Vascular endothelium as a contributor of plasma sphingosine 1-phosphate

Sphingosine 1-phosphate (S1P), an abundant lipid mediator in plasma, regulates vascular and immune cells by activating S1P receptors. In this report, we investigated the mechanisms by which high plasma S1P levels are maintained in mice. We found that plasma S1P turns over rapidly with a half-life of approximately 15 minutes, suggesting the existence of a high-capacity biosynthetic source(s). Transplantation of bone marrow from wild-type to Sphk1(-/-)Sphk2(+/-) mice restored plasma S1P levels, suggesting that hematopoietic cells are capable of secreting S1P into plasma. However, plasma S1P levels were not appreciably altered in mice that were thrombocytopenic, anemic, or leukopenic. Surprisingly, reconstitution of Sphk1(-/-)Sphk2(+/-) bone marrow cells into wild-type hosts failed to reduce plasma S1P, suggesting the existence of an additional, nonhematopoietic source for plasma S1P. Adenoviral expression of Sphk1 in the liver of Sphk1(-/-) mice restored plasma S1P levels. In vitro, vascular endothelial cells, but not hepatocytes, secreted S1P in a constitutive manner. Interestingly, laminar shear stress downregulated the expression of S1P lyase (Sgpl) and S1P phosphatase-1 (Sgpp1) while concomitantly stimulating S1P release from endothelial cells in vitro. Modulation of expression of endothelial S1P lyase with small interfering RNA and adenoviral expression altered S1P secretion, suggesting an important role played by this enzyme. These data suggest that the vascular endothelium, in addition to the hematopoietic system, is a major contributor of plasma S1P.     

Roles of iron and HFE mutations on severity and response to therapy during retreatment of advanced chronic hepatitis C

BACKGROUND & AIMS: Iron overload may cause or contribute to hepatic injury and fibrosis. Mutations in the HFE gene may influence development or progression of chronic liver disease by increasing iron stores or modulating immune responses. The aim of this work was to assess the influence of HFE mutations and serum and hepatic measures of iron status on baseline features and response to lead-in therapy in subjects with advanced chronic hepatitis C enrolled in the Hepatitis C Anti-viral Long-term Treatment to prevent Cirrhosis (HALT-C) Trial. METHODS: Entry criteria included an Ishak fibrosis score >2 and lack of iron overload (Scheuer iron grade <3+) according to local study pathologists. All baseline biopsy specimens were rescored by consensus of study pathologists, and detailed assessment of stainable iron was performed. Hepatic iron concentrations were measured on portions of 144 liver biopsy specimens. A total of 1051 out of 1145 subjects agreed to HFE mutational testing (C282Y, H63D, S65C). RESULTS: Thirty-five percent carried at least one HFE gene mutation. There were no significant differences in the prevalence of HFE gene mutations among subjects with fibrosis (35.5%) versus cirrhosis (32.9%). Thirty-three percent of subjects had end-of-treatment and 16% sustained virologic responses. Presence of HFE mutations, in particular the H63D variation, was associated with increased end-of-treatment (40% vs 29%, P = .0078) and sustained virologic responses (20% with HFE mutation vs 14% sustained virologic response without HFE mutation; P = .009). CONCLUSIONS: Although HFE mutations (especially the most frequent H63D mutation) are associated with increased iron loading, they are also associated with increased sustained virologic responses in US patients with advanced chronic hepatitis C.     

IFN-alpha receptor mRNA expression in a United States sample with predominantly genotype 1a/I chronic hepatitis C liver biopsies correlates with response to IFN therapy.

