Myotubularin-related-protein-7 inhibits mutant (G12V) K-RAS by direct interaction.

Inhibition of K-RAS effectors like B-RAF or MEK1/2 is accompanied by treatment resistance in cancer patients via re-activation of PI3K and Wnt signaling. We hypothesized that myotubularin-related-protein-7 (MTMR7), which inhibits PI3K and ERK1/2 signaling downstream of RAS, directly targets RAS and thereby prevents resistance. Using cell and structural biology combined with animal studies, we show that MTMR7 binds and inhibits RAS at cellular membranes. Overexpression of MTMR7 reduced RAS GTPase activities and protein levels, ERK1/2 phosphorylation, c-FOS transcription and cancer cell proliferation in vitro. We located the RAS-inhibitory activity of MTMR7 to its charged coiled coil (CC) region and demonstrate direct interaction with the gastrointestinal cancer-relevant K-RASG12V mutant, favouring its GDP-bound state. In mouse models of gastric and intestinal cancer, a cell-permeable MTMR7-CC mimicry peptide decreased tumour growth, Ki67 proliferation index and ERK1/2 nuclear positivity. Thus, MTMR7 mimicry peptide(s) could provide a novel strategy for targeting mutant K-RAS in cancers.

PPARγ induces PD-L1 expression in MSS+ colorectal cancer cells.

Only a small subset of colorectal cancer (CRC) patients benefits from immunotherapies, comprising blocking antibodies (Abs) against checkpoint receptor "programmed-cell-death-1" (PD1) and its ligand (PD-L1), because most cases lack the required mutational burden and neo-antigen load caused by microsatellite instability (MSI) and/or an inflamed, immune cell-infiltrated PD-L1+ tumor microenvironment. Peroxisome proliferator-activated-receptor-gamma (PPARγ), a metabolic transcription factor stimulated by anti-diabetic drugs, has been previously implicated in pre/clinical responses to immunotherapy. We therefore raised the hypothesis that PPARγ induces PD-L1 on microsatellite stable (MSS) tumor cells to enhance Ab-target engagement and responsiveness to PD-L1 blockage. We found that PPARγ-agonists upregulate PD-L1 mRNA/protein expression in human gastrointestinal cancer cell lines and MSS+ patient-derived tumor organoids (PDOs). Mechanistically, PPARγ bound to and activated DNA-motifs similar to cognate PPARγ-responsive-elements (PPREs) in the proximal -2 kb promoter of the human PD-L1 gene. PPARγ-agonist reduced proliferation and viability of tumor cells in co-cultures with PD-L1 blocking Ab and lymphokine-activated killer cells (LAK) derived from the peripheral blood of CRC patients or healthy donors. Thus, metabolic modifiers improved the antitumoral response of immune checkpoint Ab, proposing novel therapeutic strategies for CRC.

Myotubularin-related protein 7 activates peroxisome proliferator-activated receptor-gamma.

Peroxisome proliferator-activated receptor-gamma (PPARγ) is a transcription factor drugable by agonists approved for treatment of type 2 diabetes, but also inhibits carcinogenesis and cell proliferation in vivo. Activating mutations in the Kirsten rat sarcoma viral oncogene homologue (KRAS) gene mitigate these beneficial effects by promoting a negative feedback-loop comprising extracellular signal-regulated kinase 1/2 (ERK1/2) and mitogen-activated kinase kinase 1/2 (MEK1/2)-dependent inactivation of PPARγ. To overcome this inhibitory mechanism, we searched for novel post-translational regulators of PPARγ. Phosphoinositide phosphatase Myotubularin-Related-Protein-7 (MTMR7) was identified as cytosolic interaction partner of PPARγ. Synthetic peptides were designed resembling the regulatory coiled-coil (CC) domain of MTMR7, and their activities studied in human cancer cell lines and C57BL6/J mice. MTMR7 formed a complex with PPARγ and increased its transcriptional activity by inhibiting ERK1/2-dependent phosphorylation of PPARγ. MTMR7-CC peptides mimicked PPARγ-activation in vitro and in vivo due to LXXLL motifs in the CC domain. Molecular dynamics simulations and docking predicted that peptides interact with the steroid receptor coactivator 1 (SRC1)-binding site of PPARγ. Thus, MTMR7 is a positive regulator of PPARγ, and its mimicry by synthetic peptides overcomes inhibitory mechanisms active in cancer cells possibly contributing to the failure of clinical studies targeting PPARγ.

