Biomedical subjects
Publications and source records attributed to P J Middleton.
We have previously reported oligodeoxynucleotides (ODNs) containing 4'-C- and 5'-C-substituted thymidines, which demonstrated certain favorable biophysical and biochemical properties. In this communication, the hybridization and nuclease stability data of the ODNs along with their capability to induce RNase H activity are presented.
OBJECTIVES: To determine the prevalence of hepatitis C virus (HCV) infection in patients with B-cell non-Hodgkin's lymphoma (NHL) in British Columbia. DESIGN: A cross-sectional analysis. SETTING: The British Columbia Cancer Agency (BCCA), a Canadian provincial tertiary oncology referral center. SUBJECTS: Consecutive patients with B-cell NHL registered onto the BCCA lymphoma database in 1996 and part of 1997 and a control group of patients with T-cell NHL registered on the database from 1995 through 1997. Patients with HIV infection were excluded from the analysis. A second control group (n = 1085) consisted of health-care workers tested for HCV infection following a needle-stick injury. INTERVENTIONS: Stored sera from patients with B-cell NHL (n = 88) and T-cell NHL (n = 37), identified from the database, were tested for the presence of HCV infection with commercially available serologic tests. MAIN OUTCOME MEASURES: HCV seropositivity in the B-cell lymphoma group compared to the control groups (T-cell NHL and health-care workers). RESULTS: 2.3% of the B-cell NHL group, none of the T-cell NHL group and 1% of the health-care worker control group were positive for HCV infection. These results were not statistically significantly different. CONCLUSION: Patients in British Columbia with B-cell NHL do not have an increased prevalence of HCV infection. These data suggest that the lymphotrophism of HCV may differ by regional, racial and genotypic variations around the world.
We previously showed that inhibition of the expression of CD28 (an essential immune receptor on T cells) mediated by a phosphorothioate (PS)-modified aptameric oligodeoxynucleotide (ODN) sequence, GR1, resulted in reduced T cell responses in vitro and in vivo. Using GR1 sequences differing only in the amount of terminal PS linkages (chimeric SO-ODN), the present study demonstrated that even after a substantial reduction in PS linkages, this 18-mer ODN sequence could still confer functionality in the ODN-mediated inhibition of CD28 expression. We showed that secondary structure and full retention of the ability to form a specific protein-ODN complex and to increase cellular uptake in activated Jurkat T cells were critical parameters in the determination of the magnitude of bioactivity of chimeric SO-ODN. We report that a chimeric SO-ODN with terminal PS linkages that total 9 (ICN 17221) or 12 (ICN 17263) was sufficient to inhibit CD28 expression and suppress in vivo inflammatory ear responses to contact allergen in mice with similar potency to the 17-thioate S-ODN (ICN 16064). Interestingly, all chimeric SO-ODN showed similar in vitro nuclease resistance. These data suggest alternate functional properties for PS linkages, unrelated to nuclease resistance, in enhancing the bioactivity of a G-rich aptamer.
BACKGROUND: The objectives of this study were to assess the effect of British Columbia's June 1994 guidelines for prenatal HIV screening on the rate of maternal-fetal HIV transmission and to estimate the cost-effectiveness of such screening. METHODS: The authors conducted a retrospective review of pregnancy and delivery statistics, HIV screening practices, laboratory testing volume, prenatal and labour management decisions of HIV-positive women, maternal-fetal transmission rates and associated costs. RESULTS: Over 1995 and 1996, 135,681 women were pregnant and 92,645 carried to term. The rate of HIV testing increased from 55% to 76% of pregnancies on chart review at one hospital between November 1995 and November 1996. On the basis of seroprevalence studies, an estimated 50.2 pregnancies and 34.3 (95% confidence interval 17.6 to 51.0) live births to HIV-positive women were expected. Of 42 identified mother-infant pairs with an estimated date of delivery during 1995 or 1996, 25 were known only through screening. Of these 25 cases, there were 10 terminations, 1 spontaneous abortion and 14 cases in which the woman elected to carry the pregnancy to term with antiretroviral therapy. There was one stillbirth. One instance of maternal-fetal HIV transmission occurred among the 13 live births. The net savings attributable to prevented infections among babies carried to term were $165,586, with a saving per prevented case of $75,266. INTERPRETATION: A routine offer of pregnancy screening for HIV in a low-prevalence setting reduces the rate of maternal-fetal HIV transmission and may rival other widely accepted health care expenditures in terms of cost-effectiveness.
