A Hybrid Insulin Epitope Maintains High 2D Affinity for Diabetogenic T Cells in the Periphery.

ß-Cell antigen recognition by autoreactive T cells is essential in type 1 diabetes (T1D) pathogenesis. Recently, insulin hybrid peptides (HIPs) were identified as strong agonists for CD4 diabetogenic T cells. Here, using BDC2.5 transgenic and NOD mice, we investigated T-cell recognition of the HIP2.5 epitope, which is a fusion of insulin C-peptide and chromogranin A (ChgA) fragments, and compared it with the WE14 and ChgA29 -42 epitopes. We measured in situ two-dimensional affinity on individual live T cells from thymus, spleen, pancreatic lymph nodes, and islets before and after diabetes. Although preselection BDC2.5 thymocytes possess higher affinity than splenic BDC2.5 T cells for all three epitopes, peripheral splenic T cells maintained high affinity only to the HIP2.5 epitope. In polyclonal NOD mice, a high frequency (∼40%) of HIP2.5-specific islet T cells were identified at both prediabetic and diabetic stages comprising two distinct high- and low-affinity populations that differed in affinity by 100-fold. This high frequency of high- and low-affinity HIP2.5 T cells in the islets potentially represents a major risk factor in diabetes pathogenesis.

Catestatin as a Target for Treatment of Inflammatory Diseases.

It is increasingly clear that inflammatory diseases and cancers are influenced by cleavage products of the pro-hormone chromogranin A (CgA), such as the 21-amino acids long catestatin (CST). The goal of this review is to provide an overview of the anti-inflammatory effects of CST and its mechanism of action. We discuss evidence proving that CST and its precursor CgA are crucial for maintaining metabolic and immune homeostasis. CST could reduce inflammation in various mouse models for diabetes, colitis and atherosclerosis. In these mouse models, CST treatment resulted in less infiltration of immune cells in affected tissues, although in vitro monocyte migration was increased by CST. Both in vivo and in vitro, CST can shift macrophage differentiation from a pro- to an anti-inflammatory phenotype. Thus, the concept is emerging that CST plays a role in tissue homeostasis by regulating immune cell infiltration and macrophage differentiation. These findings warrant studying the effects of CST in humans and make it an interesting therapeutic target for treatment and/or diagnosis of various metabolic and immune diseases.

Effective preparation of a monoclonal antibody against human chromogranin A for immunohistochemical diagnosis.

BACKGROUND: Human chromogranin A (CgA) is a ~ 49 kDa secreted protein mainly from neuroendocrine cells and endocrine cells. The CgA values in the diagnosis of tumor, and in the potential role in prognostic and predictive tumor as a biomarker. RESULTS: The synthesized gene of CgA coding area was cloned and expressed as fusion protein CgA-His in procaryotic system. Then the purified CgA-His protein was mixed with QuickAntibody-Mouse5W adjuvant, and injected into mice. The CgA-His protein was also used as coating antigen to determine the antiserum titer. By screening, a stable cell line named 4E5, which can generate anti-CgA monoclonal antibody (mAb), was obtained. The isotype of 4E5 mAb was IgG2b, and the chromosome number was 102 ± 4. Anti-CgA mAb was purified from ascites fluid, and the affinity constant reached 9.23 × 109 L/mol. Furthermore, the specificity of the mAb was determined with ELISA, western blot and immunohistochemistry. Results indicated that the mAb 4E5 was able to detect chromogranin A specifically and sensitively. CONCLUSIONS: A sensitive and reliable method was successfully developed for rapid production of anti-CgA mAb for immunohistochemistry diagnosis in this study, and the current study also provides conclusive guidelines for preparation of mAbs and implements in immunohistochemistry diagnosis.

A novel mimovirus encoding ChgA10-19 peptide with PD-L1 induces T cell tolerance and ameliorates the severity of diabetes.

Related studies demonstrate that type 1 diabetes (T1D) is caused by ß-cell antigen specific autoreactive CD8+ T cells. ChgA has recently been identified as the autoantigen in NOD mice and T1D patients. Therefore, attenuating the activation of ChgA specific CD8+ T cells might be a promising target for T1D therapy. The negative co-stimulatory PD-L1 inhibits T cell mediated alloimmunity and induces tolerance. In this experiment, a novel mimovirus encoding ChgA10-19 peptide with PD-L1 was constructed. The NOD.ß2m null HHD mice were administrated with mimovirus transduced DCs. After immunization, the activation and proliferation of CD8+ T cells were detected, diabetes incidence and pancreatic tissue destruction were also analyzed. The results demonstrated that transduced DCs attenuated CD8+ T cell activation and proliferation. In addition, transduced DCs inhibited CD8+ T response to ChgA stimulation, and ameliorated the severity of diabetes. These data suggested that mimovirus transduced DCs might provide novel clues for T1D therapy.

