Mitchell P. Fink

Cytopathic hypoxia in sepsis

Diminished availability of oxygen at the cellular level might account for organ dysfunction in sepsis. Although the classical forms of tissue hypoxia due to hypoxemia, anemia, or inadequate perfusion all might be important under some conditions, it seems increasingly likely that a fourth mechanism, namely cytopathic hypoxia, might play a role as well. The term cytopathic hypoxia is used to denote diminished production of adenosine triphosphate (ATP) despite normal (or even supranormal) PO2 values in the vicinity of mitochondria within cells. At least in theory, cytopathic hypoxia could be a consequence of several different (but mutually compatible) pathogenic mechanisms, including diminished delivery of a key substrate (e.g., pyruvate) into the mitochondrial tricarboxylic acid (TCA) cycle, inhibition of key mitochondrial enzymes involved in either the TCA cycle or the electron transport chain, activation of the enzyme, poly‐(ADP)‐ribosylpolymerase (PARP), or collapse of the protonic gradient across the inner mitochondrial membrane leading to uncoupling of oxidation (of NADH and FADH) from phosphorylation of ADP to form ATP. Tantalizing, but limited, data support the view that cytopathic hypoxia occurs in both animals and patients with sepsis or endotoxemia.

Delayed Treatment with Recombinant Human Tissue Factor Pathway Inhibitor Improves Survival in Rabbits with Gram-Negative Peritonitis

To determine whether treatment with recombinant human tissue factor pathway inhibitor (TFPI), an inhibitor of the extrinsic coagulation pathway, can improve survival in a clinically relevant model of gram-negative sepsis, rabbits were given an intraperitoneal inoculation of a suspension containing hemoglobin (40 microg/mL), porcine mucin (150 microg/mL), and viable Escherichia coli O18:K1 (1.0 +/- 0.5 x 10(5) cfu/kg). Treatment with gentamicin (5 mg/kg every 12 h for five doses) was instituted 4 h after induction of peritonitis. At the same time point, rabbits were randomized to receive a 24-h infusion of vehicle or one of three different doses of TFPI. Treatment groups, 7-day survival rates, and significance versus control were as follows: control, 1 of 20; TFPI(LOW DOSE) (0.1 mg/kg, then 1 microg/kg/min), 3 of 12 (P = .14); TFPI(MID DOSE), (0.5 mg/kg, then 5 microg/kg/min), 7 of 12 (P = .002); TFPI(HIGH DOSE) (10 mg/kg, then 10 microg/kg/min), 4 of 13 (P = .04). Thus, delayed treatment with TFPI improves survival in septic rabbits.

Nitric oxide donor-induced hyperpermeability of cultured intestinal epithelial monolayers: role of superoxide radical, hydroxyl radical, and peroxynitrite.

Many of the cytopathic effects of nitric oxide (NO*) are mediated by peroxynitrite (PN), a product of the reaction between NO* and superoxide radical (O2*-). In the present study, we investigated the role of PN, O2*- and hydroxyl radical (OH*) as mediators of epithelial hyperpermeability induced by the NO* donor, S-nitroso-N-acetylpenicillamine (SNAP), and the PN generator, 3-morpholinosydnonimine (SIN-1). Caco-2BBe enterocytic monolayers were grown on permeable supports in bicameral chambers. Epithelial permeability, measured as the apical-to-basolateral flux of fluorescein disulfonic acid, increased after 24 h of incubation with 5.0 mM SNAP or SIN-1. Addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an NO* scavenger, or Tiron, an O2*- scavenger, reduced the increase in permeability induced by both donor compounds. The SNAP-induced increase in permeability was prevented by allopurinol, an inhibitor of xanthine oxidase (a source of endogenous O2*-). Diethyldithiocarbamate, a superoxide dismutase inhibitor, and pyrogallol, an O2* generator, potentiated the increase in permeability induced by SNAP. Addition of the PN scavengers deferoxamine, urate, or glutathione, or the OH* scavenger mannitol, attenuated the increase in permeability induced by both SNAP and SIN-1. Both donor compounds decreased intracellular levels of glutathione and protein-bound sulfhydryl groups, suggesting the generation of a potent oxidant. These results support a role for PN, and possibly OH*, in the pathogenesis of NO* donor-induced intestinal epithelial hyperpermeability.

