Significant increase in natural-killer T cells in patients with tuberculosis complicated by type 2 diabetes mellitus.

This study examined the frequency of Vα24(+)/Vß11(+) natural-killer T (NKT) cells from peripheral blood and bronchoalveolar lavage fluid in pulmonary tuberculosis (TB) patients with or without diabetes mellitus (DM). The clinical grade of TB was significantly higher among diabetic patients. NKT cells from both peripheral blood and bronchoalveolar lavage were significantly increased in diabetic TB patients compared with non-diabetic TB patients. This may be due to the generally higher bacillary burden in diabetic TB patients. NKT cells from peripheral blood mononuclear cells in TB patients with or without DM were significantly increased, compared with levels in non-TB diabetic patients and healthy controls. The measurement of NKT cells from peripheral blood has the potential to be a reliable, non-invasive, practical diagnostic marker for active TB.

Significant reduction of granulomas in Nrf2-deficient mice infected with Mycobacterium tuberculosis.

OBJECTIVE: We have reported previously that mice deficient in nuclear erythroid 2 p45-related factor 2 (Nrf2), which regulates the expression of antioxidant and detoxification genes, showed significant susceptibility to airway inflammatory responses when exposed to diesel exhaust particles for eight weeks. As disruption of Nrf2 promotes immune cells that stimulate Th2-like immunoresponsiveness, Nrf2-deficient mice may be resistant to M. tuberculosis infection. SETTING: Nrf2-deficient mice were infected with M. tuberculosis aerially, and the size of their granulomas and cytokine mRNA expression were compared with those of wild-type mice. RESULTS: Significant reduction of granuloma formation and tubercle bacilli in granulomas was noted in the deficient mice 27 weeks after infection, concurrently with higher expression of IL-2 and IL-13 mRNA. CONCLUSION: It is concluded that Nrf2 inversely regulates M. tuberculosis-induced granuloma development at the late stage.

Beijing family Mycobacterium tuberculosis isolated from throughout Japan: phylogeny and genetic features.

To estimate the current population genetic structure of Mycobacterium tuberculosis in Japan, phylogenetic traits were analysed for 237 Beijing family strains isolated from tuberculosis patients throughout the country. Unlike previous reports from other countries, the ancient Beijing sublineage was predominant throughout Japan. Clustering analysis based on JATA-VNTR (Japan Anti-Tuberculosis Association variable numbers of tandem repeats), a specialised set of VNTR for the discrimination of Japanese M. tuberculosis strains, revealed high similarity of the modern Beijing sublineage strains, irrespective of their geographic origin. JATA-VNTR might be useful for the phylogenetic classification in populations where ancient Beijing strains are frequently isolated.

Descriptive analysis of the prevalence and the molecular epidemiology of Mycobacterium avium complex-infected pigs that were slaughtered on the main island of Okinawa.

Recent genetic studies have revealed that several epidemiological factors affect Mycobacterium avium complex (MAC) infection in pig populations. However, mechanisms underlying the spread of MAC infection among hog farms have not been clarified. In consideration of this situation, we cross-sectionally investigated the mechanisms underlying the spread of MAC on the island of Okinawa. Pigs slaughtered (n=706,763) and 331 hog farms on Okinawa were surveyed during the years 2002-2004. Two outbreaks of MAC infection were occurred in several farms during survey period. Bacteria were isolated from randomly selected pigs and genotype of isolates was determined by using genetic finger printing methods with the insertion sequence (IS) 1245 restriction fragment length polymorphism (RFLP). Most isolates had large numbers of IS1245 copies, while strains with low copy numbers of IS1245 and isolates without IS1245 were seen in few farms. MACs strains were repeatedly isolated from pigs of the affected farms during the survey period. Those farms with an identical pig rearing systems showed synchronic changes in the prevalence of MAC infection. An industrial farm without an outbreak had an independent pig flow, but maintained distinct MAC strains. Multivariate analysis did not reveal independent factors for the prevalence of the MAC infection. These findings suggest that there were three clusters distinguished genetically in the main island of Okinawa, which were potentially spread by common pig flow. However, the outbreaks occurred because of unspecified conditions on each farm environment.

