Wnt-11 promotes neuroendocrine-like differentiation, survival and migration of prostate cancer cells.

BACKGROUND: Wnt-11 is a secreted protein that modulates cell growth, differentiation and morphogenesis during development. We previously reported that Wnt-11 expression is elevated in hormone-independent prostate cancer and that the progression of prostate cancer from androgen-dependent to androgen-independent proliferation correlates with a loss of mutual inhibition between Wnt-11- and androgen receptor-dependent signals. However, the prevalence of increased expression of Wnt-11 in patient tumours and the functions of Wnt-11 in prostate cancer cells were not known. RESULTS: Wnt-11 protein levels in prostate tumours were determined by immunohistochemical analysis of prostate tumour tissue arrays. Wnt-11 protein was elevated in 77/117 of tumours when compared with 27 benign prostatic hypertrophy specimens and was present in 4/4 bone metastases. In addition, there was a positive correlation between Wnt-11 expression and PSA levels above 10 ng/ml. Androgen-depleted LNCaP prostate cancer cells form neurites and express genes associated with neuroendocrine-like differentiation (NED), a feature of prostate tumours that have a poor prognosis. Since androgen-depletion increases expression of Wnt-11, we examined the role of Wnt-11 in NED. Ectopic expression of Wnt-11 induced expression of NSE and ASCL1, which are markers of NED, and this was prevented by inhibitors of cyclic AMP-dependent protein kinase, consistent with the known role of this kinase in NED. In contrast, Wnt-11 did not induce NSE expression in RWPE-1 cells, which are derived from benign prostate, suggesting that the role of Wnt-11 in NED is specific to prostate cancer. In addition, silencing of Wnt-11 expression in androgen-depleted LNCaP cells prevented NED and resulted in apoptosis. Silencing of Wnt-11 gene expression in androgen-independent PC3 cells also reduced expression of NSE and increased apoptosis. Finally, silencing of Wnt-11 reduced PC3 cell migration and ectopic expression of Wnt-11 promoted LNCaP cell invasion. CONCLUSIONS: These observations suggest that the increased level of Wnt-11 found in prostate cancer contributes to tumour progression by promoting NED, tumour cell survival and cell migration/invasion, and may provide an opportunity for novel therapy in prostate cancer.

Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins.

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.

Combined use of Paracoccidioides brasiliensis recombinant 27-kilodalton and purified 87-kilodalton antigens in an enzyme-linked immunosorbent assay for serodiagnosis of paracoccidioidomycosis.

The diagnosis of paracoccidioidomycosis (PCM) has relied on the identification of the host's humoral response by using a variety of immunological methods, such as complement fixation and immunodiffusion. Although these approaches are useful, historically their sensitivity and specificity have often been compromised by the use of complex mixtures of undefined antigens. The use of combinations of purified, well-characterized antigens appears preferable and may yield optimum results. Accordingly an indirect enzyme-linked immunosorbent assay (ELISA) using combinations of the previously described 27-kDa recombinant antigen and the 87-kDa heat shock protein were used for diagnosis and follow-up of patients with PCM. A total of 37 patients classified according to their clinical presentations (7 with the acute or subacute form of the disease, 22 with the chronic form of the disease, and 8 with the chronic unifocal form) were studied. Eighteen of these patients were also evaluated at every follow-up appointment. Forty serum samples from patients with other diseases and 50 serum samples from healthy individuals were also studied. Detection of anti-27-kDa and anti-87-kDa antibodies in sera of patients with PCM by ELISA using a combination of the two purified proteins showed a sensitivity of 92% with a specificity of 88% in comparison with normal human sera and 90% in comparison with the heterologous sera. These results demonstrated a significant increase in sensitivity and specificity compared to results when the antigens were used separately. Thus, the use of combinations of well-defined antigens appears to offer clear advantages over the use of single antigens when diagnosing PCM.

Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with L-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

Paracoccidioides brasiliensis 87-kilodalton antigen, a heat shock protein useful in diagnosis: characterization, purification, and detection in biopsy material via immunohistochemistry.

The 87-kDa antigen derived from the fungal pathogen Paracoccidioides brasiliensis can be detected in the sera of infected patients, and its levels have been shown to correlate well with response to treatment and with clinical cure. Despite its potential importance, the antigen has been poorly characterized. The 87-kDa antigen was purified to homogeneity via preparative gel electrophoresis; N-terminal amino acid sequencing revealed substantial homology with heat shock proteins (hsps) from a variety of organisms. A monoclonal antibody (MAb) raised against a Histoplasma capsulatum 80-kDa hsp showed cross-reactivity to the purified 87-kDa antigen via Western blotting, and the 87-kDa-specific MAb P1B demonstrated that the antigen was expressed at higher levels in yeast than in mycelia by the same technique. Enzyme-linked immunosorbent assay and immunofluorescence reactivity using P1B confirmed increased expression of the 87-kDa antigen during the temperature-induced transformation of mycelia to yeast. Yeast-to-mycelium transformation was accompanied by a fall in expression, although the 87-kDa antigen was clearly constitutively expressed in both phases. Immunochemical staining of tissues from patients with MAb P1B who were infected with P. brasiliensis confirmed in vivo expression of the 87-kDa antigen by yeasts, and identification of this antigen via this method appears to be a useful adjunct to other methods used to diagnose paracoccidioidomycosis.

Micosis cutáneas y mucosas en niños: estudio retrospectivo de 314 casos

Se presentan 314 casos de micosis cutáneas en niños, que fueran diagnosticados en la Corporación para Investigaciones Biológicas (CIB), durante los años de 1987 a 1993. Se hace énfasis en la frecuencia de dermatofitosis, candidiasis y micosis superficiales. Las dermatofitosis fueron diagnosticadas en 210 pacientes (67 por ciento), con mayor frecuencia de tinea capitis y tinea corporis, 88 (42 por ciento) y 81 (38.5 por ciento) casos, respectivamente. La tinea pedis también ocupó lugar importante con 27 casos (13 por ciento). Entre los agentes etiológicos, Microsporum canis fue aislado de 94 pacientes con predominio de las lesiones de cuero cabelludo, 75 casos (80 por ciento). El M.gypseum ocupó el segundo lugar con 52 casos (25 por ciento) y fué además el principal causante de tinea corporis (20.7 por ciento) en este grupo de pacientes. La candidiasis se diagnosticó en 89 pacientes (28 por ciento); la localización más común fue la de la piel glabra (35 por ciento). Candida albicans fue la especie aislada con mayor frecuencia (50.5 por ciento), seguido por C.parapsilosis, 19 casos (21 por ciento). Entre las micosis superficiales, la pitiriasis versicolor se observó en 14 casos (4 por ciento), mientras que la piedra negra fue encontrada sólo en una oportunidad. Los resultados anotados permiten señalar la importancia de las micosis dérmicas en la población pediátrica