Relationship between T-lymphocyte cytokine levels and sero-response to hepatitis B vaccines

AIM: To investigate the cellular defects by analyzing the (Th1/Th2) cytokine levels in vaccine responders and non-responders. METHODS: Peripheral blood mononuclear cell (PBMC) from responders and non-responders were stimulated with or with out recombinant HBsAg or PHA. Broad spectrum of cytokines viz (Th1) IFN-γ, IL-2, TNF-α, IL-12 and (Th2) IL-10, IL-4 were measured after in vitro stimulation with recombinant HBsAg and were compared with respective antibody titers. RESULTS: A significant decrease (P = 0.001) in Th1 and Th2 cytokines namely, IL-2, INF-γ, TNF-α and IL-10in non-responders was observed. The level of IL-4 was not significant between the three groups. Furthermore, despite a strong Th1 and Th2 cytokine response, the level of IL-12 was elevated in high-responders compared to other groups (P = 0.001) and demonstrated a positive correlation with anti-HBs titers and Th1 cytokine response. CONCLUSION: Our findings suggest that unrespon-siveness to recombinant hepatitis B vaccines (rHB) is multifactorial, including specific failure of antigen presentation or the lack of both T helper Th1 and Th2 response.

Consumption of purple sweet potato leaves modulates human immune response: T-lymphocyte functions, lytic activity of natural killer cell and antibody production

AIM： To study the immunological effects of physiologica doses of purple sweet potato leaves （PSPL）. METHODS： The randomized crossover study （two periods, each lasting for 2 wk） involved 16 healthy non-smoking adults of normal weight. The 6-wk study consisted of a run-in （wk 1） PSPL diet （daily consumption of 200 g PSPL） or a control diet （low polyphenols, with the amount of carotenoids adjusted to the same level as that of PSPL） （wk 2-3）, washout diet （wk 4）, and switched diet （wk 5-6）. Fasting blood was collected weekly in the morning. T-lymphocyte function was assessed via the proliferation and secretion of immunoreactive cytokines. Salivary IgA secretion and the specific cytotoxic activities of cytotoxic T lymphocytes and natural killer （NK） cells were determined. RESULTS： The plasma 13-carotene level increased with time in both groups, while the plasma polyphenol level decreased in the control group, and no significant difference was detected between the two groups. Although p^asma polyphenol levels did not significantly increase in the PSPL group at the end of the study, they were significantly elevated in urine. PSPL consumption produced a significant increase in proliferation responsiveness of peripheral blood mononuclear cells （PBMC） and their secretion of immunoreactive IL-2 and IL-4. As well, lytic activity in NK cells was elevated in a time-dependent fashion. Salivary IgA secretion significantly decreased in control group after 2 wk, and returned to baseline following dietary switch to PSPL. CONCLUSION： Consumption of PSPL modulates various immune functions including increased proliferation responsiveness of PBMC, secretion of cytokines IL-2 and IL-4, and the lytic activity of NK cells. The responsible determinants of PSPL remain to be elucidated, as does the biological significance of the present observations.

T cell immune response is correlated with fibrosis and inflammatory activity in hepatitis B cirrhotics

AIM： TO explore the relationship among interferon-γ （IFN-γ） activity, fibrogenesis, T cell immune responses and hepatic inflammatory activity. METHODS： Peripheral blood samples from a total of 43 hepatitis B cirrhotic patients （LC） and 19 healthy controls （NC） were collected to measure their serum levels of IFN-γ, interleukin-2 （IL-2）, soluble interleukin-2 receptor （sIL-2R）, interleukin-10 （IL-10） and three serological markers of fibrosis including hyaluronic acid （HA）, procollagen type III peptide （PIIIP）, and type iV collagen were measured using a double antibody sandwich ELISA. Also, serum total bilirubin （TB） and alanine aminotransferase （ALT） were measured by routine measures. RESULTS： The concentrations of serological markers of fibrosis in patients with active cirrhosis （ALC） were significantly higher than those in stationary liver cirrhosis （SLC） or NC groups. The levels of serological markers in HBeAg-positive patients were significantly higher than those in HBeAg-negative patients. In SLC and ALC patients, a negative linear correlation was found between IFN-γ levels and the serological markers of fibrosis. IFN-γ and IL-2 levels in the ALC group were significantly higher than those in the SLC and NC groups, but the statistical difference was not significant between the latter two. In contrast, IL-10 levels in the SLC group were significantly higher than that in the NC group, but no significant difference was found between SLC and ALC groups. The sIL-2R level was elevated gradually in all these groups, and the differences were significant. Positive linear correlations were seen between IFN-γ activity and ALT levels （r = 0.339, P 〈 0.05）, and IL-2 activity and TB levels （r = 0.517, P 〈 0.05）. sIL-2R expression was positively correlated with both ALT and TB levels （r = 0.324, 0.455, P 〈 0.05）, whereas there was no statistically significant correlation between IL-10 expression and serum ALT and TB levels （r = -0.102, -0.093, P 〉 0.05）. Finally, there was a positive correlation between IFN-γ and IL-2 levels. CONCLUSION： T cell immune responses are correlated with fibrosis and hepatic inflammatory activity and may play an important role in liver cirrhosis.

