Sylvie Ricard-Blum

Transglutaminase-mediated cross-linking is involved in the stabilization of extracellular matrix in human liver fibrosis

BACKGROUND/AIMS Lysyl oxidase-mediated cross-linking contributes to the stabilization of collagen in liver fibrosis. We have investigated transglutaminase-mediated cross-linking, to determine if it participates in the stabilization of extracellular matrix in human liver fibrosis. METHODS Transglutaminase activity was assessed in vitro by incorporation of biotinylated amine into liver proteins. The product of the transglutaminase-catalyzed cross-linking reaction, Nepsilon(gamma-glutamyl)lysine, and the extracellular proteins cross-linked by it, were localized by immunohistochemistry in fibrotic livers. The cross-linked complexes were extracted from liver tissue, immunopurified and characterized by Western blot. RESULTS Transglutaminase, detected by immunohistochemistry, Western blot and by enzymatic activity, was found in higher amounts in fibrotic than in normal liver. The Nepsilon(gamma-glutamyl)lysine cross-link, undetectable in normal liver, was present extracellularly in fibrotic liver, where it was co-distributed with osteonectin, mostly in inflammatory areas submitted to an intense remodeling. Cross-linking of osteonectin by transglutaminase was confirmed by Western blot. In parasitic fibrosis transglutaminase also originates from the parasite. CONCLUSIONS Transglutaminase-mediated cross-linking occurs in liver extracellular matrix during the early, inflammatory, stage of liver fibrosis, whereas cross-linking by pyridinoline occurs mostly later in the fibrotic process. This could lead to the development of new anti-fibrotic treatments targeted to a specific stage of fibrosis.

Biochemical properties and immunolocalization of minor collagens in foetal calf cartilage

Type II collagen is the major collagen of cartilage. However, new collagenous chains have been described in different cartilaginous tissues, recently. The la, 2a and 3a chains were extracted from human and bovine hyaline cartilage [1]. Other minor collagenous components were extracted from neonatal pig and human cartilage noted M, CFI, CF2 [2-4], from bovine nasal cartilage and human intervertebral disc noted CPS1, CPS2 [5,6] and from chicken sternal cartilage noted HMW, LMW [7,8] and M 1, M 2 [9]. Here, we report the partial characterization of 3 collagenous fractions obtained after limited pepsin treatment of foetal calf cartilage and isolated according to their solubility properties. The 1.2 M NaC1 fraction contains the la, 2t~ and 3a chains. The 2.0 M NaC1 and 3.0 M NaCl fractions contain at least 7 collagenous chains, which are related to the disulfide-bonded new chains enumerated above, but show some major differences, Antibodies against the 1.2 M and 2.0 M NaC1 fractions were raised in rabbits and their specificity tested by radioimmunoprecipitation. The localization of the corresponding collagenous chains was achieved by indirect immunofluorescence in epiphyseal proper and growth cartilage from foetal calf cartilage. 2. MATERIALS AND METHODS

Comparative analysis of collagens solubilized from human foetal, and normal and osteoarthritic adult articular cartilage, with emphasis on type VI collagen

The different collagen types were extracted sequentially, by 4 M guanidinium chloride and pepsin, from human foetal and normal and osteoarthritic adult articular cartilage. They were characterized by electrophoresis and immunoblotting. Most of the collagenous proteins present in articular cartilage from young human foetuses were solubilized: almost 40% of the total collagen was extracted in the native form with 4 M guanidinium chloride. Type VI collagen was detected in this fraction as high-molecular-mass chains (185-220 kDa) and a low-molecular-mass chain (140 kDa). Type II, IX and XI collagens were also present, but were extracted more extensively by pepsin digestion. Comparative analysis of normal and osteoarthritic cartilage from adults reveals some major differences: an increase in the solubility of the collagen and modifications of soluble collagen types in osteoarthritic cartilage. Furthermore, type VI collagen was present at a higher concentration in guanidinium chloride extracts of osteoarthritic cartilage than those of normal tissue. This finding was corroborated by electron microscopic observations of the same samples: abundant (100 nm) periodic fibrils were observed in the disorganized pericellular capsule of cloned cells in osteoarthritic cartilage. In normal tissues the pericellular zone was more compact and contained only a few such banded fibrils. The differences in the collagen types solubilized from normal and osteoarthritic cartilage, although corresponding to a minor proportion of the total collagen, demonstrate that important modifications in chondrocyte metabolism and in the collagenous network do occur in degenerated cartilage.