Neuroendocrine differentiation in basal cell carcinoma.

Basal cell carcinoma (BCC) is the prototypical basaloid tumor of the skin. It may show various patterns simulating other cutaneous tumors due to its pleomorphism. It may have an unusal pattern of differentiation such as squamous, sebaceous, apocrine, eccrine, pilar, and endocrine differentiation. In order to establish the relative frequency of neuroendocrine differentiation in BCC, we performed a retrospective study of 33 consecutive BCCs using conventional immunohistochemistry with two neuroendocrine antibodies: Chromogranine A and synaptophysine. The age of the patients ranged from 17-83 years with mean of 65 years. The male to female ratio was 16:17. In immunohistochimestry, Chromogranine A was seen in 72.2% (24/33) while Synaptophysine was positive in 9.09% (3/33). Their expression was cytoplasmic and membranous and was seen in the periphery of these tumors in the overlying cells. Positive staining of chromogranine A was high (75-100% of tumors cells) in 9%, intermediate (25-75% of tumors cells) in 33% of cases and relatively low (<25%) in 30.3% of cases.

Label-Free Detection of Human Glycoprotein (CgA) Using an Extended-Gated Organic Transistor-Based Immunosensor.

Herein, we report on the fabrication of an extended-gated organic field-effect transistor (OFET)-based immunosensor and its application in the detection of human chromogranin A (hCgA). The fabricated OFET device possesses an extended-gate electrode immobilized with an anti-CgA antibody. The titration results of hCgA showed that the electrical changes in the OFET characteristics corresponded to the glycoprotein recognition ability of the monoclonal antibody (anti-CgA). The observed sensitivity (detection limit: 0.11 µg/mL) and selectivity indicate that the OFET-based immunosensor can be potentially applied to the rapid detection of the glycoprotein concentration without any labeling.

Inherent ER stress in pancreatic islet ß cells causes self-recognition by autoreactive T cells in type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic ß cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in ß cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that ß cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4(+) diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This ß cell ER stress increased activity of the calcium (Ca(2+))-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, ß cell ER stress induced by chemical and physiological triggers leads to ß cell immunogenicity through Ca(2+)-dependent PTM. These findings elucidate a mechanism of how ß cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing ß cell recognition by autoreactive T cells.

Detection of vasostatin-1-specific CD8(+) T cells in non-obese diabetic mice that contribute to diabetes pathogenesis.

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4(+) T cells in a non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin-1 is a naturally processed fragment of ChgA. We have now identified a novel H2-K(d) -restricted epitope of vasostatin-1, ChgA 36-44, which elicits CD8(+) T cell responses in NOD mice. By using ChgA 36-44/K(d) tetramers we have determined the frequency of vasostatin-1-specific CD8(+) T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin-1-specific CD4(+) and CD8(+) T cells constitute a significant fraction of islet-infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36-44 peptide-primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin-1-specific CD8(+) T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.

Changes in small intestinal chromogranin A-immunoreactive cell densities in patients with irritable bowel syndrome after receiving dietary guidance.

Chromogranin A (CgA) is a common marker for enteroendocrine cells in the gut, and CgA-immunoreactive cell densities are abnormal in patients with irritable bowel syndrome (IBS). The majority of patients with IBS report that their symptoms develop after consuming certain foodstuffs. In the present study, we investigated the effects of dietary guidance on the total enteroendocrine cell densities in the small intestine, as detected by CgA. A total of 14 patients with IBS underwent a gastroscopy with duodenal biopsies and 11 of them also underwent a colonoscopy, with biopsy samples obtained from the ileum. Fourteen control subjects were also included. Each patient received 3 sessions of dietary guidance. Gastroscopies and colonoscopies were performed on both the controls and patients with IBS (at baseline and at 3-9 months after receiving guidance). Biopsy samples obtained from the duodenum and ileum were immunostained for CgA using the avidin-biotin complex (ABC) method and were quantified using computerized image analysis. The density of CgA-immunoreactive cells in the duodenum (mean ± SEM values) in the control subjects was 235.9 ± 31.9 cells/mm2; in the patients with IBS, the density was 36.9 ± 9.8 and 103.7 ± 16.9 cells/mm2 before and after they received dietary guidance, respectively (P=0.007). The density of CgA-immunoreactive cells in the ileum in the control subjects was 47.4 ± 8.3 cells/mm2; in the patients with IBS, the density was 48.4 ± 8.1 and 17.9 ± 4.4 cells/mm2, before and after they received dietary guidance, respectively (P=0.0006). These data indicate that changes in CgA-immunoreactive cell densities in patients with IBS after receiving dietary guidance may reflect a change in the densities of the small intestinal enteroendocrine cells, which may contribute to an improvement in the IBS symptoms.

