S-nitrosation of proteins relevant to Alzheimer's disease during early stages of neurodegeneration

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein's function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25-inducible mouse model of Alzheimer's disease-like neurodegeneration. The approach-SNOTRAP (SNO trapping by triaryl phosphine)-is a direct tagging strategy that uses phosphinebased chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer's disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.National Institutes of Health (U.S.) (Grant CA26731)National Institutes of Health (U.S.) (Grant R01 NS051874

S-nitrosation of proteins relevant to Alzheimer’s disease during early stages of neurodegeneration

Protein S-nitrosation (SNO-protein), the nitric oxide-mediated posttranslational modification of cysteine thiols, is an important regulatory mechanism of protein function in both physiological and pathological pathways. A key first step toward elucidating the mechanism by which S-nitrosation modulates a protein’s function is identification of the targeted cysteine residues. Here, we present a strategy for the simultaneous identification of SNO-cysteine sites and their cognate proteins to profile the brain of the CK-p25–inducible mouse model of Alzheimer’s disease-like neurodegeneration. The approach—SNOTRAP (SNO trapping by triaryl phosphine)—is a direct tagging strategy that uses phosphine-based chemical probes, allowing enrichment of SNO-peptides and their identification by liquid chromatography tandem mass spectrometry. SNOTRAP identified 313 endogenous SNO-sites in 251 proteins in the mouse brain, of which 135 SNO-proteins were detected only during neurodegeneration. S-nitrosation in the brain shows regional differences and becomes elevated during early stages of neurodegeneration in the CK-p25 mouse. The SNO-proteome during early neurodegeneration identified increased S-nitrosation of proteins important for synapse function, metabolism, and Alzheimer’s disease pathology. In the latter case, proteins related to amyloid precursor protein processing and secretion are S-nitrosated, correlating with increased amyloid formation. Sequence analysis of SNO-cysteine sites identified potential linear motifs that are altered under pathological conditions. Collectively, SNOTRAP is a direct tagging tool for global elucidation of the SNO-proteome, providing functional insights of endogenous SNO proteins in the brain and its dysregulation during neurodegeneration.National Institutes of Health (U.S.) (NIH Grant CA26731)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Grant ES002109)Simons FoundationNational Institutes of Health (U.S.) (NIH Grant R01 NS051874

Metabolite profiling and pharmacokinetic evaluation of hydrocortisone in a perfused 3D human liver bioreactor

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t1/2), rate of elimination (kel), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h-1, 6.6x10-5 L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver. Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a 3D human microphysiological hepatocyte-Kupffer-cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and assessed by the release of pro-inflammatory cytokines, IL6 and TNFα. A sensitive and specific RP-UHPLC-QTOF-MS method was used to evaluate hydrocortisone disappearance and metabolism at near physiological levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for two days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10 % of the loss, and 45-52 % was phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life (t[subscript 1/2]), rate of elimination (k[subscript el]), clearance (CL), and area under the curve (AUC), were 23.03 h, 0.03 h[superscript -1], 6.6x10[superscript -5] L. h-1 and 1.03 mg/L*h respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically-relevant tool for investigating hepatic function in an inflamed liver.United States. Defense Advanced Research Projects Agency (DARPA-BAA-11-73 Microphysiological Systems W911NF-12-2-0039)National Institutes of Health (U.S.) (5-UH2-TR000496)Massachusetts Institute of Technology. Center for Environmental Health Sciences (P30-ES002109

A single immunization with HA DNA vaccine by electroporation induces early protection against H5N1 avian influenza virus challenge in mice

