High-throughput single strand conformation polymorphism mutation detection by automated capillary array electrophoresis: validation of the method

Capillary array electrophoresis (CAE) is a novel technique, which allows for high throughput analysis of DNA fragments. When screening for mutations in whole populations or large patient groups it is necessary to have robust and well-characterized setups for high throughput analysis. For large-scale mutation screening, we have developed procedures for single strand conformation polymorphism (SSCP) assays using CAE (CAE-SSCP) whereby we may increase both the sensitivity and the throughput compared to conventional SSCP analysis. In this study we have validated CAE-SSCP by 1) comparing detection by slab-gel based SSCP with CAE-SSCP of mutations in the MYH7, MYL2, and MYL3 genes encoding sarcomere proteins from patients suffering from hypertrophic cardiomyopathy; and 2) by constructing a series of 185 mutants having substitution mutations, as well as insertion/deletion mutations, or some combinations of these, in different sequence contexts in four exons and different positions relative to the end of the amplicon (three from the KCNQ1 gene, encoding a cardiac potassium channel, and one from the TNNI3 gene encoding cardiac troponin I). The method identified 181 out of 185 mutations (98%), and the data suggest that the position of mutation in the fragment had no effect on the sensitivity. Analysis of the specificity of the method showed that only very few mutants could not be distinguished from each other and there were no false positives.     

The effect of anti-IgE treatment on in vitro leukotriene release in children with seasonal allergic rhinitis

BACKGROUND: Binding of allergens with IgE to the IgE receptors on mast cells and basophils results in the release of inflammatory mediators as sulfidoleukotrienes (SLTs), triggering allergic cascades that result in allergic symptoms, such as asthma and rhinitis. OBJECTIVE: We sought to investigate whether anti-IgE (Oma-lizumab), a humanized monoclonal anti-IgE antibody, in addition to specific immunotherapy (SIT) affects the leukotriene pathway. METHODS: Ninety-two children (age range, 6-17 years) with sensitization to birch and grass pollens and with seasonal allergic rhinitis were included in a phase III, placebo- controlled, multicenter clinical study. All subjects were randomized to one of 4 treatment groups. Two groups subcutaneously received birch SIT and 2 groups received grass SIT for at least 14 weeks before the start of the birch pollen season. After 12 weeks of SIT titration, placebo or anti-IgE was added for 24 weeks. The primary clinical efficacy variable was symptom load (ie, the sum of daily symptom severity score and rescue medication score during pollen season). Blood samples taken at baseline and at the end of study treatment after the grass pollen season were used for separation of leukocytes in this substudy. After in vitro stimulation of the blood cells with grass and birch pollen allergens, SLT release (LTC4, LTD4, and LTE4) was quantified by using the ELISA technique. RESULTS: Before the study treatment, SLT release to birch and grass pollen exposure did not differ significantly among the 4 groups. Under treatment with anti-IgE + SIT-grass (n = 23), a lower symptom load occurred during the pollen season compared to placebo + SIT-grass (n = 24, P =.012). The same applied to both groups receiving birch SIT (n = 23 and n = 22, respectively; P =.03). At the end of treatment, the combination of anti-IgE plus grass SIT, as well as anti-IgE plus birch SIT, resulted in significantly lower SLT release after stimulation with the corresponding allergen (416 ng/L [5th-95th percentile, 1-1168] and 207 ng/L [1-860 ng/L], respectively) compared with placebo plus SIT (2490 ng/L [384-6587 ng/L], P =.001; 2489 ng/L [1-5670 ng/L], P =.001). In addition, treatment with anti-IgE was also followed by significantly lower SLT releases to the allergens unrelated to SIT (grass SIT: 300 ng/L [1-2432 ng/L] in response to birch allergen; birch SIT: 1478 ng/L [1-4593 ng/L] in response to grass pollen) in comparison with placebo (grass SIT: 1850 ng/L [1-5499 ng/L], P =.001; birch SIT: 2792 ng/L [154-5839 ng/L], P =.04]. CONCLUSION: Anti-IgE therapy reduces leukotriene release of peripheral leukocytes stimulated with allergen in children with allergic rhinitis undergoing allergen immunotherapy independent of the type of SIT allergen used.     

Platelets and anticoagulant capacity in patients with inflammatory bowel disease

Patients with inflammatory bowel disease (IBD) are susceptible to thromboembolic complications. Several mechanisms can be responsible, including abnormal regulation of coagulation activity, disturbances of fibrinolysis, inflammatory reactions and thrombocytosis. The aim of this study was to assess hemostatic alterations in these parameters during exacerbation of disease. We studied disease activity in 99 IBD patients receiving anti-inflammatory therapy, in relation to: procoagulant markers, i.e. prothrombin fragment F1 + 2 (F1 + 2), D-dimer and platelet count, anticoagulant markers, i.e. protein C, protein S and antithrombin, and a mediator of inflammation (IL-6). Coagulation activity and platelet count were increased during active disease in IBD patients compared with those in a state of remission. The IL-6 concentrations were positively correlated with disease activity and thrombocytosis in patients with ulcerative colitis, but no association with the anticoagulant capacity could be demonstrated except for a decrease in protein C during high disease activity.     

