Analysis of influencing factors of serum HBeAg loss in patients with chronic hepatitis B / 中国热带医学

@#Abstract: Objective To explore the influencing factors of serum HBeAg loss in patients with chronic hepatitis B (CHB) and and provide evidence for effective treatment of CHB. Methods A follow-up cohort of HBeAg-positive CHB patients was established in the the Infectious Diseases Outpatient Clinic of hospital. Regular follow-up and laboratory test indicators were collected to analyze the changes of serum HBeAg in HBeAg-positive CHB patients during the follow-up period. The subjects were divided into the case group (serum HBeAg loss) and the control group (serum HBeAg not loss) according to whether serum HBeAg loss occurred. The baseline data characteristics of the two groups were analyzed and compared, and the influencing factors of serum HBeAg loss were analyzed by Cox univariate and multivariate regression. Results A total of 634 HBeAg-positive CHB patients were enrolled, with a total follow-up of 2 570.01 person-years. Among them, 237 cases of serum HBeAg loss occurred, with the mean follow-up time of 40.92 months, and the rate of HBeAg loss was 9.22/100 person-years. There were significant differences in HBV family history, antiviral therapy, baseline WBC, PLT, ALT, AST, T˗Bil, GGT, AFP, quantitative HBsAg and quantitative HBeAg between serum HBeAg loss group and serum HBeAg not loss group (P<0.05). Cox regression analysis showed that family history of HBV (HR 0.68, 95％CI:0.50-0.92, P=0.012), ALT (HR2.06, 95％CI:1.52-2.79, P<0.001), quantitative HBsAg (HR 0.68, 95％CI:0.48-0.95, P=0.024), quantitative HBeAg (HR 0.48, 95％CI:0.31-0.74, P=0.001) were independent influencing factors for HBeAg loss in HBeAg-positive CHB patients. Conclusions HBeAg-positive CHB patients without family history of HBV, initial ALT≥80 U/L, quantitative HBsAg<1 000 IU/ml, quantitative HBeAg<1 000 C.O.I are more likely to have serum HBeAg loss.

A human immunodeficiency virus-seronegative acquired immunodeficiency syndrome patient with opportunistic infections: A case report.

BACKGROUND: In rare cases, people living with chronic human immunodeficiency virus (HIV) infection do not develop antibodies despite demonstrable infection. Delayed or missed diagnosis of HIV infection leads to a lack of timely therapy, resulting in rapid disease progression with opportunistic infections or malignancies. CASE REPORT: A 44-year-old Chinese man presented with sore throat, oral leukoplakia, fever, dyspnoea and diffuse ground glass-like lesions in both lungs. Serum cytomegalovirus DNA was detectable, and CD4+ T-cell count was low. The patient was suspected of being a person living with HIV despite of the repeatedly negative HIV antibody tests using enzyme-linked immunsorbent assay and Western blot. Subsequently, high-plasma HIV RNA viral load was found on two repeated tests, while HIV DNA was also positive. Thus, the patient was confirmed as presenting with HIV-seronegative acquired immunodeficiency syndrome (AIDS). The symptoms improved in response to effective anti-fungal and anti-retroviral therapy after diagnosis. CONCLUSION: This is the third reported case of an HIV-seronegative AIDS patient in China, which are also rarely reported globally. HIV nucleic acid testing is important to screen out HIV infection, especially in those who present with severe immunodeficiency but remain HIV serogenative.

Modulation of Tim-3 Expression by Antigen-Dependent and -Independent Factors on T Cells from Patients with Chronic Hepatitis B Virus Infection.

