Boris Rogachev

Involvement of adenosine in the antiinflammatory action of ketamine

Background:Ketamine is an anesthetic drug. Subanesthetic doses of ketamine have been shown to reduce interleukin-6 concentrations after surgery and to reduce mortality and the production of tumor necrosis factor α and interleukin 6 in septic animals. Similarly, adenosine was shown to reduce tumor necrosis factor α and mortality of septic animals. The aim of this study was to determine whether adenosine mediates the antiinflammatory effects of ketamine. Methods:Sepsis was induced in mice by lipopolysaccharide or Escherichia coli inoculation. Leukocyte recruitment and cytokine concentrations were used as inflammation markers. Adenosine concentrations were assayed by high-performance liquid chromatography, and the involvement of adenosine in the effects of ketamine was demonstrated by adenosine receptor agonists and antagonists. Results:Ketamine markedly reduced mortality from sepsis, leukocyte recruitment, and tumor necrosis factor-α and interleukin-6 concentrations. Ketamine administration in mice and rats was associated with a surge at 20–35 min of adenosine in serum (up to 5 &mgr;m) and peritoneal fluid. The adenosine A2A receptor agonist CGS-21680 mimicked the effect of ketamine in peritonitis, whereas the A2A receptor antagonists DMPX and ZM 241385 blocked its antiinflammatory effects. In contrast, A1 and A3 receptor antagonists had no effect. ZM 241385 reversed the beneficial effect of ketamine on survival from bacterial sepsis. Conclusions:The current data suggest that the sepsis-protective antiinflammatory effects of ketamine are mediated by the release of adenosine acting through the A2A receptor.

The effect of a high partial pressure of carbon dioxide environment on metabolism and immune functions of human peritoneal cells—Relevance to carbon dioxide pneumoperitoneum☆☆☆

OBJECTIVE Our purpose was to evaluate in vitro the effect of a high partial pressure of carbon dioxide environment used in laparoscopy on metabolic and immune response of various human peritoneal cells. STUDY DESIGN Polymorphonuclear leukocytes were obtained from 5 healthy volunteers, peritoneal macrophages were obtained from the effluent of 8 patients undergoing continuous ambulatory peritoneal dialysis, and human peritoneal mesothelial cell cultures were prepared from omentum derived from 5 patients undergoing elective surgery. The cells were exposed to a laparoscopy-like environment (1 atmosphere carbon dioxide and 0.2 atmosphere oxygen), to a control gas mixture (1 atmosphere helium and 0.2 atmosphere oxygen), or air for 3 hours. After exposure to gas mixtures, cell functions were tested at various recovery periods. RESULTS Three hours of exposure to a high partial pressure of carbon dioxide had no effect on viability of peritoneal macrophages and human peritoneal mesothelial cells, tested by trypan blue dye uptake and lactate dehydrogenase release. A high partial pressure of carbon dioxide decreased the mitochondrial dehydrogenases activity of peritoneal macrophages and human peritoneal macrophage cells by 60%, assayed by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction. High partial pressure of carbon dioxide blocked the superoxide release from activated polymorphonuclear leukocytes and the secretion of interleukin 1beta from stimulated peritoneal macrophages, and human peritoneal macrophage cells were decreased by 15% and 30% and the secretion of tumor necrosis factor-alpha from peritoneal macrophages was suppressed by 85%. Mitochondrial activity, polymorphonuclear leukocyte function, and interleukin 1beta and tumor necrosis factor-alpha secretion returned to normal after a recovery period of 12 to 24 hours, 4.5 hours, and 24 hours, respectively. In the control experiments exposure of cells to helium had no suppressive effect. CONCLUSIONS Exposure of cells to a high partial pressure of carbon dioxide environment suppresses the inflammatory and metabolic responses of peritoneal cells. We suggest that this suppressive effect may contribute to the low postsurgery adhesion formation and the reduction in postoperative pain observed in laparoscopy. Nevertheless, the suppression of the immune response should also be taken into account for operations involving a high risk of bacterial dissemination.

The in vitro effects of ketamine at large concentrations can be attributed to a nonspecific cytostatic effect.

