BNT162b vaccines protect rhesus macaques from SARS-CoV-2.

A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates (BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4+ and IFNγ+CD8+ T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA1-3, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).

Safety and immunogenicity of a highly attenuated rVSVN4CT1-EBOVGP1 Ebola virus vaccine: a randomised, double-blind, placebo-controlled, phase 1 clinical trial.

BACKGROUND: The safety and immunogenicity of a highly attenuated recombinant vesicular stomatitis virus (rVSV) expressing HIV-1 gag (rVSVN4CT1-HIV-1gag1) was shown in previous phase 1 clinical studies. An rVSV vector expressing Ebola virus glycoprotein (EBOV-GP) in place of HIV-1 gag (rVSVN4CT1-EBOVGP1) showed single-dose protection from lethal challenge with low passage Ebola virus in non-human primates. We aimed to evaluate the safety and immunogenicity of the rVSVN4CT1-EBOVGP1 vaccine in healthy adults. METHODS: We did a randomised double-blind, placebo-controlled, phase 1 dose-escalation study at a single clinical site (Optimal Research) in Melbourne, FL, USA. Eligible participants were healthy men and non-pregnant women aged 18-60 years, with a body-mass index (BMI) of less than 40 kg/m2, no history of filovirus infection, VSV infection, or receipt of rVSV in previous studies, and who had not visited regions where Ebola virus outbreaks have occurred. Three cohorts were enrolled to assess a low (2·5 × 104 plaque forming units [PFU]), intermediate (2 × 105 PFU), or high dose (1·8 × 106 PFU) of the vaccine. Participants within each cohort were randomly allocated (10:3) to receive vaccine or placebo by intramuscular injection in a homologous prime and boost regimen, with 4 weeks between doses. All syringes were masked with syringe sleeves; participants and study site staff were not blinded to dose level but were blinded to active vaccine and placebo. The primary outcomes were safety and tolerability; immunogenicity, assessed as GP-specific humoral immune response (at 2 weeks after each dose) and cellular immune response (at 1 and 2 weeks after each dose), was a secondary outcome. All randomised participants were included in primary and safety analyses. This trial is registered with ClinicalTrials.gov, NCT02718469. FINDINGS: Between Dec 22, 2015, and Sept 15, 2016, 39 individuals (18 [46%] men and 21 [54%] women, mean age 51 years [SD 10]) were enrolled, with ten participants receiving the vaccine and three participants receiving placebo in each of three cohorts. One participant in the intermediate dose cohort was withdrawn from the study because of a diagnosis of invasive ductal breast carcinoma 24 days after the first vaccination, which was considered unrelated to the vaccine. No severe adverse events were observed. Solicited local adverse events occurred in ten (26%) of 39 participants after the first dose and nine (24%) of 38 participants after the second dose; the events lasted 3 days or less, were predominantly injection site tenderness (17 events) and injection site pain (ten events), and were either mild (19 events) or moderate (ten events) in intensity. Systemic adverse events occurred in 13 (33%) of 39 participants after the first dose and eight (21%) of 38 participants after the second dose; the events were mild (45 events) or moderate (11 events) in severity, and the most common events were malaise or fatigue (13 events) and headache (12 events). Arthritis and maculopapular, vesicular, or purpuric rash distal to the vaccination site(s) were not reported. A GP-specific IgG response was detected in all vaccine recipients after two doses (and IgG response frequency was 100% after a single high dose), and an Ebola virus neutralising response was detected in 100% of participants in the high-dose cohort. INTERPRETATION: The rVSVN4CT1-EBOVGP1 vaccine was well tolerated at all dose levels tested and was immunogenic despite a high degree of attenuation. The combined safety and immunogenicity profile of the rVSVN4CT1-EBOVGP1 vaccine vector support phase 1-2 clinical evaluation. FUNDING: US Department of Defense Joint Program Executive Office for Chemical, Biological, Radiological and Nuclear Defense: Joint Project Manager for Chemical, Biological, Radiological and Nuclear Medical.

