Study protocol P-MAPS: microbiome as predictor of severity in acute pancreatitis-a prospective multicentre translational study.

BACKGROUND: Acute pancreatitis (AP) is an inflammatory disorder that causes a considerable economic health burden. While the overall mortality is low, around 20% of patients have a complicated course of disease resulting in increased morbidity and mortality. There is an emerging body of evidence that the microbiome exerts a crucial impact on the pathophysiology and course of AP. For several decades multiple clinical and laboratory parameters have been evaluated, and complex scoring systems were developed to predict the clinical course of AP upon admission. However, the majority of scoring systems are determined after several days and achieve a sensitivity around 70% for early prediction of severe AP. Thus, continued efforts are required to investigate reliable biomarkers for the early prediction of severity in order to guide early clinical management of AP patients. METHODS: We designed a multi-center, prospective clinical-translational study to test whether the orointestinal microbiome may serve as novel early predictor of the course, severity and outcome of patients with AP. We will recruit 400 AP patients and obtain buccal and rectal swabs within 72 h of admission to the hospital. Following DNA extraction, microbiome analysis will be performed using 3rd generation sequencing Oxford Nanopore Technologies (ONT) for 16S rRNA and metagenomic sequencing. Alpha- and beta-diversity will be determined and correlated to the revised Atlanta classification and additional clinical outcome parameters such as the length of hospital stay, number and type of complications, number of interventions and 30-day mortality. DISCUSSION: If AP patients show a distinct orointestinal microbiome dependent on the severity and course of the disease, microbiome sequencing could rapidly be implemented in the early clinical management of AP patients in the future. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT04777812.

Experimental evidence of mechanical lumpy skin disease virus transmission by Stomoxys calcitrans biting flies and Haematopota spp. horseflies.

Lumpy skin disease (LSD) is a devastating disease of cattle characterized by fever, nodules on the skin, lymphadenopathy and milk drop. Several haematophagous arthropod species like dipterans and ticks are suspected to play a role in the transmission of LSDV. Few conclusive data are however available on the importance of biting flies and horseflies as potential vectors in LSDV transmission. Therefore an in vivo transmission study was carried out to investigate possible LSDV transmission by Stomoxys calcitrans biting flies and Haematopota spp. horseflies from experimentally infected viraemic donor bulls to acceptor bulls. LSDV transmission by Stomoxys calcitrans was evidenced in 3 independent experiments, LSDV transmission by Haematopota spp. was shown in one experiment. Evidence of LSD was supported by induction of nodules and virus detection in the blood of acceptor animals. Our results are supportive for a mechanical transmission of the virus by these vectors.

Plasma levels of the cardiovascular protective endogenous nucleoside adenosine are reduced in patients with primary aldosteronism without affecting ischaemia-reperfusion injury: A prospective case-control study.

BACKGROUND: Patients with primary aldosteronism (PA) experience more cardiovascular events compared to patients with essential hypertension (EHT), independent from blood pressure levels. In animals, mineralocorticoid receptor antagonists limit ischaemia-reperfusion (IR) injury by increasing extracellular adenosine formation and adenosine receptor stimulation. Adenosine is an endogenous compound with profound cardiovascular protective effects. Firstly, we hypothesized that patients with PA have lower circulating adenosine levels which might contribute to the observed increased cardiovascular risk. Secondly, we hypothesized that by this mechanism, patients with PA are more susceptible to IR compared to patients with EHT. DESIGN: In our prospective study in 20 patients with PA and 20 patients with EHT, circulating adenosine was measured using a pharmacological blocker solution that halts adenosine metabolism after blood drawing. Brachial artery flow-mediated dilation (FMD) before and after forearm IR was used as a well-established method to study IR injury. RESULTS: Patients with PA had a 33% lower adenosine level compared to patients with EHT (15.3 [13.3-20.4] vs 22.7 [19.4-36.8] nmol/L, respectively, P < .01). The reduction in FMD after IR, however, did not differ between patients with PA and patients with EHT (-1.0 ± 2.9% vs -1.6 ± 1.6%, respectively, P = .52). CONCLUSIONS: As adenosine receptor stimulation induces various powerful protective cardiovascular effects, its lower concentration in patients with PA might be an important novel mechanism that contributes to their increased cardiovascular risk. We suggest that modulation of the adenosine metabolism is an exciting novel pharmacological opportunity to limit cardiovascular risk in patients with PA that needs further exploration.

