Silencing cardiomyocyte TLR4 reduces injury following hypoxia.

Toll-like receptor 4 (TLR4), the receptor for lipopolysaccharide (LPS) of gram-negative pathogens expressed in the heart, is activated by several endogenous ligands associated with tissue injury in response to myocardial infarction (MI). The aim of this study was to investigate the involvement of TLR4 signaling in cardiomyocytes dysfunction following hypoxia (90min) using multiple methodologies such as knocking down TLR4 and small interfering RNA (siTLR4). Cardiomyocytes of C57Bl/6 mice (WT) subjected to hypoxic stress showed increased cardiac release of LDH, HMGB1, IκB, TNF-α and myocardial apoptotic and necrotic markers (BAX, PI) compared to TLR4 knock out mice (TLR4KO). Treating these cardiomyocytes with siRNA against TLR4 decreased the damage markers (LDH, IκB, TNF-α). TLR4 silencing during hypoxic stress resulted in the activation of the p-AKT and p-GSK3ß (by ∼25%). The latter is an indicator that there is a reduction of mitochondrial permeability transition pore (mPTP) opening following hypoxic myocardial induced injury leading to preserved mitochondrial membrane potential. Silencing TLR4 in cardiomyocytes improved cell survival following hypoxic injury through activation of the AKT/GSK3ß pathway, reduced inflammatory and apoptotic signals. These findings suggest that TLR4 may serve as a potential target in the treatment of ischemic myocardial injury. Moreover, RNA interfering targeting TLR4 expression represents a therapeutic strategy.

A new search tool for familial searches in the Israel Criminal DNA database.

The Israel DNA database has recently started to conduct familial searches (FS). We adopted the CODIS pedigree strategy for FS, which is used in the Unidentified Human Remains (UHR) database and implemented it into the criminal forensic database. This strategy is based on Kinship analysis performed in pedigrees containing DNA profiles from the crime scene designated "unknown," that are then searched against the entire suspects database. A list of candidates is produced and ranked by Joint Pedigree Likelihood Ratio (JPLR). Traditional Y-STR characterizing and mitochondrial sequencing can be performed in order to further minimize the list. Our novel strategy consists of an additional pedigree analysis aimed at prioritizing potential candidates from the candidate list: a Test Pedigree Tree (TPT). Candidates ranked high on the JPLR list can be verified or eliminated from the list by using other close family members included in the database. To further validate this novel strategy, we describe two cases where implementation of this strategy led to a successful match and solved the crime.

Reduced hepatic injury in Toll-like receptor 4-deficient mice following D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure.

Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), D-galactosamine (GalN)-induced FHF is a well established model of liver injury in mice. Toll-Like Receptor 4 (TLR4) has been identified as a receptor for LPS. The aim of this study was to investigate the role of TLR4 in FHF induced by D-GalN/LPS administration in mice. Wild type (WT) and TLR4 deficient (TLR4ko) mice were studied in vivo in a fulminant model induced by GalN/LPS. Hepatic TLR4 expression, serum liver enzymes, hepatic and serum TNF-α and interleukin-1ß levels were determined. Apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor-kappaß (NF-κ ß) and phosphorylated c-Jun hepatic expression were studied using Western blot analysis. All WT mice died within 24 hours after administration of GalN/LPS while all TLR4ko mice survived. Serum liver enzymes, interleukin-1ß, TNF-α level, TLR4 mRNA expression, hepatic injury and hepatocyte apoptosis all significantly decreased in TLR4ko mice compared with WT mice. A significant decrease in hepatic c-Jun and IκB signaling pathway was noted in TLR4ko mice compared with WT mice. In conclusion, following induction of FHF, the inflammatory response and the liver injury in TLR4ko mice was significantly attenuated through decreased hepatic c-Jun and NF-κB expression and thus decreased TNF-α level. Down-regulation of TLR4 expression plays a pivotal role in GalN/LPS induced FHF. These findings might have important implications for the use of the anti TLR4 protein signaling as a potential target for therapeutic intervention in FHF.

Concordance testing between Powerplex ESI 16 Fast System and VeriFiler Express.

DNA profiles generated by different STR kits may show different alleles for identical amplified loci. This well-known phenomenon affects the smooth transition of data generated by new STR kits to a database or casework laboratory or cross-laboratory comparison of STR profiles. As in other DNA databases throughout the world, it has become clear that the number of the analyzed loci should be expanded for a variety of reasons, such as partial profiles resulting from low copy-number DNA template or degraded samples, working with mixtures or when prevalence of familial inbreeding. In the course of introducing a new STR kit, VeriFiler™ Express (Applied Biosystems, Foster City, CA, USA), we compared genotyping data of 1568 samples amplified by the VeriFiler™ Express with the data generated on the same samples by the Powerplex™ ESI FAST (Promega, Madison WI, USA) kit. Discordance was noted in 20 samples (1.27%), 14 (0.89%) of them showing allele dropout mismatch and six (0.38%) showing an additional fixed-size third allele. These rates are well above the reported rates of 0.4% for this kit. Since correct genotyping and accurate consistent allele assignment is of paramount importance, it seems timely to recommend for DNA laboratories and genetic-match search systems to take these possible inconsistencies into account.

