The Thr to Met substitution of amino acid 118 in hepatitis B virus surface antigen escapes from immune-assay-based screening of blood donors.

Hepatitis B surface antigen (HBsAg) is the main diagnosis marker for hepatitis B virus (HBV) infection. In this study, a novel HBV mutant from an HBV-positive blood donor with false-negative results during HBsAg screening was identified. DNA sequencing discovered two mutations at nt 353 (A to T) and nt 349 (T to A), leading to Thr to Met and Ser to Thr substitutions at aa 118 and 117 of HBsAg, respectively. Further analysis showed that eight of ten HBsAg ELISA kits failed to detect this HBsAg mutant. A mutagenesis assay indicated that the Thr to Met substitution at aa 118 was the determinant for escape from HBsAg ELISA detection. A small-scale screening of blood donors identified two individuals infected by this unique HBV mutant, suggesting a certain level of prevalence among the general population. In conclusion, our study identified the aa 118 mutation in HBV surface antigen and provided information for improvement of HBV diagnosis products.

Prevalence and associated factors of knee osteoarthritis in a rural Chinese adult population: an epidemiological survey.

BACKGROUND: The exact pathogenic mechanism of knee osteoarthritis (OA) is still unknown. With the exception of clinical treatment to alleviate symptoms, or total knee replacement, there is currently no effective treatment method. Consequently, an in-depth etiological and epidemiological study of knee OA can provide clues for diagnosis, treatment and scientific research, and will ultimately have a beneficial effect on public health. METHODS: A cross-sectional community study in the rural village of Gaoyou was conducted in 3428 Chinese adults (aged ≥ 40 years). Subjects completed an interviewer-administered questionnaire, evaluating knee pain and associated disability, analgesia, use of health services, past medical history, walking, income, smoking, and use of oral contraceptives, and standardized weight-bearing knee radiographs were obtained. Patient demographic characteristics and biochemical parameters were recorded. RESULTS: Single-factor regression analysis indicated that age, overweight, central adiposity, high low-density lipoprotein cholesterol (LDLC), high total cholesterol (TC), high triglycerides (TG), dyslipidemia, hypertension and low income were the associated factors for knee OA in females; age, high LDLC, hypertension, low income and frequent walking were the associated factors for knee OA in males. Interestingly, male heavy smokers were less likely to develop severe knee OA compared with non-smokers. Stepwise logistic regression analysis indicated that age and overweight were the associated factors for knee OA for all individuals. Although central adiposity, high LDLC, high TC, high TG, dyslipidemia, hypertension and low income appeared to be related to knee OA in females according to univariate analysis, these factors were not identified in stepwise logistic regression analysis. In addition although age, high LDLC, hypertension and frequent walking were also the associated factors for knee OA in males by stepwise logistic regression analysis, smoking as a protective factor was not identified in this analysis. CONCLUSIONS: In this study, aging, obesity, frequent walking, low income and relevant multiple metabolic disorders were the associated factors for knee OA. Smoking might be associated with a lower prevalence of OA in male smokers according to univariate analysis. A retrospective association of smoking with OA may constitute an important etiologic clue, but further well-designed, large-scale prospective controlled trials are required to confirm these findings.

N-Linked Glycosylation at an Appropriate Position in the Pre-S2 Domain Is Critical for Cellular and Humoral Immunity against Middle HBV Surface Antigen.