Neutrophils as Suppressors of T Cell Proliferation: Does Age Matter?

Whereas, neutrophils have long been considered to mainly function as efficient innate immunity killers of micro-organisms at infected sites, they are now recognized to also be involved in modulation of adaptive immune responses. Immature and mature neutrophils were reported to have the capacity to suppress T cell-mediated immune responses as so-called granulocyte-myeloid-derived suppressor cells (g-MDSCs), and thereby affect the clinical outcome of cancer patients and impact the chronicity of microbial infections or rejection reactions in organ transplantation settings. These MDSCs were at first considered to be immature myeloid cells that left the bone marrow due to disease-specific signals. Current studies show that also mature neutrophils can exert suppressive activity. In this study we investigated in a robust T cell suppression assay whether immature CD11b+ myeloid cells were capable of MDSC activity comparable to mature fully differentiated neutrophils. We compared circulating neutrophils with myeloid cell fractions from the bone marrow at different differentiation stages. Our results indicate that functional MDSC activity is only becoming detectable at the final stage of differentiation, depending on the procedure of cell isolation. The MDSC activity obtained during neutrophil maturation correlated with the induction of the well-known highly mobile and toxic effector functions of the circulating neutrophil. Although immature neutrophils have been suggested to be increased in the circulation of cancer patients, we show here that immature neutrophils are not efficient in suppressing T cells. This suggests that the presence of immature neutrophils in the bloodstream of cancer patients represent a mere association or may function as a source of mature neutrophils in the tumor environment but not a direct cause of enhanced MDSC activity in cancer.

Glycophorin-C sialylation regulates Lu/BCAM adhesive capacity during erythrocyte aging.

Lutheran/basal cell adhesion molecule (Lu/BCAM) is a transmembrane adhesion molecule expressed by erythrocytes and endothelial cells that can interact with the extracellular matrix protein laminin-α5. In sickle cell disease, Lu/BCAM is thought to contribute to adhesion of sickle erythrocytes to the vascular wall, especially during vaso-occlusive crises. On healthy erythrocytes however, its function is unclear. Here we report that Lu/BCAM is activated during erythrocyte aging. We show that Lu/BCAM-mediated binding to laminin-α5 is restricted by interacting, in cis, with glycophorin-C-derived sialic acid residues. Following loss of sialic acid during erythrocyte aging, Lu/BCAM is released from glycophorin-C and allowed to interact with sialic acid residues on laminin-α5. Decreased glycophorin-C sialylation, as observed in individuals lacking exon 3 of glycophorin-C, the so-called Gerbich phenotype, was found to correlate with increased Lu/BCAM-dependent binding to laminin-α5. In addition, we identified the sialic acid-binding site within the third immunoglobulin-like domain within Lu/BCAM that accounts for the interaction with glycophorin-C and laminin-α5. Last, we present evidence that neuraminidase-expressing pathogens, such as Streptococcus pneumoniae, can similarly induce Lu/BCAM-mediated binding to laminin-α5, by cleaving terminal sialic acid residues from the erythrocyte membrane. These results shed new light on the mechanisms contributing to increased adhesiveness of erythrocytes at the end of their lifespan, possibly facilitating their clearance. Furthermore, this work may contribute to understanding the pathology induced by neuraminidase-positive bacteria, because they are especially harmful to patients suffering from sickle cell disease and are associated with the occurrence of vaso-occlusive crises.

Plasma galectin-3 concentrations in patients with primary aldosteronism.