Quadrivalent VesiculoVax vaccine protects nonhuman primates from viral-induced hemorrhagic fever and death.

Recent occurrences of filoviruses and the arenavirus Lassa virus (LASV) in overlapping endemic areas of Africa highlight the need for a prophylactic vaccine that would confer protection against all of these viruses that cause lethal hemorrhagic fever (HF). We developed a quadrivalent formulation of VesiculoVax that contains recombinant vesicular stomatitis virus (rVSV) vectors expressing filovirus glycoproteins and that also contains a rVSV vector expressing the glycoprotein of a lineage IV strain of LASV. Cynomolgus macaques were vaccinated twice with the quadrivalent formulation, followed by challenge 28 days after the boost vaccination with each of the 3 corresponding filoviruses (Ebola, Sudan, Marburg) or a heterologous contemporary lineage II strain of LASV. Serum IgG and neutralizing antibody responses specific for all 4 glycoproteins were detected in all vaccinated animals. A modest and balanced cell-mediated immune response specific for the glycoproteins was also detected in most of the vaccinated macaques. Regardless of the level of total glycoprotein-specific immune response detected after vaccination, all immunized animals were protected from disease and death following lethal challenges. These findings indicate that vaccination with attenuated rVSV vectors each expressing a single HF virus glycoprotein may provide protection against those filoviruses and LASV most commonly responsible for outbreaks of severe HF in Africa.

Antibody Fab-Fc properties outperform titer in predictive models of SIV vaccine-induced protection.

Characterizing the antigen-binding and innate immune-recruiting properties of the humoral response offers the chance to obtain deeper insights into mechanisms of protection than revealed by measuring only overall antibody titer. Here, a high-throughput, multiplexed Fab-Fc Array was employed to profile rhesus macaques vaccinated with a gp120-CD4 fusion protein in combination with different genetically encoded adjuvants, and subsequently subjected to multiple heterologous simian immunodeficiency virus (SIV) challenges. Systems analyses modeling protection and adjuvant differences using Fab-Fc Array measurements revealed a set of correlates yielding strong and robust predictive performance, while models based on measurements of response magnitude alone exhibited significantly inferior performance. At the same time, rendering Fab-Fc measurements mathematically independent of titer had relatively little impact on predictive performance. Similar analyses for a distinct SIV vaccine study also showed that Fab-Fc measurements performed significantly better than titer. These results suggest that predictive modeling with measurements of antibody properties can provide detailed correlates with robust predictive power, suggest directions for vaccine improvement, and potentially enable discovery of mechanistic associations.

Safety and tolerability of HIV-1 multiantigen pDNA vaccine given with IL-12 plasmid DNA via electroporation, boosted with a recombinant vesicular stomatitis virus HIV Gag vaccine in healthy volunteers in a randomized, controlled clinical trial.

BACKGROUND: The addition of plasmid cytokine adjuvants, electroporation, and live attenuated viral vectors may further optimize immune responses to DNA vaccines in heterologous prime-boost combinations. The objective of this study was to test the safety and tolerability of a novel prime-boost vaccine regimen incorporating these strategies with different doses of IL-12 plasmid DNA adjuvant. METHODS: In a phase 1 study, 88 participants received an HIV-1 multiantigen (gag/pol, env, nef/tat/vif) DNA vaccine (HIV-MAG, 3000 µg) co-administered with IL-12 plasmid DNA adjuvant at 0, 250, 1000, or 1500 µg (N = 22/group) given intramuscularly with electroporation (Ichor TriGrid™ Delivery System device) at 0, 1 and 3 months; followed by attenuated recombinant vesicular stomatitis virus, serotype Indiana, expressing HIV-1 Gag (VSV-Gag), 3.4 ⊆ 107 plaque-forming units (PFU), at 6 months; 12 others received placebo. Injections were in both deltoids at each timepoint. Participants were monitored for safety and tolerability for 15 months. RESULTS: The dose of IL-12 pDNA did not increase pain scores, reactogenicity, or adverse events with the co-administered DNA vaccine, or following the VSV-Gag boost. Injection site pain and reactogenicity were common with intramuscular injections with electroporation, but acceptable to most participants. VSV-Gag vaccine often caused systemic reactogenicity symptoms, including a viral syndrome (in 41%) of fever, chills, malaise/fatigue, myalgia, and headache; and decreased lymphocyte counts 1 day after vaccination. CONCLUSIONS: HIV-MAG DNA vaccine given by intramuscular injection with electroporation was safe at all doses of IL-12 pDNA. The VSV-Gag vaccine at this dose was associated with fever and viral symptoms in some participants, but the vaccine regimens were safe and generally well-tolerated. TRIAL REGISTRATION: Clinical Trials.gov NCT01578889.

