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Find video protocols related to scientific articles indexed in Pubmed.
A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection.
Mol. Pharm.
PUBLISHED: 11-04-2014
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As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4 × 10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8(+) and CD4(+) T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8(+) T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0 × 10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4(+) and CD8(+) T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4(+) T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.
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Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion.
Clin J Am Soc Nephrol
PUBLISHED: 08-01-2014
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Renal nitrogen metabolism primarily involves urea and ammonia metabolism, and is essential to normal health. Urea is the largest circulating pool of nitrogen, excluding nitrogen in circulating proteins, and its production changes in parallel to the degradation of dietary and endogenous proteins. In addition to serving as a way to excrete nitrogen, urea transport, mediated through specific urea transport proteins, mediates a central role in the urine concentrating mechanism. Renal ammonia excretion, although often considered only in the context of acid-base homeostasis, accounts for approximately 10% of total renal nitrogen excretion under basal conditions, but can increase substantially in a variety of clinical conditions. Because renal ammonia metabolism requires intrarenal ammoniagenesis from glutamine, changes in factors regulating renal ammonia metabolism can have important effects on glutamine in addition to nitrogen balance. This review covers aspects of protein metabolism and the control of the two major molecules involved in renal nitrogen excretion: urea and ammonia. Both urea and ammonia transport can be altered by glucocorticoids and hypokalemia, two conditions that also affect protein metabolism. Clinical conditions associated with altered urine concentrating ability or water homeostasis can result in changes in urea excretion and urea transporters. Clinical conditions associated with altered ammonia excretion can have important effects on nitrogen balance.
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An optimized, synthetic DNA vaccine encoding the toxin A and toxin B receptor binding domains of Clostridium difficile induces protective antibody responses in vivo.
Infect. Immun.
PUBLISHED: 07-14-2014
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Clostridium difficile-associated disease (CDAD) constitutes a large majority of nosocomial diarrhea cases in industrialized nations and is mediated by the effects of two secreted toxins, toxin A (TcdA) and toxin B (TcdB). Patients who develop strong antitoxin antibody responses can clear C. difficile infection and remain disease free. Key toxin-neutralizing epitopes have been found within the carboxy-terminal receptor binding domains (RBDs) of TcdA and TcdB, which has generated interest in developing the RBD as a viable vaccine target. While numerous platforms have been studied, very little data describes the potential of DNA vaccination against CDAD. Therefore, we created highly optimized plasmids encoding the RBDs from TcdA and TcdB in which any putative N-linked glycosylation sites were altered. Mice and nonhuman primates were immunized intramuscularly, followed by in vivo electroporation, and in these animal models, vaccination induced significant levels of both anti-RBD antibodies (blood and stool) and RBD-specific antibody-secreting cells. Further characterization revealed that sera from immunized mice and nonhuman primates could detect RBD protein from transfected cells, as well as neutralize purified toxins in an in vitro cytotoxicity assay. Mice that were immunized with plasmids or given nonhuman-primate sera were protected from a lethal challenge with purified TcdA and/or TcdB. Moreover, immunized mice were significantly protected when challenged with C. difficile spores from homologous (VPI 10463) and heterologous, epidemic (UK1) strains. These data demonstrate the robust immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in preclinical models of acute toxin-associated and intragastric, spore-induced colonic disease.
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Multivalent TB vaccines targeting the esx gene family generate potent and broad cell-mediated immune responses superior to BCG.
Hum Vaccin Immunother
PUBLISHED: 06-24-2014
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Development of a broad-spectrum synthetic vaccine against TB would represent an important advance to the limited vaccine armamentarium against TB. It is believed that the esx family of TB antigens may represent important vaccine candidates. However, only 4 esx antigens have been studied as potential vaccine antigens. The challenge remains to develop a vaccine that simultaneously targets all 23 members of the esx family to induce enhanced broad-spectrum cell-mediated immunity. We sought to investigate if broader cellular immune responses could be induced using a multivalent DNA vaccine representing the esx family protein members delivered via electroporation. In this study, 15 designed esx antigens were created to cross target all members of the esx family. They were distributed into groups of 3 self-processing antigens each, resulting in 5 trivalent highly optimized DNA plasmids. Vaccination with all 5 constructs elicited robust antigen-specific IFN-? responses to all encoded esx antigens and induced multifunctional CD4 Th1 and CD8 T cell responses. Importantly, we show that when all constructs are combined into a cocktail, the RSQ-15 vaccine, elicited substantial broad Ag-specific T cell responses to all esx antigens as compared with vaccination with BCG. Moreover, these vaccine-induced responses were highly cross-reactive with BCG encoded esx family members and were highly immune effective in a BCG DNA prime-boost format. Furthermore, we demonstrate the vaccine potential and immunopotent profile of several novel esx antigens never previously studied. These data highlight the likely importance of these novel immunogens for study as preventative or therapeutic synthetic TB vaccines in combination or as stand alone antigens.
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Strong HCV NS3/4a, NS4b, NS5a, NS5b-specific cellular immune responses induced in Rhesus macaques by a novel HCV genotype 1a/1b consensus DNA vaccine.
Hum Vaccin Immunother
PUBLISHED: 06-21-2014
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Chronic HCV is a surreptitious disease currently affecting approximately 3% of the world's population that can lead to liver failure and cancer decades following initial infection. However, there are currently no vaccines available for the prevention of chronic HCV. From patients who acutely resolve HCV infection, it is apparent that a strong and broad cytotoxic T lymphocyte (CTL) response is important in HCV clearance. DNA vaccines are naked plasmid DNA molecules that encode pathogen antigens to induce a pathogen-specific immune response. They are inexpensive to produce and have an excellent safety profile in animals and humans. Additionally, DNA vaccines are able to induce strong CTL responses, making them well-suited for an HCV vaccine. We aimed to maximize vaccine recipients' opportunity to induce a broad T cell response with a novel antigenic sequence, multi-antigen vaccine strategy. We have generated DNA plasmids encoding consensus sequences of HCV genotypes 1a and 1b non-structural proteins NS3/4a, NS4b, NS5a, and NS5b. Rhesus macaques were used to study for immunogenicity of these constructs. Four animals were immunized 3 times, 6 weeks apart, at a dose of 1.0mg per antigen construct, as an intramuscular injection followed by in vivo electroporation, which greatly increases DNA uptake by local cells. Immune responses were measured 2 weeks post-immunization regimen (PIR) in immunized rhesus macaques and showed a broad response to multiple HCV nonstructural antigens, with up to 4680 spot-forming units per million PBMCs as measured by Interferon-? ELISpot. In addition, multiparametric flow cytometry detected HCV-specific CD4+ and CD8+ T cell responses by intracellular cytokine staining and detected HCV-specific CD107a+/GrzB+ CD8+ T cells indicating an antigen specific cytolytic response 2 weeks PIR compared with baseline measurements. At the final study time point, 6 weeks PIR, HCV-specific CD27-/CD45RA- effector memory-like T cells remained detectable in peripheral blood. Data presented in this manuscript support the notion that vaccine immunogenicity studies using a macaque model can be used to depict key anti-HCV nonstructural antigenic cellular immune responses and support the development of DNA-based prophylactic HCV vaccines.
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Anti-inflammatory loaded poly-lactic glycolic acid nanoparticle formulations to enhance myocardial gene transfer: an in-vitro assessment of a drug/gene combination therapeutic approach for direct injection.
J Transl Med
PUBLISHED: 03-31-2014
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Cardiac gene therapy for heart disease is a major translational research area with potential, yet problems with safe and efficient gene transfer into cardiac muscle remain unresolved. Existing methodology to increase vector uptake include modifying the viral vector, non-viral particle encapsulation and or delivery with device systems. These advanced methods have made improvements, however fail to address the key problem of inflammation in the myocardium, which is known to reduce vector uptake and contribute to immunogenic adverse events. Here we propose an alternative method to co-deliver anti-inflammatory drugs in a controlled release polymer with gene product to improve therapeutic effects.
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Ammonia transport in the kidney by Rhesus glycoproteins.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 03-19-2014
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Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4(+) with a new model in which specific and regulated transport of both NH3 and NH4(+) across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport.
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Protective immunity to H7N9 influenza viruses elicited by synthetic DNA vaccine.
Vaccine
PUBLISHED: 03-12-2014
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Despite an intensive vaccine program influenza infections remain a major health problem, due to the viruses' ability to change its envelope glycoprotein hemagglutinin (HA), through shift and drift, permitting influenza to escape protection induced by current vaccines or natural immunity. Recently a new variant, H7N9, has emerged in China causing global concern. First, there have been more than 130 laboratory-confirmed human infections resulting in an alarmingly high death rate (32.3%). Second, genetic changes found in H7N9 appear to be associated with enabling avian influenza viruses to spread more effectively in mammals, thus transmitting infections on a larger scale. Currently, no vaccines or drugs are effectively able to target H7N9. Here, we report the rapid development of a synthetic consensus DNA vaccine (pH7HA) to elicit potent protective immunity against the H7N9 viruses. We show that pH7HA induces broad antibody responses that bind to divergent HAs from multiple new members of the H7N9 family. These antibody responses result in high-titer HAI against H7N9. Simultaneously, this vaccine induces potent polyfunctional effector CD4 and CD8T cell memory responses. Animals vaccinated with pH7HA are completely protected from H7N9 virus infection and any morbidity associated with lethal challenge. This study establishes that this synthetic consensus DNA vaccine represents a new tool for targeting emerging infection, and more importantly, its design, testing and development into seed stock for vaccine production in a few days in the pandemic setting has significant implications for the rapid deployment of vaccines protecting against emerging infectious diseases.
