Oral health-related quality of life depending on dental and periodontal health in different patients before and after liver transplantation.

BACKGROUND: The aim of this study was to evaluate the oral health-related quality of life (OHRQoL) depending on dental and periodontal health of different patients before and after liver transplantation (pre- and postLTx) compared to a healthy control group (HC). METHODS: OHRQoL was rated using the German short form of Oral Health Impact Profile (OHIP G14). To estimate dental health, the decayed (D-T), missing (M-T), and filled (F-T) teeth index (DMF-T) was used. Periodontal health was classified as healthy/mild, moderate, or severe periodontitis. The following statistics are used: Mann-Whitney U test, Kruskal-Wallis test, chi-square test, and Fisher test (α = 5%). RESULTS: A total of 24 preLTx, 47 postLTx, and 75 HC patients were included. Significant differences in DMF-T, D-T, M-T, and F-T scores were detected between groups (p < 0.001). Prevalence of periodontitis was comparable between groups (p = 0.340). OHRQoL was reduced in pre- and postLTx (OHIP G14 preLTx 4.2 [1.5; 0-4.0], postLTx 4.1 [1; 0-5.0], HC 1.4 [0; 0-2.0]; p = 0.003), without associations to their oral status (p > 0.05). CONCLUSIONS: These preliminary findings show a reduced OHRQoL without associations to their oral status, which might indicate an influence of potential disease-related factors on OHRQoL. Further studies with larger groups are necessary to verify this observation. CLINICAL RELEVANCE: A special dental care of patients before and after LTx is needed, including a comprehensive assessment of the individual patient's OHRQoL.

Periodontal pathogenic bacteria and aMMP-8 findings depending on periodontal conditions of patients before and after liver transplantation.

BACKGROUND: The aim of this single-center cross-sectional study was to detect the prevalence of selected periodontal pathogenic bacteria and active matrix metalloproteinase-8 (aMMP-8) level in patients before (preLTx) and after liver transplantation (postLTx). METHODS: Periodontal pocket depth (PPD) and clinical attachment loss (CAL) were assessed. Subgingival biofilm samples were analyzed using polymerase chain reaction (PCR) to detect 11 common periodontal pathogens. Gingival crevicular fluid (GCF) samples were analyzed with enzyme-linked immunosorbent assay (ELISA) to determine aMMP-8 level and assigned to a scoring system: score 0: 0-8 ng/ml, score 1: 8-20 ng/ml, and score 2: >20 ng/ml. The following were used for the statistical analysis: t test, Mann-Whitney U test, Fishers test (α = 5 %). RESULTS: In total, 110 patients (preLTx: n = 35, postLTx: n = 75) could be included in the study. Periodontal findings were not significantly different between groups. In microbiological analysis, a significantly higher prevalence of Campylobacter rectus in preLTx group was detected (p = 0.03). Significantly more patients with score 0 in postLTx group (p = 0.024) and significantly more patients with score 1 in preLTx group were found (p = 0.004). Furthermore, aMMP-8 concentrations for patients with moderate periodontitis were significantly lower in postLTx group compared to preLTx group (p = 0.045). Additionally, in postLTx group, aMMP-8 concentration was significantly higher in patients with severe periodontitis compared to those with no/mild periodontitis (p = 0.016). CONCLUSION: LTx appears to affect aMMP-8 level, but not bacterial findings in patients after LTx. CLINICAL RELEVANCE: Determination of aMMP-8 level in patients after LTx with immunosuppressive medication might lead to wrong interpretation of the results.

Oral behavior, dental, periodontal and microbiological findings in patients undergoing hemodialysis and after kidney transplantation.

