Gil Benard

Anti-TNF-α agents in the treatment of immune-mediated inflammatory diseases: mechanisms of action and pitfalls

TNF-α is a potent inducer of the inflammatory response, a key regulator of innate immunity and plays an important role in the regulation of Th1 immune responses against intracellular bacteria and certain viral infections. However, dysregulated TNF can also contribute to numerous pathological situations. These include immune-mediated inflammatory diseases (IMIDs) including rheumatoid arthritis, Crohns disease, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis and severe chronic plaque psoriasis. Animal and human studies concerning the role of TNF-α in IMIDs have led to the development of a therapy based on TNF blockage. This article focuses first on the potential mechanisms by which the three currently licensed agents, adalimumab, etarnecept and infliximab, decrease the inflammatory activity of patients with different IMIDs. Second, it focuses on the risks, precautions and complications of the use of TNF-α inhibitors in these patients.

Paracoccidioidomycosis: A Model for Evaluation of the Effects of Human Immunodeficiency Virus Infection on the Natural History of Endemic Tropical Diseases

The interaction of human immunodeficiency virus (HIV) infection with endemic tropical diseases has become a major concern, but its mechanisms are still poorly understood. Paracoccidioidomycosis (PCM), a South America endemic deep mycosis, may provide an interesting model to investigate this interaction, as clinical-epidemiological features of most HIV-PCM-coinfected patients are difficult to classify into the standard acute and chronic forms of PCM. Such patients have presented clinical features indicative of an uncontrolled infection with lymphohematogenous dissemination, similar to the more severe, acute form. However, this infection probably resulted from reactivated latent foci that, in nonimmunocompromised hosts, leads to the less severe chronic form, characterized by mucosal lesions. We propose that a new outcome of the Paracoccidioides brasiliensis-host interaction is induced by concomitant HIV infection. This outcome probably reflects an impaired anti-P. brasiliensis immune response during coinfection that is similar to that seen in the acute form, although the patients have a chronic P. brasiliensis infection.

Immunosuppression in Paracoccidioidomycosis: T Cell Hyporesponsiveness to Two Paracoccidioides brasiliensis Glycoproteins that Elicit Strong Humoral Immune Response

To assess human cellular immune response to paracoccidioidomycosis (PCM), lymphocyte proliferative responses to purified antigens from Paracoccidioides brasiliensis were determined in healthy persons previously infected by the fungus (positive donors), in healthy noninfected persons (controls), and in PCM patients. Affinity-purified gp70 and gp43, the two major antigens in humoral immune responses, were used. Both induced lymphocyte proliferation (gp43 species-specific) in positive donors but not in controls; healthy persons previously infected by Histoplasma capsulatum reacted to gp70 and not to gp43. A similar cross-reactivity in antibody response to gp70 was previously reported; however, antibody response to gp43 has been considered specific. Lymphocytes from PCM patients, who, unlike positive donors, have high levels of anti-gp43 and anti-gp70 antibodies, proliferated poorly with gp70 and gp43 but better with other stimuli. This dichotomy between humoral and cellular antigen-specific responses suggests a Th2 immune response in PCM, which may be related to failure to control the infection.

Enolase from Paracoccidioides brasiliensis: isolation and identification as a fibronectin-binding protein

Paracoccidioides brasiliensis yeast cells can enter mammalian cells and may manipulate the host cell environment to favour their own growth and survival. Moreover, fibronectin and several other host extracellular matrix proteins are recognized by various components of the yeast cell extracts. The present study was designed to isolate and characterize a fibronectin-binding protein from P. brasiliensis. We also compared P. brasiliensis strain 18, tested before (Pb18a) and after (Pb18b) animal passage, in relation to its adhesion and invasion processes. Extracts from both samples, when cultured on blood agar solid medium, showed higher levels of protein expression than when the same samples were cultured on Fava-Netto solid medium, as demonstrated by two-dimensional electrophoresis and SDS-PAGE. Also, both Pb18a and Pb18b exhibited stronger adhesion to A549 epithelial cells when cultured on blood agar medium than when cultured on Fava-Netto medium. Ligand affinity binding assays revealed a protein of 54 kDa and pI 5.6 in P. brasiliensis cell-free extracts with the properties of a fibronectin-binding adhesin, which was characterized by tryptic digestion and mass spectroscopy as a homologue of enolase from P. brasiliensis. Antibody raised against this 54 kDa protein abolished 80 % of P. brasiliensis adhesion to A549 epithelial cells. Our results demonstrate that P. brasiliensis produces a fibronectin-binding adhesin, irrespective of the culture medium, and that this activity can be inhibited by a specific antibody and is involved in the adhesion of the fungus to pulmonary epithelial cells.