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Interleukin 33: a switch-hitting cytokine.
Curr. Opin. Immunol.
PUBLISHED: 02-07-2014
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For many years IL-33 has been widely studied in the context of T helper type 2 (Th2)-driven inflammatory disorders. Interestingly, IL-33 has now emerged as a cytokine with a plethora of pleiotropic properties. Depending on the immune cells targeted by IL-33, it is reported to not only promote Th2 immunity, but also to induce T helper type 1 (Th1) immunity. Furthermore, recent studies have revealed that IL-33 can activate CD8(+) T cells. These new studies provide evidence for its beneficial role in antiviral and antitumor immunity. Here we review the evidence of IL-33 to drive protective T cell immunity plus its potential use as an adjuvant in vaccination and tumor therapy.
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VGX-1027 modulates genes involved in lipopolysaccharide-induced Toll-like receptor 4 activation and in a murine model of systemic lupus erythematosus.
Immunology
PUBLISHED: 02-04-2014
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VGX-1027 [(S,R)-3-phenyl-4,5-dihydro-5-isoxasole acetic acid] is a small molecule compound with immunomodulatory properties, which favourably influences the development of immuno-inflammatory and autoimmune diseases in different animal models such as type 1 diabetes mellitus, pleurisy, rheumatoid arthritis and inflammatory bowel disease. However, the precise mechanism of action of VGX-1027 remains to be ascertained. With this aim, we have studied the immunomodulatory effects of VGX-1027 in vitro, using a genome-wide oligonucleotide microarray approach, and in vivo, using the NZB/NZW F1 model of systemic lupus erythematosus. Microarray data revealed that the administration of VGX-1027 profoundly affected the immune response to exogenous antigens, by modulating the expression of genes that are primarily involved in antigen processing and presentation as well as genes that regulate immune activation. When administered in vivo VGX-1027 ameliorated the course of the disease in the NZB/NZW F1 mice, which correlated with higher per cent survival and improved clinical and histopathological signs. The data presented herein support the theory that VGX-1027 modulates immunity, probably by inhibiting inflammatory antigen presentation and so limiting immune cell expansion.
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Alarmin IL-33 acts as an immunoadjuvant to enhance antigen-specific tumor immunity.
Cancer Res.
PUBLISHED: 01-21-2014
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Studies of interleukin (IL)-33 reveal a number of pleiotropic properties. Here, we report that IL-33 has immunoadjuvant effects in a human papilloma virus (HPV)-associated model for cancer immunotherapy where cell-mediated immunity is critical for protection. Two biologically active isoforms of IL-33 exist that are full-length or mature, but the ability of either isoform to function as a vaccine adjuvant that influences CD4 T helper 1 or CD8 T-cell immune responses is not defined. We showed that both IL-33 isoforms are capable of enhancing potent antigen-specific effector and memory T-cell immunity in vivo in a DNA vaccine setting. In addition, although both IL-33 isoforms drove robust IFN-? responses, neither elevated secretion of IL-4 or immunoglobulin E levels. Further, both isoforms augmented vaccine-induced antigen-specific polyfunctional CD4(+) and CD8(+) T-cell responses, with a large proportion of CD8(+) T cells undergoing plurifunctional cytolytic degranulation. Therapeutic studies indicated that vaccination with either IL-33 isoform in conjunction with an HPV DNA vaccine caused rapid and complete regressions in vivo. Moreover, IL-33 could expand the magnitude of antigen-specific CD8(+) T-cell responses and elicit effector-memory CD8(+) T cells. Taken together, our results support the development of these IL-33 isoforms as immunoadjuvants in vaccinations against pathogens, including in the context of antitumor immunotherapy.
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Effect of Collecting Duct-Specific Deletion of Both Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg) on Renal Response to Metabolic Acidosis.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 12-11-2013
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The Rhesus (Rh) glycoproteins, Rh B and Rh C Glycoprotein (Rhbg and Rhcg, respectively), are ammonia-specific transporters expressed in renal distal nephron and collecting duct sites that are necessary for normal rates of ammonia excretion. The purpose of the current studies was to determine the effect of their combined deletion from the renal collecting duct (CD-Rhbg/Rhcg-KO) on basal and acidosis-stimulated acid-base homeostasis. Under basal conditions, urine pH and ammonia excretion and serum HCO3(-) were similar in control (C) and CD-Rhbg/Rhcg-KO mice. After acid-loading for seven days, CD-Rhbg/Rhcg-KO mice developed significantly more severe metabolic acidosis than did C mice. Acid-loading increased ammonia excretion, but ammonia excretion increased more slowly in CD-Rhbg/Rhcg-KO and it was significantly less than in C mice on days 1-5. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice on days 1, 3, and 5 of acid-loading. Metabolic acidosis increased phosphenolpyruvate carboxykinase (PEPCK) and NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that: 1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis; 2) the significantly greater changes in PEPCK, NHE-3 and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared to acid-loaded C mice cause the role of Rhbg and Rhcg to be underestimated quantitatively ; and 3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3 and GS expression despite the presence of persistent metabolic acidosis.
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Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.
Hum Vaccin Immunother
PUBLISHED: 09-17-2013
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Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse HIV isolates. Importantly, this delivery strategy resulted in a rapid increase (i.e., in as little as 48 h) in Fab levels when compared with protein-based immunization. The active generation of functional Fab molecules in vivo has important conceptual and practical advantages over conventional ex vivo generation, purification and passive delivery of biologically active antibodies. Further study of this technique for the rapid generation and delivery of immunoglobulin and immunoglobulin like molecules is highly relevant and timely.
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Endocrine and hypertensive disorders of potassium regulation: primary aldosteronism.
Semin. Nephrol.
PUBLISHED: 08-20-2013
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The identification of primary aldosteronism as a common cause of resistant hypertension is a significant advance in our ability to care for patients with hypertension. Primary aldosteronism is common, and when unrecognized is associated with an increased incidence of adverse cardiovascular outcomes. Identification of primary aldosteronism is based on use of the plasma aldosterone level, plasma renin activity, and the aldosterone:renin ratio. Differentiation between unilateral and bilateral autonomous adrenal aldosterone production then guides further therapy, with use of mineralocorticoid-receptor blockers for patients with bilateral autonomous adrenal aldosterone production and laparoscopic adrenalectomy for patients with unilateral autonomous aldosterone production. In this review, we discuss in detail the pathogenesis of primary aldosteronism-induced hypertension and potassium disorders, the evaluation of the patient with suspected primary aldosteronism, and the management of primary aldosteronism, both through medications and surgery.
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Expression of glutamine synthetase in the mouse kidney: localization in multiple epithelial cell types and differential regulation by hypokalemia.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 06-26-2013
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Renal glutamine synthetase catalyzes the reaction of NH4+ with glutamate, forming glutamine and decreasing the ammonia available for net acid excretion. The purpose of the present study was to determine glutamine synthetases specific cellular expression in the mouse kidney and its regulation by hypokalemia, a common cause of altered renal ammonia metabolism. Glutamine synthetase mRNA and protein were present in the renal cortex and in both the outer and inner stripes of the outer medulla. Immunohistochemistry showed glutamine synthetase expression throughout the entire proximal tubule and in nonproximal tubule cells. Double immunolabel with cell-specific markers demonstrated glutamine synthetase expression in type A intercalated cells, non-A, non-B intercalated cells, and distal convoluted tubule cells, but not in principal cells, type B intercalated cells, or connecting segment cells. Hypokalemia induced by feeding a nominally K+ -free diet for 12 days decreased glutamine synthetase expression throughout the entire proximal tubule and in the distal convoluted tubule and simultaneously increased glutamine synthetase expression in type A intercalated cells in both the cortical and outer medullary collecting duct. We conclude that glutamine synthetase is widely and specifically expressed in renal epithelial cells and that the regulation of expression differs in specific cell populations. Glutamine synthetase is likely to mediate an important role in renal ammonia metabolism.
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Renal ammonia metabolism and transport.
Compr Physiol
PUBLISHED: 05-31-2013
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Renal ammonia metabolism and transport mediates a central role in acid-base homeostasis. In contrast to most renal solutes, the majority of renal ammonia excretion derives from intrarenal production, not from glomerular filtration. Renal ammoniagenesis predominantly results from glutamine metabolism, which produces 2 NH4(+) and 2 HCO3(-) for each glutamine metabolized. The proximal tubule is the primary site for ammoniagenesis, but there is evidence for ammoniagenesis by most renal epithelial cells. Ammonia produced in the kidney is either excreted into the urine or returned to the systemic circulation through the renal veins. Ammonia excreted in the urine promotes acid excretion; ammonia returned to the systemic circulation is metabolized in the liver in a HCO3(-)-consuming process, resulting in no net benefit to acid-base homeostasis. Highly regulated ammonia transport by renal epithelial cells determines the proportion of ammonia excreted in the urine versus returned to the systemic circulation. The traditional paradigm of ammonia transport involving passive NH3 diffusion, protonation in the lumen and NH4(+) trapping due to an inability to cross plasma membranes is being replaced by the recognition of limited plasma membrane NH3 permeability in combination with the presence of specific NH3-transporting and NH4(+)-transporting proteins in specific renal epithelial cells. Ammonia production and transport are regulated by a variety of factors, including extracellular pH and K(+), and by several hormones, such as mineralocorticoids, glucocorticoids and angiotensin II. This coordinated process of regulated ammonia production and transport is critical for the effective maintenance of acid-base homeostasis.
