Ethical considerations in the collection of genetic data from critically ill patients: what do published studies reveal about potential directions for empirical ethics research?

Critical illness trials involving genetic data collection are increasingly commonplace and pose challenges not encountered in less acute settings, related in part to the precipitous, severe and incapacitating nature of the diseases involved. We performed a systematic literature review to understand the nature of such studies conducted to date, and to consider, from an ethical perspective, potential barriers to future investigations. We identified 79 trials enrolling 24 499 subjects. Median (interquartile range) number of participants per study was 263 (116.75-430.75). Of these individuals, 16 269 (66.4%) were Caucasian, 1327 (5.4%) were African American, 1707 (7.0%) were Asian Pacific Islanders and 139 (0.6%) were Latino. For 5020 participants (20.5%), ethnicity was not reported. Forty-eight studies (60.8%) recruited subjects from single centers and all studies examined a relatively small number of genetic markers. Technological advances have rendered it feasible to conduct clinical studies using high-density genome-wide scanning. It will be necessary for future critical illness trials using these approaches to be of greater scope and complexity than those so far reported. Empirical research into issues related to greater ethnic inclusivity, accuracy of substituted judgment and specimen stewardship may be essential for enabling the conduct of such trials.

Mammalian liver contains an activity which mimics bacterial chloramphenicol acetyltransferase.

Rat liver extracts contain an activity which mimics Escherichia coli chloramphenicol acetyltransferase (CAT); the latter is commonly used to report transcriptional activation of chimeric genes transfected into cultured cells. Although the activities are indistinguishable by the standard thin-layer chromatography assay, alternate methods can discriminate between them. The rat CAT-like activity appears to be an integral membrane protein. It was observed in the microsomal fraction of both liver and kidney. Similarly CAT-like activities were detected in mouse, rabbit and pig liver. In addition, liver homogenates which contain the CAT-like activity also contain a heat-labile inhibitor of (authentic) bacterial CAT.

Heat shock-induced cell death in murine microvascular endothelial cells depends on priming with tumor necrosis factor-alpha or interferon-gamma.

We previously reported that sequential exposure of cultured porcine aortic endothelial cells to lipopolysaccharide (LPS) and then to inducers of the heat shock response resulted in lethal injury by programmed cell death. In these present experiments, we evaluated the ability of other mediators of sepsis to prime endothelial cells for subsequent injury upon heat shock induction. We used SVEC4-10 microvascular endothelial cells, a cloned SV40-transformed cell line derived from LPS-resistant C3H/HeJ mice. When these endothelial cells were treated first with tumor necrosis factor-alpha followed by induction of the heat shock response with either heat or sodium arsenite (As), a standard chemical inducer of the heat shock response in vitro, a tumor necrosis factor dose-dependent cytotoxicity was observed, similar to that which we had seen previously in porcine endothelial cells primed with LPS. Subsequent experiments found that priming with interferon-gamma produced a similar dose-dependent toxicity upon heat shock with either heat or As. The reducing agents dithiothreitol and n-acetylcysteine, which we have previously shown to inhibit heat shock-induced programmed cell death in LPS-primed porcine endothelial cells, were also effective in protecting against heat shock-induced death following cytokine-priming in this microvascular cell line, suggesting that the intracellular signaling pathways of these priming agents are somewhere convergent. These data suggest that both exogenous and endogenous mediators of inflammation can prime endothelial cells for subsequent injury upon induction of the heat shock response, and are consistent with the emerging concept of redundancy in inflammatory signaling.

Shock at the millennium. I. Walter B. Cannon and Alfred Blalock.

Present management of shock derives, in part, from the classic investigations of Walter B. Cannon and Alfred Blalock. The intersections of their professional lives as recorded in the professional literature and in personal correspondence suggest that Blalock's pivotal studies of experimental shock were fueled, at least in part, by Cannon's inability to resolve the inconsistencies of the then-popular toxic theory of shock. Cannon appears to have substantially shaped Blalock's thought and work, initially as authority and competitor and later as colleague and friend. Blalock's experimental proof that injury precipitated obligatory locoregional fluid losses, the effects of which could be ameliorated by vigorous restoration of plasma volume, became a cornerstone of shock theory and therapy.

