Marcello Franco

Host-parasite relationships in paracoccidioidomycosis

A viewpoint of host-parasite relationships in paracoccidioidomycosis is presented. The characteristics of the fungus which are important to the host-parasite interaction are discussed. Aspects of inhibition of mycelium-to-yeast transformation by estrogens acting at receptors on the fungal wall and in the cytoplasm, and the role of polysaccharide components of the cell wall in virulence are reviewed. The natural mechanisms of host defense are also examined, including phagocytosis, complement system, natural-killer cells and genetic control of resistance and susceptibility. Finally, a discussion of granuloma morphogenesis and its relationship to the humoral and cellular anti-P. brasiliensis immune response is presented.

HER3 and downstream pathways are involved in colonization of brain metastases from breast cancer

IntroductionMetastases to the brain from breast cancer have a high mortality, and basal-like breast cancers have a propensity for brain metastases. However, the mechanisms that allow cells to colonize the brain are unclear.MethodsWe used morphology, immunohistochemistry, gene expression and somatic mutation profiling to analyze 39 matched pairs of primary breast cancers and brain metastases, 22 unmatched brain metastases of breast cancer, 11 non-breast brain metastases and 6 autopsy cases of patients with breast cancer metastases to multiple sites, including the brain.ResultsMost brain metastases were triple negative and basal-like. The brain metastases over-expressed one or more members of the HER family and in particular HER3 was significantly over-expressed relative to matched primary tumors. Brain metastases from breast and other primary sites, and metastases to multiple organs in the autopsied cases, also contained somatic mutations in EGFR, HRAS, KRAS, NRAS or PIK3CA. This paralleled the frequent activation of AKT and MAPK pathways. In particular, activation of the MAPK pathway was increased in the brain metastases compared to the primary tumors.ConclusionsDeregulated HER family receptors, particularly HER3, and their downstream pathways are implicated in colonization of brain metastasis. The need for HER family receptors to dimerize for activation suggests that tumors may be susceptible to combinations of anti-HER family inhibitors, and may even be effective in the absence of HER2 amplification (that is, in triple negative/basal cancers). However, the presence of activating mutations in PIK3CA, HRAS, KRAS and NRAS suggests the necessity for also specifically targeting downstream molecules.

Evaluation of the pathogenicity and immunogenicity of seven Paracoccidioides brasiliensis isolates in susceptible inbred mice.

In this investigation the pathogenicity and immunogenicity of seven isolates of Paracoccidioides brasiliensis were compared. The pathogenicity of each isolate was determined by 50% lethal dose estimations and histopathology analysed during a 24-week-period. Four basic patterns of virulence could be defined after intraperitoneal infection of susceptible, genetically homogenous, B10.A mice, namely, slightly virulent (isolates Pb 265 and IVIC Pb 267), intermediate (isolates Pb 192, IVIC Pb 9 and Pb SN), virulent (isolate Pb 2052) and highly virulent (isolate Pb 18). The granulomas induced by the individual isolates were similar although the evolution of inflammation and the organs affected varied according to the isolate used. The immunogenicity of each P. brasiliensis isolate was evaluated by measuring IgG titres with an ELISA method. The intermediate and the slightly virulent isolates induced weak antibody production whereas isolates Pb 18 and Pb 2052 induced stronger specific humoral responses. Differences in the kinetics of antibody production elicited by the different fungal isolates were also observed.

Human Pythiosis, Brazil

Pythiosis, caused by Pythium insidiosum, occurs in humans and animals and is acquired from aquatic environments that harbor the emerging pathogen. Diagnosis is difficult because clinical and histopathologic features are not pathognomonic. We report the first human case of pythiosis from Brazil, diagnosed by using culture and rDNA sequencing.

Molecular detection of Paracoccidioides brasiliensis in soil

The objective of the present study was to evaluate different techniques for the detection of Paracoccidioides brasiliensis in soil, e.g., culture, animal inoculation and specific DNA amplification by Nested PCR. We designed species-specific inner primers derived from rDNA regions (ITS, 5.8S gene) and found their sensitivity to be higher than culture and animal inoculation. In addition, the sensitivity of these primers was higher than p27-gene primers developed for detection of P. brasiliensis in soil in a previous study. DNA from P. brasiliensis was detected in soil artificially seeded with the fungus (positive soil control) and from environmental samples collected in an armadillo burrow.

Left ventricular adaptation to chronic pressure overload induced by inhibition of nitric oxide synthase in rats

