Fibrin, Bone Marrow Cells and Macrophages Interactively Modulate Cardiomyoblast Fate.

Interactions between macrophages, cardiac cells and the extracellular matrix are crucial for cardiac repair following myocardial infarction (MI). We hypothesized that cell-based treatments might modulate these interactions. After validating that bone marrow cells (BMC) associated with fibrin lowered the infarct extent and improved cardiac function, we interrogated the influence of fibrin, as a biologically active scaffold, on the secretome of BMC and the impact of their association on macrophage fate and cardiomyoblast proliferation. In vitro, BMC were primed with fibrin (F-BMC). RT-PCR and proteomic analyses showed that fibrin profoundly influenced the gene expression and the secretome of BMCs. Consequently, the secretome of F-BMC increased the spreading of cardiomyoblasts and showed an alleviated immunomodulatory capacity. Indeed, the proliferation of anti-inflammatory macrophages was augmented, and the phenotype of pro-inflammatory switched as shown by downregulated Nos2, Il6 and IL1b and upregulated Arg1, CD163, Tgfb and IL10. Interestingly, the secretome of F-BMC educated-macrophages stimulated the incorporation of EdU in cardiomyoblasts. In conclusion, our study provides evidence that BMC/fibrin-based treatment improved cardiac structure and function following MI. In vitro proofs-of-concept reveal that the F-BMC secretome increases cardiac cell size and promotes an anti-inflammatory response. Thenceforward, the F-BMC educated macrophages sequentially stimulated cardiac cell proliferation.

Severe portal and systemic acidosis during CO2-laparoscopy compared to helium or gasless laparoscopy and laparotomy in a rodent model: an experimental study.

BACKGROUND AND AIMS: This experimental study assesses the influence of different gases and insufflation pressures on the portal, central-venous and peripheral-arterial pH during experimental laparoscopy. METHODS: Firstly, 36 male WAG/Rij rats were randomized into six groups (n = 6) spontaneously breathing during anaesthesia: laparoscopy using carbon dioxide or helium at 6 and 12 mmHg, gasless laparoscopy and laparotomy. 45 and 90 min after setup, blood was sampled from the portal vein, vena cava and the common femoral artery with immediate blood gas analysis. Secondly, 12 animals were mechanically ventilated at physiological arterial pH during 90 min of laparotomy (n = 6) or carbon dioxide laparoscopy at 12 mmHg (n = 6) with respective blood gas analyses. RESULTS: Over time, in spontaneously breathing rats, carbon dioxide laparoscopy caused significant insufflation pressure-dependent portal acidosis (pH at 6 mmHg, 6.99 [6.95-7.04] at 45 min and 6.95 [6.94-6.96] at 90 min, pH at 12 mmHg, 6.89 [6.82-6.90] at 45 min and 6.84 [6.81-6.87] at 90 min; p < 0.05) compared to laparotomy (portal pH 7.29 [7.23-7.30] at 45 min and 7.29 [7.20-7.30] at 90 min; p > 0.05). Central-venous and peripheral-arterial acidosis was significant but less severely reduced during carbon dioxide laparoscopy. Laparotomy, helium laparoscopy and gasless laparoscopy showed no comparable acidosis in all vessels. Portal and central-venous acidosis during carbon dioxide laparoscopy at 12 mmHg was not reversible by mechanical hyperventilation maintaining a physiological arterial pH (pH portal 6.85 [6.84-6.90] (p = 0.004), central-venous 6.93 [6.90-6.99] (p = 0.004), peripheral-arterial 7.29 [7.29-7.31] (p = 0.220) at 90 min; Wilcoxon-Mann-Whitney test). CONCLUSION: Carbon dioxide laparoscopy led to insufflation pressure-dependent severe portal and less severe central-venous acidosis not reversible by mechanical hyperventilation.

Monthly measurement of high-sensitivity cardiac troponins T and creatine kinase in asymptomatic chronic hemodialysis patients: A one-year prospective study.

