Biomedical subjects

G Buchan

Publications and source records attributed to G Buchan.

At least 19 recordsLinked to original sources

PubMed2001

IL-2 linked to a peptide from influenza hemagglutinin enhances T cell activation by affecting the antigen-presentation function of bone marrow-derived dendritic cells.

Chimeric proteins containing antigen linked to cytokines have shown some promise as vaccine candidates but little is known of their mechanism of action, particularly at the level of the antigen-presenting cell. We have investigated this using a chimeric protein in which an immunodominant T cell epitope from influenza hemagglutinin peptide (HA), recognized in the context of I-E(d), was fused to IL-2. Immature murine dendritic cells (DC) derived from bone marrow (BMDC) were used to present the chimeric protein to a T cell hybridoma with TCR specific for the HA peptide (A5 cell line). HA-IL-2 was found to induce significantly higher T cell activation than HA alone. Although the inclusion of IL-2 and HA separately did increase the response of A5 cells compared to HA alone, they were not as effective as the HA-IL-2 chimeric protein. When an antibody known to block IL-2 receptor alpha chain (CD25) was included, A5 activation was reduced, suggesting a role for the receptor in this process. Expression of CD25 on A5 cells was low during activation, implying that the effect was mediated by CD25(+) BMDC. Antigen uptake and processing of HA-IL-2 by BMDC was required since fixing BMDC, prior to antigen exposure, greatly reduced their ability to activate A5 cells. The function of CD25 on DC is currently unknown. Our results suggest this receptor may play a role in antigen uptake and subsequent T cell activation by receptor-mediated endocytosis of antigen attached to IL-2. This finding that may have implications for the development of a new generation of vaccines.

Adjuvants, Immunologic↗

PubMed2000

Prostate cancer risk. Medical history, sexual, and hormonal factors.

PURPOSE: Various medical conditions, infectious agents, sexual, and hormonal factors have been investigated in relation to prostate cancer risk. Given inconsistent results these factors were examined in this study.METHODS: This population-based case-control study was conducted in northeastern Ontario from 1995 to 1999. Cases (n = 760), aged 45 to 84 at the time of diagnosis, were identified through the Ontario Cancer Registry and diagnosed between January 1995 and December 1998. Controls (n = 1,634) were age-frequency matched and were selected from the northeastern Ontario population using published telephone listings. Mail and telephone questionnaires were used for data collection. Logistic regression was used to investigate risk associated with: 1) particular medical conditions and 2) hormonal and sexual factors. Cases were subdivided into those with symptoms of prostate disease and those with few or no such symptoms.RESULTS: Symptomatic cases who reported a history of venereal disease (age-adjusted odds ratio (OR) = 2.11, 95% confidence interval (CI) 1.18-3.80) and vasectomy (age-adjusted OR = 1.49, 95% CI 1.14-1.95) were at significantly increased risk of prostate cancer. Asymptomatic cases who reported a check-up at least once a year were at increased risk (age-adjusted OR = 1.46, 95% CI 1.08-1.98). Asymptomatic and symptomatic cases who reported a history of prostate cancer in a first degree relative were at increased risk (age-adjusted OR = 2.41, 95% CI 1.64-3.54; age-adjusted OR = 3.18, 95% CI 2.28-4.45, respectively). Symptomatic cases with a history of urinary tract infection were at non-significantly increased risk (age-adjusted OR = 1.31, 95% CI 0.98-1.76). Heart disease, mumps, allergies, and height were generally not associated with prostate cancer.CONCLUSIONS: A history of venereal disease, family history of prostate cancer, and vasectomy were positively associated with prostate cancer. Further investigation of selected medical conditions, sexual, and hormonal factors in prostate cancer development is warranted.

Journal Article↗

PubMed2000

The production and biological assessment of cervine interferon gamma.

