Nitric oxide production and iNOS mRNA expression in mice induced by repeated stimulation with live Fusobacterium nucleatum

There have been few studies on the detection of direct nitric oxide (NO) production and interferon-gamma (IFN-gamma) in vivo without using animal cell culture. We questioned whether NO and IFN-gamma could be produced at the site of infection. The peritoneal cavity of mice was used as the local infection model. NO and IFN-gamma in abdominal washings from these mice were measured directly at various times after injection of Fusobacterium nucleatum, a gram-negative rod periodontal pathogen. The mice were divided into three groups: those treated with live bacteria (LB), those treated with heat-killed bacteria (HKB) and those untreated: normal (N). These mice were compared on the basis of cell filtration, NO and IFN-gamma production by injection of live bacteria (LFn) or heat-killed bacteria (HKFn). In the LB group, the total cell number increased corresponding to an increase in neutrophils after injection of both LFn and HKFn. A low level of NO was constantly produced in abdominal washings, but a significant amount of NO was synthesized in the LB group only 12 hr to 24 hr after injection of LFn. At the same time iNOS enzyme activity and iNOS mRNA expression were detected. IFN-gamma, which may contribute to enhance NO production, was also secreted at a high level from peritoneal exudate cells (PEC) at 12 hr and 24 hr in the LB group by stimulation of LFn. At 12 hr and 24 hr, iNOS positive cells in the LB group by infection of LFn were identified and shown to contain mostly macrophages. These findings indicate that live bacteria play important roles in NO production by macrophages. It is suggested that NO may contribute to the inflammatory response during F. nucleatum infection in periodontitis.     

Adherence-dependent increase in human monocyte PDGF(B) mRNA is associated with increases in c-fos, c-jun, and EGR2 mRNA

Adherence is an important initial step in the transition of a circulating monocyte to a tissue macrophage. This differentiation is accompanied by an augmented capacity to generate growth factors. We hypothesized that adherence itself might be an important trigger for a sequence of gene activation culminating in cells with increased mRNA encoding profibrotic growth factors such as platelet-derived growth factor B subunit (PDGF[B]) and transforming growth factor-beta (TGF- beta). After in vitro adherence, human monocytes had a biphasic increase in PDGF(B) mRNA with peaks at 6 h and 13 d. No increase in TGF- beta mRNA was observed. The 6-h increase in PDGF(B) mRNA was adherence dependent, and in addition, was abrogated when the cytoskeletal integrity was compromised by cytochalasin D. The 6-h increase in PDGF(B) mRNA was unaltered by adherence in the presence of the monocyte stimulus lipopolysaccharide. Adherence to either fibronectin or collagen-coated plastic had little consistent effect on PDGF(B) mRNA accumulation. The increased PDGF(B) mRNA observed in adherent monocytes was accompanied by increases in mRNAs of the early growth response genes c-fos (maximal at 20 min), c-jun, and EGR2 (maximal at 6-24 h). The increase in c-jun and EGR2, but not c-fos, mRNA was also abrogated by cytochalasin D. These observations suggest that adherence results in increases of c-fos, c-jun, EGR2, and PDGF(B) mRNA. In addition, the increases in c-jun, EGR2, and PDGF(B) may depend on cytoskeletal rearrangement. Modulation of these events at the time of adherence offers a mechanism by which differential priming of the cells may be accomplished.     

Cell-free antigens from Paracoccidioides brasiliensis drive IL-4 production and increase the severity of paracoccidioidomycosis

The thermally dimorphic fungus Paracoccidioides brasiliensis (Pb) is the causative agent of paracoccidioidomycosis (PCM), one of the most frequent systemic mycosis that affects the rural population in Latin America. PCM is characterized by a chronic inflammatory granulomatous reaction, which is consequence of a Th1-mediated adaptive immune response. In the present study we investigated the mechanisms involved in the immunoregulation triggered after a prior contact with cell-free antigens (CFA) during a murine model of PCM. The results showed that the inoculation of CFA prior to the infection resulted in disorganized granulomatous lesions and increased fungal replication in the lungs, liver and spleen, that paralleled with the higher levels of IL-4 when compared with the control group. The role of IL-4 in facilitating the fungal growth was demonstrated in IL-4-deficient- and neutralizing anti-IL-4 mAb-treated mice. The injection of CFA did not affect the fungal growth in these mice, which, in fact, exhibited a significant diminished amount of fungus in the tissues and smaller granulomas. Considering that in vivo anti-IL-4-application started one week after the CFA-inoculum, it implicates that IL-4-CFA-induced is responsible by the mediation of the observed unresponsiveness. Further, the characterization of CFA indicated that a proteic fraction is required for triggering the immunosuppressive mechanisms, while glycosylation or glycosphingolipids moieties are not. Taken together, our data suggest that the prior contact with soluble Pb antigens leads to severe PCM in an IL-4 dependent manner.     

