Timothy G. Buchman

Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.

OBJECTIVESnThe purpose of this study was to determine whether apoptosis is a major mechanism of cell death in patients with sepsis. The activities of caspase-3 and the antiapoptotic protein, BCL-2, were investigated also.nnnDESIGNnA prospective study of 20 patients who died of sepsis and multiple organ dysfunction was performed. The control group of 16 patients consisted of critically ill, nonseptic patients who were evaluated either prospectively (7) or retrospectively (9). In addition, normal colon sections from seven patients who had bowel resections were included. Apoptosis was evaluated in hematoxylin and eosin-stained specimens by deoxyuridine triphosphate nick end-labeling (TUNEL) and by DNA gel electrophoresis.nnnSETTINGnTwo academic medical centers.nnnPATIENTSnCritically ill patients.nnnMEASUREMENTS AND MAIN RESULTSnIn septic patients, apoptosis was detected in diverse organs by all three methods with a predominance in lymphocytes and intestinal epithelial cells. Hematoxylin and eosin-stained specimens from septic patients demonstrated at least focal apoptosis in 56.3% of spleens, 47.1% of colons, and 27.7% of ileums. Indirect evidence of lymphocyte apoptosis in septic patients included extensive depletion of lymphocytes in white pulp and a marked lymphocytopenia in 15 of 19 patients. Hematoxylin and eosin from nonseptic patients tissues revealed a low level of apoptosis in one patient only. The TUNEL method increased in positivity with a delay in tissue fixation and was highly positive in many tissues from both septic and nonseptic patients. Immunohistochemical staining for active caspase-3 showed a marked increase in septic vs. nonseptic patients (p < .01), with >25% to 50% of cells being positive focally in the splenic white pulp of six septic but in no nonseptic patients.nnnCONCLUSIONSnWe conclude that caspase-3-mediated apoptosis causes extensive lymphocyte apoptosis in sepsis and may contribute to the impaired immune response that characterizes the disorder.

Guidelines for the Selection of Anti-infective Agents for Complicated Intra-abdominal Infections

Joseph S. Solomkin, John E. Mazuski, Ellen J. Baron, Robert G. Sawyer, Avery B. Nathens, Joseph T. DiPiro, Timothy Buchman, E. Patchen Dellinger, John Jernigan, Sherwood Gorbach, Anthony W. Chow, and John Bartlett Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio; Department of Surgery, Washington University School of Medicine, St. Louis, Missouri; Department of Microbiology, Stanford University School of Medicine, Palo Alto, California; Department of Surgery, University of Virginia, Charlottesville; Department of Surgery, University of Washington, Seattle; University of Georgia College of Pharmacy, Department of Surgery, Medical College of Georgia, Augusta, and Centers for Disease Control and Prevention, Atlanta; Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts; Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland; and Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada

Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms

Caspases mediate essential key proteolytic events in inflammatory cascades and the apoptotic cell death pathway. Human caspases functionally segregate into two distinct subfamilies: those involved in cytokine maturation (caspase-1, -4 and -5) and those involved in cellular apoptosis (caspase-2, -3, -6, -7, -8, -9 and -10). Although caspase-12 is phylogenetically related to the cytokine maturation caspases, in mice it has been proposed as a mediator of apoptosis induced by endoplasmic reticulum stress including amyloid-β cytotoxicity, suggesting that it might contribute to the pathogenesis of Alzheimers disease. Here we show that a single nucleotide polymorphism in caspase-12 in humans results in the synthesis of either a truncated protein (Csp12-S) or a full-length caspase proenzyme (Csp12-L). The read-through single nucleotide polymorphism encoding Csp12-L is confined to populations of African descent and confers hypo-responsiveness to lipopolysaccharide-stimulated cytokine production in ex vivo whole blood, but has no significant effect on apoptotic sensitivity. In a preliminary study, we find that the frequency of the Csp12-L allele is increased in African American individuals with severe sepsis. Thus, Csp12-L attenuates the inflammatory and innate immune response to endotoxins and in doing so may constitute a risk factor for developing sepsis.

Apoptosis in lymphoid and parenchymal cells during sepsis : Findings in normal and T- and B-cell-deficient mice

