Showing papers in "Research in Experimental Medicine in 1995"

Treatment of patients with severe head injury by triamcinolone: a prospective, controlled multicenter clinical trial of 396 cases

Abstract: The present studies were conducted to test whether the outcome of severe head injury is improved by early administration of the synthetic corticosteroid triamcinolone. In a prospective, double-blind, multicenter clinical trial, 396 patients with severe head injury were randomized to a steroid group (n = 187) receiving 200 mg triamcinolone acetonide (Volon A soluble) i.v. within 4 h after trauma, followed by 3 x 40 mg/day i.v. for 4 days, and 3 x 20 mg/day i.v. for a further 4 days, and a placebo group (n = 209) receiving injections which did not contain any active drug. The placebo group was subjected to the same standard treatment procedures. Clinical features were not different between the groups upon admission to hospital. Subdural hematoma, epidural hematoma, and focal supratentorial contusion were among the most frequent diagnoses. The result of treatment with triamcinolone was assessed at discharge from the hospital and at 1 year after trauma, using the Glasgow Outcome Scale. Differences in favor of steroid treatment could be detected with regard to the patients' condition at discharge (P = 0.0634). More patients with steroids had a good recovery (49.2% vs 40.7%), and fewer died (16.0% vs 21.5%). Differences in outcome were even more pronounced (P < 0.0145) in patients with a focal lesion and a Glasgow Coma Score on admission of < 8 (n = 93). In this group, 34.8% of the patients made a good recovery, as against 21.3% of the placebo group; mortality was also lower in the verum group (19.6% vs 38.3%). The results indicate that a major subgroup of patients with severe head injury benefits from early administration of triamcinolone. Efficacy of the treatment can be expected, in particular, in patients with a focal cerebral lesion and a Glasgow Coma Score of < 8 on admission. Administration of steroids beginning at the scene of an accident would therefore be beneficial in these cases.

Ischemia reperfusion of the pancreas: A new in vivo model for acute pancreatitis in rats

Abstract: Based on the concept that ischemia is an important factor in the pathogenesis of acute pancreatitis, we developed a new model of complete ischemia/reperfusion of the pancreas in the rat. The aim of this study was to investigate the microcirculation of the pancreas after complete and reversible ischemia at different times after reperfusion by using intravital fluorescence microscopy. In addition, the effect of ischemia/reperfusion on the pancreas was assessed by means of light and electron microscopy and measurement of serum pancreas amylase concentration. In 35 adult Sprague-Dawley rats ischemia of the pancreas was induced by temporary occlusion of the four supplying arteries. Sham-operated animals served as controls (group A). After periods of 30 min (group B), 60 min (group C) or 120 min (group D) of ischemia the organ was reperfused. To exclude the influence of hypovolemia on microcirculation in group E (120 min ischemia) hydroxyethylstarch (HES) was given i.v. to maintain central venous pressure at baseline values. For intravital fluorescence microscopy the pancreas was exteriorized on a stage and quantitative analysis of microcirculation, including functional capillary density and leukocyte-endothelium interaction, was performed after 30 min, 1 h and 2 h of reperfusion. Serum pancreas-amylase was measured at control (prior ischemia) and at 2 h after reperfusion. Tissue samples for light and electron microscopy were taken 2 h after reperfusion. In sham-operated animals, functional capillary density (FCD) remained within baseline values (FCD 407.7±9 cm−1) during reperfusion. Dependent on the time of ischemia and time of reperfusion a gradual reduction in functional capillary density was observed; after 2 h of ischemia only 35% of capillaries were perfused (FCD 140.9±28.3 cm−1). Reduced functional capillary density was associated with an increase of perfusion heterogeneity to a maximum of 0.65±0.12, as against 0.13±0.02 in control animals. With a 2 h ischemia leukocyte-endothelium interaction was enhanced after 0.5 h of reperfusion (8-fold increase of adherent leukocytes in comparison to control) followed by a further significant increase until 2 h after the beginning of reperfusion. Amylase concentration after ischemia of 2 h (2967±289 U/l) was significantly higher as compared to controls (1857±99 U/l). Differences between group E and D were not observed. Pancreatic tissue injury was ascertained by histopathological studies. These results indicate that complete ischemia/reperfusion of the pancreas induces pancreatic microvascular failure. The severity of changes depends on duration of ischemia and duration of reperfusion. The morphological and biochemical changes suggest that ischemia/reperfusion causes an inflammatory reaction as observed in acute pancreatitis.

Semi-quantitative analysis of cytokine gene expression in blood and cerebrospinal fluid cells by reverse transcriptase polymerase chain reaction

