Longitudinal analysis of microcirculatory parameters in gingival tissues after tooth extraction in patients with different risk profiles for wound healing disorders - a pilot study.

OBJECTIVES: We aimed to establish a risk profile for intraoral wound healing disorders based on measurements of microcirculation in gingival tissues. MATERIALS AND METHODS: Oxygen saturation (SO2) and blood flow in gingival tissues were measured with tissue spectrometry and laser doppler spectroscopy in 37 patients before/after tooth extractions. Patients were assigned to four groups: anamnestically and periodontally healthy patients (n = 7), anamnestically healthy but suffering from periodontitis (n = 10), anamnestically healthy but smoking and suffering from periodontitis (n = 10) and suffering from diabetes and periodontitis (n = 10). Measurements were performed at three different time points: Baseline measurement (T0), one day post extractionem (p.e.) (T1) and seven days p.e. (T2). RESULTS: Baseline SO2 values were higher in control patients (p = .038). This effect was most evident in comparison to smokers suffering from periodontitis (p = .042), followed by diabetics suffering from periodontitis (p = .09). An opposite trend was seen for blood flow. Patients suffering from periodontitis demonstrated higher blood flow values (p = .012). Five patients, which belonged to the group of smokers suffering from periodontitis, showed clinically a delayed wound healing. CONCLUSION: Differences in SO2 and blood flow of gingival tissue could be detected in different groups of patients with existing periodontitis compared to control patients. CLINICAL RELEVANCE: Lower baseline SO2 values could be a warning signal for possible wound healing disorders after oral surgery.

Tetracycline ameliorates silica-induced pulmonary inflammation and fibrosis via inhibition of caspase-1.

BACKGROUND: Inhalation of dust containing silica particles is associated with severe pulmonary inflammation and lung injury leading to chronic silicosis including fibrotic remodeling of the lung. Silicosis represents a major global health problem causing more than 45.000 deaths per year. The inflammasome-caspase-1 pathway contributes to the development of silica-induced inflammation and fibrosis via IL-1ß and IL-18 production. Recent studies indicate that tetracycline can be used to treat inflammatory diseases mediated by IL-1ß and IL-18. Therefore, we hypothesized that tetracycline reduces silica-induced lung injury and lung fibrosis resulting from chronic silicosis via limiting IL-1ß and IL-18 driven inflammation. METHODS: To investigate whether tetracycline is a therapeutic option to block inflammasome-caspase-1 driven inflammation in silicosis, we incubated macrophages with silica alone or combined with tetracycline. The in vivo effect of tetracycline was determined after intratracheal administration of silica into the mouse lung. RESULTS: Tetracycline selectively blocks IL-1ß production and pyroptotic cell death via inhibition of caspase-1 in macrophages exposed to silica particles. Consistent, treatment of silica-instilled mice with tetracycline significantly reduced pulmonary caspase-1 activation as well as IL-1ß and IL-18 production, thereby ameliorating pulmonary inflammation and lung injury. Furthermore, prolonged tetracycline administration in a model of chronic silicosis reduced lung damage and fibrotic remodeling. CONCLUSIONS: These findings suggest that tetracycline inhibits caspase-1-dependent production of IL-1ß in response to silica in vitro and in vivo. The results were consistent with tetracycline reducing silica-induced pulmonary inflammation and chronic silicosis in terms of lung injury and fibrosis. Thus, tetracycline could be effective in the treatment of patients with silicosis as well as other diseases involving silicotic inflammation.

Characterization of Transverse Aortic Constriction in Mice Based on the Specific Recruitment of Leukocytes to the Hypertrophic Myocardium and the Aorta Ascendens.

Transverse aortic constriction (TAC) is a model that mimics pressure overload-induced left ventricular (LV) hypertrophy in mice. Alterations in immune cell functionality can promote cardiac and vascular remodeling. In the present study, we characterized the time course in innate immune cell dynamics in response to TAC in the different tissues of mice. It was determined whether TAC induces a characteristic leukocyte-driven immune response in the myocardium, aorta ascendens and descendens, spleen, blood, and draining lymph nodes supported by cytokine-driven chemotaxis in mice at 3, 6, and 21 days following surgery. We used complex flow cytometry staining combinations to characterize the various innate immune cell subsets and a multiplex array to determine cytokine concentrations in the serum. The results of the current study indicated that leukocytes accumulate in the myocardium and aorta ascendens in response to TAC. The leukocyte dynamics in the myocardium were dominated by the Ly6Clow macrophages with an early accumulation, whereas the response in the aorta ascendens was characterized by a long-lasting proinflammatory phenotype driven by Ly6Chigh macrophages, neutrophils, and activated DCs. In contrast to the high-pressure environment of the aorta ascendens, the tissue of the aorta descendens did not react to TAC with any leukocyte increase. The levels of proinflammatory cytokines in the blood were elevated in response to TAC, indicating a systemic reaction. Moreover, our findings strongly suggest that cardiac macrophages could origin from splenic pools and reach the site of the inflammation via the blood. Based on the current findings, it can be concluded that the high-pressure conditions in the aorta ascendens cause a characteristic immune response, dominated by the accumulation of leukocytes and the activation of DCs that varies in comparison to the immune cell dynamics in the myocardium and the aorta descendens.

