Novel Mechanism For Protection Research Articles

Melatonin protects against diclofenac induced oxidative stress mediated myocardial toxicity in rats: A mechanistic insight

Diclofenac, a traditional non-steroidal anti-inflammatory drug, is commonly used for treating chronic pain and inflammation. Recently, a number of articles have highlighted the toxicities associated with diclofenac. The current study explores the molecular mechanism of diclofenac induced cardiac toxicity following oxidative stress. Diclofenac inhibits catalase, disrupts the redox balance in cardiac tissue, accelerates the monoamine oxidase induced hydroperoxide generation and eventually inhibits crucial mitochondrial enzyme, viz., aldehyde dehydrogenase, thereby causing myocardial injury. Melatonin, the pineal indoleamine with high antioxidative efficacy, is well known for its cardio-protective properties and its dietary consumption has profound impact on cardiac health. The present study demonstrates perhaps for the first time, that apart from ameliorating oxidative load in the cardiac tissue, melatonin also attenuates the inhibition of catalase and aldehyde dehydrogenase, and prevents stress mediated stimulation of monoamine oxidase. Moreover, favourable binding of diclofenac with melatonin may protect the myocardium from the deleterious effects of this drug. The results indicate toward a novel mechanism of protection by melatonin, having future therapeutic relevance.

Role of ERK1/2 Signaling in Cinnabarinic Acid-Driven Stanniocalcin 2-Mediated Protection against Alcohol-Induced Apoptosis.

We have previously shown that a bona fide aryl hydrocarbon receptor (AhR) agonist, cinnabarinic acid (CA), protects against alcohol-induced hepatocyte apoptosis via activation of a novel AhR target gene, stanniocalcin 2 (Stc2). Stc2 translates to a secreted disulfide-linked hormone, STC2, known to function in cell development, calcium and phosphate regulation, angiogenesis, and antiapoptosis-albeit the comprehensive mechanism by which the CA-AhR-STC2 axis confers antiapoptosis is yet to be characterized. In this study, using RNA interference library screening, downstream antiapoptotic molecular signaling components involved in CA-induced STC2-mediated protection against ethanol-induced apoptosis were investigated. RNA interference library screening of kinases and phosphatases in Hepa1 cells and subsequent pathway analysis identified mitogen-activated protein kinase (MAPK) signaling as a critical molecular pathway involved in CA-mediated protection. Specifically, phosphorylation of ERK1/2 was induced in response to CA treatment without alterations in p38 and JNK signaling pathways. Silencing Stc2 in Hepa1 cells and in vivo experiments performed in Stc2-/- (Stc2 knockout) mice, which failed to confer CA-mediated protection against ethanol-induced apoptosis, showed abrogation of ERK1/2 activation, underlining the significance of ERK1/2 signaling in CA-STC2-mediated protection. In conclusion, activation of ERK1/2 signaling in CA-driven AhR-dependent Stc2-mediated protection represents a novel mechanism of protection against acute alcohol-induced apoptosis. SIGNIFICANCE STATEMENT: Previous studies have shown the role of stanniocalcin 2 (Stc2) in cinnabarinic acid (CA)-mediated protection against alcohol-induced apoptosis. Here, using RNA interference library screening and subsequent in vivo studies, the functional significance of ERK1/2 activation in CA-induced Stc2-mediated protection against acute ethanol-induced apoptosis was identified. This study is thus significant as it illustrates a comprehensive downstream mechanism by which CA-induced Stc2 protects against alcoholic liver disease.

The novel anti-phage system Shield co-opts an RmuC domain to mediate phage defense across Pseudomonas species.

Competitive bacteria-bacteriophage interactions have resulted in the evolution of a plethora of bacterial defense systems preventing phage propagation. In recent years, computational and bioinformatic approaches have underpinned the discovery of numerous novel bacterial defense systems. Anti-phage systems are frequently encoded together in genomic loci termed defense islands. Here we report the identification and characterisation of a novel anti-phage system, that we have termed Shield, which forms part of the Pseudomonas defensive arsenal. The Shield system comprises the core component ShdA, a membrane-bound protein harboring an RmuC domain. Heterologous production of ShdA alone is sufficient to mediate bacterial immunity against several phages. We demonstrate that Shield and ShdA confer population-level immunity and that they can also decrease transformation efficiency. We further show that ShdA homologues can degrade DNA in vitro and, when expressed in a heterologous host, can alter the organisation of the host chromosomal DNA. Use of comparative genomic approaches identified how Shield can be divided into four subtypes, three of which contain additional components that in some cases can negatively affect the activity of ShdA and/or provide additional lines of phage defense. Collectively, our results identify a new player within the Pseudomonas bacterial immunity arsenal that displays a novel mechanism of protection, and reveals a role for RmuC domains in phage defense.

