Human tumor necrosis factor receptor (p55) and interleukin 10 gene transfer in the mouse reduces mortality to lethal endotoxemia and also attenuates local inflammatory responses.

Anticytokine therapies have been promulgated in gram-negative sepsis as a means of preventing or neutralizing excessive production of proinflammatory cytokines. However, systemic administration of cytokine inhibitors is an inefficient means of targeting excessive production in individual tissue compartments. In the present study, human gene transfer was used to deliver to organs of the reticuloendothelial system antagonists that either inhibit tumor necrosis factor-alpha (TNF-alpha) synthesis or block its interactions with cellular receptors. Mice were treated intraperitoneally with cationic liposomes containing 200 micrograms of either a pCMV (cytomegalovirus)/p55 expression plasmid that contains the extracellular domain and transmembrane region of the human p55 TNF receptor, or a pcD-SR-alpha/hIL-10 expression plasmid containing the DNA for human interleukin 10. 48 h later, mice were challenged with lipopolysaccharide (LPS) and D-galactosamine. Pretreatment of mice with p55 or IL-10 cDNA-liposome complexes improved survival (p < 0.01) to LPS-D-galactosamine. In additional studies, intratracheal administration of IL-10 DNA-liposome complexes 48 h before an intratracheal LPS challenge reduced pulmonary TNF-alpha levels by 62% and decreased neutrophil infiltration in the lung by 55% as measured by myeloperoxidase activity (both p < 0.05). Gene transfer with cytokine inhibitors is a promising option for the treatment of both the systemic and local sequelae of septic shock.

A human tumor necrosis factor p75 receptor agonist stimulates in vitro T cell proliferation but does not produce inflammation or shock in the baboon.

Tumor necrosis factor (TNF) is a potentially useful adjunct to anticancer therapies. However, the clinical utility of TNF has been limited by generalized toxicity and hypotension. Recently, studies have begun to dissect the individual proinflammatory and immunologic responses that result from TNF binding to its two cellular receptors, p55 and p75, in an attempt to develop TNF receptor agonists with reduced systemic toxicity. To evaluate a p75 receptor selective TNF mutant (p75TNF), TNF and p75TNF were administered to healthy anesthetized baboons. Intravenous infusion of the p75TNF produced none of the hemodynamic changes seen after the infusion of TNF. Infusion of p75TNF also failed to induce the plasma appearance of interleukins 6 and 8. However, p75TNF enhanced in vitro baboon thymocyte proliferation to concanavalin A, and infusion of p75TNF resulted in increased soluble p55 and p75 receptor plasma concentrations. Local skin necrosis and tissue neutrophil infiltration were seen after subcutaneous injections of TNF and p55TNF. Subcutaneous injection of p75TNF did not result in skin necrosis but did result in a modest dermal infiltration of lymphocytes and macrophages. The findings suggest that p75TNF may stimulate T cell proliferation without the systemic and local toxicity seen with TNF.

Enhanced hepatic amino acid transport in tumor-bearing rats is partially blocked by antibody to tumor necrosis factor.

The liver of the host with cancer exhibits an enhanced requirement for amino acids to support tumor-induced increases in hepatic protein synthesis and gluconeogenesis. To address the mechanism by which the liver ensures adequate delivery of these substrates for intracellular utilization during cancer, we studied the activities of several amino acid transporters in hepatic plasma membrane vesicles prepared from rats implanted with a rapidly growing s.c. fibrosarcoma. The presence of the tumor resulted in a generalized stimulation of concentrative (Na(+)-dependent) glucogenic (small neutral) amino acid uptake via System A (3.4-fold), System N (2.3-fold), and System ASC (1.7-fold), as well as in the facilitative (Na(+)-independent) uptake of arginine via System y+ (1.7-fold). Kinetic analysis revealed that the tumor-induced enhancement of transport activity was due to increases in the maximum transport velocity (Vmax), whereas transporter substrate affinities (Km) did not change significantly. Administration of antibody to tumor necrosis factor-alpha to tumor-bearing rats attenuated the increase in hepatic amino acid transport activity by 60-100%. Treatment of nontumor-bearing control rats with tumor necrosis factor-alpha mAb did not alter basal transport activity. The results from these studies suggest that the tumor elicits a generalized increase in hepatic plasma membrane amino acid transport activity via a pathway that involves the cytokine tumor necrosis factor.

Parenteral nutrition techniques in cancer patients.

