Identification of early-induced broadly neutralizing activities against transmitted founder HIV strains.

OBJECTIVES: Broadly neutralizing antibodies have been proposed as key actors for HIV vaccine development. However, they display features of highly matured antibodies, hampering their induction by vaccination. As protective broadly neutralizing antibodies should be induced rapidly after vaccination and should neutralize the early-transmitted founder (T/F) viruses, we searched whether such antibodies may be induced following HIV infection. DESIGN: Sera were collected during acute infection (Day 0) and at viral set point (Month 6/12) and the neutralizing activity against T/F strains was investigated. Neutralizing activity in sera collected from chronic progressor was analyzed in parallel. METHODS: We compared neutralizing activity against T/F strains with neutralizing activity against non-T/F strains using the conventional TZM-bL neutralizing assay. RESULTS: We found neutralizing antibodies (nAbs) preferentially directed against T/F viruses in sera collected shortly after infection. This humoral response evolved by shifting to nAbs directed against non-T/F strains. CONCLUSION: Although features associated with nAbs directed against T/F viruses need further investigations, these early-induced nAbs may display lesser maturation characteristics; therefore, this might increase their interest for future vaccine designs.

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion.

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.

Extremely Thermostabilizing Core Mutations in Coiled-Coil Mimetic Proteins of HIV-1 gp41 Produce Diverse Effects on Target Binding but Do Not Affect Their Inhibitory Activity.

A promising strategy to neutralize HIV-1 is to target the gp41 spike subunit to block membrane fusion with the cell. We previously designed a series of single-chain proteins (named covNHR) that mimic the trimeric coiled-coil structure of the gp41 N-terminal heptad repeat (NHR) region and potently inhibit HIV-1 cell infection by avidly binding the complementary C-terminal heptad repeat (CHR) region. These proteins constitute excellent tools to understand the structural and thermodynamic features of this therapeutically important interaction. Gp41, as with many coiled-coil proteins, contains in core positions of the NHR trimer several highly conserved, buried polar residues, the role of which in gp41 structure and function is unclear. Here we produced three covNHR mutants by substituting each triad of polar residues for the canonical isoleucine. The mutants preserve their helical structure and show an extremely increased thermal stability. However, increased hydrophobicity enhances their self-association. Calorimetric analyses show a marked influence of mutations on the binding thermodynamics of CHR-derived peptides. The mutations do not affect however the in vitro HIV-1 inhibitory activity of the proteins. The results support a role of buried core polar residues in maintaining structural uniqueness and promoting an energetic coupling between conformational stability and NHR-CHR binding.

Exposure to four chemical UV filters through contaminated sediment: impact on survival, hatching success, cardiac frequency, and aerobic metabolic scope in embryo-larval stage of zebrafish.

UV filters are widely used in many pharmaceutical and personal care products such as sunscreen and cosmetics to protect from UV irradiation. Due to their hydrophobic properties and relative stability, they have a high capacity to accumulate in sediment. Little information is available on their ecotoxicity on fish. In aquatic ecosystems, fish eggs could be directly affected by UV filters through contact with contaminated sediment. The aim of this study was to investigate the individual toxicity of four UV filters: benzophenone-3 (BP3), butyl methoxydibenzoylmethane (BM), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), and methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT), in embryo-larval stages of zebrafish Danio rerio. Fish eggs were exposed to single UV filters by contact with spiked sediment during 96 h at a concentration of 10 µg g-1. Among the four UV filters tested, BP3 was the more toxic, reducing cardiac frequency and increasing standard metabolic rate of larvae.

Assessing Climate Change Impact on Ecosystems and Infectious Disease: Important Roles for Genomic Sequencing and a One Health Perspective.

