PubMed Health⌕ Search

Biomedical subjects

I Montfort

Publications and source records attributed to I Montfort.

At least 19 recordsLinked to original sources
PubMed2004

Inflammation, complement, ischemia and amoebic survival in acute experimental amoebic liver abscesses in hamsters.

We have examined the role of inflammatory cells, ischemia and serum complement on the development of acute experimental amoebic liver abscess in hamsters (AEALAH). In hamsters made leukopenic by whole body radiation (800 rad) and daily intraperitoneal glycogen injections, the absence of inflammatory cells and liver tissue damage surrounding the parasites resulted in their rapid (24 h) disappearance from the liver, which showed no lesions. Focal liver ischemia, always present in control AEALAH with inflammation and tissue destruction, was reproduced in radiated hamsters by injection of amoebae mixed with Superdex microspheres, but again in the absence of inflammation, amoebae caused no liver damage and disappeared in 24 h. In hamsters made hypocomplementemic by injection of purified cobra venom factor (CVF), amoebae caused AEALA indistinguishable from controls, but in leukopenic + hypocomplementemic hamsters, amoebae were unable to produce lesions and disappeared from the liver in 48 h. We conclude that inflammation and tissue damage are required for the survival of amoebae in AEALAH and for the progression of the experimental disease.

Acute Disease↗
PubMed2004

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 parasite's virulence.

Animals↗
PubMed2000

Galactose-specific adhesin and cytotoxicity of Entamoeba histolytica.

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.

Animals↗
PubMed1996

Entamoeba histolytica: localization of a 30-kDa cysteine proteinase using a monoclonal antibody.

We produced a monoclonal antibody against a major cysteine proteinase of 30kDa from trophozoites of Entamoeba histolytica strain HM1:IMSS. The specificity of the monoclonal antibody was confirmed by specific inhibition of azocasein digestion and by electrophoretic analysis, in the presence of sodium dodecyl sulfate or on a substrate gel, of the antigen precipitated by the antibody. Immunofluorescent staining of trophozoites with the monoclonal antibody revealed heterogeneity in the intensity of whole cell fluorescence and subcellular localization of the stain. The latter was also observed in trophozoites, which were stained by conventional immunohistochemical methods, from experimental liver abscesses in hamsters. Ultrastructural analysis showed antigen distributed mainly in clear amorphous zones in the cytoplasm, which were not limited by a visible membrane. Proteinases are translocated from these compartments to phagocytic vacuoles after trophozoites ingest erythrocytes, suggesting that these regions might be a lysosomal equivalent of this primitive eukaryotic cell.

Animals↗
PubMed1995

Extraction and partial characterization of a low molecular weight collagenase from normal rat kidney.

A matrix metalloproteinase with selective affinity for collagen was identified and partially purified from normal rat kidney. A two-step purification procedure consisting of gel filtration and affinity chromatography on Sepharose 4B-collagen resulted in an increase in specific activity of more than 3,000 times. The partially purified collagenase cleaved type I collagen and also showed low gelatinolytic activity. The enzyme required Ca2+ and revealed a neutral pH optimum; it was not inhibited by thiol or serineprotease inhibitors. Its activity was fully blocked using recombinant tissue inhibitor of metalloproteinases-1. Using SDS-PAGE and zymography, the estimated Mr of the collagenase was 16.5 x 10(3).

Animals↗
PubMed1994

Purification and immunologic characterization of a 30-kDa cysteine proteinase of Entamoeba histolytica.

A 30-kDa cysteine proteinase was purified from extracts of axenically grown trophozoites of Entamoeba histolytica strain HM1:IMSS. The purification procedure involved two consecutive chromatographic steps. Sequence analysis revealed high similarity with histolysin and with other 27-kDa cysteine proteinase. Western-blot analysis using F(ab')2 fragments of a polyclonal antibody raised against the purified enzyme revealed that when the amebic extract was prepared in the absence of proteinase inhibitors there were many positive bands ranging in relative molecular weight from 115 to 12.5 kDa, but when the extract was prepared in the presence of proteinase inhibitors there was only a single 30-kDa positive band. Similar results were obtained with immunoprecipitates. This phenomenon would suggest the formation of multimer aggregates of the 30-kDa cysteine proteinase after partial proteolysis.