From bench to bar side: Evaluating the red wine storage lesion.

Vitally essential red fluids like packed cells and red wine are seriously influenced in quality when stored over prolonged periods. In the case of red cell concentrates, the resulting storage lesion has particular significance in perioperative medicine. We hypothesized that, in contrast, aging rather improves the properties of red wine in several ways. A translational approach, including (I) in vitro experiments, (II) a randomized, blinded crossover trial of acute clinical effects, and (III) a standardized red wine blind tasting was used. Three monovarietal wines (Cabernet Sauvignon, Chianti, Shiraz) in three different vintages (range 2004-2016), each 5 years different, were assessed. Assessments were performed at a German university hospital (I, II) and on a garden terrace during a mild summer evening (III). Young wines induced cell stress and damage while significantly reducing cytoprotective proteins in HepG2 hepatoma cells. Sympathetic activity and multitasking skills were altered depending on wines' ages. Hangovers tended to be aggravated by young red wine. Aged variants performed better in terms of aroma and overall quality but worse in optical appearance. We found no evidence for a red wine storage lesion. However, we plead for consensus-based guidelines for proper storage, as it is common in clinical medicine.

Vancomycin and daptomycin modulate the innate immune response in a murine model of LPS-induced sepsis.

Sepsis is a leading cause of death worldwide, despite the use of multimodal therapies. Common antibiotic regimens are being affected by a rising number of multidrug-resistant pathogens, and new therapeutic approaches are therefore needed. Antibiotics have immunomodulatory properties which appear to be beneficial in the treatment of sepsis. We hypothesized that the last-resort antibiotics vancomycin (VAN) and daptomycin (DMC) modulate cell migration, phagocytosis, and protein cytokine levels in a murine model of lipopolysaccharide (LPS)-induced sepsis. Ten to twelve-week-old C57BL/6 mice (n = 4-6 animals per group) were stimulated with LPS for 20 h, followed by the administration of VAN or DMC. The outcome parameters were leukocyte accumulation and effector function. Quantification of the immune cells in the peritoneal lavage was performed using flow cytometry analysis. Phagocytosis was measured using pHrodo E. coli BioParticles. The response of the cytokines TNFα, IL-6, and IL-10 was measured in vitro using murine peritoneal macrophages stimulated with LPS and VAN or DMC. VAN decreased both the peritoneal macrophage and the dendritic cell populations following LPS stimulation. DMC reduced the dendritic cell population in the peritoneal cavity in LPS-infected mice. Both antibiotics increased the phagocytic activity in peritoneal macrophages, but this effect was diminished in response to LPS. Phagocytosis of dendritic cells was increased in LPS-infected animals treated with VAN. VAN and DMC differently modulated the levels of pro-and anti-inflammatory cytokines. In a murine model of LPS-induced sepsis, VAN and DMC exhibit immunomodulatory effects on cells involved in innate immunity. The question of whether these antibiotics exhibit synergistic effects in the treatment of septic patients, beyond their bactericidal properties, should be further evaluated in future studies.

Perioperative Vascular Biomarker Profiling in Elective Surgery Patients Developing Postoperative Delirium: A Prospective Cohort Study.

