Isamu Sugawara

Mycobacterial infection in TLR2 and TLR6 knockout mice.

To investigate the role of TLR in the development of murine tuberculosis in vivo, TLR2 and TLR6 knockout (KO) mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne infection apparatus. Both TLR2 and TLR6 KO mice survived until sacrifice at 12 weeks after infection. Infected TLR2 KO mice developed granulomatous pulmonary lesions with neutrophil infiltration, which were slightly larger in size than those in wild‐type mice. Pulmonary levels of the mRNAs for inducible nitric oxide synthase (iNOS), TNF‐α, TGF‐β, IL‐1β, and IL‐2 were significantly lower, but levels of the mRNAs for IL‐4 and IL‐6 were higher, than in wild‐type (WT) mice. No significant difference was recognized in cytokine mRNA expression between TLR2 KO and WT mice at 12 weeks after infection. DNA binding by NF‐κB was low in TLR2 KO mice. On the other hand, TLR6 KO mice were not different from WT mice in terms of pulmonary histopathology, mRNA expression and CFU assay. Therefore, TLR2 does not play an essential role in the pathogenesis of murine tuberculosis, although it is important for defense against mycobacterial infection.

Protective role of interleukin-1 in mycobacterial infection in IL-1 α/β double-knockout mice

To understand the role of the proinflammatory cytokine interleukin-1 (IL-1) in mycobacterial inflammation, IL-1 α/β double-knockout (KO) mice were produced. These mice were infected with either Mycobacterium tuberculosis H37Rv by the airborne route using an airborne infection apparatus, and their capacities to control mycobacterial growth, granuloma formation, cytokine, and nitric oxide (NO) production were examined. The IL-1 α/β mice developed significantly larger (p < 0.01) granulomatous, but not necrotic, lesions in their lungs than wild-type (WT) mice after infection with H37Rv. Inflammatory lesions, but not granulomas, were observed in spleen and liver tissues from both IL-1 α/β KO and wild-type mice. Granulomatous lesion development in IL-1 α/β KO mice was not significantly inhibited by treatment with exogenous recombinant IL-1α/β. Compared with wild-type mice, splenic IFN-γ and IL-12 levels were within the normal range. NO production by cultured alveolar macrophages from IL-1 α/β KO mice was lower than in wild-type mice but were increased by the addition of recombinant IL-1 α/β. Our data clearly indicate that IL-1 is important for the generation of early-phase protective immunity against mycobacterial infection.

Lung resistance protein (LRP) expression in human normal tissues in comparison with that of MDR1 and MRP

MDR1 (P-glycoprotein), multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) are associated with multidrug resistance in various cancer cells. It is known that P-glycoprotein and MRP are also expressed in several normal tissues. However, the exact location of LRP in normal tissues is still unclear. In order to obtain more insight into the physiological role of LRP, its expression in human normal tissues was examined by an immunohistochemical technique, using one monoclonal antibody, LRP-56. Reverse transcriptase-polymerase chain reaction (RT-PCR) was also utilized for several cell lines and fresh-frozen tissues. P-glycoprotein was found to be expressed in the kidney, adrenal, brain vessels, muscle, lung, pancreas, liver, intestine, placenta and testis. MRP was expressed in the kidney, adrenal, lung, pancreas, muscle, intestine, thyroid and prostate, and its distribution mostly overlapped with that of P-glycoprotein. Interestingly, MRP was not expressed in the liver. LRP at 110 kDa was expressed in the kidney, adrenal, heart, lung, muscle, thyroid, prostate, bone marrow and testis. These findings suggest that LRP as well as P-glycoprotein and MRP plays distinct roles in the physiology of various organs.

Potential Action of IL-4 and IL-13 as Fibrogenic Factors on Lung Fibroblasts in vitro

