Natural killer cell activity: age, estrous- and circadian-stage dependence and inverse correlation with metastatic potential.

The timing within the estrous cycle of surgical removal of a transplanted murine mammary tumor profoundly influences the frequency of pulmonary metastases. We investigated the potential role of the immune response in this phenomenon by measuring splenic natural killer (NK) cell activity and interleukin-2 (IL-2) production in syngeneic tumor-free mice of two age groups at each of two circadian times and in each of four estrous stages. Estrous stage was determined by assessment of vaginal smear cellularity immediately prior to killing and spleen harvest. In a single-cell splenocyte preparation, NK cytotoxicity against a standard tumor cell target was assessed using a radiolabeled chromium release assay while IL-2 activity was determined in a bioassay utilizing the IL-2-dependent CTLL-2 cell line. Mice from the younger group were found to have eight-fold higher NK activity and 35% greater IL-2 production. After normalization of NK and IL-2 values for age, a highly statistically significant difference in NK activity was found among the four estrous and between the two circadian stages of sacrifice. NK activity was greater during the daily resting span across every estrous stage. IL-2 values were highest in diestrus and proestrus when sampled in the light span and in estrus-metestrus when sampled in the dark. The stages within the fertility cycle associated with lowest metastatic potential (proestrus/estrus) correspond precisely with those of highest splenocyte NK activity. These results indicate that an important component of the cellular immune response varies rhythmically both during the fertility and circadian cycles of the host. The rhythmic changes in NK activity may be in part responsible for the similarly rhythmic frequency of postsurgical metastatic dissemination.

Modulation of macrophage and B cell function by glycosaminoglycans.

There is increasing evidence that the behavior of antigen-presenting cells may be regulated, in part, by the surrounding microenvironment. Components of the microenvironment of solid tissues that might influence antigen-presenting cell functions include glycosaminoglycans. We previously showed that heparan sulfate glycosaminoglycans activate macrophages, leading to profound alterations in T cell responses. Here we demonstrate the functional changes that occur in murine antigen-presenting cells induced by heparan sulfate and other glycosaminoglycans, and postulate how these functional changes influence the nature of local immune responses. Heparan sulfate triggered up-regulation of ICAM-1 and I-A, caused the release by antigen-presenting cells of interleukin (IL)-1, IL-6, tumor necrosis factor, IL-12, transforming growth factor beta, and prostaglandin E2 (PGE2), and (in macrophages) induced cytotoxic capability. Heparin induced IL-12 and interferon-gamma production but did not promote the release of other cytokines. Chondroitin sulfate and dermatan sulfate, although not stimulating the production of cytokines or of PGE2, elicited the production by macrophages of nitric oxide. These findings support a model in which the glycosaminoglycan composition of a given tissue, which may be altered by inflammatory processes, helps to regulate the behavior of antigen-presenting cells, which in turn determines the characteristics of the immune response that ensues.

Alpha1-acid glycoprotein concentration and protein binding in trauma.

Alpha 1-Acid glycoprotein (AAG) concentrations were measured every 2 to 3 days in eight trauma patients and seven healthy subjects for approximately 3 wk. Mean AAG concentrations in the trauma patients rose from 100 mg/dl to a peak value of 243 mg/dl at 10 to 14 days. AAG levels averaged more than 200 mg/dl at 15 to 21 days. Mean AAG concentration in healthy subjects was 70 mg/dl with little inter- or intraindividual variability. Lidocaine was added to all serum samples from four of the patients and to selected samples from all of the healthy subjects and protein binding was determined. The binding ratio (bound concentration/free concentration) correlated strongly with AAG concentration in the trauma patients (r = 0.92), in the healthy subjects (r = 0.84), and in both groups combined (r = 0.96). AAG concentration and binding ratio for each of the four patients individually also correlated (P less than 0.05 in all cases). The change in free fraction associated with this increase in AAG was approximately doubled in each patient. Similar findings with drugs commonly used in trauma patients would be expected to alter serum concentration-response relationships significantly.

