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Biomedical subjects

P D Smith

Publications and source records attributed to P D Smith.

At least 19 recordsLinked to original sources
PubMed1999

Poliovirus replicons encoding the B subunit of Helicobacter pylori urease elicit a Th1 associated immune response.

The development of a vaccine for Helicobacter pylori is a key strategy for reducing the worldwide prevalence of H. pylori infection. Although immunization with recombinant B subunit of H. pylori urease (ureB) has yielded promising results, for the most part, these studies relied on the use of strong adjuvant, cholera toxin, precluding the use in humans. Thus, the development of new vaccine strategies for H. pylori is essential. Previous studies from our laboratory have described a vaccine vector based on poliovirus in which foreign genes are substituted for the poliovirus capsid genes. The genomes encoding foreign proteins (replicons) are encapsidated into authentic poliovirions by providing the capsids in trans. To test the utility of replicons as a vaccine vector for H. pylori, a replicon was constructed which encodes ureB. Expression of ureB in cells from the replicon was demonstrated by metabolic labeling followed by immunoprecipitation with anti-urease antibodies. To investigate the immunogenicity of the replicons, mice containing the transgene for the receptor for poliovirus were immunized via the intramuscular route. Mice given three doses of replicons did not develop substantial antibodies to ureB as determined by Western blot analysis using lysates from H. pylori. In contrast, mice given two doses of replicon followed by a single injection of recombinant ureB developed serum antibodies to ureB which were predominately IgG2a. Splenic lymphocytes from mice immunized with replicons alone, or replicons plus recombinant ureB produced abundant interferon-gamma and no detectable interleukin-4 upon stimulation with recombinant ureB. These results establish that poliovirus replicons encoding H. pylori ureB are immunogenic and induce primarily a T helper 1 associated immune response.

Animals↗
PubMed1999

Novel p53 mutants selected in BRCA-associated tumours which dissociate transformation suppression from other wild-type p53 functions.

Inheritance of germ-line mutant alleles of BRCA1 and BRCA2 confers a markedly increased risk of breast cancer and we have previously reported a higher incidence of p53 mutations in these tumours than in grade matched sporadic tumours. We have now characterized these p53 mutants. The results of these studies identify a novel class of p53 mutants previously undescribed in human cancer yet with multiple occurrences in BRCA-associated tumours which retain a profile of p53-dependent activities in terms of transactivation, growth suppression and apoptosis induction which is close or equal to wild-type. However, these mutants fail to suppress transformation and exhibit gain of function transforming activity in rat embryo fibroblasts. These mutants therefore fall into a novel category of p53 mutants which dissociate transformation suppression from other wild-type functions. The rarity of these mutants in human cancer and their multiple occurrence in BRCA-associated breast tumours suggests that these novel p53 mutants are selected during malignant progression in the unique genetic background of BRCA1- and BRCA2-associated tumours.

Animals↗
PubMed1999

Intestinal macrophages display reduced permissiveness to human immunodeficiency virus 1 and decreased surface CCR5.

BACKGROUND & AIMS: Because the role of intestinal mononuclear cells in the pathogenesis of human immunodeficiency virus 1 (HIV-1) disease has not been elucidated, we determined the biological properties of HIV-1 infection in primary intestinal macrophages. METHODS: Mucosal macrophages purified from normal human jejunum were infected with well-characterized macrophage-tropic isolates of HIV-1 (ADA, DJV, and Ba-L). RESULTS: Productive HIV-1 infection of intestinal macrophages was demonstrated by the release of p24 antigen, the presence of proviral DNA, and zidovudine inhibition of infection. Surprisingly, the titer of virus needed to establish infection of intestinal macrophages was 100-1000-fold higher than that required to infect peripheral blood derived macrophages. This marked reduction in the permissiveness of intestinal macrophages to HIV-1 was not caused by the isolation procedure or differences in CD4 expression. Instead, intestinal macrophages expressed almost no CCR5, the principal coreceptor for macrophage-tropic HIV-1, compared with blood-derived macrophages, although both cell types contained comparable levels of CCR5 messenger RNA. Exposure of blood-derived but not intestinal macrophages to HIV-1 or gp120 led to increased surface expression of CCR5. CONCLUSIONS: Intestinal macrophages express reduced levels of HIV-1, probably because of impaired permissiveness to HIV-1 entry associated with the near absence of cell surface CCR5.

