The Cell Wall Fraction from Fonsecaea pedrosoi Stimulates Production of Different Profiles of Cytokines and Nitric Oxide by Murine Peritoneal Cells In Vitro

Chromoblastomycosis is a chronic, suppurative and granulomatous mycosis whose main etiologic agent is the fungus Fonsecaea pedrosoi. The severity of chromoblastomycosis clinical manifestations correlates with the Th1 or Th2 immune response, and an efficient cellular immune response depends on the interaction between immune cells and the cell wall of the fungi, which is able to promote this activation. The objective of this study was to analyze the influence of cell wall fractions of Fonsecaea pedrosoi on the activation of peritoneal phagocytes obtained from mice. Our results revealed that after 4 h of inoculation with fungal cell wall components, there was a cell migration predominantly comprised of neutrophils followed, after 72 h, by migration of the macrophages. After 4 h, the F2 fraction caused increased production of nitric oxide in phagocytes, but this effect was not observed in the phagocytes after 72 h. The F1 fraction stimulated production of IL-12 in cells that migrated after 72 h, while the inactivated fungus and the F2 fraction led to production of IL-10. The F2 fraction decreased the rate of phagocytosis and increased the production of IL-10. Our results suggest that the F2 fraction and its components caused an important disruption of microbicidal mechanisms negatively modulating the immune response and favoring the persistence of the fungus.     

Murine splenocytes produce inflammatory cytokines in a MyD88-dependent response to Bacillus anthracis spores

Bacillus anthracis is a sporulating Gram-positive bacterium that causes the disease anthrax. The highly stable spore is the infectious form of the bacterium that first interacts with the prospective host, and thus the interaction between the host and spore is vital to the development of disease. We focused our study on the response of murine splenocytes to the B. anthracis spore by using paraformaldehyde-inactivated spores (FIS), a treatment that prevents germination and production of products associated with vegetative bacilli. We found that murine splenocytes produce IL-12 and IFN-gamma in response to FIS. The IL-12 was secreted by CD11b cells, which functioned to induce the production of IFN-gamma by CD49b (DX5) NK cells. The production of these cytokines by splenocytes was not dependent on TLR2, TLR4, TLR9, Nod1, or Nod2; however, it was dependent on the signalling adapter protein MyD88. Unlike splenocytes, Nod1- and Nod2-transfected HEK cells were activated by FIS. Both IL-12 and IFN-gamma secretion were inhibited by treatment with B. anthracis lethal toxin. These observations suggest that the innate immune system recognizes spores with a MyD88-dependent receptor (or receptors) and responds by secreting inflammatory cytokines, which may ultimately aid in resisting infection.     

Semisynthetic neoboutomellerone derivatives as ubiquitin-proteasome pathway inhibitors

The interesting pharmacological properties of neoboutomellerones 1 and 2 were the basis for the assembly of a small library of analogues consisting of natural products isolated from the plant Neoboutonia melleri and of semisynthetic derivatives. As the two enone systems (C23-C24a and C1-C3) and the two hydroxyls groups (C22 and C26) of neoboutomellerones are required for activity, modifications were focused on these functional groups. Biological evaluation by using a cellular assay for proteasome activity provided clues regarding the mechanism of action of these natural products and synthetic derivatives. Certain neoboutomellerone derivatives inhibited the proliferation of human WM-266-4 melanoma tumor cells at submicromolar concentration and warrant evaluation as anticancer agents.     

Epigenetics of major psychosis: progress, problems and perspectives

Understanding the origins of normal and pathological behavior is one of the most exciting opportunities in contemporary biomedical research. There is increasing evidence that, in addition to DNA sequence and the environment, epigenetic modifications of DNA and histone proteins may contribute to complex phenotypes. Inherited and/or acquired epigenetic factors are partially stable and have regulatory roles in numerous genomic activities, thus making epigenetics a promising research path in etiological studies of psychiatric disease. In this article, we review recent epigenetic studies examining the brain and other tissues, including those from individuals with schizophrenia (SCZ) and bipolar disorder (BPD). We also highlight heuristic aspects of the epigenetic theory of psychiatric disease and discuss the future directions of psychiatric epigenetics.     