Cervine interferon gamma (IFN-gamma) was cloned and expressed using an Escherichia coli expression system pET-32. The expressed protein contained a 6 histidine purification tag and an 11 kDa thioredoxin fusion partner 5' to the IFN-gamma molecule. The ability of IFN-gamma to inhibit the killing of Madin-Darby bovine kidney cells by Semliki forest virus was used as a measure of the bioactivity of the recombinant cervine IFN-gamma (rIFN-gamma). It was shown that the presence of the thioredoxin fusion partner 5' to the IFN-gamma molecule did not affect its biological activity. As in the mouse model, it was shown that cervine rIFN-gamma was able to down-regulate the transcription of interleukin 10 mRNA while up-regulating the transcription of interleukin 12 mRNA in lipopolysaccharide-sensitized, peripheral blood mononuclear cells. A prototype ELISA was tested for its ability to detect both recombinant and native IFN-gamma. The ELISA was able to detect rIFN-gamma at concentrations greater than 100 pg/ml. It was also used to detect native IFN-gamma produced by peripheral blood lymphocytes from Mycobacterium bovis infected or vaccinated deer after in vitro restimulation with antigen. The rIFN-gamma and the cervine IFN-gamma specific ELISA provide valuable tools with which to study important zoonotic infections in farmed and wild deer.

Animals↗

PubMed2000

Interleukin-10 does not affect phagocytosis of particulate antigen by bone marrow-derived dendritic cells but does impair antigen presentation.

Dendritic cells (DC) are important initiators of an immune response so understanding the factors controlling antigen acquisition and presentation has important consequences for the use of these cells in vaccines and other forms of immunotherapy. We investigated the factors that influence phagocytosis by immature bone marrow-derived DC (BMDC) and the effect of interleukin-10 (IL-10) on this process. Two sizes of fluorescent particles and recombinant bacillus Calmette-Guèrin expressing the green fluorescent protein (rBCG) were used as particulate antigens. The percentage of cells taking up the antigen was found to be dependent on the size and dose of the particles, and the length of exposure to them. BMDC exposed to IL-10 at various concentrations for different periods exhibited no distinguishable change in antigen uptake. However, if BMDC treated with IL-10 and rBCG were then exposed to a second dose of particulate antigen, uptake was increased compared with those BMDC not treated with IL-10. The expression of major histocompatibility complex class II, CD80, CD86 and CD11c by BMDC after phagocytosing rBCG or inert beads, was inhibited when the BMDC were pretreated with IL-10. In contrast, the expression of CD25 was increased. BMDC that had taken up BCG or purified protein derivative (PPD) were able to stimulate primed T-cell proliferation but this was severely inhibited if the BMDC were cultured with IL-10 before exposure to the antigen. This work suggests that although IL-10 does not affect the phagocytic capacity of BMDC, it does inhibit maturation of the cells and consequently, T-cell activation.

Animals↗

PubMed2000

The expression and biologic effects of ovine interleukin-4 on T and B cell proliferation.

Using the reverse-transcriptase polymerase chain reaction (RT-PCR), cDNA encoding ovine (Ov) interleukin-4 (OvIL-4) was generated from mitogen-stimulated peripheral blood mononuclear cells (PBMC). Two identical clones generated from separate RT-PCR reactions differed from a published OvIL-4 sequence, although they had a high degree of identity with the bovine and human homologs. We show by sequence analysis that the OvIL-4 cDNA retained the four alpha-helix structure and disulfide bonds identified in human IL-4 (HuIL-4). Moreover, the cDNA encoding OvIL-4 was expressed in insect cells using the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) as a vector. Supernatants from insect cells infected with the recombinant virus secreted an additional protein with a relative molecular mass of 17,000. This protein was recognized by an anticervine IL-4 monoclonal antibody (mAb) in a Western blot and did not react with any proteins in supernatants from uninfected insect cells or cells infected with the wild-type AcMNPV. Supernatants from insect cells infected with the recombinant virus induced the proliferation of activated B cells in a dose-dependent manner and typically demonstrated 5 x 105 dilution U/ml of activity. However, OvIL-4 had no effect on the proliferation of resting T cells isolated from efferent lymph and actually inhibited the ability of a mitogen to stimulate these resting lymphocytes. In contrast, OvIL-4 induced the proliferation of mitogen-activated lymphoblast, demonstrating the complex role(s) OvIL-4 plays in the regulation of B and T cells.

Adjuvants, Immunologic↗

PubMed2000

Ontario familial colon cancer registry: methods and first-year response rates.