Shock at the millennium II. Walter B. Cannon and Lawrence J. Henderson.

Walter B. Cannon and Lawrence J. Henderson, students of shock in the early twentieth century, were contemporaries for four decades in the Harvard Department of Physiology. While their discoveries continue to have important clinical applications, both men established complementary methods of scientific investigation and description. Both men were inspired by Claude Bernard, hewing to his principle of the stability of the milieu intérieur. Cannon, the traditional experimentalist, employed a reductionist approach by holding constant the confounding variables of his experiments; in contrast, Henderson, the strategist of theoretical analysis, deduced patterns and relationships from less constrained models, focusing on complexity using mathematics and graphs. In delineation, Cannon described mechanisms; Henderson described the organization of systems. Cannon's emphasis on homeostasis with the identification of feedback arcs dominated shock research for the balance of the twentieth century. Henderson's perspective designating the importance of organization to those restorative mechanisms could well reemerge to dominate the twenty-first.

Thiol reducing agents modulate induced apoptosis in porcine endothelial cells.

When cultured porcine endothelial cells are exposed first to endotoxin (lipopolysaccharide (LPS)) followed by standard inducers of the heat shock response in vitro (heat or sodium arsenite), these cells aberrantly execute programmed cell death. This cell death is dependent upon two distinct events: the LPS-priming step and the heat shock-induced activation step. Prior work demonstrated that the LPS-priming step could be blocked by the prior application of cell-permeable hydroxyl radical scavengers, suggesting a role for this reactive oxygen species as an important intracellular signal mediating the first step. In these present experiments, we evaluated the potential role of reduction-oxidation mechanisms in the heat shock activation step. The thiol reducing agents reduced glutathione (GSH), n-acetylcysteine (NAC), and dithiothreitol (DTT) were evaluated for their ability to block programmed cell death in LPS-primed porcine aortic endothelial cells. Both DTT and NAC, agents that augment intracellular reduced glutathione levels, were protective against cell death when applied prior to heat shock induction with sodium arsenite (As) in endothelial cells treated previously with LPS. The less cell permeable agent GSH was not protective. Delayed application of DTT or NAC could block progression to cell death for up to 1.5 h after initiation of the heat shock response with As. These data show that heat shock-induced programmed cell death in LPS-primed endothelial cells can be arrested, at least in its early stages, by agents that augment or stabilize the reducing potential of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Cecal ligation and puncture (CLP) induces apoptosis in thymus, spleen, lung, and gut by an endotoxin and TNF-independent pathway.

Two challenges (intraperitoneal lipopolysaccharide (LPS) administration and cecal ligation and puncture (CLP)) and two strains of mice (LPS-normoresponder (C3H/HeN) and LPS-hyporesponder (C3H/HeJ)) were used to investigate pathways of cell injury. After intraperitoneal administration of LPS, endotoxin was absorbed into the bloodstream (HeN, 10.4 +/- 9.4 x 10(4) EU/mL; HeJ, 14.7 +/- 6.0 x 10(4) EU/mL), but as expected, only C3H/HeN mice produced serum tumor necrosis factor (TNF) (HeN, 2.5 +/- 2.0 x 10(3)pg/mL; HeJ, 87.0 +/- 38.7 pg/mL). Gel electrophoretic analysis of DNA extracted from six organs demonstrated the apoptotic "ladder" only in the thymus and only in the HeN mice. When the mice were challenged with CLP, both HeN and HeJ produced a small amount of serum TNF (HeN, 5.8 +/- 3.5 x 10(2) pg/mL; HeJ, 2.2 +/- 2.5 x 10(2) pg/mL) and both strains had very mild endotoxemia (HeN, 23.4 +/- 3.8 EU/mL; HeJ, 27.9 +/- 10.1 EU/mL). The DNA fragmentation pattern characteristic of apoptosis was observed not only in thymus but also in spleen, lung, and Peyer's patch of gut of both strains. This organ-specific pattern was more pronounced in the thymus of HeN mice; otherwise, the organ-specific patterns were similar for HeN and HeJ mice challenged by CLP but absent in those same organs when those same mice were challenged with LPS. The data suggest the existence not only of an endotoxin-driven activation for thymic apoptosis, but also of an endotoxin-independent, TNF-independent pathway activating widespread apoptosis in the murine CLP model of sepsis.