T-cell immunoglobulin domain and mucin domain-containing molecule-3 (Tim-3) was up-regulated on viral specific T cells and contributed to T cells exhaustion during chronic hepatitis B virus (HBV) infection. However, modulation of Tim-3 expression was still not fully elucidated. To evaluate the potential viral and inflammatory factors involved in the inductor of Tim-3 expression on T cells, 76 patients with chronic HBV infection (including 40 chronic hepatitis B [CHB] and 36 asymptomatic HBV carriers [AsC]) and 40 of normal controls (NCs) were enrolled in this study. Tim-3 expressions on CD4+ and CD8+ T cells were assessed in response to HBV-encoding antigens, HBV peptide pools, and common γ-chain (γc) cytokines stimulation by flow cytometry. HBV peptides and anti-CD3/CD28 directly induced Tim-3 expression on T cells. γc cytokines also drive Tim-3 up-regulations on both CD4+ and CD8+ T cells in patients with chronic HBV infection. However, γc cytokines did not enhance the Tim-3 inductions by either anti-CD3/CD28 or HBV peptides stimulation. Furthermore, γc cytokines-mediated Tim-3 induction could not be abrogated by γc cytokine receptor-neutralizing antibodies. The current results suggested that elevation of Tim-3 expression on T cells could be regulated by both antigen-dependent and -independent manner in patients with chronic HBV infection. The role of γc cytokines in modulation of inhibitory pathway might be evaluated as immunotherapies in humans.

Effect of 48-week pegylated interferon α-2a or nucleos(t)ide analogue therapy on renal function in Chinese patients with chronic hepatitis B.

BACKGROUND: Controversy remains as to whether antiviral agents contribute to renal dysfunction in patients with chronic hepatitis B virus (HBV) infection. Thus, the aim of study was to analyze the changes in renal function of chronic hepatitis B (CHB) patients in response to anti-HBV therapy and the association with treatments. METHOD: We performed a retrospective observational cohort study to investigate factors associated with renal function in 249 Chinese CHB patients who were treated with pegylated interferon α-2a (PEG-IFN-α-2a) or nucleos(t)ide analogues for 48 weeks. Changes of estimated glomerular filtration rate (eGFR), which was computed with both the Chronic Kidney Disease Epidemiology Collaboration and the Modification of Diet in Renal Disease formulas, were tested by repeated measures One-way analysis of variance within groups. A linear mixed effects model for repeated measures was also used to evaluate the association between baseline information and eGFR changes over time in all enrolled patients. The model considered the baseline age, sex, HBV DNA, aminotransferase, blood urea nitrogen, treatment group, time, and group-by-time interaction as fixed effects and incorporated random effects for individual subjects. RESULTS: The eGFR increased in patients given PEG-IFN-α-2a, decreased in patients given adefovir, but remained stable in patients given entecavir. Age and blood urea nitrogen were significant negative predictive factors for eGFR changes. CONCLUSION: In real-life study, PEG-IFN-α-2a therapy in CHB patients increased eGFR, thus may associate with renoprotective effects when compared with adefovir or entecavir therapies.

Notch Signaling Contributes to Liver Inflammation by Regulation of Interleukin-22-Producing Cells in Hepatitis B Virus Infection.

The mechanism of hepatitis B virus (HBV) induced liver inflammation is not fully elucidated. Notch signaling augmented interleukin (IL)-22 secretion in CD4+ T cells, and Notch-IL-22 axis fine-tuned inflammatory response. We previously demonstrated a proinflammatory role of IL-22 in HBV infection. Thus, in this study, we analyzed the role of Notch in development of IL-22-producing cells in HBV infection by inhibition of Notch signaling using γ-secretase inhibitor DAPT in both hydrodynamic induced HBV-infected mouse model and in peripheral blood cells isolated from patients with HBV infection. mRNA expressions of Notch1 and Notch2 were significantly increased in livers and CD4+ T cells upon HBV infection. Inhibition of Notch signaling in vivo leaded to the reduction in NKp46+ innate lymphoid cells 22 (ILC22) and lymphoid tissue inducer 4 (LTi4) cells in the liver. This process was accompanied by downregulating the expressions of IL-22 and related proinflammatory cytokines and chemokines in the liver, as well as blocking the recruitment of antigen-non-specific inflammatory cells into the liver and subsequent liver injury, but did not affect HBV antigens production and IL-22 secretion in the serum. Furthermore, IL-22 production in HBV non-specific cultured CD4+ T cells, but not HBV-specific CD4+ T cells, was reduced in response to in vitro inhibition of Notch signaling. In conclusion, Notch siganling appears to be an important mediator of the liver inflammation by modulating hepatic ILC22. The potential proinflammatory effect of Notch-mediated ILC22 may be significant for the development of new therapeutic approaches for treatment of hepatitis B.

Toll-like receptor 2 promotes T helper 17 cells response in hepatitis B virus infection.

