Robert L. Danner

Septic Shock in Humans: Advances in the Understanding of Pathogenesis, Cardiovascular Dysfunction, and Therapy

Septic shock is the commonest cause of death in intensive care units. Although sepsis usually produces a low systemic vascular resistance and elevated cardiac output, strong evidence (decreased ejection fraction and reduced response to fluid administration) suggests that the ventricular myocardium is depressed and the ventricle dilated. In survivors, these abnormalities are reversible. Failure to develop ventricular dilatation in nonsurvivors suggests that dilatation is a compensatory mechanism needed to maintain adequate cardiac output. With a canine model of septic shock that is very similar to human sepsis, myocardial depression was confirmed using load-independent measures of ventricular performance. Endotoxin administration to humans simulates the qualitative, cardiovascular abnormalities of sepsis. The pathogenesis of septic shock is extraordinarily complex. Diverse microorganisms can generate toxins, stimulating release of potent mediators that act on vasculature and myocardium. A circulating myocardial depressant substance has been closely associated with the myocardial depression of human septic shock. Therapy has emphasized early use of antibiotics, critical care monitoring, aggressive volume resuscitation, and, if shock continues, use of inotropic agents and vasopressors. Pharmacologic or immunologic antagonism of endotoxin or other mediators may prove to enhance survival in this highly lethal syndrome.

Selected Treatment Strategies for Septic Shock Based on Proposed Mechanisms of Pathogenesis

