Genetic susceptibility to in vivo and in vitro chemical transformation.
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Animals↗
Biomedical subjects
C Walker
Publications and source records attributed to C Walker.
Inflammatory determinants of asthma severity: mediator and cellular changes in bronchoalveolar lavage fluid of patients with severe asthma.
Cellular and mediator profiles in bronchoalveolar lavage have not been compared systematically between patients with asthma of different severities, mainly because the patients with more severe asthma have an increased need for antiinflammatory medication. Information is limited to comparisons of allergic and intrinsic asthma, which can be distinguished clinically. When patients from these two groups with similar degrees of bronchial hyperresponsiveness were compared, both groups showed increased numbers of activated T-helper lymphocytes; those in the allergic group expressed the IL-2 receptor (CD25+), whereas in patients with intrinsic asthma there was also an increased number of T-suppressor cells with the activation markers CD25, class II histocompatibility antigen, and very late activation antigen-I, as well as T-helper cells class II histocompatibility antigen and very late activation antigen-I. This pattern is compatible with a more chronic T-cell activation in patients with intrinsic asthma. In patients with allergic asthma the cytokine pattern is compatible with a pure TH2 response (elevated IL-4 and IL-5); however, intrinsic asthma is characterized by elevated IL-5 and IL-2 but not IL-4. Our own findings show similar concentrations of IL-1, IL-8, and granulocyte-macrophage colony-stimulating factor in bronchoalveolar lavage fluid of patients with allergic and intrinsic asthma, whereas IL-6 and interferon-gamma tended to be higher in patients with intrinsic asthma. There are probably fundamental differences in the pathogenesis of allergic and intrinsic asthma. These findings suggest that asthma does not depend on the presence of IgE or IL-4, although both may contribute to the pathogenesis of atopic asthma. The only common pathway in the different presentations of asthma that has been related to clinical symptoms appears to be IL-5-mediated activation of eosinophils; therapies aimed at this mechanism may be promising.
Asthma↗
Microsatellite-centromere mapping in the zebrafish (Danio rerio).
Ten (CA)n microsatellite simple sequence repeat (SSR) markers, 1, 2, 12, 14, 16, 18, 20, 22, 26, and 29, were used to show high chiasma interference and to determine centromere-marker map distances in the zebrafish (Danio rerio). Of these, SSR 12 exhibited no recombinant tetratypes among 175 half-tetrad embryos, placing this marker within 1 cM of the centromere of Linkage Group XVII. Fractions of heterozygous half-tetrads for the remaining nine markers ranged from 0.64 to 0.89. Of these, six recombinant fractions were more than 0.67 (P < 0.05), indicating strong chiasma interference during female meiosis in the zebrafish. Consistent with previous mapping data, SSRs 2 and 20 of Linkage Group VI were tightly linked. Half-tetrad analysis will allow the mapping of the remaining centromeres and may be useful in the mapping of new genes and mutations in the zebrafish.
Animals↗
Topology of prostaglandin H synthase-1 in the endoplasmic reticulum membrane.
Prostaglandin H synthase-1 is an integral endoplasmic reticulum membrane protein which catalyzes a key control step in prostaglandin biosynthesis. The overall arrangement of the prostaglandin H synthase-1 polypeptide with respect to the endoplasmic reticulum membrane was examined in transiently transfected COS-1 cells, using immunofluorescence microscopy. A bacterial toxin, streptolysin-O, was used for selective plasma membrane permeabilization and a detergent, saponin, for general membrane permeabilization. Treated cells were probed with six antibodies specific for particular prostaglandin H synthase-1 peptide segments and one antibody specific for an inserted viral reporter epitope. Control experiments established that actin, a cytoplasmic marker, was accessible to fluorescein-labeled phalloidin after streptolysin-O treatment, whereas antibodies against protein disulfide isomerase, an endoplasmic reticulum lumenal marker, bound only after saponin treatment, Using this approach to investigate prostaglandin H synthase-1, it was found that streptolysin-O treatment was sufficient to obtain staining of intracellular membranes by antibodies specific for the endogenous C-terminal segment, for the viral reporter inserted at the C-terminus, and for the protease-sensitive region near arg277. In contrast, saponin treatment was necessary for staining by antibodies specific for peptides spanning residues 51-66, 156-170, and 377-390. Antibodies targeted against residues 483-496 did not stain transfected cells even after saponin permeabilization, although they did bind to detergent-solubilized prostaglandin H synthase-1. These results indicate that the C-terminus and arg277 regions of the synthase can be exposed on the cytoplasmic side of the endoplasmic reticulum membrane, whereas regions near N-glycosylation sites are confined to the endoplasmic reticulum lumen and residues 483-496 are inaccessible from either side of the endoplasmic reticulum membrane.
