Lipoteichoic acids from Lactobacillus strains elicit strong tumor necrosis factor alpha-inducing activities in macrophages through Toll-like receptor 2

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-alpha) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-kappaB activation and TNF-alpha production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall > polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-alpha secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2(-/-) mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-kappaB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.     

Ultrastructural localization of laminin-5 (gamma2 chain) in the rat peri-implant oral mucosa around a titanium-dental implant by immuno-electron microscopy

Laminin-5 (Ln-5) is an important molecule associated with epithelial cell adhesion and migration. In the gingiva around the tooth, Ln-5 localizes within basement membranes between the junctional epithelium (JE) and the tooth or connective tissue. Recently, we reported that in the oral mucosa around a dental implant, Ln-5 is expressed within the basement membranes at the implant-peri-implant epithelium (PIE) interface, and at the PIE-connective tissue interface. However, the ultrastructural localization of Ln-5 within or along the PIE has not yet been reported. Therefore, peri-implant oral mucosa was treated with anti-Ln-5 (gamma2 chain) antibody and examined using immuno-electron microscopy. Ln-5 was localized in the cells of the innermost-third layer and basal layer of the PIE. A 100-nm-wide Ln-5-positive internal basal lamina (basement membrane) and hemidesmosomes as adhesion structures were formed at the apical portion of the implant-PIE interface. However, at the upper-middle portion of the interface, these adhesion structures were not observed. Furthermore, at the PIE-connective tissue interface, the Ln-5-positive external basal lamina (basement membrane) and hemidesmosomes were partially deficient. Judging from these findings, we concluded that Ln-5 contributes to the attachment of the PIE to the titanium surface, and that PIE attached to titanium at the apical portion of the dental implant-PIE interface.     

Pneumocystis carinii carriage in immunocompromised patients with and without human immunodeficiency virus infection

Eighty-one bronchoalveolar lavage (BAL) specimens obtained from 26 HIV-infected, 45 non-HIV immunosuppressed and 10 immunocompetent patients with primary pulmonary diseases were analysed for the presence of Pneumocystis carinii by staining and by P. carinii 5S rDNA determined by PCR. P. carinii was observed by staining of BAL specimens from HIV-infected patients significantly more frequently than those from immunocompromised hosts without HIV infection (57.7% versus 20.0%, respectively). P. carinii 5S rDNA was detected by PCR assay in seven (26.9%) HIV-infected individuals, which was significantly more frequent than for four (8.9%) immunosuppressed patients without HIV infection, for whom staining was negative. None of these patients developed P. carinii pneumonia (PCP) within the follow-up period. BAL specimens from 10 immunocompetent patients with pulmonary disorders were negative for PCP by both staining and PCR assay.     

Depletion of mitochondrial DNA in HIV-1-infected patients and its amelioration by antiretroviral therapy

Mitochondrial DNA (mtDNA) of peripheral blood mononuclear cells (PBMCs) collected from Human immunodeficiency virus 1 (HIV-1)-infected patients and healthy controls were measured longitudinally using real-time polymerase chain reaction to evaluate the effects of antiretroviral agents on mtDNA synthesis in vivo and to assess the value of monitoring mtDNA in PBMCs to predict adverse events amongst these patients. MtDNA levels in PBMCs were significantly decreased in treatment-naive HIV-1-infected patients compared with healthy people. MtDNA levels were not only significantly correlated with CD4(+) T-cell count, but also inversely correlated with HIV-1 viral load. MtDNA levels in untreated patients and healthy controls were stable during the period of observation. On the other hand, amongst patients treated with regimens containing AZT/3TC or d4T/3TC, mtDNA increased during treatment and recovered to levels comparable to healthy controls. In contrast, mtDNA decreased immediately after the initiation of an AZT/ddC-containing regimen. We did not find a correlation between mtDNA levels and changes in clinical parameters. There was no significant difference in mtDNA levels between patients with and those without lipoatrophy. Furthermore, there was no obvious difference in mtDNA levels amongst those patients exhibiting signs and symptoms of peripheral neuropathy. In conclusion, the decrease in mtDNA levels in PBMCs amongst HIV-1-infected patients and its amelioration by antiretroviral therapy may suggest the influence of direct effects on mitochondria or mtDNA by HIV-1 infection. Further investigations are needed to elucidate the mechanisms contributing to decreased mtDNA and the value of mtDNA measurement in the care of HIV-1-infected individuals.     