BACKGROUND: The incidence of cardiovascular events is higher in patients with primary aldosteronism than in patients with essential hypertension (EHT), despite similar blood pressure levels. This suggests detrimental cardiovascular effects of aldosterone. Amongst others, it has been suggested that galectin-3 (Gal-3) is a key mediator in aldosterone-induced myocardial fibrosis. OBJECTIVE: We studied whether patients with primary aldosteronism have higher plasma Gal-3 concentrations than patients with EHT and evaluated its reversibility after adrenalectomy. METHODS: In a retrospective cohort from our tertiary referral centre, we measured plasma Gal-3 concentrations in 78 patients with primary aldosteronism, 39 cured primary aldosteronism patients after adrenalectomy and 56 patients with EHT. Paired samples were available in 11 patients (preadrenalectomy and postadrenalectomy). We compared plasma Gal-3 levels by univariate analysis of covariance with correction for cardiovascular risk factors, plasma creatinine concentration, plasma potassium levels and alcohol intake. RESULTS: Adjusted plasma Gal-3 concentrations in patients with primary aldosteronism, patients after adrenalectomy and patients with EHT were 11.39 ±â€Š0.60, 11.64 ±â€Š0.81 and 11.41 ±â€Š0.73 ng/ml, respectively (mean ±â€ŠSD; P = 0.95). In 11 patients of whom paired samples were available, mean Gal-3 concentrations increased from 10.03 ±â€Š1.67 ng/ml preadrenalectomy to 14.36 ±â€Š2.07 ng/ml postadrenalectomy (P < 0.01). CONCLUSION: In patients with primary aldosteronism, plasma Gal-3 concentrations are not elevated when compared with patients with EHT, and levels do not decrease after adrenalectomy. These results are in contrast to previous studies and do not support a pathophysiological role of plasma Gal-3 in the increased cardiovascular risk in patients with primary aldosteronism.

Characterization of buffy coat-derived granulocytes for clinical use: a comparison with granulocyte colony-stimulating factor/dexamethasone-pretreated donor-derived products.

BACKGROUND AND OBJECTIVES: Buffy coat-derived granulocytes have been described as an alternative to the apheresis product from donors pretreated with dexamethasone and granulocyte colony-stimulating factor (G-CSF). The latter is - dependent on the local and national settings - obtained following a demanding and time-consuming procedure, which is undesirable in critically ill septic patients. In contrast, buffy coat-derived products have a large volume and are often heavily contaminated with red cells and platelets. We developed a new pooled buffy coat-derived product with high purity and small volume, and performed a comprehensive functional characterization of these granulocytes. MATERIALS AND METHODS: We pooled ten buffy coats following the production of platelet concentrates. Saline 0·9% was added to decrease the viscosity and the product was split into plasma, red cells and a 'super' buffy coat. Functional data of the granulocytes were compared to those obtained with granulocytes from healthy controls and G-CSF/dexamethasone-pretreated donors. RESULTS: Buffy coat-derived granulocytes showed adhesion, chemotaxis, reactive oxygen species production, degranulation, NETosis and in vitro killing of Staphylococcus aureus, Escherichia coli and Aspergillus species comparable to control and G-CSF/dexamethasone-derived granulocytes. Candida killing was superior compared to G-CSF/dexamethasone-derived granulocytes. Immunophenotyping was normal; especially no signs of activation in the buffy coat-derived granulocytes were seen. Viability was reduced. Buffy coats are readily available in the regular blood production process and would take away the concerns around the apheresis product. CONCLUSION: The product described appears a promising alternative for transfusion purposes.

Bispectral Index Guided Target Controlled Midazolam Sedation: a new advanced technique for dental procedures.

Objectives: To assess the efficacy and safety of Bispectral Index (BIS) guided Target Controlled Infusion (TCI) of midazolam for anxiolysis or minimal sedation during extensive periodontal or implant surgery in a single operator/sedationist model. Methods: Retrospective analysis of thirty adult ASA 1 or ASA 2 patients undergoing periodontal surgery or dental implant surgery under local anaesthesia were included. The calculated effect site concentration (Ce) of midazolam applied by TCI, BIS, heart rate (HR), and peripheral oxygen saturation (SpO2) were monitored continuously. Non-invasive blood pressure (NIBP) and mean arterial pressure (MAP) were measured every 10 minutes. All peri-operative parameters were recorded every 10 minutes. All patients were interviewed 1 week after the procedure to explore their experience of sedation and the periodontal or implant surgery procedure. Results: Extensive periodontal or implant surgery treatment in all 30 patients was completed in a mean time of 120 min (range 50-180 min). The calculated mean effect site concentration for midazolam was 50 ng/ml (range 24-80). The mean BIS was 85 (74-100) during induction and was maintained between 80 and 90 during the oral surgical procedure by adjusting TCI Ce. There were no clinically significant cardiopulmonary changes during midazolam infusion with regard to SpO2, NIBP, MAP and heart rate. Patients experienced profound anterograde amnesia and were very satisfied with the sedation and the surgical procedure. Conclusions: BIS guided TCI sedation with midazolam facilitates predictable minimal sedation enabling long periodontal or implant surgery procedures by a single operator/sedationist within safe physiological limits.