The Ontario Familial Colon Cancer Registry (OFCCR) is a novel registry that collects family history information, epidemiologic data, blood samples and tumour specimens from a population-based sample of colorectal cancer patients and their families. Families are classified as either high familial risk, intermediate familial/other risk or low (sporadic) risk for colorectal cancer. Obtaining high response rates in genetic family studies is especially challenging because of both the time commitment required and issues of confidentiality. The first-year response rate was 61%, resulting in 1,395 participating probands. In an attempt to assess potential response bias, we compared participants with non-participants. The age and sex of participants did not differ from non-participating probands; however, cases in rural areas were somewhat more likely to participate. To date, 57% of 1,587 relatives participated; females were more likely to participate, and relatives of low familial risk were least likely to participate. The OFCCR is an excellent resource that will facilitate the study of genetic and environmental factors associated with colorectal cancer.

Adult↗

PubMed1999

An in vivo comparison of bacillus Calmette-Guérin (BCG) and cytokine-secreting BCG vaccines.

A recombinant bacillus Calmette-Guérin (BCG) vaccine has been developed, which constitutively secretes interleukin (IL)-2. Groups of deer were immunized with either normal BCG (Pasteur 1173 P2 strain) or recombinant BCG (rBCG/IL-2) and their immune responses were monitored over 3 months. Animals gained weight over this period and showed no signs of adverse reactions to either vaccine. Lymphocyte transformation responses did not differ significantly between the two groups. No antibody that was specific for BCG was detected in any animal. Intradermal skin-test responses to BCG antigens showed that the rBCG/IL-2 induced a smaller delayed-type hypersensitivity response than the normal BCG. Cytokine transcription was determined by reverse transcription-polymerase chain reaction (RT-PCR). While IL-2 and interferon-gamma (IFN-gamma) levels did not differ significantly between the two groups, the level of IL-4 was found to be lower in the group given rBCG/IL-2. This resulted in a strong interferon-gamma:IL-4 ratio, suggesting a skewing of the immune response towards a Type 1 response. The rate at which the vaccine was eliminated from the host was the same regardless of whether BCG or rBCG was used. At autopsy (3 months after vaccination) 99.99% of the organisms had been eliminated. The small number of organisms isolated from the draining lymph node of animals given rBCG/IL-2 were grown in antibiotic-containing media. They were shown to still contain the shuttle plasmid and to secrete biologically active IL-2, indicating that the plasmid was stably maintained despite the host's immune response and in the absence of antibiotic selection.

Animals↗

PubMed1999

The characterisation of a cervine immunoregulatory cytokine, interleukin 12.

The cloning, sequencing, and production of cervine interleukin-12 is described. The cervine IL-12 p35 subunit coding sequence is 666 bp long and has highest homology to bovine p35 (94%), followed by human (79%), then murine (57%). The cDNA codes for a 221 aa long protein with predicted molecular weight of 24,902 Da. The cervine p40 subunit has a coding sequence of 984 bp and shows 96% homology to bovine, 85% homology to human, and 65% homology to murine p40 respectively. Cervine p40 cDNA codes for a 327 aa long protein with a predicted molecular weight of 37,461. Both subunits were inserted into a recombinant baculovirus that was then used to produce cervine IL-12 in Trichoplusia ni cells. Interleukin-12 was secreted into the culture medium and was biologically active as measured by proliferation of mitogen sensitised peripheral blood lymphocytes and the induction of interferon-gamma transcription in peripheral blood lymphocytes.

Amino Acid Sequence↗

PubMed1997

Toward a stepped care approach to treating problem drinkers: the predictive utility of within-treatment variables and therapist prognostic ratings.

AIMS: Cost containment, a central issue in current health planning, encourages the use of brief interventions. Although brief interventions for problem drinkers have proved successful, a portion of such individuals do not change their alcohol use during treatment. DESIGN: Repeated measures design (pre-treatment, within-treatment and 6 months post-treatment). SETTING AND PARTICIPANTS: To identify individuals at risk for continued problem drinking, predictors of post-treatment drinking were examined for 212 problem drinkers who presented for treatment in an outpatient treatment clinic. INTERVENTION: All participants completed a brief cognitive behavioral motivational intervention. MEASUREMENTS: At the pre-treatment assessment demographic, drinking pattern, severity of dependence and other cognitive variables (e.g. self-efficacy, goal choice) were collected. Within-treatment, drinking pattern and cognitive variables such as self-efficacy and goal choice were again measured. FINDINGS: Regression analyses showed that therapist prognosis ratings contributed significantly to the prediction of outcome even when pre-treatment variables were controlled. However, when within-treatment variables were included in the prediction, variables such as within treatment drinking eliminated the predictive utility of therapist prognosis ratings. This pattern held for both percentage of days abstinent and drinks per drinking day at a 6-month follow-up. CONCLUSIONS: It is suggested that a stepped care approach based on prediction models that include clients' within-treatment response can be applied to the treatment of problem drinkers who show little initial response to treatment.

