Sodium hypochlorite is more effective than chlorhexidine for eradication of bacterial biofilm of staphylococci and Pseudomonas aeruginosa.

PURPOSE: Periprosthetic infection is a common reason for surgical revision. Given the increasing resistance of bacteria to antibiotics (e.g., VRE, 4-MRGN) local antiseptic treatment is gaining in importance. However, no standard guideline-based treatment recommendation is yet available. The aim of this study was to investigate the effectiveness of sodium hypochlorite and chlorhexidine against bacterial biofilms. Furthermore, the toxicity of both antiseptics towards human chondrocytes was examined. METHODS: Human chondrocytes were isolated, cultivated and treated with sodium hypochlorite and chlorhexidine. The viability of cultures was assessed by determination of cell count, XTT and MTT ELISAs, and fluorescent staining with propidium iodide. Bacterial strains of Staphylococcus aureus, Staphylococcus epidermidis and Pseudomonas aeruginosa were added to liquid media and incubated overnight. After determination of bacterial concentrations polyethylene (PE) devices were inoculated with bacteria for 48 h until biofilms formed. The devices were then washed, treated with antiseptics for 2 and 5 min and subsequently spread on agar plates. RESULTS: Sodium hypochlorite is more effective than chlorhexidine in penetrating biofilms of S. aureus, S. epidermidis and P. aeruginosa. Both antiseptics are chondrotoxic, but sodium hypochlorite damages human chondrocytes less than chlorhexidine in vitro. CONCLUSIONS: The findings confirm the effectiveness of sodium hypochlorite and chlorhexidine against bacterial biofilms. Both antiseptics can be recommended for the treatment of periprosthetic infections. The toxic effects of sodium hypochlorite and chlorhexidine towards chondrocytes may mean there is a risk of damage to cartilage tissue. LEVEL OF EVIDENCE: Controlled experimental study.

Dual antiretroviral therapy with tenofovir (TDF) and darunavir/ritonavir (DRV/RTV) in an HIV-1 positive patient: a case report, review, and meta-analysis of the literature on dual treatment strategies using protease inhibitors in combination with an NRTI.

BACKGROUND: Here, we report the case of an HIV positive patient under a dual antiretroviral drug regimen with tenofovir disoproxil and darunavir/ritonavir with stable clinical, virological, and immunological response over 126 weeks. Dual antiretroviral therapy has the advantage of reduced toxicity and lower health care costs, treatment failure and fostering drug resistance are perceived risks. Optimal drug combinations and indication criteria for dual treatment remain controversial. Nevertheless, first clinical trials indicate non-inferiority for combinations of nucleoside reverse transcriptase inhibitors and protease inhibitors. This case presents the combination of tenofovir disoproxil in combination with a protease inhibitor as a new potential dual treatment regimen. METHOD: We performed a systematic literature search and meta-analysis of trials comparing dual to triple ART. RESULTS: Literature review revealed nine studies in which dual therapy with a protease inhibitor and an NRTI was compared to triple therapy. We performed a meta-analysis of six trials that reported a 48-week follow-up. In treatment-naïve patients as well when ART switch was assessed, there was no difference in the treatment success in patients with dual ART versus triple. CONCLUSION: We conclude that dual therapy with a protease inhibitor and NRTI is safe and effective. The use of tenofovir in dual treatment as described in our case needs to be assessed in future clinical trials.