Myocardial heat shock gene expression in pigs is dependent on superoxide anion generated at reperfusion.

The heat shock response is a conserved response to cell injury. We sought to determine if ischemia alone versus events at reperfusion stimulated expression of the major heat shock protein (hsp-72) in a clinically relevant model of global myocardial ischemia in pigs. Pigs were placed on nonpulsatile cardiopulmonary bypass. Serial transmural cardiac biopsies were taken at baseline following 20 min of normothermic global ischemia (induced by crossclamping the aorta) and at 20, 40, and 60 min of reperfusion. Test animals received a bolus and subsequent aortic root infusion of superoxide dismutase (total 7,500 U/kg) beginning just prior to reperfusion. Hsp-72 mRNA abundance was estimated from Northern blots. We found that hsp-72 mRNA was not induced following 20 min of ischemia but accumulated to high levels within 20 min of reperfusion. Intravascular administration of superoxide dismutase at reperfusion eliminated hsp-72 mRNA induction. We conclude that in the postischemic myocardium, hsp-72 gene expression is dependent on superoxide anion generation at reperfusion. In this setting, hsp-72 gene expression may reflect a specific response to oxidative injury rather than a more general response to metabolic stress associated with ischemia.

Complex systems analysis: a tool for shock research.

For the past century, students of shock have focused research efforts to illuminate specific mechanisms that cause, or fail as a consequence of, circulatory collapse. Although clinical strategies aimed at supporting or restoring individual organ systems have proven effective, many patients succumb to more generalized multiple organ system failure. We suggest that general biological systems failure cannot be interpreted through reliance on reductionist science. We propose that complex systems analysis is an essential tool for shock research and we evaluate its application to genomic technologies.

Injection of iron compounds followed by induction of the stress response causes tissue injury and apoptosis.

To determine whether iron-laden tissue subsequently stimulated to produce the stress ("heat shock") response-sustained injury, hindlimbs of male ND4 mice were injected with iron salts, hemin, or hemoglobin. The stress response was induced with sodium arsenite or with heat. Ulcers appeared at the injection site. Tissues were analyzed by three distinct techniques-electron microscopy, TUNEL stain, and agarose gel electrophoresis of low molecular weight DNA-which collectively suggest that the tissue injury is, at least in part, the consequence of accelerated apoptosis. The data suggest that the toxicity of free iron is amplified by induction of the stress (heat shock) response to signal a programmed response. This model and mechanism may have implications in pathological processes ranging from the cutaneous wounds of venous stasis disease to the tissue failure of multiple organ dysfunction.

Endotoxin induces organ-specific endothelial cell injury.

Endothelial cell (EC) injury is observed in clinically important pathological processes, including bacterial endotoxemia. We hypothesized that such pathological processes may exhibit target organ heterogeneity due to organ-specific heterogeneity of endothelial cells. To test this hypothesis, endothelial cells of aorta (AO), pulmonary artery (PA), left ventricle (LV), and right ventricle (RV) were cultured from individual sheep and exposed to bacterial endotoxin. Marked heterogeneity in endotoxin-induced cytotoxicity was observed. AOEC were the most sensitive, followed by PAEC, LVEC, and RVEC. This cytotoxicity was manifested as programmed cell death (apoptosis). All cells were able to express both interleukin-6 and endothelin-1 (ET-1) transcripts. Following exposure to bacterial endotoxin, interleukin-6 transcripts accumulated in all cells, whereas ET-1 expression was constant or slightly decreased. These data suggest that organ-specific heterogeneity of EC responsiveness to endotoxin is a potential determinant of organ-specific resistance to endotoxin and other mediators of injury.

Caspases -2, -3, -6, and -9, but not caspase-1, are activated in sepsis-induced thymocyte apoptosis.

