Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.
Better biomarkers are needed to identify, characterize, and/or monitor drug-induced vascular injury (DIVI) in nonclinical species and patients. The Predictive Safety Testing Consortium (PSTC), a precompetitive collaboration of pharmaceutical companies and the U.S. Food and Drug Administration (FDA), formed the Vascular Injury Working Group (VIWG) to develop and qualify translatable biomarkers of DIVI. The VIWG focused its research on acute DIVI because early detection for clinical and nonclinical safety monitoring is desirable. The VIWG developed a strategy based on the premise that biomarkers of DIVI in rat would be translatable to humans due to the morphologic similarity of vascular injury between species regardless of mechanism. The histomorphologic lexicon for DIVI in rat defines degenerative and adaptive findings of the vascular endothelium and smooth muscles, and characterizes inflammatory components. We describe the mechanisms of these changes and their associations with candidate biomarkers for which advanced analytical method validation was completed. Further development is recommended for circulating microRNAs, endothelial microparticles, and imaging techniques. Recommendations for sample collection and processing, analytical methods, and confirmation of target localization using immunohistochemistry and in situ hybridization are described. The methods described are anticipated to aid in the identification and qualification of translational biomarkers for DIVI.
Over the last few years, numerous ligand binding assay technologies that utilize real-time measurement have been introduced; however, an assemblage and evaluation of these technologies has not previously been published. Herein, we describe six emerging real-time measurement technologies: Maverick™, MX96 SPR™, NanoDLSay™, AMMP®/ViBE®, SoPrano™, and two Lab-on-a-Chip (LoC) microfluidic devices. The development stage gate of these technologies ranges from pre-commercial to commercially available. Due to the novelty, the application and utility of some of the technologies regarding bioanalysis are likely to evolve but it is our hope that this review will provide insight into the direction the development of real-time measurement technologies is moving and the vision of those that are taking us there. Following the technology discussions, a comprehensive summary table is presented.
Nonsteroidal human pharmaceuticals are prevalent in domestic wastewater and may find their way into the environment at low concentrations. Since most pharmaceuticals are designed to be biologically active at low concentrations, there is a risk that these compounds may affect aquatic wildlife. Of particular concern is the occurrence of pharmaceutical mixtures, which may lead to increased adverse effects compared to individual compounds. Interactive effects were previously demonstrated for amphibians exposed to pesticide mixtures, but no such studies investigating responses of amphibians to pharmaceutical mixtures are apparently available. Results demonstrated increased toxicity (loss of tactile response) of striped marsh frog (Limnodynastes peronii) tadpoles exposed to a mixture of naproxen, carbamazepine, and sulfamethoxazole, compared to exposures to the individual compounds. Significant time?×?treatment interactions were observed for tadpole development following chronic exposures to 10 or 100 ?g/L of each compound and the mixture; however, responses were weak and main treatment effects were not significant. Despite minor effects at low exposure concentrations, results demonstrated a potential for mixtures of nonsteroidal pharmaceuticals commonly occurring in wastewater to influence amphibian development. With the vast numbers of pharmaceuticals that exist and are found in the environment, this work highlights a need for further research into mixtures of pharmaceutically active wastewater contaminants. Further, since pharmaceuticals exert extremely varied biological actions, it is suggested that future investigations would benefit from inclusion of endpoints that are indicative of physiological or metabolic performance, as well as assessment of sensitive behavioral responses.
CD4+ T cells are critically important in HIV infection, being both the primary cells infected by HIV and likely playing a direct or indirect role in helping control virus replication. Key areas of interest in HIV vaccine research are mechanisms of viral escape from the immune response. Interestingly, in HIV infection it has been shown that peptide sequence variation can reduce CD4+ T cell responses to the virus, and small changes to peptide sequences can transform agonist peptides into antagonist peptides.