Abstract: An easy, reproducible and semi-quantitative, non-radioactive method for the analysis of mRNA expression for various cytokines, (i.e., Interleukin (IL)-1β, IL-4, IL-6, tumor necrosis factor (TNF)-α, lymphotoxin (LT), transforming growth factor (TGF)-β, interferon (IFN)-γ and endothelin-1 (ET-1)) in cells from cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) has been established. By means of polymerase chain reaction primers that cover a splice junction, amplification of contaminating DNA was omitted. Densitometric scanning of ethidium bromide-stained agarose gels proved to be very sensitive for semiquantitative analysis of PCR products. Serial tenfold dilutions of cDNA revealed a log-linear regression from 106 to 102 cells under optimal cycle conditions. The intra- and inter-assay variability of the method was below 10%. With this assay, the cytokine expression pattern of as few as 104 mononuclear cells from blood or CSF was determined. This method made it possible to detect differences in the cytokine gene expression pattern of mononuclear cells from patients with different neurological diseases. CSF cells from 43 patients with various neurological diseases were analyzed. TNF-α, LT, and IL-1 mRNA were prominent in the CSF cells of most patients with bacterial meningitis. TNF-α, LT, IFN-γ and IL-6 mRNAs were detected in patients with active multiple sclerosis, whereas TNF-α, IL-6, and endothelin-1 mRNA expression was found frequently in patients with HIV encephalitis. Pro-inflammatory cytokines were rarely detected in CSF cells from patients with non-inflammatory diseases of the central nervous system. In blood mononuclear cells from patients with multiple sclerosis, TNF-α mRNA expression was associated with disease activity. The sensitivity, specificity, velocity and reliability of this assay considerably facilitates the analysis of cytokine production in mononuclear cells even in conditions where only a limited number of cells is available for analysis.

Effects of oxytocin on the function and morphology of the rat adrenal cortex: in vitro and in vivo investigations.

Abstract: The effects of oxytocin (OX) on the function and morphology of the rat adrenal cortex were studied in vivo and in vitro. OX exerted a potent stimulatory action on basal, but not 10(-8) M ACTH-stimulated corticosterone (B) secretion of dispersed rat inner (zona fasciculata and zona reticularis) adrenocortical cells (maximal effective concentration: 10(-9) M); in contrast, at higher concentrations (10(-7)/10(-6) M) OX inhibited maximally ACTH-stimulated B output. A single subcutaneous (s.c.) injection of 1.2 nmol/100 g body weight OX resulted in a long-lasting (up to 12 h) rise in plasma B concentration (PBC). The prolonged administration of OX (daily s.c. injections of 0.6 or 1.2 nmol/100 g for 10 days) caused a marked lowering in the adrenal weight and volume of all adrenocortical zones, that in turn was due to a decrease in the number of their parenchymal cells; however, the average volume of inner adrenocortical cells was significantly increased. Basal PBC was lowered, but its response to ether stress was unchanged in comparison with control rats. Prolonged OX treatment did not change B secretion by adrenal slices, but it markedly raised that of dispersed inner adrenocortical cells. Our present findings clearly show that the effects of OX on the adrenal cortex depend on the experimental model employed (in vitro versus in vivo) and the duration of treatment (acute versus chronic). Taken together they allow us to conclude that OX exerts an acute direct stimulatory effect on the rat adrenal cortex, and a chronic inhibitory one, that at least in part could be due to the interference of OX with the mechanism(s) of intracellular transduction of the ACTH secretagogue signal.

Lipid peroxidation in experimental spinal cord injury. Comparison of treatment with Ginkgo biloba, TRH and methylprednisolone.

Abstract: Ischaemia-induced lipid peroxidation is one of the most important factors producing tissue damage in spinal cord injury. In our study, the protective effects ofGinkgo biloba, thyroid releasing hormone (TRH) and methylprednisolone (MP) on compression injury of the rat spinal cord were investigated. For this study 45 rats in four groups, including control, MP, TRH andGingko biloba, were used to determine the formation of malondialdehyde (MDA). All the animals were made paraplegic by the application clip method of Rivlin and Tator. Rats were divided randomly and blindly to one of four treatment groups (ten animals in each). MP andGinkgo biloba treatments significantly decreased MDA levels (F=54.138,P<0.01). These results suggest that MP andGinkgo biloba may have a protective effect against ischaemic spinal cord injury by the antioxidant effect.

Inhibition of tumor necrosis factor production and ICAM-1 expression by pentoxifylline: beneficial effects in sepsis syndrome

Abstract: Tumor necrosis factor (TNF) has a pivotal role in the pathogenesis of sepsis and septic shock. Suppression of its biosynthesis might therefore be one of the strategies in the treatment of sepsis. When peripheral white blood cells were stimulated with eitherE. coli lipopolysaccharide (LPS) orStaphylococcus aureus, pentoxifiline (PTX) inhibited TNF production. In contrast, only a moderate inhibitory effect was observed on the induction of interleukin 6 (IL-6). PTX inhibited not only the TNF production of monocytes, but also the TNF and IL-6 producing capacities were higher in septic patients (n=31) than in healthy blood donors (n=15). Administration of PTX (400 mg/day) to 20 of the septic patients resulted in TNF production similar to that found in healthy controls. It also subsequently led to an improvement of the clinical status classified by the APACHE II score. The soluble intercellula adhesion molecule-1 (sICAM-1) level was significantly higher in the sera of septic patients before PTX treatment (800–1200 ng/ml) than in normal individuals (50–150 ng/ml), but it decreased following PTX therapy. Cytofluorometric analysis revealed that the expression of ICAM-1 on stimulated mononuclear cells was inhibited by PTX. It is presumed that the suppressive effect of pentoxifylline on TNF production may be of clinical importance, improving the therapeutic strategies in septic syndrome.

Delayed gastric emptying in conscious male rats following chronic estrogen and progesterone treatment.