Changes in the Earth's climate and weather continue to impact the planet's ecosystems, including the interface of infectious disease agents with their hosts and vectors. Environmental disasters, natural and human-made activities raise risk factors that indirectly facilitate infectious disease outbreaks. Subsequently, changes in habitat, displaced populations, and environmental stresses that affect the survival of species are amplified over time. The recurrence and spread of vector-borne (e.g., mosquito, tick, aphid) human, animal, and plant pathogens to new geographic locations are also influenced by climate change. The distribution and range of humans, agricultural animals and plants, wildlife and native plants, as well as vectors, parasites, and microbes that cause neglected diseases of the tropics as well as other global regions are also impacted. In addition, genomic sequencing can now be applied to detect signatures of infectious pathogens as they move into new regions. Molecular detection assays complement metagenomic sequencing to help us understand the microbial community found within the microbiomes of hosts and vectors, and help us uncover mechanistic relationships between climate variability and pathogen transmission. Our understanding of, and responses to, such complex dynamics and their impacts can be enhanced through effective, multi-sectoral One Health engagement coupled with applications of both traditional and novel technologies. Concerted efforts are needed to further harness and leverage technology that can identify and track these impacts of climate changes in order to mitigate and adapt to their effects.

Effect of 10 UV Filters on the Brine Shrimp Artemia salina and the Marine Microalga Tetraselmis sp.

The presence of pharmaceutical and personal care product (PPCP) residues in the aquatic environment is an emerging issue due to their uncontrolled release through gray water, and accumulation in the environment that may affect living organisms, ecosystems and public health. The aim of this study is to assess the toxicity of benzophenone-3 (BP-3), bis-ethylhexyloxyphenol methoxyphenyl triazine (BEMT), butyl methoxydibenzoylmethane (BM), methylene bis-benzotriazolyl tetramethylbutylphenol (MBBT), 2-ethylhexyl salicylate (ES), diethylaminohydroxybenzoyl hexyl benzoate (DHHB), diethylhexyl butamido triazone (DBT), ethylhexyl triazone (ET), homosalate (HS) and octocrylene (OC) on marine organisms from two major trophic levels, including autotrophs (Tetraselmis sp.) and heterotrophs (Artemia salina). In general, results showed that both HS and OC were the most toxic UV filters for our tested species, followed by a significant effect of BM on Artemia salina due to BM-but only at high concentrations (1 mg/L). ES, BP3 and DHHB affected the metabolic activity of the microalgae at 100 µg/L. BEMT, DBT, ET, MBBT had no effect on the tested organisms, even at high concentrations (2 mg/L). OC toxicity represents a risk for those species, since concentrations used in this study are 15-90 times greater than those reported in occurrence studies for aquatic environments. For the first time in the literature, we report HS toxicity on a microalgae species at concentrations complementing those found in aquatic environments. These preliminary results could represent a risk in the future if concentrations of OC and HS continue to increase.

Development and validation of a Luminex assay for detection of a predictive biomarker for PROSTVAC-VF therapy.

Cancer therapies can provide substantially improved survival in some patients while other seemingly similar patients receive little or no benefit. Strategies to identify patients likely to respond well to a given therapy could significantly improve health care outcomes by maximizing clinical benefits while reducing toxicities and adverse effects. Using a glycan microarray assay, we recently reported that pretreatment serum levels of IgM specific to blood group A trisaccharide (BG-Atri) correlate positively with overall survival of cancer patients on PROSTVAC-VF therapy. The results suggested anti-BG-Atri IgM measured prior to treatment could serve as a biomarker for identifying patients likely to benefit from PROSTVAC-VF. For continued development and clinical application of serum IgM specific to BG-Atri as a predictive biomarker, a clinical assay was needed. In this study, we developed and validated a Luminex-based clinical assay for measuring serum IgM specific to BG-Atri. IgM levels were measured with the Luminex assay and compared to levels measured using the microarray for 126 healthy individuals and 77 prostate cancer patients. This assay provided reproducible and consistent results with low %CVs, and tolerance ranges were established for the assay. IgM levels measured using the Luminex assay were found to be highly correlated to the microarray results with R values of 0.93-0.95. This assay is a Laboratory Developed Test (LDT) and is suitable for evaluating thousands of serum samples in CLIA certified laboratories that have validated the assay. In addition, the study demonstrates that discoveries made using neoglycoprotein-based microarrays can be readily migrated to a clinical assay.

