CCR9+ T cells contribute to the resolution of the inflammatory response in a mouse model of intestinal amoebiasis.

Analysis of the mechanisms underlying the inflammatory response in amoebiasis is important to understand the immunopathology of the disease. Mucosal associated effector and regulatory T cells may play a role in regulating the inflammatory immune response associated to Entamoeba histolytica infection in the colon. A subpopulation of regulatory T cells has recently been identified and is characterized by the expression of the chemokine receptor CCR9. In this report, we used CCR9 deficient (CCR9(-/-)) mice to investigate the role of the CCR9(+) T cells in a murine model of E. histolytica intestinal infection. Intracecal infection of CCR9(+/+), CCR9(+/-) and CCR9(-/-) mice with E. histolytica trophozoites, revealed striking differences in the development and nature of the intestinal inflammatory response observed between these strains. While CCR9(+/+) and CCR9(+/-) mice were resistant to the infection and resolved the pathogen-induced inflammatory response, CCR9(-/-) mice developed a chronic inflammatory response, which was associated with over-expression of the cytokines IFN-γ, TNF-α, IL-4, IL-6 and IL-17, while IL-10 was not present. In addition, increased levels of CCL11, CCL20 and CCL28 chemokines were detected by qRT-PCR in CCR9(-/-) mice. E. histolytica trophozoites were identified in the lumen of the cecum of CCR9(-/-) mice at seven days post infection (pi), whereas in CCR9(+/+) mice trophozoites disappeared by day 1 pi. Interestingly, the inflammation observed in CCR9(-/-) mice, was associated with a delayed recruitment of CD4(+)CD25(+)FoxP3(+) T cells to the cecal epithelium and lamina propria, suggesting that this population may play a role in the early regulation of the inflammatory response against E. histolytica, likely through IL-10 production. In support of these data, CCR9(+) T cells were also identified in colon tissue sections obtained from patients with amoebic colitis. Our data suggest that a population of CCR9(+)CD4(+)CD25(+)FoxP3(+) T cells may participate in the control and resolution of the inflammatory immune response to E. histolytica infection.

Polymorphonuclear neutrophil infiltration intensity as consequence of Entamoeba histolytica density in amebic colitis.

BACKGROUND: It has been suggested that the damage observed in invasive amebiasis is related to interactions between polymorphonuclear leukocytes (PMN) and Entamoeba histolytica trophozoites. We analyzed the relation between infiltrating inflammatory cell populations and E. histolytica density in intestinal amebic lesions. METHODS: Biopsies obtained endoscopically from patients with amebic colitis were analyzed to describe their morphologic abnormalities. Cellular populations and E. histolytica trophozoites were measured quantitatively in order to assess the correlation between infiltrating inflammatory cell populations and parasite density. RESULTS: Amebic lesions were most often located in the colon (55%). The histopathologic diagnoses were colitis in 31%, erosive colitis in 26%, and ulcerated colitis in 24%. The predominant cellular populations found in the lesions were lymphocytes (59.8%) (3,672 +/- 2,413/mm(2)) followed by PMN (17%) (1,038 +/- 1,171 PMN/mm(2)) (p < 0.01). A higher density of PMN infiltration was observed in severe cases. Cellular populations predictive of the presence of E. histolytica trophozoites (p = 0.047) were PMN (p = 0.019) and lymphocytes (p = 0.033). The highest association was found between E. histolytica trophozoites and PMN (p = 0.0221). CONCLUSIONS: Neutrophils and lymphocytes, particularly the former, are associated significantly with the density of parasites. Our findings support the theory that PMN interaction with E. histolytica contributes to the pathogenesis of amebic intestinal lesions.

Entamoeba histolytica and Entamoeba dispar: differentiation by Enzyme-Linked Immunosorbet Assay (ELISA) and its clinical correlation in pediatric patients

Studies were carried out at a mexican pediatric hospital to determine the ratio between the pathogenic species Entamoeba histolytica and non-pathogenic species E. dispar using an enzyme-linked immunosorbent assay (ELISA) to detect the lectin (1 galactose N-acetyl D-galactosamine) of E. histolytica in feces. A close correlation was noted between the presence of the E. histolytica lectin and clinical symptoms. In the study, amebas were detected by microscopy in 120 children (either E. histolytica or E. dispar). But while almost all (13/14) of the children with E. histolytica had clinical symptoms, dysentery-feces with mocus and blood, diarrhea, cramping abdominal pain, tenesmus rectal, flatulence, vomiting and headache, almost none (1/106) of the children infected with the non-pathogenic ameba E. dispar had signs and symptoms. This suggests that much of the amebiasis diagnoses made in Mexico are, in fact, due to non-pathogenic E. dispar.