Induction of hepatitis C virus-specific cytotoxic T and B cell responses by dendritic cells expressing a modified antigen targeting receptor

AIM: To find a novel antigen (Ag) presentation strategy to improve the immune responses induced by dendritic cell (DC)vaccine expressing hepatitis C virus (HCV) core antigen (pcDNA3HCV C-Fc) in Balb/c mice (H-2d).METHODS: pcDNA3HCV C-Fc plasmid and eukaryotic expression vector pcDNA3 were injected into mice sc. Immune responses to pcDNA3HCV C-Fc were studied. Meanwhile the effect of pcDNA3HCV C-Fc on anti-translated subcutaneous tumor of SP2/0 cells stably expressing HCV C Ag (SP2/0-HCV C-FC) was also studied. Anti-HCV C in serum was detected by enzyme-linked immunoadsordent assay (ELISA) and HCV specific cytotoxic T lymphocyte (CTL) activity was measured by LDH release assay. After 3 wk of DNA immunization,the cells of SP2/0-HCV C-FC were inoculated into mice subcutaneously and tumor growth was measured every 5 d.The survival rate and living time of mice were also calculated.RESULTS: After 4 wk of DC immunization, the A450 nm values of sera in mice immunized with pcDNA3HCV C-Fc-DC and pcDNA3-DC were 0.56±0.17 and 0.12±0.03 respectively. The antibody titres in mice codeliveried with pcDNA3HCV C-Fc with DC were significantly higher than those of mice injected with pcDNA3-DC. The HCV specific CTL activities in mice coinjected with DC and pcDNA3HCV C-Fc or empty expression vectors were(73.2±3.1) % and (24.4±8.8) %, which were significantly higher than those of mice injected with water.The DC vaccine could evidently inhibit tumor growth, prolong the survival time of mice and improve the survival rate of mice and these effects could be improved by HCV C-Fc (pcDNA3HCV C-Fc) gene codelivered.CONCLUSION: DC vaccine has a strong antigenicity in humoral and cellular immunities, which can be promoted by transduced pcDNA3HCV C-Fc expressing HCV C or Fc.Thus, pcDNA3HCV C-Fc-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.

Dendritic cells engineered to secrete anti-Dc R3 antibody augment cytotoxic T lymphocyte response against pancreatic cancer in vitro