S. aureus endocarditis: Clinical aspects and experimental approaches.

Infective endocarditis (IE) is a life-threatening disease, caused by septic vegetations and inflammatory foci on the surface of the endothelium and the valves. Due to its complex and often indecisive presentation the mortality rate is still about 30%. Most frequently bacterial microorganisms entering the bloodstream are the underlying origin of the intracardiac infection. While the disease was primarily restricted to younger patients suffering from rheumatic heart streptococci infections, new at risk categories for Staphylococcus (S.) aureus infections arose over the last years. Rising patient age, increasing drug resistance, intensive treatment conditions such as renal hemodialysis, immunosuppression and long term indwelling central venous catheters but also the application of modern cardiac device implants and valve prosthesis have led to emerging incidences of S. aureus IE in health care settings and community. The aetiologic change has impact on the pathophysiology of IE, the clinical presentation and the overall patient management. Despite intensive research on appropriate in vitro and in vivo models of IE and gained knowledge about the fundamental mechanisms in the formation of bacterial vegetations and extracardiac complications, improved understanding of relevant bacterial virulence factors and triggered host immune responses is required to help developing novel antipathogenic treatment strategies and pathogen specific diagnostic markers. In this review, we summarize and discuss the two main areas affected by the changing patient demographics and provide first, recent knowledge about the pathogenic strategies of S. aureus in the induction of IE, including available experimental models of IE used to study host-pathogen interactions and diagnostic and therapeutic targets. In a second focus we present diagnostic (imaging) regimens for patients with S. aureus IE according to current guidelines as well as treatment strategies and surgical recommendations.

Virulence patterns of Staphylococcus aureus strains from nasopharyngeal colonization.

BACKGROUND: The prevalence of nasopharyngeal colonization with Staphylococcus aureus can reach 20-30% among the population, which can lead to invasive infection. AIM: To investigate the prevalence of colonization among different age groups, and analyse S. aureus strain-specific virulence patterns. METHOD: For analysis of the prevalence of colonization, groups consisting of newborns, healthy volunteers aged 5-60 years, and nursing home residents aged >80 years were examined with nasopharyngeal swabs. After S. aureus was cultured, genetic analysis and phenotypic virulence testing were performed by cell-based assays. FINDINGS: Among 924 volunteers, the overall colonization rate was approximately 30%, with a peak in subjects aged 5-10 years (49%). Neonates and subjects aged >80 years showed different distributions of clonal clusters. Overall, the strains of all age groups exhibited virulence characteristics that can contribute to the development of infection. In particular, the neonatal strains exhibited a high incidence of toxin genes that resulted in increased cytotoxic effects compared with the other strains tested. CONCLUSIONS: Colonizing strains showed a virulence profile in all age groups, which may lead to the establishment of invasive infection. Consequently, decolonization measures could be considered for selected patients depending on the risk of infection.

Cardiac 4D phase-contrast CMR at 9.4 T using self-gated ultra-short echo time (UTE) imaging.

BACKGROUND: Time resolved 4D phase contrast (PC) cardiovascular magnetic resonance (CMR) in mice is challenging due to long scan times, small animal ECG-gating and the rapid blood flow and cardiac motion of small rodents. To overcome several of these technical challenges we implemented a retrospectively self-gated 4D PC radial ultra-short echo-time (UTE) acquisition scheme and assessed its performance in healthy mice by comparing the results with those obtained with an ECG-triggered 4D PC fast low angle shot (FLASH) sequence. METHODS: Cardiac 4D PC CMR images were acquired at 9.4 T in healthy mice using the proposed self-gated radial center-out UTE acquisition scheme (TE/TR of 0.5 ms/3.1 ms) and a standard Cartesian 4D PC imaging sequence (TE/TR of 2.1 ms/5.0 ms) with a four-point Hadamard flow encoding scheme. To validate the proposed UTE flow imaging technique, experiments on a flow phantom with variable pump rates were performed. RESULTS: The anatomical images and flow velocity maps of the proposed 4D PC UTE technique showed reduced artifacts and an improved SNR (left ventricular cavity (LV): 8.9 ± 2.5, myocardium (MC): 15.7 ± 1.9) compared to those obtained using a typical Cartesian FLASH sequence (LV: 5.6 ± 1.2, MC: 10.1 ± 1.4) that was used as a reference. With both sequences comparable flow velocities were obtained in the flow phantom as well as in the ascending aorta (UTE: 132.8 ± 18.3 cm/s, FLASH: 134.7 ± 13.4 cm/s) and pulmonary artery (UTE: 78.5 ± 15.4 cm/s, FLASH: 86.6 ± 6.2 cm/s) of the animals. Self-gated navigator signals derived from information of the oversampled k-space center were successfully extracted for all animals with a higher gating efficiency of time spent on acquiring gated data versus total measurement time (UTE: 61.8 ± 11.5%, FLASH: 48.5 ± 4.9%). CONCLUSIONS: The proposed self-gated 4D PC UTE sequence enables robust and accurate flow velocity mapping of the mouse heart in vivo at high magnetic fields. At the same time SNR, gating efficiency, flow artifacts and image quality all improved compared to the images obtained using the well-established, ECG-triggered, 4D PC FLASH sequence.