Age, disease, and cimetidine disposition in healthy subjects and chronically ill patients.

Cimetidine induces reversible dose-related central nervous system (CNS) toxicity. Trough serum concentrations and the development of CNS toxicity correlate. We compared cimetidine kinetics in 12 healthy subjects and 31 patients. Six of the latter had normal renal and liver function, five had renal disease only, 12 had liver disease only, and eight had both renal and liver disease. Postmortem tissue distribution was assessed in 11 patients, and expressed as tissue:serum ratio. Average cimetidine total clearance (ClB) in milliliters per minute for each group was as follows: patients with renal and liver disease (182 +/- 105), renal disease only (193 +/- 24), liver disease only (463 +/- 145), normal patients (510 +/- 93), and healthy subjects (583 +/- 140). Renal function was the major determinant for ClB, and the relationship was described by ClB = 4.2(CCr) + 140, r = 0.87, where CCr is creatinine clearance. Cimetidine clearance was affected little by age. Tissue:serum ratios from highest to lowest were as follows: kidney greater than stomach greater than liver greater than bone greater than brain greater than fat. Central and steady-state distribution volumes were not influenced by age or disease. There was enchanced CNS penetration in liver disease patients; their cerebrospinal fluid (CSF):serum ratio was twice the normal. Our kinetic studies identify patient characteristics likely to result in elevated blood levels, and suggest that the greatest risk of CNS toxicity is in those with liver disease.

Modulation of cytolytic T cell responses by heparan sulfate.

Accumulating evidence suggests that the functional properties of alloactivated T cells may depend upon the microenvironment in which the T cells reside. For instance, we showed previously that heparan sulfate, a biologically active polysaccharide present on cell surfaces and extracellular matrices, modulates the proliferative responses of splenocytes through enhancement of cytokine and prostaglandin production by macrophages. Here we report that under conditions of suboptimal stimulation, heparan sulfate causes discrete alterations in the functional responses of murine cytolytic T cells. When present in a 5-day mixed leukocyte culture (MLC), heparan sulfate mediates an increase, from 3- to 10-fold, in T cell-mediated cytotoxicity. This increase is dose dependent and most pronounced when heparan sulfate is present in the highest concentration during the first 24 hr of the culture period. On the other hand, when added during the last 48-72 hr of an MLC, heparan sulfate decreases cytotoxicity by 3- to 30-fold. Neutralizing antibodies against IL-1 alpha, but not antibodies against IL1 beta, IL-6, or TNF alpha/beta, abrogate the heparan sulfate-mediated increase in cytotoxicity, suggesting that the increase depended in part upon the production of IL-1 alpha. However, studies in which exogenous IL-1 was added to MLC showed that increased cytotoxicity was not due only to increased cytokine production. Augmentation of cytotoxicity was in part independent of T cell help, as depletion of CD4+ cells from the responder population before MLC, or addition of neutralizing anti-murine IL-2 antibodies plus human IL-2 to the MLC, did not abrogate the stimulatory effect of heparan sulfate. Heparan sulfate-treated CD8+ lymphoblasts isolated after 7 days in MLC demonstrated an increased cytotoxicity, elevated intracellular serine esterase, and perforin levels compared with lymphoblasts from control MLC. The decrease in cytotoxicity observed when heparan sulfate was present during the last several days of an MLC was likely mediated by PGE2, as elevated levels of PGE2 were detected in MLC supernatants of heparan sulfate-treated cultures, and because the decrease was not observed in the presence of indomethacin. Our results are consistent with the idea that the metabolism of heparan sulfate, an endogenous component of parenchymal tissues, may regulate the tempo and magnitude of alloreactive cytotoxic T cell responses.

Regulation by heparan sulfate and interleukin 1 alpha of the ontogenic expression of T-cell receptor, CD4, and CD8 in developing thymus.