Mesenteric Dysfunction After Cardiopulmonary Bypass: Role of Complement C5a

BACKGROUNDnWe investigated the effects of cardiopulmonary bypass (CPB) on ileal homeostasis, and the influence of functional inhibition of complement C5a on CPB-induced mesenteric injury.nnnMETHODSnPigs were perfused on CPB for 1 hour and then perfused off CPB for an additional 2 hours. Antiporcine C5a monoclonal antibody (C5a MAb) was administered 20 minutes before onset of CPB to 6 pigs; 6 controls received saline vehicle. Total complement activity, ileal myeloperoxidase, and indices of ileal integrity were examined.nnnRESULTSnTreatment with C5a MAb ameliorated CPB-induced abnormalities in endothelium-dependent relaxation to ADP and substance P, and the hypercontractile response to phenylephrine of ileal microvessels (88 to 168 microm). Ileal myeloperoxidase activity [units/g protein] was 41 +/- 11 in the C5a MAb group, compared to 83 +/- 13 in the saline group (19 +/- 10 base line). Total hemolytic complement activity was similar in the C5a MAb and saline groups (0.6 +/- 0.2 and 0.7 +/- 0.2 CH50 units). During CPB, ileal mucosal blood flow and mucosal pH, edema formation, and epithelial permeability deteriorated similarly in saline and C5a MAb groups. Inducible nitric oxide synthase (iNOS) mRNA expression was similar before and after CPB.nnnCONCLUSIONSnCPB is associated with significant physiologic alterations in mucosal perfusion, epithelial permeability, edema formation, and blood flow regulation. Inhibition of C5a limits neutrophil-mediated impairment of ileal microvascular regulation after bypass, but does not improve extravascular mesenteric dysfunction after CPB.

The role of nitric oxide in sepsis and ARDS: Synopsis of a roundtable conference held in Brussels on 18–20 March 1995

M. Fink (N~) Departments of Surgery and Anesthesiology, Beth Israel Hospital and Harvard Medical School, 330 Brookline Avenue, Boston, MA 02215, USA D. Payen Department of Anesthesiology, Lariboisiere Hospital, Paris, France characteristic of septic shock and the acute respiratory syndrome (ARDS). Because of its central role as a modulator of vasomotor tone and a mediator of inflammation, NO has become a focus of intense investigation for clinicians and basic scientists interested in problems related to critical care medicine. In addition, because it is a potent pulmonary vasodilator (and weak bronchodilator), the administration of NO gas is attracting considerable interest as a pharmacologic agent for the management of persistent pulmonary hypertension in neonates (PPHN) as well as various forms of acute lung injury in adults. Prompted by these considerations, a Roundtable Conference on the Role of Nitric Oxide in Sepsis and ARDS was held recently in Brussells in conjunction with the 15th Anniversary of the International Symposium on Intensive Care and Emergency Medicine. The following is a concise synopsis of the Roundtable Conference proceedings.

INTESTINAL EPITHELIAL HYPERPERMEABILITY: Mechanisms and Relevance to Disease

Pathologic increases in intestinal permeability to hydrophilic macromolecules has been identified in a number of clinical conditions. The significance of gut barrier dysfunction as a clinical issue remains to be delineated, although it seems likely that alterations in intestinal epithelial permeability play a causative role in a number of conditions ranging from inflammatory bowel disease to the development of complications after cardiopulmonary bypass. It is unlikely that any one mechanism can account for all cases of intestinal hyperpermeability. Rather, it is more probable that myriad factors or combinations of factors, including mesenteric ischemia and cytokine-induced phenomena, lead to alterations in permeability in different clinical entities. Nevertheless, from a purely mechanistic standpoint, some common themes, notably the role of ATP depletion, increases in [Ca2+]i, and cytoskeletal derangements in enterocytes, have emerged as being particularly important.

Acquired interferon gamma responsiveness during Caco-2 cell differentiation: effects on iNOS gene expression.