Structure and mechanism of action of the hydroxy-aryl-aldehyde class of IRE1 endoribonuclease inhibitors.

Endoplasmic reticulum (ER) stress activates the unfolded protein response and its dysfunction is linked to multiple diseases. The stress transducer IRE1α is a transmembrane kinase endoribonuclease (RNase) that cleaves mRNA substrates to re-establish ER homeostasis. Aromatic ring systems containing hydroxy-aldehyde moieties, termed hydroxy-aryl-aldehydes (HAA), selectively inhibit IRE1α RNase and thus represent a novel chemical series for therapeutic development. We solved crystal structures of murine IRE1α in complex with three HAA inhibitors. HAA inhibitors engage a shallow pocket at the RNase-active site through pi-stacking interactions with His910 and Phe889, an essential Schiff base with Lys907 and a hydrogen bond with Tyr892. Structure-activity studies and mutational analysis of contact residues define the optimal chemical space of inhibitors and validate the inhibitor-binding site. These studies lay the foundation for understanding both the biochemical and cellular functions of IRE1α using small molecule inhibitors and suggest new avenues for inhibitor design.

Aerobic metabolism and cardiac activity in the descendants of zebrafish exposed to pyrolytic polycyclic aromatic hydrocarbons.

The increase of anthropogenic activities on coastal areas induces discharges of polycyclic aromatic hydrocarbons (PAHs) in aquatic ecosystem. PAH effects depend not only on their concentration and the way of contamination but also on the different developmental stages of the organism. Zebrafish were exposed to relevant concentration of pyrolytic PAHs from the first meal (i.e., 5-day post fertilization, dpf) to mature adults. Parental effect of this type of exposure was evaluated through the assessment of aerobic metabolic scope, cardiac frequency, and cardiac mRNA expression on larval and/or embryo progeny of contaminated fish. Our results suggest that cardiac frequency increased in larval descendants of fish exposed to the environmental concentration of pyrolytic PAHs (i.e., 5 ng.g(-1) of food), while a lack of effect on aerobic metabolism in 5 dpf larvae was highlighted. A surexpression of mRNA related to the cardiac calcium transporting ATPase atp2a2a, a protein essential for contraction, is in accordance with this increasing cardiac frequency. Even if cardiac development genes cmlc1 and tnnt2a were not affected at early life stages tested, complementary work on cardiac structure could be interesting to better understand PAHs action.

Chronic dietary exposure to pyrolytic and petrogenic mixtures of PAHs causes physiological disruption in zebrafish--part I: Survival and growth.

The release of polycyclic aromatic hydrocarbons (PAHs) into the environment has increased very substantially over the last decades leading to high concentrations in sediments of contaminated areas. To evaluate the consequences of long-term chronic exposure to PAHs, zebrafish were exposed, from their first meal at 5 days post fertilisation until they became reproducing adults, to diets spiked with three PAH fractions at three environmentally relevant concentrations with the medium concentration being in the range of 4.6-6.7 µg g(-1) for total quantified PAHs including the 16 US-EPA indicator PAHs and alkylated derivatives. The fractions used were representative of PAHs of pyrolytic (PY) origin or of two different oils of differing compositions, a heavy fuel (HO) and a light crude oil (LO). Fish growth was inhibited by all PAH fractions and the effects were sex specific: as determined with 9-month-old adults, exposure to the highest PY inhibited growth of females; exposure to the highest HO and LO inhibited growth of males; also, the highest HO dramatically reduced survival. Morphological analysis indicated a disruption of jaw growth in larvae and malformations in adults. Intestinal and pancreatic enzyme activities were abnormal in 2-month-old exposed fish. These effects may contribute to poor growth. Finally, our results indicate that PAH mixtures of different compositions, representative of situations encountered in the wild, can promote lethal and sublethal effects which are likely to be detrimental for fish recruitment.

