Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Ham's F12 medium on polycarbonate filters. DNA was labeled by incubation with [³H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H₂O₂). Cells incubated in H₂O₂ demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H₂O₂-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H₂O₂-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.This work was supported by NIEHS grant ES-00628 and California Primate Research Center Base grant. Portions of this work were presented at the American Thoracic Society annual meeting, May 1992, Miami, Florida.     

Methicillin-resistant Staphylococcus aureus: laboratory detection methods in use in the Republic of Ireland and Northern Ireland

There is no universally agreed laboratory protocol for the detection of methicillin-resistant Staphylococcus aureus (MRSA) and hence a variety of approaches are used. As part of an all-island survey of MRSA in the Republic of Ireland (the South) and Northern Ireland (the North), a questionnaire was circulated to 14 participating laboratories in the North and 49 in the South, to determine the methods used to isolate MRSA from clinical specimens, identify S. aureus and test for susceptibility to methicillin. Almost two-thirds (64%) of laboratories in the North but only 16% of laboratories in the South use enrichment culture. There is heavy reliance on commercial kits to confirm the identification of S. aureus in the South but all laboratories in the North use the staphylocoagulase test. More than 90% of all laboratories use a disc method for susceptibility testing and 71% of laboratories in the North supplement this with the E-test; however, a range of methicillin disk concentrations are in use. There is a need to review current laboratory methods used to detect MRSA, with follow-up audit on their implementation. Additional resources may be needed in some laboratories to comply with revised guidelines, and reference facilities are required to assess new commercially available techniques and to confirm the identification of unusual or difficult strains.     

Nuclear expression of p53, p21 and cyclin D1 is increased in bronchioloalveolar carcinoma

AIMS: The objectives of this study were: (1) to determine, using immunohistochemistry, the level of expression of the cell cycle factors p53, p21 and cyclin D1 in a group of bronchioloalveolar carcinomas (BACs), and to compare these data to relevant published data for lung carcinoma; (2) to determine if higher expression rates for these factors in BAC were associated statistically with advanced clinical stage, greater tumour size, tobacco abuse, and/or BAC subtype; (3) to seek, using Fisher's exact t-test and paired data groups, any significant associations within the expression data for p53, p21 and cyclin D1. METHODS AND RESULTS: A panel of monoclonal antibodies against p53, p21 and cyclin D1 was applied to 19 bronchioloalveolar carcinomas (17 surgical pathology cases and two autopsies) from the tissue archives of St. Louis University. These immunohistochemical stains were graded on a semiquantitative scale according to the prevalence of nuclear staining within the tumour (< 10% positive cells = 0, 10-25% = 1+, 25-50% = 2+, 50-75% = 3+ and 75-100% = 4+). Six of 19 (32%) of BACs showed 1+ or greater p53 positivity, six of 19 (32%) showed 1+ or greater nuclear cyclin D1 positivity, and nine of 19 (47%) of BACs showed 1+ or greater p21 nuclear positivity. A statistically significant correlation was found between p53 and cyclin D1 expression (P = 0.046, Fisher's exact t-test), but not between p53 and p21, or between p21 and cyclin D1. No statistically significant association was found between the cell cycle factor expression data and subtype of BAC (mucinous vs. nonmucinous), tumour diameter, clinical stage or tobacco-use history. CONCLUSIONS: BACs show p53 immunostain positivity at a frequency similar to that published for p53 mutations in lung adenocarcinomas in general. Cyclin D1 and p21 nuclear expression characterizes a significant proportion of BACs, with cyclin D1 and p53 expression showing a statistically significant association. Aberrations in p53, p21, and cyclin D1 expression may be important in the development of a significant proportion of BACs.     

