Amebic liver abscess in rat: morphological evidence of innate immune modulation by the sympathetic nervous system / Absceso hepático amebiano en rata: evidencia morfológica de la modulación de la respuesta inmune innata por el sistema nervioso simpático

All organs of the immune system are innervated and almost all neurotransmitter receptors are present on immune cells. We studied the effects of sympathetic innervation in the development of amebic liver abscess (ALA) in rats. Our results showed that lack of sympathetic innervation promote a decrease in size of ALA. We found scarce amoebas, increased the number of neutrophils and a few collagen fibers surrounding the abscess, meanwhile in control group, we observed abscesses areas with typical necrosis including trophozoites and neutrophils. Macrophages were differentially distributed surrounding abscess area in control and vehicle groups, but equally located in and outside of the abscesses in sympathectomized rat. No significant differences were observed on NK cells in analysed groups. In cytokines quantification studies, we observed down-expression of IFN-g and TNF-a, moreover, we found overexpression of IL-10 in sympathectomized and ALA group. In conclusion, our results suggest that elimination of sympathetic nerve fibers in a model rat of amebic liver abscess induces reduction of the innate immune response and presence of amebas through the liver at seven days post-inoculation.

Todos los órganos del sistema inmune están inervados y casi todos los receptores para neurotransmisores están presentes en las células de la respuesta inmune. Nosotros estudiamos el efecto de la inervación simpática en el desarrollo del Absceso Hepático Amebiano (AHA) en ratas. Nuestros resultados muestran que la inervación simpática promueve una disminución en el tamaño del AHA. Nosotros encontramos áreas fibróticas bien definidas con algunas amibas, mayor número de neutrófilos y pocas fibras de colágena rodeando el área de daño, mientras que en el grupo control, nosotros observamos áreas con necrosis, trofozoítos y pocos neutrófilos en el área fibrótica. Los macrófagos se observaron distribuidos en el área fibrótica en los animales simpatectomizados, mientras que en los controles encontramos a los macrófagos distribuidos en la periferia del absceso. No se encontró diferencia significativa en la distribución y cantidad de células NK. En el estudio de citocinas nosotros observamos una disminución de IFN-g y TNF-a y un incremento de IL-10 en animales simpatectomizados. En conclusión, nuestros resultados sugieren que la eliminación de las fibras del sistema nervioso simpático en el modelo de AHA en rata, reduce la respuesta inmune innata y persisten amebas en el tejido dañados a los 7 días post-inoculación.

Participación del óxido nítrico durante el desarrollo del absceso hepático amebiano / Nitric oxide participation during amoebic liver abscess development

El óxido nítrico participa en funciones fisiológicas y fisiopatológicas, así como en el mecanismo de defensa del sistema inmunológico de mamíferos contra parásitos, virus y bacterias. La Entamoeba histolytica es un parásito protozoario causante de la amebiasis, la cual se caracteriza por el daño intestinal y la formación del absceso hepático amebiano (AHA). El desarrollo del absceso hepático amebiano en el hámster es similar al que desarrolla el humano, mientras que el ratón es resistente a la formación de este absceso, debido a un incremento en la producción de óxido nítrico. A diferencia del ratón, el desarrollo del absceso hepático amebiano en el hámster es debido a un exceso en la producción de óxido nítrico o posiblemente a una mayor susceptibilidad del hámster al daño producido por el óxido nítrico. Por lo tanto, sería importante realizar más estudios para determinar si en el humano, un exceso en la producción de óxido nítrico favorece la formación del absceso hepático amebiano.

Nitric oxide participates in both physiological and pathophysiological functions, and it plays an important role in the mammalian immune system in killing or inhibiting the growth of many pathogens, including parasites, viruses and bacteria. Entamoeba histolytica is a protozoan parasite that causes amoebiasis, which is characterized by intestinal damage and amoebic liver abscess development. The development of amoebic liver abscess in hamsters is similar to that in humans, whereas mice are resistant to amoebic liver abscess development due to an increase in nitric oxide production. Unlike in mice, amoebic liver abscess development in hamsters is due to an excess in nitric oxide production or possibly to a greater susceptibility of the hamster to damage caused by nitric oxide. Therefore, it could be important to elucidate if, in humans, an excess in nitric oxide production favors amoebic liver abscess development.

