The role of EF-Tu in the expression of tufA and tufB genes

Microbial Biotechnolog

tuf Gene dosage effects on the intracellular concentration of EF-TuB

In this paper we have studied the effect of raising the intracellular EF‐Tu concentration on the expression of tufB. To this aim cells were transformed with multicopy plasmids carrying either tufA or tufB. The intracellular EF‐Tu concentrations were determined by the specific immunoelectrophoresis assay described in the preceding paper in this journal. We have cloned the tufA gene in a plasmid, containing the powerful major leftward promoter (Pl) of phage λ Transcription from Pl can be repressed at low temperature by a temperature‐sensitive repressor and acitvated by heat induction. Cloning occurred in two orientations in a single EcoRI site about 150 base pairs downstream of Pl. Cells carrying either plasmid were shown to contain an almost doubled amount of EF‐Tu at temperatures from 28°C to 37°C. This indicates that transcription of tufA can proceed from a possible binding site for RNA polymerase on these cloned fragments. The EF‐Tu level was further increased to about 30% of total cellular protein after a temperature shift from 37°C to 43°C. The multicopy plasmid pTuB1 described by Miyajimaet al. [FEBS Lett. 102, 207–210 (1979)] and a derivative (pTuBo, compare preceding paper in this journal) were used to study the expression of both chromosomal and plasmid‐borne tufB. Transformation with either plasmid raised the intracellular EF‐Tu concentration by 30–60% depending on the nutritional conditions. Suppression of tufB expression was observed when the intracellular level of EF‐Tu increased after transformation with all plasmids mentioned above. The results are in accord with the concept that EF‐Tu acts as an autogenous feedback inhibitor involved in the regulation of tufB.Microbial Biotechnolog

Tumor necrosis factor α promotes replication and pathogenicity of rat cytomegalovirus

We investigated the role of tumor necrosis factor alpha (TNF-α) in the pathogenesis of rat cytomegalovirus (RCMV) infection. TNF-α levels found in the sera of radiation-immunosuppressed rats in the course of infection (> 350 pg/ml) correlated with the development of RCMV disease. Administration of anti-TNF-α antibodies strongly reduced the severity of pneumonia and led to a reduction in virus titers. In immunocompetent rats, anti-TNF-α antibodies also significantly suppressed viral replication. Conversely, administration of TNF-α augmented RCMV replication and aggravated the disease signs. In vitro, TNF-α enhanced RCMV replication in the macrophage, whereas a reduction of viral replication was observed in fibroblasts, indicating that the effect on viral replication is cell type specific. Besides activation of viral replication and exacerbation of RCMV disease, TNF-α also favored lymphoid and hematopoietic tissue reconstitution after irradiation, which may contribute to antiviral resistance and survival. This finding demonstrates the protean nature of TNF-α, with both beneficial and adverse effects for the host. Our results suggest that TNF-α plays an important role in modulating the pathogenesis of RCMV infection

Suppression of rat cytomegalovirus replication by antibodies against γ interferon

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma

Suppression of rat cytomegalovirus replication by antibodies against γ interferon

The role of gamma interferon (IFN-gamma) in the resolution of rat cytomegalovirus (RCMV) infection was investigated. In the spleen, IFN-gamma-producing cells reached maximum numbers on day 7 after infection. Prophylactic treatment with high doses of recombinant rat IFN-gamma exerted antiviral activity in fibroblasts and protected immunosuppressed rats against a lethal RCMV challenge. Remarkably, in immunocompetent rats, neutralization of endogenous IFN-gamma activity significantly reduced the numbers of RCMV antigen-expressing cells in the spleen, the predominant site of viral replication. Moreover, protection of radiation-immunosuppressed infected rats by transferred immune T cells was enhanced by coinjection of IFN-gamma neutralizing antibodies. The observations were paralleled by in vitro findings: low concentrations of IFN-gamma enhanced viral replication in both macrophages and fibroblasts. These data suggest that IFN-gamma can play different and even opposite roles in the regulation of RCMV replication in vivo; T lymphocytes may contribute to the progression of RCMV infection by secreting IFN-gamma

