Effect of staining procedures on the results of micronucleus assays with exfoliated oral mucosa cells.

Micronuclei in exfoliated epithelial cells are widely used as biomarkers of cancer risk in humans. To elucidate the effect of different staining procedures on the outcome of such investigation, we conducted a study in which the micronuclei frequencies in oral mucosa cells of heavy smokers (n = 20) and nonsmokers (n = 10) were evaluated with nonspecific (Giemsa, May-Grünwald-Giemsa) and DNA-specific (4',6-diamidino-2-phenylindole, Feulgen, acridine orange) stains, whereas with Giemsa-based stains, the frequencies of micronuclei in smokers were significantly (4- to 5-fold) higher in the smokers group, no significant increase was observed with any of the DNA-specific stains. Furthermore, the evaluation of cells of the two study groups with Feulgen stain showed that oral mucosa cells from smokers had significantly increased levels of nuclear anomalies other than micronuclei. These anomalies are consequences of cell injury found in epithelial cells and are paralleled by formation of keratin bodies in the cytoplasm that resemble micronuclei. Correlation analyses showed that micronuclei frequencies scored in Giemsa-stained slides correlated significantly with karyorrhexis, karyolysis, condensed chromatin, and binucleates, whereas no such correlations were found with DNA-specific stains. These findings indicate that nuclear anomalies (and possibly keratin bodies) may be misinterpreted as micronuclei with nonspecific DNA stains and lead to false-positive results in studies with cells of epithelial origin. Furthermore, our results show that exposure of oral mucosa cells to genotoxic carcinogens contained in tobacco smoke does not lead to induction of micronuclei in these cells.     

Clinical Experience with Very High-Pressure Dilatation for Resistant Coronary Lesions

BackgroundCalcific coronary lesions can be so resistant to prevent symmetric stent dilatation with high risk of ISR/thrombosis. The aim of the current study is to evaluate the safety and efficacy of super high-pressure dilatation (>30-to-45Atm) using a dedicated NC-balloon (OPN, SIS-Medical-AG, Winterthur-Switzerland).MethodsWe retrospectively evaluated 326 consecutive undilatable lesions in which conventional NC-balloons failed to achieve adequate post-dilatation luminal gain. After the failed attempt an OPN-balloon was inflated up to achieve a uniform balloon expansion (maximal dilatation pressure of 45–50 Atm). Lesions were divided into two groups according to the final inflation pressure: Group-I: lesion responsive to 30-40Atm and Group-2:>40 Atm. Angiographic success was defined as residual angiographic stenosis<30% assessed by visual estimation with TIMI3-flow. Procedural success was defined as the achievement of angiographic success without any MACE.ResultsAngiographic success was achieved in 97.5%, procedural success in 96.6%; 53% of the lesions were responsive to a slower inflation pressure (Group I) while in the remaining 47%, the optimal expansion required a pressure > 40ATM (Group II). In 3 patients coronary rupture occurred after balloon inflation and was successfully treated with stent implantation with a final TIMI3-flow. The OPN alone was able to achieve adequate expansion in >90%. 0.9% days MACE were reported.ConclusionThe OPN-dedicated high-pressure balloon provides an effective and safe strategy for treatment of severe resistant coronary lesions.     

Update in the Percutaneous Management of Coronary Chronic Total Occlusions

Percutaneous coronary intervention (PCI) for chronic total occlusions (CTOs) has been rapidly evolving during recent years. With improvement in equipment and techniques, high success rates can be achieved at experienced centers, although overall success rates remain low. Prospective, randomized-controlled data regarding optimal use and indications for CTO PCI remain limited. CTO PCI should be performed when the anticipated benefit exceeds the potential risk. New high-quality studies of the clinical outcomes and techniques of CTO PCI are needed, as is the expansion of expert centers and operators that can achieve excellent clinical outcomes in this challenging patient and lesion subgroup. In the current review the authors summarize the latest publications in CTO PCI and provide an overview of the current state of the field.     

Vaccination against tick-borne encephalitis virus tests specific IgG production ability in patients under immunoglobulin substitution therapy

To assess B-cell function in patients under immunoglobulin (IgG)-replacement therapy, the non-licensed artificial bacteriophage (ΦX174)-neo-antigen may be used despite limited availability and experience. Active immunization against tick-borne encephalitis (TBE) is performed in few European countries. To test the feasibility of using licensed TBE vaccination as (neo-)antigen to determine residual or restored B-cell function in patients under regular IgG substitution, TBE-IgG levels were analyzed in 18 patients with ≥1–2 years of regular intravenous or subcutaneous IgG substitution and in pharmaceutical IgG-preparations (n = 21 batches, 10 products). Six individuals were boosted against TBE. Although TBE-specific IgG was detectable in concentrates (281–57,100 VieU/0.5 μL), levels were only borderline in patient sera (n = 31, 18 individuals; median 132 VieU; positive >155). Thus, TBE vaccination may be used to test B-cell function under IgG replacement therapy because IgG substitution appears insufficient to yield protective TBE-specific antibody levels in children.     