Background: Asthma is characterized by chronic inflammation of the airway with the presence of Th2 cytokines. Airway remodeling in asthma is closely related to clinical manifestations. Lung myofibroblasts play a critical role in the airway remodeling and Th2 cytokines may modulate their behavior. We examined the effect of two major Th2 cytokines, IL-4 and IL-13, on differentiation of lung fibroblasts to myofibroblasts. We hypothesized that these cytokines would stimulate fibroblast proliferation in association with decreased prostaglandin E2 (PGE2). Methods: Lung fibroblasts were incubated with IL-4 and IL-13 with or without Th1 cytokine interferon-γ (IFN-γ) in vitro. Differentiation of lung fibroblasts to myofibroblasts was characterized by the expression of α-smooth muscle actin (α-SMA) as well as a morphological and immunohistochemical analysis. Fibroblast proliferation stimulated by IL-4 and IL-13 was assessed with the MTT assay. We also investigated the effect of these cytokines on cyclooxygenase (COX) gene expression and PGE2 production. Results: IL-4 and IL-13 increased α-SMA expression and myofibroblastic differentiation. This effect was attenuated by IFN-γ and dexamethasone failed to have an influence on differentiation. IL-4 and IL-13 stimulated fibroblast proliferation. These cytokines downregulated the expression of both COX-1 and COX-2 genes and decreased the production of PGE2. Conclusions: IL-4 and IL-13 induce differentiation of fibroblasts to myofibroblasts and this response is attenuated by IFN-γ. IL-4 and IL-13 stimulate fibroblast proliferation and this effect is at least partly due to suppressed COX gene expressions and subsequently decreased PGE2 production. These findings suggest that IL-4 and IL-13 directly act on lung fibroblast to induce a fibrogenic response.

Effect of nanoparticles on the male reproductive system of mice

The effects of nanoparticles toward on the male reproductive system of mice were investigated. Three sizes (14, 56 and 95 nm) of carbon black nanoparticles were intratracheally administered (0.1 mg/mouse for 10 times every week) to ICR male mice to investigate their adverse effects on the reproductive function. The serum testosterone levels were elevated significantly in the 14- and 56-nm carbon nanoparticles-exposed groups. Histological examination showed partial vacuolation of the seminiferous tubules. In addition, the effects of particle number towards the male reproductive system were investigated. The particle number controlled 14-nm nanoparticles-exposed group (14 N group, which has approximately the same particle number per unit volume as the 56-nm nanoparticles) showed fewer effects than did the 56-nm nanoparticles-exposed groups. These results suggest that carbon nanoparticle-exposure has adverse effects on the mouse male reproductive function. Furthermore, the effects of nanoparticles on the male reproductive system depend on particle mass rather than particle number.

In utero exposure to a low concentration of diesel exhaust affects spontaneous locomotor activity and monoaminergic system in male mice

BackgroundEpidemiological studies have suggested that suspended particulate matter (SPM) causes detrimental health effects such as respiratory and cardiovascular diseases, and that diesel exhaust particles from automobiles is a major contributor to SPM. It has been reported that neonatal and adult exposure to diesel exhaust damages the central nervous system (CNS) and induces behavioral alteration. Recently, we have focused on the effects of prenatal exposure to diesel exhaust on the CNS. In this study, we examined the effects of prenatal exposure to low concentration of diesel exhaust on behaviour and the monoaminergic neuron system. Spontaneous locomotor activity (SLA) and monoamine levels in the CNS were assessed.MethodsMice were exposed prenatally to a low concentration of diesel exhaust (171 μg DEP/m3) for 8 hours/day on gestational days 2-16. SLA was assessed for 3 days in 4-week-old mice by analysis of the release of temperature-associated infrared rays. At 5 weeks of age, the mice were sacrificed and the brains were used for analysis by high-performance liquid chromatography (HPLC).Results and DiscussionMice exposed to a low concentration of diesel exhaust showed decreased SLA in the first 60 minutes of exposure. Over the entire test period, the mice exposed prenatally to diesel exhaust showed decreased daily SLA compared to that in control mice, and the SLA in each 3 hour period was decreased when the lights were turned on. Neurotransmitter levels, including dopamine and noradrenaline, were increased in the prefrontal cortex (PFC) in the exposure group compared to the control group. The metabolites of dopamine and noradrenaline also increased in the PFC. Neurotransmitter turnover, an index of neuronal activity, of dopamine and noradrenaline was decreased in various regions of the CNS, including the striatum, in the exposure group. The serum corticosterone level was not different between groups. The data suggest that decreased SLA in mice exposed prenatally to diesel exhaust is due to facilitated release of dopamine in the PFC.ConclusionsThese results indicate that exposure of mice in utero to a low concentration of diesel exhaust decreases SLA and alters the neurochemical monoamine metabolism of several regions of the brain.

Effect of prenatal exposure to diesel exhaust on dopaminergic system in mice.

