Showing papers on "Intraperitoneal injection published in 1997"

Time course of increased plasma cytokines, cortisol, and urea nitrogen in pigs following intraperitoneal injection of lipopolysaccharide

Abstract: The emerging view is that reduced feed intake, lean muscle accretion, and growth in immunologically challenged pigs is the result of increased cytokine activity, but this has not been directly tested. To begin addressing this issue, 72 crossbred barrows and gilts (11.55 +/- .19 kg BW) were not fed for 12 h and then injected i.p. with 0, .5, or 5 micrograms/kg of Escherichia coli lipopolysaccharide (LPS). Blood was collected by jugular puncture at 0, 2, 4, 8, 12, and 24 h after injection. Plasma levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), cortisol, plasma urea nitrogen (PUN), NEFA, and triglycerides were determined. Immunological stress was induced by LPS as indicated by increased secretion of TNF-alpha, IL-6, and cortisol. In pigs receiving 5 micrograms/kg of LPS, plasma TNF-alpha was increased 10-fold at 2 h after injection and was still elevated (P < .01) at 4 h. In these same pigs, plasma concentration of IL-6 was increased at 2 h and peaked at 4 h with levels exceeding baseline values by 200-fold (P < .01). Cortisol was elevated at 2, 4, and 8 h after injection (P < .01). The increased secretion of cytokines and cortisol in pigs injected with 5 micrograms/kg of LPS was followed by an increase in protein degradation, as evidenced by PUN values that were increased two- and threefold at 8 and 12 h after injection, respectively. However, unlike previous reports in laboratory animal species, plasma glucose, NEFA, and triglycerides were not altered by LPS. Nonetheless, as the period of feed deprivation progressed from 12 to 36 h, plasma NEFA and triglycerides increased (P < .05) and plasma glucose tended to decrease. We believe that immunological challenge induces cytokine synthesis and secretion in swine which, in turn, may induce protein catabolism.

Neutrophil Emigration in the Skin, Lungs, and Peritoneum: Different Requirements for CD11/CD18 Revealed by CD18-deficient Mice

Abstract: To determine the role of CD11/CD18 complexes in neutrophil emigration, inflammation was induced in the skin, lungs, or peritoneum of mutant mice deficient in CD18 (CD18−/− mutants). Peripheral blood of CD18−/− mutants contained 11-fold more neutrophils than did blood of wild-type (WT) mice. During irritant dermatitis induced by topical application of croton oil, the number of emigrated neutrophils in histological sections of dermis was 98% less in CD18−/− mutants than in WT mice. During Streptococcus pneumoniae pneumonia, neutrophil emigration in CD18−/− mutants was not reduced. These data are consistent with expectations based on studies using blocking antibodies to inhibit CD11/CD18 complexes, and on observations of humans lacking CD11/CD18 complexes. The number of emigrated neutrophils in lung sections during Escherichia coli pneumonia, or in peritoneal lavage fluid after 4 h of S. pneumoniae peritonitis, was not reduced in CD18−/− mutants, but rather was greater than the WT values (240 ± 30 and 220 ± 30% WT, respectively). Also, there was no inhibition of neutrophil emigration during sterile peritonitis induced by intraperitoneal injection of thioglycollate (90 ± 20% WT). These data contrast with expectations. Whereas CD11/CD18 complexes are essential to the dermal emigration of neutrophils during acute dermatitis, CD18−/− mutant mice demonstrate surprising alternative pathways for neutrophil emigration during pneumonia or peritonitis.

Influence of the route of administration and the chemical form (MnCl2, MnO2) on the absorption and cerebral distribution of manganese in rats.