Quantitative real-time polymerase chain reaction for the accurate detection of Toxoplasma gondii in amniotic fluid

Infection with Toxoplasma gondii during pregnancy is often asymptomatic and may cause severe fetal damage. A quantitative TaqMan minor groove binder real-time polymerase chain reaction (PCR) assay was developed for the specific and sensitive detection of the previously described 529-bp repeat element occurring up to 200 to 300 times in T. gondii genome. The qualitative and quantitative detection limits determined were 6 and 20 marker copies (1/30 to 1/50 of 1 parasite) per PCR, respectively. In addition to standard PCR cycling conditions, 3 different fast PCR protocols were evaluated to minimize run time. A higher variability but no loss of specificity was observed. For the evaluation of clinical applicability, a total of 135 amniotic fluid samples were analyzed targeting both 529-bp and B1 gene. The sensitivity and specificity were 88.0% and 100.0% for B1, and 100.0% and 98.2% for 529-bp PCR assay (positive predictive value and negative predictive value: 100.0% and 97.4%, and 92.6% and 100.0%, respectively). Our results demonstrated an increased sensitivity of the 529-bp PCR assay even in a faster protocol.     

Immaturity of infection control in preterm and term newborns is associated with impaired toll-like receptor signaling

The impaired infection control related to the functional immaturity of the neonatal immune system is an important cause of infection in preterm newborns. We previously reported that constitutive Toll-like receptor (TLR) 4 expression and cytokine secretion on lipopolysaccharide (LPS) stimulation increases with gestational age. Here, we analyzed constitutive monocyte TLR2 expression and evaluated the expression profiles of the proximal downstream adapter molecule myeloid differentiation factor 88 (MyD88). We further investigated activation of protein kinases p38 and extracellular regulated kinsase (ERK) 1/2 in CD14 monocytes after ex vivo stimulation with bacterial TLR ligands (LPS and lipoteichoic acid [LTA]). The functional outcome of the stimulation was determined by cytokine secretion. Monocytes from 31 preterm newborns (<30 weeks of gestation, n=16; 30-37 weeks of gestation, n=15), 10 term newborns, and 12 adults were investigated. In contrast to TLR4 expression, TLR2 levels did not differ between age groups. However, MyD88 levels were significantly lower in preterm newborns. Activation of p38 and ERK1/2 was impaired in all newborn age groups after stimulation with TLR-specific ligands. Accordingly, after LTA stimulation, the levels of interleukin (IL)-1 beta , IL-6, and IL-8 cytokine production were substantially lower (P<.001) in preterm newborns than in adults. The reduced functional response to bacterial cell wall components appears to be part of the functional immaturity of the neonatal immune system and might predispose premature newborns to bacterial infection.     

Short-term creatine supplementation improves maximum quadriceps contraction in women

Creatine monohydrate (CrH2O) supplementation has been demonstrated to increase skeletal muscle power output in men. However, its effect upon women is not as clearly defined. This study investigated the effect of oral creatine supplementation upon muscle function, thigh circumference, and body weight in women. Twenty-two consenting college-age women were assigned to 1 of 2 groups matched for dietary and exercise habits, phase of menstrual cycle, and fat-free mass (FFM). After familiarization with testing procedures, pretrial measures of muscle function (5 repetitions 60 deg x s(-1) and 50 repetitions 180 deg x s(-1) were conducted during maximal voluntary concentric contraction of the preferred quadriceps muscle using an isokinetic dynamometer. Subjects then ingested 0.5 g x kg(-1) FFM of either CrH2O or placebo (one fourth dosage 4 times daily) in a double-blind design for 5 days. Resistance exercise was prohibited. After the ingestion phase was completed, all measures were repeated at the same time of day as during pretrials. Statistical analysis revealed time to peak torque in quadriceps extension decreased from pre-test values of 255 +/- 11 ms (mean +/- SEM) to post-test values of 223 +/- 3 ms; average power in extension increased from 103 +/- 7 W pre-test to 112 +/- 7 W post-test; and, during flexion, average power increased from 59 +/- 5 W pre-test to 65 +/- 5 W post-test in the creatine group as compared to controls (p .05). FFM, percent body fat, mid-quadriceps circumference, skinfold thickness of the measured thigh, and total body weight did not change for both groups between trials. We conclude that CrH2O improves muscle performance in women without significant gains in muscle volume or body weight.