Cross-resistance of Mycobacterium tuberculosis isolates among streptomycin, kanamycin and amikacin.

Seventy-four streptomycin (SM)-resistant M. tuberculosis clinical isolates were subjected to cross-resistance drug testing against two major aminoglycosides, kanamycin (KM) and amikacin (AMK). Among them, 15 clinical isolates (20.3%) were resistant to both KM and AMK. Fifteen (80%) of 19 KM-resistant isolates were AMK-resistant. Fifteen SM, KM, and AMK resistant isolates harbored rrs mutation, but only two had rrs and rpsL double mutations. Low-level SM resistance was associated with rpsL mutation, whereas high-level SM resistance was linked to rrs mutation.

Protective efficacy of recombinant BCG Tokyo (Ag85A) in rhesus monkeys (Macaca mulatta) infected intratracheally with H37Rv Mycobacterium tuberculosis.

We have reported previously that recombinant BCG Tokyo (Ag85A) (rBCG-Ag85A[Tokyo]) shows promise as a tuberculosis vaccine, demonstrating protective efficacy in cynomolgus monkeys. As a next step, rhesus monkeys were utilized because they are also susceptible to Mycobacterium tuberculosis and show a continuous course of infection resembling human tuberculosis. The recombinant BCG vaccine (5x10(5) CFU per monkey) was administered once intradermally into the back skin to three groups of rhesus monkeys, and its protective efficacy was compared for 4months with that of its parental BCG Tokyo strain. Eight week vaccination of the monkeys with rBCG-Ag85A[Tokyo] resulted in a reduction of tubercle bacilli CFU (p<0.01) and lung pathology in animals infected intratracheally with 3000 CFU H37Rv M. tuberculosis. Vaccination prevented an increase in the old tuberculin test after challenge with M. tuberculosis and reaction of M. tuberculosis-derived antigen. Thus, it was shown that even in rhesus monkeys rBCG-Ag85A[Tokyo] induced higher protective efficacy than BCG Tokyo.

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.

Recombinant BCG Tokyo (Ag85A) protects cynomolgus monkeys (Macaca fascicularis) infected with H37Rv Mycobacterium tuberculosis.

One tuberculosis vaccine candidate that has shown a promising degree of protective efficacy in guinea pigs is recombinant BCG Tokyo (Ag85A)(rBCG-Ag85A[Tokyo]). As a next step, cynomolgus monkeys were utilized because they are susceptible to Mycobacterium tuberculosis and develop a continuous course of infection that resembles that in humans both clinically and pathologically. The recombinant BCG vaccine was administered once intradermally in the back skin to three groups of cynomolgus monkeys, and its protective efficacy was compared for 4 months with that of its parental BCG Tokyo strain. Vaccination of the monkeys with the rBCG-Ag85A[Tokyo] resulted in a reduction of tubercle bacilli CFU (p<0.01) and lung pathology in animals challenged intratracheally with 3000 CFU H37Rv M. tuberculosis. Vaccination prevented an increase in the old tuberculin test after challenge with M. tuberculosis and reaction of M. tuberculosis-derived antigen. Thus, it was shown in monkeys that rBCG-Ag85A[Tokyo] induced higher protective efficacy than BCG Tokyo. This warrants further clinical evaluation.

Protective efficacy of recombinant (Ag85A) BCG Tokyo with Ag85A peptide boosting against Mycobacterium tuberculosis-infected guinea pigs in comparison with that of DNA vaccine encoding Ag85A.

