Structural polymorphism of glycophorins demonstrated by immunoblotting techniques

We have explored the polymorphism of the glycophorin system in the human erythrocyte membrane using the immunoblotting techniques and examining 52 individuals selected without prior bias as to their serologic state and ten documented serologic variants of M, N, S, s blood group system. Polyclonal antisera to alpha glycophorin and to alpha glycophorin CNBr carboxyl terminal fragment C (residues 82-131) and M and N specific monoclonal antibodies (MoAbs) were used. The first two reagents detect specific regions of the alpha glycophorin molecule and all electrophoretically resolved species of glycophorins immunologically related to alpha and delta glycophorins (delta glycophorin, [alpha-delta] hybrids and other glycophorins with an alteration in the carboxyl terminal segment); the M and N MoAbs identified the glycophorin species containing or lacking the M or N determinant in the amino terminal octapeptide structures. We find that immunoblotting confirmed in all cases the serologically determined phenotype; we also find that polymorphic forms of the glycophorin system are relatively infrequent; immunoblotting, independent from serologic testing, was capable of detecting five mutants, two most likely S-s-U-phenotypes; a new glycophorin species was detected in normal red cells with both antiglycophorin and antipeptide C sera, which is not evident with MoAbs; immunoblots of known glycophorin variants (En(a-), U-, Mg, Mi I, II, III, V, and Sta) confirmed but also extended our knowledge of the abnormal glycophorins involved; and the He+ and Wrb(-) cells showed normal patterns.     

Universal nevirapine upon presentation in labor to prevent mother-to-child HIV transmission in high prevalence settings

OBJECTIVE: To assess the uptake of and adherence to nevirapine to prevent mother-to-child HIV transmission among women of unknown HIV serostatus presenting in labor. We also assessed preliminary efficacy of the approach. DESIGN: Women of unknown HIV serostatus presenting in labor were offered single-dose nevirapine in a prospective cohort study. Two additional contemporaneous comparison populations were also studied. METHODS: We measured uptake by counting the number of women that accepted enrollment when offered. We measured adherence with cord blood nevirapine assay. We measured preliminary efficacy with HIV DNA polymerase chain reaction of infant blood spots at 4-6 weeks of life. RESULTS: Of 1591 women approached in labor, 634 (40%) took up the intervention and received nevirapine, of whom 185 (29%) were HIV infected. Of 179 cord blood specimens from HIV-exposed infants that could be evaluated, 178 (99.4%) had nevirapine detected. This was higher than the 73 of 98 (74%) adherence rate observed in a comparison cohort in which women self-administered nevirapine before presenting to the labor ward (P < 0.001). Of 145 available infant specimens, 17 (11.7%) showed evidence of infection at 4-6 weeks, compared with 12 of 60 (20%) infants born immediately prior to study commencement whose HIV-infected mothers did not receive nevirapine (P < 0.05). CONCLUSIONS: Nevirapine without HIV testing upon presentation in labor was accepted by two-fifths of women. Because therapy is directly observed, adherence is nearly perfect. Labor ward dosing to enhance nevirapine coverage should be considered as an adjunct to antenatal nevirapine administration for prevention of mother-to-child transmission of HIV.     

Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.     

Establishment of a Maternal Newborn Health Registry in the Belgaum District of Karnataka,India

Background

Pregnancy-related vital registration is important to inform policy to reduce maternal, fetal and newborn mortality, yet few systems for capturing accurate data are available in low-middle income countries where the majority of the mortality occurs. Furthermore, methods to effectively implement high-quality registration systems have not been described. The goal of creating the registry described in this paper was to inform public health policy makers about pregnancy outcomes in our district so that appropriate interventions to improve these outcomes could be undertaken and to position the district to be a leader in pregnancy-related public health research.

Methods

We created a prospective maternal and newborn health registry in Belgaum, Karnataka State, India. To initiate this registry, we worked with the Ministry of Health to first establish estimated birth rates and define the catchment areas of the clusters, working within the existing health system and primary health centers. We also undertook household surveys to identify women likely to become pregnant. We then implemented monitoring measures to ensure high quality and completeness of the maternal newborn health registry. All pregnant women in the catchment area were identified, consented and enrolled during pregnancy, with follow-up visits to ascertain pregnancy outcomes and mother/infant status at 42-days postpartum.

Results

From 2008 through 2014, we demonstrated continued improvements in both the coverage for enrollment and accuracy of reporting pregnancy outcomes within the defined catchment area in Belgaum, India. Nearly 100% of women enrolled had follow-up at birth and 99% had 42-day follow-up. Furthermore, we facilitated earlier enrollment of women during pregnancy while achieving more timely follow-up and decreased time of reporting from the date of the pregnancy event.

Conclusions

We created a pregnancy-related registry which includes demographic data, risk factors, and outcomes allowing for high rates of ascertainment and follow-up while working within the existing health system. Understanding the elements of the system used to create the registry is important to improve the quality of the results. Tracking of pregnancies and their outcomes is an important step toward reducing maternal and perinatal mortality.

     

Postpartum contraceptive use and unmet need for family planning in five low-income countries

Background

During the post-partum period, most women wish to delay or prevent future pregnancies. Despite this, the unmet need for family planning up to a year after delivery is higher than at any other time. This study aims to assess fertility intention, contraceptive usage and unmet need for family planning amongst women who are six weeks postpartum, as well as to identify those at greatest risk of having an unmet need for family planning during this period.