Serum Cytokines in Patients with Legionella Pneumonia: Relative Predominance of Th1-Type Cytokines

Serum samples from 14 patients with Legionella pneumonia were examined for the presence of cytokines. In spite of high levels of serum C-reactive protein in all patients during the acute phase in only four cases (one involving interleukin-1β [IL-1β], three involving IL-6, and none involving tumor necrosis factor alpha) was the concentration of cytokines more than 100 pg/ml. Th2 cytokines IL-4 and IL-10 were detected in only one patient each. In contrast, significant increases of serum gamma interferon (IFN-γ) and IL-12 levels were observed during the acute phase in 6 and 11 cases, respectively. Interestingly, although serum IFN-γ levels diminished thereafter, in seven cases IL-12 levels remained high or increased further during the convalescent phase. In an additional 22 cases clinically suspected to be but not diagnosed as Legionella pneumonia, increases of serum IL-12 levels were observed in 16 cases, whereas the remaining 6 cases showed no detectable IL-12. Our results demonstrate the relative predominance of Th1 cytokine production in Legionella pneumonia. Although the role and significance of prolonged increases in IL-12 levels in Legionella disease are unknown, our results should prompt further investigation of the host immune response in terms of Th1 and Th2 balance in legionellosis.     

Kupffer cell-mediated cytotoxicity against hepatoma cells occurs through production of nitric oxide and adhesion via ICAM-1/CD18

Rat Kupffer cell (KC)-mediated cytotoxicity against both thesyngeneic hepatoma cell line AH70 and hepatocytes was evaluatedby changes in mitochondrial function, and the possible roleof ICAM-1/CD18 in the interaction between the cells was studied.Rhodamine 123 fluorescence, a marker of the mitochondrial membranepotential, decreased in AH70 cells after co-culture with KC,while that in hepatocytes was unchanged by co-culture. Thisdecrease was blocked by anti-ICAM-1, anti-CD18 and the Inhibitionof nitric oxide synthesis. Cytometric studies demonstrated thatICAM-1 expression on AH70 cells increased after addition ofIFN-, IL-1ß, tumor necrosis factor (TNF)- or KC, whilein hepatocytes ICAM-1 was not increased. Anti-ICAM-1 pretreatmentinhibited the increase in ICAM-1 expression and the decreasein rhodamine 123 fluorescence on AH70 cells after co-culturewith KC. CD18 on KC was increased only after co-culture withAH70. TNF- but not IFN- was detected in the supernatant of co-culturebetween KC and AH70 cells, and this production was partiallyinhibited by anti-ICAM-1 and anti-CD18. The activity of Induciblenitric oxide synthase in Kupffer cells and the levels of nitritesand nitrates in the co-culture supernatant increased over time,and this increase was attenuated either by addition of NO synthesisinhibitors, anti-ICAM-1 or anti-CD18. These results indicatethat the rat KC causes mitochondrial dysfunction in cancer cellsvia the production of NO and cell-to-cell adhesion via ICAM-1/CD18has an Important role in this cytotoxic process.     

