Genetic basis for conversion of rough-to-smooth colony morphology in Actinobacillus actinomycetemcomitans

The basis of the rough-to-smooth conversion of Actinobacillus actinomycetemcomitans was examined. Smooth variants often contained mutations at the flp promoter region. Replacing the mutated flp promoter with the wild-type promoter restored the rough phenotype. The expression level of the flp promoter was approximately 100-fold lower in smooth than in rough strains. Mutations of the flp promoter are a cause of the rough-to-smooth conversion.     

Practical Evaluation of Methods for Detection and Specificity of Autoantibodies to Extractable Nuclear Antigens

Detection and specificity of autoantibodies against extractable nuclear antigens (ENA) play a critical role in the diagnosis and management of autoimmune disease. Historically, the detection of these antibodies has employed double immunodiffusion (DID). Autoantibody specificity was correlated with diagnoses by this technique. Enzyme immunoassays have been developed by multiple manufacturers to detect and identify the specificity ENA autoantibodies. To address the relationship of ENA detection by DID and enzyme immunoassay, the performances of five immunoassays were compared. These included two DID and three enzyme-linked immunoassays (ELISA) (both screening and individual antigen profile kits). The sample set included 83 ENA-positive, antinuclear-antibody (ANA)-positive specimens, 77 ENA-negative, ANA-positive specimens, and 20 ENA- and ANA-negative specimens. Sensitivity and specificity were calculated by two methods: first, by using the in-house DID result as the reference standard, and second, by using latent class analysis, which evaluates each kit result independently. Overall, the results showed that the ELISA methods were more sensitive for detection of ENA autoantibodies than DID techniques, but presence and/or specific type of ENA autoantibody did not always correlate with the patient''s clinical presentation. Regardless of the testing strategy an individual laboratory uses, clear communication with the clinical staff regarding the significance of a positive result is imperative. The laboratory and the clinician must both be aware of the sensitivity and specificity of each testing method in use in the clinical laboratory.A diagnosis of autoimmune disease in patients is based upon clinical history, physical examination, and laboratory detection of antinuclear antibodies (ANAs). A particular class of ANAs specific for extractable nuclear antigens (ENA) was initially described in 1959 (3). Since that time, many different anti-ENA antibodies have been described. The detection of these autoantibodies and identification of their specificity have become well-established tools for the laboratory diagnosis of several autoimmune diseases. Studies of patients with ENA antibodies have shown that detection of these autoantibodies may have both diagnostic and prognostic significance, and the detection of anti-ENA antibodies has assumed an important role in the management of these patients (5, 16, 22). In most cases, ENA testing is ordered after an initial ANA screen. The indications for use are to establish a diagnosis in patients with suggestive clinical symptoms, to exclude a diagnosis of autoimmune disease in patients with few or uncertain clinical signs, to subclassify patients with a known diagnosis, and to monitor disease activity.Testing for anti-ENA antibodies has historically relied on gel-based immunoprecipitation techniques such as double immunodiffusion (DID) and counterimmunoelectrophoresis (2, 14). The associations of specific types of ENA autoantibodies with rheumatological diseases were established by using these gel-based immunoassay techniques (15). In the last decade, enzyme-linked immunoassay (ELISA) systems have been developed to detect and determine the specificity of anti-ENA antibodies. ELISA systems permit more rapid processing of more specimens with a faster turnaround time than gel-based assays. ELISA-based methods may also have increased sensitivity for detection of ENA antibodies. However, the increased sensitivity of these ELISAs may influence the clinical relevance of their detection because diagnostic specificity may be reduced (10, 12, 17, 24). As yet, a set of reference standards with known antibody specificities against defined antigen preparations is not available for evaluation of various methods or kits. Serum reference panels are available from the Association of Medical Laboratory Immunologists (4), but the specificities of these sera were determined by consensus results from multiple laboratories. The purpose of this study was to address the relationship between DID and ELISA methods for the detection and identification of anti-ENA antibodies by evaluating and comparing two DID kits and three ELISA kits. We evaluated both screening ELISAs and monospecific antigen ELISAs to determine anti-ENA specificity.     

Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.     

