Stability and Expression Levels of HLA-C on the Cell Membrane Modulate HIV-1 Infectivity.

HLA-C expression is associated with a differential ability to control HIV-1 infection. Higher HLA-C levels may lead to better control of HIV-1 infection through both a higher efficiency of antigen presentation to cytotoxic T lymphocytes and the triggering of activating killer immunoglobulin-like receptors on NK cells, whereas lower levels may provide poor HIV-1 control and rapid progression to AIDS. We characterized the relative amounts of HLA-C heterotrimers (heavy chain/ß2 microglobulin [ß2m]/peptide) and HLA-C free heavy chains on peripheral blood mononuclear cells (PBMCs) from healthy blood donors harboring both alleles with stable or unstable binding to ß2m/peptide. We analyzed the stability of HLA-C heterotrimers of different allotypes and the infectivity of HIV-1 virions produced by PBMCs with various allotypes. We observed significant differences in HLA-C heterotrimer stability and in expression levels. We found that R5 HIV-1 virions produced by PBMCs harboring unstable HLA-C alleles were more infectious than those produced by PBMCs carrying the stable variants. We propose that HIV-1 infectivity might depend both on the amounts of HLA-C molecules and on their stability as trimeric complex. According to this model, individuals with low-expression HLA-C alleles and unstable binding to ß2m/peptide might have worse control of HIV-1 infection and an intrinsically higher capacity to support viral replication.IMPORTANCE Following HIV-1 infection, some people advance rapidly to AIDS while others have slow disease progression. HLA-C, a molecule involved in immunity, is a key determinant of HIV-1 control. Here we reveal how HLA-C variants contribute to the modulation of viral infectivity. HLA-C is present on the cell surface in two different conformations. The immunologically active conformation is part of a complex that includes ß2 microglobulin/peptide; the other conformation is not bound to ß2 microglobulin/peptide and can associate with HIV-1, increasing its infectivity. Individuals with HLA-C variants with a predominance of immunologically active conformations would display stronger immunity to HIV-1, reduced viral infectivity and effective control of HIV-1 infection, while subjects with HLA-C variants that easily dissociate from ß2 microglobulin/peptide would have a reduced immunological response to HIV-1 and produce more infectious virions. This study provides new information that could be useful in the design of novel vaccine strategies and therapeutic approaches to HIV-1.

The Stochastic Complexity of Spin Models: Are Pairwise Models Really Simple?

Models can be simple for different reasons: because they yield a simple and computationally efficient interpretation of a generic dataset (e.g., in terms of pairwise dependencies)-as in statistical learning-or because they capture the laws of a specific phenomenon-as e.g., in physics-leading to non-trivial falsifiable predictions. In information theory, the simplicity of a model is quantified by the stochastic complexity, which measures the number of bits needed to encode its parameters. In order to understand how simple models look like, we study the stochastic complexity of spin models with interactions of arbitrary order. We show that bijections within the space of possible interactions preserve the stochastic complexity, which allows to partition the space of all models into equivalence classes. We thus found that the simplicity of a model is not determined by the order of the interactions, but rather by their mutual arrangements. Models where statistical dependencies are localized on non-overlapping groups of few variables are simple, affording predictions on independencies that are easy to falsify. On the contrary, fully connected pairwise models, which are often used in statistical learning, appear to be highly complex, because of their extended set of interactions, and they are hard to falsify.

Efficacy of a Standardized Extract of Prunus mume in Liver Protection and Redox Homeostasis: A Randomized, Double-Blind, Placebo-Controlled Study.

The antioxidant, anti-inflammatory and hepatoprotective effects of Prunus mume (PM) have previously been demonstrated. This double-blind, placebo-controlled study was designed to evaluate the influence of two doses of a food supplement, made of 150 mg of a standardized PM extract on liver transaminases, lipid profile, glycemia, neopterin and reduced and oxidized thiols in plasma and erythrocytes, during a 3-month treatment period, in healthy subjects with transaminases levels between 20 and 40 UI/L. Forty-five subjects (56.0 ± 11.6 years) were enrolled. The results showed a beneficial and statistically significant effect versus placebo of PM extract on liver function, with a decrease versus baseline in alanine aminotransferase (47%), aspartate aminotransferase (7%), gamma-glutamyl transpeptidase (15%) and glycemia (11%). The lipid profile modification was also positive with an increase versus baseline in HDL cholesterol (13%), and a decrease in LDL/HDL ratio (12%) and triglycerides (8%). The antioxidant action of PM translated into a decrease in oxidized glutathione, reduced/oxidized cysteine-glycine, oxidized cysteine (intracellular pro-oxidant) and neopterin (inflammation biomarker), was associated with an increase in reduced glutathione. These results are in favor of the use of a standardized extract of P. mume for the support of liver health and prevention of common metabolic and inflammation-based diseases. Copyright © 2016 John Wiley & Sons, Ltd.

