Stefan Weber

Induction of Bim and Bid gene expression during accelerated apoptosis in severe sepsis

IntroductionIn transgenic animal models of sepsis, members of the Bcl-2 family of proteins regulate lymphocyte apoptosis and survival of sepsis. This study investigates the gene regulation of pro-apoptotic and anti-apoptotic members of the Bcl-2 family of proteins in patients with early stage severe sepsis.MethodsIn this prospective case-control study, patients were recruited from three intensive care units (ICUs) in a university hospital. Sixteen patients were enrolled when they fulfilled the criteria of severe sepsis. Ten critically ill but non-septic patients and 11 healthy volunteers served as controls. Blood samples were immediately obtained at inclusion. To confirm the presence of accelerated apoptosis in the patient groups, caspase-3 activation and phosphatidylserine externalisation in CD4+, CD8+ and CD19+ lymphocyte subsets were assessed using flow cytometry. Specific mRNAs of Bcl-2 family members were quantified from whole blood by real-time PCR. To test for statistical significance, Kruskal-Wallis testing with Dunns multiple comparison test for post hoc analysis was performed.ResultsIn all lymphocyte populations caspase-3 (p < 0.05) was activated, which was reflected in an increased phosphatidylserine externalisation (p < 0.05). Accordingly, lymphocyte counts were decreased in early severe sepsis. In CD4+ T-cells (p < 0.05) and B-cells (p < 0.001) the Bcl-2 protein was decreased in severe sepsis. Gene expression of the BH3-only Bim was massively upregulated as compared with critically ill patients (p < 0.001) and 51.6-fold as compared with healthy controls (p < 0.05). Bid was increased 12.9-fold compared with critically ill patients (p < 0.001). In the group of mitochondrial apoptosis inducers, Bak was upregulated 5.6-fold, while the expression of Bax showed no significant variations. By contrast, the pro-survival members Bcl-2 and Bcl-xl were both downregulated in severe sepsis (p < 0.001 and p < 0.05, respectively).ConclusionsIn early severe sepsis a gene expression pattern with induction of the pro-apoptotic Bcl-2 family members Bim, Bid and Bak and a downregulation of the anti-apoptotic Bcl-2 and Bcl-xl proteins was observed in peripheral blood. This constellation may affect cellular susceptibility to apoptosis and complex immune dysfunction in sepsis.

A MIF haplotype is associated with the outcome of patients with severe sepsis: a case control study.

BackgroundMacrophage migration inhibitory factor (MIF) plays an important regulatory role in sepsis. In the promoter region a C/G single nucleotide polymorphism (SNP) at position -173 (rs755622) and a CATT5-8 microsatellite at position -794 are related to modified promoter activity. The purpose of the study was to analyze their association with the incidence and outcome of severe sepsis.MethodsGenotype distributions and allele frequencies in 169 patients with severe sepsis, 94 healthy blood donors and 183 postoperative patients without signs of infection or inflammation were analyzed by real time PCR and Sequence analysis. All included individuals were Caucasians.ResultsGenotype distribution and allele frequencies of severe sepsis patients were comparable to both control groups. However, the genotype and allele frequencies of both polymorphisms were associated significantly with the outcome of severe sepsis. The highest risk of dying from severe sepsis was detectable in patients carrying a haplotype with the alleles -173 C and CATT7 (p = 0.0005, fisher exact test, RR = 1,806, CI: 1.337 to 2.439).ConclusionThe haplotype with the combination of the -173 C allele and the -794 CATT7 allele may not serve as a marker for susceptibility to sepsis, but may help identify septic patients at risk of dying.

Topical α-tocotrienol supplementation inhibits lipid peroxidation but fails to mitigate increased transepidermal water loss after benzoyl peroxide treatment of human skin

Benzoyl peroxide (BPO) is a commonly used drug in the treatment of acne vulgaris, but it induces unwanted side effects related to stratum corneum (SC) function. Since it has been recently shown to oxidize SC antioxidants, it was hypothesized that antioxidant supplementation may mitigate the BPO-induced SC changes. To test this, 11 subjects were selected to be topically supplemented with alpha-tocotrienol (5% w/vol) for 7 d on defined regions of the upper back, while the contralateral region was used for vehicle-only controls. Starting on day 8, all test sites were also treated with BPO (10%) for 7 d; the alpha-tocotrienol supplementation was continued throughout the study. A single dose of BPO depleted 93.2% of the total vitamin E. While continuing the BPO exposure for 7 d further depleted vitamin E in both vehicle-only and alpha-tocotrienol-treated sites, significantly more vitamin E remained in the alpha-tocotrienol-treated areas. Seven BPO applications increased lipid peroxidation. Alpha-tocotrienol supplementation significantly mitigated the BPO-induced lipid peroxidation. The transepidermal water loss was increased 1.9-fold by seven BPO applications, while there was no difference between alpha-tocotrienol treatment and controls. The data suggest that alpha-tocotrienol supplementation counteracts the lipid peroxidation but not the barrier perturbation in the SC induced by 10% BPO.