Abstract: The absorption and cerebral distribution of manganese (Mn) have been studied with respect to the route of administration and the chemical form of the Mn compound. Different groups of adult male rats received either MnCl2, 4H2O or MnO2 once a week for 4 weeks at a dose of 24.3 mg Mn/kg body wt. (b.w.) by oral gavage (g.) or 1.22 mg Mn/kg b.w. by intraperitoneal injection (i.p.) or intratracheal instillation (i.t.). Control rats were treated with 0.9% saline. Four days after the last administration the rats were killed and the concentration of Mn measured in blood, hepatic and cerebral tissues (cortex, cerebellum, and striatum). The liver Mn concentration was not affected by the treatments whatever the chemical form or the route of administration of the Mn compound. Administration of MnCl2 by g., i.p., and i.t. routes produced equivalent steady-state blood Mn concentrations (about 1000 ng Mn/100 ml), representing increases of 68, 59, and 68% compared with controls, respectively. Mn concentrations were significantly increased in the cortex but to a lesser extent (g., 22%; i.p., 36%; i.t., 48%) and were higher in the cerebellum after i.p. and i.t. administrations than after oral gavage. Rats treated i.t. with MnCl2 showed an elective increase of the striatal Mn concentration (205%). In contrast, MnO2 given orally did not significantly increase blood and cerebral tissue Mn concentrations; the low bioavailability is most likely due to the lack of intestinal resorption. Administration of MnO2 i.p. and i.t., however, led to significant increases of Mn concentrations in blood and cerebral tissues. These increments were not significantly different from those measured after MnCl2 administration, except for striatal Mn after i.t. which was markedly less (48%) after MnO2 administration. A comparison of the blood Mn kinetics immediately after g. and i.t. treatment with MnCl2 or MnO2 indicated that the higher elevation of blood Mn concentration (> 2000 ng Mn/100 ml) after i.t. administration of MnCl2 could account for the elective uptake of Mn in the striatum observed in repeated dosing experiments. It is concluded that the modulation of Mn distribution in brain regions according to the route of administration and the chemical form of the Mn compound may be explained on the basis of different blood Mn kinetics and regional anatomic specificities of the striatal region.

Effect of L-Dopa and the catechol-O-methyltransferase inhibitor Ro 41-0960 on sulfur amino acid metabolites in rats

Abstract: L-Dopa is the most effective drug known for the treatment of Parkinson's disease. However, the large doses required to treat this neurodegenerative disorder can significantly affect tissue concentrations of sulfur amino acid metabolites due to peripheral and central O-methylation. These effects include decreases in tissue concentrations of the biochemical methyl donor S-adenosylmethionine (SAM), increases in tissue concentrations of the methylation inhibitor S-adenosylhomocysteine (SAH), and increases in plasma concentrations of homocysteine, recently identified as an independent risk factor for vascular disease. In the present study, the ability of the catechol-O-methyltransferase inhibitor Ro 41-0960 to prevent L-Dopa-induced changes in SAM, SAH, and homocysteine concentrations was determined in rats. Rats were injected intraperitoneally with Ro 41-0960 or vehicle 30 min prior to an intraperitoneal injection of L-Dopa or vehicle. One hour after the second injection, the rats were killed and their brains, livers, spleens, kidneys, and plasma collected. SAM and SAH concentrations were then determined in discrete brain regions and peripheral tissues, and total homocysteine concentrations were determined in plasma. In the rats treated with only L-Dopa, decreased SAM concentrations and increased SAH concentrations were found in all brain regions and peripheral tissues measured, and increased homocysteine concentrations were found in plasma, consistent with previous reports. In rats pretreated with Ro 41-0960, however, these L-Dopa-induced effects on sulfur amino acid metabolite concentrations were attenuated or prevented entirely. It remains to be determined if this sparing effect of Ro 41-0960 on sulfur amino acid metabolites has clinical significance.

Intraperitoneal injection of tetracyclines protects mice from lethal endotoxemia downregulating inducible nitric oxide synthase in various organs and cytokine and nitrate secretion in blood.

Abstract: We have tested whether tetracyclines (TETs) are able to protect mice from lipopolysaccharide (LPS)-induced shock, a cytokine-mediated inflammatory reaction. Mice, injected with a single dose of tetracycline base (TETb; 1.5, 10 and 20 mg/kg of body weight) or doxycycline (DOXY; 1.5 mg/kg), were significantly protected from a lethal intraperitoneal injection of LPS (500 micrograms per mouse). TETs acted in early events triggered in response to LSP; in fact, they were no longer significantly protective if injected more than 1 h after the injection of endotoxin. LPS-treated mice protected by TETs showed a significant inhibition of tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and nitrate secretion in the blood, events that were directly related with the survival. In mice treated with TETs a significant decrease of inducible nitric oxide synthase (iNOS) activity was observed in spleen and peritoneal cells compared with that detected in mice treated with LPS alone. Furthermore, TETs were found to inhibit NO synthesis by peritoneal macrophages stimulated in vitro with LPS. On the contrary, TETs were unable to decrease the ability of the macrophages to synthesize IL-1 alpha and TNF-alpha in vitro. These results indicate that TETs are not able to act directly on the synthesis of these cytokines, but they may modulate other pathways that could in turn be responsible for the inhibition of IL-1 alpha and TNF-alpha synthesis. Altogether, these results indicate that TETs are advantageous candidates for the prophylaxis and treatment of septic shock in mice, having both antimicrobial activity and the ability to inhibit endogenous TNF-alpha, IL-1 alpha, and iNOS, hence, exerting, potent anti-inflammatory effects.

