OBJECTIVE

Presenting twins are less likely to develop respiratory complications than non-presenting twins. The precise reason for this difference is not well understood, although it is known that the presence of inflammation reduces the risk of respiratory morbidity at birth. To further investigate this association, we compared the concentrations of inflammatory biomarkers in mid-trimester amniotic fluid (AF) of asymptomatic twin pairs.

METHODS

The study population consisted of women with twin pregnancies who underwent mid-trimester amniocentesis (15-20 weeks) for routine clinical indications and delivered at term. AF was analyzed for pro-inflammatory cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-15, IFN-γ, TNF-α), matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12), and chemokines (Complement Factor D/Adipsin, Serpin E1/PAI-1, Adiponectin/Acrp30, CRP, CCL2/MCP-1, Leptin, Resistin) using Luminex Performance Assay multiplex kits. Data were analyzed using Wilcoxon signed rank test.

RESULTS

A total of 82 twin pairs were enrolled. Mid-trimester AF concentrations of IL-8, MMP-8, CRP, MCP-1, leptin, and resistin were significantly higher in the presenting twin compared with the non-presenting twin (p<0.05 for each). Differences in AF concentrations of IL-8, MMP-8, and CRP persisted after adjustment for the fetal growth restriction at the time of birth and chorionicity.

CONCLUSIONS

These data suggest that, as early as the mid-trimester, the presenting fetus in an otherwise uncomplicated twin pregnancy is exposed to higher levels of pro-inflammatory mediators (especially IL-8, MMP-8, and CRP) than its non-presenting co-twin. Whether this pro-inflammatory milieu reduces the risk of neonatal respiratory morbidity at birth or has other functional implications needs to be further evaluated.

OBJECTIVE

To construct the recombinant adenovirus vector co-expressing the heterodimer of human interkeukin-12.

METHODS

The full-length cDNA encoding human IL-12 subunits p40 or p35 was cloned by RT-PCR separately. The cDNA was ligated with encephalomyocarditis internal ribosome entry site (IRES), placed under the control of CMV promoter, and inserted into E1-substituted adenovirus vector pAx1cw to produce the bicistronic coexpression vector. Subsequently, the hIL-12 recombinant adenovirus vector was cotransfected into 293 cells together with EcoT22I-digested Ad5 DNA-TPC, and the replication-deficient hIL-12 recombinant adenoviruses was generated efficiently by homologous recombination.

RESULTS

The human IL-12 recombinant adenoviruses were obtained with the titers of 2.1 x 10(9) pfu/ml. 48 hours after the infection of the 293 cells, HepG2 cells and human primary skin fibroblasts with hIL-12 recombinant adenoviruses, the hIL-12 expressions were detected by ELISA (30-50 ng/10(6) cells/24 h). The expressed hIL-12 could stimulate the in vitro proliferation and IFN-gamma production of human PMNC.

CONCLUSIONS

Prepared hIL-12 recombinant adenovirus vector can express biologically active hIL-12 and can be potentially used in cancer gene therapy.

Evidence has shown that the p7, NS2 and NS3 genes affect the outcome of pegylated-IFN-α/ribavirin (PEG-IFN/RBV) combination therapy in different populations with HCV infections. Here, we test the hypothesis that diversity in the p7, NS2 and NS3 genes influences the probability of obtaining either a sustained (SVR) or non-sustained (non-SVR) viral response in Chinese patients with genotype 1b chronic hepatitis C. There were significantly more unique variations in the p7, NS2 and NS3 genes in the sequences from SVR than non-SVR patients. Inter-patient variations related to treatment outcome in NS3 were concentrated in the protease domain. There were no significant differences in the frequency of variations in the core, E1 and E2 proteins between the groups. In conclusion, increased amino acid sequence diversity in the p7, NS2 and NS3 genes is associated with an SVR to PEG-IFN/RBV therapy in Chinese patients with genotype 1b chronic hepatitis C.