The RIG-I like receptor pathway is stimulated during RNA virus infection by interaction between cytosolic RIG-I and viral RNA structures that contain short hairpin dsRNA and 5 triphosphate (5ppp) terminal structure. In the present study, an RNA agonist of RIG-I was synthesized in vitro and shown to stimulate RIG-I-dependent antiviral responses at concentrations in the picomolar range. In human lung epithelial A549 cells, 5pppRNA specifically stimulated multiple parameters of the innate antiviral response, including IRF3, IRF7 and STAT1 activation, and induction of inflammatory and interferon stimulated genes - hallmarks of a fully functional antiviral response. Evaluation of the magnitude and duration of gene expression by transcriptional profiling identified a robust, sustained and diversified antiviral and inflammatory response characterized by enhanced pathogen recognition and interferon (IFN) signaling. Bioinformatics analysis further identified a transcriptional signature uniquely induced by 5pppRNA, and not by IFN?-2b, that included a constellation of IRF7 and NF-kB target genes capable of mobilizing multiple arms of the innate and adaptive immune response. Treatment of primary PBMCs or lung epithelial A549 cells with 5pppRNA provided significant protection against a spectrum of RNA and DNA viruses. In C57Bl/6 mice, intravenous administration of 5pppRNA protected animals from a lethal challenge with H1N1 Influenza, reduced virus titers in mouse lungs and protected animals from virus-induced pneumonia. Strikingly, the RIG-I-specific transcriptional response afforded partial protection from influenza challenge, even in the absence of type I interferon signaling. This systems approach provides transcriptional, biochemical, and in vivo analysis of the antiviral efficacy of 5pppRNA and highlights the therapeutic potential associated with the use of RIG-I agonists as broad spectrum antiviral agents.
Adjuvants are critical for the success of vaccines. Agonists of microbial pattern recognition receptors (PRRs) are promising new adjuvant candidates. A mechanism through which adjuvants enhance immune responses is to stimulate innate immunity. We studied the innate immune response in humans to synthetic double-stranded RNA (polyinosinic:polycytidylic acid [poly IC] stabilized with poly-L-lysine [poly ICLC]), an agonist for toll-like receptor (TLR) 3, and the cytosolic RNA helicase MDA-5. Transcriptional analysis of blood samples from eight volunteers, after subcutaneous administration of poly ICLC, showed up-regulation of genes involved in multiple innate immune pathways in all subjects, including interferon (IFN) and inflammasome signaling. Blocking type I IFN receptor ex vivo significantly dampened the response to poly IC. Comparative transcriptional analysis showed that several innate immune pathways were similarly induced in volunteers immunized with the highly efficacious yellow fever vaccine. Therefore, a chemically defined PRR agonist like poly ICLC can be a reliable and authentic microbial mimic for inducing innate immune responses in humans.
The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1?, TNF-?, Il-12, IL-10, IFN-? and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease.
A practical enantioselective synthesis of renin inhibitor MK-1597 (ACT-178882), a potential new treatment for hypertension, is described. The synthetic route provided MK-1597 in nine steps and 29% overall yield from commercially available p-cresol (7). The key features of this sequence include a catalytic asymmetric hydrogenation of a tetrasubstituted ene-ester, a highly efficient epimerization/saponification sequence of 4 which sets both stereocenters of the molecule, and a short synthesis of amine fragment 2.
Insulin-producing ? cells can partially regenerate in adult pancreatic tissues, both in human and animal models of type 1 diabetes (T1D). Previous studies have shown that treatment with mycobacterial adjuvants such as CFA and bacillus Calmette-Guérin prevents induction and recurrence of T1D in NOD mice with partial recovery of ? cell mass. In this study, we investigated factors involved in the regeneration of ? cells in the pancreas of NOD mice during diabetes development and after treatment with adjuvants. The Regeneration (Reg) gene family is known to be involved in regeneration of various tissues including ? cells. Reg2 expression was found to be upregulated in pancreatic islets both during diabetes development and as a result of adjuvant treatment in diabetic NOD mice and in C57BL/6 mice made diabetic by streptozotocin treatment. The upregulation of Reg2 by adjuvant treatment was independent of signaling through MyD88 and IL-6 because it was not altered in MyD88 or IL-6 knockout mice. We also observed upregulation of Reg2 in the pancreas of diabetic mice undergoing ? cell regenerative therapy with exendin-4 or with islet neogenesis-associated protein. Reg2 expression following adjuvant treatment correlated with a reduction in insulitis, an increase in insulin secretion, and an increase in the number of small islets in the pancreas of diabetic NOD mice and with improved glucose tolerance tests in streptozotocin-treated diabetic C57BL/6 mice. In conclusion, adjuvant immunotherapy regulates T1D in diabetic mice and induces Reg2-mediated regeneration of ? cells.
