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Serum cachectin/tumor necrosis factor (TNF), a cytokine implicated in the pathogenesis of septic shock, may appear in the circulation during serious infection, but the frequency of detection of elevated serum levels during protracted critical burn injury is unknown. Serial serum samples taken from 43 critically ill patients with burns with and without sepsis were analyzed for TNF using an enzyme-linked immunosorbent assay (ELISA). TNF was detectable (greater than 34 picograms per milliliter) at one or more time points in 69 per cent of the patients with sepsis versus 33 per cent of those without sepsis, in 71 per cent of the patients who died versus only 31 per cent of the survivors and in only one healthy normal control patient (n = 21). The frequency of the appearance of TNF correlated with both infection and mortality rate. Moreover, all three patients with TNF levels greater than 540 picograms per milliliter died. Neither the size of the burn nor injury from inhalation correlated with detection of TNF. A subset of 16 patients was studied longitudinally from admission until resolution of injury and these data demonstrate that TNF appears transiently and repetitively in the circulation of patients during infection and protracted critical burn injury. Also, serum cortisol levels were significantly higher during sepsis and death in the absence of serum TNF, compared with sepsis and death with detectable cachectin, suggesting that cortisol may interact with the production or detection of this cytokine during ongoing infection and lethal injury. In this study, we have demonstrated that serum TNF is detectable with greater frequency and in higher concentration in patients with sepsis and in those who ultimately succumb to the burn injury, that serum TNF appears transiently and repetitively in the circulation during injury and that higher serum cortisol levels are correlated with the absence of serum TNF during sepsis and lethal injury.

C57BL/6 mice bearing either a transplantable methylcholanthrene-induced sarcoma or Lewis lung adenocarcinoma were passively immunized every other day with a rabbit immunoglobulin fraction raised against murine cachectin/tumor necrosis factor-alpha. Mice bearing methylcholanthrene-induced sarcoma developed tumor-associated hypophagia that was attenuated by anticachectin immunoglobulin treatment. In the same tumor-bearing animals, anticachectin treatment also significantly reduced the extent of carcass protein and fat loss, and reduced tumor weight. Mice bearing Lewis lung adenocarcinoma did not develop significant anorexia or carcass lean tissue depletion as tumor growth progressed, but they lost carcass lipid. Treatment of Lewis lung adenocarcinoma bearing mice with anticachectin antibodies diminished the degree of carcass lipid depletion and prevented plasma hypertriglyceridemia. However, in both tumor models, anticachectin treatment did not affect either the development of anemia, hypoalbuminemia or the increase in serum amyloid P concentrations seen with increasing tumor burden. We conclude that an endogenous cachectin response, inhibitable by exogenously administered antibody, contributes to anorexia and to changes in body fat and protein metabolism in these tumor-bearing animals. Neutralizing endogenous cachectin production with antibodies offers the potential to reduce tissue wasting that is frequently associated with neoplastic disease, but it does not appear to affect all of the hematologic and acute phase responses in these murine tumor models.

Chronic inflammatory diseases are often associated with decreased red blood cell (RBC) mass. The cytokines cachectin/tumor necrosis factor-alpha (TNF) and interleukin 1 (IL 1) are produced by monocytes/macrophages in response to many inflammatory stimuli and have been implicated in the anemia of chronic disease. This study was undertaken to evaluate the mechanisms by which cachectin/TNF, IL 1, or endotoxin induce anemia. Hematologic parameters and RBC kinetics were quantitated in rats given chronic sublethal quantities of either recombinant human cachectin/TNF, recombinant human IL 1 alpha, or Salmonella endotoxin for 7 days. Cachectin/TNF or endotoxin treatment resulted in a 25 or 31% decrease, respectively, in total RBC mass, whereas RBC mass was unchanged by IL 1 administration. Anemia associated with either chronic cachectin or endotoxin administration was characterized by normal mean corpuscular volume, mean corpuscular hemoglobin content, and reticulocyte numbers. [59Fe]RBC survival was significantly shortened in animals given cachectin, IL 1 or endotoxin, but the magnitude of the response was greatest in cachectin/TNF-or endotoxin-treated rats. Although cachectin/TNF-IL 1-, or endotoxin treatment resulted in similar hypoferremia and shortened plasma iron half-life, endotoxin or cachectin/TNF treatment (but not IL 1) significantly reduced the incorporation of plasma 59Fe into newly synthesized RBCs. We conclude that chronic cachectin/TNF administration produces anemia by decreasing RBC synthesis and reducing the life span of circulating RBCs. An endogenous cachectin/TNF response during inflammatory disease may contribute to an associated anemic state, whereas the modestly reduced red cell life span induced by IL 1 does not lead to a net reduction in RBC mass, presumably owing to a preserved RBC synthetic rate.