Inmunología del absceso hepático amebiano / Immunology of the amebic liver abscess

La infección por el parásito Entamoeba histolytica es causa de disentería y absceso hepático amebianos, enfermedades asociadas con alta morbi-mortalidad. En el caso de la amebiosis hepática, el papel de la inmunidad humoral en la protección no ha sido del todo establecido pero tiene importancia en estudios seroepidemiológicos y métodos diagnósticos. La inmunidad celular es importante para el control de la infección y desarrollo de inmunidad protectora luego de amebiosis invasiva. Las técnicas de genética molecular y los nuevos modelos de experimentación han enseñado mucho acerca de la patogénesis de la amebiosis, su entendimiento adecuado junto con el conocimiento de la biología molecular y genética del parásito y los mecanismos inmunes del huésped permitirá no sólo el desarrollo de nuevas opciones diagnósticas y de tratamiento sino también de una vacuna efectiva y segura para eliminar la enfermedad.(AU)

Infection by the parasite Entamoeba histolytica causes amebic dysentery and amebic liver abscess, diseases associated with a high morbidity and mortality. In the case of amebic liver infection, the role of humoral immunity in protection has not been well established but it is important in seroidemiological studies and for diagnostic methods. Cellular immunity is important for the control of infection and the development of protective immunity after an invasive infection. Molecular genetic techniques and new experimental models have increased the knowledge on the pathogenesis of amebiasis. Its adequate understanding, together with the knowledge on the molecular biology and genetics of the parasite and of the host's immune mechanisms will allow the development of new diagnostic and therapeutic tools as well as the development of an effective and safe vaccine for eliminating the disease.(AU)

Importancia de las prostaglandinas en la amibiasis hepática / The importance of prostaglandins in hepatic amebiasis

Las prostaglandinas son importantes mediadores inflamatorios, pero también desempeñan un papel importante como reguladoras de las funciones de los linfocitos y los macrófagos. La inoculación por vía intrahepática o intraportal de trofozoitos viables de Entamoeba histolytica en hámsteres se caracteriza por una rápida respuesta inflamatoria aguda, en la cual los trofozoitos amibianos se ven rodeados sucesivamente por leucocitos polimorfonucleares, linfocitos y macrófagos. La incapacidad de estas células para contrarrestar la invasión amibiana ha sido demostrada en varios estudios. La prostaglandina E2 (PGE2) tiene potentes efectos sobre las células de la respuesta inmune; su participación durante la formación del absceso hepático se reportó recientemente. En este artículo hacemos una revisión de los hallazgos de los últimos años en relación con el estudio de los mediadores bioquímicos de la inflamación durante la infección con E. histolytica, y su posible participación en el establecimiento de la respuesta inmune en el huésped.

Prostaglandins are important mediators of inflammation; they also play a role in the regulation of both lymphocyte and macrophage functions. Hamster's liver lesions resulting from intraportal or intrahepatic inoculation of living Entamoeba histolytica trophozoites are characterized by an acute inflammatory response, where trophozoites are successively surrounded by polymorphonuclear leukocytes, lymphocytes, and macrophages. Incapability of these cells to counteract amebic invasion has been demonstrated in some studies. Prostaglandin E2 (PGE2) has potent effects on immune cells; its participation in amebic liver abscess has been reported recently. This paper presents a review of recent discoveries on biochemical mediators produced during inflammation due to Entamoeba histolytica infection, and their possible role in establishing the host's immune response.

Does the eosinophil have a protective role in amebiasis?

