Search PubMed⌕ Search

Biomedical subjects

P Walsh

Publications and source records attributed to P Walsh.

At least 73 records · Page 4Linked to original sources
PubMed1995

Macrophage colony-stimulating factor complementary DNA: a candidate for gene therapy in metastatic melanoma.

BACKGROUND: At present, there is no highly effective treatment for metastatic melanoma. Innovative approaches aimed at inducing a more effective immune response against tumors have shown promising results in animal models. One approach involves the genetic modification of tumor cells so that they produce cytokines that stimulate an immune response. PURPOSE: The aim of this study was to determine the effectiveness of cytokine gene therapy for metastatic melanoma in a murine melanoma model. METHODS: B16F10 murine melanoma cells, which readily metastasize to the lungs, were transduced with a retroviral vector containing genes encoding neomycin resistance and human macrophage colony-stimulating factor (M-CSF). The presence of M-CSF messenger RNA in transduced cells was examined by coupled reverse transcription and polymerase chain reaction. Concentrations of soluble M-CSF in cell culture supernatants were determined by enzyme-linked immunosorbent assays (ELISAs). A clonal cell line, designated N+/CSF+, that expressed and secreted M-CSF was identified. Another clonal cell line, designated N+/CSF-, did not secrete M-CSF at levels detectable by ELISA. B16F10, N+/CSF-, and N+/CSF+ cells, individually or in combination, were injected intravenously or subcutaneously into C57BL/6 mice; we then evaluated the tumorigenicity and metastatic behavior of the cells, as well as the immune responses and survival of the mice. The immune responses assayed were the cytotoxic T lymphocyte (CTL) and peritoneal exudate cell (PEC) tumoricidal activities. RESULTS: Injection of B16F10 cells into the tail vein of C57BL/6 mice led to the establishment of lung metastases by week 2 and death by week 8. Injection of the N+/CSF+ or N+/CSF- cells led to the establishment of lung metastases that were detected at 2 and 3 weeks, respectively; however, these metastatic lesions were eliminated, and the animals had survival rates similar to those of the noninjected control mice. Injection of mice with a mixture of B16F10 and N+/CSF- cells resulted in the development of metastatic disease and 0% survival at 8 weeks, whereas mice that had been given an injection of a mixture of B16F10 and N+/CSF+ cells had an 80% survival rate at 8 weeks and survived at least two times longer (P = .007). The CTL and PEC tumoricidal activities in animals given an injection of N+/CSF+ cells suggested that monocytes and lymphocytes were responsible for the observed antitumor response. CONCLUSION: These findings suggest that the expression of M-CSF by genetically modified melanoma cells caused an effective antitumor immune response in host C57BL/6 mice and, thus, prolonged survival over that observed in the control mice.

Animals↗
PubMed1995

Isolation of genomic DNA from the earthworm species Eisenia fetida.

Our interest in detecting genotoxic exposure in earthworms led us to isolate high quality DNA from the Eisenia fetida species. For that, we compared a modification of the conventional phenol-chloroform extraction procedure, usually referred to as the Maniatis procedure, to two commercially available kits reportedly eliminating multiple partitions in phenol and chloroform, namely the Qiagen and Nucleon protocols. From the 260 nm optical density values, the commercial kits extracts hinted toward higher DNA recovery with those procedures. However, the 260/280 nm ratios indicated that the quality of the DNA isolated with the modified Maniatis procedure was purer than that isolated with the commercial kits, the latter being most probably contaminated by proteins and/or RNA. The Maniatis procedure was slightly modified by the introduction of a potassium acetate step for protein precipitation and by shortening the proteinase K treatment from 12-18 h to only 2 h. The higher quality of the DNA isolated by phenol-chloroform extraction was confirmed by quantification with the fluorescent 3,5-diaminobenzoic acid assay. Preliminary results suggest that the modified Maniatis procedure herein described is not only applicable for DNA adducts studies using 32P-postlabelling techniques but is also suitable for DNA extraction from other earthworm species such as Lumbricus terrestris.

Animals↗
PubMed1995

Cross-national comparisons of ageing mothers of adults with intellectual disabilities.

Ageing (55+ years) mothers of adults with intellectual disabilities in the Republic of Ireland, Northern Ireland and the United States were compared with respect to three general issues. Firstly, to what extent do the adults in these three countries differ in their level of reliance on their mothers? Secondly, do the mothers differ in the extent to which they have made plans for the future care of their son or daughter with intellectual disabilities? Thirdly, do the mothers differ in physical, social and psychological well-being? These cross-national comparisons were undertaken to examine the extent to which lifelong caregiving has either a common influence on mothers across national boundaries, or, alternatively, whether the cultural context exerts a unique influence on mothers in each country. Findings supported the latter explanation, even when background characteristics were statistically controlled.