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Synthetic DNA vaccine strategies against persistent viral infections.
Expert Rev Vaccines
PUBLISHED: 05-11-2013
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The human body has developed an elaborate defense system against microbial pathogens and foreign antigens. However, particular microbes have evolved sophisticated mechanisms to evade immune surveillance, allowing persistence within the human host. In an effort to combat such infections, intensive research has focused on the development of effective prophylactic and therapeutic countermeasures to suppress or clear persistent viral infections. To date, popular therapeutic strategies have included the use of live-attenuated microbes, viral vectors and dendritic-cell vaccines aiming to help suppress or clear infection. In recent years, improved DNA vaccines have now re-emerged as a promising candidate for therapeutic intervention due to the development of advanced optimization and delivery technologies. For instance, genetic optimization of synthetic plasmid constructs and their encoded antigens, in vivo electroporation-mediated vaccine delivery, as well as codelivery with molecular adjuvants have collectively enhanced both transgene expression and the elicitation of vaccine-induced immunity. In addition, the development of potent heterologous prime-boost regimens has also provided significant contributions to DNA vaccine immunogenicity. Herein, the authors will focus on these recent improvements to this synthetic platform in relation to their application in combating persistent virus infection.
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HIV-1 p24(gag) derived conserved element DNA vaccine increases the breadth of immune response in mice.
PLoS ONE
PUBLISHED: 02-24-2013
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Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24(gag) region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24(gag) DNA immunogens that express 7 highly Conserved Elements (CE) of 12-24 amino acids in length and differ by only 1 amino acid in each CE (toggle site), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55(gag) DNA induced poor, CD4(+) mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4(+) and CD8(+) T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55(gag), which recognize the virus encoded p24(gag) protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4(+) and CD8(+) T cells to additional regions of Gag compared to vaccination with p55(gag) DNA, achieving maximal cross-clade reactive cellular and humoral responses.
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Nonstructural protein 2 (nsP2) of Chikungunya virus (CHIKV) enhances protective immunity mediated by a CHIKV envelope protein expressing DNA Vaccine.
Viral Immunol.
PUBLISHED: 02-16-2013
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Chikungunya virus (CHIKV) is an important emerging mosquito-borne alphavirus, indigenous to tropical Africa and Asia. It can cause epidemic fever and acute illness characterized by fever and arthralgias. The epidemic cycle of this infection is similar to dengue and urban yellow fever viral infections. The generation of an efficient vaccine against CHIKV is necessary to prevent and/or control the disease manifestations of the infection. In this report, we studied immune response against a CHIKV-envelope DNA vaccine (pEnv) and the role of the CHIKV nonstructural gene 2 (nsP2) as an adjuvant for the induction of protective immune responses in a relevant mouse challenge model. When injected with the CHIKV pEnv alone, 70% of the immunized mice survived CHIKV challenge, whereas when co-injected with pEnv+pnsP2, 90% of the mice survived viral challenge. Mice also exhibited a delayed onset signs of illness, and a marked decrease in morbidity, suggesting a nsP2 mediated adjuvant effect. Co-injection of the pnsP2 adjuvant with pEnv also qualitatively and quantitatively increased antigen specific neutralizing antibody responses compared to vaccination with pEnv alone. In sum, these novel data imply that the addition of nsP2 to the pEnv vaccine enhances anti-CHIKV-Env immune responses and maybe useful to include in future CHIKV clinical vaccination strategies.
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Induction of broad cytotoxic T cells by protective DNA vaccination against Marburg and Ebola.
Mol. Ther.
PUBLISHED: 02-12-2013
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Marburg and Ebola hemorrhagic fevers have been described as the most virulent viral diseases known to man due to associative lethality rates of up to 90%. Death can occur within days to weeks of exposure and there is currently no licensed vaccine or therapeutic. Recent evidence suggests an important role for antiviral T cells in conferring protection, but little detailed analysis of this response as driven by a protective vaccine has been reported. We developed a synthetic polyvalent-filovirus DNA vaccine against Marburg marburgvirus (MARV), Zaire ebolavirus (ZEBOV), and Sudan ebolavirus (SUDV). Preclinical efficacy studies were performed in guinea pigs and mice using rodent-adapted viruses, whereas murine T-cell responses were extensively analyzed using a novel modified assay described herein. Vaccination was highly potent, elicited robust neutralizing antibodies, and completely protected against MARV and ZEBOV challenge. Comprehensive T-cell analysis revealed cytotoxic T lymphocytes (CTLs) of great magnitude, epitopic breadth, and Th1-type marker expression. This model provides an important preclinical tool for studying protective immune correlates that could be applied to existing platforms. Data herein support further evaluation of this enhanced gene-based approach in nonhuman primate studies for in depth analyses of T-cell epitopes in understanding protective efficacy.
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Functional enhancement of AT1R potency in the presence of the TP?R is revealed by a comprehensive 7TM receptor co-expression screen.
PLoS ONE
PUBLISHED: 02-07-2013
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Functional cross-talk between seven transmembrane (7TM) receptors can dramatically alter their pharmacological properties, both in vitro and in vivo. This represents an opportunity for the development of novel therapeutics that potentially target more specific biological effects while causing fewer adverse events. Although several studies convincingly have established the existence of 7TM receptor cross-talk, little is known about the frequencey and biological significance of this phenomenon.
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Expression of the ammonia transporter family member, Rh B Glycoprotein, in the human kidney.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 01-16-2013
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The ammonia transporter family member, Rh B Glycoprotein (RhBG/Rhbg), is essential for ammonia transport by the rodent kidney, but in the human kidney mRNA but not protein expression has been reported. Because ammonia transport is fundamental for acid-base homeostasis, the current study addressed RhBG expression in the human kidney. Two distinct RhBG mRNA sequences have been reported, with different numbers of consecutive cytosines at nt1265 and thus encoding different carboxy-tails. Sequencing the region of difference in both human kidney and liver mRNA showed eight sequential cytosines, not seven as in some reports. Knowing the correct mRNA sequence for RhBG, we then assessed RhBG protein expression using antibodies against the correct amino acid sequence. Immunoblot analysis demonstrated RhBG protein expression in human kidney and immunohistochemistry identified basolateral RhBG in connecting segment (CNT) and the cortical and outer medullary collecting ducts. Colocalization of RhBG with multiple cell-specific markers demonstrated that that CNT cells and collecting duct type A intercalated cells express high levels of RhBG, and type B intercalated cells and principal cells do not express detectable RhBG. Thus, these studies identify the correct mRNA and thus protein sequence for human RhBG and show that the human kidney expresses basolateral RhBG protein in CNT, type A intercalated cells, and non-A, non-B cells. We conclude that RhBG can mediate an important role in human renal ammonia transport.
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In vivo electroporation of constitutively expressed HIF-1? plasmid DNA improves neovascularization in a mouse model of limb ischemia.
J. Vasc. Surg.
PUBLISHED: 01-15-2013
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Hypoxia-inducible factor-1 alpha (HIF-1?) is a transcription factor that stimulates angiogenesis during tissue ischemia. In vivo electroporation (EP) enhances tissue DNA transfection. We hypothesized that in vivo EP of plasmid DNA encoding a constitutively expressed HIF-1? gene enhances neovascularization compared with intramuscular (IM) injection alone.
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Synthetic DNA Vaccines: Improved Vaccine Potency by Electroporation and Co-Delivered Genetic Adjuvants.
Front Immunol
PUBLISHED: 01-01-2013
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In recent years, DNA vaccines have undergone a number of technological advancements that have incited renewed interest and heightened promise in the field. Two such improvements are the use of genetically engineered cytokine adjuvants and plasmid delivery via in vivo electroporation (EP), the latter of which has been shown to increase antigen delivery by nearly 1000-fold compared to naked DNA plasmid delivery alone. Both strategies, either separately or in combination, have been shown to augment cellular and humoral immune responses in not only mice, but also in large animal models. These promising results, coupled with recent clinical trials that have shown enhanced immune responses in humans, highlight the bright prospects for DNA vaccines to address many human diseases.
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Expression of the rhesus glycoproteins, ammonia transporter family members, RHCG and RHBG in male reproductive organs.
Reproduction
PUBLISHED: 01-01-2013
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The rhesus glycoproteins, Rh B glycoprotein (RHBG) and Rh C glycoprotein (RHCG), are recently identified ammonia transporters. Rhcg expression is necessary for normal male fertility, but its specific cellular expression is unknown, and Rhbg has not been reported to be expressed in the male reproductive tract. This study sought to determine the specific cellular expression of Rhcg, to determine whether Rhbg is expressed in the male reproductive tract, and, if so, to determine which cells express Rhbg using real-time RT-PCR, immunoblot analysis, and immunohistochemistry. Both Rhbg and Rhcg were expressed throughout the male reproductive tract. In the testis, high levels of Rhbg were expressed in Leydig cells, and Rhcg was expressed in spermatids during the later stages of their maturation (steps 13-16) in stages I-VIII of the seminiferous epithelium cycle. In the epididymis, basolateral Rhbg was present in narrow cells in the initial segment, in principal cells in the upper corpus, and in clear cells throughout the epididymis. Apical Rhcg immunolabel was present in principal cells in the caput and upper corpus epididymidis and in clear cells in the middle and lower corpus and cauda epididymidis. In the vas deferens, apical Rhcg immunolabel and basolateral Rhbg immunolabel were present in some principal cells and colocalized with H(+)-ATPase immunolabel. We conclude that both Rhbg and Rhcg are highly expressed in specific cells in the male reproductive tract where they can contribute to multiple components of male fertility.