Abstract: Several clinical observations and animal experiments have led to speculation concerning the possible effects of pregnancy and pregnancy-associated sex steroids on gastrointestinal function. It was reported that estrogen increases intestinal contractile activity, while progesterone or the combination of estrogen and progesterone decreases it. In order to measure gastric emptying, a methylcellulose test meal was given orally into the stomach of conscious rats. In progesteronetreated rats, at the dose of 0.2 mg/kg, gastric emptying was not significantly different from that of the control, but it was found to be significantly delayed at the dose of 10 mg/kg (P<0.05). Estrogen treatment at doses of 20 μg/kg and 600 μg/kg significantly delayed gastric emptying, when compared with controls (P<0.001). Combined therapy of estrogen and progesterone induced a significant delay in gastric emptying rate compared with the control group (P<0.001). In the animals with pseudopregnancy treatment (100 μg/kg estrogen+ 15 mg/kg progesterone; 7–12 days) the gastric empying rate was significantly different from that of the control (P<0.05). We conclude that both estrogen and progesterone exert inhibitory effects on gastric emptying, and this may account for the disturbances in gastrointestinal function that pregnant women frequently experience.

Electrical properties of extracted rat liver tissue

Abstract: We attempted to investigate the process of ischemia-induced disturbances in the rat liver, employing the electrical bio-impedance technique. The electrical bio-impedance was measured continuously over 6 h by the 4-electrode method, at various incubation temperatures, in six liver samples extracted from male Wistar rats. The electrical properties of biological tissues can be expressed in terms of three parameters: extracellular resistance (Re), intracellular resistance (Ri) and cell membrane capacitance (Cm). These three parameters were calculated from the measured values of the electrical impedance by the curve-fitting technique, using a computer program. The Re value increased rapidly after the rat livers were extracted, and then decreased slowly. The Re value reached a peak after about 13 min at 36 degrees C, and then decreased slowly, becoming constant after 3 h. There was a negative correlation between the Tmax of Re (the time when Re reached a maximum) and the incubation temperature (R = -0.973, P < 0.001). The Ri value decreased once in the early stage after extraction, followed by almost no change and then an increase after 4 h at 36 degrees C. The Cm showed a similar pattern of change to the Re value, and a negative correlation was also found between the Tmax of Cm and the incubation temperature (R = -0.969, P < 0.001). The increase in the Re and Cm values, and the decrease in the Ri value for quite long periods after the blood flow has stopped, suggest an increase in the resistance of extracellular fluid due to a decrease in its volume, an increase in cell membrane capacitance due to cell swelling, and a decrease in cellular fluid resistance due to an increase in its volume. The time when the Cm value decreases rapidly after an initial gradual decrease after the peak corresponds well with the time when the Ri value begins to increase, from which it is estimated that cell lysis proceeds and that the flow of extracellular fluid into the cell begins at this time. The findings of this study suggest the possibility of estimating the changes in liver tissue or the tissue structure due to ischemia.

Photodynamic therapy decreases cancer colonic cell adhesiveness and metastatic potential

Abstract: Plasma membrane damage induced in various cell targets by hematoporphyrin (HPD) photodynamic therapy (PDT) could modify cancer cell adhesiveness, an important parameter in cancer metastasis. We investigated the effect of HPD or HPD incubation followed by argon laser light on the adhesiveness of progressive (PROb) or regressive (REGb) cancer cells of the same colonic origin but with a different in vivo metastatic potential. Adhesiveness was studied on plastic or endothelial cell monolayers (ECM). In the absence of treatment, both PROb and REGb cells adhered better on plastic than on ECM. HPD alone or HPD-PDT induced toxicity proportional to the HPD dose. HPD-PDT increased the adhesiveness rate of both cell lines on plastic and decreased it on ECM. HPD-PDT of ECM increased adhesiveness, but only at HPD doses causing at least 50% cell death. With HPD treatment alone or HPD-PDT of culture media, there was no significant decrease in cell adhesiveness to ECM. We also studied the effect of HPD or HPD incubation followed by argon laser light on the metastatic potential of cancer cells, which was decreased for PROb with HPD alone or HPD-PDT. Decreased adhesiveness of colonic cancer cells to ECM after HPD-PDT was thus correlated with decreased metastatic potential. REGb cells did not acquire a progressive phenotype either in vitro or in vivo after HPD-PDT.

Prevention of experimental hepatic metastasis with thromboxane synthase inhibitor.

Abstract: To investigate the effectiveness of thromboxane (Tx) synthase inhibitor in the prevention of experimental hepatic metastasis, an in vivo study was designed. Hepatic metastasis was brought about by injection of 1 x 10(5) cells of colon 38 tumor into the portal vein of C57 B1/65 mice. Seven groups (n = 16 in each group) received different treatments: with TxA2 synthase inhibitor (sodium ozagrel), 5, 10 or 15 mg/kg BW before tumor inoculation, and daily for the following 3 days, (groups A, B and C, respectively); with acetyl salicylic acid (aspirin), 1.0, 1.5 or 2.0 mg/kg BW (groups C, D, and E, respectively); a control group, inoculated with vehicle only. Serum TxB2, a stable metabolite of TxA2, and prostaglandin F1 alpha were measured. Labeling index for tumor proliferation by bromodeoxy-uridine radioimmuno-assay was also studied. Incidence of metastasis in groups A (60.5%), B (49.5%), C (43.0%), D (80.5%), E (66.0%) and F (58.4%) was less than that in the control group (100%). Tumor size, number of labeling index did not differ among the groups. Serum TxB2 (pg/ml) levels were significantly lower in all of the groups than in the control. Serum PGF1 alpha levels in the groups with aspirin were lower than those in sodium ozagrel. Tx synthase inhibitor is effective in the prevention of experimental hepatic metastasis when it is given before and immediately after tumor inoculation. As Tx synthase inhibitor leaves metabolic pathway to PGI2 production intact, it is more effective in the prevention of metastasis than aspirin since aspirin inhibits both thromboxane and PGI2.

