Helminth Infections: Recognition and Modulation of the Immune Response by Innate Immune Cells

The survival of helminths in the host over long periods of time is the result of a process of adaptation or dynamic co-evolution between the host and the parasite. However, infection with helminth parasites causes damage to the host tissues producing the release of danger signals that induce the recruitment of various cells, including innate immune cells such as macrophages (Mo), dendritic cells (DCs), eosinophils, basophils, and mast cells. In this scenario, these cells are able to secrete soluble factors, which orchestrate immune effector mechanisms that depend on the different niches these parasites inhabit. Here, we focus on recent advances in the knowledge of excretory-secretory products (ESP), resulting from helminth recognition by DCs and Mo. Phagocytes and other cells types such as innate lymphocyte T cells 2 (ILC2), when activated by ESP, participate in an intricate cytokine network to generate innate and adaptive Th2 responses. In this review, we also discuss the mechanisms of innate immune cell-induced parasite killing and the tissue repair necessary to assure helminth survival over long periods of time.Fil: Motran, Claudia Cristina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Silvane, Leonardo Micael. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Chiapello, Laura Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Theumer, Martín Gustavo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Ambrosio, Laura Fernanda. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Volpini, Ximena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Celias, Daiana Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Cervi, Laura Alejandra. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

The Phospholipase B Response in Rats Infected with Fasciola Hepatica and Demonstration of the Enzyme-Parasitic Interaction

Fasciola hepatica was studied in outbred rats to determine of infection would cause an increase phospholipase B activity and the percentage of periperal blood eosinophilis. A second experiment was performed to investigate eosinophilis

Immune responses to Fasciola hepatica infection, and Fasciola hepatica derived antigens

The aim of this study was to investigate immune responses as a result of infection with the parasitic helminth, Fasciola hepatica. Analysis of IL-4 and Interferon-y cytokines described a predominant type 2 immune response in BALB/c mice infected with metacercana of F hepatica Levels of IL-4 mRNA assessed by reverse transcnption-polymerase chain reaction (RT-PCR) provide the first evidence that the immune response becomes polarised 1 day post infection. We also investigated immune responses to F hepatica derived antigens IL-4 and Interferon-y cytokine production revealed a polarised type 2 response m BALB/c mice immunised with F hepatica excretory/secretory (ES) products. As type 1 immune responses have been associated with protection against infection with F hepatica (Mulcahy et a l , 1998), we established a polarised type 1 immune response in BALB/c mice by immunising with cathepsm L in combination with various adjuvants

Fasciola hepatica: isolation and characterisation of a cathepsin L proteinase

Fasciola hepatica, a parasitic trematode, is the causative agent of liver fluke disease. It has been shown previously, that both the migratory and adult worm stage of the parasite secrete multiple cysteine proteinases when they are cultured overnight (Dalton & Heffernan, 1989). In this study, one of these proteinases has been purified by standard chromatographic techniques. The purified enzyme was characterised as a cathepsin L-like proteinase using synthetic substrates, inhibition studies, N-terminal sequencing and immunolocalisation studies. This is the first cathepsin L-like proteinase to be identified in a parasitic trematode. This cathepsin L-like proteinase is capable of cleaving immunoglobulin molecules, and is able to protect newly excysted juveniles from destruction by immune-effector cells when it is included in an eosinophil adherence assay. Antibodies to the purified proteinase are able to neutralise its proteolytic activity in vitro. A partial gene fragment encoding the cathepsin L-like proteinase has been obtained using PCR and subcloning techniques. The cathepsin L-like proteinase is present in all stages of F. hepatica and, hence, is considered an ideal target molecule at which to design a vaccine and/or drug, for use in the control of this agriculturally important parasitic disease

Eosinophil and Tissue-invasive Parasitic Helminth

Eosinophils are primarily tissue resident cells, and play important roles in host’s immune responses and maintenance of chronic infection during infection with tissue-invasive parasitic helminth. Such parasite secretes particular molecules to evade eosinophil-mediated helminthotoxicity. Continuous competition between eosinophil and parasite leads to stable equilibria between them. Recent evidence provides a concept that not only eosinophils contribute to parasite’s survival but also parasite modulates host’s immune response. Therefore, it is important to know complex interrelationship between eosinophil and parasite to understand how gently parasite talk to eosinophils and how carefully eosinophils listen to parasite’s voice. In this regard, this review examin papers about eosinophil-mediated tissue inflammatory responses in response to helminthic parasiteope

