detection of this protein in vivo. Here, we report on the development of a highly sensitive detection system and its use in cartilage proteoglycan (aggrecan)-induced arthritis, an autoimmune murine model of rheumatoid arthritis. We found significant correlation between serum concentrations of TSG-6 and arthritis severity throughout the disease process, making TSG-6 a better biomarker of inflammation than any of the other arthritis-related cytokines measured in this study. TSG-6 was present in arthritic joint tissue extracts together with the heavy chains of inter-α-inhibitor (IαI). Whereas TSG-6 was broadly detectable in arthritic synovial tissue, the highest level of TSG-6 was co-localized with tryptases in the heparin-containing secretory granules of mast cells. In vitro, TSG-6 formed complexes with the tryptases murine mast cell protease-6 and -7 via either heparin or HA. In vivo TSG-6-tryptase association could also be detected in arthritic joint extracts by co-immunoprecipitation. TSG-6 has been reported to suppress inflammatory tissue destruction by enhancing the serine protease-inhibitory activity of IαI against plasmin. TSG-6 achieves this by transferring heavy chains from IαI to HA, thus liberating the active bikunin subunit of IαI. Because bikunin is also present in mast cell granules, we propose that TSG-6 can promote inhibition of tryptase activity via a mechanism similar to inhibition of plasmin.     

Effect of hTIMP-1 overexpression in human umbilical cord mesenchymal stem cells on the repair of pancreatic islets in type-1 diabetic mice

Mesenchymal stem cells (MSCs) have been suggested for pancreatic islet repair in Type 1 diabetes mellitus (T1DM). This study aimed to investigate the effect of human umbilical cord MSCs (hUC-MSCs) transfected with tissue inhibitors of matrix metalloproteinase (TIMP)-1 on the regeneration of β-cell islets in vitro and in vivo. hUC-MSCs were isolated, cultured, and transfected with lentiviruses for the overexpression of hTIMP-1. An in vitro coculture system of hUC-MSCs and streptozotocin-induced islets was established to examine the morphology, apoptosis, and insulin secretion of the cocultured islets. Diabetic mouse models were injected with lenti-TIMP-1-enhanced green fluorescent protein (EGFP)-hUC-MSCs to test the effect of hTIMP-1 on insulin levels and glucose tolerance in vivo. The expression of insulin and glucagon was evaluated by immunofluorescence staining. The results showed that coculture with hUC-MSCs or Lenti-TIMP-1-EGFP-hUC-MSCs improved islet viability rates. Lenti-TIMP-1-EGFP-hUC-MSC coculture increased the insulin and C-peptide secretion function of the cultured islets and increased the secretion of tumor necrosis factor-β1, interleukin-6, IL-10, and hTIMP-1. hUC-MSCs, especially those transfected with Lenti-hTIMP-1-EGFP, showed a strong protective effect in diabetic mice by alleviating weight loss and improving glucose and insulin metabolism. In addition, transplantation rescued islet histology and function in vivo. The overexpression of TIMP-1 by hUC-MSCs seems to exert beneficial effects on pancreatic islet cells. In conclusion, this study may provide a new perspective on the development of hUC-MSC-based cell transplantation therapy for T1DM.     

TSG-6 promotes Cancer Cell aggressiveness in a CD44-Dependent Manner and Reprograms Normal Fibroblasts to create a Pro-metastatic Microenvironment in Colorectal Cancer

Tumor necrosis factor α stimulated gene 6 (TSG-6), a 30-KD secretory protein, plays an essential role in modulating inflammatory responses and extracellular matrix remodeling. However, little is known regarding the role of TSG-6 in human cancers. Here, we investigated the mechanism of action and the role of TSG-6 in colorectal cancer (CRC) metastasis. We found that TSG-6 was highly expressed in tumor tissues and was associated with poor prognosis and metastasis in CRC. Mechanistically, TSG-6 overexpression in CRC cells resulted in ERK activation and epithelial-mesenchymal transition by means of stabilizing CD44 and facilitating the CD44-EGFR complex formation on the cell membrane. Consequently, this resulted in the promotion of tumor migration and invasion both in vitro and in vivo. Notably, our data showed that CRC cells secreted TSG-6 could trigger a paracrine activation of JAK2-STAT3 signaling and reprogram normal fibroblasts into cancer-associated fibroblasts, which exhibited upregulation of pro-metastatic cytokines (CCL5 and MMP3) and higher movement ability. In animal models, the co-injection of cancer cells and TSG6-reprogrammed fibroblasts led to a significant increase in tumor metastasis. Our findings indicated that TSG-6 overexpression in CRC cells could promote cancer metastasis in both an autocrine and paracrine manner. Therefore, targeting TSG-6 might be a potential therapeutic strategy for the treatment of metastatic CRC.     