Cutting Edge: Nonobese Diabetic Mice Deficient in Chromogranin A Are Protected from Autoimmune Diabetes.

T cells reactive to ß cell Ags are critical players in the development of autoimmune type 1 diabetes. Using a panel of diabetogenic CD4 T cell clones derived from the NOD mouse, we recently identified the ß cell secretory granule protein, chromogranin A (ChgA), as a new autoantigen in type 1 diabetes. CD4 T cells reactive to ChgA are pathogenic and rapidly transfer diabetes into young NOD recipients. We report in this article that NOD.ChgA(-/-) mice do not develop diabetes and show little evidence of autoimmunity in the pancreatic islets. Using tetramer analysis, we demonstrate that ChgA-reactive T cells are present in these mice but remain naive. In contrast, in NOD.ChgA(+/+) mice, a majority of the ChgA-reactive T cells are Ag experienced. Our results suggest that the presence of ChgA and subsequent activation of ChgA-reactive T cells are essential for the initiation and development of autoimmune diabetes in NOD mice.

N-terminal additions to the WE14 peptide of chromogranin A create strong autoantigen agonists in type 1 diabetes.

Chromogranin A (ChgA) is an autoantigen for CD4(+) T cells in the nonobese diabetic (NOD) mouse model of type 1 diabetes (T1D). The natural ChgA-processed peptide, WE14, is a weak agonist for the prototypical T cell, BDC-2.5, and other ChgA-specific T-cell clones. Mimotope peptides with much higher activity share a C-terminal motif, WXRM(D/E), that is predicted to lie in the p5 to p9 position in the mouse MHC class II, IA(g7) binding groove. This motif is also present in WE14 (WSRMD), but at its N terminus. Therefore, to place the WE14 motif into the same position as seen in the mimotopes, we added the amino acids RLGL to its N terminus. Like the other mimotopes, RLGL-WE14, is much more potent than WE14 in T-cell stimulation and activates a diverse population of CD4(+) T cells, which also respond to WE14 as well as islets from WT, but not ChgA(-/-) mice. The crystal structure of the IA(g7)-RLGL-WE14 complex confirmed the predicted placement of the peptide within the IA(g7) groove. Fluorescent IA(g7)-RLGL-WE14 tetramers bind to ChgA-specific T-cell clones and easily detect ChgA-specific T cells in the pancreas and pancreatic lymph nodes of NOD mice. The prediction that many different N-terminal amino acid extensions to the WXRM(D/E) motif are sufficient to greatly improve T-cell stimulation leads us to propose that such a posttranslational modification may occur uniquely in the pancreas or pancreatic lymph nodes, perhaps via the mechanism of transpeptidation. This modification could account for the escape of these T cells from thymic negative selection.

Autoantibodies to chromogranin A are potential diagnostic biomarkers for non-small cell lung cancer.

Cancer-associated autoantibodies show promise as sensitive biomarkers for the early detection of cancer. To test the immunogenicity of chromogranin A (ChgA) as a B cell autoantigen and to assess the potential applications of ChgA autoantibodies as novel biomarkers for the diagnosis of non-small cell lung cancer (NSCLC), we developed a high-content peptide microarray using ChgA peptides. Autoantibody profiling was carried out using sera from 168 individuals with NSCLC and 97 healthy controls. We present evidence for the occurrence of autoantibodies to ChgA peptides in patient sera and identified five highly responsive peptides in the NSCLC group using significance analysis of microarray (SAM). Receiver operating characteristic analyses showed that ChgA autoantibodies are valuable in the predictive diagnosis of NSCLC, suggesting that serum autoantibodies to ChgA-derived peptides are promising novel markers of NSCLC. Moreover, the high-content peptide microarray antibody profiling reported in this work provides a powerful tool to visualize the overall B cell response to ChgA peptides and should enable the rapid development of in-depth research into ChgA.

The T Cell Repertoire-Diversifying Enzyme TSSP Contributes to Thymic Selection of Diabetogenic CD4 T Cell Specificities Reactive to ChgA and IAPP Autoantigens.