Genetic variants of HCV may have different degrees of resistance to IFN and may therefore influence the outcome of IFN therapy. However, selection of HCV variants by IFN has not been investigated in detail. In this paper, heteroduplex analysis was used to monitor major changes of HCV populations in 4 chronically infected patients under IFN therapy. We found that a major variant of the HCV 5' non-coding region (5' NCR) emerged in a responder. In other patients although no new variant of the 5' NCR was identified, significant changes occurred within the core and E1 region of the HCV genome. Disappearance and emergence of HCV variants may reflect their different susceptibilities to IFN. Our results indicate that responses of HCV populations to IFN are complex and need to be characterized by analysis of multiple HCV genome regions.

Osteoblasts are bone-forming mesenchymal cells, while macrophages are cells of hematopoietic origin responsible for innate immunity. Lipopolysaccharide (LPS) can induce tolerance in macrophages, whereas interferon (IFN)-gamma can activate macrophages to produce cytokines, exert bactericidal effects, and present antigens. In this study, we examined such macrophagic phenotypes regulated by LPS and IFN-gamma in murine osteoblasts. In both primary calvarial osteoblasts and osteoblastic MC3T3-E1 cells, LPS pretreatment resulted in reduced production of IL-6 in response to a subsequent LPS stimulation or to Salmonella infection, indicating the existence of LPS-induced tolerance in osteoblasts. Furthermore, IFN-gamma treatment of MC3T3-E1 cells resulted in both enhanced IL-6 production in response to LPS and upregulation of major histocompatibility complex class II (MHC II). Following infection, Salmonella-containing vacuoles (SCVs) were formed in MC3T3-E1 cells, and IFN-gamma pretreatment enhanced bactericidal effects on intracellular Salmonella. Taken together, these observations indicate that osteoblasts can exhibit a subset of phenotypes reminiscent of macrophages in the course of bacterial infection.

Many tumor cells escape from cancer immunosurveillance and resist treatment with interferons (IFNs). Although the mechanism underlying IFN resistance is mostly attributed to a deficiency of components of the IFN-signaling pathway, some types of tumor cells resist IFN-mediated cell growth arrest despite the presence of an intact JAK/STAT signaling pathway. However, the molecular mechanisms underlying the unresponsiveness to IFNs independent of the defective JAK/STAT pathway remain to be clarified. To elucidate the mechanisms underlying IFNγ resistance, we examined the anti-proliferative effect of IFNγ on mouse tumor cell lines. Mouse squamous cell carcinoma (SCCVII) cells were resistant to IFNγ-mediated cell growth arrest despite the presence of the IFNγ-induced STAT1-dependent signaling pathway, whereas IFNγ inhibited cell growth of B16/F1 cells, a well-known IFNγ-sensitive mouse melanoma cell line, at the G1 phase of the cell cycle. Treatment of SCCVII cells with IFNγ neither downregulated the expression of cyclin D1, cyclin A2, and cyclin E1 nor induced a hypo-phosphorylated, active form of retinoblastoma protein (pRb). Interestingly, the hyper-phosphorylated, inactive form of pRb was exclusively localized in the cytoplasm in SCCVII cells. The IFN-inducible 204 gene (Ifi204), whose gene product, p204, binds to pRb and exerts an anti-proliferative effect, was repressed in SCCVII cells. p204 overexpression in SCCVII significantly inhibited cell growth, and mutation of a pRb-binding LXCXE motif decreased the anti-proliferative effect. These results suggest that silencing of Ifi204/p204 induces resistance to IFNγ-mediated cell growth arrest in SCCVII cells.

Though purported to be identical cells (or in identical populations of cells), the natural killer (NK) cell mediating spontaneous natural cytotoxicity and the killer (K) cell mediating antibody-dependent cellular cytotoxicity (ADCC) may not be totally identical, at least in susceptibility to regulation by the immunomodulators prostaglandin E1 (PGE1) and interferon (IFN). We demonstrate here that NK cells are always enhanced by IFN, while K cells are inhibited from binding targets, resulting in fewer effectors at optimal concentrations of antibody. Only at 10- to 100-fold suboptimal concentrations of antibody is ADCC activity enhanced. As measured by magnitude of inhibition and dose-response titration, ADCC activity is less sensitive to the effects of PGE1 than is NK activity in the 51Cr release assay and single-cell assay. After overnight incubation with or without PGE1, whatever sensitivity ADCC activity had to PGE1 is lost. However, NK cells incubated in the presence of PGE1 overnight are still sensitive to inhibition. Indomethacin boosts NK activity without having any effect on ADCC activity. Finally, NK activity is substantially reduced by overnight incubation of cells at room temperature, which has no effect on K cells.

