Extracellular Cysteine Proteases of Key Intestinal Protozoan Pathogens-Factors Linked to Virulence and Pathogenicity.

Intestinal diseases caused by protistan parasites of the genera Giardia (giardiasis), Entamoeba (amoebiasis), Cryptosporidium (cryptosporidiosis) and Blastocystis (blastocystosis) represent a major burden in human and animal populations worldwide due to the severity of diarrhea and/or inflammation in susceptible hosts. These pathogens interact with epithelial cells, promoting increased paracellular permeability and enterocyte cell death (mainly apoptosis), which precede physiological and immunological disorders. Some cell-surface-anchored and molecules secreted from these parasites function as virulence markers, of which peptide hydrolases, particularly cysteine proteases (CPs), are abundant and have versatile lytic activities. Upon secretion, CPs can affect host tissues and immune responses beyond the site of parasite colonization, thereby increasing the pathogens' virulence. The four intestinal protists considered here are known to secrete predominantly clan A (C1- and C2-type) CPs, some of which have been characterized. CPs of Giardia duodenalis (e.g., Giardipain-1) and Entamoeba histolytica (EhCPs 1-6 and EhCP112) degrade mucin and villin, cause damage to intercellular junction proteins, induce apoptosis in epithelial cells and degrade immunoglobulins, cytokines and defensins. In Cryptosporidium, five Cryptopains are encoded in its genome, but only Cryptopains 4 and 5 are likely secreted. In Blastocystis sp., a legumain-activated CP, called Blastopain-1, and legumain itself have been detected in the extracellular medium, and the former has similar adverse effects on epithelial integrity and enterocyte survival. Due to their different functions, these enzymes could represent novel drug targets. Indeed, some promising results with CP inhibitors, such as vinyl sulfones (K11777 and WRR605), the garlic derivative, allicin, and purified amoebic CPs have been obtained in experimental models, suggesting that these enzymes might be useful drug targets.

What about the Cytoskeletal and Related Proteins of Tapeworms in the Host's Immune Response? An Integrative Overview.

Recent advances have increased our understanding of the molecular machinery in the cytoskeleton of mammalian cells, in contrast to the case of tapeworm parasites, where cytoskeleton remains poorly characterized. The pertinence of a better knowledge of the tapeworm cytoskeleton is linked to the medical importance of these parasitic diseases in humans and animal stock. Moreover, its study could offer new possibilities for the development of more effective anti-parasitic drugs, as well as better strategies for their surveillance, prevention, and control. In the present review, we compile the results of recent experiments on the cytoskeleton of these parasites and analyze how these novel findings might trigger the development of new drugs or the redesign of those currently used in addition to supporting their use as biomarkers in cutting-edge diagnostic tests.

Extracellular vesicles in COVID-19 prognosis, treatment, and vaccination: an update.

The lethality of the COVID 19 pandemic became the trigger for one of the most meteoric races on record in the search for strategies of disease control. Those include development of rapid and sensitive diagnostic methods, therapies to treat severe cases, and development of anti-SARS-CoV-2 vaccines, the latter responsible for the current relative control of the disease. However, the commercially available vaccines are still far from conferring protection against acquiring the infection, so the development of more efficient vaccines that can cut the transmission of the variants of concerns that currently predominate and those that will emerge is a prevailing need. On the other hand, considering that COVID 19 is here to stay, the development of new diagnosis and treatment strategies is also desirable. In this sense, there has recently been a great interest in taking advantage of the benefits offered by extracellular vesicles (EVs), membrane structures of nanoscale size that carry information between cells participating in this manner in many physiological homeostatic and pathological processes. The interest has been focused on the fact that EVs are relatively easy to obtain and manipulate, allowing the design of natural nanocarriers that deliver molecules of interest, as well as the information about the pathogens, which can be exploited for the aforementioned purposes. Studies have shown that infection with SARS-CoV-2 induces the release of EVs from different sources, including platelets, and that their increase in blood, as well as some of their markers, could be used as a prognosis of disease severity. Likewise, EVs from different sources are being used as the ideal carriers for delivering active molecules and drugs to treat the disease, as well as vaccine antigens. In this review, we describe the progress that has been made in these three years of pandemic regarding the use of EVs for diagnosis, treatment, and vaccination against SARS-CoV-2 infection. KEY POINTS: • Covid-19 still requires more effective and specific treatments and vaccines. • The use of extracellular vesicles is emerging as an option with multiple advantages. • Association of EVs with COVID 19 and engineered EVs for its control are presented.