Infection with hepatitis B virus (HBV) remains a worldwide health problem, and DNA-based vaccines against HBV have been tested for therapeutic applications. HBV possesses three envelope lipoproteins that are translated from a single reading-frame: large, middle, and small HBV surface antigens. Among these envelope proteins, the middle HBV surface antigen (MHBs) contains a constitutive N-linked glycosylation site at position 4 (Asn4) in the amino-terminal portion (MQWNSTTFHQ) of pre-S2 domain. Asn4 (shown in bold) is essential for secretion of viral particles and conserved among all serotypes of HBV, but its influence on the immunogenicity of MHBs remains unknown. Here, we constructed four MHBs genes carrying mutations, underlined, in the amino-terminal portion of pre-S2 domain. One mutant protein contains Q at position 4 (MQWQSTTFHQ). In addition, each of three mutant MHBs proteins contains a N-linked glycosylation site (N-X-S/T), relocated to position 5 (MQWQNTTFHQ), 6 (MQWQSNTSHQ) or 7 (MQWQSTNFTQ) in pre-S2 domain. The expression and immunogenic properties of mutant DNA vaccines were examined in 293T human renal epithelial cells and in BALB/c mice, respectively. We showed that Asn4 was critical for secretion and immunogenicity of MHBs. Moreover, the MHBs protein that carries a N-linked glycosylation site at position 5 or 7 retained the properties similar to wild-type MHBs. In contrast, the secretion-defective mutant protein carrying Asn at position 6 induced only marginal humoral and cellular immune responses in mice, despite the N-linked glycosylation. In conclusion, N-linked glycosylation at an appropriate position in pre-S2 domain is an essential requirement for DNA vaccine expressing MHBs.

Azithromycin attenuates pulmonary inflammation and emphysema in smoking-induced COPD model in rats.

INTRODUCTION: The role of inflammation and immunity in COPD treatment is increasingly being recognized. The relationship between anti-inflammation/immunoregulation and emphysema in COPD lungs remains to be elucidated. The aim of this study was to investigate the effects of azithromycin (Azm) on the development of emphysema in smoking-induced COPD in rats. METHODS: Sprague-Dawley rats (n = 50) were randomly assigned to normal, COPD, saline-treated, Azm-treated, and levofloxacin-treated (Lev) groups. The effects of treatment were assessed by measuring the levels of vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay and measuring the numbers of neutrophil and macrophage in bronchoalveolar lavage fluid, vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR2) protein expression by western blotting. Lung function measurements and histopathological evaluations (mean linear intercept and destructive index) were performed. RESULTS: FEV0.3/FVC and peak expiratory flow were lower in the COPD group than in the normal group. Mean linear intercept and destructive index were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. The numbers of neutrophil and macrophage in bronchoalveolar lavage fluid were lower in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. As confirmed by western blotting, the levels of VEGF in lung homogenates were higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. VEGFR2 protein expression was higher in the Azm-treated group than in the COPD, saline-treated, and Lev-treated groups. CONCLUSIONS: Azm attenuates pulmonary emphysema by partly reversing the decrease in the numbers of inflammatory cells (neutrophil and macrophage) and VEGF secretion and VEGFR2 protein expression in smoking-induced COPD in rats.

Activation of α7 nicotinic acetylcholine receptors protects astrocytes against oxidative stress-induced apoptosis: implications for Parkinson's disease.

Astrocytes have been implicated in the immune responses associated with Parkinson's disease (PD). Inhibition of astrocyte apoptosis is a novel strategy for the treatment of PD. Recent studies suggest that α7 nicotinic acetylcholine receptors (α7-nAChRs) expressed in glial cells are critical links between inflammation and neurodegeneration in PD. However, little is known about their contribution to astrocyte apoptosis during the development of this disorder. In the present study, we showed that nicotine exerts a protective effect on H2O2-induced astrocyte apoptosis and glial cell-derived neurotrophic factor (GDNF) downregulation, and this effect was abolished by an α7-nAChR-selective antagonist. The underlying mechanisms might involve alleviation of mitochondrial membrane potential loss, stabilization of the Bax/Bcl-2 balance, and inhibition of cleaved caspase-9 activity through α7-nAChR activation. Systemic administration of nicotine dramatically alleviated MPTP-induced symptoms, protected dopaminergic neurons against degeneration, inhibited astrocytes and microglia activation in the substantia nigra pars compacta (SNpc) and blocked the decrease of GDNF in the striatum by activating α7-nAChRs. Taken together these findings demonstrate, for the first time, that nicotine suppresses H2O2-induced astrocyte apoptosis via the mitochondrial pathway through the stimulation of α7-nAChRs. Targeting α7-nAChRs expressed in astrocytes may be a novel therapeutic strategy for the treatment of neurodegenerative disorders.

Optimal designs of an HA-based DNA vaccine against H7 subtype influenza viruses.

