Obesity, macrophage migration inhibitory factor, and weight loss.

OBJECTIVE: Elevated macrophage migration inhibitory factor (MIF) has been implicated as a causal mechanism in a number of disease conditions including cardiovascular disease (CVD), diabetes, and cancer. Excess body fat is associated with an increased risk of numerous health conditions including CVD, diabetes, and cancer. To our knowledge, the association between MIF and obesity status and the effect of weight loss on serum MIF concentrations have not been reported. In this study, we examined the effects of participation in a behavior-based weight loss program on MIF concentrations in obese individuals. SUBJECTS: Study participants were 71 men and women enrolled in The Cooper Institute Weight Management Program. Participants were predominantly female (68%, n=48), middle-aged (46.5+/-9.8 y), and severely obese (BMI=43.0+/-8.6). METHOD: Plasma MIF concentrations and other standard risk factors were measured before and after participation in a diet and physical activity based weight management program. RESULTS: The mean follow-up was 8.5+/-3.0 months with an average weight loss of 14.4 kg (P<0.001). The majority of clinical risk factors significantly improved at follow-up. Median levels of plasma MIF concentration were significantly lower at follow-up (median [IQR]; 5.1[3.6-10.3]) compared to baseline (8.4 [4.3-48.8]; P=0.0005). The percentage of participants with plasma MIF concentration > or =19.5 mg/nl (highest tertile at baseline) decreased from 33.8 to 5.6% (P<0.001). Further, elevated baseline plasma MIF concentration was associated with markers of beta-cell dysfunction and reductions in MIF were associated with improvements in beta-cell function. CONCLUSIONS: Circulating MIF concentrations are elevated in obese but otherwise healthy individuals; however, this elevation in MIF is not uniform across individuals. In obese individuals with elevated circulating MIF concentrations, participation in physical activity and a dietary-focused weight management program resulted in substantial reduction in MIF.

Human coronary endothelial cell activation by endotoxin is characterized by NF-kappa B activation and TNF-alpha synthesis.

Tumor necrosis factor-alpha (TNF-alpha), an early inflammatory mediator typically regulated by nuclear factor kappa B (NF-kappa B), plays a critical role in the development of cardiovascular dysfunction in sepsis. While several myocardial cell types synthesize TNF-alpha, the importance of the myocardial endothelium in sepsis-related cardiac cytokine production is unclear. To determine the role of the human coronary artery endothelial cell (HCA-EC) in the cytokine response to endotoxin we measured in vitro TNF-alpha synthesis, TNF-alpha mRNA, and the associated NF-kappa B response to LPS. To determine the magnitude of the HCA-EC response we assessed the TNF-alpha and NF-kappa B response to LPS in a human monocytic cell line (THP-1) as well. We observed an increase in supernatant TNF-alpha from LPS-stimulated HCA-EC (12 h) that was ablated by the proteosome inhibitor, ALLN (N-acetyl-Leu-Leu-norleucinal). Similarly, ALLN-sensitive TNF-alpha was produced by monocytes following LPS, although at concentrations 100-fold higher than HCA-EC. TNF-alpha mRNA from HCA-EC was detected at 60 min in LPS-stimulated cells, but not in unstimulated cells or cells pretreated with ALLN. NF-kappa B p50/p65 subunits were detectable in endothelial nuclear protein 60 min following LPS. In contrast, NF-kappa B subunits from monocytes were detected at 15 min. Also, while ALLN only attenuated endothelial NF-kappa B translocation, monocyte NF-kappa B translocation was completely inhibited. These data suggest endotoxin-stimulated human coronary endothelial cells express TNF-alpha, which is regulated in part by NF-kappa B activation, in a manner and degree distinct from human monocytes.

Development of an acute burn model in adult mice for studies of cardiac function and cardiomyocyte cellular function.