Increasing expression by T-cell precursors of the T-cell antigen receptor and of the CD4 and CD8 glycoproteins during thymus development enables specific interactions between primitive T cells and thymic epithelial cells, which initiate the development of the T-cell repertoire. Given the importance of GAGs, such as HS, in cell-cell interactions in other organs, we asked whether such molecules might participate in the cellular interactions essential for the development of the thymus. Using an organ culture model in which fetal murine thymuses explanted on gestational day 14 undergo phenotypic maturation from CD3-CD4-CD8- to CD3+CD4+/CD8+, the consequences of inhibiting GAG synthesis with 6-diazo-5-oxo-L-norleucine (DON) were explored. Inhibition of GAG synthesis prevented elaboration of cortical thymocyte-associated HS, IL-1, and de novo expression of the TCR, CD4, and CD8. DON did not alter the expression of TCR or CD4/CD8 by mature thymocytes. Synthesis of IL-1 alpha, TCR, CD4, and CD8 was restored by addition of HS to the cultured organs. These findings support the concept that the ontogenic (but not the constitutive) expression of the TCR and of CD4 and CD8 depends on the synthesis in the thymus of IL-1, and that IL-1 synthesis is in turn regulated by the metabolism of GAGs. Our findings suggest that components of the thymic microenvironment, particularly HS, play a critical role in the maturation of T-cell precursors.

Cardiovascular response to acute and chronic endotoxemia in an awake, volume-resuscitated, canine model.

The cardiovascular response to endotoxemia was evaluated in an awake, intravascular volume-resuscitated canine model. The animals were chronically instrumented for ultrasonic crystal dimension analysis and pressure measurements of the left ventricle (LV), aorta, right atrium (RA), and pulmonary artery (PA) and for cardiac output (CO) measurement. Lipopolysaccharide (Escherichia coli 011:B4) (LPS) was administered intravenously either as an acute, high dose bolus (5 mg/kg; n = 5), a high dose bolus after complete beta-blockade with propranolol (n = 3), or a chronic, low dose infusion (5 micrograms/kg/h; n = 7). Relative to baseline values, cardiac contractility was increased after acute high dose LPS bolus, however this effect was negated by beta-blockade. Chronic, low dose LPS infusion produced an increase in cardiac contractility at 1 h, a return to baseline by 4 h, and maximal contractile depression by 24 h. No change was seen in LV compliance after the high dose LPS bolus. The LV end diastolic volume was decreased by the high dose LPS bolus. This change was blocked by propranolol administration. Chronic LPS administration was accompanied by a decrease in LV compliance and an increase in LV end diastolic volume. Other cardiovascular indices (heart rate, CO, systemic vascular resistance) changed in a fashion similar to human sepsis. These findings confirm that endotoxemia in conscious canine subjects causes changes in cardiovascular function similar to that seen in human and animal models of sepsis. This study also allows us to explain some of the discrepancies between earlier studies of human sepsis and animal models in which the appropriate clinical conditions and an intact neuro-endocrine axis were not maintained.

Near-infrared and nuclear magnetic resonance spectroscopic assessment of tissue energetics in an isolated, perfused canine hind limb model of dysoxia.

This controlled laboratory study examined the efficacy of near-infrared spectroscopy (NIRS) and 31P-nuclear magnetic resonance (NMR) spectroscopy in measuring regional tissue oxygenation in a isolated, perfused hind limb model of tissue dysoxia. Isolated hind limb perfusion was carried out in 20 mongrel dogs and oxygen delivery was varied by manipulating either hemoglobin concentration, oxygen saturation, or flow. Hind limbs from anesthetized mongrel dogs (n = 20) were separated and isolated perfusion performed. NIRS probes for recording relative O2 saturation of tissue hemoglobin (HbO2) and cytochrome a,a3 and NMR probes for measuring 31P-high energy phosphates were placed over the limb. Measurements of physiologic parameters, blood gases, lactate, NIRS values for HbO2 and cytochrome a,a3 redox state, and 31P-phosphate levels were recorded at set intervals throughout the experiment. Measures of tissue oxygen consumption (VO2) correlated with tissue oxygenation as measured by HbO2 and cytochrome a,a3 redox state (NIRS), as well as by 31P-high energy phosphate levels (NMR) throughout the experiment. Delivery-dependent tissue oxygenation was detected at a higher DO2 by NIRS than by VO2 or NMR. Tissue oxygenation as measured by NIRS and NMR shows excellent correlation with oxygen delivery in an isolated, perfused model of shock. NIRS may allow early detection of tissue dysoxia using rapid non-invasive techniques.

Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine.

An American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference was held in Northbrook in August 1991 with the goal of agreeing on a set of definitions that could be applied to patients with sepsis and its sequelae. New definitions were offered for some terms, while others were discarded. Broad definitions of sepsis and the systemic inflammatory response syndrome were proposed, along with detailed physiologic parameters by which a patient may be categorized. Definitions for severe sepsis, septic shock, hypotension, and multiple organ dysfunction syndrome were also offered. The use of severity scoring methods when dealing with septic patients was recommended as an adjunctive tool to assess mortality. Appropriate methods and applications for the use and testing of new therapies were recommended. The use of these terms and techniques should assist clinicians and researchers who deal with sepsis and its sequelae.

Entrapment of hepatocyte spheroids in a hollow fiber bioreactor as a potential bioartificial liver.

A bioartificial liver (BAL) employing xenogeneic hepatocytes has been developed as a potential interim support for patients in hepatic failure. For application in human therapy, the BAL requires a substantial increase in liver-specific functions. Cultivation of hepatocytes as spheroids leads to enhanced liver specific functions. We explored the possibility of entrapping spheroids into the BAL in order to improve device performance. Rat hepatocyte spheroids were entrapped in collagen gel within the lumen fibers of the BAL. The morphology and ultrastructure of collagen-entrapped spheroids resembled those of suspended spheroids formed on petri dishes. Albumin synthesis and P-450 enzyme activity were measured as markers of liver specific functions of spheroids entrapped in the BAL. At least a 4-fold improvement in these functions was observed compared to BAL devices entrapped with dispersed hepatocytes in collagen gels.

Hypermetabolism, organ failure, and metabolic support.

The hypermetabolism organ failure complex remains the predominant reason for both prolonged stay and death in the surgical intensive care unit. What was perceived as isolated organ failure, such as adult respiratory distress syndrome, is now seen as part of the systemic response to injury and repair. Sepsis has become the systemic inflammatory response due to invading microorganisms. What was once perceived as diagnostic of sepsis has been recognized after severe perfusion deficits and in the presence of continuing sources of dead and injured tissue. The transition to organ failure is usually a distinct clinical event and probably represents the onset of clinical hepatic failure. Once present, the organ failure syndrome has a high mortality rate. From a treatment perspective, it is recognized that there is probably no "magic bullet"; that regimens will probably be time dependent and "multiple drug"; and that the best treatment is prevention. Malnutrition, as opposed to changes in body composition that occur as a result of disease process, has become a recognized cofactor in morbidity and death in patients with persistent hypermetabolism and organ failure. The metabolic processes of hypermetabolism have become increasingly categorized and understood. The result has been the development of metabolic support principles that are distinct from those of nutritional support and are designed to prevent the end-organ changes of malnutrition and the development of substrate-limited metabolism, to support organ structure and function, and to attempt to arrest the metabolic processes. The initial problem was to learn to do no harm, an outcome reasonably achieved. In addition, several beneficial results have been recognized including new techniques to better support total body protein synthesis, hepatic protein synthesis, and energy production. Techniques to better support organ structure and function are being tested. No techniques are currently available to control proteolysis and the redistribution of skeletal muscle nitrogen. A great deal of research is still necessary in this field, which is still in its infancy.

Hemorrhagic infarction: A reperfusion injury following prolonged myocardial ischemic anoxia.

