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Biomedical subjects

B Irvine

Publications and source records attributed to B Irvine.

At least 19 recordsLinked to original sources
PubMed2002

Upregulation of matrix metalloproteinases in a model of T cell mediated tissue injury in the gut: analysis by gene array and in situ hybridisation.

BACKGROUND AND AIM: Matrix metalloproteinases (MMPs) have been implicated in tissue remodelling and ulceration in inflammatory bowel disease and coeliac disease. Studies to date have concluded that stromelysin 1 is functionally involved in mucosal degradation. However, there are many other MMPs whose function in the gut is currently unknown. This work had two aims: firstly, to use gene array technology to measure changes in MMP and tissue inhibitor of metalloproteinase (TIMP) expression in a model of T cell mediated injury in the gut, and secondly, to correlate data from gene arrays with that generated by in situ hybridisation. METHODS: T cells in explants of human fetal gut were activated with pokeweed mitogen or anti-CD3 plus interleukin 12. Gene array analysis and in situ hybridisation were performed to investigate changes in MMP gene expression. RESULTS: Both gene array analysis and in situ hybridisation indicated marked upregulation of stromelysin 2 and macrophage metalloelastase expression in the explants associated with mucosal destruction. The arrays also confirmed our previous observation that interstitial collagenase (MMP-1), stromelysin 1 (MMP-3), and gelatinase B (MMP-9) are upregulated but there was no change in MMP-2, -7, -8, -9, -11, -13, -14-17, or -19. Following T cell activation, transcripts for TIMPs were reduced. CONCLUSIONS: These results show that there is differential upregulation of MMPs during T cell responses in the gut and suggest that further studies on the role of stromelysin 2 and macrophage metalloelastase may show that they have a functional role. In addition, the increase in MMPs and reduction in TIMPs suggest that the protease/antiprotease balance in the mucosa may determine the extent of mucosal degradation.

Collagenases↗
PubMed2001

Waiting times from abnormal breast screen to diagnosis in 7 Canadian provinces.

BACKGROUND: Delay to breast cancer diagnosis following an abnormal screening result is associated with anxiety and personal disruption. We assessed the patterns and timeliness of diagnostic follow-up after breast cancer screening for women with abnormal results who attended organized screening programs in 7 provinces. METHODS: Using data from the Canadian Breast Cancer Screening Database, we identified 203,141 women aged 50-69 years who underwent screening in 1996 through provincially organized breast cancer screening programs in British Columbia, Alberta, Saskatchewan, Manitoba, Ontario, Nova Scotia and Newfoundland. We prospectively followed women with an abnormal screening result through to the completion of the assessment process. We evaluated the waiting times from screening examination to first assessment, from screening examination to first imaging, from screening examination to diagnosis and from first assessment to diagnosis for 13,958 women, stratified according to screening program, mode of detection, whether a biopsy was performed and whether cancer was diagnosed. RESULTS: We observed considerable variations between and within programs in the time to diagnosis. The median time from screening examination to first assessment was 2.6 weeks. The median time from screening examination to diagnosis was 3.7 weeks; this time increased to 6.9 weeks for women undergoing biopsy. Even when no biopsy was performed, 10% of the women waited 9.6 weeks or longer for a diagnosis, as compared with 15.0 weeks or longer for 10% of the women undergoing biopsy. Among the women who had a biopsy, the use of core biopsy was associated with a shorter median time to diagnosis than was open biopsy, and those found to have cancer had shorter waiting times than women with benign biopsy findings. INTERPRETATION: Women undergoing assessment of an abnormal breast cancer screening result waited many weeks for a diagnosis, especially when a biopsy was performed. To ensure that targets for timeliness, adopted nationally in 1999, are realized, improved models of care or dissemination of existing efficient techniques to reach a diagnosis will be needed.

Aged↗
PubMed2001

Electronic detection of nucleic acids: a versatile platform for molecular diagnostics.

A novel platform for the electronic detection of nucleic acids on microarrays is introduced and shown to perform well as a selective detection system for applications in molecular diagnostics. A gold electrode in a printed circuit board is coated with a self-assembled monolayer (SAM) containing DNA capture probes. Unlabeled nucleic acid targets are immobilized on the surface of the SAM through sequence-specific hybridization with the DNA capture probe. A separate signaling probe, containing ferrocene-modified nucleotides and complementary to the target in the region adjoining the capture probe binding site, is held in close proximity to the SAM in a sandwich complex. The SAM allows electron transfer between the immobilized ferrocenes and the gold, while insulating the electrode from soluble redox species, including unbound signaling probes. Here, we demonstrate sequence-specific detection of amplicons after simple dilution of the reaction product into hybridization buffer. In addition, single nucleotide polymorphism discrimination is shown. A genotyping chip for the C282Y single nucleotide polymorphism associated with hereditary hemochromatosis is used to confirm the genotype of six patients' DNA. In addition, a gene expression-monitoring chip is described that surveys five genes that are differentially regulated in the cellular apoptosis response. Finally, custom modification of individual electrodes through sequence-specific hybridization demonstrates the potential of this system for infectious disease diagnostics. The versatility of the electronic detection platform makes it suitable for multiple applications in diagnostics and pharmacogenetics.