BACKGROUND: Postoperative delirium (POD) ranks among the most common complications in surgical patients. Blood-based biomarkers might help identify the patient at risk. This study aimed to assess how serum biomarkers with specificity for vascular and endothelial function and for inflammation are altered, prior to or following surgery in patients who subsequently develop POD. METHODS: This was a study on a subcohort of consecutively recruited elective non-cardiac as well as cardiac surgery patients (age > 60 years) of the single-center PROPDESC trial at a German tertiary care hospital. Serum was sampled prior to and following surgery, and the samples were subjected to bead-based multiplex analysis of 17 serum proteins (IL-3, IL-8, IL-10, Cripto, CCL2, RAGE, Resistin, ANGPT2, TIE2, Thrombomodulin, Syndecan-1, E-Selectin, VCAM-1, ICAM-1, CXCL5, NSE, and uPAR). Development of POD was assessed during the first five days after surgery, using the Confusion Assessment Method for ICU (CAM-ICU), the CAM, the 4-'A's test (4AT), and the Delirium Observation Scale (DOS). Patients were considered positive if POD was detected at least once during the visitation period by any of the applied methods. Non-parametric testing, as well as propensity score matching were used for statistical analysis. RESULTS: A total of 118 patients were included in the final analysis; 69% underwent non-cardiac surgery, median overall patient age was 71 years, and 59% of patients were male. In the whole cohort, incidence of POD was 28%. The male gender was significantly associated with the development of POD (p = 0.0004), as well as a higher ASA status III (p = 0.04). Incidence of POD was furthermore significantly increased in cardiac surgery patients (p = 0.002). Surgery induced highly significant changes in serum levels of almost all biomarkers except uPAR. In preoperative serum samples, none of the analyzed parameters was significantly altered in subsequent POD patients. In postoperative samples, CCL2 was significantly increased by a factor of 1.75 in POD patients (p = 0.03), as compared to the no-POD cohort. Following propensity score matching, CCL2 remained the only biomarker that showed significant differences in postoperative values (p = 0.01). In cardiac surgery patients, postoperative CCL2 serum levels were more than 3.5 times higher than those following non-cardiac surgery (p < 0.0001). Moreover, after cardiac surgery, Syndecan-1 serum levels were significantly increased in POD patients, as compared to no-POD cardiac surgery patients (p = 0.04). CONCLUSIONS: In a mixed cohort of elective non-cardiac as well as cardiac surgery patients, preoperative serum biomarker profiling with specificity for vascular dysfunction and for systemic inflammation was not indicative of subsequent POD development. Surgery-induced systemic inflammation-as evidenced by the significant increase in CCL2 release-was associated with POD, particularly following cardiac surgery. In those patients, postoperative glycocalyx injury might furthermore contribute to POD development.

Inhibition of Caspase-1 with Tetracycline Ameliorates Acute Lung Injury.

Rationale: Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with a mortality of up to 40%. Precision medicine approaches targeting patients on the basis of their molecular phenotypes of ARDS might help to identify effective pharmacotherapies. The inflammasome-caspase-1 pathway contributes to the development of ARDS via IL-1ß and IL-18 production. Recent studies indicate that tetracycline can be used to treat inflammatory diseases mediated by IL-1ß and IL-18, although the molecular mechanism by which tetracycline inhibits inflammasome-caspase-1 signaling remains unknown. Objectives: To identify patients with ARDS characterized by IL-1ß and IL-18 expression and investigate the ability of tetracycline to inhibit inflammasome-caspase-1 signaling in ARDS. Methods: IL-1ß and IL-18 concentrations were quantified in BAL fluid from patients with ARDS. Tetracycline's effects on lung injury and inflammation were assessed in two mouse models of direct (pulmonary) acute lung injury, and its effects on IL-1ß and IL-18 production were assessed by alveolar leukocytes from patients with direct ARDS ex vivo. Murine macrophages were used to further characterize the effect of tetracycline on the inflammasome-caspase-1 pathway. Measurements and Main Results: BAL fluid concentrations of IL-1ß and IL-18 are significantly higher in patients with direct ARDS than those with indirect (nonpulmonary) ARDS. In experimental acute lung injury, tetracycline significantly diminished lung injury and pulmonary inflammation by selectively inhibiting caspase-1-dependent IL-1ß and IL-18 production, leading to improved survival. Tetracycline also reduced the production of IL-1ß and IL-18 by alveolar leukocytes from patients with direct ARDS. Conclusions: Tetracycline may be effective in the treatment of direct ARDS in patients with elevated caspase-1 activity. Clinical Trial registered with www.clinicaltrials.gov (NCT04079426).

Gene expression in the Angiopoietin/TIE axis is altered in peripheral tissue of ovarian cancer patients: A prospective observational study.