While normal human eosinophils are destroyed in vitro by virulent Entamoeba histolytica, notwhistanding the presence of antibodies and complement, activated eosinophils promptly destroy the parasite although dying also at the end of the process. To study the possible in vivo participation of eosinophils in evasive amebiasis, we compared the induction of experimental amebic abscess of the liver (AAL) in gerbils (Meriones unguiculatus) previously made eosinophilic through Toxocara canis antigen injection and in normal control gerbils. After intraportal inoculation of 105 ameba trophozoites (6 and 24 hr), the ratio of gerbils with AAL, as well as the number and size of the microabscesses was comparable in eosinophilic and control gerbils. However, at 9 hr the number and size of the microabscesses were significantly smaller (p<0.05) in eosinophilic gerbils. On the other hand, the actuarial AAL survival curve up to 45 days post-amebic inoculation was sugnificantly (p<0.05) shifted to the right in controls. These results suggest that antigen-induced eosinophilia may exert a protective effect agaisnt AAL in gerbils.

Correlación Clínico-Inmunológica en Absceso Hepático Amebiano / Clinico-Immunological Correlation in Amebic Liver Abscess

INTRODUCCION: El curso de un Absceso Hepático Amebiano (AHA) con tratamiento adecuado presenta una etapa aguda con niveles altos de IgG, IgM, IgA, IgE y respuesta de hipersensibilidad retardada específica negativa, y una etapa de recuperación IgM, IgE e IgA no detectables y respuesta de hipersensibilidad retardada positiva; la IgG continúa positiva por largos períodos de tiempo. Existen muchas evidencias de que la protección en AHA es mediada por la respuesta inmune celular. En la etapa aguda predomina una respuesta humoral probablemente no protectora y en la fase de recuperación una respuesta celular protectora. En este estudio se pruebe la hipótesis de que la severidad del absceso es directamente proporcional al grado de respuesta inmune humoral. MATERIALES Y METODOS: Se estudiaron 10 pacientes con diagnóstico clínico, ecográfico y serológico compatible con AHA, tratados con metronidazol 750 mg/día vía oral por 15 días. Se determinió el tamaño del absceso, velocidad de sedimentación globular, niveles de bilirrubina, transaminasas, fosfatasa alcalina e IgG. Luego se realizaron análisis de regresión lineal relacionando los niveles de IgG con los indicadores clásicos de severidad en AHA (tamaño del absceso, niveles de transaminasas y bilirrubina). RESULTADOS: Los niveles de IgG estuvieron significativamente asociados con: Tamaño del absceso (P=0.0248), ALT (P=0.033) y bilirrubina indirecta (P=0.05); se observó una tendencia de correlación, aunque sin significancia estadística, con la AST (P=0.1529). El tamaño del absceso se correlacionó directamente con el tiempo de evolución (P=0.018); la AST (R=0.595, P=0.210) y ALT (R=0.761, P=0.135) se correlacionaron inversamente. Las diferencias entre los pacientes crónicos y agudos no fueron estadísticamente significativas para la ALT (P=0.0710) y AST (P=0.213). CONCLUSION: La respuesta humoral parece no tener efecto protector en AHA y los niveles de anticuerpos pudieran ser un buen indicador de severidad

A comparative study of different isolated fractions of Entamoeba histolytica in amoebic liver abscess.

Fifty six patients with amoebic liver ubscess were assessed for antibody production and lymphokine release using different antigenic fractions such as sephadex G-200 eluted fraction-I (F-I), shed of bile salt treated amoebae (SBSTA), amoebic membrane glycoprotein (AMG), amoebic RNA and whole amoebic lysate (WAL). Antibody production in response to WAL, F-I, SBSTA, AMG and RNA was assessed by indirect haemagglutination assay. Five and 53 fold increased titres of IHA was observed with F-I and SBSTA respectively compared to WAL. The difference between mean titres of F-I, SBSTA and WAL was found to be highly significant (P < 0.001). Amoebic RNA did not show pronounced antibody titres. Lymphokine release by T lymphocytes in response to F-I, SBSTA, AMG, RNA and WAL was assessed by leukocyte migration inhibition (LMI) test. A highly increased release of LMIF by RNA, AMG, F-I and SBSTA was observed compared to whole amoebic lysate. The difference between the means of the purified fractions and WAL, to release of LMIF was found to be highly significant (P 0.001). These findings suggest that SBSTA, F-I and AMG fractions might have an important role as a potent antigen in inducing humoral and cell mediated immunity in patients with amoebic liver abscess and these findings also confirm the multiple antigenicity of E. histolytica.

