TNF tolerance and cytotoxicity in the liver: the role of interleukin-1beta, inducible nitric oxide-synthase and heme oxygenase-1 in D-galactosamine-sensitized mice.

OBJECTIVE AND DESIGN: Pretreatment with tumor necrosis factor (TNF)-alpha induces tolerance towards itself in experimental liver injury. MATERIAL AND TREATMENT: To study mechanisms of TNF tolerance we used knockout mice for either TNF-receptor-2 (TNFR-2), inducible nitric oxide (NO)-synthase (iNOS) or caspase-1 (ICE) or inhibited heme oxygenase-1 (HO-1) by treatment with zinc-protoporphyrin 9. Liver damage was induced by administration of TNF to mice sensitized with D-galactosamine (GalN). Tolerance was induced by pretreatment with low doses of TNF. METHODS: Severity of liver injury was assessed by determination of plasma transaminases and apoptosis. Time courses of intra-hepatic iNOS, interleukin-1beta (IL-1beta) and HO-1 expression after TNF treatment were measured by reverse transcription polymerase chain reaction (RT-PCR). TNF-receptor-1 (TNFR-1) expression was determined by immunofluorescent staining. RESULTS: TNF-pretreatment did not affect TNFR-1 expression in the liver and resulted in time dependent up-regulation of iNOS, IL-1beta and HO-1. TNF- pretreated TNFR-2, iNOS or ICE knockout mice were as sensitive towards GalN/TNF as the wild type, while mice with impaired HO-1 activity were even more sensitive, but tolerance was inducible in all TNF-pretreated mice. CONCLUSIONS: TNF tolerance towards GalN/TNF treatment is mediated by TNFR-1. IL-1beta, iNOS and HO-1 neither mediated TNF-tolerance nor TNF cytotoxicity.

Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.

Liver resident NK1.1+ T cells are supposed to play a pivotal role in the onset of inflammatory liver injury in experimental mouse models such as concanavalin A (Con A)-induced hepatitis. These cells, expressing the adhesion receptor, CD44, are largely depleted from the liver by a single intravenous injection of low-molecular-weight fragments of hyaluronic acid (LMW-HA). Here, we report that LMW-HA pretreatment protected mice from liver injury in several models of T-cell- and macrophage-dependent, tumor necrosis factor alpha (TNF-alpha)-mediated inflammatory liver injury, i.e., from liver injury induced by either Con A or Pseudomonas exotoxin A (PEA) or PEA/lipopolysaccharide (LPS). Interestingly, apart from inhibition of cellular adhesion, pretreatment of mice with LMW-HA was also capable of preventing hepatocellular apoptosis and activation of caspase-3 induced by direct administration of recombinant murine (rmu) TNF-alpha to D-galactosamine (GalN)-sensitized mice. LMW-HA-induced hepatoprotection could be neutralized by pretreatment with the nuclear factor-kappaB (NF-kappaB) inhibitor, pyrrolidine dithiocarbamate (PDTC), demonstrating the involvement of NF-kappaB in the observed protective mechanism. Indeed, injection of LMW-HA rapidly induced the production of TNF-alpha by Kupffer cells and the translocation of NF-kappaB into hepatocellular nuclei. Both LMW-HA-induced TNF-alpha production and NF-kappaB translocation were blocked by pretreatment with PDTC. Our findings provide evidence for an unknown mechanism of LMW-HA-dependent protection from inflammatory liver disease, i.e., induction of TNF-alpha- and NF-kappaB-dependent cytoprotective proteins within the target parenchymal liver cells.

Inducible nitric oxide synthase is critical for immune-mediated liver injury in mice.

