Characterization of low molecular weight protein tyrosine phosphatases of Entamoeba histolytica.

Entamoeba histolytica is an intestinal protozoan parasite of humans and is endemic in developing countries. E. histolytica has two low molecular weight protein tyrosine phosphatase (LMW-PTP) genes, EhLMW-PTP1 and EhLMW-PTP2, which are expressed in cultured trophozoites, clinical isolates, and cysts. The amino acid sequences of proteins EhLMW-PTP1 and EhLMW-PTP2 showed only one amino acid difference between them at position A85V, respectively. Both genes are expressed in cultured trophozoites, mainly EhLMW-PTP2, and in trophozoites recovered from amoebic liver abscess, the expression of EhLMW-PTP1 is downregulated. We cloned the two genes and purified the corresponding recombinant (rEhLMW-PTPs) proteins. Antibodies anti-rEhLMW-PTP2 showed that during red blood cells uptake by E. histolytica, the EhLMW-PTPs were found in the phagocytic cups based on analysis of fluorescence signals. On the other hand, rEhLMW-PTPs showed an optimum phosphatase activity at pH 6.0 with p-nitrophenyl phosphate as the substrate. They dephosphorylate phosphotyrosine and 3-O-methylfluorescein phosphate, but not phosphoserine or phosphothreonine, and the enzymatic activity is inhibited by orthovanadate. rEhLMW-PTP1 and rEhLMW-PTP2 exhibited optimum temperatures of activities at 60 °C and 58 °C, respectively, with high thermal stability at 50 °C. Also, the rEhLMW-PTPs showed high specific activities and specific km value with pNPP or OMFP as the substrates at the physiological temperature (37 °C).

Different behavior of myeloperoxidase in two rodent amoebic liver abscess models.

The protozoan Entamoeba histolytica is the etiological agent of amoebiasis, which can spread to the liver and form amoebic liver abscesses. Histological studies conducted with resistant and susceptible models of amoebic liver abscesses (ALAs) have established that neutrophils are the first cells to contact invasive amoebae at the lesion site. Myeloperoxidase is the most abundant enzyme secreted by neutrophils. It uses hydrogen peroxide secreted by the same cells to oxidize chloride ions and produce hypochlorous acid, which is the most efficient microbicidal system of neutrophils. In a previous report, our group demonstrated that myeloperoxidase presents amoebicidal activity in vitro. The aim of the current contribution was to analyze in vivo the role of myeloperoxidase in a susceptible (hamsters) and resistant (Balb/c mice) animal models of ALAs. In liver samples of hamsters and mice inoculated intraportally with Entamoeba histolytica trophozoites, the number of neutrophils in ALAs was determined by enzymatic activity. The presence of myeloperoxidase was observed by staining, and its expression and activity were quantified in situ. A significant difference existed between the two animal models in the number of neutrophils and the expression and activity of myeloperoxidase, which may explain the distinct evolution of amoebic liver abscesses. Hamsters and mice were treated with an MPO inhibitor (4-aminobenzoic acid hydrazide). Hamsters treated with ABAH showed no significant differences in the percentage of lesions or in the percentage of amoebae damaged compared with the untreated hamsters. ABAH treated mice versus untreated mice showed larger abscesses and a decreased percentage of damaged amoebae in these lesion at all stages of evolution. Further studies are needed to elucidate the host and amoebic mechanisms involved in the adequate or inadequate activation and modulation of myeloperoxidase.

Two different chitinase genotypes in a patient with an amebic liver abscess: a case report.

The present work deals with the identification of a patient with two liver abscesses containing two different strains of Entamoeba histolytica, as defined by chitinase gene plymorphisms.

Entamoeba histolytica: inflammatory process during amoebic liver abscess formation involves cyclooxygenase-2 expression in macrophages and trophozoites.

It has been demonstrated that expression of cyclooxygenase-2 (COX-2) isoform is induced by Entamoeba histolytica in macrophages and polymorphonuclear cells during amoebic liver abscess (ALA) formation in hamsters. Trophozoites present in the lesion were also positive for COX-2 signal. However, no cross reactivity of the anti-COX-2 antibody with protein extract of cultivated trophozoites was found. To clarify if trophozoites are involved in PGE(2) production during ALA development, COX-2 expression was detected by in situ hybridization and RT-PCR in liver tissue from intrahepatically infected hamsters. COX-2 mRNA was in polymorphonuclear cells since 4h postinfection, and subsequently, local macrophages expressed COX-2 mRNA in a similar way. Additionally, a positive signal for COX-2 mRNA expression was detected in E. histolytica trophozoites, suggesting that, in vivo, parasite COX expression may be an important mechanism to promote inflammation.