CD4 Antigens↗
PubMed1999

The efficacy of 5% Sulfamylon solution for the treatment of contaminated explanted human meshed skin grafts.

Large TBSA burns have a deficiency of skin graft donor sites necessitating meshed skin autografts, cultured epithelial autografts or biosynthetic skin substitutes. Because these do not effect immediate complete biological closure of the wound, the burn victim remains at risk for life-threatening infection. Topical antimicrobials can protect colonization of these grafts from becoming invasive sepsis. However, many of these agents are cytotoxic to new partially keratinized epithelial cells. This study using a model of epithelialization kinetics of human meshed skin grafts explanted to athymic 'nude' rats evaluated: (1) the effect of bacterial colonization on the rate of closure of meshed graft interstices; (2) the efficacy of 5% Sulfamylon solution for bacterial control and (3) the effect on interstitial closure rates caused by control of bacterial proliferation. Results showed the rate of interstitial closure was progressive over 7 days in noncontaminated grafts treated with moistened saline dressings. Areas of total closure of a 1:1.5 meshed graft were seen as early as 5 days. When grafts were inoculated with 10(2) or 10(3) Pseudomonas aeruginosa organisms and treated with saline moistened dressings, the resultant bacterial load rose to 10(6) organisms, less than 3% of the interstices closed and grafts were destroyed. With the same organism level of contamination, bacterial levels were eradicated with topical 5% Sulfamylon solution, interstitial closure rates returned to normal and areas of total meshed graft closure were seen by day 4. These data demonstrate the efficacy of 5% Sulfamylon solution on epithelialization kinetics of contaminated meshed skin grafts.

Animals↗
PubMed1999

Cytotoxic response of ovarian cancer cell lines to IFN-gamma is associated with sustained induction of IRF-1 and p21 mRNA.

Interferon-gamma (IFN-gamma) has some anti-tumour activity in human ovarian cancer. This cytokine inhibited proliferation in three of four ovarian cancer cell lines in vitro. We then compared the action of IFN-gamma in two cell lines, one sensitive and one resistant to its growth inhibitory effects. IFN-gamma signalling appeared normal in both cell lines, with stat1 DNA binding activity detectable at 30 min. Continuous exposure to IFN-gamma for 2-3 days was necessary for an irreversible effect on cell growth and apoptosis in cells sensitive to growth inhibition. During this time there was an increase in mRNA for the CKI p21, but no alterations in mRNA levels for other members of the CKI family. Maintenance of p21 mRNA required continuous mRNA synthesis. mRNA for the transcription factor IRF-1 was also induced in growth inhibited cells with similar kinetics to those observed for p21. Maximal induction of both p21 and IRF-1 mRNA was observed after 2-3 days IFN-gamma exposure as the cells became committed to cell death. There was also a rapid increase in p21 and IRF-1 mRNA in cells resistant to the growth inhibitory effects of IFN-gamma, but this increase was not maintained. Thus, continuous interaction with the IFN-gamma receptor, together with a sustained induction of p21 and IRF-1, is associated with growth inhibitory and apoptotic effects of IFN-gamma in ovarian cancer cells.

Adenocarcinoma↗
PubMed1999

Mucosal events in the pathogenesis of human immunodeficiency virus type 1 infection.