Amino Acid Sequence↗
Cloning and characterization of murine p16INK4a and p15INK4b genes.
Progression through the G1 phase of the cell cycle is regulated in part by the D-type cyclin-dependent kinases, cdk4 and cdk6. Genes encoding two specific inhibitors of these kinases, human p16(INK4a/MTS1) and p15(INK4b/MTS2), map to a region of common cytogenetic abnormalities on chromosome 9p21. The murine cognates of these genes were isolated and identified as mouse p16INK4a and p15INK4b based on their homology to their human counterparts and their selective transcriptional induction by SV40T-antigen and TGF-beta, respectively. Both genes map to position C3-C6 on mouse chromosome 4, in a region syntenic with human chromosome 9p. Amplification of polyadenylated mRNA by polymerase chain reactions revealed no expression of mouse p16INK4a in many normal tissues, whereas p15INK4b was expressed ubiquitously. Like human p16INK4a, mouse p16INK4a binds specifically to cdk4 and cdk6 in vitro and inhibits the phosphorylation of the retinoblastoma protein, pRb, by each of these cyclin D-dependent kinases. In mouse MEL erythroleukemia cells, p16INK4a associates preferentially with cdk6 under conditions where cdk4 and cdk6 are coexpressed at equivalent levels. Expression vectors encoding human or mouse p16INK4a caused G1 phase arrest in NIH3T3 fibroblasts, and cyclin D1- and cdk4-dependent pRb kinase activities were inhibited in the p16INK4a-arrested cells.
3T3 Cells↗
Estrogen/antiestrogen responsiveness in an in vivo/in vitro model for myometrial tumorigenesis.
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Animals↗
A nuclear factor of activated T cell-like transcription factor in mast cells is involved in IL-5 gene regulation after IgE plus antigen stimulation.
IL-5, which is produced mainly by activated T cells and allergically triggered mast cells, is a major survival and differentiation factor for eosinophils, and therefore, is of relevance to diseases associated with this type of cell infiltration, most importantly asthma. In this study, we have examined the transcriptional regulation of human IL-5 in a mouse mast cell line, CPII, stimulated with IgE and Ag. We report that an inducible activity in the region between -177 and -80, and a constitutive activity between -80 and -70, in the promoter of the human gene, are both necessary for the allergically triggered activation. A computer-assisted search for transcription factor binding motifs revealed a nuclear factor of activated T cell (NF-AT) and a GATA consensus site in the two regions. Corresponding binding activities were detected to be present in nuclear extracts from the mouse mast cell line by defined NF-AT and GATA binding sites as probes for a gel shift analysis. Competition analysis, in combination with probes from the human IL-5 promoter, confirmed that these factors indeed bind to the consensus sequences identified by computer analysis. An oligonucleotide spanning the IL-5 NF-AT consensus site is shown to confer allergic stimulation to a basal IL-5 promoter only in conjunction with the GATA site downstream, indicating that an inducible NF-AT-like factor cooperates with a constitutive member of the GATA transcription factor family in mediating the allergic stimulation of the human IL-5 gene.