The increasing availability of mice with gene supplementation (transgenic), site-specific inactivation mutations (gene "knock-outs"), or site-specific genetic modification mutations (gene "knock-ins") has spurred interest in the development of murine trauma models. In this study, C57 BL/6 mice (28 g) were given a cutaneous burn over 40% total body surface area by applying brass probes (1 x 2 x 0.003 cm) heated to 100 degrees C in boiling water to the animals side and back for 5 s. Shams received anesthesia alone and not burn. Mice were killed 24 h post-burn to determine presence of partial-thickness or full-thickness burn injury, cardiac contractile function (Langendorff perfusion, n = 7 or 8 mice/group) or to examine cardiac myocyte cytokine secretion in isolated cardiomyocytes (collagenase perfusion, n = 4 or 5 mice/group). All mice were killed 24 h post-burn for subsequent cardiac or cardiomyocyte studies. Our studies confirm that this murine model of burn trauma produced mixed partial- or full-thickness burn injury, whereas there was no necrosis or inflammation in sham burn mice. Baseline hematocrits were similar in all mice (44+/-1) but decreased after burn trauma (37+/-1), likely because of the volume of fluid resuscitation and hemodilution. Burn trauma impaired cardiac contraction and relaxation as indicated by the lower left ventricular pressure (LVP) measured in burn (56+/-4) compared to that measured in shams (84+/-1 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dt max, 1393+/-10 vs. 2000+/-41 mmHg/s, P < 0.002), and reduced LVP fall (-dP/dt max, 1023 - 40 vs. 1550+/-50, P < 0.001). These differences occurred despite similar coronary perfusion pressures and heart rates in both sham and burn mice. Ventricular function curves were shifted downward in the burn mice in the direction of contractile failure; in addition, hearts from burn mice had reduced LVP and +dP/dt responses to increases in coronary flow rate, increases in perfusate Ca2+, and to isoproterenol challenge (P < 0.05). Burn trauma promoted cardiac myocyte secretion of tumor necrosis factor (TNFalpha) (175+/-6 pg/mL) compared to that measured in shams (72+/-9 pg/mL, P < 0.05); burn trauma also increased cardiac myocyte secretion of interleukin 1beta (IL-1beta) (sham: 2+/-0.5; burn: 22+/-1 pg/mL, P < 0.05) and IL-6 (sham: 70+/-6; burn: 148+/-16 pg/mL, P < 0.05). Anti-TNFalpha strategies prevented burn-mediated cardiac contractile deficits. Burn trauma altered Ca2+ homeostasis in murine cardiomyocytes (Fura-2 AM loading). [Ca2+]i in myocytes from burns (185+/-4 nM) was higher than values measured in myocytes from shams (86+/-nM, P < 0.05). These data confirm that the murine burn model provides a reasonable approach to study the molecular and cell biology of inflammation in organ dysfunction after burn trauma.

Differential regulation of myocardial NF kappa B following acute or chronic TNF-alpha exposure.

Tumor necrosis factor alpha (TNF-alpha) is a critical mediator of myocardial dysfunction during acute inflammatory states. TNF-alpha is also present in the serum of patients with chronic cardiac diseases. In monocytes, TNF-alpha stimulates cells by activating distinct signaling pathways that involve nuclear translocation of NF kappa B. Since NF kappa B may also regulate the expression of genes that could contribute to myocardial dysfunction, the cardiomyocyte NF kappa B activation following acute or chronic TNF-alpha challenges was investigated. To accomplish this, the authors either acutely administered TNF-alpha to healthy mice, or used transgenic mice which chronically overexpress TNF-alpha exclusively in cardiac myocytes. Following acute administration of TNF-alpha, cardiac NF kappa B translocation was detected from 15 min to 2 h post-challenge. The time course of I kappa B alpha degradation was consistent with the kinetics of NF kappa B translocation. I kappa B beta degradation was slower and less dramatic. In transgenic mice chronically overexpressing TNF-alpha, myocardial NF kappa B activation was detected at all ages tested (21, 40, and 75 days). In contrast to acutely challenged animals, two distinct NF kappa B proteins were activated in chronically challenged animals, p50--65 heterodimers as well as p50 homodimers. Activation of both could be transiently blocked by administration of a recombinant chimeric TNF-alpha receptor antagonist (rhTNFR:Fc). I kappa B alpha, but not I kappa B beta, levels were elevated in transgenics when compared to wild-type animals. These data indicate that following acute TNF-alpha administration, which simulates bacterial sepsis, myocardial p50-p65 translocates within minutes. Chronic TNF-alpha exposure, which is thought to occur in long-standing congestive heart failure, results in translocation of transcriptionally inactive p50 homodimers in addition to transcriptionally active p50--65 heterodimers. It is speculated that activation of p50 homodimers constitutes an adaptive response to minimize the inflammatory consequences of chronic cardiac TNF-alpha exposure.

Antioxidant vitamin therapy alters burn trauma-mediated cardiac NF-kappaB activation and cardiomyocyte cytokine secretion.