Apoptosis↗
PubMed2000

Bioelectronic detection of point mutations using discrimination of the H63D polymorphism of the Hfe gene as a model.

BACKGROUND: A bioelectronic detection platform has recently been developed that facilitates the detection and characterization of nucleic acids. The DNA chip platform is compatible with homogeneous assays because separate labeling and wash steps are not required. A one-step, bioelectronic detection assay was developed to genotype patient samples with respect to the H63D polymorphism of the Hfe gene, associated with hereditary hemochromatosis. METHODS AND RESULTS: Electrode arrays were modified with DNA capture probes that were perfectly matched to the wild-type or mutant allele of H63D. Amplicons containing the polymorphic site were hybridized with the capture probes on the electrode arrays in the presence of electronically labeled reporter (signaling) probes. Voltammetric analysis of the electrode arrays was conducted first at ambient temperature and then at elevated temperature. The electronic signal was preferentially diminished at elevated temperature from electrodes that hybridized with mismatched target amplicons. CONCLUSION: An assay for bioelectronic genotyping of the H63D polymorphism was developed and used with six patient specimens to show the feasibility of this system as a model for point mutation detection.

Amino Acid Substitution↗
PubMed1997

A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.

Adenosine↗
PubMed1995

Rapid and precise quantification of HIV-1 RNA in plasma using a branched DNA signal amplification assay.

The level of human immunodeficiency virus type 1 (HIV-1) RNA in human plasma has been quantitated directly with use of a solid-phase nucleic acid hybridization assay, based on branched DNA (bDNA) signal amplification technology with chemiluminescent detection. Signal amplification is accomplished by the incorporation of sites for 1,755 alkaline phosphatase-labeled probes per genome of HIV-1, after successive hybridization of target-specific oligonucleotides and bDNA amplifier molecules. The assay is performed in microwells, much like an immunoassay, and is amenable to routine laboratory use. Reproducibility and specificity studies indicated that the bDNA method was precise and showed no reactivity with seronegative donors. HIV-1 RNA levels were quantitated for 348 seropositive specimens, with a detection rate of 83% for those specimens from patients with < 500 CD4+ T-cell counts. Plasma RNA levels were found to change with disease stage, and in response to antiviral therapy. Quantitation of HIV-1 RNA in the plasma of HIV-1-infected patients, with use of the bDNA assay, may be a useful method for monitoring HIV-1 disease progression and therapeutic response.

Acyclovir↗
PubMed1994

Effect of sheep and human follicular fluid on the maturation of sheep oocytes in vitro.

This study examines the effect of sheep and human follicular fluid on the in vitro maturation (IVM) of sheep follicular oocytes. Oocyte cumulus complexes recovered post mortem were matured for 24 to 26 h at 38.6 degrees C, 5% CO(2) in air, in TCM-199 bicarbonate medium supplemented with 20% fetal calf serum (FCS) and, where stated, with maturation hormones, including FSH (5.0 microg/ml), LH (5.0 microg/ml) and estradiol (1 microg/ml), or with sheep follicular fluid recovered from large (>5 mm) or small (2 to 5 mm) ovarian follicles post mortem, or with human periovular follicular fluid obtained during routine IVF procedures. The matured oocytes were then denuded, and their maturation stage and developmental capacity were assessed by in vitro fertilization (IVF) and culture (IVC). It was found that inclusion of sheep or human follicular fluid or hormone supplements in the IVM media more than doubled the number of oocytes completing maturation (FCS alone 33%, compared with 76.2% for maturation hormones, 84.2% for fluid from large and 69.6% for fluid from small sheep follicles and 82.6% for human follicular fluid), and significantly increased fertilization rates (FCS alone 51.6%, compared with 71.9% for maturation hormones, 78.4% for fluid from the large and 75.7% for fluid from small sheep follicles and 73.1% for human follicular fluid) without discernible adverse effects on the development of the cleaving embryos to the morula or blastocyst stage in culture. Omission of FCS and supplements from the IVM medium resulted in a marked reduction (56%) in the number of oocytes maturing. This reduction could be offset to a large part, but not completely, by inclusion of human follicular fluid or human follicular fluid plus LH (5 microg/ml) in the medium. The results of this study show that addition of sheep or human follicular fluid to maturation medium can enhance rather than inhibit the maturation and fertilizability of sheep follicular oocytes in vitro.

