Sexual Dimorphisms of Adrenal Steroids, Sex Hormones, and Immunological Biomarkers and Possible Risk Factors for Developing Rheumatoid Arthritis.

Innate immunity and immunological biomarkers are believed to be interrelated with sex hormones and other neuroendocrine factors. Sexual dimorphism mechanisms may be operating in certain rheumatic and inflammatory diseases which occur more frequently in women than men, as rheumatoid arthritis (RA). Less data have been available on altered interrelations of the combined neuroendocrine and immune (NEI) systems as risk factors for development of certain diseases. In this study, serological interrelations of NEI biomarkers are analyzed before symptomatic onset of RA (pre-RA) versus control (CN) subjects, stratified by sex. Sexual dimorphism was found in serum levels of acute serum amyloid A (ASAA), soluble interleukin-2 receptor alpha (sIL-2Rα), and soluble tumor necrosis factor receptor 1 (sTNF-R1). Multiple steroidal and hormonal (neuroendocrine) factors also showed highly (p < 0.001) significant sexual dimorphism in their assayed values, but less for cortisol (p = 0.012), and not for 17-hydroxyprogesterone (p = 0.176). After stratification by sex and risk of developing RA, differential NEI correlational patterns were observed in the interplay of the NEI systems between the pre-RA and CN groups, which deserve further investigation.

Automated computational screening of the thiol reactivity of substituted alkenes.

Electrophilic olefins can react with the S-H moiety of cysteine side chains. The formation of a covalent adduct through this mechanism can result in the inhibition of an enzyme. The reactivity of an olefin towards cysteine depends on its functional groups. In this study, 325 reactions of thiol-Michael-type additions to olefins were modeled using density functional theory. All combinations of ethenes with hydrogen, methyl ester, amide, and cyano substituents were included. An automated workflow was developed to perform the construction, conformation search, minimization, and calculation of molecular properties for the reactant, carbanion intermediate, and thioether products for a model reaction of the addition of methanethiol to the electrophile. Known cysteine-reactive electrophiles present in the database were predicted to react exergonically with methanethiol through a carbanion with a stability in the 30-40 kcal mol(-1) range. 13 other compounds in our database that are also present in the PubChem database have similar properties. Natural bond orbital parameters were computed and regression analysis was used to determine the relationship between properties of the olefin electronic structure and the product and intermediate stability. The stability of the intermediates is very sensitive to electronic effects on the carbon where the anionic charge is centered. The stability of the products is more sensitive to steric factors.

The role of host gender in the pathogenesis of Cryptococcus neoformans infections.

Cryptococcus neoformans (Cn) is a pathogenic yeast and the cause of cryptococcal meningitis. Prevalence of disease between males and females is skewed, with males having an increased incidence of disease. Based on the reported gender susceptibility differences to Cn in the literature, we used clinical isolates from Botswanan HIV-infected patients to test the hypothesis that different gender environments exerted different selective pressures on Cn. When we examined this data set, we found that men had significantly higher risk of death despite having significantly higher CD4(+) T lymphocyte counts upon admittance to the hospital. These observations suggested that Cn strains are uniquely adapted to different host gender environments and that the male immune response may be less efficient in controlling Cn infection. To discriminate between these possibilities, we tested whether there were phenotypic differences between strains isolated from males and females and whether there was an interaction between Cn and the host immune response. Virulence phenotypes showed that Cn isolates from females had longer doubling times and released more capsular glucoronoxylomannan (GXM). The presence of testosterone but not 17-ß estradiol was associated with higher levels of GXM release for a laboratory strain and 28 clinical isolates. We also measured phagocytic efficiency, survival of Cn, and amount of killing of human macrophages by Cn after incubation with four isolates. While macrophages from females phagocytosed more Cn than macrophages from males, male macrophages had a higher fungal burden and showed increased killing by Cn. These data are consistent with the hypothesis that differential interaction between Cn and macrophages within different gender environments contribute to the increased prevalence of cryptococcosis in males. This could be related to differential expression of cryptococcal virulence genes and capsule metabolism, changes in Cn phagocytosis and increased death of Cn-infected macrophages.