Adult↗
PubMed1995

Subcellular localization and biochemical properties of the enzymes of carbamoyl phosphate and urea synthesis in the batrachoidid fishes Opsanus beta, Opsanus tau and Porichthys notatus

The subcellular localization and biochemical properties of the enzymes of carbamoyl phosphate and urea synthesis were examined in three representatives of fishes of the family Batrachoididae, the gulf toadfish (Opsanus beta), the oyster toadfish (Opsanus tau) and the plainfin midshipman (Porichthys notatus). The primary objective of the study was to compare the biochemical characteristics of these fishes, which represent a range between ammoniotelism and ureotelism (O. beta being facultatively ureotelic), with previous patterns observed for an ammoniotelic teleost (Micropterus salmoides, the largemouth bass) and an obligate ureogenic elasmobranch (Squalus acanthias, the dogfish shark). The present study documents the expression of mitochondrial carbamoyl phosphate synthetase (CPSase) III and cytosolic CPSase II (and its associated enzymes of pyrimidine synthesis, dihydro-orotase and aspartate carbamoyltransferase) in the livers of all three batrachoidid species. Both mitochondrial and cytosolic activities of arginase were present in the livers of all three species, as were cytosolic glutamine synthetase and argininosuccinate synthetase and lyase. However, O. beta also showed mitochondrial glutamine synthetase activity and higher total hepatic levels of glutamine synthetase than either O. tau or P. notatus. Taken together, these observations confirm that the arrangement of these enzymes in the batrachoidid fishes has greater similarity to that of M. salmoides than to that of S. acanthias. However, differences within the family appear to coincide with the different nitrogen excretion strategies. O. tau and P. notatus are primarily ammoniotelic and most closely resemble the ammoniotelic M. salmoides, whereas ureotelism in O. beta is correlated with the presence of a mitochondrial glutamine synthetase and the ability to induce higher total glutamine synthetase activities than O. tau or P. notatus. Additionally, isolated mitochondria from O. beta were able to generate citrulline from glutamine, whereas those from O. tau were not. Also in contrast to S. acanthias, glutamine synthetase activities in the mitochondria of O. beta are consistently lower than those of CPSase III. This and other kinetic observations lend support to the hypothesis that glutamine synthetase may be an important regulatory control point in determining rates of ureogenesis in O. beta.

Journal Article↗
PubMed1995

Effects of feeding and confinement on nitrogen metabolism and excretion in the gulf toadfish Opsanus beta

In order to elucidate further the cues for, and the biochemical mechanisms of, the transition to ureogenesis in the gulf toadfish Opsanus beta, experiments on the effects of feeding (i.e. nitrogen loading) were carried out. Baseline nitrogen excretion rates were first measured on solitary toadfish in large water volumes (i.e. unconfined conditions). These nitrogen excretion rates were higher, and had a higher proportion as ammonia (61 %), than previously published 'control' measurements. Feeding of unconfined toadfish elevated total nitrogen excretion approximately threefold, with little change in the proportion of urea versus ammonia. During the first 24 h of confinement of unfed toadfish, absolute levels of urea excretion remained constant while ammonia excretion rates fell to near zero, so that toadfish became 90 % ureotelic. When fed prior to confinement, urea excretion rates remained constant for the first 24 h, and the bulk of the nitrogen was excreted as ammonia (80 %); excretion of the excess dietary nitrogen took up to 48 h to complete. If pre-adapted to confinement and then fed, toadfish excreted only about 55 % of their nitrogenous waste as ammonia, and excretion of excess dietary nitrogen was completed by 24 h. Elevations of hepatic glutamine synthetase (GNS) activities accompanied confinement and were shown to be almost exclusively in the cytosolic compartment and to be correlated with a decrease in the ratio of hepatic levels of glutamate:glutamine. These GNS activity increases also appear to account in part for the decrease in the percentage of ammoniotely in toadfish under conditions of nitrogen loading after confinement. However, additional means of regulating total nitrogen excretion (e.g. changes in protein turnover rates) and the degree of ureogenesis versus ammoniogenesis (e.g. N-acetylglutamate stimulation of carbamoylphosphate synthetase) must be postulated to account fully for changes in nitrogen excretion rates and activation of ureogenesis under some circumstances.

