Irmgard Montfort

Collagenolytic activity in experimental cirrhosis of the liver.

Collagenolytic activity has been measured in insoluble sediments of normal and CCl4-induced cirrhotic rat liver. Two techniques were used: in one assay, the insoluble liver sediments were incubated with a radioactively labeled exogenous substrate; in the other assay, the endogenous collagen present in the insoluble liver sediments served as the substrate. Animals in various stages of development of CCl4-induced cirrhosis were used. Results suggest that in rat liver cirrhosis collagenolytic activity assayed with both techniques appears to increase when expressed as weight of collagen degraded in 24 hr. On the other hand, when the data are corrected for the variable amounts of collagen present in the insoluble liver sediments and expressed as weight of collagen degraded in 24 hr per unit weight of total collagen incubated, collagenolytic activity is shown to be significantly decreased below the normal level and to remain so for prolonged periods.

Collagenase of hepatocytes and sinusoidal liver cells in the reversibility of experimental cirrhosis of the liver

SummaryIn order to explore the cellular source(s) and the behaviour of the collagenolytic activity previously described in rat liver homogenates, in the reversibility of experimental cirrhosis of the liver, enriched suspensions of hepatocytes and of sinusoidal liver cells were obtained by a procedure which employs low EDTA concentrations and no bacterial collagenase. Cell suspensions were prepared from three different groups of animals: 1) normal controls, 2) rats with CCl4-induced cirrhosis of the liver, and 3) rats with swine serum-induced cirrhosis of the liver. Animals were sacrificed in each group upon completion of treatment and also after 3, 6 and 12 months. In each liver wet weight and collagen concentration were determined, and collagenolytic activity of both enriched cell suspensions was measured separately. In addition, histological studies of liver tissue and ultrastructural examination of cell suspensions were performed by standard procedures. Enriched suspensions of both normal hepatocytes and sinusoidal liver cells display Ca2+-dependent collagenolytic activities. Both cell suspensions obtained from each of the two types of cirrhotic livers show normal or slightly increased average levels of collagenase activity at the time of treatment discontinuation, when average liver collagen content ranges from 6 to 10-fold over normal, suggesting that the normal collagenase/collagen ratio is disturbed and that collagenolytic activity is deeply decreased in relation to the actual liver collagen load. In addition, and despite wide individual variations, our data suggest that through a 12-month involution period of both types of experimental cirrhosis, there is an inversely proportional relation between liver collagen concentration and the collagenase/collagen ratio of both hepatocytes and sinusoidal liver cells, suggesting that the decrease in liver collagen is related to the increase in both collagenases. Morphologic and biochemical involution of fibrosis in both models of experimental liver cirrhosis was striking, although in the CCl4-treated group the parenchymal nodular pattern was still quite apparent at the end of the experiment.

Catalytic classes of proteinases of Entamoeba histolytica.

Endopeptidase inhibitors were used to determine the catalytic classes of proteinases present in extracts of Entamoeba histolytica (strain HM 1:IMSS) axenically grown in vitro. Cysteine proteinases account for most of the proteolytic activity; one or more proteinases with different catalytic mechanisms are also present but could not be unambiguously assigned to a particular catalytic class. Proteinases in amebic lysates were resolved by polyacrylamide gel electrophoresis with sodium dodecyl sulfate. The detergent was exchanged with Triton X-100 and the proteolytic activity in the gels was demonstrated by overlaying it on another gel containing the substrate. Four lysis zones were observed corresponding to molecular weights of 66,000, 56,000, 40,000 and 27,000. The first cannot be classified yet, but the last three showed properties consistent with those of cysteine proteinases. Finally, a novel technique is described which uses purified human alpha-2-macroglobulin to trap, purify and characterize proteases from amebic lysates. The results obtained with this technique confirm those of the overlay technique, since both methods reveal four distinct proteinases in the two different amebic preparations examined.

Entamoeba histolytica : Oxygen resistance and virulence

Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O(2). The only difference observed was a higher in vitro susceptibility of nvEh to O(2). The molecular mechanism of that difference was analyzed in both groups of amoebae after high O(2) exposure. vEh O(2) resistance correlated with: (i) higher O(2) reduction (O(2)(-) and H(2)O(2) production); (ii) increased H(2)O(2) resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O(2) exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O(2) and H(2)O(2) as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.

Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica

Abstract We examined the molecular mechanisms of the cytotoxicity of Entamoeba histolytica, using the loss of transepithelial electrical resistance (TER) of monolayers of Madin-Darby canine-kidney (MDCK) cells on their incubation with axenic trophozoites of the HM1-IMSS strain. Such loss of TER occurs very early (in 2–5 min) and is caused by the opening of tight junctions and the detachment of cells. We used specific inhibitors for three of the four molecules currently accepted as being responsible for cytotoxicity: galactose-specific adhesin(s), phospholipase A, and cysteine proteinases. We also used inhibitors of calcium channels. Axenic trophozoites of E. histolytica strain HM1-IMSS were preincubated with the different inhibitors for 1 h prior to their coincubation with MDCK-cell monolayers. The only inhibitor that effectively blocked the loss of TER caused by the parasite was galactose. We suggest that in this experimental model, galactose-specific adhesin(s) are essential for amebic cytotoxicity.

