Colchicine treatment of cirrhosis: answers.
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Colchicine↗
Biomedical subjects
M Rojkind
Publications and source records attributed to M Rojkind.
Serum type III procollagen peptide and laminin (Lam-P1) detect alcoholic hepatitis in chronic alcohol abusers.
The diagnosis of alcoholic hepatitis is difficult to establish by conventional clinical and laboratory methods, and a firm diagnosis relies on liver histology. Since there are severe limitations in following patients with repeated liver biopsies, noninvasive procedures are needed to assess the presence of alcoholic hepatitis in chronic alcohol abusers. It has been suggested that serum Type III procollagen peptide levels correlates with the degree of inflammation in chronic liver disease. Since inflammation is a major histological finding in alcoholic hepatitis, we therefore studied the usefulness of measuring serum Type III procollagen peptide and laminin values in 45 consecutive chronic alcohol abusers, with or without cirrhosis, in detecting those with alcoholic hepatitis. The results showed that both Type III procollagen peptide and laminin values were elevated in all of the patients with established liver damage. However, the values were highest in those with liver cirrhosis plus alcoholic hepatitis (Type III procollagen peptide 50.4 +/- 36.4 ng per ml vs. 8.1 +/- 2.6 in controls, p less than 0.01; laminin 4.50 +/- 1.49 units per liter vs. 1.24 +/- 0.26 units per liter in controls, p less than 0.01), followed by subjects with alcoholic hepatitis alone (Type III procollagen peptide 23.5 +/- 17.6 ng per ml, p less than 0.01; laminin 2.60 +/- 1.09 units per liter, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Kupffer cells from CC1(4)-treated rat livers induce skin fibroblast and liver fat-storing cell proliferation in culture.
Conditioned media of Kupffer cells from normal rat liver produce in culture two factors that inhibit fibroblast proliferation. The inhibitory factors have molecular masses of approximately 25 and 5 kDa. In contrasts to these results, the conditioned media of Kupffer and mononuclear macrophagic cells, obtained 48 hours after CC1(4) administration to rats, contains a 17 kDa factor that stimulates fibroblast proliferation (FSF). FSF also stimulates [3H]-thymidine incorporation into DNA of cultured rat liver fat-storing cells. Two peaks with FSF activity were demonstrated after isoelectrofocusing; one with a pI of 6.1 and a second with a pI of 7.5. The fraction containing FSF is devoid of interleukin-1 (IL-1) activity and no inhibitory activity is detected in this conditioned media. Production of FSF is inhibitable by colchicine but not by indomethacin, it is thermolabile and trypsin-sensitive. In animals treated chronically with CC1(4) to produce cirrhosis, FSF activity is demonstrable from the first to the 8th week of treatment. However, the activity is lower at 8 weeks post-CC1(4) as compared with 2 weeks. The results suggest that homeostasis of cells in the liver is controlled by factors produced locally, that act by autocrine and paracrine mechanisms. When homeostasis is altered, fibroblast proliferation occurs, and excess collagen deposition leads to fibrosis. We propose that the antifibrogenic activity of colchicine is associated, in part, with its capacity to inhibit the release of FSF by Kupffer cells and liver mononuclear macrophage cells.
Animals↗
Resistance to reinfection in experimental murine schistosomiasis: role of porto-hepatic hemodynamics.
Experiments were conducted to evaluate critically, and independently of the immune system, the possible role of hemodynamic mechanisms in resistance to schistosomal reinfection. The effects of a challenge schistosomal infection were compared in groups of mice which were either previously infected with schistosomiasis, vaccinated with irradiated cercariae, or underwent partial portal vein ligation for the induction of portal hypertension and porto-systemic shunting. Following infection with 60 cercariae, the appearance of portal hypertension preceded by approximately 2 weeks the development of porto-systemic shunting, which reached maximal values 11 weeks postinfection. Such a primary infection conferred on C3H mice an estimated 90% protection to a 2nd infection, measured by the reduction of worm burden. Worm burdens were also reduced in vaccinated and ligated animals as compared to normal controls. The protection amounted to 30% and 56%, respectively, in the C3H strain and 63% and 75-85%, respectively, in the C57Bl/6 strain. Reduction in worm burden in the ligated animals is believed to be due to the extrahepatic porto-systemic vascular shunts. Hemodynamic as well as immunological factors may account for the resistance to reinfection observed in chronic murine schistosomiasis.