PURPOSE: Innate and adaptive immune responses play vital roles in initiating and maintaining the immunological homeostasis in both physiological and pathological processes. However, the expression and function of the important cells and molecules as well as their interaction in hepatitis B virus (HBV) infection has not been well elucidated. The aim of the current study was to determine the pattern of Toll-like receptor 2 (TLR2) in T cells in HBV infection and the function of TLR2 in regulation of T helper 17 (Th17) cells response. METHODS: Thirty-four patients with HBV infection (ten acute and twenty-four chronic) were enrolled. HBV-specific and -nonspecific Th17 cells and TLR2 expression in T cells were analyzed by flow cytometry. The function of TLR2 agonist for induction of IL-17 production was also determined. RESULTS: HBV-specific and -nonspecific IL-17 secretion in CD4(+) (Th17 cells) and CD8(+) T cells was significantly elevated in chronic HBV infection. Viral-specific TLR2 expression in CD4(+), CD8(+), and Th17 cells was also remarkably increased in patients with chronic hepatitis B. Moreover, TLR2 agonist Pam3Csk4 directly activated Th17 cells response without antigen stimulation in HBV infection. CONCLUSION: TLR2, which traditionally associated with innate immunity, might also promote Th17 cells response in HBV infection. The function of TLRs in regulation of adaptive immune response in HBV infection, which might play an important role in persistent HBV infection.

A fusion DNA vaccine encoding middle version of HBV envelope protein fused to interleukin-21 did not enhance HBV-specific immune response in mice.

DNA vaccination can generate both humoral and cellular immunity, resulting in potential prophylactic and therapeutic vaccines in variety of conditions, including hepatitis B virus (HBV) infection. Fusion of cytokine gene is one of the ways to increase the immunogenicity of DNA vaccine. Interleukin (IL)-21 has been demonstrated to play an immunomodulatory role in HBV infection. Thus, we aimed to investigate the ability of IL-21 in the regulation of middle version of HBV envelop protein (MS) DNA vaccine. Fusion plasmid encoding IL-21 linked with MS was constructed. Normal and HBV transgenic mice were immunized by plasmid. pcDNA-IL-21/S2S induced a comparable level of anti-HBs antibody and HBsAg-specific CD8+ T-cell response with pcDNA-S2S. Furthermore, the level of circulating HBsAg was decreased by induction of anti-HBs antibody and HBsAg-specific CD8+ T-cell response to both pcDNA-IL-21/S2S and pcDNA-S2S vaccination in HBV transgenic mice. Thus, immunization with DNA vaccine encoding HBV MS protein induced both T- and B-cell response by targeting the specific antigen. Furthermore, it was also revealed that MS DNA vaccination could break immune tolerance in HBV transgenic mice. But IL-21 did not strengthen immune response induced by HBV DNA immunization. Our study suggested that MS-expressing plasmid may be useful for both preventive and therapeutic methods in HBV infection. However, IL-21 does not improve the immunogenicity and efficacy of MS DNA vaccination, and thus may not be used as a therapeutic marker for chronic hepatitis B.

Expression profiles of circulating cytokines, chemokines and immune cells in patients with hepatitis B virus infection.

BACKGROUND: Immune cells and molecules play a vital role in initiating, maintaining, regulating immunological homeostasis and inflammation in many pathological and physiological processes; however, the changes on expressions and functions of these cells and molecules in hepatitis B virus (HBV) infection have not been elucidated well. OBJECTIVES: The current study aimed to determine the expression pattern of different cytokines, chemokines, immune cells in HBV infection and their association with disease progression. PATIENTS AND METHODS: Sixty-nine patients with chronic HBV infection were enrolled. Five immune cell subsets and 46 cytokines and chemokines were analyzed by flow cytometry and Luminex 200. RESULTS: In comparison to healthy individuals and asymptomatic HBV carriers, expression of CXCL9, CXCL10, CXCL11, and IL-10 were elevated in patients with chronic active HBV and had positive correlation with ALT levels. In contrast, G-CSF, MCP-3, and IFN-γ levels were significantly decreased in patients with chronic active HBV infection in contrast to carriers and healthy individuals; however, these down regulations did not show any correlation with either virological findings or liver inflammation. Although the proportion of CD4(+) CD25 (high) regulatory T cells (Tregs) was higher in patients with HBV infection than in healthy controls, no correlations were found between Tregs and other cytokines or chemokines. CONCLUSIONS: CXCR3-associated chemokines might contribute to liver inflammation in chronic hepatitis B, while MCP-3 and G-CSF were inhibited by HBV infection. Host immune response was suppressed as manifested by an increase in CD4(+) CD25(high) Tregs and IL-10 as well as a decrease in IFN-γ. Exploiting the expression pattern of cytokine and chemokine may help to develop a better understanding of chronic HBV infection pathogenesis.