Journal Article↗
PubMed1993

Naso-labial flap in the reconstruction of floor of the mouth defects.

Defects caused by tumour excision in the floor of the mouth can be closed by different methods. We present thirteen cases treated in our department over the last seven years in whom naso-labial flaps were used. Twelve patients had squamous cell carcinoma of the floor of the mouth and one had fibro-sarcoma of the right lower mandible. Five received post-operative radiotherapy, one was resected following radiotherapy and seven were treated with surgery alone. Patient selection is important to give good functional results without compromising tumour clearance.

Aged↗
PubMed1993

Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region.

Hepatitis C virus (HCV) showed substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68 to 79% overall sequence similarity to one another. Phylogenetic analysis of nucleotide sequences derived from part of the gene encoding a non-structural protein (NS-5) has provided evidence for six major genotypes of HCV amongst a worldwide collection of 76 samples from HCV-infected blood donors and patients with chronic hepatitis. Many of these HCV types comprised a number of more closely related subtypes, leading to a current total of 11 genetically distinct viral populations. Phylogenetic analysis of other regions of the viral genome produced relationships between published sequences equivalent to those found in NS-5, apart from the more highly conserved 5' non-coding region in which only the six major HCV types, but not subtypes, could be differentiated. A new nomenclature for HCV variants is proposed in this communication that reflects the two-tiered nature of sequence differences between different viral isolates. The scheme classifies all known HCV variants to date, and describes criteria that would enable new variants to be assigned within the classification as they are discovered.

Base Sequence↗
PubMed1992

At least five related, but distinct, hepatitis C viral genotypes exist.

Hepatitis C virus, the major causative agent of blood-borne non-A, non-B hepatitis in the world, has been the subject of considerable nucleic acid sequence analysis. Although all reported hepatitis C sequences from the United States have been represented by the prototype hepatitis C virus type 1 sequence, two groups of variant sequences have been reported in Japan. However, we have noted five distinct, but related, genotypes (I-V) throughout the world, based on detailed sequence determination and analysis of the first 1700 nucleotides and part of the nonstructural region 5 at the C terminus of the open reading frame. The nucleotide sequence for a large number of hepatitis C virus isolates spanning six continents was obtained by direct sequence analysis of PCR products after reverse transcription. Genotype was classified by using several distinct sequence motifs. We observed that most genotypes coexist in several geographic regions, including the United States, Japan, Germany, and Italy. So far, genotype V has been found only in South Africa. Interestingly, each distinct genotype seems to be maintained throughout the genome in the segments studied. These genotype distinctions should be considered when designing specific diagnostic tests, developing potential vaccines, and studying viral transmission.

Amino Acid Sequence↗
PubMed1992

Long-term outcome and complications of children born with meningomyelocele.

The long-term functional outcome of 101 children born with meningomyelocele between 1971 and 1981 was assessed, by a combination of retrospective chart review and follow-up assessments. The children had been managed at birth using a process of nonstandardized selection. Eighty-three of the 101 patients survived after a minimum follow-up of 8.6 years, for a mortality rate of 18%. Forty-four of 83 children (53%) were community ambulators, and this correlated well with the presence of intact quadriceps function. Forty-eight children (58%) attended normal school and were grade-appropriate. Sixty-two of 83 patients (75%) were socially continent of urine, and 71/83 (86%) were socially continent of stool. Hydrocephalus was present in 93 of the 101 children in the study, and 85 children were shunted. Half of the shunted children required a shunt revision in the first year of life, and thereafter the rate of revision decreased, so that after 2 years the risk of revision was approximately 10% per year.

Activities of Daily Living↗
PubMed1992

Pregnancies and live birth from in vitro fertilization of calf oocytes collected by laparoscopic follicular aspiration.