A population often contains distinct sub-populations, thereby increasing the complexity of the overall heterogeneity. However, the cellular origin and biological relevance of sub-populations in cell population have not been clearly identified. Here we demonstrated the novel roles of ISGylation, which is an IFN-induced post-translational modification, controlling heterogeneity at the population level in cultured adherent cells. Without UBE1L, an E1 enzyme of ISGylation, mouse embryonic fibroblasts (MEF) exhibited low viral resistance despite high STAT1 and ISG expression compared with the wild-type MEF. We observe that Ube1l-/- MEF populations consist of two behaviorally distinguishable sub-populations with distinct basal STAT1 activity, while wild-type MEF populations are unimodal. This population heterogeneity in Ube1l knock-out cells was perturbed by tyrosine kinase inhibitors, AG490 and PF431396. In contrast, the neutralization of type I IFN did not affect population heterogeneity. Based on these results, we concluded that UBE1L functions to adjust basal immunological states with the regulation of population heterogeneity.

Daily parenteral administration of exogenous interferon-gamma (IFN-gamma) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-gamma was constructed. The complete coding region of IFN-gamma was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-gamma minigene (dAv-IFN-gamma) was isolated from 293 cells co-transfected with the linearized plasmid and an E1 region-deleted fragment of adenovirus genome. Following in vitro infection with dAv-IFN-gamma, dose-dependent and time-dependent expression of IFN-gamma, mRNA and production of soluble protein were demonstrated in human diploid fibroblat and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for>> = 4 weeks following initial transfection, and secreted IFN-gamma induced both antiviral activity (8000-25,000 U/ml) and macrophage activation with killing of intracellular Toxoplasma gondii and leishmania donovani. These results establish that dAv-IFN-gamma generates long-term secretion of biologically active IFN-gamma in vitro and suggest that this vector may be a useful delivery system for cytokine therapy.

OBJECTIVE

To assess the evolution of in vitro T cell response to hepatitis C virus (HCV) Core, E1, E2 and NS3 antigens in 10 patients with chronic hepatitis C, before, during and after a high dose interferon alpha (IFN-alpha) therapy, and to evaluate the influence of IFN-alpha on the in vivo and in vitro production of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma).

METHODS

T cell response to HCV antigens was evaluated by lymphoproliferation assays. In vivo and in vitro cytokine production was evaluated at weeks 0, 4, 8, and 12 of IFN-alpha therapy by enzyme immunoassays.

RESULTS

In general, of all HCV antigens tested throughout the follow-up, those belonging to the Core region were the most immunostiumlatory. This observation was valid in IFN-alpha responders as well as IFN-alpha non-responders. The lymphoproliferative response to HCV antigens increased during IFN-alpha therapy. Serum levels of TNF-alpha were significantly increased in HCV patients, and six out of ten patients showed increased IFN-gamma serum levels. A significant decrease of IFN-gamma levels was observed during therapy and the same trend was seen for TNF-alpha. Mitogen-stimulated TNF-alpha and IFN-gamma production before therapy did not differ from that of normal controls, however, the cytokine production was reduced at week 4 of therapy, corresponding with a clinical improvement. A return to pretreatment values was observed after 8 weeks of therapy.

CONCLUSIONS

a) Core antigens are the most immunostimulatory HCV antigens at the T cell level in chronic hepatitis C patients; b) High dose IFN-alpha therapy induces an increase in lymphoproliferative response to HCV antigens; c) Serum levels of TNF-alpha are increased in HCV patients; d) High dose IFN-alpha therapy induces a decrease in serum levels of IFN-gamma; e) High dose IFN-alpha therapy induces a transiently decreased mitogen-induced TNF-alpha and IFN-gamma production.

