1142280946Effect of estradiol and PAMPs on class II mediated antigen presentation and immunomodulatory molecule expression in the mouse female reproductive tract

Problem:  Antigen presenting cells (APC) in the female reproductive tract play important roles in innate immune defense and activation of the adaptive immune responses. The objective of this study was to examine the effects of estradiol and PAMP on antigen presentation in the female reproductive tract.
Method of Study:  DO11.10 T cell antigen receptor transgenic mice specific for the MHC class II-restricted OVA323–339peptide were used to study the effects of estradiol and PAMP on antigen presentation of OVA by uterine epithelial (EC) and stromal cells as well as vaginal cells to OVA specific memory-T cells.
Results:  Estradiol inhibited antigen presentation of OVA by uterine EC, uterine stromal cells and vaginal cells to OVA specific memory-T cells. When ovariectomized animals were treated with estradiol for 1 or 3 days, antigen presentation decreased by 20–80%. In contrast, incubation with TLR agonists increased antigen presentation by EC (Poly (I:C), Pam3Cys), stromal cells (PGN, Pam3Cys) and vaginal cells (LPS, Pam3Cys). Analysis of mRNA expression by real time RT-PCR indicated that estradiol inhibited CD40, CD80/86 and class II in the uterus and vagina. In contrast, stimulation of antigen presentation by PAMP did not correlate with changes in costimulatory molecule mRNA expression.
Conclusions:  These results indicate that APC in the uterus and vagina are responsive to estradiol, which inhibits antigen presentation and costimulatory molecule expression. These findings suggest that whereas APC in the uterus and vagina respond to TLR agonists with increased antigen presentation, which initiates an adaptive immune response, their effects appear to be at levels other than the expression of costimulatory molecules.
Acknowledgement:  Supported by AI-13541 from NIH.     

Reliable confirmation and quantitation of human immunodeficiency virus type 1 antibody using a recombinant-antigen immunoblot assay

The recombinant DNA-derived, human immunodeficiency virus (HIV) antigen-based immunoblot assay (RIBA-HIV216) is a new supplemental (confirmatory) test developed to detect antibodies to HIV-1. The assay employs four recombinant viral antigens, corresponding to HIV-1 p24, p31, p41 and gp120 proteins, in an immunoblot format. With this assay, HIV-1 antigen reactivity was detected in all 683 infected patient serum or plasma specimens evaluated; 665 (97.6%) of these sera met the criteria for a positive interpretation, and 18 (2.6%) were classified as indeterminate. All 683 samples reacted with the recombinant gp41-equivalent protein. The first sequential enzyme immunoassay (EIA)-reactive samples collected from 33 seroconverting homosexual men reacted on RIBA-HIV216. Eleven (1.1%) of 999 EIA-negative blood donor sera reacted weakly with a single recombinant antigen (p24 or p31), whereas 13 to 48 percent had indeterminate reactions on viral lysate Western blots. One (1.5%) of 66 EIA-positive, Western blot-negative blood donor samples and 19 (29%) of 66 EIA-positive, Western blot-indeterminate blood donor samples scored indeterminate on RIBA-HIV216. Nonspecific reactivity was seen with only 1 (0.8%) of 114 patient sera containing possible interfering antibodies, whereas 33 percent of these samples had indeterminate reactions on Western blot and 35 percent had equivocal reactions on immunofluorescence assay (IFA). We conclude that the RIBA-HIV216 is approximately as sensitive as and significantly more specific than virus-derived Western blot and IFA. The RIBA-HIV216 also allows for semiquantitation of specific antibodies that may be of value in clinical staging and therapeutic monitoring.     

An unusual pattern of mutation in the duplicated portion of PKD1 is revealed by use of a novel strategy for mutation detection

The gene for the most common and severe form of autosomal dominant polycystic kidney disease, PKD1, encodes a 14 kb mRNA that is predicted to result in an integral membrane protein of 4302 amino acids. The major challenge faced by researchers attempting to complete mutation analysis of the PKD1 gene has been the presence of several homologous loci also located on chromosome 16. Because the sequence of PKD1 and its homologs is nearly identical in the 5' region of the gene, most traditional approaches to mutation analysis cannot distinguish sequence variants occurring uniquely in PKD1. Therefore, only a small number of mutations have been identified to date and these have all been found in the 3', unique portion of the gene. In order to begin analysis of the duplicated region of PKD1, we have devised a novel strategy that depends on long-range PCR and a single gene-specific primer from the unique region of the gene to amplify a PKD1-specific template that spans exons 23-34. This 10 kb template, amplified from genomic DNA, can be employed for mutation analysis using a wide variety of sequence- based approaches. We have used our long-range PCR strategy to begin screening for sequence variants with heteroduplex analysis, and several affected individuals were discovered to have clusters of base pair substitutions in exons 23 and 25. In two patients, these changes, identified in exon 23, would be predicted to result in multiple amino acid substitutions in a short stretch of the protein. This clustering of base pair substitutions is unusual and suggests that mutation may result from unique structural features of the PKD1 gene.      

Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanding CAG repeat coding for polyglutamine in the huntingtin protein. Recent data have suggested the possibility that an N-terminal fragment of huntingtin may aggregate in neurons of patients with HD, both in the cytoplasm, forming dystrophic neurites, and in the nucleus, forming intranuclear neuronal inclusion bodies. An animal model of HD using the short N-terminal fragment of huntingtin has also been found to have intranuclear inclusions and this same fragment can aggregate in vitro . We have now developed a cell culture model demonstrating that N-terminal fragments of huntingtin with expanded glutamine repeats aggregate both in the cytoplasm and in the nucleus. Neuroblastoma cells transiently transfected with full-length huntingtin constructs with either a normal or expanded repeat had diffuse cytoplasmic localization of the protein. In contrast, cells transfected with truncated N-terminal fragments showed aggregation only if the glutamine repeat was expanded. The aggregates were often ubiquitinated. The shorter truncated product appeared to form more aggregates in the nucleus. Cells transfected with the expanded repeat construct but not the normal repeat construct showed enhanced toxicity to the apoptosis- inducing agent staurosporine. These data indicate that N-terminal truncated fragments of huntingtin with expanded glutamine repeats can aggregate in cells in culture and that this aggregation can be toxic to cells. This model will be useful for future experiments to test mechanisms of aggregation and toxicity and potentially for testing experimental therapeutic interventions.      

Silencing women’s sexuality: global AIDS policies and the case of the female condom

Introduction

The female condom is the only evidence-based AIDS prevention technology that has been designed for the female body; yet, most women do not have access to it. This is remarkable since women constitute the majority of all HIV-positive people living in sub-Saharan Africa, and gender inequality is seen as a driving force of the AIDS epidemic. In this study, we analyze how major actors in the AIDS prevention field frame the AIDS problem, in particular the female condom in comparison to other prevention technologies, in their discourse and policy formulations. Our aim is to gain insight into the discursive power mechanisms that underlie the thinking about AIDS prevention and women’s sexual agency.

Methods

We analyze the AIDS policies of 16 agencies that constitute the most influential actors in the global response to AIDS. Our study unravels the discursive power of these global AIDS policy actors, when promoting and making choices between AIDS prevention technologies. We conducted both a quantitative and qualitative analysis of how the global AIDS epidemic is being addressed by them, in framing the AIDS problem, labelling of different categories of people for targeting AIDS prevention programmes and in gender marking of AIDS prevention technologies.

Results

We found that global AIDS policy actors frame the AIDS problem predominantly in the context of gender and reproductive health, rather than that of sexuality and sexual rights. Men’s sexual agency is treated differently from women’s sexual agency. An example of such differentiation and of gender marking is shown by contrasting the framing and labelling of male circumcision as an intervention aimed at the prevention of HIV with that of the female condom.

Conclusions

The gender-stereotyped global AIDS policy discourse negates women’s agency in sexuality and their sexual rights. This could be an important factor in limiting the scale-up of female condom programmes and hampering universal access to female condoms.     

IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF GROWTH FACTORS AND OTHER MARKERS IN HUMAN LONG－TERM BONE MARROW CULTURES

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A novel missense mutation in exon 4 of the factor VIII:C gene resulting in moderately severe hemophilia A

A new point mutation due to C----T transition at codon 189 (TCA) of the factor VIII:C gene was found in a Chinese patient with moderately severe hemophilia A. This mutation abolishes the EcoRI site (GAATTC) in exon 4 and can be directly detected by polyacrylamide gel electrophoresis of amplified genomic DNA.     

The C5a receptor antagonist PMX205 ameliorates experimentally induced colitis associated with increased IL-4 and IL-10

Background and Purpose

Anti-complement therapies have not been advanced for treating the inflammatory bowel diseases (IBDs) despite a growing body of evidence that blocking C5a protects against induced colitis in rodents. The purpose of this study was to further build on this evidence by examining the efficacy, mechanism and specificity of a potent, non-competitive and orally active C5a receptor (CD88) antagonist, PMX205, in the dextran sulphate sodium (DSS) model of murine innate colitis.

Experimental Approach

Mice with DSS added to their drinking water were orally administered 100 or 200 μg day⁻¹ PMX205 in prophylactic and therapeutic regimens. Clinical illness, colon histology and local generation of inflammatory mediators were measured to evaluate the impact of PMX205 on disease.

Key Results

PMX205 significantly prevented DSS-induced colon inflammation in both regimens, associated with lower pro-inflammatory cytokine production and nitrotyrosine staining in colon sections. Additionally, the levels of anti-inflammatory cytokines IL-4 and IL-10 were increased. PMX205 had no significant effect on C5a levels. The beneficial effect of PMX205 was seen in two strains of mice of differing sensitivities to DSS inflammation, but was inactive in mice lacking CD88.

Conclusions and Implications

Pharmacological inhibition of C5a activity by PMX205 is efficacious in preventing DSS-induced colitis, providing further evidence that targeting CD88 in IBD patients could be a valuable therapeutic option.