Enterococcal prosthetic valve infective endocarditis: report of 45 episodes from the International Collaboration on Endocarditis-merged database

Enterococcal prosthetic valve infective endocarditis (PVE) is an incompletely understood disease. In the present study, patients with enterococcal PVE were compared to patients with enterococcal native valve endocarditis (NVE) and other types of PVE to determine differences in basic clinical characteristics and outcomes using a large multicenter, international database of patients with definite endocarditis. Forty-five of 159 (29%) cases of definite enterococcal endocarditis were PVE. Patients with enterococcal PVE were demographically similar to patients with enterococcal NVE but had more intracardiac abscesses (20% vs. 6%; p=0.009), fewer valve vegetations (51% vs. 79%; p<0.001), and fewer cases of new valvular regurgitation (12% vs. 45%; p=0.01). Patients with either enterococcal PVE or NVE were elderly (median age, 73 vs. 69; p=0.06). Rates of in-hospital mortality, surgical intervention, heart failure, peripheral embolization, and stroke were similar in both groups. Patients with enterococcal PVE were also demographically similar to patients with other types of PVE, but mortality may be lower (14% vs. 26%; p=0.08). Notably, 93% of patients with enterococcal PVE came from European centers, as compared with only 79% of patients with enterococcal NVE (p=0.03). Thus, patients with enterococcal PVE have higher rates of myocardial abscess formation and lower rates of new regurgitation compared to patients with enterococcal NVE, but there are no differences between the groups with regard to surgical or mortality rates. In contrast, though patients with enterococcal PVE and patients with other types of PVE share similar characteristics, mortality is higher in the latter group. Importantly, the prevalence of enterococcal PVE was higher in the European centers in this study.     

Gastrointestinal graft-versus-host disease in recipients of autologous hematopoietic stem cells: incidence, risk factors, and outcome.

Graft-versus-host disease (GVHD) is seen in skin, intestinal mucosa, and liver after autologous stem cell transplantation. We reviewed 681 consecutive patients to estimate the probability of gastrointestinal (GI) GVHD, response to treatment, risk factors for development, and effect on survival. GI GVHD was defined by persistent symptoms, mucosal abnormalities at endoscopy, and histology showing apoptotic crypt cells with or without lymphoid infiltrates. The proportion of patients with GI GVHD was 90/681 (13%). Nausea and vomiting occurred in 90% and diarrhea in 40%. The mean time to developing symptoms was day +15, that to histologically proven diagnosis was day +42, and that to starting prednisone treatment was day +45 after stem cell infusion. Treatment with a short course of prednisone effected durable responses in 79% of patients, and an additional 18% responded to a second course of prednisone. A multivariable logistic regression model demonstrated that the combined factor of a diagnosis of breast cancer or hematologic malignancy and female sex was statistically significantly associated with the probability of GI GVHD (P = .003). Survival in patients with GI GVHD was not statistically different than that in those without GVHD. We conclude that women with breast cancer or hematologic malignancy are more likely to develop GI GVHD after autologous transplantation, and that treatment with prednisone was effective.     

High Throughput Fingerprint Analysis of Large-Insert Clones

As part of the Human Genome Project, the Washington University Genome Sequencing Center has commenced systematic sequencing of human chromsome 7. To organize and supply the effort, we have undertaken the construction of sequence-ready physical maps for defined chromosomal intervals. Map construction is a serial process composed of three main activities. First, candidate STS-positive large-insert PAC and BAC clones are identified. Next, these candidate clones are subjected to fingerprint analysis. Finally, the fingerprint data are used to assemble sequence-ready maps. The fingerprinting method we have devised is key to the success of the overall approach. We present here the details of the method and show that the fingerprints are of sufficient quality to permit the construction of megabase-size contigs in defined regions of the human genome. We anticipate that the high throughput and precision characteristic of our fingerprinting method will make it of general utility.     

Tri-ministry study: correlates of school-based parenting course utilization

This study examined factors associated with the utilization of universally available school-based parent training. In a randomly selected, prospectively screened, unreferred community sample of 1,498 5- to 8-year-olds, 28% to 46% of families of children with high parent-reported externalizing problems enrolled. Externalizing problems, first-child status, and a high school education were associated with increased enrollment. Single-parent status, immigrant background, and limited extracurricular child activities were associated with lower enrollment. Economic disadvantage, stress, family dysfunction, and parental depressive symptoms were not associated with participation. Most families attributed nonparticipation to busy personal schedules, inconvenient times, and logistical difficulties.     

Clinical importance of increased sensitivity of BacT/Alert FAN aerobic and anaerobic blood culture bottles.