Potent Anti-amoebic Effects of Ibogaine, Voacangine and the Root Bark Alkaloid Fraction of Tabernaemontana arborea.

Plants of Tabernaemontana species have several pharmacological activities including antimicrobial effects. Amoebiasis continues to be a public health problem, with increasing evidence of resistance to metronidazole. In this study, we assessed the effect of the alkaloid fraction of T. arborea root bark and the alkaloids ibogaine and voacangine on the viability and infectivity of Entamoeba histolytica trophozoites. Cultures were exposed to 0.1 - 10 µg/mL for 24, 48 and 72 h, and viability was then determined using a tetrazolium dye reduction assay and type of cellular death analyzed by flow cytometry. Results showed that the alkaloid fraction, but mainly ibogaine and voacangine alkaloids, exhibited potent dose-dependent anti-amoebic activity at 24 h post-exposure (IC50 4.5 and 8.1 µM, respectively), comparable to metronidazole (IC50 6.8 µM). However, the effect decreased after 48 and 72 h of exposure to concentrations below 10 µg/mL, suggesting that the alkaloids probably were catabolized to less active derivatives by the trophozoites. The treatment of trophozoites with the IC50 s for 24 h induced significant morphological changes in the trophozoites, slight increase in granularity, and death by apoptonecrosis. The capacity of T. arborea alkaloids to inhibit the development of amoebic liver abscesses in hamsters was evaluated. Results showed that even when the treatments reduced the number of amoebic trophozoites in tissue sections of livers, they were unable to limit the formation of abscesses, suggesting their rapid processing to inactive metabolites. This work leaves open the possibility of using Tabernaemontana alkaloids as a new alternative for amoebiasis control.

Field study of parasitic contamination of fruits, vegetables and leafy greens in the Ecuadorian Andes.

Background: Raw vegetables have been considered vehicles of enteroparasites. South American countries are among the most important exporters of fresh vegetables, including Ecuador, which has a tropical climate and soils rich in organic matter that allow it to harvest throughout the year for sale to different countries. The aim of the study was to assess the occurrence of the parasitic contamination of fruits, vegetables and leafy greens grown in an agricultural area of the Ecuadorian Andes. Methods: A cross-sectional field study was conducted with snowball sampling on 1,416 samples (516 fruits, 488 vegetables, and 412 leafy greens). Each sample were washed with water, and the resulting solution after removing the vegetables, was subjected to 24-hour sedimentation. The concentrated sediment underwent microscopic analysis. Results: The overall positivity for parasitic contamination was 63.4%, with leafy greens having the highest contamination rate (76.9%) (P<0.0001), surpassing vegetables (67.8%) and fruits (48.4%). Cabbage (100%), onions (84%), and strawberries (60.2%) emerged as the most contaminated within their respective groups. Protozoa were more prevalent (49.6%) than helminths (15.5%) (P<0.0001). Blastocystis sp. (33.5%) ranked highest, followed by Eimeria spp. (26.3%), Entamoeba spp. (10.3%), Giardia spp. (8.3%), Balantidium spp. (6.9%), Cryptosporidium spp. (6.6%), Cyclospora spp. (4.4%), Cystoisospora spp. (0.5%), Strongylida (15.5%), and Ascaris spp. (0.4%). Conclusions: The study reveals that vegetables and fruits for human consumption from this area of the Ecuadorian Andes are highly contaminated with various parasites, constituting a possible source of infection for humans and animals in this area, or in non-endemic areas where these products are marketed. The finding emphasizes the need for strict hygienic measures in agricultural crops, which will be properly achieved through the treatment of soil, manure and water used for cultivation.