Telbivudine plus adefovir therapy for chronic hepatitis B patients with virological breakthrough or genotypic resistance to telbivudine.

BACKGROUND/AIM: There is very limited experience in the management of telbivudine (LdT)-associated virological breakthrough (VBT) and resistance in the treatment of chronic hepatitis B (CHB) patients, and the guideline recommendations are primitively based on the general principles of rescue therapy to nucleos(t)ide analog resistance. The aim of this study is to determine the effect of the addition of adefovir (ADV) in hepatitis B e antigen (HBeAg)-positive CHB patients with VBT or resistance to LdT. METHODS: Thirty-seven CHB patients with confirmed VBT and 31 patients with genotypic resistance to LdT were enrolled and thereafter treated with a combination of LdT and ADV for 12 months. RESULTS: Combination therapy was safe and the majority of patients tolerated the therapy. LdT+ADV led to rapid decreases in viral loads, and viral replications were persistently suppressed, with 2.17 (VBT) and 2.31 (resistance) log(10) copies/ml reductions 12 months after rescue therapy, respectively. The rates corresponding to virological and biochemical responses were similar between the two groups at the end of observations (70.3 vs. 74.2% for virological response, P=0.720; 64.0 vs. 65.5% for biochemical response, P=0.907). The cumulative rates of serological responses were higher in patients with VBT than in those with resistance (35.1 vs. 9.67% for HBeAg loss, P=0.014; 10.8 vs. 3.23% for HBeAg/anti-HBe seroconversion, P=0.233). CONCLUSION: LdT and ADV combination therapy led to significant decreases in serum hepatitis B virus DNA levels and normalization of alanine aminotransferase levels in patients with VBT or genotypic resistance to LdT. This rescue strategy was also associated with a higher rate of HBeAg serological outcomes in patients with confirmed LdT-related VBT.

Dysregulation of the ß3 integrin-VEGFR2 complex in Hantaan virus-directed hyperpermeability upon treatment with VEGF.

Hantaviruses infect human endothelial cells (ECs) and are known to cause vascular-permeability-based diseases, including hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (HPS). The αvß3 integrins, which are highly expressed on the surface of ECs, serve as hantavirus receptors. Specifically, the ß3 integrin and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) form a functional complex and interact with each other. Signaling through this complex causes cytoskeletal reorganization, which is one of the most important mechanisms underlying hyperpermeability. In this study, we show that VEGF dramatically enhances Hantaan virus (HTNV)-directed permeability and increases the reorganization of the cytoskeleton and the disruption of junctional organizations in an EC monolayer at 3 days postinfection. HTNV infection reduced the effect of VEGF on adhesion, migration, and the upregulation of ß3 expression, but the infection alone upregulated the expression of ß3 and VEGFR2. These results indicate that in addition to its role in blocking ß3 integrin activation as reported previously, HTNV blocks the function of the complex of VEGFR2 and ß3 integrin, and the dysfunction of the complex may contribute to cytoskeletal reorganization in an HTNV-directed hyperpermeability response to VEGF.

[Immune response to HBsAg can not be enhanced with B7-H1 protein vaccine].