HLA-C and HIV-1: friends or foes?

The major histocompatibility complex class I protein HLA-C plays a crucial role as a molecule capable of sending inhibitory signals to both natural killer (NK) cells and cytotoxic T lymphocytes (CTL) via binding to killer cell Ig-like receptors (KIR). Recently HLA-C has been recognized as a key molecule in the immune control of HIV-1. Expression of HLA-C is modulated by a microRNA binding site. HLA-C alleles that bear substitutions in the microRNA binding site are more expressed at the cell surface and associated with the control of HIV-1 viral load, suggesting a role of HLA-C in the presentation of antigenic peptides to CTLs. This review highlights the role of HLA-C in association with HIV-1 viral load, but also addresses the contradiction of the association between high cell surface expression of an inhibitory molecule and strong cell-mediated immunity. To explore additional mechanisms of control of HIV-1 replication by HLA-C, we address specific features of the molecule, like its tendency to be expressed as open conformer upon cell activation, which endows it with a unique capacity to associate with other cell surface molecules as well as with HIV-1 proteins.

SARS-CoV-2 vaccination elicits unconventional IgM specific responses in naïve and previously COVID-19-infected individuals.

BACKGROUND: Currently, evaluation of the IgG antibodies specific for the SARS-CoV-2 Spike protein following vaccination is used worldwide to estimate vaccine response. Limited data are available on vaccine-elicited IgM antibodies and their potential implication in immunity to SARS-CoV-2. METHODS: We performed a longitudinal study to quantify anti-S SARS-CoV-2 IgG and IgM (IgG-S and IgM-S) in health care worker (HCW) recipients of the BNT162b2 vaccine. Samples were collected before administration (T0), at the second dose (T1) and three weeks after T1 (T2). The cohort included 1584 immunologically naïve to SARS-CoV-2 (IN) and 289 with history of previous infection (PI). FINDINGS: IN showed three patterns of responses: (a) IgG positive/IgM negative (36.1%), (b) coordinated IgM-S/IgG-S responses appearing at T1 (37.4%) and (c) IgM appearing after IgG (26.3%). Coordinated IgM-S/IgG-S responses were associated with higher IgG titres. In IgM-S positive PI, 64.5% were IgM-S positive before vaccination, whereas 32% and 3.5% developed IgM-S after the first and second vaccine dose, respectively. IgM-S positive sera had higher pseudovirus neutralization titres compared to the IgM-S negative. INTERPRETATION: Coordinated expression of IgG-S and IgM-S after vaccination was associated with a significantly more efficient response in both antibody levels and virus-neutralizing activity. The unconventional IgG-S positive/IgM-S negative responses may suggest a recruitment of cross coronaviruses immunity by vaccination, warranting further investigation. FUNDING: Italian Ministry of Health under "Fondi Ricerca Corrente"- L1P5 and "Progetto Ricerca Finalizzata COVID-2020-12371675"; FUR 2020 Department of Excellence 2018-2022, MIUR, Italy; The Brain Research Foundation Verona.

Serology study after BTN162b2 vaccination in participants previously infected with SARS-CoV-2 in two different waves versus naïve.

Background: The antibody response to SARS-CoV-2 mRNA vaccines in individuals with waning immunity generated by a previous SARS-CoV-2 infection, as well as the patterns of IgA and IgM responses in previously infected and in naïve individuals are still poorly understood. Methods: We performed a serology study in a cohort of BTN162b2 mRNA vaccine recipients who were immunologically naïve (N, n = 50) or had been previously infected with SARS-CoV-2 (P.I., n = 51) during the first (n = 25) or second (n = 26) pandemic waves in Italy, respectively. We measured IgG, IgM and IgA antibodies against the SARS-CoV-2 Spike (S) and IgG against the nucleocapsid (N) proteins, as well as the neutralizing activity of sera collected before vaccination, after the first and second dose of vaccine. Results: Most P.I. individuals from the first pandemic wave who showed declining antibody titres responded to the first vaccine dose with IgG-S and pseudovirus neutralization titres that were significantly higher than those observed in N individuals after the second vaccine dose. In all recipients, a single dose of vaccine was sufficient to induce a potent IgA response that was not associated with serum neutralization titres. We observed an unconventional pattern of IgM responses that were elicited in only half of immunologically naïve subjects even after the second vaccine dose. Conclusions: The response to a single dose of vaccine in P.I. individuals is more potent than that observed in N individuals after two doses. Vaccine-induced IgA are not associated with serum neutralization.