The 2009 H1N1 influenza pandemic has prompted a significant need for the development of efficient, single-dose, adjuvanted vaccines. Here we investigated the adjuvant potential of CpG oligodeoxynucleotide (ODN) when used with a human seasonal influenza virus vaccine in ferrets. We found that the CpG ODN-adjuvanted vaccine effectively increased antibody production and activated type I interferon (IFN) responses compared to vaccine alone. Based on these findings, pegylated IFN-alpha2b (PEG-IFN) was also evaluated as an adjuvant in comparison to CpG ODN and complete Freunds adjuvant (CFA). Our results showed that all three vaccines with adjuvant added prevented seasonal human A/Brisbane/59/2007 (H1N1) virus replication more effectively than did vaccine alone. Gene expression profiles indicated that, as well as upregulating IFN-stimulated genes (ISGs), CpG ODN enhanced B-cell activation and increased Toll-like receptor 4 (TLR4) and IFN regulatory factor 4 (IRF4) expression, whereas PEG-IFN augmented adaptive immunity by inducing major histocompatibility complex (MHC) transcription and Ras signaling. In contrast, the use of CFA as an adjuvant induced limited ISG expression but increased the transcription of MHC, cell adhesion molecules, and B-cell activation markers. Taken together, our results better characterize the specific molecular pathways leading to adjuvant activity in different adjuvant-mediated influenza virus vaccinations.
Type I interferons (IFNs) are essential to the clearance of viral diseases, however, a clear distinction between genes upregulated by direct virus-cell interactions and genes upregulated by secondary IFN production has not been made. Here, we investigated differential gene regulation in ferrets upon subcutaneous administration of IFN-?2b and during SARS-CoV infection. In vivo experiments revealed that IFN-?2b causes STAT1 phosphorylation and upregulation of abundant IFN response genes (IRGs), chemokine receptors, and other genes that participate in phagocytosis and leukocyte transendothelial migration. During infection with SARS-CoV not only a variety of IRGs were upregulated, but also a significantly broader range of genes involved in cell migration and inflammation. This work allowed dissection of several molecular signatures present during SARS-CoV which are part of a robust IFN antiviral response. These signatures can be useful markers to evaluate the status of IFN responses during a viral infection and specific features of different viruses.
Direct practice social workers today are challenged to address the requirements of the complex array of professional, organizational, institutional, and regulatory demands placed on them in the broader socioeconomic context of fewer resources and diminished public support for social welfare services in the United States. The common factors model provides an accessible, transtheoretical, empirically supported conceptual foundation for practice that may help to resolve this conundrum and support effective practice. Common factors are conditions and processes activated and facilitated by strategies and skills that positively influence practice outcomes across a range ofpractice theories. The model provides an expanded conceptualization of the "active ingredients" required for change to include a focus on conditions and processes as well as practice strategies and to focus on all who are involved in the work. The model is described and implications for practice are discussed.
Immune responses during infection with pandemic H1N1 2009 influenza A virus (2009-H1N1) are still poorly understood. Using an experimental infection model in ferrets, we examined the pathological features and characterized the host immune responses by using microarray analysis, during infection with 2009-H1N1 A/California/07/2009 and seasonal A/Brisbane/59/2007. Chemokines CCL2, CCL8, CXCL7 and CXCL10 along with the majority of interferon-stimulated genes were expressed early, correlated to lung pathology, and abruptly decreased expression on day 7 following infection of A/California/07/2009. Interestingly, the drop in innate immune gene expression was replaced by a significant increase of the adaptive immune genes for granzymes and immunoglobulins. Serum anti-influenza antibodies were first observed on day 7, commensurate with the viral clearance. We propose that lung pathology in humans occurs during the innate phase of host immunity and a delay or failure to switch to the adaptive phase may contribute to morbidity and mortality during severe 2009-H1N1 infections.
Reminiscent of the Severe Acquired Respiratory Syndrome (SARS) outbreak during the spring and fall of 2003, an outbreak of Severe Respiratory Illness (SRI) in Mexico has world health officials in a state of concern. The concern centers on the fact that as of April 24, 2009 over 134 cases have been reported in Mexico with an accompanying high mortality rate in healthy young adults. With new information surfacing from public health investigations of recent cases of swine influenza in the United States, here we discuss putative links between the outbreak of SRI in Mexico and the emergence of a novel swine influenza A (H1N1) strain in several southwestern U.S. states.
