Expression of haem oxygenase in cirrhotic rat liver

The haem oxygenase (HO)/carbon monoxide (CO) system has been implicated as a modulator of hepatobiliary function. This study investigated HO expression in the process of cirrhosis development, as well as its relationship to nitric oxide synthase (NOS). Liver cirrhosis was induced in rats by chronic bile duct ligation (BDL). HO mRNA expression was evaluated by competitive RT-PCR, while protein expression was determined by immunoblotting and immunohistochemistry. In liver tissue where cirrhosis had fully developed, the expression levels of HO-1 were greatly enhanced at both mRNA and protein level compared with sham livers. Immunohistochemistry showed that HO-1 was induced in hepatocytes and enhanced in some of the Kupffer-like cells in BDL livers. In contrast, there was no difference between the sham and the BDL livers for the expression levels of HO-2. Interestingly, administration of the NOS inhibitor aminoguanidine (AG) or N(G)-nitro-L-arginine methyl ester inhibited HO-1 expression. To study further the role of HO-1 in the development of liver cirrhosis, hepatocytes were isolated from the rats at different time points after BDL operation. HO-1 was expressed in hepatocytes at high levels during the early onset of cirrhosis but dropped slightly at a later stage of cirrhosis. Zinc protoporphyrin IX (ZnPP), an HO inhibitor, blocked HO-1 expression in hepatocytes from BDL cirrhotic rats, but enhanced the activity of inducible NOS (iNOS) in BDL hepatocytes. In conclusion, HO-1 was induced in the hepatocytes of rats undergoing cirrhosis, suggesting that HO-1 plays a role in the development of liver cirrhosis. Induction of HO-1 may be mediated partially by iNOS. However, once it is induced, HO-1 may be important in modulating iNOS activity, thus playing a protective role in liver cirrhosis.     

Role of liver tryptophan oxygenase activity in mice on outcome of endotoxin poisoning

Injection of cortisone into mice simultaneously with typhoid endotoxin prevents the decrease in tryptophan oxygenase activity in the liver and prevents death of the animals. Tryptophan also maintained a normal level of activity of the enzyme but did not prevent death of the animals poisoned with endotoxin. The role of the level of tryptophan oxygenase activity in the pathogenesis of poisoning produced by endotoxin is discussed.Presented by Academician of the Academy of Medical Sciences of the USSR Kh. Kh. Planel'es.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 70, No. 12, pp. 34–36, December, 1970.     

Reduction of myocardial leukocyte accumulation and myocardial infarct size following administration of BAY u3405, a thromboxane A2 receptor antagonist,in myocardial ischaemia-reperfusion injury

We investigated the effect of BAY u3405, a thromboxane A₂ receptor antagonist in pentobarbital anaesthetized rats subjected to left main coronary artery ligation (1 h) followed by reperfusion (1 h; MI/R). Sham operated rats were used as controls (Sham MI/R). Survival rate, myocardial necrosis, myocardial myeloperoxidase activity (investigated as an index of leukocyte adhesion and accumulation) and serum creatine phosphokinase activity were studied. Ischaemia-reperfusion injury significantly reduced the survival rate (45%), caused a marked myocardial necrosis, increased serum creatine phosphokinase activity (Sham MI/R=26±10.2 U/ml; MI/R=213±19 U/ml) and produced a rise in myocardial myeloperoxidase activity in the area-at-risk and in the necrotic area (6.1±0.4 U×10^–3/g tissue and 6.7±0.9 U×10^–3/g of tissue, respectively).The administration of BAY u3405 (30 and 60 mg/kg/i.v., 30 min before occlusion) significantly increased survival rate, lowered the area of myocardial necrosis, blunted the increase in serum creatine phosphokinase activity and reduced the increase in myeloperoxidase activity in both the area-at-risk and the necrotic area. Furthermore, the protective effect of BAY u3405 was dose-dependent.These data are consistent with an involvement of TXA₂ in myocardial ischaemia-reperfusion injury and suggest that BAY u3405 may represent a novel therapeutic approach to the treatment of acute ischaemia-reperfusion injury.     

