Biomedical subjects
Publications and source records attributed to K O'Rourke.
Physician-assisted suicide is a form of euthanasia. As such, it is contrary to one's ethical responsibility to self, community, and God. Religious tradition, medical tradition, and modern psychology attest that physician-assisted suicide does not solve any human problem.
We have expressed human alpha and beta-globin cDNA clones from separate, synthetic galactose-regulated hybrid promoters contained on a single plasmid in Saccharomyces cerevisiae. Co-expression of the alpha and beta-globin chains in S. cerevisiae results in the assembly of these proteins into soluble tetrameric hemoglobin that accumulates to 3-5 percent of the total cell protein. Endogenously produced heme is incorporated into the tetramer and the protein produced is functionally and structurally indistinguishable from human Ao hemoglobin. This expression system has been used to produce both wild type hemoglobin and a low O2-affinity hemoglobin mutant that has oxygen binding and dissociation characteristics similar to human whole blood. The yeast expression system we describe may be suitable for the production of a recombinant hemoglobin based blood substitute as well as for detailed structure-activity studies of human hemoglobin.
In recent years the Ontario government has been concerned that the proportion of public expenditures devoted to health care is at an all-time high. In addition, the media have devoted considerable attention to specific incidents that may represent inadequate funding of hospital services. To shed light on the debate on health care expenditures we analysed the trend in expenditures of Ontario's hospital sector in the 1980s in terms of the amount of inputs (e.g., labour) used to produce hospital services (e.g., a patient-day or admission) and after adjustment for general inflation. As in the 1970s the number of inputs grew relatively slowly during the 1980s. Inputs per patient-day grew at an annual rate of 0.46% and inputs per admission at an annual rate of 2.4%. Cost increases were largely accounted for by hospital wage increases; this could have been due to Ontario's rapidly expanding economy. These findings indicate that Ontario has continued to be successful in containing the number of inputs used in the hospital sector. However, after two decades of substantial success with publicly acceptable cost control, the government faces increased scrutiny as the media and the public focus attention on several areas of perceived inadequate funding in health care services.
Cytokines such as tumor necrosis factor alpha (TNF) profoundly affect endothelial cell function, promoting for example interaction with leukocytes and inducing a procoagulant phenotype. Changes of this nature are likely to be central to the proinflammatory effects of TNF. In order to elucidate molecular mechanisms by which TNF alters endothelial cell function we utilized differential plaque hybridization to identify TNF-responsive genes. Forty TNF-inducible cDNAs were identified which on cross-hybridization were found to arise from six unique genes. DNA sequencing of these cDNAs revealed two encoded known cytokine-induced genes, endothelial leukocyte adhesion molecule 1 and neutrophil chemotactic factor. One of the cDNAs encodes a recently described monocyte-specific chemotactic factor not previously associated with endothelium. The production of a monocyte chemotaxin by cytokine-activated endothelium has important implications for understanding the role of the vessel wall in disease states such as atherosclerosis and may also in part explain the indirect angiogenic activity of TNF. The three other cDNAs are completely novel as judged by data bank searches of partial DNA sequences and remain unidentified. On exposure of endothelial cells to TNF there is a rapid and substantial increase in levels of mRNA encoding the six genes, which are further superinduced by cycloheximide. Thus these represent primary response genes as their induction does not depend on protein synthesis. Interleukin-1 beta and lipopolysaccharide are also potent inducers. Nuclear run-on studies revealed that in most cases induction by TNF is mediated largely at the transcriptional level.
A 17-year-old boy is brought involuntarily by his parents for admission to an adolescent unit after running away from home to join a cult. The adolescent is admitted to the hospital for a multidisciplinary assessment. Follow-up 6 months after discharge shows the family to have continued in family therapy with the patient remaining at home with no additional contact with the cult. The case raises important ethical and dynamic issues about whom the child psychiatrist serves--the parents, the adolescent, or society. Discussion by an ethicist, lawyer, and child and adolescent psychoanalyst follows the case presentation.
