Biomedical subjects
Publications and source records attributed to A Allard.
In an investigation of 201 prostate tissue samples from patients with benign prostate hyperplasia that later progressed to prostate cancer and 201 matched controls that did not, there were no differences in the prevalence of adenovirus, herpesvirus, papilloma virus, polyoma virus and Candida albicans DNA.
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A new apparatus has been developed that integrates an animal restrainer arrangement for small animals with an actively tunable/detunable dual radio-frequency (RF) coil system for in vivo anatomical and functional magnetic resonance imaging of small animals at 4.7 T. The radio-frequency coil features an eight-element microstrip line configuration that, in conjunction with a segmented outer copper shield, forms a transversal electromagnetic (TEM) resonator structure. Matching and active tuning/detuning is achieved through fixed/variable capacitors and a PIN diode for each resonator element. These components along with radio-frequency chokes (RFCs) and blocking capacitors are placed on two printed circuit boards (PCBs) whose copper coated ground planes form the front and back of the volume coil and are therefore an integral part of the resonator structure. The magnetic resonance signal response is received with a dome-shaped single-loop surface coil that can be height-adjustable with respect to the animal's head. The conscious animal is immobilized through a mechanical arrangement that consists of a Plexiglas body tube and a head restrainer. This restrainer has a cylindrical holder with a mouthpiece and position screws to receive and restrain the head of the animal. The apparatus is intended to perform anatomical and functional magnetic resonance imaging in conscious animals such as mice, rats, hamsters, and marmosets. Cranial images acquired from fully conscious rats in a 4.7 T Bruker 40 cm bore animal scanner underscore the feasibility of this approach and bode well to extend this system to the imaging of other animals.
A database was examined using artificial neural network (ANN) models to investigate the efficacy of predicting PCR-identified Norwalk-like virus presence and absence in shellfish. The relative importance of variables in the model and the predictive power obtained by application of ANN modelling methods were compared with previously developed logistic regression models. In addition, two country-specific datasets were analysed separately with ANN models to determine if the relative importance of the input variables was similar for geographically diverse regions. The results of this analysis found that ANN models predicted Norwalk-like virus presence and absence in shellfish with equivalent, and better, precision than logistic regression models. For overall classification performance, ANN modelling had a rate of 93%, vs 75% for the logistic regression. ANN models were able to illuminate the site-specific relationships between indicators and pathogens.
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We have developed a system for rapid typing of adenoviruses (Ads) based on a combination of PCR and restriction endonuclease (RE) digestion (PCR-RE digestion). Degenerated consensus primers were designed, allowing amplification of DNA from all 51 human Ad prototype strains and altogether 44 different genome variants of Ad serotypes 1, 3, 4, 5, 7, 11, 19, 40, and 41. The 301-bp amplimer of 22 prototype strains representing all six subgenera and the genome variant was selected as a target for sequencing to look for subgenus and genome type variabilities. The sequences obtained were used to facilitate the selection of specific REs for discrimination purposes in a diagnostic assay by following the concept of cleavage or noncleavage of the 301-bp amplimer. On the basis of these results, a flowchart was constructed, allowing identification of subgenus B:2 and D serotypes and almost complete distinction of subgenus A, B:1, C, E, and F serotypes. Application of the PCR-RE digestion system to clinical samples allowed typing of 34 of 40 clinical samples positive for Ad. The genome type determined by this method was identical to that obtained by traditional RE typing of full-length Ad DNA. The remaining six samples were positive only after a nested PCR. Therefore, to reduce the risk of false-negative results, samples scored negative by the PCR-RE digestion system should be evaluated by the described nested PCR. Used in combination, the PCR-RE digestion method and the nested PCR provide a reliable and sensitive system that can easily be applied to all kinds of clinical samples when rapid identification of adenoviruses is needed.