Animals↗
Autocrine growth stimulation by transforming growth factor alpha in asbestos-transformed rat mesothelial cells.
Although the association between asbestos exposure and mesothelioma development has been established for decades, very little is known regarding the molecular mechanism(s) by which asbestos fibers induce this disease. In this series of experiments, the potential for transforming growth factor alpha (TGF-alpha) to act as an autocrine growth factor in transformed mesothelial cells was examined in rats, a model system frequently used to assess the tumorigenic potential of fibrous particulates. Both asbestos-transformed cells and spontaneously transformed cells expressed functional EGF receptors, although only the asbestos-transformed cells expressed TGF-alpha. Expression of TGF-alpha transcripts was correlated with secretion of picogram amounts of growth factor into conditioned medium by the asbestos-transformed cells. In addition, whereas TGF-alpha inhibited the growth of spontaneously transformed mesothelial cells, it stimulated the growth of asbestos-transformed cells. Neutralizing antibody that recognized TGF-alpha secreted by the asbestos-transformed cells was able to inhibit the growth of these cells. Taken together, these data indicate that TGF-alpha acts as an autocrine growth factor for asbestos-transformed rat mesothelial cells. Therefore, in asbestos-transformed mesothelial cells, altered production and responsiveness to TGF-alpha distinguish these cells from spontaneously transformed mesothelial cells. These data suggest that differences in mesothelioma etiology may be reflected in differences in the molecular alterations present in these tumors.
Animals↗
[Immunopathogenesis of allergic bronchial asthma. Detection of activated CD25-CD4 lymphocytes and release of cytokines in the bronchoalveolar space following segmental allergen challenge].
The aim of this study was to investigate the fundamental processes underlying the inflammatory response to allergen in mild non-symptomatic asthmatics, using a new model entailing endobronchial segmental provocation. Ten asymptomatic asthmatic volunteers (8 male, 2 female; mean age 28.2 [24-41] years) were challenged employing the segmental allergen provocation technique. 250 PNU in 5 ml 0.9% NaCl solution of either birch (n = 8) or grass (n = 2) pollen were instilled into one segment. As control, only the solvent was instilled into a segment of the contralateral lung. Bronchoalveolar lavage (BAL) with 100 ml prewarmed saline was performed 10 min and 18 h after allergen provocation. The cellular distribution and activation state in BAL fluid and peripheral blood was analysed by immunofluorescence and flow cytometry. The concentration of various cytokines was determined in the BAL fluid using ELISA and bioassays. In blood, segmental allergen provocation led to an increase both in numbers of neutrophils (3833 cells/microliters vs 6830 cells/microliters; P < 0.005) and activated IL-2R expressing (CD25+) CD4+ T cells (3.6% vs 4.8% of all CD4+ lymphocytes; P < 0.05). No change was observed in eosinophils and other leukocytes and lymphocyte subsets. In contrast, a significant 30-fold increase in eosinophils (0.39 x 10(6) vs 11.5 x 10(6) cells/100 ml; P < 0.01) and a twofold increase in CD25+ CD4+ T cells (P < 0.05) were found in BAL samples 18 h after segmental allergen challenge, when compared to the control segment. Analysis of the cytokine profile revealed significantly increased levels of several cytokines. Allergen challenge of extrinsic asthmatic subjects causes differentiation of activated CD25+ CD4+ lymphocytes which may contribute to the pathogenesis of the asthmatic inflammation through the release of various cytokines.
Adult↗
p53 status in spontaneous and dimethylnitrosamine-induced renal cell tumors from rats.
Rats carrying the Eker tumor-susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT-->CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non-p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer.
Adenoma↗
Human non-small cell lung cancer: p53 protein accumulation is an early event and persists during metastatic progression.
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Carcinoma, Non-Small-Cell Lung↗
Genetic control of adult pigment stripe development in zebrafish.