A pancreas-preserving technique for the management of symptomatic pancreatic anastomotic insufficiency refractory to conservative treatment after pancreas head resection.

BACKGROUND AND AIM: Management of symptomatic pancreatic anastomotic insufficiency after pancreas head resection remains controversial. Completion pancreatectomy as one frequently performed option is associated with poor prognosis. PATIENTS AND METHODS: During a 4-year period, a two-step strategy was applied in four consecutive patients suffering from pancreatic anastomotic insufficiency refractory to conservative management after a pancreas head resection. In the first step, sepsis was overbridged by meticulous debridement and resection of the pancreaticojejunostomy, leaving the biliary anastomosis untouched, and selective drainage of the pancreatic duct as well as the peripancreatic area. In the second step, after recovery, the procedure was completed with a novel pancreaticojejunostomy. RESULTS: The surgical procedure was completed in three patients after a mean of 164 (range: 112-213) days. One patient died from cardiac arrest 54 days after the reoperation with resolved abdominal sepsis. No pancreatic anastomotic insufficiency occurred after the new pancreaticojejunostomy had been performed. Three patients are alive and tumor-free with normal exocrine and endocrine pancreatic function after a mean follow-up of 20.3 (3-38) months following the definitive reconstruction. CONCLUSION: The two-step pancreas-preserving strategy can be used as an alternative to completion pancreatectomy for patients suffering from severe pancreatic anastomotic insufficiency.

Treatment with tigecycline of recurrent urosepsis caused by extended-spectrum-beta-lactamase-producing Escherichia coli.

A 25-year-old female was admitted to our intensive care unit with septic shock and multiorgan failure caused by extended-spectrum beta-lactamase-producing Escherichia coli originating from the right renal pelvis. A 16-day course of treatment with meropenem reversed the septic condition, but the infection recurred thereafter. The patient recovered fully after therapy was changed to tigecycline.

Activity of the lipoxygenase inhibitor 1-phenyl-3-pyrazolidinone (phenidone) and derivatives on the inhibition of adhesion molecule expression on human umbilical vascular endothelial cells.

Leukocyte adhesion contributes to perfusion abnormalities and tissue damage during trauma, shock or overwhelming inflammation. This study was performed to determine whether the lipoxygenase inhibitor phenidone and derivatives decrease the expression of adhesion molecules on tumor necrosis factor-alpha (TNF-alpha) stimulated endothelial cells and attenuate leukocyte-endothelial interactions under flow in vitro. TNF-alpha stimulated human umbilical venous endothelial cells (HUVECs) were incubated with phenidone, 4-methyl-phenidone, 4-4-dimethyl-phenidone, 5-methyl-phenidone, 5-phenyl-phenidone, and 5-methyl-1,(2,5-di-chloro-phenyl)-3-pyrazolidone. We tested the inhibition of adhesion molecule expression at different inhibitor concentrations before, during, and after the stimulation of HUVECs. The inhibition of endothelial cell expression on HUVECs was measured by flow cytometry. Rolling and firm adhesion of leukocytes to pretreated endothelium was examined in a parallel plate flow chamber. Phenidone inhibited the expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial-leukocyte adhesion molecule-1 on HUVECs when added prior to HUVEC stimulation. The inhibitory effect of phenidone was still observed when added simultaneously, but not when added after HUVEC stimulation. 4-4-dimethyl-phenidone and 5-phenyl-phenidone inhibited the expression of adhesion molecules more effectively than phenidone. The attenuation of leukocyte rolling under flow conditions was also significantly more effective with 4-4-dimethyl-phenidone than with phenidone. Lipoxygenase inhibitors might be of therapeutically interest for the treatment of overwhelming systemic inflammation during shock, trauma, and sepsis.

Intervention at the level of the neuroendocrine-immune axis and postoperative pneumonia rate in long-term alcoholics.