AIM To investigate the enhanced cytotoxic T lymphocyte responses against pancreatic cancer (PC) in vitro induced by dendritic cells (DCs) engineered to secrete anti-DcR3 monoclonal antibody (mAb). METHODS DCs, T lymphocytes and primary PC cells were obtained from PC patients. DCs were transfected with a designed humanized anti-DcR3 monoclonal antibody heavy and light chain mRNA and/or total tumor RNA (DC-tumor-anti-DcR3 RNA or DC-total tumor RNA) by using electroporation technology. The identification, concentration and function of anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA were determined by western blotting and enzyme-linked immunosorbent assay. After co-culturing of autologous isolated PC cells with target DCs, the effects of secreting anti-DcR3 mAb on RNA-DCs' viability and apoptosis were assessed by MTT assay and flow cytometry. Analysis of enhanced antigen-specific immune response against PC induced by anti-DcR3 mAb secreting DCs was performed using a Cr-51 releasing test. T cell responses induced by RNAloaded DCs were analyzed by measuring cytokine levels, including IFN-gamma, IL-10, IL4, TNF-alpha and IL-12. RESULTS The anti-DcR3 mAb secreted by DCs reacted with recombinant human DcR3 protein and generated a band with 35 kDa molecular weight. The secreting mAb was transient, peaking at 24 h and becoming undetectable after 72 h. After co-incubation with DCtumor- anti-DcR3 RNA for designated times, the DcR3 level in the supernatant of autologous PC cells was significantly down-regulated (P < 0.05). DCs secreting anti-DcR3 mAb could improve cell viability and slow down the apoptosis of RNA-loaded DCs, compared with DC-total tumor RNA (P < 0.01). The anti-DcR3 mAb secreted by DC-tumor-anti-DcR3 RNA could enhance the induction of cytotoxic T lymphocytes (CTLs) activity toward RNA-transfected DCs, primary tumor cells, and PC cell lines, compared with CTLs stimulated by DC-total tumor RNA or control group (P < 0.05). Meanwhile, the antigen-specific CTL responses were MHC class I-restricted. The CD4+ T cells and CD8+ T cells incubated with anti-DcR3 mAb secreting DCs could produce extremely higher level IFN-gamma and lower level IL4 than those incubated with DC-total tumor RNA or controls (P < 0.01). CONCLUSION DCs engineered to secrete anti-DcR3 antibody can augment CTL responses against PC in vitro, and the immune-enhancing effects may be partly due to their capability of down-regulating DC apoptosis and adjusting the Th1/Th2 cytokine network.

New perspectives in occult hepatitis C virus infection

Occult hepatitis C virus (HCV) infection, defined as the presence of HCV RNA in liver and in peripheral blood mononuclear cells (PBMCs) in the absence of detectable viral RNA in serum by standard assays, can be found in anti-HCV positive patients with normal serum levels of liver enzymes and in anti-HCV negative patients with persistently elevated liver enzymes of unknown etiology. Occult HCV infection is distributed worldwide and all HCV genotypes seem to be involved in this infection. Occult hepatitis C has been found not only in anti-HCV positive subjects with normal values of liver enzymes or in chronic hepatitis of unknown origin but also in several groups at risk for HCV infection such as hemodialysis patients or family members of patients with occult HCV. This occult infection has been reported also in healthy populations without evidence of liver disease. Occult HCV infection seems to be less aggressive than chronic hepatitis C although patients affected by occult HCV may develop liver cirrhosis and even hepatocellular carcinoma. Thus, anti-HCV negative patients with occult HCV may benefit from antiviral therapy with pegylated-interferon plus ribavirin. The persistence of very low levels of HCV RNA in serum and in PBMCs, along with the maintenance of specific T-cell responses against HCV-antigens observed during a long-term follow-up of patients with occult hepatitis C, indicate that occult HCV is a persistent infection that is not spontaneously eradicated. This is an updated report on diagnosis, epidemiology and clinical implications of occult HCV with special emphasis on anti-HCV negative cases.

CD4+ T cell responses in hepatitis C virus infection

Hepatitis C virus (HCV) infection is a major cause of liver damage, with virus-induced end-stage disease such as liver cirrhosis and hepatocellular carcinoma resulting in a high rate of morbidity and mortality worldwide. Evidence that CD4+ T cell responses to HCV play an important role in the outcome of acute infection has been shown in several studies. However, the mechanisms behind viral persistence and the failure of CD4+ T cell responses to contain virus are poorly understood. During chronic HCV infection, HCV-specific CD4+ T cell responses are rela- tively weak or absent whereas in resolved infection these responses are vigorous and multispecific. Persons with a T-helper type I profile, which promotes cellular effec- tor mechanisms are thought to be more likely to experi- ence viral clearance, but the overall role of these cells in the immunopathogenesis of chronic liver disease is not known. To define this, much more data is required on the function and specificity of virus-specific CD4+ T cells, especially in the early phases of acute disease and in the liver during chronic infection. The role and possible mechanisms of action of CD4+ T cell responses in deter- mining the outcome of acute and chronic HCV infection will be discussed in this review.

Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice

AIM： To study the induction of T cellular immune responses in BALB/c mice immunized with uric acid and dendritic cells （DCs） pulsed with hepatitis B virus surface antigen （HBsAg）. METHODS： DCs were generated from bone-marrow cells of BABL/c mice, and then pulsed or unpulsed with HBsAg protein （HBsAg-pulsed-DCs or unpulsed-DCs） in vitro. BABL/c mice were immunized with HBsAg-pulsed- DCs （1 × 10^6） and uric acid, injected through the tail vein of each mouse. The mice in control groups were immunized with HBsAg-pulsed-DCs alone, unpulsed- DCs alone or 200 μg uric acid alone or PBS alone. The immunization was repeated 7 d later. Cytotoxic T lymphocytes （CTLs） in vivo were determined by the CFSE labeled spleen lysis assay. Spleen cells or spleen T cells were isolated, and re-stimulated in vitro with HBsAg for 120 h or 72 h. Production of IFN-γ, and IL-4 secreted by spleen cells were determined by ELISA method; proliferation of spleen T cells were detected by flow cytometry. RESULTS： The cytotoxicities of HBsAg-specific-CTLs, generated after immunization of HBsAg-pulsed-DCs and uric acid, were 68.63% ±11.32% and significantly stronger than that in the control groups （P 〈 0.01）. Compared with control groups, in mice treated with uric acid and HBsAg-pulsed-DCs, the spleen T cell proliferation to HBsAg re-stimulation was stronger （1.34 ± 0.093 vs 1.081±0.028, P 〈 0.01）, the level of IFN-t, secreted by splenocytes was higher （266.575 ± 51.323 vs 135.223 ±32.563, P 〈 0.01） , and IL-4 level wasower （22.385 ± 2.252 vs 40.598 ± 4.218, P 〈 0.01）. CONCLUSION： Uric acid can strongly enhance T cell immune responses induced by HBsAg-pulsed-DCs vaccine. Uric acid may serve as an effective adjuvant of DC vaccine against HBV infection.

Immunization with autologous T cells enhances in vivo anti-tumor immune responses accompanied by up-regulation of GADD45β

Immunization with inactivated autoreactive T cells may induce idiotype anti-idiotypic reactions to deplete autoreactive T cells, which are involved in autoimmune diseases. However, it is unknown whether attenuated activated healthy autologous T-cell immunization could increase anti-tumor immune responses. To this end, C57B1/6 mice were immunized with attenuated activated autologous T cells. The splenocytes from immunized mice showed a higher proliferative ability than that from naive mice. The special phenotype analysis showed that there were more CD8+ T cells and CD62L+ T cells in immunized mice after 24 h of culture with 10% fetal calf serum complete medium in vitro (P〈0.01). These results demonstrated that this immunization may activate T cells in vivo. Furthermore, the splenocytes from immunized mice revealed resistance to activation-induced cell death (AICD) in vitro. To further study the relative genes that are responsible for the higher proliferation and resistance to AICD, the expression of Fas/Fas ligand (FasL) and GADD4513 was measured by real-time PCR. The results indicated that GADD45β transcription was higher in the splenocytes from immunized mice than that in the naive mice. In addition, the Fas expression showed a parallel higher, but FasL did not change obviously. To investigate the biologic functions induced by immunization in vivo, a tumor model was established by EL-4 tumor cell inoculation in C57/B1 mice. Mice receiving autologous T-cell immunization had significantly inhibited tumor growth in vivo (P〈0.01). This study implicated that immunization with attenuated activated autologous T cells enhances anti-tumor immune responses that participate in tumor growth inhibition.

A novel heterodimeric cytokine consisting of IL-17 and IL-17F regulates inflammatory responses

CD4＋ helper T （TH） cells play crucial roles in immune responses. Recently a novel subset of TH cells, termed THIL-17, TH 17 or inflammatory TH （THi）, has been identified as critical mediators of tissue inflammation. These cells produce IL-17 （also called IL-17A） and IL-17F, two most homologous cytokines sharing similar regulations. Here we report that when overexpressed in 293T cells, IL-17 and IL-17F form not only homodimers but also heterodimers, which we name as IL-17A/F. Fully differentiated mouse THi cells also naturally secrete IL-17A/F as well as IL-17 and IL-17F homodimeric cytokines. Recombinant IL-17A/F protein exhibits intermediate levels of potency in inducing IL-6 and KC （CXCL 1） as compared to homodimeric cytokines. IL-17A/F regulation of IL-6 and KC expression is dependent on IL-17RA and TRAF6. Thus, IL-17A/F cytokine represents another mechanism whereby T cells regulate inflammatory responses and may serve as a novel target for treating various immune-mediated diseases.