AIMS: Clinical studies suggest altered systemic vascular biology in cancer patients. We assessed expression patterns of endothelial activation- and vascular leakage-related genes in tumor as well as in tumor-free peripheral tissues from patients with and without ovarian cancer (OC). MAIN METHODS: Patients being scheduled for laparotomy for either gynecologic benign diagnosis (n = 10) or for advanced-stage OC (n = 22) were prospectively recruited to this observational study. Serum samples were taken preoperatively, and tissue samples were taken from peripheral abdominal wall musculature, tumor-free peritoneum and the tumor itself. KEY FINDINGS: Patients in OC group received significantly more fluid per time intraoperatively (p = 0.01). IL-8 and MCP-1/CCL2, VCAM-1 (CD 106) and ICAM-1 (CD 54) as well as Thrombomodulin were significantly increased in cancer patients' serum at baseline (p = 0.03). Expression of distinct vascular leakage-related genes (Angiopoietin-1 (ANG-1), ANG-2, TIE2, VEGFR1, VEGFR2) was significantly altered in tumor tissue of OC patients (p = 0.003), while in tumor-free peritoneal tissue, ANG-2/1 expression ratio was more than doubled in OC group (p = 0.03). In peripheral musculature, particularly genes from the ANG/TIE axis were significantly changed in OC patients (p = 0.005), suggesting a distinct vascular leakage-related genotype. Gene expression changes in OC patients were significantly associated with the postoperative fluid balance (p = 0.03). SIGNIFICANCE: Altered expression of barrier dysfunction- and angiogenesis-associated genes from the ANG/TIE axis was detected not only in tumor but also in peripheral tissues of cancer patients. This may contribute to a systemic vascular leakage-related genotype.

CX3CR1 is a prerequisite for the development of cardiac hypertrophy and left ventricular dysfunction in mice upon transverse aortic constriction.

The CX3CL1/CX3CR1 axis mediates recruitment and extravasation of CX3CR1-expressing subsets of leukocytes and plays a pivotal role in the inflammation-driven pathology of cardiovascular disease. The cardiac immune response differs depending on the underlying causes. This suggests that for the development of successful immunomodulatory therapy in heart failure due to chronic pressure overload induced left ventricular (LV) hypertrophy, the underlying immune patterns must be examined. Here, the authors demonstrate that Fraktalkine-receptor CX3CR1 is a prerequisite for the development of cardiac hypertrophy and left ventricular dysfunction in a mouse model of transverse aortic constriction (TAC). The comparison of C57BL/6 mice with CX3CR1 deficient mice displayed reduced LV hypertrophy and preserved cardiac function in response to pressure overload in mice lacking CX3CR1. Moreover, the normal immune response following TAC induced pressure overload which is dominated by Ly6Clow macrophages changed to an early pro-inflammatory immune response driven by neutrophils, Ly6Chigh macrophages and altered cytokine expression pattern in CX3CR1 deficient mice. In this early inflammatory phase of LV hypertrophy Ly6Chigh monocytes infiltrated the heart in response to a C-C chemokine ligand 2 burst. CX3CR1 expression impacts the immune response in the development of LV hypertrophy and its absence has clear cardioprotective effects. Hence, suppression of CX3CR1 may be an important immunomodulatory therapeutic target to ameliorate pressure-overload induced heart failure.

CpG postconditioning after reperfused myocardial infarction is associated with modulated inflammation, less apoptosis, and better left ventricular function.

