Monoclonal antibodies to phosphatidylinositol phosphate neutralize human immunodeficiency virus type 1: role of phosphate-binding subsites

Both a murine monoclonal antibody to phosphatidylinositol phosphate (PIP) and a human monoclonal antibody (4E10) that is known to have broadly neutralizing capabilities against primary isolates of human immunodeficiency virus type 1 (HIV-1) bound to PIP, as determined by enzyme-linked immunosorbent assay. Each of the antibodies had antigen subsite binding specificities in aqueous medium for small phosphate-containing molecules and for inositol. The anti-PIP monoclonal antibody inhibited infection by two HIV-1 primary isolates in neutralization assays employing primary human peripheral blood mononuclear cells. The data suggest that PIP or related lipids having free phosphates could serve as targets for the neutralization of HIV-1.     

Glucose-Raising Polymorphisms in the Human Clock Gene Cryptochrome 2 (CRY2) Affect Hepatic Lipid Content

Circadian rhythms govern vital functions. Their disruption provokes metabolic imbalance favouring obesity and type-2 diabetes. The aim of the study was to assess the role of clock genes in human prediabetes. To this end, genotype-phenotype associations of 121 common single nucleotide polymorphisms (SNPs) tagging ARNTL, ARNTL2, CLOCK, CRY1, CRY2, PER1, PER2, PER3, and TIMELESS were assessed in a study population of 1,715 non-diabetic individuals metabolically phenotyped by 5-point oral glucose tolerance tests. In subgroups, hyperinsulinaemic-euglycaemic clamps, intravenous glucose tolerance tests, and magnetic resonance imaging/spectroscopy were performed. None of the tested SNPs was associated with body fat content, insulin sensitivity, or insulin secretion. Four CRY2 SNPs were associated with fasting glycaemia, as reported earlier. Importantly, carriers of these SNPs’ minor alleles revealed elevated fasting glycaemia and, concomitantly, reduced liver fat content. In human liver tissue samples, CRY2 mRNA expression was directly associated with hepatic triglyceride content. Our data may point to CRY2 as a novel switch in hepatic fuel metabolism promoting triglyceride storage and, concomitantly, limiting glucose production. The anti-steatotic effects of the glucose-raising CRY2 alleles may explain why these alleles do not increase type-2 diabetes risk.     

Selection of H-2 molecules for the context of antigen recognition by T lymphocytes

T lymphocytes recognize the synthetic polypeptides GA and GLT and the natural antigen LDH_B and are thereby stimulated to proliferate in vitro. Simultaneously with the antigen, T cells recognize class II MHC molecules of the antigen-presenting cell and the T-cell proliferation can therefore be inhibited by the addition of monoclonal antibodies specific for either A (AA ) or E (EF ) molecules. Antibody blocking of in vitro responses thus provides an opportunity to test the rules governing the selection of class II molecules (A versus E) in the recognition of different antigens. To determine these rules we tested T cells for some 40 strains (classical inbred strains and B10.W lines) carrying H-2 haplotypes derived from wild mice) for their proliferative response to GA, GLT, and LDH_B. Strains that responded were then tested in the antibody-blocking assay to determine the class II context of the response. The response to GA occurred always in the context of the A molecule; no single instance was found of the response being channelled through the E molecule. Of the 19 different A molecules (A allomorphs) that could be tested, nine (47 percent) were able to provide the context for GA recognition (and hence conferred responsiveness), while the rest failed to do so (conferred nonresponsiveness). Of the 17 informative cases tested for the response to LDH_B, 14 channelled the response through the A molecule, while, in the remaining cases, the cells failed to respond altogether. And again, there was no case where the response was channelled through the E molecule. However, in two instances (of 14) the E molecule provided the context for the stimulation of suppressor T cells, which then suppressed the response of helper T cells occurring in the context of the A molecule. Of the 19 cases tested for the response to GLT, eight channelled the response through the E molecule and two through the A molecule. The two cases of an E A switch were those in which the strains failed to express cell-surface E molecules as a result of a mutation in one of the E-encoding loci. These data indicate a remarkable but puzzling consistency in the channelling of the response to a given antigen via either A or E molecules. This consistency may be a hint that there is a link between the specificity of antigen (nonself) and MHC (self) recognition by T lymphocytes.Abbreviations used in this paper APC antigen-presenting cell - GA poly (Glu⁴⁰Ala⁶⁰) - GLT poly (Glu⁵¹Lys³⁴Tyr¹⁵) - Ir immune response - LDH_B lactate dehydrogenase 134 - MHC major histocompatibility complex - T_H T helper (cell) - T_S T suppressor (cell)     

Biochemical interactions of carbamates and ecothiophate with the activated conformation of nicotinic acetylcholine receptor