Effect of prior portosystemic shunt on early hepatic hemodynamics and sinusoids following 84% hepatectomy in dogs

Abstract: The effects of a prior portosystemic shunt (PSS) on the hepatic hemodynamics and sinusoids shortly after an 84% hepatectomy (Hx) were investigated in dogs. Fifteen mongrel dogs were divided into three groups, a 70% Hx group (n=5), an 84% Hx group (n=5) and an 84% Hx+PSS group (n=5). In the last group, a shunt was inserted between the splenic and femoral veins prior to the hepatectomy. The systemic and hepatic hemodynamics were measured, before and 180 min after the hepatectomy, and the remaining liver tissue was then examined immuno-histochemically by light microscopy using the thrombomodulin (TM) staining method. The postoperative portal vein pressure and the vascular resistance were significantly lower in the PSS group than in the 84% non-PSS group. The total postoperative hepatic blood flow was higher in the 84% non-PSS group than in the other two groups. Immunohistochemical observation after TM staining indicated that the sinusoidal endothelial cells in the 84% non-PSS group were markedly damaged 3 h after surgery. We conclude that a prior PSS improves the hepatic hemodynamics and is beneficial to the sinusoids within the first few hours of an 84% hepatectomy in dogs.

Changes in levels of glutathione and related compounds and activities of glutathione-related enzymes during rat liver regeneration.

Abstract: The levels of glutathione and glutathione disulfide increased during the regeneration process of rat liver, reaching a maximum (about twice the control value) on day 2 and reverting to the normal level within 5 days. During this regeneration process, changes in the hepatic level of cysteine, glycine and glutamate, the substrates for glutathione synthesis, were determined. The cysteine level in liver increased, reaching a maximum on day 2 and returned to the normal level after 5 days. The levels of glycine and glutamate did not change. The enzyme activities of cystathionine-beta synthase and gamma-cystathionase for cysteine synthesis, and of gamma-glutamylcysteine synthetase, which is a limiting enzyme for glutathione synthesis, were clearly increased in regenerating liver. The increase of glutathione level could be clearly accounted for by the elevation of these enzyme activities.

Glutathione in plasma, liver, and kidney in the development of CCl4-induced cirrhosis of the rat

Abstract: Plasma glutathione is markedly decreased in human cirrhosis of the liver. This decrease is said to be caused by reduced concentrations of liver glutathione. However, several studies on hepatic glutathione have revealed its concentrations to be unchanged, decreased, or even elevated. To test these inconsistencies we investigated the glutathione status of plasma, liver, and kidney in rats chronically exposed to carbon tetrachloride (CCl4). After 14 weeks of CCl4 treatment, histological examination revealed progressive cirrhotic transformation. After 20 weeks, complete micro-nodular cirrhosis was present and distinct ascites had developed. Plasma reduced glutathione (GSH) decreased by 34% in the early and by 44% in the late group, paralleled by a 65% and 76% decrease of plasma oxidized glutathione (GSSG). Liver GSH in early stages of cirrhosis was reduced by 49%, but in late cirrhosis it did not differ from controls. In contrast, liver GSSG increased by 35% in the early and by 191% in the late group. Kidney GSH increased by 14% in early and 44% in late stage cirrhosis. Kidney GSSG was unchanged in the early group, but increased by 18% in the late group. The decrease of plasma GSH and GSSG is closely related to the severity of experimental cirrhosis and inversely related to an increase of hepatic oxidized glutathione. The hepatic content of reduced glutathione, however, is decreased in early cirrhosis only. According to these results the inconsistent findings in man could be due to differences in the stages of cirrhosis in the patients. The increase in kidney glutathione is a new finding that needs further investigation, but it may probably be related to kidney dysfunction in liver disease.

Carbohydrate-binding proteins (plant/human lectins and autoantibodies from human serum) as mediators of release of lysozyme, elastase, and myeloperoxidase from human neutrophils.

Abstract: Analysis of cell surface glycosylation not only provides information about cell properties such as their state of differentiation or histogenetic lineage. The carbohydrate chains also provide potentially functional binding sites to endogenous carbohydrate-binding proteins. This interaction can elicit consequent signalling processes. Because of the importance of neutrophils in the host defence system, we monitored the effect of the binding of such sugar receptors to their cell surface on the release of the enzymatic activities of lysozyme, elastase, and myeloperoxidase. Besides the mannose-binding lectin concanavalin A and the immunomodulatory α/β-galactoside-binding lectin fromViscum album L., three preparations of human sugar receptors—β-galactoside-binding lectin (Mr 14 kDa) and two affinity-purified polyclonal IgG fractions from serum with the capacity to recognize α- or β-galactosides, respectively—were used. Two animal lectins from chicken liver and intestine that bind β-galactosides, as well as the lectin-like human serum amyloid P component, were included in order to assess the importance of slight differences in ligand recognition. Cytochalasin B-enhanced enzyme release was invariably seen with the two plant lectins and the chicken liver β-galactoside-binding lectin, but the related intestinal lectin did not increase enzyme release. The mammalian homologue of these avian lectins triggered lysozyme secretion, and the lactoside-binding IgG fraction enhanced the amount of extracellular elastase activity slightly but significantly. Thus, the actual lectin, not the nominal specificity of sugar receptors, is crucial for elucidation of responses. Due to the highly stimulatory activity of the two plant lectins, neutrophils from patients with non-cancerous diseases and from patients with lung cancer were monitored for the extent of lectin-mediated enzyme release. Only the concanavalin A-mediated reactivity of the neutrophils was associated with the type of disease.

