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.

The Danger Signal Extracellular ATP Is Involved in the Immunomediated Damage of α-Sarcoglycan-Deficient Muscular Dystrophy.

In muscular dystrophies, muscle membrane fragility results in a tissue-specific increase of danger-associated molecular pattern molecules (DAMPs) and infiltration of inflammatory cells. The DAMP extracellular ATP (eATP) released by dying myofibers steadily activates muscle and immune purinergic receptors exerting dual negative effects: a direct damage linked to altered intracellular calcium homeostasis in muscle cells and an indirect toxicity through the triggering of the immune response and inhibition of regulatory T cells. Accordingly, pharmacologic and genetic inhibition of eATP signaling improves the phenotype in models of chronic inflammatory diseases. In α-sarcoglycanopathy, eATP effects may be further amplified because α-sarcoglycan extracellular domain binds eATP and displays an ecto-ATPase activity, thus controlling eATP concentration at the cell surface and attenuating the magnitude and/or the duration of eATP-induced signals. Herein, we show that in vivo blockade of the eATP/P2X purinergic pathway by a broad-spectrum P2X receptor-antagonist delayed the progression of the dystrophic phenotype in α-sarcoglycan-null mice. eATP blockade dampened the muscular inflammatory response and enhanced the recruitment of forkhead box protein P3-positive immunosuppressive regulatory CD4+ T cells. The improvement of the inflammatory features was associated with increased strength, reduced necrosis, and limited expression of profibrotic factors, suggesting that pharmacologic purinergic antagonism, altering the innate and adaptive immune component in muscle infiltrates, might provide a therapeutic approach to slow disease progression in α-sarcoglycanopathy.

Human Natural Killer Receptors, Co-Receptors, and Their Ligands.

In the last 20 years, the study of human natural killer (NK) cells has moved from the first molecular characterizations of very few receptor molecules to the identification of a plethora of receptors displaying surprisingly divergent functions. We have contributed to the description of inhibitory receptors and their signaling pathways, important in fine regulation in many cell types, but unknown until their discovery in the NK cells. Inhibitory function is central to regulating NK-mediated cytolysis, with different molecular structures evolving during speciation to assure its persistence. More recently, it has become possible to characterize the NK triggering receptors mediating natural cytotoxicity, unveiling the existence of a network of cellular interactions between effectors of both natural and adaptive immunity. This unit reviews the contemporary history of molecular studies of receptors and ligands involved in NK cell function, characterizing the ligands of the triggering receptor and the mechanisms for finely regulating their expression in pathogen-infected or tumor cells. © 2018 by John Wiley & Sons, Inc.

HHV-8 DNA replication correlates with the clinical status in AIDS-related Kaposi's sarcoma.

BACKGROUND: The value of plasma levels of human herpesvirus 8 (HHV-8) DNA as a marker of clinical status in acquired immunodeficiency syndrome-related Kaposi's sarcoma (AIDS-KS) remains to be elucidated. OBJECTIVES: To investigate the relationship between the plasma HHV-8 DNA viral load and the clinical status of AIDS-KS. STUDY DESIGN: A total of 378 blood samples were obtained from 62 patients with AIDS-KS followed longitudinally. All patients received antiretroviral therapy (ART) or anti-neoplastic therapy. The patients were divided into four groups according to their clinical status: onset disease (OD), progressive disease (PD), stable or partial remission (S/PR) and complete remission (CR). RESULTS: Plasma HHV-8 DNAaemia was detected in all samples obtained from patients with OD or PD (100%); in contrast, HHV-8 DNAaemia was found only in a minority of patients with CR (8%) and was invariably undetectable in patients with stable CR. HHV-8 DNA detection in plasma was strongly associated with an unfavourable outcome (odds ratio=231.9; p<0.0001). Conversely, neither the HIV-1 viral load nor peripheral CD4(+) T-cell counts were associated with the KS clinical status, though both parameters did affect HHV-8 DNAaemia levels (p<0.0001). Multivariate analysis confirmed that HHV-8 DNAaemia was strongly and independently correlated with both clinical status (p<0.05) and HIV-1 plasma viraemia (p=0.027). CONCLUSIONS: The strong association of plasma HHV-8 DNAaemia with onset or progressive disease is compatible with an active role of replicating virus in clinically active AIDS-KS. An accurate evaluation of the plasma HHV-8 load might be useful for monitoring AIDS-KS under antiretroviral or antineoplastic therapy.