Haplotypes produced from rare variants in the promoter and coding regions of angiotensinogen contribute to variation in angiotensinogen levels

The most efficient study design to map genes underlying complex traits will be determined by assumptions about whether the genetic effects are likely to be due to relatively few common variants or multiple rare variants. To examine the possibility that rare variants may influence blood pressure, we sequenced a 6.8 kb region of the angiotensinogen (AGT) gene in 29 male Nigerians with high plasma AGT levels and 28 with low levels. The frequency of haplotypes produced from rare variants in the promoter and coding regions was significantly different between the two groups, and it is unlikely that this difference was due to the manner in which the rare variants were selected. Further analysis suggested that most of the haplotypes produced by these rare variants are found on a haplotype background created by three common SNPs. Our study confirms in an additional trait that rare variants can influence the distribution of complex traits; whether these variants can be captured by common SNPs or haplotypes requires further investigation.     

Comparison of complement fixation and hemagglutination inhibition assays for detecting antibody responses following influenza virus vaccination

Complement fixation (CF) was compared to hemagglutination inhibition (HI) as a method for identifying antibody responses to influenza virus vaccination. CF assays were performed at two different laboratories using paired (pre- and postvaccination) sera from 38 vaccinated laboratory employees; HI assays were performed at a third laboratory. As expected, most vaccinees (31/38 = 82%) responded to at least one of three influenza virus antigens as measured by HI. In contrast, only 21% (8/38) of vaccinees showed a response by CF at laboratory 1, and only 29% (11/38) showed a response by CF at laboratory 2. These findings indicate that due to low sensitivity, CF assays should not be used to assess the antibody response to influenza virus vaccination.     

Myocardial disease and catecholamine metabolism in JCR:LA-corpulent rat

The JCR:LA-cp rat is a strain carrying the mutant cp (corpulent) gene. Animals that are homozygous cp are hyperphagous, hyperinsulinemic, hyperlipidemic, and obese. Corpulent male rats, but not females or lean rats, develop atherosclerotic lesions and myocardial lesions. Since the myocardial lesions are apparently of ischemic origin, the noradrenergic system and vascular hyperactivity and vasospasm may play a role in the pathogenesis. To test this we have studied the brain contents of the amines norepinephrine, dopamine, and 5-hydroxtryptamine and their breakdown products and depleted the peripheral sympathetic terminals with 6-hydroxydopamine. Only 5-hydroxytryptamine and 5 hydroxyindole-3-acetic acid were present at higher concentrations in the corpulent rats with depressed levels of dopamine in very young or old lean rats. The activity of monoamine oxidase may provide an indication of nonadrenergic activity in tissue. The activity in the heart increased with age and was higher in the corpulent rats than in the lean at all ages. Activity in aorta was independent of age or genotype. Long term treatment with 6-hydroxydopamine caused marked depletion of norepinephrine in the heart with only a slight decrease in brain concentration. There were no effects on the hyperlipidemia or hyperinsulinemia that are strongly associated with vascular and myocardial disease. The myocardial lesion frequency in corpulent rats was not altered by the chemical sympathectomy. The results suggest that norepinephrine and the sympathetic nervous system are probably not involved in the generation of the myocardial lesions or metabolic abnormalities in this strain of rat.     

Examining Trichophyton tonsurans genotype and biochemical phenotype as determinants of disease severity in tinea capitis.

Trichophyton tonsurans infections occur in various host populations, on various body sites and with varying degrees of inflammation. This investigation was undertaken to determine whether fungal factors could explain the degree of severity in clinical symptomatology among infected children. Otherwise healthy children (n=54) presenting with tinea capitis were enrolled in this study. A thorough history was performed, the extent and severity of infection graded and a fungal specimen collected from each child. Strain type was determined by genotyping for 11 sequence variations in the rDNA and ALP1 loci. Secreted protease activity was quantitated after 5 days of growth in aqueous medium. Forty participants were evaluable. Infection duration ranged from 1 day to 3 years and clinical severity score (CSS) from 4-19. Seventeen unique fungal genotypes were present. Keratinase, collagenase and elastase activity varied 32.7-fold, 64.9-fold and 303.3-fold, respectively. A significant association was observed between genotype and disease severity with the rDNA sequence variations accounting for over 50% of the variation observed in CSS (r2=0.539; P<0.001). Phylogenetic analyses appear to suggest that the ancestral strain types of T. tonsurans cause more severe disease. These observations are consistent with reports that recently diverge anthropophilies are associated with diminished inflammatory involvement.