Phenome-wide association analysis of LDL-cholesterol lowering genetic variants in PCSK9

Abstract: Background: We characterised the phenotypic consequence of genetic variation at the PCSK9 locus and compared findings with recent trials of pharmacological inhibitors of PCSK9. Methods: Published and individual participant level data (300,000+ participants) were combined to construct a weighted PCSK9 gene-centric score (GS). Seventeen randomized placebo controlled PCSK9 inhibitor trials were included, providing data on 79,578 participants. Results were scaled to a one mmol/L lower LDL-C concentration. Results: The PCSK9 GS (comprising 4 SNPs) associations with plasma lipid and apolipoprotein levels were consistent in direction with treatment effects. The GS odds ratio (OR) for myocardial infarction (MI) was 0.53 (95% CI 0.42; 0.68), compared to a PCSK9 inhibitor effect of 0.90 (95% CI 0.86; 0.93). For ischemic stroke ORs were 0.84 (95% CI 0.57; 1.22) for the GS, compared to 0.85 (95% CI 0.78; 0.93) in the drug trials. ORs with type 2 diabetes mellitus (T2DM) were 1.29 (95% CI 1.11; 1.50) for the GS, as compared to 1.00 (95% CI 0.96; 1.04) for incident T2DM in PCSK9 inhibitor trials. No genetic associations were observed for cancer, heart failure, atrial fibrillation, chronic obstructive pulmonary disease, or Alzheimer’s disease – outcomes for which large-scale trial data were unavailable. Conclusions: Genetic variation at the PCSK9 locus recapitulates the effects of therapeutic inhibition of PCSK9 on major blood lipid fractions and MI. While indicating an increased risk of T2DM, no other possible safety concerns were shown; although precision was moderate

Klonierung und pharmakologische Charakterisierung des humanen 5-HT_2C-Rezeptors und seiner 5-HT_2CCys23Ser-Variante