Amino Acid Sequence↗
PubMed1993

Phagocytosis and proteinase activity are not related to pathogenicity of E. histolytica.

To examine the relationship between phagocytosis, proteinase activity and pathogenicity of axenically grown trophozoites of E. histolytica strain HM-1:IMSS four different cultures were used: (1) a culture preserved in our laboratory for over 4 years, which lost its pathogenicity 3 years ago; (2) a culture passaged several times through hamster liver, which lost its pathogenicity recently; (3) a highly virulent culture supplied by another laboratory; and (4) amebas recovered from hamster liver abscesses caused by culture 3. Phagocytosis was measured as erythrophagocytosis. Proteinase activity was determined on azocasein. Pathogenicity was defined as the capacity to cause liver abscesses in hamsters. A negative correlation was found between phagocytic activity and pathogenicity, since amebas unable to cause liver abscesses had the highest phagocytic activity, whereas those recovered from liver abscesses had the lowest phagocytic activity. The percent of phagocytic amebas showed wide variations through a 2-month observation period, with no change in amebic pathogenicity. No correlation was found between the level of proteinase activity and pathogenicity. It is concluded that neither phagocytosis nor proteinase activity is an adequate marker of amebic pathogenicity.

Animals↗
PubMed1993

Role of cysteine proteinases of Entamoeba histolytica on the cytopathogenicity of axenic trophozoites on rat and hamster hepatocytes in vitro.

The role of amebic cysteine proteinase(s) on the in vitro cytotoxic and cytolytic effects of axenic trophozoites of a virulent strain (HM-1) of Entamoeba histolytica has been studied using freshly isolated rat and hamster hepatocytes as target cells. The cytotoxic effect was defined as cell killing without loss of cell structure and the cytolytic effect as cell disintegration. Incubation experiments of axenic trophozoites with rat or hamster liver cells in the presence and absence of bovine serum, of several proteinase inhibitors, and of galactose, galactosamine, and cysteine, at various ameba:liver cell ratios, different temperatures, and for several time periods, suggest that amebic cysteine proteinase is not involved in the cytotoxic effect but is essential for the cytolytic effect. This suggestion is supported by additional observations made with Transwell chambers, which effectively prevent contact between amebas and target cells, and by experiments with a fraction of an extract of lysed amebas that contained most of their cysteine proteinase activity.

Animals↗
PubMed1992

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.

Acute Disease↗
PubMed1992

Phagocytosis and proteinase activity are not related to pathogenicity of Entamoeba histolytica.

To examine the relationship between phagocytosis, proteinase activity and pathogenicity of axenically grown trophozoites of E. histolytica of strain HM-1:IMSS four different cultures were used: 1) a culture preserved in our laboratory for over 4 years, that lost its pathogenicity 3 years ago; 2) a culture passaged several times through hamster liver, that lost its pathogenicity recently; 3) a highly virulent culture supplied by another laboratory; and 4) amebas recovered from hamster liver abscesses caused by the latter culture. Phagocytosis was measured as erythrophagocytosis. Proteinase activity was determined on azocasein. Pathogenicity was defined as the capacity to cause liver abscesses in hamsters. A negative correlation was found between phagocytic activity and pathogenicity, since amebas unable to cause liver abscesses had the highest phagocytic activity whereas those recovered from liver abscesses had the lowest phagocytic activity. The percent of phagocytic amebas increased progressively in all cultures through a 2-month observation period. No correlation was found between the level of proteinase activity and pathogenicity. It is concluded that neither phagocytosis nor proteinase activity are adequate markers of amebic pathogenicity.

Animals↗
PubMed1992

Phenotypic heterogeneity in the expression of a 30 kDa cysteine proteinase in axenic cultures of Entamoeba histolytica.