Hepatitis C virus (HCV) entry into hepatocytes is a multistep process that represents a promising target for antiviral intervention. The viral envelope protein E1E2 plays a critical role in HCV entry. In this study, we sought to identify peptide inhibitors of HCV by screening a library of overlapping peptides covering E1E2. Screening the peptide library identified several novel anti-HCV peptides. Four peptides from glycoprotein E2 were selected for further investigation. The 50% effective dose (ED50) was approximately 5 nM for each peptide. Our data indicated that these peptides inhibited HCV entry at the post-attachment step. Moreover, these peptides blocked cell-to-cell transmission of HCVcc and had broad-spectrum antiviral effects on HCVcc. These peptides exhibited combination inhibitory effects on HCVcc infection when combined with IFN-α2b or anti-CD81 antibody. Interestingly, we observed that E2-42 associated with E1 and E2. Our results indicate that E2-42 inhibits HCV entry via E1 and E2. These findings suggest a new avenue for HCV therapeutic development.

This study addresses the fabrication of an extracellular matrix material of acellular sheep periosteum, and systematic evaluation of its biocompatibility to explore its potential application in guided bone regeneration. Sheep periosteum was harvested and decellularized by a combined decellularization protocol. The effectiveness of cell removal was proved and residual α-Gal antigen was also quantitively detected. Then, mouse MC3T3-E1 cells were seeded onto the acellular periosteum. Scanning electron microscope (SEM) was used to record the whole process of cell adhesion. CCK-8 assay suggested that the acellular periosteum not only had zero toxic effect on pre-osteoblasts, but played a positive role in cell proliferation. It was also tested that whether the acellular periosteum possess favorable osteogenesis induction activity attributed to ALP assay and quantitative real-time PCR (Col I, Runx2, OCN) assay. In vivo study of subcutaneous implantation test using SD rats was performed to detect the changes of IL-2, IFN-γ, IL-4 in serum and elucidate the host's local response to acellular periosteum through HE and immunohistochemical staining. The results show that acellular sheep periosteum did not elicit a severe immunogenic response via Th1 pathway unlike fresh sheep periosteum. In conclusion, acellular sheep periosteum possesses favorable biocompatibility to be employed for guided bone regeneration.

Integrin-mediated cell-matrix interactions play an important role in osteogenesis. Here, we constructed a novel osteoinductive fibronectin matrix protein (oFN) for bone tissue engineering, designed to combine the integrin-binding modules from fibronectin (iFN) and a strong osteoinductive growth factor, bone morphogenetic protein-2. Compared with iFN, the purified oFN matrix protein caused a significant increase in cell adhesion and osteogenic differentiation of pre-osteoblast MC3T3-E1 cells (p < 0.05).

Vascular hypertrophy may increase the blood pressure by its effect on vascular resistance. In this study, adenoviral gene transfer of IFN-beta was analysed in a porcine model of balloon injury to determine whether a secreted growth inhibitory protein might affect the regrowth of vascular smooth muscle cells (VSMC) in vitro and in arteries. An adenoviral vector encoding IFN-beta (ADV-IFN-beta) was constructed by homologous recombination between sub360 genomic DNA, an ADV 5 derivative with a deletion in the E3 region and a porcine IFN-beta expression plasmid. Its antiproliferative effect was analysed using cell proliferation assays, and used in a porcine model of balloon injury. After injury, arteries were immediately transfected with 7 x 10(9) plaques forming units of either ADV-IFN-beta or a control E1A deficient adenovirus that does not encode a recombinant protein, ADV-delta E1. The intima/media (I/M) area ratio was determined by quantitative morphometry 21 days after artery injury and gene transfer. Expression of recombinant porcine IFN-beta in VSMC reduced cell proliferation significantly in vitro, and supernatants derived from IFN-beta vector infected cells inhibited VSMC proliferation relative to controls. When introduced into porcine arteries after balloon injury, a reduction in I/M ratio of 30% was found. I/M ratio in the IFN-beta transduced arteries was 0.54 +/- 0.03 vs 0.69 +/- 0.06 in ADV-delta E1 transfected arteries and 0.702 +/- 0.05 in the non-transfected arteries. Gene transfer of an adenoviral vector encoding IFN-beta to VSMC and injured arteries reduced cell proliferation and vascular thickening. This approach is potentially applicable to vascular proliferative diseases.

BACKGROUND

Numerous evidences indicate that in some HIV-1 positive patients, the humoral and cellular immune responses are induced against HIV-1 proteins and this is inversely related to the progress of infection.

OBJECTIVE

The aim of this study was the evaluation of the Adenovectors containing HIV genes in induction of immune responses in mice.

