Sexual Risk Behavior in HIV-Uninfected Pregnant Women in Western Uganda.

Our aim was to identify sexual risk behavior among HIV-negative pregnant women in Kabarole District, Uganda, by conducting a cross-sectional study among 1610 women within three healthcare settings. One in six women engaged in HIV-specific risk behaviors including multiple sexual partners or alcohol abuse; 80% of the pregnant women reported to generally abstain from using condoms. In multivariate analysis, predictors of sexual risk behavior included being a client of the public health facilities as compared to the private facility (AOR 3.6 and 4.8, p < 0.001), being single, widowed or divorced or not cohabiting with the partner (AOR 4.7 and 2.3, p < 0.001), as well as higher household wealth (AOR 1.8, p < 0.001) and lack of partner status knowledge (AOR 1.6, p = 0.008). Self-estimated risk perception was linked with engagement in HIV-related risk behaviors except for alcohol abuse. Our findings indicate that reducing risky behaviors in pregnancy in order to prevent HIV should be a high-priority public health concern.

The incidence of HIV and associated risk factors among pregnant women in Kabarole District, Uganda.

OBJECTIVES: The study attempted to determine the incidence of HIV among pregnant women in Kabarole District, Uganda, and to identify socio-demographic and behavioral risk factors for seroconversion during pregnancy. METHODS: We carried out a retrospective cohort study among women for whom a documented HIV-negative test result from the first pregnancy trimester could be confirmed using available records, and who were HIV-retested in the third trimester or during delivery. In total, 1610 pregnant women from three different healthcare settings took part in the study. We captured the results of repeated HIV tests and conducted semi-structured interviews to explore participants' socio-demographic characteristics and sexual risk behavior. For HIV incidence rates, we calculated the number of seroconversions per 100 person-years. We used Fisher's exact test to test for potential associations. Penalized maximum likelihood logistic regression and Poisson regression were applied to adjust for potential confounders. RESULTS: The overall HIV incidence rate among participants was 2.9/100 women-years. Among socio-demographic characteristics, the multivariable analysis showed a significant association of marital status with HIV incidence in pregnancy (IRR 8.78, 95%CI [1.13-68.33]). Risky sexual behaviors including higher number of sexual partners in pregnancy (IRR 2.78 [1.30-5.94]), unprotected sex with unknown persons (IRR 14.25 [4.52-44.93]), alcohol abuse (IRR 12.08 [4.18-34.90]) and sex under the influence of drugs or alcohol (IRR 6.33 [1.36-29.49]) were significantly associated with seroconversion in pregnancy (similar results in logistic regression). CONCLUSIONS: HIV incidence was three times higher among our pregnant study population compared to the general female population in Uganda. This underlines the importance of HIV prevention and repeat testing during pregnancy. Identified risk groups should be considered for pre-exposure prophylaxis.

Membrane assembly of aquaporin-4 autoantibodies regulates classical complement activation in neuromyelitis optica.

Neuromyelitis optica (NMO) is an autoimmune CNS disorder mediated by pathogenic aquaporin-4 (AQP4) water channel autoantibodies (AQP4-IgG). Although AQP4-IgG-driven complement-dependent cytotoxicity (CDC) is critical for the formation of NMO lesions, the molecular mechanisms governing optimal classical pathway activation are unknown. We investigated the molecular determinants driving CDC in NMO using recombinant AQP4-specific autoantibodies (AQP4 rAbs) derived from affected patients. We identified a group of AQP4 rAbs targeting a distinct extracellular loop C epitope that demonstrated enhanced CDC on target cells. Targeted mutations of AQP4 rAb Fc domains that enhance or diminish C1q binding or antibody Fc-Fc interactions showed that optimal CDC was driven by the assembly of multimeric rAb platforms that increase multivalent C1q binding and facilitate C1q activation. A peptide that blocks antibody Fc-Fc interaction inhibited CDC induced by AQP4 rAbs and polyclonal NMO patient sera. Super-resolution microscopy revealed that AQP4 rAbs with enhanced CDC preferentially formed organized clusters on supramolecular AQP4 orthogonal arrays, linking epitope-dependent multimeric assembly with enhanced C1q binding and activation. The resulting model of AQP4-IgG CDC provides a framework for understanding classical complement activation in human autoantibody-mediated disorders and identifies a potential new therapeutic avenue for treating NMO.

CNS Aquaporin-4-specific B cells connect with multiple B-cell compartments in neuromyelitis optica spectrum disorder.

OBJECTIVES: Neuromyelitis optica spectrum disorder (NMOSD) is a severe inflammatory disorder of the central nervous system (CNS) targeted against aquaporin-4 (AQP4). The origin and trafficking of AQP4-specific B cells in NMOSD remains unknown. METHODS: Peripheral (n = 7) and splenic B cells (n = 1) recovered from seven NMOSD patients were sorted into plasmablasts, naïve, memory, and CD27-IgD- double negative (DN) B cells, and variable heavy chain (VH) transcriptome sequences were generated by deep sequencing. Peripheral blood (PB) VH repertoires were compared to the same patient's single-cell cerebrospinal fluid (CSF) plasmablast (PB) VH transcriptome, CSF immunoglobulin (Ig) proteome, and serum Ig proteome. Recombinant antibodies were generated from paired CSF heavy- and light chains and tested for AQP4 reactivity. RESULTS: Approximately 9% of the CSF VH sequences aligned with PB memory B cells, DN B cells, and plasmablast VH sequences. AQP4-specific VH sequences were observed in each peripheral B-cell compartment. Lineage analysis of clonally related VH sequences indicates that CSF AQP4-specific B cells are closely related to an expanded population of DN B cells that may undergo antigen-specific B-cell maturation within the CNS. CSF and serum Ig proteomes overlapped with the VH sequences from each B-cell compartment; the majority of matches occurring between the PB VH sequences and serum Ig proteome. INTERPRETATION: During an acute NMOSD relapse, a dynamic exchange of B cells occurs between the periphery and CNS with AQP4-specific CSF B cells emerging from postgerminal center memory B cells and plasmablasts. Expansion of the PB DN B-cell compartment may be a potential biomarker of NMOSD activity.

