Biomedical subjects
Publications and source records attributed to A Doyle.
The increasing diversity of new cell cultures is seriously stretching the capabilities of traditional methods of identification. DNA fingerprinting is set to play an important role in increasing confidence in the authenticity of cultures in research and industry.
DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic "snap-shot" of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.
The technique of multilocus DNA fingerprinting has great potential for the authentication of animal cell cultures and in identification of cross-contamination. The Alec Jeffreys probes 33.6 and 33.15 were used as multilocus probes to demonstrate the consistent DNA fingerprint profiles in human peripheral blood and its derivative Epstein-Barr virus (EBV) transformed B-lymphoblastoid cultures maintained by repeated subculture for six months. However, fingerprint analysis of EBV transformed cultures generated from small numbers of cells showed that the majority (seven of eight cultures) had anomalous profiles. Some of these altered profiles shared common features not seen in the peripheral blood pattern. Analysis of seven murine hybridoma clones from a single fusion experiment revealed only two clones which could not be distinguished using probe 33.15. Further studies of master and distribution cell banks for eleven cell lines demonstrated consistent fingerprint profiles in all cases except one (U937). However, this cell line showed only minor differences in the master and distribution bank profiles. These data indicate that, while changes in fingerprint profile may be identified in exceptional instances, the multilocus fingerprinting method using probes 33.6 and 33.15 is a powerful and reliable tool in the quality control of animal cell cultures.
Studies have investigated associations between the Child Behavior Checklist (CBCL) and clinical diagnoses without assessing the impact of comorbidity on these results. This study evaluates associations between parental reports from the CBCL and a structured diagnostic interview in children with attention deficit disorder with hyperactivity (ADDH) stratified by the presence (ADDH+) or absence (ADDH-) of psychiatric comorbidity. Interview-defined ADDH children scored significantly worse on all scales of the CBCL compared with scores from interview-defined non-ill comparisons. However, these findings were accounted for by the subgroup of children with ADDH+. The results indicate a good correspondence between CBCL-based ratings and interview-defined diagnoses. These findings also suggest that the CBCL may be a good screening instrument, not only for ADDH but also for comorbid psychiatric disorders.
Reproducibility in animal cell culture technology requires careful preparation and characterisation of banks of cell cultures. The two standard techniques used in the quality control of such banks are isoenzyme analysis and cytogenetics which require complex and time-consuming procedures to enable cell line identification. However, DNA fingerprinting is potentially a more powerful method of analysis which can detect mutation and intra-species cross-contamination. At the European Collection of Animal Cell Cultures (ECACC) multilocus fingerprint analysis using probes 33.6 and 33.15 has been assessed in the quality control of cell banks. This method has confirmed consistency between master and working banks, has proven useful over a wide species range and can differentiate closely related cell lines. The key advantage of this method is its ability to detect cross-contamination by cell lines from a wide range of species using a straightforward and economical test. In addition the reproducibility of DNA fingerprints indicates their possible role in cell line authentication procedures which are important for patent and product licence applications.
A phase I clinical trial was conducted to evaluate the toxicology and biological activity of a new liposome-incorporated lipophilic disaccharide tripeptide, ImmTher. Twelve patients with advanced nonhematological malignant disease received 13 courses of therapy at dose levels of 200-1,200 micrograms/m2. A course of therapy consisted of once-weekly administration of the drug for 2-12 weeks. The major clinical toxicities observed were chills and hypotension. No renal, hepatic, cardiac, or hematological toxicity was observed. A small decrease in pulmonary diffusion capacity was observed. Biological activity was demonstrated by changes in plasma cytokine levels, changes in in vitro monocyte cytotoxicity, and by a decrease in tumor size. Improvement was observed in three of three patients with metastatic disease to the liver. Response in these three patients correlated with an increase in their tumor necrosis factor and neopterin levels compared with nonresponders. These preliminary indications of biological and clinical activity of a liposome-incorporated lipophilic disaccharide tripeptide in patients with advanced metastatic hepatic disease suggest a potential new therapeutic approach to this common problem.