Animals↗
Colchicine in the treatment of cirrhosis of the liver.
There is preliminary evidence that colchicine, an inhibitor of collagen synthesis, may be beneficial in the treatment of cirrhosis of the liver. To evaluate the use of colchicine (1 mg per day, five days per week) in the treatment of hepatic cirrhosis, we performed a randomized, double-blind, placebo-controlled trial in which 100 patients were followed for up to 14 years. Forty-five patients had alcoholic cirrhosis, 41 had posthepatitic cirrhosis, and the remaining 14 had cirrhosis with various other causes. Histologic studies were available for 92 percent of patients. Seventy-three patients were in Child-Turcotte class A, 26 were in class B, and one was in class C. Fifty-four patients received colchicine, and 46 received placebo. The overall survival in the colchicine group was markedly better than in the placebo group (median survival, 11 and 3.5 years, respectively; P less than 0.001). The cumulative 5-year survival rates were 75 percent in the colchicine group and 34 percent in the placebo group; the corresponding 10-year survival rates were 56 percent and 20 percent. Among the 30 patients treated with colchicine who underwent repeated liver biopsies, histologic improvement was seen in 9; the liver appeared normal in 2, and 7 had minimal portal fibrosis. No histologic improvement was observed in the 14 members of the placebo group who had two or more biopsies. Few side effects were observed in either group.
Clinical Trials as Topic↗
Immunohistochemical micromethods for the measurement of specific collagen types in human liver biopsies.
Micromethods are needed for measuring the amount of collagen and other matricial proteins in human liver biopsies. Recently, a colorimetric method for the quantitative estimation of total collagen and protein has been developed. This method is sensitive, can be applied to fresh and fixed tissues and provides the absolute value of total collagen per section. Results are expressed as microgram of collagen per mg of proteins or per section. We now report two micromethods for the determination of specific collagen types in fresh frozen liver sections. The first method is a direct method utilizing the tissue section as an ELISA plate. Results are expressed as the amount of color reaction read at 450 nm per slide. After cleaving the antigen antibody complex with dilute acetic acid, the amount of total collagen can be measured by the colorimetric microchemical method and the amount of specific collagen type is expressed as the amount of color reaction per microgram of total collagen. The second method is an indirect method: the floating tissue section is incubated with an optimal amount of specific anti-collagen antibody and the residual amount of antibody present in the supernatant is measured in an ELISA plate coated with the specific collagen type. The amount of specific collagen type present in the tissue section is determined by comparing the optical deviation obtained with a standard curve prepared with known amounts of specific collagen, and values are expressed as microgram of collagen per tissue sections. As with the direct method, the amount of total collagen per section can be measured by the colorimetric method and then, the amount of specific collagen type can be expressed as microgram of specific collagen type per microgram of total collagen.
Biopsy, Needle↗
Lipid quantitation in formalin-fixed liver sections.
We describe a simple, sensitive, and quantitative procedure for measurement of triglycerides and protein contents in formalin-fixed liver sections. The method can detect as little as 0.27 microgram of triglycerides per mg of protein. It is based on selective binding of Sudan IV and Fast Green FCF to fat and total proteins, respectively, and their sequential elution with solvents. Sudan IV is eluted readily with acetone and Fast Green with NaOH-methanol, and the absorbances obtained at 500 and 610 nm can be used to determine the amount of triglycerides and total protein. The color equivalence for Fast Green was obtained after destaining the sections and measuring the protein contents by micro-Kjeldahl analysis. The color equivalence of Sudan IV was estimated by determining the triglyceride content in liver homogenates by an enzymatic procedure and then measuring the amount of dye bound to multiple fixed sections. There was a strong linear correlation between the triglyceride content as determined chemically and that obtained using the equivalence colors (r2 = 0.98). This method is useful to measure fat content in tissue samples and could be applied to evaluate the progression of liver disease.
Animals↗
Isolation of a 220-kilodalton protein with lectin properties from a virulent strain of Entamoeba histolytica.