The interaction between human immunodeficiency virus type 1 (HIV-1) and primary mucosal cells isolated from normal human small intestine was investigated. Purified primary intestinal epithelial cells could transport cell-free HIV-1 to mononuclear cells, although the epithelial cells did not support viral replication. An unexpected finding was that primary intestinal macrophages were markedly less permissive to HIV-1 than were blood monocytes. The reduced permissiveness appeared to be due to the near absence of surface CCR5 on resident intestinal macrophages. Surface CCR5 could be up-regulated on the monocytes but not the intestinal macrophages by HIV-1 and gp120. Impaired permissiveness of intestinal macrophages to HIV-1 may play an important role in the low prevalence of HIV-1 mRNA-expressing macrophages in the lamina propria during HIV-1 infection in vivo. Characterization of the biologic properties of HIV-1 transport and infection in primary mucosal cells will be key to elucidating the pivotal role of mucosal surfaces in HIV-1 disease.

Disease Reservoirs↗
PubMed1999

A geographical information system technique for record-matching in a study of cancer deaths in welders.

Retrospective studies are frequently complicated by incomplete worker identifiers. We encountered this problem when evaluating the risk of cancer death in a welding cohort. We dealt with it by developing birth-date ranges for each welder with unknown birth dates and using geographical information system techniques in conjunction with last name, gender, and birth-date range to assign death certificates to welders on the basis of residential proximity to the worksite. Deaths for total malignant cancers and lung/tracheobronchial/pleural cancers among these welders were not significantly different from those in county, state, and US populations, using standardized mortality ratios. The ratios in our study subjects were consistent with ratios found in other published welder cohorts.

Adult↗
PubMed1999

No relationship between gastric pH, small bowel bacterial colonisation, and diarrhoea in HIV-1 infected patients.

BACKGROUND/AIMS: Conclusive studies of small bowel bacterial overgrowth in patients with HIV-1 infection are limited. The relation was therefore determined between the quantity and species of bacteria in the proximal small intestine of HIV-1 infected patients and the presence of diarrhoea, gastric acidity, severity of immune deficiency, and clinical outcome. METHODS: Bacteria in the duodenal fluids obtained endoscopically from 32 HIV-1 infected patients, 21 of whom had diarrhoea, and seven control subjects without HIV-1 risk factors were quantified and speciated. Gastric pH was determined at the time of endoscopy. Clinical follow up was performed to assess outcome. RESULTS: Oropharyngeal Gram positive cocci were present in fluids from 28 patients (88%). Gram negative aerobic or facultatively anaerobic bacteria were present in fluids from 12 patients (38%), and strict anaerobes were detected in six patients (19%), but for both groups colony counts infrequently exceeded 10(4) colony forming units/ml. The number and species of bacteria did not correlate with the presence of diarrhoea, gastric pH, or CD4 lymphocyte count. CONCLUSIONS: Small bowel bacterial overgrowth is not common in HIV-1 infected patients, regardless of the presence of diarrhoea, and is not associated with hypochlorhydria

AIDS-Related Opportunistic Infections↗
PubMed1999

Neutrophil activation and mediators of inflammation in chronic venous insufficiency.

The effect of venous hypertension on the state of activation of leucocytes has been investigated in patients with venous disease and control subjects. Leucocytes become 'trapped' in the circulation of the leg during periods of venous hypertension produced by sitting or standing. This is greater in the limbs of patients with chronic venous disease than controls. Studies of the plasma levels of neutrophil granule enzymes show that these are increased during periods of venous hypertension, suggesting that this causes activation of the neutrophils. Investigation of the leucocyte surface ligand CD11b shows that the more activated neutrophils and monocytes are sequestered during venous hypertension. Measurement of plasma levels of the soluble parts of the vascular (VCAM), intercellular (ICAM) and endothelial leucocyte (ELAM) adhesion molecules show that these are all elevated in patients with chronic venous disease compared to controls. Following 30 min of venous hypertension produced by standing, these levels are further increased. These data suggest that venous hypertension causes neutrophil and monocyte activation, which in turn causes injury to the endothelium. I believe that this may be the mechanism that initiates the pathological processes which lead to venous ulceration. It has recently been shown that the venotonic drug Daflon 500 mg (450 mg diosmin, 50 mg hesperidin, Servier, France) influences these processes. Surface expression of CD62L is reduced in neutrophils and monocytes, and plasma levels of soluble endothelial adhesion molecules are reduced. These observations may explain the anti-inflammatory effects of Daflon 500 mg.