Somatostatin reduces the levels of tumor necrosis factor alpha in a rat model of endotoxemia induced by lipopolysaccharide.

Abstract: The role of tumor necrosis factor alpha (TNF) in the toxic and lethal effects of the endotoxemia associated with septic shock is well known. This study was designed to establish whether natural somatostatin (SS-14) is capable of modifying the production of TNF in a model of septic shock induced in the rat by bacterial lipopolysaccharide (LPS), and its theoretical relationship to prostaglandin E2 (PGE2). An experimental study was carried out in 80 Wistar rats subjected to intravenous LPS injection. Perfusion of SS-14 at 2 micrograms/h or continuous isotonic saline (IS) at 0.1 ml/h started 30 min prior to LPS injection and continued until 90 min after. All the animals were primed 15 days earlier with on intraperitoneal dose of BCG (2.2 x 10(7) CFU). ELISA assays were used to measure TNF levels after 90 min of perfusion and those of PGE2 at 30 and 90 min. The effects of two different doses of LPS (0.5 mg/kg of body weight and 5 mg/kg bw) were compared. SS-14 administration was associated with a decrease in TNF levels (1130.0 +/- 272.4 vs 4720.0 +/- 1278.1 pg/ml, P = 0.013), and an increase in serum PGE2 basally (255.7 +/- 94.2 vs 62.0 +/- 10.6 pg/ml, P = 0.04) and after 90 min of perfusion (1872.7 +/- 1250.6 vs 1009.7 +/- 612.0 pg/ml, P = NS), there being a statistically significant correlation between the basal PGE2 levels and these TNF after 90 min when compared using a regression model (r = -0.88, P = 0.04 for the 0.5 mg/kg dose; r = -0.47, P = 0.07 for 5 mg/kg). At 90 min, the level of TNF also depended on the PGE2 values (r = 0.84, P = 0.07 for 0.5 mg/kg; r = 0.55, P = 0.03 for 5 mg/kg). Multiple regression permitted TNF levels to be estimated on the basis of basal and 90 min PGE2 levels (P = 0.03). Pretreatment with SS-14 led to a significant reduction of TNF and an increase of PGE2, there being an apparent correlation between the two.

Experimental evaluation of peritoneum and pericardium as dural substitutes

Abstract: Although many substances have been tested in the search for an ideal dural substitute, an entirely satisfactory material has still not been found. The authors report an experimental study involving the closure of dural defects in rabbits with biomaterials developed from pig peritoneum and pericardium. Macroscopic and histologic examination, performed over a period between 15 and 45 days after implantation showed slight or no adhesion between the graft material and the cortex. No infection, CSF leakage, fistula or toxicity was noticed. The results demonstrated that these biomaterials could be used as satisfactory dural substitutes.

Verapamil and flunarizine protect the isolated perfused rat liver against warm ischemia and reperfusion injury.

Abstract: Using the model of the isolated perfused rat liver, we investigated the influence of the two pharmacologically different calcium channel blockers, verapamil and flunarizine, on changes of ion homeostasis, liver weights, pH deviations and enzyme activities during warm ischemia (37°C) and reperfusion. The LDH and GLDH activities were determined and the calcium, potassium, and sodium concentrations were measured in the effluent. Warm ischemia (180 min) caused an increased enzyme release, a high influx of calcium and sodium into the liver and a massive potassium efflux current. Normoxic reperfusion led to a further increase in hepatic enzyme release and although the loss of potassium ceased, the calcium influx into the liver continued. By the end of reperfusion the liver weight had increased significantly (P<0.01) in the control group. The two calcium entry blockers were added to the perfusate in various concentrations. Both substances protected the liver against warm ischemia and normoxic reperfusion damage, but they did not inhibit calcium inflow. However, the potassium efflux was significantly reduced by all concentration tasted (P<0.001). After reperfusion the liver weights were significantly lower in the treated groups (P<0.001) than in control animals. Thus, the calcium entry blockers verapamil and flunarizine protect liver cells against damage caused by warm ischemia and reperfusion. Furthermore, they prevent the disruption of intracellular potassium homeostasis, which seems to be related to improved volume regulation of liver cells.

Amylin immunoreactivity in the rat trachea and characterization of the interaction of amylin and somatostatin on airway mucus secretion.

