Sabin IPV基础免疫后不同时间的中和抗体水平研究

目的 探讨不同剂量配比的Sabin株脊髓灰质炎灭活疫苗(Sabin IPV)基础免疫后中和抗体的持续时间及加强免疫前后中和抗体水平的变化.方法 用不同配比的Ⅰ、Ⅲ型脊髓灰质炎病毒原液制备两批Sabin IPV,并使用GSK制备的DTaP-w IPV作为阳性对照组,对18只Wistar大鼠进行3针基础免疫后,每间隔3个月采血直到加强免疫前,并同时于加强免疫后1个月采血,并对血清中抗脊髓灰质炎病毒3个型别的中和抗体效价进行初步研究.结果 大鼠采用0、1、2月免疫程序进行3针免疫后,Ⅰ、Ⅱ、Ⅲ型脊髓灰质炎病毒中和抗体的几何平均滴度随着时间的变化均有所下降,但绝大部分组大鼠的血清中和抗体阳转率仍维持在100％,在加强免疫后,各组的三个型别的中和抗体水平在短期内明显升高.结论 Sabin IPV有良好的免疫持久性,并在加强免疫后,可产生更高水平的中和抗体.     

Temperature and density dependent induction of a cytopathic effect following infection with non-cytopathic HAV strains

Hepatitis A virus infection and growth in cultured cells is protracted, cell-type restricted, and generally not accompanied by the appearance of a cytopathic effect, with the exception of some culture-adapted strains. We demonstrate that the non-cytopathic HAV strain HM175/clone 1 can be induced to exhibit a cytopathic phenotype in both persistently or acutely infected cells under co-dependent conditions of lower incubation temperature (<34 °C) and reduced cell density in both monkey (FRhK-4) and human (A549) cells. This phenotype is not virus-strain restricted, as it was also observed in cells infected with HAV strains, HAS-15 and LSH/S. Cytopathic effect was accompanied by rRNA cleavage, indicating activation of the RNase L pathway, viral negative strand synthesis, caspase-3 activation, and apoptosis. The results indicate that a cytopathic phenotype may be present in some HAV strains that can be induced under appropriate conditions, suggesting the potential for development of a plaque assay for this virus.     

Increased sensitivity of early apoptotic cells to complement-mediated lysis

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.     

人博卡病毒感染支气管上皮细胞生物学特征的研究

目的 研究人博卡病毒(HBoV)在呼吸道感染中的致病性及机制.方法采用人博卡病毒阳性标本感染健康人支气管上皮细胞,观察HBoV感染支气管上皮细胞后的生物学特征,并进行病毒空蚀斑、红细胞吸附抑制及免疫荧光测定.结果 75%感染细胞变圆并堆积成葡萄状,伴随有坏死、破碎、脱落.病毒蚀斑测定出典型"猫头鹰眼"现象.红细胞吸附实验观察到90%以上红细胞附着感染支气管上皮细胞.荧光检测出在细胞核内具有典型荧光块.结论支气管上皮细胞可用于分离HBoV,HBoV感染支气管上皮细胞后能够在该细胞内增殖,并导致特征性生物学改变.本研究为今后人博卡病毒致病机制研究奠定基础.     

In vitro susceptibility to infection with SIVcpz and HIV-1 is lower in chimpanzee than in human peripheral blood mononuclear cells

This study was undertaken to evaluate and compare the susceptibility of chimpanzee versus human peripheral blood mononuclear cells (PBMCs) to infection with SIVcpz and HIV-1 non-syncitium inducing primary isolates. The results demonstrate clearly that chimpanzee PBMCs have a lower capacity to support viral replication as compared to human PBMCs. There was no experimental evidence that this difference was due to a lower availability of target cells for viral infection (PBMCs positive for CD4 and CCR5 molecules) or to a differential susceptibility to apoptosis (PBMCs positive for CD4 and CD95 molecules). A lower capacity of chimpanzee PBMCs to support SIVcpz and HIV-1 replication in vitro is related to a post-entry barrier to virus replication.     