BACKGROUND: Cardiology guidelines recommend measuring high-sensitivity cardiac troponin (hs-cTn) for the diagnostic work-up of acute coronary syndromes (ACS). Many hospitals measure hs-cTnT, but preliminary data have shown that hs-cTnT is higher than normal in many hemodialysis patients without evidence of ACS. The purpose of this study was therefore to determine the hs-cTnT levels every month for 1 year in asymptomatic hemodialysis patients, in order to assess their changes over time relative to creatine kinase. METHODS: Fourty-four hemodialysis patients (mean age 67 ± 14 years) were included. The predialysis levels of fifth-generation hs-cTnT, CK, and CK-MB were measured every month for 1 year using a Cobas® 6000 analyzer (Roche Diagnostics, Switzerland). RESULTS: Almost 100% of hs-cTnT measurements were higher than normal (N < 14 ng/L); the mean ± SD annual level was 84 ± 59 ng/L, ranging from a minimum of 24 ± 2 to 241 ± 28 ng/L in individual patients. The mean levels of CK and CK-MB were normal. Thirteen myocardial infarctions were analyzed, which were all associated with an initial elevation in hs-cTnT >45% from the individual baseline value. By comparison, CK and CK-MB only increased in 38% and 31% of these myocardial infarctions, respectively. DISCUSSION: hs-cTnT is persistently higher than normal in chronic hemodialysis patients. Standard algorithms for diagnosing ACS can obviously not be used and alternative diagnostic strategies need to be developed. According to our data, and given the huge variation in baseline hs-cTnT levels among patients, the use of higher cut-offs as proposed in the literature cannot be recommended. Instead, we consider that hs-cTnT should be checked at regular intervals (e.g., every 3-6 months) in order to establish individual baseline levels for hs-cTnT. This approach, in most instances, not only makes it possible to more rapidly rule-in but also to rapidly rule-out, cases of ACS in hemodialysis patients who develop cardiac symptoms.

The "EPiQ"-Study (Evaluation of preanalytical quality): S-Monovette® in manual aspiration mode drastically reduces hemolytic samples in head-to-head study.

BACKGROUND: Hemolytic blood samples are the number one cause for specimen rejection at emergency departments. Triggered by unsuitable blood sampling material or incorrect handling and a related strong vacuum force, hemolytic samples often must be retaken. The objective of this study was to assess whether correct manual aspiration using S-Monovette® could reduce the number of hemolytic samples. METHODS: Between January and April 2019, a head-to-head study was conducted. Whereas in the first eight weeks, all specimens were collected using Vacutainer®, in the second eight weeks, blood was taken using S-Monovette® in aspiration mode. Specimens were categorized into five classes (0-30, 31-50, 51-75, 76-100, and 101+ mg/dl of cell-free hemoglobin) and for the statistical analyses, all samples exceeding 30 mg/dl were classified as hemolytic. RESULTS: Data were collected on 4794 blood specimens (Vacutainer®: 2634 samples, S-Monovette®: 2160 samples). While the percentage of non-hemolytic samples (HI of 0-30 mg/dl) was substantially higher for specimens drawn by S-Monovette® (95.7 %) than Vacutainer® (83.0 %), the opposite was true for all HI categories above 30 mg/dl. Importantly, the reduction of hemolytic samples took place immediately following the imposition of S-Monovette® and remained stable at a low level until the end of the study. CONCLUSIONS: Based on our results, we conclude that switching to S-Monovette® in manual aspiration mode in the blood sampling process could be highly beneficial, not only from a financial point of view, but also with regards to reducing unnecessary tasks and stress for nursing staff and improving patient outcome overall.

Circulating immune cell populations related to primary breast cancer, surgical removal, and radiotherapy revealed by flow cytometry analysis.

BACKGROUND: Advanced breast cancer (BC) impact immune cells in the blood but whether such effects may reflect the presence of early BC and its therapeutic management remains elusive. METHODS: To address this question, we used multiparametric flow cytometry to analyze circulating leukocytes in patients with early BC (n = 13) at the time of diagnosis, after surgery, and after adjuvant radiotherapy, compared to healthy individuals. Data were analyzed using a minimally supervised approach based on FlowSOM algorithm and validated manually. RESULTS: At the time of diagnosis, BC patients have an increased frequency of CD117+CD11b+ granulocytes, which was significantly reduced after tumor removal. Adjuvant radiotherapy increased the frequency of CD45RO+ memory CD4+ T cells and CD4+ regulatory T cells. FlowSOM algorithm analysis revealed several unanticipated populations, including cells negative for all markers tested, CD11b+CD15low, CD3+CD4-CD8-, CD3+CD4+CD8+, and CD3+CD8+CD127+CD45RO+ cells, associated with BC or radiotherapy. CONCLUSIONS: This study revealed changes in blood leukocytes associated with primary BC, surgical removal, and adjuvant radiotherapy. Specifically, it identified increased levels of CD117+ granulocytes, memory, and regulatory CD4+ T cells as potential biomarkers of BC and radiotherapy, respectively. Importantly, the study demonstrates the value of unsupervised analysis of complex flow cytometry data to unravel new cell populations of potential clinical relevance.