A 220-kilodalton (kDa) protein with lectin properties was isolated from Entamoeba histolytica strain HM1:IMSS and was purified by Sepharose 4B chromatography and electroelution from 5% SDS-polyacrylamide gels. The protein contains 9% carbohydrate by weight; is rich in hydrophobic residues; and is very immunogenic in mice, hamsters, and rabbits. The protein binds to fixed monolayers of MDCK cells and inhibits trophozoite attachment to the cultured cells. The 220-kDa protein agglutinates human erythrocytes, and agglutination is inhibited by micromolar concentrations of hyaluronic acid, chitin, chitin-derived products (chitotriose), and antibodies to the purified protein. The 220-kDa protein is recognized by an antibody to the membrane but not by antibodies to other subcellular fractions. We therefore suggest that this 220-kDa protein with lectin properties is a component of the plasma membrane and could be one of the putative "receptor" molecules involved in cell and/or matrix attachment.
Adhesiveness↗
Use of antibodies to characterize a 220-kilodalton surface protein from Entamoeba histolytica.
Antibodies were prepared against a 220-kilodalton (kDa) protein partially purified by Sepharose 4B chromatography of Entamoeba histolytica strain HM38:IMSS homogenates, and the protein was found to have lectin properties. The antibodies specifically recognized this protein in trophozoite homogenates. Immunologically related molecules with the same molecular weight were identified by polyclonal antibodies in strains HM1:IMSS, Entamoeba invadens, and Entamoeba histolytica Laredo. Six monoclonal antibodies recognized only the 220-kDa protein present in strains HM38 and HM1, a result indicating the presence of similar epitopes in the proteins from virulent strains isolated from humans. All the antibodies against the 220-kDa protein have the following properties: (1) they bind to the plasma membrane of live or fixed trophozoites, (2) they partially inhibit the adhesion of trophozoites to erythrocytes and cultured cells, and (3) they inhibit erythrophagocytosis.
Adhesiveness↗
Lactate and pyruvate increase the incorporation of [3H]proline into collagen [3H]hydroxyproline in liver slices of CCl4 cirrhotic rats.
Lactate and pyruvate enhanced the incorporation of [3H]proline into collagen [3H]hydroxyproline when added to liver slices of CCl4-treated rats. In addition, pyruvate stimulated the accumulation of cAMP, reaching maximum values after 10 minutes of incubation. Similar results were obtained with newborn rat calvariae, a tissue that normally produces large amounts of type I collagen. In normal liver, which produces relatively small amounts of collagen, lactate had no effect on cAMP levels or collagen synthesis. Pyruvate stimulated the accumulation of cAMP, but had no effect on collagen synthesis. These results indicate that different control mechanisms are involved in regulation of collagen biosynthesis in normal as compared with cirrhotic liver; the latter resembling mesenchymal tissues specialized in collagen production such as newborn rat calvariae.
Animals↗
Effect of colchicine on in vivo and in vitro ethanol metabolism in the rat.
Since colchicine has been used in the treatment of alcoholic cirrhosis of the liver, the effect of this drug on ethanol metabolism in the rat has been investigated. The acute intraperitoneal injection of colchicine (500 micrograms per kg body weight) did not significantly influence in vivo ethanol elimination rate nor the ethanol peak blood concentrations following an acute dose of ethanol (3 g per kg body weight). Cytoplasmic hepatic alcohol dehydrogenase activity was not changed by in vitro colchicine. However, an approximate 30% inhibition of hepatic microsomal ethanol oxidation was observed by colchicine at a concentration of 4 microM and more. In addition, when colchicine was given chronically in a daily dose of 50 micrograms per kg body weight for 3 weeks, again in vivo ethanol disappearance from the blood was not affected by the drug. These data indicate that neither acute nor chronic colchicine administration alters in vivo ethanol elimination. But the question is raised whether colchicine can decrease hepatic acetaldehyde concentration following a therapeutic dose in vivo by inhibiting the hepatic ethanol metabolizing enzyme system. This could be a possible explanation for the observation that alcoholics improve their liver function under colchicine therapy although they continue to drink.
Alcohol Dehydrogenase↗
Increased stimulation of alkaline phosphatase by small doses of colchicine entrapped in liposomes. A biochemical test to detect effective liposome-hepatocyte interaction.