Study on the utility of a statewide counselling programme for improving mortality outcomes of patients with Staphylococcus aureus bacteraemia in Thuringia (SUPPORT): a study protocol of a cluster-randomised crossover trial.

INTRODUCTION: Staphylococcus aureus bacteraemia (SAB) is a frequent infection with high mortality rates. It requires specific diagnostic and therapeutic management such as prolonged intravenous administration of antibiotics and aggressive search for and control of infectious sources. Underestimation of disease severity frequently results in delayed or inappropriate management of patients with SAB leading to increased mortality rates. According to observational studies, patient counselling by infectious disease consultants (IDC) improves survival and reduces the length of hospital stay as well as complication rates. In many countries, IDC are available only in some tertiary hospitals. In this trial, we aim to demonstrate that the outcome of patients with SAB in small and medium size hospitals that do not employ IDC can be improved by unsolicited ID phone counselling. The SUPPORT trial will be the first cluster-randomised controlled multicentre trial addressing this question. METHODS AND ANALYSIS: SUPPORT is a single-blinded, multicentre interventional, cluster-randomised, controlled crossover trial with a minimum of 15 centres that will include 250 patients with SAB who will receive unsolicited IDC counselling and 250 who will receive standard of care. Reporting of SAB will be conducted by an electronic real-time blood culture registry established for the German Federal state of Thuringia (ALERTSNet) or directly by participating centres in order to minimise time delay before counselling. Mortality, disease course and complications will be monitored for 90 days with 30-day all-cause mortality rates as the primary outcome. Generalised linear mixed modelling will be used to detect the difference between the intervention sequences. We expect improved outcome of patients with SAB after IDC. ETHICS AND DISSEMINATION: We obtained ethics approval from the Ethics committee of the Jena University Hospital and from the Ethics committee of the State Chamber of Physicians of Thuringia. Results will be published in a peer-reviewed journal and additionally disseminated through public media. TRIAL REGISTRATION NUMBER: DRKS00010135.

Evaluation of loop-mediated isothermal amplification for the rapid identification of bacteria and resistance determinants in positive blood cultures.

The use of molecular assays to rapidly identify pathogens and resistance genes directly from positive blood cultures (BCs) contribute to shortening the time required for the diagnosis of bloodstream infections. In this work, loop-mediated isothermal amplification (LAMP) assays have been examined for their potential use in BC diagnosis. Three different assays were applied. The commercially available eazyplex® MRSA test detects Staphylococcus aureus, S. epidermidis, mecA, and mecC. Two in-house assays [Gram-positive (GP) and Gram-negative (GN)] have been developed for the detection of streptococci, enterococci, vanA, vanB, Pseudomonas spp., Enterobacteriaceae, and the bla CTX-M family. A total of 370 positive BCs were analyzed. LAMP test results were obtained within 30 min, including sample preparation. Amplification was measured by real-time fluorescence detection. The threshold time for fluorescence intensity values ranged from 6.25 to 13.75 min. The specificity and sensitivity of the assays varied depending on the target. Overall, from 87.7% of BCs, true-positive results were obtained, compared to routine standard diagnosis. Twenty-one tests were true-negative because of the lack of an appropriate target (5.7%). The concordance of positive test results for resistance genes with subsequent antibiotic susceptibility testing was 100%. From 15 BC bottles with mixed cultures, eazyplex® assays produced correct results in 73% of the cases. This study shows that LAMP assays are fast and cost-saving tools for rapid BC testing in order to expedite the diagnostic report and improve the antibiotic stewardship for sepsis patients.