Effect of dispersed crude oil exposure upon the aerobic metabolic scope in juvenile golden grey mullet (Liza aurata).

This study evaluated the toxicity of dispersant application which is, in nearshore area, a controversial response technique to oil spill. Through an experimental approach with juveniles of Liza aurata, the toxicity of five exposure conditions was evaluated: (i) a chemically dispersed oil simulating dispersant application; (ii) a single dispersant as an internal control of chemically dispersed oil; (iii) a mechanically dispersed oil simulating natural dispersion of oil; (iv) a water soluble fraction of oil simulating an undispersed and untreated oil slick and (v) uncontaminated seawater as a control exposure condition. The relative concentration of PAHs (polycyclic aromatic hydrocarbons) biliary metabolites showed that the incorporation of these toxic compounds was increased if the oil was dispersed, whether mechanically or chemically. However, toxicity was not observed at the organism level since the aerobic metabolic scope and the critical swimming speed of exposed fish were not impaired.

Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease.

Inositol-requiring enzyme 1 (IRE1) is the most highly conserved signaling node of the unfolded protein response (UPR) and represents a potential therapeutic target for a number of diseases associated with endoplasmic reticulum stress. IRE1 activates the XBP-1 transcription factor by site-specific cleavage of two hairpin loops within its mRNA to facilitate its nonconventional splicing and alternative translation. We screened for inhibitors using a construct containing the unique cytosolic kinase and endoribonuclease domains of human IRE1α (hIRE1α-cyto) and a mini-XBP-1 stem-loop RNA as the substrate. One class compounds was salicylaldehyde analogs from the hydrolyzed product of salicylaldimines in the library. Salicylaldehyde analogs were active in inhibiting the site-specific cleavage of several mini-XBP-1 stem-loop RNAs in a dose-dependent manner. Salicyaldehyde analogs were also active in inhibiting yeast Ire1 but had little activity inhibiting RNase L or the unrelated RNases A and T1. Kinetic analysis revealed that one potent salicylaldehyde analog, 3-ethoxy-5,6-dibromosalicylaldehyde, is a non-competitive inhibitor with respect to the XBP-1 RNA substrate. Surface plasmon resonance studies confirmed this compound bound to IRE1 in a specific, reversible and dose-dependent manner. Salicylaldehydes inhibited XBP-1 splicing induced pharmacologically in human cells. These compounds also blocked transcriptional up-regulation of known XBP-1 targets as well as mRNAs targeted for degradation by IRE1. Finally, the salicylaldehyde analog 3-methoxy-6-bromosalicylaldehyde strongly inhibited XBP-1 splicing in an in vivo model of acute endoplasmic reticulum stress. To our knowledge, salicylaldehyde analogs are the first reported specific IRE1 endoribonuclease inhibitors.

Toll-like receptor-3 as a target to enhance bioactivity of cancer immunotherapy.

OBJECTIVE: The purpose of this study was to explore the potential of toll-like receptor-3 stimulation, with polyI:C(12)U (poly[l].poly[C(12),U]; rintatolimod [Ampligen; Hemispherx Biopharma, Philadelphia, PA]) to enhance bioactivity of cancer immunotherapies. STUDY DESIGN: Several models of immune activation were assessed with polyI:C(12)U at concentrations that were achieved clinically. Dendritic cell maturation and antigen-specific immune responses were evaluated in vitro and in a murine model. The potential for polyI:C(12)U to enhance antibody-dependent cellular cytotoxicity against tumor was also evaluated. RESULTS: Dendritic cells are matured and T-cell stimulation is enhanced in the presence of polyI:C(12)U. In addition, polyI:C(12)U induced the release of proinflammatory chemokines and cytokines. Prostate-specific antigen-specific T-cell and antibody responses were enhanced significantly in a BALB/c prostate-specific antigen transgenic mouse model. Finally, rituximab-mediated antibody-dependent cellular cytotoxicity against tumor targets was improved significantly by the addition of polyI:C(12)U. CONCLUSION: PolyI:C(12)U shows promise as a potential agent for selective enhancement of effect with currently available and future cancer immunotherapies.