Immunomodulatory effect of extracellular vesicles from Entamoeba histolytica trophozoites: Regulation of NETs and respiratory burst during confrontation with human neutrophils.

Parasites release extracellular vesicles (EVs) which, in some cases, modulate the host's immune response contributing to the establishment of the infection. In this work we have isolated and characterized the EVs released by trophozoites of the human protozoan parasite Entamoeba histolytica, the causal agent of amoebiasis, when alone or in coculture with human neutrophils, and determined their effect on neutrophil NETs and ROS production. Nanoparticle tracking analysis showed that amoebic EVs are variable in size, ranging from less than 50 nm to nearly 600 nm in diameter (average of 167 nm), whereas neutrophil EVs are more uniform in size, with an average of 136 nm. In cocultures amoeba:neutrophil (1:100) most EVs are 98 nm in size, which is the typical size of exosomes. EVs from amoebae and neutrophils showed almost equal levels of ROS, which were considerably increased in EVs from cocultures. Uptake of amoebic EVs by neutrophils was demonstrated by fluorescence and resulted in a significant reduction in the oxidative burst and NET release triggered by PMA, ionophore A23187, or the amoebae itself used as stimuli. Interestingly, uptake of EVs from cocultures did not affect ROS production, but instead caused a greater delay in the onset of NETs release and in their quantity. A comparative proteomic analysis between the EVs of amoebae and neutrophils separately vs the cocultures showed a similar distribution of protein categories in the GO analysis, but differences in the expression and abundance of proteins such as the N-acetyl-D-galactosamine (GalNAc) inhibitable surface lectin and calreticulin in amoeba EVs, and various antimicrobial molecules in neutrophil EVs, such as lactoferrin and myeloperoxidase. These results highlight the importance of EVs in the immunomodulatory effects exerted by amoeba on human neutrophils.

Towards the development of an epitope-focused vaccine for SARS-CoV-2.

The rapid spread of COVID-19 on all continents and the mortality induced by SARS-CoV-2 virus, the cause of the pandemic coronavirus disease 2019 (COVID-19) has motivated an unprecedented effort for vaccine development. Inactivated viruses as well as vaccines focused on the partial or total sequence of the Spike protein using different novel platforms such us RNA, DNA, proteins, and non-replicating viral vectors have been developed. The high global need for vaccines, now and in the future, and the emergence of new variants of concern still requires development of accessible vaccines that can be adapted according to the most prevalent variants in the respective regions. Here, we describe the immunogenic properties of a group of theoretically predicted RBD peptides to be used as the first step towards the development of an effective, safe and low-cost epitope-focused vaccine. One of the tested peptides named P5, proved to be safe and immunogenic. Subcutaneous administration of the peptide, formulated with alumina, induced high levels of specific IgG antibodies in mice and hamsters, as well as an increase of IFN-γ expression by CD8+ T cells in C57 and BALB/c mice upon in vitro stimulation with P5. Neutralizing titers of anti-P5 antibodies, however, were disappointingly low, a deficiency that we will attempt to resolve by the inclusion of additional immunogenic epitopes to P5. The safety and immunogenicity data reported in this study support the use of this peptide as a starting point for the design of an epitope restricted vaccine.

An RBD-Based Diagnostic Method Useful for the Surveillance of Protective Immunity against SARS-CoV-2 in the Population.

After more than two years, the COVID-19 pandemic is still ongoing and evolving all over the world; human herd immunity against SARS-CoV-2 increases either by infection or by unprecedented mass vaccination. A substantial change in population immunity is expected to contribute to the control of transmission. It is essential to monitor the extension and duration of the population's immunity to support the decisions of health authorities in each region and country, directed to chart the progressive return to normality. For this purpose, the availability of simple and cheap methods to monitor the levels of relevant antibodies in the population is a widespread necessity. Here, we describe the development of an RBD-based ELISA for the detection of specific antibodies in large numbers of samples. The recombinant expression of an RBD-poly-His fragment was carried out using either bacterial or eukaryotic cells in in vitro culture. After affinity chromatography purification, the performance of both recombinant products was compared by ELISA in similar trials. Our results showed that eukaryotic RBD increased the sensitivity of the assay. Interestingly, our results also support a correlation of the eukaryotic RBD-based ELISA with other assays aimed to test for neutralizing antibodies, which suggests that it provides an indication of protective immunity against SARS-CoV-2.

