Access to medical records for research purposes: varying perceptions across research ethics boards.

INTRODUCTION: Variation across research ethics boards (REBs) in conditions placed on access to medical records for research purposes raises concerns around negative impacts on research quality and on human subject protection, including privacy. AIM: To study variation in REB consent requirements for retrospective chart review and who may have access to the medical record for data abstraction. METHODS: Thirty 90-min face-to-face interviews were conducted with REB chairs and administrators affiliated with faculties of medicine in Canadian universities, using structured questions around a case study with open-ended responses. Interviews were recorded, transcribed and coded manually. RESULTS: Fourteen sites (47%) required individual patient consent for the study to proceed as proposed. Three (10%) indicated that their response would depend on how potentially identifying variables would be managed. Eleven sites (38%) did not require consent. Two (7%) suggested a notification and opt-out process. Most stated that consent would be required if identifiable information was being abstracted from the record. Among those not requiring consent, there was substantial variation in recognising that the abstracted information could potentially indirectly re-identify individuals. Concern over access to medical records by an outside individual was also associated with requirement for consent. Eighteen sites (60%) required full committee review. Sixteen (53%) allowed an external research assistant to abstract information from the health record. CONCLUSIONS: Large variation was found across sites in the requirement for consent for research involving access to medical records. REBs need training in best practices for protecting privacy and confidentiality in health research. A forum for REB chairs to confidentially share concerns and decisions about specific studies could also reduce variation in decisions.

The precursor pro-nerve growth factor is the predominant form of nerve growth factor in brain and is increased in Alzheimer's disease.

Nerve growth factor (NGF) is important for regulation, differentiation, and survival of peripheral and central nervous system neurons, including basal forebrain cholinergic neurons (BFCN) which degenerate in Alzheimer's disease (AD). Mature NGF protein is processed from a larger precursor, proNGF. We demonstrate that proNGF is the predominant form of NGF in mouse, rat, and human brain tissue, whereas little or no mature NGF is detected. Previous reports showed NGF protein, measured by ELISA, is increased in AD BFCN target regions such as hippocampus and cortex. Using Western blotting, we demonstrate a twofold increase in proNGF in AD parietal cortex compared to controls, indicating that it is this precursor form, proNGF, that accumulates in AD. This increase may reflect either a role for biologically active proNGF or posttranslational disturbances in NGF biosynthesis that decrease the processing of proNGF to mature NGF in AD.

Treatment of PC12 cells with nerve growth factor increases iron uptake.

Phaeochromocytoma PC12 cells treated with nerve growth factor (NGF) differentiate into a neuronal phenotype. Here we compare the uptake of transferrin-bound and non-transferrin-bound iron in NGF-treated (neuronal phenotype) and control (proliferating) PC12 cells. The non-transferrin-bound iron uptake was greater in the NGF-treated cells than in the control, independently of the uptake time, the iron-chelating agents used, the oxidation state of iron (Fe(2+) or Fe(3+)) and the iron concentration tested. The NGF-treated cells expressed L-type and N-type voltage-operated Ca(2+) channels. Nitrendipine (an L-type inhibitor) and possibly omega-conotoxin (an N-type inhibitor) inhibited the iron uptake by 20%. Thapsigargin inhibits the endoplasmic reticulum Ca(2+) pump and allowed Mn(2+) entry into cells. Preincubating PC12 cells with thapsigargin increased the iron uptake. The rate of transferrin-bound iron uptake was less than 1% of the non-transferrin-bound iron uptake and the maximum transferrin-bound iron uptake was also very low. We conclude that an increase in the iron uptake by multiple pathways accompanies the transition of PC12 cells from the proliferating to the neuronal phenotype.

Equipoise and the ethics of the Canadian Lung Volume Reduction Surgery Trial study: should there be a randomized, controlled trial to evaluate lung volume reduction surgery?