Post-invasion events after infection with Staphylococcus aureus are strongly dependent on both the host cell type and the infecting S. aureus strain.

Host cell invasion is a major feature of Staphylococcus aureus and contributes to infection development. The intracellular metabolically active bacteria can induce host cell activation and death but they can also persist for long time periods. In this study a comparative analysis was performed of different well-characterized S. aureus strains in their interaction with a variety of host cell types. Staphylococcus aureus (strains 6850, USA300, LS1, SH1000, Cowan1) invasion was compared in different human cell types (epithelial and endothelial cells, keratinocytes, fibroblasts, osteoblasts). The number of intracellular bacteria was determined, cell inflammation was investigated, as well as cell death and phagosomal escape of bacteria. To explain strain-dependent differences in the secretome, a proteomic approach was used. Barrier cells took up high amounts of bacteria and were killed by aggressive strains. These strains expressed high levels of toxins, and possessed the ability to escape from phagolysosomes. Osteoblasts and keratinocytes ingested less bacteria, and were not killed, even though the primary osteoblasts were strongly activated by S. aureus. In all cell types S. aureus was able to persist. Strong differences in uptake, cytotoxicity, and inflammatory response were observed between primary cells and their corresponding cell lines, demonstrating that cell lines reflect only partially the functions and physiology of primary cells. This study provides a contribution for a better understanding of the pathomechanisms of S. aureus infections. The proteomic data provide important basic knowledge on strains commonly used in the analysis of S. aureus-host cell interaction.

Should daptomycin-rifampin combinations for MSSA/MRSA isolates be avoided because of antagonism?

PURPOSE: There is increasing clinical evidence from observational studies, that combination therapy of daptomycin with rifampin is a valuable treatment option for biofilm-associated difficult to treat Staphylococcus aureus infections such as osteomyelitis, prosthetic joint infection and endocarditis. However, two studies analyzing a limited number of S. aureus isolates reported an antagonism of those two drugs questioning the benefit of this combination. METHODS: To estimate the frequency of this possible antagonism, we performed in vitro checkerboard assays on 58 consecutive clinical isolates of S. aureus (MSSA n = 9, MRSA n = 49). We determined the fractional inhibitory concentration index (FICI) and the susceptible breakpoint index (SBPI). All isolates were characterized by a microprobe array detecting 336 different genes/alleles to ensure their non-clonal origin. RESULTS: For all isolates, the FICI was between 1.00 and 1.25 indicating additive effects for the daptomycin/rifampin combination. Neither antagonism nor synergism as defined by the FICI was found for any of the isolates. CONCLUSION: Based on these data, there is no evidence to advise against the daptomycin/rifampin combination therapy.

Staphylococcus aureus develops increased resistance to antibiotics by forming dynamic small colony variants during chronic osteomyelitis.

OBJECTIVES: Staphylococcus aureus osteomyelitis often develops to chronicity despite antimicrobial treatments that have been found to be susceptible in in vitro tests. The complex infection strategies of S. aureus, including host cell invasion and intracellular persistence via the formation of dynamic small colony variant (SCV) phenotypes, could be responsible for therapy-refractory infection courses. METHODS: To analyse the efficacy of antibiotics in the acute and chronic stage of bone infections, we established long-term in vitro and in vivo osteomyelitis models. Antibiotics that were tested include ß-lactams, fluoroquinolones, vancomycin, linezolid, daptomycin, fosfomycin, gentamicin, rifampicin and clindamycin. RESULTS: Cell culture infection experiments revealed that all tested antibiotics reduced bacterial numbers within infected osteoblasts when treatment was started immediately, whereas some antibiotics lost their activity against intracellular persisting bacteria. Only rifampicin almost cleared infected osteoblasts in the acute and chronic stages. Furthermore, we detected that low concentrations of gentamicin, moxifloxacin and clindamycin enhanced the formation of SCVs, and these could promote chronic infections. Next, we treated a murine osteomyelitis model in the acute and chronic stages. Only rifampicin significantly reduced the bacterial load of bones in the acute phase, whereas cefuroxime and gentamicin were less effective and gentamicin strongly induced SCV formation. During chronicity none of the antimicrobial compounds tested showed a beneficial effect on bone deformation or reduced the numbers of persisting bacteria. CONCLUSIONS: In all infection models rifampicin was most effective at reducing bacterial loads. In the chronic stage, particularly in the in vivo model, many tested compounds lost activity against persisting bacteria and some antibiotics even induced SCV formation.