Introduction to the immunology of pregnancy.

Introduction to the immunology of pregnancy.

A novel mechanism of protection against isoproterenol-induced cardiac inflammation via regulation of the SIRT1/NRF2 signaling pathway with a natural SIRT1 agonist

Stress-induced cardiomyopathy (SIC) is associated with high mortality rates, potentially due to a lack of available therapies. To facilitate the identification of therapeutic targets for SIC, we explored the detailed mechanisms of disease onset and progression using a mouse model. Over-activation of the β-adrenergic receptor (β-AR) upon stress leads to inflammasome activation, cytokine cascades, macrophage infiltration, and pathological cardiac remodeling in mice, mimicking SIC. However, the detailed mechanisms by which acute β-AR stimulation induces cardiac inflammation remain elusive. We found that resveratrol (RSV) could attenuate isoproterenol-induced cardiac inflammation in mice, suggesting that RSV might be a promising therapeutic option in SIC. Mechanistically, we revealed that the SIRT1/NRF2 signaling pathway is the bona fide target of RSV and plays a significant role in the RSV-induced protective effect in cardiac inflammation.

Pejvakin-mediated pexophagy protects auditory hair cells against noise-induced damage

Noise overexposure causes oxidative stress, leading to auditory hair cell damage. Adaptive peroxisome proliferation involving pejvakin, a peroxisome-associated protein from the gasdermin family, has been shown to protect against this harmful oxidative stress. However, the role of pejvakin in peroxisome dynamics and homeostasis remains unclear. Here we show that sound overstimulation induces an early and rapid selective autophagic degradation of peroxisomes (pexophagy) in auditory hair cells from wild-type, but not pejvakin-deficient (Pjvk -/-), mice. Noise overexposure triggers recruitment of the autophagosome-associated protein MAP1LC3B (LC3B; microtubule-associated protein 1 light chain 3β) to peroxisomes in wild-type, but not Pjvk -/-, mice. We also show that pejvakin-LC3B binding involves an LC3-interacting region within the predicted chaperone domain of pejvakin. In transfected cells and in vivo transduced auditory hair cells, cysteine mutagenesis experiments demonstrated the requirement for both C328 and C343, the two cysteine residues closest to the C terminus of pejvakin, for reactive oxygen species-induced pejvakin-LC3B interaction and pexophagy. The viral transduction of auditory hair cells from Pjvk -/- mice in vivo with both Pjvk and Lc3b cDNAs completely restored sound-induced pexophagy, fully prevented the development of oxidative stress, and resulted in normal levels of peroxisome proliferation, whereas Pjvk cDNA alone yielded only a partial correction of the defects. Overall, our results demonstrate that pexophagy plays a key role in noise-induced peroxisome proliferation and identify defective pexophagy as a cause of noise-induced hearing loss. They suggest that pejvakin acts as a redox-activated pexophagy receptor/adaptor, thereby identifying a previously unknown function of gasdermin family proteins.

MG 53 Protein Protects Aortic Valve Interstitial Cells From Membrane Injury and Fibrocalcific Remodeling.

BackgroundThe aortic valve of the heart experiences constant mechanical stress under physiological conditions. Maladaptive valve injury responses contribute to the development of valvular heart disease. Here, we test the hypothesis that MG53 (mitsugumin 53), an essential cell membrane repair protein, can protect valvular cells from injury and fibrocalcific remodeling processes associated with valvular heart disease.Methods and ResultsWe found that MG53 is expressed in pig and human patient aortic valves and observed aortic valve disease in aged Mg53−/− mice. Aortic valves of Mg53−/− mice showed compromised cell membrane integrity. In vitro studies demonstrated that recombinant human MG53 protein protects primary valve interstitial cells from mechanical injury and that, in addition to mediating membrane repair, recombinant human MG53 can enter valve interstitial cells and suppress transforming growth factor‐β‐dependent activation of fibrocalcific signaling.ConclusionsTogether, our data characterize valve interstitial cell membrane repair as a novel mechanism of protection against valvular remodeling and assess potential in vivo roles of MG53 in preventing valvular heart disease.

