Intercellular adhesion molecule 1 (ICAM-1) expression and its role in neutrophil-induced ischemia-reperfusion injury in rat liver.

The potential role of intercellular adhesion molecule-1 (ICAM-1) in the pathogenesis of reperfusion injury was investigated in male Fischer rats subjected to 45 min of hepatic ischemia and 24 h of reperfusion. ICAM-1 mRNA levels increased during ischemia in the ischemic liver lobes; however, during reperfusion mRNA levels increased in both the ischemic and nonischemic lobes. Immunohistochemical evaluation indicated ICAM-1 expression only on sinusoidal lining cells in controls; ischemia-reperfusion enhanced ICAM-1 expression in the sinusoids and induced some expression on hepatocytes. The monoclonal anti-ICAM-1 antibody 1A29, but not an immunoglobulin G control antibody, administered at 1 h and 8 h of reperfusion (2 mg/kg) significantly attenuated liver injury as indicated by 51% lower plasma alanine aminotransferase activities and 32-36% less hepatic necrosis at 24 h without affecting reactive oxygen formation by Kupffer cells and hepatic neutrophils. Although 1A29 reduced neutrophil extravasation in a glycogen peritonitis by 60%, the antibody had no significant effect on hepatic neutrophil infiltration during reperfusion. These data suggest that ICAM-1 plays a significant role during the neutrophil-dependent injury phase after hepatic ischemia and reperfusion and therefore blocking this adhesion molecule may have therapeutic potential against postischemic acute liver failure.

Neutrophil-induced lung damage after hepatic ischemia and endotoxemia.

Administration of Salmonella enteritidis endotoxin (0.5 mg ET/kg) during reperfusion (RP) after short-term hepatic ischemia (20 min) caused severe liver injury induced by Kupffer cells and neutrophils and a high mortality rate. To investigate potential lung damage in this model, lung wet-to-dry weight ratios (W/D) and broncho-alveolar lavage (BAL) protein content were determined after 4 h of reperfusion. Both parameters increased significantly during RP/ET (W/D: 4.4 +/- 0.1; BAL: 639 +/- 30 micrograms/ml) compared to controls (W/D: 3.5 +/- 0.1; BAL: 332 +/- 17). The antioxidants Trolox or tirilazad mesylate (U-74006F) effectively reduced the BAL protein increase. Alveolar macrophages were not activated; however, neutrophils isolated from the lung microvasculature of RP/ET animals showed a 300% increase of spontaneous and PMA-induced superoxide formation compared to controls (spontaneous: 1.4 +/- 0.5 nmol O2-/h/10(6) cells; PMA: 2.2 +/- 0.4). Complement factors and TNF-alpha injection induced a similar priming of vascular neutrophils for superoxide generation. Vascular neutrophil activation highly correlated with the severity of lung injury. It is concluded that neutrophils accumulated in the lung microvasculature were the major source of the oxidant stress and mainly responsible for lung injury under these conditions. Antioxidants such as tirilazad mesylate (U-74006F) may have therapeutic potential for attenuating lung injury induced by remote organ trauma and a systemic inflammatory response.

Activation of Kupffer cells and neutrophils for reactive oxygen formation is responsible for endotoxin-enhanced liver injury after hepatic ischemia.

The potential role of reactive oxygen species generated by Kupffer cells and neutrophils was investigated in a model of endotoxin-enhanced liver injury after hepatic ischemia. Male Fischer rats were subjected to 20 min ischemia and reperfusion of up to 24 h; .5 mg/kg Salmonella enteritidis endotoxin was injected at 30 min of reperfusion. The animals developed severe liver injury resulting in 50% hepatocellular necrosis at 24 h. Isolated Kupffer cells and neutrophils from the postischemic liver generated 10-fold more superoxide than cells from control livers. Treatment with gadolinium chloride (GdCl3) selectively reduced the capacity of Kupffer cells to generate superoxide by 65% and attenuated liver injury by 73% at 4 h and 58-69% at 24 h. Monoclonal antibodies against neutrophil adhesion molecules (CD11/CD18) had no effect on the early injury but reduced hepatocellular necrosis by 90-95% at 24 h. The antioxidant Trolox and the iron-chelator deferoxamine attenuated liver injury by 71 and 80%, respectively. It is concluded that Kupffer cells are mainly responsible for the initial injury, and neutrophils are the dominant cytotoxic cell type during the later phase. Reactive oxygen generated by both cell types is critical for this pathogenesis.