Postconditioning attenuates inflammation and fibrosis in myocardial infarction (MI). The aim of this study was to investigate whether postconditioning with the cytosine-phosphate-guanine (CpG)-containing Toll-like receptor-9 (TLR9) ligand 1668-thioate (CpG) can modulate inflammation and remodeling in reperfused murine MI. Thirty minutes of left descending coronary artery (LAD) occlusion was conducted in 12-wk-old C57BL/6 mice. Mice were treated with CpG intraperitoneally 5 min before reperfusion. The control group received PBS; the sham group did not undergo ischemia. M-mode echocardiography (3, 7, and 28 days) and Millar left ventricular (LV) catheterization were performed (7 and 28 days) before the hearts were excised and harvested for immunohistochemical (6 h, 24 h, 3 days, 7 days, and 28 days), gene expression (6 h, 24 h, and 3 days; Taqman RT-qPCR), protein, and FACS analysis (24 h and 3 days). Mice treated with CpG showed significantly better LV function after 7 and 28 days of reperfusion. Protein and mRNA expressions of proinflammatory and anti-inflammatory cytokines were significantly induced after CpG treatment. Histology revealed fewer macrophages in CpG mice after 24 h, confirmed by FACS analysis with a decrease in both classically M1- and alternative M2a-monocytes. CpG treatment reduced apoptosis and cardiomyocyte loss and was associated with induction of adaptive mechanisms, e.g., of heme-oxigenase-1 and ß-/α-myosin heavy chain (MHC) ratio. Profibrotic markers collagen type Iα (Col-Ια) and Col-III induction was abrogated in CpG mice, accompanied by fewer myofibroblasts. This led to the formation of a smaller scar. Differential matrix metalloproteinase (MMP)/tissue inhibitor of metalloproteinase (TIMP) expression contributed to attenuated remodeling in CpG, resulting in preserved cardiac function in a Toll-like receptor 1- and TLR9-dependent manner. Our study suggests a cardioprotective mechanism of CpG postconditioning, involving Toll-like receptor-driven modulation of inflammation. This is followed by attenuated remodeling and preserved LV function.NEW & NOTEWORTHY Cytosine-phosphate-guanine (CpG) postconditioning seems to mediate inflammation via Toll-like receptor-1 and Toll-like receptor-9 signaling. Enhanced cytokine and chemokine expressions are partly attenuated by IL-10 and matrix metalloproteinase-8 (MMP8) induction, being associated with lower macrophage infiltration and M1-monocyte differentiation. Furthermore, switch from α- to ß-MHC and balanced MMP/TIMP expression led to lesser cardiomyocyte apoptosis, smaller scar size, and preserved cardiac function. Data of pharmacological postconditioning have been widely disappointing to date. Our study suggests a new pathway promoting myocardial postconditioning via Toll-like receptor activation.

Sustained Immunoparalysis in Endotoxin-Tolerized Monocytic Cells.

Sepsis is associated with a strong inflammatory reaction triggering a complex and prolonged immune response. Septic patients have been shown to develop sustained immunosuppression due to a reduced responsiveness of leukocytes to pathogens. Changes in cellular metabolism of leukocytes have been linked to this phenomenon and contribute to the ongoing immunological derangement. However, the underlying mechanisms of these phenomena are incompletely understood. In cell culture models, we mimicked LPS tolerance conditions to provide evidence that epigenetic modifications account for monocyte metabolic changes which cause immune paralysis in restimulated septic monocytes. In detail, we observed differential methylation of CpG sites related to metabolic activity in human PBMCs 18 h after septic challenge. The examination of changes in immune function and metabolic pathways was performed in LPS-tolerized monocytic THP-1 cells. Passaged THP-1 cells, inheriting initial LPS challenge, presented with dysregulation of cytokine expression and oxygen consumption for up to 7 days after the initial LPS treatment. Proinflammatory cytokine concentrations of TNFα and IL1ß were significantly suppressed following a second LPS challenge (p < 0.001) on day 7 after first LPS stimulation. However, the analysis of cellular metabolism did not reveal any noteworthy alterations between tolerant and nontolerant THP-1 monocytes. No quantitative differences in ATP and NADH synthesis or participating enzymes of energy metabolism occurred. Our data demonstrate that the function and epigenetic modifications of septic and tolerized monocytes can be examined in vitro with the help of our LPS model. Changes in CpG site methylation and monocyte function point to a correlation between epigenetic modification in metabolic pathways and reduced monocyte function under postseptic conditions.

Differential modulation of endothelial cell function by fresh frozen plasma.

AIMS: In vivo studies suggest a positive influence of fresh frozen plasma (FFP) on endothelial properties and vascular barrier function, leading to improved outcomes in animal sepsis models as well as in major abdominal surgery. However, those effects are incompletely described. It was our aim to evaluate in vitro effects of FFP on endothelial key functions and to identify underlying mechanisms. MATERIALS AND METHODS: Human pulmonary microvascular endothelial cells (HPMECs) were prestimulated with LPS, followed by incubation with FFP. Permeability for FITC-dextran was assessed, and intercellular gap formation was visualized. NF-κB nuclear translocation and expression of pro-inflammatory, pro-adhesion, and leakage-related genes were evaluated, and monocyte adhesion to ECs was assessed. Intracellular cAMP levels as well as phosphorylation of functional proteins were analyzed. In patients undergoing major abdominal surgery, Syndecan-1 serum levels were assessed prior to and following FFP transfusion. KEY FINDINGS: Post-incubation of HPMVECs with FFP increased intracellular cAMP levels that had been decreased by preceding LPS stimulation. On one hand, this reduced endotoxin-mediated upregulation of IL-8, ICAM-1, VCAM-1, VEGF, and ANG-2. Impaired phosphorylation of functional proteins was restored, and intercellular cohesion and barrier function were rescued. On the other hand, NF-κB nuclear translocation as well as monocyte adhesion was markedly increased by the combination of LPS and FFP. Syndecan-1 serum levels were lower in surgery patients that were transfused with FFP compared to those that were not. SIGNIFICANCE: Our data provide evidence for a differential modulation of crucial endothelial properties by FFP, potentially mediated by elevation of intracellular cAMP levels.