Tumor Necrosis Factor-stimulated Gene-6 (TSG-6) Amplifies Hyaluronan Synthesis by Airway Smooth Muscle Cells

We tested the hypothesis that the artificial addition of heavy chains from inter-α-inhibitor to hyaluronan (HA), by adding recombinant TSG-6 (TNF-stimulated gene-6) to the culture medium of murine airway smooth muscle (MASM) cells, would enhance leukocyte binding to HA cables produced in response to poly(I:C). As predicted, the addition of heavy chains to HA cables enhanced leukocyte adhesion to these cables, but it also had several unexpected effects. (i) It produced thicker, more pronounced HA cables. (ii) It increased the accumulation of HA in the cell-associated matrix. (iii) It decreased the amount of HA in the conditioned medium. Importantly, these effects were observed only when TSG-6 was administered in the presence of poly(I:C), and TSG-6 did not exert any effect on its own. Increased HA synthesis occurred during active, poly(I:C)-induced HA synthesis and did not occur when TSG-6 was added after poly(I:C)-induced HA synthesis was complete. MASM cells derived from TSG-6^−/−, HAS1/3^−/−, and CD44^−/− mice amplified HA synthesis in response to poly(I:C) + TSG-6 in a manner similar to WT MASM cells, demonstrating that they are expendable in this process. We conclude that TSG-6 increases the accumulation of HA in the cell-associated matrix, partially by preventing its dissolution from the cell-associated matrix into the conditioned medium, but primarily by inducing HA synthesis.     

IL-6抗体对银屑病小鼠的治疗作用及其对外周血辅助性T细胞和树突状细胞影响的研究

摘要 目的：研究白介素-6（Interleukin-6,IL-6）抗体对银屑病小鼠的治疗作用,并探讨IL-6抗体治疗对银屑病小鼠外周血辅助性T细胞和树突状细胞影响。方法：30只6-8周龄SPF级BALB/c小鼠按照随机数字表法分为Normal组、Model组和IL-6组,Model组和IL-6组小鼠通过在背部脱毛区涂抹咪喹莫特建立银屑病小鼠模型。IL-6组小鼠通过在背部脱毛区皮下注射IL-6抗体进行治疗。比较三组小鼠皮损组织银屑病皮损面积及严重程度指数（psoriasis area and severity index,PASI）、表皮厚度和血管数、血清TNF-α、IL-17和IL-23含量、外周血树突状细胞、Th17细胞和Th22细胞比例以及皮损组织IL-6、IL-21和STAT3蛋白表达。结果：IL-6抗体治疗后,Model组和IL-6组小鼠皮损组织PASI、表皮厚度和血管数,血清TNF-α、IL-17和IL-23含量,外周血树突状细胞、Th17细胞和Th22细胞比例,以及皮损组织IL-6、IL-21和STAT3蛋白表达均显著高于Normal组小鼠（P<0.05）;与Model组相比,IL-6组小鼠皮损组织PASI、表皮厚度和血管数,血清TNF-α、IL-17和IL-23含量,外周血树突状细胞、Th17细胞和Th22细胞比例,以及皮损组织IL-6、IL-21和STAT3蛋白表达均显著降低（P<0.05）。结论：IL-6抗体对银屑病小鼠模型具有较好的治疗作用,其机制可能与通过抑制IL-6/STAT3通路影响Th17/Th22和树突状细胞水平有关。     

Prevention of autoimmune diabetes in NOD mice by troglitazone is associated with modulation of ICAM-1 expression on pancreatic islet cells and IFN-gamma expression in splenic T cells