[Persistent and recurrent skin and soft tissue infections by Staphylococcus aureus. Impact of the small colony-variant (SCV) phenotype and of Panton-Valentine leukocidin (PVL)-positive S. aureus isolates]. / Chronisch rezidivierende Infektionen der Haut und Weichgewebe durch Staphylococcus aureus. Klinische Bedeutung des Small-colony-variant (SCV)-Phänotyps und von Panton-Valentine-Leukozidin (PVL)-positiven S.-aureus-Isolaten.

Staphylococcus aureus is one of the major pathogens causing chronic skin and soft tissue infections. Particularly isolates producing Panton-Valentine leukocidin (PVL) comprising methicillin-susceptible and community-associated methicillin-resistant S. aureus (CA-MRSA) have been associated with more aggressive and persistent or relapsing courses. Beyond classical resistance mechanisms, functional resistance as shown by the small colony-variant (SCV) phenotype could be also responsible for treatment failures, despite the administration of antibiotics tested in vitro as susceptible. Also this phenotype has been associated with chronic courses of infections often with multiple exacerbations. Due to their ability to persist intracellularly, SCVs are protected from host defense and antibiotic treatment if only extracellularly active agents are administered. Reduced growth, abnormal colony morphology and changes in the metabolism of the SCVs aggravate drastically their identification, differentiation and susceptibility testing. The diagnostic and therapeutic challenges of PVL-positive and SCV isolates necessitate close collaboration with microbiological and infectious disease specialists.

[Incidents of malfunction of mechanical heart valves as reported to the German Medical Devices Vigilance System]. / Vorkommnismeldungen bei mechanischen Herzklappenprothesen. Auswertung des deutschen Medizinprodukte-Beobachtungs- und Meldesystems im Bundesinstitut für Arzneimittel und Medizinprodukte.

BACKGROUND: The purpose of this study was to collect the experience gained from risk assessment of incidents associated with mechanical heart valves as reported to the German Medical Devices Vigilance System. METHODS: All events in connection with the use of prosthetic heart valves which had been reported to the Federal Institute for Drugs and Medical Products were identified in the Institute's anonymized database. Subsequently all information that had been obtained and the reports of investigations by the various responsible manufacturer of medical products were retrospectively analysed. RESULTS: Of 53 incident reports the aortic valve position was involved 32 times, the mitral valve position 21 times. 17 cases of leaflet breakage, 12 of leaflet dysfunction, 6 of valve thrombosis, 4 of paravalvular leakage, 4 of damage to the suture ring, 3 of endocarditis and 7 of various other singular defects were reported. The outcome was mostly very serious (re-surgery, prolongation of surgery, death). Corrective actions taken by the manufacturers consisted of recalls, advisory notices and stoppage of production. CONCLUSIONS: The Medical Devices Vigilance System is able to detect product failures of mechanical heart valves and to initiate corrective action. As the reporting rate over more than 10 years is relatively low, it can be assumed that in general mechanical heart valves are safe medical devices. The quality of incident reporting by manufactures and users of medical devices should be improved to reduce the number of undetected and unsolved incidents.

Staphylococcus aureus induces caspase-independent cell death in human peritoneal mesothelial cells.