Meta-analytic methods were applied to review clinical trials published in English that have assessed the efficacy of parenteral nutrition for cirrhotic patients with acute hepatic encephalopathy. Modified amino acid solutions containing increased amounts of branched-chain amino acids were used as part of the treatment regimen in all studies. Pooled analysis of five randomized controlled studies showed a highly significant improvement in mental recovery (p less than 0.001) from high-grade encephalopathy over follow-up times varying from 5 to 14 days. The significance level of the treatment effect did not change when the analysis was repeated using alternative methods of counting and attributing events in these trials. Sharp differences in direction of treatment effect precluded pooling case fatality data. Two studies reported an increased risk of death in the treatment group. Two others showed a clear benefit from administration of parenteral nutrition: the aggregate relative risk reduction was 0.59 (95% confidence interval: 0.23-0.80, p = 0.002). Addition of unpublished data from a third positive study increased the relative risk reduction to 0.82 (p less than 0.0001), and the most conservative interpretation of the published data still yielded a significant reduction in mortality (p = 0.023). However, given the uncertainty about effects on mortality and short follow-up times in all studies, a confirmatory randomized controlled trial with longer follow-up periods is warranted.
In this article, we counter some criticism regarding the desirability of performing meta-analysis in clinical research. These criticisms, we argue, are based mainly on current difficulties in deriving firm conclusions based on meta-analysis, resulting from poor methodology and reporting of primary studies. This is not a fault of meta-analysis. In fact, with a better understanding of meta-analysis in the context of the full scientific research process, meta-analysis is seen as a key element for improving individual research efforts and their reporting in the literature. This in turn will further enhance the role of meta-analysis in helping clinicians and policy makers answer clinical questions.
Thrombospondin (TSP) is a homotrimeric extracellular glycoprotein with a subunit molecular mass of 140 kD. The subunits have a modular or domain-like structure and are held together by interchain disulphide bonds. A number of domains have been identified including those for the binding of collagen, fibrinogen, and heparin. Due to the trimeric form of the TSP molecule, the various domains are trivalent in nature and this contributes to the ability of TSP to mediate cell-substrate interactions. Indeed, TSP has recently been shown not only to promote cell adhesion but also to be intimately involved in cell growth and migration. The adhesive function of TSP is attributable to the "solid-phase" or matrix-bound form of the molecule. There is some evidence that the heparin-binding domain mediates incorporation of soluble TSP into the insoluble matrix form. The heparin-binding domain of TSP is a compact globular amino-terminal moiety that contains two clusters of basic amino acids and a single intrachain disulphide bond. To delineate the role of the heparin-binding domain in matrix assembly and to define further the precise region of interchain disulphide bonding that results in trimer formation, we have expressed deleted forms of the cDNA encoding TSP in SV-40-transformed. African green monkey kidney cells. The proteins synthesized from the various deleted TSP cDNAs were examined for (a) secretion into the culture medium and incorporation into the extracellular matrix; (b) binding to heparin-Sepharose; (c) immunoprecipitability by a conformation-specific monoclonal antibody; and (d) ability to form trimers. This analysis allowed us to draw the following conclusions. (a) A 218 amino acid NH2-terminal protein that preserves the intrachain disulphide bridge of the heparin-binding domain is capable of binding to heparin-Sepharose and incorporating into the extracellular matrix. (b) A shorter 164 amino acid NH2-terminal peptide that does not contain the intrachain disulphide bridge of the heparin-binding domain is neither able to bind to heparin-Sepharose nor able to incorporate into the extracellular matrix. (c) The region of interchain disulphide bridging necessary for trimer assembly resides within a cluster of seven cysteine residues immediately adjacent to the heparin-binding domain.
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Human squamous carcinoma cells attach and spread on thrombospondin (TSP)-coated culture dishes but exhibit significant variability among individual cell lines in their degree of responsiveness. Using a highly responsive squamous carcinoma line and a cell line which is much less responsive (as well as a human melanoma cell line which does not respond at all in the adhesion assay), we have examined binding of exogenous radiolabeled TSP. The cells which were the most responsive to TSP in the adhesion assay bound the greatest amount of radiolabeled ligand. Binding was time- and dose-dependent, saturable, inhibitable with excess unlabeled TSP, reversible, and specific. The less-responsive squamous carcinoma cells bound only 25-30% of the amount of TSP bound by the highly responsive cells while the nonresponsive melanoma cells bound less than 10% of the amount bound by the highly responsive squamous carcinoma cells. Our previous studies (J. Varani et al. (1986) Exp. Cell Res. 167, 376) have shown that the highly responsive squamous carcinoma cells also synthesized the greatest amount of TSP as indicated by biosynthetic labeling studies. The less-responsive squamous carcinoma cells were intermediate in synthetic activity and no synthetic activity was seen with the melanoma cells. These findings suggest that the amount of ligand bound may determine the degree of biological responsiveness and that endogenously synthesized TSP may be the source of that ligand.