Journal Article↗
PubMed1995

Pulsatile urea excretion in the ureagenic toadfish Opsanus beta: an analysis of rates and routes

This study focused on the rates and routes of urea-N and ammonia-N excretion in the ureagenic toadfish and on the possibility that urea-N excretion occurs in pulses. Experimental approaches included the following: confinement in small individual containers with automated hourly sampling of water to follow temporal excretion patterns; divided chambers to separate excretion from the anterior and posterior parts of the fish; collection of urine and rectal fluid via chronic indwelling catheters; and gavage with [14C]-labelled polyethylene glycol 4000 to detect regurgitation of gastrointestinal fluids. When a standardized 'crowding' pre-treatment was employed to induce ureotelic behaviour, the fish exhibited significant elevations in the activity of glutamine synthetase in liver, kidney and gills, elevated plasma and bile urea-N levels, but unchanged ammonia-N and urea-N levels in most other body fluids. Unencumbered ureotelic fish confined in small containers excreted 82 % of their waste-N as urea-N and 18 % as ammonia-N; almost all (94 %) of this urea-N excretion occurred in a single pulse of less than 3 h duration about once every 24 h. This daily pulse did not occur by regurgitation of gut fluids, by excretion through prominent pores behind the pectoral fins or by discharge of rectal fluid or urine. Intestinal and urinary excretion accounted for less than 10 % of whole-body urea-N excretion and a negligible fraction of ammonia-N excretion. Pulsatile urea-N excretion occurred at the head end across the gills and/or body surface. Ammonia-N excretion, which was not pulsatile, also occurred largely through the head end. However, once the toadfish had been placed in divided chambers, urea-N excretion became continuous rather than pulsatile, and ammonia-N excretion increased greatly. A severe stress response was indicated by high levels of plasma cortisol, and the skin, which lacks scales, became a significant route of both ammonia-N and urea-N excretion. We speculate that the normal adaptive significance is that ureotelism facilitates cryptic behaviour, allowing the toadfish to virtually eliminate N-waste excretion during long periods while it remains sheltered in burrows. However, during severe stress, the effects of extremely high cortisol levels overwhelm the ammonia and urea retention mechanisms, and both substances leak across the general body surface.

Journal Article↗
PubMed1994

Effect of finasteride on prostate-specific antigen density.

OBJECTIVE: Prostate-specific antigen density (PSAD) has been proposed as a diagnostic marker for prostate cancer. Because treatment with finasteride may affect PSAD differently in men with BPH compared with men with BPH plus prostate cancer, we evaluated the diagnostic utility of PSAD in men treated with finasteride for twelve months. METHODS: Data for this analysis were obtained from 895 men with BPH enrolled in a twelve-month placebo-controlled North American study. Prostate volume was measured by magnetic resonance imaging and PSA was measured by the Hybritech immunoradiometric assay. RESULTS: Treatment with finasteride for twelve months increased the positive predictive value of PSAD for identifying the presence of prostate cancer from 14 percent to 30 percent. Using PSA values alone with a cutoff of 10 ng/mL at baseline and 5 ng/mL after twelve months of treatment, similar specificity and sensitivity could be achieved. CONCLUSION: The data suggest that adjustment of PSA values during treatment with finasteride can be used to maintain the diagnostic accuracy for prostate cancer while PSAD can be used to provide additional reassurance without requiring adjustment for treatment.

Adult↗
PubMed1994

UREA PRODUCTION, ACID-BASE REGULATION AND THEIR INTERACTIONS IN THE LAKE MAGADI TILAPIA, A UNIQUE TELEOST ADAPTED TO A HIGHLY ALKALINE ENVIRONMENT

The Lake Magadi tilapia, Oreochromis alcalicus grahami, thrives in highly alkaline geothermal springs and pools surrounding Lake Magadi, Kenya (control pH=9.9, CCO2=173 mmol l-1), has a functional hepatic ornithine­urea cycle (OUC) and excretes all nitrogenous waste as urea-N at variable rates (JUrea) related to O2 consumption (M·O2). The mean value of JUrea/M·O2 (N/O2=0.183) was high for fish but below the theoretical maximum (approximately 0.27) for 100 % aerobic respiration of protein, so an exogenous source of substrates is not required to explain the observed JUrea. JUrea was insensitive to thiourea. Urea excretion occurred largely (80 %) through the gills, but urea-N was also present in bile and urine. Control blood pHe, pHi and [HCO3-] (approximately 8.1, 7.6 and 15 mmol l-1, respectively, at approximately 32°C) were extremely high. When fish were exposed to lake water titrated with HCl and aerated to remove CO2, N/O2 progressively declined. At a lake water pH of 7.05 and CCO2 of 0 mmol l-1, N/O2 was reduced by 80 % and an intense metabolic acidosis occurred (pHe=7.04, [HCO3-]=1.5 mmol l-1). Restoration of control water pH 9.9 at a CCO2 of 0 mmol l-1 resulted in intermediate levels of N/O2 and internal acid­base status. Additional experiments confirmed that urea production was inhibited by low pHe, was dependent on blood [HCO3-] with a Km of 3.06 mmol l-1 and was insensitive to acetazolamide. While metabolic acidosis clearly inhibited OUC ureagenesis, the system appeared to be saturated with HCO3- under control conditions so that additional basic equivalent loading would not stimulate ureagenesis. Urea production in the Lake Magadi tilapia does not appear to remove exogenous HCO3- or to play a role in normal acid­base regulation.