Adult zebrafish stripes are formed from stripes of gold iridophores alternating with stripes of black melanocytes lying beneath silver stripes. Analysis of defects in pigment pattern development caused by sparse (spa, rose (ros), and leopard (leo) single and double mutant combinations suggests that spa+ and ros+ functions are required for development of separate populations of pigment cells in the adult and that leo+ functions to control assembly of melanocytes into stripes. Thus, between 2 and 3 weeks of zebrafish development, spa-dependent melanocytes differentiate throughout the flank, followed by leo-dependent assembly of these cells into stripes. Beginning at 3 weeks of development, a distinct ros-dependent population of melanocytes differentiates in the stripe. Both early and late differentiating melanocytes then affect the formation of the silver stripes, ensuring registration of melanocyte and iridophore stripes.
Animals↗
Contemporary issues in fiber toxicology.
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Administration, Inhalation↗
Analysis of feline dual lymphocyte populations observed by flow cytometry.
Two discrete lymphocyte populations were observed commonly on flow cytometric analysis (FCM) of feline lymphocyte subsets. The identity of these populations as small and large lymphocytes was established by correlating data from FCM with that from peripheral blood films. Dual lymphocyte populations were more likely to be seen in feline immunodeficiency virus-positive (FIV(-)+ ve) cats but their occurrence was not affected by health status, age, gender or breed. FIV(-)+ ve cats had a significantly higher proportion of large lymphocytes than FIV-negative (FIV(-)- ve) cats. However, FIV(-)+ ve cats had significantly fewer small lymphocytes than FIV(-)- ve cats but similar numbers of large lymphocytes. Lymphocyte subset analysis revealed that small lymphocytes had a greater proportion of CD4+ cells than large lymphocytes, regardless of the FIV or health status of the cat. In FIV(-)- ve cats, small lymphocytes had a greater proportion of Pan T + lymphocytes than large lymphocytes, but the converse was seen in FIV(-)+ ve cats. The proportion of CD8 + cells was higher in small lymphocytes than large lymphocytes in well FIV(-)- ve cats but this distinction was not seen in sick FIV(-)- ve cats or FIV(-)+ ve cats of any health status. Regardless of health status, FIV(-)+ ve cats had a lower absolute count of small lymphocytes which were T cells (due to lower numbers of both CD4 + and CD8 + cells) than FIV(-)- ve cats. The numbers of small B cells were similar for both FIV(-)+ ve and FIV(-)- ve cats. However, there were no differences between FIV(-)+ ve and FIV(-)- ve cats in the absolute values of any subset of the large lymphocytes, which suggested that FIV may affect only small lymphocytes. Statistically, the inclusion or exclusion of the large lymphocyte population for routine lymphocyte subset analysis did not affect the overall results. However, because there were significant differences in subset distribution between small and large lymphocytes, analysis of both groups should be included in studies examining the role of lymphocytes in disease.
Animals↗
Expression of the BCL-2 protein in normal and dysplastic bronchial epithelium and in lung carcinomas.
Although expression of the bcl-2 protein has been investigated in a number of non-haematological malignancies, little is known of its distribution in premalignant lesions. Expression of bcl-2 was investigated immunohistochemically in archival biopsies of normal (n = 8) and dysplastic bronchial epithelium (n = 56) and in 31 bronchial resection margins and their corresponding carcinomas. All dysplasias had lost the prominent basal staining pattern seen in histologically normal epithelium. Two were negative and six had occasional basal positive cells. In 37 cases up to 66% of the epithelial cells throughout the full epithelial thickness were bcl-2 positive with weak to moderate staining intensity. In 11 cases, all severe dysplasias, strong expression was observed in > 90% of the epithelial cells. Four patterns of bcl-2 expression in dysplasias were identified and an increasingly aberrant pattern of bcl-2 expression correlated with an increasing grade of dysplasia (Spearman's rank correlation, P < or = 0.0001). Sixty-five per cent of the carcinomas contained bcl-2-positive cells. Patients with non-small-cell lung carcinomas (n = 27) in which > 50% of the tumour cells were bcl-2 positive showed a survival advantage compared with those with 0-25% bcl-2-positive cells (P = 0.02). No correlation was found between p53 expression (Walker et al., 1994) and bcl-2 expression in dysplasias or carcinomas.