BACKGROUND: Aim of this single center cross-sectional study was to investigate oral behavior, dental, periodontal and microbiological findings in patients undergoing hemodialysis (HD) and after kidney transplantation (KT). METHODS: Patients undergoing HD for end-stage renal failure and after KT were investigated. Oral health behavior was recorded using a standardized questionnaire, e.g. dental behavior, tooth brushing, oral hygiene aids. Oral investigation included screening of oral mucosa, dental findings (DMF-T) and periodontal situation (Papilla bleeding index [PBI] periodontal probing depth [PPD] and clinical attachment loss [CAL]). Additionally, microbiological analysis of subgingival biofilm samples (PCR) was performed. STATISTICAL ANALYSIS: Student's t-test or Mann-Whitney-U-test, Fisher's exact test (α = 5 %). RESULTS: A total of 70 patients (HD: n = 35, KT: n = 35) with a mean age of 56.4 ± 11.1 (HD) and 55.8 ± 10.9 (KT) years were included. Lack in use of additional oral hygiene (dental floss, inter-dental brush) was found. KT group presented significantly more gingivial overgrowth (p = 0.01). DMF-T was 19.47 ± 5.84 (HD) and 17.61 ± 5.81 (KT; p = 0.21). Majority of patients had clinically moderate and severe periodontitis; showing a need for periodontal treatment of 57 % (HD) and 71 % (KT; p = 0.30). Significantly higher prevalence of Parvimonas micra and Capnocytophaga species in the HD group were found (p < 0.01). CONCLUSION: Periodontal treatment need and lack in oral behavior for both groups indicate the necessity of an improved early treatment and prevention of dental and periodontal disease, e.g. in form of special care programs. Regarding microbiological findings, no major differences between KT and HD patients were found.

Distinct patterns of leukocyte recruitment in the pulmonary microvasculature in response to local and systemic inflammation.

The mechanisms of leukocyte recruitment in the pulmonary microvasculature in response to local and systemic inflammation remain elusive. Male C57BL/6 mice received lipopolysaccharide (LPS) intrapulmonary (intratracheally, it) or systemically (intravenously, iv) for 1-18 h. Leukocyte responses in lung were analyzed by use of intravital fluorescence microscopy. Plasma and lung levels of CXC chemokines as well as Mac-1 and F-actin expression in leukocytes and bronchoalveolar leukocytes were quantified. Venular leukocyte rolling was markedly increased in response to local LPS but only marginally after systemic LPS. Leukocyte adhesion in venules was enhanced in both groups although adhesion was higher in mice receiving LPS intratracheally compared with LPS intravenously. Systemic LPS caused more leukocytes trapping in capillaries compared with local LPS. The ratio of adherent leukocytes in venules compared with capillaries was higher in response to local LPS, suggesting that leukocytes were more prone to accumulate in venules in local inflammation and in capillaries in systemic inflammation. Systemic LPS triggered higher F-actin formation and Mac-1 expression in leukocytes compared with local LPS. Local and systemic LPS caused similar increases in CXC chemokines in the lung whereas intravenous endotoxin provoked higher levels of CXC chemokines in the circulation. Interestingly, intratracheal LPS increased recruitment of leukocytes in the alveolar space whereas intravenous LPS was ineffective in promoting leukocyte accumulation in the bronchoalveolar space. In conclusion, our data demonstrate that pulmonary microvascular recruitment of leukocytes differs in local and systemic inflammation, which might be related to premature activation and stiffening of circulating leukocytes in endotoxemia.

Digital droplet PCR for rapid quantification of donor DNA in the circulation of transplant recipients as a potential universal biomarker of graft injury.

BACKGROUND: Cell-free DNA (cfDNA) from grafts in the circulation of transplant recipients is a potential biomarker of rejection. Its usefulness was investigated after heart transplantation during the maintenance phase by use of microarrays and massive parallel sequencing of donor and recipient DNA. Disadvantages of these methods are high costs, long turnaround times, and need for donor DNA. Therefore, we sought to develop a rapid and cost-effective method using digital droplet PCR (ddPCR). METHODS: Plasma samples were collected from stable recipients after liver (LTx, n = 10), kidney (KTx, n = 9), and heart (HTx, n = 8) transplantation as well as from 7 additional patients directly after LTx. Known single-nucleotide polymorphisms were selected for high minor allelic frequencies, of which 41 hydrolysis probe assays were established. Plasma cfDNA was preamplified, followed by conventional real-time PCR to define informative (heterologous) SNPs, which were then used for quantification (percentage) of graft-derived cfDNA (GcfDNA) using ddPCR. RESULTS: Mean recovery was 94% (SD, 13%) with an imprecision of 4%-14% with the use of controls with 2% minor allele. GcfDNA in stable patients was <6.8% (LTx), <2.5% (KTx), and <3.4% (HTx). On the day of LTx, GcfDNA was approximately 90% and by day 10 it was <15% in complication-free LTx recipients. In 2 patients with biopsy-proven rejection, GcfDNA increased to >60%, whereas in 1 patient with cholestasis no increase was found. CONCLUSIONS: A novel, cost-effective, rapid technique was developed to quantify GcfDNA in transplant recipients. This technique embodies a promising, potentially universal biomarker for early detection of rejection, which could enable more effective therapeutic interventions.