METHODS

The HIV-1 genes including gag p24, rev, nef and exon-1 of tat were amplified from HIV-1 RNA (clade-A). The cDNA of each gene was cloned into a transfer vector. The transfer vector was then co-transformed into E. coli strain BJ5183 together with pAdenovector ∆E1/E3. The recombinant adenoviral construct was transfected into QBI-293A cells. Recombinant viruses were purified and titrated on 293 cell plates. Expression of transgenes was evaluated using western blotting. Then 10(12) viral particles were injected into 15 groups of 5 mice and all patterns of combination of these 4 HIV-1 genes were evaluated. After 2 weeks, humoral and cellular immune responses were evaluated using ELISA, cell proliferation and ELISpot (IL-2, IL-4 and IFN-γ) assays, consecutively.

RESULTS

It was demonstrated that each gene was expressed. The response targets were mostly toward Th1, though several Th2 responses were also observed. Single injection in our study induced a good cellular response but the humoral responses were not as strong as the cellular ones.

CONCLUSIONS

Considering and comparing all results and evaluating the various possible interactions revealed that simultaneous injection of tat and gag has enhanced the humoral and cellular responses.

The sensitivity of a library of adenovirus-transformed rat cell lines to lysis with highly enriched populations of rat NK cells and LAK cells activated in vitro by culture with recombinant human IL-2 was studied and correlated with the tumorigenic potential of these cell lines. The cell lines studied express the transforming E1 region of highly oncogenic Ad12 or nononcogenic Ad2. Two cell lines express recombinant E1A regions. In one the E1A genes were of Ad12 origin and the E1B region was derived from nononcogenic Ad5. In the other, the E1A region was from Ad5 and the E1B genes from Ad12. All cell lines tested which express the early region E1 of Ad12 are tumorigenic in syngeneic rats. The two cell lines which express only the E1A or the E1B genes of Ad12, and the Ad2-transformed cells did not induce tumors. Transformed cell lines which express the E1A region of nononcogenic Ad2 or Ad5 are efficiently killed by rat NK cells, but cells which express the Ad12 E1A genes are resistant to lysis by NK-enriched cell fractions even at high effector:target ratio; cells containing the Ad12 E1 region are also resistant to IFN-activated NK cells. Although such NK-resistant cells have a uniformly low level of class I MHC antigen, their resistance is not affected by MHC antigen level modulation by rat IFN. Ad12-transformed cells resistant to endogeneous NK cells, however, are efficiently lysed by LAK cells stimulated in vitro by recombinant IL-2. Sensitivity to LAK killing is unaffected by IFN treatment of target cells. These results show that expression of the E1A region of highly oncogenic Ad12 in the transformed cells, which confers resistance to endogeneous NK cells, fails to protect against lysis by LAK cells.

One of the prominent features of hepatitis C virus (HCV) is persistent infection, which is assumed to be a crucial event as a result of evading host defense system. Type I interferon beta (IFN- beta) system is induced rapidly after viral infection and plays a central role in innate immunity. Upon immediate induction of type I IFN as host first defense line, interferon regulatory factor-3 (IRF-3) is phosphorylated, formed of homodimer and translocates to nucleus. IFN-beta induction due to new castle disease virus (NDV) was significantly decreasd after the expression of full HCV genome (HCR6-Rz). Similar modification was observed in cell line expressing core to the NS2 protein region (HCR6-Fse). However, this decreasing was not observed in cell line expressing NS2 to the NS5B region (HCR6-Age). IRF-3 dimer formation induced by NDV infection was also suppressed after the expression of HCR6-Rz and HCR6-Fse, but not HCR6-Age. We further analyzed using transiently expressed HCV core, E1 or E2 in HepG2 cells. The suppression of IRF-3 dimer formation was caused by HCV core protein alone. These results indicated that a new crucial biological function of HCV core protein that may be related to persistence and pathogenesis of HCV.

