Jun Fukushima

Macrophage inflammatory protein-1α (MIP-1α) expression plasmid enhances DNA vaccine-induced immune response against HIV-1

CD8+ cell‐secreted CC‐chemokines, MIP‐1α, and MIP‐β have recently been identified as factors which suppress HIV. In this study we co‐inoculated MIP‐1α expression plasmid with a DNA vaccine constructed from HIV‐1 pCMV160IIIB and pcREV, and evaluated the effect of the adjuvant on HIV‐specific immune responses following intramuscular and intranasal immunization. The levels of both cytotoxic T lymphocyte (CTL) activity and DTH showed that HIV‐specific cell‐mediated immunity (CMI) was significantly enhanced by co‐inoculation of the MIP‐1α expression plasmid with the DNA vaccine compared with inoculation of the DNA vaccine alone. The HIV‐specific serum IgG1/IgG2a ratio was significantly lowered when the plasmid was co‐inoculated in both intramuscular and intranasal routes, suggesting a strong elicitation of the T helper (Th) 1‐type response. When the MIP‐1α expression plasmid was inoculated intramuscularly with the DNA vaccine, an infiltration of mononuclear cells was observed at the injection site. After intranasal administration, the level of mucosal secretory IgA antibody was markedly enhanced. These findings demonstrate that MIP‐1α expression plasmid inoculated together with DNA vaccine acts as a strong adjuvant for eliciting Th1‐derived immunity.

Intranasal administration of human immunodeficiency virus type-1 (HIV-1) DNA vaccine with interleukin-2 expression plasmid enhances cell-mediated immunity against HIV-1

DNA vaccine against human immunodeficiency virus type‐1 (HIV‐1) can induce substantial levels of HIV‐1‐specific humoral and cell‐mediated immunity. To develop more potent HIV‐1 DNA vaccine formulations, we used a murine model to explore the immunomodulatory effects of an interleukin‐2 (IL‐2) expression plasmid on an HIV‐1 DNA vaccine following intranasal administration of the combination. When the vaccine and expression plasmid were incorporated into cationic liposomes and administered to mice, the HIV‐1‐specific delayed‐type hypersensitivity response and cytotoxic T lymphocyte activity were significantly increased. Restimulated immune lymphoid cells showed enhanced production of both IL‐2 and interferon‐γ and reduced secretion of IL‐4. The level of total antibody to HIV‐1 antigen was not greatly changed by coadministration of the DNA vaccine and IL‐2 expression plasmid. An analysis of serum HIV‐1‐specific IgG subclasses showed a significant drop in the IgG1/IgG2a ratio in the group that received the plasmid–vaccine combination. These results demonstrate that the IL‐2 expression plasmid strongly enhances the HIV‐1‐specific immune response via activation of T helper type‐1 cells.

Differential Gene Expression of TGF-β Isoforms and TGF-β Receptors During the First Trimester of Pregnancy at the Human Maternal-Fetal Interface

PROBLEM: The transforming growth factor (TGF)‐βs are multifunctional cytokines, and they play a role in the controlled growth of trophoblasts. Moreover they are thought to be important in maternal‐fetal interaction during early gestation.

Induction of HIV-1 specific mucosal immune responses by DNA vaccination

DNA vaccination has been shown to induce immunity against several different pathogens including HIV‐1. The authors demonstrate here that administration of DNA vaccines via the intranasal route is sufficient to induce immune responses both at distal mucosal sites and systemically. Since transmission of HIV‐1 occurs largely across mucosal surfaces, the intranasal route provides a further means of application for DNA immunization.

