Staphylococcus aureus flavohaemoglobin contributes to early stage biofilm development under nitrosative stress.

Staphylococcus aureus is a Gram-positive bacterium with capacity to form biofilms, which constitute an important resistance mechanism and virulence factor. Flavohaemoglobin (Hmp) is a major nitric oxide (NO) detoxifier of several bacteria, including S. aureus. Although Hmp has a well-known physiological role linked to response of planktonic cells to nitrosative stress, its contribution to biofilm formation remains unaddressed. Hence, in this work, we investigated the role of Hmp in biofilm development of a methicillin-resistant S. aureus strain. For this purpose, we exposed the hmp mutant to nitrosative stress and examined its behaviour along biofilm development. We observed that cells inactivated in hmp and grown under nitrosative stress conditions have significantly impaired capacity to develop early stage biofilms. Furthermore, the wild-type biofilm phenotype was fully restored by trans-complementation of hmp in the hmp mutant. Coculture studies of NO-producing macrophages with S. aureus revealed that the hmp mutant has significantly lower capacity to develop biofilm biomass when compared with the wild type. Thus, we concluded that the pathogen S. aureus relies on Hmp to establish viable biofilms in the presence of cells of the host innate immune system.

A modified assay for measuring thymocyte co-stimulatory activity

The observation that murine thymocytes increase their proliferation to interleukin 1 (IL-1) in the presence of phytohemagglutinin (PHA) when pre-incubated with interleukin 2 (IL-2) allowed the introduction of a modified assay for the measurement of IL-1 or the search of thymocyte-inducing proliferative activities in biological samples. Pre-incubation of thymocytes for 24 hr with 50 u/ml IL-2, followed by washings, elicited their maximal response to IL-1 in the usual lymphocyte activating factor (LAF) assay. This suggests that sequential events lead to thymocyte activation. The responsiveness is three to five fold greater than, and the total time of assay is the same as that of the LAF assay. Interestingly, pre-incubation with IL-2 renders thymocytes more sensitive than responsive to crude monocyte conditioned media. The use of the MTT colorimetric method for the assessment of thymocyte proliferation, and of the lectin jacalin as a co-mitogen are suggested as alternatives to be used in co-stimulatory assays

Hematopoese: I. Citocinas envolvidas em sua regulaçäo / Hematopoiesis: I. Cytokines involved in its regulation

O estudo da participaçäo direta de citocinas, tais como os fatores estimuladores do crescimento de colônias, as interleucinas, e outros, no controle das etapas da hematopoese, vem sendo prejudicado pela presença in vitro de células näo-hematopoéticas, capazes de intermediar os efeitos das citocinas sobre os precursores hematopoéticos. Recentemente pôde-se verificar que o antígeno CD34 se expressa na membrana de essencialmente todas as células pluripotenciais, mas näo da maioria das células diferenciadas do sangue ou do estroma da medula óssea, o que veios a permitir experimentos in vitro com populaçöes ricas em células pluripotenciais, purificadas com base na expressäo deste antígeno. Tais experimentos permitiram uma reavaliaçäo dos resultados obtidos previamente com populaçöes menos puras e ampliaram o conhecimento sobre a participaçäo das diferentes citocinas nos processos de diferenciaçäo das distintas linhagens hematopoéticas, tema desta revisäo. O papel de interferons, fatores necrosantes de tumor e fatores de transformaçäo de crescimento ß na regulaçäo negativa da hematopoese säo também analisados