Sumio Arai

Mycoplasma can enhance HIV replication in vitro: a possible cofactor responsible for the progression of AIDS.

Mycoplasma membrane protein (MMP) augmented HIV production from MOLT-4 cells chronically infected by HIV. A nearly 3-fold increase of HIV p24 antigen was detected in MMP-treated culture as compared to control culture at 100 micrograms/ml. MMP also augmented HIV production in different T cells chronically infected by HIV-1 and ARV-1. Kinetic experiment showed that HIV production was maximally elevated 24 hr after exposure to MMP. Similarly, MMP also augmented HIV-induced cell fusion and virus production in coculture. Hybridization experiment revealed that this augmentation was due to enhancement of HIV transcription.

Enhancement of Cytotoxicity of Active Macrophages by Mycoplasma: Role of Mycoplasma‐Associated Induction of Tumor Necrosis Factor‐α (TNF‐α) in Macrophages

Tumor necrosis factor‐alpha (TNF‐α) ‐inducing activity of several mycoplasmas including Mycoplasma pneumoniae, a causative agent in human respiratory infectious diseases, was investigated. Purified peritoneal macrophages from BALB/c mice markedly enhanced their cytotoxic activity to Meth A cells, when cultured with either viable or non‐viable mycoplasmas. The supernatants of the macrophage culture with mycoplasmas, M. pneumoniae and Acholeplasma laidlawii, showed the potent cytotoxic activity to TNF‐α‐sensitive L cells but not to TNF‐α‐insensitive L cells. Addition of anti‐TNF‐α antiserum inhibited completely the cytotoxic activity of these supernatants, indicating that a major part of the cytotoxic activity might be due to TNF‐α. Various other mycoplasmas, either glucose‐ or arginine‐utilizing species, as far as tested showed also the potent activity to produce TNF‐α. These results strongly suggest the possibility that mycoplasmas possess the activity of TNF‐α induction which might be responsible for a part of enhancement of cytotoxic activity of macrophages and resistance to infection with mycoplasmas in vivo.

Gene Expression of Tumor Necrosis Factorα and Interferonγ in the Lungs of Mycoplasma pulmonis-Infected Mice

ICR mice were infected intranasally with Mycoplasma pulmonis isolated freshly from the lungs of a rat with pneumonia. We demonstrated with high reproducibility the expressions of messenger RNAs of cytokines, tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ) in the lung tissue of M. pulmonis‐infected mice by the reverse transcriptase‐polymerase chain reaction and confirmed specific mRNA of the cytokines by restriction endonuclease digestion. Both the viable population of M. pulmonis in the lung tissue and the titers of the neutralizing antibody in the serum increased between 7 and 21 days, and reached their maximum 35 days after infection. The pneumonia in mice progresses with the development of lung lesions after 7 days of infection. The early lesions are characterized primarily by neutrophils and edema in the alveolar spaces. mRNAs prepared from the lung tissue of M. pulmonis‐infected and ‐uninfected mice were also tested for the presence of messages specific to TNFα and IFNγ by the reverse transcriptase‐polymerase chain reaction. The expression of the genes encoding TNFα and IFNγ was constitutively demonstrated from 24 hr through 35 days after the intranasal inoculation of M. pulmonis. Furthermore, cells of two types, adherent and nonadherent cells, in bronchoalveolar lavage fluids obtained from the mice 3 weeks after inoculation of M. pulmonis were also found to express the genes of TNFα and IFNγ respectively. These data suggest that these cytokines would play a role in both stimulation in the development of pathological changes in mycoplasmal infection, affecting the inflammatory responses.

Acholeplasma laidlawii up-regulates granulysin gene expression via transcription factor activator protein-1 in a human monocytic cell line, THP-1

An antimicrobial protein granulysin is constitutively expressed in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. However, little is known about the precise regulatory mechanisms underlying granulysin gene expression. In this study, we examined the regulatory mechanisms underlying granulysin gene expression using a human monocytic cell line, THP‐1, treated with Acholeplasma laidlawii. The level of granulysin mRNA expression in THP‐1 cells was significantly augmented in response to stimulation with A. laidlawii. The transfection of reporter gene constructs into THP‐1 cells indicated that DNA sequences between residues −329 and −239, relative to the transcriptional start site of the granulysin gene, are responsible for mediating gene induction. In addition, mutagenesis of a putative activator protein‐1 (AP‐1)‐binding site between residues −277 and −271 in the granulysin promoter resulted in the reduction of granulysin promoter activity. Electrophoretic mobility shift assays (EMSA) demonstrated that nuclear extract prepared from A. laidlawii‐treated THP‐1 cells can generate specific binding to DNA oligonucleotides encompassing the AP‐1‐binding site, whereas unstimulated nuclear extract from the cells failed to do so. Furthermore, competition and supershift assays confirmed that A. laidlawii can induce the activation of AP‐1. These results indicate that AP‐1 dominantly participates in the regulation of inducible granulysin gene expression in THP‐1 cells. Therefore, the finding of inducible granulysin gene expression by A. laidlawii suggests that inducible granulysin in macrophages may function as a protective weapon when microbial invasion occurs.

Antimycoplasmal activities of new quinolones, tetracyclines, and macrolides against Mycoplasma pneumoniae.

Fifty strains of Mycoplasma pneumoniae were tested for susceptibility to new quinolones, tetracyclines, and macrolides. Temafloxacin, ofloxacin, and ciprofloxacin possessed the most mycoplasmacidal activity against these organisms. The MBC for 50% of the strains (MBC50)-to-MIC50 ratio for each of these drugs was 4. The MBC50-to-MIC50 ratios for the tetracyclines and macrolides were markedly higher, within a range of 32 to 2,000. On the basis of these results, temafloxacin and ofloxacin might be promising antimicrobial agents for the treatment of mycoplasmal infection.