Popular belief meets surgical reality: impact of lunar phases, Friday the 13th and zodiac signs on emergency operations and intraoperative blood loss.

BACKGROUND: The influence of superstition, moon calendars, and popular belief on evidence-based medicine is stunning. More than 40% of medical staff is convinced that lunar phases can affect human behavior. The idea that Friday the 13th is associated with adverse events and bad luck is deep-rooted in the population of Western industrial countries. The aim of the present study was to test the hypothesis that these myths are transferable to real-life surgery. METHODS: We analyzed the extent to which moon phases, zodiac signs, and Friday the 13th influence blood loss, emergency frequency, and intestinal perforations by evaluating the operation records of all 27,914 consecutive patients of our institution undergoing general, visceral, or vascular surgery between August 2001 and August 2010. Dates of surgery were allocated to lunar phases and to zodiac signs, as well as to Friday the 13th. RESULTS: A total of 111 lunar cycles and 15 Fridays the 13th occurred within the 3,281-day observation period. Patients' characteristics did not differ in lunar phases, zodiac signs, or Fridays the 13th. Full moon phases, the presence of Friday the 13th, and zodiac signs influenced neither intraoperative blood loss nor emergency frequency. No statistical peaks regarding perforated aortic aneurysms and gastrointestinal perforations were found on full moon or Friday the 13th. CONCLUSIONS: Scientific analysis of our data does not support the belief that moon phases, zodiac signs, or Friday 13th influence surgical blood loss and emergency frequency. Our data indicate that such beliefs are myths far beyond reality.

Simvastatin attenuates hepatic sensitization to lipopolysaccharide after partial hepatectomy.

BACKGROUND: Hepatic resection may be curative in patients with hepatobiliary malignancies but excessive loss of liver volume may cause hepatic dysfunction and increase susceptibility to subsequent postoperative infections and sepsis. Herein, we hypothesized that pretreatment with simvastatin may protect against lipopolysaccharide (LPS)-induced liver damage after partial hepatectomy. MATERIALS AND METHODS: Male C57Bl/6 mice underwent 68% hepatectomy and exposed to LPS after 24h. Animals were pretreated with simvastatin (0.02 and 0.2mg/kg). Serum alanine aminotransferase (ALT) and tumor necrosis factor-alpha (TNF-alpha) as well as leukocyte infiltration and hepatocyte apoptosis were examined 6h after LPS challenge. An in vitro endothelial cell adhesion assay was used to study the mechanisms of simvastatin on leukocyte adhesion and the role of P-selectin and lymphocyte function antigen-1 (LFA-1). RESULTS: Partial hepatectomy increased the sensitivity of the remnant liver tissue to LPS-provoked tissue injury. Simvastatin pretreatment reduced the LPS-induced increase in serum levels of ALT by 65% in hepatectomized animals. Moreover, simvastatin decreased leukocyte infiltration and hepatocyte apoptosis in the liver remnant of endotoxemic mice. LPS-provoked serum levels of TNF-alpha were decreased by 83% in hepatectomized animals treated with simvastatin. TNF-alpha-induced leukocyte adhesion as well as P-selectin expression in endothelial cells and LFA-1 function were inhibited by simvastatin in vitro. CONCLUSIONS: These novels findings demonstrate that simvastatin protects the remnant liver against endotoxemic injury following major hepatectomy. Thus, simvastatin reduced LPS-induced leukocyte recruitment and hepatocyte apoptosis in the liver remnant. One key mechanism appears to be related to the inhibition of TNF-alpha formation and function (P-selectin expression) as well as to direct actions on LFA-1 function. Thus, simvastatin may represent a novel therapeutic approach to prevent septic liver damage after partial liver resection.

