OBJECTIVETo investigate the pathogenesis and immunogenicity of H9N2 influenza virus A/Guangzhou/333/99 (a reassortant of G1 and G9 viruses isolated from a female patient in 1999) in a mouse model of infection.

METHODSMice were infected with increasing virus titers. Viral load in the lungs and trachea was determined by EID50 assay. Pulmonary histopathology was assessed by hematoxylin-eosin staining. Anti-HI antibody titers and T-cell responses to viral HA were determined by ELISPOT and confirmed by flow cytometry.

RESULTSMice presented a mild syndrome after intranasal infection with A/Guangzhou/333/99 (H9N2) influenza virus. Virus was detected in the trachea and lungs of mice harvested on days 3, 6, and 9 post-infection. A T-cell response to viral HA was detected on day 6 and H9 HA-specific CD(4+) T-cells predominated. Seroconversion was detected after 14 days and antibody persisted for at least 28 weeks.

CONCLUSIONOur results suggest that H9N2 (A/Guangzhou/333/99) can replicate in the murine respiratory tract without prior adaptation, and both humoral and cell-mediated immunity play an important role in the immune response.

Animals ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Cell Line ; Dogs ; Enzyme-Linked Immunospot Assay ; Female ; Hemagglutination Inhibition Tests ; Hemagglutinins, Viral ; immunology ; Humans ; Infant ; Influenza A Virus, H9N2 Subtype ; immunology ; isolation & purification ; pathogenicity ; Interferon-gamma ; immunology ; Lung ; virology ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections ; immunology ; virology ; Spleen ; immunology ; Trachea ; virology ; Viral Load ; Virulence

Studies were performed to determine the effects of Bcell suppression on the pathogenesis of Subgroup J avian leukosis virus (ALV-J) in broiler chickens. Neonatal chickens were treated with cyclophosphamide (CY) or PBS, and then infected with ALV-J (ADOL-7501) at 2 weeks of age. CY treatment induced B cell specific immunosuppression throughout the experiment confirmed by decreased bursal weight, intact lymphocyte mitogenetic activity stimulated by Con A and increased relative subpopulation of CD3-positive cells as measured by flow cytometry. Chickens in this experiment had Mareks disease virus exposure prior to three weeks of age as determined by the presence of lymphocytic infiltration and antibody. Virus neutralizing antibody against ALV-J was first observed at 6 weeks post-infection in some of the infected chickens in the PBS group. As expected, none of the chickens from the CY group and uninfected chickens developed virus-neutralizing antibody. The viremic status was measured by real time RT-PCR using SYBR green I dye. The percentage of viremic chickens was significantly higher, and more chickens had high titered viremia, in the CY treated group. No neoplastic foci consistent with ALVJ infection were observed in any of the experimental chickens. The frequency and intensity of viral antigen expression determined by immunohistochemistry was significantly higher in tissues from CY treated birds than those of PBS treated chickens at 3 weeks post-infection. This study showed that B cell specific immunosuppression with CY treatment in chickens resulted in increase in viremia and viral antigen load in tissues.
Animals ; Avian Leukosis/*immunology/virology ; Avian leukosis virus/genetics/*immunology ; Body Weight/physiology ; Bursa of Fabricius/immunology ; *Chickens ; Concanavalin A/immunology ; Cyclophosphamide/*pharmacology ; Flow Cytometry/veterinary ; Immunocompromised Host ; Immunohistochemistry/veterinary ; Immunophenotyping/veterinary ; Immunosuppressive Agents/*pharmacology ; Lymphocyte Activation/drug effects/immunology ; Organic Chemicals/chemistry ; Poultry Diseases/immunology/*virology ; RNA, Viral/chemistry/genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Spleen/immunology/virology ; Statistics, Nonparametric ; Viremia/veterinary

OBJECTIVETo investigate the effect of Dureping Injection (DRP) on the T-cells function of mice and the function of T-cells in killing MF infected by influenza virus subtype A mice-lung adaptive strain FM1 in vitro.

METHODSNumber of splenic normal and FM1 infected T-cells in mice were measured by MTT and double-antibody sandwich ELISA, after being treated with DRP at different concentrations (2.1, 8.5 and 17.0 microg/mL), and the effect of DRP on interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) production as well as on splenic T-cell killing FM1 infected Mphi/Ana-1 function were detected.

RESULTSDRP inhibited the multiplication of normal spleen T cells induced by concanavalin A in vitro, suppressed Th2 cell factor IL-10 production, and maintained Th1 cell factor IFN-y at a definite level, moreover, it directly enhanced the power of T-cells in killing FM1 infected Mphi (P < 0.05, P < 0.01).