Gas chromatographic-mass spectrometric analysis of lipoxygenase products in post-ischemic rabbit myocardium.

Leukotriene B4 (LTB4) is a potent chemotactic compound for neutrophils and is thought to be an important mediator of myocardial ischemia-reflow injury. We have measured LTB4 in rabbit cardiac tissue following ischemia-reflow using a sensitive and specific gas chromatographic-mass spectrometric (GC-MS) assay. The concentration of LTB4 in rabbit myocardium following 45 min ischemia and 3 h reflow was 48.7 +/- 12.5 pg/g, significantly higher than in non-ischemic tissue from the same animal (17.5 +/- 3.9 pg/g). These concentrations were at least an order of magnitude lower than previously reported values assessed by radioimmunoassay (RIA). Compared with the GC-MS method, RIA greatly overestimated LTB4 concentrations in cardiac tissue. The capacity of post-ischemic myocardium to produce lipoxygenase products, LTB4, 5-, 12- and 15-HETEs was also assessed following incubation of myocardium ex vivo with calcium ionophore. In all animals ischemic cardiac tissue produced greater amounts of LTB4, 5-, and 12-HETEs than non-ischemic myocardium and 12-HETE was the major product. Neutrophils that have accumulated in the injured tissue may be a major source of these products. However, in contrast to cardiac tissue, isolated rabbit neutrophils stimulated with A23187 produced 5-HETE as the major product with very little 12-HETE formed. These latter findings suggest that cells other than neutrophils may contribute to the production of lipoxygenase products during myocardial ischemia-reflow injury.

PAF formation by H2O2-stimulated perfused canine carotid arteries.

Perfusion of noncytotoxic concentrations of hydrogen peroxide (H2O2) through canine carotid arteries potentiates neutrophil adhesion to vessel endothelium. Platelet-activating factor (PAF) receptor antagonists block neutrophil adhesion to vessels pretreated with low millimolar concentrations of H2O2. We have used a specific gas chromatographic-mass spectrometric (GC-MS) assay for PAF and applied this to studies of canine carotid arteries perfused with H2O2. Vessels perfused with 1 and 10 mM H2O2 for 20 min produced PAF in a dose-dependent manner, 331 +/- 67 pg/g tissue with 1 mM H2O2 and 1160 +/- 194 pg/g with 10 mM. Vessels that had been denuded of endothelium with a balloon catheter prior to H2O2 perfusion produced similar quantities of PAF in response to H2O2 (220 +/- 72 pg/g and 960 +/- 210 pg/g with 1 and 10 mM, respectively). Cultured canine jugular venous endothelial cells produced PAF in response to 10 mM H2O2 (809 +/- 117 pg/10(7) cells) but carotid arterial smooth muscle cells did not. These results suggest that vascular cells other than endothelial cells may produce PAF following H2O2 perfusion of canine carotid arteries.

Priming of phagocytes for reactive oxygen production during hepatic ischemia-reperfusion potentiates the susceptibility for endotoxin-induced liver injury.