The outbreak of a novel H7N9 influenza virus in 2013 has raised serious concerns for the potential of another avian-source pandemic influenza. Effective vaccines against H7N9 virus are important in the prevention and control of any major outbreak. Novel vaccination technologies are useful additions to existing approaches. In the current report, DNA vaccine studies were conducted to identify the optimal design of an H7 HA antigen using the HA gene from a previously reported H7N7 virus that is lethal in humans as the model antigen. New Zealand White rabbits were immunized with DNA vaccines expressing 1 of 3 forms of H7 HA antigen inserts encoding the HA gene from the same H7N7 virus. High-level H7 HA-specific IgG was detected by ELISA, and functional antibodies were confirmed by hemagglutination inhibition assay and pseudotyped virus-based neutralization assay against viruses expressing HA antigens from either the previous H7N7 virus or the novel H7N9 virus. HA antigen design under the tissue plasminogen activator leader (tPA) was the most immunogenic. The data presented in the current report confirm the immunogenicity of the H7 HA antigen and provide useful guidance to prepare for an optimized H7 HA DNA vaccine to help to control the emerging H7N9 virus if and when it is needed.

Profiles of acute cytokine and antibody responses in patients infected with avian influenza A H7N9.

The influenza A H7N9 virus outbreak in Eastern China in the spring of 2013 represented a novel, emerging avian influenza transmission to humans. While clinical and microbiological features of H7N9 infection have been reported in the literature, the current study investigated acute cytokine and antibody responses in acute H7N9 infection. Between March 27, 2013 and April 23, 2013, six patients with confirmed H7N9 influenza infection were admitted to Drum Tower Hospital, Nanjing, China. Acute phase serum cytokine profiles were determined using a high-throughput multiplex assay. Daily H7 hemagglutinin (HA)-specific IgG, IgM, and IgA responses were monitored by ELISA. Neutralizing antibodies specific for H7N9 viruses were determined against a pseudotyped virus expressing the novel H7 subtype HA antigen. Five cytokines (IL-6, IP-10, IL-10, IFNγ, and TNFα) were significantly elevated in H7N9-infected patients when compared to healthy volunteers. Serum H7 HA-specific IgG, as well as IgM and IgA responses, were detected within 8 days of disease onset and increased in a similar pattern during acute infection. Neutralizing antibodies developed shortly after the appearance of binding antibody responses and showed similar kinetics as a fraction of the total H7 HA-specific IgG responses. H7N9 infection resulted in hallmark serum cytokine increases, which correlated with fever and disease persistence. The novel finding of simultaneous development of IgG, IgM, and IgA responses in acute H7N9 infection points to the potential for live influenza viruses to elicit fast and potent protective antibodies to limit the infection.

Concurrent measurement of dynamic changes in viral load, serum enzymes, T cell subsets, and cytokines in patients with severe fever with thrombocytopenia syndrome.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infection caused by a novel Bunyavirus. Analysis on the dynamic changes of clinical, laboratory, and immunological abnormalities associated with SFTS in a concurrent study is lacking. Thirty-three SFTS patients were admitted to Jiangsu People's Hospital, Nanjing, China, and diagnosis was made based on the clinical symptoms and positive viral RNA detected by RT-PCR. Four patients deceased and twenty-nine survived. Blood samples were collected every other day between Day 5 and Day 15 from the onset of fever. Samples from healthy volunteers were used as normal controls. Peak viral RNA load, serum enzymes, IL-6, and IL-10 were significantly higher in deceased patients compared to survivors. Viral load, serum enzymes, and cytokines declined in survivors within 2 weeks from onset of fever. CD69+ T cells were elevated early after infection while HLA-DR+ and CTLA4+ T cells were elevated during the recovery phase of those who survived. High level SFTSV viral load was concurrently observed with reduced PLT, elevated serum enzymes, elevated pro-inflammatory and anti-inflammatory cytokines, and activation of CD69+ T cells. The degree and pattern of changes in these parameters may indicate the clinical outcome in SFTSV-infected patients.

A case of brucellosis displaying Parkinsonian-like tremor.