If a patient is expected to respond optimally to one or more forms of oncologic therapy, he should simultaneously be in the best possible nutritional and metabolic condition. When the alimentary tract cannot be used effectively for feeding cancer patients, parenteral nutrition can be lifesaving. Moreover, patients who are poor candidates or noncandidates for any antineoplastic therapy because of their debility or cachexia can be converted to reasonable candidates following a course of i.v. hyperalimentation. This i.v. hyperalimentation can significantly reduce the morbidity and mortality of cancer patients without stimulating tumor growth when applied conscientiously according to the established principles and techniques and when integrated with specific tumor therapy. With the use of ambulatory or home hyperalimentation techniques, normal nutritional status can be restored or maintained during prolonged periods of antineoplastic therapy on a practical and relatively economical outpatient basis. It is anticipated that specific nutrient substrate formulas and parenteral therapy techniques will be developed to maintain optimal host nutrition while adversely affecting the neoplasm.

Conservative treatment of rectal adenocarcinoma with endocavitary irradiation or wide local excision and postoperative irradiation.

PURPOSE: To evaluate the role of endocavitary irradiation and wide local excision followed by irradiation in the treatment of early-stage rectal adenocarcinoma. MATERIALS AND METHODS: Sixty-five patients with early-stage adenocarcinoma of the rectum were treated with endocavitary irradiation (n = 20) or wide local excision followed by external-beam irradiation (n = 45) between 1974 and 1994 at the University of Florida. All patients were monitored for a minimum of 2 years or until death. RESULTS: The rates of local-regional control at 5 years were 80% after endocavitary irradiation and 86% after wide local excision and radiotherapy. The ultimate 5-year local-regional control rates were 85% and 92%, respectively. Multivariate analysis of local-regional control with sphincter preservation showed that tumor configuration (exophytic v ulcerative) significantly influenced this end point; local-regional control was decreased in patients with ulcerated cancers. Five-year cause-specific survival rates were 84% after endocavitary irradiation and 88% after wide local excision and radiotherapy. Multivariate analysis revealed that tumor configuration significantly influenced cause-specific survival; patients with ulcerated tumors had a worse prognosis. CONCLUSION: Endocavitary irradiation is a highly effective treatment for properly selected patients with early-stage rectal adenocarcinoma. Patients with less favorable lesions that appear to be limited to the muscularis propria have a high chance of cure with sphincter preservation after wide local excision and external-beam irradiation.

Total parenteral nutrition in mice bearing a metastatic carcinoma: tumor growth, metastasis and immunologic parameters.

The role of dietary manipulation of tumor growth, metastasis and immunologic parameters was studied in mice bearing Lewis lung carcinoma. Fourteen days following subcutaneous tumor implant, groups with tumor and their non-tumor bearing counterparts were assigned to one of the following feeding protocols: total parenteral nutrition (TPN), per oral (PO) intake of the parenteral diet, an oral casein diet (CAS), or electrolyte infusion plus the casein diet (ELECT). Intakes of energy and nitrogen were similar among all groups. Mice were killed 12 days later and peritoneal macrophages were tested for phagocytic activity. Tumor growth and metastasis were decreased from both infusion regimens with minimal loss of body weight as compared with casein fed mice. PO mice also showed lower tumor weight but metastasis was as great as in the casein group. Non-tumor-bearing infused mice showed depressed thymic weight, but thymic weight was not further reduced in tumor-bearing infused mice. PO feeding afforded no such protection in the presence of the carcinoma. Splenomegaly was observed in tumor-bearing mice on all regimens, but mice maintained on the parenteral diet demonstrated the largest proportion of macrophages containing nuclear debris. Analysis of free macrophages indicated no effect of diet regimen on non-immune phagocytic activity in both tumor-free and tumor-bearing mice. Possible alteration of splenic macrophage intracellular digestive capacity or phagocytic activity was suggested as a result of TPN.

Isolated local-regional recurrence following mastectomy for adenocarcinoma of the breast treated with radiation therapy alone or combined with surgery and/or chemotherapy.

The results of radiation therapy alone or combined with surgery and/or chemotherapy are reported for 47 patients who presented with local and/or regional recurrence without evidence of distant metastases following initial management of adenocarcinoma of the breast with radical or modified radical mastectomy (43) or simple mastectomy (4). Patients were treated between October 1964 and March 1983 at the University of Florida; all have a 2-year minimum follow-up and 42/47 (89%) have had follow-up for greater than or equal to 5 years. The overall actuarial local-regional control rates were 80% at 2 years, 68% at 5 years, and 61% at 10 years. The 5-year actuarial local-regional control rates by site and extent of disease were as follows: single chest wall nodule, 92%; multiple chest wall nodules, 49%; regional lymph nodes, 66%; and multiple sites, 64%. The 5- and 10-year actuarial determinate disease-free survival rates for all patients were 41 and 17%, respectively. The 5- and 10-year actuarial survival rates for all patients were 50 and 34%, respectively.

