Jan E. Slotta

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.

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 α (TNF‐α); 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‐α 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‐α 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.

Fasudil, a Rho-kinase inhibitor, inhibits leukocyte adhesion in inflamed large blood vessels in vivo

Abstract.Objective and DesignEmerging data suggest that Rho-kinase signaling may regulate numerous aspects of inflammatory reactions. Herein, we investigated the role of Rho-kinase in inflammatory interactions between leukocytes and the endothelium in femoral arteries and veins in vivo.Material and methodsMice were injected with lipopolysaccharide (LPS) and Rho-kinase was inhibited by pre-treatment with fasudil, which is a highly selective inhibitor of Rho-kinase. Six hours after LPS challenge, intravital fluorescence microscopy of the femoral vessels was performed and leukocyte-endothelium interactions were visualized after in vivo staining with rhodamine 6G.ResultsLPS increased leukocyte rolling and adhesion in femoral arteries and veins. Pre-treatment with fasudil had no effect on leukocyte rolling but significantly decreased venular leukocyte adhesion by 85% and completely abrogated leukocyte adhesion in femoral arteries in endotoxin-treated mice.ConclusionsWe conclude that Rho-kinase signaling regulates LPS-induced leukocyte adhesion in femoral arteries and veins in vivo and that inhibition of Rho-kinase may be useful in the treatment of pathological inflammation in large blood vessels of the vascular system.

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.

Incisional hernia after abdominal closure with slowly absorbable versus fast absorbable, antibacterial-coated sutures

BACKGROUND Incisional hernia remains among the most common complications after midline incision of the abdominal wall. The role of the suture material used for abdominal wall closure remains controversial. To decrease bacterial adherence to surgical sutures, braided suture materials with antibacterial activity (Vicryl plus, Ethicon, Inc) were developed. This is the first study to analyze long-term results using an antibacterial-braided suture material for abdominal wall closure in a large clinical trial. METHODS To analyze the effects of Triclosan-coated suture material (Vicryl plus) on the development of incisional hernia, we performed a 36-month follow-up of 1,018 patients who had a primary midline incision for elective abdominal surgery. In the first time period, a PDS II loop suture was used. In the second observation period, we used Vicryl plus. All variables were recorded prospectively in a database. The primary outcome was the number of incisional hernias. Risk factors for the development of incisional hernias were collected prospectively to compare the 2 groups. RESULTS The overall incisional hernia rate in the 36-month follow-up period was 14.6%. Analyzing the influence of the suture material used on the development of incisional hernia, we did not find differences between the 2 groups (PDS II, 14%; Vicryl plus, 15.2%). In the multivariate analysis of possible factors in the study population, only body mass index (BMI) showed a significant influence on the development of incisional hernias. Despite the incidence of wound infections being less in the Vicryl plus group (6.1% vs 11.9%; P < .05), there were no difference in incidence of incisional hernia between the 2 groups. CONCLUSION Fast absorbable sutures with antibacterial coating (Tricosan) do not increase the hernia rate after midline abdominal incision compared with slowly absorbable sutures, when wound infection rates are decreased by coating the fast absorbable suture with Triclosan. The development of incisional hernia is significantly increasing in patients with a BMI >30 kg/m(2).

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.

Popular Belief Meets Surgical Reality: Impact of Lunar Phases, Friday the 13th and Zodiac Signs on Emergency Operations and Intraoperative Blood Loss

BackgroundThe 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.MethodsWe 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.ResultsA 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.ConclusionsScientific 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.

Capture of platelets to the endothelium of the femoral vein is mediated by CD62P and CD162

Platelets contribute to blood coagulation at sites of vascular injury and to the recruitment of leukocytes at sites of inflammation. Under pathological conditions, platelets are involved in numerous diseases and clinical complications, such as deep venous thrombosis, embolism and atherosclerosis. But so far, little is known about the mechanisms of inflammation in large veins and the role of platelets in inflamed large veins. For this purpose, we investigated primary and secondary interactions between platelets, leukocytes and endothelial cells in the femoral vein in vivo with special regard to the role of CD62P (P-selectin) and CD162 (PSGL-1). Mice were challenged with lipopolysaccharide (LPS)/D-galactosamine (D-gal) and either CD162 or CD62P was blocked by intravenous administration of a corresponding antibody at the time point of LPS/D-gal injection. Four hours after LPS/gal injection, intravital fluorescence microscopy of the femoral vein was performed and primary and secondary platelet-leukocyte-endothelial cell-interactions were visualized after in vivo platelet and leukocyte staining with rhodamine 6G. Analysis of intravital fluorescence microscopy revealed that LPS/D-gal caused a strong inflammatory reaction of the venous endothelium with significant induction of platelet and leukocyte tethering, rolling and adhesion. Secondary interactions of platelets to adherent or rolling platelets or leukocytes were also increased after LPS/D-gal-injection. Immunoneutralization of either CD162 or CD62P significantly decreased platelet primary and secondary capture as well as leukocyte rolling and adhesion. CD162 and CD62P play a central role in mediating inflammatory primary and secondary interactions of platelets and leukocytes to the endothelium in inflamed large veins in vivo. Thus, blocking CD162 or CD62P might be an attractive tool for preventing platelet and leukocyte-driven venous diseases.

Rho-kinase signalling mediates endotoxin hypersensitivity after partial hepatectomy.

Excessive loss of functional liver mass results in hepatic dysfunction and is associated with an increased sensitivity to infection. This experimental study investigated the role of Rho‐kinase in hepatectomy‐induced sensitization to endotoxin.

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.