Oocytes were recovered by laparoscopic aspiration from 3- to 8-week-old calves treated with follicle-stimulating hormone (FSH) followed by human chorionic gonadotropin (hCG) to induce follicular growth and oocyte maturation in vivo. Most of the recovered oocytes either had resumed meiotic maturation at the time of aspiration or were competent to undergo maturation during subsequent culture in vitro. Oocytes matured in vivo following FSH and hCG treatment underwent in vitro fertilization (70%) at rates not significantly different from those of control oocytes recovered from adult cow ovaries at abattoirs and matured in vitro (75%). Calf oocytes that were immature at aspiration exhibited lower fertilization rates after in vitro maturation (36%) but their rate of development to morulae and blastocysts did not differ from that of mature oocytes at aspiration. A total of 91% of the zygotes produced from calf oocytes developed to morula and 27% to blastocyst stages during 6 days of culture. The proportion developing to morulae was significantly higher (P<0.05) than that observed for zygotes resulting from in vitro maturation and fertilization of oocytes recovered from cow ovaries obtained at an abattoir and processed concomitantly (59% to morulae and 18% to blastocysts). Morulae or blastocysts developed from oocytes from 5 to 6-week-old calves, when transferred to synchronized recipient heifers, resulted in 2 confirmed pregnancies, one of which produced a single full-term live calf. The ability to produce embryos from oocytes recovered from newborn or prepubertal calves offers the potential for markedly reducing the generation interval in cattle, thereby substantially accelerating the rate of genetic gain that can be achieved through embryo transfer.

Journal Article↗
PubMed1992

Comparison of hepatitis C virus markers in patients with NANB hepatitis.

10 different HCV-specific assays and RT-PCR of the 5' untranslated region of HCV RNA were used to analyze sixty-four patients with chronic NANB liver disease. Po, CP-9 and C22 antigens are located in the putative core; C33c in the putative NS3; C100-3 in the putative NS3/4; KCL in the putative NS4/5 and C825 is located in the putative NS5. GOR protein is not part of the HCV genome, but antibodies to it appear to be present in response to a hepatitis C infection. Positive rates were 91% for Po, 89% for CP-9, 94% for C22, 97% for C33c, 88% for C100-3 (Ortho, EIA), 86% for C100-3 (Abbott, EIA), 84% for C100-3 (Ohtsuka, RIA), 88% for KCL, 59% for C825, 58% for GOR, and 83% for RT-PCR. There were 8 cases which were negative by all anti-C100 tests. 7 of these cases were positive by other anti-HCV markers and/or PCR suggesting the need for improved blood screening assays. There is a variation in the relative reactivity for different markers with different samples. Of the tests employed, anti C33c shows the highest positivity rate.

Adult↗
PubMed1991

Characterization of the terminal regions of hepatitis C viral RNA: identification of conserved sequences in the 5' untranslated region and poly(A) tails at the 3' end.

We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses of these sequences show (i) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical origin, (ii) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (iii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. These data differentiate HCV from the flaviviruses and indicate a closer evolutionary relationship of HCV with the pestiviruses. However, HCV also appears to be substantially different from other known pestiviruses. These data are consistent with the assignment of HCV to a separate viral genus.

Amino Acid Sequence↗
PubMed1991

Use of a signature nucleotide sequence of hepatitis C virus for detection of viral RNA in human serum and plasma.

The nucleic acid sequence of the putative 5'-untranslated (5PUT) region of hepatitis C virus (HCV), determined for samples obtained from a variety of geographic origins, was found to be over 98% conserved among all isolates. On the basis of this signature sequence for HCV, a viral RNA assay was developed by using cDNA synthesis with reverse transcriptase, followed by polymerase chain reaction (PCR). The new assay was compared with the Ortho-Chiron C100-3 HCV enzyme-linked immunosorbent assay to research radioimmunoassays for antibodies to the C33c and C22 HCV antigens and to the first reported set of HCV PCR primers designed from the NS3 domain. Plasma samples from 16 Japanese patients with non-A, non-B hepatitis (NANBH) and 16 immunoassay-positive blood donors from the United States were investigated. The 5PUT PCR primers were found to be superior to the NS3 primers in sensitivity and specificity (15 of 25 versus 3 of 25 of the C100 enzyme-linked immunosorbent assay-positive samples, respectively). Samples from two C100-negative patients with acute NANBH were found to react with the 5PUT primers but not with the NS3 primers. Also, two of three patients with chronic NANBH converted from reverse transcriptase PCR positive to negative after interferon treatment. Although the clinical significance of the presence or absence of HCV RNA in samples from patients is not fully understood, the use of probes and primers from the 5PUT region (as opposed to primers from other segments) should not lead to false-negative results due to nucleic acid sequence variations in viral isolates.

Base Sequence↗
PubMed1989

Bone metastases in carcinoma of the rectum: a clinical and pathological review.

Bone metastases were confirmed in 48 patients after treatment of rectal cancer and occurred predominantly in the lumbar spine and pelvis. Most of the deposits were lytic and coexisted with widespread metastatic disease. Correspondingly, the median survival from diagnosis was only 4 months. The new prognostic classification for rectal cancer was applied to the original tumour specimens and categorized 23 of these tumours into group IV which confidently predicts a poor outcome compared with only eight by the method of Dukes.

Adult↗