Cardiovascular actions of cannabinoids and their generation during shock

Marijuana is a widely abused recreational drug well known for its psychoactive properties. Cannabinoids, the active ingredients of marijuana, elicit their neurobehavioral effects by interacting with the CB₁ cannabinoid receptor subtype, expressed primarily in the brain but also present in some peripheral tissues. A second receptor subtype, the CB₂ receptor, is expressed on cells of the immune system and is thought to be responsible for the immunosuppressant effects of cannabinoids. Recently, endogenous lipidlike substances have been identified, including arachidonyl ethanolamide (anandamide) and 2-arachidonyl glyceride, that bind to cannabinoid receptors and mimic many of the neurobehavioral effects of plant-derived cannabinoids. Both plant-derived cannabinoids and the endogenous ligands have been shown to elicit hypotension and bradycardia via activation of peripherally located CB₁ receptors. Possible underlying mechanisms include presynaptic CB₁ receptor mediated inhibition of norepinephrine release from peripheral sympathetic nerve terminals, and/or direct vasodilation via activation of vascular cannabinoid receptors. The latter may also be the target of endocannabinoids of vascular endothelial origin. Recent studies indicate that a peripheral endogenous cannabinoid system in circulating macrophages and platelets is activated in hemorrhagic and septic shock and may contribute to the hypotension associated with these conditions via activation of vascular cannabinoid receptors. The potential role of this mechanism in human shock conditions is under investigation. Received: 20 May 1998 / Accepted: 24 August 1998     

The Importance of Ventilation in Exercise-Induced Asthma

The degree of post treadmill-running decrease in pulmonary function (Exercise-Induced Asthma) in 11 adult asthmatics was compared with the decrease in pulmonary function followed by resting isocapnic hyperventilation. It was checked that ventilation during the hyperventilation was kept identical to the ventilation during treadmill-running by continuous recording of respiratory frequency, minute ventilation, tidal volume and accumulated ventilation. The temperature of the inspired air was identical in the two situations and the relative humidity was 40% during treadmill-running and 15% during hyperventilation. The average accumulated ventilation during treadmill-running and hyperventilation was 411 1/6 min in both events. The decrease in peak expiratory flow after treadmill-running was 25% and after isocapnic hyperventilation 24%. It is concluded that the ventilation is of more importance for the decrease in pulmonary function after exercise, than the work load.     

Development of a Cell Patterning Technique Using Poly(Ethylene Glycol) Disilane

A simple technique for controlling cell adhesion on glass substrates by surface modification using a commercially available poly(ethylene glycol) (PEG) disilane, which can bind directly to glass in a single step, in combination with photolithographic micropatterning, was developed, characterized, and evaluated for patterning of HepG2 hepatoma cells and 3T3 fibroblasts. The optimal concentration of PEG-disilane for surface modification was 5 mM, and patterning of strongly adherent cells such as HepG2 required the chelation of divalent metal cations in order to inhibit nonspecific binding and cell aggregation. Whereas the average thickness of the PEG-disilane layer was 18±3.5 nm, the perimeters of patterned areas of exposed glass exhibited ridges of average height 857±50 nm, which may have aided in constraining cell spreading and migration. Although unpatterned PEG-treated substrates were hydrophilic (contact angle 46±1°), micropatterned surfaces behaved as if they were somewhat hydrophobic (contact angle 90°), necessitating special protocols for preventing deleterious dewetting of cells. For optimized protocols, the probability of adhesion of HepG2 cells to a patterned area of exposed glass was almost 15 times higher than the probability of adhesion to a PEG-treated background region of equal area. Our technique is useful for short- to intermediate-term patterning of cell or tissue morphology, e.g., for investigation of the effects of cell–cell interactions or cell geometry.     

Association between pituitary adenylate cyclase-activating polypeptide and thyrotropin-releasing hormone in the rat hypothalamus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is present in many regions of the hypothalamus including the paraventricular nucleus (PVN). In this study the anatomical relationship between PACAP- and thyrotropin-releasing hormone (TRH)-immunoreactive neuronal elements was investigated in the rat hypothalamus. Using a well-characterized mouse monoclonal antibody against PACAP and a rabbit polyclonal antiserum against TRH, we found numerous nerve fibers with PACAP-immunoreactivity (ir) closely apposed to TRH neurons in the PVN suggesting synaptic contacts. Electron microscopy confirmed the presence of synapses between PACAP-ir terminals and TRH-ir perikarya and various dendritic profiles as well as between PACAP-ir terminals and unlabeled perikarya and small- to medium-sized dendrites. Coexistence of the two peptides in perikarya of the PVN was limited to only a few neurons in the periventricular subdivision, but PACAP-ir and TRH-ir extensively coexisted in perikarya of the perifornical cell group, medial preoptic area, lateral hypothalamus and dorsomedial nucleus. The interactions between PACAP-containing neuronal processes and TRH neurons in the PVN raise the possibility that PACAP modulates the secretion of TRH destined for regulation of anterior pituitary TSH. The more general association between PACAP and TRH in other regions of the hypothalamus suggests a further role for PACAP as a cofactor in the function of TRH neurons.     