Protocatechuic acid-mediated DJ-1/PARK7 activation followed by PI3K/mTOR signaling pathway activation as a novel mechanism for protection against ketoprofen-induced oxidative damage in the gastrointestinal mucosa

Oxidative stress contributes to the progression of non-steroidal anti-inflammatory drug (NSAID)-induced gastrointestinal (GI) cell apoptosis. In our previous study, we reported that nuclear factor erythroid 2-related factor 2 (Nrf2) plays a protective role against ketoprofen-induced GI mucosal oxidative injury. Recent reports suggest that Nrf2 could exhibit antioxidative and antiapoptosis responses through up-regulation of DJ-1 (PARK7). In the current study, we proposed that induction of DJ-1 expression by protocatechuic acid (PCA) might provide a potential therapeutic approach for treating oxidative stress-associated GI ulcer diseases. The results indicated that PCA increased mRNA expression of glutathione peroxidase and heme oxygenase-1 through up-regulation of DJ-1 followed by Nrf2 translocation. Furthermore, PCA protected Int-407 cells against ketoprofen-induced oxidative stress by regulating the DJ-1, PI3K, and mTOR pathways. Pretreatment with PCA inhibited mitochondrial ROS generation, up-regulated the mitochondrial membrane potential, and down-regulated pro-apoptotic Bax as well as downstream caspase-8, caspase-9, and caspase-3 activity, and reversed impaired DJ-1 and anti-apoptotic Bcl-2 protein expression in Int-407 cells induced by ketoprofen. Similar to the in vitro results, SD rats treated with PCA before administration of ketoprofen exhibited decreased caspase-3 protein expression as well as oxidative damage, and impairment of the antioxidant system and DJ-1 protein expression in the GI mucosa were reversed. The administration of lansoprazole, a type of proton pump inhibitor (PPI), strongly inhibited ketoprofen-induced GI mucosal injuries via up-regulation of DJ-1, indicating that DJ-1 is essential for the dietary antioxidant- and PPI drug-mediated mechanism of ulcer therapy. These results suggest that DJ-1 could be a novel target for protection against ketoprofen-induced GI ulcers due to its antioxidant and anti-apoptosis characteristics.

A Biofilm Matrix-Associated Protease Inhibitor Protects Pseudomonas aeruginosa from Proteolytic Attack.

ABSTRACTPseudomonas aeruginosa produces an extracellular biofilm matrix that consists of nucleic acids, exopolysaccharides, lipid vesicles, and proteins. In general, the protein component of the biofilm matrix is poorly defined and understudied relative to the other major matrix constituents. While matrix proteins have been suggested to provide many functions to the biofilm, only proteins that play a structural role have been characterized thus far. Here we identify proteins enriched in the matrix of P. aeruginosa biofilms. We then focused on a candidate matrix protein, the serine protease inhibitor ecotin (PA2755). This protein is able to inhibit neutrophil elastase, a bactericidal enzyme produced by the host immune system during P. aeruginosa biofilm infections. We show that ecotin binds to the key biofilm matrix exopolysaccharide Psl and that it can inhibit neutrophil elastase when associated with Psl. Finally, we show that ecotin protects both planktonic and biofilm P. aeruginosa cells from neutrophil elastase-mediated killing. This may represent a novel mechanism of protection for biofilms to increase their tolerance against the innate immune response.

Cognitive benefits of lithium chloride in APP/PS1 mice are associated with enhanced brain clearance of β-amyloid