Abstract: Amylin is a peptide containing 37 amino acids that is mainly expressed in pancreatic B-cells and cosecreted with insulin. It is the major component of the islet amyloid typically found in non-insulin-dependent diabetes mellitus. The amylin mRNA is present in RNA isolated from lung, and amylin receptors have been detected in lung membranes. Recently, amylin was shown to be a potent stimulator of airway mucus secretion. In this study, we characterized the site of amylin expression in rat trachea using a highly specific antiserum and the functional interaction of amylin with somatostatin-14 in mucus secreting cells. Amylin-like immunoreactivity is present in epithelial cells of submucous gland acini. The expression pattern varies, since some acini showed strong staining while others were negative. In addition, some columnar cells of the tracheal lining epithelium are strongly stained. Amylin applied submucosally is a potent stimulator of airway mucus secretion. Somatostatin inhibits this effect. Amylin may influence airway mucus secretion by paracrine and endocrine mechanisms, and our data suggest that amylin and somatostatin belong to the increasing number of peptides that are known to influence airway function.

The effect of parenteral indomethacin on T-lymphocyte subpopulations and cytokine production in patients under major surgical operations

Abstract: Major surgical trauma has been considered as a cause of immunosuppression mainly through the production of prostaglandin E2 from activated monocytes/macrophages. In the present study we investigated the effect of parenteral indomethacin—a cyclo-oxygenase inhibitor—on T-lymphocyte subsets and cytokine production in patients under major operations. We studied 20 patients undergoing major surgical procedures, 10 of whom were randomly treated pre-and postoperatively with indomethacin (group 2) and 10 were not (group 2). We measured total T-cells T-helper, T-suppressor, T-helper/T-suppressor (Th/Ts) cell ratio, NK-cells and interleukin (IL-1) and tumor necrosis factor production by endotoxin-or phytohemagglutinin-stimulated peripheral blood mononuclear cells, before operation and at days 1, 3 and 7 postoperatively. We detected a significant increase in Th/Ts cell ratio and an improvement in delayed type hypersensitivity response in the treated group at day 3. We believe that the above immunomodulating effect of in vivo cyclo-oxygenase inhibition may be beneficial in patients under major surgical procedures with a high susceptibility to postoperative infections.

Quantitative analysis of angiogenesis and growth of bone: effect of indomethacin exposure in a combined in vitro-in vivo approach.

Abstract: Nonsteroidal anti-inflammatory agents have been used experimentally and clinically to suppress a variety of physiological events, including angiogenesis and formation of bone. The exact mechanisms by which indomethacin alters skeletal tissue generation are unknown, due in part to methodological limitations. By the use of an organ culture assay and an animal model using intravital microscopy in mice bearing dorsal skinfold chambers, the effect of indomethacin on growth and angiogenesis of neonatal femora was characterized over 16 days. In both assays, femora significantly elongated with time (P<0.05). The in vitro growth rate was more rapid than in vivo and dependent on the serum concentration, culture medium and age of mice. Although enthancing the serum content promoted cellular proliferation in organ culture, it dose-dependently suppressed femoral elongation, leading at 20% fetal calf serum to growth rates identical to those observed in vivo. Indomethacin supplementation (2 and 10 mg l−1) significantly accelerated longitudinal femoral growth in organ culture (P<0.05), whereas in vivo indomethacin (2 mg kg−1) did not modulate either angiogenesis or elongation of bone. Our in vitro data propose a central role of serum in the regulation of bone formation. Although indomethacin altered femoral gowth in vitro, our findings do not suggest that indomethacin suppresses angiogenesis or growth of bone in vivo. The complexity of physiological events in vivo may be obscuring a detectable effect.

Dose-dependent stimulation/inhibition effects of cyclosporin A on lysosomal cathepsin activities in cultured proximal tubule cells

Abstract: The effects of cyclosporin A on the activities of lysosomal cysteine proteinases (cathepsin B, H, L+B) in LLC-Pk1 cells were investigated to elucidate their potential role in cyclosporin A-induced nephrotoxicity. Cyclosporin A at lower doses (0.1–1,000 ng/ml) stimulated cathepsin B, H, L+B. In contrast, at a higher dose (10,000 ng/ml), it inhibited these proteinase activities associated with a reduction in protein degradation. In line with the altered proteinase activities, cellular protein content was decreased at the lower dose (10 ng/ml) and increased at the higher dose. The higher dose of cyclosporin A also enhanced cellular lipid peroxide content after an exposure of 4 and 10h. Co-incubation with superoxide dismutase (40 U/ml) did not ameliorate the inhibition of cathepsin B activity induced by the high dose of cyclosporin A. On the contrary, the calcium channel blocker verapamil (10−6 M) prevented this inhibition. In conclusion, cyclosporin A exerts a dose-dependent biphasic effect on lysosomal cysteine proteinase activities. A rise in cytosolic Ca2+ concentration, but not an enhanced lipid peroxidation, may be involved in the suppression of cathepsin B activity induced by the higher dose of cyclosporin A. These studies raise the possibility that alterations of tubular proteinase activity may play a role in the cyclosporin A-induced nephrotoxicity.

The effect of methylprednisolone on cisplatin-induced nephrotoxicity in rat renal cortical slices.