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BACKGROUND: Acute infectious diarrhea in young children is a leading cause of morbidity and mortality in developing countries. Even in developed countries, infectious enteritis is second only to respiratory infections as a cause of morbidity in early childhood. OBJECTIVE: To nominate the various viral agents that cause enteritis, discuss the pathogenesis, clinical features, epidemiology and diagnostic procedures employed. STUDY DESIGN: Pertinent literature was reviewed and the findings of investigations carried out on viral enteritis by various colleagues recalled. RESULTS: The viruses causing gastroenteritis include: Rotaviruses; Adenoviruses-especially Ad 31, Ad 40 and Ad 41; members of the Caliciviridae, e.g. Norwalk virus, Hawaii virus, Snow Mountain virus, Taunton virus, Southampton virus, Toronto virus (formerly mini-reovirus) and others; Astrovirus; Coronavirus; Torovirus; Cytomegalovirus (CMV) and possibly Picobirnavirus. Enteritis-producing viruses replicate in columar epithelial cells in the distal parts of villi of the small intestine. Two mechanisms are addressed to explain why diarrhea occurs. Clinically, the main expression of illness is a watery diarrhea that lasts 24 h to about 7 days. Vomiting is of shorter duration and may not always accompany the diarrhea. Fever is generally 38.5 degrees C. Virus is shed in the stool for about 3-7 days. Diagnostic procedures employ electron microscopy (EM), immune electron microscopy (IEM), enzyme-linked immunosorbent assay (ELISA), time-resolved fluoroimmunoassay (TR-FIA), latex agglutination, polyacrylamide gel electrophoresis (PAGE) and the polymerase chain reaction (PCR). CONCLUSION: In developed countries viral enteritis among young children may be up to three times more common than bacterial gut disease. With the exception of CMV enteric involvement, the stool is characteristically not bloody and white blood cells are not found. Patient management may involve the employment of IV replacement therapy to counter dehydration and electrolyte imbalance. Milder cases may be managed with oral rehydration.
The accuracy of a saliva collection and testing protocol for determination of HIV-1 antibody status was assessed under realistic field conditions. The 1,256 study participants came from two lower-prevalence settings--a self-referral testing clinic (478) and a street-based outreach program (431)--and two high-prevalence clinics--an HIV/AIDS treatment clinic (337) and a hemophilia clinic (10). Saliva was collected using the Omni-Sal collection device and was tested by technicians blind to serum status using a modified protocol that employed the Recombigen HIV-1 EIA. Serum was tested using standard methods. A single saliva enzyme immunoassay (EIA) correctly identified 358 of 368 seropositive individuals, for a sensitivity of 97.3% (95% CI: 95.1%-98.7), and 888 of 888 seronegative individuals, for a specificity of 100% (95% CI: 99.6-100). Confirmation of saliva EIA positives with a repeat saliva EIA and an in-house saliva radioimmunoprecipitation assay resulted in a substantial drop in sensitivity to 85.3% (95% CI: 81.0-89.0). We conclude that a single saliva EIA using the modified test protocol described is sufficiently accurate for surveillance purposes, but we do not recommend it for diagnostic purposes or screening.