Fever suppression by subdiaphragmatic vagotomy in guinea pigs depends on the route of pyrogen administration

Abstract: It has recently been shown that subdiaphragmatic vagotomy blocks some of the effects of inflammatory stimuli on brain-controlled functions. Therefore, vagal afferent fibers have been proposed to play a prominent role in the communication pathways between the immune system and the brain. In the present study, we investigated the effect of subdiaphragmatic vagotomy on fever induced by intraperitoneal or intramuscular injection of lipopolysaccharide (LPS) or muramyl dipeptide (MDP), which are both cytokine-inducing agents in guinea pigs. Intraperitoneal and intramuscular injections of LPS or MDP were tested in the same animal with an interval of 1 wk. In one experiment, intraperitoneal injections of LPS or MDP were performed at the beginning, followed by intramuscular injections 1 wk later. In another experiment, intramuscular injections of LPS or MDP were performed at the beginning, followed by intraperitoneal injections 1 wk later. The febrile response to intraperitoneal injection of LPS was almost completely abrogated in vagotomized animals compared with sham-operated controls (from 30-330 min after intraperitoneal injection of LPS). The suppression of LPS fever was quantitatively the same in both experiments. In contrast, the response to intramuscular injection of LPS was the same in vagotomized and sham-operated guinea pigs in each of the experiments. Fever induced by intraperitoneal injection of MDP was partly attenuated by subdiaphragmatic vagotomy (between 150 and 300 min after intraperitoneal injection of MDP) in both experiments. Again, the febrile response to intramuscular injections of MDP was not significantly altered by subdiaphragmatic vagotomy. In conclusion, the suppressive effect of subdiaphragmatic vagotomy on fever induced by peripheral immune stimuli depends on the route of pyrogen administration. Because the febrile response to intraperitoneal injection of bacterial pyrogens is strongly diminished in vagotomized guinea pigs, it is suggested that vagal afferent fibers play a crucial role in the transduction of immune signals from the abdominal cavity to the brain.

Cell Cycle-Dependent Chronotoxicity of Irinotecan Hydrochloride in Mice

Abstract: The mechanisms underlying the circadian rhythm of the toxicity induced by irinotecan hydrochloride (CPT-11; 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin) were investigated from the viewpoint of the sensitivity of living organisms and the pharmacokinetics of the drug. ICR male mice were housed under standardized light-dark cycle conditions (lights on at 0700, off at 1900) with food and water ad libitum. The loss of body weight after an intraperitoneal injection of CPT-11 (100 mg/kg) was more serious in the late dark and the early light and milder in the late light and the early dark. The CPT-11-induced leukopenia was more serious in the late dark and milder in the late light. The lower toxicity of CPT-11 was observed when DNA synthesis and type I DNA topoisomerase activity in bone marrow cells decreased and the higher toxicity was observed when these activities began to increase. There were circadian stage-dependent changes in the concentrations of CPT-11 and its major metabolite (SN-38; 7-ethyl-10-hydroxycamptothecin) in plasma. The higher concentrations of CPT-11 and SN-38 in plasma were observed when the level of CPT-11-induced toxicity increased. The present study suggests that the toxicity of CPT-11 is influenced by circadian rhythm-dependent processes.

Tolerance Induction and Autoimmune Encephalomyelitis Amelioration After Administration of Myelin Basic Protein–derived Peptide

Abstract: Experimental autoimmune encephalomyelitis (EAE), a demyelinating disease of the central nervous system, is an animal model of paralyzing human disease, multiple sclerosis. EAE is readily induced by immunization with myelin basic protein (MBP) in mice transgenic for an αβ T cell receptor (TCR) that is specific for MBP. Subcutaneous injection of p17 (a peptide consisting of 17 NH2-terminal aminoacids of MBP) in complete Freund's adjuvant (CFA) causes paralysis. Induction of paralysis is inhibited by prior intraperitoneal injection of the same peptide in incomplete Freund's adjuvant (IFA). In addition, ongoing paralysis is ameliorated by subsequent intraperitoneal injection of p17 in IFA. Tolerance induction is equally efficient in Fas-deficient and IL-4–deficient TCR-transgenic mice, suggesting that neither activation-induced cell death nor differentiation into Th2 type cells plays a role in the tolerance induction. Tolerance induction by p17 seems to be based on reduction in the responsiveness of anti-MBP T cells, as documented by lower overall antigen-induced lymphokine production and proliferation, as well as diminished upregulation of early activation marker CD69 by tolerized T cells. We propose that continuous encounters of MBP-specific T cells with p17 play a critical role in the induction and maintenance of tolerance.