Dr. Charles Natanson (Critical Care Medicine Department, Clinical Center, National Institutes of Health [NIH], Bethesda, Maryland): Sepsis and septic shock are heterogeneous clinical syndromes that can be triggered by many microorganisms, including gram-negative bacteria, gram-positive bacteria, and fungi [1-3]. Participants in a recent consensus conference tried to define sepsis and septic shock. Sepsis was characterized as a systemic response to infection manifested by tachycardia, tachypnea, change in temperature, and leukopenia or leukocytosis. Septic shock was defined as severe sepsis accompanied by hypotension [4]. However, patients with sepsis may have one or several signs and symptoms, and no single physiologic or laboratory parameter can universally identify this syndrome. Advances in molecular biology and immunology during the past decade have increased our understanding of the pathogenesis of septic shock (Figure 1). In particular, we now believe that the hosts inflammatory response to infection contributes substantially to the development of septic shock [5-7]. Infections begin when microorganisms circumvent or penetrate host barriers such as skin and mucosa. Depending on the infecting agents virulence and the patients immunocompetence, local host defenses may be overwhelmed, resulting in microbial invasion of the bloodstream. Toxic bacterial products present in the circulation activate systemic host defenses, including plasma factors (complement and clotting cascades) and cellular components (neutrophils, monocytes, macrophages, and endothelial cells). In turn, activated cells produce potentially toxic host mediators (cytokines such as tumor necrosis factor [TNF] and interleukin-1 [IL-1], kinins, eicosinoids, platelet-activating factor, and nitric oxide) that augment the inflammatory response. This escalating immune response, in concert with microbial toxins, can lead to shock, multiple organ failure, and death. Figure 1. The pathogenesis and treatment of septic shock. Standard sepsis treatment strategies include use of antibiotics to kill invading bacteria, surgical procedures to eradicate the nidus of infection, and intensive life-support procedures such as dialysis, mechanical ventilation, and use of vasoactive drugs. Despite these approaches, the mortality rate from septic shock is high, ranging from 25% to 75% [1, 8-12]. In addition, the reported incidence of sepsis syndrome in U.S. hospitals increased 139%, from 73.6 to 175.9 per 100 000 persons discharged between 1979 and 1987 [13]. This increase may be caused by several medical trends: improved life-support technology that keeps patients who have a high risk for infection alive at the extremes of age; increased use of invasive medical procedures; advances in cancer chemotherapy and immunotherapy; and the prevalence of acquired immunodeficiency syndrome. The increasing incidence of sepsis and its high mortality rate have mobilized a search for new therapies. Development of new drugs to treat sepsis has been based in part on the premise that neutralizing bacterial toxins and potentially harmful host mediators could stop or slow this syndrome. The discussants in this conference review several of these new therapies that are directed at different elements of the inflammatory cascade, including a bacterial toxin (endotoxin), host proteins that mediate the inflammatory response (TNF and IL-1), an inflammatory cell (the neutrophil), and a low-molecular-weight messenger (nitric oxide) that causes hypotension. Other targets (eicosinoids, platelet-activating factor, bradykinin, and so on) are being evaluated to treat this syndrome. Each target selected for discussion was studied in our laboratories or evaluated in human clinical trials. Each discussant offers unique insight into the pathogenesis of septic shock and into the difficulties inherent in inhibiting a potentially toxic inflammatory mediator that may also play a role in host defense. Antiendotoxin Therapies in Septic Shock Dr. William D. Hoffman (Critical Care Medicine Department, Clinical Center, NIH, Bethesda, Maryland): The outer membrane of gram-negative bacteria contains lipopolysaccharides called endotoxin [14]. Endotoxin induces an inflammatory response that may protect the host from infection but may also cause multiple-organ failure and death when present in excess amounts. Specific immunochemical properties have been associated with different components of the endotoxin molecule. The O-polysaccharide chain (O-side chain) of endotoxin is exposed on the outside surface of gram-negative bacteria. The O-side chain is not toxic when injected into animals and has a molecular structure that varies among gram-negative bacteria. The core sugar and lipid A regions of endotoxin are embedded deeply in the outer bacterial membrane, and their molecular structures are similar for all gram-negative bacteria. In contrast to the O-side chain, lipid A is toxic when given to animals [14]. Effects of Endotoxin Challenge-Endotoxemia in Sepsis Experimental observations have supported and challenged the concept that endotoxin-directed therapies can benefit patients with septic shock. Reversible organ dysfunction and hemodynamic changes that are qualitatively similar to those seen in patients with septic shock develop in animals injected with endotoxin [15] and healthy human volunteers injected with safe doses of endotoxin [16]. In addition, development of endotoxemia in patients with septic shock has been associated with severe organ damage [9]. However, neither induced tolerance to endotoxin in humans [17] nor genetic resistance to endotoxin in mice [18] is protective during gram-negative infections. In addition, increased sensitivity to endotoxin does not alter the course of gram-negative infection in animals [19]. Finally, endotoxin and endotoxemia are not necessary to produce the septic shock syndrome, and endotoxin may be only one of many bacterial products that can trigger the septic response [3, 15]. Approaches to Antiendotoxin Therapy Although no antiendotoxin therapy is in clinical use, several are being investigated (Table 1). Antibodies to the O-side chain produce serotype-specific [20], complement-dependent bactericidal activity [21]. However, serotype specificity limits the clinical utility of O-side chain therapies because treating patients empirically with an effective dose of antibody for every probable infecting bacterial strain would be difficult. This problem led to investigation of antibodies directed at core and lipid A structures of endotoxin, because these antibodies might cross-protect against diverse gram-negative bacteria [22]. Although core or lipid A antibodies were thought to mediate antiendotoxin [23] or endotoxin-clearing effects [24], the function of these antibodies is unknown and controversial [25-28]. Nevertheless, core-directed antibodies are the only antiendotoxin therapies studied in clinical trials. Other antiendotoxin agents listed in Table 1 may reduce the host inflammatory response by directly neutralizing endotoxin, increasing its clearance, antagonizing its effects on host cells, or inducing tolerance. Controlled therapeutic trials of agents that reduce the bioactivity of endotoxin and have no antibacterial effect may determine whether circulating endotoxin is a useful therapeutic target in septic shock. Table 1. Approaches to Antiendotoxin Therapies for Septic Shock* Polyclonal Antibodies Directed at Core Epitopes and Lipid A The first clinical trial of core-directed antibodies studied patients with gram-negative bacteremia treated with control (n = 100) or J5 antiserum (n = 91) [8]. In that study, 21 of 39 patients with localized gram-negative infection but no bacteremia were included in the gram-negative bacteremia group because they had been given appropriate antibiotics before blood cultures were obtained [8]. The sepsis-related mortality rate for patients with gram-negative bacteremia given J5 antiserum was 22% (compared with 39% with control serum). In a subgroup of patients who required vasopressor drugs for more than 6 hours, the mortality rate was 44% (compared with 77% with control serum). The effect of J5 antiserum on mortality from all causes or in patients with gram-negative infection was not reported [8]. Five subsequent clinical trials (Table 2) using polyclonal core-reactive antiserum or immunoglobulin to prevent or treat gram-negative sepsis showed essentially no survival benefit [29-34]. Table 2. Summary of 10 Clinical Trials with Lipopolysaccharide Core-Directed Antibodies* Monoclonal Antibodies Directed at Core Epitopes and Lipid A: E5 and HA-1A Monoclonal antibodies were developed to produce a more specific antiendotoxin therapy with less risk for transmission of infection. E5, a murine IgM, protected mice injected with bacteria [35], and HA-1A, a human IgM, protected mice and rabbits injected with bacteria [36]. However, E5 did not protect sheep given endotoxin [37], and other researchers subsequently could not reproduce the beneficial effects of HA-1A in mice and rabbits [38]. E5 Clinical Trials E5 was tested in two multicenter, randomized, placebo-controlled clinical trials (Table 2). In the first trial of 468 patients, E5 provided no significant benefit to patients with gram-negative infection. The antibody improved survival in a retrospectively identified subgroup of 137 patients with gram-negative infection without refractory shock (30% compared with 43%; P = 0.01) [39]. A second trial of 847 patients was conducted to confirm this favorable effect (Table 2). However, in the second study, E5 did not significantly improve survival in the 530 patients who had gram-negative infection without refractory shock (E5, 30% mortality compared with control, 26%; P = 0.21) [40]. Using a meta-analysis and combining data from the two trials, researchers found that E5 substantially decreased the time to recovery from organ dysfunction and improv