CONCLUSIONDRP could act on mice T-cells to enhance the immune response for antiinfluenza viral FM1 in vitro.

Adjuvants, Immunologic ; pharmacology ; Animals ; Antiviral Agents ; pharmacology ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Influenza A virus ; drug effects ; Interferon-gamma ; immunology ; metabolism ; Interleukin-10 ; immunology ; metabolism ; Mice ; Mice, Inbred BALB C ; Orthomyxoviridae Infections ; immunology ; Spleen ; cytology ; T-Lymphocytes ; cytology ; immunology ; virology

OBJECTIVETo investigate role of CD4-CD8- T cells in murine hepatitis virus type 3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice and to identify their surface markers.

METHODSThirty C3H/Hej mice received 10 Pfu MHV-3 intraperitoneally, the CD4-CD8- T cells were isolated using magnetic bead sorting on 0, 4, 15, 30, 40 days post MHV-3 infection. The cytotoxic effects of CD4-CD8- T cells on normal and infected hepatocytes, CD8+ T cells and unrelated-virus (murine cytomegalovirus, MCMV) infected CD8+ T cells were examined by non-radioactive cytotoxicity assay. The surface markers of CD4-CD8- T cells were determined by flow cytometry.

RESULTSMHV-3 infected CD4-CD8- T cells showed significant cytotoxic effect on CD8+ T cells, but not on infected hepatocytes or MCMV infected CD8+ T cells. The analysis of cell surface markers demonstrated that the CD4-CD8- T cells are a completely new T cell subset.

CONCLUSIONSCD4-CD8- T cells have significant cytotoxic effect on virus specific CD8+ T cells in MHV-3 infected C3H/Hej mice, which suggests that CD4-CD8- T cells have immune modulatory functions in the development of chronic viral hepatitis. The phenotype of these CD4-CD8- T cells detected by flow cytometry is TCR alpha beta +CD3+CD4- CD8- CD25- CD28- CD30- CD44+.

Animals ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; Coronavirus Infections ; immunology ; pathology ; virology ; Female ; Flow Cytometry ; Hepatitis, Viral, Animal ; immunology ; pathology ; virology ; Liver ; immunology ; pathology ; Mice ; Mice, Inbred C3H ; Murine hepatitis virus ; Spleen ; immunology ; pathology ; T-Lymphocyte Subsets ; immunology ; Time Factors

This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Chitosan ; administration & dosage ; immunology ; Dendritic Cells ; immunology ; virology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; genetics ; immunology ; Humans ; Immunity, Cellular ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; administration & dosage ; Spleen ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

The critical time of avian leukosis virus subgroup J (ALV-J)-mediated immunosuppression was determined by body weight, relative immune organ weight, histopathology, and presence of group specific antigen and antibodies in specific pathogen-free (SPF) chickens. CD4+ and CD8+ cell activity in the spleen, total and differential leukocyte counts in blood, and viral RNA levels in spleen were measured. Significant growth suppression was observed in the two ALV-J-infected groups. A strong immune response by infected groups was present in spleen at 2-weeks-of-age, but after 4-weeks-of-age, the response decreased quickly. The thymus and bursa showed persistent immunosuppression until 4-weeks-of-age. Proliferation of fibroblasts and dendritic cells were observed in immune organs at 4- and 5-weeks-of-age. However, the granulocyte cell number was markedly lower in the infected groups than in the control group. In group 1 (day 1 infection) CD4+ cells increased during the second week but significantly decreased during the fourth week, while group 2 (day 7 infection) showed the opposite effect. Viral RNA increased significantly by the fourth week. These data identify 3~4 weeks post-infection as the key time at which the ALV-J virus exerts its immunosuppressive effects on the host.
Animals ; Antibodies, Viral/blood ; Antigens, CD4/blood ; Antigens, CD8/blood ; Avian Leukosis/*immunology/transmission/virology ; Avian leukosis virus/classification/*immunology ; Body Weight ; *Chickens ; China ; Enzyme-Linked Immunosorbent Assay/veterinary ; Immune Tolerance ; Leukocyte Count/veterinary ; Poultry Diseases/*immunology/transmission/virology ; RNA, Viral/genetics ; Real-Time Polymerase Chain Reaction/veterinary ; Reverse Transcriptase Polymerase Chain Reaction/veterinary ; Specific Pathogen-Free Organisms ; Spleen/immunology