AIM: To observe the immunization effect to HBsAg by B7-H1 protein vaccine in transgenic mice, and to explore new methods for the treatment of chronic hepatitis B. METHODS: After the joint immunization in HBV transgenic mice with different doses of hepatitis B virus (HBV) vaccine and B7-H1 protein, the anti-B7-H1 antibody titors, Th1 type of cytokines (IFN-γ and IL-2) from the spleen cells of mice, the number of the T cells secreting IFN-γ and the number of the mice lymphocyte proliferation were measrued by ELISA, ELISPOT and MTT technique respectively, to compare the immune effect of different immune methods and regimen. RESULTS: The immune plans were completed successfully. The anti-B7-H1 antibody was detected in the fifth week after immunization with B7-H1 vaccine, at the same time no obvious difference of antibodies titors between groups were found. IL-2 and the number of T cells secreting IFN-γ were significantly reduced(P<0.05)in joint immunization group with B7-H1 protein and HBsAg, but no difference in other immune tests, such as IFN-γ, lymphocyte proliferation. CONCLUSION: A lower doses of HBsAg can cause the secretion of Th1 type of cytokines and lymphocyte proliferation. B7-H1 protein vaccines have a better immunogetic effect for HBV transgenic mice, but can notupregulate the immune response to HBsAg.

A proinflammatory role for interleukin-22 in the immune response to hepatitis B virus.

BACKGROUND & AIMS: T-helper (Th)17 cells that secrete interleukin (IL)-22 have immunomodulatory and protective properties in the liver and other tissues. IL-22 induces expression of proinflammatory genes but is also mitogenic and antiapoptotic in hepatocytes. Therefore, it could have multiple functions in the immune response to hepatitis B virus (HBV). METHODS: We examined the role of IL-22 in regulating liver inflammation in HBV transgenic mice and measured levels of IL-22 in HBV-infected patients. RESULTS: In HBV transgenic mice, injection of a single dose of IL-22 increased hepatic expression of proinflammatory genes but did not directly inhibit virus replication. When splenocytes from HBV-immunized mice were transferred into HBV transgenic mice, the severity of the subsequent liver damage was ameliorated by neutralization of IL-22. In this model, IL-22 depletion did not affect interferon gamma-mediated noncytopathic inhibition of virus replication initiated by HBV-specific cytotoxic T cells, but it significantly inhibited recruitment of antigen-nonspecific inflammatory cells into the liver. In patients with acute HBV infections, the percentage of Th17 cells in peripheral blood and concentration of IL-22 in serum were significantly increased. CONCLUSIONS: IL-22 appears to be an important mediator of the inflammatory response following recognition of HBV by T cells in the liver. These findings might be relevant to the development of cytokine-based therapies for patients with HBV infection.

[The induced IL-21 levels in CD4(+) T cells of peripheral blood from the different clinical types of patients infected with hepatitis B virus].

AIM: To investigate the levels of interleukin (IL)-21 in CD4(+);T cells of peripheral blood from the different types of patients infected with hepatitis B virus (HBV) and elucidate its role in the hepatitis B pathogenesis. METHODS: Peripheral blood mononuclear cells (PBMCs) from the patients infected with HBV and healthy individuals were stimulated with or without PMA coupled with ionomycin. The levels of IL-21 in CD4(+) T cells and Th17 cells were analyzed by flow cytometry. RESULTS: PMA and ionomycin induced the expression of IL-21, and IL-21 was mainly produced by CD4(+); T cells, but IL-17A(+) IL-21(+) CD4(+) T cells were not detected. The frequencies of IL-21(+) CD4(+) T cells in the patients of acute hepatitis B and chronic asymptomatic HBV carriers were higher than in healthy controls and severe chronic hepatitis B patients; there were no remarkable differences in the proportion of Th17 cells among the different groups of patients. Furthermore, the proportion of IL-21(+) CD4(+) T cells correlated with Th17 cells in all groups except for the acute hepatitis B patients. CONCLUSION: IL-21 levels, which correlated with Th17 cells, were different in CD4(+);T cells from the different types of patients infected with hepatitis B virus (HBV). The results showed that IL-21 may play a role in the hepatitis B pathogenesis.

Overexpression of Toll-like receptor 2/4 on monocytes modulates the activities of CD4(+)CD25(+) regulatory T cells in chronic hepatitis B virus infection.