Dietary sulphur amino acid adequacy influences glutathione synthesis and glutathione-dependent enzymes during the inflammatory response to endotoxin and tumour necrosis factor-alpha in rats.

Abstract: 1 Glutathione concentrations in liver and lung fall when food intake or sulphur amino acid intake is inadequate However, concentrations may be restored during inflammation, despite anorexia, provided that prior sulphur amino acid intake is adequate 2 We studied the mechanisms of these changes by measuring the effect of sulphur amino acid and protein intake on hepatic glutathione synthesis and gamma-glutamylcysteine synthetase activity, hepatic and lung glutathione concentrations, glutathione reductase and glutathione peroxidase activities in young rats given an inflammatory challenge by intraperitoneal injection of tumour necrosis factor-alpha or endotoxin (lipopolysaccharide) 3 Diets containing 200 g of casein and 8 g of L-cysteine/kg (normal-protein diet), or 80 g of casein and 8 g of L-cysteine, or isonitrogenous amounts of L-methionine or L-alanine (low-protein diets) were fed ad libitum to young Wistar rats for 8 days Dietary groups were subdivided into three: one subgroup continued feeding ad libitum, a second was given tumour necrosis factor or lipopolysaccharide and killed 24 h thereafter, while the third was pair-fed to the intakes of the second subgroup for 24 h before being killed 4 Glutathione concentrations in liver and lung were reduced in rats fed the low-protein diet containing alanine, and in all dietary groups when food intake was restricted The inflammatory challenges restored hepatic glutathione concentrations in all groups but the diet supplemented with alanine, which had an inadequate sulphur amino acid content In lung, restoration occurred only in animals fed the normal-protein diet 5 The activity of gamma-glutamylcysteine synthetase, which is rate limiting for glutathione synthesis, was unaffected by dietary or sulphur amino acid intake or by the inflammatory response Substrate supply may therefore be a major determinant in glutathione synthesis in vivo 6 Total hepatic glutathione synthesis was affected by food intake, the type and amount of sulphur amino acids in the diet and by inflammation Total synthesis was 207, 137, 421 and 90 mumol/day for animals fed ad libitum the normal-protein diet, or low-protein diets supplemented with cysteine, methionine or alanine respectively, ad libitum Pair-feeding resulted in values of 76, 31, 71, and 0 mumol/day respectively After lipopolysaccharide injection, rates increased to 200, 117, 151 and 56 mumol/day respectively 8 Reductase and peroxidase activities increased in liver and lung, when low-protein diets which contained supplemental methionine or alanine were consumed ad libitum A reduction in food intake resulted in enzyme activity changes, which suggested that recycling of glutathione increased in lung and decreased in liver Injection of tumour necrosis factor reversed this effect 9 The restoration of glutathione concentrations in liver after an inflammatory challenge is closely associated with an enhanced rate of synthesis and increased recycling The former is impaired when inadequate sulphur amino acid is consumed before the challenge In lung, increased recycling of glutathione may help maintain concentrations when food intake is restricted, but not during inflammation

Leptin-induced decrease in food intake is not associated with changes in gastric emptying in lean mice

Abstract: Chronic treatment with leptin regulates body weight and energy balance and reduces food intake in obese and lean mice. In 18- to 20-h fasted lean mice (C57BL/6, +/+), we examined the acute effect of a single intraperitoneal injection of recombinant mouse leptin (0.12 mg/kg) on food intake and gastric emptying. Leptin reduced food intake, with a peak inhibition at the 5th h postinjection (69 +/- 12%/h), although there was no change in food consumption at the 1st h. Leptin did not alter the 4-h rate of gastric emptying of a solid nutrient meal (free access to Purina chow for either 1-, 2-, or 4-h period). In normal Sprague-Dawley rats fasted for 18-20 h, a single intraperitoneal injection of recombinant mouse leptin (0.2 or 1.2 mg/kg) did not modify the 7-h cumulative or hourly food intake. These results show that a single intraperitoneal injection of recombinant mouse leptin reduces food intake within 5 h while not influencing gastric emptying of ingested food in lean mice. Sprague-Dawley rats are unresponsive to the food intake-reducing effect of a single intraperitoneal injection of mouse leptin at a dose 10-fold higher than that shown to be effective in mice within the first 4-7 h postinjection.