Interferon gamma (IFN-γ) is the major immune mediator that prevents toxoplasmic encephalitis in murine models. The lack of IFN-γ secretion causes reactivation of latent T. gondii infection that may confer a risk for severe toxoplasmic encephalitis. We analyse the effect of IFN-γ on immune mediator production and parasite multiplication in human nerve cells infected by tachyzoites of two T. gondii strains (RH and PRU). IFN-γ decreased the synthesis of MCP-1, G-CSF, GM-CSF and Serpin E1 in all cell types. It decreased IL-6, migration inhibitory factor (MIF) and GROα synthesis only in endothelial cells, while it increased sICAM and Serpin E1 synthesis only in neurons. The PRU strain burden increased in all nerve cells and in contrast, RH strain replication was controlled in IFN-γ-stimulated microglial and endothelial cells but not in IFN-γ-stimulated neurons. The proliferation of the PRU strain in all stimulated cells could be a specific effect of this strain on the host cell.

Hepatitis C virus (HCV) has a devastating impact on human health, and infections can progress into liver fibrosis, cirrhosis, and hepatocellular carcinoma. There is no effective HCV vaccine. In this study, we rescued a recombinant PR8 influenza viral vector, called rgFLU-HCVCE1E2, carrying the core and envelope glycoprotein (C/E1/E2) epitopes of HCV inserted into the influenza nonstructural protein 1 gene. The morphological characteristics of rgFLU-HCVCE1E2 and the expression of the C/E1/E2 epitopes of HCV were examined. rgFLU-HCVCE1E2 replicated in various cell lines, including MDCK, A549, and Huh7.5 cells. More importantly, in BALB/c mice immunized intranasally twice at a 21-day interval with 104, 105, or 106 TCID50 rgFLU-HCVCE1E2, the viral vector induced a robust antibody response to influenza and HCV and potent IFN-γ and IL-4 secretion in response to HCV antigens in a dose-dependent manner. The rgFLU-HCVCE1E2 virus also stimulated IFN-γ production by virus-specific peripheral blood mononuclear cells in patients with chronic HCV infection. The study demonstrated that rgFLU-HCVCE1E2 carrying HCV antigens is immunogenic in vivo and has potential for the development of a HCV vaccine.

Hepatitis C virus (HCV) genotype 1b comprises mainly two subtypes in Japan, each named for its geographic prevalence (Japan-specific, J type; worldwide, W type). Because the newly identified subtypes have not been fully characterized, the present study directed this issue from virological viewpoints such as hypervariable region (HVR)-1 as well as interferon (IFN) sensitivity-determining region (ISDR). Fifty chronic hepatitis patients with HCV 1b (31 men and 19 women; mean age 50.5 years) were enrolled, and J/W type was determined according to envelope 1 (E1) sequence as described previously (23 J type and 27 W type). Correlations between age, number of HVR-1 clones, HVR-1 diversity, and ISDR mutations were analyzed in J and W type patients independently. In addition, the sequences of the three HCV regions obtained for the determination of the above genetic factors were studied phylogenetically. The number of HVR-1 clones was significantly higher for J type in comparison with W type (P = 0.044). In the J type-infected patients, the ISDR mutation number was correlated inversely with HVR-1 clone number (P = 0.0001, r = -0.734) and HVR-1 diversity (P = 0.0001, r = -0.722). However, this correlation was not observed in the W type patients. W type patients showed a significant correlation between age and HVR-1 clone number (P = 0.015, r = 0.462). Phylogenetic study revealed that the nonstructural (NS) 5A sequence, which is obtained for ISDR type determination, can distinguish between J and W types. The inverse correlation in J type patients between ISDR mutations and HVR-1 complexity may explain the usefulness of the ISDR for prediction of IFN response only in Japanese patients. This suggests that the ISDR is not directly related to IFN responsiveness, but the degree of HVR-1 complexity may be more important.

An E1-deleted adenoviral vector, pAdl2/RSV-IFN-bpA, harboring human IFN-gamma cDNA, and plasmid pJM17 were cotransfected into a human embryo kidney cell line 293 by the calcium-phosphate precipitation method. Six strains of recombinant adenovirus (rAd) were obtained after 11-day incubation. PCR analysis indicated that all six rAds contained human IFN-gamma cDNA. The biological activity of IFN-gamma was detected in the supernatant of the cultured 293 cells following rAd infection, and it could be blocked by the antibody against IFN-gamma. The B16F10 cells infected with the purified rAd (RSV/IFN-01) didn't show cytopathic effect (CPE), and more biological activity of IFN-gamma was detected in the supernatant of the infected B16F10 cells with increased MOI (multiple of infection) values. Therefore, the recombinant adenovirus constructed in this study can express and secrete human IFN-gamma.

