Antibody Specificities Associated with Neutralization Breadth in Plasma from Human Immunodeficiency Virus Type 1 Subtype C-Infected Blood Donors

Defining the specificities of the anti-human immunodeficiency virus type 1 (HIV-1) envelope antibodies able to mediate broad heterologous neutralization will assist in identifying targets for an HIV-1 vaccine. We screened 70 plasmas from chronically HIV-1-infected individuals for neutralization breadth. Of these, 16 (23%) were found to neutralize 80% or more of the viruses tested. Anti-CD4 binding site (CD4bs) antibodies were found in almost all plasmas independent of their neutralization breadth, but they mainly mediated neutralization of the laboratory strain HxB2 with little effect on the primary virus, Du151. Adsorption with Du151 monomeric gp120 reduced neutralizing activity to some extent in most plasma samples when tested against the matched virus, although these antibodies did not always confer cross-neutralization. For one plasma, this activity was mapped to a site overlapping the CD4-induced (CD4i) epitope and CD4bs. Anti-membrane-proximal external region (MPER) (r = 0.69; P < 0.001) and anti-CD4i (r = 0.49; P < 0.001) antibody titers were found to be correlated with the neutralization breadth. These anti-MPER antibodies were not 4E10- or 2F5-like but spanned the 4E10 epitope. Furthermore, we found that anti-cardiolipin antibodies were correlated with the neutralization breadth (r = 0.67; P < 0.001) and anti-MPER antibodies (r = 0.6; P < 0.001). Our study suggests that more than one epitope on the envelope glycoprotein is involved in the cross-reactive neutralization elicited during natural HIV-1 infection, many of which are yet to be determined, and that polyreactive antibodies are possibly involved in this phenomenon.The generation of an antibody response capable of neutralizing a broad range of viruses remains an important goal of human immunodeficiency virus type 1 (HIV-1) vaccine development. Despite multiple efforts in the design of immunogens capable of inducing such humoral responses, little progress has been made (18, 20, 39). The sequence variability of the virus, as well as masking mechanisms exhibited by the envelope glycoprotein, has further hindered this pursuit (6, 22). It is known that while the majority of HIV-infected individuals mount a strong neutralization response against their own virus within the first 6 to 12 months of infection, breadth is observed in only a few individuals years later (5, 10, 15, 26, 33, 40, 41). However, very little is known about the specificities of the antibodies that confer this broad cross-neutralization. It is plausible that broadly cross-neutralizing (BCN) plasmas contain antibodies that target conserved regions of the envelope glycoprotein, as exemplified by a number of well-characterized broadly neutralizing monoclonal antibodies (MAbs). The b12 MAb recognizes the CD4 binding site (CD4bs), and 2G12 binds to surface glycans (7, 42, 44, 56). The 447-52D MAb recognizes the V3 loop, and 17b, E51, and 412d bind to CD4-induced (CD4i) epitopes that form part of the coreceptor binding site (13, 21, 51, 54). Finally, the MAbs 2F5, 4E10, and Z13e1 recognize distinct linear sequences in the gp41 membrane-proximal external region (MPER) (36, 57). The targets of these neutralizing MAbs provide a rational starting point for examining the complex nature of polyclonal plasma samples.Several groups have addressed the need to develop methodologies to elucidate the presence of certain neutralizing-antibody specificities (1, 8, 9, 29, 30, 43, 55). A number of these studies reported that the BCN antibodies in plasma can in some cases be adsorbed using gp120 immobilized on beads (1, 9, 29, 30, 43). Furthermore, the activities of some of these anti-gp120 neutralizing antibodies could be mapped to the CD4bs, as the D368R mutant gp120 failed to adsorb them (1, 29, 30, 43).Antibodies to CD4i epitopes are frequently found in HIV-1-infected individuals and are thought to primarily target the coreceptor binding site, which includes the bridging sheet and possibly parts of the V3 region. Decker and colleagues (8) showed that MAbs to HIV-1 CD4i epitopes can neutralize HIV-2 when pretreated with soluble CD4 (sCD4), indicating that the CD4i epitope is highly conserved among different HIV lineages. The poor accessibility of CD4i epitopes, however, has precluded this site from being a major neutralizing-antibody target (24), although a recent study suggested that some of the cross-neutralizing activity in polyclonal sera mapped to a CD4i epitope (30).Another site that has attracted considerable attention as a target for cross-neutralizing antibodies is the MPER, a linear stretch of 34 amino acids in gp41. Anti-MPER antibodies have been detected in the plasma of HIV-infected individuals by using chimeric viruses with HIV-1 MPER grafted into a simian immunodeficiency virus or an HIV-2 envelope glycoprotein (15, 55). These studies concluded that 2F5- and 4E10-like antibodies were rarely found in HIV-1-infected plasmas; however, other specificities within the MPER were recognized by around one-third of HIV-1-infected individuals (15). More recently, 4E10-like and 2F5-like antibodies (30, 43), as well as antibodies to novel epitopes within the MPER (1), have been shown to be responsible for neutralization breadth in a small number of plasma samples. The anti-MPER MAb 4E10 has been shown to react to autoantigens, leading to the suggestion that their rarity in human infection is due to the selective deletion of B cells with these specificities (17, 35). Furthermore, a recent study found an association between anti-MPER and anti-cardiolipin (CL) antibodies, although an association with neutralization was not examined (31).A recent study by Binley and coworkers used an array of methodologies to determine the antibody specificities present in subtype B and subtype C plasma samples with neutralization breadth (1). While antibodies to gp120, some of which mapped to the CD4bs, and to MPER were identified, most of the neutralizing activity in the BCN plasma could not be attributed to any of the known conserved envelope epitopes. Furthermore, it is not clear how common these specificities are among HIV-1-positive plasmas and whether they are only associated with BCN activity.In this study, we investigated a large collection of HIV-1-infected plasmas obtained from the South African National Blood Services. We aimed to determine if there is a relationship between the presence of certain antibody specificities, such as those against CD4i epitopes, MPER, or the CD4bs, and the neutralizing activities present in these plasmas. Furthermore, we evaluated the presence of various autoreactive antibodies and analyzed whether they might be associated with neutralization breadth.     