IL-15 up-regulates iNOS expression and NO production by gingival epithelial cells

To investigate the biological activity of epithelial cells in view of host defense, we analyzed the mRNA expression of inducible NOS (iNOS) as well as NO production by human gingival epithelial cells (HGEC) stimulated with IL-15. RT-PCR analysis revealed that HGEC expressed IL-15 receptor alpha-chain mRNA. In addition, stimulation with IL-15 enhanced iNOS expression by HGEC through an increase of both mRNA and protein levels. Moreover, IL-15 up-regulated the production of NO(2)(-)/NO(3)(-), a NO-derived stable end product, from HGEC. The enhanced NO production by IL-15 was inhibited by AMT, an iNOS-specific inhibitor. These results suggest that IL-15 is a potent regulator of iNOS expression by HGEC and involved in innate immunity in the mucosal epithelium.     

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.     

Pulmonary immune responses induced in BALB/c mice by Paracoccidioides brasiliensis conidia

Knowledge concerning the host–Paracoccidioides brasiliensis interactions is abundant. Yet, most of the experimental studies have used yeast cells to prepare the corresponding inoculum. As these cells do not represent the naturally infecting propagules, the corresponding experiments by-pass the earlier stages of such interactions. Studies done in patients, who also harbour yeast cells, suffer from the same bias. The review presented below focuses on the immune responses of BALB/c mice infected with conidia obtained from P. brasiliensis mycelial form cultures, the fungal stage most probably existing in nature. As such, the corresponding experiments would copy the onset and course of the human infection. A large number of experimental studies done by the CIB Medical and Experimental Mycology Unit in a period of almost 25 years have been revised and extracted so as to present a comprehensive record on the immune responses induced when mice are infected intranasally with the conidia. The establishment of this mouse model has permitted the analysis of the immune responses taking place during the early and late stages post-challenge. This unique model has made possible to characterize the course of the experimental disease including the inflammatory reaction, the expression of cytokines and of the various molecules associated to these responses, all of which lead to granuloma formation. The latter structure serves as a nest for the development of fibrosis. Thus, we have also obtained a glimpse on the complexity that accompanies the fibrosis, the most common sequelae of paracoccidioidomycosis. Additionally, a concerted effort has been made to appraise the whole gamut of immune factors and related molecules that directly or indirectly, contribute to shape the pathogenesis of this Latin American mycosis.     

Serological Evidence of Infection by Paracoccidioides brasiliensis in Dogs with Leishmaniasis

Paracoccidioidomycosis is a systemic mycosis prevalent in Latin American countries, caused by the dimorphic fungi Paracoccidioides brasiliensis and P. lutzii. The habitat of these fungi in nature remains undefined, although it is believed that infection occurs by inhalation of infective propagules present in soil. Sentinel animals, such as dogs, can be valuable epidemiological markers of paracoccidioidomycosis. Taking into account that paracoccidioidomycosis and visceral leishmaniasis may occur in the same area, the objective of this study was to evaluate the occurrence of P. brasiliensis infection in dogs positive for Leishmania sp. Serum samples of dogs positive (n = 199) and negative (n = 101) for Leishmania sp. were analyzed by the immunodiffusion test using P. brasiliensis exoantigen, and 22 samples (7.3%) were positive. The serum samples positive in the immunodiffusion test were also analyzed by Western blotting using the P. brasiliensis gp43 recombinant protein, and 86% of the samples were positive. A high positive correlation (r = 0.96) between positivity for Leishmania sp. and P. brasiliensis was observed. These data suggest an association between leishmaniasis and paracoccidioidomycosis in dogs.

     

Small forms and hyphae of Paracoccidioides brasiliensis in human tissue

A 53 year-old man had a three-year recurrent respiratory infection. No fungi was detected in sputum examinations. Immunodiffusion test with paracoccidioidin revealed two precipitin bands. Very small forms and hyphae of a fungus were seen on silver methenamine stained serial sections from lung's lesion. P. brasiliensis was identified on the basis of the rare multibudding small forms.     

Carboxylic activity of giant cells of human granuloma produced by Paracoccidioides brasiliensis.