Nested polymerase chain reaction (nPCR) demonstrated the presence of Chlamydia pneumoniae-specific DNA in peripheral blood mononuclear cells (PBMC). PBMC samples were obtained from 103 consecutive patients (62 male, 41 female) aged 22-85 years (mean, 64) admitted for coronary angiography because of suspected coronary heart disease and from 52 blood donors (43 male, 9 female) aged 40-64 years (mean, 49). Of the 101 evaluable patients, 60 (59%) were identified by nPCR assay as C. pneumoniae DNA carriers; C. pneumoniae-specific microimmunofluorescence (MIF) serology confirmed exposure to the bacterium in 57 (95%) of the 60 nPCR-positive patients. Among the 52 blood donors, the nPCR assay identified 24 (46%) C. pneumoniae DNA carriers, all of whom were positive by C. pneumoniae-specific serology. Thirty-two patients (32%) and 23 blood donors (44%) were MIF antibody-positive but repeatedly nPCR-negative; Bartonella henselae- or Bartonella quintana-specific antibodies were not detected among any of these subjects. In this study, C. pneumoniae DNA was common in PBMC of patients with coronary heart disease and in middle-aged blood donors.
Chlamydia pneumoniae is a common cause of respiratory tract infection and community-acquired pneumonia. During an extensive outbreak of C. pneumoniae in northern Sweden, 319 respiratory samples from 129 persons were collected. Sputum, throat, and nasopharyngeal samples were obtained and analyzed by nested touchdown polymerase chain reaction (PCR), EIA, and culture in Hep-2 and McCoy cells. Serology was performed by complement fixation and microimmunofluorescence tests. By PCR, 30 patients were diagnosed with C. pneumoniae compared with 26 positive by EIA and 23 by culture. The finding of C. pneumoniae in the respiratory samples was accompanied by serology indicating acute infection in 26 (96%) of 27 patients for whom adequate sera were available. Nested PCR was sensitive and reliable for diagnosing acute respiratory C. pneumoniae infection. Sputum samples had the highest diagnostic efficacy, and the nested type of PCR was superior to one-step PCR. EIA and culture were less sensitive than nested PCR.
A primer pair originally designed for the universal detection of human adenovirus (HAV) serotypes of all subgenera was modified then tested and found feasible for the detection of different bovine, ovine, and porcine adenovirus (BAV, OAV, and PAV, respectively) serotypes. Apparently, in the examined viruses, parts of the DNA sequence coding for the basal part of the hexon protein are conserved enough for being applicable in polymerase chain reaction (PCR) as primers. Positive amplification could be obtained even from the so-called subgroup 2 BAVs, which viruses do not cross react with HAVs or subgroup 1 BAVs in Southern hybridisation.
A procedure was developed for specific discrimination and typing of the enteric human adenoviruses, Ad40 and Ad41, after polymerase chain reaction (PCR) amplification of a sequence in the hexon gene highly conserved among all 47 serotypes recognised. By Taq I restriction of the 300 bp amplimers, subgenus F DNA could be discriminated from DNAs of adenoviruses belonging to all other subgenera. Discrimination between Ad40 and Ad41 was subsequently achieved by cleavage with either Cfo I, HinP I, Mae III, Mvn I, and/or Rsa I. Thus, PCR detection of viral DNA combined with restriction analysis of amplified products provides a valuable tool for use in epidemiological studies of diarrhoea of adenoviral aetiology.
A procedure has been developed for the rapid detection of enteroviruses and adenoviruses in environmental samples. Several systems for virus concentration and extraction of nucleic acid were tested by adding adenovirus type 2 and poliovirus type 1 to different sewage samples. The most promising method for virus recovery involved the concentration of viruses by centrifugation and elution of the virus pellets by treatment with 0.25 N glycine buffer, pH 9.5. Nucleic acid extraction by adsorption of RNA and DNA to silica particles was the most efficient. One aliquot of the extracted nucleic acids was used for a nested two-step PCR, with specific primers for all adenoviruses; and another aliquot was used to synthesize cDNA for a nested two-step PCR with specific primers for further detection of seeded polioviruses or all enteroviruses in the river water and sewage samples. The specificity and sensitivity were evaluated, and 24 different enterovirus strains and the 47 human adenovirus serotypes were recognized by the primers used. The sensitivity was estimated to be between 1 and 10 virus particles for each of the species tested. Twenty-five samples of sewage and polluted river water were analyzed and showed a much higher number of positive isolates by nested PCR than by tissue culture analysis. The PCR-based detection of enteroviruses and adenoviruses shows good results as an indicator of possible viral contamination in environmental wastewater.