Adult↗

PubMed1996

The cloning, expression and purification of cervine interleukin 2.

The cloning, sequencing and expression of cervine interleukin 2 (IL-2) is described. Cervine IL-2 cDNA is 489 base pairs long and shows high homology to bovine and ovine IL-2 (approximately 94%) with lower homologies to human (50%) and mouse (53%). The predicted protein sequence is 162 amino acids long with a signal sequence containing 20 amino acids. A molecular weight of 16273 Da was predicted for the mature protein. The expression plasmid pTRXFUS was redesigned to allow recombinant proteins to be expressed at high levels in a soluble form and subsequently affinity purified. This new plasmid, pTRXHIS, has been used to express the first cervine cytokine, IL-2. The fusion of the cervine IL-2 gene to the thioredoxin gene (TRX) stabilizes the recombinant product allowing the high expression of soluble IL-2. A polyHis (6 x Histidines) tag has been inserted between the two fusion partners which allows the fusion product to be affinity purified on a nickel-nitrilo-tri-acetic acid (Ni-NTA) column. The purified cervine IL-2 fusion protein was shown to be biologically active despite the presence of the TRX at the amino terminus. The TRX can be removed enzymatically with enterokinase releasing the biologically active IL-2 molecule. This expression system has several features that are useful in producing and purifying large quantities of biologically active cytokines.

Amino Acid Sequence↗

PubMed1996

Aftercare telephone contacts with problem drinkers can serve a clinical and research function.

Research staff typically gather treatment outcome data, whereas clinicians perform aftercare contacts. To date no alcohol treatment outcome study has examined the utility of therapists collecting outcome data through aftercare contacts. Using the Alcohol Timeline Followback (TLFB) method modified for clinical aftercare contacts, 154 problem drinkers who were part of a cognitive-behavioral intervention completed the modified TLFB with their primary therapist during aftercare telephone contacts conducted 1 and 3 months after their last treatment session. Clients reported their daily alcohol use over the past 30 days using four consumption categories (i.e. 0 drinks, 1-4 drinks, 5-9 drinks and 10+ drinks). At a 6-month follow-up research interview, a trained research assistant gathered standard TLFB data from the clients that included the time period for aftercare contacts. Correlations between the two TLFB formats showed good alternate form reliability, especially for frequency of alcohol use. Discrepancies between reports were positively associated with heavier pre-treatment and post-treatment drinking, suggesting possible memory biases among heavier drinkers. Subject reports also closely paralleled collateral reports of the subjects' drinking. These results support the utility of a brief TLFB instrument for use by therapists in assessing clients' outcomes by telephone during aftercare contacts.

Adolescent↗

PubMed1996

Activation of an interleukin-4 mRNA-producing population of peripheral blood mononuclear cells after infection with Mycobacterium bovis or vaccination with killed, but not live, BCG.

This study examines the expression of mRNA for the Th2 cytokine, interleukin-4 (IL-4). Peripheral blood mononuclear cells from deer infected with Mycobacterium bovis or vaccinated with live or killed M. bovis bacillus Calmette-Guérin (BCG) were cultured with mycobacterial antigens. IL-4 mRNA production was assayed using the polymerase chain reaction. Elevated levels of IL-4 mRNA were detected in response to at least one antigen preparation in all animals infected with M. bovis as compared with none of the non-infected control animals. After a primary immunization, elevated levels of IL-4 mRNA were detected in only a proportion of vaccinated animals and this did not correlate with whether the vaccine was live BCG or killed BCG in oil. After boosting, all the animals vaccinated with killed BCG in oil exhibited elevated IL-4 mRNA production whereas none of the animals vaccinated with live BCG showed elevated levels. The data suggest that IL-4 is turned off during the immune response to live BCG, that boosting of low-dose live BCG vaccine may be required to 'imprint' this signal and that this may be important in the development of protective immunity to tuberculosis. Killed BCG in adjuvant is not protective and as with experimental infection with virulent M. bovis it failed to switch off the IL-4 response. IL-4 may be useful as a diagnostic tool and as an in vitro marker of vaccine efficacy.