Abstract: To investigate the protective action of methylprednisolone against cisplatin nephrotoxicity, the effect of in vivo pretreatment with methylprednisolone on the cisplatin-induced reduction inp-aminohippurate (PAH) accumulation and gluconeogenesis was examined using renal cortical slices prepared from Sprague-Dawley rats. The PAH accumulation in the kidney slices prepared from methylprednisolone-pretreated rats was significantly reduced following in vitro incubation with 2 mM cisplatin, to a degree equal to that observed in the slices prepared from untreated rats. However, the inhibitory effect of cisplatin on gluconeogenesis in the renal cortical slices obtained from methylpredimisolone-pretreated rats was significantly smaller than that seen in the slices from untreated rats. Our present studies suggest that in vivo pretreatment with methylprednisolone may contribute to its protective effect against cisplatin nephrotoxicity through the process of gluconeogenesis in renal epithelial cells.

The role of the vascular endothelium in the contractile responses of human chorionic plate artery in pre-eclampsia to prostaglandin F2alpha, 5-hydroxytryptamine, and potassium chloride

Abstract: This study characterises the reactivity of chorionic plate artery in preeclampsia to prostaglandin F2alpha (PGF2alpha), 5-hydroxytryptamine (5-HT), and potassium chloride (KCl) and examines the role of the vascular endothelium in these responses. Ring segments of the chorionic plate arteries of women after normal and pre-eclamptic pregnancies were contracted by PGF2alpha, 5-HT, and KCl. The experiments were carried out in the presence and absence of endothelium, and on intact rings treated with 10−6M indomethacin. The maximal contractile responses of rings from pre-eclamptic women to 5-HT, PGF2alpha, or KCl were significantly greater than those of rings from normotensive pregnant women. The EC50 values of responses were significantly lower in rings from pre-eclamptic subjects. Endothelium removal and treatment of the rings with indomethacin had no effect on the contractile responses of rings from normotensive pregnant women to all the agents, but significantly increased the EC50 value and decreased the maximal contractile responses of rings from pre-eclamptic women to 5-HT and PGF2alpha. While de-endothelialisation increased the EC50 value for responses of the rings from pre-eclamptic women to KCl, pretreatment with indomethacin did not significantly affect the KCl-induced responses. The results of the study suggest that pre-eclampsia enhanced the reactivity of human chorionic plate artery to 5-HT, PGF2alpha, and KCl through the involvement of endothelial derived contracting factors. The increased responses to 5-HT and PGF2alpha were inhibited by indomethacin, but those to KCl were not.

Gastrin stimulates gastric mast cells in rabbits

Abstract: In a previous study we demonstrated that in human gastric mucosa tryptase was localized only in mast cells and that its levels were correlated with serum gastrin, suggesting a link between gastrin action and mucosal mast cell function. The aim of the present study was to discover whether pentagastrin injection could stimulate gastric mucosal mast cells in rabbits. Ten female rabbits (group S) were injected s.c. with pentagastrin (10 μg/kg); another group of ten animals (group C) was injected s.c. with an equal volume of saline solution. One hour after the injection the rabbits were sacrificed and their stomachs removed. Antrum (A), corpus (C) and fundus (F) mucosal homogenates were assayed for total protein, tryptase, pepsinogen A (PGA), histamine and gastrin. Histamine tissue levels were significantly lower in group S than in group C in the antrum (Mann-Whitney test: U=82,P<0.01) and in the corpus (U=83,P<0.005). Tryptase levels were significantly higher in group S than in group C in all gastric areas (antrum: U=95,P<0.001; corpus: U=85,P<0.005 and fundus: U=75,P<0.05). Total protein PGA and gastrin did not vary significantly between groups. In group C, no signficant correlations were found among the five parameters. In group S, corpus tryptase was correlated with fundus tryptase (Spearman'sr=0.831,P<0.01). The same relationship was observed for histamine (r=0.672,P<0.05). In group S, antrum gastrin was inversely correlated with antrum tryptase (r=0.903,P<0.001), and with corpus PGA (r=−0.806,P<0.05). This study demonstrates that bolus pentagastrin administration stimulates gastric mucosal mast cells in the rabbit.

Liver regeneration following partial hepatectomy and stimulation by hepatic stimulatory substance in cirrhotic and non-cirrhotic rats

Abstract: The effects of hepatic stimulatory substance (HSS) on cirrhotic and noncirrhotic rats were studied after 70% partial hepatectomy. Liver cirrhosis was produced by weekly intragastric infusion of chloroform for 12–16 weeks. The HSS was prepared by extraction from the livers of weanling mice. Rats in the experimental group were injected with 5 ml HSS after 70% partial hepatectomy, and those in the control group received normal saline. The results showed that the3H-thymidine incorporation was higher in the HSS group 24 h after partial hepatectomy in both cirrhotic and non-cirrhotic rats, and persistently higher in the non-cirrhotic rats at 48h. Total DNA was significantly higher in the HSS group of non-cirrhotic rats 24 and 48 h after partial hepatectomy. The restituted liver volume and weight was significantly higher in non-cirrhotic rats 48 h after partial hepatectomy, while there was no significant difference between the HSS and the control groups in the cirrhotic rats. The HSS induced significant effects on3H-thymidine incorporation in the non-cirrhotic liver, resulting in increasing liver weight, volume and total DNA 48 h after partial hepatectomy. In cirrhotic rats, the3H-thymidine incorporation was higher in the HSS group at 24 h after partial hepatectomy, though not showing any increase at 48 h, but the regeneration of liver weight, volume and total DNA at 48 h showed no difference between the HSS group and the control group.