Thiazolidinediones acting as PPAR-gamma agonists are a new generation of oral antidiabetics addressing insulin resistance as a main feature of type-2 diabetes. In accordance to our results, pre-clinical studies have demonstrated that the thiazolinedione troglitazone prevents the development of insulin-dependent autoimmune type-1 diabetes. To investigate whether TGZ acts by affecting the ICAM-1/LFA-1 pathway and/or the Th1/Th2 cytokine balance in NOD mice, we analysed the IL-1beta-induced ICAM-1 expression on islet-cells and the LFA-1, CD25, IL-2, IFN-gamma, IL-4, and IL-10 expression on splenocytes. After 200 days of oral TGZ administration, islet cells from TGZ-treated NOD mice showed a reduced ICAM-1 expression in response to the pro-inflammatory cytokine IL-1beta. The expression of the ligand LFA-1 on CD4(+) and CD8(+) T-cells was comparable to that of placebo- and untreated controls. Also, the expression of Th1/Th2 cytokines was comparable in groups receiving TGZ or Placebo. Nevertheless, the investigated NOD mice segregated into IFN-gamma low- and IFN-gamma high producers as revealed by cluster analysis. Interestingly, the majority of TGZ-treated mice belonged to the cluster of IFN-gamma low producers. Thus, the prevention of autoimmune diabetes in NOD mice by TGZ seems to be associated with suppression of IL-1beta-induced ICAM-1 expression leading to a reduced vulnerability of pancreatic beta-cells during the effector stage of beta-cell destruction. In addition, IFN-gamma production was modulated, implicating that alteration of the Th1/Th2 cytokine balance might have contributed to diabetes prevention. The findings of this study suggest that TGZ exerts its effects by influencing both the beta-cells as the target of autoimmune beta-cell destruction and the T-cells as major effectors of the autoimmune process.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Plasticity of the TSG-6 HA-binding loop and mobility in the TSG-6-HA complex revealed by NMR and X-ray crystallography

Tumour necrosis factor-stimulated gene-6 (TSG-6) is a glycosaminoglycan-binding protein expressed during inflammatory and inflammation-like processes. Previously NMR structures were calculated for the Link module of TSG-6 (Link_TSG6) in its free state and when bound to an octasaccharide of hyaluronan (HA(8)). Heparin was found to compete for HA binding even though it interacts at a site that is distinct from the HA-binding surface. Here we present crystallography data on the free protein, and (15)N NMR relaxation data for the uncomplexed and HA(8)-bound forms of Link_TSG6. Although the Link module is comparatively rigid overall, the free protein shows a high degree of mobility in the beta4/beta5 loop and at the Cys47-Cys68 disulfide bond, both of which are regions involved in HA binding. When bound to HA(8), this dynamic behaviour is dampened, but not eliminated, suggesting a degree of dynamic matching between the protein and sugar that may decrease the entropic penalty of complex formation. A further highly dynamic residue is Lys54, which is distant from the HA-binding site, but was previously shown to be involved in heparin binding. When HA is bound, Lys54 becomes less mobile, providing evidence for an allosteric effect linking the HA and heparin-binding sites. A mechanism is suggested involving the beta2-strand and alpha2-helix. The crystal structure of free Link_TSG6 contains five molecules in the asymmetric unit that are highly similar to the NMR structure and support the dynamic behaviour seen near the HA-binding site: they show little or no electron density for the beta4/beta5 loop and display multiple conformations for the Cys47-Cys68 disulfide bond. The crystal structures were used in docking calculations with heparin. An extended interface between a Link_TSG6 dimer and heparin 11-mer was identified that is in excellent agreement with previous mutagenesis and calorimetric data, providing the basis for further investigation of this interaction.     