1. The contribution of Li+ reabsorption in the loop of Henle to lithium clearance (CLi) and the possible mechanism(s) involved were assessed in healthy volunteers. Four mechanisms were considered: (a) passive reabsorption in the thin ascending limb, (b) solvent drag in the thin descending limb, (c) the Na+, K+, 2Cl- transporter in the thick ascending limb and (d) paracellular movement in the thick ascending limb. 2. Since alterations in the corticomedullary osmolal concentration gradient produced by fluid restriction (500 ml day-1) and subsequent water loading (15 ml kg-1) did not affect either CLi (28.5 +/- 2.1 vs. 28.2 +/- 1.9 ml min-1) or fractional lithium clearance (FELi; 23.5 +/- 2.0 vs. 23.0 +/- 1.9%), it is unlikely that substantial Li+ reabsorption occurs in the thin limbs by either passive movement or solvent drag. 3. Increasing plasma Li+ with unchanged plasma Na+ in salt-replete volunteers was associated with only small reductions in CLi (32.8 +/- 1.3 ml min-1, P less than 0.05) and FELi (27.3 +/- 1.8 vs. 25.3 +/- 2.0%, P less than 0.05). This suggests that substantial Li+ reabsorption on the Na+, K+, 2Cl- transporter does not occur. 4. Bumetanide increased FELi in salt-depleted (LS) and salt-replete (HS) volunteers and abolished the pre-diuretic difference in FELi between salt intakes (LS, 16.6 +/- 1.5 vs. 38.7 +/- 2.3%, P less than 0.001; HS, 30.1 +/- 1.5 vs. 40.5 +/- 2.0%, P less than 0.001). Changes in CPO4 and CHCO3 were not detected. Acetazolamide produced comparable increases in FELi (LS, 16.6 +/- 1.5 vs. 38.7 +/- 2.2%, P less than 0.001; HS, 30.1 +/- 1.5 vs. 43.1 +/- 2.4%, P less than 0.01); and CPO4 and CHCO3 were increased. When tubular flow to the loop of Henle was increased by acetazolamide, the bumetanide-induced increases in FELi were reduced (LS, 38.7 +/- 2.2 vs. 48.7 +/- 2.3%, P less than 0.001; HS, 43.1 +/- 2.4 vs. 48.1 +/- 2.6%, P less than 0.001). 5. These data are consistent with the view that (a) Li+ is reabsorbed by a bumetanide-sensitive mechanism in the loop of Henle, (b) approximately 20 and 10% of the filtered load, respectively, is reabsorbed in the loop in salt-depleted and salt-replete volunteers, (c) flow-dependent, voltage-driven paracellular movement in the thick ascending limb is the likely mechanism and (d) this mechanism could account for the difference in Li+ reabsorption between low and high salt intakes.
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The human CD4 positive cell lines JM, CCRF, CEM, U937, HL60 and THP-1 have been cleared of mycoplasma contamination and defined by DNA fingerprinting and cell surface phenotype marker analysis. These cells have been banked and are now available as a source of standardized cell lines for HIV related research.
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Aggregating platelets release serotonin, which induces contraction of most vascular smooth muscle by activation of S2-serotoninergic receptors. Serotonin released in the circulation may contribute to the increase in peripheral resistance of hypertension as the responsiveness of blood vessels from hypertensive animals and humans to the vasoconstrictor action of the monoamine is augmented. The data obtained with the new antihypertensive agent ketanserin may favor that interpretation. Ketanserin is a selective S2-serotoninergic antagonist with additional alpha 1-adrenergic blocking properties. In humans, it has a terminal half-life of 12 to 25 hours and is eliminated predominantly by the liver. The hemodynamic profile of ketanserin is that of a vasodilator drug with actions on both resistance and capacitance vessels. On short-term intravenous administration, it lowers blood pressure in hypertensive patients with minimal reflex changes in cardiovascular function. When given orally long term to hypertensive patients, ketanserin causes a sustained reduction in arterial blood pressure, comparable to that obtained with either beta-adrenergic blockers or diuretics. Several studies have shown a greater efficacy in older (greater than 60 years of age) than in younger patients independent of starting pressure. Side effects mainly consist of dizziness, somnolence, and dry mouth, but they are usually not severe. The mechanism underlying the antihypertensive effect of ketanserin is unclear. It cannot be attributed to either S2-serotoninergic or alpha 1-adrenergic blockade alone, but an interaction between the two effects appears to be required.