The significance of TLR expression and Tregs in HBV infection has not been clearly described. In this report, flow cytometry was performed to assess TLR2/4 expression on monocytes and circulating CD4(+)CD25(+)CD127(low/-) Tregs frequency of 16 acute hepatitis B (AHB), 42 chronic hepatitis B (CHB), 22 asymptomatic HBV carriers (AsC), and 20 normal controls (NC). We found that TLR2 and TLR4 were overexpressed on CD14(+) monocytes in HBV-infected patients as compared with NCs. Upregulation of TLR2 in NCs and TLR4 in CHBs was observed following HBeAg incubation. However, TLR2 and TLR4 expression decreased after HBcAg stimulation. The difference in the proportion of Tregs between NCs and CHBs was significant. Both Pam3Csk4 (TLR2 agonist)- and lipopolysaccharide (TLR4 agonist)-activated CD4(+)CD25(+) Tregs showed enhanced suppression function in CHBs. These results suggest that overexpression of TLR2 and TLR4 may modulate the suppressive function of Tregs, which contribute to the immunotolerance of chronic HBV infection.

Correlation of circulating TLR2/4 expression with CD3+/4+/8+ T cells and Treg cells in HBV-related liver cirrhosis.

Toll-like receptors (TLRs) 2 and 4 play a key role in chronic hepatitis B virus (HBV) infection. However, the role of TLRs in the pathogenesis of HBV-related liver cirrhosis and their regulation of the innate immune response of patients with liver cirrhosis remain unknown. To assess the contribution of TLR2/4 in HBV-related liver cirrhosis, we examined the expression of circulating TLR2 and TLR4 on peripheral blood mononuclear cells (PBMCs), CD4(+)CD25(+)CD127(low/-) Treg proportions, and CD3(+)/CD4(+)/CD8(+) T-cell counts in 30 liver cirrhosis patients, 21 chronic hepatitis B (CHB) patients, and 16 normal controls (NC). Furthermore, we analyzed the relationship between TLR2/4 expression and Treg proportions and T-cell counts. We show that the expression of TLR2 and TLR4 was significantly upregulated in patients with liver cirrhosis compared to NC. TLR4 expression was significantly increased in patients with liver cirrhosis compared to patients with CHB, and for TLR2 expression there were no differences between them. TLR4 expression showed a significant positive correlation with the frequency of Tregs in liver cirrhosis patients. TLR2 expression negatively correlated with CD3(+)/CD4(+)/CD8(+) T-cell counts and HBV viral load in patients with liver cirrhosis. These findings indicate that TLR may be involved in the pathogenesis of HBV-related liver cirrhosis, and may interact with Tregs and CD3(+)/CD4(+)/CD8(+) T cells in the immune response during HBV-related liver cirrhosis.

Analysis of the immune response to Hantaan virus nucleocapsid protein C-terminal-specific CD8(+) T cells in patients with hemorrhagic fever with renal syndrome.

Hantaan virus (HTNV), the prototype member of the Hantavirus genus in the family Bunyaviridae, causes hemorrhagic fever with renal syndrome (HFRS), which is characterized by capillary leakage, hemorrhage, and renal injury, and is an important public health problem in China. Some kinds of immune cells, particularly CD8(+) T cells, are involved in the pathogenesis of Hantavirus infection. The nucleocapsid protein (NP) of the Hantavirus is the most conserved structural protein and the most abundant viral protein produced during infection. It is one of the important target antigens that induce the CD8(+) T-cell response. In this study, we examined the CD8(+) T-cell response to HTNV NP C-terminal polypeptides. We synthesized 23 overlapping C-terminal polypeptides and detected the antigen-specific CD8(+) T cell response in 15 patients with HFRS. The results demonstrated that there were NP-specific T-cell responses in bulk cultures of peripheral blood mononuclear cells (PBMCs) from 9 of 15 patients. The peptide 51 (aa 301-315: SPSSIWVFAGAPDRC), peptide 60 (aa 355-369: LRKKSSFYQSYLRRT), and peptide 70 (aa 415-429: DVKVKEISNQEPLKL) induced strong CD8(+) T-cell responses. Among them, peptide 70 induced CTL responses in donors 7, 9, and 11, and the strongest responses were seen in donor 11. Depletion of CD8(+) T cells from PBMCs completely abrogated the peptide-specific T-cell response, while depletion of CD4(+) T cells did not diminish the number of IFN-gamma spot-forming cells. These data suggest that infection with HTNV results in CTL responses to immunodominant regions on the NP.