GM-CSF induces bone marrow precursors of NOD mice to skew into tolerogenic dendritic cells that protect against diabetes

We have reported that GM-CSF treatment of NOD mice suppressed diabetes by increasing the number of tolerogenic dendritic cells (tDCs) and Tregs in the periphery. Here, we have investigated whether GM-CSF acted on NOD bone marrow DCs precursors to skew their differentiation to tDCs. DCs were generated from the bone marrow of GM-CSF-treated (GM.BMDCs) and PBS-treated (PBS.BMDCs) NOD mice and were assessed for their ability to acquire tolerogenic properties. Upon LPS stimulation, GM.BMDCs became fully mature, expressed high levels of PD-L1 and produced more IL-10 and less IL-12p70 and IFN-γ than PBS.BMDCs. In addition, LPS-stimulated GM.BMDCs possessed a reduced capacity to activate diabetogenic CD8⁺ T cells in a PD-1/PD-L1-dependent manner. A single injection of LPS-stimulated GM.BMDCs in NOD mice resulted in long-term protection from diabetes, in contrast to LPS-stimulated PBS.BMDCs. Our results showed that GM-CSF-treatment acted on bone marrow precursors to skew their differentiation into tDCs that protected NOD mice against diabetes.     

Metal Ion-dependent Heavy Chain Transfer Activity of TSG-6 Mediates Assembly of the Cumulus-Oocyte Matrix

The matrix polysaccharide hyaluronan (HA) has a critical role in the expansion of the cumulus cell-oocyte complex (COC), a process that is necessary for ovulation and fertilization in most mammals. Hyaluronan is organized into a cross-linked network by the cooperative action of three proteins, inter-α-inhibitor (IαI), pentraxin-3, and TNF-stimulated gene-6 (TSG-6), driving the expansion of the COC and providing the cumulus matrix with its required viscoelastic properties. Although it is known that matrix stabilization involves the TSG-6-mediated transfer of IαI heavy chains (HCs) onto hyaluronan (to form covalent HC·HA complexes that are cross-linked by pentraxin-3) and that this occurs via the formation of covalent HC·TSG-6 intermediates, the underlying molecular mechanisms are not well understood. Here, we have determined the tertiary structure of the CUB module from human TSG-6, identifying a calcium ion-binding site and chelating glutamic acid residue that mediate the formation of HC·TSG-6. This occurs via an initial metal ion-dependent, non-covalent, interaction between TSG-6 and HCs that also requires the presence of an HC-associated magnesium ion. In addition, we have found that the well characterized hyaluronan-binding site in the TSG-6 Link module is not used for recognition during transfer of HCs onto HA. Analysis of TSG-6 mutants (with impaired transferase and/or hyaluronan-binding functions) revealed that although the TSG-6-mediated formation of HC·HA complexes is essential for the expansion of mouse COCs in vitro, the hyaluronan-binding function of TSG-6 does not play a major role in the stabilization of the murine cumulus matrix.     

A Refined Model for the TSG-6 Link Module in Complex with Hyaluronan: USE OF DEFINED OLIGOSACCHARIDES TO PROBE STRUCTURE AND FUNCTION*

Tumor necrosis factor-stimulated gene-6 (TSG-6) is an inflammation-associated hyaluronan (HA)-binding protein that contributes to remodeling of HA-rich extracellular matrices during inflammatory processes and ovulation. The HA-binding domain of TSG-6 consists solely of a Link module, making it a prototypical member of the superfamily of proteins that interacts with this high molecular weight polysaccharide composed of repeating disaccharides of d-glucuronic acid and N-acetyl-d-glucosamine (GlcNAc). Previously we modeled a complex of the TSG-6 Link module in association with an HA octasaccharide based on the structure of the domain in its HA-bound conformation. Here we have generated a refined model for a HA/Link module complex using novel restraints identified from NMR spectroscopy of the protein in the presence of 10 distinct HA oligosaccharides (from 4- to 8-mers); the model was then tested using unique sugar reagents, i.e. chondroitin/HA hybrid oligomers and an octasaccharide in which a single sugar ring was ¹³C-labeled. The HA chain was found to make more extensive contacts with the TSG-6 surface than thought previously, such that a d-glucuronic acid ring makes stacking and ionic interactions with a histidine and lysine, respectively. Importantly, this causes the HA to bend around two faces of the Link module (resembling the way that HA binds to CD44), potentially providing a mechanism for how TSG-6 can reorganize HA during inflammation. However, the HA-binding site defined here may not play a role in TSG-6-mediated transfer of heavy chains from inter-α-inhibitor onto HA, a process known to be essential for ovulation.     