Inhibition of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase reduces leukocyte recruitment and hepatocyte apoptosis in endotoxin-induced liver injury.

BACKGROUND: Endotoxemia is well known to be associated with an excessive host response to bacteria or microbial compounds, resulting in systemic inflammation and organ injury. The aim of the present study was to examine the effects of simvastatin on endotoxemic liver injury. METHODS: Male C57BL/6J mice were challenged intraperitoneally with 0.5 mg/kg Escherichia coli-lipopolysaccharide (LPS) and 0.9 g/kg d-galactosamine (Gal). Mice were pretreated with 0.2 mg/kg simvastatin. Lipopolysaccharide/d-Gal-injected mice without simvastatin served as endotoxemic controls, and sham mice served as negative controls. Additional mice were challenged with LPS/d-Gal and co-treated with simvastatin and 10 mg/kg mevalonate to determine the role of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase. After 6 hours of endotoxemia serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities as well as caspase-3 and myeloperoxidase activity were determined. RESULTS: Endotoxemia caused a substantial hepatocellular injury as indicated by significantly elevated serum ALT and AST levels and hepatocellular apoptosis. Leukocyte infiltration in the liver was significantly elevated in endotoxemic mice. Simvastatin significantly reduced endotoxin-induced hepatocellular damage and apoptosis. Moreover, hepatic accumulation of leukocytes was attenuated by simvastatin in endotoxemic animals. Co-administration of mevalonate abolished protective effects of simvastatin on endotoxin-provoked increases in ALT, AST, and hepatocellular apoptosis as well as leukocyte recruitment. CONCLUSIONS: Simvastatin has the capacity to prevent endotoxemic liver injury by inhibiting leukocyte infiltration and hepatocellular apoptosis. These protective effects exerted by simvastatin are dependent on the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase pathway. Thus, simvastatin may represent a potential approach to prevent endotoxemia-associated liver dysfunction.

Primary and secondary capture of platelets onto inflamed femoral artery endothelium is dependent on P-selectin and PSGL-1.

Platelets constitute a key role in vascular injuries, however, the detailed mechanisms behind platelet-endothelial cell and platelet-leukocyte interactions in the femoral artery are not yet fully elucidated. We used intravital fluorescence microscopy of the femoral artery in C57BL/6 mice to study primary and secondary capture of platelets onto endothelial cells as well as onto adherent platelets and leukocytes in vivo. By use of monoclonal antibodies, the role of P-selectin and P-selectin glycoprotein ligand 1 (PSGL-1) in these adhesive interactions in mice exposed to endotoxin was determined. Intravenous injection of endotoxin significantly increased gene expression of P-selectin as well as platelet tethering, rolling and adhesion in the femoral artery. Pretreatment with the anti-PSGL-1 antibody decreased platelet tethering by 85%, platelet rolling by 88% and platelet adhesion by 96%. Immunoneutralization of P-selectin reduced platelet tethering by 91%, platelet rolling by 98%, and platelet adhesion by 97%. In addition, inhibition of P-selectin and PSGL-1 completely abolished secondary capture of platelets onto adherent platelets and leukocytes. Our data show that P-selectin and PSGL-1 mediate early interactions between platelets and other cells, including endothelial cells and leukocytes, in inflamed arteries. These novel results suggest that interference with P-selectin and PSGL-1 may be a useful target in strategies aiming to protect the vascular wall during arterial inflammation.

Central role of rho kinase in lipopolysaccharide-induced platelet capture on venous endothelium.