A recombinant form of BCG Tokyo with an Ag85A gene insert was administered once subcutaneously to guinea pigs and its protective efficacy was compared with that of a DNA vaccine encoding Ag85A from Mycobacterium tuberculosis administered twice to guinea pigs by epidermal gene gun bombardment. Vaccination with either the recombinant BCG Tokyo or Ag85A DNA significantly reduced the severity of pulmonary pathology and the number of pulmonary and splenic colony-forming units (cfu) (p<0.001). The recombinant BCG Tokyo was better than Ag85A DNA in terms of protective efficacy against M. tuberculosis. When immunogenic synthetic Ag85A peptide was further used as a booster together with recombinant BCG Tokyo (Ag85A) or Ag85A DNA, lung pathology was improved significantly and the number of pulmonary CFU was reduced significantly. Neither recombinant BCG Tokyo, Ag85A DNA, nor the parental BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. The spleen tissues from guinea pigs vaccinated with recombinant BCG Tokyo or Ag85A DNA expressed IFN-gamma and IL-2 mRNA at significantly high levels (p<0.001) as evaluated by reverse transcription polymerase chain reaction. It is concluded that peptide boosting is important for the induction of higher protective efficacy by recombinant BCG Tokyo or a tuberculosis DNA vaccine and both recombinant BCG Tokyo (Ag85A) and Ag85A DNA vaccine induce Th2 cytokine mRNA expression significantly.

Vaccination of guinea pigs with DNA encoding Ag85A by gene gun bombardment.

A DNA vaccine encoding Ag85A from Mycobacterium tuberculosis was administered to guinea pigs by epidermal gene gun bombardment and its protective efficacy was determined. Vaccination with Ag85A DNA twice significantly reduced the severity of pulmonary pathology and number of pulmonary colony-forming units (CFU) (p<0.01). When immunogenic synthetic Ag85A peptide was used as a booster, lung pathology was improved significantly and pulmonary CFU were reduced dramatically. Neither Ag85A DNA nor BCG Tokyo protected the guinea pigs from hematogenous spread of tubercle bacilli to the spleen because splenic granulomas without central necrosis were recognized. When the vaccinated guinea pigs were followed up for 7 months, the pulmonary lesions became fibrotic in guinea pigs vaccinated with Ag85A DNA plus Ag85A peptide, or BCG Tokyo, and no tubercle bacilli were detected. The protective efficacy of the tuberculosis Ag85A DNA vaccine was improved significantly by peptide boosting. It is concluded that dosage and peptide boosting are important in the induction of higher protective efficacy by a tuberculosis DNA vaccine.

Relative importance of STAT4 in murine tuberculosis.

This study was designed to determine the roles of STAT proteins in defence against mycobacterial infection. Airborne infection of STAT4 knockout (KO) mice with a Mycobacterium tuberculosis strain induced large granulomas with massive neutrophil infiltration over time, while that in STAT6 KO mice did not. The STAT4 KO mice succumbed to mycobacterial infection by the 80th day after infection. Compared with the levels in wild-type (WT) and STAT6 KO mice, pulmonary inducible nitric oxide synthase, interferon-alpha, -beta and -gamma mRNA levels were significantly lower in STAT4 KO mice, but expression of interleukin-2, -6, -12 and -18 mRNAs was slightly higher up to the fifth week after aerial infection. Therefore, STAT4, but not STAT6, appears to be a critical transcription factor in mycobacterial regulation.

Mycobacterial infection in natural killer T cell knockout mice.

To gain a better understanding of the pathological role of natural killer (NK) T cells in murine tuberculosis, NKT knockout (KO) mice (J(alpha)281(-/-)mice) were utilized. Eight-week-old NKT KO mice of BALB/c origin and wild-type (WT) mice were infected with Mycobacterium tuberculosis Kurono strain by the airborne route using an airborne infection apparatus, and their capacity to control mycobacterial growth, granuloma formation, and cytokine production were examined. The NKT KO mice developed granulomatous lesions in the lungs; there was no statistically significant difference in pulmonary granuloma size between NKT KO and WT mice (p<0.01). The average CFU values increased 3 weeks post-infection, but decreased 9 and 11 weeks post-infection, in the lungs of NKT KO mice. When stimulated with Kurono strain in vitro, splenic cells from NKT KO mice produced less IFN-gamma than did those from WT mice. Expression of mRNA for IL-2, IL-4, IL-6, IL-10 and IL-12 p40 was slightly lower in NKT KO mice compared with WT mice. Our data indicate that NKT cells play a detrimental role in late-phase mycobacterial infection, although Th1 cells are essential in early-phase mycobacterial infection.