Analysis of T-cell responses in metastatic melanoma patients vaccinated with dendritic cells pulsed with tumor lysates

In melanoma patients, CD8⁺ cytotoxic T cells have been found recognizing self-proteins of which the expression is restricted to the melanocytic lineage. These melanocyte differentiation antigens are expressed in normal melanocytes as well as in 80–100% of primary and metastatic melanoma. In this report, six HLA-A*0201–subtyped metastatic melanoma patients vaccinated with dendritic cells (DCs) pulsed with autologous tumor lysates and keyhole limpet hemocyanin (KLH) were screened for the presence of CD8⁺ T cells specific for three HLA-A*0201–binding peptides derived from the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase. For this purpose, nonstimulated as well as in vitro peptide-stimulated peripheral blood mononuclear cells (PBMCs) were tested for peptide-specific IFN- release by enzyme-linked immunosorbent spot (ELISpot) assays. Furthermore, expression of the melanosomal antigens MART-1/Melan-A, gp100, and tyrosinase in tumor lesions was analyzed by immunohistochemistry before and after vaccination. We also used the ELISpot technique to investigate whether KLH-specific T cells were induced and whether these cells released type 1 (IFN-) and/or type 2 (IL-13) cytokines. Our data show induction of CD8⁺ T cells specific for the melanosomal peptides MART-1/Melan-A_27–35 or tyrosinase_1–9, as well as IFN-–releasing KLH-specific T cells, in two of six vaccinated melanoma patients, but do not support an association between the induction of these T cells and clinical responses.     

Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing β cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2rγ^null mice. The selective destruction of pancreatic islet β cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total β-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the β cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet β cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4⁺ T cell infiltration and clonal expansion, and the mouse islet β-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet β cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.     

How viral infections affect the autoimmune process leading to type 1 diabetes

Despite a large body of evidence describing associations between viruses and the development of type 1 diabetes (T1D) in genetically prone individuals, clearly defining causative infectious agents has not been successful. A likely explanation is that the link between infections and autoimmunity is more multifaceted than we initially assumed. Viral footprints might be hard to detect systemically or in the target organ once autoimmunity has been initiated, and several infections might have to act in concert to precipitate clinical autoimmunity. Furthermore, cells cross-reactive between viral and self-antigens might express low avidity T cell receptors and only be present transiently in the blood of affected individuals. In addition, there are two new observations from animal models that we should take into account at this point: first, viral infections alone might not be able to induce disease in the absence of other inflammatory factors (supporting the "fertile field hypothesis" [M.G. von Herrath et al., Microorganisms and autoimmunity: making the barren field fertile? Nat. Rev. Microbiol. 1 (2003) 151-157, ]). Second, increasing evidence indicates that viruses can play a role in preventing rather than enhancing T1D development (supporting the "hygiene hypothesis" [J.F. Bach, Protective role of infections and vaccinations on autoimmune diseases. J. Autoimmun. 16 (2001) 347-353]). In this article we will present an overview of the early events and requirements that could account for T1D predisposition and development, and explain how these can be modulated by viral infections. Focusing on coxsackie B and lymphocytic choriomeningitis virus infections, we will discuss new data that can hopefully help us understand how virus-induced inflammation can positively or negatively affect the clinical outcome of islet-autoimmunity and T1D.     

Ablation of Elovl6 protects pancreatic islets from high-fat diet-induced impairment of insulin secretion

ELOVL family member 6, elongation of very long-chain fatty acids (Elovl6) is a microsomal enzyme that regulates the elongation of C12–16 saturated and monounsaturated fatty acids and is related to the development of obesity-induced insulin resistance via the modification of the fatty acid composition. In this study, we investigated the role of systemic Elovl6 in the pancreatic islet and β-cell function. Elovl6 is expressed in both islets and β-cell lines. In mice fed with chow, islets of the Elovl6^−/− mice displayed normal architecture and β-cell mass compared with those of the wild-type mice. However, when fed a high-fat, high-sucrose (HFHS) diet, the islet hypertrophy in response to insulin resistance observed in normal mice was attenuated and glucose-stimulated insulin secretion (GSIS) increased in the islets of Elovl6^−/− mice compared with those of the wild-type mice. Enhanced GSIS in the HFHS Elovl6^−/− islets was associated with an increased ATP/ADP ratio and the suppression of ATF-3 expression. Our findings suggest that Elovl6 could be involved in insulin secretory capacity per β-cell and diabetes.