Acute Ethanol Intoxication Suppresses the Pulmonary Inflammatory Response in Rats Challenged with Intrapulmonary Endotoxin

Abstract: The effects of acute ethanol intoxication on the functional activities of circulating and lung-recruited polymorphonuclear leukocytes (PMNs) and alveolar macrophages (AMs) were determined in rats challenged with intratracheal endotoxin to elucidate the mechanisms underlying the defects of pulmonary host defenses caused by acute ethanol intoxication. Acute ethanol Intoxication was induced by an intraperitoneal injection of 20% ethanol at a dose of 5.5 g of ethanol/kg. The control animals were injected with an equal amount of saline. Thirty min after intraperitoneal injection, rats were challenged with intratracheal endotoxin (300 micrograms/kg in 0.5 ml of saline) or saline. The rats were killed 3 h after intratracheal injection. CD11b/c expression on PMNs and phagocytosis and hydrogen peroxide generation of PMNs and AMs were determined by flow cytometry. Cytokine-Induced neutrophil chemoattractant (CINC) level in bronchoalveolar lavage fluid was measured with a specific ELISA. Intratracheal endotoxin caused a significant PMN recruitment into the lung in control animals. Acute ethanol intoxication completely suppressed the endotoxin-induced pulmonary recruitment of PMNs. Pulmonary-recruited PMNs exhibited a significant upregulation (8-fold) of CD11b/c expression when compared with circulating PMNs. This upregulation of CD11b/c expression was abolished by ethanol intoxication. Ethanol intoxication suppressed hydrogen peroxide generation by AMs and lung-recruited PMNs, and the phagocytosis of circulating PMNs. In contrast, acute ethanol intoxication did not affect pulmonary CINC production. These data indicate that the antiinflammatory effects of alcohol seem to be primarily based on the effects of ethanol on the PMNs themselves and not on the generation of certain chemotactic stimuli. In addition to the impairment of PMN recruitment, the suppression of AM and PMN activities also contributes to the mechanisms underlying ethanol-induced defects of pulmonary host defenses.

Treatment of surgically induced acute liver failure by transplantation of conditionally immortalized hepatocytes.

Abstract: The shortage of human livers available for hepatocyte isolation limits its clinical application. The availability of cloned, conditionally immortalized hepatocytes that could be grown in culture but would lose their transformed phenotype and provide metabolic support upon transplantation would greatly facilitate the treatment of acute liver failure. Toward this goal, we transduced isolated Lewis rat hepatocytes using a replication-defective recombinant retrovirus capable of transferring a gene encoding a thermolabile mutant simian virus 40 T antigen (SV40ts). The cloned, immortalized hepatocytes proliferate at 33 degrees C. At the nonpermissive temperatures (37-39 degrees C), they stop growing and exhibit characteristics of differentiated hepatocytes. These cells did not produce tumors when transplanted in mice with severe combined immunodeficiency disease or in syngeneic rats. To induce acute liver failure, Lewis rats were subjected to 90% hepatectomy (Hpx) and given 5% oral dextrose. All rats that did not undergo hepatocyte transplantation died within 96 hr. Fifty percent of rats that received intrasplenic injection of 10 x 10(6) primary Lewis rat hepatocytes (G2, n=6) or 10 x 10(6) SV40ts-conditionally immortalized (SV40ts-ci) hepatocytes (G3, n=8) 1 day before 90% hepatectomy survived, whereas 80% of rats that received an intraperitoneal injection of 200 x 10(6) primary Lewis rat hepatocytes (G4, n=10) or 200 x 10(6) SV40ts-ci hepatocytes (G5, n=10) on the day of hepatectomy survived. Survival after intraperitoneal injection of a cellular homogenate of 200 x 10(6) primary Lewis rat (G7, n=9) or SV40ts-ci hepatocytes (G8, n=10) on the day of Hpx was 33% and 40%, respectively, whereas survival after intraperitoneal injection of 200 x 10(6) Lewis rat bone marrow cells (G6, n=7) was 29%. Thus, transplanted, conditionally immortalized hepatocytes can be as effective as primary hepatocytes in supporting life during acute liver insufficiency. This work represents the first step in developing an hepatocyte cell line that would partially alleviate the organ-donor shortage and could be of potential clinical value.