Correlation between antiamoebic IgG and autoreactive anti-IgG in amoebic liver abscess cases.

IgG isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography was used for detection of its own antibodies and correlated with the antiamoebic antibody titres in amoebic liver abscess cases. The mean titres with standard deviation of the self reactive antibodies to serum IgG both ALA cases and healthy controls show a highly significant difference, and antiamoebic antibody titres (IgG) are very much correlated with the autoreactive anti IgG titres in amoebic liver abscess cases. This correlation suggests that as antiamoebic IgG levels reach to its maximum, autoreactive anti IgG are produced to switch off antiamoebic anti IgG production in amoebic liver abscess cases.

Rendimiento diagnóstico de las pruebas inmunológicas en absceso hepático amibiano a través de curvas de características operantes del receptor / Diagnostic performance of immunologic tests for amebic liver abscess using ROC curves

OBJETIVOS. 1)Identificar las pruebas de diagnóstico inmunológico con mayor rendimiento diagnóstico en absceso hepático amibiano. 2)Determinar el punto de corte ideal para dichas pruebas. 3)Identificar el grado de influencia del antígeno utilizado sobre el rendimiento de la prueba. DISEÑO. Encuesta comparativa. UNIDADES DE ESTUDIO. Análisis de 24 artículos publicados en la literatura médica sobre pruebas de diagnóstico inmunológico en absceso hepático amibiano. MEDICIONES. A partir de los artículos se construyeron curvas de características operantes (ROC) derivadas de la aplicación de la prueba a pacientes con absceso hepático amibiano. RESULTADOS. Se identificó una gran variación en el rendimiento diagnóstico entre las diferentes pruebas aún cuando el análisis se centró sobre las investigaciones que abordaban una misma prueba. No fue posible concluir respecto a las pruebas con mayor grado de rendimiento debido a dicha variabilidad. El nivel de corte considerado como clínicamente relevante fue mayor que el tradicionalmente considerado en hemaglutinación indirecta, y concordó con el actualmente aceptado en la prueba de anticuerpos fluorescentes. Manteniendo constantes el espectro de enfermos estudiados y el tipo de prueba, persistió la variabilidad entre las pruebas atribuible al empleo de diferentes antígenos. CONCLUSIONES. Se identificó una amplia variación en el rendimiento diagnóstico de las pruebas analizadas. Las fuentes de variación fueron atribuibles al tipo de prueba, al antígeno utilizado y probablemente al espectro de enfermedad.

Role of pure and biologically active amoebal RNA in assessment of lymphokines: a report of 55 ALA cases.

Amoebic liver abscess cases (55) were assessed for release of lymphokines (LMIF) using pure and biologically active amoebal RNA of axenic Entamoeba histolytica (NIH: 200) obtained with cesium chloride centrifugation. Lymphokines released by T lymphocytes in response to both amoebal RNA and whole amoebic lysate (WAL) were tested by leukocyte migration inhibition test (LMIT) on blood samples from amoebic liver abscess cases. A significant increase was observed in the release of lymphokine and 100% positivity was observed with amoebal RNA compared to whole amoebic extract with a positivity of only 78%. The difference between means leukocyte migration inhibition of the above two with regards to release of lymphokine was highly significant (P less than 0.001). This shows that patients had high degree of leukocyte sensitization to amoebal RNA of E. histolytica compared to whole amoebic lysate. These findings suggest that the amoebal RNA plays an important role as a potent antigen in the elicitation of cell mediated immune responses in amoebic liver abscess cases.