Interleukin-1 and interleukin-1 antagonism in sepsis, systemic inflammatory response syndrome, and septic shock.

Interleukin-1 (IL-1) is one of several proinflammatory cytokines produced during infection, sepsis, and the systemic inflammatory response syndrome (SIRS) that serves to initiate the host inflammatory response and to integrate nonspecific immunity. Many of IL-1's biologic effects are beneficial to the host in times of stress, but when produced for extended periods of time or in excessive quantities, IL-1 contributes to morbidity and mortality. In fact, excessive IL-1 production has been directly linked to the development of hypotension, shock, multi-organ system failure, hematologic dyscrasia, and death in patients and animals with sepsis, SIRS, and septic shock. Recent research interest has focused on IL-1 inhibition to improve outcome in sepsis and septic shock. This article will review the role for IL-1 in sepsis and septic shock, and the function and status of the IL-1 receptors and IL-1 receptor antagonist in modulating IL-1 actions. The results of investigations of IL-1 inhibition in animal models and in human subjects with sepsis and septic shock will also be reviewed.

Application of gene therapy to acute inflammatory diseases.

The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.

A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor alpha (TNF alpha) and abrogates endotoxin-induced lethality.

Excessive tumor necrosis factor alpha (TNF alpha) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNF alpha production rely on monoclonal antibodies or immunoadhesins that bind TNF alpha and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNF alpha processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 microgram/mL endotoxin, GM-6001 at concentrations > 5 micrograms/mL blocked release of TNF alpha, but did not affect the release of either IL-1 beta or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNF alpha. To confirm the role of secreted TNF alpha in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 micrograms/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNF alpha response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1 beta and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNF alpha processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNF alpha both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

Endotoxin stimulates arginine transport in pulmonary artery endothelial cells.

BACKGROUND: The pulmonary endothelium plays an important role in the metabolism of the amino acid arginine, the exclusive precursor molecule for nitric oxide (NO). Despite decreased circulating arginine levels, endothelial NO production is elevated during endotoxemia. However, the regulation of pulmonary artery endothelial arginine transport has not been studied. We hypothesized that endotoxin stimulates carrier-mediated arginine transport by the pulmonary endothelium. METHODS: The relative contributions of the various transport systems to total arginine transport by porcine pulmonary artery endothelial cells (PAECs) was determined by assaying the uptake of 3H-L-arginine in the presence or absence of Na+. PAECs were then incubated with various concentrations of Escherichia coli endotoxin, and y(+)-mediated arginine transport was measured at different time points thereafter. Kinetic studies were performed over a range of arginine concentrations to determine changes in transport affinity and maximum rate of metabolism. To address the role of RNA and protein synthesis in the increased transport, uptake was measured after exposure of cells to the transcriptional inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide. RESULTS: Most (75%) of arginine transport by PAECs was mediated by the high-affinity Na(+)-independent transport system y+. Endotoxin stimulated y(+)-mediated arginine transport by PAECs twofold to fivefold, a response that was time and dose dependent. The accelerated transport was detectable within 2 hours and maximal at 12 hours. Kinetic studies revealed that the accelerated arginine transport was the result of a 68% increase in the maximal transport velocity (1519 +/- 65 pmol/mg protein/30 sec in endotoxin-treated cells vs 903 +/- 96 in control cells; p < 0.01) without a change in transport affinity. The endotoxin-mediated increase in arginine uptake was abrogated by actinomycin D and cycloheximide. CONCLUSIONS: Endotoxin stimulates Na(+)-independent arginine transport by PAECs through a process that requires de novo RNA and protein synthesis, possibly of the transporter itself. This response may be designed to support arginine-dependent biosynthetic pathways in the lung during septic states.

Stimulation of hepatocyte System y(+)-mediated L-arginine transport by an inflammatory agent.