Epidemiological evidence suggests that people with bipolar disorder prescribed lithium exhibit a lower risk of Alzheimer’s disease (AD) relative to those prescribed other mood-stabilizing medicines. Lithium chloride (LiCl) reduces brain β-amyloid (Aβ) levels, and the brain clearance of Aβ is reduced in AD. Therefore, the purpose of this study was to assess whether the cognitive benefits of LiCl are associated with enhanced brain clearance of exogenously-administered Aβ. The brain clearance of intracerebroventricularly (icv) administered 125I-Aβ42 was assessed in male Swiss outbred mice administered daily oral NaCl or LiCl (300 mg/kg for 21 days). LiCl exhibited a 31% increase in the brain clearance of 125I-Aβ42 over 10 min, which was associated with a 1.6-fold increase in brain microvascular expression of the blood–brain barrier efflux transporter low density lipoprotein receptor-related protein 1 (LRP1) and increased cerebrospinal fluid (CSF) bulk-flow. 8-month-old female wild type (WT) and APP/PS1 mice were also administered daily NaCl or LiCl for 21 days, which was followed by cognitive assessment by novel object recognition and water maze, and measurement of soluble Aβ42, plaque-associated Aβ42, and brain efflux of 125I-Aβ42. LiCl treatment restored the long-term spatial memory deficit observed in APP/PS1 mice as assessed by the water maze (back to similar levels of escape latency as WT mice), but the short-term memory deficit remained unaffected by LiCl treatment. While LiCl did not affect plaque-associated Aβ42, soluble Aβ42 levels were reduced by 49.9% in APP/PS1 mice receiving LiCl. The brain clearance of 125I-Aβ42 decreased by 27.8% in APP/PS1 mice, relative to WT mice, however, LiCl treatment restored brain 125I-Aβ42 clearance in APP/PS1 mice to a rate similar to that observed in WT mice. These findings suggest that the cognitive benefits and brain Aβ42 lowering effects of LiCl are associated with enhanced brain clearance of Aβ42, possibly via brain microvascular LRP1 upregulation and increased CSF bulk-flow, identifying a novel mechanism of protection by LiCl for the treatment of AD.

Abstract 5257: Upregulation of glutathione S-transferase P1 by green tea polyphenols: A novel transcriptional target of p53 tumor suppressor gene

Abstract Green tea polyphenols (GTPs) and its major constituent (-)-epigallocatechin-3-gallate (EGCG) reactivate epigenetically silenced genes in cancer cells. Glutathione S-transferase P1 (GSTP1), an enzyme involved in detoxification process, is frequently inactivated in prostate cancer due to epigenetic modifications. The mechanisms underlying the re-expression of GSTP1 expression by GTP/EGCG in prostate cancer is currently not well understood. Earlier we demonstrated the ability of GTP/EGCG to increase p53 transcriptional activity through suppression of class I histone deacetylases (Carcinogenesis 33(2):377-84, 2012). Here we report that the GSTP1 gene is an unrecognized downstream transcriptional target of the tumor suppressor p53, and that GTP/EGCG has ability to increase GSTP1 expression mediated through p53. Treatment of human prostate cancer LNCaP cells with 5μg/ml GTP and 20μM EGCG, activate p53 through acetylation at the Lys373 and Lys382 residues along with consequent increase in GSTP1 protein expression in time-dependent manner. To study the possible interaction between GSTP1 and p53, LNCaP cells stably-transfected with short hairpin-RNA against p53 (LNCaPshp53) and control vector (LNCaPshV) for generation of p53 knockout cells. GTP/EGCG treatment induced p53 activation and acetylation at p53 Lys373 and Lys382 specifically in LNCaPshV cells in time-dependent manner, but not in LNCaPshp53. This increase in p53 acetylation led to activation of GSTP1 in LNCaPshV cells both at protein and message levels. GTP/EGCG-mediated increase in GSTP1 expression is caused by increase binding of p53 to its consensus binding site on the intron 4 of GSTP1 gene as analyzed by electrophoretic mobility shift binding assay. Furthermore, this association was validated by ChIP assay where GTP/EGCG treatment led to an increase in the amount of acetylated p53-Lys373 associated within the intron site of GSTP1, compared to untreated controls. Taken together, our findings indicate the ability of p53 to transcriptionally activate the human GSTP1 gene, which defines a novel mechanism of protection of the genome by green tea polyphenols. Citation Format: Vijay S. Thakur, Gauri Deb, Mark W. Jackson, Sanjay Gupta. Upregulation of glutathione S-transferase P1 by green tea polyphenols: A novel transcriptional target of p53 tumor suppressor gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5257.

Brief Report: Enhanced Allogeneic Cellular Responses to Mismatched HLA-B Antigens Results in More Efficient Killing of HIV Infected Cells.