Induction of CD4＋CD25＋Foxp3＋ regulatory T cell response by glatiramer acetate in type 1 diabetes

Glatiramer acetate （GA） is an immunomodulatory peptide drug used to treat multiple sclerosis. Its treatment effect has been expanded to other autoimmune conditions such as uveoretinitis, inflammatory bowel disease, graft re- jection and hepatic fibrosis. Here, we report that GA was effective in altering the clinical course of diabetes in cyclo- phosphamide （CY）-potentiated non-obese diabetic （CY-NOD） mice. Treatment with GA significantly reduced the dia- betic rate in the mice and ameliorated insulitis, which coincided with increased CD4＋CD25＋Foxp3＋ T cell response in treated mice. GA treatment led to increased expression of transcription factor Foxp3 and elevated production of interleukin-4 （IL-4） both in vivo and in vitro. It was evident that the effect of GA on up-regulation of Foxp3 was me- diated partially through IL-4. IL-4 was found to maintain Foxp3 expression and regulatory function of CD4＋CD25＋ regulatory T cells （Tregs）. This study provides new evidence that GA has treatment potential for type 1 diabetes through the induction of Tregs and that increased IL-4 production is partially responsible for the enhanced Treg＇s function in GA treatment.

Characterization of a Putative Filovirus Vaccine:Virus-Like Particles

Filoviruses are hemorrhagic fever viruses endemic to parts of Africa and the Philippines. Infection carries with it a mortality rate of up to 90% and currently there are no effective vaccines or therapeutics available to combat infection. However, the filovirus virus-like particles (VLP), which are currently under development, have been shown to be a promising vaccine candidate. They provide protection from infection in the mouse, guinea pig, and nonhuman primate models of infection, eliciting high anti-glycoprotein antibody titers and T cell responses to viral proteins. In this review, we will highlight the development of the filovirus VLP and describe the current understanding of VLP immunogenicity and correlates of protection.

Potent T cell Responses Induced by Single DNA Vaccine Boosted with Recombinant Vaccinia Vaccine

Plasmid DNA, an effective vaccine vector, can induce both cellular and humoral immune responses. However, plasmid DNA raises issues concerning potential genomic integration after injection. This issue should be considered in preclinical studies. Tiantan vaccinia virus (TV) has been most widely utilized in eradicating smallpox in China. This virus has also been considered as a successful vaccine vector against a few infectious diseases. Potent T cell responses through T-cell receptor (TCR) could be induced by three injections of the DNA prime vaccine followed by a single injection of recombinant vaccinia vaccine. To develop a safer immunization strategy, a single DNA prime followed by a single recombinant Tiantan vaccinia (rTV) AIDS vaccine was used to immunize mice. Our data demonstrated that one DNA prime/rTV boost regimen induced mature TCR activation with high functional avidity, preferential T cell Vβ receptor usage and high sensitivity to anti-CD3 antibody stimulation. No differences in T cell responses were observed among one, two or three DNA prime/rTV boost regimens. This study shows that one DNA prime/rTV boost regimen is sufficient to induce potent T cell responses against HIV.

Interaction between antigen presenting cells and autoreactive T cells derived from BXSB mice with murine lupus

