Late experimental amebic liver abscess in hamster is inhibited by cyclosporine and N-acetylcysteine.

During early experimental amebic liver abscess in hamsters (EALAH), acute inflammation is primarily responsible for tissue damage. However, during the late stages of this process, the relative contribution to tissue destruction of both parasite factors and host response is unknown. In the present work, the role of the cellular immune response in tissue damage during EALAH is explored by using the immunosuppressor drug cyclosporine A (CsA). CsA treatment inhibits tissue damage after 72 h (but not at 24 h). Also, many well-preserved parasite clusters with minimal or no leukocyte influx and with minimal or no tissue destruction characterize the late stage of the process (7 days). The same results are observed with the immunosuppressor tacrolimus, but not with sirolimus; the latter drug does not cause immunosuppression in hamsters. On the other hand, similar results are observed with the antioxidant and anti-inflammatory N-acetylcysteine, with minimal immunosuppression in hamsters. These results suggest that, as in the early EALAH (24 h), during the late stages of the process (7 days), inflammation is also primarily responsible for tissue damage. However, lysosomal and cationic proteins are responsible for the early lesions, whereas reactive oxygen and nitrogen species are primarily involved in late stages.

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.

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.

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.

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.

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.

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.

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.

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.

Immunohistochemical identification of collagenase in carrageenin granuloma.

The collagenase present in experimental carrageenin granuloma in the guinea pig has been purified to homogeneity in acrylamide gel electrophoresis by a combination of ammonium sulfate salting out and affinity chromatography on Sepharose 4B--collagen-packed columns. The single protein band thus obtained was used as an antigen to obtain a monospecific antibody in heterologous conditions. Several immunodiffusion, immunoaffinity chromatography, and immunoinhibition tests of the antibody against the specific antigen and various possible serum and tissue contaminants suggested that the antibody was specifically directed against the enzyme protein collagenase. Indirect immunohistochemical staining of carrageenin granulomas, samples at different developmental phases with this specific anti-collagenase antibody, revealed that the specific antigenic protein (the enzyme collagenase) is universally present on the extracellular structures at both the collagen-deposition and the collagen-resorption stages. A hypothesis is proposed to account for these findings, namely, that the enzyme collagenase is bound to its substrate (collagen) under both normal and pathological conditions, and that the critical point of control of collagen degradation must be the activation of the collagen-bound enzyme.

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.