Impact of Age, Season, and Flowing vs. Stagnant Water Habitat on Avian Influenza Prevalence in Mute Swan (Cygnus olor) in Belgium.

Due to their probable role in the spread of Asian highly pathogenic avian influenza (HPAI) H5N1 virus, and in order to explore its implication in the low pathogenic avian influenza (LPAI) virus epidemiology, mute swans represent one particular wild bird species specifically targeted in the avian influenza (AI) surveillance elaborated in Belgium. A total of 640 individual mute swans have been sampled during a 4-yr AI surveillance program (2007-2010) to determine the AI seroprevalence and viroprevalence in this species; all were analyzed through age, temporal, and habitat (flowing and stagnant water) factors. Using a nucleoprotein (NP)-based ELISA, a global antibody prevalence of 35% has been found and was characterized by two peaks in the winter and the summer that might be indicative of a greater LPAI virus circulation in the autumn than in the spring. A significantly higher antibody prevalence was detected in adult swans (53.8%) as compared to juveniles (15.5%). In contrast, a low prevalence of infection (2.7%) was found, mainly in juvenile mute swans and only during the autumn migration period. Interestingly, an impact of water habitat was observed based on the comparison of the antibody prevalence and prevalence of infection from swan populations living on stagnant water vs. flowing water, suggesting that stagnant water provides a more-favorable environment for LPAI persistence and transmission.

Ticagrelor Does Not Inhibit Adenosine Transport at Relevant Concentrations: A Randomized Cross-Over Study in Healthy Subjects In Vivo.

BACKGROUND AND PURPOSE: In patients with myocardial infarction, ticagrelor reduces cardiovascular and sepsis-related mortality, and can cause dyspnea. It is suggested that this is caused by adenosine receptor stimulation, because in preclinical studies, ticagrelor blocks the nucleoside transporter and increases cellular ATP release. We now investigated the effects of ticagrelor on the adenosine system in humans in vivo. EXPERIMENTAL APPROACH: In a double-blinded, placebo-controlled cross-over trial in 14 healthy subjects, we have tested whether ticagrelor (180 mg) affects adenosine- and dipyridamole-induced forearm vasodilation, as surrogates of nucleoside uptake inhibition and adenosine formation, respectively. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was measured. Primary endpoint was adenosine-induced vasodilation. KEY RESULTS: Ticagrelor did not affect adenosine- or dipyridamole-induced forearm vasodilation. Also, ex vivo uptake of adenosine and uridine in isolated red blood cells was not affected by ticagrelor. In vitro, ticagrelor dose-dependently inhibited nucleoside uptake, but only at supra-physiological concentrations. CONCLUSION AND IMPLICATIONS: In conclusion, at relevant plasma concentration, ticagrelor does not affect adenosine transport, nor adenosine formation in healthy subjects. Therefore, it is unlikely that this mechanism is a relevant pleiotropic effect of ticagrelor. TRIAL REGISTRATION: ClinicalTrials.gov NCT01996735.

Study on the protective effect of the KIR3DL1 gene in ankylosing spondylitis.

