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Biomedical subjects

P Clarke

Publications and source records attributed to P Clarke.

At least 73 records · Page 4Linked to original sources
PubMed2000

Human T lymphocyte genetic modification with naked DNA.

Endowing T lymphocytes with novel functional attributes by genetic modification is under development for a broad range of clinical cellular immunotherapy applications. To circumvent many of the limitations associated with viral vector systems, a plasmid-based electroporation system that reliably generates G418-resistant primary human T lymphocyte clones was developed. TCR alpha/beta+ CD4+CD8-, and CD4-CD8+ T lymphocyte clones can be routinely isolated from OKT3-stimulated peripheral blood mononuclear cells electroporated with linear plasmid DNA in a limiting dilution drug selection format. Fluorescence in situ hybridization (FISH) studies performed on T cell metaphase spreads using a probe specific for plasmid sequence demonstrated a single FISH signal doublet that varied in chromosomal location from clone to clone. Southern blot analysis using a Neo-specific probe verified chromosomal integration of plasmid vector at a single site. Band intensity quantitation of blots developed with a zeta-specific probe capable of annealing to both endogenous TCR-zeta and the introduced chimeric zeta sequence demonstrated that integrated plasmid was present at a single copy number. Expression levels of the CD20-specific chimeric immunoreceptor construct from a CMV immediate/early promoter present in the plasmid vector varied widely from clone to clone but remained stable during ex vivo expansion to cell numbers in excess of 10(10). This T lymphocyte genetic modification strategy is currently being piloted in a FDA-sanctioned adoptive therapy trial for recurrent lymphoma.

CD4-Positive T-Lymphocytes↗
PubMed2000

More or less equal? Comparing Australian income-related inequality in self-reported health with other industrialised countries.

OBJECTIVE: To measure the distribution of self-reported health by income in order to compare the level of health inequality in Australia with other industrialized countries. METHOD: Using data from the two National Health Surveys undertaken in 1989-90 and 1995, concentration indexes were calculated to quantify the distribution of self-reported health by equivalent income. The concentration index for Australia was compared with those reported for nine industrialized countries in Europe and North America. RESULTS: The estimated income-related concentration indexes were -0.1172 in 1989-90 and -0.1094 in 1995. CONCLUSION: The level of health inequality is not significantly different from the US or the UK, but significantly greater than seven other European nations. IMPLICATIONS: Australia has significant income related health inequalities and the distribution of health appears to be more unequal than in many other industrialized nations. There is a need to further investigate and quantify those features of the anglophone societies that set them apart from some other industrialized nations.

Adolescent↗
PubMed2000

Reovirus-induced apoptosis is mediated by TRAIL.

Members of the tumor necrosis factor (TNF) receptor superfamily and their activating ligands transmit apoptotic signals in a variety of systems. We now show that the binding of TNF-related, apoptosis-inducing ligand (TRAIL) to its cellular receptors DR5 (TRAILR2) and DR4 (TRAILR1) mediates reovirus-induced apoptosis. Anti-TRAIL antibody and soluble TRAIL receptors block reovirus-induced apoptosis by preventing TRAIL-receptor binding. In addition, reovirus induces both TRAIL release and an increase in the expression of DR5 and DR4 in infected cells. Reovirus-induced apoptosis is also blocked following inhibition of the death receptor-associated, apoptosis-inducing molecules FADD (for FAS-associated death domain) and caspase 8. We propose that reovirus infection promotes apoptosis via the expression of DR5 and the release of TRAIL from infected cells. Virus-induced regulation of the TRAIL apoptotic pathway defines a novel mechanism for virus-induced apoptosis.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Reovirus-induced apoptosis requires activation of transcription factor NF-kappaB.

Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-kappaB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-kappaB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-kappaB DNA-binding activity correlated with the onset of NF-kappaB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-kappaB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-kappaB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IkappaB that constitutively represses NF-kappaB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-kappaB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-kappaB signaling pathway. These findings indicate that NF-kappaB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.