The use of the polymerase chain reaction (PCR) for detection of human adenoviruses in diluted stool samples was investigated. Two sets of nested primers, including primers specific for the hexon-coding region and for the E1B region of enteric adenoviruses (EAd), were assessed by two-step amplification. The primers constitute two different PCR systems designed for the detection of adenoviruses belonging to all six subgenera (A-F), and the two EAds Ad40 and Ad41, respectively. In a two-step PCR mediated amplification a single virus particle was detected when the two sets of general hexon primers or EAd specific primers were used. Earlier results from PCR detection of adenoviruses in stool from children suffering from diarrhea gave indications that adenovirus particles are commonly shed in stools without being identified as the cause of illness [Allard et al.: Journal of Clinical Microbiology 28:2659-2667, 1990]. Therefore, the general and the EAd specific PCR assays were assessed on 150 stool specimens from three groups including 50 healthy children, 50 healthy adults, and 50 adults suffering from diarrhea. When the two sets of general hexon primers were used, 25 of the 50 specimens from the healthy children (mean age 21 months) were found positive by two-step PCR amplification. Nine of the 50 specimens from the healthy adults (mean age 32 years) were found positive whereas 12 of the 50 specimens from sick adults (mean age 31 years) gave amplification products, using the two sets of general hexon primers in a nested fashion. None of the 150 specimens were found to be positive by two-step PCR amplification using the two sets of EAd-specific primers.(ABSTRACT TRUNCATED AT 250 WORDS)
Enteric adenovirus type 41 (Ad41) is defective for growth in conventional established cell lines. Ad41 is dependent on the Ad5 early regions E1A/E1B since it cannot grow in HEK cells but only in 293 HEK cells transformed by Ad5 E1 region. However, Hep-2 cells have also been shown to support the growth of Ad41 to some extent. The nucleotide sequence of the E1B region of the Ad41 strain D389 has been determined. When compared to the corresponding region of the Ad41 prototype strain (Tak) the degree of homology in the DNA sequences was close to 100%. The mRNAs from the E1B region of the Ad41 strain D389 have been studied by Northern blot, primer extension, and polymerase chain reaction-cDNA analysis. E1B transcripts corresponding to Ad2 14 S, 22 S, and 9 S mRNAs were identified but no 13 S mRNA equivalent was detected, a pattern similar to that seen in the Ad40 and Ad12 transcription maps. However, the Ad41 E1B 14S mRNA equivalent has one additional small exon of 23 nucleotides, created by a donor and an acceptor splice site located at positions not seen in other E1B transcripts of human adenoviruses analyzed so far. The coding potential for E1B 19K, 55K, and 15K proteins and for pIX is retained in the Ad41 transcripts. In contrast to other adenoviruses, except for the closely related Ad40, the ORF of pIX starts in the intron of the 22 S mRNA.
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The usefulness of the polymerase chain reaction (PCR) method for diagnosing adenovirus infections was investigated. Several primers, including primers specific for the hexon-coding region and for enteric adenovirus types 40 and 41, were evaluated. The PCR method was validated against cell culturing in routine diagnostic work and against restriction enzyme analysis of viral DNA. Sixty diagnostic specimens were selected for evaluation by the PCR method. Twenty of the 60 specimens were found positive on the basis of cytopathic effects and latex agglutination (Adenolex [Orion Diagnostica, Helsinki, Finland]), and 16 were identified and typed as adenoviruses by polyacrylamide gel electrophoresis. PCR was performed on all 60 specimens in parallel directly on diluted stool samples and on viral DNA extracted from cells inoculated with the same stool samples. When the general hexon primers were used 51 of the 60 specimens from infected cell cultures were found positive by PCR, whereas only 13 specimens were found positive when PCR was performed directly on stool samples. With the use of selective primers for enteric adenoviruses 16 of the 60 cell cultures were found to exhibit amplification products by PCR, whereas 4 were detected in stool samples. None of the 60 specimens were found positive by PCR when an adenovirus type 40-specific primer pair was used. PCR was found to be a fast, sensitive, and reliable method for the detection of adenoviruses in diarrheal disease, provided the amplifications were performed directly on diluted stool samples.