Sixteen canine myocardial pedicle preparations were divided into four groups. Group I received 60 minutes of reversible ischemic anoxia, Group II 90 minutes, Group III 120 minutes, and Group IV 180 minutes. Transmural ischemic infarction was present in Group I. With longer periods of anoxia, capillary disruption occurred, first in the subendocardium (90 minutes) and then transmurally (180 minutes). Reperfusion after ischemic, anoxic time periods of 90 minutes or greater produced hemorrhagic necrosis, the extent of which was directly proportional to the duration of the preceding ischemic, anoxic period. One hundred and twenty patients with aortic valve replacement surgery then were analyzed retrospectively. Of the 25 deaths, seven were due to immediate postinfarction, five from subendocardial hemorrhagic necrosis. The common factor in the patients with hemorrhagic infarction was a markedly reduced coronary flow (45 to 55 ml. per minute) for more than 70 minutes during the interval of cardiopulmonary bypass. Although other factors may be involved, it is hypothesized that the main cause is a degree of localized ischemia of sufficient duration to induce capillary disruption and subsequent postbypass hemorrhagic necrosis when normal coronary artery perfusion is resumed.

The endothelial damage of pulsatile renal preservation and its relationship to perfusion pressure and colloid osmotic pressure.

To evaluate the effects of pulsatile preservation, 34 heterotopic renal autografts with immediate contralateral nephrectomy were divided into four groups with either the pulsatile preservation or cold storage technique. Group I had pulsatile preservation with plasma-albumin perfusate at a mean pressure of 60 mm. Hg and a colloid osmotic pressure (COP) of 20 to 50 cm. H2O. Group II had pusatile preservation with the same plasma-albumin perfusates as in Group I, but at a mean pressure of 30 mm. Hg. Group III had pulsatile preservation with albumin-phosphate buffer solution at a mean pressure of 30 mm. Hg. Group IV used cold storage technique. Group I demonstrated severe endothelial destruction and denuding of basement membrane. The changes were inversely related to COP. None of these kidneys functioned and, following transplantation, had persistent severe tubular damage and plugging of glomerular capillary loops with platelets, fibrinogen, and white cells. In Groups II and III, there were minimal changes, consisting of widening of the endothelial pores and focal endothelial damage. All of these kidneys functioned following reimplantation with minimal ultrastructural alterations. Group IV had no glomerular changes following preservation or following transplantation. All preservation techniques produced proximal tubular damage. There is a definite mechanical lesion associated with pulsatile preservation, which can be minimized by utilizing low perfusion pressures and solutions of high colloid osmotic pressure and which has the potential of producing a picture resembling hyperacute rejection following transplantation.

Efficacy of type-specific and cross-reactive murine monoclonal antibodies directed against endotoxin during experimental sepsis.

To study the role of antibodies in promoting survival during gram-negative bacterial sepsis, we have developed several murine monoclonal antibodies (MAbs). One MAb (5B10) reacted in an enzyme-linked immunosorbent assay with only a single organism (Escherichia coli 0111:B4), while the other (8A1) reacted to all gram-negative whole-cell and lipopolysaccharide (LPS) antigens examined. Either 5B10 MAb, 8A1 MAb, or sterile saline solution was administered intravenously to outbred male Swiss-Webster mice immediately before one of three challenges: (1) viable bacteria intravenously, (2) viable bacteria with hemoglobin intraperitoneally, or (3) intravenous actinomycin D plus LPS. 5B10 MAb provided significant protection against either an E. coli 0111:B4 bacterial or LPS challenge but not against any other organism or type of LPS. 8A1 MAb provided protection against several challenge bacteria (intravenously or intraperitoneally) and against all types of LPS studied except Pseudomonas aeruginosa LPS. A higher dose (2 mg) of cross-reactive antibody (8A1 MAb) was required to produce protection when compared with the type-specific protection produced with 5B10 MAb (0.1 mg). Although ideal antibody therapy would consist of directing a specific MAb against a single microorganism, the acute nature of the disease process and time required to prepare reagents may preclude the use of type-specific MAbs. We believe that the cross-reactive and cross-protective capacity of 8A1 MAb or a similar MAb may be useful in averting the lethal effects of clinical gram-negative bacterial sepsis and warrants testing in the clinical setting.

