Detection of Bacteroides fragilis Enterotoxin Gene by PCR

Bacteroides fragilis constitutes about 1% of the bacterial flora in intestines of normal humans. Enterotoxigenic strains of B. fragilis have been associated with diarrheal diseases in humans and animals. The enterotoxin produced by these isolates induces fluid changes in ligated intestinal loops and an in vitro cytotoxic response in HT-29 cells. We developed a nested PCR to detect the enterotoxin gene of B. fragilis in stool specimens. After DNA extraction, a 367-bp fragment was amplified with two outer primers. The amplicon from this reaction was subjected to a second round of amplification with a set of internal primers. With these inner primers, a 290-bp DNA fragment was obtained which was confirmed as part of the B. fragilis enterotoxin gene by Southern blotting with a nonradioactive internal probe and a chemiluminescence system. By this approach, B. fragilis enterotoxin gene sequences were detected in eight known enterotoxigenic human isolates and nine enterotoxigenic horse isolates. No amplification products were obtained from DNA extracted from 28 nonenterotoxigenic B. fragilis isolates or B. distasonis, B. thetaiotaomicron, B. uniformis, B. ovatus, Escherichia coli, or Clostridium difficile. The sensitivity of this assay allowed us to detect as little as 1 pg of enterotoxin DNA sequences or 100 to 1,000 cells of enterotoxigenic B. fragilis/g of stool. Enterotoxin production of all isolates was confirmed in vitro in HT-29 cells. A 100% correlation was obtained between enterotoxin detection by cytotoxin assay and the nested PCR assay. This rapid and sensitive assay can be used to identify enterotoxigenic B. fragilis and may be used clinically to determine the role of B. fragilis in diarrheal diseases.     

In VivoStudies of Adenovirus-Based p53 Gene Therapy for Ovarian Cancer

Objectives.To test the safety, efficacy, and toxicity of gene therapy using wild-type p53-expressing adenovirus (Ad-CMV-p53) in a nude mouse model with intraperitoneal (ip) 2774 human ovarian cancer cell line that contains a p53 mutation.Study design.An initial study of adenovirus tolerance was determined in nude mice by a single ip injection of increasing doses of Ad-CMV-p53. Nude mice were implanted with an LD₁₀₀dose of 1 × 10⁷cells. To study the efficacy and specificity of Ad-CMV-p53 treatment, the mice received treatment with different adenovirus constructs. One group received Ad-CMV-p53 and another group received a control adenovirus construct, Ad-CMV-βgal. To study the treatment response to Ad-CMV-p53, the mice were divided into groups and received various treatment schedules of 1 × 10⁸pfu of Ad-CMV-p53.Results.The mice tolerated Ad-CMV-p53 without adverse effects at doses of 1 × 10⁸pfu. The response to Ad-CMV-p53 showed significant survival duration in each dose regimen, with a survival time greater than that of untreated animals (P= 0.0173). However, no statistically significant survival advantage was observed between Ad-CMV-p53- and Ad-CMV-βgal-treated mice.Conclusions.These studies show that at the adenovirus dose and administration regimen used, there is effective but not specific 2774 tumor growth inhibitionin vivo.Efficient introduction of biologically active genes into tumor cells would greatly facilitate cancer therapy. Thus, although promising, these results caution that much effort will be required to realize the potential for clinical application of adenovirus-based ovarian cancer gene therapy.     

Serum and Cerebrospinal Fluid Pharmacokinetics of Intravenous and Oral Lamivudine in Human Immunodeficiency Virus-Infected Children

We studied the pharmacokinetics of intravenously and orally administered lamivudine at six dose levels ranging from 0.5 to 10 mg/kg of body weight in 52 children with human immunodeficiency virus infection. A two-compartment model with first-order elimination from the central compartment was simultaneously fitted to the serum drug concentration-time data obtained after intravenous and oral administration. The maximal concentration at the end of the 1-h intravenous infusion and the area under the concentration-time curve after oral and intravenous administration increased proportionally with the dose. The mean clearance of lamivudine (± standard deviation) in the children was 0.53 ± 0.19 liter/kg/h (229 ± 77 ml/min/m² of body surface area), and the mean half-lives at the distribution and elimination phases were 0.23 ± 0.18 and 2.2 ± 2.1 h, respectively. Clearance was age dependent when normalized to body weight but age independent when normalized to body surface area. Lamivudine was rapidly absorbed after oral administration, and 66% ± 25% of the oral dose was absorbed. Serum lamivudine concentrations were maintained above 1 μM for ≥8 h of 24 h on the twice daily oral dosing schedule with doses of ≥2 mg/kg. The cerebrospinal fluid drug concentration measured 2 to 4 h after the dose was 12% (range, 0 to 46%) of the simultaneously measured serum drug concentration. A limited-sampling strategy was developed to estimate the area under the concentration-time curve for concentrations in serum at 2 and 6 h.     