K etamine seems to promote a favorable outcome for critically ill patients, including those with septic shock (1). It has been proposed that the nonanesthetic effects of ketamine may be attributed to antiinflammatory mechanisms. This hypothesis has been supported by in vivo findings, such as decreased levels of interleukin (IL)-6 in patients treated with a single IV small dose of ketamine during postoperative stress (2) or during abdominal hysterectomy (3). Notably, the doses that produce the antiinflammatory effects in these studies reach a plasma concentration of 0.1 mg/mL, whereas the plasma ketamine concentration required for anesthesia is 0.6–2.0 mg/mL (1,4,5). In vitro attempts to uncover the antiinflammatory mechanisms of ketamine revealed widespread inhibitory activities, but they used large ketamine concentrations. For example, inhibition of tumor necrosis factor-a and nitric oxide production in lipopolysaccharide (LPS)-stimulated mouse-activated macrophagelike cells was attained at ketamine concentrations of 7.1–142.6 mg/mL (6); inhibition of action and production of nitric oxide synthase in LPS-treated rat alveolar macrophages, at 2.38 mg/mL ketamine concentration (7); and inhibition of IL-6, IL-8, and tumor necrosis factor-a production by LPS-stimulated human whole blood, at ketamine concentrations of 100– 500 mg/mL (8). We believe that the in vitro responses with large concentrations of ketamine are misleading. To verify this, we sought to clarify whether these large concentrations of ketamine exert a broad nonspecific cytostatic effect that includes both the arrest of cell proliferation and a blockade of cytokine production. We introduced varying concentrations of ketamine to both growth factor-dependent and independent human and mouse cell cultures and examined cell proliferation. Our data indicate that large doses of ketamine arrest cytokine production and cell proliferation in a strikingly similar pattern.

The effects of peritoneal dialysis on blood glutamate levels: implementation for neuroprotection.

Background: Previous study has demonstrated the efficacy of hemodialysis in reducing blood glutamate levels. The purpose of the present study is to investigate whether peritoneal dialysis (PD) may be effective in lowering blood glutamate levels, which may serve as a potential tool for improving neurological function after brain injury. Methods: Two liters of dialysis solution were infused over 10 minutes into 18 patients with stage V chronic kidney disease. Blood samples were collected immediately before initiation of PD, and hourly for a total of 5 blood samples. Blood samples were sent for determination of glutamate, creatinine, urea, glucose, glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase. PD samples were collected and analyzed for glutamate, creatinine, urea, and glucose at the same time points as the blood samples. Results: Blood glutamate concentrations were significantly reduced by 60 minutes after the infusion of dialysis solution (P<0.0001), whereas levels of glutamate in the dialysis solution were increased significantly by 60 minutes (P<0.0001). Conclusions: We demonstrated that PD is an effective modality in reducing blood glutamate concentrations. This method may be potentially utilized for the treatment of acute and chronic brain disorders that are accompanied by elevated glutamate in the brain’s extracellular fluid. Considering the rapid saturation of the PD solution with glutamate, we recommend frequent dwelling of the PD solution in order to maintain low concentrations of blood glutamate.

Blocking adenosine A2A receptor reduces peritoneal fibrosis in two independent experimental models

BACKGROUND Long-term peritoneal dialysis (PD) is associated with peritoneal fibrosis and loss of function. It has been shown that activation of the adenosine A(2A) receptor (A(2A)R) promotes tissue repair, wound healing and extracellular matrix (ECM) production. We have previously shown that adenosine is a potent regulator of inflammation in the peritoneum. In the current study, we explored the role of adenosine and the A(2A)R in two experimental models. METHODS Collagen deposition was evaluated in primary peritoneal fibroblasts following treatment with an A(2A)R agonist and antagonist. In addition, peritoneal fibrosis was induced by i.p. injection of either chlorhexidine gluconate for 2 weeks or 4.25% glucose peritoneal dialysis fluid (PDF) for 1 month. The development of fibrosis was compared between wild-type (WT) and WT mice treated with caffeine (an A(2A)R antagonist) in drinking water or between (A(2A)R(+/+)) mice and A(2A)R-deficient mice (A(2A)R(-/-)). RESULTS Adenosine or the A(2A)R agonist CGS21680 stimulated collagen production by peritoneal fibroblasts in vitro and A(2A)R antagonists (ZM241385 and caffeine) blocked this effect. Consistent with these results, caffeine-treated WT or A(2A)R(-/-) mice had reduced submesothelial thickness, collagen deposition and mRNA levels of fibroblast-specific protein (FSP-1) and connective tissue growth factor (CTGF). In addition, treatment with caffeine in vitro and in vivo diminished A(2A)R and A(2B)R mRNA levels induced by CG or PDF while it upregulated A(1)R levels. CONCLUSION Our data suggest that adenosine through its A(2A)R promotes peritoneal fibrosis and therefore should be considered as a target for pharmacological intervention.

