beta-estradiol attenuates the anti-HIV-1 efficacy of Stavudine (D4T) in primary PBL

<p>Abstract</p> <p>Background</p> <p>Female hormones are known to play an important role in predisposition for many infectious diseases. Recent work suggests there are gender effects in HIV/AIDS progression. Here we ask whether the sex steroid hormone β-estradiol affects the replication of HIV-1 or the efficacy of a common anti-retroviral drug, Stavudine (D4T).</p> <p>Results</p> <p>Human PBL were infected with HIV-1 in the presence or absence of combinations of sex steroid hormones and the anti-retroviral drug, D4T. After seven days in culture, viral supernatants were assayed for HIV-1 p24 protein. β-estradiol resulted in a modest inhibition of HIV-1 replication of ~26%. However, 2 nM β-estradiol increased the amount of HIV-1 replication in the presence of 50 nM D4T from a baseline of 33% (+/- SE = 5.4) to 74% (+/- SE = 5.4) of control virus levels in the absence of drug. Both results were statistically highly significant (p < 0.001). β-estradiol did not increase the replication of a D4T-resistant strain of HIV in the presence of D4T. The effects were unlikely to be due to general cell inhibition or toxicity because these concentrations of drug and hormone cause no cytotoxicity in PBL as measured by trypan blue exclusion.</p> <p>Conclusion</p> <p>β-estradiol inhibited both HIV-1 replication in primary human PBL and the antiretroviral efficacy of D4T in PBL cultures. To optimize antiretroviral drug therapy, it may be necessary to monitor patient hormonal status.</p

Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps that may Compromise Product Quality

Therapeutic protein products provide unique and effective treatments for numerous human diseases and medical conditions. In many cases, these treatments are used chronically to slow disease progression, reduce morbidity and/or to replace essential proteins that are not produced endogenously in patients. Therefore, any factor that reduces or eliminates the effectiveness of the treatment can lead to patient suffering and even death. One means by which efficacy of therapeutic proteins can be compromised is by an immune response, resulting in antibody-mediated neutralization of the protein’s activity or alterations in bioavailability.1,2 For example, in the case of treatment of hemophilia A, neutralizing antibodies to Factor VIII can cause life-threatening bleeding episodes, resulting in significant morbidity and necessitating treatment with a prolonged course of a tolerance-inducing therapy to reverse immunity.3,4 In other cases, drug-induced antibodies to a therapeutic version of an endogenous protein can cross-react with and neutralize the patient’s endogenous protein. If the endogenous protein serves a non-redundant biological function, such an immune response can have devastating results. For example, pure red cell aplasia can result from neutralizing antibodies to epoetin alpha. 1,2 It is well established that protein aggregates in therapeutic protein products can enhance immunogenicity2, and such an effect is therefore an important risk factor to consider when assessing product quality. The purpose of this commentary is to accomplish the following: i. provide brief summaries on the factors affecting protein aggregation and the key aspects of protein aggregates that are associated with immunogenicity; ii. emphasize the current scientific gaps in understanding and analytical limitations for quantitation of species of large protein aggregates that are referred to as subvisible particles, with specific consideration of those particles 0.1–10 μm in size; iii. offer a rationale for why these gaps may compromise the safety and/or efficacy of a product; iv. provide scientifically sound, risked based recommendations/conclusions for assessment and control of such aggregate species

A combined functional and structural genomics approach identified an EST-SSR marker with complete linkage to the Ligon lintless-2 genetic locus in cotton (Gossypium hirsutum L.)

<p>Abstract</p> <p>Background</p> <p>Cotton fiber length is an important quality attribute to the textile industry and longer fibers can be more efficiently spun into yarns to produce superior fabrics. There is typically a negative correlation between yield and fiber quality traits such as length. An understanding of the regulatory mechanisms controlling fiber length can potentially provide a valuable tool for cotton breeders to improve fiber length while maintaining high yields. The cotton (<it>Gossypium hirsutum </it>L.) fiber mutation Ligon lintless-2 is controlled by a single dominant gene (<it>Li₂</it>) that results in significantly shorter fibers than a wild-type. In a near-isogenic state with a wild-type cotton line, <it>Li₂</it>is a model system with which to study fiber elongation.</p> <p>Results</p> <p>Two near-isogenic lines of Ligon lintless-2 (<it>Li₂</it>) cotton, one mutant and one wild-type, were developed through five generations of backcrosses (BC₅). An F₂population was developed from a cross between the two <it>Li₂</it>near-isogenic lines and used to develop a linkage map of the <it>Li₂</it>locus on chromosome 18. Five simple sequence repeat (SSR) markers were closely mapped around the <it>Li₂</it>locus region with two of the markers flanking the <it>Li₂</it>locus at 0.87 and 0.52 centimorgan. No apparent differences in fiber initiation and early fiber elongation were observed between the mutant ovules and the wild-type ones. Gene expression profiling using microarrays suggested roles of reactive oxygen species (ROS) homeostasis and cytokinin regulation in the <it>Li₂</it>mutant phenotype. Microarray gene expression data led to successful identification of an EST-SSR marker (NAU3991) that displayed complete linkage to the <it>Li₂</it>locus.</p> <p>Conclusions</p> <p>In the field of cotton genomics, we report the first successful conversion of gene expression data into an SSR marker that is associated with a genomic region harboring a gene responsible for a fiber trait. The EST-derived SSR marker NAU3991 displayed complete linkage to the <it>Li₂</it>locus on chromosome 18 and resided in a gene with similarity to a putative plectin-related protein. The complete linkage suggests that this expressed sequence may be the <it>Li₂</it>gene.</p

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal

Heme oxygenase-1 induction alters chemokine regulation and ameliorates human immunodeficiency virus-type-1 infection in lipopolysaccharide-stimulated macrophages

We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed by transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1β) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1β, and LD78β chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM. © 2013

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production