Adoptively transferred effector cells derived from naïve rather than central memory CD8+ T cells mediate superior antitumor immunity

Effector cells derived from central memory CD8+ T cells were reported to engraft and survive better than those derived from effector memory populations, suggesting that they are superior for use in adoptive immunotherapy studies. However, previous studies did not evaluate the relative efficacy of effector cells derived from naïve T cells. We sought to investigate the efficacy of tumor-specific effector cells derived from naïve or central memory T-cell subsets using transgenic or retrovirally transduced T cells engineered to express a tumor-specific T-cell receptor. We found that naïve, rather than central memory T cells, gave rise to an effector population that mediated superior antitumor immunity upon adoptive transfer. Effector cells developed from naïve T cells lost the expression of CD62L more rapidly than those derived from central memory T cells, but did not acquire the expression of KLRG-1, a marker for terminal differentiation and replicative senescence. Consistent with this KLRG-1− phenotype, naïve-derived cells were capable of a greater proliferative burst and had enhanced cytokine production after adoptive transfer. These results indicate that insertion of genes that confer antitumor specificity into naïve rather than central memory CD8+ T cells may allow superior efficacy upon adoptive transfer.

Th17 Cells Are Long Lived and Retain a Stem Cell-like Molecular Signature

Th17 cells have been described as short lived, but this view is at odds with their capacity to trigger protracted damage to normal and transformed tissues. We report that Th17 cells, despite displaying low expression of CD27 and other phenotypic markers of terminal differentiation, efficiently eradicated tumors and caused autoimmunity, were long lived, and maintained a core molecular signature resembling early memory CD8(+) cells with stem cell-like properties. In addition, we found that Th17 cells had high expression of Tcf7, a direct target of the Wnt and β-catenin signaling axis, and accumulated β-catenin, a feature observed in stem cells. In vivo, Th17 cells gave rise to Th1-like effector cell progeny and also self-renewed and persisted as IL-17A-secreting cells. Multipotency was required for Th17 cell-mediated tumor eradication because effector cells deficient in IFN-γ or IL-17A had impaired activity. Thus, Th17 cells are not always short lived and are a less-differentiated subset capable of superior persistence and functionality.

Bundled care for septic shock: An analysis of clinical trials

Context: Sepsis bundles have been developed to improve patient outcomes by combining component therapies. Valid bundles require effective components with additive benefits. Proponents encourage evaluation of bundles, both as a whole and based on the performance of each component. Objective: Assess the association between outcome and the utilization of component therapies in studies of sepsis bundles. Data Source: Database searches (January 1980 to July 2008) of PubMed, Embase, and the Cochrane Library, using the terms sepsis, bundles, guidelines, and early goal directed therapy. Data Extraction: Inclusion required comparison of septic adults who received bundled care vs. nonprotocolized care. Survival and use rates for individual interventions were abstracted. Main Results: Eight unblinded trials, one randomized and seven with historical controls, were identified. Sepsis bundles were associated with a consistent (I2 = 0%, p = .87) and significant increase in survival (odds ratio, 1.91; 95% confidence interval, 1.49–2.45; p < .0001). For all studies reporting such data, there were consistent (I2 = 0%, p ≥ .64) decreases in time to antibiotics, and increases in the appropriateness of antibiotics (p ≤ .0002 for both). In contrast, significant heterogeneity was seen across trials for all other treatments (antibiotic use within a specified time period; administration of fluids, vasopressors, inotropes, and packed red blood cells titrated to hemodynamic goals; corticosteroids and human recombinant activated protein C use) (all I2 ≥ 67%, p < .002). Except for antibiotics, sepsis bundle components are still being investigated for efficacy in randomized controlled trials. Conclusion: Bundle use was associated with consistent and significant improvement in survival and antibiotic use. Use of other bundle components changed heterogeneously across studies, making their impact on survival uncertain. However, this analysis should be interpreted cautiously as these studies were unblinded, and only one was randomized.