A rapid immunoblotting technique based on the IgM response to a major immunogenic protein is described for the diagnosis of Mycoplasma pneumoniae infection. Using results of the complement fixation test as the criterion for diagnosis, the rapid immunoblot method was positive in 95.7% of patients. The sensitivity was reduced to 81.9% if the test was performed on either single sera or acute sera only from serum pairs. Although the few sera that failed to demonstrate a positive IgM response were more likely to be from older patients, there was a consistent IgM response recorded for both younger (less than 20 years) and older (greater than or equal to 20 years) patients.
A time-resolved fluoroimmunoassay (TR FIA) was developed for the direct detection of adenovirus types 40 (Ad40) and 41 (Ad41) in stool specimens by using a monoclonal antibody (5D8/2C2) which recognizes both Ad40 and Ad41 but does not cross-react with other adenovirus serotypes. In this assay, the detector antibody is biotinylated directly rather than labeled with europium, and the fluorescent signal is generated on a solid phase in the presence of excess europium (Eu3+). The strength of the signal is dependent on the amount of a Eu3+ chelator [4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA)]-streptavidin complex bound by the biotinylated detector antibody (5D8/2C2). In a pilot study with 41 specimens, this TR FIA demonstrated a maximum sensitivity and specificity of 88% compared with SmaI restriction analysis of adenovirus isolates from the same specimens. TR FIA using the europium chelator BCPDA represents a feasible approach for the direct identification of specific adenovirus serotypes in stool specimens.
We retrospectively analyzed the clinical features of 127 hospitalized pediatric patients whose fecal samples were positive for adenovirus (Ad) by electron microscopy during an 18-month period. Serotyping results obtained by microneutralization tests and restriction endonuclease analysis were available for 105 of 127 cases. There were 69 males and 58 females and 94% of patients were less than 4 years of age. The average body temperature was 38 degrees C rectal (range, 36.2-40.8 degrees C) with an average duration of fever of 1.6 days. The average duration of clinical illness was 8.8 days (range, 1 to 32 days). Although Ad 40 and Ad 41 were isolated in the majority of cases (59 of 105 (56%], Ad 31 was associated with 18 of 105 cases (17%). Of the 18 cases associated with Ad 31, 14 were nosocomial and associated with diarrhea. Our survey confirms the importance of fastidious enteric Ad in infantile diarrhea (Ad 40, Ad 41) and suggests that Ad 31 produces a clinical syndrome indistinguishable from that caused by Ad 40 and Ad 41. The occurrence of Ad enteritis in patients admitted for unrelated illnesses well after initial hospitalization suggests that Ad is also an important cause of nosocomial enteritis in our hospital.
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The association of Mycoplasma pneumoniae with arthropathy in children and young adults has been established essentially on the basis of complement fixation (CF) serology. Because the CF test may be nonspecific for M. pneumoniae, we have confirmed the association by determination of IgM ELISA and IgM and IgG immunoblotting responses in 2 children. Mycoplasma pneumoniae should be considered in any patient with acute respiratory illness who subsequently develops arthropathy.
After two cases of acute hepatitis B infection occurred in phlebotomists at The Hospital for Sick Children in 1985, a seroprevalence survey of hepatitis B virus markers was undertaken. Directors in high-risk areas were advised by phone and memorandum to screen employees. Participation was entirely voluntary, and employees who did not respond were contacted six weeks after initial notification. Information obtained from each participant through a self-administered questionnaire included age, duration of employment in current pediatric occupation, history of blood transfusions, immune globulin prophylaxis, needlestick injury, and country of birth (North America/United Kingdom or other). Sera identified by code were tested by radioimmunoassay. Interactions were analyzed by using a multiple logistic regression model. A total of 10% of the personnel in high-risk areas, in which there was frequent exposure to blood or blood products, had hepatitis B markers in their blood, compared with 2% who did not have this exposure. Birthplace and occupation have independently significant effects on the likelihood of having hepatitis B markers. There appears to be an increased risk to employees in pediatric units, depending on the patient (and parent) population being served, although the risk may be lower than in adult hospitals.