Animals↗
PubMed1999

Demonstration of new sites of expression of the CCK-B/gastrin receptor in pancreatic acinar AR42J cells using immunoelectron microscopy.

The CCK-B/gastrin receptor has been characterised in both normal and tumour tissues. Endocytosis of the CCK-B/gastrin receptor has recently been demonstrated and this has similarly been described for other peptide receptors. In addition, ligand and ligand-receptor translocation to the nucleus has been demonstrated for other peptides. The aim of this study was to identify the sites of expression of the CCK-B/gastrin receptor in the known CCK-B/gastrin receptor bearing pancreatic acinar AR42J cells. The specificity of the CCK-B/gastrin receptor antibody (alpha-CCKBR-Ser antibody) was demonstrated by inhibition ELISA studies, radioligand inhibition studies and immunofluorescence binding studies on AR42J cells. Western blotting and immunogold electron microscopy techniques were used to identify the receptor in AR42J cell preparations. The affinity purified alpha-CCKBR-Ser antibody was shown to be specific for the CCK-B/gastrin receptor. The receptor was expressed on the cell membrane, in the cytoplasm and within the nucleus. Isoforms of the receptor protein identified in extra-nuclear and nuclear extracts ranged in molecular weight from 58 to 66 kDa. We conclude that the CCK-B/gastrin receptor is not only expressed on the cell membrane, but also in the cytoplasm and nucleus of AR42J pancreatic acinar cells.

Animals↗
PubMed1999

Screening of sulphonamides in egg using gas chromatography-mass-selective detection and liquid chromatography-mass spectrometry.

A rapid extraction and clean-up procedure for sulphonamide antibiotics in eggs suitable for both GC-MSD and LC-MS end determinations has been developed. The drugs were extracted using acetonitrile, acidified using acetic acid and cleaned-up using cation- and anion-exchange. For determination by GC-MSD, extracts were derivatised with diazomethane followed by pentafluoropropionic acid anhydride. For LC-MS extracts were taken up in water and used directly. The methodology developed was validated at the 100 and 25 microg kg(-1) levels, equivalent to the MRL and one quarter of the MRL. Results for the GC-MS procedure were quantitated against deuterated sulphadiazine with relative recoveries ranging from 54% for sulphachloropyridazine to 135.5% for sulphamethazine. Recoveries for the LC-MS procedure ranged from 33% for sulphaguanidine to 92% for sulphamethazine and sulphadimethoxine.

Chromatography, Liquid↗
PubMed1999

Bcl-2 regulates a caspase-3/caspase-2 apoptotic cascade in cytosolic extracts.

Apoptosis is accompanied by the activation of a number of apoptotic proteases (caspases) which selectively cleave specific cellular substrates. Caspases themselves are zymogens which are activated by proteolysis. It is widely believed that 'initiator' caspases are recruited to and activated within apoptotic signalling complexes, and then cleave and activate downstream 'effector' caspases. While activation of the effector caspase, caspase-3, has indeed been observed as distal to activation of several different initiator caspases, evidence for a further downstream proteolytic cascade is limited. In particular, there is little evidence that cellular levels of caspase-3 that are activated via one pathway are sufficient to cleave and activate other initiator caspases. To address this issue, the ability of caspase-3, activated upon addition to cytosolic extracts of cytochrome c, to cause cleavage of caspase-2 was investigated. It was demonstrated that cleavage of caspase-2 follows, and is dependent upon, activation of caspase-3. Moreover, the activation of both caspases was inhibited by Bcl-2. Together, these data indicate that Bcl-2 can protect cells from apoptosis by acting at a point downstream from release of mitochondrial cytochrome c, thereby preventing a caspase-3 dependent proteolytic cascade.

Apoptosis↗
PubMed1999

Antibodies raised by gastrimmune inhibit the spontaneous metastasis of a human colorectal tumour, AP5LV.