Concanavalin A (Con A) causes severe TNF-alpha-mediated and IFN-gamma-mediated liver injury in mice. In addition to their other functions, TNF-alpha and IFN-gamma both induce the inducible nitric oxide (NO) synthase (iNOS). Using different models of liver injury, NO was found to either mediate or prevent liver damage. To further elucidate the relevance of NO for liver damage we investigated the role of iNOS-derived NO in the Con A model. We report that iNOS mRNA was induced in livers of Con A-treated mice within 2 hours, with iNOS protein becoming detectable in hepatocytes as well as in Kupffer cells within 4 hours. iNOS-/- mice were protected from liver damage after Con A treatment, as well as in another TNF-alpha-mediated model that is inducible by LPS in D-galactosamine-sensitized (GalN-sensitized) mice. iNOS-deficient mice were not protected after direct administration of recombinant TNF-alpha to GalN-treated mice. Accordingly, pretreatment of wild-type mice with a potent and specific inhibitor of iNOS significantly reduced transaminase release after Con A or GalN/LPS, but not after GalN/TNF-alpha treatment. Furthermore, the amount of plasma TNF-alpha and of intrahepatic TNF-alpha mRNA and protein was significantly reduced in iNOS-/- mice. Our results demonstrate that iNOS-derived NO regulates proinflammatory genes in vivo, thereby contributing to inflammatory liver injury in mice by stimulation of TNF-alpha production.

Cloning and expression of the dog mast cell alpha-chymase gene.

Chymases are chymotrypsin-like serine proteinases secreted by mast cells. Alpha- and beta-chymases differ in structure, function, and mast cell subset- and species-specific expression. Seeking genetic regulatory elements shared by alpha-chymases, we sequenced the dog alpha-gene. Extensive homology was found in intronic and flanking sequences of the dog, human, and mouse alpha-chymase genes, but little in corresponding beta-chymase sequences. Repetitive elements probably derived from retroposons are unique features of the dog flank. DNA blots suggest that the dog alpha-gene, like its human counterpart, may be the genome's sole chymase, unlike in rodents, in which beta-chymases predominate. Nuclear runoff studies predict that transcriptional mechanisms explain differences in steady state chymase and tryptase mRNA levels between mastocytoma and non-mast cells. In dog BR mastocytoma cells incubated with phorbol ester, high steady state levels of alpha-chymase mRNA drop dramatically with little change in tryptase mRNA, whereas dexamethasone decreases expression of both mRNAs. Portions of the dog or human gene 5' flank transfected into BR cells drive expression of a reporter gene and define regions with active promoters. Thus, BR cells express high levels of alpha-chymase mRNA regulated independently of tryptase and support transcription using dog or human promoters. These studies reinforce the alphabeta-chymase dichotomy and suggest the utility of BR cells in probing regulation of alpha-chymase expression.

Long-term persistence of AZT-resistance mutations in the plasma HIV-1 of patients removed from AZT therapy.

Zidovudine (3'-azido-2',3'-dideoxythymidine) resistant isolates of human immunodeficiency virus type I (HIV-1) were previously demonstrated in zidovudine-treated AIDS patients. The genetic linkage of multiple mutations characteristic of zidovudine-resistance as well as dideoxyinosine-resistance were demonstrated by examining clones of viral reverse transcriptase after polymerase chain reaction (PCR) amplification of plasma culture DNA. The zidovudine-resistance mutations persisted in seven timepoints from four patients for 5 to 22 months despite cessation of zidovudine therapy (and while patients underwent ddI therapy). One patient's plasma virus isolate at 14 months possessed a genotype doubly resistant to ZDV and ddI. Virus recovered from four timepoints showed Intermediate to high levels of zidovudine-resistance. As these genotypes were mainly derived from plasma culture, the zidovudine resistant virus appears to persist and replicate well in vivo after cessation of zidovudine therapy.

Long-term persistence of zidovudine resistance mutations in plasma isolates of human immunodeficiency virus type 1 of dideoxyinosine-treated patients removed from zidovudine therapy.

Zidovudine (3'-azido-2',3'-dideoxythymidine)-resistant isolates of human immunodeficiency virus type 1 (HIV-1) were previously demonstrated in zidovudine-treated AIDS patients. The genetic linkage of multiple mutations characteristic of zidovudine resistance as well as dideoxyinosine resistance were demonstrated by examining clones of viral reverse transcriptase after polymerase chain reaction amplification of plasma culture DNA. The zidovudine resistance mutations persisted at seven time points from 4 patients for 5-22 months despite cessation of zidovudine therapy (and while patients underwent dideoxyinosine therapy). One patient's plasma virus isolate at 14 months possessed a genotype doubly resistant to zidovudine and dideoxyinosine. Virus recovered from four time points showed intermediate to high levels of zidovudine resistance. As these genotypes were mainly derived from plasma culture, the zidovudine-resistant virus appears to persist and replicate well in vivo after cessation of zidovudine therapy.

