Masao Mitsuyama

Multistage Regulation of Th1-Type Immune Responses by the Transcription Factor IRF-1

Eradication of a given pathogen is dependent on the selective differentiation of T helper (Th) cells into Th1 or Th2 types. We show here that T cells from mice lacking the transcription factor IRF-1 fail to mount Th1 responses and instead exclusively undergo Th2 differentiation in vitro. Compromised Th1 differentiation is found to be associated with defects in multiple cell types, namely impaired production of interleukin-12 by macrophages, hyporesponsiveness of CD4+ T cells to interleukin-12, and defective development of natural killer cells. These results indicate the involvement of IRF-1 in multiple stages of the Th1 limb of the immune response.

Conversion of the CD4+ T cell profile from TH2-dominant type to TH1-dominant type after varicella-zoster virus infection in atopic dermatitis☆☆☆★

Skin lesions of atopic dermatitis were examined for cytokine expression by reverse transcription-polymerase chain reaction. The profile of mRNA for various cytokines revealed that both T(H1) and T(H2) types of CD4+ T cells, probably including T(H0) type, infiltrate into the skin lesion. We observed that atopic skin lesions improved after varicella infection. In such lesions, expression of T(H1) type cytokines predominated. The peripheral blood T cells from atopic patients exhibited a differentiation into T(H2) type cells upon in vitro stimulation with mite antigen. In contrast they differentiated into T(H1) type cells upon stimulation by varicella antigen. Since IL-12 has been reported to switch the in vitro recall response of allergen-specific T cells of atopic donors from a T(H2)- to a T(H1)-like phenotype, we examined its local production in varicella lesions. IL-12 p35 and p40 mRNA were expressed in fresh lesions. Peripheral blood mononuclear cells from atopic patients expressed p40 mRNA upon in vitro stimulation with live varicella zoster virus, but they did not show p40 mRNA without stimulation. This finding suggested that in atopic skin lesions containing the virus, IL-12 was produced and the cell type was changed to T(H1) type-predominance. These results suggested that patients with atopic dermatitis always have highly reactive CD4+ T cells infiltrating into their skin, and that the switch to T(H1) or T(H2) dominance is related to whether the lesion is improved or exacerbated.

Protective effect of an acidic glycoprotein obtained from culture of Chlorella vulgaris against myelosuppression by 5-fluorouracil

Abstractu2003An acidic glycoprotein prepared from a culture of Chlorella vulgaris (CVS) was examined for its protective effect on 5-fluorouracil(5FU)-induced myelosuppression and indigenous infection in mice. Subcutaneous administration of CVS greatly reduced the mortality of non-tumor-bearing mice given a high dose of 5FU, and could increase the LD50 value of 5FU for these mice. After 5FU treatment, indigenous infection developed probably as a result of the impairment of the host defense system. CVS reduced the incidence of indigenous infections and this effect was attributable to the acceleration of recovery from 5FU-induced myelosuppression. Early recovery of hematopoietic stem cells, or cells responding to interleukin-3 or granulocyte/macrophage-colony-stimulating factor, was especially observed in the bone marrow of CVS-treated mice on days 4u200a–u200a9 after the injection of 5FU. When tumor-bearing mice were given CVS during treatment with 5FU, CVS prolonged the survival of mice without affecting the antitumor activity of 5FU. In addition, CVS was itself shown to exert an antitumor effect. These results suggested that CVS may be beneficial for the alleviation of side-effects in cancer chemotherapy without affecting the antitumor activity of the chemotherapeutic agent.

Correlation between the presence of virulence-associated genes as determined by PCR and actual virulence to mice in various strains of Listeria Spp.

Five chromosomal genes, prfA, plcA, hlyA, mpl and plcB, are implicated in the virulence of Listeria monocytogenes and some of these genes have been used for the identification of bacteria by polymerase chain reaction (PCR). Using 6 strains of L. monocytogenes and 3 L. innocua strains, the relationship was examined between the presence of five virulence‐associated genes and actual virulence to mice in terms of 50% lethal dose (LD50), bacterial viability in the organ of infected mice and the intracellular growth in cultured macrophages. None of the five genes could be amplified by PCR in all the L. innocua strains and they were actually avirulent to mice. All L. monocytogenes strains were shown to be virulent and to have intact virulence‐associated genes except for the strain ATCC15313. This particular strain was revealed to be avirulent and defective in hlyA and plcA in PCR amplification. It was suggested that PCR detection of genes prfA, mpl, or plcB may not be sufficient to detect virulent strains of L. monocytogenes. It appeared that the ability to produce listeriolysin O (LLO), which is encoded by hlyA, was critical for the expression of virulence regardless of the amount of LLO produced.

Mycolic acid-containing glycolipid as a possible virulence factor of Rhodococcus equi for mice.

By the use of various Rhodococcus equi strains differing in the length of carbon chains of glycolipid, we examined whether the glycolipid, glucose mono‐mycolate, was contributing to the virulence of R. equi for mice. R. equi strains with longer carbon chain mycolic acid showed a higher virulence as determined by lethality and granuloma formation in mice than those with shorter ones. When purified glycolipid was injected into mice, granuloma formation and liver damage were most prominent with the glycolipid having longer carbon chain mycolic acid. Only a representative strain with longer carbon chain mycolic acid persisted in the spleen of mice after intravenous injection, while a strain with shorter carbon chain mycolic acid was readily eliminated. These results suggested that glycolipid was at least one of the virulence factors of R. equi and that the carbon chain length of mycolic acid might be critical in the expression of virulence.