Increased Expression of Regulatory T Cells and Down-Regulatory Molecules in Lepromatous Leprosy

T regulatory cells (Tregs) play an important role in the mechanism of hosts failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-γ (IFN-γ) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-β (TGF-β), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-β. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Evaluation of tests for antibody response in the follow-up of patients with acute and chronic forms of paracoccidioidomycosis

Several serological tests have been used successfully in the diagnosis of paracoccidioidomycosis (PCM). In contrast, data about the use of these tests in the follow-up of PCM patients have been heterogeneous. In this study, serum samples from 43 PCM patients with different clinical forms were analysed by counter-immuno-electrophoresis (CIE), complement fixation (CF) and ELISA before treatment. With CIE and ELISA, the chronic unifocal form showed significantly lower antibody levels compared with chronic multifocal and acute forms. Acute form patients had significantly higher titres than patients with multifocal disease by CIE but not by ELISA. No significant differences were observed with CF. Twenty-seven of these patients were followed-up for 2 years and showed a decline in antibody levels by all three tests, paralleling clinical improvement. However, only patients with unifocal disease cleared their antibodies after 1 year of treatment as analysed by CF and ELISA and after 2 years by CIE, suggesting that these patients may need shorter courses of therapy. Patients with the other clinical form of the disease needed > or =2 years of therapy to clear their antibodies. Sera from a further five patients who presented with a relapse were analysed. At the time of relapse all showed increases in antibody levels by CIE and ELISA, but only three showed increases by CF tests. Therefore, CIE and ELISA demonstrated a better clinical correlation than CF, probably reflecting the fungal burden of PCM patients more accurately.

Climate and acute/subacute paracoccidioidomycosis in a hyper-endemic area in Brazil

BACKGROUND Paracoccidioidomycosis (PCM) is Latin Americas most prevalent systemic mycosis, carrying an important social burden. Its agent, Paracoccidioides brasiliensis, has rarely been identified in nature. Studies characterizing acute/subacute PCM incidence and their relationship with climate variables are not available. This work analysed a series of acute/subacute cases that occurred in the Botucatu area, São Paulo State, Brazil, from 1969 to 1999, as an outcome of weather variability. METHODS Stepwise regression of annual data was applied to model incidence, calculated based on 91 cases, from lagged variables: antecedent precipitation, air temperature, soil water storage, absolute and relative air humidity, and Southern Oscillation Index (SOI). RESULTS Multiple regression analyses resulted in a model, which explains 49% of the incidence variance, taking into account the absolute air humidity in the year of exposure, soil water storage and SOI of the previous 2 years. CONCLUSIONS The correlations may reflect enhanced fungal growth after increase in soil water storage in the longer term and greater spore release with increase in absolute air humidity in the short term.

Severe acute paracoccidioidomycosis in children.

&NA; This report describes clinical and immunologic features of five illustrative cases of paracoccidioidomycosis in previously healthy children. All had disseminated disease and two of them died despite treatment. The major clinical presentation in four patients was fever and diffuse superficial and intraabdominal adenopathy, with or without hepatosplenomegaly. Other sites were also affected: three patients had multiple osteoarticular lesions, occasionally with intense tissue destruction; two had cutaneous eruptions; two had pericardial effusions; and two had pulmonary involvement, once considered an organ spared in the young. We detected variable lymphocyte responses to mitogens and to Candida albicans antigen and nonresponsiveness to Paracoccidioides brasiliensis cell wall antigen. High concentrations of serum immunoglobulins and anti‐P. brasiliensis antibodies were present. These immune alterations tended to resolve with treatment, suggesting a reversible nature of the immune defect. We conclude that this mycosis has a high morbidity and mortality in children, which is probably related to an antigen‐specific immunodeficiency. Further studies are needed to increase knowledge of this mycosis in children.

Topical Application of Imiquimod as a Treatment for Chromoblastomycosis

Chromoblastomycosis is a subcutaneous mycosis that remains a therapeutic challenge, with no standard treatment and high rates of relapse. On the basis of our recent discoveries in mouse models, we tested the efficacy of topical applications of imiquimod to treat patients afflicted with this chronic fungal infection. We report results of treatment for the first 4 recipients of topical imiquimod, all of whom displayed a marked improvement of their lesions, both with and without concurrent oral antifungal therapy.

First description of a cluster of acute/subacute paracoccidioidomycosis cases and its association with a climatic anomaly.

Background Identifying clusters of acute paracoccidioidomycosis cases could potentially help in identifying the environmental factors that influence the incidence of this mycosis. However, unlike other endemic mycoses, there are no published reports of clusters of paracoccidioidomycosis. Methodology/Principal Findings A retrospective cluster detection test was applied to verify if an excess of acute form (AF) paracoccidioidomycosis cases in time and/or space occurred in Botucatu, an endemic area in São Paulo State. The scan-test SaTScan v7.0.3 was set to find clusters for the maximum temporal period of 1 year. The temporal test indicated a significant cluster in 1985 (P<0.005). This cluster comprised 10 cases, although 2.19 were expected for this year in this area. Age and clinical presentation of these cases were typical of AF paracccidioidomycosis. The space-time test confirmed the temporal cluster in 1985 and showed the localities where the risk was higher in that year. The cluster suggests that some particularities took place in the antecedent years in those localities. Analysis of climate variables showed that soil water storage was atypically high in 1982/83 (∼2.11/2.5 SD above mean), and the absolute air humidity in 1984, the year preceding the cluster, was much higher than normal (∼1.6 SD above mean), conditions that may have favored, respectively, antecedent fungal growth in the soil and conidia liberation in 1984, the probable year of exposure. These climatic anomalies in this area was due to the 1982/83 El Niño event, the strongest in the last 50 years. Conclusions/Significance We describe the first cluster of AF paracoccidioidomycosis, which was potentially linked to a climatic anomaly caused by the 1982/83 El Niño Southern Oscillation. This finding is important because it may help to clarify the conditions that favor Paracoccidioides brasiliensis survival and growth in the environment and that enhance human exposure, thus allowing the development of preventive measures.