Lactobacillus-dominated cervicovaginal microbiota associated with reduced HIV/STI prevalence and genital HIV viral load in African women

Cervicovaginal microbiota not dominated by lactobacilli may facilitate transmission of HIV and other sexually transmitted infections (STIs), as well as miscarriages, preterm births and sepsis in pregnant women. However, little is known about the exact nature of the microbiological changes that cause these adverse outcomes. In this study, cervical samples of 174 Rwandan female sex workers were analyzed cross-sectionally using a phylogenetic microarray. Furthermore, HIV-1 RNA concentrations were measured in cervicovaginal lavages of 58 HIV-positive women among them. We identified six microbiome clusters, representing a gradient from low semi-quantitative abundance and diversity dominated by Lactobacillus crispatus (cluster R-I, with R denoting ‘Rwanda'') and L. iners (R-II) to intermediate (R-V) and high abundance and diversity (R-III, R-IV and R-VI) dominated by a mixture of anaerobes, including Gardnerella, Atopobium and Prevotella species. Women in cluster R-I were less likely to have HIV (P=0.03), herpes simplex virus type 2 (HSV-2; P<0.01), and high-risk human papillomavirus (HPV; P<0.01) and had no bacterial STIs (P=0.15). Statistically significant trends in prevalence of viral STIs were found from low prevalence in cluster R-I, to higher prevalence in clusters R-II and R-V, and highest prevalence in clusters R-III/R-IV/R-VI. Furthermore, only 10% of HIV-positive women in clusters R-I/R-II, compared with 40% in cluster R-V, and 42% in clusters R-III/R-IV/R-VI had detectable cervicovaginal HIV-1 RNA (P_trend=0.03). We conclude that L. crispatus-dominated, and to a lesser extent L. iners-dominated, cervicovaginal microbiota are associated with a lower prevalence of HIV/STIs and a lower likelihood of genital HIV-1 RNA shedding.     

Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagasdisease and the proximity of parasitised glial cells and neurons in damaged myentericganglia is a frequent finding. Glial cells have crucial roles in manyneuropathological situations and are potential sources of NO. Here, we investigateperipheral glial cell response to Trypanosoma cruzi infection toclarify the role of these cells in the neuronal lesion pathogenesis of Chagasdisease. We used primary glial cell cultures from superior cervical ganglion toinvestigate cell activation and NO production after T. cruziinfection or lipopolysaccharide (LPS) exposure in comparison to peritonealmacrophages. T. cruzi infection was greater in glial cells, despitesimilar levels of NO production in both cell types. Glial cells responded similarlyto T. cruzi and LPS, but were less responsive to LPS thanmacrophages were. Our observations contribute to the understanding of Chagas diseasepathogenesis, as based on the high susceptibility of autonomic glial cells toT. cruzi infection with subsequent NO production. Moreover, our findingswill facilitate future research into the immune responses and activation mechanismsof peripheral glial cells, which are important for understanding the paradoxicalresponses of this cell type in neuronal lesions and neuroprotection.     

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.     

An A alpha Ser-434 to N-glycosylated Asn substitution in a dysfibrinogen, fibrinogen Caracas II, characterized by impaired fibrin gel formation.

We have identified a unique N-glycosylated Asn substitution for a Ser at position 434 of the A alpha chain of an abnormal fibrinogen designated fibrinogen Caracas II. This dysfibrinogen was characterized by impaired fibrin monomer aggregation. Since there were 4 Thr residues immediately following the mutation, a new Asn-X-Thr/Ser-type consensus sequence, Asn-Thr-Thr arose for N-glycosylation of the Asn. The extra oligosaccharide was found to consist mainly of a disialylated biantennary structure comprising 81.9%, while a neutral and a monosialylated biantennary oligosaccharide represented only 3.6% and 14.5%, respectively. The mutation resides in the carboxyl-terminal region of the A alpha chain, which could fold back to form an extra small globular region located near the central region of the molecule (Erickson, H.P., and Fowler, W.E. (1983) Ann. N. Y. Acad. Sci. 408, 146-163; Weisel, H.P., Stauffacher, C.V., Bullitt, E., and Cohen, C. (1985) Science 230, 3124-3133). Therefore, the participation of this region, referred to as an additional central domain or an alpha domain, in fibrin gel formation is strongly implicated.     

Structural and Functional Properties of Glycerol-3-Phosphate Dehydrogenase from a Mammalian Hibernator

Glycerol-3-phosphate dehydrogenase (G3PDH; E.C.1.1.1.8) was purified from liver and skeletal muscle of black-tailed prairie dogs (Cynomys ludivicianus), a hibernating species. Native and subunit molecular masses of the dimeric enzyme were 77 and 40 kD, respectively, and both tissues contained a single isozyme with a pI of 6.4. Kinetic parameters of purified G3PDH from prairie dog liver and muscle were characterized at 22 and 5 °C and compared with rabbit muscle G3PDH. Substrate affinities for hibernator muscle G3PDH were stable (NAD) or increased significantly (K_m G3P and DHAP decreased) at low temperature whereas K_m NAD and DHAP of rabbit G3PDH increased. Prairie dog G3PDH showed greater conservation of K_m G3P over a wide temperature range as well as greater thermal stability and resistance to chemical denaturation by guanidine hydrochloride than the rabbit enzyme. In addition, using the protein sequence of the hibernating thirteen-lined ground squirrel (Ictidomys tridecemlineatus) and bioinformatics tools, the deduced protein structure of G3PDH was compared between heterothermic and homeothermic mammals. Structural and functional characteristics of G3PDH from the hibernating species would support enzyme function over a wide range of core body temperatures over cycles of torpor and arousal.     