An essential role for endogenous interferon‐γ in the generation of protective T cells against Mycobacterium bovis BCG in mice

Protective CD4+ Tu2003cells against Mycobacterium bovis bacillus Calmette–Guérin(BCG), which are characterized by the ability to produce interferon‐γ (IFN‐γ), could be induced by immunization of mice with viable BCG but not with killed BCG. A high level of IFN‐γ mRNA was observed when normal spleen cells were stimulated with viable but not killed BCG. By comparing mice immunized with either viable or killed M. bovis BCG, it was found that a high level of IFN‐γ mRNA was expressed only after immunization with viable BCG. This finding prompted us to investigate the effect of neutralizing the IFN‐γ on the final generation of protective Tu2003cells against M. bovis BCG. When endogenous IFN‐γ was neutralized by administration of anti‐IFN‐γ monoclonal antibody in mice immunized with viable BCG, the generation of protective Tu2003cells was significantly impaired, as revealed by the adoptive transfer of spleen Tu2003cells. The generation of BCG‐specific, IFN‐γ‐producing Tu2003cells was also abolished. These results clearly demonstrate that endogenous IFN‐γ actually plays a critical role in the generation of protective Tu2003cells against M. bovis BCG in vivo. Moreover, this study suggests that the lack of IFN‐γ‐inducing ability is responsible for the inability of killed M. bovis BCG to induce protective Tu2003cells in mice.

Characteristic infectivity of Sporothrix schenckii to mice depending on routes of infection and inherent fungal pathogenicity

Isolates of Sporothrix schenckii were examined for their infectivity in BALB/c mice. The mice were injected with yeast forms of S. schenckii isolates differing in clinical source (human cutaneous lesions and pulmonary lesions), and fungal growth was determined at intervals in the footpad and visceral organs. After subcutaneous injection of approximately 10 colony forming units (cfu) of S. schenckii into the footpad, locally restricted fungal infection developed gradually. At the peak of the infection (3-4 weeks post-inoculation), viable fungal counts reached 102-106 cfu/footpad. Dissemination to other tissues and visceral organs was not observed. After intravenous or intraperitoneal injection of 106 cfu of yeast forms, three of four isolates from cutaneous sporotrichosis were unable to establish infection and were eliminated from blood and visceral organs. The development of systemic infection was observed only with S. schenckii isolates obtained from the human lung lesion. Thus, inherent properties of each clinical isolate and routes of infection were shown to be critical for the establishment of systemic infection in spite of the remarkably strong infectivity of S. schenckii to the cutaneous tissue.

Killing mechanism of Listeria monocytogenes in activated macrophages as determined by an improved assay system

Exposure of Listeria monocytogenes to gentamicin 5 mg/L for 4 h resulted in the killing of most extracellular bacteria, but had no effect on the survival of bacteria inside macrophages. Higher concentrations of gentamicin caused a reduction in the number of intracellular bacteria. This effect was associated with cellular uptake of gentamicin, but was unaffected by activation of macrophages by interferon-gamma and lipopolysaccharide. In experiments in which exposure to gentamicin 5 mg/L for 4 h was used to kill extracellular bacteria, killing by activated macrophages was impaired when O2- production was inhibited by superoxide dismutase, but not when nitric oxide production was blocked by NG-monomethyl-L-arginine. These data suggest that the reactive oxygen intermediates are more important than nitric oxide in the killing of L. monocytogenes, at least in macrophages activated in vitro.

Administration of killed bacteria together with listeriolysin O induces protective immunity against Listeria monocytogenes in mice.

It is known that only listeriolysin O (LLO)‐producing Listeria monocytogenes strains are able to induce protective imm_unity, but the underlining relationship between LLO produced by virulent strains and generation of protective imm_unity in the infected host remains poorly understood. In the present study, it was found that LLO gene expression was only detected in the mice infected with virulent strain which was able to induce protective imm_unity, while non‐virulent strains or killed bacteria were not able to generate protective imm_unity. When mice were imm_unized with LLO plus killed bacteria in the presence of incomplete Freund’s adjuvant, the protective imm_unity was partially generated, and adoptive transfer experiment confirmed that this protection was antigen specific. Reverse‐transcription polymerase chain reaction revealed that LLO plus killed bacteria induced the expression of interferon‐γ (IFN‐γ) and interleukin‐12 (IL‐12). Our results also showed CD4+ Tu2003cells were the principal cells constituting protective imm_unity. Taken together, it may be concluded that LLO produced from virulent strains of L. monocytogenes was essential for the generation of protective imm_unity, and that LLO plus killed bacteria induced IFN‐γ and IL‐12 expression which resulted in the generation of protective imm_unity.

Ultraviolet-B irradiation alters cytokine production by immune lymphocytes in herpes simplex virus infected mice

Previous studies from our laboratories have shown that UV-B irradiation at the site of an intradermal infection of herpes simplex virus (HSV) resulted in a higher incidence of zosteriform lesions and suppressed cellular immune responses to HSV in mice. In order to determine whether the production of T-cell-derived cytokine (IFN-gamma, IL-2 and IL-4) by immune cells from irradiated mice is also suppressed, we examined the production of cytokines by lymph node cells and spleen cells taken from UV-B irradiated, HSV type 1 (HSV-1)-infected mice. UV-B irradiation (120 mJ/cm2) prior to HSV-1 infection was found to markedly suppress IFN-gamma production compared to that of the non-irradiated control. IL-4 production was enhanced compared to IL-2 in the UV-B irradiated mice. These results suggest that alteration(s) in the cytokine production profile may therefore be involved in the development of severe skin lesions caused by HSV infection in UV-B irradiated mice.