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.

An outbreak of post-acupuncture cutaneous infection due to Mycobacterium abscessus

BACKGROUND: Despite the increasing popularity of acupuncture, the importance of infection control is not adequately emphasized in Oriental medicine. In December 2001, an Oriental medical doctor in Seoul, South Korea, encountered several patients with persistent, culture-negative skin lesions on the trunk and extremities at the sites of prior acupuncture treatment. We identified and investigated an outbreak of Mycobacterium abscessus cutaneous infection among the patients who attended this Oriental medicine clinic. METHODS: Patients were defined as clinic patients with persistent cutaneous infections at the acupuncture sites. Medical records for the previous 7 months were reviewed. Clinical specimens were obtained from the patients and an environmental investigation was performed. M. abscessus isolates, cultured from patients, were compared by pulsed-field gel electrophoresis (PFGE). RESULTS: Forty patients who attended the Oriental medicine clinic and experienced persistent cutaneous wound infections were identified. Cultures from five of these patients proved positive, and all other diagnoses were based on clinical and histopathologic examinations. All environmental objects tested were negative for M. abscessus, however, most were contaminated by various nosocomial pathogens. Molecular analysis using PFGE found all wound isolates to be identical. CONCLUSION: We have identified a large outbreak of rapidly growing mycobacterial infection among patients who received acupuncture at a single Oriental medicine clinic. Physicians should suspect mycobacterial infections in patients with persistent cutaneous infections following acupuncture, and infection control education including hygienic practice, should be emphasized for Oriental medical doctors practicing acupuncture

Prediction of social structure and genetic relatedness in colonies of the facultative polygynous stingless bee Melipona bicolor (Hymenoptera, Apidae)

Stingless bee colonies typically consist of one single-mated mother queen and her worker offspring. The stingless bee Melipona bicolor (Hymenoptera: Apidae) shows facultative polygyny, which makes this species particularly suitable for testing theoretical expectations concerning social behavior. In this study, we investigated the social structure and genetic relatedness among workers from eight natural and six manipulated colonies of M. bicolor over a period of one year. The populations of M. bicolor contained monogynous and polygynous colonies. The estimated genetic relatedness among workers from monogynous and polygynous colonies was 0.75 ± 0.12 and 0.53 ± 0.16 (mean ± SEM), respectively. Although the parental genotypes had significant effects on genetic relatedness in monogynous and polygynous colonies, polygyny markedly decreased the relatedness among nestmate workers. Our findings also demonstrate that polygyny in M. bicolor may arise from the adoption of related or unrelated queens

Cell Therapy of Congenital Corneal Diseases with Umbilical Mesenchymal Stem Cells: Lumican Null Mice

BACKGROUND: Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation. METHODOLOGY/PRINCIPAL FINDINGS: Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice. CONCLUSIONS/SIGNIFICANCE: Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need