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Peripheral immunization induces functional intrahepatic hepatitis C specific immunity following selective retention of vaccine-specific CD8 T cells by the liver.
Hum Vaccin
PUBLISHED: 12-01-2011
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It is believed that an effective HCV vaccine must induce strong HCV-specific cytotoxic IFN-?? CD8? T cells able to migrate into and become fully activated within the liver, an organ known to suppress T cell responses and induce tolerance. Given the importance of intrahepatic HCV-specific T cells in the clearance of acute infection, the goal of this present study was to determine if peripheral immunization was able to induce functional intrahepatic HCV-specific T cell based immunity both in the presence and absence of HCV antigen expression within the liver. Using a novel HCV NS3/NS4A DNA vaccine, we show that peripheral immunization of C57BL/6 mice results in the formation of a large pool of fully functional HCV-specific cytotoxic IFN-?? CD8? T cells within the liver and that these cells were highly enriched within the liver as compared to the spleen. Following hepatic expression of cognate HCV antigen using a previously described liver transfection method, we show that this pool of vaccine-induced HCV-specific CD8? T cells retained its ability to become highly activated as shown by the upregulation of IFN-? and CCR5 expression, as well as by the clearance of HCV NS3 expressing hepatocytes. Taken together, these findings suggest that T cell effector function is preserved within the liver and that selective recruitment of antigen-specific T cells to the liver may play a previously unappreciated role in the process of immune surveillance, which may be exploited for future T cell based HCV vaccines.
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Renal ischemia-reperfusion injury causes intercalated cell-specific disruption of occludin in the collecting duct.
Histochem. Cell Biol.
PUBLISHED: 10-23-2011
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Renal ischemic events open tight junctions and disrupt epithelial polarity. The purpose of this study was to examine the effects of ischemia-reperfusion (IR) injury on expression and distribution of the tight junction proteins, occludin and ZO-1, in the rat kidney. IR injury was induced by clamping both renal pedicles for 30 min and animals were killed at 6 h after the reperfusion. IR injury decreased blood bicarbonate level, but did not persistently alter pH, Na(+), K(+), or Cl(-). In control kidneys, occludin immunoreactivity was intense in the tight junctions in the thick ascending limb, distal convoluted tubule, and collecting duct, moderate in the thin limbs of the loop of Henle, and was not detected in the proximal tubule, glomerulus, and blood vessels. ZO-1 was expressed in the same sites in which occludin was expressed, and additionally was also expressed in the proximal tubule, glomerulus, and vascular endothelial cells. IR kidneys exhibited damaged renal tubular epithelial cells in both proximal tubule and collecting duct segments in the outer medulla. In the collecting duct, the response of intercalated cells and principal cells differed. Following IR injury, intercalated cells, but not principal cells, lost their normal epithelial polarity and were frequently extruded into the tubule lumen. Occludin, instead of being localized to tight junctions, was localized diffusely in the cytoplasm in intercalated cells of IR kidneys. Principal cells, in contrast, were not detectably affected and neither occludin nor ZO-1 expression were altered in response to IR injury. The normal localization of ZO-1 expression to tight junction sites in both the proximal tubule and collecting duct was altered in response to IR, and, instead, ZO-1 expression was present diffusely in the cytoplasm. IR injury did not alter detectably either occludin or ZO-1 localization to the tight junction of the thick ascending limb cells. The abundance of total occludin protein by immunoblot analysis was not changed with IR injury. These results demonstrate that renal IR injury causes tight junction disruptions in both the proximal tubule and the collecting duct, and that altered distribution of the tight junction protein, occludin, may play a critical role in the collecting duct dysfunction which IR induces.
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Hepatitis C virus NS3/NS4A DNA vaccine induces multiepitope T cell responses in rhesus macaques mimicking human immune responses [corrected].
Mol. Ther.
PUBLISHED: 09-27-2011
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Numerous studies have suggested that an effective hepatitis C virus (HCV) vaccine must induce a strong T helper 1 (Th1) T cell response. While several therapeutic vaccine candidates have shown promise in clinical trials, response rates have been low suggesting that further optimization is important. However, such optimization has been hindered by a lack of a benchmark animal model in which to test vaccine-induced immune responses before clinical evaluation. The goal of this study was to analyze the utility of the rhesus macaque vaccination model in assessing HCV vaccine-induced T cell responses. To test this, we employed the use of a novel HCV genotype 1a/1b consensus DNA vaccine encoding both HCV nonstructural protein 3 (NS3) and nonstructural protein 4A (NS4A) proteins. Following immunization, rhesus macaques mounted HCV-specific responses strikingly similar to those reported in resolving patients, including strong NS3-specific interferon-? (IFN-?) responses, robust CD4(+) and CD8(+) T cell proliferation, and induction of polyfunctional T cells. Additionally, fine epitope mapping revealed one animal that mounted a T cell response against a known HCV NS3 human leukocyte antigen A2 (HLA-A2) epitope previously identified in humans. Taken together our findings suggest that the rhesus macaque vaccination model is a useful tool in the evaluation of immune responses induced by HCV immunogens.
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Angiotensin II acts through the angiotensin 1a receptor to upregulate pendrin.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 09-14-2011
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Pendrin is an anion exchanger expressed in the apical regions of B and non-A, non-B intercalated cells. Since angiotensin II increases pendrin-mediated Cl(-) absorption in vitro, we asked whether angiotensin II increases pendrin expression in vivo and whether angiotensin-induced hypertension is pendrin dependent. While blood pressure was similar in pendrin null and wild-type mice under basal conditions, following 2 wk of angiotensin II administration blood pressure was 31 mmHg lower in pendrin null than in wild-type mice. Thus pendrin null mice have a blunted pressor response to angiotensin II. Further experiments explored the effect of angiotensin on pendrin expression. Angiotensin II administration shifted pendrin label from the subapical space to the apical plasma membrane, independent of aldosterone. To explore the role of the angiotensin receptors in this response, pendrin abundance and subcellular distribution were examined in wild-type, angiotensin type 1a (Agtr1a) and type 2 receptor (Agtr2) null mice given 7 days of a NaCl-restricted diet (< 0.02% NaCl). Some mice received an Agtr1 inhibitor (candesartan) or vehicle. Both Agtr1a gene ablation and Agtr1 inhibitors shifted pendrin label from the apical plasma membrane to the subapical space, independent of the Agtr2 or nitric oxide (NO). However, Agtr1 ablation reduced pendrin protein abundance through the Agtr2 and NO. Thus angiotensin II-induced hypertension is pendrin dependent. Angiotensin II acts through the Agtr1a to shift pendrin from the subapical space to the apical plasma membrane. This Agtr1 action may be blunted by the Agtr2, which acts through NO to reduce pendrin protein abundance.
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HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.
J. Immunol.
PUBLISHED: 08-19-2011
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Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-?, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.
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Clinical applications of DNA vaccines: current progress.
Clin. Infect. Dis.
PUBLISHED: 07-19-2011
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It was discovered almost 20 years ago that plasmid DNA, when injected into the skin or muscle of mice, could induce immune responses to encoded antigens. Since that time, there has since been much progress in understanding the basic biology behind this deceptively simple vaccine platform and much technological advancement to enhance immune potency. Among these advancements are improved formulations and improved physical methods of delivery, which increase the uptake of vaccine plasmids by cells; optimization of vaccine vectors and encoded antigens; and the development of novel formulations and adjuvants to augment and direct the host immune response. The ability of the current, or second-generation, DNA vaccines to induce more-potent cellular and humoral responses opens up this platform to be examined in both preventative and therapeutic arenas. This review focuses on these advances and discusses both preventive and immunotherapeutic clinical applications.
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Effect of hypokalemia on renal expression of the ammonia transporter family members, Rh B Glycoprotein and Rh C Glycoprotein, in the rat kidney.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 07-13-2011
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Hypokalemia is a common electrolyte disorder that increases renal ammonia metabolism and can cause the development of an acid-base disorder, metabolic alkalosis. The ammonia transporter family members, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), are expressed in the distal nephron and collecting duct and mediate critical roles in acid-base homeostasis by facilitating ammonia secretion. In the current studies, the effect of hypokalemia on renal Rhbg and Rhcg expression was examined. Normal Sprague-Dawley rats received either K(+)-free or control diets for 2 wk. Rats receiving the K(+)-deficient diet developed hypokalemia and metabolic alkalosis associated with significant increases in both urinary ammonia excretion and urine pH. Rhcg expression increased in the outer medullary collecting duct (OMCD). In OMCD intercalated cells, hypokalemia resulted in more discrete apical Rhcg expression and a marked increase in apical plasma membrane immunolabel. In principal cells, in the OMCD, hypokalemia increased both apical and basolateral Rhcg immunolabel intensity. Cortical Rhcg expression was not detectably altered by immunohistochemistry, although there was a slight decrease in total expression by immunoblot analysis. Rhbg protein expression was decreased slightly in the cortex and not detectably altered in the outer medulla. We conclude that in rat OMCD, hypokalemia increases Rhcg expression, causes more polarized apical expression in intercalated cells, and increases both apical and basolateral expression in the principal cell. Increased plasma membrane Rhcg expression in response to hypokalemia in the rat, particularly in the OMCD, likely contributes to the increased ammonia excretion and thereby to the development of metabolic alkalosis.
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Selected approaches for increasing HIV DNA vaccine immunogenicity in vivo.