Determining the Spatial Relationship of Membrane-Bound Aquaporin-4 Autoantibodies by STED Nanoscopy.

Determining the spatial relationship of individual proteins in dense assemblies remains a challenge for superresolution nanoscopy. The organization of aquaporin-4 (AQP4) into large plasma membrane assemblies provides an opportunity to image membrane-bound AQP4 antibodies (AQP4-IgG) and evaluate changes in their spatial distribution due to alterations in AQP4 isoform expression and AQP4-IgG epitope specificity. Using stimulated emission depletion nanoscopy, we imaged secondary antibody labeling of monoclonal AQP4-IgGs with differing epitope specificity bound to isolated tetramers (M1-AQP4) and large orthogonal arrays of AQP4 (M23-AQP4). Imaging secondary antibodies bound to M1-AQP4 allowed us to infer the size of individual AQP4-IgG binding events. This information was used to model the assembly of larger AQP4-IgG complexes on M23-AQP4 arrays. A scoring algorithm was generated from these models to characterize the spatial arrangement of bound AQP4-IgG antibodies, yielding multiple epitope-specific patterns of bound antibodies on M23-AQP4 arrays. Our results delineate an approach to infer spatial relationships within protein arrays using stimulated emission depletion nanoscopy, offering insight into how information on single antibody fluorescence events can be used to extract information from dense protein assemblies under a biologic context.

Mutagenesis of the aquaporin 4 extracellular domains defines restricted binding patterns of pathogenic neuromyelitis optica IgG.

Neuromyelitis optica-immunoglobulin G (NMO-IgG) binds to aquaporin-4 (AQP4) water channels in the central nervous system leading to immune-mediated injury. We have previously demonstrated that a high proportion of CSF plasma cells of NMO patients produce antibody to the extracellular domains of the AQP4 protein and that recombinant IgG (rAb) derived from these cells recapitulate pathogenic features of disease. We performed a comprehensive mutational analysis of the three extracellular loops of the M23 isoform of human AQP4 using both serial and single point mutations, and we evaluated the effects on binding of NMO AQP4-reactive rAbs by quantitative immunofluorescence. Whereas all NMO rAbs required conserved loop C ((137)TP(138) and Val(150)) and loop E ((230)HW(231)) amino acids for binding, two broad patterns of NMO-IgG recognition could be distinguished based on differential sensitivity to loop A amino acid changes. Pattern 1 NMO rAbs were insensitive to loop A mutations and could be further discriminated by differential sensitivity to amino acid changes in loop C ((148)TM(149) and His(151)) and loop E (Asn(226) and Glu(228)). Alternatively, pattern 2 NMO rAbs showed significantly reduced binding following amino acid changes in loop A ((63)EKP(65) and Asp(69)) and loop C (Val(141), His(151), and Leu(154)). Amino acid substitutions at (137)TP(138) altered loop C conformation and abolished the binding of all NMO rAbs and NMO-IgG, indicating the global importance of loop C conformation to the recognition of AQP4 by pathogenic NMO Abs. The generation of human NMO rAbs has allowed the first high resolution mapping of extracellular loop amino acids critical for NMO-IgG binding and identified regions of AQP4 extracellular structure that may represent prime targets for drug therapy.

Mechanism of platelet adhesion to von Willebrand factor and microparticle formation under high shear stress.

We describe here the mechanism of platelet adhesion to immobilized von Willebrand factor (VWF) and subsequent formation of platelet-derived microparticles mediated by glycoprotein Ibalpha (GPIbalpha) under high shear stress. As visualized in whole blood perfused in a flow chamber, platelet attachment to VWF involved one or few membrane areas of 0.05 to 0.1 microm(2) that formed discrete adhesion points (DAPs) capable of resisting force in excess of 160 pN. Under the influence of hydrodynamic drag, membrane tethers developed between the moving platelet body and DAPs firmly adherent to immobilized VWF. Continued stretching eventually caused the separation of many such tethers, leaving on the surface tube-shaped or spherical microparticles with a diameter as low as 50 to 100 nm. Adhesion receptors (GPIbalpha, alphaIIbbeta3) and phosphatidylserine were expressed on the surface of these microparticles, which were procoagulant. Shearing platelet-rich plasma at the rate of 10,000 s(-1) in a cone-and-plate viscosimeter increased microparticle counts up to 55-fold above baseline. Blocking the GPIb-VWF interaction abolished microparticle generation in both experimental conditions. Thus, a biomechanical process mediated by GPIbalpha-VWF bonds in rapidly flowing blood may not only initiate platelet arrest onto reactive vascular surfaces but also generate procoagulant microparticles that further enhance thrombus formation.