Plasma levels of glutathione disulfide (GSSG) as an indicator of a vascular oxidant stress, tumor necrosis factor-alpha (TNF-alpha) formation, and liver injury (alanine aminotransferase activity, histology) were monitored in male Fischer rats after 30 min of hepatic ischemia followed by up to 4 hr of reperfusion. The injection of 1 mg/kg Salmonella enteritidis endotoxin at 30 min of reflow potentiated the postischemic oxidant stress and liver injury. TNF-alpha levels increased from 10 +/- 7 pg/ml (baseline) to 3,553 +/- 738 pg/ml after ischemia-reperfusion followed by endotoxin, or to 3,670 +/- 508 pg/ml after endotoxin alone. Depletion of serum complement before ischemia attenuated the endotoxin-mediated increase of reactive oxygen formation by 70% but did not affect TNF-alpha levels. Complement activation with cobra venom factor (CVF) during reperfusion had an effect similar to that of endotoxin on the oxidant stress and liver injury. CVF did not increase TNF-alpha formation during reperfusion. Kupffer cells and neutrophils isolated from the postischemic liver 2.5 hr after endotoxin injection generated 600% and 400% more superoxide, respectively, than cells isolated from control livers. The results demonstrate a substantial priming of hepatic phagocytes for reactive oxygen production but not TNF-alpha formation, even after short periods of hepatic ischemia, and the vulnerability of the postischemic liver to severe endotoxin-induced injury. Activated complement seems to be mainly responsible for the effects. These results may explain the high risk for hepatic failure after extensive liver resection and hypovolemic shock.

Differential induction of mRNA for ICAM-1 and selectins in hepatocytes, Kupffer cells and endothelial cells during endotoxemia.

Intercellular adhesion molecule-1 (ICAM-1) and selectins (E- and P-selectin) mRNAs were determined in individual liver cell types by Northern blot analysis before and after injection of endotoxin. A constitutive expression of ICAM-1 mRNA was found in endothelial cells and Kupffer cells but not in hepatocytes. All three cell types showed upregulation of ICAM-1 mRNA after endotoxin. No constitutive selectin expression could be detected in any liver cell, but endotoxin induced massive synthesis of E- and P-selectin mRNA in endothelial cells and Kupffer cells. The differential expression of cellular adhesion molecules in the liver is consistent with the involvement of selectins in neutrophil rolling in the vasculature and ICAM-1 in transendothelial migration and adherence to parenchymal cells.

Endotoxin-induced activation of the nuclear transcription factor kappa B and expression of E-selectin messenger RNA in hepatocytes, Kupffer cells, and endothelial cells in vivo.

Endotoxin causes neutrophil sequestration in the liver and severe vascular and parenchymal cell injury. Inducible adhesion molecules such as intercellular adhesion molecule-1 and selectins are involved in neutrophil recruitment to an inflammatory site in vivo. The objective of our study was to characterize the translocation of the nuclear factor kappa B (NF-kappa B) from the cytoplasm to the nucleus (NF-kappa B activation) during endotoxemia using the electrophoretic mobility shift assay and to investigate its role in regulation of E-selectin gene transcription in liver cells in vivo. Administration of 0.5 mg/kg Salmonella enteritidis endotoxin to male Fischer rats induced a drastic but transient activation of NF-kappa B at the whole liver level. Major subunits identified were p50 (NF-kappa B1), p65 (RelA), and c-Rel, but not p52 (NF-kappa B2). Isolation of liver cells from control animals induced substantial NF-kappa B activation in Kupffer cells and endothelial cells and minor activation in hepatocytes. One hour after endotoxin treatment in vivo, NF-kappa B was uniformly activated in all isolated cells. At 5 h, NF-kappa B activation was reduced to various degrees in all cell types, with hepatocytes showing only a moderate decrease. Northern blot analysis indicated no E-selectin mRNA in all control cells; however, at 1 h, endotoxin induced E-selectin gene transcription transiently in endothelial cells and in Kupffer cells but not in hepatocytes. These data support the hypothesis that NF-kappa B activation is important for E-selectin gene transcription in hepatic vascular lining cells. However, the fact that hepatic parenchymal cells, despite NF-kappa B activation do not express E- selectin mRNA, suggests that NF-kappa B activation alone is not sufficient for E-selectin gene transcription in vivo.

The effects of chronic carbamazepine treatment of haem biosynthesis in man and rat.