Inhibition of responder cell activity in mixed leukocyte culture reactions

An HLA-B7 antiserum showed cross-reactivity with HLA-B8, Bw41, a split of B40 (Bw60) and possibly Bw22 and B27, thus detecting one or more determinants on these antigens similar to an antigenic site on HLA-B7 usually not detected by other B7-antisera. The cross-reactions were demonstrable by adsorption of antibodies on lymphocytes followed by release at 37 degrees C, which enabled the detection of weak and otherwise hardly detectable reactivity. Released antibody molecules were detected in two different assays: (1) Antibody-dependent cellular cytotoxicity (ADCC) with HLA-B7 positive target cells (fluorochromasia micro ADCC). (2) Inhibition of MLC reactions with B7 positive stimulator cells. The B7-antibody, as detected in both assays, was released in decreasing activity from Bw41 greater than B8 greater than Bw60 much greater than B7 greater than (B27 = Bw22) positive cells. The order of sensitivity in which the various antigens were detected in ADCC assays in which the antiserum activity was measured directly on various target cells was different, viz. HLA-B7 greater than Bw60 = B27 greater than Bw41 greater than B8. Bw22 was not detected. Absorption studies demonstrated that HLA-B7 positive cells bound more B7 antibody activity than B8 positive cells. However, antibody molecules bound to B7 positive cells were mainly released as immune complexes, which could be dissociated by treatment with acid. In contrast, B7 antibody molecules bound to B8 positive cells were released as free antibody molecules. This marked difference in shedding properties further explained the previously described B7 specific unresponsiveness in MLC of HLA-B8 (and also Bw41) positive responder cells after sensitization with the B7 antiserum (de Rooij et al. 1980).     

Chemiluminescence of neutrophil accumulation in liver following ischemia-reperfusion

The median and left lateral lobes of rat liverin situ were rendered ischemic for 30 min, then blood flow re-instituted. After 1, 3, 6, 24, or 48 h livers were removed and set up for isolated perfused organ study. Luminol enhanced chemiluminescence (LEC) was recorded from the surface of the median and left lateral lobe before and for 90 min following phorbol myristate acetate PMA (1.6×10⁻⁸ M) perfusion. An increase in PMA induced luminol enhanced chemiluminescence was evident at 1 h, and continued to increase up to 6 h. By 24 h the magnitude of the PMA response had returned to within control values. This indicates that a large influx of inflammatory cells had occurred in the liver following thein vivo ischemia-reperfusion insult, and that these cells gave a very large and sustained burst of radical production on stimulation with PMA.

     

Inhibition of macrophage activity by mitogen-induced goldfish leukocyte deactivating factor

Macrophage activating and deactivating cytokines have been characterized in mammalian systems but little is known about these immunoregulatory molecules in fish. Using gel permeation and chromatofocusing fast performance liquid chromatography (GP-FPLC and C-FPLC) we partially purified a macrophage deactivating factor (MDF) from mitogen-induced goldfish kidney leukocytes. Inhibition of the macrophage-derived nitric oxide (NO) response induced by this MDF was time-, dose- and temperature-dependent. Macrophages pre-treated for 6 or 24 h with MDF before activation with macrophage activating factors (MAF) and/or bacterial lipopolysaccharide (LPS) exhibited a down-regulation in their NO response, while those treated with MDF 24 h after activation with MAF and LPS did not. MDF treatment also impaired the NO response of goldfish macrophages infected with the mammalian protozoan parasite Leishmania major. These results suggest that MDF exhibits its inhibitory effect downstream of the converging intracellular pathways induced by LPS and/or L. major. The novel teleost MDF has an approximate M_r of 15 kD and a pI<4, and is the first endogenous molecule of teleosts known to down regulate macrophage antimicrobial responses.     