Plasma amino acid clearance as an indicator of hepatic function and high-energy phosphate in hepatic ischemia.

A major function of the liver is to maintain normal levels of plasma amino acids. This function may depend in part on tissue levels of high-energy phosphate. Experiments were performed in adult mongrel dogs to assess the relationship between plasma amino acid clearance (PAAC) and tissue high-energy phosphate after 90 minutes of warm hepatic ischemia. In addition, when PAAC was assessed in the anhepatic dog, PAAC fell to low levels after hepatectomy. After 90 minutes of warm ischemia, both tissue adenosine triphosphate (ATP) and PAAC fell to low levels, with PAAC similar to those levels observed in anhepatic dogs. Recovery of ATP and PAAC progressed over a 48-hour period but did not reach control values. Mortality rate was 33% in a group of 12 animals at 48 hours after ischemia. At 24 hours after ischemia, total free plasma amino acid levels were significantly higher in those animals that were dying as compared with those that were surviving (4352 mumol/L versus 2850 mumol/L; p less than 0.05). There was a strong correlation between PAAC and ATP (r = 0.81). PAAC appears to be an indicator of hepatic functional recovery and tissue ATP levels after ischemia.

Further characterization of Kupffer cell/macrophage-mediated alterations in hepatocyte protein synthesis.

Alterations in hepatic function are seen in sepsis or multiple-system organ failure. We have hypothesized that Kupffer cells (KCs) within the liver alter the function of contiguous hepatocytes after exposure to septic stimuli. Using an in vitro coculture system, we have found that lipopolysaccharide (LPS) induced a 40% to 60% decrease in cocultured hepatocyte protein synthesis but had no effect on hepatocytes alone. Coculture in the absence of LPS resulted in enhanced hepatocyte protein synthesis proportional to the number of KCs or macrophages (M0s). Conditioned medium (CM) from LPS-triggered coculture or KC alone decreased protein synthesis of hepatocytes whereas CM from hepatocytes alone had no effect. Gel filtration of active M0-CM showing maximal hepatocyte inhibitory activity was present in fractions between 15,000 and 30,000 daltons. This inhibitory activity was inactivated by exposure to 65 degrees C for 30 minutes. Although CM from untriggered M0 did not inhibit hepatocyte protein synthesis, lysates from both LPS-triggered and control M0 were inhibitory. These results show that M0s/KCs exposed to septic stimuli decrease hepatocyte protein synthesis via heat labile, soluble mediator(s) that may already be synthesized within untriggered cells. We hypothesize that soluble M0/KC mediators normally modulate hepatocyte function and that this normal homeostatic control is profoundly altered during sepsis.

Enteral nutrition does not prevent multiple organ failure syndrome (MOFS) after sepsis.

Gut malnutrition in patients with persistent hypermetabolism is hypothesized to be an important factor in postseptic multiple organ failure syndrome (MOFS). The hypothesis was made that enteral nutrition (EN) started at the onset of hypermetabolism could reduce the incidence of MOFS. Sixty-six patients with persistent hypermetabolism 4 to 6 days after onset of sepsis were prospectively randomized to receive either parenteral nutrition (PN) or enteral nutrition (EN) at 1.5 gm protein/kg/day and 30 nonprotein calories/kg/day; the EN and TPN were of the same composition. There was no reduction in either the incidence of MOFS or mortality attributable to the route of nutrition administration. The PN group tended to have better visceral protein support; the EN group had more gut complications. When analyzed, the type of formula given did have an effect on the nutritional outcome but not on the mortality rate. A formula with a nonprotein-calorie-to-nitrogen ratio of 100:1 was associated with more nitrogen retention, higher levels of visceral proteins, and better gut tolerance. The route of nutrition administration does not seem to affect the incidence of postseptic MOFS or mortality when hypermetabolism is already present and when commercially available nutritional formulas are used. The relationships among the route of nutrition, the type of enteral formula, and the disease process of hypermetabolism and MOFS appear to be complex and require much more investigation before the role of the gut and enteral nutrition can be defined.