Epidemiologic studies have demonstrated that HIV-1 discordant couples who share HLA-B alleles were more likely to transmit HIV-1. These data lead us to hypothesize that individuals who match at both HLA-B alleles should have a reduced allogeneic response than those who are not matched. We observed diminished killing of CD4 target cells only when HLA-B alleles were matched. We propose that for cell-associated HIV-1 transmission, the ability of the recipient to eliminate infected cells from a donor partner may be enhanced when HLA-B alleles are different between partners. These findings suggest a novel mechanism for protection against HIV infection.

A Mechanistic Review of Mitophagy and Its Role in Protection against Alcoholic Liver Disease.

Alcoholic liver disease (ALD) is a major health problem worldwide, and alcohol is well-known to cause mitochondrial damage, which exacerbates alcohol-induced liver injury and steatosis. No successful treatments are currently available for treating ALD. Therefore, a better understanding of mechanisms involved in regulation of mitochondrial homeostasis in the liver and how these mechanisms may protect against alcohol-induced liver disease is needed for future development of better therapeutic options for ALD. Mitophagy is a key mechanism for maintaining mitochondrial homeostasis by removing damaged mitochondria, and mitophagy protects against alcohol-induced liver injury. Parkin, an E3 ubiquitin ligase, is well-known to induce mitophagy in in vitro models although Parkin-independent mechanisms for mitophagy induction also exist. In this review, we discuss the roles of Parkin and mitophagy in protection against alcohol-induced liver injury and steatosis. We also discuss Parkin-independent mechanisms for mitophagy induction, which have not yet been evaluated in the liver but may also potentially have a protective role against ALD. In addition to mitophagy, mitochondrial spheroid formation may also provide a novel mechanism of protection against ALD, but the role of mitochondrial spheroids in protection against ALD progression needs to be further explored. Targeting removal of damaged mitochondria by mitophagy or inducing formation of mitochondrial spheroids may be promising therapeutic options for treatment of ALD.

Sphingosine Kinase 1 Protects Hepatocytes from Lipotoxicity via Down-regulation of IRE1α Protein Expression

Aberrant deposition of fat including free fatty acids in the liver often causes damage to hepatocytes, namely lipotoxicity, which is a key pathogenic event in the development and progression of fatty liver diseases. This study demonstrates a pivotal role of sphingosine kinase 1 (SphK1) in protecting hepatocytes from lipotoxicity. Exposure of primary murine hepatocytes to palmitate resulted in dose-dependent cell death, which was enhanced significantly in Sphk1-deficient cells. In keeping with this, expression of dominant-negative mutant SphK1 also markedly promoted palmitate-induced cell death. In contrast, overexpression of wild-type SphK1 profoundly protected hepatocytes from lipotoxicity. Mechanistically, the protective effect of SphK1 is attributable to suppression of ER stress-mediated pro-apoptotic pathways, as reflected in the inhibition of IRE1α activation, XBP1 splicing, JNK phosphorylation, and CHOP induction. Of note, SphK1 inhibited the IRE1α pathway by reducing IRE1α expression at the transcriptional level. Moreover, S1P mimicked the effect of SphK1, suppressing IRE1α expression in a receptor-dependent manner. Furthermore, enforced overexpression of IRE1α significantly blocked the protective effect of SphK1 against lipotoxicity. Therefore, this study provides new insights into the role of SphK1 in hepatocyte survival and uncovers a novel mechanism for protection against ER stress-mediated cell death.

Agglutinating Secretory IgA Preserves Intestinal Epithelial Cell Integrity during Apical Infection by Shigella flexneri

Shigella flexneri, by invading intestinal epithelial cells (IECs) and inducing inflammatory responses of the colonic mucosa, causes bacillary dysentery. Although M cells overlying Peyer's patches are commonly considered the primary site of entry of S. flexneri, indirect evidence suggests that bacteria can also use IECs as a portal of entry to the lamina propria. Passive delivery of secretory IgA (SIgA), the major immunoglobulin secreted at mucosal surfaces, has been shown to protect rabbits from experimental shigellosis, but no information exists as to its molecular role in maintaining luminal epithelial integrity. We have established that the interaction of virulent S. flexneri with the apical pole of a model intestinal epithelium consisting of polarized Caco-2 cell monolayers resulted in the progressive disruption of the tight junction network and actin depolymerization, eventually resulting in cell death. The lipopolysaccharide (LPS)-specific agglutinating SIgAC5 monoclonal antibody (MAb), but not monomeric IgAC5 or IgGC20 MAbs of the same specificity, achieved protective functions through combined mechanisms, including limitation of the interaction between S. flexneri and epithelial cells, maintenance of the tight junction seal, preservation of the cell morphology, reduction of NF-κB nuclear translocation, and inhibition of proinflammatory mediator secretion. Our results add to the understanding of the function of SIgA-mediated immune exclusion by identifying a mode of action whereby the formation of immune complexes translates into maintenance of the integrity of epithelial cells lining the mucosa. This novel mechanism of protection mediated by SIgA is important to extend the arsenal of effective strategies to fight against S. flexneri mucosal invasion.