Elevated basal activity of tryptophan oxygenase in alcohol-preferring C57Bl mice

Tryptophan oxygenase activity in alcohol-preferring C57Bl mice and control CBA and DBA/2 mice was studied under nonstressful conditions and after glucocorticoid-induced stress. Elevated basal tryptophan oxygenase activity in C57Bl mice is probably responsible for reduced brain content of tryptophan and serotonin associated with alcohol preference. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 4, pp. 408–410, April, 2000     

Inhibition of the accumulation of macrophages and the generation of macrophage chemotactic activity by dexamethasone in concanavalin A-induced peritonitis of mice

A delayed-type inflammatory response was evoked in mice using concanavalin A (Con A) as a stimulus, and the effect of various anti-inflammatory agents on the inflammations was examined. The intraperitoneal injection of Con A in the mouse resulted in the marked accumulation of leukocytes, especially macrophages, in the peritoneal cavity between 16 and 48 hr after the injection. Prior to the accumulation of macrophages, the chemotactic activity for macrophages appeared in the peritoneal fluid, and was associated with protein(s) in the molecular weight range from 10000 to 100000 daltons. When the effect of various agents on Con A-induced peritonitis was examined, neither anticomplementary agents (FUT-175 and K-76 COONa), bromophenacyl bromide, nordihydroguaiaretic acid nor indomethacin affected the generation of chemotactic activity and the accumulation of macrophages, suggesting that C5a, prostaglandins and leukotriene B₄ are hardly involved in the Con A-induced macrophage accumulation. On the other hand, dexamethasone suppressed both the generation of chemotactic activity and the accumulation of macrophages. Taking into consideration the observation that the synthesis of macrophage chemotactic factors by mitogen-stimulated lymphocytes is inhibited by glucocorticoids these results suggest that the macrophage chemotactic lymphokines might be involved in the accumulation of macrophages in Con A-induced peritonitis.     

Correlation of leukocyte accumulation with chemotactic activity in the exudate of an allergic air-pouch inflammation

Chemotactic activity at the site of an allergic air-pouch inflammation induced with azobenzene-arsonate-conjugated acetyl bovine serum albumin as an antigen was studied and a close correlation of the chemotactic activity with the intensity of leukocyte migration was demonstrated. In the period of vigorous leukocyte immigration into the fluid in the allergic air-pouch, chemotactic activity of the exudate was maintained at a high level, while no significant activity was detected after the number of leukocytes in the pouch fluid reached maximum.     

Inhibition of activin receptor type IIB increases strength and lifespan in myotubularin-deficient mice

X-linked myotubular myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in myotubularin deficiency.     

Inhibition of pregnancy viability in mice following IL-2 administration.

The influence of administration of interleukin-2 (IL-2) on syngeneic and allogeneic murine pregnancy has been investigated. Human or mouse recombinant IL-2 (rhIL-2 and rmIL-2), or partially purified rat IL-2, was inoculated i.p. into C57B1 mice following syngeneic mating but before embryo implantation. This inhibited subsequent fetal development in up to 100% of cases, compared with mice inoculated with control material, including recombinant human interleukin-1 alpha (IL-1 alpha), where no inhibition of pregnancy viability was observed. Similar data were obtained in both syngeneic and allogeneic matings when rhIL-2 was administered on Day 1 of pregnancy. Administration of rhIL-2 during the second pregnancy, rather than a first pregnancy, was less effective. Administration of rhIL-2 during the first pregnancy does not induce a permanent sterility. Histological examination of the endometrium further demonstrated that mice injected with rhIL-2 on Day 1 of their first pregnancy showed a complete absence of embryonic tissue.     