Dynamic changes of angiopoietins and endothelial nitric oxide supply during fluid resuscitation for major gyn-oncological surgery: a prospective observation.

BACKGROUND: Despite goal-directed hemodynamic therapy, vascular function may deteriorate during surgery for advanced abdominal tumor masses. Fluid administration has been shown to be associated with distinct changes in serum levels of functional proteins. We sought to determine how serum total protein and angiopoietin (ANG) levels change during major abdominal tumor surgery. In addition, ex vivo endothelial nitric oxide synthase (eNOS) activation as well as NO bioavailability in vivo were assessed. METHODS: 30 patients scheduled for laparotomy for late-stage ovarian or uterine cancer were prospectively included. Advanced hemodynamic monitoring as well as protocol-driven goal-directed fluid optimization were performed. Total serum protein, ANG-1, -2, and soluble TIE2 were determined pre-, intra-, and postoperatively. Phosphorylation of eNOS was assessed in microvascular endothelial cells after incubation with patient serum, and microvascular reactivity was determined in vivo by near-infrared spectroscopy and arterial vascular occlusion. RESULTS: Cardiac output as well as preload gradually decreased during surgery and were associated with a median total fluid intake of 12.8 (9.7-15.4) mL/kg*h and a postoperative fluid balance of 6710 (4113-9271) mL. Total serum protein decreased significantly from baseline (66.5 (56.4-73.3) mg/mL) by almost half intraoperatively (42.7 (36.8-51.5) mg/mL, p < 0.0001) and remained at low level. While ANG-1 showed no significant dilutional change (baseline: 12.7 (11.9-13.9) ng/mL, postop.: 11.6 (10.8 -13.5) ng/mL, p = 0.06), serum levels of ANG-2 were even increased postoperatively (baseline: 2.2 (1.6-2.6) ng/mL vs. postop.: 3.4 (2.3-3.8) ng/mL, p < 0.0001), resulting in a significant shift in ANG-2 to ANG-1 ratio. Ex vivo phosphorylation of eNOS was decreased depending on increased ANG-2 levels and ANG-2/1 ratio (Spearman r = - 0.37, p = 0.007). In vivo, increased ANG-2 levels were associated with impaired capillary recruitment and NO bioavailability (Spearman r = - 0.83, p = 0.01). CONCLUSIONS: Fluid resuscitation-associated changes in serum vascular mediator profile during abdominal tumor surgery were accompanied by impaired eNOS activity ex vivo as well as reduced NO bioavailability in vivo. Our results may explain disturbed microvascular function in major surgery despite goal-directed hemodynamic optimization.

Network of Mediators for Vascular Inflammation and Leakage Is Dysbalanced during Cytoreductive Surgery for Late-Stage Ovarian Cancer.

BACKGROUND: Cytoreductive surgery (CS) in late-stage ovarian cancer patients is often challenging due to extensive volume shifts, and high fluid intake may provoke postoperative complications. Expression of vasoactive mediators is altered in cancer patients, which may affect systemic vascular function. We sought to assess how serum levels of vasoactive markers and mediators change during CS in ovarian cancer. METHODS: Following IRB approval and informed consent, pre- and postoperative serum samples were analyzed in 26 late-stage ovarian cancer patients using multiplex protein arrays and ELISA. RESULTS: The proinflammatory cytokines and chemokines IL-6, IL-8, and CCL2 were significantly elevated after 24 hrs compared to the baseline values, with IL-6 and IL-8 being most prominently increased. While ANGPT1 remained unchanged after surgery, its competitive antagonist ANGPT2 was significantly increased. In contrast, serum levels of the ANGPT receptor TIE2 were decreased to 0.6 of the baseline values. While VEGF-D, E-selectin, P-selectin, ICAM-1, and PECAM-1 remained unchanged, serum activity of both thrombomodulin and syndecan-1 was significantly increased following surgery. CONCLUSION: We identified a regulatory network of acute-phase reaction during CS in late-stage ovarian cancer. This suggests that IL-6 exerts positive regulation of other proinflammatory mediators and, by upregulating ANGPT2 and suppressing ANGPT1, induces a serum profile that promotes vascular leakage. This may contribute to the observed hemodynamic alterations during CS procedures.