OBJECTIVESnTo determine if apoptosis (programmed cell death) occurs systemically in lymphoid and parenchymal cells during sepsis. To examine the potential role of T and B cells in the apoptotic process using knockout mice deficient in mature T and B lymphocytes.nnnDESIGNnProspective, randomized, controlled trial.nnnSETTINGnAnimal laboratory in a university medical setting.nnnINTERVENTIONSnCecal ligation and puncture (CLP) (n = 34) or sham surgery (n = 13) was performed in female ND4 mice and, 15 to 22 hrs postoperatively, thymus, lung, heart, spleen, ileum, colon, liver, kidney, brain, and muscle were obtained and examined for apoptosis. A second group of mice (Rag-1) which are totally deficient in mature T and B cells also underwent CLP (n = 14) or sham surgery (n = 14) and had examination of tissues for apoptosis.nnnMEASUREMENTS AND MAIN RESULTSnFour methods with varying sensitivities and specificities were used to detect apoptosis, including: a) DNA agarose gel electrophoresis; b) terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL); c) electron microscopy; and d) light microscopy. In CLP mice, multiple methods demonstrated apoptosis in lymphocytes in thymus, spleen, ileum, colon, lung, and skeletal muscle. In addition to lymphocytes, parenchymal cells in ileum, colon, lung, and to a lesser extent, in skeletal muscle and kidney were apoptotic in CLP mice. There was no evidence of apoptosis by any method of detection in liver, brain, or heart. Results in Rag-1 mice which are deficient in T and B cells demonstrated extensive apoptosis in thymus, spleen, and ileum with less degrees of apoptosis in colon and lung. Both lymphoid cells and parenchymal cells were apoptotic. Rag-1 mice which underwent CLP did not die prematurely and there were no apparent observable differences in the physical response (tachypnea, piloerection, lethargy, etc), or intra-abdominal bowel inflammation/adhesions compared with CLP mice with normal T and B cells.nnnCONCLUSIONSnApoptosis is an important mechanism of cell death in lymphocytes and parenchymal cells in sepsis and occurs systemically in many organs. Apoptosis may be an important cause of immunologic suppression in sepsis by inducing widespread lymphocyte depletion. Alternately, apoptosis may be beneficial to host survival by down-regulating the inflammatory response which accompanies sepsis. The degree to which parenchymal cell apoptosis is contributing to multiple organ failure cannot be determined from the present study. Findings in Rag-1 mice demonstrate that mature T and B cells and their secretory products are not necessary for apoptosis to occur during sepsis and that apoptotic cell death is not restricted to T or B cells. Apoptosis may be a key regulator of the balance between the pro- and anti-inflammatory process.

The community of the self

Good health, which reflects the harmonious integration of molecules, cells, tissues and organs, is dynamically stable: when displaced by disease, compensation and correction are common, even without medical care. Physiology and computational biology now suggest that healthy dynamic stability arises through the combination of specific feedback mechanisms and spontaneous properties of interconnected networks. Todays physicians are already testing to see if the network is right; tomorrows physicians may well use therapies to make the network right.

Rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patients with trauma and shock.

ObjectiveApoptosis is a cellular suicide program that can be activated by cell injury or stress. Although a number of laboratory studies have shown that ischemia/reperfusion injury can induce apoptosis, few clinical studies have been performed. The purpose of this study was to determine whether apoptosis is a major mechanism of cell death in intestinal epithelial cells and lymphocytes in patients who sustained trauma, shock, and ischemia/reperfusion injury. DesignIntestinal tissues were obtained intraoperatively from 10 patients with acute traumatic injuries as a result of motor vehicle collisions or gun shot wounds. A control population consisted of six patients who underwent elective bowel resections. Apoptosis was evaluated by conventional light microscopy, laser scanning confocal microscopy using the nuclear staining dye Hoechst 33342, immunohistochemical staining for active caspase-3, and immunohistochemical staining for cytokeratin 18. SettingAcademic medical center. PatientsPatients with trauma or elective bowel resections. Measurements and Main ResultsExtensive focal crypt epithelial and lymphocyte apoptosis were demonstrated by multiple methods of examination in the majority of trauma patients. Trauma patients having the highest injury severity score tended to have the most severe apoptosis. Repeat intestinal samples obtained from two of the trauma patients who had a high degree of apoptosis on initial evaluation were negative for apoptosis at the time of the second operation. Tissue lymphocyte apoptosis was associated with a markedly decreased circulating lymphocyte count in 9 of 10 trauma patients. ConclusionsFocal apoptosis of intestinal epithelial and lymphoid tissues occurs extremely rapidly after injury. Apoptotic loss of intestinal epithelial cells may compromise bowel wall integrity and be a mechanism for bacterial or endotoxin translocation into the systemic circulation. Apoptosis of lymphocytes may impair immunologic defenses and predispose to infection.

Surgeons, intensivists, and the covenant of care: administrative models and values affecting care at the end of life--Updated.

ContextEnd-of-life care remains a challenging and complex activity in critical care units. There is little information concerning the influence of administrative models of care delivery on end-of-life care. ObjectiveTo compare and contrast end-of-life care delivery in intensive care units using “semiclosed,” “open,” and “closed” administrative models. DesignEthnographic study of three critical care units. SettingUniversity hospitals in the United States and New Zealand. SubjectsApproximately 600 physicians, nurses, allied health personnel, patients, family members, and friends. Measurements and Main ResultsEthnographic observations were made at three sites for 75, 3, and 10 wks, respectively. Eighty end-of-life care episodes were observed. The interactions among care personnel and families varied according to the administrative model, depending on whether surgeons or intensivists had primary patient responsibility. This led to differential timing on the shift from “cure” to “comfort,” and differential decision-making power for families. ConclusionsEnd-of-life care varies according to the administrative model. When surgeons have primary responsibility for the patient, the most important goal is defeating death. When intensivists have sole patient responsibility, scarce resources are considered and quality of life is a significant variable. Discussions about improving the way end-of-life decisions are carried out in intensive care units rarely consider the administrative models and personal, professional, and national values affecting such decisions. To improve care at the end of life, we must critically examine these features.