Preclinical Development of Oncolytic Immunovirotherapy for Treatment of HPVPOS Cancers.

Immunotherapy for HPVPOS malignancies is attractive because well-defined, viral, non-self tumor antigens exist as targets. Several approaches to vaccinate therapeutically against HPV E6 and E7 antigens have been adopted, including viral platforms such as VSV. A major advantage of VSV expressing these antigens is that VSV also acts as an oncolytic virus, leading to direct tumor cell killing and induction of effective anti-E6 and anti-E7 T cell responses. We have also shown that addition of immune adjuvant genes, such as IFNß, further enhances safety and/or efficacy of VSV-based oncolytic immunovirotherapies. However, multiple designs of the viral vector are possible-with respect to levels of immunogen expression and method of virus attenuation-and optimal designs have not previously been tested head-to-head. Here, we tested three different VSV engineered to express a non-oncogenic HPV16 E7/6 fusion protein for their immunotherapeutic and oncolytic properties. We assessed their profiles of efficacy and toxicity against HPVPOS and HPVNEG murine tumor models and determined the optimal route of administration. Our data show that VSV is an excellent platform for the oncolytic immunovirotherapy of tumors expressing HPV target antigens, combining a balance of efficacy and safety suitable for evaluation in a first-in-human clinical trial.

Single-Dose Trivalent VesiculoVax Vaccine Protects Macaques from Lethal Ebolavirus and Marburgvirus Challenge.

Previous studies demonstrated that a single intramuscular (i.m.) dose of an attenuated recombinant vesicular stomatitis virus (rVSV) vector (VesiculoVax vector platform; rVSV-N4CT1) expressing the glycoprotein (GP) from the Mayinga strain of Zaire ebolavirus (EBOV) protected nonhuman primates (NHPs) from lethal challenge with EBOV strains Kikwit and Makona. Here, we studied the immunogenicities of an expanded range of attenuated rVSV vectors expressing filovirus GP in mice. Based on data from those studies, an optimal attenuated trivalent rVSV vector formulation was identified that included rVSV vectors expressing EBOV, Sudan ebolavirus (SUDV), and the Angola strain of Marburg marburgvirus (MARV) GPs. NHPs were vaccinated with a single dose of the trivalent formulation, followed by lethal challenge 28 days later with each of the three corresponding filoviruses. At day 14 postvaccination, a serum IgG response specific for all three GPs was detected in all the vaccinated macaques. A modest and balanced cell-mediated immune response specific for each GP was also detected in a majority of the vaccinated macaques. No matter the level of total GP-specific immune response detected postvaccination, all the vaccinated macaques were protected from disease and death following lethal challenge with each of the three filoviruses. These findings indicate that vaccination with a single dose of attenuated rVSV-N4CT1 vectors each expressing a single filovirus GP may provide protection against the filoviruses most commonly responsible for outbreaks of hemorrhagic fever in sub-Saharan Africa.IMPORTANCE The West African Ebola virus Zaire outbreak in 2013 showed that the disease was not only a regional concern, but a worldwide problem, and highlighted the need for a safe and efficacious vaccine to be administered to the populace. However, other endemic pathogens, like Ebola virus Sudan and Marburg, also pose an important health risk to the public and therefore require development of a vaccine prior to the occurrence of an outbreak. The significance of our research was the development of a blended trivalent filovirus vaccine that elicited a balanced immune response when administered as a single dose and provided complete protection against a lethal challenge with all three filovirus pathogens.

