Dissociation of hepatitis A virus antigen-anti-HAV antibody complexes by 2-mercaptoethanol and dithiothreitol

Intravenous inoculation of two marmosets and one chimpanzee with hepatitis A virus (HAV) resulted in the replication of virus in liver, excretion of HAV particles in stool, and the appearance of circulating antibodies specific for hepatitis A. The development of an early antibody response in the chimpanzee and in one of the two infected marmosets was shown to interfere with the serologic detection of HAV antigen (HAV Ag) in homogenates of acute phase liver tissue obtained from these animals. Treatment of HAV Ag-positive and IgM anti-HAV-positive liver homogenates with thiol reducing compounds was shown to release HAV Ag from in vitro formed immune complexes. The increased RIA response for HAV Ag in homogenates treated with 2-mercaptoethanol (2-ME) or dithiothreitol (DTT) was further shown not to be due to activation of HAV Ag itself or to a nonspecific effect on the RIA coating antibody, radiolabeled probe, or homogenized liver tissue. IgG and IgM double-antibody sandwich RIAs for HAV Ag were also compared for their ability to detect HAV Ag under reducing and nonreducing conditions. Application of the 2-ME or DTT treatment procedure to the serologic detection of other viral antigens or viruses whose presence in blood, stool, tissue macerate, or other milieu may be masked by specific antibody appears to be feasible.     

Regulated activity of the IgH intron enhancer (E{micro}) in the T lymphocyte lineage

The activity of the IgH (E_µ) enhancer in the T lymphocytelineage has been investigated using both transgenic mice andtransfection studies. Thymocyte fractionation experiments indicatethat a transgene consisting of the bacterial chloramphenicolacetyl transferase (CAT) gene, linked to Eµ and the SV40early promoter (E_µ–CAT), is expressed only in thymocyteswith a mature medullary phenotype and not in immature cells.Transfection of this same construct into two thymoma cell linesrepresenting different stages of thymocyte development mimicsthe pattern of activity observed in vivo. Further transfectionexperiments suggest that this pattern of expression might beattributed to the differential activity of the E2E3 and octanucleotidemotifs of E_µ during development. In contrast, an Ig transgene(linked to E_µ and an Ig V promoter) is expressed in themajority of thymocytes. We envisage that the different patternsof expression of the two transgenes reflect interactions betweentheir respective promoters and the factors which are bound toE_µ at different stages of thymocyte development. Althoughdiffering in their pattern of expression within the thymus,the two transgenes share the property of extinction in peripheralT lymphocytes. These results indicate that the expression ofE_µ-linked transgenes in the thymus cannot simply be explainedby activation of the enhancer in a lymphoid progenitor cellprior to B/T lineage divergence. Rather, the enhancer (or componentsof it) must be independently activated (and inactivated) duringT lymphocyte development. Furthermore, this activity is consistentwith the developmental timing of Ig D_H–J_H rearrangementsin these cells.     

Conventional methods versus 16S ribosomal DNA sequencing for identification of nontuberculous mycobacteria: cost analysis

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.     

Genome-wide linkage analysis of families with obsessive-compulsive disorder ascertained through pediatric probands

The goal of this study was to identify chromosomal regions likely to contain susceptibility alleles for early-onset obsessive-compulsive disorder (OCD). A genome scan was done in 56 individuals from seven families ascertained through pediatric OCD probands; 27 of the 56 subjects had a lifetime diagnosis of definite OCD. Denser mapping of regions on chromosomes 2, 9, and 16 was subsequently done with those subjects and ten additional subjects from the largest family in the study. Direct interviews were completed with 65 of the 66 genotyped individuals. Relatives were interviewed blind to proband status. Of the 65 interviewed individuals, 32 had a lifetime diagnosis of definite OCD. Three of the seven probands had a history of Tourette disorder. Two of the 25 relatives with OCD had a tic history, whereas none of the 33 relatives without OCD had tics. The genome scan consisted of 349 microsatellite markers with an average between-marker distance of 11.3 centiMorgan (cM). Fine mapping was done with 24 additional markers at an average spacing of 1.6 cM. Parametric and nonparametric linkage analyses were conducted using GENEHUNTER(+). The maximum multipoint LOD score with a dominant model was 2.25 on 9p. However, with fine mapping and additional subjects, that LOD score decreased to 1.97. The maximum multipoint nonparametric LOD* score was 1.73 on 19q. The maximum multipoint LOD score with a recessive model was 1.40 on 6p. The results provide suggestive evidence for linkage on 9p and identify regions requiring further study with much larger samples.     

Morphologic Development of Fetal Rabbit Lung and Its Acceleration with Cortisol

The influence of cortisol on the maturation of fetal lungs of rabbits has been studied. The normal developmental pattern of the various lobes is also documented and it is shown that there is a definite sequence of maturation among the five lobes of the lung. At a given gestational age, both upper lobes are consistently most mature. The right middle lobe is somewhat less mature while right and left lower lobes are least mature. The difference in the rate of maturation between the most advanced right apex and the least advanced base of lower lobes is as much as 1½ days out of a total gestation of 30 days. Cortisol, injected into the fetuses 2 days prior to delivery, accelerated the overall maturation of the lung by about 1½ days. The maturation of the type II epithelial cells also was accelerated and their inclusion bodies were more numerous than those of the controls. As reported elsewhere, prematurely delivered cortisol-injected rabbits of 26 to 28 days' gestation are more viable, breathe better and their lungs are better aerated when compared with control littermates. The surface activity of the alveolar lining of these animals is greater than that of the controls as indicated by higher ratios of bubble stability. These findings are apparently the results of accelerated maturation of the type II alveolar epithelial cells, which contain surfactant or its precursors as inclusions within the cytoplasm.     