We report a rare case of brucellosis with Parkinsonian-like tremor and simple partial motor seizure. This patient worked as a sheep butcher and the sheep were imported from brucellosis-endemic areas. He presented with classical manifestations of brucellosis; infection was confirmed using the Rose Bengal Plate and Standard Tube Agglutination tests. The patient also suffered from headache, partial seizures, changes of personality and static tremor of both upper limbs. After anti-infection therapy, but without the use of anti-Parkinson drugs, the patient fully recovered and remained free of Parkinsonian-like tremor. Brucellosis can present with atypical symptoms, clinicians should widen their diagnostic view of brucella infection.

[Hepatitis B e antigen from chronic hepatitis B patients induces Th1/Th2 cytokine imbalance in vitro].

OBJECTIVE: To study the immunoregulatory effect of hepatitis B virus (HBV) e antigen (HBeAg) on peripheral blood monocytes (PBMCs). METHODS: PBMCs were isolated from patients with chronic hepatitis B (CHB; both HBeAg- and HBeAg+) and healthy controls, and cultured with recombinant HBeAg. The HBeAg-induced changes in expression of PD-1/PD-L1 were measured by flow cytometry of the cells and in secreted cytokines were measured by enzyme-linked immunosorbent assay of the supernatants. Comparisons between two groups were made by the independent-samples t-test; the relationship between PD-1/B7-H1 level and HBV DNA copy number was evaluated by Spearman's correlation analysis. RESULTS: Exposure to HBeAg led to a significant decrease in CD3+CD4+ T lymphocyte-specific expression of IFNa for both the CHB patients' and healthy controls' samples (t = 2.382 and -4.190 respectively, P less than 0.01). For the HBeAg- CHB patients' and healthy controls' samples, the HBeAg exposure led to increased levels of secreted cytokines IL-6, IL-10 and TNFa (t = 2.504, 3.583 and 4.324, P less than 0.01 and t = 3.542, 6.246 and 5.273, P less than 0.01 respectively) and of CD14+ PBMC-specific expression of PD-L1 (t = 4.815 and 3.454, P less than 0.05 respectively). Compared to the HBeAg-negative CHB patients' and healthy controls' samples, the HBeAg+ CHB patients' samples had significantly lower CD3+CD4+ T cell-specific expression of IFNa (t = -3.177 and -4.541, P less than 0.01 respectively), but significantly higher levels of secreted IL-4 (t = 3.382 and 4.393, P less than 0.01 respectively), of CD3+ T cells-specific expression of PD-1/PD-L1 (t = 4.755, 2.942 and 4.518, 4.595, P less than 0.01 respectively), and of CD14+ T cells-specific expression of PD-L1 (t = 5.092 and 5.473, P less than 0.01 respectively). The CD3+ T cells-specific expression of PD-L1 was significantly higher in the samples from HBeAg- CHB patients than from the healthy controls (t = 3.214, P less than 0.01). CONCLUSION: HBeAg was able to down-regulate the production of Th1-type cytokines (IFNgamma), and up-regulate the secretion of Th2-type cytokines (IL-6, IL-10) and the expression of PD-1/PD-L1on monocytes. These changes are conducive to the formation of immune tolerance to HBV. Therefore, HBeAg may play an important role in immune tolerance to chronic HBV infection.

Decreased antigenicity profiles of immune-escaped and drug-resistant hepatitis B surface antigen (HBsAg) double mutants.