Serologic responses by immunoblot following natural infection with rotavirus serotypes G1 and G4 in children

Serologic responses to proteins of rotavirus serotypes G1, P1A[8]; G2, P1B[4]; G3, P1A[8]; and G4, P2A[6] were evaluated by immunoblotting paired sera from 17 children with primary rotavirus infection. Ten children were infected with G1, P1A[8]; five with G4, P1A[8]; and two with G4, P2A[6] viruses. Anti-VP6 and anti-VP2 were seen in most responses. Homotypic anti-VP7 developed following G1 and G4 infections in 8 (80%) and 6 (86%) cases, respectively. Homotypic anti-VP4 developed in 9 (60%) cases following P1A[8] infection and in 0 of 2 cases following P2A[6] infection. Heterotypic anti-VP7 appeared against G4 (20%) and G3 (20%) following the 10 G1 infections, and against G3 (86%) and G1 (57%) following the 7 G4 infections. Heterotypic anti-VP4 occurred in only 3 (18%) children. The data show the antigenic predominance of internal proteins VP6 and VP2. Homotypic antibodies developed against VP7 but not against VP4 in most cases, while heterotypic antibodies were infrequent. J. Med. Virol. 56:52–57, 1998. © 1998 Wiley-Liss, Inc.     

Fueling influenza and the immune response: Implications for metabolic reprogramming during influenza infection and immunometabolism

Recent studies support the notion that glycolysis and oxidative phosphorylation are rheostats in immune cells whose bioenergetics have functional outputs in terms of their biology. Specific intrinsic and extrinsic molecular factors function as molecular potentiometers to adjust and control glycolytic to respiratory power output. In many cases, these potentiometers are used by influenza viruses and immune cells to support pathogenesis and the host immune response, respectively. Influenza virus infects the respiratory tract, providing a specific environmental niche, while immune cells encounter variable nutrient concentrations as they migrate in response to infection. Immune cell subsets have distinct metabolic programs that adjust to meet energetic and biosynthetic requirements to support effector functions, differentiation, and longevity in their ever-changing microenvironments. This review details how influenza coopts the host cell for metabolic reprogramming and describes the overlap of these regulatory controls in immune cells whose function and fate are dictated by metabolism. These details are contextualized with emerging evidence of the consequences of influenza-induced changes in metabolic homeostasis on disease progression.     

Caspase inhibition reduces lymphocyte apoptosis and improves host immune responses to Trypanosoma cruzi infection

In experimental Chagas' disease, lymphocytes from mice infected with Trypanosoma cruzi show increased apoptosis in vivo and in vitro. Treatment with a pan-caspase blocker peptide inhibited expression of the active form of effector caspase-3 in vitro and rescued both B and T cells from cell death. Injection of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone, but not a control peptide, reduced parasitemia and lymphocyte apoptosis in T. cruzi-infected mice. Moreover, treatment with caspase inhibitor throughout acute infection increased the absolute numbers of B and T cells in the spleen and lymph nodes, without affecting cell infiltrates in the heart. Following treatment, we found increased accumulation of memory/activated CD4 and CD8 T cells, and secretion of IFN-gamma by splenocytes stimulated with T. cruzi antigens. Caspase inhibition in the course of infection reduced the intracellular load of parasites in peritoneal macrophages, and increased the production of TNF-alpha and nitric oxide upon activation in vitro. Our results indicate that inhibition of caspases with a pan-caspase blocker peptide improves protective type-1 immune responses to T. cruzi infection. We suggest that mechanisms of apoptosis are potential therapeutic targets in Chagas' disease.     