BACKGROUND: Platelet-endothelial cell interactions play a key role in hemostasis and pathological coagulation and are dependent on different surface molecules that are expressed upon activation (eg, mediated by lipopolysaccharide [LPS]). Recently, we have shown that Rho kinase plays a central role in LPS-mediated leukocyte-endothelial cell interactions. We investigated the role of Rho-kinase in inflammatory interactions between platelets and the endothelium in femoral veins in vivo. METHODS: Mice were injected intravenously with LPS (0.5 mg/kg)/D-galactosamine (900 mg/kg), and Rho kinase was blocked with fasudil 15 minutes before LPS application. Four hours after LPS administration, intravital fluorescence microscopy of the femoral vein was performed, and primary and secondary platelet-endothelial cell interactions were visualized after in vivo platelet staining with rhodamine 6G. RESULTS: Intravital microscopy showed a significant increase in platelet tethering, rolling, and firm adhesion as well as platelet secondary capture in LPS-treated mice. Rho-kinase inhibition by fasudil significantly reduced platelet tethering, rolling, and firm adhesion. Interestingly, functional blockade of Rho kinase was also able to diminish secondary platelet capture by 79%. CONCLUSIONS: From our data, we conclude that Rho-kinase signaling plays a central role in the regulation of LPS-induced platelet-endothelial cell interactions in large veins in vivo. Thus, Rho-kinase inhibition might be useful in prevention or treatment of pathological inflammation and endotoxin-mediated hypercoagulation.

Post-hypoxic cellular disintegration in glycine-preserved renal tubules is attenuated by hydroxyl radical scavengers and iron chelators.

BACKGROUND: Cellular stress during reoxygenation is a common phenomenon in solid organ transplantation and is characterized by production of reactive oxygen species. Herein, we studied in isolated tubular segments of rat kidney cortex the impact of oxygen radical scavengers and an iron chelator on post-hypoxic recovery. METHODS: Tubules, suspended in Ringer's solution containing 5 mM glycine, underwent 30 min hypoxia and 60 min reoxygenation. Untreated tubules served as controls. Hypoxia-reoxygenation injury was measured by membrane leakage, lipid peroxidation and cellular functions. In hypoxia-reoxygenated-isolated tubular segments, protective effects of different scavengers and of the iron chelator deferoxamine on hypoxia-reoxygenation injury were analyzed. RESULTS: Scavengers protected isolated tubular segments from hypoxia-reoxygenation-induced cellular disintegration and dysfunction. Deferoxamine was found to exert the most distinct protection. It was further found to exert a dose-dependent protection on hypoxia-reoxygenation damage in isolated tubular segments, which was critically mediated by chelating tissue and bond iron. CONCLUSIONS: Our data demonstrate that radical scavengers effectively protect from hypoxia-reoxygenation injury in isolated tubular segments and that the iron chelator deferoxamine is especially a potent inhibitor of iron ion-mediated hypoxia-reoxygenation damage. Thus, inclusion of this iron chelator in organ storage solutions might improve post-transplant organ function and protect from reperfusion injury.

Protective effect of fasudil, a Rho-kinase inhibitor, on chemokine expression, leukocyte recruitment, and hepatocellular apoptosis in septic liver injury.

Rho-kinase signaling regulates important features of inflammatory reactions. Herein, we investigated the effect and mechanisms of action of the Rho-kinase inhibitor fasudil in endotoxemic liver injury. C57/BL/6 mice were challenged with lipopolysaccharide (LPS) and D-galactosamine, with or without pretreatment with the Rho-kinase inhibitor fasudil. Six hours after endotoxin challenge, leukocyte-endothelium interactions in the hepatic microvasculature were studied by use of intravital fluorescence microscopy and tumor necrosis factor alpha (TNF-alpha); CXC chemokines as well as liver enzymes and apoptosis were determined. Administration of fasudil reduced LPS-induced leukocyte adhesion in postsinusoidal venules and sequestration in sinusoids. Moreover, we found that fasudil abolished extravascular infiltration of leukocytes as well as production of TNF-alpha and CXC chemokines in the liver of endotoxemic mice. Liver enzymes and hepatocellular apoptosis were markedly reduced, and sinusoidal perfusion was improved significantly in endotoxemic mice pretreated with fasudil. Our novel data document that fasudil is a potent inhibitor of endotoxin-induced expression of TNF-alpha and CXC chemokines as well as leukocyte infiltration and hepatocellular apoptosis in the liver. Based on the present findings, it is suggested that inhibition of the Rho-kinase signaling pathway may be a useful target in the treatment of septic liver injury.

Oral findings and dental behaviour before and after liver transplantation - a single-centre cross-sectional study.