Differentiation of entamoeba histolytica/entamoeba dispar by PCR and their correlation with humoral and cellular immunity in individuals with clinical variants of amoebiasis.

To correlate a particular state of immunity with Entamoeba spp., we used colorimetric PCR to differentiate E. histolytica from E. dispar in individuals with amoebiasis and to associate its presence with the clinical profile, including humoral and cellular immune responses to E. histolytica. Our results showed high levels of antibody in acute amoebiasis and elevation of IL-4 production, a cytokine related to Th2 profile, associated with E. histolytica. In chronic amoebiasis, even with anti-E. histolytica seropositivity, intestinal symptoms were associated with E. dispar in all the cases, without differences in level of antibodies, BTI, CD4+/CD8+ ratio, INF-gamma, and IL-4. Among asymptomatic carriers, E. dispar was more frequently found; however, identification of E. histolytica in two asymptomatic carriers associated with high levels of INF-gamma, a cytokine related to Th1 profile, demonstrate the importance of making specific diagnosis of Entamoeba spp., to establish the clinical and epidemiological behavior in both intestinal and extra-intestinal amoebiasis.

Entamoeba histolytica: acute granulomatous intestinal lesions in normal and neutrophil-depleted mice.

To study the role of neutrophils in the innate resistance to Entamoeba histolytica intestinal infection in mice, animals were treated with anti-neutrophil monoclonal antibodies prior to intracecal parasite inoculation and the resulting lesions were compared with normal mice that had been equally infected. In contrast to our previous finding that neutrophils are critical in eliminating E. histolytica infection in the liver, we show here that neutrophils are not absolutely required to eliminate E. histolytica infection from the intestine. Although the neutrophils are not critical for resolution of the E. histolytica infection, neutrophils do appear to provide some measure of protection as the intestinal amoeba burden was higher at early timepoints after infection in the neutropenic animals. In addition, we found that while both the normal and the neutrophil-depleted mice developed ulcerative lesions in the colon, the neutropenic mice had an increased frequency of granulomas that formed around the amoeba. Thus, our findings appear to be the first evidence showing that granulomatous inflammation can occur after intestinal infection in mice using axenically cultured amoeba.

[Diagnosis of intestinal amebiasis using coproscopic and immunological methods in a population sample in greater metropolitan Belém, Pará, Brazil]. / Diagnóstico de amebíase intestinal utilizando métodos coproscópicos e imunológicos em amostra da população da área metropolitana de Belém, Pará, Brasil.

We compare diagnostic methods for Entamoeba histolytica in fecal samples from the city of Belém, Pará, Brazil. We analyze stool samples from children and adults (Group I); stool and serum samples from adults (Group II); and stool samples from children (Group III). In groups I and III, we used direct examination with lugol (DM), Faust et al (FM), and ELISA (detection of E. histolytica anti-GIAP coproantigen) and in group II, DM, iron hematoxylin staining (IHS), FM, ELISA, and the indirect immunofluorescence test (IFAT) for detection of IgG antibodies. Positivity was 10.50% by DM plus FM and 28.99% by ELISA. There was no correlation between positivity and age group. In Group II (n = 87), the positive rate was 4.59% by DM plus FM, 8.04% by IHS, 4.59% by IFAT, and 21.83% by ELISA. The ELISA test was the most sensitive for all groups. IFAT alone is still not a useful tool for diagnosis of E. histolytica infection. The ELISA test is simple, performed in one-third of cases used for IHS and IFAT, and greatly improves quality of diagnosis. We recommend this as the method of choice for diagnosis of suspected E. histolytica infection.

Increased frequency of HLA-DR3 and complotype SCO1 in Mexican mestizo children with amoebic abscess of the liver.

The increase of HLA-DR3 and complotype SCO1 previously found in Mexican mestizo adults with E. histolytica amoebic abscess of the liver, was also found in Mexican mestizo children of either sex with the same disease, when compared to the healthy control population (adults and/or children) of the same ethnic and socioeconomic background. This HLA and complotype pattern was not found in Mexican Mestizo patients with amoebic rectocolitis. No linkage disequilibrium was found between these and the other MHC determinants tested in this survey. Thus, HLA-DR3 and SCO1 may constitute primary, independent risk factors, not for any kind of amoebic tissue invasion (i.e. amoebic rectocolitis), but specifically for amoebic liver abscess, irrespective of age or sex. The possibility of linkage disequilibrium with other factors (i.e. the TNF family) within or close to the MHC that were not tested in this study, is discussed. Children with amoebic liver abscess revealed a significant increase in HLA-DR5, and the absence of HLA-DR6 when compared to adults with amoebic liver abscess, suggesting that at least in this ethnic group these class II HLA traits may contribute to some of the peculiarities of pediatric amoebic liver abscess as opposed to the adult version of this disease. HLA-DR3, SCO1, but also HLA-DR5 and HLA-DR6 have all been associated with certain forms of immune-dysfunction, and may thus contribute to some of the clinical and immunological features of this parasitic disease.