DNA Priming Increases Frequency of T-Cell Responses to a Vesicular Stomatitis Virus HIV Vaccine with Specific Enhancement of CD8+ T-Cell Responses by Interleukin-12 Plasmid DNA.

The HIV Vaccine Trials Network (HVTN) 087 vaccine trial assessed the effect of increasing doses of pIL-12 (interleukin-12 delivered as plasmid DNA) adjuvant on the immunogenicity of an HIV-1 multiantigen (MAG) DNA vaccine delivered by electroporation and boosted with a vaccine comprising an attenuated vesicular stomatitis virus expressing HIV-1 Gag (VSV-Gag). We randomized 100 healthy adults to receive placebo or 3 mg HIV-MAG DNA vaccine (ProfectusVax HIV-1 gag/pol or ProfectusVax nef/tat/vif, env) coadministered with pIL-12 at 0, 250, 1,000, or 1,500 µg intramuscularly by electroporation at 0, 1, and 3 months followed by intramuscular inoculation with 3.4 × 107 PFU VSV-Gag vaccine at 6 months. Immune responses were assessed after the prime and boost and 6 months after the last vaccination. High-dose pIL-12 increased the magnitude of CD8+ T-cell responses postboost compared to no pIL-12 (P = 0.02), while CD4+ T-cell responses after the prime were higher in the absence of pIL-12 than with low- and medium-dose pIL-12 (P ≤ 0.05). The VSV boost increased Gag-specific CD4+ and CD8+ T-cell responses in all groups (P < 0.001 for CD4+ T cells), inducing a median of four Gag epitopes in responders. Six to 9 months after the boost, responses decreased in magnitude, but CD8+ T-cell response rates were maintained. The addition of a DNA prime dramatically improved responses to the VSV vaccine tested previously in the HVTN 090 trial, leading to broad epitope targeting and maintained CD8+ T-cell response rates at early memory. The addition of high-dose pIL-12 given with a DNA prime by electroporation and boosted with VSV-Gag increased the CD8+ T-cell responses but decreased the CD4+ responses. This approach may be advantageous in reshaping the T-cell responses to a variety of chronic infections or tumors. (This study has been registered at ClinicalTrials.gov under registration no. NCT01578889.).

An Interleukin 12 Adjuvanted Herpes Simplex Virus 2 DNA Vaccine Is More Protective Than a Glycoprotein D Subunit Vaccine in a High-Dose Murine Challenge Model.

Vaccination is a proven intervention against human viral diseases; however, success against Herpes Simplex Virus 2 (HSV-2) remains elusive. Most HSV-2 vaccines tested in humans to date contained just one or two immunogens, such as the virion attachment receptor glycoprotein D (gD) and/or the envelope fusion protein, glycoprotein B (gB). At least three factors may have contributed to the failures of subunit-based HSV-2 vaccines. First, immune responses directed against one or two viral antigens may lack sufficient antigenic breadth for efficacy. Second, the antibody responses elicited by these vaccines may have lacked necessary Fc-mediated effector functions. Third, these subunit vaccines may not have generated necessary protective cellular immune responses. We hypothesized that a polyvalent combination of HSV-2 antigens expressed from a DNA vaccine with an adjuvant that polarizes immune responses toward a T helper 1 (Th1) phenotype would compose a more effective vaccine. We demonstrate that delivery of DNA expressing full-length HSV-2 glycoprotein immunogens by electroporation with the adjuvant interleukin 12 (IL-12) generates substantially greater protection against a high-dose HSV-2 vaginal challenge than a recombinant gD subunit vaccine adjuvanted with alum and monophosphoryl lipid A (MPL). Our results further show that DNA vaccines targeting optimal combinations of surface glycoproteins provide better protection than gD alone and provide similar survival benefits and disease symptom reductions compared with a potent live attenuated HSV-2 0ΔNLS vaccine, but that mice vaccinated with HSV-2 0ΔNLS clear the virus much faster. Together, our data indicate that adjuvanted multivalent DNA vaccines hold promise for an effective HSV-2 vaccine, but that further improvements may be required.