Innate and adaptive immune response to apoptotic cells

The immune system is constantly exposed to dying cells, most of which arise during central tolerance and from effete circulating immune cells. Under homeostatic conditions, phagocytes (predominantly macrophages and dendritic cells) belonging to the innate immune system, rapidly ingest cells and their debris. Apoptotic cell removal requires recognition of altered self on the apoptotic membrane, a process which is facilitated by natural antibodies and serum opsonins. Recognition, may be site and context specific. Uptake and ingestion of apoptotic cells promotes an immunosuppressive environment that avoids inflammatory responses to self-antigens. However, it does not preclude a T cell response and it is likely that constant exposure to self-antigen, particularly by immature dendritic cells, leads to T cell tolerance. Tolerance occurs by several different mechanisms including anergy and deletion (for CD8+T cells) and induction of T regulatory cells (for CD4+T cells). Failed apoptotic cell clearance promotes immune responses to self-antigens, especially when the cellular contents are leaked from the cell (necrosis). Inflammatory responses may be induced by nucleic acid stimulation of Toll like receptors and other immune sensors, specific intracellular proteins and non-protein (uric acid) stimulation of inflammasomes.     

Matrix protein of VSV New Jersey serotype containing methionine to arginine substitutions at positions 48 and 51 allows near-normal host cell gene expression

The matrix (M) protein of vesicular stomatitis virus (VSV) plays significant roles in the replication of VSV through its involvement in the assembly of virus particles as well as by facilitating the evasion of innate host cell defense mechanisms. The presence of methionine at position 51 (M51) of the matrix (M) protein of the VSV Indiana serotype (VSV(Ind)) has been proven to be crucial for cell rounding and inhibition of host cell gene expression. The M protein of VSV(Ind) with the substitution of M51 with arginine (R:M51R) results in the loss of inhibitory effects on host cell gene expression. The VSV(Ind) expressing the M(M51R) protein became the attractive oncolytic virus which is safer and more tumor-specific because the normal cells can clear the mutant VSV(Ind) easily but tumor cells are susceptible to the virus because a variety of tumor cells lack innate antiviral activities. We have studied the role of the methionines at positions 48 and 51 of the M protein of the New Jersey serotype of VSV (VSV(NJ)) in the induction of cytopathic effects (CPE) and host cell gene expression. We have generated human embryonic kidney 293 cell lines inducibly expressing M proteins with M to R mutations at positions 48 and 51, either separately or together as a double mutant, and examined expression of heat shock protein 70 (HSP70) as an indicator of host cell gene expression. We have also generated recombinant VSV(NJ) encoding the mutant M proteins M(M48R) or M(M48R+M51R) for the first time and tested for the expression of HSP70 in infected cells. Our results demonstrated that the M51 of VSV(NJ) M proteins has a major role in cell rounding and in suppressing the host cell gene expression either when the M protein was expressed alone in inducible cell lines or when expressed together with other VSV proteins by the recombinant VSV(NJ). Amino acid residue M48 may also have some role in cell rounding and in the inhibitory effects of VSV(NJ) M, which was demonstrated by the fact that the cell line expressing the double substitution mutant M(M48R+M51R) exhibited the least cytopathic effects and the least inhibitory effect on host cell gene expression.     

Cultured early goat embryos and cells are susceptible to infection with caprine encephalitis virus

Zona-pellucida-free embryos at 8-16 cell stage were co-cultured for 6 days in an insert over a mixed cell monolayer infected with CAEV-pBSCA. Embryos were washed and transferred to an insert on CAEV indicator goat synovial membrane cells for 6 h, then they were washed and cultivated in B2 Ménézo for 24 h, finally, embryo cells were dissociated and cultivated on a feeder monolayer for 8 days. After 5 weeks, multinucleated giant cells typical of CAEV infection were observed in indicator GSM cell monolayers. In the acellular medium, the early embryonic cells produced at least 10(3.25) TCID50/ml over 24 h. The monolayer of cultivated embryonic cells developed cytopathic lesions within 8 days, and CAEV RNA, CAEV proviral DNA and protein p28 of the capsid were detected. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with CAEV and that infection with this virus is productive.     

