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M M Kelly

Publications and source records attributed to M M Kelly.

At least 19 recordsLinked to original sources
PubMed2004

Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets.

Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry. Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20 C after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing. Compared to immediate processing, percentages of total lymphocytes and T-suppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used. In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects.

Adult↗
PubMed2003

A method to preserve sputum for delayed examination.

Examination of sputum cell counts is limited by the need to process samples within hours of expectoration. The validity and repeatability of a method to preserve sputum for delayed processing and examination were investigated in this study. Portions of selected sputum from 39 subjects were dispersed with dithiothreitol (routine method), or were fixed in a dithiothreitol-formaldehyde mixture for 48-72 h before dispersal with trypsin (preservation method). Total and differential cell counts using the two methods were compared; within-method repeatability of the preservation method was also examined. The intraclass correlation coefficient (ICC) for total cell counts and percentage of eosinophils, neutrophils and macrophages in sputum processed by the two methods was 0.89, 0.86, 0.91 and 0.90, respectively. Within-method repeatability (ICC) of the preservation method for the same cellular indices was 0.99, 0.94, 0.97 and 0.97, respectively. The interobserver repeatability for eosinophils, neutrophils and macrophages was 0.96, 0.97 and 0.97 using the preservation method, and 0.96, 0.99 and 0.99 using the routine method, respectively. This method of sputum preservation and dispersal is valid, reliable and convenient, and may be used for delayed processing and examination.

Cell Count↗
PubMed2001

Phospholipase C-gamma mediates the hydrolysis of phosphatidylinositol, but not of phosphatidylinositol 4,5-bisphoshate, in carbamylcholine-stimulated islets of langerhans.

In pancreatic islets the activation of phospholipase C (PLC) by the muscarinic receptor agonist carbamyolcholine (carbachol) results in the hydrolysis of both phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) and phosphatidylinositol (PtdIns). Here we tested the hypothesis that PtdIns hydrolysis is mediated by PLCgamma1, which is known to be regulated by activation of tyrosine kinases and PtdIns 3-kinase. PtdIns breakdown was more sensitive than that of PtdInsP(2) to the tyrosine kinase inhibitor, genistein. Conversely, the tyrosine phosphatase inhibitor, vanadate, alone promoted PtdIns hydrolysis and acted non-additively with carbachol. Vanadate did not stimulate PtdInsP(2) breakdown. Carbachol also stimulated a rapid (maximal at 1-2 min) tyrosine phosphorylation of several islet proteins, although not of PLCgamma1 itself. Two structurally unrelated inhibitors of PtdIns 3-kinase, wortmannin and LY294002, more effectively attenuated the hyrolysis of PtdIns compared with PtdInsP(2). Adenovirally mediated overexpression of PLCgamma1 significantly increased carbachol-stimulated PtdIns hydrolysis without affecting that of PtdInsP(2). Conversely overexpression of PLCbeta1 up-regulated the PtdInsP(2), but not PtdIns, response. These results indicate that the hydrolysis of PtdIns and PtdInsP(2) are independently regulated in pancreatic islets and that PLCgamma1 selectively mediates the breakdown of PtdIns. The activation mechanism of PLCgamma involves tyrosine phosphorylation (but not of PLCgamma directly) and PtdIns 3-kinase. Our findings point to a novel bifurcation of signaling pathways downstream of muscarinic receptors and suggest that hydrolysis of PtdIns and PtdInsP(2) might serve different physiological ends.

Adenoviridae↗
PubMed2001

A tale of three blind men on the proper subject matter of clinical science and practice: commentary on Plaud's behaviorism vs. Ilardi and Feldman's cognitive neuroscience.

Plaud (J Clin Psychol 57, 1089-1102, 1109-1111, 1119-1120) and Ilardi and Feldman (J Clin Psychol 57, 1067-1088, 1103-1107, 1113-1117, 1121-1124) argue for two very different approaches to clinical science and practice (i.e., behavior analysis and cognitive neuroscience, respectively). We comment on the assets and liabilities of both perspectives as presented and attempt to achieve some semblance of balance between the three protagonists embroiled in this current debate. The vision of clinical science we articulate is more ecumenical and evolutionary, rather than paradigmatic and revolutionary. As we see it, the problem clinical psychology faces is much larger than the authors let on; namely, how best to make clinical science meaningful and relevant to practitioners, consumers, the general public, and the behavioral health-care community. Clinical psychology's immediate internal problem is not pluralism with regard to subject matter, worldview, methodology, or school of thought, but pluralism in clinical psychologists' adherence to a scientific epistemology as the only legitimate form of clinical psychology. On this latter point, we still have a very long way to go.

Behaviorism↗
PubMed2001

Increased detection of interleukin-5 in sputum by addition of protease inhibitors.