BACKGROUND: This study examined the effects of antioxidant vitamins A, C, and E on nuclear transcription factor-kappa B (NF-kappaB) nuclear translocation, on secretion of inflammatory cytokines by cardiac myocytes, and on cardiac function after major burn trauma. METHODS: Adult rats were divided into four experimental groups: group I, shams; group II, shams given oral antioxidant vitamins (vitamin C, 38 mg/kg; vitamin E, 27 U/kg; vitamin A, 41 U/kg 24 hours before and immediately after burn); group III, burns (third-degree scald burn over 40% total body surface area) given lactated Ringer's solution (4 mL/kg/% burn); and group IV, burns given lactated Ringer's solution plus vitamins as described above. Hearts were collected 4, 8, 12, and 24 hours after burn to assay for NF-kappaB nuclear translocation, and hearts collected 24 hours after burn were examined for cardiac contractile function or tumor necrosis factor-alpha secretion by cardiomyocytes. RESULTS: Compared with shams, left ventricular pressure was lower in burns given lactated Ringer's solution (group III) (88 +/- 3 vs. 64 +/- 5 mm Hg, p < 0.01) as was +dP/dt max (2,190 +/- 30 vs. 1,321 +/- 122 mm Hg/s) and -dP/dt max (1,775 +/- 71 vs. 999 +/- 96 mm Hg, p < 0.01). Burn injury in the absence of vitamin therapy (group III) produced cardiac NF-kappaB nuclear migration 4 hours after burn and cardiomyocyte secretion of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 by 24 hours after burn. Antioxidant therapy in burns (group IV) improved cardiac function, producing left ventricular pressure and +/-dP/dt (82 +/- 2 mm Hg, 1,880 +/- 44 mm Hg, and 1,570 +/- 46 mm Hg/s) comparable to those measured in shams. Antioxidant vitamins in burns inhibited NF-kappaB nuclear migration at all times after burn and reduced burn-mediated cytokine secretion by cardiomyocytes. CONCLUSION: These data suggest that antioxidant vitamin therapy in burn trauma provides cardioprotection, at least in part, by inhibiting translocation of the transcription factor NF-kappaB and interrupting cardiac inflammatory cytokine secretion.

Targeted disruption of ICAM-1, P-selectin genes improves cardiac function and survival in TNF-alpha transgenic mice.

We have developed a transgenic mouse model in which tumor necrosis factor (TNF)-alpha is overexpressed exclusively in the heart under the regulation of the alpha-myosin heavy chain promoter. These animals develop chronic heart failure associated with severe leukocyte infiltration in both the atria and the ventricles. The purpose of this study was to investigate the role of adhesion molecules in mediating cardiac dysfunction in the TNF-alpha transgenic model. TNF-alpha transgenic mice were bred with mice null for intercellular adhesion molecule (ICAM)-1 and P-selectin genes to obtain a lineage of ICAM-1 and P-selectin null mice with selective overexpression of TNF-alpha in the heart. TNF-alpha transgenic animals showed marked upregulation of ICAM-1 mRNA and protein; however, P-selectin mRNA and protein remained undetectable despite chronic TNF overexpression. Cardiac function was markedly improved in the ICAM-1(-/-), P-selectin(-/-), TNF-alpha transgenic group versus the ICAM(+/+), P-selectin(+/+), TNF-alpha transgenic group. Kaplan-Meier survival curves showed statistically significant prolonged survival in the ICAM-1(-/-), P-selectin(-/-), TNF-alpha transgenic animals. These data suggest that ICAM-1 mediates at least in part the cardiac dysfunction induced by TNF-alpha expression by cardiac myocytes.

Overexpression of cardiac I-kappaBalpha prevents endotoxin-induced myocardial dysfunction.

Nuclear factor-kappa B (NF-kappaB) is an inducible transcription factor that regulates expression of many genes, such as tumor necrosis factor-alpha (TNF-alpha), which may contribute to myocardial dysfunction. We investigated whether cardiac NF-kappaB activation is involved in the development of myocardial dysfunction after lipopolysaccharide (LPS) challenge. Mice were intraperitoneally injected with LPS, and the hearts were harvested and assayed for NF-kappaB translocation. After LPS challenge, NF-kappaB activation was detected within 30 min and remained for 8 h. In transgenic mice constitutively overexpressing a nondegradable form of I-kappaBalpha (I-kappaBalphaDeltaN) in cardiomyocytes, myocardial NF-kappaB translocation was prevented after LPS challenge. Myocytes isolated from these transgenics secreted significantly less TNF-alpha than did wild-type cardiomyocytes after LPS stimulation. When whole hearts were excised, perfused in a Langendorff preparation, and challenged with endotoxin, I-kappaBalphaDeltaN transgenic hearts displayed normal cardiac function, whereas profound contractile dysfunction was observed in wild-type hearts. These data indicate that myocardial NF-kappaB translocates within minutes after LPS administration. Inhibition of myocyte NF-kappaB activation by overexpression of myocyte I-kappaBalpha is sufficient to block cardiac TNF-alpha production and prevent cardiac dysfunction after LPS challenge.