Multiple studies highlighted the overtly self-reactive T cell repertoire in the diabetes-prone NOD mouse. This autoreactivity has primarily been linked to defects in apoptosis induction during central tolerance. Previous studies suggested that thymus-specific serine protease (TSSP), a putative serine protease expressed by cortical thymic epithelial cells and thymic dendritic cells, may edit the repertoire of self-peptides presented by MHC class II molecules and shapes the self-reactive CD4 T cell repertoire. To gain further insight into the role of TSSP in the selection of self-reactive CD4 T cells by endogenous self-Ags, we examined the development of thymocytes expressing distinct diabetogenic TCRs sharing common specificity in a thymic environment lacking TSSP. Using mixed bone marrow chimeras, we evaluated the effect of TSSP deficiency confined to different thymic stromal cells on the differentiation of thymocytes expressing the chromogranin A-reactive BDC-2.5 and BDC-10.1 TCRs or the islet amyloid polypeptide-reactive TCR BDC-6.9 and BDC-5.2.9. We found that TSSP deficiency resulted in deficient positive selection and induced deletion of the BDC-6.9 and BDC-10.1 TCRs, but it did not affect the differentiation of the BDC-2.5 and BDC-5.2.9 TCRs. Hence, TSSP has a subtle role in the generation of self-peptide ligands directing diabetogenic CD4 T cell development. These results provide additional evidence for TSSP activity as a novel mechanism promoting autoreactive CD4 T cell development/accumulation in the NOD mouse.

Identification of autoreactive CD8+ T cell responses targeting chromogranin A in humanized NOD mice and type 1 diabetes patients.

ChgA has recently been identified as the autoantigen for diabetogenic CD4(+) T cells in NOD mice and T1D patients. However, autoreactive CD8(+) T-cell responses targeting ChgA haven't been studied yet. Here several HLA-A*0201-restricted peptides derived from mChgA and hChgA were selected by an integrated computational prediction approach, followed by an HLA-A*0201 binding assay. MChgA10-19 and mChgA(43-52) peptides, which bound well with HLA-A*0201 molecule, induced significant proliferation and IFN-γ-releasing of splenocytes from diabetic NOD.ß2m(null).HHD mice. Notably, flow cytometry analysis found that mChgA(10-19) and mChgA(43-52) stimulated the production of IFN-γ, perforin, and IL-17 by splenic CD8(+) T cells of diabetic NOD.ß2m(null).HHD mice. Furthermore, hChgA(10-19) and hChgA(43-52)-induced IFN-γ releasing by specific CD8(+) T cells were frequently detected in recent-onset HLA-A*0201-positive T1D patients. Thus, this study demonstrated that autoreactive CD8(+) T cells targeting ChgA were present in NOD.ß2m(null).HHD mice and T1D patients, and might contribute to pathogenesis of T1D through secreting proinflammatory cytokines and cytotoxic molecules.

[The role of chromogranin-A and its derived peptide, WE-14 in the development of type 1 diabetes mellitus]. / A kromogranin-A és a belole lehasadó WE-14 szerepe az 1-es típusú cukorbetegség kialakulásában.

Chromogranin-A is a member of the granine protein family. It is produced in neuroendocrine cells via secretory granules. Many cleavage proteins are formed from chromogranin-A, from which some have well known biological activity, while the function of others is not yet fully known. Serum chromogranin-A levels are used in neuroendocrine tumour diagnostics. Recent studies showed that one of its cleavage protein, WE-14 may also play a role in the development of type 1 diabetes. WE-14 may function as an autoantigen for T-cells involved in the destruction of ß-cells. This mechanism was previously observed only in non-obese diabetic mice. Novel results show that WE-14 also serves as a target for autoreactive cells in newly diagnosed type 1 diabetic patients as well, which reaction can be increased with transglutaminase. In this paper the authors summarize the recent knowledge about chromogranin-A and its potential role in the pathomechanism of type 1 diabetes mellitus.

Pancreastatin-dependent inflammatory signaling mediates obesity-induced insulin resistance.