Serotonin 2A receptor gene polymorphism and personality traits: no evidence for significant association

A number of studies have observed associations between the serotonin 2A (5-HT2A) receptor and mental disorders. Here, we investigated correlations between polymorphisms (-1438G/A and 102T/C) of the 5-HT2A gene and personality traits in healthy Japanese volunteers (n = 239). The personality traits were evaluated using the Revised NEO Personality Inventory (NEO PI-R). The -1438G/A and 102T/C were in complete linkage disequilibrium. There was a tendency for associations between the genotype and the scores for Agreeableness, Conscientiousness and Neuroticism of the NEO PI-R (P = 0.028, 0.039 and 0.062, respectively; analysis of variance, uncorrected for multiple testing). Subjects with the A/A of -1438G/A (or T/T of 102T/C) appeared to be lower in Neuroticism and higher in Conscientiousness than the rest of the subjects. However, the results were statistically non-significant after Bonferroni's correction for multiple testing of the five scales of the NEO PI-R. Thus, the present study provided no evidence for statistically significant associations between the 5-HT2A polymorphisms and the personality traits.     

Systemic administration of rIL-12 induces complete tumor regression and protective immunity: response is correlated with a striking reversal of suppressed IFN-{gamma} production by anti-tumor T cells

Unfractionated spleen cells taken from tumor-bearing mice 2weeks after tumor implantation contained tumor-primed T cellswhich produced cytokines including IL-2 and IFN- when culturedin vitro. With progressive tumor growth this initial lymphokine-producingcapacity decreased. Here, we investigated the ability of IL-12to (I) restore suppressed IFN- production, (II) cause tumorregression and (II) induce anti-tumor protective immunity. Additionof rIL-12 to spleen cell cultures from 4- to 10-week-old tumor-bearingmice resulted in a striking enhancement in the production ofIFN- compared with cultures of these cells in the absence ofrIL-12 or of normal spleen cells in the presence of rIL-12.Five I.p. injections of rIL-12 into mice bearing s.c. tumorsinduced complete tumor regression. This was found when rIL-12was given at early (1–2 weeks), intermediate (4–5weeks) or even late (7 weeks) stages of tumor growth. Furthermore,IL-12-treated mice which rejected the primary tumor exhibitedcomplete resistance to a rechallenge with the same tumor butdid not reject a second syngenetic tumor. Immunohistochemicalanalyses following IL-12 treatment revealed that CD4⁺ and CD8⁺T cells infiltrate the tumor. More importantly, IFN- mRNA expressionwas observed in fresh tumor masses from tumor-bearing mice receivingIL-12 treatment The importance of IFN- was further demonstratedby the observation that the systemic administration of anti-IFN-mAb prior to IL-12 treatment completely abrogated the anti-tumoreffect of IL-12. Thus, these results indicate that administrationof modest levels of rIL-12 to tumor-bearing mice results intumor regression through mechanisms involving reversal of suppressedIFN- production by anti-tumor T cells and the establishmentof a tumor-specific protective immune response.     

The assessment of cell proliferation during 9,10-dimethyl-1,2-benzanthracene-induced hamster tongue carcinogenesis by means of histone H3 mRNA in situ hybridization

The aim of this study was to investigate cell kinetics and ultrastructural changes during carcinogenesis using a hamster 9,10-dimethyl-1,2-benzanthracene (DMBA)-induced tongue cancer model. Five squamous cell carcinomas, five dysplastic epithelia, seven hyperplastic epithelia, and four normal epithelia were obtained from 21 hamster tongues by applying 1.0% acetone solution of DMBA on the left lingual mucosa after scratching with a root canal broach. Ultrastructural examination revealed that the number of microvilli increased, whereas that of desmosomes decreased during carcinogenesis. Cell proliferation was analyzed by means of 5-bromodeoxyuridine (BrdU) immunohistochemistry and in situ hybridization (ISH) for histone H3 mRNA. The BrdU and histone H3 mRNA labeling indices (LIs) were lowest for normal epithelium, higher for hyperplastic and dysplastic epithelia, and highest for squamous cell carcinoma. Cytoplasmic histone H3 mRNA and nuclear BrdU were localized in virtually identical areas of serial sections. The correlation coefficient for the relationship between these two LIs was 0.97 (P 0.001). These results suggest that the assessment of cell proliferation using H3 mRNA ISH will be a useful technique for investigating biological behavior during carcinogenesis.