Cardiomyocyte Toll-like receptor 4 is involved in heart dysfunction following septic shock or myocardial ischemia.

Toll-like receptors are expressed in immune cells and cardiac muscle. We examined whether the cardiac Toll-like receptor 4 (TLR4) is involved in the acute myocardial dysfunction caused by septic shock and myocardial ischemia (MI). We used wild type mice (WT), TLR4 deficient (TLR4-ko) mice and chimeras that underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the heart (TLR4-ko/WT) and the immunohematopoietic system (WT/TLR4-ko). Mice were injected with lipopolysaccharide (LPS) (septic shock model) or subjected to coronary artery ligation (MI model) and tested in vivo and ex vivo, for function, histopathology proinflammatory cytokine and TLR4 expression. WT mice challenged with LPS or MI displayed reduced cardiac function, increased myocardial levels of IL-1 beta and TNF-alpha and upregulation of mRNA encoding TLR4 prior to myocardial leukocyte infiltration. TLR4 deficient mice sustained significantly smaller infarctions as compared to control mice at comparable areas at risk. The cardiac function of TLR4-ko mice was not affected by LPS and demonstrated reduced suppression by MI compared to WT. Chimeras deficient in myocardial TLR4 were resistant to suppression induced by LPS and the heart function was less depressed, compared to the TLR4-ko, following MI in the acute phase (4h). In contrast, hearts of chimeras deficient in immunohematopoietic TLR4 expression were suppressed both by LPS and MI, exhibiting increased myocardial cytokine levels, similar to WT mice. We concluded that cardiac function of TLR4-ko mice and chimeric mice expressing TLR4 in the immunohematopoietic system, but not in the heart, revealed resistance to LPS and reduced cardiac depression following MI, suggesting that TLR4 expressed by the cardiomyocytes themselves plays a key role in this acute phenomenon.

The role of excessive versus acute administration of erythropoietin in attenuating hepatic ischemia-reperfusion injury.

Ischemia-reperfusion injury (I/R) is the main cause of primary graft nonfunction. Our aim was to evaluate the effect of excessive versus acute administration of erythropoietin (EPO) in attenuating the hepatic injury induced by I/R in mice. The effect of segmental (70%) hepatic ischemia was evaluated in a transgenic mouse line with constitutive overexpression of human EPO cDNA and in wild-type (WT) mice. Mice were randomly allocated to 5 main experimental groups: (i) WT-sham, (ii) WT ischemia, (iii) WT ischemia + recombinant human erythropoietin (rhEPO), (iv) transgenic-sham, and (v) transgenic ischemia. The EPO-pretreated mice showed a significant reduction in liver enzyme levels and intrahepatic caspase-3 activity and fewer apoptotic hepatocytes (p < 0.05 for all) compared with the WT untreated I/R group. EPO decreased c-Jun N-terminal kinase (JNK) phosphorylation and nuclear factor-κB (NF-κB) expression during I/R. In transgenic I/R livers, baseline histology showed diffused hepatic injury, and no significant beneficial effect was noted between the WT untreated and the transgenic I/R mice. In conclusion, acute pretreatment with EPO in WT mice attenuated in vivo I/R liver injury. However, in excessive EPO overexpression, the initial liver injury abolished the beneficial effect of EPO. These findings have important implications for the potential use of acute EPO in I/R injury during liver transplantation.

Investigation of Different Ways in Which the CODIS 7.0 May be Used in Mass Disaster Identification.

DNA analysis is a key method for the identification of human remains in mass disasters. Reference samples from relatives may be used to identify missing persons by kinship analysis. Different methods of applying the CODIS in disaster victim identification (DVI) were investigated. Two searches were evaluated: (i) relating family relatives to a pedigree tree (FPT) and (ii) relating unidentified human remains to a pedigree tree (UPT). A joint pedigree likelihood ratio (JPLR) and rank were calculated for each search. Both searches were similar in average JPLR and rank. In exceptional cases, namely the existence of a mutation different from the CODIS model, a nonbiological father, a mistake in STR, or incorrect profile association, the UPT search returned one true rank, whereas the FPT search returned no results. This paper suggests a novel strategy to overcome these limitations and increase efficiency in conducting identification of mass disaster victims.

TLR4 Expression Is Associated with Left Ventricular Dysfunction in Patients Undergoing Coronary Artery Bypass Surgery.