Cytokine Promoter Polymorphisms in Severe Sepsis

The need to develop individualized risk profiles and drug therapy regimens motivates interest in genetic studies of critically ill patients. Gene promoter variants may predict interindividual variability in response to inflammatory stimuli, such as infection and trauma. Genomic variations also may affect gene expression profiles, as well as the structure and production of proteins. The genes involved in inflammation are numerous, as are genomic variations within most of those genes. Cytokine genes involved in inflammatory cascades are important candidate genes that may determine the extent of a persons response to injury. Understanding the genetic determination of the inflammatory process includes the possibility of developing valuable diagnostic tools and new therapeutic approaches in severe sepsis. To date, specific patterns of markers of genomic variation reliably indicating at-risk populations do not exist. Evaluation of possible genomic markers for risk stratification of patients with sepsis and persons at high risk of developing organ failure has begun at a level of well-powered genetic epidemiological research. Cytokine promoter variants may contribute substantially to studies of genetic predisposition of sepsis because they operate in a gene region of high regulatory activity.

Inducibility of the endogenous antibiotic peptide β-defensin 2 is impaired in patients with severe sepsis

IntroductionThe potent endogenous antimicrobial peptide human β-defensin 2 (hBD2) is a crucial mediator of innate immunity. In addition to direct antimicrobial properties, different effects on immune cells have been described. In contrast to the well-documented epithelial β-defensin actions in local infections, little is known about the leukocyte-released hBD2 in systemic infectious disorders. This study investigated the basic expression levels and the ex vivo inducibility of hBD2 mRNA in peripheral whole blood cells from patients with severe sepsis in comparison to non-septic critically ill patients and healthy individuals.MethodsThis investigation was a prospective case-control study performed at a surgical intensive care unit at a university hospital. A total of 34 individuals were tested: 16 patients with severe sepsis, 9 critically ill but non-septic patients, and 9 healthy individuals. Serial blood samples were drawn from septic patients, and singular samples were obtained from critically ill non-septic patients and healthy controls. hBD2 mRNA levels in peripheral white blood cells were quantified by real-time polymerase chain reaction in native peripheral blood cells and following ex vivo endotoxin stimulation. Defensin plasma levels were quantified by enzyme-linked immunosorbent assay.ResultsEndotoxin-inducible hBD2 mRNA expression was significantly decreased in patients with severe sepsis compared to healthy controls and non-septic critically ill patients (0.02 versus 0.95 versus 0.52, p < 0.05, arbitrary units). hBD2 plasma levels in septic patients were significantly higher compared to healthy controls and critically ill non-septic patients (541 versus 339 versus 295 pg/ml, p < 0.05).ConclusionIn contrast to healthy individuals and critically ill non-septic patients, ex vivo inducibility of hBD2 in peripheral blood cells from septic patients is reduced. Impaired hBD2 inducibility may contribute to the complex immunological dysfunction in patients with severe sepsis.

Apoptose als Pathomechanismus in der Sepsis

ZusammenfassungDie Sepsis gehört nach wie vor zu den häufigsten Todesursachen bei Intensivpatienten. Der Pathogenese der Erkrankung liegt ein komplexes Immungeschehen zugrunde. Neueste Erkenntnisse zeigen, dass Zellen des Abwehrsystems bei der Sepsis im Rahmen der Immunantwort häufig eine Signalkaskade aktivieren, die zum programmierten Zelltod, der sog. Apoptose, führt. Apoptose von Leukozyten wurde sowohl im Tiermodell als auch bei Patienten mit Sepsis beobachtet. Bei der Regulation der Apoptose spielen das mitochondriale Protein Bcl-2 und die zytoplasmatische Enzymkaskade der Kaspasen eine wesentliche Rolle. Durch Überexprimierung von Bcl-2 oder Hemmung der Kaspasen konnte im Tiermodell der Sepsis ein deutlicher Überlebensvorteil demonstriert werden. Erste Arbeiten zeigten auch die Relevanz der Apoptose bei Patienten mit schwerer Sepsis. Ziel aktueller Forschung auf diesem Gebiet ist es, durch gezielte Beeinflussung der Apoptose neue molekulare Ansätze zur Therapie der Sepsis zu finden.AbstractSepsis is still a leading cause of death in many intensive care patients. The pathophysiology of the disease is dominated by complex immune cascades. Recent research demonstrates that immune cells respond to sepsis with an increased rate of programmed cell death. Up-regulated apoptosis of leukocytes was observed in animal models of sepsis as well as in patients suffering from severe sepsis. The mitochondrial protein Bcl-2 and the caspase cascade play an important role in the regulation of apoptosis. Overexpression of Bcl-2 or inhibition of caspases resulted in an increased survival in animal models of sepsis. Recent reports indicate the relevance of apoptosis in patients with severe sepsis. These results may spawn novel immunomodulatory strategies in the treatment of sepsis.