Burn injury and infection result in significant losses of lean tissue. The cytokine cachectin/tumor necrosis factor has been implicated in this process but is not uniformly detected during infection. We sought to determine the relationship between body composition changes and in vivo hepatic levels of pretranslational message for cachectin (messenger RNA) in a burn and infection rodent model. Adult Wistar rats were grouped as follows: (1) freely fed, (2) 30% burn, (3) 30% burn with Pseudomonas aeruginosa infection, (4) pair fed, and (5) 30% burn and infection with recombinant cachectin. Compared with controls or animals only burned, burned and infected rats had a 100% increase in hepatic cachectin messenger RNA content, lost carcass protein, and exhibited muscle loss with sparing of liver mass. Tissue production of cachectin as well as other cytokines may be sufficient to mediate several body composition changes observed in response to injury and infection.

Intestinal mucosal atrophy, as induced by total parenteral nutrition (TPN) and/or prolonged bowel rest, is hypothesized to enhance bowel endotoxin (LPS) translocation and may alter host responses to infection. To examine the effect of TPN-induced bowel atrophy on the response to LPS, 12 healthy volunteers were randomized to receive either enteral feedings (ENT, n = 6) or seven days of TPN without oral intake (TPN, n = 6). Enteral or TPN feedings were terminated 12 hours before the study period when a constant dextrose infusion (50 mg/kg/hour) was initiated and continued throughout the subsequent study period. After placement of arterial, hepatic vein, and femoral vein catheters, metabolic parameters were determined before and for six hours after an intravenous E. coli LPS challenge (20 U/kg). Subsequent peak levels of arterial glucagon (ENT, 189 +/- 39 pg/mL; TPN, 428 +/- 48; p less than 0.01), arterial epinephrine (ENT, 236 +/- 52 pg/mL; TPN, 379 +/- 49; p less than 0.05) and hepatic venous cachectin/tumor necrosis factor (cachectin/TNF) (ENT, 250 +/- 56 pg/mL; TPN, 479 +/- 136; p less than 0.05) were significantly higher in the TPN group than in the ENT group. The extremity efflux of lactate (ENT, -16 +/- 4 micrograms/min-100cc tissue; TPN, -52 +/- 13; t = 2 hours; p less than 0.05) and of amino acids (ENT, -334 +/- 77 nmol/min-100cc tissue; TPN, -884 +/- 58; t = 4 hours; p less than 0.05) were higher in the TPN subjects after the endotoxin challenge. Circulating C-reactive Protein (CRP) levels measured 24 hours postendotoxin were also significantly higher in the TPN subjects (ENT, 1.7 +/- 0.2 mg/dL; TPN, 3.2 +/- 0.3; p less than 0.01). Hence the counter-regulatory hormone and splanchnic cytokine responses to LPS were enhanced after TPN and bowel rest. This is associated with a magnified acute-phase response, peripheral amino acid mobilization, and peripheral lactate production. Thus antecedent TPN may influence the metabolic alterations seen in infection and sepsis via both an exaggerated counter-regulatory hormone response as well as an enhanced systemic and splanchnic production of cytokines.

Hepatocellular membrane dysfunction, as indicated by depolarization of the membrane potential, occurs after acute injury and early bacteremia. To determine whether hepatocellular membrane dysfunction occurs in the setting of ongoing thermal injury and infection, Wistar rats were divided into four groups: (1) sham-burned, freely fed controls (FF); (2) rats sustaining approximately 30% total body surface area dorsal full-thickness scald burn (Burn); (3) rats sustaining burns as in group 2 followed by immediate inoculation of 1 x 10(8) CFU Pseudomonas aeruginosa (Burn/Inf); and (4) sham-burned rats pair-fed to the food intake of the Burn/Inf group (PF). On the third and seventh days postburn, body and liver weights were determined. In vivo hepatocellular transmembrane potentials were measured and hepatic ATP, RNA, DNA, and protein contents were assayed. By Day 7, despite greater weight loss in the Burn/Inf group than due to starvation alone (P less than 0.01 Burn/Inf vs FF and PF), hepatic mass was conserved. This was associated with hyperpolarization of the hepatic transmembrane potential (-46.6 +/- 1.5 vs -32.1 +/- 0.6 mV, Burn/Inf vs FF, P less than 0.01) and increases in RNA (141 +/- 9 vs 91 +/- 4 mg/liver, Burn/Inf vs FF, P less than 0.01) and DNA (37 +/- 5 vs 22 +/- 2 mg/liver, Burn/Inf vs FF, P less than 0.05) contents, with no change in ATP or hepatic protein contents. There was a significant hypercorticosteronemia observed in the Burn/Inf group (43 +/- 9 vs 2.8 +/- 0.7 micrograms/dl, Burn/Inf vs FF, P less than 0.01). This hepatic membrane hyperpolarization and augmented RNA content were not secondary to burn or starvation alone as the response in these groups was significantly less than that of the Burn/Inf group. It is suggested that this hepatic membrane hyperpolarization is one mechanism by which hepatic function is maintained during ongoing burn infection in the rat.