We showed previously that transplantation of 10(7) unmanipulated C57BL/6 marrow cells to irradiated LP mice yields healthy (B6-LP) chimeras showing no signs of rejection or graft-versus-host disease (GVHD). The aim of this work was to gain more insight into the mechanism(s) responsible for tolerance to host minor histocompatibility antigens following allogeneic bone marrow transplantation (BMT). (B6-LP) chimeras showed very good immune reconstitution when studied in vitro for proliferative response to mitogens and alloantigens and generation of T cell cytotoxic activity. In co-culture experiments their spleen cells showed no natural suppressor activity. When used as cell donors, their capacity to initiate GVHD in four strains of mice presenting H-2 differences was normal when compared to C57BL/6 donors. However, they provoked no GVHD in the three strains of H-2 compatible mice studied. Re-irradiated (B6-LP) chimeras rapidly died of GVHD following injection of C57BL/6 marrow + spleen cells. (B6-LP.R111) chimera cells appeared tolerant to LP minor antigens presented in the context of H-2r or H-2b. No anamnestic anti-idiotypic suppressor response was noted when stable (B6-LP) chimeras were stimulated with naive C57BL/6 cells. These findings suggest that in BMT chimeras transplanted across minor histocompatibility barriers: (1) both host and donor-derived antigen-presenting cells can present host antigens to donor T cells whose numbers in the marrow inoculum will determine if GVHD or tolerance will ensue, (2) GVHD can be triggered by only a limited number of 'dominant' minor antigens, and (3) we found no evidence for the presence of natural suppressors, veto cells or anti-idiotypic suppressor T cells.
A better understanding of the mechanism(s) involved in graft-host-tolerance following allogeneic bone marrow transplantation is needed to develop new strategies to prevent graft-versus-host disease (GVHD). Based on previous studies, mainly in MHC-mismatched donor-recipient pairs, three hypotheses have been proposed: clonal deletion, active suppression and lack of adequate antigen-presenting cells. Our goal was to identify the mechanism(s) by which tolerance is achieved and maintained in radiation chimeras transplanted across minor histocompatibility barriers. Healthy (B6----LP) chimeras were obtained following injection of 10(7) C57BL/6 marrow cells to irradiated (9.5 Gy) LP hosts and used experimentally 100 days after chimerization. The tolerance state of (B6----LP) chimeras could not be abrogated after i.v. transfer of 5 x 10(7) donor-type spleen cells alone or with repeated i.p. injection of host-type antigen-presenting cells. No GVHD was observed when 10(7) marrow cells plus 5 x 10(7) spleen cells from (B6----LP) chimeras were injected to irradiated LP recipients. Chimera spleen cells suppressed GVHD when adoptively transferred to LP recipients of a C57BL/6 graft. These results suggest that in this model the presence of suppressor cells is both necessary and sufficient to maintain graft-host-tolerance.
Enteric adenovirus types 40 and 41 (Ad40 and Ad41), representing subgenus F, differ from all other human adenoviruses by being so fastidious that productive replication does not occur in conventional established cell lines. They are dependent of the Ad5 early regions E1A and E1B since they can not grow in HEK cells, only in 293 HEK cells transformed by Ad5 E1. The overall genetic organization of Ad41 E1A is similar to the E1A region of other characterized human adenoviruses but it is slightly shorter, comprising 1350 bp. The inverted terminal repeat (ITR) at the 5' end of both Ad40 and AD41 consists of 163 nucleotides, being similar to the ITR of Ad12 (subgenus A) and longer than the ITRs of adenoviruses of subgenera B, C, and E. The early mRNA products (12 and 13 S) can be translated into a 222-amino acid (aa) and a 251-aa tentative protein, respectively. In a comparison of the Ad41 251-aa protein with corresponding peptides of Ad12, Ad7, Ad5, and Ad4, three conserved amino acid sequences CS1-CS3 can be found. In the second conserved domain CS2, which is particularly acidic, the homology is very high within all five serotypes compared. Only one among eight conserved amino acids differs in the Ad41 251-aa protein. Within CS1 and CS3 which exhibit a hydrophilic and a hydrophobic character, respectively, the amino acid composition of the Ad41 protein is less conserved than the corresponding regions in all other analyzed adenovirus types. Ten of 16 conserved amino acids in CS1 are shared by Ad41 and 18 of 23 conserved amino acids in CS3 are shared by Ad41.