Is Cross-Reactive Immunity Triggering COVID-19 Immunopathogenesis?

The serological responses to both SARS-CoV-1 and SARS-CoV-2 virus have some unique characteristics that suggest cross-reactive priming by other human coronaviruses (hCoVs). The early kinetics and magnitude of these responses are, in some cases, associated with worse clinical outcomes in SARS and COVID-19. Cross-reactive hCoV antibody responses have been detected in both SARS and COVID-19 patients. There is also evidence that pre-existing T cell immunity to common cold coronaviruses can prime the response to SARS-CoV-2. Studies in non-human primates show that SARS-CoV-1 S-protein vaccine-induced antibodies are associated with acute lung injury in macaques challenged with SARS-CoV-1. Here we discuss the potential of cross-reactive immunity to drive the immunopathogenesis of COVID-19 and its implications for current efforts to develop immune-based therapies and vaccines.

Microbial community dynamics in phyto-thermotherapy baths viewed through next generation sequencing and metabolomics approach.

Phyto-thermotherapy is a treatment consisting in immersing oneself in baths of self-heating alpine grass, to benefit of the heat and rich aromatic components released by the process. The aim of this study was to characterize the bacterial and fungal diversity of three phyto-thermal baths (PTB) performed in three different months, and to compare the data with the profile of the volatile organic compounds (VOCs) of the process. All the data collected showed that PTBs were structured in two stages: the first three days were characterised by an exponential rise of the temperature, a fast bacterial development, higher microbial diversity and higher concentrations of plant aliphatic hydrocarbons. The second stage was characterised by a stable high temperature, shrinkage of the microbial diversity with a predominance of few bacterial and fungi species and higher concentrations of volatiles of microbial origin. Erwinia was the dominant microbial species during the first stage and probably responsible of the self-heating process. In conclusion, PTBs has shown both similarities with common self-heating processes and important peculiarities such as the absence of pathogenic bacteria and the dominance of plant terpenoids with health characteristics among the VOCs confirming the evidence of beneficial effects in particular in the first three days.

Antioxidant Activity with Increased Endogenous Levels of Vitamin C, E and A Following Dietary Supplementation with a Combination of Glutathione and Resveratrol Precursors.

The effects of two different dietary supplements on the redox status of healthy human participants were evaluated. The first supplement (GluS, Glutathione Synthesis) contains the precursors for the endogenous synthesis of glutathione and the second (GluReS, Glutathione and Resveratrol Synthesis) contains in addition polydatin, a precursor of resveratrol. To assess the influence of GluS and GluReS on the redox status, ten thiol species and three vitamins were measured before (t0) and after 8 weeks (t1) of dietary supplementation. An inflammatory marker, neopterin, was also assessed at the same time points. Both supplements were highly effective in improving the redox status by significantly increasing the reduced-glutathione (GSH) content and other reduced thiol species while significantly decreasing the oxidized species. The positive outcome of the redox status was most significant in the GluRes treatment group which also experienced a significant reduction in neopterin levels. Of note, the endogenous levels of vitamins C, E and A were significantly increased in both treatment groups, with best results in the GluReS group. While both dietary supplements significantly contributed to recognized antioxidant and anti-inflammatory outcomes, the effects of GluReS, the combination of glutathione and resveratrol precursors, were more pronounced. Thus, dietary supplementation with GluReS may represent a valuable strategy for maintaining a competent immune status and a healthy lifespan.

HLA-C increases HIV-1 infectivity and is associated with gp120.