Curr Opin Virol
PUBLISHED: 07-05-2011
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The safety, stability, and ability for repeat homologous vaccination makes the DNA vaccine platform an excellent candidate for an effective HIV-1 vaccine. However, the immunogenicity of early DNA vaccines did not translate from small animal models into larger non-human primates and was markedly lower than viral vectors. In addition to improvements to the DNA vector itself, delivery with electroporation, the inclusion of molecular adjuvants, and heterologous prime-boost strategies have dramatically improved the immunogenicity of DNA vaccines for HIV and currently makes them a leading platform with many areas warranting further research and clinical development.
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Immune modulation through 4-1BB enhances SIV vaccine protection in non-human primates against SIVmac251 challenge.
PLoS ONE
PUBLISHED: 06-09-2011
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Costimulatory molecules play a central role in the development of cellular immunity. Understanding how costimulatory pathways can be directed to positively influence the immune response may be critical for the generation of an effective HIV vaccine. Here, we evaluated the ability of intravenous administration of a blocking monoclonal antibody (mAb) directed against the negative costimulatory molecule CTLA-4, and an agonist mAb directed against the positive costimulatory molecule 4-1BB, either alone or in combination, to augment intramuscular SIV DNA immunizations. We then tested the ability these of these responses to impact a high-dose SIVmac251 challenge. Following immunization, the groups infused with the anti-4-1BB mAb exhibited enhanced IFN-? responses compared to the DNA vaccine only group. Interestingly, although CTLA-4 blockade alone did not enhance IFN-? responses it did increase the proliferative capacity of the CD4(+) and CD8(+) T cells. The combination of both mAbs enhanced the magnitude of the polyfunctional CD8(+) T cell response. Following challenge, the group that received both mAbs exhibited a significant, ?2.0 log, decrease in plasma viral load compared to the naïve group the included complete suppression of viral load in some animals. Furthermore, the use of the CTLA-4 blocking antibody resulted in significantly higher viral loads during chronic infection compared to animals that received the 4-1BB mAb, likely due to the higher CD4(+) T cell proliferative responses which were driven by this adjuvant following immunization. These novel studies show that these adjuvants induce differential modulation of immune responses, which have dramatically different consequences for control of SIV replication, suggesting important implications for HIV vaccine development.
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Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine.
Vaccine
PUBLISHED: 06-07-2011
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DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding suggests that such synthetic immunogens could be important for examination of their potential as part of an efficient HIV DNA vaccine.
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Priorities in Parkinsons disease research.
Nat Rev Drug Discov
PUBLISHED: 05-03-2011
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The loss of dopaminergic neurons in the substantia nigra pars compacta leads to the characteristic motor symptoms of Parkinsons disease: bradykinesia, rigidity and resting tremors. Although these symptoms can be improved using currently available dopamine replacement strategies, there is still a need to improve current strategies of treating these symptoms, together with a need to alleviate non-motor symptoms of the disease. Moreover, treatments that provide neuroprotection and/or disease-modifying effects remain an urgent unmet clinical need. This Review describes the most promising biological targets and therapeutic agents that are currently being assessed to address these treatment goals. Progress will rely on understanding genetic mutations or susceptibility factors that lead to Parkinsons disease, better translation between preclinical animal models and clinical research, and improving the design of future clinical trials.
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Long-term programming of antigen-specific immunity from gene expression signatures in the PBMC of rhesus macaques immunized with an SIV DNA vaccine.
PLoS ONE
PUBLISHED: 04-05-2011
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While HIV-1-specific cellular immunity is thought to be critical for the suppression of viral replication, the correlates of protection have not yet been determined. Rhesus macaques (RM) are an important animal model for the study and development of vaccines against HIV/AIDS. Our laboratory has helped to develop and study DNA-based vaccines in which recent technological advances, including genetic optimization and in vivo electroporation (EP), have helped to dramatically boost their immunogenicity. In this study, RMs were immunized with a DNA vaccine including individual plasmids encoding SIV gag, env, and pol alone, or in combination with a molecular adjuvant, plasmid DNA expressing the chemokine ligand 5 (RANTES), followed by EP. Along with standard immunological assays, flow-based activation analysis without ex vivo restimulation and high-throughput gene expression analysis was performed. Strong cellular immunity was induced by vaccination which was supported by all assays including PBMC microarray analysis that identified the up-regulation of 563 gene sequences including those involved in interferon signaling. Furthermore, 699 gene sequences were differentially regulated in these groups at peak viremia following SIVmac251 challenge. We observed that the RANTES-adjuvanted animals were significantly better at suppressing viral replication during chronic infection and exhibited a distinct pattern of gene expression which included immune cell-trafficking and cell cycle genes. Furthermore, a greater percentage of vaccine-induced central memory CD8+ T-cells capable of an activated phenotype were detected in these animals as measured by activation analysis. Thus, co-immunization with the RANTES molecular adjuvant followed by EP led to the generation of cellular immunity that was transcriptionally distinct and had a greater protective efficacy than its DNA alone counterpart. Furthermore, activation analysis and high-throughput gene expression data may provide better insight into mechanisms of viral control than may be observed using standard immunological assays.
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Electroporation delivery of DNA vaccines: prospects for success.
Curr. Opin. Immunol.
PUBLISHED: 02-07-2011
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A number of noteworthy technology advances in DNA vaccines research and development over the past few years have led to the resurgence of this field as a viable vaccine modality. Notably, these include--optimization of DNA constructs; development of new DNA manufacturing processes and formulations; augmentation of immune responses with novel encoded molecular adjuvants; and the improvement in new in vivo delivery strategies including electroporation (EP). Of these, EP mediated delivery has generated considerable enthusiasm and appears to have had a great impact in vaccine immunogenicity and efficacy by increasing antigen delivery upto a 1000 fold over naked DNA delivery alone. This increased delivery has resulted in an improved in vivo immune response magnitude as well as response rates relative to DNA delivery by direct injection alone. Indeed the immune responses and protection from pathogen challenge observed following DNA administration via EP in many cases are comparable or superior to other well studied vaccine platforms including viral vectors and live/attenuated/inactivated virus vaccines. Significantly, the early promise of EP delivery shown in numerous pre-clinical animal models of many different infectious diseases and cancer are now translating into equally enhanced immune responses in human clinical trials making the prospects for this vaccine approach to impact diverse disease targets tangible.
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Pimavanserin tartrate, a 5-HT(2A) receptor inverse agonist, increases slow wave sleep as measured by polysomnography in healthy adult volunteers.
Sleep Med.
PUBLISHED: 01-21-2011
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Determine the effects of pimavanserin tartrate [ACP-103; N-(4-flurophenylmethyl)-N-(1-methylpiperidin-4-yl)-N-(4-(2-methylpropyloxy)phenylmethyl)carbamide], a selective serotonin 5-HT(2A) receptor inverse agonist, on slow wave sleep (SWS), other sleep parameters, and attention/vigilance.
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A highly optimized DNA vaccine confers complete protective immunity against high-dose lethal lymphocytic choriomeningitis virus challenge.
Vaccine
PUBLISHED: 01-14-2011
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Protection against infection is the hallmark of immunity and the basis of effective vaccination. For a variety of reasons there is a great demand to develop new, safer and more effective vaccine platforms. In this regard, while first-generation DNA vaccines were poorly immunogenic, new genetic optimization strategies and the application of in vivo electroporation (EP) have dramatically boosted their potency. We developed a highly optimized plasmid DNA vaccine that expresses the lymphocytic choriomeningitis virus (LCMV) nucleocapsid protein (NP) and evaluated it using the LCMV challenge model, a gold standard for studying infection and immunity. When administered intramuscularly with EP, robust NP-specific cellular and humoral immune responses were elicited, the magnitudes of which approached those following acute LCMV infection. Furthermore, these responses were capable of providing 100% protection against a high-dose, normally lethal virus challenge. This is the first non-infectious vaccine conferring complete protective immunity up to 8 weeks after vaccination and demonstrates the potential of next-generation DNA vaccines.
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Multivalent smallpox DNA vaccine delivered by intradermal electroporation drives protective immunity in nonhuman primates against lethal monkeypox challenge.
J. Infect. Dis.
PUBLISHED: 01-12-2011
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The threat of a smallpox-based bioterrorist event or a human monkeypox outbreak has heightened the importance of new, safe vaccine approaches for these pathogens to complement older poxviral vaccine platforms. As poxviruses are large, complex viruses, they present technological challenges for simple recombinant vaccine development where a multicomponent mixtures of vaccine antigens are likely important in protection. We report that a synthetic, multivalent, highly concentrated, DNA vaccine delivered by a minimally invasive, novel skin electroporation microarray can drive polyvalent immunity in macaques, and offers protection from a highly pathogenic monkeypox challenge. Such a diverse, high-titer antibody response produced against 8 different DNA-encoded antigens delivered simultaneously in microvolumes has not been previously described. These studies represent a significant improvement in the efficiency of the DNA vaccine platform, resulting in immune responses that mimic live viral infections, and would likely have relevance for vaccine design against complex human and animal pathogens.
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A DNA vaccine against chikungunya virus is protective in mice and induces neutralizing antibodies in mice and nonhuman primates.
