Maria Aparecida de Souza

Increased IgG1, IFN-γ, TNF-α and IL-6 responses to Mycobacterium tuberculosis antigens in patients with Tuberculosis are lower after chemotherapy

Detection of specific antibodies may represent an additional tool in diagnosis of tuberculosis (TB). Herein, levels of serum IgG antibodies against early secreted antigenic target (ESAT-6), culture filtrate antigen-10 (CFP-10) and 16 kDa Mycobacterium tuberculosis antigens were measured in 33 active pulmonary TB patients (0M-TB), in 47 patients after 1-3 months of treatment (3M-TB) and in 22 patients who had completed 6 months of chemotherapy (6M-TB). The control group consisted of 38 BCG-vaccinated healthy controls (HC). In addition, IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-6, IL-2, IL-4 and IL-10 production in PBMC cultures from 20 patients were measured following stimulation with the M. tuberculosis-specific fusion protein ESAT-6/CFP-10. Elevated levels of IgG against ESAT-6, CFP-10 and 16 kDa antigens were detected in 0M-TB and 3M-TB patients in comparison to the HC and 6M-TB groups. Receiver operating characteristic analysis indicated sensitivity of 85, 94 and 61% and specificity of 89, 87 and 89% for serum IgG against ESAT-6, CFP-10 and 16 kDa, respectively. A predominant IgG1 response to ESAT-6 and CFP-10 was observed in 0M-TB patients, together with ESAT-6/CFP-10-specific IFN-gamma, TNF-alpha and IL-6 that were produced at lower levels in the 6M-TB group. These data indicate that a T(h)1 phenotype against early phase Mtb antigens appears to be dominant in the peripheral blood of patients with active pulmonary TB that is reduced after chemotherapy. Taken together, ESAT-6/CFP-10 cytokine tests together with detecting IgG antibodies specific to ESAT-6 and CFP-10 may be the useful TB disease biomarkers in monitoring treatment success.

Genistein down-modulates pro-inflammatory cytokines and reverses clinical signs of experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is the most common non-traumatic, disabling neurological human inflammatory demyelinating disease of the central nervous system (CNS). Experimental autoimmune encephalomyelitis (EAE) models MS and is characterized as a CD4+ T-helper type 1 (Th1) cell-mediated autoimmune disease. It is characterized by an influx of activated leukocytes into the CNS. Genistein, occurring abundantly in soy products, has apoptotic, antioxidant, and anti-inflammatory properties. In the present report, we investigated the use of genistein for the treatment of the murine model of MS. After induction of EAE with myelin oligodendrocyte glycoprotein 35-55 peptide (MOG(35-55)), we observed that genistein treatment ameliorated significantly the clinical symptoms, modulating pro- and anti-inflammatory cytokines. Moreover, we analyzed the leukocyte rolling and adherence in the CNS by performing intravital microscopy. Genistein treatment resulted in decreased rolling and adhering of leukocytes as compared to the untreated group. Our data suggest that genistein might be a potential therapy for MS.

Apoptosis of macrophages during pulmonary Mycobacterium bovis infection: correlation with intracellular bacillary load and cytokine levels

Apoptosis of macrophages infected with pathogenic mycobacteria is an alternative host defence capable of removing the environment supporting bacterial growth. In this work the influence of virulence and bacterial load on apoptosis of alveolar macrophages during the initial phase of infection by Mycobacterium bovis was investigated. BALB/c mice were infected intratracheally with high or low doses of the virulent (ATCC19274) or attenuated (bacillus Calmette–Guérin Moreau) strains of M. bovis. The frequency of macrophage apoptosis, the growth of mycobacteria in macrophages, and the in situ levels of the cytokines tumour necrosis factor‐α (TNF‐α), interleukin‐10 (IL‐10) and IL‐12 and of the anti‐apoptotic protein Bcl‐2 were measured at day 3 and day 7 post‐infection. An increase of macrophage apoptosis was observed after infection with both strains but the virulent strain induced less apoptosis than the attenuated strain. On the 3rd day after infection with the virulent strain macrophage apoptosis was reduced in the high‐dose group, while on the 7th day post‐infection macrophage apoptosis was reduced in the low‐dose group. Inhibition of apoptosis was correlated with increased production of IL‐10, reduced production of TNF‐α and increased production of Bcl‐2. In addition, the production of IL‐12 was reduced at points where the lowest levels of macrophage apoptosis were observed. Our results indicate that virulent mycobacteria are able to modulate macrophage apoptosis to an extent dependent on the intracellular bacterial burden, which benefits its intracellular growth and dissemination to adjacent cells.

