Interlaboratory validation of a CD71-based flow cytometric method (Microflow) for the scoring of micronucleated reticulocytes in mouse peripheral blood

An interlaboratory study was performed to validate an anti-CD71/flow cytometry-based technique for enumerating micronucleated reticulocytes (MN-RETs) in mouse peripheral blood. These experiments were designed to address International Workshop on Genotoxicity Test Procedures validation criteria by evaluating the degree of correspondence between MN-RET measurements generated by flow cytometry (FCM) with those obtained using traditional microscopy-based methods. In addition to these cross-methods data, flow cytometric MN-RET measurements for each blood sample were performed at two separate sites in order to evaluate the reproducibility of data between laboratories. In these studies, groups of male CD-1 mice were treated with vehicle (saline or vegetable oil), a negative control (saline or vegetable oil), or four dose levels of five known genotoxicants (clastogens: cyclophosphamide, benzo[a]pyrene, 5-fluorouracil, methotrexate; aneugen: vincristine sulfate). Exposure occurred on 3 consecutive days via intraperitoneal injection, and blood samples were obtained approximately 24 hr after the final treatment. MN-RET frequencies were determined for each sample based on the analysis of 2,000 (microscopy) and 20,000 (FCM) reticulocytes. Regardless of the method utilized, each genotoxic agent was observed to cause statistically significant increases in the frequency of MN-RETs, and each response occurred in a dose-dependent manner. Spearman's correlation coefficient (rs) for FCM versus microscopy-based MN-RET measurements (nine experiments, 252 paired measurements) was 0.740, indicating a high degree of correspondence between methods. The rs value for all flow cytometric MN-RET measurements performed at the two independent sites was 0.857 (n = 248), suggesting that the automated method is highly transferable between laboratories. Additionally, the flow cytometric system offered advantages relative to microscopy-based scoring, including a greater number of cells analyzed, much faster analysis times, and a greater degree of objectivity. Collectively, data presented in this report suggest that the overall performance of mouse peripheral blood micronucleus tests is enhanced by the use of the flow cytometric scoring procedure.     

Anxiety Treatment and Targeted Sleep Enhancement to Address Sleep Disturbance in Pre/Early Adolescents with Anxiety

Sleep disturbance is prevalent in anxious youth and prospectively predicts poor emotional adjustment in adolescence. Study 1 examined whether anxiety treatment improves subjective and objective sleep disturbance in anxious youth. Study 2 examined whether a sleep intervention called Sleeping TIGERS can further improve sleep following anxiety treatment. Study 1 examined 133 youth (ages 9–14; 56% female; 11% ethnic/racial minority) with generalized, social, or separation anxiety over the course of anxiety treatment (cognitive behavioral treatment or client-centered treatment). Sleep-related problems (parent-, child-report) and subjective (diary) and objective (actigraphy) sleep patterns were assessed across treatment in an open trial design. Study 2 included 50 youth (ages 9–14; 68% female; 10% ethnic/racial minority) who continued to report sleep-related problems after anxiety treatment and enrolled in an open trial of Sleeping TIGERS. Pre- and postassessments duplicated Study 1 and included the Focal Interview of Sleep to assess sleep disturbance. Study 1 demonstrated small reductions in sleep problems and improvements in subjective sleep patterns (diary) across anxiety treatment, but outcomes were not deemed clinically significant, and 75% of youth stayed above clinical cutoff. Study 2 showed clinically significant, large reductions in sleep problems and small changes in some subjective sleep patterns (diary). Anxiety treatment improves, but does not resolve, sleep disturbance in peri-pubertal youth, which may portend risk for poor emotional adjustment and mental health. The open trial provides preliminary support that Sleeping TIGERS can improve sleep in anxious youth to a clinically significant degree.     

Computational simulations of mitral regurgitation quantification using the flow convergence method: Comparison of hemispheric and hemielliptic formulae

Mitral regurgitation results from the incomplete closure of the mitral valve, and the noninvasive diagnosis of this disease remains an important clinical goal. In this study, steady flow computer simulations were used to evaluate flow convergence method for flow rate estimation. The hemispheric and hemielliptic formulae were compared for accuracy in the presence of complicating factors such, as ventricular confinement, orifice shape, and aortic outflow. Results showed that in the absence of aortic outflow and ventricular confinement, there was a plateau zone where the hemispheric formula approximated the true flow rate, independent of orifice shape. However, in the presence of complicating factors such as aortic outflow and ventricular confinement, there was no clear zone where the hemispheric formula could be applied. The hemielliptic formula, however, worked in, all cases, regardless of chamber size or magnitude of aortic outflow. Therefore, application of the hemielliptic formula shold be considered in future clinical studies.     

Clinical importance of Campylobacter pyloridis and associated serum IgG and IgA antibody responses in patients undergoing upper gastrointestinal endoscopy.