Pulmonary granulomas of guinea pigs induced by inhalation exposure of heat-treated BCG Pasteur, purified trehalose dimycolate and methyl ketomycolate.

This study was designed to determine the identity of granulomatogenic substances in Mycobacterium bovis BCG Pasteur. When heat-treated BCG Pasteur bacilli were introduced into the lungs of guinea-pigs by an inhalation exposure apparatus, pulmonary granulomas without necrosis developed. Furthermore, when four kinds of mycolates derived from M. tuberculosis Aoyama B strain were introduced into the lungs by the same method, only trehalose 6,6'-dimycolate (TDM) and methyl ketomycolate induced pulmonary granulomas without central necrosis. The pulmonary granulomas consisted of epithelioid macrophages and lymphocytes. When a mixture of TDM and anti-TDM antibody was introduced into the lungs, development of granulomatous lesions was reduced. These data indicate that TDM and methyl ketomycolate are potent granulomatogenic reagents.

Biological characteristics and chemosensitivity profile of four human anaplastic thyroid carcinoma cell lines.

Anaplastic thyroid carcinoma is a rapidly growing, aggressive neoplasm affecting the elderly which does not respond to most of the therapies. We established cultured cell lines from four untreated tumors. The cultures grew in a monolayer of spindle-shaped cells in three cell lines and of small polygonal cells in one line, having relatively long doubling times and chromosomal abnormalities. The xenotransplantation of the lines in athymic nude mice produced tumors with a histology similar to the original tumors. The immunocytochemical staining showed the expression of PCNA, HLA-class 1, cytokeratin, vimentin and FAS (fatty acid synthase) but not CEA, desmin or P-glycoprotein. The lines secreted TPA, IL-6, IL-8 and few or no thyroid-related hormones in the culture supernatant. One cell line produced G-CSF. The chemosensitivity assay revealed intrinsic drug resistance to nine out of 11 antineoplastic agents. The reverse transcriptase-polymerase chain reaction (RT-PCR) detected MRP (multidrug resistance-associated protein) mRNA but not mdr (multidrug resistance protein)-1 and mdr-3 mRNAs. This finding indicates that the multidrug resistance of these lines is mediated by a P-glycoprotein-unrelated mechanism. The RT-PCR also presented FAS mRNA in all the lines, and IL-6 and IL-8 mRNAs in some of the lines.

Relative importance of NF-kappaB p50 in mycobacterial infection.

To understand the role of NF-kappaB in the development of murine tuberculosis in vivo, NF-kappaB p50 knockout mice were infected with Mycobacterium tuberculosis by placing them in the exposure chamber of an airborne-infection apparatus. These mice developed multifocal necrotic pulmonary lesions or lobar pneumonia. Compared with the levels in wild-type mice, pulmonary inducible nitric oxide synthase, interleukin-2 (IL-2), gamma interferon, and tumor necrosis factor alpha mRNA levels were significantly low but expression of IL-10 and transforming growth factor beta mRNAs were within the normal ranges. The pulmonary IL-6 mRNA expression level was higher. Therefore, NF-kappaB and its interaction with host cells play an important role in the pathogenesis of tuberculosis.

Disruption of nuclear factor-interleukin-6, a transcription factor, results in severe mycobacterial infection.