The measurement of interleukin (IL)-5 in sputum is problematic, with interfering factors affecting immunoassay. The authors investigated whether sputum proteases could be acting as interfering factors by studying the effect of protease inhibitors (PI) on sputum IL-5 measurement. Induced sputa from 20 subjects with asthma were divided into aliquots, processed with and without protease inhibitors (in low and high concentrations) and the levels of IL-5 (spiked and endogenous) measured by enzyme immunoassay were compared. The concentration of sputum IL-5 was significantly increased by PI, with median (interquartile range) levels processed with no, low and high PI concentrations being 0 (0), 41.8 (75.6) and 66.1 (124.4) pg x mL(-1), respectively. There was also a significant increase in percentage recovery of spiked IL-5. Although high concentrations of PI reduced cell viability, there was no effect on total or differential cell counts and low concentrations of PI had no effect on cell counts or viability. Levels of endogenous interleukin-5 in sputum of asthmatic subjects can be significantly increased by the addition of protease inhibitors, and samples which would be regarded as negative for interleukin-5 without protease inhibitors may instead have considerable amounts of interleukin-5 detected.

Asthma↗
PubMed2000

Induced sputum: validity of fluid-phase IL-5 measurement.

BACKGROUND: IL-5 measurement in the fluid phase of induced sputum is considered to be important in the assessment of asthma, but the validity of these measurements is uncertain. OBJECTIVE: We investigated the validity of sputum IL-5 measurements through a series of spiking experiments and examined the effect of dithiothreitol (DTT) on these measurements. METHODS: Induced sputum from 26 asthmatic subjects was spiked with IL-5 and processed, and the percentage of recovery was measured by means of immunoassay. In 6 of the 26 samples the effect of adding albumin to the processing fluids was studied. In 3 separate samples radiolabeled IL-5 was added, and the recovery measured by means of gamma counting and immunoassay were compared. In addition, the effect of DTT on the immunoassay was examined. RESULTS: The mean +/- SD recovery of spiked IL-5 was 26.1% +/- 14.6% measured by means of immunoassay; adding albumin increased the recovery to 47.7% +/- 8.0% (P <.001). The mean recovery measured by means of gamma counting was 84.8% +/- 5.7% (P <.001); adding albumin had no effect on recovery. DTT had no significant effect on IL-5 measurement. CONCLUSION: The validity of IL-5 measurement by means of current methods is poor. The discrepancy in recovery as measured by gamma counting compared with immunoassay suggests that there is a problem with the recognition of IL-5 epitopes by immunoassay in induced sputum. This cannot be attributed to DTT but may be due to other interfering substances present in sputum, such as sputum proteases, soluble receptors, or autoantibodies.

Dithiothreitol↗
PubMed2000

C/EBPalpha inhibits cell growth via direct repression of E2F-DP-mediated transcription.

Using an inducible transcription system which allows the regulated expression of C/EBP isoforms in tissue culture cells, we have found that the ectopic expression of C/EBPalpha, at a level comparable to that found in normal liver tissue, has a pronounced antimitogenic effect in mouse L cells and NIH 3T3 cells. The inhibition of cell division by C/EBPalpha in mouse cells cannot be reversed by simian virus 40 T antigen, by oncogenic ras, or by adenovirus E1a protein. When expressed in thymidine kinase-deficient L cells or 3T3 cells, C/EBPalpha is detected in a protein complex which binds to the E2F binding sites found in the promoters of the genes for E2F-1 and dihydrofolate reductase (DHFR). Bacterially expressed C/EBPalpha has no affinity for these E2F sites, but when recombinant C/EBPalpha is added to nuclear extracts from mouse fibroblasts, a new E2F binding activity appears, which contains the C/EBPalpha protein. Using an E2F-DP1-responsive promoter linked to a reporter gene, it can be shown that C/EBPalpha directly inhibits the induction of this promoter by E2F-DP1 in transient-transfection assays. Furthermore, C/EBPalpha can be shown to inhibit the S-phase induction of the E2F and DHFR promoters in permanent cell lines. These findings delineate a straightforward mechanism for C/EBPalpha-mediated cell growth arrest through repression of E2F-DP-mediated S-phase transcription.

Animals↗
PubMed2000

Induced sputum examination: diagnosis of pulmonary involvement in Fabry's disease.

Fabry's disease is a rare inherited metabolic disorder caused by a deficiency in the enzyme alpha-galactosidase A. It can affect almost every organ, including the lungs. Confirmation of lung involvement has depended on invasive bronchial biopsy specimens or brushings to confirm the presence of typical lamellar inclusion bodies within bronchial epithelial cells. We report a patient with known Fabry's disease in whom these inclusion bodies were identified by examination of induced sputum.

Adult↗
PubMed2000

Enhanced epithelial gene transfer by modulation of tight junctions with sodium caprate.