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)-γ producing cells determined by enzyme-linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN-γ producing cells reactive to this pathway increased significantly (P = 0.04) during AR and the number decreased (P = 0.005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN-γ producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR

Induction of the trypanosome lymphocyte-triggering factor (TLTF) and neutralizing antibodies to the TLTF in experimental African trypanosomiasis

We have demonstrated that African trypanosomes secrete a novel trypanokine, the trypanosome-derived lymphocyte-triggering factor (TLTF), which activates CD8+ cells to produce interferon-γ (IFN-γ) that in turn stimulates parasite growth. The gene for TLTF was recently cloned, and recombinant TLTF (rTLTF) showed bioactivity that was similar to native TLTF. In this work, we employed two anti-TLTF monoclonal antibodies (mAbs) to detect levels of TLTF during Trypanosoma brucei brucei (T. b. brucei) infections in mice. Furthermore, rTLTF was utilized to assess levels of anti-TLTF antibodies. Mice with intact genes (wild type), and knockout mice with disrupted IFN-γ (IFN-γ−/−) or IFN-γR (IFN-γR−/−) genes were studied. The knockout mice were used in order to illustrate the role of IFN-γ in the production of antibodies to TLTF. While wild-type mice showed high parasitaemia accompanied by high TLTF levels and low anti-TLTF antibodies at day 3 postinfection (p.i.), low TLTF was measured together with increased anti-TLTF antibodies at day 21 p.i. IFN-γ−/− mice exhibited very low parasitaemia, TLTF and anti-TLTF antibody levels. In contrast, IFN-γR−/− mice revealed very high parasitaemia, increased TLTF levels, but decreased anti-TLTF antibodies. In a biological assay for TLTF, Fab′ fragments of anti-TLTF antibodies dose dependently inhibited the TLTF-induced IFN-γ production by splenocytes, suggesting a regulatory importance of these antibodies. Our data demonstrate a role of IFN-γ in the generation of neutralizing antibodies to TLTF. Furthermore, the induction of TLTF and its antibodies may constitute a new approach for future diagnosis of African trypanosomiasis

Inhibitory effects of endogenous and exogenous interferon-γ on bronchial hyperresponsiveness, allergic inflammation and T-helper 2 cytokines in Brown–Norway rats

Interferon-γ (IFN-γ) is an important cytokine involved in the regulation of allergen-induced immune responses. We examined the role of IFN-γ in a Brown–Norway rat model of bronchial hyperresponsiveness (BHR) and airway eosinophilia, and its effects on the mRNA expression of T helper type 1 (Th1)/Th2 cytokine. Ovalbumin (OA)-sensitized animals were given either exogenous IFN-γ (105 U/rat over 3 days, intraperitoneally) or anti-IFN-γ blocking antibody (DB-1 0·3 mg/rat, intravenously) prior to exposure to OA aerosol and were studied 18–24 hr later. In sensitized animals, OA induced significant BHR, accumulation of eosinophils, T lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also increased eosinophils and CD8+ T cells in the airways. Exogenous IFN-γ attenuated allergen-induced BHR (P<0·02, compared with sham-treated animals) together with a significant reduction in eosinophil and neutrophil numbers in BAL fluid (P<0·005), and eosinophils and CD8+ T cells in airways (P<0·05). By contrast, anti-IFN-γ antibody increased airway CD4+ T cells and BHR. Using reverse transcriptase–polymerase chain reaction, significant increases in Th2 [interleukin-4 (IL-4), IL-5 and IL-10], and IFN-γ cytokine mRNA were found in the lungs of sensitized and OA-exposed animals, while exogenous IFN-γ significantly suppressed IL-4, IL-5 and IL-10 mRNA expression, and anti-IFN-γ antibody increased IL-4 and IL-5 mRNA expression. These results indicate that Th1 effects, such as those mediated by IFN-γ, play a down-regulatory role to suppress the Th2 responses associated with allergen-induced BHR and eosinophilic inflammation