Preoperative and postoperative endotoxemia in children with congenital heart disease.

STUDY OBJECTIVES: Recent data indicate that increases in inflammatory cytokines are seen in patients with diverse cardiac diseases. However, the primary stimulus for cytokine secretion during cardiac illness remains unknown. Since bacterial endotoxin is a potent inducer of cytokines, we determined the incidence, magnitude, and clinical relevance of endotoxemia in children with congenital heart disease before and after surgical repair. DESIGN: A prospective, observational study. SETTING: A large, urban, university-affiliated, tertiary-care children's hospital. PATIENTS: Thirty children with a variety of congenital heart defects (median age, 59 days; median weight, 4.0 kg) were sequentially enrolled. INTERVENTIONS: Blood was sampled prior to surgery, and at 1, 8, 24, 48, and 72 h following cardiopulmonary bypass. Assays included plasma endotoxin, lipopolysaccharide-binding protein (LBP), and interleukin-6 (IL-6). MEASUREMENTS AND RESULTS: Twenty-nine of 30 patients (96%) had evidence of endotoxemia during the study period. Twelve of the 30 patients (40%) were significantly endotoxemic prior to surgery. LBP, a plasma marker that responds to bacteria and endotoxin, rose significantly following cardiopulmonary bypass, as did the plasma levels of IL-6. Fifteen of 30 patients met prospectively defined criteria for experiencing a severe hemodynamic disturbance in their postoperative course. These patients had significantly higher preoperative plasma LBP (p < 0.02) and plasma endotoxin levels (p < 0.05), compared to patients with less-severely disturbed hemodynamics. Mortality was 25% in patients with preoperative endotoxemia, compared with no mortality in patients who were not endotoxemic before surgery (p = 0.05). CONCLUSIONS: These data demonstrate that endotoxemia in children with congenital heart disease is more common than previously suspected, and is associated with clinical outcomes. We conclude that clinical trials targeting endotoxin will be necessary to determine if endotoxin is a causal, etiologic agent in the disease process.

Magnetic resonance imaging and invasive evaluation of development of heart failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha.

BACKGROUND: Transgenic mice expressing tumor necrosis factor-alpha (TNF-alpha) in cardiac myocytes develop dilated cardiomyopathy, but the temporal progression to cardiac dysfunction is not well characterized. We asked (1) Does magnetic resonance imaging (MRI) provide a reproducible assessment of cardiac output in mice that correlates with invasive measurements obtained with thermodilution? (2) What is the time course of left ventricular (LV) remodeling in transgenic mice with myocardial expression of TNF-alpha? METHODS AND RESULTS: Transgenic mice from 2 different lineages with differing amounts of myocardial TNF-alpha expression [lineage 1 (L1) and lineage 2 (L2)] and littermate controls (LC) were studied. In protocol 1, cardiac output (CO) and stroke volume (SV) were measured by MRI and thermodilution (TD) in 15 mice (3 L1, 4 L2, 8 LC). In protocol 2, 23 mice (7 L1, 8 L2, 8 LC) were scanned at 1 month of life and every 4 weeks thereafter. In both protocols, cine-MRI was performed with the use of a 1.5-T clinical system (1.5-mm slices, 195x195 microm in-plane resolution). MRI CO and SV correlated well with TD [COTD (mL/min)=0.94*COMRI+0.72, r=0.84; SVTD( microL)=1. 01*SVMRI-1.07, r=0.94]. Serial MRI studies showed significant increase in LV mass and volumes over time and a significant decrease in ejection fraction in transgenic mice when compared with littermate controls. Compared with lineage 2, lineage 1 showed significantly larger LV mass and volumes and significantly lower ejection fraction. CONCLUSIONS: MRI assessment of cardiac function in mice correlates well with invasive measurements. Serial MRI studies in the TNF-alpha mouse model demonstrate that the rate of progression and severity of LV dysfunction are dependent on the degree of TNF-alpha overexpression.