BACKGROUND: Arginine participates in several distinct metabolic pathways, including polyamine and nitric oxide biosynthesis. Normally, arginine is effectively sequestered from the hepatocyte intracellular space by the low basal activity of membrane transport system y+. This has implications for the subsequent metabolism of arginine and for hepatic arginine requirements during a septic insult. We investigated the influence of tumor necrosis factor (TNF) on the activity of System y(+)-mediated hepatocyte arginine transport employing hepatic plasma membrane vesicles (HPMVs). METHODS: Rats were treated with a single intraperitoneal injection of TNF (50 or 150 micrograms/kg body weight) for 2, 4, or 24 hours, and HPMVs were prepared by Percoll density gradient centrifugation. Vesicle purity was established by assay of enzyme markers. Vesicle arginine transport activity was evaluated by measurement of tritiated arginine uptake employing a rapid mixing-filtration technique. RESULTS: Arginine transport by HPMVs was entirely independent of sodium and consisted of saturable and nonsaturable components. Prior treatment with TNF resulted in a time- and dose-dependent stimulation of saturable transport within 2 hours and a return to basal levels after 24 hours. Nonsaturable uptake was unchanged. Inhibition analysis indicated that the TNF-induced increase in saturable arginine transport activity was mediated by an increase in System y+. Kinetic analysis revealed that accelerated transport was caused by a 78% increase in the maximal velocity of transport without alteration in transport affinity. CONCLUSIONS: In vivo treatment with TNF results in a rapid stimulation of saturable, System y(+)-mediated arginine transport in the liver. This TNF-induced stimulation of hepatic arginine transport may serve to increase the normally restricted availability of extrahepatic arginine to the hepatocyte intracellular space during a septic insult to support important arginine-dependent pathways in the liver.

Effects of intravenous nutrition on tumor growth and host immunocompetence in malnourished animals.

To evaluate the effects of oral and intravenous nutritional repletion on tumor growth and host immunocompetence in malnourished animals, 60 adult purified protein derivative (PPD) positive Buffalo rats were inoculated with Morris hepatoma 5123 and were fed a regular diet for 14 days. All animals then were switched to a high carbohydrate, protein-free diet for the next 14 days, at which time only 30% of the animals remained PPD positive. Rats then were divided into three groups: group I underwent superior vena cava catheterization and received a constant infusion of 25% dextrose--4.25% amino acid solution; group II was switched to the regular protein diet orally ad libitum; and group III remained on the oral protein-free diet. PPD reactivities were measured prior to death 7 days later. Group I animals gained an average of 14 gm of body weight, and 91% of the animals were PPD positive. Group II animals lost an average of 17 gm of body weight, but 78% of the animals were PPD positive. Group III animals lost an average of 23 gm of body weight, and only 12% of the animals remained PPD positive. Absolute tumor weight and tumor weight: body weight ratios were not significantly different among the three groups of animals. Provision of adequate nutrition intravenously to malnourished tumor-bearing animals restores body weight and host immunocompetence without adversely stimulating tumor growth out of proportion to growth of the host.

Cytokines regulate endotoxin stimulation of endothelial cell arginine transport.

BACKGROUND: Endotoxin (lipopolysaccharide) stimulates transmembrane L-arginine transport in pulmonary artery endothelial cells (PAECs). The proinflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) mediate many of the pathophysiologic effects of endotoxemia and sepsis. Endothelial cells secrete TNF and IL-1 in response to endotoxin. We hypothesize that lipopolysaccharide stimulation of plasma membrane L-arginine transport is mediated via an autocrine cytokine loop involving TNF and IL-1. METHODS: Confluent porcine PAECs were incubated with various concentrations of lipopolysaccharide, TNF, or IL-1, and arginine uptake was determined by assaying the uptake of 3H-L-arginine in the presence or absence of Na+ at different time points. PAECs were then incubated with lipopolysaccharide or saline solution after pretreatment with either anti-TNF antibody or IL-1-receptor antagonist, and transport was measured 12 hours later. RESULTS: Lipopolysaccharide, IL-1, and TNF all increased both Na+-dependent and Na+-independent carrier-mediated L-arginine transport in a fashion that was both time and dose dependent. Maximal increases in stimulated arginine uptake occurred 8 hours after exposure to the cytokines and 12 hours after exposure to lipopolysaccharide. Pretreatment of endothelial cells with anti-TNF antibody blocked lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 100% and 90%, respectively. In addition, IL-1-receptor antagonist inhibited lipopolysaccharide stimulation of both Na+-independent and Na+-dependent transport by 65% and 85%, respectively. CONCLUSIONS: The marked increase in carrier-mediated L-arginine transport activity produced by lipopolysaccharide, IL-1, and TNF may represent an adaptive response by the pulmonary endothelium to support arginine-dependent biosynthetic pathways during sepsis. Furthermore, lipopolysaccharide stimulation of arginine transport is mediated in part through an autocrine mechanism involving IL-1 and TNF.

