HIV envelope proteins are bound by human epidermal Langerhans cells by a binding site which differs from the site on the CD4 molecule, and are internalized by receptor endocytosis

Langerhans cells (LC) are epidermal dendritic cells which express several surface antigens among them the CD4 antigens. We investigated the fate of HIV envelope glycoproteins (gp 120 and gp 160) incubated with healthy human trypsinized LC in suspension. After trypsin treatment only the epitope for OKT4 appeared to be resistant. In absence of antigenic sites identified by OKT4A, Leu 3a or BL4, LC fixed and internalized gp 120 or gp 160 recombinant HIV proteins. This finding support the hypothesis that there exists at the surface of LC a second molecule which may act as a HIV receptor.     

Functional role of the glycan cluster of the human immunodeficiency virus type 1 transmembrane glycoprotein (gp41) ectodomain.

To examine the role of the glycans of human immunodeficiency virus type 1 transmembrane glycoprotein gp41, conserved glycosylation sites within the env sequence (Asn-621, Asn-630, and Asn-642) were mutated to Gln. The mutated and control wild-type env genes were introduced into recombinant vaccinia virus and used to infect BHK-21 or CD4+ CEM cells. Mutated gp41 appeared as a 35-kDa band in a Western blot (immunoblot), and it comigrated with the deglycosylated form of wild-type gp41. Proteolytic cleavage of the recombinant wild-type and mutant forms of the gp160 envelope glycoprotein precursor was analyzed by pulse-chase experiments and enzyme-linked immunosorbent assay: gp160 synthesis was similar whether cells were infected with control or mutated env-expressing recombinant vaccinia virus, but about 10-fold less cleaved gp120 and gp41 was produced by the mutated construct than the control construct. The rates of gp120-gp41 cleavage at each of the two potential sites appeared to be comparable in the two constructs. By using a panel of antibodies specific for gp41 and gp120 epitopes, it was shown that the overall immunoreactivities of control and mutated gp41 proteins were similar but that reactivity to epitopes at the C and N termini of gp120, as present on gp160 produced by the mutated construct, was enhanced. This was no longer observed for cleaved gp120 in supernatants. Both gp120 proteins, from control and mutated env, were expressed on the cell surface under a cleaved form and could bind to membrane CD4, as determined by quantitative immunofluorescence assay. In contrast, and despite sufficient expression of env products at the cell membrane, gp41 produced by the mutated construct was unable to induce membrane fusion. Therefore, while contradictory results reported in the literature suggest that gp41 individual glycosylation sites are dispensable for the bioactivity and conformation of env products, it appears that such is not the case when the whole gp41 glycan cluster is removed.     

Proximo-distal pattern regulation in deficient avian limb buds

Summary Deficient limb buds composed of prospective stylopod and autopod are able to regulate the missing intercalary zeugopod, the origin of which was investigated by heterospecific quail/chick recombinants. The associations of quail prospective autopod and chick prospective stylopod failed to regulate. The reverse combination of chick prospective autopod grafted onto a quail prospective stylopod gave rise to a three-segmented limb. In 13 out of 16 cases the regulated zeugopod was made up of both chick and quail cells. Chick cells were located predominantly along the postaxial half of the zeugopod, while the quail cells made up most of its preaxial half. In two cases, the intercalary zeugopod consisted exclusively of chick cells originating from the tip and in one case of quail cells originating from the base.These results demonstrate that during the regulative processes, the prospective values of some of the original stylopodial and autopodial cells have been shifted along the proximo-distal axis, towards the expression of more distal as well as of more proximal structures.Heteropolar stylo-autopodial or zeugo-autopodial recombinants, in which the proximo-distal axis of the base was reversed with respect to that of the tip, were unable to regulate the pattern defects and thus revealed the importance of concordant p-d polarity for regulative processes to take place between abutted tissues.     

The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells

Using chimeras consisting of chick embryos that had received substitution grafts of quail somites, we have determined the distalmost extension of the myogenic primordia in the outgrowing wing bud at 5 days of incubation. At Hamburger-Hamilton stage 25 the most distal premuscle cell is consistently 300 mum or more from the apex of the wing mesoblast. The stage 25 wing tip resembles very early whole limb buds in not having proceeded beyond the mesenchymal state or having expressed markers of terminal differentiation. However, unlike early whole limb buds it is free of a myogenic subpopulation. We therefore propose that the stage 25 wing tip is the appropriate system for in vitro and molecular studies of cartilage differentiation.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998     

Protein-protein interactions between hepatitis C virus nonstructural proteins

Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.     

Comparative vaccine studies in HLA-A2.1-transgenic mice reveal a clustered organization of epitopes presented in hepatitis C virus natural infection

A polyepitopic CD8(+)-T-cell response is thought to be critical for control of hepatitis C virus (HCV) infection. Using transgenic mice, we analyzed the immunogenicity and dominance of most known HLA-A2.1 epitopes presented during infection by using vaccines that carry the potential to enter clinical trials: peptides, DNA, and recombinant adenoviruses. The vaccines capacity to induce specific cytotoxic T lymphocytes and interferon gamma-producing cells revealed that immunogenic epitopes are clustered in specific antigens. For two key antigens, flanking regions were shown to greatly enhance the scope of epitope recognition, whereas a DNA-adenovirus prime-boost vaccination strategy augmented epitope immunogenicity, even that of subdominant ones. The present study reveals a clustered organization of HCV immunogenic HLA.A2.1 epitopes and strategies to modulate their dominance.     