Role of endotoxemia in cardiovascular dysfunction and mortality. Escherichia coli and Staphylococcus aureus challenges in a canine model of human septic shock.

Using different types of bacteria and a canine model simulating human septic shock, we investigated the role of endotoxin in cardiovascular dysfunction and mortality. Either Escherichia coli (a microorganism with endotoxin) or Staphylococcus aureus (a microorganism without endotoxin) were placed in an intraperitoneal clot in doses of viable or formalin-killed bacteria. Cardiovascular function of conscious animals was studied using simultaneous radionuclide heart scans and thermodilution cardiac outputs. Serial plasma endotoxin levels were measured. S. aureus produced a pattern of reversible cardiovascular dysfunction over 7-10 d that was concordant (P less than 0.01) with that of E. coli. Although this cardiovascular pattern was not altered by formalin killing (S. aureus and E. coli), formalin-killed organisms produced a lower mortality and less myocardial depression (P less than 0.01). S. aureus, compared to E. coli, produced higher postmortem concentrations of microorganisms and higher mortality (P less than 0.025). E. coli produced significant endotoxemia (P less than 0.01), though viable organisms (versus nonviable) resulted in higher endotoxin blood concentrations (P less than 0.05). Significant endotoxemia did not occur with S. aureus. Thus, in the absence of endotoxemia, S. aureus induced the same cardiovascular abnormalities of septic shock as E. coli. These findings indicate that structurally and functionally distinct microorganisms, with or without endotoxin, can activate a common pathway resulting in similar cardiovascular injury and mortality.

Eradication of Solid Human Breast Tumors in Nude Mice with an Intravenously Injected Light-Emitting Oncolytic Vaccinia Virus

Previously, we reported that a recombinant vaccinia virus (VACV) carrying a light-emitting fusion gene enters, replicates in, and reveals the locations of tumors in mice. A new recombinant VACV, GLV-1h68, as a simultaneous diagnostic and therapeutic agent, was constructed by inserting three expression cassettes (encoding Renilla luciferase-Aequorea green fluorescent protein fusion, beta-galactosidase, and beta-glucuronidase) into the F14.5L, J2R (encoding thymidine kinase) and A56R (encoding hemagglutinin) loci of the viral genome, respectively. I.v. injections of GLV-1h68 (1x10(7) plaque-forming unit per mouse) into nude mice with established (approximately 300-500 mm3) s.c. GI-101A human breast tumors were used to evaluate its toxicity, tumor targeting specificity, and oncolytic efficacy. GLV-1h68 showed an enhanced tumor targeting specificity and much reduced toxicity compared with its parental LIVP strains. The tumors colonized by GLV-1h68 exhibited growth, inhibition, and regression phases followed by tumor eradication within 130 days in 95% of the mice tested. Tumor regression in live animals was monitored in real time based on decreasing light emission, hence demonstrating the concept of a combined oncolytic virus-mediated tumor diagnosis and therapy system. Transcriptional profiling of regressing tumors based on a mouse-specific platform revealed gene expression signatures consistent with immune defense activation, inclusive of IFN-stimulated genes (STAT-1 and IRF-7), cytokines, chemokines, and innate immune effector function. These findings suggest that immune activation may combine with viral oncolysis to induce tumor eradication in this model, providing a novel perspective for the design of oncolytic viral therapies for human cancers.

An Official ATS/ERS/ESICM/SCCM/SRLF Statement: Prevention and Management of Acute Renal Failure in the ICU Patient: an international consensus conference in intensive care medicine.