Role of Furin in Delivery of a CTL Epitope of an Anthrax Toxin-Fusion Protein

Anthrax toxin lethal factor (LF) in combination with anthrax toxin protective antigen (PA) was endocytosed and translocated to the cytosol of mammalian cells. Residues 1–255 of anthrax toxin lethal factor (LFn) was fused to a cytotoxic T lymphocyte (CTL) epitope of an influenza virus. For processing the toxins, PA must be cleaved into a 63‐kDa fragment (PA63) by furin, which is a subtilisin‐like processing endoprotease expressed by many eukaryotic cells. To test the ability of cells treated with the LFn fusion protein plus PA to deliver the epitope, CTL assay was performed. Two types of cell lines were identified, one was able to deliver CTL epitope while the other failed to efficiently deliver the epitope. To further elucidate the differences between these cells, the role of furin in these cells was examined. Disruption of the furin gene reduced its ability to deliver the CTL epitope. Furin expression in cells capable of efficiently delivering CTL epitope was quantitatively higher than in cells unable to deliver the epitope. The results suggest that furin plays a critical role in delivery of the CTL epitope of LFn fusion protein.

Mycoplasma stimulates HIV-1 expression from acutely- and dormantly-infected promonocyte/monoblastoid cell lines

SummaryTreatment of a myelo-monocyte cell line, J22HL-60, dormantly infected with human immunodeficiency virus type 1 (HIV-1) with heat-inactivated extracts ofAcholeplasma (A) laidlawii (250 µg/ml) enhanced virus production more than 45-fold as assessed by p24 viral core antigen assay. When treated with a suboptimal dose of TPA or TNF-α, Acholeplasma extracts further augmented virus production in J22HL-60 cells. H7, an inhibitor of protein kinase C(PKC), almost completely abrogated HIV-1-inducing ability of Acholeplasma extracts in the cells.A. laidlawii and several other mycoplasmas also enhanced acute infection of U937 cells as shown by increased virus-positive cells and augmentation of HIV-1 production in the culture supernatant independent of their pathogenicity to humans.

In Vitro Activities of Moxifloxacin and Other Fluoroquinolones against Mycoplasma pneumoniae

ABSTRACT A total of 105 isolates of Mycoplasma pneumoniae were evaluated for susceptibility to moxifloxacin, sparfloxacin, levofloxacin, and ciprofloxacin. Moxifloxacin, a newly synthesized compound, showed the greatest activity. The MICs and MBCs at which 50 and 90% of isolates were affected were 0.15 (MIC50 and MBC50) and 0.3 μg/ml (MIC90 and MBC90) respectively. The results indicate that moxifloxacin might be promising an antimycoplasmal agent.

Induction of Tumor Necrosis Factor Alpha (TNFα) and Enhancement of HIV‐1 Replication in the J22HL60 Cell Line by Mycoplasma penetrans

Mycoplasma penetrans isolated from clinical specimens of AIDS patients showed potent activity in tumor necrosis factor alpha (TNFα) production in THP‐1, U937 and J22HL60 cell lines, and in the enhancement of HIV‐1 replication in a dormantly‐infected J22HL60 cell line as compared with the activities of other mycoplasmas. Both activities were found in the methanol layer but not in the chloroform layer of the membrane extracted by the Bligh‐Dyer method. TNFα production was observed in the peritoneal macrophages from both lipopolysaccharide‐responsive and ‐unresponsive mouse strains, and was not inhibited by polymyxin B. The induction of TNFα production and enhancement of HIV‐1 replication were strongly inhibited by Concanavalin A‐Sepharose. The inhibitory effect of Concanavalin A‐Sepharose was partially prevented by sugars in the order methyl‐α‐D‐mannopyranoside and methyl‐α‐D‐glucopyranoside but not methyl‐α‐D‐galactopyranoside. Anti‐human TNFα antibody, however, did not reduce the activity of the methanol layer to enhance HIV‐1 replication, suggesting that the methanol layer could enhance HIV‐1 replication directly. These results suggest that the carbohydrate derived from M. penetrans might be responsible for the progression of HIV‐1 infection.

Effects of new quinolones on Mycoplasma pneumoniae-infected hamsters.

The efficacies of the new quinolones temafloxacin, ofloxacin, and ciprofloxacin were investigated against Mycoplasma pneumoniae in an experimental hamster pneumonia model. Hamsters were infected intratracheally with M. pneumoniae and sacrificed 18 h after the final medication, and their lungs were aseptically removed, homogenized, and cultured quantitatively. The efficacies of these drugs were determined by the CFU of M. pneumoniae in lungs. Temafloxacin and ofloxacin, but not ciprofloxacin, were active when the oral administration of 200 mg/kg of body weight per day (once per day) for 5 days was initiated 24 h after infection. Although no effect on the elimination of M. pneumoniae was observed after the administration of these drugs at 200 mg/kg/day at 5 days after infection, the continuous administration for 15 days of temafloxacin, but not ofloxacin or ciprofloxacin, significantly reduced viable M. pneumoniae in the lungs. These results suggest that temafloxacin and ofloxacin are effective in the acute phase of infection and, moreover, that temafloxacin is effective in the late stage of infection during which progressive lung alterations and continuous increases in mycoplasmal growth occurred. The peak levels of temafloxacin in sera and lungs after oral administration were similar to those of ofloxacin and higher than those of ciprofloxacin. The areas under the curve of temafloxacin in the lung tissue, however, were higher than those of ofloxacin and ciprofloxacin. On the basis of these results, temafloxacin and ofloxacin might be promising antimicrobial agents for the treatment of mycoplasmal infection. Images