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Myocardial maladaptation to pressure overload in CB2 receptor-deficient mice.

BACKGROUND: Adaptation to aortic valve stenosis leads to myocardial hypertrophy, which has been associated with inflammation, fibrosis and activation of the endocannabinoid system. Since the endocannabinoid system and the CB2 receptor provide cardioprotection and modulate immune response in experimental ischemia, we investigated the role of CB2 in a mouse model of cardiac pressure overload. METHODS: Transverse aortic constriction was performed in CB2 receptor-deficient (Cnr2-/-) mice and their wild-type littermates (Cnr2+/+). After echocardiography and Millar left heart catheter hemodynamic evaluation hearts were processed for histological, cellular and molecular analyses. RESULTS: The endocannabinoid system showed significantly higher anandamide production and CB2 receptor expression in Cnr2+/+ mice. Histology showed non-confluent, interstitial fibrosis with rare small areas of cardiomyocyte loss in Cnr2+/+ mice. In contrast, extensive cardiomyocyte loss and confluent scar formation were found in Cnr2-/- mice accompanied by significantly increased apoptosis and left ventricular dysfunction when compared with Cnr2+/+ mice. The underlying cardiac maladaptation in Cnr2-/- mice was associated with significantly reduced expression of myosin heavy chain isoform beta and less production of heme oxygenase-1. Cnr2-/- hearts presented after 7 days with stronger proinflammatory response including significantly higher TNF-alpha expression and macrophage density, but lower density of CD4+ and B220+ cells. At the same time, we found increased apoptosis of macrophages and adaptive immune cells. Higher myofibroblast accumulation and imbalance in MMP/TIMP-regulation indicated adverse remodeling in Cnr2-/- mice. CONCLUSIONS: Our study provides mechanistic evidence for the role of the endocannabinoid system in myocardial adaptation to pressure overload in mice. The underlying mechanisms include production of anandamide, adaptation of contractile elements and antioxidative enzymes, and selective modulation of immune cells action and apoptosis in order to prevent the loss of cardiomyocytes.

Temporal profile of serum mitochondrial DNA (mtDNA) in patients with aneurysmal subarachnoid hemorrhage (aSAH).

Aneurysmal subarachnoid hemorrhage (aSAH) is a highly complex disease. Majority of aSAH survivors confront post-SAH complications including cerebral vasospasm (CVS) and delayed cerebral ischemia (DCI) that mainly influence the clinical outcome. Tissue damage during early brain injury may lead to release of damage associated molecular pattern molecules (DAMPs) that may initiate and sustain inflammation during the course of aSAH through activation of pattern recognition receptors. Mitochondrial DNA (mtDNA) due to unmethylated CpG motifs acts as a DAMP via binding to toll-like receptor-9. The aim of this study was to investigate the cell free circulating mtDNA in the systemic circulation of aSAH patients and its association with post-SAH complications and clinical outcome. The DNA was extracted from the serum of 80 aSAH patients at days 1, 3, 5, 7, 9, 11, 13 and from 18 healthy controls. Three representative mitochondrial gene fragments including Cytochrome B (CytB), D-Loop and Cytochrome c oxidase subunit-1 (COX-1) were quantified using a Taqman-probes based qPCR. Levels of mtDNA were quantified from standard curves generated using mtDNA extracted from HepG2 cell mitochondria. Clinical outcome of the patients was assessed by Glasgow outcome scale (GOS) and modified Rankin scale (mRS). Clinical data and post-SAH complications were recorded from patient's record file. Serum D-Loop and COX-1 were significantly elevated early after aSAH and remained high over first 2 weeks. CytB levels were however, initially unchanged but elevated later at day 7 as compared to healthy controls. Cumulative levels measured over two weeks showed significant correlations with post-SAH complications including a negative correlation of D-Loop with pneumonia infection, hydrocephalus and occurrence of epilepsy, a positive correlation of Cyt B with occurrence of CVS and a negative correlation of COX-1 with occurrence of systemic infections and seizures. Cumulative D-Loop values negatively correlated with clinical outcome. Our data suggest that mtDNA may directly or indirectly influence post-SAH complications and clinical outcome.

Experimental murine acute lung injury induces increase of pulmonary TIE2-expressing macrophages.