Range-Separated DFT Functionals are Necessary to Model Thio-Michael Additions.

The textbook mechanism for the addition of a thiol to an olefin is the Michael-type addition, which involves a nucleophilic attack of a thiolate anion on an alkene to form a carbanion intermediate. Previous computational models of these reactions have proposed alternative mechanisms, as no minimum corresponding to the carbanion intermediate was present on the potential energy surface. We show that many popular pure and hybrid DFT functionals, such as PBE and B3LYP, erroneously predict that the carbanion is not an intermediate, favoring a noncovalent charge-transfer complex stabilized spuriously by delocalization error. Range-separated DFT functionals correct this problem and predict stable carbanion structures and energies. In particular, calculations using the ωB97X-D functional are in close agreement with CCSD(T) data for the structures and energies of a series of thio-carbanions. Range-separated functionals will make it possible to model the reaction mechanisms of Michael-type additions that occur in biochemistry, such as the covalent modification of a cysteine side chain by drugs containing an electrophilic double bond.

Pathogen recognition in the human female reproductive tract: expression of intracellular cytosolic sensors NOD1, NOD2, RIG-1, and MDA5 and response to HIV-1 and Neisseria gonorrhea.

PROBLEM: Expression patterns and regulation of cytosolic pattern recognition receptors (PRR) NOD-1, NOD-2, RIG-1, and MDA5 have not been elucidated in the human female reproductive tract (FRT). METHOD OF STUDY: Primary epithelial cells (EC) isolated from Fallopian tube (FT), endometrium (EM), cervix (Cx), and ectocervix (Ecx) were treated with estradiol, poly(I:C), Neisseria gonorrhea (GC), and HIV-1. PRR mRNA expressions were analyzed by Real-time RT-PCR. Conditioned media were analyzed for IL-8 by ELISA. RESULTS: EC from all FRT compartments constitutively expressed NOD1, NOD2, RIG-1, and MDA5 with highest levels expressed by FT. Stimulation with poly(I:C) resulted in upregulation of NOD2, RIG-1, and MDA5 in all FRT compartments and correlated with increased secretion of IL-8, whereas estradiol treatment had no effects. Exposure to GC and HIV-1 IIIB but not BaL resulted in selective upregulation of NOD2 and MDA5. CONCLUSION: PRR are expressed throughout the FRT and differentially regulated by poly(I:C), GC and HIV-1.

Decreased transcription-coupled nucleotide excision repair capacity is associated with increased p53- and MLH1-independent apoptosis in response to cisplatin.

BACKGROUND: One of the most commonly used classes of anti-cancer drugs presently in clinical practice is the platinum-based drugs, including cisplatin. The efficacy of cisplatin therapy is often limited by the emergence of resistant tumours following treatment. Cisplatin resistance is multi-factorial but can be associated with increased DNA repair capacity, mutations in p53 or loss of DNA mismatch repair capacity. METHODS: RNA interference (RNAi) was used to reduce the transcription-coupled nucleotide excision repair (TC-NER) capacity of several prostate and colorectal carcinoma cell lines with specific defects in p53 and/or DNA mismatch repair. The effect of small inhibitory RNAs designed to target the CSB (Cockayne syndrome group B) transcript on TC-NER and the sensitivity of cells to cisplatin-induced apoptosis was determined. RESULTS: These prostate and colon cancer cell lines were initially TC-NER proficient and RNAi against CSB significantly reduced their DNA repair capacity. Decreased TC-NER capacity was associated with an increase in the sensitivity of tumour cells to cisplatin-induced apoptosis, even in p53 null and DNA mismatch repair-deficient cell lines. CONCLUSION: The present work indicates that CSB and TC-NER play a prominent role in determining the sensitivity of tumour cells to cisplatin even in the absence of p53 and DNA mismatch repair. These results further suggest that CSB represents a potential target for cancer therapy that may be important to overcome resistance to cisplatin in the clinic.

The contribution of transactivation subdomains 1 and 2 to p53-induced gene expression is heterogeneous but not subdomain-specific.