BACKGROUND: Selective pressure from either the immune response or the use of nucleoside analogs in antiviral therapy could be driving the emergence of HBV mutants. Because of the overlap of the open reading frame (ORF) S for the HBsAg and ORF P for viral polymerase, rtM204I and rtM204V mutations in the polymerase would produce sI195M and sW196S in the HBsAg. The combined effects of immune-escaped mutations (sT118M, sG145K, sG145R) and drug-resistant mutations (rtM204I, rtM204V) on the antigenicity profiles of HBsAg has not been widely explored. METHODS: To determine the combined effects of immune-escaped and drug-resistant mutants on the antigenicity profiles of HBsAg, recombinant plasmids encoding HBsAg double mutants were constructed using site-directed mutagenesis. The supernatant from each plasmid transfection was analyzed for HBsAg in the western-blotting and five of the most commonly used commercial ELISA kits in China. RESULTS: Western-blotting assay showed the successful expression of each HBsAg mutant. All five ELISA kits manifested similar avidity, which were demonstrated by the slope of the curves, for the sT118M mutant, and sT118M-rtM204I (sT118M-sI195M) and sT118M-rtM204V (sT118M-sW196S) double mutants, suggesting that drug-resistant YMDD mutants caused negligible losses in the antigenicity of immune-escaped sT118M HBsAg. In contrast, the presence of the rtM204I (sI195M) mutation, but not rtM204V (sW196S) in combination with the sG145K mutation significantly reduced the avidity of sG145K HBsAg. The rtM204I (sI195M) mutation also decreased the antigenicity profiles for sG145R HBsAg. CONCLUSIONS: Drug-resistant mutations rtM204I (sI195M) and rtM204V (sW196S) caused significant reduction in antigenicity for the immune-escaped HBsAg mutants sG145K and sG145R, which may hamper HBV diagnosis and disease control from HBV blood-transfusion transmissions in China. The development of ELISA kits with a greater sensitivity for drug-resistant and immune-escaped HBsAg warrants further consideration.

Potent monoclonal antibodies against Clostridium difficile toxin A elicited by DNA immunization.

Recent studies have demonstrated that DNA immunization is effective in eliciting antigen-specific antibody responses against a wide range of infectious disease targets. The polyclonal antibodies elicited by DNA vaccination exhibit high sensitivity to conformational epitopes and high avidity. However, there have been limited reports in literature on the production of monoclonal antibodies (mAb) by DNA immunization. Here, by using Clostridium difficile (C. diff) toxin A as a model antigen, we demonstrated that DNA immunization was effective in producing a panel of mAb that are protective against toxin A challenge and can also be used as sensitive reagents to detect toxin A from various testing samples. The immunoglobulin (Ig) gene usage for such mAb was also investigated. Further studies should be conducted to fully establish DNA immunization as a unique platform to produce mAb in various hosts.

Regulation of B7-H1 expression on peripheral monocytes and IFN-γ secretion in T lymphocytes by HBeAg.

This study is to observe the expression of B7-H1, PD-1 and TLR2 on peripheral blood monocytes (PBMCs) regulated by HBeAg in chronic hepatitis B (CHB), and to illustrate the relation between HBeAg and persistent infection of HBV. In both CHB patients and healthy controls, the expression of B7-H7 was significantly increased on CD14(+) monocytes incubated with HBeAg, while that of TLR2 was significantly reduced; the expression of specific IFN-γ was significantly decreased in CD3(+)CD4(+) T lymphocytes incubated with HBeAg, while IL-6 and IL-10 in conditioned media were significantly increased. HBeAg is able to significantly up-regulate B7-H1, down-regulate TLR2 on monocytes, reduce IFN-γ produced by T lymphocytes and increase Th2-type cytokines secretion. These findings suggest that HBeAg suppresses the specific cellular immunity to clear the virus, and eventually lead to immune tolerance to HBV infection. Therefore, HBeAg plays an important role in immune suppression in chronic HBV patients.

[Role of T lymphocyte activation in the development of severe fever accompanied by thrombocytopenia syndrome].

OBJECTIVE: To explore the effect of peripheral blood T lymphocyte activation on the blood cells, tissue injury and the development of disease in patients with severe fever with thrombocytopenia syndrome (SFTS). METHODS: The expressions of CD69, HLA-DR, CD28 and CTLA-4 on peripheral blood T lymphocytes were determined dynamically by flow cytometry and the relationships between the above immune molecules and ALT, AST, leukocytes, platelets were analyzed respectively. RESULTS: The expressions of CD69 and HLA-DR on peripheral blood T lymphocytes in patients with SFTS were elevated significantly during the whole course of disease (P<0.05). CD28 expression on CD4(+); lymphocyte subset decreased in the early stage and gradually increased to the normal range. Meanwhile, CTLA-4 expression on T lymphocytes went up in the late stage of viral infection. The levels of serum ALT, AST, LDH and CK were significantly higher than the upper limit of the normal and the counts of WBC and PLT dropped to the lowest at the outset. But all of them returned back to the normal range gradually with the down-regulation of CD69 and HLA-DR and the up-regulation of CTLA-4 on T lymphocyte. CONCLUSION: The overactivation of T lymphocytes may contribute to tissue injury and the high expression of CTLA-4 may be a negative feedback regulation to the overactivation of T lymphocytes, which plays an important role in immunoregulation of SFTS patients.