RATIONALE: Postoperative pneumonia is three to four times more frequent in patients with alcohol use disorders followed by prolonged intensive care unit (ICU) stay. Long-term alcohol use leads to an altered perioperative hypothalamus-pituitary-adrenal (HPA) axis and immunity. OBJECTIVES: The aim of this study was to evaluate HPA intervention with low-dose ethanol, morphine, or ketoconazole on the neuroendocrine-immune axis and development of postoperative pneumonia in long-term alcoholic patients. METHODS: In this randomized, double-blind controlled study, 122 consecutive patients undergoing elective surgery for aerodigestive tract cancer were included. Long-term alcohol use was defined as consuming at least 60 g of ethanol daily and fulfilling the Diagnostic and Statistical Manual of Mental Disorders IV criteria for either alcohol abuse or dependence. Nonalcoholic patients were included but only as a descriptive control. Perioperative intervention with low-dose ethanol (0.5 g/kg body weight per day), morphine (15 mug/kg body weight per hour), ketoconazole (200 mg four times daily), and placebo was started on the morning before surgery and continued for 3 d after surgery. Blood samples to analyze the neuroendocrine-immune axis were obtained on the morning before intervention and on Days 1, 3, and 7 after surgery. MEASUREMENTS AND MAIN RESULTS: In long-term alcoholic patients, all interventions decreased postoperative hypercortisolism and prevented impairment of the cytotoxic T-lymphocyte type 1:type 2 ratio. All interventions decreased the pneumonia rate from 39% to a median of 5.7% and shortened intensive care unit stay by 9 d (median) compared with the placebo-treated long-term alcoholic patients. CONCLUSIONS: Intervention at the level of the HPA axis altered the immune response to surgical stress. This resulted in decreased postoperative pneumonia rates and shortened intensive care unit stay in long-term alcoholic patients.

Small-dose epoprostenol decreases systemic oxygen consumption and splanchnic oxygen extraction during normothermic cardiopulmonary bypass.

Normothermic, nonpulsatile cardiopulmonary bypass (CPB) impairs systemic and splanchnic oxygen transport and increases gastrointestinal permeability. It is an important therapeutic goal to avoid splanchnic dysoxia during CPB. Small-dose prostacyclin therapy improves splanchnic oxygen transport and microcirculation in septic patients. In this study, we sought to determine if during cardiac surgery, the prostacyclin analog epoprostenol improves the balance of systemic and splanchnic oxygen transport. Eighteen patients undergoing cardiac valve replacement were randomized to receive either epoprostenol (3 ng x kg(-1) x min(-1)) or placebo during, and for 1 hour after, surgery. Systemic and splanchnic oxygen delivery, consumption, and extraction and arterial, mixed venous, and hepato-venous lactate concentrations were measured before, during, and after CPB. Gastrointestinal permeability was measured 1 day before and 1 day after surgery using the triple sugar permeability test. During CPB, the epoprostenol group had decreased systemic oxygen consumption and splanchnic oxygen extraction (P = 0.024). These effects were not present 1 hour after the end of epoprostenol infusion. The study was not adequately powered to determine whether epoprostenol altered the trend towards increased lactate metabolism and increased postoperative gastrointestinal permeability, nor could we demonstrate any differences between groups in clinically relevant end-points. In conclusion, these findings suggest that during normothermic CPB, small-dose epoprostenol therapy may reduce systemic oxygen consumption and splanchnic oxygen extraction.

Recombinant activated factor VII for refractory bleeding after cardiac surgery--a retrospective analysis of safety and efficacy.

OBJECTIVE: Analysis of safety and efficacy of recombinant activated factor VII (rFVIIa) used as the last resort for refractory bleeding after cardiac surgery. DESIGN: Retrospective cohort analysis and matched pairs analysis with historic controls were performed. In the rFVIIa group, which also received conventional hemostatic therapy, data were collected for a median of 14 hrs from admission to the intensive care unit (ICU) to the administration of rFVIIa and for the following 24 hrs. In the control group, which received only conventional hemostatic therapy, data were collected for 14 and then for 24 hrs after admission to the ICU. SETTING: University hospital. PATIENTS: Twenty-four patients matched with historic controls. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: No thromboembolic complications were observed in the rFVIIa group. Blood loss and transfusion requirements were significantly reduced in the period after the administration of rFVIIa. However, in the 24-hr period after rFVIIa administration, blood loss (p = .140) and transfusion of packed red blood cells (p = .442) and fresh frozen plasma (p = .063) were not different between the rFVIIa and control groups. Platelet concentrates (p = .004) were transfused less in the control group. Mortality and 6-month survival rates were not different between the groups. CONCLUSIONS: When used as a last resort, rFVIIa was safe but not incrementally efficacious over conventional hemostatic therapy.