The physical improvement is so great following lung volume reduction surgery that there is growing opinion that a randomized, controlled trial is unnecessary. A randomized, controlled trial, it is argued, would deprive those patients randomly assigned to the nonsurgical treatment arm the 'benefit' of lung volume reduction surgery. Entering a trial in which one arm leads to a surgical intervention and the other to best medical management also poses a variety of ethical difficulties. If one is to be offered surgery, there must be perceived benefit because the physician has an obligation to offer the best possible treatment for his or her patient. If a patient agrees to have surgery, the expectation is that surgery would help. Thus, a patient randomly assigned to the medical arm of a trial may easily believe that he or she is being deprived of surgery that may help them. This paper illustrates this dilemma using the Canadian Lung Volume Reduction Surgery Trial. The authors discuss the concept of 'equipoise' in three dimensions, adding community equipoise to theoretical equipoise and clinical equipoise earlier described by Freedman. The paper concludes that the Canadian Lung Volume Reduction Surgery Trial needs to continue because of the clinical equipoise that drives it.

On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells.

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca(2+)-dependent and Ca(2+)-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca(2+) with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe(2+) to NTA with and without Ca(2+). We observed Ca(2+) activation of NTBI uptake in PC12 cells with the characteristics reported for other cells upon using 1 mm ascorbate and a constant ratio of total Fe(2+) to NTA with or without Ca(2+). However, Ca(2+) did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca(2+) and Fe(2+) by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe(2+), and total Ca(2+) concentrations). In solutions based on these constants and taking Ca(2+) chelation into account, Ca(2+) did not affect NTBI uptake over a range of free Fe(2+) concentrations. Thus, the Ca(2+) activation of NTBI uptake observed using the constant total Fe(2+) to NTA ratio was because of Ca(2+)-NTA chelation rather than an activation of the NTBI transporter itself. It is suggested that the previously reported Ca(2+) dependence of NTBI uptake be re-evaluated.

A descriptive study of healthcare ethics consultants in Canada: results of a national survey.

As part of a project to examine health care ethics consultation in Canada, we surveyed individuals who were considered by themselves or others to play a significant role in health care ethics consultation. Since one goal of the project was to examine the education and abilities necessary for consultants, we sought to determine the qualifications and skills currently possessed by persons considered to be ethics consultants. For the purposes of the questionnaire, 'health care ethics consultation' was defined broadly to include consultation on ethical issues in clinical care or in clinical research, ethics consultation to Clinical Ethics Committees, Research Ethics Committees, and policy formulation committees in health care institutions; 'clinical ethics work' was defined more broadly still to include, in addition to the above, ethics education, administration, research and writing on bioethics other than the above, and public speaking. Three hundred and fifty questionnaires were sent to individuals and institutions across Canada that were thought to have some involvement in health care ethics consultation. Two hundred and fifty-three questionnaires were returned for a response rate of 72%. This report presents initial findings of the study and attempts to provide a comprehensive overview of the current state of ethics consultation within Canada. The survey examines demographics, educational background, time spent on ethics, institutional affiliations, approaches to the role of consultation, research related issues, and attitudes toward certification. Of the 253 questionnaires returned, 162 were completed by individuals who indicated that they provided some kind of ethics consultation. Of these, 43 indicated that they spent 30% or more of their time in clinical ethics work. These individuals are quite heterogeneous in background, training and activities, and while the great majority of them are based in an academic setting (university or teaching hospital), many act as resources to community hospitals, long-term care facilities and other organizations. Finally, the survey found that respondents' views on the advisability of certification for those offering ethics consultation were split evenly between those in favour of and those against certification. This report serves, then, as a reference point for studying the roles, responsibilities, training and accreditation of ethics consultants in health care.

Expression and modulation of nerve growth factor in murine keratinocytes (PAM 212).

Nerve growth factor (NGF) is a polypeptide that is required for normal development and maintenance of the sympathetic and sensory nervous systems. Skin has been shown to contain relatively high amounts of NGF, which is in keeping with the finding that the quantity of NGF in a tissue is proportional to the extent of sympathetic innervation of that organ. Since the keratinocyte, a major cellular constituent of the skin, is known to produce other growth factors and cytokines, our experiments were designed to determine whether keratinocytes are a source of NGF. Keratinocyte-conditioned media from the keratinocyte cell line PAM 212 contained NGF-like activity, approximately 2-3 ng/ml, as detected by the neurite outgrowth assay. Freshly isolated BALB/c keratinocytes contained approximately 0.1 ng/ml. Using a cDNA probe directed against NGF, we demonstrated the presence of a 1.3-kb NGF mRNA in both PAM 212 and BALB/c keratinocytes. Since ultraviolet radiation (UV) is a potentially important modulating factor for cytokines in skin, we examined the effect of UV on NGF mRNA expression. Although UV initially inhibited the expression of keratinocyte NGF mRNA (4 h), by 24 h an induction of NGF mRNA was seen. The NGF signal could also be induced by phorbol esters. Thus, keratinocytes synthesize and express NGF, and its expression is modulated by UVB and phorbol esters.