Animals↗
PubMed1999

MRI volumes of the hippocampus and amygdala in adults with Down's syndrome with and without dementia.

OBJECTIVE: This study sought to determine whether volumes of the hippocampus and amygdala are disproportionately smaller in subjects with Down's syndrome than in normal comparison subjects and whether volume reduction is greater in Down's syndrome subjects with dementia. METHOD: The subjects were 25 adults with Down's syndrome (eight with dementia) and 25 cognitively normal adults who were individually matched on age, sex, and race. Magnetic resonance imaging measures included volumes of the hippocampus, amygdala, and total brain. Nineteen of the Down's syndrome subjects had follow-up scans (interscan interval = 9-41 months). RESULTS: Nondemented Down's syndrome subjects had significantly smaller volumes of the hippocampus, but not the amygdala, than their comparison subjects, even when total brain volume was controlled for. Volumes of both the hippocampus and the amygdala were smaller in the demented Down's syndrome subjects than in their comparison subjects, even when total brain volume was controlled for. Age was not correlated with volume of the hippocampus or amygdala among the nondemented Down's syndrome subjects and the comparison subjects; age was correlated with volume of the amygdala, but not the hippocampus, among the Down's syndrome subjects with dementia. Changes in volume over time were not statistically significant for either the demented or the nondemented subjects. CONCLUSIONS: Hippocampal volume, while disproportionately small for brain size in individuals with Down's syndrome, remains fairly constant through the fifth decade of life in those without dementia. All subjects over age 50 who had Down's syndrome demonstrated volume reduction in the hippocampus as well as clinical signs of dementia. Dementia was also associated with volume reductions in the amygdala that exceeded reductions in total brain volume.

Adult↗
PubMed1999

Laser capture microdissection of single cells from complex tissues.

Laser capture microdissection (LCM) is a new method used to select and procure cell clusters from tissue sections. Once captured, the DNA, RNA or protein can be easily extracted from the isolated cells and analyzed by conventional PCR, reverse transcription (RT)-PCR or polyacrylamide gel electrophoresis, including protein zymography for specific macromolecular changes. In LCM, a thermoplastic polymer coating [ethylene vinyl acetate (EVA)] attached to a rigid support is placed in contact with a tissue section. The EVA polymer over microscopically selected cell clusters is precisely activated by a near-infrared laser pulse and then bonds to the targeted area. Removal of the EVA and its support from the tissue section procures the selected cell aggregates for molecular analysis. This initial NIH LCM approach using a flat transfer EVA film has been recently commercialized and has proven to be an effective routine microdissection technique for subsequent macromolecular analysis in many laboratories around the world. However, reliable and precise capture of individual cells from tissue sections has been difficult to perform with the current LCM instruments. In this report, we describe the capture of individual cells with a new NIH LCM microscope, which epi-irradiates the EVA polymer overlying individual cells with 1-ms laser pulses focused to 6 microns. A computer-controlled arm precisely positions a 40-micron-wide strip of a cylindrical EVA surface onto a sample with a light contact force (ca. 0.1 g). The small contact force and contact area on the film on the sample diminishes nonspecific transfer to negligible levels. By slightly rotating the cylinder to provide a renewable transfer surface, concentration of a distinct cell type on a single cylinder is possible. Using this novel adaptation, we demonstrate the rapid and practical capture of single cells from different types of tissue sections, including immunostained cells.