Ausência de amebíase invasiva em aidéticos homossexuais masculinos, no Recife / Absence of invasive amoebiasis in male homosexual AIDS patients in Recife

The purpose of this paper was to determine if among male homosexual AIDS [correction of SIDA] patients, the Entamoeba histolytica was able to product invasive illness. From the 77 investigated patients only one (1.3 per cent) has cysts of E. histolytica in his stools. The electrophoresis of the only isolated strain has shown it was from zimodeme I, non pathogenic. The research of antiamebic antibodies was negative in the serum of the totality of patients. Those results showed that even in immunocompromised patients with AIDS [correction of SIDA], E. histolytica strain found in Recife was not able to produce disease. The utilization of metronidazol is not indicated in amoebic cysts carriers.

[The absence of invasive amebiasis in male homosexual AIDS patients in Recife]. / Ausência de amebíase invasiva em aidéticos homossexuais masculinos, no Recife.

The purpose of this paper was to determine if among male homosexual AIDS [correction of SIDA] patients, the Entamoeba histolytica was able to product invasive illness. From the 77 investigated patients only one (1.3%) has cysts of E. histolytica in his stools. The electrophoresis of the only isolated strain has shown it was from zimodeme I, non pathogenic. The research of antiamebic antibodies was negative in the serum of the totality of patients. Those results showed that even in immunocompromised patients with AIDS [correction of SIDA], E. histolytica strain found in Recife was not able to produce disease. The utilization of metronidazol is not indicated in amoebic cysts carriers.

Amibiasis, aspectos de interés para el internista / Amebiasis. interesting aspects for the internist

La amibiasis es una enfermedad parasitaria común, de distribución mundial, que tiene gran impacto en la salud. Entamoeba histolytica es el protozario causante de este padecimiento. Puede vivir en forma comensal en el intestino humano, pero en ocasiones invade la mucosa intestinal y otros tejidos, principalmente hígado y pulmón. La infección depende de factores provenientes del parásito y del huésped y se encuentra estrechamente relacionada con el nivel socioeconómico de las comunidades. A pesar de que la amibiasis responde al tratamiento, en ocasiones puede ser letal; es por ello muy importante contar con un diagnóstico oportuno y un tratamiento eficaz. Para lograrlo se han estudiado ampliamente los diferentes aspectos de esta enfermedad, así como la biología de Entamoeba histolytica. El objetivo de esta revisión es proporcionar un panorama generalizado y actualizado sobre los aspectos de esta parasitosis

Identification of immunogenic epitopes of the 170-kDa subunit adhesin of Entamoeba histolytica in patients with invasive amebiasis.

Entamoeba histolytica causes amebic dysentery (AD) and liver abscess (ALA). Little is known about protective immunity to amebiasis, and studies in this area have been complicated by the paucity of defined ameba antigens. We examined the proliferative responses of peripheral blood mononuclear cells (PBMC) from patients with AD and ALA to a recombinant protein containing a portion of the 170 kDa adhesin of E. histolytica (170CR), and to two synthetic peptides (1 and 2) derived from the 170 kDa sequence that were predicted to contain T cell epitopes. A significant number of patients with AD and ALA had PBMC that proliferated to 170CR molecule, and several individuals with ALA and AD had T cells that recognized one or both peptides. Contrarily, individuals from a non-endemic region for amebiasis did not respond to 170CR protein, or to both peptides. In regard to antibody response, nine of fifteen patients with ALA showed antibodies to 170CR protein. These same patients had antibodies to peptide 2. We identified peptides from 170-kDa adhesin that may contain both T and B cell epitopes recognized by some patients with invasive amebiasis. These peptides may be valuable reagents in studies of the immune response to amebiasis.

[In vivo experimental models of amebiasis]. / Los modelos experimentales in vivo en la amibiasis.

In vivo experimental models for amebiasis have provided important information about the mechanisms of host-parasite interaction which determine the production of disease. In the laboratory, several species of rodents have been used to study the intestinal and hepatic amebiasis. For the former, the model of "washed-closed cecal loop" in guinea-pig and hamster has been useful to study the early invasive events. In gerbils we also produced early intestinal lesions by intracecal inoculation with monoxenically cultured amebas. Hamsters and gerbils have been used as susceptible animals for hepatic amebiasis, and rats and guinea-pigs as resistant animals. Morphological analysis of hepatic lesions of susceptible animals showed the role of host inflammatory cells in the process of liver damage. The resistance in the production of liver abscess in rats and guinea-pigs is due in part to the action of polymorphonuclear leukocytes in the rat and the macrophages in the guinea pigs. Complete characterization and standardization of the various models of amebiasis in rodents constitute the bases for other important biomedical studies aimed to the disease control.