Cirrhosis, encephalopathy, and improved results with metabolic support.

Twenty-two patients with cirrhosis and acute encephalopathy who were refractory to medical therapy were entered into a randomized, double-blind prospective trial. This trial consisted of either neomycin or a modified amino acid solution rich in branched chains and low in aromatic amino acids and methionine (F080) in the presence of isocaloric amounts of dextrose. The groups were indistinguishable from each other by clinical or laboratory criteria; they were primarily patients who had undergone operation and they would tolerate only 30 gm of oral protein or intravenous standard amino acids. The group receiving F080 had a faster and more complete improvement in encephalopathy. This improvement correlated with the plasma molar ratio and occurred with a lower mortality rate. In addition, the patients also tolerated twice the amino acid load without encephalopathy and were in positive nitrogen balance. Modified metabolic support is effective in the setting of acute liver failure in chronic cirrhosis, particularly in patients who have undergone operation.

Anticore endotoxin F(ab')2 equine immunoglobulin fragments protect against lethal effects of gram-negative bacterial sepsis.

Gram-negative bacterial sepsis and shock remain a cause of substantial morbidity and mortality in hospitalized patients despite appropriate antimicrobial therapy, fluid resuscitation, and monitoring. We sought to test the ability of equine antibody directed against core endotoxin, a portion of bacterial outer membrane lipopolysaccharide common to many gram-negative microorganisms, to bind to various gram-negative bacteria in vitro, to promote bacterial phagocytosis by leukocytes, and to protect against lethal gram-negative bacteremia in mice. The importance of the IgG Fc leukocyte attachment site was examined by comparing the ability of intact IgG and IgG F(ab')2 fragments to protect against lethality during murine sepsis. A single horse was immunized with Escherichia coli J5, an organism that expresses a portion of core endotoxin extensively on the cell surface. Preimmunization IgG and F(ab')2 possessed no titer as determined by enzyme-linked immunosorbent assay, did not promote in vitro phagocytosis, and did not protect in vivo. Postimmunization IgG and F(ab')2 possessed a significant titer to E. coli J5 whole cell and lipopolysaccharide antigens and provided significant (p less than 0.05) protection in vivo during lethal intravenous sepsis caused by either E. coli J5, E. coli 0111:B4, Klebsiella pneumoniae, or Pseudomonas aeruginosa. Only postimmunization IgG, but not F(ab')2, promoted in vitro phagocytosis of these same organisms. We therefore hypothesized that protection occurred as a result of antitoxin activity rather than opsonization and phagocytosis, as F(ab')2 fragments were as active as the intact molecule. Further studies must be done to determine the role of anticore endotoxin antibody in conjunction with antibiotics so that appropriate clinical studies may be undertaken.

Branched chains support postoperative protein synthesis.

A blinded, prospective trial of the effects of branched-chain amino acid (BcAA)-enriched total parenteral nutrition (TPN) versus standard TPN was undertaken in nonseptic noncirrhotic abdominal surgery patients and patients with multiple traumatic injuries. The study reflected data from the immediate 7-day postoperative period. With isocaloric and isonitrogenous input, the BcAA-TPN patients achieved positive nitrogen balance on day 3. Although the urinary nitrogen output was decreased by day 3 in BcAA-TPN, the plasma BcAA levels did not increase until day 6. With no change in 3-methylhistidine urinary excretion, the early nitrogen retention with BcAA-TPN probably reflects a stimulation of protein synthesis. The ability to favorably modulate the metabolic stress response with alternate fuels has become a clinical reality.