Nuclear factor-interleukin-6 (NF-IL-6) is one of several nuclear transcription factors (NF-IL-6, NF-kappaB, PU.1, interferon-regulatory factor 1, Egr-1, and Stat-1). NF-IL-6 and NF-kappaB are expressed in macrophages and is induced by bacterial lipopolysaccharides. To evaluate whether NF-IL-6 is required for the inflammatory immune response to mycobacterial infection, in which epithelioid macrophages comprise the leading cell population, we generated NF-IL-6 knockout (KO) mutant mice. Airborne infection of these mice with Mycobacterium tuberculosis strains induced disseminated tuberculosis lacking granuloma formation, although interferon-gamma, tumor necrosis factor-alpha, and interleukin-12 mRNA expression levels were within the normal range compared with those of wild-type mice. Generation of O2- and mycobacterial killing by neutrophils from these mice were impaired severely compared with wild-type mice. We conclude that NF-IL-6 is a critical transcription factor in mycobacterial control as well as in granulocyte-colony stimulating factor induction resulting in neutrophil activation.

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-y 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.

Early oxidative DNA damages and late development of lung cancer in diesel exhaust-exposed rats.

To demonstrate DNA damages in the early stage of diesel exhaust exposure, an inhalation study of 1 through 12 months was conducted. The lung burden of diesel soot increased with increase in exposure duration. Histologically, hyperplastic foci of alveolar epithelia were found at 6-month exposure and became prominent at the 12th month, with slight nuclear atypia and positive p53 staining. The level of 8-OH-hydroxyguanosine (8-OH-dG) in the exposed rat lungs showed an increase from 1 month of exposure, followed by a gradual increase, reaching almost a plateau level at the 9th month. An in vitro experiment demonstrated significant 8-OH-dG formation when diesel particles and H(2)O(2) were added to the DNA solution. The level of bulky aromatic DNA adducts peaked at the 1st month of exposure, followed by a decrease. By the end of the observation period of 30 months, lung tumors developed even in the 6-month exposure group, and the earliest lung tumors were found only in rats that survived longer than 18 months. In conclusion, persisting oxidative stress on DNA induced in the early phase of diesel exhaust exposure, together with inflammation, seems to play an important role in carcinogenesis at advance ages after a long latent period.

Interleukin-18 (IL-18) and infectious diseases, with special emphasis on diseases induced by intracellular pathogens.

Interleukin-18 (IL-18) is a novel cytokine mainly produced by activated macrophages. IL-18 was originally called interferon-gamma inducing factor, due to its action in inducing IFN-gamma secretion from Th1 cells, NK cells and NKT cells. It has been reported that IL-18 may play important roles in various diseases including cancer and infectious diseases. This review deals with the roles of IL-18 in infectious diseases, with special emphasis on IL-18 in infectious diseases caused by intracellular pathogens including Mycobacterium tuberculosis, Mycobacterium leprae, Listeria monocytogenes and Salmonella typhimurium.

Significance of lung resistance-related protein in the clinical outcome of acute leukaemic patients with reference to P-glycoprotein.

Lung resistance-related protein (LRP) overexpression in leukaemic blast cells from acute leukaemia patients and the effect of LRP or P-glycoprotein (P-gp) on the clinical outcome of acute leukaemia were investigated individually by dividing patients into four groups. The complete remission rate of group I (LRP and P-gp both negative) was 81.7%, group II (only LRP positive) 87.5%, group III (only P-gp positive) 87.1% and group IV (LRP and P-gp both positive) 40.0%. There were no statistical differences between group I and groups II or III, but a significant difference was observed between groups I, II or III and group IV. Median overall survival in group IV was significantly shorter (4.6 months) than in groups I, II or III, although no significant differences were observed between group I and groups II or III (18.9, 20.5 and 31.8 months). There was a tendency for disease-free survival in group III to be longer than that in groups I, II or IV. The reasons for these findings are discussed. Our present results indicate that the co-existence of LRP and P-gp strongly influenced the effectiveness of induction chemotherapy and long-term prognosis, whereas the isolated presence of LRP or P-gp did not.