BACKGROUND: A recently identified genetic polymorphism located in the 5' region of the HLA-C gene is associated with individual variations in HIV-1 viral load and with differences in HLA-C expression levels. HLA-C has the potential to restrict HIV-1 by presenting epitopes to cytotoxic T cells but it is also a potent inhibitor of NK cells. In addition, HLA-C molecules incorporated within the HIV-1 envelope have been shown to bind to the envelope glycoprotein gp120 and enhance viral infectivity. We investigated this last property in cell fusion assays where the expression of HLA-C was silenced by small interfering RNA sequences. Syncytia formation was analyzed by co-cultivating cell lines expressing HIV-1 gp120/gp41 from different laboratory and primary isolates with target cells expressing different HIV-1 co-receptors. Virus infectivity was analyzed using pseudoviruses. Molecular complexes generated during cell fusion (fusion complexes) were purified and analyzed for their HLA-C content. RESULTS: HLA-C positive cells co-expressing HIV-1 gp120/gp41 fused more rapidly and produced larger syncytia than HLA-C negative cells. Transient transfection of gp120/gp41 from different primary isolates in HLA-C positive cells resulted in a significant cell fusion increase. Fusion efficiency was reduced in HLA-C silenced cells compared to non-silenced cells when co-cultivated with different target cell lines expressing HIV-1 co-receptors. Similarly, pseudoviruses produced from HLA-C silenced cells were significantly less infectious. HLA-C was co-purified with gp120 from cells before and after fusion and was associated with the fusion complex. CONCLUSION: Virionic HLA-C molecules associate to Env and increase the infectivity of both R5 and X4 viruses. Genetic polymorphisms associated to variations in HLA-C expression levels may therefore influence the individual viral set point not only by means of a regulation of the virus-specific immune response but also via a direct effect on the virus replicative capacity. These findings have implications for the understanding of the HIV-1 entry mechanism and of the role of Env conformational modifications induced by virion-associated host proteins.

Applying novel approaches for GC × GC-TOF-MS data cleaning and trends clustering in VOCs time-series analysis: Following the volatiles fate in grass baths through passive diffusion sampling.

Phytothermotherapy ("grass baths") is a traditional phytotherapy for rheumatism consisting of taking baths in hot fermenting grass. Scientific studies have demonstrated its efficiency in treating several rheumatic diseases. However the efficiency and repeatability of the therapy is dependent on the wild fermentations, determining sometimes the appearance of unpleasant conditions leading to the early abandonment of the therapy. The metabolism undergoing in the grass baths is unknown and there is not an established method to evaluate and predict grass baths quality. The aim of this study is to establish a simple VOCs profiling method able to evaluate the grass baths, predicting their evolution, through the identification of marker volatiles related to the best conditions and/or the spoilage. After replicating in real scale the traditional grass baths, the volatile profiles were measured using passive diffusion samplers injected in a thermal desorption-comprehensive GC × GC-TOF-MS. The high dimensionality of the data coupled with the limited number of time points, required a rigorous method development for the analysis of the data, achieved through the development of a novel R package for variable selection in GC × GC data matrices. The further application of a fuzzy clustering approach demonstrated to be a useful tool dealing with short time series, allowing to discard un-trending volatiles and giving a clear snapshot of the main trends in the data. A broad coverage of the volatolome was provided, thus suitable to describe the main metabolic changes ongoing in the grass baths. Coupling this data with the temperature and pH, and comparing it to the data from similar processes, like silage and compost, we demonstrated that the established method can be helpful to evaluate short time series, allowing us to obtain a list of volatiles as candidate markers for the quality of the grass baths. The established method gave a list of markers applicable to real scale grass baths to predict spoilage; furthermore it provides a list of volatiles where to search for candidate markers with reported health-related effects and can be used to generate hypothesis on the mechanisms of action of the treatment.

Corrigendum: HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity.

This corrects the article DOI: 10.1038/srep40037.

HIV-1 Env associates with HLA-C free-chains at the cell membrane modulating viral infectivity.

HLA-C has been demonstrated to associate with HIV-1 envelope glycoprotein (Env). Virions lacking HLA-C have reduced infectivity and increased susceptibility to neutralizing antibodies. Like all others MHC-I molecules, HLA-C requires ß2-microglobulin (ß2m) for appropriate folding and expression on the cell membrane but this association is weaker, thus generating HLA-C free-chains on the cell surface. In this study, we deepen the understanding of HLA-C and Env association by showing that HIV-1 specifically increases the amount of HLA-C free chains, not bound to ß2m, on the membrane of infected cells. The association between Env and HLA-C takes place at the cell membrane requiring ß2m to occur. We report that the enhanced infectivity conferred to HIV-1 by HLA-C specifically involves HLA-C free chain molecules that have been correctly assembled with ß2m. HIV-1 Env-pseudotyped viruses produced in the absence of ß2m are less infectious than those produced in the presence of ß2m. We hypothesize that the conformation and surface expression of HLA-C molecules could be a discriminant for the association with Env. Binding stability to ß2m may confer to HLA-C the ability to preferentially act either as a conventional immune-competent molecule or as an accessory molecule involved in HIV-1 infectivity.