Sialic acid-binding Ig-like lectin-7 interacts with HIV-1 gp120 and facilitates infection of CD4pos T cells and macrophages.

BACKGROUND: Sialic acid-binding Ig-like lectin-7 (Siglec-7) expression is strongly reduced on natural killer (NK) cells from HIV-1 infected viremic patients. To investigate the mechanism(s) underlying this phenomenon, we hypothesized that Siglec-7 could contribute to the infection of CD4pos target cells following its interaction with HIV-1 envelope (Env) glycoprotein 120 (gp120). RESULTS: The ability of Siglec-7 to bind gp120 Env in a sialic acid-dependent manner facilitates the infection of both T cells and monocyte-derived macrophages (MDMs). Indeed, pre-incubation of HIV-1 with soluble Siglec-7 (sSiglec-7) increases the infection rate of CD4pos T cells, which do not constitutively express Siglec-7. Conversely, selective blockade of Siglec-7 markedly reduces the degree of HIV-1 infection in Siglec-7pos MDMs. Finally, the sSiglec-7 amount is increased in the serum of AIDS patients with high levels of HIV-1 viremia and inversely correlates with CD4pos T cell counts. CONCLUSIONS: Our results show that Siglec-7 binds HIV-1 and contributes to enhance the susceptibility to infection of CD4pos T cells and MDMs. This phenomenon plays a role in HIV-1 pathogenesis and in disease progression, as suggested by the inverse correlation between high serum level of sSiglec-7 and the low CD4pos T cell count observed in AIDS patients in the presence of chronic viral replication.

B-cell subset alterations and correlated factors in HIV-1 infection.

OBJECTIVES: During HIV-1 infection, the development, phenotype, and functionality of B cells are impaired. Transitional B cells and aberrant B-cell populations arise in blood, whereas a declined percentage of resting memory B cells is detected. Our study aimed at pinpointing the demographic, immunological, and viral factors driving these pathological findings, and the role of antiretroviral therapy in reverting these alterations. DESIGN: B-cell phenotype and correlating factors were evaluated. METHODS: Variations in B-cell subsets were evaluated by flow cytometry in HIV-1-infected individuals naive to therapy, elite controllers, and patients treated with antiretroviral drugs (virological control or failure). Multivariable analysis was performed to identify variables independently associated with the B-cell alterations. RESULTS: Significant differences were observed among patients' groups in relation to all B-cell subsets. Resting memory B cells were preserved in patients naive to therapy and elite controllers, but reduced in treated patients. Individuals naive to therapy and experiencing multidrug failure, as well as elite controllers, had significantly higher levels of activated memory B cells compared to healthy controls. In the multivariate analysis, plasma viral load and nadir CD4 T cells independently correlated with major B-cell alterations. Coinfection with hepatitis C but not hepatitis B virus also showed an impact on specific B-cell subsets. Successful protracted antiretroviral treatment led to normalization of all B-cell subsets with exception of resting memory B cells. CONCLUSION: Our results indicate that viremia and nadir CD4 T cells are important prognostic markers of B-cell perturbations and provide evidence that resting memory B-cell depletion during chronic infection is not reverted upon successful antiretroviral therapy.

Comparison of oncogenic HPV type-specific viral DNA load and E6/E7 mRNA detection in cervical samples: results from a multicenter study.