Evaluación del daño hepático y respuesta inmunitaria local en cabras vacunadas con Catepsina L1 e infectadas con Fasciola Hepatica

El objetivo del presente estudio ha sido estudiar la eficacia protectora, así como las lesiones hepáticas y la respuesta inmunitaria inmunitaria local en cabras inmunizadas con catepsina L1 recombinante (CL1) de F. hepática con dos adyuvantes (Montanide Isa 70 VG y Quil A), con el segundo adyuvante se estudiaron las lesiones y respuesta inmunitaria en fases tempranas y tardías de la enfermedad. Para alcanzar los objetivos propuestos se realizaron dos experiencias, en la Experiencia 1 se utilizaron 21 cabras, hembras, de raza Florida Sevillana de 4 meses de edad que fueron divididas en tres grupos: los grupos 1 y 2 correspondieron al grupo adyuvante (n=8) (Montanide) y grupo inmunizado con CL1 en Montanide (n=8), respectivamente; el grupo 3 (n=5) correspondió al grupo control negativo (no infectado y no inmunizado). Sobre los grupos 1 y 2 se realizaron tres inmunizaciones separadas entre ellas por un periodo de 3 semanas. Posteriormente, en la 10ª semana desde la primera inmunización, los animales fueron infectados con 200 metacercarias viables de F. hepatica procedentes de la Universidad de Bristol (Reino Unido), y finalmente sacrificados 15 semanas después de la infección (15 spi) para estudiar el número de parásitos, eliminación de huevos, lesiones hepáticas y respuesta inmunitaria en fases tardías. En la Experiencia 2 se utilizaron 27 cabras, machos, de raza Malagueña de 4 meses de edad que fueron dividas también en 3 grupos: los grupos 4 y 5 correspondieron al grupo adyuvante (n=10) (Quil A) y grupo inmunizado con CL1 utilizando Quil A como adyuvante (n=10) respectivamente; el grupo 6 (n=7) correspondió al grupo control negativo (no infectado y no inmunizado). En los grupos 4 y 5 se realizaron dos inmunizaciones separadas por 4 semanas y posteriormente se infectaron en la semana 10 con una dosis de 100 metacercarias de F. hepatica de origen ovino (Ridgeway Research Ltd., UK). Además, en este ensayo tres animales de cada grupo fueron sacrificados por inyección intravenosa de tiobarbital a los 7, 8 y 9 días después de la infección (dpi) para estudiar los cambios durante las primeras etapas de la misma. Las cabras restantes (7 animales en los grupos 4 y 5; 4 animales en el grupo 6) fueron eutanasiadas 15 semanas después de la infección (15 spi). Las dos experiencias fueron aprobadas por el Comité de bioética de la Universidad de Córdoba para experimentación animal, referencia N. 7119. El estudio parasitológico no mostró diferencias significativas entre los grupos vacunados y controles adyuvantes en ambas experiencias, tanto en el número de parásitos recuperados como en la tasa de implantación. El análisis estadístico llevado a cabo respecto a la longitud y anchura de los parásitos adultos tampoco mostró diferencias significativas entre los grupos de ambas experiencias, exceptuando el ancho observado en los animales pertenecientes a la experiencia 1 donde esta medida si fue significativamente menor en el grupo vacunado. Respecto a la ganancia de peso no hubo diferencias entre grupo vacunado y control adyuvante en la experiencia 1, mientras que en la experiencia 2 sí hubo una ganancia de peso significativamente mayor en el grupo vacunado respecto al control de adyuvante. El estudio anatomopatológico macroscópico en las fases tardías de ambas experiencias mostró lesiones típicas de la infección por F. hepatica y coinciden de forma genérica con las observadas por otros autores en ovinos y caprinos. Si bien los animales de ambas experiencias mostraron lesiones similares, éstas fueron más severas en los animales de la experiencia 1 respecto a los de la experiencia 2 como cabría esperar debido a la mayor dosis de infección y mayor número de parásitos recuperados. Respecto a los nódulos linfáticos hepáticos en ambas experiencias se observó un aumento significativo en el tamaño y el peso de los grupos