The airway epithelium is resistant to infection by gene transfer vectors when infected from the luminal surface. One strategy for enhancing airway epithelial gene transfer is to modify paracellular permeability, thereby permitting the diffusion of vectors to the basolateral surface, where uptake receptors are expressed. We investigated the ability of a medium-chain fatty acid known to enhance drug absorption, sodium caprate (C10), to increase airway paracellular permeability in comparison with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Apical application of C10 decreased transepithelial resistance by > 90% within minutes, whereas EGTA required an hour or more to produce a similar effect. C10 increased mannitol and dextran permeability by sevenfold, as compared with a twofold increase produced by EGTA. A greater enhancement of adenoviral lacZ gene transfer was mediated by C10 (50-fold over controls) than by EGTA (10-fold over controls). This correlated with a significant enhancement of adenoviral CFTR-mediated correction of Cl(-) transport in polarized human airway epithelial (HAE) cells from cystic fibrosis (CF) patients. Confocal microscopy revealed a redistribution of claudin-1 following C10 but not EGTA treatment as a possible mechanism of gene-transfer enhancement by C10. These data suggest that C10 may be a better agent for enhancing gene transfer than is EGTA, and that this effect occurs through disruption of claudin-1.

Decanoic Acids↗
PubMed2000

Extraction of nuclear proteins with increased DNA binding activity.

We have developed a method for the rapid extraction of nuclear proteins from cultured cells. The ammonium sulfate method described here extracts larger quantities of proteins that retain DNA binding activity than the modified Dignam method and another popular method used for the extraction of transcription factors. The ammonium sulfate method is rapid and can be used to process a large number of samples for gel shift analysis.

Animals↗
PubMed1998

Glutathione S-transferase in hormonal carcinogenesis.

Treatment with testosterone propionate (TP) and diethylstilbestrol (DES) or TP and estradiol (E2) for 8-9 months causes development of leiomyosarcomas in the vas deferens or uterus of Golden Syrian hamsters at a frequency of 100%. In males, treatment with estrogens alone results in renal tumors, fatal within 6 months. No leiomyosarcomas have been detected after treatment with estrogens alone, perhaps due to this high mortality rate. In tissue culture, treatment with the glucocorticoid (GC) triamcinolone acetonide (TA) results in an increased expression of a mu-class glutathione S-transferase (GST) (hGSTYBX). We have characterized this induction as a secondary response, i.e. requiring new protein synthesis. Here we describe homologies to known transcription factor-binding sites in the hGSTYBX promoter which may be involved in the induction event. hGSTYBX is a member of a superfamily of detoxification enzymes, induced by genotoxic compounds and reactive oxygen species (ROS). We also describe the effects of several known inducers of other GST family members on hGSTYBX. While implicated in many chemotherapeutic-resistant tumors, GST enzymes have not yet been characterized as a functional agent in hormonal carcinogenesis. This latter possibility is the focus of our investigations. To study the effects of hormone treatment on GST levels in vivo, we have developed a polyclonal antibody to hGSTYBX, and conducted immunohistochemistry on tissues from control and treated animals. Treatment with TP and E2 causes a loss of hGSTYBX expression in the epithelium of the vas deferens. We hypothesize that the loss of this protective enzyme leaves the cells vulnerable to the genotoxic effects of estrogen or estrogenic metabolites.

Animals↗
PubMed1998

Decomplexification in critical illness and injury: relationship between heart rate variability, severity of illness, and outcome.

OBJECTIVES: To determine if decomplexification of heart rate dynamics occurs in critically ill and injured pediatric patients. We hypothesized that heart rate power spectra, a measure of heart rate dynamics, would inversely correlate with measures of severity of illness and outcome. DESIGN: A prospective clinical study. SETTING: A 12-bed pediatric intensive care unit (ICU) in a tertiary care children's hospital. PATIENTS: One hundred thirty-five consecutive pediatric ICU admissions. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: We compared heart rate power spectra with the Pediatric Risk of Mortality (PRISM) score, the Pediatric Cerebral Performance Category (PCPC), and the Pediatric Overall Performance Category (POPC). We found significant negative correlations between minimum low-frequency and high-frequency heart rate power spectral values recorded during ICU stay and the maximum PRISM score (log low-frequency heart rate power vs. PRISM, r2 = .293, p < .001; and log high-frequency heart rate power vs. PRISM, r2 = .243, p < .001) and outcome at ICU discharge (log low-frequency heart rate power vs. POPC or PCPC, r2 = .429, p < .001; and log high-frequency heart rate power vs. POPC or PCPC, r2 = .271, p < .001). CONCLUSIONS: Our data support the hypothesis that measures of heart rate power spectra are inversely related and negatively correlated to severity of illness and outcome in critically ill and injured children. The phenomenon of decomplexification of physiologic dynamics may have important clinical implications in critical illness and injury.

Adolescent↗