The effect of administering equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) post artificial insemination on fertility of lactating dairy cows

The objective was to evaluate the effect of equine chorionic gonadotropin (eCG) and hCG post artificial insemination (AI) on fertility of lactating dairy cows. In Experiment 1, cows were either treated with eCG on Day 22 post AI (400 IU; n = 80) or left untreated (n = 84). On Day 29, pregnant cows were either treated with hCG (2500 IU; n = 32) or left untreated (n = 36). Pregnancy and progesterone were evaluated on Days 29 and 45. In Experiment 2, cows (n = 28) were either treated with eCG on Day 22 (n = 13) or left untreated (n = 15) and either treated with hCG on Day 29 (n = 14) or left untreated (n = 14). Blood sampling and ultrasonography were conducted between Days 22 and 45. In Experiment 3, cows were either treated with eCG on Day 22 post AI (n = 229) or left untreated (n = 241). Pregnancy was evaluated on Days 36 and 85. In Experiment 1, eCG on Day 22 increased (P < 0.02) the number of pregnant cows on Day 29 (50.0 vs. 33.3%) and on Day 45, the increase was higher (P < 0.01) in cows with timed AI (41.2 vs. 6.5%) than in cows AI at detected estrus (50.0 vs. 37.8%). Pregnancy losses were reduced by eCG and hCG, but increased in cows that did not receive eCG but were given hCG (P < 0.01). Treatment with hCG tended (P < 0.06) to increase progesterone in control cows, but not in cows treated with eCG. In Experiment 2, hCG increased (P < 0.01) the number of accessory CLs on Day 35 (28.5 vs. 0.0%) and tended (P < 0.07) to increase progesterone. In Experiment 3, eCG increased the number of pregnant cows (P < 0.05) on Days 36 and 85, but only in cows with low body condition (eCG = 45.6 and 43.5%; Control = 22.9 and 22.9%). In conclusion, eCG at 22 days post insemination increased fertility, primarily in cows with low body condition and reduced pregnancy losses when given 7 days before hCG; hCG induced accessory CLs and slightly increased progesterone, but hCG given in the absence of a prior eCG treatment reduced fertility.     

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation

The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.     

Glucocorticoid alternative effects on proliferating and differentiated mammary epithelium are associated to opposite regulation of cell-cycle inhibitor expression

Glucocorticoids influence post-natal mammary gland development by sequentially controlling cell proliferation, differentiation, and apoptosis. In the mammary gland, it has been demonstrated that glucocorticoid treatment inhibits epithelial apoptosis in post-lactating glands. In this study, our first goal was to identify new glucocorticoid target genes that could be involved in generating this effect. Expression profiling, by microarray analysis, revealed that expression of several cell-cycle control genes was altered by dexamethasone (DEX) treatment after lactation. Importantly, it was determined that not only the exogenous synthetic hormone, but also the endogenous glucocorticoids regulated the expression of these genes. Particularly, we found that the expression of cell cycle inhibitors p21CIP1, p18INK4c, and Atm was differentially regulated by glucocorticoids through the successive stages of mammary gland development. In undifferentiated cells, DEX treatment induced their expression and reduced cell proliferation, while in differentiated cells this hormone repressed expression of those cell cycle inhibitors and promoted survival. Therefore, differentiation status determined the effect of glucocorticoids on mammary cell fate. Particularly, we have determined that p21CIP1 inhibition would mediate the activity of these hormones in differentiated mammary cells because over-expression of this protein blocked DEX-induced apoptosis protection. Together, our data suggest that the multiple roles played by glucocorticoids in mammary gland development and function might be at least partially due to the alternative roles that these hormones play on the expression of cell cycle regulators.     

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.     

Protein disulfide isomerase in redox cell signaling and homeostasis

Thiol proteins may potentially act as redox signaling adaptor proteins, adjusting reactive oxygen species intermediates to specific signals and redox signals to cell homeostasis. In this review, we discuss redox effects of protein disulfide isomerase (PDI), a thioredoxin superfamily oxidoreductase from the endoplasmic reticulum (ER). Abundantly expressed PDI displays ubiquity, interactions with redox and nonredox proteins, versatile effects, and several posttranslational modifications. The PDI family contains >20 members with at least some apparent complementary actions. PDI has oxidoreductase, isomerase, and chaperone effects, the last not directly dependent on its thiols. PDI is a converging hub for pathways of disulfide bond introduction into ER-processed proteins, via hydrogen peroxide-generating mechanisms involving the oxidase Ero1α, as well as hydrogen peroxide-consuming reactions involving peroxiredoxin IV and the novel peroxidases Gpx7/8. PDI is a candidate pathway for coupling ER stress to oxidant generation. Emerging information suggests a convergence between PDI and Nox family NADPH oxidases. PDI silencing prevents Nox responses to angiotensin II and inhibits Akt phosphorylation in vascular cells and parasite phagocytosis in macrophages. PDI overexpression spontaneously enhances Nox activation and expression. In neutrophils, PDI redox-dependently associates with p47phox and supports the respiratory burst. At the cell surface, PDI exerts transnitrosation, thiol reductase, and apparent isomerase activities toward targets including adhesion and matrix proteins and proteases. Such effects mediate redox-dependent adhesion, coagulation/thrombosis, immune functions, and virus internalization. The route of PDI externalization remains elusive. Such multiple redox effects of PDI may contribute to its conspicuous expression and functional role in disease, rendering PDI family members putative redox cell signaling adaptors.