The ultrasensitive photochemical silver stain for nucleic acids, described by Beidler et al. (1982), has been applied to the detection of adenovirus restriction fragments as a relatively rapid technique for the identification of virus isolates. In this study, restriction enzyme cleavage analysis was used to characterize adenovirus isolates from what appeared to be two nosocomial outbreaks. The first outbreak was thus shown to include two clusters of patients, and involved two serotypes Ad7c and Ad40. The second outbreak was unrelated and involved Ad35. Although restriction analysis does not replace serum neutralization as a routine method for typing adenoviruses, it is a much more rapid means of discriminating between different patient isolates, providing a current rather than retrospective analysis of a nosocomial outbreak. During the first outbreak, restriction analysis identified two distinct adenovirus serotypes from one patient--Ad7c from a nasopharyngeal aspirate and Ad41 from a stool specimen. Restriction analysis is also valuable for the sub-typing of virus isolates. In this study, the Ad40 and Ad41 isolates were shown to be variants of the respective prototype strains.
The oligosaccharides of the structural glycoprotein (VP7) of calf rotavirus were characterized. The precursor of VP7 produced in infected cells in the presence of tunicamycin migrated on SDS-polyacrylamide gels with an apparent molecular weight 6000 less than the glycosylated glycoprotein. Endoglycosidase (Endo) H digestion of the mature virus resulted in a decrease of 5000 in the molecular weight of VP7 in two discrete stages. Analysis of Endo H-treated, 3H-labelled digestion products of VP7 on Bio-Gel P-4 identified an oligosaccharide of molecular weight 1350 as the predominant form. Further treatment of the digest with mannosidase and analysis on Bio-Gel P-2 columns indicated that the oligosaccharide was digested into a free mannose and an oligosaccharide of molecular weight 400 in the ratio of 6:1. This indicates that the oligosaccharides of VP7 consist of four N-linked (Man)7 residues, two of which occupy more exposed and two more cryptic positions in the VP7 molecule.
The structural glycoproteins of calf (BDV 486), human (Wa) and simian (SA11) rotaviruses were compared for sensitivity to endoglycosidase (Endo) H. The calf and human virus glycoproteins were reduced by approximately 5000 molecular weight in two equal stages. The simian rotavirus glycoprotein sustained only one 2500 molecular weight decrease. The effect of the host cell on the viral oligosaccharide composition was examined. Endo H digests of calf rotavirus 82-124 extracted from stool and its progeny propagated and [35S]methionine-labelled in MA104 cells were compared. Endo H had the same effects on the glycoproteins, indicating that the nature of the host cell does not affect the oligosaccharide composition of the virus glycoproteins.
Thirty-five stool specimens, collected over a 14-week period from pediatric gastroenteritis patients and shown to contain adenovirus by electron microscopy, were inoculated onto 293 and HeLa cells. Virus isolates were characterized by serum neutralization and restriction endonuclease cleavage analysis of viral DNA from infected cells. Adenovirus was isolated upon primary inoculation of 293 cells from all 35 specimens shown to contain adenovirus by electron microscopy. Fastidious adenoviruses 40 and 41 (Ad40 and Ad41) were found in 17 (49%) of the stool specimens, and 4 of these specimens contained a conventional species (Ad1, Ad1, Ad18, Ad31) as well as Ad40. This was first manifest by the observation that four of the isolates which initially grew only in 293 cells acquired the capacity to grow in HeLa cells upon subsequent passage. In each case, the conventional species was undetectable by DNA analysis in the original inoculum but was selected in 293 cells and became the only one detectable by the second passage. Four other specimens, containing Ad1 or Ad31 alone, failed to grow initially in HeLa cells but did grow in 293 cells. The results of this study demonstrate therefore that (i) 293 cells are more sensitive than HeLa cells for the isolation of conventional as well as fastidious enteric adenovirus species and (ii) identification of viruses from patient specimens should involve minimal passage of the virus in cell culture, as a single passage can result in misdiagnosis of the virus associated with the infection.
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