Sex-Specific HLA Alleles Contribute to the Modulation of COVID-19 Severity.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, responsible for Coronavirus Disease 2019 (COVID-19), exhibits a spectrum of clinical manifestations, ranging from asymptomatic to severe pulmonary dysfunction or death. The variability in COVID-19 severity has largely been attributed to the host's genetic characteristics, suggesting a polygenic genetic architecture, without significant strong evidence of sex-related genetic differences. In this Italian retrospective case-control study, we investigated the association between COVID-19 severity (severe vs.

Using gold nanoparticles for enhanced intradermal delivery of poorly soluble auto-antigenic peptides.

Ultra-small 1-2 nm gold nanoparticles (NP) were conjugated with a poorly-soluble peptide auto-antigen, associated with type 1 diabetes, to modify the peptide pharmacokinetics, following its intradermal delivery. Peptide distribution was characterized, in vivo, after delivery using either conventional intradermal injection or a hollow microneedle device. The poorly-soluble peptide was effectively presented in distant lymph nodes (LN), spleen and draining LN when conjugated to the nanoparticles, whereas peptide alone was only presented in the draining LN.

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PI peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy.

IntegraGen SA.

IntegraGen SA is at the stage of commercializing a series of innovative IntegraTests™ to position itself as a leader within the rapidly growing market of predictive medicine. By applying its proprietary gene-mapping technology GenomeHIP™ (Genome Hybrid Identity Profiling) in premier patient collections, IntegraGen has rapidly discovered novel genes and genetic markers associated with a variety of complex, multifactorial diseases to use in its IntegraTests - a new class of personalized medicine diagnostics. IntegraTests provide prediction, prevention, detailed diagnosis and tailored treatment of complex diseases.

Transcript imaging of the development of human T helper cells using oligonucleotide arrays.

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells at the site of inflammation. Understanding the genetic program that controls the functional properties of T helper type 1 (Th1) versus T helper type 2 (Th2) cells may provide insight into the pathophysiology of inflammatory diseases. We compared the gene-expression profiles of human Th1 and Th2 cells using high-density oligonucleotide arrays with the capacity to display transcript levels of 6,000 human genes.

Tetracycline-responsive gene expression in mouse brain after amplicon-mediated gene transfer.

Amplicon vectors incorporate genetic elements from Herpes simplex virus (HSV) in a plasmid form which is packaged into virions in the presence of a replication-defective helper virus. We constructed a new amplicon vector, pHermes-tet-lacZ, that carries the bacterial beta-galactosidase (lacZ) gene under the control of a minimal promoter preceded by a heptameric tetracycline operator. The minimal promoter element is activated by a tetracycline-responsive hybrid protein, the gene for which is also present in the vector.

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe.

Presenilins are processed by caspase-type proteases.

Presenilin 1 (PS1) and presenilin 2 (PS2) are endoproteolytically processed in vivo and in cell transfectants to yield 27-35-kDa N-terminal and 15-24-kDa C-terminal fragments. We have studied the cleavage of PS1 and PS2 in transiently and stably transfected hamster kidney and mouse and human neuroblastoma cells by immunoblot and pulse-chase experiments. C-terminal fragments were isolated by affinity chromatography and SDS-polyacrylamide gel electrophoresis and sequenced.

Emergence of resistant variants of HIV in vivo during monotherapy with the proteinase inhibitor saquinavir.

We examined the phenotypic and genotypic properties of virus from the peripheral blood mononuclear cells (PBMC) and plasma of eight HIV-1-infected asymptomatic patients before and during monotherapy with the proteinase inhibitor saquinavir. Susceptibility of primary isolates to drug was assessed in PBMC culture by deriving IC50 and IC90 values. The observed increases in IC50 and IC90 after approximately one year of therapy with a dosage of 600 mg tds suggests the presence of virus resistant to saquinavir in vivo.

Evaluation of hepatitis C virus envelope proteins expressed in E. coli and insect cells for use as tools for antibody screening.