Both precursor forms of gastrin and mature amidated gastrin peptides can enhance proliferation of colorectal tumours and may regulate growth in an autocrine manner. The purpose of this study was to evaluate the effect of neutralization of precursor and amidated gastrin on primary and secondary in vivo growth of a human colorectal tumour. The human colorectal cell line, AP5LV, when injected into the muscle layer of the abdominal wall of severe combined immunodeficient (SCID) mice, grows as a well-vascularized primary tumour and metastasis to the lung. AP5LV expressed the precursor gastrin forms; progastrin and glycine-extended gastrin and gastrin/CCKB receptors, as assessed by immunocytochemistry. Gastrimmune is a gastrin immunogen in which the amino terminus of the gastrin-17 molecule is linked to diphtheria toxoid and induces antibodies which neutralise the amidated and glycine-extended forms of gastrin-17. Rabbit antiserum, raised against Gastrimmune, was administered intravenously into SCID mice bearing AP5LV tumours. Control animals were treated with antiserum raised against diphtheria toxoid only. Antibodies raised against Gastrimmune significantly limited the growth of primary AP5LV tumours, as assessed by median cross-sectional area (controls = 244 mm2; antibody-treated = 179 mm2; P = 0.033). In addition Gastrimmune-induced antiserum limited the growth of lung metastasis as assessed by nodule number (controls = 3.5; antibody-treated = 1.0; P = 0.0001) and nodule cross-sectional as assessed by image analysis (controls = 11.9 mm2; antibody-treated = 3.75 mm2; P = 0.0064). In conclusion in vivo neutralization of gastrin forms, which may potentially be fueling growth by an autocrine pathway, inhibited both primary growth and, to a greater degree, lung metastasis of a human colorectal tumour cell line. Immunization against tumour-associated gastrin forms may provide an effective therapy for advanced colorectal cancer.

Animals↗
PubMed1999

Intermittent short courses of cyclosporin (Neoral(R)) for psoriasis unresponsive to topical therapy: a 1-year multicentre, randomized study. The PISCES Study Group.

We performed a 1-year study to determine whether intermittent short courses of the microemulsion formulation of cyclosporin (Neoral) could effectively control plaque psoriasis and whether tapering or abrupt cessation of cyclosporin therapy would influence time to relapse. Four hundred patients with plaque psoriasis were included in this open, multicentre, randomized study. All patients commenced cyclosporin at a dose of 2.5 mg/kg daily. Cyclosporin dosage could be increased to a maximum of 5 mg/kg daily. Treatment was continued until clearance of psoriasis or for a maximum of 12 weeks. Patients were then randomly assigned either to stop cyclosporin abruptly or to have the dose reduced by 1 mg/kg daily each week until cessation. On relapse, patients were given another course of cyclosporin. Patients were followed for at least 1 year, during which they could receive as many treatment courses as necessary. The number of patients who required one, two, three and four treatment courses was 400, 259, 117 and 26, respectively. The median time to relapse after the end of the first treatment period was 109 days in the group of patients randomized to stop cyclosporin abruptly and 113 days in patients randomized to taper off cyclosporin (P = 0.038). More than 30% of patients had not relapsed 6 months after having stopped treatment. After each treatment course, the Kaplan-Meier probability of achieving 75% or more reduction in disease area by day 84 of treatment was 83%, 76%, 73% and 66%, respectively. Mean serum creatinine concentration and blood pressure did not show any clinically significant changes over time. Our results show that intermittent short-course therapy with Neoral, when used in conjunction with topical therapy, is well tolerated and provides effective control of plaque psoriasis for 1 year. Tapering off cyclosporin on treatment cessation induces a slight delay in psoriasis relapse.

Adolescent↗
PubMed1999

Comparison of the immunogenicity and safety of two 17D yellow fever vaccines.