HIV‐1‐specific cell‐mediated immunity is enhanced by co‐inoculation of TCA3 expression plasmid with DNA vaccine

We developed a candidate DNA vaccine designated pCMV160IIIB with pcREV (pCMV160IIIB/REV) that encodes gp160 of human immunodeficiency virus (HIV)‐1IIIB and Rev driven by the cytomegalovirus (CMV) promotor. This vaccine induced both HIV‐1‐specific antibodies and cytotoxic T lymphocyte (CTL) activity. In the present study, we inoculated the TCA3 expression plasmid into mouse skeletal muscle with pCMV160IIIB/REV to determine whether this cytokine expression plasmid was able to modify the immune response. Results of a delayed‐type hypersensitivity (DTH) assay using footpad swelling as well as those of a CTL assay clearly demonstrated that cell‐mediated immunity (CMI) elicited by co‐inoculation of pCMV160IIIB/REV with the TCA3 expression plasmid was markedly enhanced compared with that obtained using pCMV160IIIB/REV alone. When TCA3 expression plasmid was inoculated with anti‐TCA3 antibody, enhancement of the DTH response was suppressed below the level of that obtained with pCMV160IIIB/REV alone. The titre of HIV‐1‐specific IgG2a was slightly high when pCMV160IIIB/REV was co‐inoculated with this plasmid, suggesting that T‐helper 1 (Th1) response was predominant in TCA3‐inoculated mice. Infiltration of mononuclear cells was seen in the muscles at sites where TCA3 expression plasmid had been inoculated. Our present data suggest that TCA3 expression plasmid has potent adjuvant activity that results in an augmented CMI response.

DNA vaccination followed by macromolecular multicomponent peptide vaccination against HIV-1 induces strong antigen-specific immunity.

The induction of a strong and long-lasting immunity characterized by both a humoral and cell-mediated immune (CMI) response is one of the most important considerations in developing an effective HIV vaccine. In previous studies, we have independently developed both DNA vaccine and macromolecular multicomponent peptide vaccine (VC1) candidates. In the present study, we attempted to optimize the vaccination protocol using mice, guinea pigs, rabbits and Macaca fuscata monkeys. Repeated vaccination with VC1 induced a substantial level of multivalent antibodies which neutralized various HIV-1 strains, as determined using a p24 inhibition assay. On the other hand, repeated immunization with DNA vaccine induced and sustained high levels of cytotoxic T lymphocytes (CTLs). In addition, when DNA vaccination was followed by multicomponent peptide vaccination, levels of both humoral immunity and CMI increased, and this effect continued for at least 10 months. These data clearly demonstrate that for inducing HIV-1 specific immunity, immunization with DNA vaccine followed by VC1 boosting produces better results than immunizing with either vaccine alone.

Role of MexZ and PA5471 in transcriptional regulation of mexXY in Pseudomonas aeruginosa

MexXY, a drug efflux pump in Pseudomonas aeruginosa, confers resistance to aminoglycoside antibiotics. We recently reported that MexZ binds to the promoter region of the mexXY operon. Electrophoretic mobility shift assay (EMSA) using recombinant MexZ and oligonucleotide probes prepared from the intergenic region between mexZ and mexX revealed that MexZ binds to a 20 bp palindromic sequence. Culture of P. aeruginosa in the presence of tetracycline induced higher levels of MexX and MexZ, as measured by immunoblotting and EMSA, than in the absence of antibiotics. When MexZ was expressed by a mexZ expression plasmid, the plasmid-borne MexZ repressed drug-induced MexX production, further confirming that MexZ acts as a repressor of the mexXY operon. PA5471 protein has been reported to be essential for drug-induced MexXY production. Similarly to that report, we observed that plasmid-borne PA5471 induced both MexX and MexZ production in PAO1 cells. Interestingly, interaction between MexZ and PA5471 was observed in a yeast two-hybrid assay. Furthermore, EMSA and in vitro transcription assays revealed that interaction between PA5471 and MexZ reduced MexZ DNA-binding ability, leading to mexXY transcription. These findings contribute to the understanding of the molecular mechanisms underlying the transcriptional regulation of mexZ and mexXY by drug-induced PA5471 expression.