The subcutaneous administration of colchicine encapsulated in small unilamellar vesicles reduced the initial toxicity peak and maintained for several days an adequate level of the drug in the liver. Colchicine is an excellent marker for effective liposome-hepatocyte interaction since it fulfills the following criteria: (a) When taken up by the hepatocytes within liposomes, it is active and induces the synthesis of alkaline-phosphatase two to three times over control values. The injection of at least ten times more free colchicine is necessary to attain a similar induction. (b) If released from extracellular liposomes, colchicine is cleared rapidly from the circulation. The results show that liposomes, in spite of their reduced aqueous compartment (approximately 1.0 microliter/mumole of lipid), can achieve clinical utility when administered subcutaneously because of their efficient interaction with parenchymal cells and their continual arrival from the injection site.
Alkaline Phosphatase↗
Liver fibroblast proliferation in murine schistosomiasis.
Liver fibrosis in schistosomiasis is associated with prominent accumulations of fibroblasts. Primary cell cultures were prepared from the fibrotic livers of Schistosoma mansoni-infected mice, and cells with the appearance of fibroblasts by light microscopy were isolated from these cultures. Proliferation of these cells was examined in coculture experiments with syngeneic inflammatory cells. T cell-enriched mononuclear cells from spleens of S. mansoni-infected or normal mice, and Kupffer cell/macrophages from fibrotic liver all stimulated the proliferation of liver fibroblasts, as measured by 3H-thymidine uptake. Primary cultures of mouse skin fibroblasts showed similar responses to coculture, but an established fibroblast line, 3T3, was unresponsive. Cell-free supernatant medium from coculture experiments did not affect fibroblast proliferation, perhaps because of the requirement for serum in the culture medium. Liver fibroblasts derived from this disease model may be especially suitable for study of the interaction between tissue inflammation and fibrosis.
Animals↗
Regulation of fibroblast proliferation by Kupffer cells and monocytes.
Liver cirrhosis can be considered as an alteration of the homeostatic mechanisms that maintain cell-cell and cell-matrix interactions. The mechanisms that maintain homeostasis and, therefore, that control fibroblast proliferation and collagen synthesis, are unknown. Experiments were performed to study the role of Kupffer cells in regulating fibroblast proliferation and collagen synthesis and to study the antifibrogenic properties of colchicine. Non-parenchymal cells isolated from normal and CCl4-treated rats were cultured. The cultures from normal livers contained few fibroblasts whereas those from CCl4-treated animals contained many fibroblasts. Removal of adherent cells obtained from normal liver favoured fibroblast proliferation. The fibroblasts from normal or CCl4-treated rats were similar and contained collagens type I and type III. The results obtained suggest that normal Kupffer cells control fibroblast proliferation and that incoming monocytes stimulate fibroblast proliferation. Colchicine inhibits the entry of monocytes into the injured liver and could prevent liver fibrosis by this mechanism.
Animals↗
A simple micromethod for collagen and total protein determination in formalin-fixed paraffin-embedded sections.
A simple, sensitive, and quantitative procedure is described for the measurement of collagen and protein content in tissue sections prepared from formalin-fixed paraffin-embedded samples. The method can detect as little as 5.7 micrograms of collagen per mg of protein. It is based on the selective binding of Sirius red F3BA and Fast green FCF to collagen and noncollagenous components, respectively, when the sections are stained with both dyes dissolved in aqueous saturated picric acid. Both dyes are eluted readily and simultaneously with NaOH-methanol and the absorbances obtained at 540 and 605 nm can be used to determine the amount of collagen and protein. The color equivalence of each dye was determined after destaining the sections and measuring the collagen content by hydroxyproline analysis and the amount of protein by the micro-Kjeldahl procedure. When several sections prepared from five rat tissues were analyzed first by the dye binding method and then by the chemical procedure, comparable results were obtained. This method could be of use in measuring collagen in tissue specimens and could be helpful in assessing the degree of fibrosis in tissue samples and in evaluating the effects of antifibrogenic drugs currently in use.
Animals↗
Cryptic adenosine triphosphatase activities in plasma membranes of CCl4-cirrhotic rats. Its modulation by changes in cholesterol/phospholipid ratios.