Vaccination With a Highly Attenuated Recombinant Vesicular Stomatitis Virus Vector Protects Against Challenge With a Lethal Dose of Ebola Virus.

Previously, recombinant vesicular stomatitis virus (rVSV) pseudotypes expressing Ebolavirus glycoproteins (GPs) in place of the VSV G protein demonstrated protection of nonhuman primates from lethal homologous Ebolavirus challenge. Those pseudotype vectors contained no additional attenuating mutations in the rVSV genome. Here we describe rVSV vectors containing a full complement of VSV genes and expressing the Ebola virus (EBOV) GP from an additional transcription unit. These rVSV vectors contain the same combination of attenuating mutations used previously in the clinical development pathway of an rVSV/human immunodeficiency virus type 1 vaccine. One of these rVSV vectors (N4CT1-EBOVGP1), which expresses membrane-anchored EBOV GP from the first position in the genome (GP1), elicited a balanced cellular and humoral GP-specific immune response in mice. Guinea pigs immunized with a single dose of this vector were protected from any signs of disease following lethal EBOV challenge, while control animals died in 7-9 days. Subsequently, N4CT1-EBOVGP1 demonstrated complete, single-dose protection of 2 macaques following lethal EBOV challenge. A single sham-vaccinated macaque died from disease due to EBOV infection. These results demonstrate that highly attenuated rVSV vectors expressing EBOV GP may provide safer alternatives to current EBOV vaccines.

The catalytic A1 domains of cholera toxin and heat-labile enterotoxin are potent DNA adjuvants that evoke mixed Th1/Th17 cellular immune responses.

DNA encoded adjuvants are well known for increasing the magnitude of cellular and/or humoral immune responses directed against vaccine antigens. DNA adjuvants can also tune immune responses directed against vaccine antigens to better protect against infection of the target organism. Two potent DNA adjuvants that have unique abilities to tune immune responses are the catalytic A1 domains of Cholera Toxin (CTA1) and Heat-Labile Enterotoxin (LTA1). Here, we have characterized the adjuvant activities of CTA1 and LTA1 using HIV and SIV genes as model antigens. Both of these adjuvants enhanced the magnitude of antigen-specific cellular immune responses on par with those induced by the well-characterized cytokine adjuvants IL-12 and GM-CSF. CTA1 and LTA1 preferentially enhanced cellular responses to the intracellular antigen SIVmac239-gag over those for the secreted HIVBaL-gp120 antigen. IL-12, GM-CSF and electroporation did the opposite suggesting differences in the mechanisms of actions of these diverse adjuvants. Combinations of CTA1 or LTA1 with IL-12 or GM-CSF generated additive and better balanced cellular responses to both of these antigens. Consistent with observations made with the holotoxin and the CTA1-DD adjuvant, CTA1 and LTA1 evoked mixed Th1/Th17 cellular immune responses. Together, these results show that CTA1 and LTA1 are potent DNA vaccine adjuvants that favor the intracellular antigen gag over the secreted antigen gp120 and evoke mixed Th1/Th17 responses against both of these antigens. The results also indicate that achieving a balanced immune response to multiple intracellular and extracellular antigens delivered via DNA vaccination may require combining adjuvants that have different and complementary mechanisms of action.

Single-dose attenuated Vesiculovax vaccines protect primates against Ebola Makona virus.