Genotype determining low catechol-O-methyltransferase activity as a risk factor for obsessive-compulsive disorder

In the present study, we address the role of the gene for catechol-O-methyltransferase (COMT), a key modulator of dopaminergic and noradrenergic neurotransmission, in the genetic predisposition to obsessive-compulsive disorder (OCD). We show that a common functional allele of this gene, which results in a 3- to 4-fold reduction in enzyme activity, is significantly associated in a recessive manner with susceptibility to OCD, particularly in males. This association is further supported by psychiatric evaluation of patients who carry microdeletions encompassing the comt gene. The mechanism underlying this sex-selective association remains to be defined and may include a sexual dimorphism in COMT activity, although close linkage with a nearby disease susceptibility locus cannot be excluded at this point.     

Comparison of 99m technetium hexamethylpropylene-amine oxime labelled leucocyte with 111-indium tropolonate labelled granulocyte scanning and ultrasound in the diagnosis of intra-abdominal abscess.

Fifty patients with suspected intra-abdominal abscess were investigated prospectively with ultrasound and with 99mTc-hexamethylpropylene-amine oxime (HMPAO) isotope labelled mixed leucocytes, using 111-In tropolonate granulocyte scanning as the reference standard. Twenty five patients had inflammatory bowel disease (three were postoperative): 21 of these had Crohn's disease and four had ulcerative colitis. The remainder comprised nine with postoperative fever and 16 with fever and abdominal pain. An abscess was diagnosed when focal activity on serial 111-In tropolonate and 99m-Tc-HMPOA images at one, three, and 24 hours resulted in activity at least equal to liver activity at 24 hours. Thirteen abscesses were diagnosed using each type of white cell scanning, resulting in 100% sensitivity for 99m-Tc-HMPAO compared with 111-In tropolonate. Bowel inflammation was easily distinguished from abscess on serial images. Eight of these 13 abscesses were detected by ultrasound. Altogether 17 abscesses were found. Ultrasound detected 12, including four liver abscesses which were not purulent and had not been detected by white cell scanning. Ultrasound had a sensitivity of 71% (12 of 17) and a specificity of 87% (33 of 38) using all confirmed abscesses as the reference standard. White cell scanning showed a sensitivity of 76% (13 of 17: as a result of the four non-purulent liver abscesses) and a specificity of 100%. 99m-Tc-HMPAO scanning is as accurate as 111-In tropolonate scanning, and has several advantages including simplicity, availability, superior image quality, and reduced radiation dose. Both methods are more sensitive and specific than ultrasound for intra-abdominal abscess detection but ultrasound is advisable if a neutrophil infiltrate is not suspected.     

Neuron transplantation into mice hippocampus alters sensitivity to barbital narcosis

The role of several hippocampal innervations in the sensitivity to barbital-induced narcosis was studied in selected mice strains. The outbred and inbred mouse strains HS/Ibg, SABRA/HUC, C57BL, CBA/LAC, and BALB/c were tested for barbital-induced sleep (315 mg/kg). The relatively short sleeping HS/Ibg (HS) and the longest sleeping BALE/c (BALE) were chosen for further investigation. Cholinergic (ACh), serotonergic (5-HT), and noradrenergic (NE) innervations were studied in HS strain; whereas BALE, which possesses both an unusually high sensitivity to barbital and unique NE innervations in the cortex and hippocampus, was employed in a detailed study of the NE innervations. Transplantation of embryonic NE cells from the mouse embryo into the hippocampus of adult HS mice increased barbital narcosis by 65% (p < 0.05), whereas transplantation of 5-HT cells decreased barbital narcosis by 54% (p < 0.001). Transplantation of ACh cells had no significant effect on barbital-induced narcosis. BALB mice were subjected to NE cell transplantation into the hippocampus and cortex. Similarly to HS, BALB receiving NE transplants into their hippocampus slept 34% longer than control after barbital challenge (p < 0.025). Noradrenergic cell transplantation into frontal cortex had no effect on barbital sleep. The results suggest that (a) enhancement by neural grafting of the NE innervation to the hippocampus accentuates and enhancement of the 5-HT innervations attenuates the sensitivity to barbital narcosis, whereas ACh innervations have no effect on the sensitivity to barbital narcosis, and (b) the unusually high sensitivity of BALB mice to barbital may not be related to its unique NE innervations.     