Aspiration pneumonia-induced sepsis increases cardiac dysfunction after burn trauma.

Pneumonia occurs in approximately 50% of incubated patients in burn intensive care units and carries a mortality as high as 40%. A model was developed to study altered cardiopulmonary function in burn complicated by pneumococcal pneumonia. Sprague-Dawley rats were given a 43% total body surface area scald burn or sham burn; 24 h later they were transtracheally inoculated with either 10(7) Streptococcus pneumoniae in 0.5 ml phosphate buffer solution (PBS) or 0.5 ml PBS alone. The four groups were: Sham (N = 7), Burn alone (N = 10), Pneumonia alone (N = 11), and Burn and Pneumonia ( N = 12). A fifth group of burned rats (N = 10), given an identical fluid resuscitation regimen, was sacrificed 24 h postburn to examine the early cardiac responses to burn injury alone. Shams and burned animals had normal lung histology, negative bronchoalveolar lavage (BAL) cultures, and negative blood cultures. Pneumonia and burn plus pneumonia animals had abnormal lung histology, positive BAL cultures, and positive blood cultures. Cardiac function was assessed 24 h after S.pneumoniae challenge (48 h after burn) (Langendorff preparation). Compared to the Sham group, Pneumonia group, and Burn group, the Burn plus Pneumonia group had the lowest left ventricular pressure (LVP: 94 +/- 4, 71 +/- 3, and 87 +/- 3 mm Hg vs 63 +/- 4 mm Hg, P < 0.05), the lowest maximal rate of LVP rise (+dP/dt[max]:1932 +/- 115, 1419 +/- 71, and 1772 +/- 96 mm Hg vs 1309 +/- 59 mm Hg/s, P < 0.05), and the lowest maximal rate of LVP fall (-dP/dt[max]:1704 +/- 120, 1263 +/- 73, and 1591 +/- 83 mm Hg vs 1025 +/- 98 mm Hg/s, P < 0.05). Cardiac contraction and relaxation deficits were confirmed in animals 24 h postburn (group 5), as indicated by a significantly lower LVP and +/-dP/dt(max) (62 +/- 3 mm Hg 1210 +/- 60, and 909 +/- 50 mm Hg/s, respectively, P < 0.05 compared to Sham group). Tumor necrosis factor-alpha (TNF-alpha) concentrations in serum, but not bronchoalveolar lavage, were greater in burned animals with aspiration pneumonia-induced sepsis than in animals with either burn alone or aspiration pneumonia-induced sepsis alone. While our data suggest that elevated circulating TNF-alpha levels may contribute, in part, to depressed cardiac function, further studies are needed to fully define the mechanisms underlying cardiac contractile deficits in this model. We speculate that depressed cardiopulmonary function due to burn complicated by pneumonia and sepsis contributes to the high mortality of this patient population.

Molecular biology of septic shock.

Septic shock is a complex pathophysiologic state which often leads to multiple organ dysfunction, multiple organ failure, and death. This review summarizes current views on the molecular biology of three aspects of septic shock: recognition of bacterial invasion and induction of the cytokine response; genetic variability among humans and their predispositions toward pathologic inflammatory responses; and the signal transduction mechanisms which account for the transfer of molecular signals from cytokine receptors on the plasma membrane to cytokine-responsive genes in the nucleus. In particular, the review summarizes the pathway involved in tumor necrosis factor signaling through nuclear factor-kappaB, and elucidates the molecular signals involved in inflammatory responses and apoptosis.

Cardiac failure in transgenic mice with myocardial expression of tumor necrosis factor-alpha.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that has been detected in several human cardiac-related conditions, including congestive heart failure and septic cardiomyopathy. In these conditions, the origin of TNF-alpha secretion is, at least in part, cardiac myocytes. METHODS AND RESULTS: To determine the consequences of TNF-alpha production by cardiac myocytes in vivo, we developed transgenic mice in which expression of a murine TNF-alpha coding sequence was driven by the murine alpha-myosin heavy chain promoter. Four transgenic founders developed an identical illness consisting of tachypnea, decreased activity, and hunched posture. In vivo, ECG-gated MRI of symptomatic transgenic mice documented a severe impairment of cardiac function evidenced by biventricular dilatation and depressed ejection fractions. All transgenic mice died prematurely. Pathological examination of affected animals revealed a globular dilated heart, bilateral pleural effusions, myocyte apoptosis, and transmural myocarditis in both the right and left ventricular free walls, septum, and atrial chambers. In all terminally ill animals, there was significant biventricular fibrosis and atrial thrombosis. CONCLUSIONS: This is the first report detailing the effects of tissue-specific production of TNF-alpha by cardiac myocytes in vivo. These findings indicate that production of TNF-alpha by cardiac myocytes is sufficient to cause severe cardiac disease and support a causal role for this cytokine in the pathogenesis of human cardiac disease.