Differentiation between viral and bacterial acute infections using chemiluminescent signatures of circulating phagocytes.

Oftentimes the etiological diagnostic differentiation between viral and bacterial infections is problematic, while clinical management decisions need to be made promptly upon admission. Thus, alternative rapid and sensitive diagnostic approaches need to be developed. Polymorphonuclear leukocytes (PMNs) or phagocytes act as major players in the defense response of the host during an episode of infection, and thereby undergo functional changes that differ according to the infections. PMNs functional activity can be characterized by quantification and localization of respiratory burst production and assessed by chemiluminescent (CL) byproduct reaction. We have assessed the functional states of PMNs of patients with acute infections in a luminol-amplified whole blood system using the component CL approach. In this study, blood was drawn from 69 patients with fever (>38 °C), and diagnosed as mainly viral or bacterial infections in origin. Data mining algorithms (C4.5, Support Vector Machines (SVM) and Naïve Bayes) were used to induce classification models to distinguish between clinical groups. The model with the best predictive accuracy was induced using C4.5 algorithm, resulting in 94.7% accuracy on the training set and 88.9% accuracy on the testing set. The method demonstrated a high predictive diagnostic value and may assist the clinician one day in the distinction between viral and bacterial infections and the choice of proper medication.

The effects of hemodialysis on blood glutamate levels in chronic renal failure: Implementation for neuroprotection ☆

PURPOSE The purpose of the present study is to investigate whether hemodialysis (HD) is effective in lowering blood glutamate levels. In addition, we examined the effect of HD on glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the blood and described the rate and pattern of blood glutamate clearance during HD. MATERIALS AND METHODS Blood samples were taken from 45 patients with stage V chronic kidney disease immediately after initiation of HD and hourly, for a total of 5 blood samples. Samples were sent for determination of glutamate, glucose, GOT, GPT, hemoglobin, hematocrit, urea, and creatinine levels. A blood sample from 25 healthy volunteers without chronic renal failure was used as a control for the determination of baseline blood levels of glutamate, GOT, and GPT. RESULTS Glutamate and GPT levels in patients on HD were higher at baseline compared with healthy controls (P < .001). In the first 3 hours after HD, there was a decrease in blood glutamate levels compared with baseline levels (P < .00001). At the fourth hour, there was an increase in blood glutamate levels compared with the third hour (P < .05). CONCLUSIONS Hemodialysis may be a promising method of reducing blood glutamate levels.

Classification of infectious diseases based on chemiluminescent signatures of phagocytes in whole blood