The activities of Na+,K+-, and Ca2+-ATPases were determined in plasma membranes obtained from livers of rats treated acutely and chronically with CCl4. Twenty-four hours after a single oral dose of CCl4 the ATPases decreased below 50% of control values. The activity of Ca2+-ATPase returned to normal after 4 days, and Na+,K+-ATPase activity returned to normal values after 12 days. One week after initiation of the chronic intraperitoneal treatment with CCl4, the Na,K+-ATPase decreased to 40% of control values and continued to decrease further until reaching values below 1%. Ca2+-ATPase followed a pattern similar to that obtained with Na+,K+-ATPase, except that the decrease was not as severe. Colchicine treatment prevented the modifications in ATPases when given simultaneously with CCl4 and reverted the alterations in ATPase activities of the CCl4-cirrhotic animals. Because ATPases are known to be modulated by the lipid composition of the membrane, we also determined the cholesterol to phospholipid ratio in all the isolated membranes. The ratios were increased in membranes with low ATPase activity due to an increase in the total concentration of cholesterol. Plasma membranes of cirrhotic rats treated with colchicine showed a low concentration of cholesterol, a decreased cholesterol to phospholipid ratio, and Na+,K+-ATPase activity was almost normal. When plasma membranes of cirrhotic rats were fused with phosphatidyl serine-containing liposomes, the cholesterol to phospholipid ratio decreased and the ATPase activity increased. The ATPase activity of normal plasma membranes decreased below 20% of control values when enriched with cholesterol. Our results suggest that the decrease in the plasma membrane Na+,K+-ATPase activity of the cirrhotic rat is due in part to an increase in its cholesterol concentration and in the cholesterol to phospholipid ratio.
Acute Disease↗
Some properties of the reactive hydroxylysyl residues in collagen: their possible role in nucleation during fibril formation.
Native or heat-denatured collagens were incubated under controlled conditions of temperature and pH with variable molar ratios of KCNO or 2,4,6-trinitrobenzene sulphonic acid. The results obtained suggest that a small number of free amino groups are available for reaction on the native protein, while all the free amino groups react on the denatured protein. The highly reactive free amino groups in the native protein are hydroxylysine residues and have an abnormally low pK of 8.5 which is conformation dependent; this pK becomes normalized upon denaturation of the protein. The reactive hydroxylysines appear to be located in basic regions that could be the nucleation sites needed for fiber formation in the heat-gelation assay; the modified protein does not form stable fibrils upon heating at 37 degrees C and the few fibers formed are not stabilized after reduction with NaBH4. Our results also suggest that the triple helix in collagen is heterogeneous with respect to the reactivity of free amino groups and that several discrete transition temperatures are observed with two main breaks at 30 degrees C and at 37 degrees C, respectively.
Animals↗
A simple quantitative method for collagen typing in tissue samples: its application to human liver with schistosomiasis.
Collagen chains separated on 5% SDS-acrylamide gels were stained with a 0.1% Sirius F3BA solution in saturated aqueous picric acid. After destaining the gels with methanol: acetic acid: water (30: 7 : 63), they were scanned at 540 nm and the area under each peak was determined. After that, the bands were sliced and the slices incubated overnight with trypsin at 37 degrees C. The color of the slices was eluted completely when the denatured collagen was ingested with trypsin. The absorbance of the color eluted was determined at 540 nm. The results obtained demonstrated that both procedures are reproducible and linear from 10 to at least 120 micrograms of protein. The correlation coefficient between both procedures was greater than 95%. The color is stable and the same end-point is obtained after destaining. In order to test the usefulness of the procedure in the typing of collagens from parenchymatous tissues, liver biomatrix was prepared from normal and cirrhotic human specimens obtained at autopsy. Over 95% of the collagen originally present in each liver was recovered in the corresponding biomatrix . We also showed that over 80-85% of biomatrix collagen could be solubilized with pepsin. These extracts were neutralized to pH 7.0 to inactivate pepsin and were applied onto the acrylamide gels. After electrophoresis and staining with Sirius red the types of collagen were determined by densitometric analysis. Our findings confirmed the results obtained by others using more complex and time consuming methodologies, mainly that type I and type III collagens are present in the normal liver in equal concentrations and that the ratio of I/III is 1. In all the cirrhotic livers investigated, the ratio of type I/type III collagen was greater than 1 due to an increase in type I collagen.
Azo Compounds↗