Seroepidemiología de la amebiasis / Seroepidemiological study of amebiasis

Por medio de la técnica de ELISA se estudiaron 35 sueros de personas del distrito de Sapillica (Piura) y 36 sueros de estudiantes de la Joya (Arequipa) encontrándose que el 46 por ciento de los sueros de Sapillica y el 72 por ciento de los sueros de La Joya eran positivos para anticuerpos contra Entamoeba histolytica.

IgA reactivity in patients with intestinal or hepatic amebiasis.

The antigens of E. histolytica recognized by IgA antibodies in sera from patients with either amebic liver abscess or intestinal amebiasis, as well as saliva from the latter, were determined in immunoblots of whole native trophozoite proteins. Results show that sera of patients with amebic liver abscess reacted mainly with amebic proteins of more than 200 kDa, whereas those of individuals with intestinal amebiasis recognized a protein of 145 kDa. The saliva of the latter detected a protein of 210 kDa which is not always seen when using the sera of patients with intestinal or hepatic amebiasis.

Amebiasis in Nicaragua: class specific serum antibody responses.

With the aid of the indirect hemagglutination (IHA) test and IgG ELISA the antibody profile against E. histolytica in León, Nicaragua was investigated in 562 sera from individuals belonging to various age groups. The highest reactivity was invariably recorded in the age group 6-15 years where 48% were seropositive. Several sera reactive by either one of IHA and IgG ELISA were negative by the other test. The main reason for this seems to be reactivity in different Ig classes. Treatment with 2-mercaptoethanol reduced the titre level in 63 of the 66 sera tested. Immunofluorescence using an anti-IgM conjugate showed that 26 of 43 sera contained specific IgM-antibodies, indicating that also unspecific reactions are involved in the IHA test. A comparison was made between class-specific reactivity in three population groups: healthy residents, healthy cyst carriers and patients with recent or acute liver abscess. No significant difference in the prevalence of reactions above the diagnostic significance level was recorded between cyst carriers and healthy residents. However, among the cyst carriers 33% had IgA and/or IgM antibodies but no demonstrable specific IgG. Most patients with recent and all with acute liver abscess reacted significantly above the diagnostic limit in all three tests.

Antibodies against Entamoeba histolytica in individuals with intestinal amoebiasis presenting cysts and/or trophozoites in the feces

Amostras de soros foram obtidas de 154 individuos comprovadamente parasitados pela Entamoeba histolytica (78 sintomaticos e 76 assintomaticos). Doze apresentavam trofozoitas nas fezes, enquanto 142 tinham apenas cistos. Os soros foram utilizados para testar a ocorrencia de anticorpos anti-Entamoeba histolytica, empregando-se para tal, Reacao de Hemaglutinacao Indireta (HAI), Reacao de Imunofluorescencia Indireta (RIFI), Reacao de Fixacao de Complemento (RFC) e Contraimunoeletroforese (CIEP). Entre os soros dos individuos com trofozoitas em suas fezes, 75,0 por cento foram positivos para HAI e RIFI, 83,3 por cento por RFC, e 41,7 por cento por CIEP. Nos individuos que tinham apenas cistos, resultados positivos pelos mesmos testes foram respectivamente, 5,6 por cento; 12,0 por cento; 19,0 por cento e 5,6 por cento. A diferenca em relacao aos titulos de anticorpos detectados atraves de HAI, RIFI, RFC e CIEP e em relacao a presenca de trofozoitas ou cistos nas fezes foi significativa para as quatro reacoes imunologicas, quando 'X IND. 2' foi empregado (P<0,05).

Antibodies against Entamoeba histolytica in individuals with intestinal amoebiasis presenting cysts and/or trophozoites in the feces.

Serum samples were obtained from 154 individuals infected with Entamoeba histolytica (78 symptomatic and 76 asymptomatic). Twelve had trophozoites in the feces whereas 142 had only cysts. The sera were used to test the existence of antibodies anti-Entamoeba histolytica employing the Indirect Hemagglutination (IHA), Indirect Immunofluorescence (IFAT), Complement Fixation Reaction (CFR) and Counterimmunoelectrophoresis (CIEP). For those individuals with trophozoites in their feces, 75.0 were positive by IHA and IFAT, 83.0 by CFR and 41.7 by CIEP. In individuals who had only cysts, positive results by the same tests were respectively, 5.6%, 12.0%, 19.0% and 5.6%. The difference in relation to the titers of antibodies detected through IHA, IFAT, CFR and CIEP and in relation to the presence of trophozoites or cysts in the feces was significative for four immunological reactions when chi 2, was employed (P < 0.05).