Systemic lupus erythematosus （SLE） is a typical autoimmune disease involving multiple systems and organs. Ample evidence suggests that autoreactive T cells play a pivotal role in the development of this autoimmune disorder. This study was undertaken to investigate the mechanisms of interaction between antigen presenting cells （APCs） and an autoreactive T cell （ATLI） clone obtained from lupus-prone BXSB mice. ATLI cells, either before or after 7-ray irradiation, were able to activate naive B cells, as determined by B cell proliferation assays. Macrophages from BXSB mice were able to stimulate the proliferation of resting ATL 1 cells at a responder/stimulator （R/S） ratio of 1/2.5. Dendritic cells （DCs） were much more powerful stimulators for ATLI cells on a per cell basis. The T cell stimulating ability ofmacrophages and B cells, but not DCs, was sensitive to T-ray irradiation. Monoclonal antibodies against mouse MHC-Ⅱ and CD4 were able to block DC-mediated stimulation of ATL 1 proliferation, indicating cognate recognition between ATL 1 and APCs. Our data suggest that positive feedback loops involving macrophages, B cells and autoreactive T cells may play a pivotal role in keeping the momentum of autoimmune responses leading to autoimmune diseases.

CD28/B7-MEDIATED COSTIMULATION IS REQUIRED FOR PARATHYROID GLAND ALLOGRAFT REJECTION IN RATS

The activation of T cells to differentiate and to proliferate is an essential step in the immune response to antigen, especially in cell mediated acute allograft rejection. Besides the interaction of CD3/TCR complex with Ag/MHC complex presented on antigen presenting cells, a complete T cell activation and proliferation requires a second costimulatory signal. The interaction of CD28/CTLA 4 and B7 is a major costimulatory pathway for T Cell activation. Inhibition of this pathway results in development of antigen specific unresponsiveness and clonal anergy.In present study,the biologic function of anti CD28 monoclonal antibody and its Fab fragment were investigated in vitro and in vivo.The results indicate that mAbCD28 and its Fab fragments could promote the functional recovery of allografts and prolong the graft survival,but could not reverse the acute rejection or induce transplantation tolerance in the rat PTG allograft model. We also found that peripheral TNF α level and NK cell activity were suppressed in the presence of mAbCD28 and its Fab fragments for a relatively long time after PTG transplantation.

Changes of inducible protein-10 and regulated upon activation, normal T cell expressed and secreted protein in acute rejection of pancreas transplantation in rats

AIM： To investigate the role of IFN-T inducible protein -10 （IP-10） and regulated upon activation, normal T cell expressed and secreted （RANTES） protein in acute pancreatic allograft rejection in rats. METHODS： An experimental model was established using pancreas transplantation diabetic SD rats as the recipient, induced by applying streptozocin （STZ）. Pancreas transplantation was performed with a physiologic method of portal venous and enteric drainage. Rats were divided into two groups, isograft group （group A, n = 24） and allograft group （group B, n = 24） in which either healthy SD rats or Wistar rats served as donors, respectively. Twelve diabetic or healthy SD rats were used as controls. At d 1, 4, 7, and 10 post transplantation, serum IP-10 and RANTES were assessed by ELISA and their expression in the allografts was determined by immunohistochemistry. RESULTS： In group B （allograft group）, the development of acute rejection was significantly correlated with increased serum concentration and tissue expression of IP-10 and RANTES, with a peak level at d 7 post transplantation. In contrast, there was no obvious change before and after transplantation in group A （isograft group）. CONCLUSION： Our study suggests a possible role of IP-10 and RANTES in acute rejection and early monitoring of chemokines may be helpful in predicting the outcome of pancreas transplantation.

Potential role of Th17 cells in the pathogenesis of inflammatory bowel disease

The etiopathology of inflammatory bowel disease （IBD） remains elusive. Accumulating evidence suggests that the abnormality of innate and adaptive immunity responses plays an important role in intestinal inflam- mation. IBD including Crohn＇s disease （CD） and ulcerative colitis （UC） is a chronic inflammatory disease of the gastrointestinal tract, which is implicated in an inappropriate and overactive mucosal immune response to luminal flora. Traditionally, CD is regarded as a Thl- mediated inflammatory disorder while UC is regarded as a Th2-1ike disease. Recently, Th17 cells were identified as a new subset of T helper cells unrelated to Thl or Th2 cells, and several cytokines [e.g. interleukin （IL）-21, IL-23] are involved in regulating their activation and differentiation. They not only play an important role in host defense against extracellular pathogens, but are also associated with the development of autoimmunity and inflammatory response such as IBD. The identification of Th17 cells helps us to explain some of the anomalies seen in the Thl/Th2 axis and has broadened our understanding of the immunopathological effects of Th17 cells in the development of IBD.