Cytokines decrease glutaminase expression in human fibroblasts.

BACKGROUND: Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. METHODS: Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-alpha, or gamma-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a beta-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. RESULTS: IL-1, IL-6, tumor necrosis factor-alpha, and gamma-interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. CONCLUSIONS: A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine-mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

Effects of glucocorticoids on lung glutamine and alanine metabolism.

The role of the glucocorticoid hormones as possible mediators of the accelerated lung glutamine and alanine release that occurs during critical illness was investigated. Studies were done in adult rats receiving dexamethasone (0.6 mg intramuscularly/100 gm body weight/day for 2 consecutive days; n = 24) or saline solution (controls; n = 20). Measurements were made in the postabsorptive state and amino acid flux was calculated by multiplying pulmonary blood flow by the right ventricular-arterial concentration difference for glutamine and alanine. Lung glutamine release was 703 +/- 184 nmol/100 gm body weight/min in control rats. This release rate doubled in the dexamethasone-treated rats (1476 +/- 256; p less than 0.05). The activity of the glutamine synthetase enzyme increased by 33% in the dexamethasone-treated animals and there was a 50% decrease in lung glutamine content (p less than 0.01). Likewise, dexamethasone accelerated the release of alanine by the lungs twofold (559 +/- 173 nmol/100 gm body weight/min in controls vs 1113 +/- 184 nmol/100 gm body weight/min in dexamethasone-treated rats; p less than 0.05). The increased release of both amino acids was caused by a significant increase in the concentration difference across the lungs and not a change in pulmonary blood flow. Glucocorticoids appear to be key mediators of the accelerated lung amino acid release that characterizes catabolic diseases.

Adaptation to amino acid infusion in patients undergoing operation.

Urine ketone levels were measured in patients receiving peripheral amino acid solutions, and the results were correlated with changes in nitrogen balance. Thirty well-nourished patients who were to undergo cystectomy were placed on liquid, noncarbohydrate diets 3 days before operation, and no oral intake was allowed until 7 days after operation. Crystalline amino acid (1.3 to 1.5 gm/kg/day) solutions were infused continuously from 3 days before to 7 days after operation. Blood was obtained 3 days before and 3, 7, and 10 days after operation; 24-hour urine outputs were determined daily. Qualitative urine acetone levels were determined four times daily. During the infusion period, 14 (47%) patients developed ketonuria (group I); 16 patients did not (group II). The mean serum glucose levels ranged from 99 to 107 mg/dl in group I and from 108 to 113 mg/dl in group II (P less than 0.05). The mean serum transferrin level decreased after operation to 117 mg/dl in group I and 97 mg/dl in group II. The mean cumulative adjusted nitrogen balance was -24 +/- 8 gm in group I and -47 +/- 9 gm in group II (P less than 0.05). No patient developed sepsis. Qualitative testing of urinary ketones correlated with significant alterations in blood urea nitrogen, serum glucose, transferrin, and cumulative adjusted nitrogen balance. The bedside determination of urinary ketones may be useful in assessing a patient's adaptation to peripheral amino acid infusions.

Discordant tumor necrosis factor-alpha superfamily gene expression in bacterial peritonitis and endotoxemic shock.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a member of a large family of predominantly homotrimeric type II membrane-associated proteins with both proinflammatory and apoptosis-inducing properties. Although TNF-alpha expression has been studied extensively, little is known about the expression of other members of the TNF-alpha superfamily during acute inflammatory processes. METHODS: TNF-alpha, Fas ligand (FasL), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) messenger RNA (mRNA) expression were examined in liver, lung, spleen, and kidney after either a cecal ligation and puncture or endotoxemic shock with use of semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: Cecal ligation and puncture increased TNF-alpha mRNA in lung and liver (both P < .05) within 3 hours, which was paralleled by increased FasL mRNA. In the spleen TNF-alpha and FasL mRNA significantly declined (both P < .05). In contrast to TNF-alpha and FasL, TRAIL mRNA levels were unchanged in all organs except lung, where it was reduced at 24 hours (P < .05). Endotoxemic shock also increased lung TNF-alpha and FasL mRNA levels (both P < .05). CONCLUSIONS: In acute inflammatory processes TNF-alpha and FasL mRNA increase concordantly in several solid organs. In contrast, TRAIL mRNA levels do not consistently change during these acute inflammatory processes, suggesting that its expression is under independent and discordant regulatory control.