Interleukin 1-Dependent and -Independent Mouse Melanoma Metastases

Abstract: Background: The adhesion of cancer cells to the endothelial lining of blood vessels, which is important for metastasis, is promoted by the action of interleukin 1 (IL-1) and other cytokines. Purpose: IL-1-producing melanoma cells were used to induce metastases in mice to test whether melanoma metastasis-wherever it occurs-depends on the action of IL-1. Methods: We used the following experimental designs in this study: 1) Male C57BL/6J mice were inoculated in the left cardiac ventricle with 5 x 10 4 murine B16 melanoma cells, and no treatment was given (control animals). 2) Mice received an intraperitoneal injection of either saline (control animals) or recombinant human IL-1 receptor antagonist (rHuIL-1Ra) 2 hours before the injection of cancer cells; thereafter, they received an additional injection of saline or rHuIL-1Ra daily for 20 days. 3) Mice received an intravenous injection of either saline or rHuIL-1Ra; 15 minutes later, mice that received saline were given either a second injection of saline (control animals) or an injection of bacterial lipopolysaccharide (LPS) to stimulate host IL-1 production and endothelial cell activation. The mice that received rHuIL-1Ra were also given an injection of LPS at this time. Six hours later, all mice were inoculated with cancer cells, followed by no further treatment. In all experiments, the mice were killed 20 days after the injection of cancer cells, and metastases were counted in multiple organs and bones. Metastasis incidence values (relating to the frequency that a given site was positive for metastasis) and metastasis development index values (relating to the extent of metastasis at a given site) were calculated. A hierarchical cluster analysis was performed to determine whether groups of organs exhibited characteristic changes in their metastasis development index values in response to the three treatments given (i.e., rHuIL-1Ra, LPS, or rHuIL-1Ra plus LPS). Reported P values are two-sided. Results and Conclusions: Treatment with rHuIL-1Ra alone significantly (P<.05) reduced the occurrence of metastasis in the bone marrow, spleen, liver, lung, pancreas, skeletal muscle, adrenal gland, and heart, indicating that host- and/or melanoma-derived IL-1 promoted metastasis in these organs; treatment with rHuIL-1Ra had no effect on metastasis in the kidney, testis, brain, skin, and gastrointestinal tract, suggesting that metastasis in these latter organs was IL-1 independent. Treatment with LPS alone significantly (P<.05) enhanced metastasis in the same organs for which rHuIL-1Ra treatment reduced metastasis, except for the heart and the adrenal gland. Treatment with rHuIL-1Ra 15 minutes before LPS treatment abrogated the LPS-mediated enhancement of metastasis. Two independent organ groups for which IL-1 promoted melanoma metastasis were identified in the cluster analysis.

Induction Of Heat Shock Protein 70 By Zinc- bis -(dlhydrogenaspartate) Reduces Cytokine Liberation, Apoptosis, And Mortality Rate In A Rat Model Of Ld100 Endotoxemia

Abstract: A prospective, randomized model of LD100/24 h endotoxemia was performed in male Wistar rats (n = 26; 250–300 g). The animals were divided into four groups: Group I (n = 5; saline treatment only), Group II (n = 5; Zn2+ treatment only), Group III (n = 8; saline pretreatment, lipdpolysaccharide (LPS) treatment), and Group IV (n = 8; Zn2+ pretreatment, LPS treatment). Zn2+ pretreatment was carried out by intraperitoneal injection of 50 mg/kg zinc-bis-(DL-hydrogenaspartate) (10 mg/kg Zn2+). LD100/24 h endotoxemia was induced by intraperitoneal administration of 20 mg/kg LPS of the Escherichia coli strain WO111:B4. Tumor necrosis factor α, interleukin-1β, and interleukin-6 were detected by enzyme-linked immunosorbent assay (ELISA). HSP70 expression in the lungs, the liver, and the kidneys was determined by immunohistochemistry, Western blotting, and an HSP70 ELISA. Apoptosis was also detected by an in situ apoptosis detection kit (TUNEL) and a cell death detection ELISA, respectively. This rat model of endotoxemia proves the close relationship between HSP70 expression, cytokine liberation, and development of apoptosis. The data demonstrate that: 1) Zn2+ is a potent inducer of HSP70 expression; 2) the application of Zn2+ leads to slightly increased cytokine plasma levels; and 3) the manipulation of the heat shock response by Zn2+ significantly increases the survival rate after LD100 endotoxemia. Enhanced survival rate in animals pretreated with Zn2+ may be explained by increased tissue levels of HSP70, a subsequent significantly decreased liberation of the proinflammatory cytokines after LPS challenge, and a significantly decreased rate of apoptosis.

Dietary n-3 fatty acids differentially affect sickness behavior in mice during local and systemic inflammation