Purified Torpedo nobiliana electric organ acetylcholine receptor (AChR) was reconstituted into membranes containing natural phospholipids supplemented with cholesterol (25% w/w). The reconstituted system facilitates the study of the effects of drugs on the regulation of the AChR channel complex under both resting and carbachol (carb)-stimulated conditions. Neostigmine (Neo) was the only carbamate to induce activation of [3-H]-phencyclidine ([3-H]-PCP) binding to the channel sites, acting as a weak agonist. The activation of [3-H]-PCP binding is dependent upon the nature of the reconstituted systems, with carb/Neo activation ratios of 8, 3, and 1 for the intact purified AChR vesicles fraction (PVF), the PVF reconstituted in phospholipid/cholesterol (CRPVF), and the PVF reconstituted in phospholipid (RPVF), respectively. The carbamates Neo, physostigmine (Physo), and pyridostigmine (Pyrido) inhibited carb-activated [3-H]-PCP binding with K_i values of 10, 20, and 1,600 μM, respectively. The inhibition was mixed competitivenoncompetitive in nature. The characteristic response of CRPVF to carb-stimulated [22-Na] influx was inhibited by the three carbamates, with IC-50 values of 6,50, and 1,000 μM for Neo, Physo, and Pyrido, respectively. The quaternary ammonium organophosphate ecothiophate (Eco) inhibited carb-stimulated [22-Na] influx with potency similar to that of Neo. Preincubation of AChR preparation with the carbamates and ecothiophate caused a reduction in the binding of [125-I]-α- bungarotoxin ([125-I]-α-BGT) with the following decreasing order of potency: Neo < Physo < Eco < Pyrido. Calcium has a direct modulatory role on the time-course inhibition of [125-I]-α-BGT binding by these drugs. While we observed a high potency of Neo and Physo in inhibiting [125-I]-α-BGT binding, it was undetectable for the carbamate insecticide 2-methyl-2-(methylthio)propionaldehyde-O-(methylcarbamoyl)oxime (aldicarb). These data suggest that the potent anticholinesterase carbamate agents interact differently with the AChR and its ionic channel. Their interactions with the nicotinic AChR channel system can be described as (a) weakly agonist, (b) directly acting on the open conformation of the channel, and (c) blocking the AChR-binding sites.     

Cross-inhibition of SR-BI- and ABCA1-mediated cholesterol transport by the small molecules BLT-4 and glyburide

Scavenger receptor class B type I (SR-BI) and ABCA1 are structurally dissimilar cell surface proteins that play key roles in HDL metabolism. SR-BI is a receptor that binds HDL with high affinity and mediates both the selective lipid uptake of cholesteryl esters from lipid-rich HDL to cells and the efflux of unesterified cholesterol from cells to HDL. ABCA1 mediates the efflux of unesterified cholesterol and phospholipids from cells to lipid-poor apolipoprotein A-I (apoA-I). The activities of ABCA1 and other ATP binding cassette superfamily members are inhibited by the drug glyburide, and SR-BI-mediated lipid transport is blocked by small molecule inhibitors called BLTs. Here, we show that one BLT, [1-(2-methoxy-phenyl)-3-naphthalen-2-yl-urea] (BLT-4), blocked ABCA1-mediated cholesterol efflux to lipid-poor apoA-I at a potency similar to that for its inhibition of SR-BI (IC(50) approximately 55-60 microM). Reciprocally, glyburide blocked SR-BI-mediated selective lipid uptake and efflux at a potency similar to that for its inhibition of ABCA1 (IC(50) approximately 275-300 microM). As is the case with BLTs, glyburide increased the apparent affinity of HDL binding to SR-BI. The reciprocal inhibition of SR-BI and ABCA1 by BLT-4 and glyburide raises the possibility that these proteins may share similar or common steps in their mechanisms of lipid transport.     

Distinct composition and metabolic functions of human gut microbiota are associated with cachexia in lung cancer patients

Cachexia is associated with decreased survival in cancer patients and has a prevalence of up to 80%. The etiology of cachexia is poorly understood, and limited treatment options exist. Here, we investigated the role of the human gut microbiome in cachexia by integrating shotgun metagenomics and plasma metabolomics of 31 lung cancer patients. The cachexia group showed significant differences in the gut microbial composition, functional pathways of the metagenome, and the related plasma metabolites compared to non-cachectic patients. Branched-chain amino acids (BCAAs), methylhistamine, and vitamins were significantly depleted in the plasma of cachexia patients, which was also reflected in the depletion of relevant gut microbiota functional pathways. The enrichment of BCAAs and 3-oxocholic acid in non-cachectic patients were positively correlated with gut microbial species Prevotella copri and Lactobacillus gasseri, respectively. Furthermore, the gut microbiota capacity for lipopolysaccharides biosynthesis was significantly enriched in cachectic patients. The involvement of the gut microbiome in cachexia was further observed in a high-performance machine learning model using solely gut microbial features. Our study demonstrates the links between cachectic host metabolism and specific gut microbial species and functions in a clinical setting, suggesting that the gut microbiota could have an influence on cachexia with possible therapeutic applications.Subject terms: Microbiome, Metagenomics, Next-generation sequencing, Metabolomics     

Isolation of the pyrrolizidine alkaloid 1-epialexine from Castanospermum australe

The Australian leguminous tree Castanospermum australe contains the anti-viral glucose analogue indolizidine alkaloid castanospermine. As the result of a search for new bioactive carbohydrate-like compounds we now report the isolation of the novel polyhydroxylated pyrrolizidine alkaloid, 1-epialexine, from the leaves and stems of C. australe. 1-Epialexine is a weak inhibitor of β-mannosidase from Cellullomonas fimi.     