Four short peptides from rubella virus proteins E1 and E2, predicted to contain B cell epitopes, were used to vaccinate BALB/c mice. Sera from peptide-vaccinated animals reacted with viral antigens in ELISA and three of the four induced virus-neutralising antibody (nAb) responses. Peptide PY4, in contrast to the others, induced IgG2a responses upon vaccination and stimulated spleen cells in vitro produced IFN gamma in the absence of IL-5. It was reasoned that vaccination with PY4 caused Th1 subset activation, the appropriate type of response for anti-viral immunity and hence the efficient neutralising antibody response. Presentation of peptide for vaccination proved to be as important as the sequence. Similar profiles of IgG1 and IgG2a were detected in the sera of mice vaccinated with PY4 in Freund's complete adjuvant or alum; however nAb responses were not found when alum was used.

OBJECTIVE

To investigate the effect of Escherichia coil heat-labile enterotoxin B subunit (LTB) as a genetic adjuvant in enhancing the immune response induced by Toxoplasma gondii tachyzoite compound gene vaccine.

METHODS

The eukaryotic expression plasmids of pcDNA3.1-SAG1-ROP2 and pEASY-E1-LTB were constructed. Eighty-eight BALB/c mice were randomly divided into four groups: PBS (group A), pcDNA3.1(B), pcDNA3.1-SAG1-ROP2 (C) and pcDNA3.1-SAG1-ROP2+pEASY-E1-LTB (D). Fifteen mice in each group were randomly selected, and intranasally immunized weekly with 20 microg plasmid or 20 microl PBS, respectively. Each mouse received four immunizations with the same dose of antigen. Two weeks after the final immunization, the antibodies and cytokines were detected, including the specific IgG and IgA antibodies in serum, sIgA in mucosa douche, IFN-gamma and IL-4 in splenocyte culture supernatant. The remaining mice in each group were immunized three times weekly with 20 microg plasmid or 20 microl PBS, respectively, and challenged by T. gondii tachyzoites at four weeks after the final vaccination (1 x 10(3) per mouse). The survival time of mice was recorded.

RESULTS

The recombinant plasmids pEASY-E1-LTB were constructed. The specific IgG (0.626/- 0.100) and IgA antibodies (1.086 +/- 0.138) in serum, sIgA (0.886 +/- 0.164) in mucosa douche, cytokines IFN-gamma [(2017 +/- 266) pg/ml] and IL-4 [(203.31) pg/ml] in splenocyte culture supernatant in group D were all higher than those in other groups (P < 0.05). After challenged with T. gondii tachyzoites, the median survival time of mice in groups A, B, C, and D were 3, 4, 6, and 10 d, respectively. The survival time of mice in group D was longest (P < 0.05).

CONCLUSIONS

E. coil heat-labile enterotoxin can enhance the immune response induced by the compound gene vaccine of T. gondii tachyzoites.

HCV genomes are considerable heterogeneities in nucleotide and amino acid sequences among individual isolates. The primary structure of the putative core and NS3 protein regions are relatively conserved among HCV isolates, while those of envelope proteins (E1 and E2) and NS4 are variable. On the basis of nucleotide sequence homology of parts of HCV genomes, several research groups have reported the possible existence of multiple subtypes of HCV isolates. From comparative sequence studies on HCV cDNA clones corresponding to NS3 and NS4 regions followed by diagnostic studies using these clones, have shown that there are at least two groups of HCV, group I and group II. The peptide produced in E. coli, carrying group I and II cDNA clones (A.A. positions 1676-1736 of NS4 region) are recognized by circulating antibodies specific to group I and II HCV. These group specific antigens of NS4 region are highly reliable in detecting (over 90%) circulating antibodies against either groups of HCV. In this study, most of the HCV isolates used were shown to be classified into group I or II HCV based on their amino acid differences and phylogenic analysis within the putative 5'UTR, core and NS3-4 regions. Biological significance of these two groups of HCV is suggested by the observation that the group I HCV was resistance to the IFN therapy (10-20% showing a good response), on the other hand the group II HCV showed a good response (70-90%).