Hypoxia modulates phenotype, inflammatory response, and leishmanial infection of human dendritic cells

Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA-DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL-12p70, the bioactive interleukin-12 (IL-12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro-inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.

Analysis of Intracellular Substrates and Products of Thimet Oligopeptidase in Human Embryonic Kidney 293 Cells

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) is an intracellular enzyme that has been proposed to metabolize peptides within cells, thereby affecting antigen presentation and G protein-coupled receptor signal transduction. However, only a small number of intracellular substrates of EP24.15 have been reported previously. Here we have identified over 100 peptides in human embryonic kidney 293 (HEK293) cells that are derived from intracellular proteins; many but not all of these peptides are substrates or products of EP24.15. First, cellular peptides were extracted from HEK293 cells and incubated in vitro with purified EP24.15. Then the peptides were labeled with isotopic tags and analyzed by mass spectrometry to obtain quantitative data on the extent of cleavage. A related series of experiments tested the effect of overexpression of EP24.15 on the cellular levels of peptides in HEK293 cells. Finally, synthetic peptides that corresponded to 10 of the cellular peptides were incubated with purified EP24.15 in vitro, and the cleavage was monitored by high pressure liquid chromatography and mass spectrometry. Many of the EP24.15 substrates identified by these approaches are 9-11 amino acids in length, supporting the proposal that EP24.15 can function in the degradation of peptides that could be used for antigen presentation. However, EP24.15 also converts some peptides into products that are 8-10 amino acids, thus contributing to the formation of peptides for antigen presentation. In addition, the intracellular peptides described here are potential candidates to regulate protein interactions within cells.

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts over-expressed in T rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-beta-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Human cytomegalovirus reinfection is associated with intrauterine transmission in a highly cytomegalovirus-immune maternal population

OBJECTIVE: To determine contribution of reinfection with new strains of cytomegalovirus in cytomegalovirus seromimmune women to incidence of congenital cytomegalovirus infection. STUDY DESIGN: In 7848 women studied prospectively for congenital cytomegalovirus infection from a population with near universal cytomegalovirus seroimmunity, sera from 40 mothers of congenitally infected infants and 109 mothers of uninfected newborns were analyzed for strain-specific anticytomegalovirus antibodies. RESULTS: All women were cytomegalovirus seroimmune at first prenatal visit. Reactivity for 2 cytomegalovirus strains was found in 14 of 40 study mothers and in 17 of 109 control mothers at first prenatal visit (P=.009). Seven of 40 (17.5%) study women and 5 of 109 (4.6%) controls (P=.002) acquired antibodies reactive with new cytomegalovirus strains during pregnancy. Evidence of infection with more than 1 strain of cytomegalovirus before or during current pregnancy occurred in 21 of 40 study mothers and 22 of 109 controls (P<.0001). CONCLUSION: Maternal reinfection by new strains of cytomegalovirus is a major source of congenital infection in this population.

Ovarian hyperstimulation syndrome: pathophysiology and prevention

To review and discuss the pathophysiology and prevention strategies for ovarian hyperstimulation syndrome (OHSS), which is a condition that may occur in up to 20% of the high risk women submitted to assisted reproductive technology cycles. The English language literature on these topics were reviewed through PubMed and discussed with emphasis on recent data. The role of estradiol, luteinizing hormone, human chorionic gonadotropin (hCG), inflammatory mediators, the renin-angiotensin system and vascular endothelial growth factor is discussed in the pathophysiology of OHSS. In addition we consider the prevention strategies, including coasting, administration of albumin, renin-angiotensin system blockage, dopamine agonist administration, non-steroidal anti-inflammatory administration, GnRH antagonist protocols, reducing hCG dosage, replacement of hCG and in vitro maturation of oocytes (IVM). Among the many prevention strategies that have been discussed, the current evidence points to the replacement of hCG by GnRH agonists in antagonist cycles and the performance of IVM procedures as the safest approaches.

Supplemented tissue culture medium 199 is a better medium for in vitro maturation of oocytes from women with polycystic ovary syndrome women than human tubal fluid

Objective: To compare oocyte maturation, fertilization and cleavage rates, and embryonic developmental quality after culture of human immature oocytes from polycystic ovary syndrome (PCOS) patients in human tubal fluid (HTF) or tissue culture medium (TCM) 199. Design: Prospective, randomized, controlled trial. Setting: University hospital. Patient(s): Thirteen women undergoing 23 in vitro maturation cycles, from whom 119 oocytes were retrieved. Intervention(s): Cumulus-enclosed germinal vesicle-stage oocytes matured in TCM-199-supplemented or HTF-supplemented media. Main Outcome Measure(s): Oocyte maturation and fertilization rates, embryonic developmental quality. Result(s): Significant differences were observed between TCM 199 and HTF regarding maturation rate (82% vs. 56.9%), fertilization rate (70% vs. 39.4%), and embryo quality (81.3% vs. 41.7%). Conclusion(S): Human tubal fluid medium, although widely used for embryo fertilization and maintenance in IVF techniques, is not,in appropriate medium for the maturation of oocytes obtained from PCOS patients in nonstimulated cycles. (Fertil Steril(R) 2009;91:509-13. (C)2009 by American Society for Reproductive Medicine.)

Effect of image analysis software on neurofunctional activation during processing of emotional human faces

Functional brain imaging techniques such as functional MRI (fMRI) that allow the in vivo investigation of the human brain have been exponentially employed to address the neurophysiological substrates of emotional processing. Despite the growing number of fMRI studies in the field, when taken separately these individual imaging studies demonstrate contrasting findings and variable pictures, and are unable to definitively characterize the neural networks underlying each specific emotional condition. Different imaging packages, as well as the statistical approaches for image processing and analysis, probably have a detrimental role by increasing the heterogeneity of findings. In particular, it is unclear to what extent the observed neurofunctional response of the brain cortex during emotional processing depends on the fMRI package used in the analysis. In this pilot study, we performed a double analysis of an fMRI dataset using emotional faces. The Statistical Parametric Mapping (SPM) version 2.6 (Wellcome Department of Cognitive Neurology, London, UK) and the XBAM 3.4 (Brain Imaging Analysis Unit, Institute of Psychiatry, Kings College London, UK) programs, which use parametric and non-parametric analysis, respectively, were used to assess our results. Both packages revealed that processing of emotional faces was associated with an increased activation in the brain`s visual areas (occipital, fusiform and lingual gyri), in the cerebellum, in the parietal cortex, in the cingulate cortex (anterior and posterior cingulate), and in the dorsolateral and ventrolateral prefrontal cortex. However, blood oxygenation level-dependent (BOLD) response in the temporal regions, insula and putamen was evident in the XBAM analysis but not in the SPM analysis. Overall, SPM and XBAM analyses revealed comparable whole-group brain responses. Further Studies are needed to explore the between-group compatibility of the different imaging packages in other cognitive and emotional processing domains. (C) 2009 Elsevier Ltd. All rights reserved.