infectados respecto al control no infectado...The aim of this study was to investigate the protective efficacy as well as liver damage and local immune response in goats immunized with recombinant cathepsin L1 (CL1) of Fasciola hepatica with two adjuvants (Montanide ISA 70 VG and Quil A). Hepatic damage and local immune response in early and late stages of the infection were studied in the second adjuvant trial. To achieve these objectives two experiments were conducted, in the first one 21 females, Florida Sevillana, 4-months of age goats were divided into three groups: groups 1 and 2 correspond to the adjuvant group (n = 8 ) (Montanide) and CL1 in Montanide (n = 8), respectively; Group 3 (n = 5) corresponded to the negative control group (not infected and not immunized). On groups 1 and 2 three immunizations were administered subcutaneously 3 weeks apart. Subsequently, at week 10 from the first immunization, the animals were infected with 200 F. hepatica metacercariae (University of Bristol,UK), and finally animals were sacrificed 15 weeks post infection (15 wpi) to study the number of parasites, egg output, liver damage and local immune response. In second trial 27 male, Malagueña, 4-months of age goats were also divided into 3 groups: Groups 4 and 5 corresponded to the adjuvant group (n = 10) (Quil A) and immunized group CL1 (n=10), respectively; Group 6 (n = 7) corresponded to the negative control group (not infected and not immunized). In groups 4 and 5 two subcutaneous immunizations were administered 4 weeks apart, and 10 weeks after first immunization animals were orally infected with a dose of 100 F. hepatica metacercariae of ovine origin (Ridgeway Research Ltd., UK). Moreover, in this trial three animals from each group were sacrificed by intravenous injection of thiobarbital at 7, 8 and 9 days after infection (dpi) to study liver damage and local immune response in the early stages of infection. The remaining goats (7 animals in groups 4 and 5; 4 animals in Group 6) were euthanized 15 weeks after infection (15 wpi). The two experiments were approved by the Bioethics Committee of the University of Cordoba to animal experiments, reference N. 7119. Parasitological study showed no significant difference between vaccinated groups and adjuvants groups in the two experiences, for both the number of parasites recovered as the implantation rate. The statistical analysis carried out with respect to the length and width of the adult parasites also showed no significant differences between vaccinated and adjuvant controls, except the fluke width which was lower in the vaccinated group respect to the adjuvant group. Animal weight gain did not differ between adjuvant and vaccinated group in vaccine trial 1, while significantly greater in the vaccinated group compared to the adjuvant control group the second vaccine trial The macroscopic pathologic examination at 15 wpi in both experiences showed lesions typical of chronic fasciolosis such as whitish tortuous scars over the liver surface, mainly involving the left lobe, enlarged bile ducts and gallbladder. The severity of gross hepatic lesions was higher in animals from the first vaccine trial which was correleated with the higher dose administered and higher number of parasite recovered. In the two vaccine trial the severity of gross hepatic lesions was similar in vaccinated and their adjuvant control groups. Regarding the hepatic lymph nodes in both experiences a significant increase in the size and weight of the infected compared to uninfected control groups, were noted. Liver microscopic lesions observed at 15 wpi of both experiences were similar to those described by other authors in chronic infections of F. hepatica. Comparison between vaccinated and adjuvant control groups showed no significant differences, except for the increased number of eosinophils and globular leukocytes in the first trial CL1 vaccinated group, while the Montanide group showed higher number of granulomas..