Summary Recent studies have indicated that chronic inhibition of nitric oxide synthase induces arterial hypertension without myocardial hypertrophy. We investigated the mechanisms of left ventricular (LV) adaptation to this condition, Also, we analyzed the effect of the angiotensin-converting enzyme inhibitor (ACEI), lisinopril, in this experimental model of ventricular pressure overload. Fiftyeight Wistar rats received eight weeks of treatment with either Nw-nitro-L-arginine-methyl ester (L-NAME group, n=19), lisinopril (LISINOPRIL group, n=19) or the combination of both drugs (LNAMELIS group, n=20). All results were compared to age and sex matched untreated rats (CONTROL group, n=18). Tail-cuff blood pressure rose significantly in L-NAME treated rats (195±29 mm Hg) compared to the CONTROL (141±12 mm Hg), LISINOPRIL (97±13 mm Hg), and LNAMELIS (113±16 mm Hg) groups. There was no myocardial hypertrophy in the chronically hypertensive rats. The ventricular unstressed volume was significantly reduced in the L-NAME group (0.119±0.027 mL) compared to the CONTROL (0.158±0.026 mL) indicating a disproportional reduction in ventricular volume related to the myocardial mass. The chamber size modification resulted in a systolic stress which was comparable to the CONTROL even though the isovolumetric systolic pressure was higher. The systolic functional data indicated preserved myocardial contractility in L-NAME. LV compliance was increased in the LISINOPRIL group and myocardial passive stiffness was lower in all treated rats compared to CONTROL. We conclude that LV adaptation to chronic pressure overload without hypertrophy involves changes in chamber geometry and myocardial diastolic mechanical properties. Also, ACEI fully prevents L-NAME induced hypertension, reduces myocyte cross-sectional area, and myocardial passive stiffness. The combination of L-NAME plus lisinopril decreases the load independent index of myocardial contractility.

A comparative histopathological, immunological, and biochemical study of experimental intravenous paracoccidioidomycosis induced in mice by three Paracoccidioides brasiliensis isolates

We carried out a comparative study of the histopathology (lung, liver, spleen, kidney and adrenals) and the anti-P. brasiliensis humoral (immunodiffusion test) and cellular (footpad test) immune response of mice intravenously inoculated with yeast forms of three P. brasiliensis isolates (Pb 18, Pb 192, Pb 265). Pb 265 (avirulent strain) did not evoke specific lesions or antibody production; the levels of cellular immunity were significantly lower than those of the two other isolates. Lung granulomas induced by strain Pb 18 were richer in fungi and neutrophils and poorer in mononuclear cells when compared to those induced by strain Pb 192. Extrapulmonary lesions were more frequent in mice infected with strain Pb 18. Strains Pb 18 and Pb 192 raised similar humoral and cellular anti-P. brasiliensis responses. Cell wall analysis did not suggest striking differences among the strains. Slightly higher levels of the water soluble fraction 3 (which contains the immunogenic galactomannan and protein) were detected in strain Pb 265.

Monitoring of intragraft and peripheral blood TIRC7 expression as a diagnostic tool for acute cardiac rejection in humans

T-cell immune response cDNA 7 (TIRC7) is a recently described T-cell costimulatory molecule that exhibits a central role in T-cell activation in vitro and in vivo. The present study was undertaken to investigate association between intragraft and peripheral blood mononuclear cell (PBMC) TIRC7 mRNA levels and cardiac allograft rejection in humans. TIRC7 gene expression levels were determined by a quantitative-competitive reverse transcriptase-polymerase chain reaction (QC-RT-PCR) in endomyocardial biopsies and in PBMC from cardiac transplant recipients. Biopsies collected during rejection or up to 15 days before rejection showed heightened TIRC7 mRNA expression in comparison with biopsies without rejection. All prerejection and rejection biopsies showed TIRC7 mRNA upregulation, while this was present in only 30% of the biopsies without rejection. Regarding TIRC7 mRNA in PBMC, transplant recipients showed lower levels than healthy individuals and, in contrast to the results obtained in biopsies, the levels were lower during rejection than in rejection-free periods. In summary, TIRC7 mRNA expression levels increase in biopsies and decrease in peripheral blood during acute cardiac rejection. We conclude that intragraft detection of TIRC7 transcripts is a useful tool not only for the diagnosis but also for the prediction of acute heart allograft rejection episodes.

DELAYED-TYPE HYPERSENSITIVITY RESPONSE IN AN ISOGENIC MURINE MODEL OF PARACOCCIDIOIDOMYCOSIS

The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265)Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses againstP. brasiliensis depend on both the hosts genetically determined resistance and the virulence of the fungal isolate.

Pre- and post-transplant anti-myosin and anti–heat shock protein antibodies and cardiac transplant outcome

BACKGROUND The purpose this study was to investigate the relationship of anti-myosin and anti-heat shock protein immunoglobulin G (IgG) serum antibodies to the original heart disease of cardiac transplant recipients, and also to rejection and patient survival after cardiac transplantation. METHODS Anti-myosin and anti-heat shock protein (anti-hsp) IgG antibodies were evaluated in pre-transplant sera from 41 adult cardiac allograft recipients and in sequential post-transplant serum samples from 11 recipients, collected at the time of routine endomyocardial biopsies during the first 6 months after transplantation. In addition, the levels of these antibodies were determined from the sera of 28 healthy blood donors. RESULTS Higher anti-myosin antibody levels were observed in pre-transplant sera than in sera from normal controls. Moreover, patients with chronic Chagas heart disease showed higher anti-myosin levels than patients with ischemic heart disease, and also higher levels, although not statistically significant, than patients with dilated cardiomyopathy. Higher anti-hsp levels were also observed in patients compared with healthy controls, but no significant differences were detected among the different types of heart diseases. Higher pre-transplant anti-myosin, but not anti-hsp, levels were associated with lower 2-year post-transplant survival. In the post-transplant period, higher anti-myosin IgG levels were detected in sera collected during acute rejection than in sera collected during the rejection-free period, whereas anti-hsp IgG levels showed no difference between these periods. CONCLUSIONS The present findings are of interest for post-transplant management and, in addition, suggest a pathogenic role for anti-myosin antibodies in cardiac transplant rejection, as has been proposed in experimental models of cardiac transplantation.