Our aim was to assess whether, in the United States, with the predominant hepatitis C viral (HCV) genotypes 1a/I and 1b/II, hepatic interferon-alpha receptor (IFNAR) mRNA expression correlated with response to IFN therapy, levels of HCV RNA, or histologic activity index (HAI). Nine of 24 patients (38%) had an initial response to IFN treatment, 5 of whom (21%) had a sustained response. The corrected hepatic IFNAR mRNA expression (measured by RT-PCR) for the sustained responder group (mean +/- SE, 0.16 +/- 0.06, n = 5) was significantly higher than for the nonresponding group (0.059 +/- 0.01, n = 15) (p < 0.02). Patients who relapsed had an intermediate value (0.092 +/- 0.029, n = 4). Higher IFNAR expression was inversely correlated with a lower serum HCV RNA titer (p < 0.01), and responders to IFN treatment tended to have a lower titer of HCV RNA (p = 0.056). We found no significant correlation between the amounts of IFNAR with (1) the total HAI (low HAI < or = 7, IFNAR 0.076 +/- 0.013, n = 10; high HAI > or = 8, IFNAR 0.092 +/- 0.027, n = 14, ns) or (2) individual inflammation, necrosis, or fibrosis components of the HAI. As with Japanese HCV patients with genotypes 1b/II-2b/IV, higher hepatic IFNAR mRNA expression in the United States with predominant genotypes 1a/I and 1b/II appears to correlate with response to IFN therapy and a low HCV RNA titer but not with the total HAI or its components.     

Hepatic heme synthesis in a new model of experimental hemochromatosis: studies in rats fed finely divided elemental iron

Rats fed chow containing finely divided elemental iron (from carbonyl-iron) develop hepatic iron overload resembling human hereditary hemochromatosis in that deposition of iron is primarily in periportal hepatocytes and with hepatic iron concentrations sufficiently high to be associated in the human disease with hepatic fibrosis or cirrhosis. In recent studies using this model, we reported changes in hepatic hemoproteins and heme oxygenase, the rate-controlling enzyme of heme breakdown. We now report effects of iron-loading on three enzymes of heme synthesis: 5-aminolevulinate synthase; the first and rate-controlling enzyme of the pathway, 5-aminolevulinate dehydrase (or porphobilinogen synthase), and uroporphyrinogen decarboxylase, the activity of which is decreased in porphyria cutanea tarda, a liver disease in which iron is known to play an important but still poorly understood role. Of the three enzymes, only activity of the dehydrase was altered by iron-loading: it was decreased significantly as early as 1 week after starting iron feeding, and with marked iron overload was 30 to 32% of control values. The degree of decrease was inversely related (r = -0.77 to -0.88) to the degree of iron overload and was partially reversed within 1 to 3 days when feeding of the iron-supplemented diet was stopped. The decrease in dehydrase activity was not attributable to lack of reduced glutathione or other disulfide-reducing agents or to zinc deficiency; nor was evidence found for inhibition by iron compounds or other possible inhibitors present in iron-loaded livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies against mitochondrial dehydrogenase complexes in primary biliary cirrhosis

Antimitochondrial antibodies, serological hallmarks of primary biliary cirrhosis, recently were found to be directed against the E2 subunits of mitochondrial dehydrogenase complexes (pyruvate, branched-chain ketoacid, and alpha-ketoglutarate dehydrogenases). The objectives of this study were to extend these findings and to determine whether purified immunoglobulin from the sera of patients with primary biliary cirrhosis inhibit activity of these dehydrogenase complexes in vitro. Sera were examined from 14 patients with primary biliary cirrhosis (13 mitochondrial antibody positive), 23 with rheumatic diseases and 30 with chronic active hepatitis (all 53 positive for mitochondrial antibodies by indirect immunofluorescence), 10 with alcoholic liver disease, and 5 normal controls. Antibodies against pyruvate dehydrogenase, branched-chain alpha-ketoacid dehydrogenase and alpha-ketoglutarate dehydrogenase complexes were detected by immunoblot and quantified by enzyme-linked immunosorbent assay. Of the 14 serum samples obtained from patients with primary biliary cirrhosis, 13, 11, and 2 samples tested positive by immunoblot for the E2 subunits of pyruvate, branched-chain ketoacid, and alpha-ketoglutarate dehydrogenase, respectively. In contrast, samples from subjects with rheumatic diseases, chronic active hepatitis, and alcoholic liver disease and control subjects tested negative for these antibodies. Serum immunoglobulin G with high titers of mitochondrial antibodies showed concentration-dependent inhibition of activity of the dehydrogenase complexes, and close correlation (r = 0.917, n = 13) was observed between inhibitory activity against pyruvate dehydrogenase complex and the reciprocal titer of immunoglobulin against this complex. These data suggest that such autoantibodies, besides serving as diagnostic markers for primary biliary cirrhosis, may have a pathogenic role by their ability to inhibit important mitochondrial enzymes.     