Wheel-running activity increases with social stress in male DBA mice

Social affiliation-avoidance behaviors are essential indices of sociality. We examined changes in social affiliation-avoidance behaviors in an open-field apparatus while simultaneously measuring wheel-running activity. Recent studies suggest that mice increase wheel-running activity in stressful situations; thus, we hypothesized that wheel-running activity would reflect a state of social stress and avoidance. Mean duration of wheel-running increased significantly when mice were confronted with unfamiliar mice compared to cage mates. There were negative correlations between the amount of wheel-running and social affiliation indices. We also examined the effect of social defeat on wheel-running activity. Mice that had experienced social defeat significantly increased their wheel-running when an aggressor mouse was present. This social defeat-induced wheel-running activity was ameliorated by the administration of diazepam. Our results indicate that wheel-running activity is relevant to social affiliation-avoidance behaviors and may be a reliable index of anxiety induced by social stress.     

The size of a microsatellite polymorphism of the haem oxygenase 1 gene is associated with idiopathic recurrent miscarriage

Endothelial damage, impaired microvascularization and immune maladaptation have been described as aetiological factors in recurrent miscarriages. We investigated the relationship between idiopathic recurrent miscarriage (IRM) and a (GT)(n) repeat microsatellite polymorphism of the gene encoding haem oxygenase 1 (HO-1), known to modulate immune functions such as T-helper (TH) cell function and to be associated with cardiovascular disease. We investigated 162 women with IRM and 129 healthy, post-menopausal controls. The length of the HO-1 (GT)(n) microsatellite was assessed by PCR and direct sequencing in all women. Results were correlated with clinical data. The distribution of genotypes was in Hardy-Weinberg equilibrium. The HO-1 (GT)(n) microsatellite repeat numbers ranged from 13 to 37, with (GT)(23) and (GT)(30) being the most common alleles in both groups. We compared alleles consisting of < or =27 GT repeats, termed class S (short) alleles and alleles consisting of >28 GT repeats, termed class L (long) alleles. Seventy per cent of women with IRM had an S allele either in heterozygous (L/S) or homozygous (S/S) form, compared to 56% of controls (P = 0.02; OR 0.54; 95% CI 0.32-0.90). With respect to S allele frequencies, we found no significant difference among women with IRM and controls [P = 0.3; odds ratio (OR) 1.23, 95% confidence interval (CI) 0.86-1.76]. Comparing women with primary and secondary IRM, no difference with respect to the length of the HO-1 (GT)(n) microsatellite was ascertained. In summary, this is the first report on a HO-1 (GT)(n) microsatellite polymorphism among women with IRM, demonstrating that the investigated polymorphism is associated with IRM in a relatively large Caucasian population.     

Changes in hippocampal cholinergic activity following learning in mice

A short bar-press operant conditioning acquisition session with food reward on continuous reinforcement was shown to induce a decrease (13.5%) of hippocampal choline acetyltransferase activity in mice. Such an effect seems to be specific to this kind of learning since no change was observed in several control groups, including a group of mice submitted to another type of conditioning in the same apparatus. It is suggested that these enzymatic modifications might be responsible for the delayed improvement of performance observed on retention of this task.     

Enalapril increases mitochondrial nitric oxide synthase activity in heart and liver

Heart and liver mitochondria isolated from rats treated with enalapril, 3-30 mg/kg/day in the drinking water for 7-120 days, showed a time- and dose-dependent increased nitric oxide (NO) production in the range of 14-250%. Heart and liver mitochondria from control rats produced 0.69 and 0.50 nmol of NO/min/mg of protein, respectively, as determined by dual wavelength spectrophotometry (577-591 nm) following hemoglobin oxidation to methemoglobin. The response to enalapril treatment, attributed to a gene-mediated up-regulation of mitochondrial nitric oxide synthase (mtNOS) activity, was half-maximal at 5-6 days and was maintained up to 120 days. Enalapril-treated animals showed an increased mtNOS functional activity in heart mitochondria that inhibited state 3 O(2) uptake (from 22% in control rats to 43%) and increased state 4 hydrogen peroxide (H(2)O(2)) production (from 30% in control rats to 52%). Calculated heart intramitochondrial NO and H(2)O(2) steady-state concentrations were increased 66% and 20%, respectively, by enalapril treatment. Signaling pathways dependent on mitochondrial NO and H(2)O(2) may account for the beneficial effects of enalapril in aging mammals.