A class I histone deacetylase is implicated in the encystation of Entamoeba invadens.

Epigenetic mechanisms such as histone acetylation and deacetylation participate in regulation of the genes involved in encystation of Entamoeba invadens. However, the histones and target residues involved, and whether the acetylation and deacetylation of the histones leads to the regulation of gene expression associated with the encystation of this parasite, remain unknown. In this study, we found that E. invadens histone H4 is acetylated in both stages of the parasite and is more highly acetylated during the trophozoite stage than in the cyst. Histone hyperacetylation induced by Trichostatin A negatively affects the encystation of E. invadens, and this inhibition is associated with the downregulation of the expression of genes implicated in the synthesis of chitin, polyamines, gamma-aminobutyric acid pathways and cyst wall proteins, all of which are important in the formation of cysts. Finally, in silico analysis and activity assays suggest that a class I histone deacetylase (EiHDAC3) could be involved in control of the expression of a subset of genes that are important in several pathways during encystation. Therefore, the identification of enzymes that acetylate and/or deacetylate histones that control encystation in E. invadens could be a promising therapeutic target for preventing transmission of other amoebic parasites such as E. histolytica, the causative agent of amoebiasis in humans.

Anti-amoebic Activity of Leaf Extracts and Aporphine Alkaloids Obtained from Annona purpurea.

Annona purpurea has been traditionally used by indigenous and socioeconomically disadvantaged people to treat infectious and parasitic diseases, including amoebiasis. The goal of this study was to assess the effect of a crude methanolic extract, an alkaloid extract, and aporphine alkaloids from leaves of A. purpurea on the viability of Entamoeba histolytica trophozoite cultures and to identify the mechanism of action. Different concentrations of the extracts and alkaloids purpureine (1: ), 3-hydroxyglaucine (2: ), norpurpureine (3: ) glaziovine (4: ), and oxopurpureine (5: ) were added to the cultures, and dead parasites were counted after 24 h using a tetrazolium dye reduction assay and analyzed by flow cytometry. The crude extract did not affect the viability of amoebae, but the alkaloid extract and the derived alkaloid glaziovine (4: ) had important anti-amoebic activity with an IC50 of 33.5 µM compared to that shown by metronidazole (6.8 µM). The treatments induced significant morphological changes in the trophozoites, and most parasites killed by the alkaloid extract were positive for Annexin V, suggesting that apoptosis was the main mechanism of action. In contrast, glaziovine (4: ) induced less apoptosis with more amoebic lysis. This study supports the idea that aporphine alkaloids from A. purpurea, mainly (+)-(R)-glaziovine (4: ), could contribute to the development of new formulations for the treatment of amoebiasis. In addition, X-ray diffraction structural analysis and complete 1H and 13C NMR assignments of (+)-(R)-glaziovine (4: ) were performed and reported for the first time.

The Impact of Lactoferrin on the Growth of Intestinal Inhabitant Bacteria.

Lactoferrin (Lf) is an iron-binding milk glycoprotein that promotes the growth of selected probiotic strains. The effect of Lf on the growth and diversification of intestinal microbiota may have an impact on several issues, including (i) strengthening the permeability of the epithelial cell monolayer, (ii) favoring the microbial antagonism that discourages the colonization and proliferation of enteric pathogens, (iii) enhancing the growth and maturation of cell-monolayer components and gut nerve fibers, and (iv) providing signals to balance the anti- and pro-inflammatory responses resulting in gut homeostasis. Given the beneficial role of probiotics, this contribution aims to review the current properties of bovine and human Lf and their derivatives in in vitro probiotic growth and Lf interplay with microbiota described in the piglet model. By using Lf as a component in pharmacological products, we may enable novel strategies that promote probiotic growth while conferring antimicrobial activity against multidrug-resistant microorganisms that cause life-threatening diseases, especially in neonates.