Ten novel MSH2 and MLH1 germline mutations in families with HNPCC

Hereditary nonpolyposis colorectal cancer (HNPCC) is one of the most common hereditary cancer-susceptibility syndromes. Germline mutations in mismatch repair genes are associated with the clinical phenotype of HNPCC. We report ten novel germline mutations, three in MSH2 and seven in MLH1. All but one mutation have been found in families fulfilling criteria of the Bethesda guidelines; four of them additionally fulfilled the Amsterdam criteria I or II. Eight mutations were considered pathogenic and predictive diagnostics in healthy family members at risk shall be undertaken; these include five frameshift mutations leading to premature stop codons, in MSH2: c.1672delT (p.S558Xfs) and c.2466_2467delTG (p.C822X) and in MLH1: c.1023delG (p.R341Xfs), c.1127_1128dupAT (p.K377Xfs) and c.1310delC (p.P437Xfs); three mutations leading to splice aberrations, in MSH2: c.1661G>C (r.1511_1661del) and in MLH1: c.677+3A>C (r.589_677del) and c.1990-2A>G predicted to result in a splice site defect. The remaining two mutations are unclassified variants with assumed pathogenicity: one missense mutation in the highly conserved ATPase domain of MLH1 (c.122A>G [p.D41G]) and one in-frame insertion of twelve nucleotides in MLH1 (c.2155_2156insATGTGTTCCACA [p.I719delinsNVFHI]). These two mutations were not found in 102 alleles of healthy control individuals. The corresponding tumors from all patients showed a high level of microsatellite instability (MSI-H). Immunohistochemistry (IHC) revealed complete loss of expression of the affected protein in the tumor cells from all but three patients. The tumors from the patients with the mutations c.1127_1128dupAT and c.1990-2A>G showed a reduction of expression of the MLH1-protein, rather than complete loss. In the tumor from the patient with the missense mutation c.122A>G [p.D41G] a normal expression of the proteins coded by MLH1 and MSH2 was noticed.     

Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles

A new fluorescence in situ hybridization (FISH) method that uses peptide nucleic acid (PNA) probes for identification of Candida albicans directly from positive-blood-culture bottles in which yeast was observed by Gram staining (herein referred to as yeast-positive blood culture bottles) is described. The test (the C. albicans PNA FISH method) is based on a fluorescein-labeled PNA probe that targets C. albicans 26S rRNA. The PNA probe is added to smears made directly from the contents of the blood culture bottle and hybridized for 90 min at 55 degrees C. Unhybridized PNA probe is removed by washing of the mixture (30 min), and the smears are examined by fluorescence microscopy. The specificity of the method was confirmed with 23 reference strains representing phylogenetically related yeast species and 148 clinical isolates covering the clinically most significant yeast species, including C. albicans (n = 72), C. dubliniensis (n = 58), C. glabrata (n = 5), C. krusei (n = 2), C. parapsilosis (n = 4), and C. tropicalis (n = 3). The performance of the C. albicans PNA FISH method as a diagnostic test was evaluated with 33 routine and 25 simulated yeast-positive blood culture bottles and showed 100% sensitivity and 100% specificity. It is concluded that this 2.5-h method for the definitive identification of C. albicans directly from yeast-positive blood culture bottles provides important information for optimal antifungal therapy and patient management.     

Cytomegalovirus transmission to preterm infants during lactation.

Breastfeeding has a major impact on HCMV epidemiology. The incidence of postnatal HCMV reactivation during lactation equals the maternal seroprevalence. Infectious virus, viral DNA and RNA can be isolated easily from cell and fat-free milk whey. Early onset of viral DNAlactia and virolactia as well as high viral load in milk whey are maternal risk factors for virus transmission. The dynamics of HCMV reactivation can be described by unimodal kinetics with interindividual variation. Virus reactivation during lactation is a self-limiting local process in the absence of systemic HCMV infection. Preterm infants below 1000g birthweight and a gestational age below 30 weeks may be at high risk of acquiring a symptomatic HCMV infection. Several recent studies described low transmission rates and mostly asymptomatically infected neonates using frozen milk. Despite different freeze-storing procedures, HCMV transmissions occurred, and severe HCMV infections were observed. Few data exist on the long-term outcome of postnatally acquired HCMV infection via breast milk. To substantiate the international debate on the use of native or inactivated milk for feeding of preterm infants, additional data are necessary for better identification of mother-infant-pairs at risk for viral transmission and symptomatic infection early after birth.