Role of TGF-ß in Self-Peptide Regulation of Autoimmunity.

Transforming growth factor (TGF)-ß has been implicated in regulation of the immune system, including autoimmunity. We have found that TGF-ß is readily produced by T cells following immunization with self-peptide epitopes that downregulate autoimmune responses in type 1 diabetes (T1D) prone nonobese diabetic (NOD) mice. These include multiple peptide epitopes derived from the islet ß-cell antigens GAD65 (GAD65 p202-221, GAD65 p217-236), GAD67 (GAD67 p210-229, GAD67 p225-244), IGRP (IGRP p123-145, IGRP p195-214) and insulin B-chain (Ins. B:9-23) that protected NOD mice from T1D. Immunization of NOD mice with the self-MHC class II I-Ag7 ß-chain-derived peptide, I-Aßg7 p54-76 also induced large amounts of TGF-ß and also protected these mice from diabetes development. These results indicate that peptides derived from disease related self-antigens and MHC class II molecules primarily induce TGF-ß producing regulatory Th3 and Tr1-like cells. TGF-ß produced by these cells could enhance the differentiation of induced regulatory iTreg and iTreg17 cells to prevent induction and progression of autoimmune diseases. We therefore suggest that peripheral immune tolerance could be induced and maintained by immunization with self-peptides that induce TGF-ß producing T cells.

Enhanced Synthesis of Curculigoside by Stress and Amino Acids in Static Culture of Curculigo orchioides Gaertn (Kali Musli).

BACKGROUND: Curculigo orchioides Gaertn (Kali musli; Family: Hypoxidaceae) is an endangered medicinal plant used for many medicinal purposes such as impotency, aphrodisiac, tonic, jaundice, and skin ailments. Its hepatoprotective, antioxidant, and anti-cancerous potential have also been evaluated by many scientists. OBJECTIVE: The objective of this study is to enhance the curculigoside content in tissue culture of C. orchioides. MATERIALS AND METHODS: The present study deals with the enhancement of an active compound of C. orchioides by incorporating various concentration of phenylalanine (Phe), tyrosine, (20, 40, 60, and 80 mg/100 ml), chromium (Cr) and nickel (Ni) (1, 2, 3, 4, and 5 ppm) into Zenk media in controlled and aseptic conditions. RESULTS: Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and industrially important biochemicals. Accumulation of such metabolites often occurs in plants subjected to stresses including various elicitors or signal molecules. A significantly remarkable enhancement in all induced samples was noted. Curculigoside content was maximum in the 6-week-old tissue induced with 3 ppm of Cr (7.63%) followed by 4 weeks tissue of tissue fed with 4 ppm of Ni (5.66%) and 4-week-old tissue fed with tyrosine 7.5 mg/100 ml (2.38%) among all samples used. These results suggest that tyrosine is better enhancer than Phe in the biosynthetic pathway of curculigoside. The presence of curculigoside in all extracts was confirmed by Fourier transform infrared spectroscopy, high-performance thin layer chromatography analysis with standard compound of curculigoside and histology of treated samples. CONCLUSION: This investigation was carried out for the 1(st) time, and it is a significant step in understanding the biochemistry of curculigoside. The developed protocol will be beneficial for marketing in pharmaceutical industries. SUMMARY: Curculigo orchioides Gaertn (Kali musli; Family: Hypoxidaceae) is an endangered medicinal plant used for many medicinal purposes such as impotency, aphrodisiac, tonic, jaundice, and skin ailments.It was observed that dry matter % was maximum in 6-week-old tissue fed with 2.5 mg/100 ml of tyrosine and diminished beyond this concentration among all samples usedThe nickel (Ni) and chromium (Cr) stress has enhanced the curculigoside in considerable amount in nontoxic range, in tissue culture of C. orchioides.Curculigoside content was maximum in 6-week-old tissue induced with 3 ppm of Cr (7.63%; 11-fold enhancement) followed by 4 weeks tissue of tissue fed with 4 ppm of Ni (5.66%) and 4-week-old tissue fed with tyrosine 7.5 mg/100 ml (2.38%) among all samples used. Histological studies confirmed the enhanced production of curculigoside. Abbreviations Used: Phe: Phenylalanine, PAL: Phenylalanine ammonia-lyase, mM: mille Molar, Cr: Chromium, Ni: Nickel, HPTLC: High-performance thin layer chromatography.

Dendritic cell differentiation induced by a self-peptide derived from apolipoprotein E.

Dendritic cells (DCs) are professional APCs and potent stimulators of naive T cells. Since DCs have the ability to immunize or tolerize T cells they are unique candidates for use in immunotherapy. Our laboratory has discovered that a naturally processed self-peptide from apolipoprotein E, Ep1.B, induces DC-like morphology and surface marker expression in a murine monocytic cell line (PU5-1.8), human monocytic cell line (U937), murine splenocytes, and human peripheral blood monocytes. Microscopy and flow cytometric analysis revealed that Ep1.B-treated cells display decreased adherence to plastic and increased aggregation, dendritic processes, and expression of DC surface markers, including DEC-205, CD11c, B7.1, and B7.2. These effects were observed in both PU5-1.8 cells and splenocytes from various mouse strains including BALB/c, C57BL/6, NOD/Lt, and C3H/HeJ. Coadministration of Ep1.B with OVA antigenic peptide functions in dampening specific immune response to OVA. Ep1.B down-regulates proliferation of T cells and IFN-gamma production and stimulates IL-10 secretion in immunized mice. Ep1.B-induced differentiation resulted in the activation of PI3K and MAPK signaling pathways, including ERK1/2, p38, and JNK. We also found that NF-kappaB, a transcription factor essential for DC differentiation, is critical in mediating the effects of Ep1.B. Ep1.B-induced differentiation is independent of MyD88-dependent pathway of TLR signaling. Cumulatively, these findings suggest that Ep1.B acts by initiating a signal transduction cascade in monocytes leading to their differentiation into DCs.