Role of tubular epithelial arginase-II in renal inflammaging.

The aging kidney undergoes complex changes and is vulnerable to injury and development of chronic kidney disease (CKD) with preponderance affecting more women than men. Evidence has been presented that the type-II L-arginine:ureohydrolase, arginase-II (Arg-II) plays a role in the acceleration of aging. Arg-II is highly expressed in the kidney. However, the role of Arg-II in renal aging is not known. This study is to investigate whether Arg-II is involved in the kidney aging process dependently on sex. Arg-II level in the kidney of wild type (WT) mice is significantly elevated with aging, which is accompanied by an increase in expression of the inflammatory cytokines/chemokines, tissue macrophages, factors involved in fibrosis, and tubulointestitial fibrosis in both males and females. This renal aging phenotype is significantly suppressed in arg-II-/- mice, mainly in the females in which Arg-II level is higher than in the males. Importantly, numerous factors such as IL-1ß, MCP1, VCAM-1, and TGFß1 are mainly localized in the proximal tubular S3 segment cells expressing Arg-II in the aging kidney. In human proximal tubular cells (HK-2), TNF-α enhances adhesion molecule expression dependently on Arg-II upregulation. Overexpression of Arg-II in the cells enhances TGFß1 levels which is prevented by mitochondrial ROS inhibition. In summary, our study reveals that renal proximal tubular Arg-II plays an important role in the kidney aging process in females. Arg-II could be a promising therapeutic target for the treatment and prevention of aging-associated kidney diseases.

γδ T Cells Kill Plasmodium falciparum in a Granzyme- and Granulysin-Dependent Mechanism during the Late Blood Stage.

Plasmodium spp., the causative agent of malaria, have a complex life cycle. The exponential growth of the parasites during the blood stage is responsible for almost all malaria-associated morbidity and mortality. Therefore, tight immune control of the intraerythrocytic replication of the parasite is essential to prevent clinical malaria. Despite evidence that the particular lymphocyte subset of γδ T cells contributes to protective immunity during the blood stage in naive hosts, their precise inhibitory mechanisms remain unclear. Using human PBMCs, we confirmed in this study that γδ T cells specifically and massively expanded upon activation with Plasmodium falciparum culture supernatant. We also demonstrate that these activated cells gain cytolytic potential by upregulating cytotoxic effector proteins and IFN-γ. The killer cells bound to infected RBCs and killed intracellular P. falciparum via the transfer of the granzymes, which was mediated by granulysin in a stage-specific manner. Several vital plasmodial proteins were efficiently destroyed by granzyme B, suggesting proteolytic degradation of these proteins as essential in the lymphocyte-mediated death pathway. Overall, these data establish a granzyme- and granulysin-mediated innate immune mechanism exerted by γδ T cells to kill late-stage blood-residing P. falciparum.

Substantial Inter-Subject Variability in Blood Pressure Responses to Glucose in a Healthy, Non-obese Population.

Aim: A large inter-subject variability in the blood pressure (BP) response to glucose drinks has been reported. However, the underlying factors remain elusive and we hypothesized that accompanying changes in glucose metabolism affect these BP responses. Methods: Cardiovascular and glycemic changes in response to a standard 75 g oral-glucose-tolerance-test were investigated in 30 healthy, non-obese males. Continuous cardiovascular monitoring, including beat-to-beat BP, electrocardiographically deduced heart rate and impedance cardiography, was performed during a 30 min baseline and continued up to 120 min after glucose ingestion. Blood samples were taken at baseline, 30, 60, 90, and 120 min for the assessment of glucose, insulin and c-peptide. Additionally, we evaluated body composition by using validated bioelectrical impedance techniques. Results: Individual overall changes (i.e., averages over 120 min) for systolic BP ranged from -4.9 to +4.7 mmHg, where increases and decreases were equally distributed (50%). Peak changes (i.e., peak averages over 10 min intervals) for systolic BP ranged from -1.3 to +9.5 mmHg, where 93% of subjects increased systolic BP above baseline values (similar for diastolic BP) whilst 63% of subjects increased peak systolic BP by more than 4 mmHg. Changes in peak systolic BP were negatively associated with the calculated Matsuda-index of insulin sensitivity (r = -0.39, p = 0.04) but with no other evaluated parameter including body composition. Moreover, besides a trend toward an association between overall changes in systolic BP and total fat mass percentage (r = +0.32, p = 0.09), no association was found between other body composition parameters and overall BP changes. Conclusion: Substantial inter-subject variability in BP changes was observed in a healthy, non-obese subpopulation in response to an oral glucose load. In 63% of subjects, peak systolic BP increased by more than a clinically relevant 4 mmHg. Peak systolic BP changes, but not overall BP changes, correlated with insulin sensitivity, with little influence of body composition.