<p>Abstract</p> <p>Background</p> <p>Developing vaccines for the prevention of human infection by H5N1 influenza viruses is an urgent task. DNA vaccines are a novel alternative to conventional vaccines and should contribute to the prophylaxis of emerging H5N1 virus. In this study, we assessed whether a single immunization with plasmid DNA expressing H5N1 hemagglutinin (HA) could provide early protection against lethal challenge in a mouse model.</p> <p>Methods</p> <p>Mice were immunized once with HA DNA at 3, 5, 7 days before a lethal challenge. The survival rate, virus titer in the lungs and change of body weight were assayed to evaluate the protective abilities of the vaccine. To test the humoral immune response induced by HA DNA, serum samples were collected through the eye canthus of mice on various days after immunization and examined for specific antibodies by ELISA and an HI assay. Splenocytes were isolated after the immunization to determine the antigen-specific T-cell response by the ELISPOT assay.</p> <p>Results</p> <p>Challenge experiments revealed that a single immunization of H5N1 virus HA DNA is effective in early protection against lethal homologous virus. Immunological analysis showed that an antigen-specific antibody and T-cell response could be elicited in mice shortly after the immunization. The protective abilities were correlated with the amount of injected DNA and the length of time after vaccination.</p> <p>Conclusion</p> <p>A single immunization of 100 μg H5 HA DNA vaccine combined with electroporation was able to provide early protection in mice against homologous virus infection.</p

A Porcine Adenovirus with Low Human Seroprevalence Is a Promising Alternative Vaccine Vector to Human Adenovirus 5 in an H5N1 Virus Disease Model

Human adenovirus 5 (AdHu5) vectors are robust vaccine platforms however the presence of naturally-acquired neutralizing antibodies may reduce vector efficacy and potential for re-administration. This study evaluates immune responses and protection following vaccination with a replication-incompetent porcine adenovirus 3 (PAV3) vector as an alternative vaccine to AdHu5 using an avian influenza H5N1 disease model. Vaccine efficacy was evaluated in BALB/c mice following vaccination with different doses of the PAV3 vector expressing an optimized A/Hanoi/30408/2005 H5N1 hemagglutinin antigen (PAV3-HA) and compared with an AdHu5-HA control. PAV3-HA rapidly generated antibody responses, with significant neutralizing antibody titers on day 21, and stronger cellular immune responses detected on day 8, compared to AdHu5-HA. The PAV3-HA vaccine, administered 8 days before challenge, demonstrated improved survival and lower virus load. Evaluation of long-term vaccine efficacy at 12 months post-vaccination showed better protection with the PAV3-HA than with the AdHu5-HA vaccine. Importantly, as opposed to AdHu5, PAV3 vector was not significantly neutralized by human antibodies pooled from over 10,000 individuals. Overall, PAV3-based vector is capable of mediating swift, strong immune responses and offer a promising alternative to AdHu5

Multivalent HA DNA Vaccination Protects against Highly Pathogenic H5N1 Avian Influenza Infection in Chickens and Mice

Sustained outbreaks of highly pathogenic avian influenza (HPAI) H5N1 in avian species increase the risk of reassortment and adaptation to humans. The ability to contain its spread in chickens would reduce this threat and help maintain the capacity for egg-based vaccine production. While vaccines offer the potential to control avian disease, a major concern of current vaccines is their potency and inability to protect against evolving avian influenza viruses.The ability of DNA vaccines encoding hemagglutinin (HA) proteins from different HPAI H5N1 serotypes was evaluated for its ability to elicit neutralizing antibodies and to protect against homologous and heterologous HPAI H5N1 strain challenge in mice and chickens after DNA immunization by needle and syringe or with a pressure injection device. These vaccines elicited antibodies that neutralized multiple strains of HPAI H5N1 when given in combinations containing up to 10 HAs. The response was dose-dependent, and breadth was determined by the choice of the influenza virus HA in the vaccine. Monovalent and trivalent HA vaccines were tested first in mice and conferred protection against lethal H5N1 A/Vietnam/1203/2004 challenge 68 weeks after vaccination. In chickens, protection was observed against heterologous strains of HPAI H5N1 after vaccination with a trivalent H5 serotype DNA vaccine with doses as low as 5 microg DNA given twice either by intramuscular needle injection or with a needle-free device.DNA vaccines offer a generic approach to influenza virus immunization applicable to multiple animal species. In addition, the ability to substitute plasmids encoding different strains enables rapid adaptation of the vaccine to newly evolving field isolates