Campylobacter pyloridis was isolated from 77% of 220 (35%) unselected adults undergoing gastroscopy. Isolation was significantly associated with histological gastritis (p less than 0.0001), duodenal ulcer (p less than 0.0001), and to a much lesser extent, with gastric ulcer (p less than 0.05). The relation between the isolation of C pyloridis and peptic ulcer seemed to be independent of coexisting gastritis. In those with no endoscopic or histological evidence of disease there was no relation between isolation and increasing age. Antibody responses to a whole cell sonicate of a strain of C pyloridis were measured by means of an enzyme linked immunosorbent assay (ELISA). Increased IgA (p less than 0.0001) and IgG (p less than 0.0001) antibody titres were found in patients with C pyloridis. Peptic ulceration or gastritis were present in 78% and 100% of patients with a high concentration of IgG and IgA, respectively, but in only 9% and 18% of those with low titres. These results provide further evidence for a possible pathogenic role of these organisms in gastric disease and suggest that immunological markers of their presence might be useful non-invasive indicators of disease.     

Inverted repeats direct repair of adenovirus minichromosome ends

Adenovirus DNA initiates strand-displacement replication from origins located in identical inverted terminal repetitions (ITRs). Panhandle structures, formed by base pairing between ITRs on the displaced strands, have been proposed as replication intermediates for complementary strand synthesis. We have used a model system, which separates adenovirus replication origin sequences from those involved in panhandle formation, to study the length and sequence integrity of panhandles. By making a series of unidirectional deletion in the panhandle sequence, we show that 31 bp are necessary for panhandle formation. Removal of long stretches of 3'-unpaired nucleotides distal to the panhandle is extremely efficient. Our results argue for the formation of panhandles during adenovirus DNA replication and provide a mechanism for maintaining sequence identity between distantly located inverted repetitions. The size constraint may explain why the adenovirus ITRs are larger than the viral DNA replication origins.     

In vitro erosion of 20 commercial glass ionomer cements measured using the lactic acid jet test.

The lactic acid jet test has been proposed for incorporation in international standards for dental cements, since results correlate well with in vivo erosion. In the literature, jet test results have been reported on only six glass ionomer cements. This study examines three types of glass ionomer, restorative, luting and metal reinforced, twenty materials from six manufacturers. A wide range of erosion rates was found particularly for restorative cements. The hypotheses put forward by previous workers to explain differences in erosion rates found on their limited range of glass ionomers do not apply to the wider range that we tested.     

Semiautomated determination of minimum bactericidal concentrations of antibiotics

Using a newly devised 50-channel photometer which records the opacity of growing bacterial cultures, it was shown that the time taken by cultures diluted 1/1000 in fresh broth to reach 50% of the opacity of a fully grown culture was inversely related to the concentration of organisms in the original culture. This relation was used to determine the numbers of survivors after exposure to benzylpenicillin and gentamicin alone and in combination. The procedure is commended as a labour-saving and potentially rapid method of obtaining comprehensive information on the bactericidal action and interaction of antibiotics.     

Cytotoxic potential of different lymphoid cell populations against chromium-51 labelled tumour cells

Release of chromium-51 (⁵¹Cr) from pre-labelled target cells was used as an assay of cytotoxicity. Different immune and non-immune lymphoid cell populations were tested for in vitro cytotoxicity against pre-labelled tissue culture target cells (Ha 3) which originated from a tumour induced by murine sarcoma virus (MSV) in a CBA mouse. Anti-Ha 3 peritoneal exudate and spleen cells showed significant specific cytotoxicity against these target cells. This was true for cells taken from animals immunized against either strong (H-2 plus non-H-2) or weak (non-H-2) antigenic determinants on the Ha 3 cells. Exogenous complement was not required for this cytotoxic activity. Tissue culture cells derived from a methylcholanthrene-induced tumour (MBE) of CBA origin were not damaged by peritoneal exudate cells from mice immunized against non-H-2 antigens on the Ha 3 cells. Anti-Ha 3 lymph node cells were relatively ineffective in these experiments although some cytotoxic activity was detected with lymph node cells sensitized against strong antigens. Direct morphological observation confirmed the cytotoxicity of anti-Ha 3 peritoneal exudate cells measured by ⁵¹Cr release.     

Scleromyxedema myopathy. Histochemical and electron microscopic observations

A biopsy-proven case of scleromyxedema (papular mucinosis) with IgG lambda light chain paraproteinemia, eosinophilia and severe proximal myopathy is presented. Muscle biopsy revealed an atypical necrotizing vacuolar myopathy. Histochemical studies of cryostat sections revealed fiber necrosis, severe type II fiber atrophy, and fiber vacuolization with NADH tetrazolium reductase hyperactivity. Electron microscopy showed myocytolysis, reduplication of the basement membrane, and unit membrane-lined vacuoles negative for acid mucopolysaccharide.