Synthetic colloids attenuate leukocyte-endothelial interactions by inhibition of integrin function.

BACKGROUND: It has been suspected that synthetic colloids may interfere with leukocyte adhesion by down-regulation of endothelial cell adhesion molecules. Although inhibition of endothelial inflammation might reduce leukocyte-related tissue injury, the same mechanism may be detrimental for host defense during severe infection. Regarding the widespread use of colloids, the authors performed a laboratory investigation to determine the mechanisms by which synthetic colloids interfere with leukocyte-endothelial interactions. METHODS: Adhesion molecule expression on native and cytokine-activated endothelium from umbilical veins was measured after pretreatment with gelatin and various preparations of dextran or hydroxyethyl starch. Inhibition of neutrophil adhesion to activated endothelium was examined in a flow chamber by perfusion of untreated and colloid-treated neutrophils over colloid-pretreated endothelium at 2 dyn/cm. Comparisons were made between untreated controls, colloid-pretreated endothelium, and colloid-cotreated neutrophils. RESULTS: Intercellular adhesion molecule 1, vascular cell adhesion molecule 1, E-selectin, and P-selectin were not attenuated by any colloid. Accordingly, colloid pretreatment of endothelium alone did not reduce neutrophil adhesion. In contrast, when neutrophils were cotreated by addition of colloids to the perfusate immediately before perfusion, adhesion decreased by 31-51% (P < 0.05) regardless of the colloid type. As indicated by the twofold increased rolling fractions, this reduction was due to an inhibition of neutrophil integrins. CONCLUSIONS: This study shows that synthetic colloids inhibit neutrophil adhesion by a neutrophil-dependent mechanism rather than interfering with endothelial cell activation. This suggests that inhibition of leukocyte sequestration by volume support is a common and transient phenomenon depending on the colloid concentration in plasma.

Effects of smoking on renal function in patients with type 1 and type 2 diabetes mellitus.

BACKGROUND: Smoking increases the risk of end-stage renal failure in patients with primary renal disease. Whether and to what extent smoking affects the kidneys in diabetic patients with normal renal function and variable degrees of proteinuria has not been fully studied. METHODS: We followed 185 patients with type 1 or 2 diabetes mellitus and with or without signs of overt renal disease for at least 3 years, median 5.1 (3-6.8) years. Each patient had a baseline visit and at least four follow-up visits (average 4.8+/-0.3). Cases were patients who were smoking (n = 44) at the time the survey was started. Controls were patients who had never smoked (n = 141). Glomerular filtration rate (GFR) was estimated using the MDRD formula. Multiple logistic regression was used to correct for confounding factors. RESULTS: At baseline, smokers were younger (47+/-14 vs 54+/-16 years, P < 0.01), and had a lower GFR (95+/-26 ml/min) than non-smokers (107+/-33 ml/min, P < 0.05). Mean GFR remained constant during follow-up in non-smokers (106+/-31 ml/min), but decreased significantly in smokers (83+/-22 ml/min, P < 0.0001), and this relationship persisted when adjusted for retinopathy, glycaemic control, age, body habitus, ACE-inhibitor treatment, blood pressure control or severity of proteinuria. The effect of smoking on GFR decline was stronger in patients with type 1 diabetes or male gender. CONCLUSIONS: Cigarette smoking causes a decrease in GFR in diabetic patients with normal or near-normal renal function, independent of confounding factors including severity of proteinuria. The latter finding suggests a mechanism independent of glomerular damage.

Effects of fresh versus old stored blood in the priming solution on whole blood lactate levels during paediatric cardiac surgery.