Recognition of the neural chemoattractant Netrin-1 by integrins alpha6beta4 and alpha3beta1 regulates epithelial cell adhesion and migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.     

Strategic Focus on 3R Principles Reveals Major Reductions in the Use of Animals in Pharmaceutical Toxicity Testing

The principles of the 3Rs, Replacement, Reduction and Refinement, are being increasingly incorporated into legislations, guidelines and practice of animal experiments in order to safeguard animal welfare. In the present study we have studied the systematic application of 3R principles to toxicological research in the pharmaceutical industry, with particular focus on achieving reductions in animal numbers used in regulatory and investigatory in vivo studies. The work also details major factors influencing these reductions including the conception of ideas, cross-departmental working and acceptance into the work process. Data from 36 reduction projects were collected retrospectively from work between 2006 and 2010. Substantial reduction in animal use was achieved by different strategies, including improved study design, method development and project coordination. Major animal savings were shown in both regulatory and investigative safety studies. If a similar (i.e. 53%) reduction had been achieved simultaneously within the twelve largest pharmaceutical companies, the equivalent reduction world-wide would be about 150,000 rats annually. The results point at the importance of a strong 3R culture, with scientific engagement, collaboration and a responsive management being vital components. A strong commitment in leadership for the 3R is recommended to be translated into cross-department and inter-profession involvement in projects for innovation, validation and implementation. Synergies between all the three Rs are observed and conclude that in silico-, in vitro- and in vivo-methods all hold the potential for applying the reduction R and should be consequently coordinated at a strategic level.     

Defining a land boundary for sustainable livestock consumption

The need for more sustainable production and consumption of animal source food (ASF) is central to the achievement of the sustainable development goals: within this context, wise use of land is a core challenge and concern. A key question in feeding the future world is: how much ASF should we eat? We demonstrate that livestock raised under the circular economy concept could provide a significant, nonnegligible part (9–23 g/per capita) of our daily protein needs (~50–60 g/per capita). This livestock then would not consume human‐edible biomass, such as grains, but mainly convert leftovers from arable land and grass resources into valuable food, implying that production of livestock feed is largely decoupled from arable land. The availability of these biomass streams for livestock then determines the boundaries for livestock production and consumption. Under this concept, the competition for land for feed or food would be minimized and compared to no ASF, including some ASF in the human diet could free up about one quarter of global arable land. Our results also demonstrate that restricted growth in consumption of ASF in Africa and Asia would be feasible under these boundary conditions, while reductions in the rest of the world would be necessary to meet land use sustainability criteria. Managing this expansion and contraction of future consumption of ASF is essential for achieving sustainable nutrition security.     