Journal Article↗
PubMed1994

EFFECTS OF CONFINEMENT/CROWDING ON UREOGENESIS IN THE GULF TOADFISH OPSANUS BETA

In order to elucidate the cues for, and the biochemical mechanisms of, the transition to ureogenesis in the gulf toadfish Opsanus beta, experiments on the effects of confinement/crowding were carried out. Confinement of toadfish to small volumes of water initiated a switch to nearly complete reliance on ureogenesis for nitrogen excretion within 24­48 h. Further experiments suggested that this switch was probably due to the physical confinement per se, rather than to a measurable build-up of ammonia in the water. However, the possibility of the response being triggered by a very low concentration of a pheromone-like substance was not excluded by our experimental design. The activities of several enzymes of ureogenesis, ornithine­citrulline transcarbamoylase, aspartate amino transferase and glutamine synthetase, increased in the liver of toadfish during confinement. Notably, glutamine synthetase activity increased almost fourfold within 24 h, and this increase preceded increases in urea excretion. A number of plasma and liver amino acid concentrations changed during confinement: there were declines in plasma asparagine, glutamate and glycine levels and an increase in plasma valine, as well as a decline in liver alanine and an increase in liver arginine concentrations. Liver glutamine was not detectable. When the amino acid data are taken together with the enzyme activity changes, it appears that the switch to ureogenesis occurs primarily upstream of the ornithine­urea cycle, at the level of supply of nitrogen to the pathway. The results are discussed in the context of the habitat of toadfish.

Journal Article↗
PubMed1994

UREA TRANSPORT BY HEPATOCYTES AND RED BLOOD CELLS OF SELECTED ELASMOBRANCH AND TELEOST FISHES

Although urea transport is receiving increased attention in mammalian systems, very little is known about urea transport in fish tissues. This study examined mechanisms of urea transport in red blood cells and hepatocytes from the lesser spotted dogfish (Scyliorhinus canicula), Atlantic stingray (Dasyatis sabina), turbot (Scopthalmus maximus), redfish (Scianops ocellatus), gulf toadfish (Opsanus beta) and oyster toadfish (Opsanus tau). Urea appeared to be passively distributed in both tissues (i.e. there was no difference between plasma and tissue urea concentrations). Additionally, a number of in vitro experiments examining [14C]urea flux were performed. In red blood cells from all species except redfish, urea transport occurred via simple passive diffusion, but redfish red blood cells showed a small (25 %) phloretin-sensitive uptake component. In hepatocytes of the two elasmobranch species (dogfish and stingray), urea efflux was also by simple passive diffusion. However, urea efflux in toadfish (both O. beta and O. tau) hepatocytes exhibited a marked phloretin-sensitivity, and O. beta hepatocytes were used in further experiments with other inhibitors and treatments. Urea transport in O. beta had a relatively high specificity for urea compared with the urea analogues acetamide, thiourea and N-methylurea, was unaffected by phloridzin and extracellular Na+ removal, and was not inhibited by physiological levels of glucose (0.5­10 mmol l-1). A phloretin-sensitive glucose transport, that was not inhibited by physiological levels of urea, was discovered in O. beta hepatocytes. The results are discussed in terms of patterns of species distribution and similarities between urea and glucose transport.

Journal Article↗
PubMed1993

A potential role for superantigens in the pathogenesis of psoriasis.

Psoriasis is a complex inflammatory skin disease in which local vascular changes, T-cell activation, abnormal keratinocyte proliferation and differentiation, and neutrophil activation all contribute to the ongoing disease process. Because of recent interest in T-cell activation as a trigger for psoriatic lesions, we hypothesized that psoriasis may be triggered by superantigens, e.g., toxins of microbial origin that stimulate T cells expressing particular T-cell receptor (TCR) beta chain variable (V beta) gene segments. Lesional skin biopsies and peripheral blood from two patients with acute exacerbations of their psoriasis that appeared to be triggered by infection were analyzed for TCR V beta gene expression using monoclonal antibodies directed against V beta 5.1, 5.2, 6.7, 8.1, and 12. Skin biopsies from both patients demonstrated a different pattern of V beta expansion that correspond to the V beta pattern expected to be induced by the type of superantigen expressed during the infection. In contrast, using immunofluorescence and flow cytometry, peripheral blood T cells from these patients did not demonstrate any expansion of the 5 V beta subsets studied. These observations support the hypothesis that local activation of cutaneous T cells in psoriasis may be caused by a superantigen and provides a new direction for investigating the pathogenesis of this complex and fascinating skin disorder.

Adult↗