CD14 expression is increased on monocytes in patients with anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis and correlates with the expression of ANCA autoantigens.

Monocyte subsets with differing functional properties have been defined by their expression of CD14 and CD16. We investigated these subsets in anti-neutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and determined their surface expression of ANCA autoantigens. Flow cytometry was performed on blood from 14 patients with active AAV, 46 patients with AAV in remission and 21 controls. The proportion of classical (CD14(high) CD16(neg/low)), intermediate (CD14(high) CD16(high)) and non-classical (CD14(low) CD16(high)) monocytes and surface expression levels of CD14 and CD16 were determined, as well as surface expression of proteinase 3 (PR3) and myeloperoxidase (MPO) on monocyte subsets. There was no change in the proportion of monocytes in each subset in patients with AAV compared with healthy controls. The expression of CD14 on monocytes from patients with active AAV was increased, compared with patients in remission and healthy controls (P < 0.01). Patients with PR3-ANCA disease in remission also had increased monocyte expression of CD14 compared with controls (P < 0.01); however, levels in patients with MPO-ANCA disease in remission were lower than active MPO-ANCA patients, and not significantly different from controls. There was a correlation between CD14 and both PR3 and MPO expression on classical monocytes in AAV patients (r = 0.79, P < 0.0001 and r = 0.42, P < 0.005, respectively). In conclusion, there was an increase in monocyte CD14 expression in active AAV and PR3-ANCA disease in remission. The correlation of CD14 expression with ANCA autoantigen expression in AAV may reflect cell activation, and warrants further investigation into the potential for increased CD14 expression to trigger disease induction or relapse.

Inter-observer agreement in athletes ECG interpretation using the recent international recommendations for ECG interpretation in athletes among observers with different levels of expertise.

INTRODUCTION: International criteria for the interpretation of the athlete's electrocardiogram (ECG) have been proposed. We aimed to evaluate the inter-observer agreement among observers with different levels of expertise. METHODS: Consecutive ECGs of Swiss elite athletes (≥14 years), recorded during routine pre-participation screening between 2013 and 2016 at the Swiss Federal Institute of Sports were analysed. A medical student (A), a cardiology fellow (B) and an electrophysiologist (C) interpreted the ECG's independently according to the most recent criteria. The frequencies and percentages for each observer were calculated. An inter-observer reliability analysis using Cohen Kappa (κ) statistics was used to determine consistency among observers. RESULTS: A total of 287 ECGs (64.1% males) were analysed. Mean age of the athletes was 20.4±4.9 years. The prevalence of abnormal ECG findings was 1.4%. Both, normal and borderline findings in athletes showed moderate to good agreement between all observers. κ scores for abnormal findings resulted in excellent agreement (κ 0.855 in observer A vs C and B vs C to κ 1.000 in observer A vs B). Overall agreement ranged from moderate (κ 0.539; 0.419-0.685 95% CI) between observer B vs C to good agreement (κ 0.720; 0.681-0.821 95% CI) between observer A vs B. CONCLUSIONS: Our cohort of elite athletes had a low prevalence of abnormal ECGs. Agreement in abnormal ECG findings with the use of the recently published International recommendations for ECG interpretation in athletes among observers with different levels of expertise was excellent. ECG interpretation resulted in moderate to good overall agreement.

Specificities mediated by neighboring nucleotides appear to underlie mutation induced by antifolates in E. coli.

The antifolate, trimethoprim (TRMP, 5 microM) caused a 10-fold increase in mutation frequency and primarily induced base substitution and deletion mutations in wild-type E. coli. Base-substitutions induced by antifolates were equally divided between transition and transversion mutations. When mutations consistent with expected antifolate-induced deoxynucleotide pool imbalances were considered, 29 out of 32 base-substitution mutations in the i-d region of the lacI gene were followed 3' by the same nucleotide substituted at the base mismatch site and all but one mutation occurred at sites consistent with next nucleotide effects resulting from antifolate-induced deoxynucleotide pool alterations. 66% of the TRMP-induced base-substitutions were also found at sites of frequent mutation identified in the spontaneous spectrum of a mutator D5 strain of E. coli which is deficient in the 3'-exonucleolytic proofreading function of DNA polymerase III holoenzyme. These results suggest that the pool imbalances induced by the antifolate trimethoprim compromise the proofreading activity of polymerase III holoenzyme and lead to mutation at specific sites. The results also imply that not all DNA sequence environments encountered by DNA polymerase III holoenzyme and its accompanying exonuclease are handled with equal facility at the level of nucleotide insertion and exonucleolytic proofreading when the enzyme is faced with an intracellular nucleotide pool imbalance. A number of small deletion and duplication mutations were also induced by the antifolate trimethoprim. In most cases these mutations were flanked by at least two A:T base pairs which could facilitate DNA-strand breakage at deoxyuridine misincorporation sites.