Role of matrix metalloproteinases in failure to re-epithelialize after corneal injury.

Delayed re-epithelialization of the cornea after injury usually precedes stromal ulceration. Previous findings using a rat thermal injury model suggested that re-epithelialization is impeded by products of resident corneal cells, which destroy adhesive structures at the basement membrane zone. In this study, we provide additional evidence for this concept. Failure to re-epithelialize was found to correlate with an increase in the amounts of gelatinolytic matrix metalloproteinases present in the rat cornea. One of these gelatinases, gelatinase B, is synthesized by the resident corneal cells, and inhibitions of its synthesis correlated with inhibition of basement membrane dissolution. The matrix metalloproteinases collagenase and stromelysin are also synthesized by resident corneal cells in thermally injured corneas of rabbits, but the timing of bulk enzyme synthesis correlated more closely with deposition of repair tissue in the stroma than with failure to re-epithelialize. Nevertheless, in human corneas with repair defects, gelatinase B and collagenase are synthesized by cells in the basal layer of the epithelium directly adjacent to the basement membrane, suggesting that both could participate in dissolution of this structure. Importantly, treatment of thermally injured corneas with a synthetic inhibitor of matrix metalloproteinases significantly improved basement membrane integrity. These data support the concept that over-expression of matrix metalloproteinases by resident corneal cells impedes re-epithelialization after some types of corneal injury.     

Ionic basis of the resting membrane potential and action potential in the pharyngeal muscle of Caenorhabditis elegans

The pharynx of C. elegans is a rhythmically active muscle that pumps bacteria into the gut of the nematode. This activity is maintained by action potentials, which qualitatively bear a resemblance to vertebrate cardiac action potentials. Here, the ionic basis of the resting membrane potential and pharyngeal action potential has been characterized using intracellular recording techniques. The resting membrane potential is largely determined by a K(+) permeability, and a ouabain-sensitive, electrogenic pump. As previously suggested, the action potential is at least partly dependent on voltage-gated Ca(2+) channels, as the amplitude was increased as extracellular Ca(2+) was increased, and decreased by L-type Ca(2+) channel blockers verapamil and nifedipine. Barium caused a marked prolongation of action potential duration, suggesting that a calcium-activated K(+) current may contribute to repolarization. Most notably, however, we found that action potentials were abolished in the absence of external Na(+). This may be due, at least in part, to a Na(+)-dependent pacemaker potential. In addition, the persistence of action potentials in nominally free Ca(2+), the inhibition by Na(+) channel blockers procaine and quinidine, and the increase in action potential frequency caused by veratridine, a toxin that alters activation of voltage-gated Na(+) channels, point to the involvement of a voltage-gated Na(+) current. Voltage-clamp analysis is required for detailed characterization of this current, and this is in progress. Nonetheless, these observations are quite surprising in view of the lack of any obvious candidate genes for voltage-gated Na(+) channels in the C. elegans genome. It would therefore be informative to re-evaluate the data from these homology searches, with the aim of identifying the gene(s) conferring this Na(+), quinidine, and veratridine sensitivity to the pharynx.     

Serotonin transporter promoter polymorphism, peripheral indexes of serotonin function, and personality measures in families with alcoholism

A functional polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) is considered to be a plausible candidate gene for anxiety-related personality traits and for alcoholism. Empirical support for the association between 5-HTTLPR and psychological traits has been somewhat inconsistent; however, observations of the functional dominance of the low-activity s-allele over the l-allele have been more consistent. When studying the influence of particular genes on psychological traits, it seems useful also to assess more biological intermediate traits that may mediate the effects of those genes on the traits of interest. The present study examined relationships between 5-HTTLPR genotype, whole blood serotonin (5-HT) level, and platelet 5-HT binding in 150 Caucasian subjects from 50 biological families. Individuals with the s-allele had lower average platelet 5-HT binding availability than those with the l/l genotype (P<0.025). Whole blood 5-HT level was not associated with 5-HTTLPR genotype. In adult men, those with the s-allele had higher mean scores on the NEO-FFI personality trait of openness than did those with the l/l genotype (P=0.002). The effect was not statistically significant in women (P=0.42), although it was in the same direction. Our findings do not support an association of 5-HTTLPR genotype with alcoholism diagnosis, alcoholism subtype, or the personality trait of neuroticism. The results of this pilot study suggest that further work should examine the mediation of the genetic effects on personality traits by biochemical measures and their moderation by gender.     

Local and systemic antibody class responses to an infectious laryngotracheitis virus vaccine strain

Chickens infected with infectious laryngotracheitis virus (ILTV) responded by producing virus-specific IgG in their sera, which increased steadily in concentration, but with slight fluctuations, until peak titres were reached 40 days post-inoculation (pi), immediately prior to the second challenge. Thereafter, following an initial lag, concentrations continued to increase for 21 days before falling slightly at the end of the experiment. In contrast, peak concentrations of ILTV-specific IgM were reached 6 days pi falling to their lowest levels by day 16, before increasing to a second peak and trough on days 26 and 32, respectively. This cyclical production of ILTV-specific IgM was confirmed in a second experiment. The pattern of production of ILTV-specific IgG, IgM and IgA, detected in tracheal washings, occurred in the same cyclical manner. IgM was produced first, peak concentrations being detected 5 days pi, whereas IgG and IgA did not peak until 10 days pi, with second peaks of each class being detected 25-30 days pi. The possibility that the cyclical antibody class response to ILTV infection is related to the previously reported intermittent pattern of re-excretion of the virus is discussed.