Uninephrectomy-Induced Lipolysis and Low-Grade Inflammation Are Mimicked by Unilateral Renal Denervation.

Uninephrectomy (UniNX) in rats on a fixed food intake leads to increased lipolysis and a low-grade inflammation with an increased subset of circulating cytokines. Because UniNX ablates renal nerves on the side of the removed kidney, we tested the contribution of unilateral renal denervation in the phenotype of UniNX. We compared Sham-operated controls, left nephrectomy (UniNX) and unilateral left kidney denervation (uDNX) in rats 4 weeks after surgery. uDNX did not affect kidney weight and function. In general, the uDNX phenotype was similar to the UniNX phenotype especially for lipolysis in fat pads and increased low-grade inflammation. uDNX led to decreased fat pad weight and increased hormone sensitive lipase and adipocyte triglyceride lipase mRNA levels in epididymal and inguinal adipose tissue, as well as increased circulating lipolysis markers ß-hydroxybutyrate and glycerol. Measured circulating hormones such as leptin, T3 and insulin were similar amongst the three groups. The lipolytic cytokines interferon-gamma and granulocyte macrophage colony stimulating factor were increased in the circulation of both uDNX and UniNX groups. These two cytokines were also elevated in the spleen of both groups, but contrastingly they were decreased in fat pads, liver, and kidneys. Both uDNX and UniNX similarly increased noradrenaline content in fat pads and spleen. Melanocortin 4 receptor mRNA levels were increased in the brains of both uDNX and UniNX compared to Sham and may contribute to increased tissue noradrenaline levels. In addition, the farnesoid x receptor (FXR) may contribute to changes in tissue metabolism and inflammation, as anti-inflammatory FXR was decreased in the spleen but increased in other tissues in uDNX and UniNX compared to Sham. In summary, both uDNX and UniNX in rats promote metabolic and immunological alterations by mechanisms that seem to implicate modification of unilateral renal nerve pathways as well as central and peripheral neural pathways.

Targeting arginase-II protects mice from high-fat-diet-induced hepatic steatosis through suppression of macrophage inflammation.

Nonalcoholic fatty liver disease (NAFLD) associates with obesity and type 2 diabetes. Hypoactive AMP-activated protein kinase (AMPK), hyperactive mammalian target of rapamycin (mTOR) signaling, and macrophage-mediated inflammation are mechanistically linked to NAFLD. Studies investigating roles of arginase particularly the extrahepatic isoform arginase-II (Arg-II) in obesity-associated NAFLD showed contradictory results. Here we demonstrate that Arg-II(-/-) mice reveal decreased hepatic steatosis, macrophage infiltration, TNF-α and IL-6 as compared to the wild type (WT) littermates fed high fat diet (HFD). A higher AMPK activation (no difference in mTOR signaling), lower levels of lipogenic transcription factor SREBP-1c and activity/expression of lipogenic enzymes were observed in the Arg-II(-/-) mice liver. Moreover, release of TNF-α and IL-6 from bone marrow-derived macrophages (BMM) of Arg-II(-/-) mice is decreased as compared to WT-BMM. Conditioned medium from Arg-II(-/-)-BMM exhibits weaker activity to facilitate triglyceride synthesis paralleled with lower expression of SREBP-1c and SCD-1 and higher AMPK activation in hepatocytes as compared to that from WT-BMM. These effects of BMM conditioned medium can be neutralized by neutralizing antibodies against TNF-α and IL-6. Thus, Arg-II-expressing macrophages facilitate diet-induced NAFLD through TNF-α and IL-6 in obesity.

Bevacizumab specifically decreases elevated levels of circulating KIT+CD11b+ cells and IL-10 in metastatic breast cancer patients.