Bacterial peritonitis remains a serious complication of peritoneal dialysis. Although Staphylococcus epidermidis is the most common pathogen involved, infections with Staphylococcus aureus lead to severe peritoneal damage and are often associated with a dramatic loss of mesothelial cells. Induction of cell death appears to be involved in peritoneal damage and mesothelial cell loss during bacterial infections. Using cultured human peritoneal mesothelial cells (HMCs), we investigated the ability of different S. epidermidis and S. aureus strains to damage the HMC monolayer and to trigger cell death. We show that only a subgroup of live S. aureus isolates, characterized by an invasive and alpha-hemolysin-producing phenotype, induces cell death. None of the tested S. epidermidis strains, which were not invasive or hemolytic, had a cytotoxic effect. After host cell invasion, S. aureus resided within phagocytic vacuoles, and HMCs were apparently able to degrade staphylococci. However, even after prolonged infection, a high percentage of S. aureus remained alive within HMCs and might be released after host cell death. Cell death induced by S. aureus was accompanied by apoptotic alterations, such as DNA fragmentation, but was independent of endogenous FasL and tumor necrosis factor-alpha death ligand expression. Moreover, caspases were not involved in S. aureus-induced mesothelial cell death. In conclusion, our data indicate that mesothelial cell death might represent a major mechanism of S. aureus-induced damage of the peritoneum during bacterial peritonitis.

Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector.

Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B. Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C. difficile associated diarrhea. Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis. We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector. Ten clones analyzed contained the complete toxin A gene. Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA. Toxin A expression was confirmed by Western immunoblot analysis. The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.

Pharmacological regulation of postischemic sinusoidal diameters in rats--a new approach for reducing hepatic ischemia/reperfusion injury.

UNLABELLED: Ischemia leads to profound endothelin-related constriction of the hepatic microcirculation with resulting disturbances in blood and oxygen supply. The aim of the study was to modulate hepatic microvascular diameters by blocking endothelin receptors with bosentan, and also to find the best possible vessel width (as produced by bosentan) for minimizing ischemia/reperfusion injury. METHODS: In an in vivo rat model hepatic ischemia was induced for 30 minutes by crossclamping the hepatoduodenal ligament. The endothelin receptor antagonist (ERA) bosentan was administered before ischemia in stepwise dosages of 0.1, 1.0 and 10 mg/kg bw i.v. and 10 mg/kg bw intraportally (i.p.). Vasoactive effect was assessed by in vivo microscopy. The influence on hepatic oxygen supply and hepatocellular function was evaluated by measuring local tissue pO(2) and AST levels. RESULTS: Because of ischemia sinusoidal diameters were reduced to 76.3 +/- 7.4% compared with values found in sham-operated animals. After administration of 0.1 mg/kg ERA (bosentan) the sinusoids remained constricted (89.7 +/- 9.9%). Blocking endothelin receptors with 1 mg/kg bosentan avoided sinusoidal constriction (99.0 +/- 8.8%, p<0.05) and led to the most effective reduction of AST level peak after 6 h of reperfusion (244.0 +/- 34.2 U/l vs 422.9 +/- 163.3 U/l in untreated ischemia). 10 mg/kg i.v. caused an increase in sinusoidal diameter to 109.1 +/- 6.4% and 10 mg/kg intraportally to 136.8 +/- 19.3% and even an increase in AST levels (618.9 +/- 209.3 U/l). Hepatic ischemia led to a significant decrease of local tissue pO(2) after reperfusion (9.4 +/- 1.2 mm Hg; p<0.05 vs sham: 16.8 +/- 1.8 mm Hg). The greatest improvement in postischemic oxygen supply was found in the 1.0 mg/kg group (12.9 +/- 1.0 mm Hg; p< 0.05 vs ischemia). Venular diameter changed almost to the same extent as sinusoidal diameter. Perfusion rate was significantly increased and sticking of leukocytes in sinusoids and venules was reduced after doses of 1 and 10 mg/kg bw bosentan i.v. (p<0.05). IMPLICATIONS: In this model we were able to regulate the diameters of sinusoids and postsinusoidal venules incrementally. We conclude that the avoidance of constriction, without excessive vasodilatation gives increased perfusion rates with improved hepatic oxygen supply and hepatocellular function.