The anticonvulsant drug carbamazepine has been reported to produce a condition clinically and biochemically similar to acute intermittent porphyria (AIP). We have determined the effect of chronic carbamazepine treatment on the activities of the enzymes of haem biosynthesis in circulating blood cells and on the urinary excretion of porphyrins and their precursors in 53 epileptic patients receiving monotherapy and in 42 age- and sex-matched controls. In the patients the mean activity of leucocyte 5-aminolaevulinic acid (ALA) synthase, the rate-limiting enzyme of the pathway, was 218% of control values (p less than 0.001) and ALA-dehydratase activity was reduced by 37% (p less than 0.001). Circulating carbamazepine concentrations correlated negatively with ALA dehydratase (rs = -0.45; p less than 0.01). Porphobilinogen deaminase and uroporphyrinogen decarboxylase appeared unaffected by carbamazepine treatment. Significant quantitative increases in the urinary excretion of porphobilinogen and total porphyrins (both p less than 0.05) accompanied the changes in enzyme activity. Similar dose-dependent effects on ALA synthase and ALA dehydratase were shown to occur in rats treated for 5 days with 3 different doses of carbamazepine. These findings further support the porphyrinogenicity of carbamazepine, but the pattern of enzyme alteration differs from that found in AIP.

Effects of sodium valproate on haem biosynthesis in man: implications for seizure management in the porphyric patient.

The short-term effects of sodium valproate (VPA) on haem biosynthesis were assessed in a placebo-controlled crossover trial in eight healthy male subjects who ingested VPA 500 mg t.i.d. and matched placebo for 5 days. All showed augmented activity of leucocyte 5-aminolaevulinate synthase (ALA-S) activity, the rate-limiting enzyme of the haem biosynthetic pathway, following 3 and 5 days of VPA treatment (P less than 0.001). This was accompanied by increased urinary excretion of 5-aminolaevulinic acid (ALA; P less than 0.02) and total porphyrins (P less than 0.01). Mean (+/- SD) total VPA concentrations on day 3 (89 +/- 16 mg 1-1) and day 5 (91 +/- 22 mg 1-1) were within the target range for the drug. The long-term effects of VPA administration were examined in epileptic patients on established monotherapy. Leucocyte ALA-S activity (P less than 0.001), and daily urinary excretion of porphobilinogen (P less than 0.01) and total porphyrins (P less than 0.01) were all higher than in age-matched controls. No significant differences in erythrocyte ALA-dehydratase, porphobilinogen deaminase and uroporphyrinogen decarboxylase activities were found between the groups. These data suggest that VPA is porphyrinogenic in man and cannot be recommended as safe for seizure management in the porphyric patient.

The 21-aminosteroid tirilazad mesylate protects against endotoxin shock and acute liver failure in rats.

The protective effect of the 21-aminosteroid tirilazad mesylate (U-74006F) was investigated in an experimental model of endotoxin shock and acute liver failure. In male Fischer rats subjected to 20 min of hepatic no-flow ischemia followed by reperfusion and injection of 0.5 mg/kg of Salmonella enteritidis endotoxin, severe hepatic injury developed, as indicated by a histological evaluation and liver enzyme release. Treatment with U-74006F (two bolus doses of 3 mg/kg each; the first dose was injected i.v. 30 min before ischemia and the second dose, at the time of reflow) reduced the hepatic injury by 60% at 4 hr of reperfusion, improved the survival rate from 18% to 55% and decreased the degree of hepatic injury at 48 hr of reperfusion. U-74006F treatment did not affect the extent of complement activation during reperfusion, the Kupffer cell-induced oxidant stress, or tumor necrosis factor-alpha formation in this model. U-74006F did not significantly reduce superoxide formation of Kupffer cells and neutrophils in vitro or in vivo. The substantial neutrophil infiltration in the liver during the pathogenesis was not affected at 4 hr of reperfusion but was attenuated by 70% at 48 hr. It was therefore concluded that, in the sequence of pathophysiological events, U74006F acted at a site distal to inflammatory cell activation and the generation of cytotoxic mediators. The protection against the initial endotoxin-enhanced reperfusion injury in the liver strongly inhibited the progression of the inflammatory response and subsequent liver failure.(ABSTRACT TRUNCATED AT 250 WORDS)

Antiepileptic drug monitoring at the epilepsy clinic: a prospective evaluation.