BACKGROUND: Whether bevacizumab exerts its anti-tumor properties through systemic effects beyond local inhibition of angiogenesis and how these effects can be monitored in patients, remain largely elusive. To address these questions, we investigated bone marrow-derived cells and cytokines in the peripheral blood of metastatic breast cancer patients undergoing therapy with bevacizumab. METHODS: Circulating endothelial cells (CEC), circulating endothelial progenitor (CEP) and circulating CD11b+ cells in metastatic breast cancer patients before and during therapy with paclitaxel alone (n = 11) or in combination with bevacizumab (n = 10) were characterized using flow cytometry, real time PCR and RNASeq. Circulating factors were measured by ELISA. Aged-matched healthy donors were used as baseline controls (n = 12). RESULTS: Breast cancer patients had elevated frequencies of CEC, CEP, TIE2+CD11b+ and KIT+CD11b+ cell subsets. CEC decreased during therapy, irrespective of bevacizumab, while TIE2+CD11b+ remained unchanged. KIT+CD11b+ cells decreased in response to paclitaxel with bevacizumab, but not paclitaxel alone. Cancer patients expressed higher mRNA levels of the M2 polarization markers CD163, ARG1 and IL-10 in CD11b+ cells and increased levels of the M2 cytokines IL-10 and CCL20 in plasma. M1 activation markers and cytokines were low or equally expressed in cancer patients compared to healthy donors. Chemotherapy with paclitaxel and bevacizumab, but not with paclitaxel alone, significantly decreased IL-10 mRNA in CD11b+ cells and IL-10 protein in plasma. CONCLUSIONS: This pilot study provides evidence of systemic immunomodulatory effects of bevacizumab and identified circulating KIT+CD11b+ cells and IL-10 as candidate biomarkers of bevacizumab activity in metastatic breast cancer patients.

Oral postdialysis cholecalciferol supplementation in patients on maintenance hemodialysis: a dose-response approach.

The aim of the present study was to evaluate the dose of postdialysis cholecalciferol needed to maintain the 25-hydroxyvitamin D [25(OH)D] levels in the optimal range of 75-150 nmol/L. Twenty-six patients who had low baseline 25(OH)D levels (mean 27.5 ± 14.9 nmol/L) were studied. The 25(OH)D levels were measured every 2 months for one year. During the first two months, all the patients received 2000 IU of cholecalciferol after each hemodialysis (=6000 IU/wk). Thereafter, the dose was individualized and adapted every 2 months by administering 1 to 6 cholecalciferol tablets (2000 IU each) per week (total weekly dose = 2000-12000 IU/wk). During cholecalciferol supplementation, the 25(OH)D concentrations rapidly increased from baseline to 140.1 ± 28.3 nmol/L at month 6 and 95.6 ± 20.9 nmol/L at month 12. At month twelve, 86% of the patients had 25(OH)D levels within the target range with a mean dose of 5917 ± 4106 IU/wk of cholecalciferol; however, the amount needed to maintain these levels varied widely from 0 (n = 2) to 12000 IU/wk (n = 5). In conclusion, postdialysis cholecalciferol prescription is quite effective in correcting vitamin D deficiency/insufficiency, but the amount of cholecalciferol needed to maintain the 25(OH)D levels within the optimal range over the long-term varies widely among patients and must be individualized.

The role of penicillin in benign skin rashes in childhood: a prospective study based on drug rechallenge.

BACKGROUND: Delayed-onset urticarial or maculopapular rashes are frequently observed in children treated with ß-lactams. Many are labeled "allergic" without reliable testing. OBJECTIVE: Determine the etiology of these rashes by exploring both infectious and allergic causes. METHODS: Children presenting to the emergency department with delayed-onset urticarial or maculopapular rashes were enrolled. Acute and convalescent sera were obtained for viral screening along with a throat swab. Subjects underwent intradermal and patch skin testing for ß-lactams 2 months after presentation. Anti-ß-lactam blood allergy tests were also obtained. All subjects underwent an oral challenge test (OCT) with the culprit antibiotic. RESULTS: Eighty-eight children were enrolled between 2006 and 2008. There were 11 (12.5%) positive intradermal and no positive patch tests. There were 2 (2.3%) positive blood allergy tests. There were 6 (6.8%) subjects with a positive OCT, 2 were intradermal-negative, and 4 were intradermal-positive. No OCT reactions were more severe than the index event. Most subjects had at least 1 positive viral study, 54 (65.9%) in the OCT negative group. CONCLUSION: In this situation, ß-lactam allergy is clearly overdiagnosed because the skin rash is only rarely reproducible (6.8%) by a subsequent challenge. Viral infections may be an important factor in many of these rashes. OCTs were positive in a minority of intradermal skin test-positive subjects. Patch testing and blood allergy testing provided no useful information. OCTs should be considered in all children who develop a delayed-onset urticarial or maculopapular rash during treatment with a ß-lactam.