Stimulus functions of drugs and the assessment of abuse liability

The development of a unifying framework for conceptualizing the commonalities in various forms of substance abuse must encompass the data base focused upon the stimulus functions of drugs. In the first instance, for example, the research on drug self-administration has provided convincing evidence of a remarkable concordance between laboratory animals and human substance abusers in the reinforcing stimulus functions of a range of chemical agents. The recognition of these cross-species and cross-drug generalities has radically changed conceptualizations of substance abuse from a reactive to a more active process and has encouraged the kind of functional analysis of drug-seeking and drug-taking that has proven productive and useful in the study of other behavioral interactions. In this regard as well, recent refinements in the analysis of the discriminative stimulus functions of drugs have provided a more comprehensive basis for characterizing a chemical agent's spectrum of action and evaluating its abuse liability. While the correlation between the discriminative stimulus functions and the reinforcing stimulus functions is remarkably high for some drug classes, there are notable exceptions. Finally, the assessment of abuse liability requires an analysis of the eliciting stimulus functions of drugs as reflected by the physiological and behavioral changes, both acute and chronic, that follow drug administration. The methods used to evaluate both physiological dependence and behavioral toxicity in relationship to sensory and motor effects for a range of abused drugs have depended heavily upon an assessment of the eliciting stimulus functions of such compounds.     

Development of intestinal epithelial cell lines using a transgenic mouse

The epithelial cells of the colonic mucosa of the animal have proved impossible to culture using standard tissue culture techniques. Immortalization of adult colonic epithelial cells has been unsuccessful due to the lack of DNA synthesis in these cells once they are isolated from the tissue. Recently an unique transgenic mouse bearing a temperature sensitive mutant of the known immortalizing gene, SV40 large T has become available. The advantage of this mouse is that the SV40 large T gene is expressed in every cell. Active immortalizing protein is produced in each cell at the permissive temperature. We have used colonic mucosa from these mice to initiate cultures of epithelial cells from the colon of adult mice. The cells grow readily at the permissive temperature but die within 7 days at the non-permissive temperature. The methods used to develop these cultures are described.     

Recent trends in infant mortality rates and proportions of low-birth-weight live births in Canada

OBJECTIVE: To identify spatial patterns of changes in infant mortality rates and proportions of low-birth-weight live births observed in 1994. SETTING: Canada. SUBJECTS: Live births and infant deaths in Canada between 1987 and 1994. Data for Newfoundland were unavailable for 1987 through 1990. OUTCOME MEASURES: Annual infant mortality rates (crude and after excluding live newborns weighing less than 500 g); proportion of live births by low-birth-weight category (500-2499 g). RESULTS: Nova Scotia, New Brunswick, Quebec and Manitoba had lower crude and adjusted infant mortality rates in 1994 than in 1993. Newfoundland, Saskatchewan, Alberta and British Columbia had higher rates in 1994 than in 1993. The crude rate in Ontario was lower, and the adjusted rate higher, in 1994 than in 1993. A downward trend in the proportion of low-birth-weight live births was observed in Quebec (chi(2) for trend = 29.2, p < 0.01). Conversely, an upward trend was observed in Ontario (chi(2) for trend = 241.3, p < 0.01). However, the increase may have been due to data errors, especially in 1993 and 1994, involving truncation of ounces in 2 digits to 1 digit (e.g., 5 pounds 10 ounces became 5 pounds 1 ounce). CONCLUSIONS: Although the marginal increases in infant mortality observed in several provinces could be the result of random variation, future trends should be closely monitored. The proportion of low-birth-weight live births in Canada (excluding Ontario) appears to be stable, with Quebec showing significant reductions. The errors in data for Ontario need to be corrected before trends can be estimated for that province and for Canada as a whole.