PLoS Negl Trop Dis
PUBLISHED: 01-11-2011
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Chikungunya virus (CHIKV) is an emerging mosquito-borne alphavirus indigenous to tropical Africa and Asia. Acute illness is characterized by fever, arthralgias, conjunctivitis, rash, and sometimes arthritis. Relatively little is known about the antigenic targets for immunity, and no licensed vaccines or therapeutics are currently available for the pathogen. While the Aedes aegypti mosquito is its primary vector, recent evidence suggests that other carriers can transmit CHIKV thus raising concerns about its spread outside of natural endemic areas to new countries including the U.S. and Europe. Considering the potential for pandemic spread, understanding the development of immunity is paramount to the development of effective counter measures against CHIKV. In this study, we isolated a new CHIKV virus from an acutely infected human patient and developed a defined viral challenge stock in mice that allowed us to study viral pathogenesis and develop a viral neutralization assay. We then constructed a synthetic DNA vaccine delivered by in vivo electroporation (EP) that expresses a component of the CHIKV envelope glycoprotein and used this model to evaluate its efficacy. Vaccination induced robust antigen-specific cellular and humoral immune responses, which individually were capable of providing protection against CHIKV challenge in mice. Furthermore, vaccine studies in rhesus macaques demonstrated induction of nAb responses, which mimicked those induced in convalescent human patient sera. These data suggest a protective role for nAb against CHIKV disease and support further study of envelope-based CHIKV DNA vaccines.
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Fresh MESA improved embryo fertilization, cleavage, blastula formation and implantation rates after failed TESA in couples with obstructive azoospermia.
J. Assist. Reprod. Genet.
PUBLISHED: 01-07-2011
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To determine whether our use of fresh MESA cycles improved outcomes in patients with obstructive azoospermia who had failed IVF with TESA.
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Cellular immune response in the presence of protective antibody levels correlates with protection against 1918 influenza in ferrets.
Vaccine
PUBLISHED: 01-04-2011
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The identification of immune correlates of protection against highly pathogenic human-adapted influenza is instrumental in the development of the next generation of vaccines. Towards this, ferrets received either one dose of a conventionally produced vaccine, two inoculations of a hemagglutinin (HA)-expressing DNA vaccine, or a prime-boost regimen of the DNA vaccine followed by injection of a HA-expressing adenoviral vector. In addition to the antibody response, ferret-specific interferon-gamma (IFN-?) ELISpot and flow cytometry assays were developed to follow the cellular immune response. Animals that received the conventional vaccine mounted a humoral response, while the DNA vaccinated groups also developed IFN-? producing T cells. Upon challenge with the matched highly pathogenic A/South Carolina/1/18 H1N1 influenza A virus, the conventionally vaccinated group developed moderate to severe signs of disease, whereas the DNA vaccinated animals experienced mild disease. In the presence of an antibody response within the protective range, the extent of the T cell response correlated more accurately with reduced morbidity in vaccinated ferrets.
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High antibody and cellular responses induced to HIV-1 clade C envelope following DNA vaccines delivered by electroporation.
Vaccine
PUBLISHED: 01-04-2011
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Clade C is the predominant HIV-1 strain infecting people in sub-Saharan Africa, India, and China and there is a critical need for a vaccine targeted to these areas. In this study we tested a DNA based vaccine that encodes the SIVgag, SIVpol and HIV-1 envelope clade C.
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Co-delivery of PSA and PSMA DNA vaccines with electroporation induces potent immune responses.
Hum Vaccin
PUBLISHED: 01-01-2011
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Prostate cancer (PCa) remains a significant public health problem. Current treatment modalities for PCa can be useful, but may be accompanied by deleterious side effects and often do not confer long-term control. Accordingly, additional modalities, such as immunotherapy, may represent an important approach for PCa treatment. The identification of tissue-specific antigens engenders PCa an attractive target for immunotherapeutic approaches. Delivery of DNA vaccines with electroporation has shown promising results for prophylactic and therapeutic targets in a variety of species including humans. Application of this technology for PCa immunotherapy strategies has been limited to single antigen and epitope targets. We sought to test the hypothesis that a broader collection of antigens would improve the breadth and effectiveness of a PCa immune therapy approach. We therefore developed highly optimized DNA vaccines encoding prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) as a dual antigen approach to immune therapy of PCa. PSA-and PSMA-specific cellular immunogenicity was evaluated in a mouse model for co-delivery and single antigen vaccination. Mice received 2 immunizations spaced 2 weeks apart and immunogenicity was evaluated 1 week after the second vaccination. Both the PSA and PSMA vaccines induced robust antigen-specific IFN? responses by ELISpot. Further characterization of cellular immunogenicity by flow cytometry indicated strong antigen-specific TNF? production by CD4+ T cells and IFN? and IL-2 secretion by both CD4+ and CD8+ T cells. There was also a strong humoral response as determined by PSA-specific seroconversion. These data support further study of this novel approach to immune therapy of PCa.
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A novel prototype device for electroporation-enhanced DNA vaccine delivery simultaneously to both skin and muscle.
Vaccine
PUBLISHED: 01-01-2011
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Electroporation (EP) of either muscle or skin has proven to be an efficient method for increasing DNA-based vaccine delivery and immunogenicity in small and large animals. Previous comparative studies in large animals suggest that intramuscular (i.m.) DNA EP delivery appears to favor cellular immunity, while intradermal (i.d.) EP delivery may favor humoral immunity. While current EP devices are primarily designed either for i.m. or i.d. delivery, we developed a novel prototype Dual-Depth Device (DDD) for EP-mediated simultaneous i.d. and i.m. delivery of DNA-based vaccines with an attempt to elicit superior antibody and cellular immune responses. We performed comparisons of DDD EP delivery with standard i.d. EP, standard i.m. EP, and combined delivery of i.d. and i.m. EP at separate sites, for the ability to induce antigen-specific immune responses. In a guinea pig model using a SynCon™ DNA vaccine encoding the influenza virus H5 hemaglutinin (H5HA), vaccination via DDD or combined delivery induced higher antibody titers than via either i.d. or i.m. delivery alone. In a mouse model using a DNA vaccine encoding the nucleoprotein (NP) of influenza H1N1, the resulting trend of antibody responses was similar to that detected in guinea pig study. Importantly, cellular immune responses in the DDD or combined delivery groups were significantly stronger than that in either i.d. or i.m. delivery groups. We conclude that EP-mediated DNA-based vaccine delivery to both skin and muscle is superior to delivery to either tissue alone for induction of antigen-specific antibody and cellular immunity.
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Role of NH3 and NH4+ transporters in renal acid-base transport.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 11-03-2010
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Renal ammonia excretion is the predominant component of renal net acid excretion. The majority of ammonia excretion is produced in the kidney and then undergoes regulated transport in a number of renal epithelial segments. Recent findings have substantially altered our understanding of renal ammonia transport. In particular, the classic model of passive, diffusive NH3 movement coupled with NH4+ "trapping" is being replaced by a model in which specific proteins mediate regulated transport of NH3 and NH4+ across plasma membranes. In the proximal tubule, the apical Na+/H+ exchanger, NHE-3, is a major mechanism of preferential NH4+ secretion. In the thick ascending limb of Henles loop, the apical Na+-K+-2Cl- cotransporter, NKCC2, is a major contributor to ammonia reabsorption and the basolateral Na+/H+ exchanger, NHE-4, appears to be important for basolateral NH4+ exit. The collecting duct is a major site for renal ammonia secretion, involving parallel H+ secretion and NH3 secretion. The Rhesus glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), are recently recognized ammonia transporters in the distal tubule and collecting duct. Rhcg is present in both the apical and basolateral plasma membrane, is expressed in parallel with renal ammonia excretion, and mediates a critical role in renal ammonia excretion and collecting duct ammonia transport. Rhbg is expressed specifically in the basolateral plasma membrane, and its role in renal acid-base homeostasis is controversial. In the inner medullary collecting duct (IMCD), basolateral Na+-K+-ATPase enables active basolateral NH4+ uptake. In addition to these proteins, several other proteins also contribute to renal NH3/NH4+ transport. The role and mechanisms of these proteins are discussed in depth in this review.
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Interferons as potential adjuvants in prophylactic vaccines.
Expert Opin Biol Ther
PUBLISHED: 09-15-2010
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Vaccines are still one of the best approaches to manage infectious diseases. Despite the advances in drug therapies, prophylactic medicine is still more cost efficient and minimizes the burden in the heath system. Despite all the research in vaccine development, many infectious diseases are still without an effective vaccine. The use of adjuvants in vaccines has been one successful strategy to increase efficacy. IFNs are widely expressed cytokines that have potent antiviral effects. These cytokines are the first line of defense against viral infections and have important roles in immuno surveillance for malignant cells. One of the most promising uses of IFNs is as adjuvants that are co-applied with antigen in vaccines.
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Role of the Rhesus glycoprotein, Rh B glycoprotein, in renal ammonia excretion.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 08-18-2010
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Rh B glycoprotein (Rhbg) is a member of the Rh glycoprotein family of ammonia transporters. In the current study, we examine Rhbgs role in basal and acidosis-stimulated acid-base homeostasis. Metabolic acidosis induced by HCl administration increased Rhbg expression in both the cortex and outer medulla. To test the functional significance of increased Rhbg expression, we used a Cre-loxP approach to generate mice with intercalated cell-specific Rhbg knockout (IC-Rhbg-KO). On normal diet, intercalated cell-specific Rhbg deletion did not alter urine ammonia excretion, pH, or titratable acid excretion significantly, but it did decrease glutamine synthetase expression in the outer medulla significantly. After metabolic acidosis was induced, urinary ammonia excretion was significantly less in IC-Rhbg-KO than in control (C) mice on days 2-4 of acid loading, but not on day 5. Urine pH and titratable acid excretion and dietary acid intake did not differ significantly between acid-loaded IC-Rhcg-KO and C mice. In IC-Rhbg-KO mice, acid loading increased connecting segment (CNT) cell and outer medullary collecting duct principal cell Rhbg expression. In both C and IC-Rhbg-KO mice, acid loading decreased glutamine synthetase in both the cortex and outer medulla; the decrease on day 3 was similar in IC-Rhbg-KO and C mice, but on day 5 it was significantly greater in IC-Rhbg-KO than in C mice. We conclude 1) intercalated cell Rhbg contributes to acidosis-stimulated renal ammonia excretion, 2) Rhbg in CNT and principal cells may contribute to renal ammonia excretion, and 3) decreased glutamine synthetase expression may enable normal rates of ammonia excretion under both basal conditions and on day 5 of acid loading in IC-Rhbg-KO mice.