Sepsis induces extensive lymphocyte cell death that may contribute to immune depression and morbidity/mortality in the disorder. bcl-2 is a member of a new class of oncogenes that prevents cell death from an array of noxious stimuli. Transgenic mice that overexpress BCL-2 in T lymphocytes are resistant to sepsis-induced T cell apoptosis, and mortality was decreased in sepsis. The purpose of this study was to identify key initiator and executioner "caspases" involved in sepsis-induced lymphocyte apoptosis and to determine if BCL-2 acts prior to caspase activation. Thymi were removed 5-22 h post-cecal ligation and puncture (CLP) or sham surgery. Apoptosis was evaluated in thymocytes by annexin-V FITC labeling and flow cytometry. Caspase-1 activity was determined by western blot analysis of the procaspase protein and p20 subunit of the activated caspase; activities of caspases -2, -6, and -9 were determined by colorimetric assays using specific substrates conjugated to a color reporter molecule. Caspase-3 activity was determined both by western blot and by a fluorogenic assay in which a fluorescent compound was generated. Thymocytes from CLP mice had markedly increased apoptosis and activation of caspases -2, -3, -6, and -9 in comparison with thymocytes of sham-operated mice. Caspase-1 was not activated. BCL-2 prevented sepsis-induced thymocyte apoptosis and inhibited activation of all caspases. We conclude that sepsis causes activation of multiple caspases and that BCL-2 acts upstream as an inhibitor of caspase activation. The pattern of caspase activation suggests a mitochondrial mediated pathway.

Pyrrolidine dithiocarbamate activates the heat shock response and thereby induces apoptosis in primed endothelial cells.

Transcription factor NF-kappaB is an important regulator of the cellular response to diverse stresses. Pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB activity, was used to determine the role of this transcription factor in our model of stress-induced endothelial cell apoptosis. Porcine aortic endothelial cells were treated with an inducer of the acute phase response (LPS) followed by treatment with an inducer of the heat shock response (arsenite), a sequence that produces cell death by apoptosis. Treatment with PDTC attenuated LPS-induced NF-kappaB activity and endothelial cell death when added prior to LPS. However, PDTC unexpectedly increased cell death when given after LPS priming. This time-dependent effect of PDTC on endothelial cell death was similar to that which we had observed previously for inducers of the heat shock response. Therefore, we hypothesized that PDTC could induce the heat shock response in porcine and human endothelial cells. PDTC increased heat shock protein (HSP)-70 production and heat shock factor (HSF) activity. Thus, treatment of endothelial cells with PDTC, like other inducers of the heat shock response, increased HSP-70 levels and HSF activity and had time-dependent effects on cell death by apoptosis in primed endothelial cells. We conclude that PDTC induced the heat shock response, that induction of HSF activity may be linked with inhibition of NF-kappaB activity, and that interaction between acute phase and heat shock regulatory factors may be pivotal to determining cell fate (apoptosis).

Injury in the era of genomics.

The traditional approach to the study of biology employs small-scale experimentation that results in the description of a molecular sequence of known function or relevance. In the era of the genome the reverse is true, as large-scale cloning and gene sequencing come first, followed by the use of computational methods to systematically determine gene function and regulation. The overarching goal of this new approach is to translate the knowledge learned from a systematic, global analysis of genomic data into a complete understanding of biology. For investigators who study shock, the specific goal is to increase understanding of the adaptive response to injury at the level of the entire genome. This review describes our initial experience using DNA microarrays to profile stress-induced changes in gene expression. We conclude that efforts to apply genomics to the study of injury are best coordinated by multi-disciplinary groups, because of the extensive expertise required.

A meta-analysis of prospective trials comparing percutaneous and surgical tracheostomy in critically ill patients.