Down-regulation of CXCR4 expression by SDF-KDEL in CD34(+) hematopoietic stem cells: An anti-human immunodeficiency virus strategy.

CXCR4 plays an essential role as the first discovered coreceptor for the entry of T cell tropic isolates of HIV-1. Blocking the surface expression of this receptor may be a potential strategy to prevent HIV-1 infection. A lentiviral vector, pLenti6/V5-S-K, expressing a SDF-KDEL fusion protein was constructed and a replication-incompetent lentiviral stock was produced. The lentiviral stock was transduced into CD34(+) hHSC and the transient expression of the recombinant protein, SDF-1, was assayed using indirect immunofluorescence. The surface expression of CXCR4 in CD34(+) hHSC pretreated with different amounts of recombinant lentiviral vectors was detected by flow cytometric analysis. A marked down-regulation of CXCR4 expression in the cells transduced with recombinant lentiviral vectors pLenti6/V5-S-K was observed by flow cytometry with PE-conjugated anti-human CXCR4 monoclonal antibodies which showed the percentages of the inhibition effects of CXCR4-SDF-1 mediated syncytium formation are presented by concentration. P24 antigen levels of cell culture supernatants were detected on the 4th, 7th, and 10th day, with 10(3) TCID50 HIV-1 infected CD34(+) hHSC to evaluate the inhibitory effect of pLenti6/V5-S-K transduction on HIV-1 infection. The cells transfected with pLenti6/V5-S-K had a significant reduction of HIV-1 DP27 infection compared to controls (P<0.05).

Therapeutic effect of RANTES-KDEL on inhibition of HIV-1 in CD34(+) human hematopoietic stem cells (hHSC).

Cell surface receptors, such as the CCR5 chemokine receptors, represent key determinants of the human immunodeficiency virus type 1 (HIV-1) entry into target cells. The CC-chemokine, RANTES (regulated upon activation, normal T-cell expressed and secreted), a ligand for CCR5, have been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL (ER-retention signal) fusion termed RANTES-KDEL and this construct was found to prevent effectively transport of newly synthesized CCR5 to the cell surface. Lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, an HIV-based lentiviral vector expressing RANTES-KDEL, pLenti6/V5-R-K, was constructed and then cotransfected with the ViraPower Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce a replication-incompetent lentivirus stock. The lentiviral stock was titrated using HeLa cells, and the expression of the gene of interest, RANTES, was detected by indirect immunofluorescence. Based on the above results, the lentiviral stock was transduced into CD34(+) human hematopoietic stem cells (hHSC) separated magnetically from the cord blood (the purity was 96.8% evaluated by flow cytometry). Finally, the levels of p24 in the cultures of pLenti6/V5-R-K-transduced CD34(+) hHSC were detected after infection by HIV-1 DP1 (a R5-tropic HIV-1 strain, which was isolated by the Centers for Disease Control and Prevention of China in Henan province in 2000 from a Chinese man who had asymptomatic HIV-1 infection with a history of blood transfusions). It was shown that pLenti6/V5-R-K transduction inhibited expression of the DP1 p24 antigen by 51%, 58% and 60% on the 4th, 7th and 10th day respectively (P<0.05).

Hantaan virus induces toll-like receptor 4 expression, leading to enhanced production of beta interferon, interleukin-6 and tumor necrosis factor-alpha.

Hantaan virus (HTNV) infects endothelial cells and is associated with increased vascular permeability during hemorrhagic fever with renal syndrome (HFRS). The pattern of increased vascular permeability is mediated by immune response. Therefore, it is necessary to characterize the mechanism of HTNV involvement in the host's innate immune. In this study, the expression of five toll-like receptors (TLRs) was analyzed in Endothelial vein cells (EVC-304) following HTNV infection in vitro. TLR4 showed an altered expression after HTNV infection. HTNV infection significantly increased IFN-beta, IL-6 and TNF-alpha secretion from EVC-304 cells, particularly after lipopolysaccharide stimulation. The increased IFN-beta, IL-6 and TNF-alpha production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HTNV-induced cytokine production. In conclusion, HTNV infection directly induces TLR4 expression and thereby enhanced production of IFN-beta, IL-6 and TNF-alpha, which may contribute to the host's innate immune response.