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Unique Th1/Th2 phenotypes induced during priming and memory phases by use of interleukin-12 (IL-12) or IL-28B vaccine adjuvants in rhesus macaques.
Clin. Vaccine Immunol.
PUBLISHED: 08-04-2010
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Adjuvant compounds are usually included in vaccinations in order to bolster total vaccine-specific responses or to tailor an immune response toward a desired endpoint, such as the production of gamma interferon or an increase in antibody titers. While most adjuvants are studied in regard to their impact on vaccine-specific responses during and just after the vaccination period, a detailed analysis of how adjuvants skew the Th1/Th2 axis at more distant time points is not often undertaken. In the current study, we present data that suggests that adjuvants differ in their relative abilities to bolster and skew immune responses in the short term compared with more distant time points. To that end, we have employed interleukin-12 (IL-12) and IL-28B as adjuvants for DNA vaccination of rhesus macaques. While both adjuvants were able to bolster Th1-biased responses, our analysis shows that this skewing was achieved through different mechanisms. Moreover, analysis 3 months after the final immunization revealed the activity of the IL-12 adjuvant to be short lived, while the IL-28B adjuvant continued to exert its influence on the immune system. Taken together, these data suggest that the scientific and medical communities would benefit from a more detailed analysis of adjuvant function, including the determination of long-term influences of administered adjuvants.
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Circular permutation provides an evolutionary link between two families of calcium-dependent carbohydrate binding modules.
J. Biol. Chem.
PUBLISHED: 07-21-2010
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The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a ?-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two ?-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a ?-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.
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DNA vaccines for targeting bacterial infections.
Expert Rev Vaccines
PUBLISHED: 07-14-2010
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DNA vaccination has been of great interest since its discovery in the 1990s due to its ability to elicit both humoral and cellular immune responses. DNA vaccines consist of a DNA plasmid containing a transgene that encodes the sequence of a target protein from a pathogen under the control of a eukaryotic promoter. This revolutionary technology has proven to be effective in animal models and four DNA vaccine products have recently been approved for veterinary use. Although few DNA vaccines against bacterial infections have been tested, the results are encouraging. Because of their versatility, safety and simplicity a wider range of organisms can be targeted by these vaccines, which shows their potential advantages to public health. This article describes the mechanism of action of DNA vaccines and their potential use for targeting bacterial infections. In addition, it provides an updated summary of the methods used to enhance immunogenicity from codon optimization and adjuvants to delivery techniques including electroporation and use of nanoparticles.
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IL-28B/IFN-lambda 3 drives granzyme B loading and significantly increases CTL killing activity in macaques.
Mol. Ther.
PUBLISHED: 06-22-2010
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Type III/lambda interferons (IFNs) were discovered less than a decade ago and are still in the process of being characterized. Although previous studies have focused on the function of IFN-lambda 3 (also known as interleukin (IL)-28B) in a small animal model, it is unknown whether these functions would translate to a larger, more relevant model. Thus in the present study, we have used DNA vaccination as a method of studying the influence of IFN-lambda 3 on adaptive immune responses in rhesus macaques. Results of our study show for the first time that IFN-lambda 3 has significant influence on antigen-specific CD8(+) T-cell function, especially in regards to cytotoxicity. Peripheral CD8(+) T cells from animals that were administered IFN-lambda 3 showed substantially increased cytotoxic responses as gauged by CD107a and granzyme B coexpression as well as perforin release. Moreover, CD8(+) T cells isolated from the mesenteric lymph nodes (MLN) of animals receiving IFN-lambda 3 loaded significant amounts of granzyme B upon extended antigenic stimulation and induced significantly more granzyme B-mediated cell death of peptide pulsed targets. These data suggest that IFN-lambda 3 is a potent effector of the immune system with special emphasis on CD8(+) T-cell killing functions which warrants further study as a possible immunoadjuvant.
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Comparative analysis of immune responses induced by vaccination with SIV antigens by recombinant Ad5 vector or plasmid DNA in rhesus macaques.
Mol. Ther.
PUBLISHED: 06-15-2010
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DNA vaccines have undergone important enhancements in their design, formulation, and delivery process. Past literature supports that DNA vaccines are not as immunogenic in nonhuman primates as live vector systems. The most potent recombinant vector system for induction of cellular immune responses in macaques and humans is adenovirus serotype 5 (Ad5), an important benchmark for new vaccine development. Here, we performed a head-to-head evaluation of the Merck Ad5 SIV vaccine and an optimized electroporation (EP) delivered SIV DNA vaccine in macaques. Animals receiving the Ad5 vaccine were immunized three times, whereas the DNA-vaccinated animals were immunized up to four times based on optimized protocols. We observed significant differences in the quantity of IFNgamma responses by enzyme-linked immunosorbent spot (ELISpot), greater proliferative capacity of CD8(+) T cells, and increased polyfunctionality of both CD4(+) and CD8(+) T cells in the DNA-vaccinated group. Importantly, Ad5 immunizations failed to boost following the first immunization, whereas DNA responses were continually boosted with all four immunizations demonstrating a major advantage of these improved DNA vaccines. These optimized DNA vaccines induce very different immune phenotypes than traditional Ad5 vaccines, suggesting that they could play an important role in vaccine research and development.
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Molecular physiology of the Rh ammonia transport proteins.
Curr. Opin. Nephrol. Hypertens.
PUBLISHED: 06-12-2010
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Recent studies have identified a new family of ammonia-specific transporters, Rh glycoproteins, which enable NH3-specific transport. The purpose of this review is to summarize recent evidence regarding the role of Rh glycoproteins in renal ammonia transport.
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VennVax, a DNA-prime, peptide-boost multi-T-cell epitope poxvirus vaccine, induces protective immunity against vaccinia infection by T cell response alone.
Vaccine
PUBLISHED: 06-08-2010
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The potential for smallpox to be disseminated in a bioterror attack has prompted development of new, safer smallpox vaccination strategies. We designed and evaluated immunogenicity and efficacy of a T-cell epitope vaccine based on conserved and antigenic vaccinia/variola sequences, identified using bioinformatics and immunological methods. Vaccination in HLA transgenic mice using a DNA-prime/peptide-boost strategy elicited significant T cell responses to multiple epitopes. No antibody response pre-challenge was observed, neither against whole vaccinia antigens nor vaccine epitope peptides. Remarkably, 100% of vaccinated mice survived lethal vaccinia challenge, demonstrating that protective immunity to vaccinia does not require B cell priming.
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Effect of intercalated cell-specific Rh C glycoprotein deletion on basal and metabolic acidosis-stimulated renal ammonia excretion.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 05-12-2010
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Rh C glycoprotein (Rhcg) is an NH(3)-specific transporter expressed in both intercalated cells (IC) and principal cells (PC) in the renal collecting duct. Recent studies show that deletion of Rhcg from both intercalated and principal cells inhibits both basal and acidosis-stimulated renal ammonia excretion. The purpose of the current studies was to better understand the specific role of Rhcg expression in intercalated cells in basal and metabolic acidosis-stimulated renal ammonia excretion. We generated mice with intercalated cell-specific Rhcg deletion (IC-Rhcg-KO) using Cre-loxP techniques; control (C) mice were floxed Rhcg but Cre negative. Under basal conditions, IC-Rhcg-KO and C mice excreted urine with similar ammonia content and pH. Mice were then acid loaded by adding HCl to their diet. Ammonia excretion after acid loading increased similarly in IC-Rhcg-KO and C mice during the first 2 days of acid loading but on day 3 was significantly less in IC-Rhcg-KO than in C mice. During the first 2 days of acid loading, urine was significantly more acidic in IC-Rhcg-KO mice than in C mice; there was no difference on day 3. In IC-Rhcg-KO mice, acid loading increased principal cell Rhcg expression in both the cortex and outer medulla as well as expression of another ammonia transporter, Rh glycoprotein B (Rhbg), in principal cells in the outer medulla. We conclude that 1) Rhcg expression in intercalated cells is necessary for the normal renal response to metabolic acidosis; 2) principal cell Rhcg contributes to both basal and acidosis-stimulated ammonia excretion; and 3) adaptations in Rhbg expression occur in response to acid-loading.
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Chikungunya: a potentially emerging epidemic?
PLoS Negl Trop Dis
PUBLISHED: 04-27-2010
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Chikungunya virus is a mosquito-borne emerging pathogen that has a major health impact in humans and causes fever disease, headache, rash, nausea, vomiting, myalgia, and arthralgia. Indigenous to tropical Africa, recent large outbreaks have been reported in parts of South East Asia and several of its neighboring islands in 2005-07 and in Europe in 2007. Furthermore, positive cases have been confirmed in the United States in travelers returning from known outbreak areas. Currently, there is no vaccine or antiviral treatment. With the threat of an emerging global pandemic, the peculiar problems associated with the more immediate and seasonal epidemics warrant the development of an effective vaccine. In this review, we summarize the evidence supporting these concepts.
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Expression of ammonia transporter family members, Rh B glycoprotein and Rh C glycoprotein, in the developing rat kidney.