Complementary DNA (cDNA) sequence of baboon tumor necrosis factor alpha.

Baboons are less LPS-sensitive than humans, even though their immune response mechanisms are similar. Since TNFalpha is a central mediator of the LPS-response we cloned and sequenced the baboon TNFalpha cDNA and compared the resulting sequence with the human TNFalpha sequence. Analysis of the TNFalpha protein coding region indicated 97% homology and of the 3' UTF 89%. The predicted baboon TNFalpha amino acid sequence differed at 10 positions from the human sequence. "TA" rich motifs within the 3' UTR were 100% homologous.

Regulation of cytokine gene expression: tumor necrosis factor, interleukin-1, and the emerging biology of cytokine receptors.

Cytokines are bioactive molecules which mediate host responses to inflammatory stimuli. For cytokines to exert their effects, a number of molecular processes must occur. Inflammatory cells must sense the presence of a stimulus, often by detection of that stimulus via cell-surface receptors. Information detected at the cell surface must be transduced intracellularly, and the machinery of cytokine messenger RNA and protein synthesis initiated. Once secreted, cytokines must bind specific receptors on target tissues; these receptors in turn transduce signals which alter target cell phenotypes and responses. Each one of these steps is tightly regulated and may serve as a target for therapeutic manipulation. In this article, the regulation of cytokine gene expression is summarized, with particular emphasis on the regulation of tumor necrosis factor alpha and interleukin-1 biosynthesis. Attention is focused on therapeutic agents which alter cytokine production or activity, some of which are currently used in the ICU.

Sequence analysis of the tumor necrosis factor gene in pediatric patients with autoimmunity.

Tumor necrosis factor-alpha (TNF) is a multifunctional protein hormone that contributes to host defense and perinatal immunologic development. Dysregulated TNF production, however, occurs during the pathogenesis of autoimmune diseases and may be inherent to their development. In animal models of autoimmunity, dysregulated TNF synthesis has resulted from mutations in TNF gene regulatory sequences, specifically those sequences involved in translational control of TNF gene expression. In this study, we have determined whether mutations in the TNF translational control sequences are present in pediatric patients with type I diabetes mellitus and connective tissue diseases. Blood samples were collected from 48 patients with connective tissue diseases, 32 patients with diabetes, and 29 controls. A 250-bp fragment of the translational control sequences present in the TNF 3'-untranslated region was amplified by the polymerase chain reaction, sequenced, and analyzed relative to the published TNF sequence. In this study, all patients and controls exhibited the normal sequence, with no insertions or deletions in the translational control motifs. We conclude that polymorphisms in the TNF 3'-untranslated region occur infrequently, if at all, in patients with diseases examined here.

Cytokine-induced expression of nitric oxide synthase in C2C12 skeletal muscle myocytes.

Nitric oxide (NO) is an important mediator of diverse physiological and pathological responses. To determine whether NO production can be induced in skeletal muscle, we stimulated C2C12 mouse skeletal muscle myocytes with putative inducers of nitric oxide synthase (NOS). Neither lipopolysaccharide (LPS), interleukin-1 alpha (IL-1), tumor necrosis factor-alpha (TNF), nor interferon-gamma (IFN) was able to stimulate nitrite production by C2C12 cells when administered alone. However, combinations of IFN with either TNF or IL-1 resulted in significant nitrite production; simultaneous stimulation of cells with all three cytokines resulted in significantly increased nitrite production compared with any combination of two cytokines. Northern analysis of RNA obtained from stimulated C2C12 cells revealed induction of a single mRNA band that precisely coincided with the mRNA band of mouse macrophage-inducible NOS (iNOS). Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis followed by sequencing of the 5' 765 bases of the skeletal muscle iNOS cDNA demonstrated exact homology with mouse macrophage iNOS. These findings indicate that combinations of cytokines stimulate NO production in skeletal muscle cells via induction of the macrophage-type iNOS gene.