Protective antibody responses against Clostridium difficile elicited by a DNA vaccine expressing the enzymatic domain of toxin B.

A DNA vaccination approach was used in the current study to screen for the immunogenicity of different fragments of toxin A and toxin B from Clostridium difficile. With this approach, protein antigens do not need to be produced in vitro and the immunogenicity of candidate C. difficile antigens can be identified directly in animals. Codon optimized toxin gene fragments were individually cloned into the DNA vaccine vector and tested in mice and rabbits for their ability to elicit C. difficile toxin-specific antibody responses. Only a subset of the C. difficile toxin fragments, including the C-terminal receptor binding domain of toxin A and a novel N-terminal enzymatic domain of toxin B, were able to elicit protective antibody responses as determined by protection of target cells in a cytotoxicity assay or by preventing death of mice in a passive antibody protection study. Significantly, antibodies elicited by the novel N-terminus of the toxin B DNA vaccine were able to increase the level of protection when used in combination with anti-toxin A antibodies in a toxin challenge model in mice.

DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China.

Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach.

Serum neutralizing activities from a Beijing homosexual male cohort infected with different subtypes of HIV-1 in China.

Protective antibodies play a critical role in an effective HIV vaccine; however, eliciting antibodies to block infection by viruses from diverse genetic subtypes remains a major challenge. As the world's most populous country, China has been under the threat of at least three major subtypes of circulating HIV-1 viruses. Understanding the cross reactivity and specificities of serum antibody responses that mediate broad neutralization of the virus in HIV-1 infected Chinese patients will provide valuable information for the design of vaccines to prevent HIV-1 transmission in China. Sera from a cohort of homosexual men, who have been managed by a major HIV clinical center in Beijing, China, were analyzed for cross-sectional neutralizing activities against pseudotyped viruses expressing Env antigens of the major subtype viruses (AE, BC and B subtypes) circulating in China. Neutralizing activities in infected patients' blood were most capable of neutralizing viruses in the homologous subtype; however, a subset of blood samples was able to achieve broad neutralizing activities across different subtypes. Such cross neutralizing activity took 1-2 years to develop and CD4 binding site antibodies were critical components in these blood samples. Our study confirmed the presence of broadly neutralizing sera in China's HIV-1 patient population. Understanding the specificity and breadth of these neutralizing activities can guide efforts for the development of HIV vaccines against major HIV-1 viruses in China.

Removing N-terminal sequences in pre-S1 domain enhanced antibody and B-cell responses by an HBV large surface antigen DNA vaccine.

Although the use of recombinant hepatitis B virus surface (HBsAg) protein vaccine has successfully reduced global hepatitis B infection, there are still a number of vaccine recipients who do not develop detectable antibody responses. Various novel vaccination approaches, including DNA vaccines, have been used to further improve the coverage of vaccine protection. Our previous studies demonstrated that HBsAg-based DNA vaccines could induce both humoral and CMI responses in experimental animal models. However, one form of the the HBsAg antigen, the large S antigen (HBs-L), expressed by DNA vaccine, was not sufficiently immunogenic in eliciting antibody responses. In the current study, we produced a modified large S antigen DNA vaccine, HBs-L(T), which has a truncated N-terminal sequence in the pre-S1 region. Compared to the original HBs-L DNA vaccine, the HBs-L(T) DNA vaccine improved secretion in cultured mammalian cells and generated significantly enhanced HBsAg-specific antibody and B cell responses. Furthermore, this improved HBsL DNA vaccine, along with other HBsAg-expressing DNA vaccines, was able to maintain predominantly Th1 type antibody responses while recombinant HBsAg protein vaccines produced in either yeast or CHO cells elicited mostly Th2 type antibody responses. Our data indicate that HBsAg DNA vaccines with improved immunogenicity offer a useful alternative choice to recombinant protein-based HBV vaccines, particularly for therapeutic purposes against chronic hepatitis infection where immune tolerance led to poor antibody responses to S antigens.