Inducer of heme oxygenase‐1 cobalt protoporphyrin accelerates autophagy and suppresses oxidative damages during lipopolysaccharide treatment in rat liver

Mitochondrial damage and subsequent oxidative stresses play important roles in the pathogenesis of sepsis-induced organ failure. Recently, autophagy, the major degradation pathway involved in mitochondrial quality control, was reported as a cellular adaptive response to oxidative stresses. The aim of the present study was to elucidate the molecular mechanism that underlies hepatic damage during lipopolysaccharide (LPS) treatment. We also try to determine if the damage can be attenuated by administration of cobalt protoporphyrin (CoPP), a potent heme oxygenase-1 (HO-1) inducer. Five-week-old male Sprague-Dawley rats were injected i.p. with 15 mg/kg LPS. To determine if hepatic damage following LPS administration can be attenuated by HO-1, CoPP was injected s.c. for 4 days consecutively at 24-h intervals. After treatment with LPS, the liver was obtained and analyzed. A large reduction in liver mitochondrial protein and induction of autophagy were observed in LPS-treated rats. Electron microscopic and immunohistochemical analyses demonstrated autophagic vacuoles in LPS-treated rat liver. Oxidative stress markers (4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine) were increased in LPS-treated animals; CoPP treatment ablated these alterations. An inhibitor for the opening of mitochondrial permeability transition pore, cyclosporine A, suppressed oxidative stress as well as liver damage during LPS administration. CoPP promoted autophagy and prevented rats from liver damage during LPS administration. HO-1 promotes autophagy and elimination of damaged mitochondria thereby repressing oxidative stress in LPS-treated rat liver, revealing a novel mechanism for protection by HO-1 against septic liver damage.

Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection

RhoA a small G-protein that has an established role in cell growth and in regulation of the actin cytoskeleton. Far less is known about whether RhoA can modulate cell fate. We previously reported that sustained RhoA activation induces cardiomyocyte apoptosis (Del Re, D. P., Miyamoto, S., and Brown, J. H. (2007) J. Biol. Chem. 282, 8069-8078). Here we demonstrate that less chronic RhoA activation affords a survival advantage, protecting cardiomyocytes from apoptotic insult induced by either hydrogen peroxide treatment or glucose deprivation. Under conditions where RhoA is protective, we observe Rho kinase-dependent cytoskeletal rearrangement and activation of focal adhesion kinase (FAK). Activation of endogenous cardiomyocyte FAK leads to its increased association with the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) and to concomitant activation of Akt. Treatment of isolated perfused hearts with sphingosine 1-phosphate recapitulates this response. The pathway by which RhoA mediates cardiomyocyte Akt activation is demonstrated to require Rho kinase, FAK and PI3K, but not Src, based on studies with pharmacological inhibitors (Y-27632, LY294002, PF271 and PP2) and inhibitory protein expression (FAK-related nonkinase). Inhibition of RhoA-mediated Akt activation at any of these steps, including inhibition of FAK, prevents RhoA from protecting cardiomyocytes against apoptotic insult. We further demonstrate that stretch of cardiomyocytes, which activates endogenous RhoA, induces the aforementioned signaling pathway, providing a physiologic context in which RhoA-mediated FAK phosphorylation can activate PI3K and Akt. We suggest that RhoA-mediated effects on the cardiomyocyte cytoskeleton provide a novel mechanism for protection from apoptosis.