BACKGROUND Impairment of intestinal barrier function occurs under a variety of inflammatory conditions and is mediated at least in part by interferon γ (IFN-γ) induced nitric oxide (NO) production. Previous in vivo studies have shown that systemic lipopolysaccharide treatment caused an induction of the rat inducible nitric oxide synthase (iNOS) mRNA primarily in villus cells, rather than in undifferentiated crypt cells. AIMS To examine iNOS induction by IFN-γ in vitro as a function of enterocyte differentiation. METHODS Preconfluent and postconfluent Caco-2 cells were treated with IFN-γ in the presence or absence of various inhibitors. Northern analyses were performed to assess the magnitude of iNOS mRNA induction. IFN-γ receptor mRNA and protein levels were determined. RESULTS iNOS mRNA induction by IFN-γ occurred at two hours and was not blocked by cycloheximide, indicating that it is an immediate early response. iNOS induction and nitrite/nitrate increases were inhibited by dexamethasone and pyrrolidine dithiocarbamate, supporting an important role for the NF-κB transcription factor in this process. The stimulated iNOS induction was seen almost exclusively under conditions of cellular differentiation—that is, in postconfluent Caco-2 cells. This increased IFN-γ responsiveness seen in postconfluent Caco-2 cells correlated with an increased expression of IFN-γ receptor, whereas T84 and HT-29 cells did not show any significant alterations in either iNOS induction or IFN-γ receptor levels as a function of postconfluent growth. CONCLUSIONS With regard to iNOS mRNA induction, IFN-γ responsiveness is acquired during Caco-2 cell differentiation, perhaps related to an increase in the numbers of IFN-γ receptors.

Roundtable conference on tissue oxygenation in acute medicine, Brussels, Belgium, 14-16 March 1998.

* Nutritional tissue perfusion and tissue metabolic demand are heterogeneously distributed. * Oxygen diffusion occurs preferentially at the precapillary arteriolar level. * Determination of adequacy of tissue oxygenation requires local organ measurements. * While there remains considerable variability in individual RBC transfusion practices, a recent clinical trial questioned the efficacy of RBC transfusion to hemoglobin concentrations greater than 80 gm/l in patients without heart disease [63]. * RBC substitutes, including cell-free hemoglobin solutions and PFC solutions are efficacious, yet may exhibit a number of direct vascular effects.

Extremely low doses of tissue factor pathway inhibitor decrease mortality in a rabbit model of septic shock

Abstract. Objective: We sought to determine the lowest dose of recombinant human tissue factor pathway inhibitor (TFPI) that can provide protection from lethality in a rabbit model of septic shock. Methods: Sepsis was induced in New Zealand white rabbits by intraperitoneal implantation of 7.0xa0ml of a solution containing hemoglobin (4.8xa0g/dl), porcine mucin (6xa0g/dl), and 0.8–1.4×104 viable Escherichia coli (strain O:18 K+). Gentamicin (5xa0mg/kg) was administered 4xa0h following surgery, and this dose was repeated every 12xa0h for 3xa0days. Beginning 4xa0h following the induction of sepsis, animals were treated with a bolus (1xa0ml) plus a continuous infusion (100xa0ml over 24) of either TFPI (various doses) or its vehicle. Four different doses of TFPI were studied, and each experiment included a contemporaneous control group. The primary outcome parameter was survival time. Results were analyzed using the Wilcoxen log rank test. Results: The average survival time for rabbits treated with the highest dose of TFPI tested (50xa0µg/kg bolus and 0.5xa0µg/kg per minute infusion) was 118xa0h, as compared to 81xa0h in vehicle-treated controls). The average survival time for septic rabbits treated with a much lower dose of TFPI (100xa0ng/kg bolus and 1.0xa0ng/kg per minute infusion) was 119xa0h as compared to 57xa0h in surviving vehicle-treated controls. Treatment with an even lower dose of TFPI (10xa0ng/kg bolus and 0.1xa0ng/kg per minute infusion) still produced a marginally significant prolongation of average survival time (80xa0h) relative to contemporaneously studied controls (47xa0h). When the dose of TFPI was decreased still further (1.0xa0ng/kg bolus and 0.01xa0ng/kg per minute infusion), average survival times were not significantly different between TFPI-treated and vehicle-treated rabbits (77 and 51xa0h, respectively). Conclusions: Delayed infusion with remarkably low doses of recombinant human TFPI prolongs survival in a rabbit model of antibiotic-treated Gram-negative bacterial sepsis. In planning human trials of TFPI as an adjuvant treatment for sepsis it may be reasonable to use much lower doses of the agent than were heretofore contemplated.

Intestinal Epithelial Hyperpermeability

The gut serves not only as a portal of entry for nutrients, small ions, and water, but also as a selective barrier preventing systemic contamination by lumen-derived microbes or microbial products. A key component of the gastrointestinal barrier is the epithelium itself. There are only two ways that substances (ions, molecules, or particles) can traverse the epithelium from the lumenal compartment to the basolateral compartment. Permeation can occur via the transcellular pathway (i.e. through cells) or via the paracellular pathway (i.e. between cells).