Improvement of the Trivalent Inactivated Flu Vaccine Using PapMV Nanoparticles

Commercial seasonal flu vaccines induce production of antibodies directed mostly towards hemaglutinin (HA). Because HA changes rapidly in the circulating virus, the protection remains partial. Several conserved viral proteins, e.g., nucleocapsid (NP) and matrix proteins (M1), are present in the vaccine, but are not immunogenic. To improve the protection provided by these vaccines, we used nanoparticles made of the coat protein of a plant virus (papaya mosaic virus; PapMV) as an adjuvant. Immunization of mice and ferrets with the adjuvanted formulation increased the magnitude and breadth of the humoral response to NP and to highly conserved regions of HA. They also triggered a cellular mediated immune response to NP and M1, and long-lasting protection in animals challenged with a heterosubtypic influenza strain (WSN/33). Thus, seasonal flu vaccine adjuvanted with PapMV nanoparticles can induce universal protection to influenza, which is a major advancement when facing a pandemic

Identification of a Dual-Specific T Cell Epitope of the Hemagglutinin Antigen of an H5 Avian Influenza Virus in Chickens

Avian influenza viruses (AIV) of the H5N1 subtype have caused morbidity and mortality in humans. Although some migratory birds constitute the natural reservoir for this virus, chickens may play a role in transmission of the virus to humans. Despite the importance of avian species in transmission of AIV H5N1 to humans, very little is known about host immune system interactions with this virus in these species. The objective of the present study was to identify putative T cell epitopes of the hemagglutinin (HA) antigen of an H5 AIV in chickens. Using an overlapping peptide library covering the HA protein, we identified a 15-mer peptide, H5246–260, within the HA1 domain which induced activation of T cells in chickens immunized against the HA antigen of an H5 virus. Furthermore, H5246–260 epitope was found to be presented by both major histocompatibility complex (MHC) class I and II molecules, leading to activation of CD4+ and CD8+ T cell subsets, marked by proliferation and expression of interferon (IFN)-γ by both of these cell subsets as well as the expression of granzyme A by CD8+ T cells. This is the first report of a T cell epitope of AIV recognized by chicken T cells. Furthermore, this study extends the previous finding of the existence of dual-specific epitopes in other species to chickens. Taken together, these results elucidate some of the mechanisms of immune response to AIV in chickens and provide a platform for creation of rational vaccines against AIV in this species

Tandem mass tag-based quantitative proteomic profiling identifies candidate serum biomarkers of drug-induced liver injury in humans

Diagnosis of drug-induced liver injury (DILI) and its distinction from other liver diseases are significant challenges in drug development and clinical practice. We used Tandem Mass Tag-labeled quantitative proteomics detecting 2323 proteins in a cohort comprising patients with DILI [at onset (DO) and follow-up (DF)], acute non-DILI [at onset (NDO) and follow-up (NDF)], and healthy volunteers (HV) to identify novel serum biomarkers. Thirteen candidates selected based on differential expression, liver-specific expression, and mechanistic relevance to liver pathology, were assessed in confirmatory and replication cohorts of HV (n=94), DO (n=123), DF (n=110), NDO (n=58) and NDF (n=37) using a targeted label-free SureQuant assay. Area under the receiver operating characteristic curve (AUC) ranging between 0.94 and 0.99 across cohorts for five of these biomarkers, reflected differentiation between DO and HV with high sensitivity and specificity. In addition, fructose-1,6-bisphosphatase 1 distinguished NDO from DO (AUC: 0.75 and 0.65) on its own or in combination with glutathione S-transferase A1 and leukocyte cell derived chemotaxin 2 (AUC: 0.78 and 0.68). These can potentially differentiate DILI and acute liver injury from non-drug etiologies