STUDY OBJECTIVES: Tracheostomy is one of the most commonly performed procedures in the patient receiving long-term mechanical ventilation. While percutaneous dilational tracheostomy (PDT) is becoming increasingly utilized as an alternative to conventional surgical tracheostomy, most literature evaluating these two techniques is neither prospective nor controlled. We performed a meta-analysis of available prospective controlled studies comparing PDT and surgical tracheostomy in critically ill patients to more fully understand the relative benefits and risks of these two procedures in this population. DESIGN: Meta-analysis using Mantel-Haenszel fixed effect model. INTERVENTIONS: We performed searches of MEDLINE, Current Contents, Best Evidence, Cochrane, and HealthSTAR databases from 1985 to present to identify prospective controlled studies comparing PDT and surgical tracheostomy in critically ill patients. After establishing clinical and statistical homogeneity (Q: statistic), studies were analyzed by a Mantel-Haenszel fixed effect model. For each clinical end point examined, PDT and surgical tracheostomy were compared by calculating either absolute differences or odds ratios (ORs) with 95% confidence intervals (CIs) for continuous or discrete variables, respectively. MEASUREMENTS AND RESULTS: We pooled data from five studies (236 patients) satisfying our search criteria to analyze eight clinical end points. Operative time was shorter for PDT than surgical tracheostomy: absolute difference with 95% CI, 9. 84 min (7.83 to 10.85 min). There was no difference comparing PDT and surgical tracheostomy with respect to overall operative complication rates: OR with 95% CI, 0.732 (0.05 to 9.37). However, relative to surgical tracheostomy, PDT was associated with less perioperative bleeding (OR with 95% CI, 0.14 [0.02 to 0.39]), a lower overall postoperative complication rate (OR with 95% CI, 0.14 [0.07 to 0.29]), as well as a lower postoperative incidence of bleeding (OR with 95% CI, 0.39 [0.17 to 0.88]), and stomal infection (OR with 95% CI, 0.02 [0.01 to 0.07]). No difference was identified in days intubated prior to tracheostomy (absolute difference with 95% CI, 0.16 days [- 0.9 to 1.22 days]), overall procedure-related complications (OR with 95% CI, 0.73 [0.06 to 9.37]), or death (OR with 95% CI, 0.63 [0.18 to 2.20]) comparing these two techniques. CONCLUSIONS: Despite its popularity, there are currently only a limited number of small studies prospectively evaluating PDT and surgical tracheostomy. Our meta-analysis of these studies suggests potential advantages of PDT relative to surgical tracheostomy, including ease of performance, and lower incidence of peristomal bleeding and postoperative infection. If confirmed by additional, adequately powered prospective trials, these findings support PDT as the procedure of choice for the establishment of elective tracheostomy in the appropriately selected critically ill patient.

Substance abuse-related admissions to adult intensive care.

The frequency of adult surgical and medical intensive care unit (ICU) admissions related to substance abuse was determined at a large community, trauma, and tertiary referral hospital. Of 435 ICU admissions, 14 percent (95 percent confidence interval [CI], 5 to 23 percent) were tobacco related generating 16 percent of costs, 9 percent (95 percent CI, 0 to 18 percent) were alcohol related generating 13 percent of costs, and 5 percent (95 percent CI, 0 to 14 percent) were illicit drug related generating 10 percent of costs. In all, 28 percent (95 percent CI, 20 to 36 percent) of ICU admissions generating 39 percent of costs were substance abuse related. Substance abuse-related admissions were significantly longer and more costly than admissions not related to substance abuse (4.2 days vs 2.8 days, p = 0.004; $9,610 vs $5,890, p = 0.001). Frequency of substance abuse-related admission was linked with the patient's insurance status (Medicare, private insurance, uninsured). In the uninsured group, 44 percent of admissions were substance abuse related (95 percent CI, 35 to 52 percent), significantly higher than in the private insurance and Medicare groups, and generating 61 percent of all ICU costs in the uninsured group. Large fractions of adult ICU admissions and costs are substance abuse related, particularly in uninsured patients.

Interleukin-1 receptor antagonist as therapy for inflammatory disorders.

IL-1 is a pivotal mediator of the immune response and has been implicated in inflammatory and infectious diseases. As a consequence, the administration of IL-1 receptor antagonist (IL-1ra), a recombinantly synthesised endogenous inhibitor of IL-1, has appeal as a therapeutic strategy in these conditions. To date, the largest clinical experiences with IL-1ra have been in the setting of sepsis and rheumatoid arthritis (RA). Like other anti-inflammatory agents that target a specific mediator, IL-1ra was found to lack efficacy when given in conjunction with standard therapy in patients with sepsis and septic shock. In contrast, recent studies enrolling patients with RA suggest that IL-1ra significantly ameliorates disease activity and retards joint destruction. Whether the respective lack of efficacy and success of IL-1ra in these two diseases is a result of differences in the pathologic processes involved, or reflects the nature in which the clinical studies were conducted, is unclear. Further, the effectiveness of IL-1ra compared to other anticytokine and conventional treatments in RA remains to be clarified. Nonetheless, the recent finding that IL-1ra has the ability to favourably influence a chronic inflammatory disease supports the hypothesis that inhibition of a single mediator of the immune response may have clinical impact.