Lipopolysaccharide (LPS, endotoxin) administration to human subjects elicits significant elevations in plasma cachectin/TNF, epinephrine, and cortisol. This study examined the temporal relationship between changes in blood leukocyte subsets and plasma mediators following endotoxin administration to normal human subjects. A five-minute intravenous infusion of purified LPS (20 units/kg Escherichia coli) was administered to 12 healthy volunteers. Blood samples were obtained at varying intervals after infusion and analyzed for differential cell counts and lymphocyte subsets (CD2, CD3, CD4, CD8, CD20, and HLA-DR) by flow microfluorimetry, and also assayed for plasma cachectin/TNF, epinephrine, and cortisol. Plasma cachectin/TNF was significantly elevated at 75 and 90 minutes after infusion with a peak concentration of 261 +/- 115 pg/ml noted 75 minutes after infusion. A significant plasma epinephrine elevation of 181 +/- 75 pg/ml was demonstrated one hour after infusion, while significant elevations in plasma cortisol were noted from one to five hours after infusion with a peak level of 34 +/- 3 micrograms/dl three hours after infusion. A profound monocytopenia (p less than 0.01) was noted one hour after infusion. Temporally associated with the rise in plasma cortisol was a reversal of the early granulocytopenia to a significant granulocytosis (p less than 0.01 versus preinfusion mean), whereas a marked lymphocytopenia (p less than 0.01) was observed from one to six hours after infusion. During the period of hypercortisolemia, CD2, CD3, and CD4 lymphocyte percentages were decreased (p less than 0.01) while CD20 and HLA-DR lymphocyte percentages were increased (p less than 0.01). There was a small percentage decrease in CD8 lymphocytes from one to 24 hours after infusion (p less than 0.01), although relative to the one-hour nadir, there was a significant rise in the percentage during the time of elevated plasma cortisol concentrations. A six-hour infusion of epinephrine (30 ng/kg/min) administered to six healthy volunteers resulted in a monocytosis (p less than 0.05) and granulocytosis (p less than 0.01) without a change in lymphocyte number or lymphocyte subset percentage. Previous reports have shown that in vivo corticosteroid infusion causes a prominent granulocytosis, monocytopenia, and lymphocytopenia with a decrease in the percentages of CD3 and CD4 lymphocytes. The peripheral blood leukocyte dynamics documented in the current study are similar to patterns observed following in vivo corticosteroid administration. This study suggests that the acute adrenocortical response to endotoxemia primarily mediates the subsequent changes in leukocyte subsets.

Experiments were conducted to evaluate the teratogenic potential of nickel chloride given to developing chick embryos. Nickel chloride was dissolved in saline and injected into chicken eggs at dosages ranging from 0.02 to 0.7 mg per egg. The injections were made into the air sacs of eggs at days 0, 1, 2, 3, and 4 of incubation. Control eggs were injected with an equivalent volume of saline (0.1 ml per egg); in all, 840 chicken eggs were used. All embryos were examined on day eight. The following malformations were observed: exencephaly, everted viscera, short and twisted neck, short and twisted limbs, microphthalmia, hemorrhage, and reduced body size. The dose-response relationship was observed in all of the groups tested. The toxicity and teratogenicity of nickel chloride was the highest in the embryos treated at day 2. The results of the present study indicate that nickel chloride is teratogenic.

Malnutrition is a common complicating factor in surgical illness. To investigate the cellular changes and mechanisms responsible for the protein wasting associated with nutritional deprivation, Sprague-Dawley rats were subjected to total protein-calorie starvation for 3 (n = 12) or 5 days (n = 12) and compared to freely fed animals monitored for 3 (n = 8) or 5 (n = 8) days. Gastrocnemius protein and RNA content and levels of mRNA coding for the myofibrillar proteins myosin heavy chain, myosin light chain, and alpha-actin were measured. Starvation resulted in a significant decrease in gastrocnemius mass and protein content, and was associated with decreases in mRNA levels for the three myofibrillar proteins assayed. We conclude that changes in mRNA levels for these proteins likely contribute to the loss of peripheral protein which occurs during total nutritional deprivation. In addition, the changes in mRNA levels for these three structural proteins appear to be coordinate, suggesting that transcription of no single myofibrillar protein is rate-limiting in the regulation of skeletal muscle protein content.

Using single- and two-color fluorescence flow cytometry, 10 thermally injured human subjects were assessed over time for both percentages and absolute numbers of lymphocytes comprising peripheral blood lymphocyte subpopulations. The CD3+ lymphocyte percentage decreased significantly in the early postburn period, and this decrease could be accounted for entirely by a concomitant decrease in the CD4+ lymphocyte percentage. Further, the decline in CD4+ percentage was due to a specific decrease in the suppressor-inducer subset of CD4 as defined using anti-CD45R. No change in the helper-effector subset of CD4 was noted. The percentage of CD8+ lymphocytes did not change significantly at any time postburn nor did subsets of CD8 as defined using anti-CD11. Numerical changes in lymphocyte subsets were dominated by a general lymphopenia occurring on Day 4 following injury. However, suppressor-inducer (CD4+/CD45R+) T cells also decreased significantly on postburn Day 1. These results further elucidate phenotypic changes in immunoregulatory subsets following major injury and suggest a possible basis for depressed autologous mixed lymphocyte responsiveness of burn patient T cells, one of the functional immunologic defects associated with severe injury.