Der 5-HT2C-Rezeptor gehört zur Gruppe der G-Protein-gekoppelten Rezeptoren und ist nur im Gehirn ausgebildet, wo er an wichtigen physiologischen und psychologischen Prozessen beteiligt ist. Verschiedene antipsychotische und antidepressive Medikamente wie Clozapin oder Fluoxetin binden an diesen Rezeptor. Die 5-HT2C-KO-Maus entwickelt ein enormes Übergewicht in Folge eines gestörten Ernährungsverhaltens und leidet an Krampfanfällen, sowie an Schlafstörungen. Deshalb gilt der 5-HT2C-Rezeptor als Target in der Behandlung von Schizophrenie, affektiven Störungen, Epilepsie oder Suchterkrankungen. Spezifische Agonisten werden aktuell in der Behandlung von Fettleibigkeit erprobt. Aufgrund dieser Tatsachen gelten genetische Veränderungen des Rezeptors als potentielle Ursache für neurologische oder psychiatrische Erkrankungen. Eine solche Veränderung stellt der Einzelbasenaustausch (SNP) 5-HT2CCys23Ser dar. Dieser führt an Position 68 der kodierenden Sequenz zu einem Austausch von Guanin zu Cytosin und bedingt auf Proteinebene eine Transversion der Aminosäure Cytosin zu Serin an Position 23. Die Inzidenz dieses Polymorphismus beträgt etwa 13%. In der vorliegenden Arbeit wurde dieser SNP mit Hilfe einer pharmakologischen Bindungststudie mit dem Radioliganden [³H]Mesulergin untersucht. Mit Hilfe dieser Methode gelang es, die Expressionsdichte des Rezeptors (Bmax) in der Plasmamembran und die Affinität des Radioliganden (KD) im Vergleich zum Wildtyp (WT) zu bestimmen. Das Ergebnis dieser Untersuchung zeigte eine signifikante Erhöhung der Expression des Mutations-Rezeptors um 20% im Vergleich zum WT-Rezeptor. Um diese biochemisch-pharmakologische Versuche durchführen zu können wurden zuerst mittels molekularbiologischer Methoden der 5-HT2C-Rezeptor kloniert, sowie seine 5-HT2CCys23Ser-Variante mittels gerichteter Mutagenese generiert. Anschließend erfolgte die funktionelle Expression in transient transfizierten HEK293-Zellen. Ein weiteres Ergebnis war die Analyse einer Splice-Variante, die im Rahmen der Klonierung mehrfach gefunden wurde. Diese ist durch eine Deletion von 95 Basen gekennzeichnet und führt durch eine Verschiebung des Leserasters zur Verkürzung des Proteins von 458 AS auf 248 AS. Auch diese Variante wurde in die Bindungsstudie integriert, erwies sich aber als eine pharmakologisch inaktive Form. Um die Bedeutung der Ergebnisse abzuschätzen, wurden diese mit Assoziationsstudien verglichen. So könnte eine höhere Expression des 5-HT2C-Rezeptors in Patienten mit dem vorliegenden SNP einen Prädiktor für Untergewicht als auch einen Protektionsfaktor für Übergewicht darstellen. Ferner scheint ein Einfluss des SNPs in der Pathogenese von affektiven Störungen zu bestehen, was Expressionsdichten dieses Rezeptors an post mortem untersuchten Gehirnen von Suizidenten, sowie eine große Assoziationsstudie zu diesem Thema zeigen. Auch eine Beteiligung an der Entwicklung von Schizophrenie ist durch die enge Verschaltung dieses Rezeptors mit dopaminergen und GABAergen Neuronen denkbar. Hierzu sind verschiedene Studien bekannt. Allerdings ist ein monokausaler Zusammenhang in der Pathogenese dieser Erkrankungen und dem Auftreten des SNPs aufgrund der heterogenen Studienlage eher unwahrscheinlich. Neben Umweltfaktoren und sozialen Faktoren scheinen v.a. weitere genetische Veränderungen mitverantwortlich zu sein. Das kürzlich beim 5-HT2C-Rezeptor entdeckte RNA-Editing, bei dem es im Rahmen einer posttranskriptionalen Modifikation der pre-mRNA zur Ausbildung weiterer funktionell unterschiedlicher Rezeptor-Isoformen kommt, könnte eine solche genetische Veränderung darstellen. Somit ist die genaue Bedeutung des SNP 5-HT2CCys23Ser in der Pathogenese neuronaler und psychiatrischer Erkrankungen erst durch weiterführende pharmakologische und genetische Studien möglich, die eine gleichzeitige Kopplung mehrerer genetischer Veränderungen berücksichtigen

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment

SP-D Serum Levels Reveal Distinct Epithelial Damage in Direct Human ARDS

Acute respiratory distress syndrome (ARDS) is a heterogeneous syndrome with multiple underlying diseases. Particularly epithelial damage results from direct (e.g., pneumonia) rather than indirect lung injury (e.g., nonpulmonary sepsis), which is more likely associated with endothelial damage. Hence, targeting ARDS patients based on their molecular phenotypes is a promising approach to improve outcome. With regard to distinct inflammatory responses and subsequent lung damage in direct ARDS due to the causing pathogen, we quantified markers of epithelial and endothelial damage and pro-inflammatory cytokines in patients with ARDS triggered by bacterial, viral, and atypical pathogen pneumonia or indirect ARDS. The serum levels of interleukin-6 (IL-6) and interleukin-8 (IL-8), lung epithelial injury markers surfactant protein D (SP-D), and soluble receptor for advanced glycation end-products (sRAGE) as well as endothelial injury marker angiopoietin-2 (Ang-2) from 49 patients with distinct types of ARDS were analyzed by multiplex immunoassay. Epithelial damage marker SP-D was significantly higher in direct ARDS caused by viral and atypical pathogens in contrast to ARDS caused by typical bacterial pneumonia and nonpulmonary sepsis. In contrast, sRAGE levels did not differ due to the causing pathogen. Patients with atypical pathogen pneumonia related ARDS showed significantly lower Ang-2 levels compared to patients with viral and indirect ARDS. Patients with viral and atypical pneumonia related ARDS possessed significantly lower serum IL-6 levels compared to bacterial pneumonia related ARDS and IL-6 levels in atypical pneumonia related ARDS were significantly lower than in indirect ARDS. Current findings report a potential difference in ARDS biomarkers due to the underlying disease and pathogen