The appealing story of HIV entry inhibitors : from discovery of biological mechanisms to drug development.

Current therapeutic intervention in HIV infection relies upon 20 different drugs. Despite the impressive efficacy shown by these drugs, we are confronted with an unexpected frequency of adverse effects, such as mitochondrial toxicity and lipodystrophy, and resistance, not only to individual drugs but to entire drug classes.Thus, there is now a great need for new antiretroviral drugs with reduced toxicity, increased activity against drug-resistant viruses and a greater capacity to reach tissue sanctuaries of the virus. Two different HIV molecules have been selected as targets of drug inhibition so far: reverse transcriptase and protease. Drugs that target the interactions between the HIV envelope and the cellular receptor complex are a 'new entry' into the scenario of HIV therapy and have recently raised great interest because of their activity against multidrug-resistant viruses. There are several compounds that are at different developmental stages in the pipeline to counter HIV entry, among them: (i) the attachment inhibitor dextrin-2-sulfate; (ii) the inhibitors of the glycoprotein (gp) 120/CD4 interaction PRO 542, TNX 355 and BMS 488043; (iii) the co-receptor inhibitors subdivided in those targeting CCR5 (SCH 417690 [SCH D], UK 427857 GW 873140, PRO 140, TAK 220, AMD 887) and those targeting CXCR4 (AMD 070, KRH 2731); and (iv) the fusion inhibitors enfuvirtide (T-20) and tifuvirtide (T-1249). The story of the first of these drugs, enfuvirtide, which has successfully completed phase III clinical trials, has been approved by the US FDA and by the European Medicines Agency, and is now commercially available worldwide, is an example of how the knowledge of basic molecular mechanisms can rapidly translate into the development of clinically effective molecules.

A simplified flow cytometry method of CD4 and CD8 cell counting based on thermoresistant reagents: implications for large scale monitoring of HIV-infected patients in resource-limited settings.

OBJECTIVE: To validate a simplified flow cytometry assay for CD4 and CD8 T cell counting based on monoclonal antibodies which are made resistant to high temperatures (simplified thermoresistant assay (STRA)). METHOD: The STRA employs FITC-conjugated anti-CD4 and anti-CD8 monoclonal antibodies, predispensed into test tubes and chemically treated to be resistant to high temperatures. Five correlation studies were performed in three different laboratories on a total of 560 blood samples from HIV-1 infected patients. Each study correlated the STRA with either double or single platform assays currently available. Accelerated stability tests on the FITC-conjugated monoclonal antibodies were performed to assess the resistance of the STRA to high temperatures. RESULTS: Comparison of STRA with both single platform and double platform assays gave correlation coefficients ranging 0.957-0.987 for CD4+ T cells and 0.946-0.968 for CD8+ T cells. In all correlation studies there was a perfect data overlapping in the low-pathological interval of CD4+ T cells (0-400 cells/ml). The FITC-conjugated CD4 and CD8 monoclonal antibodies maintained intact binding activity and fluorescence brightness after storage for 4 weeks at 45 degrees C and can be stored for up to 8 years in regular conditions (+4 degrees C). CONCLUSIONS: The STRA correlates well with both single-platform and double-platform flow-cytometry assays currently used to assess CD4+ T cells. The test procedure is simple, rapid, and easy to perform. The reagents can be stored under unfavorable environmental conditions for long period of time. These features should facilitate access to flow cytometry testing in resource-poor settings.

Comment on "Influence of HLA-C expression level on HIV control".

Apps et al. (Reports, 5 April 2013, p. 87) found that high human leukocyte antigen C (HLA-C) expression favors HIV-1 control. However, as noted here, HLA-C was assessed with a monoclonal antibody (DT9) that cross-reacts with HLA-E. In the context of the available evidence, this is consistent with the idea that the two leukocyte antigens collaborate to keep the HIV-1 virus at bay.