Abstract: We tested the hypothesis that increased dietary fish oil levels (via modulation of the production of inflammatory mediators) modulate sickness symptoms (i.e., anorexia, cachexia, fever, lethargy) of systemic and local inflammation. Swiss Webster mice were implanted with biotelemeters to measure body temperature and motor activity and were fed a diet high in n-3 fatty acids (17% wt/wt menhaden oil) or a reference diet (17% wt/wt hydrogenated coconut oil or normal rodent chow) for 6 wk. Local inflammation was induced by subcutaneous injection of turpentine (100 microl/mouse). Systemic inflammation was elicited by intraperitoneal injection of lipopolysaccharide (LPS; 2.5 mg/kg). Fever, lethargy, anorexia, and weight decrease during turpentine abscess were all inhibited (P < 0.05) in mice fed the fish oil diet. Indomethacin, similar to the fish oil diet, attenuated the turpentine-induced symptoms in mice fed a normal diet. Dietary n-3 fatty acids prevented fever and attenuated the decrease in body weight caused by LPS but did not affect the LPS-induced lethargy and anorexia. Within 90 min of LPS injection, the bioactivity of plasma tumor necrosis factor-alpha (TNF-alpha) increased to 98.2 +/- 5.1 ng/ml in mice fed fish oil compared with 32.6 +/- 3.6 ng/ml in those fed the reference diet (P < 0.05). Plasma prostaglandin E2 (PGE2) levels after LPS injection of mice fed the control diet increased within 90 min to 16.4 +/- 5.1 pg/ml. Mice fed the fish oil diet did not show any elevation in plasma PGE2 levels at that time (P < 0.05). We speculate that dietary n-3 fatty acids suppressed PGE2-related responses, including a PGE2-dependent negative feedback on TNF-alpha production, which resulted in differential modulation of sickness behavior depending on the locus of inflammation.

Central noradrenergic system modulates plasma interleukin-6 production by peripheral interleukin-1

Abstract: The role of the central noradrenergic system in systemic interleukin-6 (IL-6) production induced by intravenously administered recombinant human interleukin-1 beta (IL-1 beta) was examined in rats. Pretreatment of rats intracerebroventricularly with 6-hydroxydopamine (6-OHDA, 100 or 200 micrograms/rat) significantly attenuated the increase in plasma IL-6 levels caused by IL-1 beta (2 micrograms/kg i.v.). A modest inhibition of the IL-1 beta-induced plasma IL-6 production was observed following pretreatment with prazosin (20 micrograms/rat i.c.v.) but not after administration of idazoxan or propranolol. There were no significant increases in the IL-6 content in the hypothalamus, medulla oblongata, and cortex of the brain after intravenous IL-1 beta. Adrenalectomy produced an augmented plasma IL-6 response to intravenous IL-1 beta, whereas chemical sympathectomy with intraperitoneal injection of 6-OHDA (50 or 100 mg/kg) decreased the IL-1 beta-induced plasma IL-6 levels. Nor-epinephrine (NE), in the dose range 10(-6)-10(-4) M, significantly increased the IL-6 levels in the rat spleen lymphocyte culture media. At doses of 10(-9)-10(-7) M, NE enhanced the effect of IL-1 beta on the IL-6 release by spleen lymphocytes in a dose-dependent manner. These findings suggest that the plasma IL-6 response to intravenous IL-1 beta is partially mediated through the activation of the central noradrenergic system and a consequent increase in the sympathetic outflow to the peripheral tissues and that the NE released from the sympathetic terminals may function as a mediator and/or modulator to facilitate the synthesis/release of IL-6 in the sympathetic nerve-innervated organs.

Gradual development of organ damage in the murine zymosan-induced multiple organ dysfunction syndrome.

Abstract: A well defined animal model is a prerequisite to test intervention strategies aimed at preventing the development of the multiple organ dysfunction syndrome. This study compares changes in clinical parameters to histopathologic changes in tissues, observed over a 12 day period after a single intraperitoneal injection of zymosan in C57BL/6 mice. Administration of zymosan induced gradual and progressive changes in wet and dry organ weight of the liver, kidneys, and particularly, lungs and spleen that correlated with increasing histopathology. From 6 days after zymosan injection onwards, hemorrhagic spots were found in the lungs evolving into massive hemorrhages at 12 days, when thickened interstitial walls and loss of the honey comb-like structures were observed. The liver displayed progressive accumulation of macrophage-like and mononuclear cells. After 12 days, numerous granuloma-like structures were disseminated throughout the liver parenchyma. The spleen displayed great changes in red and white pulp with increasing numbers of megakaryocytes and plasma-like cells. In the kidneys, necrosis of the tubular epithelium adjacent to granulomas on the ventral (peritoneal) side was found. In mice, a single intraperitoneal challenge with zymosan leads to progressive multiple organ damage, which becomes apparent at some time after the insult. This animal model displays a number of features encountered in human multiple organ dysfunction syndrome and appears suitable to conduct intervention studies.