Background/methods: The two envelope proteins of hepatitis C virus, E1 and E2, were expressed in E. coli and, as secretory proteins, in Sf9 insect cells using recombinant baculoviruses. Co-infection of insect cells with E1 and E2-recombinant baculoviruses was performed, which has been shown to result in formation of E1-E2 dimers.

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA.

Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase.

The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns.

In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: mutations, kinetics, and frequencies.

Resistance to saquinavir (Ro 31-8959), an inhibitor of human immunodeficiency virus type I proteinase, was studied in peripheral blood mononuclear cell-derived proviral DNA from patients undergoing prolonged treatment. A Leu90-->Met exchange was the predominant resistance mutation in vivo; Gly48-->Val or doubly mutant virus was rarely observed. After 8-12 months of treatment with saquinavir alone (600 mg, 3 times/day) or in combination with zidovudine (200 mg, 3 times/day), approximately 45% of all patients carried provirus with mutant proteinase; the incidence was lower (22%) in patients treated with a combination of saquinavir, zidovudine, and dideoxycytidine.

Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inducer of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected cells. The aim of this study was to investigate the role played by the two known TNF-alpha receptors, TNFR-p55 and TNFR-p75, in the activation of HIV-1 expression. As a model system the latently infected human promonocytic cell line U1 was stimulated with wild-type TNF-alpha, with TNF-alpha muteins that specifically bind to one or the other receptor or with receptor-specific monoclonal antibodies.

Reduced sensitivity to saquinavir: an update on genotyping from phase I/II trials.

A genotypic analysis of the HIV-1 proteinase was performed on clinical specimen obtained from patients after different periods of Saquinavir (SQV) treatment. Proteinase genes of integrated proviral DNA from PBMC were isolated by PCR, cloned and individual sequences were obtained. Genotypic resistance was defined by the Gly48-->Val and Leu90-->Met exchanges.

An in vitro assay for hepatitis C virus NS3 serine proteinase.

Hepatitis C virus (HCV) encodes a polyprotein of which the majority of nonstructural proteins are matured by the viral serine proteinase located in the N terminus of NS3. Intracellular studies using recombinant vaccinia virus have shown that both NS3 and its cofactor NS4A are required to enhance processing at the NS3-dependent cleavage sites. We developed an in vitro (cell-free) assay in which the HCV serine proteinase was shown to be enzymatically active, by mixing lysates of cells expressing either the serine proteinase or a nonstructural protein substrate.

Characterization of human immunodeficiency virus type 1 mutants with decreased sensitivity to proteinase inhibitor Ro 31-8959.

A human immunodeficiency virus type 1 (HIV-1) variant with highly reduced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase, has been selected by repeated passage of wild-type virus in CEM cells in the presence of increasing concentrations of the inhibitor. Peptide sequences of the proteinase of selected virus were obtained from proviral DNA. Sequence comparison to wild-type (wt) proteinase demonstrated two amino acid substitutions in the resistant virus, a Gly to Val exchange at position 48 and a Leu to Met exchange at position 90.

Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase.

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans.

Identification of an amino acid substitution involved in the reduction of sensitivity of HIV-1 to an inhibitor of viral proteinase.

Clones derived from HIV variants previously characterized as resistant to Ro31-8959, an inhibitor of viral proteinase (PR), were sequenced. Substitution of glycine by valine at position 48 of the PR protein was found. None of the 20 clones derived from wild type HTLV-IIIB contain this mutation.

Kinetic and structural analyses of hepatitis C virus polyprotein processing.

Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites.

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency.

Conserved residues Pro-109 and Asp-116 are required for interaction of the human immunodeficiency virus type 1 integrase protein with its viral DNA substrate.

The human immunodeficiency virus type 1 integrase protein can be specifically cross-linked to viral long terminal repeat substrate oligonucleotides in vitro by using UV light. Site-directed mutagenesis and deletion analyses were used to define the domains involved in the interaction of integrase with the viral DNA substrate. Our results showed that mutation of conserved residues Pro-109 and Asp-116, which are found to be critical for the endonuclease and integration activities of IN protein, abolished the ability of the protein to cross-link to its DNA substrate.