Comparison of anticoagulation strategies for veno-venous ECMO support in acute respiratory failure

BACKGROUND Extracorporeal membrane oxygenation (ECMO) support in acute respiratory failure may be lifesaving, but bleeding and thromboembolic complications are common. The optimal anticoagulation strategy balancing these factors remains to be determined. This retrospective study compared two institutional anticoagulation management strategies focussing on oxygenator changes and both bleeding and thromboembolic events. METHODS We conducted a retrospective observational cohort study between 04/2015 and 02/2020 in two ECMO referral centres in Germany in patients receiving veno-venous (VV)-ECMO support for acute respiratory failure for > 24 h. One centre routinely applied low-dose heparinization aiming for a partial thromboplastin time (PTT) of 35-40 s and the other routinely used a high-dose therapeutic heparinization strategy aiming for an activated clotting time (ACT) of 140-180 s. We assessed number of and time to ECMO oxygenator changes, 15-day freedom from oxygenator change, major bleeding events, thromboembolic events, 30-day ICU mortality, activated clotting time and partial thromboplastin time and administration of blood products. Primary outcome was the occurrence of oxygenator changes depending on heparinization strategy; main secondary outcomes were the occurrence of severe bleeding events and occurrence of thromboembolic events. The transfusion strategy was more liberal in the low-dose centre. RESULTS Of 375 screened patients receiving VV-ECMO support, 218 were included in the analysis (117 high-dose group; 101 low-dose group). Disease severity measured by SAPS II score was 46 (IQR 36-57) versus 47 (IQR 37-55) and ECMO runtime was 8 (IQR 5-12) versus 11 (IQR 7-17) days (P = 0.003). There were 14 oxygenator changes in the high-dose group versus 48 in the low-dose group. Freedom from oxygenator change at 15 days was 73% versus 55% (adjusted HR 3.34 [95% confidence interval 1.2-9.4]; P = 0.023). Severe bleeding events occurred in 23 (19.7%) versus 14 (13.9%) patients (P = 0.256) and thromboembolic events occurred in 8 (6.8%) versus 19 (19%) patients (P = 0.007). Mortality at 30 days was 33.3% versus 30.7% (P = 0.11). CONCLUSIONS In this retrospective study, ECMO management with high-dose heparinization was associated with lower rates of oxygenator changes and thromboembolic events when compared to a low-dose heparinization strategy. Prospective, randomized trials are needed to determine the optimal anticoagulation strategy in patients receiving ECMO support

Extensive Therapeutic Drug Monitoring of Colistin in Critically Ill Patients Reveals Undetected Risks

(1) Background: With the rise of multi-/pan-drug resistant (MDR/PDR) pathogens, the less utilized antibiotic Colistin has made a comeback. Colistin fell out of favor due to its small therapeutic range and high potential for toxicity. Today, it is used again as a last resort substance in treating MDR/PDR pathogens. Although new guidelines with detailed recommendations for Colistin dosing are available, finding the right dose in critically ill patients with renal failure remains difficult. Here, we evaluate the efficiency of the current guidelines’ recommendations by using high resolution therapeutic drug monitoring of Colistin. (2) Methods: We analyzed plasma levels of Colistin and its prodrug colisthimethate sodium (CMS) in 779 samples, drawn from eight PDR-infected ICU patients, using a HPLC-MS/MS approach. The impact of renal function on proper Colistin target levels was assessed. (3) Results: CMS levels did not correlate with Colistin levels. Over-/Underdosing occurred regardless of renal function and mode of renal replacement therapy. Colistin elimination half-time appeared to be longer than previously reported. (4) Conclusion: Following dose recommendations from the most current guidelines does not necessarily lead to adequate Colistin plasma levels. Use of Colistin without therapeutic drug monitoring might be unsafe and guideline adherence does not warrant efficient target levels in critically ill patients