INTRODUCTION: Toll-like receptor 4 (TLR4) is an innate immune receptor expressed in immune cells and the heart. Activation of the immune system following myocardial ischemia causes the release of proinflammatory mediators that may negatively influence heart function. AIM: The aim of this study is to determine whether TLR4 is activated in peripheral monocytes and heart tissue taken from patients with varying degrees of myocardial dysfunction caused by coronary artery diseases and scheduled for coronary artery bypass graft (CABG) surgery before 12 months following operation. METHODS AND RESULTS: Patients (n = 44) undergoing CABG surgery having left ventricular ejection fraction ≤ 45% ('reduced EF', n = 20) were compared to patients with preserved EF >45% ('preserved EF' group, n = 24). 'Reduced EF' patients exhibited increased TLR4 expression in monocytes (2.78±0.49 vs. 1.76±0.07 rMFI, p = 0.03). Plasma levels of C-reactive protein, microRNA miR-320a, brain natriuretic peptide (pro BNP) and NADPH oxidase (NOX4) were also significantly different between the 'preserved EF' and 'reduced EF'groups. Elevated TLR4 gene expression levels in the right auricle correlated with those of EF (p<0.008), NOX4 (p<0.008) and miR320, (p<0.04). In contrast, no differences were observed in peripheral monocyte TLR2 expression. After CABG surgery, monocyte TLR4 expression decreased in all patients, reaching statistical significance in the 'reduced EF' group. CONCLUSION: TLR4 is activated in peripheral monocytes and heart tissue obtained from patients with ischemic heart disease and reduced left ventricular function. Coronary revascularization decreases TLR4 expression. We therefore propose that TLR4 plays a pathogenic role and may serve as an additional marker of ischemic myocardial dysfunction.

Bone marrow and nonbone marrow Toll like receptor 4 regulate acute hepatic injury induced by endotoxemia.

BACKGROUND: Toll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model). METHODS: Wild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later. RESULTS: Compared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1ß (IL-1ß), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1ß and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed. CONCLUSIONS: These results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury.

Toll-like receptor 4 stimulation initiates an inflammatory response that decreases cardiomyocyte contractility.

Toll-like receptors (TLRs) have been identified as primary innate immune receptors for the recognition of pathogen-associated molecular patterns by immune cells, initiating a primary response toward invading pathogens and recruitment of the adaptive immune response. TLRs, especially Toll-like receptor 4 (TLR4), can also be stimulated by host-derived molecules and are expressed in the cardiovascular system, thus acting as a possible key link between cardiovascular diseases and the immune system. TLR4 is involved in the acute myocardial dysfunction caused by septic shock and myocardial ischemia. We used wild-type (WT) mice, TLR4-deficient (TLR4-knockout [ko]) mice, and chimeras that underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the heart (TLR4-ko/WT) and the immunohematopoietic system (WT/TLR4-ko). Following lipopolysaccharide (LPS) challenge (septic shock model) or coronary artery ligation, myocardial ischemia (MI) model, we found WT/TLR4-ko mice challenged with LPS or MI displayed reduced cardiac function, increased myocardial levels of interleukin-1ß and tumor necrosis factor-α, and upregulation of mRNA encoding TLR4 prior to myocardial leukocyte infiltration. The cardiac function of TLR4-ko or WT/TLR4-ko mice was less affected by LPS and demonstrated reduced suppression by MI compared with WT. These results suggest that TLR4 expressed in the cardiomyocytes plays a key role in this acute phenomenon.

Erythropoietin increases survival and attenuates fulminant hepatic failure injury induced by D-galactosamine/lipopolysaccharide in mice.

BACKGROUND: Liver transplantation is the only therapy of proven benefit in fulminant hepatic failure (FHF). Lipopolysaccharide (LPS), d-galactosamine (GalN)-induced FHF is a well-established model of liver injury in mice. Erythropoietin has a powerful tissue-protective effect in animal models. The aim of this study was to investigate the effect and mechanism of recombinant human erythropoietin (rhEPO) administration in FHF mice. METHODS: C57BL/6 (n=42) mice were studied in vivo in a fulminant model induced by GalN/LPS. rhEPO was administered 30 min after the induction of FHF. Serum liver enzymes and hepatic tumor necrosis factor (TNF)-α and interleukin (IL)-1ß levels were determined. Histologic analysis was performed, and apoptotic cells were identified by immunohistochemistry for caspase-3. Nuclear factor (NF)-κB and c-Jun-N-terminal kinase (JNK) activation were studied using Western blot analysis. RESULTS: After the induction of FHF, all control mice died within 12 hr of GalN/LPS administration. However, 83% of mice that were administered rhEPO were alive 2 weeks later, and overall survival improved (Kaplan-Meier, P<0.001). The serum liver enzymes, hepatic TNF-α and IL-1ß levels, liver histologic injury, and apoptotic hepatocytes were significantly reduced in FHF mice that were administered rhEPO compared with untreated mice. A significant decrease in hepatic NF-κB and JNK activation was noted in FHF rhEPO-treated mice compared with FHF untreated mice. CONCLUSIONS: The administration of rhEPO brought about increased survival and attenuation of the hepatic injury. This was associated with decreased hepatic NF-κB and JNK activation and thus TNF-α and IL-1ß levels. These findings have important implications for the potential use of rhEPO in FHF.