α7 nicotinic acetylcholine receptor-mediated neuroprotection against dopaminergic neuron loss in an MPTP mouse model via inhibition of astrocyte activation.

BACKGROUND: Although evidence suggests that the prevalence of Parkinson's disease (PD) is lower in smokers than in non-smokers, the mechanisms of nicotine-induced neuroprotection remain unclear. Stimulation of the α7 nicotinic acetylcholine receptor (α7-nAChR) seems to be a crucial mechanism underlying the anti-inflammatory potential of cholinergic agonists in immune cells, including astrocytes, and inhibition of astrocyte activation has been proposed as a novel strategy for the treatment of neurodegenerative disorders such as PD. The objective of the present study was to determine whether nicotine-induced neuroprotection in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model occurs via α7-nAChR-mediated inhibition of astrocytes. METHODS: Both in vivo (MPTP) and in vitro (1-methyl-4-phenylpyridinium ion (MPP+) and lipopolysaccharide (LPS)) models of PD were used to investigate the role(s) of and possible mechanism(s) by which α7-nAChRs protect against dopaminergic neuron loss. Multiple experimental approaches, including behavioral tests, immunochemistry, and stereology experiments, astrocyte cell cultures, reverse transcriptase PCR, laser scanning confocal microscopy, tumor necrosis factor (TNF)-α assays, and western blotting, were used to elucidate the mechanisms of the α7-nAChR-mediated neuroprotection. RESULTS: Systemic administration of nicotine alleviated MPTP-induced behavioral symptoms, improved motor coordination, and protected against dopaminergic neuron loss and the activation of astrocytes and microglia in the substantia nigra. The protective effects of nicotine were abolished by administration of the α7-nAChR-selective antagonist methyllycaconitine (MLA). In primary cultured mouse astrocytes, pretreatment with nicotine suppressed MPP(+)-induced or LPS-induced astrocyte activation, as evidenced by both decreased production of TNF-α and inhibition of extracellular regulated kinase1/2 (Erk1/2) and p38 activation in astrocytes, and these effects were also reversed by MLA. CONCLUSION: Taken together, our results suggest that α7-nAChR-mediated inhibition of astrocyte activation is an important mechanism underlying the protective effects of nicotine.

[Humoral and cellular immune responses to HBV in the blood donors].

AIM: To study the magnitude and correlation of humoral and cellular immune responses to hepatitis surface antigen(HBsAg), preS1+S2 antigen, and core antigen(HBcAg) in the blood donors. METHODS: Enzyme linked immunosorbent assay (ELISA) was employed to scan the humoral immune responses to HBsAg, preS1+S2 antigen, and HBcAg. Enzyme linked immunospot assay (ELISPOT) was used to check the antigen specific IFN-γ secreting cells stimulated by HBsAg, preS1+S2 antigen or HBcAg in peripheral blood mononuclear cell from blood donors. RESULTS: The prevalence of humoral immune response to preS1+S2 antigen was as high as 85.7% (48/56), compared with the lower response to HBcAg (30.4%, 17/56). In the cellular immune response, there was no difference between preS1+S2 antigen and HBcAg, both of which were stronger than the response stimulated by HBsAg. PreS1+S2 antigen or HBsAg specific humoral immune and cellular immune responses were highly correlated, which could not be observed in the HBcAg specific humoral immune and cellular immune responses(P>0.05). CONCLUSION: Either humoral or cellular immune responses to HBsAg, preS1+S2 antigen and HBcAg could be detected in the blood donors. PreS1+S2 antigen immune responses, however, were most powerful, which may suggest that PreS1+S2 antigen may be a candidate for the further HBV vaccine.