OBJECTIVE: The aim of this single-centre, cross-sectional study was to evaluate dental, periodontal and mycological findings, as well as oral behaviour, in patients before (pre-LTx) and after (post-LTx) liver transplantation. METHODS: A total of 47 patients pre-LTx and 119 patients post-LTx were asked to participate. Oral health behaviour was assessed using a standardised questionnaire. Oral examinations included dental [decayed, missing and filled teeth (DMFT) index] and periodontal [papillary bleeding index (PBI), periodontal probing depth (PPD) and clinical attachment loss (CAL)] findings. For Candida screening, swabs from the oral mucosa were cultured. Statistical analysis was performed using the Student's t-test or the Mann-Whitney U-test, depending on whether or not the data followed a normal distribution; Fisher's exact test was also performed. The significance level was α = 5%. RESULTS: A total of 110 patients were included (pre-LTx, n = 35; post-LTx, n = 75). Different patients were investigated in the post-LTx and pre-LTx groups. Lack of use of supplemental oral-hygiene aids was noted. Between-group comparisons failed to find significant overall differences in DMFT and periodontal status. The post-LTx group showed fewer decayed teeth (P = 0.03). A total of 86% of patients pre-LTx and 84% of patients post-LTx were found to need dental treatment, and 60% of patients pre-LTx and 55% of patients post-LTx showed a need for periodontal treatment. The prevalence of Candida albicans was high; however, there were no statistically significant differences between the groups in regard to fungal infection. CONCLUSION: Improved dental care pre- and post-transplant, including screening for fungal infections, is recommended to avoid systemic infections in LTx patients. Increased attention to oral health care, and interdisciplinary collaboration to provide guidance, is needed to improve the oral health of patients before and after LTx.

Hepatic resection of non-colorectal and non-neuroendocrine liver metastases - survival benefit for patients with non-gastrointestinal primary cancers - a case-controlled study.

PURPOSE: Whereas resection of colorectal liver metastases is gold standard, there is an ongoing debate on benefit of resection of non-colorectal (NCRC) and non-neuroendocrine (NNEC) liver metastases. METHODS: The potential survival benefit of patients undergoing resection of NCRC or NNEC liver metastases was investigated. Data from a prospectively maintained database were reviewed over a 7-year period. Kaplan-Meier method was used for the evaluation of outcome following resection. RESULTS: 101 patients underwent 116 surgical procedures for synchronous and metachronous NCRC or NNEC liver metastases with a morbidity of 23% and a mortality of ∼1%. 11 patients underwent repeated liver resection procedures. Overall 5-year survival after liver resection was 30% depending on primary tumour site. Median survival was significantly increased after resection of hepatic metastases from non-gastrointestinal primaries compared to gastrointestinal primaries. Resection of hepatic metastases from non-gastrointestinal primaries resulted in significantly increased median survival compared to exploration only. Patients with hepatic metastases from gastrointestinal primaries did not benefit from hepatic surgery. CONCLUSION: Hepatic resection for liver metastases from NCRC or NNEC cancers is a save treatment procedure. However, the decision to perform surgery should depend on the primary cancer. Especially patients with liver metastases from non-gastrointestinal primaries profit from hepatic surgery.

Gastrectomy with isoperistaltic jejunal parallel pouch in a 15-year-old adolescent boy with gastric adenocarcinoma and autosomal recessive agammaglobulinemia.

A 15-year-old adolescent boy with autosomal recessive agammaglobulinemia underwent endoscopy because of unexplained growth failure and malnutrition. Esophagogastroduodenoscopy revealed antropyloric stenosis, and a biopsy showed an invasive gastric adenocarcinoma. Chronic atrophic corpus gastritis type A and Helicobacter pylori were also identified. Abdominal magnetic resonance imaging confirmed the stenosis resulting from a semicircular intramural tumor without obvious local or distant metastatic spread. Gastrectomy with an extended lymphadenectomy was performed. Esophagoduodenal continuity was restored by an interposed jejunal parallel pouch developed from the first jejunal loop. Oral feeding was supplemented by parenteral nutrition via a Broviac catheter, and the patient is well 4 months later. Several cases of gastric cancer have been reported in children with hereditary agammaglobulinemia. Thus, endoscopy is mandatory in such patients with gastrointestinal symptoms to identify and treat tumors before metastasis occurs. Total gastrectomy, extended lymphadenectomy, and reconstruction using a jejunal reservoir with maintenance of duodenal continuity should be considered.