Exogenous CLIP localizes into endocytic compartment of cells upon internalization: implications for antigen presentation by MHC class II molecules.

We have previously shown that exogenous CLIP (class II associated invariant chain peptide) downregulated MHC class II expression on antigen presenting cells (APC) and modulated T cell mediated immune responses. The present study was undertaken to investigate the mechanism of uptake of exogenously added CLIP peptide by APC. We found that exogenous CLIP is rapidly internalized by APC and it co-localize with MHC class II in intracellular compartments including early-, late-endosomes and lysosomes. We suggest that exogenous CLIP acts as an in vivo regulator of immune response by internalization and passage through the intracellular compartments where it interferes in peptide loading and recycling of MHC class II molecules to the APC surface. Therefore, exogenous CLIP regulates immune responses by modulation of antigen presentation by the APC.

In vivo apoptosis of diabetogenic T cells in NOD mice by IFN-gamma/TNF-alpha.

Immunization with mycobacterial preparation such as Bacille Calmette-Guerin (BCG) or complete Freund's adjuvant (CFA) prevents the onset and recurrence of type 1 diabetes in non-obese diabetic (NOD) mice. In this study, we explored the mechanism underlying the down-regulation of diabetogenic T cells by BCG treatment. We found that the potential of splenocytes from BCG-immunized diabetic NOD mice to adoptively transfer diabetes was significantly impaired. BCG immunization sequentially induced the production of TNF-alpha, IFN-gamma and IL-4 by splenocytes, increased the expression of Fas(high) (Apo-1/CD95), Fas ligand (FasL, CD95L) and TNF receptor (TNFR) on T cells leading to T cell apoptosis. The primary role of IFN-gamma and TNF-alpha in BCG-immunotherapy was demonstrated by (i) reversing the immune regulatory effect of BCG by in vivo treatment with neutralizing anti-cytokine antibodies, (ii) inducing effect similar to BCG by treatment with these cytokines. We show that Fas and TNF are two pathways in BCG-induced apoptosis of diabetogenic T cells, since in vitro blocking FasL or TNFR1 with antibody reduced T cell apoptosis and increased T cell proliferative response. In addition, TNF-alpha and agonistic anti-Fas antibody had a synergistic effect on the in vitro apoptosis of diabetogenic T cells. Our results suggest that BCG down-regulates destructive autoimmunity by TNF-alpha/IFN-gamma-induced apoptosis of diabetogenic T cells through both Fas and TNF pathways. These studies provide a novel mechanism for blocking disease recurrence and immune modulating effect of BCG immunization in type 1 diabetes.

CD4+CD25+ regulatory T cells generated in response to insulin B:9-23 peptide prevent adoptive transfer of diabetes by diabetogenic T cells.

NOD mice have a relative deficiency of CD4+CD25+ regulatory T cells that could result in an inability to maintain peripheral tolerance. The aim of this study was to induce the generation of CD4+CD25+ regulatory T cells in response to autoantigens to prevent type 1 diabetes (T1D). We found that immunization of NOD mice with insulin B-chain peptide B:9-23 followed by 72 h in vitro culture with B:9-23 peptide induces generation of CD4+CD25+ regulatory T cells. Route of immunization has a critical role in the generation of these cells. Non-autoimmune mice BALB/c, C57BL/6 and NOR did not show up regulation of CD4+CD25+ regulatory T cells. These cells secreted large amounts of TGF-beta and TNF-alpha with little or no IFN-gamma and IL-10. Adoptive transfer of these CD4+CD25+ regulatory T cells into NOD-SCID mice completely prevented the adoptive transfer of disease by diabetogenic T cells. Although, non-self antigenic OVA (323-339) peptide immunization and in vitro culture with OVA (323-339) peptide does result in up regulation of CD4+CD25+ T cells, these cells did not prevent transfer of diabetes. Our study for the first time identified the generation of antigen-specific CD4+CD25+ regulatory T cells specifically in response to immunization with B:9-23 peptide in NOD mice that are capable of blocking adoptive transfer of diabetes. Our results suggest the possibility of using autoantigens to induce antigen-specific regulatory T cells to prevent and regulate autoimmune diabetes.

Type 1 diabetes alters anti-hsp90 autoantibody isotype.

The 90-kDa chaperon family includes heat shock protein (hsp) 90 and glucose-regulated protein (grp) 94. These proteins play an important role in normal cellular architecture, in the etiology of some autoimmune and infectious diseases and in antigen presentation to T cells. Owing to its role in autoimmunity, we explored anti-hsp90 autoantibody (hsp90AA) response in the sera of persons with type 1 diabetes, first-degree relatives (FDR) and in normal subjects. Significant high level of hsp90AA was found in FDR, but there was no significant difference between the normal and diabetic persons. The IgG1 and IgG3 isotypes of hsp90AA were higher in persons with type 1 diabetes and FDR than in normal subjects. We found a good correlation between hsp90AA measured by ELISA and RIA. A positive correlation between serum hsp90AA and glutamic acid decarboxylase (GAD65) autoantibody (GAA) was also observed. Hsp90AA positive sera from diabetic persons immunoblotted recombinant hsp90, GAD65 and corresponding proteins in islet lysates. Our study suggests that hsp90AA are present in normal, FDR and diabetic persons. However, there is a higher level of IgG1 and IgG3 isotypes of hsp90AA in FDR and type 1 diabetic subjects. Thus, autoimmunity leading to type 1 diabetes significantly alters the autoantibody isotype to autoantigens, such as hsp90.