Equine infectious anaemia virus is related by genome sequence homology to human immunodeficiency virus, caprine arthritis-encephalitis virus and visna virus. Failure of the host to mount a strong neutralizing response detectable in vitro or to eliminate persistent infection in vivo characterizes lentivirus infections in the natural host. In this study the specificities and neutralizing activity of antibodies induced during experimental infection with equine infectious anaemia virus were investigated using antiviral ELISA, radioimmunoprecipitation and neutralization assays. ELISA antibody titres of 10(5) to 10(6) were demonstrated in samples collected 30 and 60 days after infection. Immunoprecipitation titrations demonstrated that antibody titres to the glycoproteins gp90 and gp45 were 10 to 100 times higher than titres to the internal structural protein, p24. Low levels of neutralizing antibody appeared at 23 to 46 days post-infection. The presence of low levels of neutralizing activity in the presence of high levels of anti-glycoprotein activity suggests that the major immunogenic sites on the viral surface are not sensitive to neutralization.
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Human epidermal keratinocytes obtained from normal skin attached and spread on thrombospondin (TSP)-coated plastic dishes but failed to attach and spread on untreated plastic culture dishes or dishes coated with fibronectin or laminin. These cells produced minimal amounts of immunoreactive TSP. Keratinocytes established in culture on MCDB 153 medium and maintained for one to three passages in an undifferentiated state by continued cultivation in this low Ca2+-containing medium attached and spread on plastic dishes as well as on TSP-coated dishes. These cells also secreted significant amounts of TSP into the culture medium. When the keratinocytes were incubated for one day in MCDB 153 medium supplemented with high Ca2+ or in MEM (which also contains high Ca2+), there was decreased secretion of TSP into the culture medium concomitant with a reduction in attachment and spreading on plastic culture dishes. Proteolytic fragments of TSP were examined for stimulation of keratinocyte attachment and spreading. A 140-kd fragment produced by removal of the 25-kd heparin-binding domain had similar activity to the intact molecule while the 25-kd fragment was without effect. Further proteolytic treatment of the 140-kd fragment gave rise to a fragment consisting of 120 kd and 18-D moieties held together in disulphide linkage. This fragment did not support attachment or spreading. This study reveals that normal epidermal keratinocytes grown under conditions that maintain the undifferentiated state are able to produce TSP and utilize it as an attachment factor. When keratinocytes are grown under conditions that promote differentiation, ability to produce and utilize TSP is diminished. Since TSP is present at the dermal-epidermal junction and because TSP promotes keratinocyte attachment and spreading, this molecule may play an important role in maintaining normal growth of the basal cell layer and may also participate in reepithelialization during wound repair.
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We used a meta-analysis protocol to evaluate the results of 18 controlled trials that measured the effectiveness of perioperative total parenteral nutrition. The pooled results of 11 trials that were randomized or quasi-randomized showed trends suggesting that total parenteral nutrition reduced the risk for complications from major surgery (p = 0.21) and fatalities (p = 0.21). Consideration of these pooled estimates of the effectiveness of this intervention must be offset by the poor quality of the trials' methodologies and the iatrogenic complications (pneumothorax, septicemia) that occurred at a pooled rate of 0.067. Alternately, other design flaws, such as the failure to exclude patients who were not malnourished from the trials, may have limited the ability of these trials to show the effectiveness of total parenteral nutrition. The evidence available up to August 1986 shows that the routine use of perioperative total parenteral nutrition in unselected patients having major surgery is not justified; however, this intervention may be helpful in subgroups of these patients who are at high risk.
Meta-analysis is the process of combining study results that can be used to draw conclusions about therapeutic effectiveness or to plan new studies. We review important design and statistical issues of this process. The design issues include protocol development, objectives, literature search, publication bias, measures of study outcomes, and quality of the data. The statistical issues include consistency (homogeneity) of study outcomes, and techniques for pooling results from several studies. Guidelines are provided to assess the quality of meta-analyses based on our discussion of the design and statistical issues. Limitations and areas for further development of this approach are discussed; researchers should come to a general agreement on how to conduct meta-analysis. As an explicit strategy for summarizing results, meta-analysis may help clinicians and researchers better understand the findings of clinical studies.
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