OBJECTIVES To address the issues of Prevention and Management of Acute Renal Failure in the ICU Patient, using the format of an International Consensus Conference. METHODS AND QUESTIONS Five main questions formulated by scientific advisors were addressed by experts during a 2-day symposium and a Jury summarized the available evidence: (1) Identification and definition of acute kidney insufficiency (AKI), this terminology being selected by the Jury; (2) Prevention of AKI during routine ICU Care; (3) Prevention in specific diseases, including liver failure, lung Injury, cardiac surgery, tumor lysis syndrome, rhabdomyolysis and elevated intraabdominal pressure; (4) Management of AKI, including nutrition, anticoagulation, and dialysate composition; (5) Impact of renal replacement therapy on mortality and recovery. RESULTS AND CONCLUSIONS The Jury recommended the use of newly described definitions. AKI significantly contributes to the morbidity and mortality of critically ill patients, and adequate volume repletion is of major importance for its prevention, though correction of fluid deficit will not always prevent renal failure. Fluid resuscitation with crystalloids is effective and safe, and hyperoncotic solutions are not recommended because of their renal risk. Renal replacement therapy is a life-sustaining intervention that can provide a bridge to renal recovery; no method has proven to be superior, but careful management is essential for improving outcome.

NRAS mutation causes a human autoimmune lymphoproliferative syndrome

The p21 RAS subfamily of small GTPases, including KRAS, HRAS, and NRAS, regulates cell proliferation, cytoskeletal organization, and other signaling networks, and is the most frequent target of activating mutations in cancer. Activating germline mutations of KRAS and HRAS cause severe developmental abnormalities leading to Noonan, cardio-facial-cutaneous, and Costello syndrome, but activating germline mutations of NRAS have not been reported. Autoimmune lymphoproliferative syndrome (ALPS) is the most common genetic disease of lymphocyte apoptosis and causes autoimmunity as well as excessive lymphocyte accumulation, particularly of CD4−, CD8− αβ T cells. Mutations in ALPS typically affect CD95 (Fas/APO-1)-mediated apoptosis, one of the extrinsic death pathways involving TNF receptor superfamily proteins, but certain ALPS individuals have no such mutations. We show here that the salient features of ALPS as well as a predisposition to hematological malignancies can be caused by a heterozygous germline Gly13Asp activating mutation of the NRAS oncogene that does not impair CD95-mediated apoptosis. The increase in active, GTP-bound NRAS augments RAF/MEK/ERK signaling, which markedly decreases the proapoptotic protein BIM and attenuates intrinsic, nonreceptor-mediated mitochondrial apoptosis. Thus, germline activating mutations in NRAS differ from other p21 Ras oncoproteins by causing selective immune abnormalities without general developmental defects. Our observations on the effects of NRAS activation indicate that RAS-inactivating drugs, such as farnesyltransferase inhibitors should be examined in human autoimmune and lymphocyte homeostasis disorders.

Randomization in clinical trials of titrated therapies: unintended consequences of using fixed treatment protocols.

Objective:Clinical trial designs that randomize patients to fixed treatment regimens may disrupt preexisting relationships between illness severity and level of therapy. The practice misalignments created by such designs may have unintended effects on trial results and safety. Methods:To illustrate this problem, the Transfusion Requirements in Critical Care (TRICC) trial and the Acute Respiratory Distress Syndrome Network low tidal volume (ARMA) trial were analyzed. Results:Publications before TRICC indicated that clinicians used higher transfusion thresholds in patients with ischemic heart disease compared with younger, healthier patients (p = .001). The trial, however, randomized patients (n = 838) to liberal (10 g/dL hemoglobin) or restrictive (7 g/dL) transfusion thresholds. Thirty-day mortality was different and opposite in the liberal compared with the restrictive arm depending on presence (21 vs. 26%) or absence (25 vs. 16%) of ischemic heart disease (p = .03). At baseline in ARMA, consistent with prior publications, physicians set ventilator volumes lower in patients with high airway pressures and poor compliance (8.4–10.6 mL/kg interquartile range) than patients with less severe abnormalities (9.6–12 mL/kg) (p = .0001). In the trial, however, patients (n = 861) were randomized to low (6 mL/kg) or high (12 mL/kg) tidal volumes. In patients with low compliance (<0.6 mL/kg), 28-day mortality was higher when tidal volumes were raised rather than lowered (42 vs. 29%), but this effect was reversed in patients with higher compliance (21 vs. 37%; p = .003). Conclusions:In TRICC and ARMA, randomization to fixed treatment regimens disrupted preexisting relationships between illness severity and therapy level. This created noncomparable subgroups in both study arms that received care different and opposite from titrated care, that is, practice misalignments. These subgroups reduced the interpretability and safety of each trial. Characterizing current practice, incorporating current practice controls, and using alternative trial designs to minimize practice misalignments should improve trial safety and interpretability.