To assess the value of on site therapeutic drug monitoring at the epilepsy clinic, management decisions were recorded before and immediately after antiepileptic drug (AED) concentrations became available. In the first year of this prospective study, 632 [277 carbamazepine (CBZ), 170 phenytoin (PHT), 113 valproate (VPA), and 72 phenobarbital (PB)] assays were performed during 488 clinic attendances in 182 actively managed epileptic patients. The results of drug analysis led to alterations in management at 114 patients visits, i.e., 23% of those monitored. Dosage was increased in response to the circulating AED concentration in 12% of consultations and decreased in another 7.5%. Unsuspected poor compliance was uncovered in eight patients, and in three others an AED was added or discontinued on the basis of the assay result. The time of the next appointment was rearranged in 58 attendances. Only 50% of results were in the "therapeutic" ranges for the four major AEDs. Dosage was adjusted (50 up, 16 down) after 54% of low results. "Therapeutic" levels were followed by a change in AED dose (52 up, 31 down) in 26%. Only 29% of concentrations above the "therapeutic" range persuaded the doctor to alter the dosage regimen, and in 20% of these an increase in dose was recommended. On-site AED monitoring had an immediate impact on clinical decision-making in greater than 23% of consultations but in a form more subtle than the simple quest for a therapeutic result.

Severe microcephaly with normal intellectual development: the Nijmegen breakage syndrome.

A brother and sister are described with severe microcephaly of prenatal onset, normal intellectual and motor development, chromosomal breakage and cellular immunodeficiency, which is characteristic of the autosomal recessive condition, Nijmegen breakage syndrome. The proband was a girl who presented at 15 months, with normal developmental milestones and an extremely small head circumference of 36 cm. Twenty per cent of her lymphocytes showed spontaneous translocations involving chromosome 7p13, 7q35, 14q11, and 14q32. The lymphocytes also showed excessive x ray induced chromosome damage. She had T cell lymphopenia, but normal immunoglobulins, and a normal alpha fetoprotein. A brother was born shortly after her diagnosis was made. He also had extreme microcephaly of 28 cm, with similar spontaneous and x ray induced chromosomal breakage, and T cell lymphopenia. Neither child has clinical evidence of immunodeficiency. To test the hypothesis that Nijmegen breakage syndrome and ataxia telangiectasia are allelic disorders, haplotype analysis was carried out in the family using DNA markers spanning the AT locus on chromosome 11q22. The affected boy had a different haplotype from his affected sister. Thus in this family, the Nijmegen breakage syndrome is not allelic to the ataxia telangiectasia locus on chromosome 11q, and the two conditions are genetically distinct. The normal intellect in these children raises questions about normal brain development in the presence of severe microcephaly.

Mutations revealed by sequencing the 5' half of the gene for ataxia telangiectasia.

Ataxia telangiectasia is a recessive disorder in which patients show a progressive cerebellar degeneration leading to ataxia, abnormal eye movements and deterioration of speech. Other features include ocular telangiectasia, high serum AFP levels, immunodeficiency, growth retardation and an increased predisposition to some tumours, particularly T cell leukaemia and lymphoma. We report the 1348 amino acid sequence of the N-terminal half of the A-T gene product which, together with the previously published C-terminal half, completes the sequence of the A-T protein. No homologies with other genes have been found within the N-terminal half of the A-T protein. We have also identified six mutations affecting the N-terminal half of the protein. One of these mutations was found to be associated with a haplotype that is common to four apparently unrelated families of Irish descent. All the patients so far examined for both A-T alleles were shown to be compound heterozygotes. None of these mutations affected a putative promoter region which may direct divergent transcription of both the A-T gene and a novel gene E14. The ability to recognise mutations across the entire coding sequence of the A-T gene provides a practical advantage to A-T families since a DNA based prenatal diagnosis will be possible in families where the mutations are identified irrespective of the level of radiosensitivity in these families.

The systemic absorption and disposition of levobupivacaine 0.5% after epidural administration in surgical patients: a stable-isotope study.