Safety of the Recombinant Cholera Toxin B Subunit, Killed Whole-Cell (rBS-WC) Oral Cholera Vaccine in Pregnancy

Introduction

Mass vaccinations are a main strategy in the deployment of oral cholera vaccines. Campaigns avoid giving vaccine to pregnant women because of the absence of safety data of the killed whole-cell oral cholera (rBS-WC) vaccine. Balancing this concern is the known higher risk of cholera and of complications of pregnancy should cholera occur in these women, as well as the lack of expected adverse events from a killed oral bacterial vaccine.

Methodology/Principal Findings

From January to February 2009, a mass rBS-WC vaccination campaign of persons over two years of age was conducted in an urban and a rural area (population 51,151) in Zanzibar. Pregnant women were advised not to participate in the campaign. More than nine months after the last dose of the vaccine was administered, we visited all women between 15 and 50 years of age living in the study area. The outcome of pregnancies that were inadvertently exposed to at least one oral cholera vaccine dose and those that were not exposed was evaluated. 13,736 (94%) of the target women in the study site were interviewed. 1,151 (79%) of the 1,453 deliveries in 2009 occurred during the period when foetal exposure to the vaccine could have occurred. 955 (83%) out of these 1,151 mothers had not been vaccinated; the remaining 196 (17%) mothers had received at least one dose of the oral cholera vaccine. There were no statistically significant differences in the odds ratios for birth outcomes among the exposed and unexposed pregnancies.

Conclusions/Significance

We found no statistically significant evidence of a harmful effect of gestational exposure to the rBS-WC vaccine. These findings, along with the absence of a rational basis for expecting a risk from this killed oral bacterial vaccine, are reassuring but the study had insufficient power to detect infrequent events.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00709410","term_id":"NCT00709410"}}NCT00709410     

Monitoring Progress towards Universal Health Coverage at Country and Global Levels

Universal health coverage (UHC) has been defined as the desired outcome of health system performance whereby all people who need health services (promotion, prevention, treatment, rehabilitation, and palliation) receive them, without undue financial hardship. UHC has two interrelated components: the full spectrum of good-quality, essential health services according to need, and protection from financial hardship, including possible impoverishment, due to out-of-pocket payments for health services. Both components should benefit the entire population.This paper summarizes the findings from 13 country case studies and five technical reviews, which were conducted as part of the development of a global framework for monitoring progress towards UHC.The case studies show the relevance and feasibility of focusing UHC monitoring on two discrete components of health system performance: levels of coverage with health services and financial protection, with a focus on equity. These components link directly to the definition of UHC and measure the direct results of strategies and policies for UHC. The studies also show how UHC monitoring can be fully embedded in often existing, regular overall monitoring of health sector progress and performance. Several methodological and practical issues related to the monitoring of coverage of essential health services, financial protection, and equity, are highlighted. Addressing the gaps in the availability and quality of data required for monitoring progress towards UHC is critical in most countries.

Summary Points

• The overall goal of universal health coverage (UHC) is that all people obtain the good-quality essential health services, including promotion, prevention, treatment, rehabilitation, and palliation, that they need without enduring financial hardship.
• A global UHC monitoring framework, developed by WHO and the World Bank Group in interaction with the process that led to this PLOS Collection, was used in 13 country case studies, underpinned by five technical reviews.
• The UHC monitoring framework focuses on the simultaneous monitoring of coverage of the population with essential health services and with financial protection against catastrophic out-of-pocket health payments, stratified by wealth quintile, place of residence, and sex.
• Most countries focus on regular monitoring of a set of tracer indicators for priority health services, as well as the occurrence of financial hardship and impoverishment due to out-of-pocket health expenses. The indicators generally follow international standards of measurement and can be used for global comparisons.
• Most countries do not have an explicit framework for UHC monitoring. The monitoring of UHC is, however, partially embedded in regular overall health sector progress and performance reviews which include health system inputs, service delivery, and health status indicators.
• There are major gaps in the availability and quality of data required for monitoring progress towards UHC. Countries mostly rely on international survey programs or national surveys to obtain disaggregated data on coverage and financial protection indicators, complemented by health facility data, but often the frequency and contents of these surveys are not sufficient to meet the country''s information needs.
• Monitoring progress towards the two components of UHC will be complementary and critical to achieving desirable health outcome goals, such as ending preventable deaths and promoting healthy life expectancy, and also reducing poverty and protecting household incomes.

     

New versatile cloning and sequencing vectors based on bacteriophage M13

A new pair of cloning and sequencing vectors based on bacteriophage M13mp7 has been developed. These vectors (M13tg130 and M13tg131) contain, in addition to the EcoRI, BamHI, HindIII, SmaI, SalI and PstI sites present in other vectors [cf., M13mp8 and M13mp9, Messing and Vieira, Gene 19 (1982) 269-276], unique restriction recognition sequences for the enzymes EcoRV, KpnI, SphI, SstI and XbaI. A restriction site for the enzyme BglII has been incorporated into the polylinker region of one of the vector pair to permit rapid discrimination between the two vectors.