Two adjacent regions within the transactivation domain of p53 are sufficient to support sequence-specific transactivation when fused to a heterologous DNA binding domain. It has been hypothesized that these two subdomains of p53 may contribute to the expression of distinct p53-responsive genes. Here we have used oligonucleotide microarrays to identify transcripts induced by variants of p53 with point mutations within subdomains 1, 2, or 1 and 2 (QS1, QS2, and QS1/QS2, respectively). The expression of 254 transcripts was increased in response to wild-type p53 expression but most of these transcripts were poorly induced by these variants of p53. Strikingly, a number of known p53-regulated transcripts including TNFRSF10B, BAX, BTG2, and POLH were increased to wild-type levels by p53(QS1) and p53(QS2) but not p53(QS1/QS2), indicating that either subdomain 1 or 2 is sufficient for p53-dependent expression of a small subset of p53-responsive genes. Unexpectedly, there was no evidence for p53(QS1)- or p53(QS2)-specific gene expression. Taken together, we found heterogeneity in the requirement for transactivation subdomains 1 and 2 of p53 without any subdomain-specific contribution to p53-induced gene expression.

DDB2-independent role for p53 in the recovery from ultraviolet light-induced replication arrest.

Ultraviolet light (UV light) induces helix distorting DNA lesions that pose a block to replicative DNA polymerases. Recovery from this replication arrest is reportedly impaired in nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) fibroblasts and primary fibroblasts lacking functional p53. These independent observations suggested that the involvement of p53 in the recovery from UV-induced replication arrest was related to its role in regulating the global genomic subpathway of NER (GG-NER). Using primary human fibroblasts, we confirm that the recovery from UV-induced replication arrest is impaired in cells lacking functional p53 and in primary XP fibroblasts derived from complementation groups A or C (XP-A and XP-C) that are defective in GG-NER. Surprisingly, DNA synthesis recovered normally in GG-NER-deficient XP complementation group E (XP-E) cells that carry mutations in the p53 regulated DNA repair gene DDB2 and are specifically defective in the repair of cyclobutane pyrimidine dimers (CPD) but not pyrimidine (6-4) pyrimidone photoproducts. Disruption of p53 in these XP-E fibroblasts prevented the recovery from UV-induced replication arrest. Therefore, the roles of p53 and GG-NER in the recovery from UV-induced replication are separable and DDB2-independent. These results further indicate that primary human fibroblasts expressing functional p53 efficiently replicate DNA containing CPD whereas p53-deficient cells do not, consistent with a role for p53 in permitting translesion synthesis of these DNA lesions.

Unique characteristics of NK cells throughout the human female reproductive tract.

In this study, we have analyzed the presence and subsets of NK cells throughout the tissues of the FRT. We demonstrate that there are NK cells in the various FRT tissues and that their phenotype and regulation are largely dependent upon the FRT tissue where they reside. NK cells in the Fallopian tube, endometrium, cervix, and ectocervix expressed CD9 while blood NK cells did not. We have also found that unique subsets of NK cells are in specific locations of the FRT. The NK cells in the lower reproductive tract did not express CD94, but they did express CD16. In contrast, NK cells in the upper FRT express high amounts of CD94 and CD69, but few NK cells expressed CD16. All of these FRT NK cells were able to produce IFN-gamma upon stimulation with cytokines. Furthermore, the number of NK cells varied with the menstrual cycle in the endometrium but not in the cervix or ectocervix. These data suggest that unique characteristics of the tissues may account of specific localization of different NK cell subsets.

Differential regulation of neutrophil chemotaxis to IL-8 and fMLP by GM-CSF: lack of direct effect of oestradiol.

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.

CXCL12 activation of CXCR4 regulates mucosal host defense through stimulation of epithelial cell migration and promotion of intestinal barrier integrity.