Am. J. Physiol. Renal Physiol.
PUBLISHED: 04-14-2010
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Ammonia metabolism is a primary component of acid-base homeostasis but is incompletely developed at time of birth. Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg) are recently recognized ammonia transporter family members expressed in the mammalian kidney. This studys purpose was to establish the expression and localization of Rhbg and Rhcg during kidney development. We examined kidneys from fetal days 16 (E16), 18 (E18), and 20 (E20), and from the first 21 days of postnatal development. Rhbg was expressed initially at E18, with expression only in the connecting tubule (CNT); at E20, Rhbg was expressed in both the CNT and the medullary collecting duct (MCD). In contrast, Rhcg was first expressed at E16 with basal expression in the ureteric bud; at E18, it was expressed in a subset of CNT cells with an apical pattern, followed by apical and basolateral expression in the MCD at E20. In the cortex, Rhbg and Rhcg expression increased in the CNT before expression in the cortical collecting duct during fetal development. In the MCD, both Rhbg and Rhcg expression was initially in cells in the papillary tip, with gradual removal from the tip during the late fetal period and transition during the early neonatal period to an adult pattern with predominant expression in the outer MCD and only rare expression in cells in the initial inner MCD. Double-labeling with intercalated cell-specific markers identified that Rhbg and Rhcg were expressed initially in CNT cells, CNT A-type intercalated cells and non-A, non-B intercalated cells, and in MCD A-type intercalated cells. We conclude that expression of Rhbg and Rhcg parallels intercalated cell development and that immature Rhbg and Rhcg expression at birth contributes to incomplete ammonia excretion capacity.
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Anti-cancer activity of the HIV accessory molecule viral protein R (Vpr): Delivery as a DNA expression plasmid or biologically active peptides.
Vaccine
PUBLISHED: 03-02-2010
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By virtue of its ability to induce cell cycle arrest and apoptosis, the HIV accessory protein Vpr (viral protein R) has been evaluated by us and others as an anti-proliferative/anti-cancer agent. We have demonstrated that Vpr, when delivered to established experimental B16.F10 melanoma tumors in mice as a DNA expression plasmid through in vivo electroporation, can result in complete regression of the established tumors. We have also demonstrated that Vpr peptides from the carboxy region of the protein can inhibit in vitro growth of both B16.F10 melanoma as well as human HeLa cervical carcinoma tumor cells. These findings, summarized in this report, underscore the potential of Vpr as an anti-cancer agent and warrants, we believe, further experimental as well as clinical evaluation.
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Systemic immunization with CCL27/CTACK modulates immune responses at mucosal sites in mice and macaques.
Vaccine
PUBLISHED: 03-02-2010
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Plasmid DNA is a promising vaccine platform that has been shown to be safe and able to be administered repeatedly without vector interference. Enhancing the potency of DNA vaccination through co-delivery of molecular adjuvants is one strategy currently under investigation. Here we describe the use of the novel chemokine adjuvant CCL27/CTACK to enhance immune responses to an HIV-1 or SIV antigen in mice and rhesus macaques. CCL27 has been shown to play a role in inflammatory responses through chemotaxis of CCR10+ cells, and we hypothesized that CCL27 may modulate adaptive immune responses. Immunizations in mice with HIV-1gag/CCL27 enhanced immune responses both at peripheral and, surprisingly, at mucosal sites. To confirm these findings in a large-animal model, we created optimized CCL27 and SIV antigenic plasmid constructs for rhesus macaques. 10 macaques (n=5/group) were immunized intramuscularly with 1mg/construct of antigenic plasmids+/-CCL27 with electroporation. We observed significant IFN-gamma secretion and CD8+ T-cell proliferation in peripheral blood. Interestingly, CCL27 co-immunized macaques exhibited a trend toward greater effector CD4+ T cells in the bronchiolar lavage (BAL). CCL27 co-delivery also elicited greater antigen-specific IgA at unique sites including BAL and fecal samples but not in the periphery. Future studies incorporating CCL27 as an adjuvant in vaccine or therapy models where eliciting immune responses in the lung are warranted.
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Phase I/II trial of the anti-HIV activity of mifepristone in HIV-infected subjects ACTG 5200.
J. Acquir. Immune Defic. Syndr.
PUBLISHED: 02-05-2010
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Mifepristone is a glucocorticoid receptor inhibitor shown in vitro to have anti-HIV activity and anti-simian immunodeficiency virus activity in a macaque model. A phase I/II trial was performed to assess the drugs safety and anti-HIV activity.
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Current strategies and limitations of HIV vaccines.
Curr Opin Investig Drugs
PUBLISHED: 01-30-2010
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There is currently no cure for HIV infection, and the possibility of developing a vaccine in the near future appears unlikely. With more than 33 million individuals living with HIV/AIDS worldwide, there is a distinct need for a prophylactic vaccine against HIV infection. However, conventional vaccine strategies aimed at eliciting antibody and T-cell responses have failed to protect against the virus. Current research has been directed toward the more realistic goal of controlling viral replication during the early stages of infection, thus reducing the viral setpoint, through the use of novel vaccine delivery systems and techniques. In this review, several of the key milestones achieved as a result of research efforts aimed at developing an effective HIV vaccine are identified, and future prospects are examined.
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Ki-67 staining for determination of rhesus macaque T cell proliferative responses ex vivo.
Cytometry A
PUBLISHED: 01-28-2010
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The capacity for robust proliferation upon re-infection is a hallmark of adaptive immunity and the basis of vaccination. A widely used animal model for the study of human disease is the rhesus macaque (RM), where capacity for proliferation can be assessed ex vivo using carboxyfluorescein succinimidyl ester (CFSE)-based dilution assays. However, we show over the course of the standard ex vivo proliferation assay that CFSE-labeling at commonly used dye concentrations induces significant cell death, but that this phenomenon is dose-dependent. Here, we describe an alternative semiquantitative method for estimating T cell proliferative responses that avoids the putative biases associated with chemical modification. RM peripheral blood mononuclear cells were stimulated ex vivo with cognate peptides for 5 days, immunostained for intracellular Ki-67, and then analyzed by flow cytometry. We describe a gating strategy using Ki-67 and side light scatter, also a marker of blastogenesis, which correlates strongly with data from CFSE dilution. We show that this method is a valid tool for measuring RM antigen-specific cellular proliferation ex vivo and can be used as an alternative to CFSE dilution assays.
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Monkeying around with HIV vaccines: using rhesus macaques to define gatekeepers for clinical trials.
Nat. Rev. Immunol.
PUBLISHED: 10-28-2009
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Rhesus macaques are an important animal model for the study of human disease and the development of vaccines against HIV and AIDS. HIV vaccines have been benchmarked in rhesus macaque preclinical challenge studies using chimeric viruses made up of parts of HIV and simian immunodeficiency viruses. However, the lack of efficacy in a recent clinical trial calls for a re-evaluation of the scientific assumptions regarding the predictive value of using data generated from rhesus macaques as a gatekeeper for the advancement of candidate vaccines into the clinic. In this context, there is significant consensus among HIV vaccinologists that next-generation HIV vaccines must generate better immunity in rhesus macaques than clinically unsuccessful vaccines generated using validated assays. Defining better immunity is the core challenge of HIV vaccine development in this system and is the focus of this Review.
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CD8+ cell depletion of SHIV89.6P-infected macaques induces CD4+ T cell proliferation that contributes to increased viral loads.
J. Immunol.
PUBLISHED: 09-28-2009
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Previous studies have shown that depletion of CD8(+) cells during acute and chronic simian immunodeficiency virus (SIV) infection leads to increased viral replication, morbidity, and mortality, which have been attributed to loss of CD8(+) T cell-mediated control of SIV. However, these studies did not exclude that CD8(+) cell depletion increased homeostatic proliferation of CD4(+) T cells, resulting in increased viral targets and, therefore, viral rebound. Chronically SHIV89.6P-infected cynomolgus macaques were CD8(+) cell-depleted, and the frequency, cell number, and phenotype of CD4(+) T cells and viral infection were examined using flow cytometry and quantitative real-time PCR. The frequency and number of Ki-67-expressing CD4(+) T cells were increased with CD8(+) cell depletion. This proliferation of CD4(+) T cells occurred even in animals with no rebound of viral loads. Most of the proliferating cells were effector memory CD4(+) T cells. Plasma simian HIV (SHIV) RNA copies positively correlated with proliferating CD4(+) T cells and SHIV DNA copies in Ki-67(+) CD4(+) T cells. Although this study does not exclude an important role for virus-specific CD8(+) T cells in SIV and SHIV infection, our data suggest that homeostatic proliferation is an important contributor to increases in plasma viremia that follow CD8(+) cell depletion.
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DNA vaccines in veterinary use.
Expert Rev Vaccines
PUBLISHED: 09-03-2009
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DNA vaccines represent a new frontier in vaccine technology. One important application of this technology is in the veterinary arena. DNA vaccines have already gained a foothold in certain fields of veterinary medicine. However, several important questions must be addressed when developing DNA vaccines for animals, including whether or not the vaccine is efficacious and cost effective compared with currently available options. Another important question to consider is how to apply this developing technology in a wide range of different situations, from the domestic pet to individual fish in fisheries with several thousand animals, to wildlife programs for disease control. In some cases, DNA vaccines represent an interesting option for vaccination, while in others, currently available options are sufficient. This review will examine a number of diseases of veterinary importance and the progress being made in DNA vaccine technology relevant to these diseases, and we compare these with the conventional treatment options available.
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