Controlled infection with a therapeutic virus defines the activation kinetics of human natural killer cells in vivo

Human natural killer (NK) cells play an important role in anti‐viral immunity. However, studying their activation kinetics during infection is highly problematic. A clinical trial of a therapeutic virus provided an opportunity to study human NK cell activation in vivo in a controlled manner. Ten colorectal cancer patients with liver metastases received between one and five doses of oncolytic reovirus prior to surgical resection of their tumour. NK cell surface expression of the interferon‐inducible molecules CD69 and tetherin peaked 24–48 h post‐infection, coincident with a peak of interferon‐induced gene expression. The interferon response and NK cell activation were transient, declining by 96 h post‐infection. Furthermore, neither NK cell activation nor the interferon response were sustained in patients undergoing multiple rounds of virus treatment. These results show that reovirus modulates human NK cell activity in vivo and suggest that this may contribute to any therapeutic effect of this oncolytic virus. Detection of a single, transient peak of activation, despite multiple treatment rounds, has implications for the design of reovirus‐based therapy. Furthermore, our results suggest the existence of a post‐infection refractory period when the interferon response and NK cell activation are blunted. This refractory period has been observed previously in animal models and may underlie the enhanced susceptibility to secondary infections that is seen following viral infection.     

Active caspase 3 and DNA fragmentation as markers for apoptotic cell death in primary and metastatic liver tumours

AIMS: The induction of tumour cell death by apoptosis is a major goal of cancer therapy and the in situ detection of apoptosis in tumour tissue has become an important diagnostic parameter. Different apoptosis detection methods assess distinct biochemical processes in the dying cell. Thus, their direct comparison is mandatory to evaluate their diagnostic value. The aim of this study was to compare the immunohistochemical detection of active caspase 3 and single-stranded DNA in primary and metastatic liver tumours as markers of apoptotic cell death. METHODS: We studied detection of active caspase 3 and single-stranded DNA in 20 primary hepatocellular carcinomas (HCC) and 20 liver metastases from colorectal carcinomas (CRC) using immunohistochemistry on paraffin sections. RESULTS: Our results reveal that both methods are suitable and sensitive techniques for the in situ detection of apoptosis, however, they also demonstrate that immunohistochemistry for active caspase 3 and single-stranded DNA have differential sensitivities in HCC and CRC. CONCLUSION: The sensitivity of apoptosis detection using immunohistochemistry for active caspase 3 and single-stranded DNA may be tumour cell type dependent.     

Antibodies to the Giardia lamblia double-stranded RNA virus major protein can block the viral infection

The double-stranded RNA virus-like particles, found among several independent isolates and cloned strains of Giardia lamblia, have previously been reported to be spheres of 35 nm with a genome of 7 kilobase pairs and a major protein of 100 kDa. The virus is capable of infecting certain virus-free isolates of G. lamblia. Antisera raised in mice against the intact virus did not react with the double-stranded RNA, but reacted strongly with the 100 kDa protein in Western blots. Preincubation of the virus with antisera abolished viral infectivity, whereas the antisera against double-stranded RNA showed only a weak blocking effect. Inclusion of the antiviral sera in the cultures of virus-infected G. lamblia at 10³-fold dilution resulted in elimination of the virus from the protozoa. Apparently, the 100 kDa protein is necessary for the initiation of viral infection and possibly subsequent assembly or replication of viral progeny particles.     

Herpes simplex virus infections in male and female mice following pinna inoculation: responses to primary infection and artificially induced recurrent disease

The pathogenesis of herpes simplex virus (HSV) infection is now well understood. After humans or experimental animals recover from primary infection, the virus remains latent in sensory ganglia of the peripheral nervous system. Latency, for the most part, remains unresolved, and elucidating the mechanisms involved with latency has proved difficult. The requirement for unravelling HSV latency is the availability of a reproducible animal model. Previously, the mouse ear model has been extensively characterized; however, many studies using this model have involved female mice only despite evidence that recurrent HSV infection in humans may vary by gender. We inoculated male and female mice subcutaneously in the pinna with varying amounts of one of four strains of HSV and monitored the mice for signs of primary infection. Following recovery from primary infection, mice were induced to develop recurrent disease. In addition, we attempted to isolate virus from dorsal root ganglia of mice suspected of harboring latent virus. There were no differences in the response of male and female mice to either primary infection or artificially induced recurrent disease when inoculated with the same virus. Differences were noted when female mice were inoculated with different strains of virus.