OBJECTIVE: Ankylosing spondylitis (AS) is an autoimmune disease that mainly affects the sacroiliac joints and the spine of the lower back. The disease is strongly associated with HLA-B27. Additional genes, single-nucleotide polymorphisms, and molecular components have been identified to be associated with AS, but the exact mechanism that drives disease development remains poorly understood. The killer cell immunoglobulin-like receptors (KIRs) are regulators of cytotoxicity of natural killer cells and T cell subsets and may be relevant in binding to HLA-B27 and the development of AS. We undertook this study to identify possible associations of KIR genotype with susceptibility to AS and disease characteristics including the presence of the HLA-B27 allele, disease severity, and uveitis. METHODS: We performed complete genotyping of the KIR locus in 303 Caucasian AS patients, 119 randomly selected healthy Caucasian controls, and 50 HLA-B27-positive healthy Caucasian controls by multiplex ligation-dependent probe amplification assay for detection of gene presence and copy number. RESULTS: We did not observe a significant association of any specific KIR gene or haplotype with susceptibility to AS or any other clinical manifestation. Disease severity, as measured by fulfilling the criteria for treatment with tumor necrosis factor blocking therapy, was linked to a lower number of genes for the functional variant of KIR3DL1 (P = 0.007). CONCLUSION: Our exploratory study indicates that KIR genes are not a major risk factor for susceptibility to AS. However, the data do suggest a role for KIRs in progression of the disease, whereby KIR3DL1 has a protective effect against the more severe manifestations of AS.

Nonallelic homologous recombination of the FCGR2/3 locus results in copy number variation and novel chimeric FCGR2 genes with aberrant functional expression.

The human FCGR2/3 locus, containing five highly homologous genes encoding the major IgG receptors, shows extensive copy number variation (CNV) associated with susceptibility to autoimmune diseases. Having genotyped >4000 individuals, we show that all CNV at this locus can be explained by nonallelic homologous recombination (NAHR) of the two paralogous repeats that constitute the majority of the locus, and describe four distinct CNV regions (CNRs) with a highly variable prevalence in the population. Apart from CNV, NAHR events also created several hitherto unidentified chimeric FCGR2 genes. These include an FCGR2A/2C chimeric gene that causes a decreased expression of FcγRIIa on phagocytes, resulting in a decreased production of reactive oxygen species in response to immune complexes, compared with wild-type FCGR2A. Conversely, FCGR2C/2A chimeric genes were identified to lead to an increased expression of FCGR2C. Finally, a rare FCGR2B null-variant allele was found, in which a polymorphic stop codon of FCGR2C is introduced into one FCGR2B gene, resulting in a 50% reduction in protein expression. Our study on CNRs and the chimeric genes is essential for the correct interpretation of association studies on FCGR genes as a determinant for disease susceptibility, and may explain some as yet unidentified extreme phenotypes of immune-mediated disease.

[Safety of intravenous sedation with midazolam for dental treatment]. / Veiligheid van sedatie met midazolam voor tandheelkundige behandelingen.

In the December 2014 issue of the Nederlands Tijdschrift voor Tandheelkunde, T.H. van den Berg and B. Preckel published an article entitled 'Mild intravenous sedation with midazolam by dentists'. Broers et al responded to this article arguing that the administration of intravenous sedation with midazolam by dentists is unsafe for patients. In the current article the authors, Van den Berg and Preckel, address the points of criticism.

Differential effects of platelets and platelet inhibition by ticagrelor on TLR2- and TLR4-mediated inflammatory responses.

Platelets and platelet-monocyte interaction play an important role in inflammation. Both pro- and anti-inflammatory effects of platelet inhibition have been reported in animal models. This study aimed to investigate the effect of platelets and platelet inhibition by the new P2Y12 receptor antagonist ticagrelor on monocyte function, as assessed by cytokine responses to Toll-like Receptor (TLR) ligands. In a set of in vitro experiments, peripheral blood mononuclear cells (PBMC) incubated with the TLR2 ligand Pam3CSK4 produced less cytokines in the presence of platelets, whereas platelets increased the production of cytokines when PBMC were exposed to TLR4 ligand lipopolysaccharide (LPS). These effects of platelets were dependent on direct platelet-leukocyte aggregation and for the Pam3CSK4-induced response, on phagocytosis of platelets by monocytes. In a double blind, placebo-controlled crossover trial in healthy volunteers, a single oral dosage of 180 mg ticagrelor reduced platelet-monocyte complex (PMC) formation. This was associated with an increase in pro-inflammatory cytokines in blood exposed to Pam3CSK4, but a decrease in these cytokines in blood exposed to LPS. These findings show that platelets differentially modulate TLR2- and TLR4-mediated cytokine responses of PBMC. Through inhibition of platelet-leukocyte interaction, P2Y12 receptor antagonists may either exert a pro- or anti-inflammatory effect during infections depending on the TLR primarily involved.