Diesel exhaust (DE) is composed of particles and gaseous compounds. It has been reported that DE causes pulmonary and cardiovascular disease. We have previously reported that fetal exposure to DE had deleterious effects to the reproductive system of mice offspring. However, there is still little known about the effects of prenatal exposure to DE to the central nervous system (CNS). In the present study, we found that prenatal exposure to DE induced reduction of locomotion, furthermore, dopamine (DA) turnover was significantly decreased in the striatum and nucleus accumbens. These results suggest that prenatal exposure to DE has an effect on the CNS. Hypolocomotion could be due to a decrease in DA turnover associated with DA nervous system abnormality. The present study provides the possibility that maternally inhaled DE might influence the development of central dopaminergic system and result in behavior disorder.

Role of Interleukin (IL)-1 Type 1 Receptor in Mycobacterial Infection

It is important to gain a better understanding of IL‐1‐mediated signaling events in mycobacterial infection. In order to clarify the role of IL‐1 receptor type 1 (IL‐1 R1) in IL‐1 R1, knockout (KO) mice were infected with either Mycobacterium tuberculosis H37Rv or Kurono strain by the respiratory route, and their ability to control mycobacterial growth, pulmonary granuloma formation, and cytokine mRNA expression was investigated. IL‐1 R1 KO mice developed significantly larger (P < 0.01) granulomatous lesions with neutrophil infiltration in their lungs than wild‐type mice did after infection with the M. tuberculosis Kurono strain. The number of mycobacterial colonies in lungs and spleen increased from five weeks post‐infection. Interferon‐γ production by spleen cells was low in IL‐1 R1 KO mice. It is concluded that the IL‐1 R1 is essential for IL‐1‐mediated signaling events in mycobacterial infection.

Significance of Immunological Detection of Human Telomerase Reverse Transcriptase : Re-Evaluation of Expression and Localization of Human Telomerase Reverse Transcriptase

Human telomerase reverse transcriptase (hTERT) is a catalytic subunit of telomerase and is a potentially useful diagnostic marker for cancers. There have been few studies in which immunological detection of hTERT has been attempted and its subcellular localization has not been precisely defined. In the present study, we re-evaluated expression and localization of hTERT in cancer and normal cells using a newly developed antibody. Immunohistochemistry revealed that hTERT is expressed in approximately 80% of gynecological cancers, but some premalignant lesions exhibited weak expression of hTERT. Interestingly, not only nuclei but also cytoplasm of cancer cells were positive for hTERT staining. This finding was supported by the results of Western blot analysis of cell lines, in which both nuclear and cytoplasmic extracts exhibited significant hTERT bands. Cytoplasmic hTERT in cancer cells may be functional because the telomeric repeat amplification protocol assay of cytoplasmic extracts showed high levels of telomerase activity. Unexpectedly, not all normal primary cells and telomerase-negative cancer cell lines lacked hTERT expression; some exhibited weak TERT signals. In Western analysis, hTERT signals did not always correlate with telomerase activity of the various cell types. These findings suggest that functional hTERT is expressed in both the nucleus and cytoplasm of cancer cells and that hTERT expression does not strictly reflect telomerase activity. Further analysis is needed to clarify the biological significance of cytoplasmic hTERT.

Promising loci of variable numbers of tandem repeats for typing Beijing family Mycobacterium tuberculosis

We analysed the genotypes of 325 Mycobacterium tuberculosis clinical isolates obtained during 2002 throughout Japan. The genotyping methods included insertion sequence IS6110 RFLP, spoligotyping and variable number of tandem repeat (VNTR) analyses. Clustered isolates revealed by IS6110 RFLP analysis accounted for 18.5 % (60/325) of the isolates. Beijing genotype tuberculosis (TB) accounted for 73.8 % (240/325) of the isolates. Using VNTR, we analysed 35 loci, including 12 standard mycobacterial interspersed repetitive units and 4 exact tandem repeats. The discriminatory power of these 16 loci was low. Using VNTR analyses of the 35 loci, 12 loci (VNTRs 0424, 0960, 1955, 2074, 2163b, 2372, 2996, 3155, 3192, 3336, 4052 and 4156) were selected for the genotyping of Beijing genotype strains. Comparison of the discriminatory power of the 12-locus VNTR [Japan Anti-Tuberculosis Association (JATA)] to that of the 15-locus and 24-locus VNTRs proposed by Supply et al. (2006) showed that our established VNTR system was superior to the reported 15-locus VNTR and had almost equal discriminatory power to the 24-locus VNTR. This 12-locus VNTR (JATA) can therefore be used for TB genotyping in areas where Beijing family strains are dominant.