Effect of ursodeoxycholic acid on biochemical parameters, hepatocyte proliferation and liver histology in galactosamine hepatitis in the rat

Abstract: The effect of oral administration of ursodeoxycholic acid (UDCA) on biochemical parameters, liver histology and liver cell proliferation was investigated in rats with galactosamine hepatitis. Treatment with UDCA led to a decrease of aminotransferases, but did not show any significant changes in liver histology or liver cell proliferation. The improvement of liver enzymes without change of histology in this animal model of hepatitis following treatment with UDCA is in agreement with results obtained from clinical trials with UDCA in patients with chronic viral hepatitis.

Effects of glycosaminoglycans on the glomerular changes induced by Adriamycin

Abstract: Glycosaminoglycans can stimulate the synthesis of glomerular basement membrane (GBM) protein by glomerular cells and correct biochemical alterations of the GBM. In this study we examine the effects of heparin and low-molecular-weight heparin (LMWH) on glomerular permeability to proteins and glomerular structure in Adriamycin-treated rats. One Adriamycin dose of 5 mg/kg body weight was administered i.v. to 38 female Wistar rats. Eleven animals were also treated with heparin (250 U) administered s.c. twice daily and 7 with LMWH 6 mg/kg body weight administered s.c. twice daily. Urine samples were collected before and 30 and 60 days after the beginning of treatment to quantify albumin excretion and to characterize urinary proteins by gel filtration and electrophoresis in sodium dodecyl sulfate polyacrylamide gels. Blood samples were also collected on day 60 from these rats to estimate renal permeability by gel filtration; the rats were then killed and the kidneys removed for histological analysis. Heparin administration caused a reduction in urinary albumin excretion and in the incidence of segmental glomerulosclerosis in Adriamycin-treated rats. However, heparin did not modify the selectivity of the GBM to proteins of different molecular weights. These data suggest that the effect of heparin on albuminuria may be due to changes in the negative GBM charges induced by this drug.

Endothelin-1 and in vitro evidence of renal artery vasoconstriction after liver transplantation

Abstract: Twelve pigs underwent orthotopic liver transplantation. The mean endothelin-1 (ET-1) levels in the serum samples of the recipient animals 1 h after reperfusion of the graft (6.2±1.5 pg/ml) was significantly higher (P<0.05) than in pretransplantation samples (3.2±0.6 pg/ml). Serum blood urea nitrogen (BUN) 24 h after transplantation was 13.8±5.9 mg/dl, which was significantly higher than before transplantation (6.4±2.2 mg/dl). There was a positive correlation between the serum BUn and ET-1 (r=0.62,P<0.05). An in vitro isometric tension study was performed for the contractility response rate of the intact renal artery in the bath chamber containing the serum of the corresponding recipient animals. The mean contractility response rates were higher with the serum obtained after reperfusion of the graft (66.9±32.4%) than with those obtained before transplantation (18.3±9.2%) when compared to a standard contractility rate of 100% with 40 mM KCl. Moreover, these contractility response rates were significantly reduced (32.8±21.0%) with the addition of the ET-1 receptor antagonist FR139 317. The results of the present study demonstrated that the liver transplantation was associated with elevation of ET-1 in the serum of the recipient animals. It was considered that ET-1 in the serum caused a direct vasoconstriction of the renal artery in vitro. This may help to explain the renal dysfunction that is often seen in the recipients of clinical liver transplantation.

Isolation and characterization of apical membrane vesicles of the rat distal colon

Abstract: In this study a method for the isolation of apical membrane vesicles of the rat distal colon was developed. It is based on the purification of intact membrane caps followed by separation of the vesiculated apical membranes on a discontinous sucrose gradient. Purification of the apical membrane vesicles revealed an 11-fold enrichment of the marker enzyme alkaline phosphatase compared with the homogenate, while marker enzymes of other subcellular structures showed negligible enrichments and recovery of activity. The membrane fluidity (lipid structural order) of the isolated membranes measured from the fluorescence anisotropy by several fluorophores also coincided with the typical structural order of apical membranes of the rat colon. Transport studies with the fluorescent dye acridine orange implied that a diffusion potential independent, amiloride-sensitive Na+−H+ exchange mechanism is present in the isolated apical membranes. Furthermore, the results suggest that a possible short chain fatty acid (SCFA) absorption by simple passive diffusion of the undissociated form, preceded by intraluminal protonation of the SCFA anion, is not provided by this Na+−H+ exchange transport in the luminal membrane of the absorptive cell.

Differing kinase activity of the c-yes and c-src gene proteins in TPA-induced megakaryocytic differentiation of T-33 and K562 cell lines.

Abstract: We examined the protein kinase (PK) activity of the c-yes and c-src gene proteins (c-YES, c-SRC) at an early phase of 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced megakaryocytic differentiation of T-33 and K562 cells with use of immunoprecipitation and in vitro kinase assay. We found that c-SRCPK activity of TPA-treated T-33 and K562 cell lines had been enhanced compared with the untreated ones, but in contrast, no enhancement of c-YES PK activity by the TPA treatment was observed in these cell lines. We also examined PK activity in TPA-induced monocytic differentiation of U937 monoblastic cells that exhibited no megakaryocytic markers and found that both the c-YES and c-SRC PK activity was enhanced by the TPA treatment. Our data suggest that c-YES and c-SRC play different and unique roles in TPA-induced megakaryocytic differentiation in T-33 and K562 cells.