Elevated Levels of Asymmetric Dimethylarginine (ADMA) in the Pericardial Fluid of Cardiac Patients Correlate with Cardiac Hypertrophy

Background

Pericardial fluid (PF) contains several biologically active substances, which may provide information regarding the cardiac conditions. Nitric oxide (NO) has been implicated in cardiac remodeling. We hypothesized that L-arginine (L-Arg) precursor of NO-synthase (NOS) and asymmetric dimethylarginine (ADMA), an inhibitor of NOS, are present in PF of cardiac patients and their altered levels may contribute to altered cardiac morphology.

Methods

L-Arg and ADMA concentrations in plasma and PF, and echocardiographic parameters of patients undergoing coronary artery bypass graft (CABG, n = 28) or valve replacement (VR, n = 25) were determined.

Results

We have found LV hypertrophy in 35.7% of CABG, and 80% of VR patients. In all groups, plasma and PF L-Arg levels were higher than that of ADMA. Plasma L-Arg level was higher in CABG than VR (75.7±4.6 μmol/L vs. 58.1±4.9 μmol/L, p = 0.011), whereas PF ADMA level was higher in VR than CABG (0.9±0.0 μmol/L vs. 0.7±0.0 μmol/L, p = 0.009). L-Arg/ADMA ratio was lower in the VR than CABG (VR_plasma: 76.1±6.6 vs. CABG_plasma: 125.4±10.7, p = 0.004; VR_PF: 81.7±4.8 vs. CABG_PF: 110.4±7.2, p = 0.009). There was a positive correlation between plasma L-Arg and ADMA in CABG (r = 0.539, p = 0.015); and plasma and PF L-Arg in CABG (r = 0.357, p = 0.031); and plasma and PF ADMA in VR (r = 0.529, p = 0.003); and PF L-Arg and ADMA in both CABG and VR (CABG: r = 0.468, p = 0.006; VR: r = 0.371, p = 0.034). The following echocardiographic parameters were higher in VR compared to CABG: interventricular septum (14.7±0.5 mm vs. 11.9±0.4 mm, p = 0.000); posterior wall thickness (12.6±0.3 mm vs. 11.5±0.2 mm, p = 0.000); left ventricular (LV) mass (318.6±23.5 g vs. 234.6±12.3 g, p = 0.007); right ventricular (RV) (33.9±0.9 cm² vs. 29.7±0.7 cm², p = 0.004); right atrial (18.6±1.0 cm² vs. 15.4±0.6 cm², p = 0.020); left atrial (19.8±1.0 cm² vs. 16.9±0.6 cm², p = 0.033) areas. There was a positive correlation between plasma ADMA and RV area (r = 0.453, p = 0.011); PF ADMA and end-diastolic (r = 0.434, p = 0.015) and systolic diameter of LV (r = 0.487, p = 0.007); and negative correlation between PF ADMA and LV ejection fraction (r = -0.445, p = 0.013) in VR.

Conclusion

We suggest that elevated levels of ADMA in the PF of patients indicate upregulated RAS and reduced bioavailability of NO, which can contribute to the development of cardiac hypertrophy and remodeling.     

Fully human,HLA-DR-specific monoclonal antibodies efficiently induce programmed death of malignant lymphoid cells

The Human Combinatorial Antibody Library (HuCAL) was screened for antibodies specific to human leukocyte antigen-DR (HLA-DR) that induce programmed death of lymphoma/leukemia cells expressing the target antigen. The active Fab fragments were affinity-matured, and engineered to IgG(4) antibodies of sub-nanomolar affinity. The antibodies exhibited potent in vitro tumoricidal activity on several lymphoma and leukemia cell lines and on chronic lymphocytic leukemia patient samples. They were also active in vivo in xenograft models of non-Hodgkin lymphoma. Cell death occurred rapidly, without the need for exogenous immunological effector mechanisms, and was selective to activated/tumor-transformed cells. Although the expression of HLA-DR on normal hematopoietic cells is a potential safety concern, the antibodies caused no long-lasting hematological toxicity in primates, in vivo. Such monoclonal antibodies offer the potential for a novel therapeutic approach to lymphoid malignancies.