A polyclonal antibody raised against a purified approximately 30 kDa cysteine proteinase derived from extracts of axenically grown trophozoites of E. histolytica strain HM-1:IMSS was used to stain 72 h cultures of the same amebas by indirect immunofluorescence. Fluorescence was limited to the outer membrane of the parasite and was either uniformly distributed or more condensed on a segment, at times on a single point of the membrane. In relation to the intensity of fluorescence staining, three distinct amebic populations were present: negative, weakly stained and intensely stained. The relative numbers of these three groups remained quite constant for at least one year under the same culture conditions. Flow cytometry was used to quantitate simultaneous variations in amebic size and intensity of fluorescence at various times after different treatments. Amebic size was registered in three levels: small (< 7 microns), medium (7-20 microns), and large (> 20 microns). Staining intensity was measured in arbitrary units. Exposure to 100% fresh hamster serum, phagocytosis of erythrocytes, exposure to cysteine proteinase inhibitors E-64 and cystatin, and to calmodulin antagonist W-7, resulted in various modifications of the phenotype of amebas in very short time periods. We conclude that the expression of the membrane approximately 30 kDa cysteine proteinase in axenic amebic cultures is phenotypically heterogeneous, and that such heterogeneity is modulated and not constitutive.

Animals↗
PubMed1991

Role of leukocytes and amebic proteinases in experimental rat testicular necrosis produced by Entamoeba histolytica.

To investigate the role of amebic proteinases and host leukocytes, we studied amebiasis experimentally in the rat testis. The degree of inflammation and necrosis produced by different strains was correlated with proteinase activity and with zymograms. Intratesticular injection of axenically grown trophozoites of a pathogenic strain (HM-1 of Entamoeba histolytica) produced indistinguishable lesions in normal animals and leukopenic rats (less than 1000 leukocytes/mm3), suggesting that granulocytes do not contribute to the formation of lesions in this model. Testicular lesions produced by five different strains of E. histolytica ranging from highly virulent to almost nonpathogenic were proportional to the proteinase activity of each amebic strain. Inhibition of amebic proteinases in vitro and subsequent injection into the rat testis markedly reduced the inflammatory lesions resulting from highly virulent E. histolytica. The pathogenicity of three other amebae (E. laredo, E. moshkovskii, and E. invadens) was generally proportional to their proteinase activity; however, E. laredo showed high proteinase activity and caused minimal tissue damage. These results suggest that the pathogenic potential of Entamoeba spp. in the rat testis may be related to the type as well as the level of their proteinase activity.

Animals↗
PubMed1991

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.

Acute Disease↗
PubMed1990

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

In 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

[Role of leukocytes and amebic proteases in experimental testicular necrosis produced in the rat by Entamoeba histolytica].

We examined the participation of polymorphonuclear leucocytes and amebic proteinases upon tissue damage by means of an experimental model of acute amebic lesions developed in rat's testicle. In leukopenic rats (less than 1,000 leucocytes/ml) intratesticular injection of axenic E. histolytica's trophozoites (HM-1) produced lesions undistinguishable from the normal controls. On the other hand, inhibition of 80% (average) of the proteinase activity by means of previous incubation of the trophozoites with human a2M gave way to minimal inflammatory lesions almost undistinguishable from the controls which were injected with PBS-A. Our data suggest that in this experimental model of acute amebiasis polymorphonuclear leukocytes do not participate in the tissue damage and that amebic proteinases are responsible for Entamoeba histolytica's virulence.

Animals↗
PubMed1990

[The mechanism of natural resistance in the rat to Entamoeba histolytica].

The data presented in this paper may be summarized as follows: 1) intraportal injection of a virulent strain (HM-1) of Entamoeba histolytica in Wistar rats, of both sexes, gives way to non-progressive microscopic changes, characterized by a rapid leukocytic reaction surrounding the amebas, disappearance of the parasites within 5 hours, and total lack of hepatic damage; 2) leucopenia only modifies the previous description by the fact that there are no leucocytes around the amebas, although these disappear in the same time, showing, at this moment, an early and prominent vacuolar degeneration; 3) hypocomplementaemia shows the same results as leucopenia; 4) fragments and extracts from various tissues from the rat and hamster show variable degrees of interference with the viability of axenic amebas of Entamoeba histolytica conserved in culture; only a minimal part of the interference to be due to the activity of the complement which is present in the tissue extracts.

Animals↗
PubMed1988

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.

Amebiasis↗