High-risk human papillomavirus (HR-HPV) genotype viral load and E6/E7 mRNA detection are proposed as surrogate markers of malignant cervical lesion progression. Currently, the use of commercially available DNA-based or mRNA-based tests is under investigation. In this study, the viral DNA load and E6/E7 mRNA detection of the five most common HR-HPV types detected in cervical cancer worldwide were compared in 308 cervical samples by using in-house type-specific quantitative real-time PCR assays and PreTect HPV-Proofer test, respectively. Sensitivity and negative predictive values were higher for the HPV-DNA assays combined (95.0% and 96.0%, respectively) than the RNA assays (77.0% and 88.0%, respectively); conversely, the mRNA test showed a higher specificity and higher positive predictive value (81.7% and 66.9%, respectively) than the DNA test (58.6% and 52.5%, respectively) for detecting histology-confirmed high-grade cervical intraepithelial neoplasia. A significantly higher association between viral DNA load and severity of disease was observed for HPV 16 and 31 (γ = 0.62 and γ = 0.40, respectively) than for the other HPV types screened. A good degree of association between the two assays was found for detection of HPV 16 (k = 0.83), HPV 18 (k = 0.72), HPV 33 (k = 0.66), and HPV 45 (k = 0.60) but not for HPV 31 (k = 0.24). Sequence analysis in L1 and E6-LCR regions of HPV 31 genotypes showed a high level of intra-type variation. HR-HPV viral DNA load was significantly higher in E6/E7 mRNA positive than negative samples (P < 0.001), except for HPV 31. These findings suggest that transcriptional and replicative activities can coexist within the same sample.

Molecular study of human herpesvirus 6 and 8 involvement in coronary atherosclerosis and coronary instability.

Several lines of evidence suggest the involvement of infectious agents in the pathogenesis of atherosclerosis. Furthermore, a correlation between infection-driven inflammatory burden and acute manifestation of coronary artery disease has been hypothesized. The aim of this work was to assess whether human herpesvirus (HHV)-6 and HHV-8, two DNA viruses with a distinct tropism for endothelium and lymphocytes, may be associated with coronary instability. An age- and gender-matched cross-sectional study was undertaken in 70 patients with testing of plasma HHV-6 and HHV-8 DNA load in different cardiovascular clinical settings: 29 patients with acute myocardial infarction, 21 patients with stable coronary artery disease, and 20 patients without coronary and carotid artery atherosclerosis subjected to cardiac valve replacement. In all patients, HHV-6 and HHV-8 plasma DNA was tested by using highly sensitive, calibrated quantitative real-time PCR assays which employ a synthetic DNA calibrator to adjust for DNA extraction and amplification efficiency. HHV-8 viremia was undetectable in all three groups. HHV-6 viremia was detected in a substantial fraction of the samples examined (18.6%) without significant differences among the three groups (ST segment elevation myocardial infarction: 17.2%; stable coronary artery disease: 14.3%; patients without coronary and carotid artery atherosclerosis: 25%). Furthermore, no significant differences in plasma HHV-6 load were observed amongst the three groups of patients. These findings indicate that coronary instability is not associated specifically with active HHV-6 or HHV-8 infection. However, an unusually high rate of active HHV-6 infection was documented among patients without atherosclerosis admitted to hospital with cardiac disease.

A new antigen scanning strategy for monitoring HIV-1 specific T-cell immune responses.

Delineation of the immune correlates of protection in natural infection or after vaccination is a mandatory step for vaccine development. Although the most recent techniques allow a sensitive and specific detection of the cellular immune response, a consensus on the best strategy to assess their magnitude and breadth is yet to be reached. Within the AIDS Vaccine Integrated Project (AVIP http://www.avip-eu.org) we developed an antigen scanning strategy combining the empirical-based approach of overlapping peptides with a vast array of database information. This new system, termed Variable Overlapping Peptide Scanning Design (VOPSD), was used for preparing two peptide sets encompassing the candidate HIV-1 vaccine antigens Tat and Nef. Validation of the VOPSD strategy was obtained by direct comparison with 15mer or 20mer peptide sets in a trial involving six laboratories of the AVIP consortium. Cross-reactive background responses were measured in 80 HIV seronegative donors (HIV-), while sensitivity and magnitude of Tat and Nef-specific T-cell responses were assessed on 90 HIV+ individuals. In HIV-, VOPSD peptides generated background responses comparable with those of the standard sets. In HIV-1+ individuals the VOPSD pools showed a higher sensitivity in detecting individual responses (Tat VOPSD vs. Tat 15mers or 20mers: p≤0.01) as well as in generating stronger responses (Nef VOPSD vs. Nef 20mers: p<0.001) than standard sets, enhancing both CD4 and CD8 T-cell responses. Moreover, this peptide design allowed a marked reduction of the peptides number, representing a powerful tool for investigating novel HIV-1 candidate vaccine antigens in cohorts of HIV-seronegative and seropositive individuals.