Novel insights in the genomic organization and hotspots of recombination in the human KIR locus through analysis of intergenic regions.

The Killer Immunoglobulin-like Receptor (KIR) proteins constitute a family of highly homologous surface receptors involved in the regulation of the innate cytotoxicity of natural killer (NK) cells. Within the human genome, 17 KIR genes are present, many of which show large variation across the population owing to the high number of allelic variants and copy number variation (CNV). KIR genotyping and CNV determination were used to map the KIR locus in a large cohort of >400 Caucasian individuals. Gene order and structure was determined by sequence-specific polymerase chain reaction of the intergenic regions. In this way, we could show that KIR3DL1 and KIR2DS4 gene variants are linked and that--contrary to current views--the gene KIR2DS5 is only present in the telomeric half of the KIR locus. Our study revealed novel insights in the highly organized distribution of KIR genes. Novel recombination hotspots were identified that contribute to the diversity of KIR gene distribution in the Caucasian population. Next-generation sequencing of the KIR intergenic regions allowed for a detailed single-nucleotide polymorphism analysis, which demonstrated several gene-specific as well as haplotype-specific nucleotides for a more accurate genotyping of this notoriously complex gene cluster.

Follow-up of the Schmallenberg Virus Seroprevalence in Belgian Cattle.

Schmallenberg virus (SBV), which emerged in Northwestern Europe in 2011, is an arthropod-borne virus affecting primarily ruminants. Based on the results of two cross-sectional studies conducted in the Belgian ruminant population during winter 2011-2012, we concluded that at the end of 2011, almost the whole population had already been infected by SBV. A second cross-sectional serological study was conducted in the Belgian cattle population during winter 2012-2013 to examine the situation after the 2012 transmission period and to analyse the change in immunity after 1 year. A total of 7130 blood samples collected between 1st January and 28 February 2013 in 188 herds were tested for the presence of SBV-specific antibodies. All sampled herds tested positive and within-herd seroprevalence was estimated at 65.66% (95% CI: 62.28-69.04). A statistically significant decrease was observed between the beginning and the end of 2012. On the other hand, age-cohort-specific seroprevalence stayed stable from 1 year to the other. During winter 2012-2013, calves between 6 and 12 months had a seroprevalence of 20.59% (95% CI: 15.34-25.83), which seems to be an indication that SBV was still circulating at least in some parts of Belgium during summer-early autumn 2012. Results showed that the level of immunity against SBV of the animals infected has not decreased and remained high after 1 year and that the spread of the virus has slowed down considerably during 2012. This study also indicated that in the coming years, there are likely to be age cohorts of unprotected animals.

Extended transmission of two H5/H7 low pathogenic avian influenza viruses in chickens.

Transmission experiments are useful for investigating the mechanisms of low pathogenic notifiable avian influenza virus (LPNAI) transmission. In this study, the hypothesis that inoculation-infected chickens are more infectious than contact-infected chickens was tested. To this end, extended transmission experiments with one H5N2 and one H7N1 LPAIV which had previously been characterized in a series of standard transmission experiments were conducted in specific pathogen-free (SPF) chickens. For the H5N2 LPAIV, the infectivity of contact-infected chickens was similar to the infectivity of inoculated chickens. Despite results from a previous study suggesting the H7N1 LPAIV strain to be similarly infectious to SPF chickens as the H5N2 LPAIV strain, the acquisition of contact-infected chickens proved more difficult for H7N1 LPAIV. It was assumed that this might have been a consequence of the length and timing of the exposure period. In conclusion, for LPNAIVs that first seemed equally infectious, short-term transmissibility may vary considerably.