As part of the clinical validation process of a new working seed of a licensed yellow fever vaccine (new working seed PV26, Stamaril; Pasteur Mérieux Connaught, Lyon, France), the immunogenicity and safety of two batches of this vaccine (PM-YF) were compared with those of another commercially available vaccine (Arilvax; Evans Medical-Wellcome, Liverpool, United Kingdom) in 211 healthy adults. While the geometric mean titer values at days 10-14 and day 28 after vaccination were higher in the PM-YF group, the vaccines provided equivalent seroprotection (titers > or = 1/10) one month after a single vaccine dose (100% PM-YF versus 99% W-YF; P = 0.001, by one-sided equivalence test). Both vaccines were safe. There were no serious local or systemic reactions reported, nor any clinically significant hepatic function abnormalities associated with the use of either vaccine. These two 17D yellow fever vaccines from different European vaccine manufacturers were highly immunogenic and safe, and provided equivalent seroprotection.

Adolescent↗
PubMed1998

Mice transgenic for human carcinoembryonic antigen as a model for immunotherapy.

Mice transgenic for the human carcinoembryonic antigen (CEA) gene were prepared for use as a preclinical model for immunotherapy. A 32.6-kb fragment containing the complete human CEA gene and flanking sequences was isolated from a genomic cosmid clone and used to produce transgenic C57BL/6 mice. A homozygous line was established that was designated C57BL/6J-TgN(CEAGe)18FJP. Southern blot analysis showed that this line contained intact copies of the cosmid clone, with approximately 19 integrated copies at one chromosomal location. A mouse-human chimeric anti-CEA monoclonal antibody was used to examine CEA expression by immunohistochemical staining of frozen tissue sections. In the cecum and colon, approximately 20% of the luminal epithelial cells had strong cytoplasmic staining, whereas occasional glands showed intense staining. CEA was also expressed in gastric foveolar cells, whereas small intestine villi had only a few (<1%) positive cells. CEA was not found by immunohistochemistry in other tissues of the digestive tract, nor was it found in a wide range of other tissues or organs. Concordance in results was obtained between immunohistochemistry and analysis of tissue extracts by enzyme immunoassay. The lone exception was the testis, which was positive only by enzyme immunoassay. Expression of human CEA was not observed in tissues derived from nontransgenic mice. The fecal content of CEA in transgenic mice was approximately 100-fold less than that observed for humans. Circulating CEA was not detected. A CEA-transfected syngeneic murine colon carcinoma cell line, MC-38, was prepared that had stable expression of CEA in vitro and in vivo. The molecular size of CEA produced by CEA-transfected MC-38 cells and by the colon of transgenic mice was similar to that obtained with CEA purified from human colon tumors. Anti-CEA antibody appeared in nontransgenic but not transgenic mice bearing transfected MC-38 tumors. These findings demonstrate that CEA distribution and its properties in tissues of mice transgenic for the human CEA gene are similar to that observed in human tissues. As in humans, immune responsiveness to CEA, as reflected by antibody formation, was not detectable in transgenic mice bearing CEA-positive tumors. Thus, CEA transgenic mice may serve as a useful model for studying the efficacy and safety of various immunotherapy strategies directed at this tumor self-antigen.

Adenocarcinoma↗
PubMed1998

Toxoplasma gondii-infected cells are resistant to multiple inducers of apoptosis.

Infection with certain intracellular pathogens, including viruses and bacteria, may induce host cell apoptosis. On the other hand, infection with some viruses inhibits apoptosis. Complex protozoan parasites, including Toxoplasma gondii and members of Plasmodium, Leishmania, and Microsporidia, are also obligate intracellular pathogens, yet relatively little is known regarding their subversion of host cell functions. We now report that cells infected with T. gondii are resistant to multiple inducers of apoptosis, including Fas-dependent and Fas-independent CTL-mediated cytotoxicity, IL-2 deprivation, gamma irradiation, UV irradiation, and the calcium ionophore beauvericin. Inhibition of such a broad array of apoptosis inducers suggests that a mechanism common to many, or perhaps all, apoptotic pathways is involved. The inhibitory activity requires live intracellular parasite and ongoing protein synthesis. Despite T. gondii-mediated inhibition of DNA fragmentation, infected cells can still be lysed by CTL.