It has been suggested that lactate levels may predict morbidity and mortality in paediatric patients during corrective open-heart surgery. Packed red blood cells (PRBC) are frequently necessary for priming the reservoir used in cardiopulmonary bypass (CPB). The storage of PRBC might cause a significant increase in lactate levels. The purpose of the present study was to quantify the increase in lactate levels in stored red blood cells over time and to compare lactate levels after transfusions of fresh (< or =12 days) versus old blood (>12 days) in 20 patients. We found an increase in lactate levels from 6.0 to 44.7mmol/L (mean 17.0+/-7.8 mmol/L) during storage. Lactate levels were also significantly higher after the onset of CPB in paediatric patients transfused with old blood than in patients transfused with fresh blood (1.43+0.36 versus 3.46+/-0.63, p=0.0006). Our results suggest that the higher lactate levels found after the initiation of CPB should be used with caution when assessing tissue hypoxia and predicting outcome.

Role of cross-allergies to latex in clinical routine of anesthesia.

STUDY OBJECTIVE: To determine the applicability and reliability of a screening questionnaire to detect patients at high-risk of latex allergy; to assess the importance of other allergies such as profilin allergies (pollinosis) for presence of latex sensitization; and to determine the clinical effectiveness of preemptive avoidance of latex exposure in high-risk patients. DESIGN: Prospective, clinical trial. SETTING: Operative theater of a university hospital. PATIENTS: 95 adult patients. INTERVENTIONS: Patients were preoperatively screened and classified for present latex allergy (high-risk and low-risk group) according to a specially designed screening questionnaire. Anesthesia and surgery in the high-risk group were performed strictly avoiding latex-containing materials. The low-risk group (other allergies including pollinosis) received routine treatment, without latex-avoidance. Effects of latex avoidance or exposure were evaluated by measuring specific IgE titers perioperatively. MEASUREMENTS AND MAIN RESULTS: According to the questionnaire, 45 patients at high risk were defined. Validity of classification of high-risk patients is supported by significantly higher total IgE and latex and grass profilin specific IgE compared to the low-risk group. There were no significant differences in other profilin-specific IgEs. In one case of severe anaphylactic reaction a drop of latex-specific IgE during surgery could be observed. CONCLUSION: The questionnaire allowed the identification of most patients at high risk for latex allergy. In isolated pollinosis no changes in any specific IgE levels were detectable. Strict avoidance of perioperative latex exposure in high-risk patients increases safety during anesthesia and surgery.

Correlation of meropenem plasma levels with pharmacodynamic requirements in critically ill patients receiving continuous veno-venous hemofiltration.

BACKGROUND: In patients with acute renal failure, the pharmacokinetics of meropenem depend on the operational characteristics of the renal replacement therapy. Dosage recommendations are based on the correlation of plasma levels with pharmacodynamic requirements. METHODS: Eight critically ill patients with acute renal failure were treated by continuous veno-venous hemofiltration with a filtrate flow of 1,600 ml/h and received 500 mg of meropenem every 12 h. Plasma and hemofiltrate concentrations of meropenem at steady state were determined by HPLC. RESULTS: Peak levels in plasma amounted to 39.5 +/- 10.5 mg/l (mean +/- SD) and trough levels were 2.4 +/- 1.5 mg/l. The minimal inhibitory concentration (MIC) for susceptible bacteria (4 mg/l) was covered for 40% of the dosing interval or longer in all patients. The MIC for intermediately susceptible organisms (8 mg/l) was covered for 33% in 6 of the 8 patients. The elimination half-life was prolonged to 3.63 +/- 0.77 h. The sieving coefficient of meropenem was 0.91 +/- 0.10 and the recovery in hemofiltrate amounted to 30.9 +/- 11.5% of the dose. CONCLUSIONS: A dosage of 500 mg twice daily provides appropriate serum levels for the treatment of infections caused by susceptible bacteria. A higher dosage is adequate for infections by intermediately susceptible bacteria or for renal replacement therapies with markedly higher filtrate flow rates.

Pseudomonas aeruginosa activates Cl- channels in host epithelial cells.