Measuring the gut microbiome in birds: Comparison of faecal and cloacal sampling

The gut microbiomes of birds and other animals are increasingly being studied in ecological and evolutionary contexts. Numerous studies on birds and reptiles have made inferences about gut microbiota using cloacal sampling; however, it is not known whether the bacterial community of the cloaca provides an accurate representation of the gut microbiome. We examined the accuracy with which cloacal swabs and faecal samples measure the microbiota in three different parts of the gastrointestinal tract (ileum, caecum, and colon) using a case study on juvenile ostriches, Struthio camelus, and high‐throughput 16S rRNA sequencing. We found that faeces were significantly better than cloacal swabs in representing the bacterial community of the colon. Cloacal samples had a higher abundance of Gammaproteobacteria and fewer Clostridia relative to the gut and faecal samples. However, both faecal and cloacal samples were poor representatives of the microbial communities in the caecum and ileum. Furthermore, the accuracy of each sampling method in measuring the abundance of different bacterial taxa was highly variable: Bacteroidetes was the most highly correlated phylum between all three gut sections and both methods, whereas Actinobacteria, for example, was only strongly correlated between faecal and colon samples. Based on our results, we recommend sampling faeces, whenever possible, as this sample type provides the most accurate assessment of the colon microbiome. The fact that neither sampling technique accurately portrayed the bacterial community of the ileum nor the caecum illustrates the difficulty in noninvasively monitoring gut bacteria located further up in the gastrointestinal tract. These results have important implications for the interpretation of avian gut microbiome studies.     

MRS study of glutamate metabolism in cultured neurons/glia

[U-¹³C]Glutamate metabolism was studied in primary brain cell cultures. Cell extracts as well as redissolved lyophilized media were subjected to nuclear magnetic resonance spectroscopy in order to identify¹³C labeled metabolites. Both neurons and astrocytes metabolized glutamate extensively with¹³C label appearing in aspartate in all cultures. Additionally, GABA is synthesized in the GABAergic cortical neurons. Labeling of lactate and glutamine was prominent in medium from astrocytes, but not detectable in cerebral cortical neurons. Cerebellar granule neurons showed some labeling of lactate. Glutamate derived from the first turn of the tricarboxylic acid cycle (1,2,3-¹³C₃-isotopomer) is present in all cell types analyzed. However, glutamate derived from the second turn of the cycle was only detected in granule neurons. In astrocytes, the transaminase inhibitor aminooxyacetic acid not only abolished the appearance of aspartate, but also of the 1,2,3-¹³C₃-isotopomer of glutamate, thus showing that transmination is necessary for the conversion of 2-oxoglutarate to glutamate. The entry of glutamate into the tricarboxylic acid cycle was, however, not seriously impaired. 3-nitropropionic acid abolished the appearance of aspartate, the 1,2,3-¹³C₃-isotopomer of glutamate and lactate in cerebellar granule neurons. Special issue dedicated to Dr. Herman Bachelard.     

Gastric bypass surgery reveals independency of obesity and diabetes melitus type 2

Background

Roux-en-Y gastric bypass surgery is widely applied to ameliorate morbid obesity, including diabetes in people with type 2 diabetes. The latter vanish a few days after surgery for many, but not in all patients before any weight reduction has occurred. The explanation for this change in metabolic status is poorly understood, but the observation may suggest that the fate obesity and diabetes is only partly linked after surgery.

Methods

The trajectories of weight reduction measured as reduced body mass index (BMI) in 741obese subjects with and without diabetes were evaluated. Evaluation was performed on three groups: 1) subjects that were non-diabetic before and after surgery; 2) subjects that were diabetics before surgery but non-diabetics after surgery; and 3) subjects that were diabetics before surgery and remained diabetics after surgery. The diabetic state was established at HbA1c above 48 mmol/mol.

Results

The trajectories differ significantly between groups and any sub-populations of groups, the latter identified by the distance between individual trajectories using a k-means procedure. The results suggest that different domains in the enormous genetic network governing basic metabolism are perturbed in obesity and diabetes, and in fact some of the patients are affected by two distinct diseases: obesity and diabetes mellitus type 2.

Conclusion

Although RYGB “normalized” many glycaemic parameters in some of the diabetic subjects apparently converting to a non-diabetics state, other diabetic subjects stay diabetic in the context of the new gut anatomy after surgery. Thus, the obesity part of the glycaemic derangement may have been ameliorated, but some defects of the diabetic state had not.