Intestinal epithelial cell migration plays a key role in gastrointestinal mucosal barrier formation, enterocyte development, differentiation, turnover, wound healing, and adenocarcinoma metastasis. Chemokines, through engagement of their corresponding receptors, are potent mediators of directed cell migration and are critical in the establishment and regulation of innate and adaptive immune responses. The aim of this study was to define the role for the chemokine CXCL12 and its sole cognate receptor CXCR4 in regulating intestinal epithelial cell migration and to determine its impact on barrier integrity. CXCL12 stimulated the dose-dependent chemotactic migration of human T84 colonic epithelial cells. Epithelial cell migration was inhibited by CXCR4 neutralizing antibody, pertussis toxin, LY-294002, and PD-98059, thereby implicating Galpha(i), phosphatidylinositol 3-kinase (PI3-kinase), and the ERK1/2 MAP kinase pathways in CXCR4-specific signaling. CXCL12 was also shown to increase barrier integrity, as defined by transepithelial resistance and paracellular flux across differentiating T84 monolayers. To determine whether CXCL12 regulated epithelial restitution, we used the normal nontransformed intestinal epithelial cell-6 (IEC-6) wound healing model. By using RT-PCR, immunoblot analysis, and immunofluorescence microscopy, we first showed expression of both CXCR4 and its ligand by IEC-6 cells. We then demonstrated that CXCL12 activated comparable signaling mechanisms to stimulate epithelial migration in the absence of proliferation in wounded IEC-6 monolayers. Taken together, these data indicate that CXCL12 signaling via CXCR4 directs intestinal epithelial cell migration, barrier maturation, and restitution, consistent with an important mechanistic role for these molecules in mucosal barrier integrity and innate host defense.

Involvement of protein kinase C in chitosan glutamate-mediated tight junction disruption.

Chitosan has been successfully used as an excipient for trans-epithelial drug delivery systems. It is known to transiently open intercellular tight junctions thus increasing the permeability of an epithelium. In order to investigate the possible role of protein kinases in trans-epithelial delivery, changes in trans-epithelial electrical resistance ('TEER') of epithelial (Caco-2) cell monolayers were assessed in response to chitosan glutamate treatment, in the presence and absence of specific protein kinase inhibitors. Changes in subcellular localisation of the tight junction protein ZO-1 observed by immunofluorescence and western blotting of cellular fractions were also assessed. Inhibition of protein kinase C (PKC), but not mitogen activated protein kinase (MAPK) was found to prevent the chitosan-mediated decrease in TEER, and changes in localisation of ZO-1. In order to determine which PKC isozymes were responsible for the chitosan-mediated tight junction disruption, the activation of the PKC isozymes alpha, beta and delta was investigated. A chitosan-mediated translocation of PKC alpha but not PKC beta or delta from the cytosol to the membrane fraction, indicative of PKC alpha activation was observed. Thus, treatment of Caco-2 cells with chitosan may result in the activation of PKC-dependent signal transduction pathways which affect tight junction integrity.

Regulation of ultraviolet light-induced gene expression by gene size.

UV light induces the expression of a wide variety of genes. At present, it is unclear how cells sense the extent of DNA damage and alter the expression of UV-induced genes appropriately. UV light induces DNA damage that blocks transcription, and the probability that a gene sustains transcription-blocking DNA damage is proportional to locus size and dose of UV light. Using colon carcinoma cells that express a temperature-sensitive variant of p53 and undergo p53-dependent apoptosis after UV irradiation, we found that the number of p53-induced genes identified by oligonucleotide microarray analysis decreased in a UV dose-dependent manner. This was associated with a statistically significant shift in the spectrum of p53-induced genes toward compact genes with fewer and smaller introns. Genes encoding proapoptotic proteins involved in the initiation of the mitochondrial apoptotic cascade were prominent among the compact p53 target genes, whereas genes encoding negative regulators of p53 and the mitochondrial apoptotic pathway were significantly larger. We propose that the shift in spectrum of UV-responsive gene expression caused by passive effects of UV lesions on transcription acts as a molecular dosimeter, ensuring the elimination of cells sustaining irreparable transcription-blocking DNA damage.

Mosquitoes infected with West Nile virus in the Florida Keys, Monroe County, Florida, USA.

More than 30,000 mosquitoes in 22 species or species groups were collected from the Florida Keys, Monroe County, FL, USA, in dry ice-baited light and gravid traps. Dry ice-baited traps collected more mosquitoes than did gravid traps. West Nile virus was detected in pools of Anopheles atropos Dyar & Knab, Deinocerites cancer Theobald, and Ochlerotatus taeniorhynchus (Wiedemann).