OBJECTIVES Despite medical advances, infectious diseases are still a major cause of mortality and morbidity, disability and socio-economic upheaval worldwide. Early diagnosis, appropriate choice and immediate initiation of antibiotic therapy can greatly affect the outcome of any kind of infection. Phagocytes play a central role in the innate immune response of the organism to infection. They comprise the first-line of defense against infectious intruders in our body, being able to produce large quantities of reactive oxygen species, which can be detected by means of chemiluminescence (CL). The data preparation approach implemented in this work corresponds to a dynamic assessment of phagocytic respiratory burst localization in a luminol-enhanced whole blood CL system. We have previously applied this approach to the problem of identifying various intra-abdominal pathological processes afflicting peritoneal dialysis patients in the Nephrology department and demonstrated 84.6% predictive accuracy with the C4.5 decision-tree algorithm. In this study, we apply the CL-based approach to a larger sample of patients from two departments (Nephrology and Internal Medicine) with the aim of finding the most effective and interpretable feature sets and classification models for a fast and accurate identification of several infectious diseases. MATERIALS AND METHODS Whole blood samples were collected from 78 patients (comprising 115 instances) with respiratory infections, infections associated with renal replacement therapy and patients without infections. CL kinetic parameters were calculated for each case, which was assigned into a specific clinical group according to the available clinical diagnostics. Feature selection wrapper and filter methods were applied to remove the irrelevant and redundant features and to improve the predictive performance of disease classification algorithms. Three data mining algorithms, C4.5 (J48) decision tree, support vector machines and naive Bayes classifier were applied for inducing disease classification models and their performance in classifying three clinical groups was evaluated by 10 runs of a stratified 10-fold cross-validation. RESULTS AND CONCLUSIONS The results demonstrate that the predictive power of the best models obtained with the three evaluated algorithms after feature selection was found to be in the range of 63.38 ± 2.18-70.68 ± 1.43%. The highest disease classification accuracy was reached by C4.5, which also provides the most informative model in the form of a decision tree, and the lowest accuracy was obtained with naive Bayes. The feature selection method attaining the best classification performance was the wrapper method in forward direction. Moreover, the classification models exposed biological patterns specific to the clinical states and the predictive features selected were found to be characteristic of a specific disorder. Based on these encouraging results, we believe that the CL-based data pre-processing approach combined with the wrapper forward feature selection procedure and the C4.5 decision-tree algorithm has a clear potential to become a fast, informative, and sensitive tool for predictive diagnostics of infectious diseases in clinics.

Severe Coombs'-negative autoimmune hemolytic anemia in a kidney transplant patient.

Background/Aim: Few cases are found in the literature regarding autoimmune hemolytic anemia which is Coombs’ test positive in kidney transplant patients, although hemolytic uremic syndrome due to cyclosporin and FK506 has been well described. In the following, we describe a case of severe life-threatening Coombs’ test negative autoimmune hemolytic anemia after kidney transplantation. Methods: Soon after undergoing renal transplantation, the patient presented with hemolytic anemia. Kidney biopsy, routine Coombs’ test, gel filtration and flow-cytometric assay were undertaken. Results: Kidney biopsy ruled out hemolytic uremic syndrome; although Coombs’ test and gel filtration assay were negative, flow cytometry revealed circulating antierythrocytic autoantibodies. Conclusions: Our findings indicate that flow cytometry may be an efficient method in the diagnosis of hemolysis of unknown origin in transplant patients. We further hypothesize that the underlying mechanism of autoimmune hemolytic anemia is related to the passenger B lymphocytes in the graft.

Dynamic Component Chemiluminescent Sensor for Assessing Circulating Polymorphonuclear Leukocyte Activity of Peritoneal Dialysis Patients

Recurrent bacterial peritonitis is a major complication in peritoneal dialysis (PD) patients, which is associated with polymorphonuclear leukocyte (PMN) functional changes and can be assessed by a chemiluminescent (CL) reaction. We applied a new approach of a dynamic component chemiluminescence sensor for the assessment of functional states of PMNs in a luminol-amplified whole-blood system. This method is based on the evaluation of CL kinetic patterns of stimulated PMNs, while the parallel measurements of intracellular and extracellular production of reactive oxygen species (ROS) from the same sample can be conducted. Blood was drawn from diabetic and nondiabetic patients during follow-up, and during peritonitis. Healthy medical personnel served as the control group. Chemiluminescence curves were recorded and presented as a sum of three biological components. CL kinetic parameters were calculated, and functional states of PMNs were assessed. Data mining algorithms were used to build decision tree models that can distinguish between different clinical groups. The induced classification models were used afterward for differentiating and classifying new blind cases and demonstrated good correlation with medical diagnosis (84.6% predictive accuracy). In conclusion, this novel method shows a high predictive diagnostic value and may assist in detection of PD-associated clinical states.