Induction of Foxp3+ regulatory T cells with histone deacetylase inhibitors.

Histone deacetylase inhibitors are under investigation in the clinic as a new class of anti-cancer therapeutics. While recent studies have also suggested their potential as inhibitors of a wide spectrum of inflammatory reactions, the anti-inflammatory mechanism of action of these compounds is not fully defined. We show here that the histone deacetylase inhibitors MS-275 and SAHA induce the generation of regulatory T cells (Tregs) from anti-CD3/anti-CD28-stimulated human CD4(+)CD25(-) T cells. These Tregs express the regulatory T cell-associated transcription factor Foxp3 and display suppressive activity against CD4(+)CD25(-) T cell proliferation. Topical treatment with histone deacetylase inhibitors also induces Foxp3 expression in the draining lymph nodes and the skin in the context of a murine contact hypersensitivity model. These findings suggest that Treg generation may serve as a novel mechanism by which histone deacetylase inhibitors regulate the immune response, and provide an additional rationale for the use of histone deacetylase inhibitors in the treatment of inflammation.

Sunlight triggers cutaneous lupus through a CSF-1-dependent mechanism in MRL-Fas(lpr) mice.

Sunlight (UVB) triggers cutaneous lupus erythematosus (CLE) and systemic lupus through an unknown mechanism. We tested the hypothesis that UVB triggers CLE through a CSF-1-dependent, macrophage (Mø)-mediated mechanism in MRL-Fas(lpr) mice. By constructing mutant MRL-Fas(lpr) strains expressing varying levels of CSF-1 (high, intermediate, none), and use of an ex vivo gene transfer to deliver CSF-1 intradermally, we determined that CSF-1 induces CLE in lupus-susceptible MRL-Fas(lpr) mice, but not in lupus-resistant BALB/c mice. UVB incites an increase in Møs, apoptosis in the skin, and CLE in MRL-Fas(lpr), but not in CSF-1-deficient MRL-Fas(lpr) mice. Furthermore, UVB did not induce CLE in BALB/c mice. Probing further, UVB stimulates CSF-1 expression by keratinocytes leading to recruitment and activation of Møs that, in turn, release mediators, which induce apoptosis in keratinocytes. Thus, sunlight triggers a CSF-1-dependent, Mø-mediated destructive inflammation in the skin leading to CLE in lupus-susceptible MRL-Fas(lpr) but not lupus-resistant BALB/c mice. Taken together, CSF-1 is envisioned as the match and lupus susceptibility as the tinder leading to CLE.

Programmed death ligand 1 regulates a critical checkpoint for autoimmune myocarditis and pneumonitis in MRL mice.

MRL/MpJ-Fas(lpr) (MRL-Fas(lpr)) mice develop a spontaneous T cell and macrophage-dependent autoimmune disease that shares features with human lupus. Interactions via the programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway down-regulate immune responses and provide a negative regulatory checkpoint in mediating tolerance and autoimmune disease. Therefore, we tested the hypothesis that the PD-1/PD-L1 pathway suppresses lupus nephritis and the systemic illness in MRL-Fas(lpr) mice. For this purpose, we compared kidney and systemic illness (lymph nodes, spleen, skin, lung, glands) in PD-L1 null (-/-) and PD-L1 intact (wild type, WT) MRL-Fas(lpr) mice. Unexpectedly, PD-L1(-/-);MRL-Fas(lpr) mice died as a result of autoimmune myocarditis and pneumonitis before developing renal disease or the systemic illness. Dense infiltrates, consisting of macrophage and T cells (CD8(+) > CD4(+)), were prominent throughout the heart (atria and ventricles) and localized specifically around vessels in the lung. In addition, once disease was evident, we detected heart specific autoantibodies in PD-L1(-/-);MRL-Fas(lpr) mice. This unique phenotype is dependent on MRL-specific background genes as PD-L1(-/-);MRL(+/+) mice lacking the Fas(lpr) mutation developed autoimmune myocarditis and pneumonitis. Notably, the transfer of PD-L1(-/-);MRL(+/+) bone marrow cells induced myocarditis and pneumonitis in WT;MRL(+/+) mice, despite a dramatic up-regulation of PD-L1 expression on endothelial cells in the heart and lung of WT;MRL(+/+) mice. Taken together, we suggest that PD-L1 expression is central to autoimmune heart and lung disease in lupus-susceptible (MRL) mice.