Iron‐Mediated Regulation of Alkaline Proteinase Production in Pseudomonas aeruginosa

We analyzed the regulation by iron of alkaline proteinase (AP) production in Pseudomonas aeruginosa. Extracellular AP production was detected from the mid‐logarithmic to the stationary phase by an antibody‐based assay system, and was strongly repressed by iron in the medium. This repression was shown by Northern hybridization and primer extension to occur at the level of transcription. The primer extension analysis revealed that the start point of transcription of AP gene was the nucleotide position −84 from the start point of translation. Furthermore, we investigated whether this transcriptional repression involved PvdS protein. Using the mutant strain of pvdS, the alternative σ factor gene revealed that the PvdS protein is required for the full expression of AP, and a previous study showed that expression of pvdS is also repressed by iron. Therefore, we thought that one mechanism of repression of AP production operated through reduction of the PvdS protein level. Purified AP decomposed the transferrin, and released iron from it Purified AP added to the medium containing transferrin as the only iron source enhanced the growth of P. aeruginosa. Moreover, mutation in the AP gene decreased the growth rate in the medium containing the transferrin as the only iron source. These results clearly indicated that AP expression should occur in a free‐iron‐deficient environment and emphasized the importance of AP to iron acquisition in the infection site.

DNA-plasmids of HIV-1 induce systemic and mucosal immune responses.

Abstract DNA-based immunization has been shown to induce protective immunity against several microbial pathogens including HIV-1. Several routes of DNA vaccination have been exploited. However, the properties of the immune responses seem to differ with the different routes used for DNA delivery, ultimately affecting the outcome of experimental challenge. We measured the primary immune response following one vaccination. This report presents differences associated with three different DNA delivery routes: intramuscular injection, intranasal application, and gene-gun based immunization. Induction of systemic humoral immune responses was achieved most efficiently by either intranasal or gene-gun mediated immunization, followed by intramuscular injection. Mucosal IgA was reproducibly induced by intranasal instillation of the DNA, and found in lung washings, faeces, and vaginal washings. Cytotoxic T cells were not induced by a single immunization, but were observed after three immunizations using intramuscular injections.

Activation of alveolar macrophages in acid-injured lung in rats: different effects of pentoxifylline on tumor necrosis factor-alpha and nitric oxide production.

ObjectiveTo determine whether acid instillation augments tumor necrosis factor-&agr; and nitric oxide production by alveolar macrophages in rats, and to study the effects of treatment with pentoxifylline before acid instillation on the production of these inflammatory mediators. DesignControlled laboratory investigation on tumor necrosis factor-&agr; and nitric oxide production by alveolar macrophages of rats that had acid-induced lung injury. SettingUniversity research laboratory. SubjectAlveolar macrophages of rats. InterventionsAlveolar macrophages were recovered by bronchoalveolar lavage at 4, 10, 16, 24, and 72 hrs after unilateral hydrochloric acid (pH, 1.0; volume, 0.1 mL) instillation into the lungs of rats. Alveolar macrophages then were cultured with or without lipopolysaccharide. One group of rats was pretreated with pentoxifylline before acid instillation. Measurements and Main Results Alveolar macrophages from both acid-instilled and contralateral lungs, which had recovered 24 hrs after acid instillation, produced significantly greater tumor necrosis factor-&agr; and nitric oxide. Subsequent exposure to lipopolysaccharide, as a surrogate for bacterial infection, further promoted tumor necrosis factor-&agr; and nitric oxide release. Alveolar macrophages from rats pretreated with pentoxifylline before acid instillation produced significantly less tumor necrosis factor-&agr; and did not overproduce tumor necrosis factor-&agr; when exposed to lipopolysaccharide. In contrast, pretreatment with pentoxifylline had no effect on nitric oxide production by alveolar macrophages. ConclusionsAcid instillation stimulates alveolar macrophages to produce tumor necrosis factor-&agr; and nitric oxide. Pentoxifylline preserved innate production of tumor necrosis factor-&agr; to lipopolysaccharide and did not inhibit the production of bactericidal nitric oxide. This may partly explain why pentoxifylline reduces acid aspiration-induced lung injury while maintaining the host’s ability to combat bacterial infection after acid aspiration.