Animals↗
PubMed1999

Effect of TGF-beta2 on proliferative scar fibroblast cell kinetics.

Keloids, hypertrophic scars, and burn hypertrophic scars are all forms of proliferative scarring characterized by overabundant matrix formation. Recently these dermal proliferative disorders have been linked clinically to the cytokine transforming growth factor beta (TGF-beta), and in vitro tests have shown it to be responsible for the activation of fibroblasts and their production and deposition of collagen. Using an established in vivo animal model of proliferative scarring, the effects of this cytokine, specifically the isoform TGF-beta2, on these scars were examined. Proliferative scar specimens were implanted into athymic, asplenic nude rats and isolated in sandwich island flaps based on the superficial inferior epigastric pedicle. After establishment of the transferred flap, the scars were injected with varying doses of TGF-beta2 or vehicle for 5 consecutive days and then again on days 10, 15, and 20. The specimens were measured weekly during the period of dosing, and a biopsy was acquired on days 30 and 60. Fibroblasts from the explanted biopsies and the original scars were grown in cell culture, and cell proliferation studies were performed and the results compared. There was a dose response to TGF-beta2, with 200 ng showing the greatest effect. From the original scar specimens, keloid scars demonstrated the greatest cell proliferation kinetics--significantly faster than nonburn and burn hypertrophic scars. After treatment with TGF-beta2, both keloids and burn hypertrophic scars showed an increase in their cell proliferation kinetics compared with vehicle alone. This was not demonstrated with the nonburn hypertrophic scars. Elevated levels of TGF-beta2 are a major contributing factor to the process of proliferative scars, but because nonburn hypertrophic scars do not result in an equally increased response to this cytokine, a truly causative role for this cytokine cannot be promulgated. Rather, it is the combination of the proliferative scar fibroblasts' abnormal response to TGF-beta2 stimulation and elevated levels of this cytokine that controls more accurately the process of keloid and burn hypertrophic scar formation.

Animals↗
PubMed1998

Gene expression profiling of alveolar rhabdomyosarcoma with cDNA microarrays.

Several forms of human sarcoma, lymphoma, and leukemia are characterized by somatically acquired chromosome translocations that result in fusion genes that encode chimeric transcription factors with oncogenic properties. We have used cDNA microarrays containing 1238 cDNAs to investigate the gene expression profile of a group of seven alveolar rhabdomyosarcoma (ARMS) cell lines characterized by the presence of the PAX3-FKHR fusion gene. Using the method of multidimensional scaling to represent the relationships among the cell lines in two-dimensional Euclidean space, we determined that ARMS cells show a consistent pattern of gene expression, which allows the cells to be clustered together. By searching across the seven ARMS cell lines, we found that 37 of 1238 genes were most consistently expressed in ARMS relative to a reference cell line. Only three of these genes have been previously reported to be expressed in ARMS. Among these 37 were genes related to both primary (PAX3-FKHR) and secondary (CDK4) genetic alterations in ARMS. These results in ARMS demonstrate the potential of cDNA microarray technology to elucidate tumor-specific gene expression profiles in human cancers.

Chromosomes, Human, Pair 13↗
PubMed1998

p53 mutation with frequent novel condons but not a mutator phenotype in BRCA1- and BRCA2-associated breast tumours.