Nuclear factor-kappa B is upregulated in colorectal cancer.

BACKGROUND: Chemoresistance may involve the anti-apoptotic transcriptional regulator, nuclear factor-kappa B (NF-kappa B). The purpose of this study was to determine whether chemotherapy induces NF-kappa B activation in a human colon cancer cell line (SW48) and whether NF-kappa B is constitutively activated in colorectal cancer. METHODS: SW48 cells were incubated with gemcitabine hydrochloride (Gemzar) in the presence and absence of the 26s proteasome inhibitor, MG132, and NF-kappa B binding (electrophoretic mobility shift assay), DNA synthesis (tritiated thymidine uptake), cell viability (3-[4,5-dimethylthiazol-2-yl]-diphenyl-tetrazolium bromide assay), and apoptosis (caspase-3 activity) were measured at 24 hours. NF-kappa B binding (electrophoretic mobility shift assay) was also assayed in 10 colorectal cancer tumors. RESULTS: SW48 cells demonstrated constitutive NF-kappa B binding that was enhanced by gemcitabine hydrochloride in a dose-dependent manner. MG132 inhibited NF-kappa B binding and enhanced gemcitabine hydrochloride's inhibition of DNA synthesis (gemcitabine hydrochloride = 73% +/- 1.4% vs gemcitabine hydrochloride + MG132 = 6% +/- 0.4%, P <.05), cell killing (gemcitabine hydrochloride = 87% +/- 2.0 vs gemcitabine hydrochloride + MG132 = 25% +/- 1.3%, P <.05), and caspase-3 activity (gemcitabine hydrochloride = 870 +/- 17.4 vs gemcitabine hydrochloride + MG132 = 1075 +/- 20.4, P <.05). NF-kappa B binding was increased in 8 of 10 colorectal cancer tumors compared with adjacent normal mucosa. CONCLUSIONS: Gemcitabine hydrochloride enhances NF-kappa B binding in a colorectal cancer cell line, whereas inhibition of NF-kappa B enhances gemcitabine hydrochloride's antitumor activity. NF-kappa B is also activated in human colorectal cancer. NF-kappa B may identify chemoresistant tumors, whereas inhibition of NF-kappa B may be a novel, biologically based therapy. (Surgery 2001;130:363-9).

Endotoxin-induced nitric oxide production in pulmonary artery endothelial cells is regulated by cytokines.

BACKGROUND: L-Arginine is the sole precursor of nitric oxide (NO). Bacterial lipopolysaccharide (endotoxin) (LPS) stimulates carrier-mediated L-arginine transport in porcine pulmonary artery endothelial cells (PAECs) through an autocrine pathway that involves interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF-alpha). OBJECTIVES: To determine if Escherichia coli LPS stimulates NO synthesis in PAECs and, if so, if LPS stimulation of NO production is also mediated by autocrine secretion of IL-1 alpha and TNF-alpha. DESIGN: Monolayers of PAECs were incubated with various concentrations of LPS, recombinant human TNF-alpha, or IL-1 alpha, and total nitrate-nitrite accumulation was measured at different time points with the Greiss reagent following cadmium reduction. Release of TNF-alpha and IL-1 alpha release by LPS-stimulated PAECs were measured using the WEHI (for TNF-alpha) and A375.S2 (for IL-1 alpha) bioassays. The PAECs were then incubated with saline solution or LPS in the presence or absence of either a polyclonal antibody to human TNF or IL-1 receptor antagonist, and nitrate-nitrite accumulation was measured at 48 hours. RESULTS: Production of NO by PAECs was increased 230% by LPS (1 microgram/mL), 350% by TNF-alpha (1000 U/mL), and 240% by IL-1 alpha (1000 U/mL) (P < .05 vs control). The LPS-stimulated NO production was inhibited by IL-1 receptor antagonist (100 micrograms/mL) or antibody to TNF (10 micrograms/mL) to control levels (P < .05 vs LPS; difference vs saline solution was not significant). The LPS-stimulated TNF-alpha secretion by PAECs and TNF-alpha activity were maximal at 6 hours (400 +/- 42 pg/mL). The IL-1 alpha activity was not detectable in LPS-stimulated PAECs by the A375.S2 bioassay. CONCLUSIONS: Endotoxin, TNF-alpha, and IL-1 alpha stimulated NO synthesis in PAECs. Endotoxin-stimulated NO synthesis through an autocrine pathway involving the cytokines TNF-alpha and IL-1 alpha.