The family Filoviridae contains three genera, Ebolavirus (EBOV), Marburg virus, and Cuevavirus. Some members of the EBOV genus, including Zaire ebolavirus (ZEBOV), can cause lethal haemorrhagic fever in humans. During 2014 an unprecedented ZEBOV outbreak occurred in West Africa and is still ongoing, resulting in over 10,000 deaths, and causing global concern of uncontrolled disease. To meet this challenge a rapid-acting vaccine is needed. Many vaccine approaches have shown promise in being able to protect nonhuman primates against ZEBOV. In response to the current ZEBOV outbreak several of these vaccines have been fast tracked for human use. However, it is not known whether any of these vaccines can provide protection against the new outbreak Makona strain of ZEBOV. One of these approaches is a first-generation recombinant vesicular stomatitis virus (rVSV)-based vaccine expressing the ZEBOV glycoprotein (GP) (rVSV/ZEBOV). To address safety concerns associated with this vector, we developed two candidate, further-attenuated rVSV/ZEBOV vaccines. Both attenuated vaccines produced an approximately tenfold lower vaccine-associated viraemia compared to the first-generation vaccine and both provided complete, single-dose protection of macaques from lethal challenge with the Makona outbreak strain of ZEBOV.

Differential protective effects of volatile anesthetics against renal ischemia-reperfusion injury in vivo.

BACKGROUND: Volatile anesthetics protect against cardiac ischemia-reperfusion injury via adenosine triphosphate-dependent potassium channel activation. The authors questioned whether volatile anesthetics can also protect against renal ischemia-reperfusion injury and, if so, whether cellular adenosine triphosphate-dependent potassium channels, antiinflammatory effects of volatile anesthetics, or both are involved. METHODS: Rats were anesthetized with equipotent doses of volatile anesthetics (desflurane, halothane, isoflurane, or sevoflurane) or injectable anesthetics (pentobarbital or ketamine) and subjected to 45 min of renal ischemia and 3 h of reperfusion during anesthesia. RESULTS: Rats treated with volatile anesthetics had lower plasma creatinine and reduced renal necrosis 24-72 h after injury compared with rats anesthetized with pentobarbital or ketamine. Twenty-four hours after injury, sevoflurane-, isoflurane-, or halothane-treated rats had creatinine (+/- SD) of 2.3 +/- 0.7 mg/dl (n = 12), 1.8 +/- 0.5 mg/dl (n = 6), and 2.4 +/- 1.2 mg/dl (n = 6), respectively, compared with rats treated with pentobarbital (5.8 +/- 1.2 mg/dl, n = 9) or ketamine (4.6 +/- 1.2 mg/dl, n = 8). Among the volatile anesthetics, desflurane demonstrated the least reduction in plasma creatinine after 24 h (4.1 +/- 0.8 mg/dl, n = 12). Renal cortices from volatile anesthetic-treated rats demonstrated reduced expression of intercellular adhesion molecule 1 protein and messenger RNA as well as messenger RNAs encoding proinflammatory cytokines and chemokines. Volatile anesthetic treatment reduced renal cortex myeloperoxidase activity and reduced nuclear translocation of proinflammatory nuclear factor kappaB. Adenosine triphosphate-dependent potassium channels are not involved in sevoflurane-mediated renal protection because glibenclamide did not block renal protection (creatinine: 2.4 +/- 0.4 mg/dl, n = 3). CONCLUSION: Some volatile anesthetics confer profound protection against renal ischemia-reperfusion injury compared with pentobarbital or ketamine anesthesia by attenuating inflammation. These findings may have significant clinical implications for anesthesiologists regarding the choice of volatile anesthetic agents in patients subjected to perioperative renal ischemia.

Local anesthetics worsen renal function after ischemia-reperfusion injury in rats.