Bronchi↗
Accumulation of activated CD4+ lymphocytes in the lung of individuals infected with HIV accompanied by increased virus production in patients with secondary infections.
The lung is continuously exposed to infectious and non-infectious agents causing cell activation. Activated cells in the lung such as antigen-presenting cells which harbour HIV may favour this organ as a site for virus production. To test this hypothesis, cells from blood and bronchoalveolar lavage (BAL) of HIV-infected patients and healthy controls were obtained and the activation of the cells were analysed by measuring the expression of IL-2 receptor, HLA-DR and VLA-1. The HIV-infected individuals were subdivided into 'lung symptomatic' or 'lung asymptomatic' patients, depending on the presence or absence of secondary lung diseases besides HIV. All HIV-infected individuals demonstrated a decreased number of CD4+ lymphocytes in blood; however, normal numbers of these cells were found in BAL. The activation state of CD4+ and CD8+ T lymphocytes in blood and BAL was higher in lymphocytes from HIV-infected patients compared with controls. The activation state was highest in the lung symptomatic group. Lung symptomatic patients and lung asymptomatic patients with extrapulmonary infections had increased levels of free virus in plasma. Four out of four individuals without or with only low amounts of cell-free HIV in plasma belonged to the symptom-free subgroup. These results suggest that microorganisms other than HIV may promote viral replication via antigen-driven accumulation and activation of CD4+ cells in the lung or other organs, and thus may be responsible for the loss of helper T cells and the progression of the disease.
Bronchoalveolar Lavage Fluid↗
Thick bone section preparation using a silicon-rubber-based sealant.
A method has been developed, using a silicon-rubber-based sealant, which allows 2-3-mm-thick specimens to be maintained in a protected fluid environment for a number of months, without risk of dehydration. Following this, the specimen can be retrieved, stained, embedded and sectioned further. For example, 2-mm-thick sections of fixed unstained bone are easily examined by means of epi-illuminated polarized light and fluorescence microscopies using either conventional or confocal optics. The method could easily be extended to other tissues, for example brain tissue.
Bone and Bones↗
Analysis of leucocytes and lymphocyte subsets in cats with naturally-occurring cryptococcosis but differing feline immunodeficiency virus status.
Although cryptococcosis is a well-characterised disease of cats, the factors predisposing individuals to infection are unknown. As an indication of the immune status of an individual, lymphocyte subsets can be analysed. Reference ranges for feline lymphocyte subsets (Pan T+, CD4+, CD8+ and B cells) were established using a rapid whole blood technique and flow cytometry. There were no effects of age or sex on lymphocyte subset values. The numbers of circulating leucocytes and lymphocyte subsets were determined in FIV-positive and FIV-negative cats with cryptococcosis and compared with a group of healthy control cats. There were only minor differences in the numbers of lymphocyte subsets among the subgroups of cats examined in the study and the predisposition to cryptococcosis in cats could not be explained by deficiencies in lymphocyte subsets. There was a tendency for FIV-negative cats with cryptococcosis to have reduced numbers of circulating CD4+ cells and lower CD4:CD8 ratios compared with normal cats, although the interpretation of this finding was complicated by the wide reference range for normal cats. The extent to which this is the cause of the fungal infection was not determined. The only difference in leucocyte or lymphocytes subset values between FIV-negative cats with cryptococcosis and FIV-positive cats with cryptococcosis was that the CD4+ percentage was lower in the FIV-positive cats. The absolute CD4+ count was similar however, in FIV-positive and FIV-negative cryptococcosis cases. On the basis of this and other available information, the categorisation of cryptococcosis as a disease defining the AIDS phase of FIV infection may be incorrect.
Animals↗