Immunostimulatory CpG-oligodeoxynucleotides (CpG-ODN) induce early hepatic injury, but provide a late window for protection against endotoxin-mediated liver damage.

BACKGROUND/AIMS: An impaired immunologic response to infection has been recognized as a major defect in the pathogenesis of sepsis and multi-organ failure. Sepsis-associated liver dysfunction and damage are main determinants for the course of the disease. CpG-motif-containing DNA-sequences (CpG-ODN) were previously shown to confer protection in models of infection by stimulating both innate and specific immune responses. Herein, we studied the effect of CpG-ODN in lipopolysaccharide (LPS)-associated hepatotoxicity. METHODS: Sprague Dawley rats pre-treated at day 6 with either CpG-ODN or inert DNA were challenged with E. coli LPS and subsequently studied for liver injury at 6 and 16 h using in vivo fluorescence microscopy and immunohistochemistry. Western blot protein analysis served for assessment of expression of TLR4, TNF receptor-associated factor 6 (TRAF6), NFkappaB and caspase-3. To evaluate CpG-ODN effects during non-septic conditions, additional animals were solely exposed to CpG-ODN and studied after 1 and 6 days. RESULTS: CpG-ODN application induced marked hepatic microcirculatory deterioration and liver dysfunction at day 1, however, with almost complete recovery to normal at day 6. Interestingly, CpG-ODN pre-treatment decreased LPS-induced leukocyte-endothelial cell interaction, sinusoidal perfusion failure and caspase-3-dependent apoptotic cell death. Although Kupffer cell phagocytic activity was not affected, CpG-ODN pre-treatment in LPS-challenged animals attenuated hepatic protein expression of TRAF6 and NFkappaB and increased TLR4 by almost 100%. CONCLUSIONS: CpG-containing DNA-sequences induce early hepatic injury, but mediate long-term protection against LPS hepatotoxicity. The mechanism of protection is based on the induction of cross-tolerance, probably via inhibition of the downstream TRAF6-NFkappaB signaling pathway and upregulation of the TLR4 surface receptor.

Comparative analysis of platelet isolation techniques for the in vivo study of the microcirculation.

OBJECTIVE: In vitro and in vivo studies using isolated platelets require that the cells used for testing are not activated by the isolation procedure. This ensures that the effects measured by the test are the result of the environment or the applied stimulus, but is not an artifact resulting from activation by cell isolation. METHODS: Herein, we analyzed two different platelet isolation procedures (i.e., a Sepharose column versus density gradient centrifugation) with special emphasis on cell activation, including flow cytometric analysis of P-selectin expression, functional quantification of mechanical platelet retention, light microscopic assessment of platelet aggregation, and fluorescence microscopic determination of in vivo rat liver platelet-endothelium cell interaction. RESULTS: Under resting conditions, Sepharose column-isolated platelets showed a negligible fraction of only 2.7 +/- 3.3% cells (mean +/- SEM) with P-selectin expression, and an appropriate response (i.e., a 33-fold increase) upon activation with thrombin receptor-activating peptide (TRAP). In contrast, density gradient centrifugation resulted in P-selectin expression under resting conditions of approximately 50% of the isolated cells and only a 1.6-fold increase on further TRAP stimulation. In addition, density gradient-isolated platelets, but not Sepharose column-isolated platelets, showed increased mechanical retention and agglutination/aggregation in vitro, as well as pronounced adhesion to hepatic venular endothelium in vivo. Interestingly, density gradient-isolated platelets additionally induced in vivo an increase of colocalization of platelets with adherent leukocytes, indicating a generalized microvascular inflammatory response that is comparable to that observed after a 60-minute ischemia/30-minute reperfusion insult. CONCLUSION: Density gradient centrifugation-isolated platelets, but not Sepharose column-isolated platelets, are activated already under resting conditions and induce in vivo a platelet-leukocyte-endothelial cell-associated inflammatory response. Thus, we propose that the method of platelet isolation using the Sepharose column is superior to the density gradient centrifugation technique and might therefore be preferred for in vitro and in vivo assays to study platelet function.