Local anesthetics are widely used during the perioperative period, even in patients with preexisting renal disease. However, local anesthetics have been shown to cause cell death in multiple cell lines, including human kidney proximal tubule cells. We questioned whether local anesthetics potentiate renal dysfunction after ischemia-reperfusion (I/R) injury in vivo. Rats were implanted with subcutaneous miniosmotic pumps that continuously delivered lidocaine (2 mg.kg-1.h-1), bupivacaine (0.4 mg.kg-1.h-1), tetracaine (1 mg.kg-1.h-1), or saline vehicle, and 6 h later the rats were subjected to 30 min of renal ischemia or to sham operation. Renal function was assessed by measurement of plasma creatinine at 24 and 48 h after renal I/R injury in the presence or absence of chronic infusions of local anesthetics and correlated to histological changes indicative of necrosis. The degree of renal apoptosis was assessed by three methods: 1) DNA fragmentation detected by terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling staining, 2) DNA laddering detected after agarose gel electrophoresis, and 3) morphological identification of apoptotic tubules at the corticomedullary junction. We also measured the expression of the proinflammatory markers ICAM-1 and TNF-alpha. Continuous local anesthetic infusion with renal I/R injury resulted in an increased magnitude and duration of renal dysfunction compared with the saline-infused I/R group. Additionally, both apoptotic and necrotic renal cell death as well as inflammatory changes were significantly potentiated in local anesthetic-treated rat kidneys. Local anesthetic infusion alone without I/R injury had no effect on renal function. We conclude that local anesthetics potentiated renal injury after I/R by increasing necrosis, apoptosis, and inflammation.

Oxidant preconditioning protects human proximal tubular cells against lethal oxidant injury via p38 MAPK and heme oxygenase-1.

Ischemic preconditioning protects the kidney from subsequent ischemic injury but the signal transduction pathways involved are unknown. Human proximal tubular (HK-2) cells were protected from injury with 2.5 mM H(2)O(2) by preconditioning with a single 15-min exposure to 500 microM H(2)O(2) followed by 16 h of recovery (oxidant preconditioning). To identify the signaling pathways involved in oxidant preconditioning, we utilized inhibitors of several signaling intermediates (MAPK/ERK kinase I, p38 mitogen-activated protein kinase (MAPK), protein kinase C and tyrosine kinase). A rapid but transient increase in p38 MAPK was observed following oxidant preconditioning and an inhibitor of p38 MAPK (SB203580) abolished the protection provided by oxidant preconditioning. Oxidant preconditioning was also associated with heat shock protein-27 phosphorylation (by p38 MAPK) and an increased synthesis of heme oxygenase-1 (HO-1). Stimulation or inhibition of HO-1 with hemin or Zn(II) protoporphyrin IX, respectively, mimicked or abolished oxidant preconditioning-mediated cytoprotection. Inhibitors of new protein synthesis (cycloheximide) and gene transcription (actinomycin D) also blocked the cytoprotection by oxidant preconditioning. We conclude that oxidant preconditioning protects HK-2 cells against more severe oxidant injury via activation of signaling pathways that include p38 MAPK and increased synthesis of HO-1.

A3 adenosine receptor knockout mice are protected against ischemia- and myoglobinuria-induced renal failure.

A(3) adenosine receptor (AR) activation and inhibition worsen and improve, respectively, renal function after ischemia-reperfusion (I/R) injury in rats. We sought to further characterize the role of A(3) ARs in modulating renal function after either I/R or myoglobinuric renal injury. A(3) knockout mice had significantly lower plasma creatinines compared with C57 controls 24 h after I/R or myoglobinuric renal injury. C57 control mice pretreated with the A(3) AR antagonist [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5 dicarboxylate] or agonist [0.125 mg/kg N(6)-(3-iodobenzyl)-N-methyl-5'-carbamoyladenosine (IB-MECA)] demonstrated improved or worsened renal function, respectively, after I/R or myoglobinuric renal injury. Higher doses of IB-MECA were lethal in C57 mice subjected to renal ischemia. H(1) but not H(2) histamine receptor antagonist prevented death in mice pretreated with IB-MECA before renal ischemia. Improvement in renal function was associated with significantly improved renal histology. In conclusion, preischemic A(3) AR activation (0.125 mg/kg IB-MECA) exacerbated renal I/R injury in mice. Mice lacking A(3) ARs or blocking A(3) ARs in wild-type mice resulted in significant renal protection from ischemic or myoglobinuric renal failure.