A new radioimmunoassay using a commercially available solid support for the detection of IgE antibodies against muscle relaxants

It is well established that muscle-relaxant drugs may be responsible for anaphylactoid reactions during anesthesia. In this study, we developed an in vitro test with a commercially available solid phase for the detection of specific IgE directed to the tertiary or quaternary ammonium groups of neuromuscular-blocking drugs. The solid-phase complex was P-aminophenylphosphoryl-choline (PAPPC) immobilized on agarose, and an RIA was performed with an antihuman IgE labeled with 125I. The results, expressed as the percentage of 125I-labeled anti-IgE linked to the solid phase, were at 0.41 +/- 0.19 for 34 control sera from nonallergic healthy adults, with an upper limit estimated at 1%. The values obtained with the sera of 31 allergic patients ranged from 0.6% to 41% with a sensitivity of 97%. The specificity and the positive predictive value of the PAPPC RIA were 97% and 94%, respectively. These results were compared with results of other RIAs with morphine, trimethylamine, triethylamine immobilized on epoxy-activated Sepharose, and choline hydrochloride immobilized on Sepharose (quaternary ammonium Sepharose RIA) and with Phadebas RAST succinylcholine and Phadebas RAST alcuronium. The PAPPC RIA appears to be the most efficient test to screen sera for the presence of IgE antibodies directed to neuromuscular-blocking drugs. One major advantage is that this solid phase is commercially available and ready to use. This advantage will improve the accuracy in the comparison of the results with results from different laboratories.

The carboxy-terminal cross-linked telopeptide of type I collagen (ICTP) is a potential serum marker of ongoing liver fibrosis.

We report for the first time the measurement of the serum concentration of the carboxy-terminal cross-linked telopeptide of type I collagen in patients with various liver diseases. This breakdown product of type I collagen, which is the major collagen type found in fibrotic liver, was measured by a radioimmunoassay in the serum of 149 patients with various liver diseases and in 67 controls. Its concentration is significantly elevated (P < 0.05) above reference intervals in sera from patients with liver diseases, except in patients with chronic active hepatitis of unknown origin and in patients with acute hepatitis A. In the 143 patients with liver fibrosis, the serum level of the carboxy-terminal telopeptide of type I collagen is correlated with the extent of fibrosis, as assessed by a histological scoring system (r = 0.3899, P < 0.0001), but not with inflammation and necrosis.

Kinetics of internalization and subcellular binding sites for T3 in mouse liver

Summry— The intracellular fate of radiolabeled T3 taken up by mice hepatocytes in vivo was determined at specific time intervals (2–120 min) after injection by quantitative electron microscopic radioautography. Injection of a 200‐fold excess of unlabeled T3 together with [125I]‐T3 resulted in a more than 90% inhibition of radioactivity detected in hepatocytes. A simple grain density (GD) analysis of radioautograms revealed that a specific labeling (GD > 1) was displayed by only five cell compartments: the plasma membrane, lipid droplets, mitochondria, nuclear envelope and nuclear matrix whereas other compartments were not labeled. Labeled compartments showed distinct changes in the pattern of labeling over time: the plasma membrane was labeled only 2 min after T3 injection, whereas labeling of the nuclear envelope was high at 2 min, decreased at 15 min and progressively increased to maximal measured levels at 120 min. After a lag time of 30 min, nuclear matrix labeling increased progressively with time. Mitochondrial labeling was found to be specific at any time point studied but showed no change over time. These ultrastructural data have been confirmed in vitro by the interaction of T3 with plasma membrane, nuclear membrane, nuclear matrix and mitochondria by real‐time biospecific interaction analysis in a BIAcore™ system. These results demonstrate that T3 binds to hepatocytes before internalization, is transported both to mitochondria and to the nuclear envelope and translocated into the nuclear matrix.

Histamine release assay and radioimmunoassay for the detection of IgE antibodies against neuromuscular blocking drugs.