Entamoeba histolytica: induction of cyclooxygenase-2 expression during amoebic liver abscess formation in hamsters (Mesocricetus auratus).

Experimental amoebic liver abscess in hamsters curses with an increase in both, systemic levels of prostaglandin E2 (PGE(2)) and local cyclooxygenase activity in liver microsomes. The cellular source of PGE(2) and the isoform of cyclooxygenase responsible are not completely evidenced. Cyclooxygenase-2 (COX-2) protein and gene expression were demonstrated on macrophages and polymorphonuclear cells as a result of Entamoeba histolytica infection in hamsters at 2, 4, and 7 days postinfection by immunohistochemistry and RT-PCR. E. histolytica trophozoites located in the lesion showed a strong positive signal for COX-2, however the enzyme was not detected in cultured trophozoites by Western blot. Our results indicate that the increment in PGE(2) is the result of COX-2 activity from cells of the reticuloendothelial system and reinforce the possibility that PGE(2) production by enzyme induction in macrophages may be a mechanism by which E. histolytica modulates the host immune response in this parasitic infection.

Antisense inhibition of expression of cysteine proteinases affects Entamoeba histolytica-induced formation of liver abscess in hamsters.

Trophozoites of virulent Entamoeba histolytica transfected with the antisense gene encoding cysteine proteinase 5 (CP5) have only 10% of the CP activity but retain their cytopathic activity on mammalian monolayers. In the present study we found that the transfected trophozoites with low levels of CP activity were incapable of inducing the formation of liver lesions in hamsters.

Role of the Entamoeba histolytica cysteine proteinase in amebic liver abscess formation in severe combined immunodeficient mice.

Evidence from in vitro studies suggest that the Entamoeba histolytica cysteine proteinase plays a role in the tissue lysis and cytopathic effects seen in invasive amebiasis. We used affinity-purified antibodies against a recombinant E. histolytica cysteine proteinase to demonstrate that the proteinase is present extracellularly in amebic liver abscesses in mice with severe combined immunodeficiency (SCID mice). Treatment of E. histolytica trophozoites with specific cysteine proteinase inhibitor E-64 blocked or greatly reduced liver abscess formation at 48 h in SCID mice. Our study suggests an important role for a functional cysteine proteinase in amebic liver abscess formation.

Decreased adenosine deaminase activity in peripheral lymphocytes of patients suffering from amebic liver abscess.

Amebic liver abscess (ALA) is associated with a state of transient suppression of cell-mediated immunity (CMI). T4 helper-inducer cell number is reported to be reduced. However, little is known of the reason for such reduction. Adenosine deaminase (ADA), an enzyme of salvage pathway in purine metabolism, is reported to be essential for the normal growth, differentiation and proliferation of T-lymphoid cells. In a pilot study we estimated the ADA levels in the peripheral lymphocytes of ALA patients. It was observed that the mean enzyme level in patients was 223.98 +/- 51.81 as against 405 +/- 38.12 in controls. The significantly low levels (p < 0.05) of enzyme in patients' lymphocytes may possibly explain the reduction of T4 helper-inducer cells reported in these cases. This parameter, if further evaluated, can serve as a differentiation marker between helper and suppressor T-cell subsets.

The pathogenesis of raised serum alpha-fetoprotein levels in amoebic and pyogenic hepatic abscesses.

Serum alpha-fetoprotein (AFP) concentrations may be slightly raised in patients with amoebic hepatic abscesses. In an attempt to learn more about the pathogenesis of the raised levels, we studied 74 patients with amoebic and six with pyogenic hepatic abscesses. Serum (AFP) levels were slightly elevated (24-72 ng/ml) on admission in four patients and markedly raised (2000 ng/ml) in one, who had hepatocellular carcinoma in addition to a pyogenic hepatic abscess. The pattern of the early rise in AFP levels could not be determined because these four patients were lost to follow-up. However, serial serum AFP estimations were obtained in 29 patients with a normal value on admission and in none of these did the concentration rise during recovery. Our findings do not support the prevailing hypothesis that regenerating hepatocytes are responsible for the raised serum AFP levels in non-neoplastic hepatic disorders, including hepatic abscesses.