BACKGROUND AND OBJECTIVE: Absorption and disposition kinetics can be studied with a stable-isotope method. The aim of this study was to validate a stable-isotope method for levobupivacaine and to derive the relevant pharmacokinetics after epidural administration. METHODS: Eight volunteers (18-32 yr) received approximately 23 mg of both levobupivacaine and deuterium-labelled levobupivacaine simultaneously by intravenous infusion. Venous blood samples were taken for 8 h. Fifteen patients (23-85 yr) received 19 mL levobupivacaine 0.5% (including a 3 mL test dose) epidurally and, 25 min later, approximately 25 mg deuterium-labelled levobupivacaine (D3-levobupivacaine) intravenously. Arterial blood samples were collected for 24 h. Plasma concentrations were determined using liquid chromatography-mass spectrometry. Plasma concentration-time data were analysed by compartmental and non-compartmental analysis. RESULTS: Based on the ratio of the normalized areas under the curve of unlabelled and deuterium-labelled levobupivacaine in volunteers, as determined by both compartmental (mean ratio: 1.02, 90% CI: 1.00-1.04) and non-compartmental analysis (mean ratio: 1.02, 90% CI: 1.00-1.03) the two formulations were considered equivalent. In surgical patients the elimination half-life (mean +/- SD: 196 +/- 65 min), total body clearanc (349 +/- 114 mL min(-1)) and volume of distribution at steady state (56 +/- 14 L), derived by compartmental analysis, were similar to those obtained by non-compartmental analysis. The absorption was bi-phasic. The fractio absorbed and half-life of the fast absorption process were 0.22 +/- 0.06 and 5.2 +/- 2.7 min, respectively. Th values for the slow absorption process were 0.84 +/- 0.14 and 386 +/- 91 min, respectively. CONCLUSIONS: D3-levobupivacaine is pharmacokinetically equivalent to unlabelled levobupivacaine and can be used to study the absorption and disposition kinetics after perineural administration of levobupivacaine in a single experiment.

Mutations associated with variant phenotypes in ataxia-telangiectasia.

We have identified 14 families with ataxia-telangiectasia (A-T) in which mutation of the ATM gene is associated with a less severe clinical and cellular phenotype (approximately 10%-15% of A-T families identified in the United Kingdom). In 10 of these families, all the homozygotes have a 137-bp insertion in their cDNA caused by a point mutation in a sequence resembling a splice-donor site. The second A-T allele has a different mutation in each patient. We show that the less severe phenotype in these patients is caused by some degree of normal splicing, which occurs as an alternative product from the insertion-containing allele. The level of the 137-bp PCR product containing the insertion was lowest in two patients who showed a later onset of cerebellar ataxia. A further four families who do not have this insertion have been identified. Mutations detected in two of four of these are missense mutations, normally rare in A-T patients. The demonstration of mutations giving rise to a slightly milder phenotype in A-T raises the interesting question of what range of phenotypes might occur in individuals in whom both mutations are milder. One possibility might be that individuals who are compound heterozygotes for ATM mutations are more common than we realize.

ATM mutations and phenotypes in ataxia-telangiectasia families in the British Isles: expression of mutant ATM and the risk of leukemia, lymphoma, and breast cancer.

We report the spectrum of 59 ATM mutations observed in ataxia-telangiectasia (A-T) patients in the British Isles. Of 51 ATM mutations identified in families native to the British Isles, 11 were founder mutations, and 2 of these 11 conferred a milder clinical phenotype with respect to both cerebellar degeneration and cellular features. We report, in two A-T families, an ATM mutation (7271T-->G) that may be associated with an increased risk of breast cancer in both homozygotes and heterozygotes (relative risk 12.7; P=. 0025), although there is a less severe A-T phenotype in terms of the degree of cerebellar degeneration. This mutation (7271T-->G) also allows expression of full-length ATM protein at a level comparable with that in unaffected individuals. In addition, we have studied 18 A-T patients, in 15 families, who developed leukemia, lymphoma, preleukemic T-cell proliferation, or Hodgkin lymphoma, mostly in childhood. A wide variety of ATM mutation types, including missense mutations and in-frame deletions, were seen in these patients. We also show that 25% of all A-T patients carried in-frame deletions or missense mutations, many of which were also associated with expression of mutant ATM protein.