Animals↗

PubMed1995

The cloning and sequencing of cervine interleukin 10.

We report the cloning and sequencing of the cervine interleukin-10 gene. Specific cDNA was amplified by PCR using primers based on the bovine sequence. This was cloned into pGEM 5Zf and several clones were sequenced. The 762 nucleotide product coded for a 179 amino acid protein which was 86% homologous with its bovine and 77% homologous with its human counterparts. There is a strongly hydrophobic signal sequence consisting of the first 20 amino acids and a potential glycosylation site at amino acids 134-136. There are three regions, comprising 34% of the protein, which show complete homology between the cervine, bovine and human sequences. The transcription of the gene was shown by Northern Blotting where a single, 1.8kb, mRNA transcript was detected 4-8 hours after activation of peripheral blood mononuclear cells with mitogen.

Amino Acid Sequence↗

PubMed1993

Detection of proliferating cell nuclear antigens and interleukin-2 beta receptor molecules on mitogen- and antigen-stimulated lymphocytes.

The expression of interleukin-2 receptors (IL-2R) and proliferating cell nuclear antigens (PCNA) were compared for their usefulness as markers of lymphocyte activation. Heterologous polyclonal (anti-bovine IL-2R) and monoclonal (anti-human PCNA) antibodies were used to detect the expression of these molecules on activated deer lymphocytes. Both molecules were co-expressed on blast cells which had been activated with mitogen [concanavalin A (Con A)]. There was detectable up-regulation of IL-2R expression in response to antigen [Mycobacterium bovis-derived purified protein derivative (PPD)] stimulation while PCNA expression mimicked lymphocyte transformation (LT) reactivity. PCNA expression was found to more accurately reflect both antigen- and mitogen-activated lymphocyte activation, as estimated by LT activity. The expression of PCNA was used to identify antigen reactive cells from animals exposed to M. bovis. A very low percentage (1.1 +/- 0.4%) of peripheral blood lymphocytes from non-infected animals could be stimulated to express PCNA by in vitro culture with antigen (PPD). Within the infected group both diseased and healthy, 'in-contact', animals expressed significantly higher levels of PCNA upon antigen stimulation.

Animals↗

PubMed1991

Cytokine assays: role in evaluation of the pathogenesis of autoimmunity.

Cytokines are protein mediators involved in inflammation, the immune response, cell growth, repair and fibrosis. All of these processes are ongoing in active autoimmune diseases such as rheumatoid arthritis (RA), and so it would be expected that many cytokines would be actively produced in RA joints or Graves' disease (GD) thyroid glands. The cDNA cloning of cytokines has permitted the generation of pure recombinant molecules, and of newer more sensitive assays, and spurred the rapid development of knowledge in this field. Here we review the molecular strategies devised to study the possible role of cytokines in the pathogenesis of RA and GD, and describe some of the initial results. After 'cataloguing' the relative abundance of various cytokines, we sought to discover which cytokines are of major importance in pathogenesis. For that purpose we used neutralizing anti-cytokine antibodies and found that TNF alpha is one of the major signals regulating the production of IL-1 in the RA but not in the osteoarthritic (OA) joint. In order to further understand the dynamics of the cytokine network, the localization of the cytokine-producing cells by immunostaining and in situ hybridization has also been performed. The latter techniques are particularly valuable for attempting to establish the role of the target cell, such as thyroid epithelium, in the pathogenesis of disease. Cytokines act on cells via binding to high-affinity receptors. The last two years has been the cDNA cloning of many molecules encoding cytokine receptor chains, and it is now possible to begin to evaluate the other half of the cytokine pathway. Taken together, there are now exciting opportunities for the molecular dissection of the cytokine events occurring in auto-immune tissues.