Methods

Using the NICHD Global Network for Women’s and Children’s Health Research’s multi-site, prospective, ongoing, active surveillance system to track pregnancies and births in 100 rural geographic clusters in 5 countries (India, Pakistan, Zambia, Kenya and Guatemala), we assessed fertility intention and contraceptive usage at day 42 post-partum.

Results

We gathered data on 36,687 women in the post-partum period. Less than 5% of these women wished to have another pregnancy within the year. Despite this, rates of modern contraceptive usage varied widely and unmet need ranged from 25% to 96%. Even amongst users of modern contraceptives, the uptake of the most effective long-acting reversible contraceptives (intrauterine devices) was low. Women of age less than 20 years, parity of two or less, limited education and those who deliver at home were at highest risk for having unmet need.

Conclusions

Six weeks postpartum, almost all women wish to delay or prevent a future pregnancy. Even in sites where early contraceptive adoption is common, there is substantial unmet need for family planning. This is consistently highest amongst women below the age of 20 years. Interventions aimed at increasing the adoption of effective contraceptive methods are urgently needed in the majority of sites in order to reduce unmet need and to improve both maternal and infant outcomes, especially amongst young women.

Study registration

Clinicaltrials.gov (ID# NCT01073475)

     

Preliminary Results that Assess Metformin Treatment in a Preclinical Model of Pancreatic Cancer Using Simultaneous [<Superscript>18</Superscript>F]FDG PET and acidoCEST MRI

Purpose

We sought to determine if the synergy between evaluations of glucose uptake in tumors and extracellular tumor acidosis measured with simultaneous positron emission tomography (PET)/magnetic resonance imaging (MRI) can improve longitudinal evaluations of the response to metformin treatment.

Procedures

A standard 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]FDG) PET protocol that evaluates glucose uptake in tumors, and a standard acidoCEST MRI protocol that measures extracellular pH (pHe) in tumors, were simultaneously performed to assess eight vehicle-treated (control) mice and eight metformin-treated mice 1 day before treatment, 1 day after initiating daily treatment with metformin, and 7 days after initiating treatment. Longitudinal changes in SUV_max and extracellular pH (pHe) were evaluated for each treatment group, and differences in SUV_max and pHe between metformin-treated and control groups were also evaluated.

Results

MRI acquisition protocols had little effect on the PET count rate, and the PET instrumentation had little effect on image contrast during acidoCEST MRI, verifying that [¹⁸F]FDG PET and acidoCEST MRI can be performed simultaneously. The average SUV_max of the tumor model had a significant decrease after 7 days of treatment with metformin, as expected. The average tumor pHe decreased after 7 days of metformin treatment, which reflected the inhibition of the consumption of cytosolic lactic acid caused by metformin. However, the average SUV_max of the tumor model was not significantly different between the metformin-treated and control groups after 7 days of treatment, and average pHe was also not significantly different between these groups. For comparison, the combination of average SUV_max and pHe measurements significantly differed between the treatment group and control group on Day 7.

Conclusions

[¹⁸F]FDG PET and acidoCEST MRI studies can be performed simultaneously. The synergistic combination of assessing glucose uptake and tumor acidosis can improve differentiation of a drug-treated group from a control group during drug treatment of a tumor model.

     

High and long‐term von Willebrand factor expression after Sleeping Beauty transposon‐mediated gene therapy in a mouse model of severe von Willebrand disease

Essentials

• von Willebrand disease (VWD) is the most common inherited bleeding disorder.
• Gene therapy for VWD offers long‐term therapy for VWD patients.
• Transposons efficiently integrate the large von Willebrand factor (VWF) cDNA in mice.
• Liver‐directed transposons support sustained VWF expression with suboptimal multimerization.

Summary

Background

Type 3 von Willebrand disease (VWD) is characterized by complete absence of von Willebrand factor (VWF). Current therapy is limited to treatment with exogenous VWF/FVIII products, which only provide a short‐term solution. Gene therapy offers the potential for a long‐term treatment for VWD.

Objectives

To develop an integrative Sleeping Beauty (SB) transposon‐mediated VWF gene transfer approach in a preclinical mouse model of severe VWD.

Methods

We established a robust platform for sustained transgene murine VWF (mVWF) expression in the liver of Vwf^?/? mice by combining a liver‐specific promoter with a sandwich transposon design and the SB100X transposase via hydrodynamic gene delivery.

Results

The sandwich SB transposon was suitable to deliver the full‐length mVWF cDNA (8.4 kb) and supported supra‐physiological expression that remained stable for up to 1.5 years after gene transfer. The sandwich vector stayed episomal (~60 weeks) or integrated in the host genome, respectively, in the absence or presence of the transposase. Transgene integration was confirmed using carbon tetrachloride‐induced liver regeneration. Analysis of integration sites by high‐throughput analysis revealed random integration of the sandwich vector. Although the SB vector supported long‐term expression of supra‐physiological VWF levels, the bleeding phenotype was not corrected in all mice. Long‐term expression of VWF by hepatocytes resulted in relatively reduced amounts of high‐molecular‐weight multimers, potentially limiting its hemostatic efficacy.

Conclusions

Although this integrative platform for VWF gene transfer is an important milestone of VWD gene therapy, cell type‐specific targeting is yet to be achieved.