Chromogranin A knockout (Chga-KO) mice exhibit enhanced insulin sensitivity despite obesity. Here, we probed the role of the chromogranin A-derived peptide pancreastatin (PST: CHGA(273-301)) by investigating the effect of diet-induced obesity (DIO) on insulin sensitivity of these mice. We found that on a high-fat diet (HFD), Chga-KO mice (KO-DIO) remain more insulin sensitive than wild-type DIO (WT-DIO) mice. Concomitant with this phenotype is enhanced Akt and AMPK signaling in muscle and white adipose tissue (WAT) as well as increased FoxO1 phosphorylation and expression of mature Srebp-1c in liver and downregulation of the hepatic gluconeogenic genes, Pepck and G6pase. KO-DIO mice also exhibited downregulation of cytokines and proinflammatory genes and upregulation of anti-inflammatory genes in WAT, and peritoneal macrophages from KO mice displayed similarly reduced proinflammatory gene expression. The insulin-sensitive, anti-inflammatory phenotype of KO-DIO mice is masked by supplementing PST. Conversely, a PST variant peptide PSTv1 (PST-NΔ3: CHGA(276-301)), lacking PST activity, simulated the KO phenotype by sensitizing WT-DIO mice to insulin. In summary, the reduced inflammation due to PST deficiency prevented the development of insulin resistance in KO-DIO mice. Thus, obesity manifests insulin resistance only in the presence of PST, and in its absence obesity is dissociated from insulin resistance.

Vasostatin-1 antigenic epitope mapping for induction of cellular and humoral immune responses to chromogranin A autoantigen in NOD mice.

The chromogranin A (ChgA) 29-42 sequence is the antigenic epitope for the BDC2.5 CD4(+) T-cell receptor in NOD mice (H-2(g7) ). We have now characterized the binding register of the ChgA 29-42 peptide for the I-A(g7) molecule. Truncation of the peptide demonstrated that the KCVLEVISD sequence 34-42 is the binding register and extension of this sequence by flanking residues increased its binding affinity and antigenic capacity. We employed anti-ChgA peptide antibodies generated against different fragments of ChgA for immunostaining of pancreatic islet sections from NOD mice. A strong immuno-staining pattern was observed for the ChgA 17-38 peptide antibodies that overlap with the ChgA 29-42 sequence. Moreover, sera from diabetic NOD mice showed elevated titers of autoantibodies to the ChgA 29-42 peptide. These findings indicate that peptides from the N-terminal region of ChgA are able to induce cellular and humoral immune responses in NOD mice.

Catestatin-like immunoreactivity in the rat eye.

The aim of the study was to investigate the presence and distribution of the chromogranin A-derived peptide catestatin in the rat eye and trigeminal ganglion by immunofluorescence using an antibody which recognizes not only free catestatin but also larger fragments containing the sequence of catestatin. Western blots were performed in an attempt to characterize the immunoreactivities detected by the catestatin antiserum. Sparse immunoreactive nerve fibers were visualized in the corneal stroma, in the chamber angle, in the sphincter muscle but also in association with the dilator muscle, in the stroma of the ciliary body and processes, but dense in the irideal stroma, around blood vessels at the limbus and in the choroid and in cells of the innermost retina representing amacrine cells as identified by colocalization with substance P. Furthermore, catestatin-immunoreactivity was detected in the trigeminal ganglion in small to medium-sized cells and there were abundant catestatin-positive nerve fibers stained throughout the stroma of the ganglion. Double immunofluorescence of catestatin with substance P revealed colocalization both in cells of the trigeminal ganglion as well as in nerve fibers in the choroid. The immunoreactivities are present obviously as free catestatin and/or small-sized catestatin-containing fragments in the retina and ocular nerves but as large processed fragments as well, weak in the retina and more prominent in remaining ocular tissues, possibly in endothelial cells. This indicates that this peptide is a constituent of sensory neurons innervating the rat eye and the presence in amacrine cells in the retina is typical for neuropeptides. Catestatin is biologically highly active and might be of significance in the pathophysiology of the eye.

Chromogranin A is a T cell antigen in human type 1 diabetes.

Chromogranin A (ChgA) is a beta cell secretory granule protein and a peptide of ChgA, WE14, was recently identified as a ligand for diabetogenic CD4 T cell clones derived from the NOD mouse. In this study we compared responses of human CD4 T cells from recent onset type 1 diabetic (T1D) and control subjects to WE14 and to an enzymatically modified version of this peptide. T cell responders to antigens were detected in PBMCs from study subjects by an indirect CD4 ELISPOT assay for IFN-γ. T1D patients (n = 27) were recent onset patients within one year of diagnosis, typed for HLA-DQ8. Controls (n = 31) were either 1st degree relatives with no antibodies or from the HLA-matched general population cohort of DAISY/TEDDY. A second cohort of patients (n = 11) and control subjects (n = 11) was tested at lower peptide concentrations. We found that WE14 is recognized by T cells from diabetic subjects vs. controls in a dose dependent manner. Treatment of WE14 with transglutaminase increased reactivity to the peptide in some patients. This work suggests that ChgA is an important target antigen in human T1D subjects and that post-translational modification may play a role in its reactivity and relationship to disease.