Search

Therapeutic efficacy of P277 against type 1 diabetes was extensively investigated and clinically evidenced. Clinical trials Phases I and II concluded promising results, while the data of P277 immunogenicity in Phase III trials represented weak responses that led to abolish medical use. But, a therapeutic performance of P277 cannot be forgotten. So, in order to exploit its therapeutic benefits and improve its immunogenicity, we developed a new analogue VP to optimize therapeutic efficacy and enhancing immunosuppressive modulations. However, new analogue was purified, and then used to immunize diabetic NOD mice to investigate antidiabetic effects through modulation of immunological status. So, DCs immune responses, relative TLRs, MyD88, and NF-Kβ1 mRNA expression on DCs and splenocytes under VP effect were tested. Circulating and intracellular cytokines were also evaluated at treated and non-treated mice. Splenic T lymphocytes proliferation (Th1 and Treg cells) were also determined. Results revealed that VP significantly down regulates DCs maturation through TLR2, TLR4, and MyD88 pathways. It also shifts DCs to a tolerogenic polarization through NF-Kβ1 pathway that mediates Th1 immunosuppression and enhances iTreg expanding in type1diabetes mice. Meanwhile, we noticed that VP significantly enhances iTreg CD25 + FoxP3+ proliferation. In conclusion, VP showed promising immune potential to modulate immune regulatory responses and shifts DCs to suppress autoreactive Th1 cells which ameliorated immunosuppressive potency in the type1 diabetic mice.

Therapeutic efficacy of P277 against type 1 diabetes was extensively investigated and clinically evidenced. Clinical trials Phases I and II concluded promising results, while the data of P277 immunogenicity in Phase III trials represented weak responses that led to abolish medical use. But, a therapeutic performance of P277 cannot be forgotten. So, in order to exploit its therapeutic benefits and improve its immunogenicity, we developed a new analogue VP to optimize therapeutic efficacy and enhancing immunosuppressive modulations. However, new analogue was purified, and then used to immunize diabetic NOD mice to investigate antidiabetic effects through modulation of immunological status. So, DCs immune responses, relative TLRs, MyD88, and NF-Kβ1 mRNA expression on DCs and splenocytes under VP effect were tested. Circulating and intracellular cytokines were also evaluated at treated and non-treated mice. Splenic T lymphocytes proliferation (Th1 and Treg cells) were also determined. Results revealed that VP significantly down regulates DCs maturation through TLR2, TLR4, and MyD88 pathways. It also shifts DCs to a tolerogenic polarization through NF-Kβ1 pathway that mediates Th1 immunosuppression and enhances iTreg expanding in type1diabetes mice. Meanwhile, we noticed that VP significantly enhances iTreg CD25 + FoxP3+ proliferation. In conclusion, VP showed promising immune potential to modulate immune regulatory responses and shifts DCs to suppress autoreactive Th1 cells which ameliorated immunosuppressive potency in the type1 diabetic mice. [ABSTRACT FROM AUTHOR]

Previous evidence has proved the ability of immunization with heat shock protein (HSP) 60/65 to induce atherosclerosis. P277, a 24-residue peptide of human HSP60, is a promising peptide vaccine against autoimmune diabetes. But as a fragment of HSP60, its potential ability of promoting atherosclerosis has never been investigated yet. In the present study, the rabbits fed with normal standard diet or high cholesterol diet were immunized with P277 or PBS emulsified in incomplete Freund's adjuvant 4 times at 4-week intervals. Atherosclerotic lesions of the rabbits receiving P277 treatment and fed with high cholesterol diet increased significantly compared with those of the rabbits receiving PBS treatment and the same diet. However, no obvious lesions were found in the two groups of rabbits fed with the normal standard diet. Significant expression of P277 was detected in the high cholesterol diet-induced atherosclerotic lesions and heat-stressed endothelial cells. Surface exposure of P277 was also observed in the stressed cells. In the subsequent assay of endothelial cells in vitro , the purified anti-P277 antibodies mediated a noticeable cytotoxicity to the stressed cells with the participation of complement. In conclusion, subcutaneous immunization with P277 emulsified in IFA can aggravate the atherosclerosis in high cholesterol diet-fed rabbits. Surface expression of P277 was observed on stressed endothelial cells, and were suggested to mediate the autoimmune attack and promote the disease. [ABSTRACT FROM AUTHOR]

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of pancreatic beta cells leading to inadequate glycemic control. Trials with immunomodulatory monotherapies have shown that the disease course can in principle be altered. The observed preservation of endogenous insulin secretion however is typically transient and chronic treatment is often associated with significant side effects. Here we combined anti-CD3 with the Hsp60 peptide p277, two drugs that have been evaluated in Phase 3 trials, to test for enhanced efficacy. Female NOD mice with recent onset diabetes were given 5 μg anti-CD3 i.v., on three consecutive days in combination with 100 μg of p277 peptide in IFA s.c., once weekly for four weeks. Anti-CD3 alone restored normoglycemia in 44% of the mice while combination therapy with anti-CD3 and p277 induced stable remission in 83% of mice. The observed increase in protection occurred only in part through TLR2 signaling and was characterized by increased Treg numbers and decreased insulitis. These results have important implications for the design of combination therapies for the treatment of T1D. [ABSTRACT FROM AUTHOR]

Abstract: P277 is a peptide derived from the HSP60 regions, have potent immunological effect on insulin-dependent diabetes mellitus (IDDM) and its phase III clinical trials are currently under investigation. However, we recently discovered a positive correlation between anti-P277 autoantibodies and the presence of endothelial cells damage in inducing vascular leak syndrome. Therefore, the aim of our study was to demonstrate the critical peptide epitope of P277 to IDDM and to highlight the effects of this peptide therapy on inflammation of the islets. Groups of 4-week old female non-obese diabetic (NOD) mice were immunized one time every three weeks for three times with a residue of P277, showing a significant effect of down-regulating immunity to P277 protein and preventing the development of IDDM. Immunologic results including the suppression of T-cell proliferation, the increase of interleukin-10 (IL-10) production and reduction of interferon-γ (IFN-γ) production caused immune tolerance to P277. Hence, a functional role of the key epitope in P277 peptide capable of preventing IDDM is suggested, which could be modified to develop a novel safe and effective peptide vaccine against IDDM by reconstructing P277 in the further studies. [Copyright &y& Elsevier]

Abstract: To improve the weak immunogenicity of peptide P277, the recombinant expression plasmid pET28-Hsp65-6×P277 was constructed by inserting 5×P277 which was amplified by PCR into the vector pET28-Hsp65-P277. It was transformed into Escherichia coli BL21 (DE3) and the fusion protein (Hsp65-6×P277) was expressed effectively as soluble protein after inducing by lactose. The fusion protein was purified by means of cell disruption, ammonium sulfate precipitation, double-distilled H2O dialysis, DEAE52-cellulose column chromatography, and then used to immunize female NOD mice with three i.n. inoculations in the absence of adjuvants. Serum samples from the immunized mice were collected at 3 weeks interval. Antibodies against P277 and HSP65 were detected in immunized mice sera by enzyme-linked immunosorbent assay (ELISA) and Western blot. Specific antibodies were successfully induced and lasted for more than 20 weeks in animals immunized with the fusion protein via intranasal route even in the absence of adjuvants. [Copyright &y& Elsevier]