Induction of porphyria in cultured chick-embryo hepatocytes by halogenated aromatic compounds

Chick-embryo liver cells in culture were used to study the mechanism by which hexachlorobenzene (HCB) and other chlorobenzenes cause hepatic porphyria with accumulation of uroporphyrin (URO). The actions of the chlorobenzenes were similar to those of 3,4,3',4'-tetrachlorobiphenyl (TCB), but pretreatment with 3-methylcholanthrene (MC) or hexachlorobenzene was needed for maximum accumulation of URO. HCB was as potent and almost as rapid as the biphenyl in causing URO accumulation, but tetrachlorobenzenes caused little URO accumulation. Ellipticine, an inhibitor of cytochrome P-448, stopped the accumulation of URO. A mechanism is proposed for the action of the chlorobenzenes that involves binding to cytochrome P-448 and production of active oxygen species that oxidize uroporphyrinogen.     

Twenty years of the ethics committee at the medical faculty of the University of Vienna. An interim report

The organisation and assessment procedures of the ethics committee of the University of Vienna medical faculty are described. Data concerning the work of the committee from 1993 through to 1997 are given in detail. Finally, the results of a survey among physicians on the acceptance of the committee's work are presented. In conclusion, the workload of an ethics committee in a large medical faculty can only be handled by efficient management of assessment procedures. However, it is difficult to achieve sufficient acceptance in a field governed by various interests.     

Severe liver injury due to phenelzine with unique hepatic deposition of extracellular material

Severe acute and chronic hepatic damage occurred in a white man who had taken phenelzine sulfate (Nardil) 45 mg daily for 70 days. Liver biopsy showed a mixed hepatitic and cholestatic pattern with extracellular deposition of a unique homogeneous collagenous substance. Portal cirrhosis developed and has persisted. The patient was found to have a "rapid acetylator phenotype" and high rate of metabolism of antipyrine. These innate factors may have predisposed to hepatic injury due to phenelzine.     

Ubiquitin as a marker of cell injury in nonalcoholic steatohepatitis

Ubiquitin (UB), an intracellular protein that binds to other proteins to target them for proteolysis, is associated with Mallory hyalin (MH), which supports a biopsy diagnosis of nonalcoholic steatohepatitis (NASH). We analyzed 54 liver biopsy specimens from 49 patients with a clinical diagnosis of NASH for immunoreactive UB and multiple features of necroinflammation, fibrosis, and Prussian blue-positive iron to determine whether the presence of immunoreactive UB increases detection of MH or correlates with other features of cell injury or mutations of the HFE gene. MH and UB were graded. Analysis for HFE gene mutations was performed in 48 patients. Biopsy diagnoses were distributed as follows: NASH, 42; steatosis, 10; and nonspecific changes, 2. UB was present in 20 specimens and MH in 23. Of 31 specimens with 0 MH, 6 had UB; of 14 with 1 + (questionable) MH, 7 had 1+ or 2+ UB. UB correlated positively and significantly with the diagnosis and grade of NASH, presence of MH, cell swelling, lobular inflammation, and fibrosis. Immunostaining for UB may enhance detection of MH in questionable cases, support the diagnosis of NASH, and indicate which patients may be at risk for progression of disease.