Ischemia in experimental acute amebic liver abscess in hamsters

In experimental acute amebic liver abscess, produced in hamsters by the intraportal inoculation of 1 x 10(6) axenic trophozoites of Entamoeba histolytica strain HM-1, we examined the blood perfusion of the lesions 5, 10, 24 and 72 h after injection of the parasites. India ink introduced into the portal circulation filled all liver vessels but was systematically excluded from even the earlier amebic lesions. The absence of serum proteinase inhibitors from the lesions may allow the participation of amebic proteinases in the causation of tissue necrosis.

Cysteine proteinase activity is required for survival of the parasite in experimental acute amoebic liver abscesses in hamsters.

Axenic trophozoites of Entamoeba histolytica strain HM1-IMSS grown in vitro in the presence of E-64, a potent irreversible inhibitor of cysteine proteinases, preserved their viability, their rate of replication, their resistance to complement, their haemolytic capacity and their ability to destroy target cells, despite complete inhibition of total cysteine proteinase activity. On the other hand, their erythrophagocytic capacity and their ability to decrease TER of MDCK cells was partially decreased. The same trophozoites injected into the portal vein of hamsters receiving a maintaining dose of E-64 failed to cause tissue damage and were rapidly eliminated. Our results suggest that amoebic cysteine proteinase activity is not required for amoebic functions in in vitro conditions, but that it becomes necessary for survival of trophozoites in in vivo conditions, whatever other role (if any) it may play in the parasites virulence.

Entamoeba histolytica: role of amebic proteinases and polymorphonuclear leukocytes in acute experimental amebiasis in the rat.

The injection of 1 x 10(6) trophozoites of axenically grown Entamoeba histolytica strain HM-1 in the subcutaneous tissue of the rat results in an acute and self-limited inflammatory process, characterized by the early onset of conspicuous tissue necrosis and focal hemorrhage in the vicinity of the parasites, followed by infiltration with polymorphonuclear leukocytes. The process develops for 5-10 hr but during that period amebic trophozoites progressively disappear, leukocytes undergo degenerative changes, and the lesion tends to heal in 72-96 hr. In leukopenic animals (less than 1000 white blood cells/ml) tissue necrosis and hemorrhage are equally conspicuous in the neighborhood of amebas. Inhibition of amebic proteinase activity prior to injection by heat denaturation, p-hydroxy-mercuri-benzoate (PHMB), soybean trypsin inhibitor (STI), and human alpha-2-macroglobulin (alpha 2M), alone or in various combinations, results in absence or notorious decrease in tissue necrosis as well as in clearly diminished inflammatory reaction. This effect is particularly evident when cysteine proteinases are either specifically or generally inhibited. On the other hand, amebic proteinase inhibition with alpha 2M and STI does not interfere with the cell-killing capacity of trophozoites co-incubated in vitro for 2 hr with rat peritoneal cells enriched for macrophages. We conclude that in acute experimental amebiasis produced in the subcutaneous tissue of the rat, amebic cysteine (and perhaps other) proteinases are primarily responsible for necrosis and are also important, but not essential, for inflammation. We also suggest that in this model polymorphonuclear leukocytes are not required for tissue necrosis. Finally, in an in vitro model, the cell-killing capacity of amebas is not influenced by the proteinase activity of the parasite.

The Distribution of Collagenase in the Rat Uterus During Postpartum Involution: An Immunohistochemical Study

A monospecific rabbit anti-rat uterus collagenase antibody has been prepared and used to study the distribution of the enzyme in the rat uterus during postpartum involution. Cryostat sections of rat uteri from 24 to 240 hours postpartum were stained by the indirect immunofluorescent method. Nonpregnant rat uterus revelaed positive staining in basement membranes, in endometrial stroma, in perimuscular and in vascular connective tissue. During postpartum involution of the uterus two types of changes in uterine collagenase were observed: (1) variations in the distrubiton of the enzyme, which became selectively localized in the epithelial basement membrane and in the wall of small blood vessels, and (2) variations in the overall intensity of fluorescent staining, which decreased immediately after delivery and slowly increased back to nonpregnant levels in 5 days. The significance of these findings is discussed in relation to the mechanisms of control of collagenase activity in vivo.

Pathogenesis of acute experimental amebic liver abscess in hamsters

The hypothesis was tested that tissue necrosis in acute experimental amebic liver abscess in hamsters is not caused directly by the parasite but rather, indirectly, by the destruction of closely surrounding leukocytes that release their lysosomal enzymes and damage neighboring liver cells. Axenically grown trophozoites of Entamoeba histolytica strain HM-1 were injected intraportally into normal, hypocomplementemic, and leukopenic hamsters, and the lesions were studied histologically 2, 5, 10, and 24 hr after injection. Hypocomplementemia (less than 5% of normal CH50) and leukopenia (less than 1,000 leukocytes/ml) were achieved and sustained for up to 72 hr with repeated intraperitoneal injections of goat anti-hamster C3 and anti-hamster leukocyte antibodies, respectively. Decrease or absence of polymorphonuclear leukocytes in the vicinity of intrahepatic amebas effectively blocked deleterious effects of the parasites on surrounding hepatocytes. We conclude that in acute experimental amebic liver abscess the direct effect of the parasites on hepatocytes and their stroma is not responsible for tissue necrosis, but rather it is due to their indirect action through the destruction of inflammatory cells.