Nerve growth factor enhances antigen and other secretagogue-induced histamine release from rat peritoneal mast cells in the absence of phosphatidylserine.

The effects of 2.5S nerve growth factor (NGF) and epidermal growth factor (EGF), isolated from mouse submaxillary glands, on histamine release from rat peritoneal mast cells (PMCs) were studied. In the absence of phosphatidylserine, NGF (1 ng/ml to 1 microgram/ml) did not cause histamine release from PMCs isolated from normal rats and those infected with the nematode Nippostrongylus brasiliensis. However, when PMCs (greater than 97% pure) were preincubated with NGF and then challenged with worm antigen (Ag), there was a marked enhancement of histamine release (approximately twofold with a maximum effect at 10 ng/ml of NGF [3.8 X 10(-10) mol/L]) compared with the release induced by Ag alone. EGF (1 ng/ml to 1 microgram/ml) neither produced histamine release from PMCs in the presence of phosphatidylserine nor enhanced Ag-induced histamine release. This suggests that NGF acts directly on PMCs by activation of cell-surface receptors. The early kinetics of Ag-induced histamine release were altered by NGF that increased the initial rate at 15 seconds but did not prolong the overall duration of histamine release. Simultaneous addition of Ag and NGF did not cause enhanced histamine release; thus, some preincubation time with NGF (5 minutes or less) was required for the activation of PMCs. Moreover, after PMCs were activated by NGF, that state persisted for 1 hour, even when unbound NGF was removed by washing, and thereafter subsided gradually. Further studies revealed that NGF enhanced histamine release induced by concanavalin A, compound 48/80, and ionophore A23187. These results suggest that NGF might be an important molecule in inflammatory responses through the regulation of mediator release from mast cells.

Nerve growth factor promotes human hemopoietic colony growth and differentiation.

Nerve growth factor (NGF) is a neurotropic polypeptide necessary for the survival and growth of some central neurons, as well as sensory afferent and sympathetic neurons. Much is now known of the structural and functional characteristics of NGF, whose gene has recently been cloned. Since it is synthesized in largest amounts by the male mouse submandibular gland, its role exclusively in nerve growth is questionable. NGF also causes histamine release from rat peritoneal mast cells in vitro, and we have shown elsewhere that it causes significant, dose-dependent, generalized mast cell proliferation in the rat in vivo when administered neonatally. Our experiments now indicate that NGF causes a significant stimulation of granulocyte colonies grown from human peripheral blood in standard hemopoietic methylcellulose assays. Further, NGF appears to act in a relatively selective fashion to induce the differentiation of eosinophils and basophils/mast cells. Depletion experiments show that the NGF effect may be T-cell dependent and that NGF augments the colony-stimulating effect of supernatants from the leukemic T-cell (Mo) line. The hemopoietic activity of NGF is blocked by polyclonal and monoclonal antibodies to NGF. We conclude that NGF may indirectly act as a local growth factor in tissues other than those of the nervous system by causing T cells to synthesize or secrete molecules with colony-stimulating activity. In view of the synthesis of NGF in tissue injury, the involvement of basophils/mast cells and eosinophils in allergic and other inflammatory processes, and the association of mast cells with fibrosis and tissue repair, we postulate that NGF plays an important biological role in a variety of repair processes.

Human basophilic cell differentiation promoted by 2.5S nerve growth factor.

In liquid cultures of human cord blood mononuclear cells, the activities of the 2.5S nerve growth factor (NGF) inducing basophil and eosinophil differentiation were investigated. Various concentrations of immunopurified 2.5S NGF derived from murine submaxillary glands were added to cultures with or without conditioned medium from a human T cell line (Mo-CM), which has previously been shown to produce activities stimulating granulocyte-macrophage colonies. Addition of NGF led to significant increases in differentiation of basophilic cells accompanied by histamine synthesis at 2 weeks in vitro; eosinophil differentiation was not increased in these cultures. In addition, NGF could be shown to amplify basophil differentiation induced by Mo-CM, and the activity of NGF inducing basophil differentiation was dependent on the presence of T lymphocytes. These results indicate that NGF stimulates T-lymphocyte-dependent basophilic cell differentiation from human cord blood progenitors and may in this way support differentiation of basophils or mast cells in vivo at sites of allergic tissue inflammation.