Human serum antibody reactivity towards Paracoccidioides brasiliensis antigens treated with sodium metaperiodate

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

In-vitro study of the cellular viability and nitric oxide production by J774 macrophages with ceramic, polycarbonate, and polyoxymethylene brackets

INTRODUCTIONnStudies show that ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade, releasing bisphenol-A and formaldehyde, respectively. In addition to the traditional cytotoxicity tests, the study of nitric oxide cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating its cytotoxic potential.nnnMETHODSnWe aimed to assess cellular viability by MTT (Sigma, St. Louis, Mo): 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide assay in a murine macrophage cell line J774 with esthetic brackets and quantify nitric oxide production by these macrophages. Cell cultures were evaluated at 3 times: 24, 48, and 72 hours.nnnRESULTSnCellular viability in all groups was higher at 72 hours compared with 24 hours. This increase was significant in the control and ceramic brackets groups. Final means in the bracket groups showed no significant differences compared with the control group. Nitric oxide production was significantly greater in all groups at final time. There was no significant difference between the final means of the bracket groups and the control group, although polyoxymethylene brackets showed significantly greater means at 24 and 48 hours.nnnCONCLUSIONSnFinal means in the bracket groups showed no significant differences compared with the control group.

In-vitro study of cellular viability and nitric oxide production by J774 macrophages stimulated by interferon gamma with ceramic, polycarbonate, and polyoxymethylene brackets

INTRODUCTIONnCeramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade and release bisphenol-A and formaldehyde, respectively. More reliable tests are needed to assess the potential toxicity of these materials. In addition to traditional cytotoxicity tests, the study of nitric oxide (NO) cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating cytotoxic potential. The purpose of this study was to assess, with esthetic brackets, cellular viability by 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay (Sigma, St. Louis, Mo) in the macrophage cell line J774 stimulated with interferon gamma. Interferon gamma is a key cytokine in the activation of macrophages, plays an important role in immunologic processes, and also quantifies NO production by these macrophages.nnnMETHODSnWell plates were seeded with 2 x 104 J774 cells per well, in a volume of 100 microL, resuspended in Roswell Park Memorial Institute Supplemented Medium 1640. The macrophage cell line J774 was stimulated with interferon gamma. Ceramic, polycarbonate, and polyoxymethylene brackets were added and kept in the culture for 24, 48, or 72 hours in 5% carbon dioxide at 37 degrees C; the control samples did not include brackets. At the end of each incubation period, the supernatant was collected for posterior NO quantification, and the cells were evaluated for cytotoxicity.nnnRESULTSnCellular viability in all groups was higher at 72 hours than at 24 hours. The final means in the bracket groups did not show significant differences compared with the control group. NO production was significantly greater in all groups at the final time than at the initial time. However, the brackets with the interferon gamma stimulation did not result in greater NO production than did the cells in the control group.

Increased susceptibility to Strongyloides venezuelensis in mice due to Mycobacterium bovis co-infection which modulates production of Th2 cytokines

An estimated quarter of the worlds population possesses an infection caused by gastrointestinal nematodes, which induce a Th2 type immune response. Concomitant infection of nematodes with Mycobacterium tuberculosis, which induces a predominantly Th1 type response, is very frequent in tropical and subtropical regions. This study examined immune responses of BALB/c mice infected with Strongyloides venezuelensis and then co-infected with Mycobacterium bovis. The number of worms in the intestine, eggs in feces, cytokine production in lungs and intestine and the expression of CD80, CD86, CTLA-4 and CD28 cell markers on pulmonary cells were analysed. Our results indicate that co-infected mice had an increased parasite burden, which correlates with elevated IFN-gamma and IL-10 cytokine production and decreased IL-4 and IL-13. Moreover, decreased expression of CD80 and increased expression of CTLA-4 were observed in co-infected mice. Our data point out that susceptibility to Strongyloides venezuelensis infection is increased by Mycobacterium bovis co-infection, resulting in higher parasite survival.

Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform

An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.

Cellular and humoral immune responses during intrathoracic paracoccidioidomycosis in BALB/c mice

Paracoccidioidomycosis is a chronic infection that primarily affects the lungs. Here we investigated cellular and humoral immune responses after intrathoracic Paracoccidioidesbrasiliensis infection in BALB/c mice. P. brasiliensis-colony-forming units (CFUs), fungal DNA and granulomas in lungs increased progressively, peaking at day 90 postinfection (p.i.). IFN-gamma production was highest on day 15 p.i., declining thereafter. The kinetics of the NO production was similar to that described for IFN-gamma. In contrast, IL-10 increased from day 45 p.i. reaching a peak at day 90. Levels of serum IgG1 were higher than IgG2a between days 30 and 90 p.i. 30% of mice died by day 90 p.i. These data indicate that infection with P. brasiliensis by the intrathoracic route shows high IFN-gamma and NO production at day 15 p.i., unable to control multiplication of fungi, which appears to be associated with a progressive increase in IL-10 and in the number and complexity of granulomas.

Xylitol inhibits J774A.1 macrophage adhesion in vitro

The aim of this work was to evaluate the effect of xylitol on J774A.1 macrophage adhesion. Adhesion consisted of a three-hour interval, at room temperature, followed by washing and cell incubation at 37oC/5% CO2/ 48h. Xylitol was used to treat the cells either before (for 24h) or after the cell incubation (for 48h) at 5% as final concentration in both the situations. It was found that xylitol was effective in preventing the adhesion in both the conditions in spite of the former being 100-fold greater and significant (p < 0.001). The results pointed to an important xylitol action on macrophage adhesion, which should be further investigated as an inflammatory control.