Gastrointestinal complications in heart and in heart-lung transplant patients.

Gastrointestinal complications after heart and heart-lung transplantation are being recognized and reported more frequently in the literature as a cause of significant morbidity. Between July 1983 and December 1989, 131 consecutive patients underwent 133 heart or heart-lung transplant procedures at The Johns Hopkins Hospital. Immunosuppression consisted of either cyclosporine and prednisone or cyclosporine, prednisone, and azathioprine. Twenty-eight patients (21%) had 38 gastrointestinal complications, including visceral perforations (n = 6), gastrocutaneous fistula (n = 1), retroperitoneal abscess (n = 1), cholecystitis (n = 5), gastric atony (n = 1), perianal abscess (n = 1), gastrointestinal bleeding (n = 4), esophagitis (n = 2), pancreatitis (n = 2), pancreatic abscess (n = 2), hepatitis (n = 2), cytomegalovirus infection (n = 3), and diarrhea (n = 8). Among this group of 28 patients, 17 operative procedures were needed by 13 patients (46%), for an incidence of major abdominal procedures in the entire transplant cohort of 10% (13/131). Operations included cholecystectomy (n = 5), colon resection with colostomy (n = 3), closure of perforated gastroduodenal ulcer (n = 3) and repair of gastrocutaneous fistula (n = 1), drainage of pancreatic abscess (n = 2), pyloroplasty (n = 1) and incision and drainage of perianal abscess (n = 1). The operative mortality rate was 8% (1/13). Overall survival in patients with gastrointestinal complications was no different than that in the entire transplant population. Age, gender, race, and number of rejection episodes did not correlate with the presence of gastrointestinal complications. Patients with gastrointestinal pathologic conditions necessitating surgery often had atypical presentations, with subtle clinical findings but with common general surgical problems.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular biology of circulatory shock. Part III. Human hepatoblastoma (HepG2) cells demonstrate two patterns of shock-induced gene expression that are independent, exclusive, and prioritized.

During shock and resuscitation, parenchymal cells of solid organs are exposed to a rapidly changing microenvironment, which may include a reduced oxygen tension and an increased concentration of certain cytokines including tumor necrosis factor alpha and interleukin-1. In vivo experiments that testedl iver biopsied from pigs undergoing cardiogenic shock and resuscitation demonstrated several patterns of gene expression. To study the independent and additive influences of hypoxia and of cytokines in vitro, human hepatoblastoma (HepG2) cells were perturbed by hypoxia/reoxygenation (H/R), by heat shock, and by the cytokines interleukin-1 and tumor necrosis factor alpha alone and in combination. H/R induces new patterns of protein synthesis and secretion that are indistinguishable from those induced by heat shock and independent of the acute-phase response mediated by the cytokines. The H/R (heat-shock) response has priority over and will extinguish gene expression supported by the cytokines. This previously unrecognized hierarchy of stress gene expression could well form the molecular basis of shock-related cell and organ failure.

Detection of surgical pathogens by in vitro DNA amplification. Part I. Rapid identification of Candida albicans by in vitro amplification of a fungus-specific gene.

The management of candidemia and disseminated candidiasis depends on rapid, unambiguous identification of Candida. Such identification is retarded by the slow growth of Candida from clinical specimens. Administration of effective but potentially toxic antifungal therapy is often withheld pending identification. To circumvent this slow growth and thus to expedite diagnosis and therapy, the polymerase chain reaction (PCR) was used to amplify a segment of fungal DNA coding for the cytochrome P450L1A1 (lanosterol-14 alpha-demethylase) in vitro. The technique provides unambiguous evidence of C. albicans in as few as 6 hours with a detection threshold of 10 organisms in a 100 mu specimen. Clinical specimens of urine (n = 4), sputum (n = 6), wound fluid (n = 1, and blood (n = 2) were collected from patients, and C. albicans was conventionally documented at these sites; in each case, PCR was confirmed. Of 17 additional specimens that were culture negative, PCR suggested the presence of yeast in two of the specimens. PCR-based detection of surgical pathogens may have broad application in rapid screening for the presence of organisms either indigenous to a particular surgical intensive care unit or peculiar to selected patient populations.