Structural and functional consequence of neonatal sympathectomy on the blood vessels of spontaneously hypertensive rats.

Neonatal sympathectomy of spontaneously hypertensive rats (SHR) and control Wistar-Kyoto rats (WKY) was performed by a combined treatment with antiserum to nerve growth factor and guanethidine during the first 4 weeks after birth. The development of hypertension was completely prevented in the treated SHR: at 28 to 30 weeks of age, systolic blood pressure of treated SHR was 139 +/- 2 mm Hg as compared with 195 +/- 8 mm Hg in untreated SHR. The extent of sympathectomy was verified by histofluorescence. Fluorescence histochemistry for catecholamine-containing nerves showed a complete absence of adrenergic nerves in the mesenteric arteries of treated rats. A supersensitivity to norepinephrine was exhibited by mesenteric arteries, anococcygeus muscle, and tail arteries from the treated SHR and WKY. In the mesenteric vascular bed, maximal response to norepinephrine was significantly reduced by sympathectomy. Sympathectomy also abolished the responses (e.g., generation of excitatory junctional potentials) of tail arteries to electrical stimulation of perivascular nerves. Morphometric measurements of three categories of mesenteric arteries showed that sympathectomy had no effect on the hypertrophic change of smooth muscle cells in the conducting vessels, but it prevented the hyperplastic changes of the muscle cells from reactive, muscular arteries and small resistance vessels. These results suggest that one of the primary roles of the overactive sympathetic nervous system in the development of hypertension in SHR is manifested through its trophic effect on the arteries of SHR. This trophic effect appears to cause a hyperplastic change in the smooth muscle cells in the reactive and resistance vessels, thereby contributing to the development of hypertension in older SHR.

The role of mast cells in inflammatory processes: evidence for nerve/mast cell interactions.

In the rat, there is considerable evidence of mast cell/nerve interaction both in the normal and infected intestine. Between 67 and 87% of all mast cells in the intestinal lamina propria of rats infected 22-35 days earlier with Nippostrongylus brasiliensis were touching nerves. These membrane contacts were between subepithelial mast cells and nonmyelinated nerves containing substance P, calcitonin gene-related peptide and neurone specific enolase. 2.5S nerve growth factor (NGF) has a significant enhancement effect on antigen-induced histamine release without addition of phosphatidylserine, and the in vivo administration of NGF to rats causes both connective tissue and mucosal mast cells to dramatically increase in number. All of these effects are both dose dependent and NGF specific, as evidenced by inhibition with anti-NGF. 2.5S NGF also causes in vitro increase of colonies in methylcellulose cultures of human peripheral blood. The effects of NGF in this system are synergistic with other T cell-derived growth factors and relatively specific for metachromatic cell growth. These observations support the conclusions that nerves and mast cells may constantly communicate and provide a structural and conceptual framework whereby the central nervous system may communicate with inflammatory events.

Determination of the molecular weight of neuronectin, a conditioned medium-derived, substrate-binding neurite-extension factor: comparison with laminin using radiation-inactivation analysis.

Neurite outgrowth from a wide variety of peripheral neurons is stimulated and may be directed in culture by a substrate-binding factor(s) derived from medium conditioned over numerous types of cells. This factor, or family of factors, which we shall call neuronectin by reason of its ability to serve as an attachment molecule for neurons, has been studied by target-size analysis using radiation inactivation. The radiation-inactivation method has the unique advantage of providing a means for determining the actual functional size of a biologically active molecule irrespective of its state of purification. By this method, the functional size of the major neurite outgrowth-promoting activity (neuronectin) from mouse heart cell conditioned medium has been found to be 350,000 Da. While neuronectin has not yet been purified, determination of the actual functional size provides a framework within which possible models must fit. Thus, although neurite outgrowth-promoting activity in this system is found to be associated with a complex containing laminin, fibronectin, heparan sulfate proteoglycan, and other extracellular matrix molecules, the total size of the functional molecule or molecular complex serving as the major source of activity is limited to 350,000 Da. Consequently, our results suggest that neuronectin from mouse heart cell-conditioned medium is different from laminin (Mr approximately 900,000), a molecule that also exhibits neurite-promoting activity. In addition to the difference in molecular size, neuronectin and laminin differ in that laminin, unlike neuronectin, gives rise to toxic or inhibitory products when exposed to high-energy radiation.