The heat shock paradox: does NF-κB determine cell fate?

Cellular injury induces an adaptive response whether the insult is physical (e.g., heat, radiation), chemical (e.g., reactive oxygen species), infectious (e.g., bacteria), or inflammatory (e.g., lipopolysaccharide). Recent data indicate that the interactions of these responses are not predictable and that sequence permutations can have opposite effects on outcome after injury. Our overarching hypothesis is that interactions among stress responses contribute to the fate of cells, tissues, and organisms and that modulation of these interactions can have important affects on both function and survival. For example, whereas it is well known that a prior heat shock stress can protect cells against inflammatory stress both in vitro and in vivo, we and others have shown that induction of a subsequent heat stress in cells ‘primed’ by inflammation can precipitate cell death by apoptosis. We call this seemingly paradoxical ability of heat shock to induce cytoprotection and cytotoxicity the heat shock paradox. The molecular mechanisms by which cells integrate responses to these and other stresses are poorly understood. We present data linking the heat shock paradox to the activity of the acute‐phase transcription factor nuclear factor kappa Β (identifying an ‘NF‐ΚB paradox’) and hypothesize that the mechanism is linked to the downstream effects of induction of NF‐ΚBs endogenous inhibitor, ΙκΒα, a putative heat shock protein.—DeMeester, S. L., Buchman, T. G., Cobb, J. P. The heat shock paradox: does NF‐ΚB determine cell fate? FASEB J. 15, 270–274 (2001)

Inducible nitric oxide synthase (iNOS) gene deficiency increases the mortality of sepsis in mice

BACKGROUNDnNitric oxide (NO) produced by the inducible isoform of NO synthase (iNOS or NOS2) has been implicated in the hypotension, organ failure, and death that complicate sepsis. To avoid the confounding effects and limitations of iNOS inhibitors, we used iNOS gene knockout mice to examine the effect of inducible NO production in a model of polymicrobial abdominal sepsis treated with antibiotics. We hypothesized that iNOS gene deficiency would significantly alter outcome.nnnMETHODSnC57BL6 wild-type (control) and congenic iNOS knockout mice were studied concurrently. Under halothane anesthesia, the ceca were ligated with 4-0 silk suture and punctured twice with a 26-gauge needle (cecal ligation and puncture, CLP). Survival was followed for 7 days, after which necropsies were performed in surviving animals. In an accompanying study examining the acute effects of sepsis, organ injury at 18 hours after CLP as determined by histology and the degree of cell death by apoptosis were examined with the use of hematoxylin and eosin (H&E) and TUNEL staining and two-channel fluorescence-activated cell sorter (FACS) analysis.nnnRESULTSnSham laparotomy produced no lethality in either knockout (n = 3) or wild-type (n = 3) animals. Compared with survival in controls (n = 20), survival after CLP in iNOS knockout mice (n = 21) was significantly decreased (P < .01 at 2 days, P = .080 at 7 days, Mantel-Haenszel log-rank test). CLP-induced apoptotic cell death was significantly less in the thymus of iNOS knockout mice compared with wild-type mice.nnnCONCLUSIONSnWe conclude that iNOS gene function provides a survival benefit in septic mice and is associated with increased sepsis-induced thymocyte apoptosis. To our knowledge, this is the first survival study examining the effect of iNOS gene deficiency in a clinically relevant model of sepsis.

The Impact of a Simple, Low-cost Oral Care Protocol on Ventilator-associated Pneumonia Rates in a Surgical Intensive Care Unit

Objective: The purpose of this study was to determine the effects of a simple low-cost oral care protocol on ventilator-associated pneumonia rates in a surgical intensive care unit. Design: Preintervention and postintervention observational study. Setting: Twenty-four bed surgical/trauma/burn intensive care units in an urban university hospital. Patients: All mechanically ventilated patients that were admitted to the intensive care unit between June 1, 2004 and May 31, 2005. Interventions: An oral care protocol to assist in prevention of bacterial growth of plaque by cleaning the patients teeth with sodium monoflurophosphate 0.7% paste and brush, rinsing with tap water, and subsequent application of a 0.12% chlorhexidine gluconate chemical solution done twice daily at 12-hour intervals. Measurements and main results: During the preintervention period from June 1, 2003 to May 31, 2004, there were 24 infections in 4606 ventilator days (rate = 5.2 infections per 1000 ventilator days). After the institution of the oral care protocol, there were 10 infections in 4158 ventilator days, resulting in a lower rate of 2.4 infections per 1000 ventilator days. This 46% reduction in ventilator-associated pneumonia was statistically significant (P = .04). Staff compliance with the oral care protocol during the 12-month period was also monitored biweekly and averaged 81%. The total cost of the oral care protocol was US