Animals↗
PubMed1998

A 10-year review of high-pressure injection injuries to the hand.

High-pressure injection injuries to the hand are uncommon, but often result in poor outcome or even amputation. We report a review of the 28 cases treated surgically in our department over the last 10 years and have examined the factors leading to increased morbidity. The severity of these injuries was related to the nature of the injected material, involvement of the tendon sheath and proximal spread of the injected substance. All cases in which the digit was noted to be poorly perfused from the outset resulted in amputation. We conclude that early amputation should be considered in cases in which the affected digit is initially cool or poorly perfused.

Accidents, Occupational↗
PubMed1998

Gastric carcinoid expresses the gastrin autocrine pathway.

BACKGROUND: In gastric adenocarcinoma the gastrin autocrine-paracrine pathway is activated. As enterochromaffin-like (ECL) cells originate from the same stem as epithelial cells, the aim of this study was to determine if the gastrin autocrine pathway is present in gastric carcinoid. METHODS: Samples from ten patients with gastric carcinoid were assessed by immunocytochemistry using primary antibodies directed against gastrin precursors and the gastrin/cholecystokinin B receptor and detected using the avidin-biotin immunoperoxidase system. RESULTS: A high level of expression of precursor and mature gastrin peptides, together with the gastrin receptor, was seen in all carcinoids screened. CONCLUSION: In common with the glandular epithelium of the stomach the gastrin gene is activated during the neoplastic process in ECL cells. This finding may explain why some carcinoids do not regress after surgical procedures that lower serum gastrin. Antigastrin agents may be a useful treatment for carcinoid either in their own right or as an adjunct to surgery.

Adult↗
PubMed1998

General practitioner screening for melanoma: sensitivity, specificity, and effect of training.

OBJECTIVE: To measure the performance of trained and untrained general practitioners (GPs) in screening men and women aged 50 or more for melanomas. METHODS: GPs trained in melanoma diagnosis, untrained GPs, and skin cancer specialists examined groups of volunteers, each of which included a small number of subjects with prediagnosed suspicious pigmented lesions (SPLs) that were subsequently excised for histopathological examination. RESULTS: Trained and untrained GPs achieved mean sensitivities of 0.73 and 0.71, and mean predictive values of 0.40 and 0.37, respectively, for the detection of prediagnosed SPLs. When the SPLs had been excised and examined histopathologically, reanalysis showed mean sensitivities of 0.98 and 0.95, mean specificities of 0.52 and 0.49, and mean positive predictive values of 0.24 and 0.22 for the detection of subjects with melanomas by trained and untrained GPs respectively. Trained GPs were significantly better than untrained GPs at diagnosing as melanomas SPLs that subsequently proved to be melanomas (p = 0.04). CONCLUSIONS: GPs in this study achieved high sensitivities in screening older Australian men and women for melanomas, but at the cost of low specificities and positive predictive values. Training in melanoma diagnosis had no significant effect on sensitivity, specificity, and positive predictive value for screening. Data from the study were tested in a model of population screening for melanomas, and costs per life year saved for men aged 50-70 ranged from $A11,852 to $A40,259 depending upon the screening interval and whether the GPs excised the SPLs diagnosed, or referred all patients to skin cancer specialists; this would be as cost effective as cervical cancer screening.

Female↗
PubMed1997

The brief admission unit in emergency psychiatry.

The study evaluates a Brief Admission Unit for clients of an emergency service located within a comprehensive community psychiatric program. Eighty-five clients completed the Brief Symptom inventory and a structured interview. Substance abuse disorder (n = 29) and major depression (n = 24) were the most common Axis I diagnoses, of which 30 subjects had two or more. Sixty subjects had an Axis II diagnosis. Mean duration of admission was 3.9 days, compared with the average in other acute units of 11.5 days. At discharge, half the subjects were rated as moderately to greatly improved and client satisfaction was high. The unit was crucial to the psychiatric emergency service and had a key role in relieving pressure on beds elsewhere within the system.

Adolescent↗