Role of lysosomal enzymes in tissue damage during hepatic amoebiasis. An experimental study.

Two key lysosomal enzymes, N-acetyl-beta-glucosaminidase and beta-glucuronidase of Kupffer cells and blood monocytes were studied in guinea pigs infected intramesenterically with E. histolytica. The animals were sacrificed on days 0, 2, 5 and 8 post-infection. The enzyme levels in the cell lysate and supernatants of both Kupffer cells and blood monocytes were significantly higher (P less than 0.01) in the infected animals compared with the controls. This increase was more marked on the 5th and 8th days post infection (P less than 0.001). The alteration in enzyme levels was higher in Kupffer cells than in blood monocytes (P less than 0.05). The enzyme levels increased exponentially with the severity of the hepatic lesions. A direct correlation was observed between the enzyme levels and the severity of the hepatic lesions (P less than 0.01). Hence, the role of lysosomal enzymes, released by activated mononuclear phagocytic cells, in the pathogenesis of hepatic amoebiasis is postulated.

Entamoeba histolytica: effect of growth conditions and bacterial associates on isoenzyme patterns and virulence.

In xenic culture, isolates of Entamoeba histolytica from asymptomatic carriers are characterized, with rare exception, by possession of a nonpathogenic zymodeme. During the process of axenizing such an isolate, strain CDC:0784:4, a change in the pattern of the isoenzymes from nonpathogenic zymodeme I to pathogenic zymodeme II was observed 40 days after the amebae had been transferred from a medium for xenic cultivation to one used for axenic cultivation, but before axenization of the amebae had actually occurred. Axenization was accomplished by feeding the amebae lethally irradiated bacteria while suppressing and finally eradicating with antibiotics the bacterial flora accompanying the amebae in the original xenic culture. The change in zymodeme was accompanied by a change in virulence as evidenced by the ability of the amebae to produce hepatic abscesses in hamsters and to destroy monolayers of tissue culture cells. Two explanations are offered for the observed changes in zymodeme and virulence: a zymodeme is not a stable inherent property of the ameba. Alternatively, the original isolate consisted of two zymodeme populations and the conditions of growth selected for one or the other of the populations. In either case, our results suggest that the finding of a particular zymodeme in a culture of E. histolytica isolated from an asymptomatic carrier of the parasite cannot be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Biochemical homogeneity of Entamoeba histolytica isolates, especially those from liver abscess.

The electrophoretic mobility patterns of four enzymes from trophozoites of Entamoeba histolytica were examined to determine the zymodeme of the amoebae populations involved. E. histolytica populations from aspirated liver pus belonged exclusively to types II and XI of eighteen zymodemes. E. histolytica from cases of amoebic dysentery--i.e., those patients presenting with intestinal ulceration and/or with haematophagous trophozoites in the faeces--also belonged to zymodemes II and XI. However, symptom-free cyst passers harboured E. histolytica belonging to one of several zymodemes none of which corresponded wth the zymodemes of the patients with liver abscesses or amoebic dysentery.

[Histochemical aspects of liver lesions induced in hamsters by inoculation of E. histolytica in axenic culture. II. Evidence of perinecrotic fibrosis]. / Aspectos histoquímicos de la lesión hepática en hamster producida por inoculación de de E. histolytica en cultivo axénico II. Evidencias de fibrosis perinecrótica.

The percutaneous, intrahepatic inoculation of E. histolytica trophozoites in weanling hamsters produced lesions of various shapes and sizes. The time-span between inoculation and sacrifice did not correlate with the size of the lesions. Histoenzymologic technics for acid and alkaline phosphatases, adenosinetriphosphatase (ATPase) and glucose-6-phosphate dehydrogenase (G-6-P DH) showed that the necrotic material is positive to the two former phosphatases whereas ATP-ase shows distorted bile canaliculi due to profound parenchymal alterations and the presence of G-06-P DH activity in parenchyma as well as its lack of activity in necrotic material suggests that the pentose cycle is involved in the genesis of the lesion. The presence of collagen fibers surrounding the lesions suggests that during the expansion of the lesions, there is a granulomatous inflammatory stage with fibrosis immediately followed by fibrinolysis. Four zones can be distinguished in an expanding lesion: necrosis, a zone of junction between necrosis and parenchyma which depicts a crown of invasive trophozoites surrounding the necrosis, a histiocytic halo and a zone of peripheral necrosis.