Zidovudine-resistant human immunodeficiency virus type 1 genomes detected in plasma distinct from viral genomes in peripheral blood mononuclear cells.

The emergence of zidovudine (3'-azido-2',3'-deoxythymidine)-resistant strains of human immunodeficiency virus type 1 (HIV-1) from AIDS patients treated with zidovudine has been linked to six amino acid substitutions localized within the viral polymerase gene. Here, in 2 patients, three resistance mutations were detected by polymerase chain reaction amplification of HIV-1 polymerase (reverse transcriptase) sequences from cultures of patient plasma only and not from the same patients' uncultured leukocytes. The differences in distribution of the mutant genotypes from the two sources were highly significant. Both plasma- and peripheral blood mononuclear cell (PBMC)-derived virus (and/or RNA genomes) should be studied in additional subjects to confirm the hypothesis raised by these data that zidovudine-resistant virus may be more frequent in plasma than in uncultured PBMC.

Serum concentrations of insulin-like growth factor II are not changed by short-term fasting and refeeding.

To determine the factors that regulate insulin-like growth factor II (IGF-II), we raised polyclonal antibodies to this peptide and developed a RIA that measures IGF-II in serum or plasma samples after extraction of IGF-binding proteins by C18 cartridge chromatography. The IGF-II antiserum was highly specific, exhibiting no cross-reactivity with IGF-I or insulin at the highest concentrations tested (10(-6) mol/L). As little as 0.43 micrograms/L IGF-II was detectable, and 50% displacement of tracer occurred at 1.7 microgram/L. The serum IGF-II concentrations of normal adults [mean, 634 +/- 170 (+/- SD) micrograms/L], patients with acromegaly (570 +/- 146 micrograms/L), and patients with hypopituitarism (156 +/- 58 micrograms/L) were similar to those reported by others. In eight obese subjects injected with GH (0.1 mg/kg ideal BW, im, every 48 h for 16 days), serum IGF-II concentrations did not rise significantly, whereas IGF-I concentrations increased 67%. Sixteen normal subjects, within 15% of ideal body weight, were fasted for 5 days on two to four occasions and refed diets of differing protein and calorie contents. Their mean serum IGF-II concentration before fasting (691 +/- 26 micrograms/L) was not significantly different from that after fasting (674 +/- 21 micrograms/L) or after refeeding (641 +/- 20 micrograms/L). In contrast, their mean IGF-I concentration decreased 42% with fasting and rose with refeeding. Unlike IGF-I, serum IGF-II concentrations do not appear to be regulated by short term changes in nutritional status. It is clear from this study and others that IGF-II and IGF-I are regulated differently despite their structural homology and the similarity of their actions in vitro.

[Blood glucose and insulin levels in healthy persons and diabetics after intake of coarse wholemeal preparations, especially fresh grain muesli]. / Blutglucose- und Insulinverlauf bei Gesunden und Diabetikern nach Gaberoher Vollkornzubereitungen, insbesondere Frischkornmüsli.

In a study of seven type II diabetics the effects of the usual diabetic diet were compared with those of a cereal of whole-meal (both having the same energy content and nutrients proportions). The cereal gave a more even blood-glucose curve at a significantly lower level (maximal rise after the first breakfast with the cereal was 20 mg/100 ml, after the usual diabetic breakfast 75 mg/100 ml). In addition, in 12 metabolically healthy persons comparison was made of post-prandial blood-glucose and insulin levels after intake of raw and of heated wheat and corn whole-meal preparations. The raw wheat variant produced much flatter blood-glucose and insulin curves than the heated test meals (maximal rise of blood-glucose 6 vs 27-38 mg/100 ml; blood insulin 8 vs 35-50 microU/ml), while the raw corn (oat) variant achieved only a small flattening of the curve compared with that after the heat-treated preparation. Measurement of H2 exhalation provided no evidence for differential malabsorption between raw and heated test meals. Fresh-corn muesli with unheated wheat whole-meal is suitable in the diet of type II diabetics to counteract high postprandial levels of blood-glucose and thus improve the diabetic metabolic state.