The carboxilic activity of giant cells of human granuloma produced by P. brasiliensis was studied. The enzymatic activity was revealed by reddish-brown, purple red, and indigo-blue cytoplasmic precipitate, using the substrates alpha-naphthyl-acetate, naphthol-AS acetate and 5-bromo-4-chloro-indoxyl acetate respectively. The giant cells were intensely positive in all cases studied. We believe this esterasic activity is related to the lytic, lisosomic activity of the macrophages and giant cells in response to the activity by the P. brasiliensis in tissue.     

iNOS/NO signaling regulates apoptosis induced by glycochenodeoxycholate in hepatocytes

Inducible nitric oxide synthase (iNOS) and nitric oxide (NO) can ameliorate apoptosis induced by toxic glycochenodeoxycholate (GCDC) in hepatocytes. However, the underlying molecular mechanisms are not yet understood in detail. This study is to clarify the function of iNOS/NO and its mechanisms during the apoptotic process. The apoptosis was brought about by GCDC in rat primary hepatocytes. iNOS/NO signaling was then investigated. iNOS inhibitor 1400 W enhanced the GCDC-induced apoptosis as reflected by caspase-3 activity and TUNEL assay. Exogenous NO regulated the apoptosis subsequent to NO donor S-nitroso-N-acetyl-penicillamine (SNAP) or sodium nitroprusside (SNP). The GCDC-induced apoptosis was decreased with 0.1 mM SNAP or 0.15 mM SNP, while it was increased with 0.8 mM SNAP or 1.2 mM SNP. The endogenous iNOS inhibited apoptosis, but the exogenous NO played a dual role during the GCDC-induced apoptosis. There was a potential iNOS/Akt/survivin axis that inhibited the hepatocyte apoptosis in low doses of NO donors. In contrast, high doses of NO donors activated CHOP through p38MAP-kinase (p38MAPK), upregulated TRAIL receptor DR5, and suppressed survivin. Consequently the high doses of NO donors promoted the apoptosis in hepatocytes. Our data suggest that the iNOS/NO signaling can modulate Akt/survivin and p38MAPK/CHOP pathways to mediate the GCDC-induced the apoptosis in hepatocytes. These signaling pathways may serve as targets for therapeutic intervention in cholestatic liver disease.     

Presence and expression of the mating type locus in Paracoccidioides brasiliensis isolates

Paracoccidioides brasiliensis has been classified in the phylum Ascomycota, order Onygenales, family Ajellomycetaceae, even in the absence of a known sexual cycle or mating system. The objective of this work was to determine the presence of the mating type locus in 71 P. brasiliensis isolates from various sources. A PCR assay using specific primers for the MAT 1 gene was developed and applied for the detection of such genes. Two heterothallic groups (MAT1-1 or MAT1-2) were recognized and, in some isolates, gene expression was confirmed indicating the existence of a basal gene expression. The distribution of two mating type loci in the studied population suggested that sexual reproduction might occur in P. brasiliensis. This finding points towards the possibility of applying a more precise definition of the concept of biological species to P. brasiliensis. Further studies should be conducted to confirm the sexual capacity of this fungus and its implications among phylogenetic species and geographical distribution.     

Inhibition of aldosterone production by alpha-human atrial natriuretic polypeptide is associated with an increase in cGMP production

Synthetic alpha-human atrial natriuretic polypeptide caused rapid and marked inhibition of aldosterone production in dispersed rat adrenal capsular cells. The polypeptide also slightly, but significantly, decreased cAMP production in the adrenal dispersed capsular cells, while markedly stimulating cGMP production. The cGMP production was accelerated at the concentration of alpha-human atrial natriuretic polypeptide lower than the threshold level to stimulate aldosterone production. These findings suggest that alpha-human atrial natriuretic polypeptide possibly plays a regulatory role in aldosterone production and an additional role in natriuresis through inhibition of aldosterone production. The stimulation of cGMP production by alpha-human atrial natriuretic polypeptide may be involved in the inhibitory effect of this peptide on aldosterone production.     

Experimental Model of Arthritis Induced by Paracoccidioides brasiliensis in Rats

Paracoccidioidomycosis (PCM), a disease caused by the fungus Paracoccidioides brasiliensis (Pb), is highly prevalent in Brazil, where it is the principal cause of death by systemic mycoses. The disease primarily affects men aged 30-50 year old and usually starts as a pulmonary focus and then may spread to other organs and systems, including the joints. The present study aimed to develop an experimental model of paracoccidioidomycotic arthritis. Two-month-old male Wistar rats (n = 48) were used, divided in 6 groups: test groups EG/15 and EG/45 (received one dose of 100 μl of saline containing 10(5) Pb viable yeasts in the knee); heat killed Pb-group HK/15 and HK/45 (received a suspension of 10(5) Pb nonviable yeasts in the knee) and control groups CG/15 and CG/45 (received only sterile saline in the knee). The rats were killed 15 and 45 days postinoculation. In contrast with the control rats, the histopathology of the joints of rats of the test groups (EG/15 and EG/45) revealed a picture of well-established PCM arthritis characterized by extensive sclerosing granulomatous inflammation with numerous multiple budding fungal cells. The X-ray examination revealed joint alterations in these groups. Only metabolic active fungi evoked inflammation. The experimental model was able to induce fungal arthritis in the knees of the rats infected with metabolic active P. brasiliensis. The disease tended to be regressive and restrained by the immune system. No evidence of fungal dissemination to the lungs was observed.