Two recent multicenter blood culture studies found that BacT/Alert FAN (FAN) bottles (Organon Teknika, Durham, N.C.) had increased yields in detecting bacteremia and fungemia compared with standard BacT/Alert (STD) bottles. Because the clinical importance of this increase in microbial recovery is unknown, we performed a retrospective analysis to determine the frequency with which FAN bottles were the sole means of detecting an episode of bacteremia. There were 1,047 positive blood cultures in which both study bottles were adequately filled and the organism isolated was judged to be the cause of sepsis: 240 (23%) were positive only in FAN bottles and 73 (7%) were positive only in STD bottles. Of a total of 664 episodes of bacteremia, 126 (19%) were identified only by FAN bottles and 43 (7%) were identified only by STD bottles (P < 0.0001). Episodes detected only by FAN bottles more often were recurrent events (23 of 126, or 18%) than episodes detected only by STD bottles (2 of 43, or 5%) (P < 0.05) and more commonly occurred in patients receiving theoretically effective antibiotic therapy (33 of 126 [26%] versus 4 of 43 [9%]) (P < 0.05). The medical records for patients with 127 of these episodes (92 FAN bottles only; 35 STD bottles only) were available for review. More than half of both FAN bottle-only (60 of 92, or 65%) and STD bottle-only (20 of 35, or 57%) episodes were judged to be clinically important. We conclude that FAN bottles improve the detection of bacteremia and that the majority of the additional episodes detected are clinically important. The benefits of the greater yield in specific patient populations must be balanced against the higher costs of FAN bottles.     

Detection of Campylobacter pylori DNA by hybridisation with non-radioactive probes in comparison with a 32P-labelled probe

A dot-blot hybridisation assay for the detection of Campylobacter pylori was used to compare a 32P-labelled probe with two biotinylated probes and a sulphonated probe. The minimum amount of pure C. pylori DNA that could be detected by the 32P-labelled probe was 100 pg, which corresponded to 5 x 10(4) bacteria. A biotin-labelled DNA (biotin-DNA) probe together with the BluGeneTM detection system produced by Bethesda Research Laboratories (BRL), and a sulphonated probe and ChemiprobeTM detection system (Orgenics) gave similar levels of sensitivity; nylon membranes could be used with both these non-radioactive detection systems. However, a photobiotin-labelled DNA (photobiotin-DNA) probe and detection system produced by Biotechnology Research Enterprises S.A. (BRESA) gave optimum results only with nitrocellulose membranes, and was quantitatively 100 times less sensitive than the other types of probe. The detection systems for the biotin-DNA and photobiotin-DNA probes produced non-specific reactions with crude bacterial blots of heterologous organisms; these non-specific reactions could be removed by treating the dot blots with proteinase K, but not by treatment with RNAase. The sulphonated probe and detection system did not give any reaction with heterologous organism blots.     

Effect of glycine and glycine receptor antagonists on NMDA-induced brain injury

In postnatal day 7 rats, a unilateral intrastriatal injection of 12.5 nmol of N-methyl-D-aspartate (NMDA) reproducibly injures the ipsilateral striatum, adjacent hippocampus and overlying cortex. The severity of injury can be quantified by comparing cerebral hemisphere weights in animals sacrificed 5 days after the injection. Co-injection of NMDA and the glycine receptor antagonists kynurenic acid (KYN) or 7-chlorokynurenic acid (7-CKA) reduced the severity of NMDA-induced damage in a dose-dependent fashion. One hundred nmol of KYN with 12.5 nmol of NMDA reduced average % damage from 19.3 +/- 0.9% (n = 9) to 2.3 +/- 0.5% (n = 6), P less than 0.001, ANOVA. Co-injection of 40 nmol of 7-CKA with 12.5 nmol of NMDA (n = 6) reduced average % damage from 17.1 +/- 1.6% (n = 15) to 3.0 +/- 0.6%, P less than 0.001, ANOVA. Concurrent injection of 1000 nmol glycine with 5 nmol NMDA did not increase the extent of NMDA-induced damage. Our results demonstrate that glycine receptor antagonists attenuate NMDA-induced brain injury in vivo.