Kinetics of specific IgM in patients of hepatic amoebiasis.

Entamoeba histolytica (EH) specific IgM was measured in 54 patients with diagnosed amoebic liver abscess (ALA), 13 with non-suppurative hepatic amoebiasis (NSHA) and 50 controls. The mean levels of EH specific IgM, estimated by ELISA were significantly raised in patients of invasive amoebiasis (both ALA and NSHA) compared to controls (P less than 0.05). EH specific IgG was also raised in both groups of patients. Follow up of patients with ALA showed a significant decline (P less than 0.05) in the specific IgM levels three months after treatment while the specific IgG antibodies persisted in high titres (1:160). Only four patients of NSHA could be followed up and all showed a decline in specific IgM levels. Raised specific serum IgM seems to be an indicator of active (invasive) amoebiasis.

Autoimmunity in amoebiasis.

Amoebic liver abscess (ALA) and symptomatic intestinal amoebiasis cases were assessed by indirect haemagglutination assay for auto-reactive IgG and IgA class of antibodies in response to healthy human serum IgG and IgA. The present results indicated the presence of autoreactive IgG and IgA class of antibodies in ALA and intestinal amoebiasis respectively.

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.

Colonic immunity in patients and amoebic liver abscess.

Secretory immunoglobulin A (S-IgA), coproantibody titre (antiamoebic) and IgA, IgG, IgM immunocytes in rectal mucosa were studied in 13 patients with amoebic liver abscess (ALA) prior to and 4-6 weeks after completion of antiamoebic therapy. Ten asymptomatic Entamoeba histolytica cyst passers and 17 healthy age and sex matched volunteers served as controls. Fecal S-IgA levels and counts of IgA bearing immunocytes in mucosa were significantly higher in patients with ALA and cyst passers as compared to healthy controls and showed a significant fall after treatment. Fecal antiamoebic antibodies were high in cyst passers and in cases of ALA after treatment. Raised levels of S-IgA and IgA class immunocyte counts probably indicate a local mucosal immune response directed at containing the infection.

Anti-amoebic antibody levels in gastro-intestinal disorders.

Serum anti-amoebic antibody levels were studied in 91 cases having clinical presentation of bowel disorders and 31 cases of control. Anti-amoebic antibody was positive in significant dilutions in 38 cases (31.15%) in total comprising 9 cases out of 22 cases showing presence of Ent histolytica cyst in stool, 13 cases who were positive for ova, parasite and cysts other than Ent histolytica in stool and 16 cases out of 70 cases with negative stool findings. It was positive in 5 cases out of 9 diagnosed cases of amoebic liver abscess. Assessment of immunoglobulin levels in amoebic liver abscess cases revealed high levels of immunoglobulin G and immunoglobulin M.

Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.

Immunogenicity of detergent membrane proteins of Entamoeba histolytica.

The lymphokine release and antibody production were assessed in the peripheral blood of 52 and 48 cases of amoebic liver abscess respectively, by employing detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200). Lymphokine release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was performed by leukocyte migration inhibition test. A highly increased release of LMIF and 100 per cent positivity was observed with DDMP where as the same for whole amoebic extract, was only 73 per cent. The difference between the means of the above two values with regards to release of LMIF, was found to be highly significant (P less than 0.005). Antibodies production in response to both DDMP and whole amoebic lysate was performed by indirect haemagglutination assay on blood samples from amoebic liver abscess cases. A 53 folds increased titres of IHA and cent percent positivity was observed with DDMP compared to WAL. The difference between mean titres of the above two with regards to detection of antibodies, was found to be highly significant (P less than 0.001). This shows that the patients, had high degree of leukocyte sensitization and production of antibodies which will not be assessed simply with WAL. These findings suggest that the shed material might have important role as a potent antigen in elicitation cell mediated and humoral immune response in amoebic liver abscess cases.