West Nile virus (WNV) causes asymptomatic infection in most humans, but for undefined reasons, approximately 20% of immunocompetent individuals develop West Nile fever, a potentially debilitating febrile illness, and approximately 1% develop neuroinvasive disease syndromes. Notably, since its emergence in 1999, WNV has become the leading cause of epidemic viral encephalitis in North America. We hypothesized that CD4+ Tregs might be differentially regulated in subjects with symptomatic compared with those with asymptomatic WNV infection. Here, we show that in 32 blood donors with acute WNV infection, Tregs expanded significantly in the 3 months after index (RNA+) donations in all subjects. Symptomatic donors exhibited lower Treg frequencies from 2 weeks through 1 year after index donation yet did not show differences in systemic T cell or generalized inflammatory responses. In parallel prospective experimental studies, symptomatic WNV-infected mice also developed lower Treg frequencies compared with asymptomatic mice at 2 weeks after infection. Moreover, Treg-deficient mice developed lethal WNV infection at a higher rate than controls. Together, these results suggest that higher levels of peripheral Tregs after infection protect against severe WNV disease in immunocompetent animals and humans.
The 5th GCC in Barcelona (Spain) and 6th GCC in San Antonio (TX, USA) events provided a unique opportunity for CRO leaders to openly share opinions and perspectives, and to agree upon recommendations on biomarker bioanalytical method validation.
In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)(3)-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.
Live attenuated simian immunodeficiency virus (SIV) vaccines (LAVs) remain the most efficacious of all vaccines in nonhuman primate models of HIV and AIDS, yet the basis of their robust protection remains poorly understood. Here we show that the degree of LAV-mediated protection against intravenous wild-type SIVmac239 challenge strongly correlates with the magnitude and function of SIV-specific, effector-differentiated T cells in the lymph node but not with the responses of such T cells in the blood or with other cellular, humoral and innate immune parameters. We found that maintenance of protective T cell responses is associated with persistent LAV replication in the lymph node, which occurs almost exclusively in follicular helper T cells. Thus, effective LAVs maintain lymphoid tissue-based, effector-differentiated, SIV-specific T cells that intercept and suppress early wild-type SIV amplification and, if present in sufficient frequencies, can completely control and perhaps clear infection, an observation that provides a rationale for the development of safe, persistent vectors that can elicit and maintain such responses.
CXCL10 is part of the group of interferon-stimulated genes and it plays an important role during different viral infections by inducing cell activation, chemotaxis and lymphocyte priming toward the Th1 phenotype. In this study, we investigated in vitro the effects of CXCL10 in activated human primary T lymphocytes in terms of apoptosis or survival, and delineated the signaling pathways that are involved. CXCL10, in combination with IL-2 and/or IFN?, induces apoptosis in T lymphocytes. Moreover, CXCL10-induced activation of CXCR3 also triggers pro-survival signals that can be blocked by pertussis toxin. The analysis of the downstream signaling kinases shows that apoptosis is p38 MAPK-dependent and the pro-survival signals rely on the sustained activation of PI3K and the transient activation of Akt. On the other hand, the transient activation of p44/p42 ERK did not have an impact on T lymphocyte survival. We propose an immunological model in which CXCL10, together with other co-stimulating cytokines, participates in the activation of T lymphocytes, promotes survival and expansion of certain lymphocyte subsets, and induces chemotaxis toward the infected tissues. On the other hand, CXCL10 might contribute to the triggering of apoptosis in other subsets of T lymphocytes, including those lymphocytes that were transiently activated but later lacked the appropriate sets of specific co-stimulating signals to ensure their survival.
Recombinant adenovirus (rAd) vectors are being investigated as vaccine delivery vehicles in preclinical and clinical studies. rAds constructed from different serotypes differ in receptor usage, tropism, and ability to activate cells, aspects of which likely contribute to their different immunogenicity profiles. In this study, we compared the infectivity and cell stimulatory capacity of recombinant adenovirus serotype 5 (rAd5), recombinant adenovirus serotype 28 (rAd28), and recombinant adenovirus serotype 35 (rAd35) in association with their respective immunogenicity profiles. We found that rAd28 and rAd35 infected and led to the in vitro maturation and activation of both human and mouse dendritic cells more efficiently compared with rAd5. In stark contrast to rAd5, rAd28 and rAd35 induced production of IFN-? and stimulated IFN-related intracellular pathways. However, the in vivo immunogenicity of rAd28 and rAd35 was significantly lower than that of rAd5. Deletion of IFN-? signaling during vaccination with rAd28 and rAd35 vectors increased the magnitude of the insert-specific T cell response to levels induced by vaccination with rAd5 vector. The negative impact of IFN-? signaling on the magnitude of the T cell response could be overcome by increasing the vaccine dose, which was also associated with greater polyfunctionality and a more favorable long-term memory phenotype of the CD8 T cell response in the presence of IFN-? signaling. Taken together, our results demonstrate that rAd-induced IFN-? production has multiple effects on T cell immunogenicity, the understanding of which should be considered in the design of rAd vaccine vectors.
Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.
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