Correlation of doxorubicin footprints with deletion endpoints in lacO of E. coli.

This study explored the possibility that the sequence location of doxorubicin-induced deletion endpoints might relate to DNA structural alterations caused by doxorubicin binding to DNA. The 3'-OH endpoints of doxorubicin-induced deletions terminating in the 35-bp region of lacO appear to distribute differently from spontaneous deletion endpoints. Doxorubicin-induced deletions focus in the 26-bp palindrome which is separated by a 9-bp region with no reverse complementary, whereas spontaneous deletion 3'-OH endpoints are found distributed throughout the operator region. In order to explore the mechanism of deletion induction by doxorubicin, drug footprinting studies were carried out with DNA labeled at the 5' end of each of the complementary DNA strands encompassed by lacO. Doxorubicin protected the 9-bp region between the palindromic sequences from DNase I cutting and caused enhanced DNase I cleavage at symmetrical sites in the palindrome, which were inherently resistant to the nuclease in the absence of the drug. These symmetrical sites also define regions in which the occurrence of deletion endpoints is enhanced 6-fold in the presence of doxorubicin. This enhanced cutting and mutation occur in regions of the palindrome that are flanked by expected doxorubicin binding sites, but are not themselves binding sites of the drug. Similarly, other sites where the frequency of deletion endpoints increased in response to doxorubicin occurred directly adjacent to regions where doxorubicin appeared to inhibit cutting by DNase I. These results suggest that the binding of doxorubicin in the palindrome directs both the frequency and the specificity of deletion formation in this gene region.

Variability of anti-PF4/heparin antibody results obtained by the rapid testing system ID-H/PF4-PaGIA.

BACKGROUND: Recent studies have shown that a low clinical pretest probability may be adequate for excluding heparin-induced thrombocytopenia. However, for patients with intermediate or high pretest probability, laboratory testing is essential for confirming or refuting the diagnosis. Rapid assessment of anti-PF4/heparin-antibodies may assist clinical decision-making. OBJECTIVES: To evaluate the performance of rapid ID-H/PF4-PaGIA. In particular, we verified reproducibility of results between plasma and serum specimens, between fresh and frozen samples, and between different ID-H/PF4-polymer lots (polystyrene beads coated with heparin/PF4-complexes). PATIENTS/METHODS: The samples studied were 1376 plasma and 914 corresponding serum samples from patients investigated for suspected heparin-induced thrombocytopenia between January 2000 and October 2008. Anti-PF4/heparin-antibodies were assessed by ID-H/PF4-PaGIA, commercially available ELISAs and heparin-induced platelet aggregation test. RESULTS: Among 914 paired plasma/serum samples we noted discordant results (negative vs. low-titre positive) in nine instances (1%; 95%CI, 0.4-1.6%). Overall, agreement between titres assessed in plasma vs. serum was highly significant (Spearman correlation coefficient, 0.975; P < 0.0001). Forty-seven samples tested both fresh and after freezing/thawing showed a good agreement, with one discordant positive/negative result (Spearman correlation coefficient, 0.970; P < 0.0001). Among 1376 plasma samples we noted a strikingly variable incidence of false negative results (none - 82%; 95%CI, 66-98%), depending on the employed ID-H/PF4-polymer lot. Faulty lots can be recognized by titrating commercial positive controls and stored samples of HIT-patients. CONCLUSION: Laboratories performing the assay should implement stringent internal quality controls in order to recognize potentially faulty ID-H/PF4-polymer lots, thus avoiding false negative results.

[Bleeding complication due to accumulation of low-molecular-weight heparin in a patient with renal insufficiency]. / Blutungskomplikation durch Akkumulation von niedermolekularem Heparin bei einer Patientin mit Niereninsuffizienz.

We report of a 71-year-old woman with a history of chronic analgesic nephropathy, who underwent coronary angiography. Because of anterior ventricular aneurysm, anticoagulation with nadroparine was installed. Continued ACE-inhibitor and ASA with additional intravenous contrast substance lead to acute tubular necrosis with rapid decline of the renal function. Due to accumulation of the low molecular weight heparin, the patient developed an extensive retroperitoneal haematoma with circulatory shock and temporary anuric kidney failure. Low molecular weight heparins are commonly used during percutaneous coronary interventions. They are as safe and efficient compared to unfractioned heparin. But due to their renal elimination, they have to be monitored by measuring anti-factor Xa-activity if creatinine-clearance is <30 ml/min.