Nerve growth factor-independent development of embryonic mouse sympathetic neurons in dissociated cell culture.

Although ganglia from neonatal mouse sympathetic ganglia require nerve growth factor (NGF) for survival in culture, explanted sympathetic ganglia from early embryonic stages do not require added NGF for survival and growth. To determine whether the change in growth factor requirement is due to changes in the neurons themselves, to variations in neuronal populations, or to changes in nonneuronal cells, we examined the response to growth factors by dissociated sympathetic neurons at various stages of development. Results indicate that neurons from the 14-day gestational (E14) superior cervical ganglion (SCG) do not require NGF for initial survival and neurite extension, but do require the conditioned medium neurite extension factor, CMF. By 2 to 3 days thereafter, whether in vivo or in culture, most neurons have developed a requirement for NGF for survival in culture. During the same period, there is a concomitant increase in responsiveness to NGF alone as a trophic agent. Changes in response to NGF are not due to changes in NGF content of ganglia, to interactions in culture with nonneuronal cells, or to age-related differences in NGF requirements for maximum survival. The changes in growth factor requirements may be related to mechanisms regulating specificity of nerve-target connections.

Antiserum to a new neuronal growth factor: effects on neurite outgrowth.

A new neuronal growth factor (CMF) isolated from mouse heart-cell-conditioned medium stimulates morphologic and biochemical development of mouse embryo sympathetic neurons [Coughlin et al, 1981]. Further analysis of CMF by chromatographic and electrophoretic procedures has shown that under nondissociating conditions, CMF activity is associated with very high molecular weight material. All biological activity eluted within the included volume of a Sepharose CL-2B column in a molecular weight range corresponding to approximately 5 X 10(6) daltons. Similarly, electrophoresis showed no activity and very little protein entering 3% polyacrylamide gels, whereas both protein and activity migrated through 0.6% agarose-1.2% acrylamide composite gels. To further characterize the biological effects of CMF on normal neuronal development, antibodies to CMF were employed. Rabbits immunized against CMF developed high titres of antibodies with activity specifically directed against CMF (anti-CMF). Although anti-CMF inhibited nerve growth factor (NGF)-stimulated neurite outgrowth from the neonatal superior cervical ganglion, it did not inhibit the NGF-stimulated increase in tyrosine hydroxylase activity. Moreover, ganglia incubated for 3 days in the presence of anti-CMF were subsequently capable of producing neurites when washed and cultured in medium free of antiserum. Thus, anti-CMF specifically blocked neurite extension without causing cell death or irreversible damage in ganglionic explants. Our observations suggest, therefore, that CMF or antigenically similar material is a requirement for neurite extension.

Embryologic development of a mouse sympathetic ganglion in vivo and in vitro.

The morphologic and biochemical development of the embryonic mouse superior cervical ganglion was characterized in vivo and in tissue culture. From 13 days of gestation, when the superior cervical ganglion was first visible, to birth at 19 days, tyrosine hydroxylase [tyrosine 3-monooxygenase; L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] activity increased 100-fold in vivo. Explants of ganglia from 14-day embryos exhibited abundant neurite outgrowth in basal medium without added nerve growth factor (NGF), and increases in tyrosine hydroxylase activity paralleled that observed in vivo. Ganglia from 14-day embryos elaborated neurites and exhibited 3-fold increases in enzyme activity in vitro in the presence of antiserum to NGF (anti-NGF) or NGF + anti-NGF. In direct contrast, ganglia from 18-day fetuses failed to grow without added NGF or in medium containing anti-NGF or NGF + anti-NGF: virtually no axon outgrowth occurred and tyrosine hydroxylase activity decreased by half. These observations suggest that developmental regulatory mechanisms change radically during embryologic and fetal life of mammalian superior cervical ganglion.