Programmed death 1 ligand (PD-L) 1 and PD-L2 limit autoimmune kidney disease: distinct roles.

The programmed death 1/programmed death 1 ligand (PD-L) pathway is instrumental in peripheral tolerance. Blocking this pathway exacerbates experimental autoimmune diseases, but its role in autoimmune kidney disease has not been explored. Therefore, we tested the hypothesis that the programmed death 1 ligands (PD-L1 and PD-L2), provide a protective barrier during T cell- and macrophage (Mphi)-dependent autoimmune kidney disease. For this purpose, we compared nephrotoxic serum nephritis (NSN) in mice lacking PD-L1 (PD-L1(-/-)), PD-L2 (PD-L2(-/-)), or both (PD-L1/L2(-/-)) to wild-type (WT) C57BL/6 mice. Kidney pathology, loss of renal function, and intrarenal leukocyte infiltrates were increased in each PD-L(-/-) strain as compared with WT mice. Although the magnitude of renal pathology was similar in PD-L1(-/-) and PD-L2(-/-) mice, our findings suggest that kidney disease in each strain is regulated by distinct mechanisms. Specifically, we detected increased CD68(+) cells along with elevated circulating IgG and IgG deposits in glomeruli in PD-L2(-/-) mice, but not PD-L1(-/-) mice. In contrast, we detected a rise in activated CD8(+) T cells in PD-L1(-/-) mice, but not PD-L2(-/-) mice. Furthermore, since PD-L1 is expressed by parenchymal and hemopoietic cells in WT kidneys, we explored the differential impact of PD-L1 expression on these cell types by inducing NSN in bone marrow chimeric mice. Our results indicate that PD-L1 expression on hemopoietic cells, and not parenchymal cells, is primarily responsible for limiting leukocyte infiltration during NSN. Taken together, our findings indicate that PD-L1 and PD-L2 provide distinct negative regulatory checkpoints poised to suppress autoimmune renal disease.

Subtle defects in pre-TCR signaling in the absence of the Tec kinase Itk.

alphabeta T cell development in the thymus is dependent on signaling through the TCR. The first of these signals is mediated by the pre-TCR, which is responsible for promoting pre-T cell proliferation and the differentiation of CD4(-)8(-)3(-) (DN) thymocytes into CD4(+)8(+)3(+) (DP) cells. In many cases, T cell signaling proteins known to be essential for TCR signaling in mature T cells are also required for pre-TCR signaling in DN thymocytes. Therefore, it came as a surprise to discover that mice lacking the Tec kinases Itk and Rlk, enzymes required for efficient activation of phospholipase C-gamma1 in mature T cells, showed no obvious defects in pre-TCR-dependent selection events in the thymus. In this report, we demonstrate that DN thymocytes lacking Itk, or Itk and Rlk, are impaired in their ability to generate normal numbers of DP thymocytes, especially when placed in direct competition with WT DN thymocytes. We also show that Itk is required for maximal pre-TCR signaling in DN thymocytes. These data demonstrate that the Tec kinases Itk and Rlk are involved in, but are not essential for, pre-TCR signaling in the thymus, suggesting that there is an alternative mechanism for activating phospholipase C-gamma1 in DN thymocytes that is not operating in DP thymocytes and mature T cells.