Statins Downregulate the Constitutive Expression of HLA-DR and ReduceIntracellular CD74 in the Monocyte Cell Line Mono Mac 6

Background: Statins exert immune effects and have been shown to strongly inhibit the interferon-γ induced expression of the human leukocyte antigen (HLA) DR. Monocyte HLA-DR is decreased severe in sepsis and indicates hypo inflammatory and immunosuppressive phases. The aim of the study was to investigate the statin-induced regulation of constitutive HLA-DR expression on monocytes. Method: Monocytes and the monocyte cell line Mono Mac 6 were incubated with simvastatin, mevastatin, pravastatin and fluvastatin. HMG-Coenzyme A reductase was inhibited by l-mevalonate. Protein expression of HLA-DR, Major histocompatibility complex, invariant CD74 and apoptosis were measured by flow-cytometry. mRNA expression was assessed by PCR. Results: Simvastatin dose dependently reduced the constitutive HLA-DR expression on monocytes starting from 500 nM. Bypassing statin-induced inhibition of HMG- Coenzyme A reductase reversed this effect. HLA-DR was also decreased in response to mevastatin, pravastatin, lovastatin and fluvastatin after 24 h. Simvastatin induced caspase-3 activation and DNA-fragmentation in a small subpopulation of Mono Mac 6 cells. HLA-DR expression was lower on apoptotic cells, but simvastatin also reduced HLA-DR on non-apoptotic cells. Simvastatin did no repress HLA-DR mRNA. For transferring HLADR to endosomes the chaperon CD74 is required. Intracellular CD74 was decreased by simvastatin and fluvastatin. Conclusion: Statins decrease the constitutive expression of MHC II on monocytes and Mono Mac 6 independently of apoptosis. Reduced intracellular levels of the chaperon CD74 by statins may potentially impede HLA-DR surface expression.

[Traumatic tricuspid valve insufficiency with right-to-left shunt: bridging using extracorporeal venovenous membrane oxygenation].

The case of a young male motor vehicle driver is reported who suffered multiple trauma in a car accident with pulmonary and cardiac contusions. In the course of severe pneumonia and traumatic tricuspid valve insufficiency a right-to-left shunt with refractory hypoxemia developed across a pre-existing atrial septal defect (ASD). The patient could be successfully treated by the combination of extracorporeal membrane oxygenation for bridging, interventional ASD occlusion and in the long-term by operative reconstruction of the tricuspid valve.

Traumatische Trikuspidalklappeninsuffizienz mit Rechts-links-Shunt

The case of a young male motor vehicle driver is reported who suffered multiple trauma in a car accident with pulmonary and cardiac contusions. In the course of severe pneumonia and traumatic tricuspid valve insufficiency a right-to-left shunt with refractory hypoxemia developed across a pre-existing atrial septal defect (ASD). The patient could be successfully treated by the combination of extracorporeal membrane oxygenation for bridging, interventional ASD occlusion and in the long-term by operative reconstruction of the tricuspid valve.

Serologic Follow-up of Middle East Respiratory Syndrome Coronavirus Cases and Contacts—Abu Dhabi, United Arab Emirates

Abstract Background Although there is evidence of person-to-person transmission of Middle East respiratory syndrome coronavirus (MERS-CoV) in household and healthcare settings, more data are needed to describe and better understand the risk factors and transmission routes in both settings, as well as the extent to which disease severity affects transmission. Methods A seroepidemiological investigation was conducted among MERS-CoV case patients (cases) and their household contacts to investigate transmission risk in Abu Dhabi, United Arab Emirates. Cases diagnosed between 1 January 2013 and 9 May 2014 and their household contacts were approached for enrollment. Demographic, clinical, and exposure history data were collected. Sera were screened by MERS-CoV nucleocapsid protein enzyme-linked immunosorbent assay and indirect immunofluorescence, with results confirmed by microneutralization assay. Results Thirty-one of 34 (91%) case patients were asymptomatic or mildly symptomatic and did not require oxygen during hospitalization. MERS-CoV antibodies were detected in 13 of 24 (54%) case patients with available sera, including 1 severely symptomatic, 9 mildly symptomatic, and 3 asymptomatic case patients. No serologic evidence of MERS-CoV transmission was found among 105 household contacts with available sera. Conclusions Transmission of MERS-CoV was not documented in this investigation of mostly asymptomatic and mildly symptomatic cases and their household contacts. These results have implications for clinical management of cases and formulation of isolation policies to reduce the risk of transmission.