To explore the association between T-cell receptor beta variable (TCR BV) complementarity determining region 3 (CDR3) spectratyping and CMV activation in the recipients of allogeneic hematopoietic stem cell transplantation (HSCT).Fluorescence quantitative PCR melting curve analysis was used to sequence 24 TCR BV families in 7 HSCT recipients and 3 healthy controls. CMV-pp65 antigenemia was measured by immunohistochemical staining. Plasma IgM specific for CMV was identified using ELISA. Relationship between TCR BV families and CMV activation was statistically analyzed.Twenty-four TCR BV families were expressed in 3 healthy controls, while TCR BV CDR3 sequencing results in 7 recipients turned out to be BV9, BV11, BV17, BV20 and so on. Amino acid sequence features were as follows:TCR BV9 contained "QVRGGTDTQ", TCR BV11 contained "VATDEQ" and "LGDEQ", TCR BV17 contained "IGQGNTEA", and TCR BV20 contained "VGLAANEQ". Five recipients suffered from pp65 antigenemia in 3 month after transplantation, and pp65-positive cells ranged from 2 to 15 per 5×10white blood cells. Three recipients were CMV-IgM positive. No significant differences were found in TCR BV families between pp65-positive recipients and pp65-negative recipients (all>0.05). But there was statistically significant difference in frequency of TCR BV11 between CMV-IgM negative recipients and CMV-IgM positive recipients (<0.05).T cell immune response was characterized by special TCR BV CDR3 spectratyping in HSCT recipients, and TCR BV11 expression may be associated with CMV activation.
Amino Acid Sequence ; Complementarity Determining Regions ; genetics ; immunology ; Cytomegalovirus ; genetics ; immunology ; Cytomegalovirus Infections ; genetics ; Genotype ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Lymphocyte Activation ; genetics ; Phosphoproteins ; Polymerase Chain Reaction ; Polymorphism, Genetic ; immunology ; Receptors, Antigen, T-Cell, alpha-beta ; genetics ; immunology ; Spleen ; T-Lymphocytes ; immunology ; virology ; Viral Matrix Proteins

OBJECTIVETo investigate the anti-HIV effects of ampelopsin and its interaction with HIV-1 coreceptor CXCR4.

METHODSThrough anti-virus experiments in vitro, the inhibitory effect of ampelopsin on HIV-1 infection was verified. Chemotaxis assay was performed to show the ability to induce PBMCs migration by ampelopsin, RANTES and SDF-1alpha. Fluorescence labelling monoclonal antibody was utilized to observe the interaction of ampelopsin and CXCR4. Mice immunosuppressant model was also established to detail the role ampelopsin played in regulating cellular immunological functions.

RESULTSAmpelopsin could protect sensitive cells against HIV-1 infection and dramatically reduce HIV-1 antigen P24 expression. HIV-1SF33 attaching to MT-4 cells was interfered by ampelopsin, and the EC50 was 0.175 mg/mL for cellular protection and 0.024 mg/mL for P24 inhibition. At co-cultivating phase, EC50 was 0.229 mg/mL and 0.197 mg/mL respectively. Furthermore, the EC50 was 0.179 mg/mL and 0.348 mg/mL in acute infection. Human PBMCs migration was induced after being challenged with ampelopsin or chemokines, and synergistic action was observed during co-treatment. Ampelopsin alone resulted in maximal chemotaxis at 1 mg/mL. HIV-1 co-receptor CXCR4 on the surface of PBMCs was decreased by internalization, which indicated the effect of ampelopsin on CXCR4. About 70% CXCR4 was reduced by ampelopsin at 1 mg/mL. Ampelopsin also augmented cellular immunological functions in immunosuppressive mice.

CONCLUSIONAmpelopsin displays a strong inhibitive role during HIV-1 absorption, incubation and acute infection. These results are coincident with its immune enhancement.

Ampelopsis ; chemistry ; Animals ; Anti-HIV Agents ; pharmacology ; Cell Line ; Chemokine CCL5 ; pharmacology ; Chemokine CXCL12 ; Chemokines, CXC ; pharmacology ; Chemotaxis, Leukocyte ; Down-Regulation ; Drugs, Chinese Herbal ; Flavonoids ; economics ; isolation & purification ; pharmacology ; HIV Infections ; virology ; HIV-1 ; drug effects ; metabolism ; pathogenicity ; Humans ; Interleukin-2 ; biosynthesis ; Leukocytes, Mononuclear ; drug effects ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plant Roots ; chemistry ; Receptors, CXCR4 ; antagonists & inhibitors ; drug effects ; Spleen ; immunology ; T-Lymphocytes ; immunology