BACKGROUND: Breakdown of the alveolo-capillary wall is pathognomonic for Acute Lung Injury (ALI). Angiopoietins, vascular-specific growth factors, are linked to endothelial barrier dysfunction, and elevated Angiopoietin-2 (ANG2) levels are associated with poor outcome of ALI patients. Specialized immune cells, referred to as 'TIE2-expressing monocytes and macrophages' (TEM), were shown to specifically respond to ANG2 binding. However, their involvement in acute inflammatory processes is so far completely undescribed. Thus, our aim was to assess the dynamics of TEMs in a murine model of ALI. RESULTS: Intratracheal instillation of LPS induced a robust pulmonary pro-inflammatory response with endothelial barrier dysfunction and significantly enhanced ANG2 expression. The percentage number of TEMs, assessed by FACS analysis, was more than trebled compared to controls, with TEM count in lungs reaching more than 40% of all macrophages. Such distinct dynamic was absent in all other analyzed compartments (alveolar space, spleen, blood). Incubation of the monocytic cell line THP-1 with LPS or TNF-α resulted in a dose-dependent, significant upregulation of TIE2, suggesting that not recruitment from extra-pulmonary compartments but TIE2 upregulation in resident macrophages accounts for increased lung TEM frequencies. CONCLUSIONS: For the first time, our data provide evidence that the activity of TEMs changes at sites of acute inflammation.

Synthetic CpG oligonucleotides induce a genetic profile ameliorating murine myocardial I/R injury.

We previously demonstrated that pre-conditioning with CpG oligonucleotide (ODN) 1668 induces quick up-regulation of gene expression 3 hours post-murine myocardial ischaemia/reperfusion (I/R) injury, terminating inflammatory processes that sustain I/R injury. Now, performing comprehensive microarray and biocomputational analyses, we sought to further enlighten the "black box" beyond these first 3 hours. C57BL/6 mice were pretreated with either CpG 1668 or with control ODN 1612, respectively. Sixteen hours later, myocardial ischaemia was induced for 1 hour in a closed-chest model, followed by reperfusion for 24 hours. RNA was extracted from hearts, and labelled cDNA was hybridized to gene microarrays. Data analysis was performed with BRB ArrayTools and Ingenuity Pathway Analysis. Functional groups mediating restoration of cellular integrity were among the top up-regulated categories. Genes known to influence cardiomyocyte survival were strongly induced 24 hours post-I/R. In contrast, proinflammatory pathways were down-regulated. Interleukin-10, an upstream regulator, suppressed specifically selected proinflammatory target genes at 24 hours compared to 3 hours post-I/R. The IL1 complex is supposed to be one regulator of a network increasing cardiovascular angiogenesis. The up-regulation of numerous protective pathways and the suppression of proinflammatory activity are supposed to be the genetic correlate of the cardioprotective effects of CpG 1668 pre-conditioning.

Impaired liver regeneration after hepatectomy and bleeding is associated with a shift from hepatocyte proliferation to hypertrophy.

Extensive liver resections are common, and bleeding is frequent in these operations. Impaired regeneration after partial hepatectomy (PHx) may contribute to liver failure. We thus assessed the impact of acute bleeding on the liver regeneration progress after PHx and explored possible contributing molecular mechanisms. In rats, the regeneration progress was delayed and attenuated with PHx and bleeding and was not restored with colloid resuscitation. Livers restored their initial volume by postoperative day (POD) 2 after PHx through hepatocyte proliferation vs. POD 4 in the PHx and bleeding group, primarily by hepatocyte hypertrophy. With bleeding, hepatocyte proliferation was hindered in two mechanisms: by inhibiting cells from starting proliferation and by causing hindrance in G1/S progression. Liver hypoxia was prominent, with significant prolonged up-regulation of hypoxia-inducible factors (HIF) and HIF-targeted genes only in the PHx and bleeding group. Gene expression profiling revealed alterations in numerous genes that belong to critical pathways, including cell cycle, DNA replication, PI3K-Akt, purine, and pyrimidine metabolism. Because liver surgery is frequently performed in patients with a predamaged liver, an improper regenerative process after PHx and bleeding might lead to decompensation. The results hint at specific pathways to target in order to improve liver regeneration during PHx and bleeding.-Matot, I., Nachmansson, N., Duev, O., Schulz, S., Schroeder-Stein, K., Frede, S., Abramovitch, R. Impaired liver regeneration after hepatectomy and bleeding is associated with a shift from hepatocyte proliferation to hypertrophy.