Exposure to Pseudomonas aeruginosa triggers the apoptotic cell death of Chang epithelial cells, and this depends on the expression of both the CD95 receptor and CD95 ligand. In lymphocytes CD95-mediated apoptosis is paralleled by the activation of outwardly rectifying Cl- channels. The present study was performed to explore whether P. aeruginosa-induced apoptosis of Chang epithelial cells is paralleled by activation of Cl- channels. According to whole-cell patch-clamp recordings, exposure of Chang epithelial cells to P. aeruginosa does lead to rapid activation of an outwardly rectifying Cl- -selective current. The current is inhibited by the Cl- channel blocker NPPB. Exposure of Chang epithelial cells to P. aeruginosa led to a significant decrease of cell membrane capacitance by 6%, pointing to a decrease in cell volume by 7%. Exposure to P. aeruginosa depolarized the mitochondrial membrane potential indicating apoptotic cell death. The decline of mitochondrial membrane potential was not significantly affected by NPPB. In conclusion, P. aeruginosa-induced apoptosis of Chang epithelial cells is paralleled by activation of Cl- channels. Activation of the channels participates in the alteration of cell volume but is not a prerequisite for P. aeruginosa-induced apoptosis.

The effects of fresh frozen plasma on neutrophil-endothelial interactions.

UNLABELLED: Leukocyte adhesion to endothelial cells contributes to microcirculatory disturbances during severe shock syndromes. Whereas certain plasma expanders inhibit leukocyte adhesion, contaminants of plasma protein solutions upregulate endothelial cell adhesion molecules in certain cases. We performed this study to determine whether fresh frozen plasma (FFP) affects neutrophil-endothelial interactions in cocultures of neutrophils and human umbilical vein endothelial cells (HUVEC) in vitro. HUVEC (n = 9) were incubated with either 20% FFP or 20% serum in medium for 6 h. Expression of E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule-1 was induced by tumor necrosis factor alpha (0.5 ng/mL for 4 h) and measured by flow cytometry. Neutrophil adhesion was examined in a parallel plate flow chamber in which isolated neutrophils were perfused over pretreated HUVEC under postcapillary flow conditions. Incubation with FFP decreased E-selectin and intercellular adhesion molecule 1 on activated HUVEC by 28% and 22%, respectively (P < or = 0.01; analysis of covariance). Consequently, neutrophil adhesion decreased by 20%-41% in FFP-treated cocultures (n = 4; P < or = 0.01; paired Student's t-test). We conclude that FFP attenuates the inflammatory response of endothelial cells with regard to neutrophil-endothelial interactions. Because the composition of patients' plasma is affected not only by transfusion, but more frequently by shock treatment with IV fluids, plasma dilution in critically ill patients could be important. IMPLICATIONS: During shock, fluid administration leads to a massive dilution of plasma. Apart from maintaining hemodynamics, this might affect tissue damage by influencing leukocyte accumulation in the microvasculature. Using endothelial cells, isolated neutrophils, and a parallel plate flow chamber, we studied the effects of fresh frozen plasma on neutrophil-endothelial interactions.

CFTR is a pattern recognition molecule that extracts Pseudomonas aeruginosa LPS from the outer membrane into epithelial cells and activates NF-kappa B translocation.

Immune cells are activated during cellular responses to antigen by two described mechanisms: (i) direct uptake of antigen and (ii) extraction and internalization of membrane components from antigen-presenting cells. Although endocytosis of microbial antigens by pattern recognition molecules (PRM) also activates innate immunity, it is not known whether this involves extraction and internalization of microbial surface components. Epithelial cells on mucosal surfaces use a variety of receptors that are distinct from the classical endocytic PRM to bind and internalize intact microorganisms. Nonclassical receptor molecules theoretically could act as a type of endocytic PRM if these molecules could recognize, bind, extract, and internalize a pathogen-associated molecule and initiate cell signaling. We report here that the interaction between the cystic fibrosis transmembrane conductance regulator (CFTR) and the outer core oligosaccharide of the lipopolysaccharide (LPS) in the outer membrane of Pseudomonas aeruginosa satisfies all of these conditions. P. aeruginosa LPS was specifically recognized and bound by CFTR, extracted from the organism's surface, and endocytosed by epithelial cells, leading to a rapid (5- to 15-min) and dynamic translocation of nuclear transcription factor NF-kappa B. Inhibition of epithelial cell internalization of P. aeruginosa LPS prevented NF-kappa B activation. Cellular activation depended on expression of wild-type CFTR, because both cultured Delta F508 CFTR human airway epithelial cells and lung epithelial cells of transgenic-CF mice failed to endocytose LPS and translocate NF-kappa B. CFTR serves as a critical endocytic PRM in the lung epithelium, coordinating the effective innate immune response to P. aeruginosa infection.