A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2.

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1µg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

Human leukocyte antigen-B (-Bw6/-Bw4 I80, T80) and human leukocyte antigen-C (-C1/-C2) subgrouping using pyrosequence analysis.

Specific combinations of killer immunoglobulin-like receptors (KIR) and human leukocyte antigen (HLA) class I molecules characterized by a particular residue 80 are significantly associated with outcomes in different pathologic conditions, such as autoimmunity, pathogenic infection, cancer, and reproductive failure. Thus, a simplified method for HLA typing used in association with the analysis of KIR genotype (Kirotype) is of particular interest to extend the analysis of larger series. Here, we describe a quick and inexpensive method that allows use of pyrosequencing, a helpful subtyping of HLA class I molecules, into HLA-Bw6, -Bw4 I(80) or -Bw4 T(80), HLA-C1, or -C2 groups and HLA-A allotypes sharing Bw4+ epitope or the rare HLA-B allotypes displaying the C1 motif. In particular, this analysis is focused on the amino acids around residue 80, known to be relevant in defining the affinity of KIR/HLA interaction and in the functional effects. This method was demonstrated to have good sensitivity, specificity, and reproducibility of detection and it was validated using a panel of HLA-typed International Histocompatibility Workshop (IHW) cell lines and clinical isolates. Using an allele quantitative acquisition mode, the method permitted us to obtain an accurate sequencing as required in heterozygous and/or homozygous sample definition.

Preparing for phase II/III HIV vaccine trials in Africa.

Before performing phase II/III clinical trials in Africa, preliminary studies, including assessment and building up of clinical and laboratory infrastructures, estimates of human immunodeficiency virus incidence, investigation of the background immune response, and evaluation of the cross-clade immune response, need to be done. Plans and ongoing work in the context of the AIDS Vaccine Integrated Project and some preliminary data are presented.

A relapsing inflammatory syndrome and active human herpesvirus 8 infection.

We describe an immunocompetent 61-year-old woman who was negative for human immunodeficiency virus and who had recurrent human herpesvirus 8 (HHV-8) infection associated with a relapsing systemic inflammatory syndrome characterized by fever, lymphadenopathy, splenomegaly, edema, arthrosynovitis, and rash. Kaposi's sarcoma developed 10 months after the initial clinical presentation. A correlation was documented between the recurrent clinical manifestations and the HHV-8 load in plasma and peripheral-blood mononuclear cells. Histologic examination of an enlarged lymph node heavily infected with HHV-8 revealed an atypical lymphoproliferative disorder characterized by paracortical hyperplasia and collapsed primary and secondary follicles.

Human herpesvirus 8 (HHV-8/KSHV) and hematologic malignancies.

Human herpesvirus 8 (HHV-8), also defined Kaposi's sarcoma (KS)-associated herpesvirus, was identified by Chang and colleagues in 1994 using purely molecular techniques, before any serological evidence or virus isolation in cell culture could be achieved. HHV-8 is unique among herpesviruses because its prevalence in the general population is low and because it possesses the richest weaponry of viral oncogenes and tumor-promoting factors ever described. Eleven HHV-8-specific genes are homologs of cellular genes, which were hijacked from the host during a long parallel evolution, and at least five of such genes show both in vitro and in vivo transforming ability. HHV-8 is the causative agent of KS, but it has also been associated with different hematologic malignancies, including primary effusion lymphoma (PEL), multicentric Castelman's disease (MCD), MCD-related immunoblastic/plasmablastic lymphoma and various atypical lymphoproliferative disorders. Although low-level silent infection was detected in bone marrow stromal cells from patients with multiple myeloma, a role of HHV-8 in this disease is unlikely. As seen with KS, the incidence of HHV-8-associated lymphoproliferative disorders is increased in the setting of human immunodeficiency virus infection.