Role of the astrocytic ET(B) receptor in the regulation of extracellular endothelin-1 during hypoxia.

Astrocytes are known to possess an effective endothelin (ET) eliminatory system which involves astrocytic ET(A) and ET(B) receptors and may become particularly relevant under pathophysiological conditions. The present study has therefore been designed to explore the effect of standardized hypoxia on extracellular concentrations of endothelin-1 (ET-1) and on endothelin-converting enzyme (ECE) activity in primary rat astrocytes genetically (sl/sl) or experimentally (dexamethasone) deficient in ET(B) receptors. The results revealed (1) a hypoxia-mediated decrease of extracellular ET-1 in wildtype astrocytes (+/+) that was not observed in ET(B)-deficient (sl/sl) cultures; (2) an ET receptor antagonist-induced increase in ET-1 in the media of both genotypes with further elevation upon hypoxia in +/+ cultures only; (3) augmentation of the dexamethasone-induced increase in extracellular ET-1 by hypoxia in +/+, but not in sl/sl cultures; (4) synergistic reduction of ET(B) gene transcription by hypoxia and dexamethasone; and (5) significant increases in endothelin-converting enzyme activity in the presence of hypoxia. To conclude, hypoxia stimulates astrocytic release of mature ET-1. This stimulation is (over)compensated for by increased ET-1 binding to functional ET(B) receptors. ET(B) deficiency, whether genetic or experimentally induced, impairs elimination of extracellular ET-1.

Endothelin-converting enzyme inhibitors: current status and perspectives.

Recent research has led to the discovery of potent selective or mixed endothelin-converting enzyme (ECE), ECE/neutral endopeptidase24.11 (NEP) and ECE/NEP24.11/angiotensin-converting enzyme (ACE) inhibitors. There is also increasing evidence, that the functions of the endothelin (ET), renin-angiotensin and NEP systems for the regulation of the cardiovascular homeostasis are connected by a complex regulation network. It will be the challenging task of future research with the newly available selective and mixed ECE-1 inhibitors to show whether the combined inhibition of more than one cardiovascular system is superior to selective inhibition.

Effect of bosentan on NF-kappaB, inflammation, and tissue factor in angiotensin II-induced end-organ damage.

Reports on the effectiveness of endothelin receptor blockers in angiotensin (Ang) II-induced end-organ damage are conflicting, and the mechanisms involved are uncertain. We tested the hypothesis that endothelin (ET)(A/B) receptor blockade with bosentan (100 mg/kg by gavage after age 4 weeks) ameliorates cardiac and renal damage by decreasing inflammation in rats harboring both human renin and angiotensinogen genes (dTGR). Furthermore, we elucidated the effect of bosentan on tissue factor (TF), which is a key regulator of the extrinsic coagulation cascade. We compared bosentan with hydralazine (80 mg/L in the drinking water for 3 weeks) as a blood pressure control. Untreated dTGR featured hypertension, focal necrosis in heart and kidney, and a 45% mortality rate (9 of 20) at age 7 weeks. Compared with Sprague-Dawley controls, both systolic blood pressure and 24-hour albuminuria were increased in untreated dTGR (203+/-8 versus 111+/-2 mm Hg and 67.1+/-8.6 versus 0.3+/-0.06 mg/d at week 7, respectively). Bosentan and hydralazine both reduced blood pressure and cardiac hypertrophy. Mortality rate was markedly reduced by bosentan (1/15) and partially by hydralazine (4/15). However, only bosentan decreased albuminuria and renal injury. Untreated and hydralazine-treated dTGR showed increased nuclear factor (NF)-kappaB and AP-1 expression in the kidney and heart; the p65 NF-kappaB subunit was increased in the endothelium, vascular smooth muscles cells, infiltrating cells, glomeruli, and tubules. In the heart and kidney, ET(A/B) receptor blockade inhibited NF-kappaB and AP-1 activation compared with hydralazine treatment. Macrophage infiltration, ICAM-1 expression, and the integrin expression on infiltrating cells were markedly reduced. Renal vasculopathy was accompanied by increased tissue factor expression on macrophages and vessels of untreated and hydralazine-treated dTGR, which was markedly reduced by bosentan. Thus, ET(A/B) receptor blockade inhibits NF-kappaB and AP-1 activation and the NF-kappaB- and/or AP-1-regulated genes ICAM-1, VCAM-1, and TF, independent of blood pressure-related effects. We conclude that Ang II-induced NF-kappaB and AP-1 activation and subsequent inflammation and coagulation involve at least in part the ET(A/B) receptors.