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Selected neuroendocrine tumors, such as small cell lung cancer (SCLC), and neuroblastoma express markedly diminished class I major histocompatibility complex (MHC) antigens (HLA framework and beta 2-microglobulin, beta 2m). Another neuroendocrine tumor, mid-gut carcinoid, also expresses reduced beta 2m antigen as demonstrated herein. Antigen expression is greatly enhanced on SCLC cell lines by in vitro exposure to interferon (IFN). To determine whether IFN mediates similar effects in vivo, we examined by immunoperoxidase staining beta 2m expression in paraffin-embedded tumor tissue sections obtained from 4 SCLC and 7 mid-gut carcinoid patients before and after receiving partially purified human leukocyte IFN-alpha therapy. Before IFN treatment, 3/4 SCLC tumors and 5/7 mid-gut carcinoids did not express beta 2m. By contrast, all tumors showed considerable expression of beta 2m after IFN therapy. Induction of class I antigens on tumor cells deficient in such expression may be one mechanism by which IFN exerts antitumor effects. We believe this is the first report of in vivo induction of class I MHC antigens in epithelial tumor cells in humans.
Two-hundred-and-twelve untreated subjects (mean age 52.3 +/- 0.8 years; 181 males and 31 females) with a diastolic blood pressure between 90 and 100 mmHg were recruited to the study. Subjects were seen fortnightly and, after 4 pre-diet visits, were randomized into a normal diet group (A, 55 subjects), a high-potassium diet group (B, 52 subjects receiving greater than 100 mmol K+/day) a reduced-sodium diet group (C, 52 subjects receiving 50-75 mmol Na+/day) or a high-potassium and low-sodium diet group (D, 53 subjects receiving same Na+ and K+ as groups B and C). Two-hundred subjects completed the diet phase of 12 weeks. Urine sodium fell to 86 +/- 7 mmol/day in group C and 73 +/- 6 mmol/day in group D, while daily potassium excretion rose to 96 +/- 5 mmol in group B and 87 +/- 4 mmol in group C. Systolic and diastolic blood pressure fell by 3.8 +/- 1.0 and 1.6 +/- 0.6 mmHg respectively in the normal diet group. The falls in systolic and diastolic blood pressures (mmHg) in the diet phase were 7.7 +/- 1.1 and 4.7 +/- 0.7 (B), 8.9 +/- 1.0 and 5.8 +/- 0.6 (C) and 7.9 +/- 0.9 and 4.2 +/- 0.7 (D). These falls were all greater than those in the control group on an intention-to-treat analysis (P less than 0.005) but did not differ from each other. Factorial analysis confirmed that the falls in pressure attributable to the low-sodium diet and high-potassium diet were not additive.(ABSTRACT TRUNCATED AT 250 WORDS)
We have found markedly deficient expression of the class I major histocompatibility antigens HLA-A,B,C and beta 2m on human small-cell lung cancer (SCLC) lines and fresh tumor samples. The deficit of HLA-A,B,C and beta 2-microglobulin (beta 2m) antigen expression was demonstrated with both radiobinding assays and indirect immunofluorescence assays. Immunoprecipitation of metabolically labeled cells with antibodies to class I antigens showed most SCLC lines to have synthesized almost no beta 2m and HLA-A,B,C proteins. Northern blot analysis, using human HLA-A,B, and beta 2m cDNA probes, showed that almost all SCLC lines tested had markedly decreased amounts of HLA and beta 2m mRNA, but both gene products could be induced with interferon treatment of SCLC lines. We conclude that human SCLC, in contrast to other lung cancer types, is characterized by greatly reduced transcription of HLA-A,B,C and beta 2m genes, which suggests the existence of a mechanism for evading the host immune response to the tumor and of an E1a-like product in this type of tumor cell.
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A polyclonal human lymphoblastoid cell line transformed in vitro with Epstein-Barr virus produced specific anti-Rhesus D antibody. It was repeatedly enriched by rosetting procedures and subsequently cloned. The cloning conditions employed a combination of mouse macrophage feeder layers, antimycoplasma agents, and low density passage. Formal evidence of monoclonality was obtained in one case which was of human IgG1 isotype and was secreted at the level of 15-20 micrograms/ml. All clones showed long-term stability in culture after 10 months of continuous passage. Both polyclonal and monoclonal cell lines possessed antigens characteristic of highly differentiated B cells, yet they also expressed Epstein-Barr virus nuclear antigen (EBNA). This study exemplifies a simple method for obtaining monoclonal antibody secreting plasmacytomas of human origin.