Development of production and purification processes of recombinant fragment of pneumococcal surface protein A in <Emphasis Type="Italic">Escherichia coli</Emphasis> using different carbon sources and chromatography sequences

Pneumococcal surface protein A (PspA) is essential for Streptococcus pneumoniae virulence and its use either as a novel pneumococcal vaccine or as carrier in a conjugate vaccine would improve the protection and the coverage of the vaccine. Within this context, the development of scalable production and purification processes of His-tagged recombinant fragment of PspA from clade 3 (rfPspA3) in Escherichia coli BL21(DE3) was proposed. Fed-batch production was performed using chemically defined medium with glucose or glycerol as carbon source. Although the use of glycerol led to lower acetate production, the concentration of cells were similar at the end of both fed-batches, reaching high cell density of E. coli (62 g dry cell weight/L), and the rfPspA3 production was higher with glucose (3.48 g/L) than with glycerol (2.97 g/L). A study of downstream process was also carried out, including cell disruption and clarification steps. Normally, the first chromatography step for purification of His-tagged proteins is metal affinity. However, the purification design using anion exchange followed by metal affinity gave better results for rfPspA3 than the opposite sequence. Performing this new design of chromatography steps, rfPspA3 was obtained with 95.5% and 75.9% purity, respectively, from glucose and glycerol culture. Finally, after cation exchange chromatography, rfPspA3 purity reached 96.5% and 90.6%, respectively, from glucose and glycerol culture, and the protein was shown to have the expected alpha-helix secondary structure.     

The plating of rat scar myofibroblasts on matrigel unmasks a novel phenotype; the self assembly of lumen-like structures

During tissue healing, the primary role of myofibroblasts involves the synthesis and deposition of collagen. However, it has also been reported that selective populations of myofibroblasts can acquire the phenotype and/or differentiate to other cells types. The present study tested the hypothesis that myofibroblasts isolated from the scar of the ischemically damaged rat heart can recapitulate an endothelial cell-like response when plated in a permissive in vitro environment. Scar myofibroblasts, neonatal and adult ventricular fibroblasts express smooth muscle α-actin, collagen α(1) type 1 and a panel of pro-fibrotic and pro-angiogenic peptide growth factor mRNAs. Myofibroblasts plated alone on matrigel led to the self assembly of lumen-like structures whereas neonatal and adult rat ventricular fibroblasts were unresponsive. Myofibroblasts labeled with the fluorescent cell tracker CM-DiI were injected in the viable myocardium of 3-day post-myocardial infarcted Sprague-Dawley rats and sacrificed 7 days later. Injected CM-DiI-labeled myofibroblasts were detected predominantly in the peri-infarct/infarct region, highlighting their migration to the damaged region. However, engrafted myofibroblasts in the peri-infarct/infarct region were unable to adopt an endothelial cell-like phenotype or lead to the de novo formation of CM-DiI-labeled blood vessels. The non-permissive nature of the infarct region may be attributed at least in part to the presence of growth-promoting stimuli as TGF-β and the β-adrenergic agonist isoproterenol inhibited the self assembly of lumen-like structures by myofibroblasts. Thus, when plated in a permissive in vitro environment, scar myofibroblasts can self assemble and form lumen-like structures providing an additional novel phenotype distinguishing this population from normal ventricular fibroblasts.     

Interactions between Sox10, Edn3 and Ednrb during enteric nervous system and melanocyte development

The requirement for SOX10 and endothelin-3/EDNRB signalling pathway during enteric nervous system (ENS) and melanocyte development, as well as their alterations in Waardenburg-Hirschsprung disease (hypopigmentation, deafness and absence of enteric ganglia) are well established. Here, we analysed the genetic interactions between these genes during ENS and melanocyte development. Through phenotype analysis of Sox10;Ednrb and Sox10;Edn3 double mutants, we show that a coordinate and balanced interaction between these molecules is required for normal ENS and melanocyte development. Indeed, double mutants present with a severe increase in white spotting, absence of melanocytes within the inner ear, and in the stria vascularis in particular, and more severe ENS defects. Moreover, we show that partial loss of Ednrb in Sox10 heterozygous mice impairs colonisation of the gut by enteric crest cells at all stages observed. However, compared to single mutants, we detected no apoptosis, cell proliferation or overall neuronal or glial differentiation defects in neural crest cells within the stomach of double mutants, but apoptosis was increased in vagal neural crest cells outside of the gut. These data will contribute to the understanding of the molecular basis of ENS, pigmentation and hearing defects observed in mouse mutants and patients carrying SOX10, EDN3 and EDNRB mutations.