Serial CEA and CA 15-3 measurements during follow-up of breast cancer patients.

OBJECTIVE: CEA and CA 15-3 are adequate parameters for the early diagnosis of metastases in breast cancer patients. During the last years serial marker measurements have been discussed controversely and moreover accused of frightening breast cancer patients in the sense of a tumor marker terror. Therefore we evaluated the acceptance and practicability of an intense follow-up program including monthly measurements of CEA and CA 15-3. MATERIALS AND METHODS: Between 1985 and 1995. 547 breast cancer patients were asked to give blood samples every four weeks for CEA and CA 15-3 measurements. In a field research study using a half-standardized interview by telephone we evaluated the acceptance of serial marker measurements in defined aspects: personal acceptance, blood sampling intervals, form of information, role of the home practitioner, motivation. RESULTS: 280 patients could be analyzed for the acceptance of this follow-up program. 52% of this patient group accepted serial tumor marker measurements, while 48% did not. 99% of the participating patients agreed to monthly blood sampling intervals compared to only 41% of the non-participating patients. 64% of the analyzed patients would have preferred both written and personal information about the role of CEA and CA 15-3 and this kind of follow-up program. Only in 6% of the participating patients the home practitioner had a negative attitude towards monthly tumor marker measurements compared to 36% in non-participating patients. Moreover serial marker measurements led to a feeling of security and reassurance in 85% of the patients. CONCLUSION: CEA and CA 15-3 measurements are adequate and accepted instruments for the follow-up and early diagnosis of metastases in breast cancer patients. If patients got detailed information and support from their home practitioner, serial CEA and CA 15-3 measurements could lead to a feeling of security in most of the patients.

Pharmacology of tezosentan, new endothelin receptor antagonist designed for parenteral use.

Tezosentan (Ro 61-0612) [5-isopropyl-pyridine-2-sulfonic acid 6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2-(2-1H-tetrazol-5-yl-+ ++pyri din-4-yl)-pyrimidin-4-ylamide] is a new endothelin (ET) receptor antagonist specifically designed for parenteral use. Tezosentan competitively antagonizes the specific binding of (125)I-labeled ET-1 and of the selective ET(B) receptor ligands (125)I-labeled ET-3 and (125)I-labeled sarafotoxin S6c on cells and tissues carrying ET(A) and ET(B) receptors, with inhibitory constants in the nanomolar range, and has high water solubility. Tezosentan exhibits high functional inhibitory potency for inhibiting contraction induced by ET-1 on isolated rat aorta (ET(A) receptors; pA(2) = 9.5) and by sarafotoxin S6c on rat trachea (ET(B) receptors; pA(2) = 7.7). In vivo, tezosentan inhibits the pressor effect of big ET-1 in pithed rats and increases ET-1 plasma concentrations in conscious rats in a dose-dependent fashion. In spontaneously hypertensive rats, i.v. injection of tezosentan has acute hemodynamic effects and decreases blood pressure. Tezosentan is also able to prevent the acute renal failure that complicates rhabdomyolysis in a rat model of myoglobinuric nephropathy. Finally, tezosentan exhibits an apparent elimination half-life of less than 1 h in rabbits and primates and of 2 h in rats. In conclusion, tezosentan, a potent mixed ET receptor antagonist with a short half-life, may offer a novel medical approach for the i.v. treatment of acute pathological conditions.