The status of p53 was investigated in breast tumours arising in germ-line carriers of mutant alleles of BRCA1 and BRCA2 and in a control series of sporadic breast tumours. p53 expression was detected in 20/26 (77%) BRCA1-, 10/22 (45%) BRCA2-associated and 25/72 (35%) grade-matched sporadic tumours. Analysis of p53 sequence revealed that the gene was mutant in 33/50 (66%) BRCA-associated tumours, whereas 7/20 (35%) sporadic grade-matched tumours contained p53 mutation (P<0.05). A number of the mutations detected in the BRCA-associated tumours have not been previously described in human cancer databases, whilst others occur extremely rarely. Analysis of additional genes, p16INK4, Ki-ras and beta-globin revealed absence or very low incidence of mutations, suggesting that the higher frequency of p53 mutation in the BRCA-associated tumours does not reflect a generalized increase in susceptibility to the acquisition of somatic mutation. Furthermore, absence of frameshift mutations in the polypurine tracts present in the coding sequence of the TGF beta type II receptor (TGF beta IIR) and Bax implies that loss of function of BRCA1 or BRCA2 does not confer a mutator phenotype such as that found in tumours with microsatellite instability (MSI). p21Waf1 was expressed in BRCA-associated tumours regardless of p53 status and, furthermore, some tumours expressing wild-type p53 did not express detectable p21Waf1. These data do not support, therefore, the simple model based on studies of BRCA-/- embryos, in which mutation of p53 in BRCA-associated tumours results in loss of p21Waf1 expression and deregulated proliferation. Rather, they imply that proliferation of such tumours will be subject to multiple mechanisms of growth regulation.

BRCA1 Protein↗
PubMed1998

Mesial temporal lobe measurements on magnetic resonance imaging scans.

Changes in the mesial temporal lobe, particularly in the hippocampus, amygdala, and entorhinal cortex, are reported to occur in several neuropsychiatric conditions. Neuroimaging provides a non-invasive means of studying these changes. We present a method for reliably measuring the hippocampus, amygdala, and entorhinal cortex on MRI. The advantages of our method include high reliability, the use of orthogonal views in delineating boundaries and circumscription of measurement such that no tissue of any one anatomic structure is included in the measurement of another structure.

Amygdala↗
PubMed1998

Expression of v-src in mammary epithelial cells induces transcription via STAT3.

Transgenic mouse models of mammary tumorigenesis and analyses of human breast tumour samples have indicated a role for Src proteins in the tumorigenic process. The downstream effectors of Src function in mammary epithelial cells are less well understood. STAT proteins constitute a family of transcription factors whose activation by cytokine and non-cytokine receptors leads to tyrosine phosphorylation, dimerization and translocation from the cytoplasm to the nucleus. In the nucleus they activate the transcription of specific genes by binding to consensus DNA elements. STATs 1 and 3 can be activated by both cytokine and non-cytokine receptors, and bind as homodimers or heterodimers to viral simian sarcoma virus (sis)-inducible elements such as that found in the c-fos promoter. Here we report that one of the downstream effectors of Src function in mammary epithelial cells is STAT3. We demonstrate that v-src expression in mammary epithelial cells induces Tyr-705 phosphorylation, nuclear translocation and DNA binding of STAT3. Furthermore, we demonstrate that v-src can induce STAT3-dependent transcription. These observations are the first direct evidence that v-src can regulate transcription through the activation of STAT proteins, and add a further level of complexity to the understanding of the mode of action of v-src.

Biological Transport↗
PubMed1998

Geographic variations in the composition of ivory of the African elephant (Loxodonta africana).

Tracing the source of origin of illegal ivory will contribute to the identification of poorly managed game parks and facilitate steps taken to prevent the African elephant from becoming extinct. This study was aimed at establishing a database on the composition of ivory obtained from elephant sanctuary areas in Southern Africa. Fragments of elephant ivory from seven geographically distinct areas in South Africa, Namibia and Botswana were analysed for inorganic and organic content. A total of 20 elements was detected in the inorganic fraction of ivory, some in concentrations as low as 0.25 microg/g. The concentrations of calcium, phosphate, magnesium, fluoride, cobalt and zinc showed statistically significant differences (p < 0.007) between ivory obtained from different regions. Analyses of the organic fraction identified 17 amino acids. Ivory from arid regions showed significantly lower proline plus hydroxyproline content and under-hydroxylation of lysine residues. This study indicates that chemical analyses of ivory could be beneficial in tracing the source of illegal ivory.

Africa, Southern↗