Calibrated real-time PCR assay for quantitation of human herpesvirus 8 DNA in biological fluids.

Accurate laboratory tests for the diagnosis of active human herpesvirus 8 (HHV-8) infection are becoming essential to study the pathogenesis of HHV-8-associated tumors and for the clinical management of HHV-8-infected individuals. We have developed a highly sensitive, calibrated quantitative real-time PCR assay for the measurement of cell-free HHV-8 DNA in body fluids, based on the addition of a synthetic DNA calibrator prior to DNA extraction. The calibrator controls each sample for the presence of PCR inhibitors, determines a cutoff value of sensitivity for negative samples, and normalizes positive samples for the efficiency of DNA recovery. The assay shows a wide dynamic range of detection (between 1 and 10(6) viral genome equivalents/reaction) and a high degree of accuracy even in the presence of high amounts (up to 1 micro g) of human genomic DNA. Moreover, the assay has a very high sensitivity (lower detection limit, 10 genome equivalents/ml) and a high degree of reproducibility and repeatability with a coefficient of variation (CV) of <15 and 23%, respectively. Furthermore, the use of the calibrator improves the accuracy of quantitation and decreases the intersample variability (CV, 9 and 6%, respectively). The sensitivity and specificity of the assay were tested with a series of clinical specimens obtained from patients affected by various HHV-8-related diseases, as well as from a wide number of controls. In conclusion, our calibrated real-time PCR assay provides a reliable high-throughput method for quantitation of HHV-8 DNA in clinical and laboratory specimens.

Retrospective analysis of HHV-8 viremia and cellular viral load in HIV-seropositive patients receiving interleukin 2 in combination with antiretroviral therapy.

The combination of interleukin 2 (IL-2) and antiretroviral therapy (ART) represents an emerging strategy in the treatment of patients infected with HIV. Aside from its immunomodulatory role, however, IL-2 may induce replication of human herpesvirus 8 (HHV-8)/Kaposi sarcoma (KS)-associated herpesvirus. We retrospectively evaluated HHV-8 plasma viremia and cellular load, as well as anti-HHV-8 antibody titers, in sequential samples from 84 patients receiving ART alone or in combination with IL-2. At baseline, HHV-8 plasma viremia was present only in 2 HHV-8-seropositive patients in whom KS subsequently developed during or immediately after termination of IL-2 therapy. The level of viremia increased during follow-up and peaked at the time of the clinical manifestation of KS. Moreover, transient peaks of HHV-8 viremia were temporally associated with administration of IL-2. HHV-8 plasma viremia was never detected in the other 47 patients receiving IL-2 nor in 35 controls treated only with ART. Thus, IL-2 therapy seems safe in most patients infected with both HIV and HHV-8, except for those with detectable HHV-8 viremia, who may not be eligible for IL-2 treatment.

Qualitative and quantitative polymerase chain reaction detection of the residual myeloma cell contamination after positive selection of CD34+ cells with small- and large-scale Miltenyi cell sorting system.

The purging efficacy of the Miltenyi sorting system was evaluated by qualitative and TaqMan quantitative polymerase chain reaction (PCR) in myeloma patients, using immunoglobulin genes. After small-scale selection, qualitative PCR showed that in 6 of 12 leukaphereses myeloma cells were no longer detectable. Envisaging a possible clinical application, the leukaphereses from three patients underwent large-scale selection. Qualitative PCR showed that myeloma cells were still detectable. Quantitative PCR, performed in two patients, showed a tumour depletion of 1 and 2 logs respectively. Although numbers are small, the promising results obtained with small-scale selection were not reproduced in large-scale experiments.