Involvement of endogenous kinins in the pathogenesis of peptidoglycan-induced arthritis in the Lewis rat

Abstract: Objective. To investigate the pathophysiologic roles of endogenous bradykinin (BK) and des-Arg 9 -BK on local and systemic inflammatory responses in a rat model of acute arthritis induced by peptidoglycan-polysaccharide (PG-APS). Methods. Female Lewis rats were injected intraperitoneally with PG-APS. Selective antagonists of B 1 (Lys-[Leu8]-des-Arg9-BK) and B 2 (Hoe 140) receptors were infused at 500 μg/kg and 5 mg/kg per day for 6 days, starting 3 days before induction of inflammation, with subcutaneous micro-osmotic pumps. The local inflammatory response was assessed by paw edema, joint swelling, and tissue content of BK and des-Arg 9 -BK. These peptides were measured by highly sensitive and specific chemiluminescent enzyme immunoassays. Systemic inflammatory reaction was evaluated by the hepatic concentration of the type 2 acute-phase protein T-kininogen. I Results. PG-APS induced significant paw edema and joint swelling 24-72 hours after intraperitoneal injection. The maximal responses to PG-APS observed at 72 hours were significantly reduced (31-38%) by the combination of both B 1 and B 2 receptor antagonists at 5 mg/kg per day. PG-APS induced a significant increase of BK (up to 5.3-fold) and des-Arg 9 -BK (up to 4.1-fold) 72 hours after challenge. Liver T-kininogen content was increased by 5.3-, 7.7-, and 5.8-fold at 24, 48, and 72 hours, respectively, after PG-APS injection. At 24 hours, Hoe 140 and Lys-[Leu 8 ]-des.Arg 9 -BK increased liver T-kininogen content by 43% and 45%, respectively, but they had no effect at 72 hours. Conclusion. The results indicate that endogenous kinins are involved in local and systemic acute inflammatory responses, through both B 1 and B 2 kinin receptors, in the model of PG-APS-induced arthritis.

Intraperitoneal Injection of Genetically Modified, Human Mesothelial Cells for Systemic Gene Therapy

Abstract: An ideal cell type for ex vivo gene therapy should be easy to biopsy, propagate, and genetically engineer in culture, should be transplantable using simple procedures, and should express therapeutic proteins at useful levels. The mesothelial cell appears to satisfy these criteria. Several thousand proliferative mesothelial cells were present in typical specimens of nonpathologic human peritoneal fluid obtained by needle aspiration. These divided rapidly in a specialized medium to yield pure cultures of approximately 10(7) cells within 2 weeks. The replicative lifespan of mesothelial cells cultured from adults was approximately 42-52 population doublings, permitting expansion and cryopreservation of a lifetime supply of autologous cells from one fluid sample. Cells transduced with a human growth hormone (hGH) adenoviral vector secreted 100-300 microg of hGH/10(6) cells per day for at least 6 weeks in culture when maintained at quiescence. Intraperitoneal injection of transduced cells into athymic mice resulted in rapid systemic delivery of hGH, with peak plasma levels of 0.1-1 microg/ml declining over 3 weeks to <1 ng/ml. Mice receiving a second injection of engineered cells displayed the same plasma hGH levels and duration as naive mice. Cells labeled with a beta-galactosidase vector were identifiable by in situ enzymatic staining as clusters attached to peritoneal surfaces at multiple sites for at least 19 days after injection. Cells serially passaged through about three-quarters of their lifespan before transduction and injection were as effective at hGH delivery as earlier-passage cells. These results indicate the clinical potential for ex vivo gene therapy using mesothelial cells.

Safety studies of the intraperitoneal injection of E1A–liposome complex in mice

Abstract: The HER-2/neu (also called c-erbB-2) proto-oncogene is overexpressed in many human cancer cells, including those of breast cancer and ovarian cancer. We have previously shown that adenovirus 5 E1A inhibits HER-2/neu transcription and functions as a tumor suppressor gene in HER-2/neu-overexpressing cancer cells. Liposome-mediated E1A gene transfer suppresses tumor development and prolongs survival of tumor-bearing mice. In support of a phase I clinical trial of an E1A-liposome complex administered to patients with HER-2/neu-overexpressing breast of ovarian cancer, we conducted a series of studies to evaluate the safety of intraperitoneal injection of E1A in normal mice. The cumulative doses used were from five to 40 times the DNA-lipid starting dose proposed for the phase I clinical trial. In this dosing range, the administration of the E1A-liposome complex had no adverse effects on renal, hepatic and hematological parameters studied. No major organ pathologic changes were observed. We concluded that intraperitoneal administration of E1A-liposome complex at the proposed dose would not be expected to produce significant toxicity. The E1A-liposome clinical trial was recently approved by the Recombinant DNA Advisory Committee and Food and Drug Administration for a phase I trial in patients with HER-2/neu-overexpressing breast and ovarian cancer.