Anti-Inflammatory Effects of the 70 kDa Heat Shock Protein in Experimental Stroke

The 70-kDa heat shock protein (Hsp70) is involved in protecting the brain from a variety of insults including stroke. Although the mechanism has been largely considered to be because of its chaperone functions, recent work indicates that Hsp70 also modulates inflammatory responses. To explore how and whether Hsp70 regulate immune responses in brain ischemia, mice overexpressing Hsp70 (Hsp Tg) were subjected to 2 h middle cerebral artery occlusion, followed by 24 h reperfusion. Parallel experiments were performed using a brain inflammation model. Hsp Tg microglia cocultured with astrocytes were used to evaluate the direct effects of Hsp70 on cytotoxicity of microglia. Compared with wild-type (Wt) littermates, Hsp Tg mice showed decreased infarct size and improved neurological deficits. The number of activated microglia/macrophages were also reduced in ischemic brains of Hsp Tg mice. Similar observations were made in a model of brain inflammation that does not result in brain cell death. Overexpression of Hsp70 in microglia completely prevented microglia-induced cytotoxicity to astrocytes. Activation of the inflammatory transcription factor, nuclear factor-kappaB (NF-kappaB) was inhibited significantly in Hsp Tg mice and microglia. This was associated with decreased phosphorylation of NF-kappaB inhibitor protein, IkappaBalpha, and decreased expression of several NFkappaB-regulated genes. Co-immunoprecipitation studies revealed an interaction of Hsp70 with NF-kappaB and IkappaBalpha, but not with IkappaB kinase, IKKgamma, suggesting that Hsp70 binds to the NF-kappaB:IkappaB complex preventing IkappaB phosphorylation by IKK. The findings of the present work establish an anti-inflammatory role for Hsp70 in the context of brain ischemia as a novel mechanism of protection.

Immunization with theHaemophilus ducreyiHemoglobin Receptor HgbA Protects against Infection in the Swine Model of Chancroid

The etiologic agent of chancroid is Haemophilus ducreyi. To fulfill its obligate requirement for heme, H. ducreyi uses two TonB-dependent receptors: the hemoglobin receptor (HgbA) and a receptor for free heme (TdhA). Expression of HgbA is necessary for H. ducreyi to survive and initiate disease in a human model of chancroid. In this study, we used a swine model of H. ducreyi infection to demonstrate that an experimental HgbA vaccine efficiently prevents chancroid, as determined by several parameters. Histological sections of immunized animals lacked typical microscopic features of chancroid. All inoculated sites from mock-immunized pigs yielded viable H. ducreyi cells, whereas no viable H. ducreyi cells were recovered from inoculated sites of HgbA-immunized pigs. Antibodies from sera of HgbA-immunized animals bound to and initiated antibody-dependent bactericidal activity against homologous H. ducreyi strain 35000HP and heterologous strain CIP542 ATCC; however, an isogenic hgbA mutant of 35000HP was not killed, proving specificity. Anti-HgbA immunoglobulin G blocked hemoglobin binding to the HgbA receptor, suggesting a novel mechanism of protection through the limitation of heme/iron acquisition by H. ducreyi. Such a vaccine strategy might be applied to other bacterial pathogens with strict heme/iron requirements. Taken together, these data suggest continuing the development of an HgbA subunit vaccine to prevent chancroid.

Inducible heat shock protein 70 kD and inducible nitric oxide synthase in hemorrhage/resuscitation-induced injury

Inducible head shock protein 70 kD (HSP-70i) has been shown to protect cells, tissues, and organs from harmful assaults in vivo and in vitro experimental models. Hemorrhagic shock followed by resuscitation is the principal cause of death among trauma patients and soldiers in the battlefield. Although the underlying mechanisms are still not fully understood, it has been shown that nitric oxide (NO) overproduction and inducible nitric oxide synthase (iNOS) overexpression play important roles in producing injury caused by hemorrhagic shock including increases in polymorphonuclear neutrophils (PMN) infiltration to injured tissues and leukotriene B(4) (LTB(4)) generation. Moreover, transcription factors responsible for iNOS expression are also altered by hemorrhage and resuscitation. It has been evident that either up-regulation of HSP-70i or down-regulation of iNOS can limit tissue injury caused by ischemia/reperfusion or hemorrhage/resuscitation. In our laboratory, geldanamycin, a member of ansamycin family, has been shown to induce HSP- 70i overexpression and then subsequently to inhibit iNOS expression, to reduce cellular caspase-3 activity, and to preserve cellular ATP levels. HSP-70i is found to couple to iNOS and its transcription factor. Therefore, the complex formation between HSP-70i and iNOS may be a novel mechanism for protection from hemorrhage/resuscitation-induced injury.