Expression of stage-specific Fasciola proteases and their evaluation in vaccination trials

The liver flukes Fasciola hepatica and F. gigantica cause infectious disease in ruminants and humans. The geographical range of these two parasite species (temperate and tropical respectively) ensures that infection can occur worldwide. Although anthelmintic treatment is effective against disease, emerging drug resistant strains leads to the development of a vaccine. However, despite several decades of research, there is no commercial vaccine available. The main challenge at present is to produce recombinant proteins in an immunologically active form using recombinant DNA technology. This is an essential step in Fasciola vaccine production. Cysteine proteases are probably the most important facilitators of virulence in flukes and are produced by all stages of the fluke life-cycle. Two classes of cysteine protease are found in the excretory and secretory material of liver flukes- these are cathepsin L and cathepsin B. As such, the major aims of this thesis were to investigate the expression and purification of Fasciola recombinant cysteine proteins, and characterisation by SDS-PAGE and immunoblotting using monoclonal and polyclonal antibodies. These studies demonstrate the production of functionally active cathepsin proteins in S. cerevisiae BJ3505 cells which will lead to vaccine candidate analysis. The second aim of this thesis was to determine the protective efficacy of stage specific target antigens against experimental infection. In addressing this issue, the protective efficacy of single and multivalent recombinant protein vaccinations of adult stage F. hepatica cathepsin L5, immature F. gigantica cathepsin L1g and juvenile F. hepatica cathepsin B were analysed in Sprague Dawley rats against F. hepatica infection. This study demonstrates that juvenile fluke target antigen-cathepsin B induces better immune protection than adult fluke antigen-cathepsin L5. Cocktails of juvenile and adult stage fluke recombinant proteins (cathepsin B and L5) elicited the highest protective immunity against experimental infection and this combination showed not only reduction in fluke recovery and size of flukes, but also marked diminution in the intensity of liver lesions in vaccinated rats. In order to assess the immunogenic property of an early infective stage fluke secreting cysteine protease as a vaccine candidate, DNA vaccination vectors encoding cathepsin B were analysed in BALB/c mice. In this study, the ability of four DNA vaccination strategies such as secretory, chemokine-activating, lymph node targeting vectors encoding cathepsin B were assessed by antibody titre, antibody avidity, western blotting and ELIPSOT assay. The results have further validated the immunoprophylactic potential of a cathepsin B vaccine against F. hepatica. In this study, we have expressed and attained high yields of F. gigantica cathepsin L1g from E. coli BL21, and compared this to a yeast-expressed system. This protease was over-expressed and formed insoluble inclusion bodies that were subsequently solubilised with urea or guanidine hydrochloride. In order to purify the urea-solubilised protein, step-wise urea gradient chromatography was used. For refolding of solubilised protein, a dilution and dialysis procedure was utilised. Proteolytic activity was confirmed by gelatin SDS-PAGE analysis. In conclusion, the determination of the immune potential of recombinant stage specific antigens allows the development of effective vaccines against Fasciola infection

The Phospholipase B Response in Mice Infected With Fasciola Hepatica and Histochemical Demonstration of the Enzyme Source

The phospholipase B activity was assayed in the small intestines, spleen and liver/bile duct of nonsensitized and sensitized mice infected with Fasciola hepatica. The primary infection resulted in a significant increase in phospholipase B activity in the small intestine, spleen and liver/bile duct over that of uninfected control animals. The response to the challenge infection was characterized by an earlier increase in enzyme activity with values significantly above those found for the primary infection in the same tissues. These data demonstrate that one response of mice to infection with F. hepatica is characterized by an increased phospholipase B activity. Thus, the enzyme response is not unique to cestode and nematode infections, but also is part of or associated with the inflammatory mechanisms against trematode infections. In addition, phospholipase B was demonstrated in leukocytes using electron microscopic histochemical techniques. Leukocytes were harvested from peritoneal exudates of mice. Cells were fixed in 4% calcium-forol fixative for 30 minutes at roan temperature for electron microscopy, after which they were incubated at 370C in medium at pH 6.6 containing 2 aM lysolecithin and Cal2 . The fatty acids released during the hydrolytic reaction were trapped as a calcium precipitate and were converted to a lead precipitate for electron microscopy by treatment with lead nitrate. The reaction products were observed to be present in eosinophils and absent in neutrophils, lymphocytes and macrophages. It is concluded that the eosinophilic leukocyte is the carrier cell for phospholipase B in inflanmatory reactions