It is well established that muscle-relaxant drugs may be responsible for anaphylactoid reactions during anaesthesia. In the present work we evaluated in 41 patients who had experienced anaphylactoid reactions during general anesthesia, the value of different radioimmunoassays (RIAs) and of an histamine release assay (HRA). The RIA was performed with different solid phases as PAPPC (para amino phenyl phosphoryl choline), morphine, TMA (trimethylamine) and TEA (triethylamine). The results were expressed as the percentage of 125I anti-human IgE adsorbed onto the solid phase. The sensitivity was estimated respectively at 95, 82, 93 and 64%. The results were significantly in accordance with those obtained with QAS-RIA (from Guéant) but a weak correlation was seen with Phadebas Rast succinylcholine (r = 0.85 and 0.53 respectively). The HRA gave with the NMBD incriminated a sensitivity of 88%. The correlation between HRA and IDR for the drug involved was 89% and 43% for the other NMBDs. The PAPPC RIA was apparently the most efficient test to screen sera for the presence of IgE antibodies whatever the NMBDs involved in anaphylaxis. An advantage is that this solid phase is commercially available and therefore this RIA can be used routinely with a high sensitivity, allowing the comparison of the results obtained by different laboratories.

Urinary excretion of the collagen cross-link pyridinoline increases during liver fibrogenesis.

BACKGROUND/AIMS Pyridinoline, a specific cross-link of mature collagen, increases in liver during fibrogenesis and its hepatic level is related to the degree of reversibility of the fibrotic process. Since pyridinoline is excreted in urine, we have investigated the relationship between its urinary level and liver fibrogenesis in a model of mild and reversible liver fibrosis, murine schistosomiasis. METHODS Pyridinoline was measured by HPLC in urine and in liver of Schistosoma mansoni-infected mice during the acute and the chronic phases of the infection. Collagen deposition was measured colorimetrically. Both the isolated granulomas and the surrounding liver parenchyma were analyzed. RESULTS In infected mice, pyridinoline increased mainly in the isolated granulomas, corresponding to the fibrotic lesions, and slightly in the surrounding parenchyma. The urinary excretion of pyridinoline increased during liver fibrogenesis and was correlated to the duration of infection (r=0.81) and to the collagen content of granulomas (r=0.81). The treatment of infected mice by praziquantel, an antiparasitic drug, did not lead to significant changes in liver collagen cross-linking by pyridinoline either in granulomas or in parenchyma. The major effect of the drug was targeted at the collagen content of parenchyma, which decreased by 50%, 18 weeks after treatment. The urinary level of pyridinoline of treated mice was negatively correlated to the length of the treatment follow-up (r=-0.76). CONCLUSIONS The measurement of the urinary excretion of pyridinoline could be helpful to monitor the remodeling of liver extracellular matrix occurring in fibrogenesis and the effect of chemotherapy.

The level of the collagen cross-link pyridinoline reflects the improvement of cutaneous lesions in one case of skin alveolar echinococcosis

Abstract Cutaneous parasitic lesions, associated with a dense fibrous reaction, markedly improved under albendazole treatment in one case of supraumbilical skin localization of alveolar echinococcosis. Since collagen cross-linking increases during fibrogenesis and contributes to the stability of fibrotic lesions, we monitored the level of the cross-links pyridinoline and pentosidine in skin lesions from this patient to determine if they would reflect the changes occurring during treatment. We looked at the deposition of cross-linked type I collagen by immunohistochemistry and also measured the serum concentrations of pentosidine and of a fragment of type I collagen (ICTP), which contains a site of pyridinoline formation. Albendazole treatment did not affect either the collagen content of skin lesions or the serum concentrations of ICTP and pentosidine, but it led to a pronounced decrease in pyridinoline level concomitant with the disappearance, observed by immunohistochemistry, of extensively cross-linked fibrotic type I collagen. The follow-up of collagen cross-linking by pyridinoline in skin tissue thus appears to be useful in reflecting the improvement of fibrotic skin diseases during therapy.

The Different Types of Collagen Present in Cartilaginous Tissues

It was thought, for several years that type II collagen was the only constituent of hyaline cartilage. However, since 1978, other types of collagen have been described from different cartilaginous tissues, (Table 1). We describe these collagens here, as well as their localization in the tissue, with particular reference to the so-called minor collagens (1α2α3α and type IX) which represent from 5 to 10% of hyaline cartilage whilst type II represents around 90%.