Inhibition of PbGP43 expression may suggest that gp43 is a virulence factor in Paracoccidioides brasiliensis

ABSTARCT: Glycoprotein gp43 is an immunodominant diagnostic antigen for paracoccidioidomycosis caused by Paracoccidioides brasiliensis. It is abundantly secreted in isolates such as Pb339. It is structurally related to beta-1,3-exoglucanases, however inactive. Its function in fungal biology is unknown, but it elicits humoral, innate and protective cellular immune responses; it binds to extracellular matrix-associated proteins. In this study we applied an antisense RNA (aRNA) technology and Agrobacterium tumefaciens-mediated transformation to generate mitotically stable PbGP43 mutants (PbGP43 aRNA) derived from wild type Pb339 to study its role in P. brasiliensis biology and during infection. Control PbEV was transformed with empty vector. Growth curve, cell vitality and morphology of PbGP43 aRNA mutants were indistinguishable from those of controls. PbGP43 expression was reduced 80-85% in mutants 1 and 2, as determined by real time PCR, correlating with a massive decrease in gp43 expression. This was shown by immunoblotting of culture supernatants revealed with anti-gp43 mouse monoclonal and rabbit polyclonal antibodies, and also by affinity-ligand assays of extracellular molecules with laminin and fibronectin. In vitro, there was significantly increased TNF-α production and reduced yeast recovery when PbGP43 aRNA1 was exposed to IFN-γ-stimulated macrophages, suggesting reduced binding/uptake and/or increased killing. In vivo, fungal burden in lungs of BALB/c mice infected with silenced mutant was negligible and associated with decreased lung ΙΛ-10 and IL-6. Therefore, our results correlated low gp43 expression with lower pathogenicity in mice, but that will be definitely proven when PbGP43 knockouts become available.

Stress response symptoms in adolescents during the first year after a parent's cancer diagnosis

Purpose This work aims to prospectively study stress response symptoms (SRS) in adolescents during the first year after a parent's cancer diagnosis and factors associated with SRS. Additionally, SRS in these adolescents were compared to SRS in adolescents whose parents were diagnosed 1-5 years (reference group) previously. Methods Forty-nine adolescents, 37 ill parents, and 37 spouses completed questionnaires within 4 months after diagnosis (T1) and six (T2) and 12 months (T3) later. Results Clinically elevated SRS were found in 29% of adolescents at T1,16% at T2, and 14% at T3. In contrast, in the reference group, we found 29% clinically elevated SRS. Daughters seemed more at risk than sons. Adolescents' age, patient's gender, and intensity and duration of treatment did not significantly affect SRS. Adolescents with more SRS reported having more emotional/behavioral problems. Parents observed fewer problems in those adolescents. Initial SRS affected later SRS and emotional problems. Conclusions The findings illustrate that adolescent children of cancer patients may have clinically elevated SRS that are associated with emotional and behavioral problems. The prevalence of such problems may be underestimated by the parents