Animals↗

PubMed1990

Cytokine production in the rheumatoid joint: implications for treatment.

Cytokines are protein mediators that play a part in inflammation, the immune response, cell growth, repair, and fibrosis. All of these are continuing processes in active rheumatoid arthritis (RA), and so it would be expected that many cytokines would be actively produced in RA joints. Here, the molecular strategies devised to study the possible role of cytokines in the pathogenesis of RA, are reviewed and some of the initial results described. The relative abundance of various cytokines is 'catalogued' and then attention is turned to an attempt to discover which cytokines are of major importance in the pathogenesis. Neutralising antibodies to cytokines were used for that purpose, and it was found that tumour necrosis factor alpha (TNF alpha) is one of the major signals regulating the production of interleukin-1 in the RA, but not in the osteoarthritic joint. To understand further the dynamics of the cytokine network localisation of the cytokine producing cells by immunostaining--for example, TNF alpha, is currently being established.

Arthritis, Rheumatoid↗

PubMed1989

Regulation of expression of human IL-1 alpha and IL-1 beta genes.

IL-1 gene expression was investigated in human blood mononuclear cells. IL-1 alpha and IL-1 beta mRNA were induced with LPS or TNF. Kinetic measurements on Northern blots revealed that these stimuli elicited qualitatively similar changes in IL-1 mRNA levels, and that expression of IL-1 mRNA was transient. IL-1 beta mRNA was the predominant mRNA species and remained elevated for somewhat longer than IL-1 alpha mRNA. TNF and IFN-gamma synergized to induce both species of IL-1 mRNA and IL-1 bioactivity. Transcriptional control, as measured by nuclear run on assays, partly determines the greater levels of IL-1 beta mRNA because the rate of IL-1 beta transcription was greater than that of IL-1 alpha. Cycloheximide (CHX) was able to induce IL-1 mRNA but did not induce transcription of either IL-1 gene. When added to cultures pretreated with TNF or LPS, CHX superinduced IL-1 mRNA, but IL-1 transcription was not increased. If added simultaneously CHX blocked TNF-induced IL-1 gene transcription, suggesting that TNF may induce factors required for IL-1 gene transcription. CHX increased the stability of both IL-1 alpha and IL-1 beta mRNA, demonstrating the existence of a post-transcriptional form of control. In half-life experiments IL-1 beta mRNA was more stable than IL-1 alpha mRNA, indicating that post-transcriptional control also contributes to the greater steady state levels of IL-1 beta. Taken together, the available evidence suggests IL-1 alpha and IL-1 beta mRNA are regulated differentially at both the transcriptional and post-transcriptional level.

Cycloheximide↗

PubMed1989

Intrathyroidal cytokine production in thyroid disease.

The intrathyroidal production of Interferon gamma, tumour necrosis factor alpha and beta, Interleukin-1 alpha and beta, Interleukin-6, platelet-derived growth factor A and of transforming growth factor-beta was analysed in patients with autoimmune and non-autoimmune thyroid disease. Cytokines were assessed indirectly by slot blot mRNA analysis in fresh tissue samples (unpurified cells, infiltrating mononuclear cells and thyroid follicular cells), in thyroid follicular cells in primary culture, as well as in thyroid-derived T-cell clones. The production of Interleukin-1 alpha and beta, Interleukin-6 and transforming growth factor beta was additionally measured by bioassay. Cytokine production by thyroid-infiltrating mononuclear cells generally did not differ between autoimmune and non-autoimmune samples, the whole panel of all cytokines being found in freshly purified cells as well as in thyroid-derived T-cell clones from patient with Graves' disease, as well as with multinodular non-toxic goitre. Thyroid follicular cells produced Interleukin-1 alpha, Interleukin-6 and transforming growth factor beta. Interleukin-1 and Interleukin-6 production did not differ between thyroid follicular cells from autoimmune and non-autoimmune thyroids. Transforming growth factor beta was, however, lower in non-toxic goitre than in Graves' disease and in normal thyroid tissue, but could be increased by exposure of the cells to micromolar concentrations of iodide. This seemed of special interest, as transforming growth factor beta proved to inhibit thyroid follicular cell growth in response to known growth stimuli, such as insulin-like growth factor I or epidermal growth factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoimmune Diseases↗