Inhibition of tumor necrosis factor prevents myocardial dysfunction during burn shock.

Tumor necrosis factor-alpha (TNF) is a pluripotent cytokine that mediates many of the hemodynamic manifestations of endotoxic shock. To determine whether TNF is responsible for postburn myocardial dysfunction, we compared cardiac function (Langendorff preparation) in 49 guinea pigs 18 h after thermal injury. Group 1 (n = 15) was sham burned; all remaining animals received a 43% surface area burn under anesthesia. Group 2 (n = 15) received lactated Ringer solution (LR, 4 ml.kg-1.%burn-1). Group 3 (n = 9) received LR and drug vehicle. Group 4 (n = 10) received LR plus 1 mg of TNF inhibitor consisting of the human p80 TNF receptor linked to the Fc portion of human immunoglobulin G1, which was shown to specifically bind and neutralize TNF secreted by guinea pig peritoneal macrophages in vitro. Burn injury caused a significant fall in left ventricular pressure (LVP, from 86 +/- 2 to 62 +/- 3 mmHg, P < 0.05) and maximal rate of LVP rise (+) and fall (-) (+/- dP/dtmax) [from 1,365 +/- 42 to 1,109 +/- 44 mmHg/s (P < 0.05) and from 1,184 +/- 31 to 881 +/- 40 mmHg/s (P < 0.05), respectively], a decrease in time to peak systolic LVP (from 111 +/- 2 to 102 +/- 2 ms, P < 0.05), and a decrease in time to +dP/dtmax (from 57 +/- 1 to 48 +/- 1 ms, P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A reporter transgene indicates renal-specific induction of tumor necrosis factor (TNF) by shiga-like toxin. Possible involvement of TNF in hemolytic uremic syndrome.

We have examined the hypothesis that TNF may play a pathogenetically important role in the hemolytic uremic syndrome. Specifically, we considered the possibility that shigatoxin, which eventuates this syndrome, might induce TNF biosynthesis, and/or that TNF and shigatoxin might sensitize animals, each to the toxic effects of the other agent. Shigatoxin was found to sensitize mice to the lethal effect of LPS and to the lethal effect of TNF. On the other hand, pretreatment of animals with either TNF or LPS did not noticeably sensitize mice to the lethal effect of shigatoxin. Intraperitoneal injections of shigatoxin did not induce the production of detectable quantities of TNF in the plasma of mice. When shigatoxin was injected into transgenic mice bearing a chloramphenicol acetyltransferase (CAT) reporter gene that indicates TNF synthesis, CAT activity was induced within the kidney, but not in other tissues. We therefore conclude that shigatoxin acts to induce TNF synthesis within the kidney, and at the same time increases renal sensitivity to the toxic effects of TNF. While this mouse model does not reproduce the hemolytic uremic syndrome as it occurs in humans, it does suggest that local synthesis of TNF within the kidney may contribute to renal injury induced by shigatoxin.

Mediators of septic shock: new approaches for interrupting the endogenous inflammatory cascade.

OBJECTIVES: To review the molecular pathogenesis of septic shock, with particular emphasis on the induction of cytokines by endotoxin. By understanding the mechanisms that result in the systemic inflammatory response, novel clinical interventions may be more effectively studied. DATA SOURCES: The English medical literature was reviewed, including human clinical trials, animal experiments, and in vitro studies elucidating cellular and molecular interactions. Expert testimony from the Roundtable Conference on Sepsis (Brussels, March 1992) was also used to synthesize emerging concepts and to ensure inclusion of ongoing investigations. STUDY SELECTION: Emphasis on controlled experimental studies which elucidated the molecular and cellular interactions during sepsis. DATA EXTRACTION: This study focused only on data that directly involved the induction and regulation of protein mediators of sepsis, especially tumor necrosis factor (TNF) and interleukin-1. Data concerning the role of TNF during health were extracted from the author's peer-reviewed data. DATA SYNTHESIS: Information concerning the many facets of the systemic inflammatory response was integrated into a chronological, clinically oriented model of cytokine induction during endotoxemia. CONCLUSIONS: The induction of inflammation during sepsis is a complex, but increasingly understood, biological cascade that is dependent on inter- and intracellular signaling. Novel biotherapies may improve patient outcome in sepsis by interrupting any or all points of signal transduction.