Lactoferrin and Peptide-derivatives: Antimicrobial Agents with Potential Use in Nonspecific Immunity Modulation.

Lactoferrin (Lf) is a conserved cationic non-heme glycoprotein that is part of the innate immune defense system of mammals. Lf is present in colostrum, milk and mucosal sites, and it is also produced by polymorphonuclear neutrophils and secreted at infection sites. Lf and Lf N-terminus peptide-derivatives named lactoferricins (Lfcins) are molecules with microbiostatic and microbicidal action in a wide array of pathogens. In addition, they display regulatory properties on components of nonspecific immunity, including toll-like receptors, proand anti-inflammatory cytokines, and reactive oxygen species. Mechanisms explaining the ability of Lf and Lfcins to display both up- and down-modulatory properties on cells are not fully understood but result, in part, from their interactions with membrane receptors that elicit biochemical signal pathways, whereas other receptors enable the nuclear translocation of these molecules for the modulation of target genes. The dual role of Lf and Lfcins as antimicrobials and immunomodulators is of biotechnological and pharmaceutical interest. Native Lf and its peptide-derivatives from human and bovine sources, the recombinant versions of the human protein, and their synthetic peptides have potential application as adjunctive agents in therapies to combat infections caused by multi-resistant bacteria and those caused by fungi, protozoa and viruses, as well as in the prevention and reduction of several types of cancer and response to LPS-shock, among other effects. In this review, we summarize the immunomodulatory properties of the unique multifunctional protein Lf and its N-terminus peptides.

New Entamoeba group in howler monkeys (Alouatta spp.) associated with parasites of reptiles.

Our knowledge of the parasite species present in wildlife hosts is incomplete. Protozoans such as amoebae of the genus Entamoeba infect a large variety of vertebrate species, including NHPs. However, traditionally, their identification has been accomplished through microscopic evaluation; therefore, amoeba species have not always been identified correctly. We searched for Entamoeba spp. using a fragment of the small subunit rDNA in free-ranging howler monkeys (Alouatta palliata and A. pigra) from southeast Mexico. One hundred fifty five samples were collected, with 46 from A. palliata and 109 from A. pigra and 8 of the total samples were positive. We detected a new clade of Entamoeba, which was separated from other described species but closer to E. insolita, as well as an unnamed sequence typically found in iguana species with low shared identity values (<90%). We designated this new clade as conditional lineage 8 (CL8) and we have shown that members of this group are not exclusive to reptiles.

Lactoferrin: Balancing Ups and Downs of Inflammation Due to Microbial Infections.

Lactoferrin (Lf) is a glycoprotein of the primary innate immune-defense system of mammals present in milk and other mucosal secretions. This protein of the transferrin family has broad antimicrobial properties by depriving pathogens from iron, or disrupting their plasma membranes through its highly cationic charge. Noteworthy, Lf also exhibits immunomodulatory activities performing up- and down-regulation of innate and adaptive immune cells, contributing to the homeostasis in mucosal surfaces exposed to myriad of microbial agents, such as the gastrointestinal and respiratory tracts. Although the inflammatory process is essential for the control of invasive infectious agents, the development of an exacerbated or chronic inflammation results in tissue damage with life-threatening consequences. In this review, we highlight recent findings in in vitro and in vivo models of the gut, lung, oral cavity, mammary gland, and liver infections that provide experimental evidence supporting the therapeutic role of human and bovine Lf in promoting some parameters of inflammation and protecting against the deleterious effects of bacterial, viral, fungal and protozoan-associated inflammation. Thus, this new knowledge of Lf immunomodulation paves the way to more effective design of treatments that include native or synthetic Lf derivatives, which may be useful to reduce immune-mediated tissue damage in infectious diseases.

Parasiticidal effect of synthetic bovine lactoferrin peptides on the enteric parasite Giardia intestinalis.

Giardia intestinalis is the most common infectious protozoan parasite in children. Despite the effectiveness of some drugs, the disease remains a major worldwide problem. Consequently, the search for new treatments is important for disease eradication. Biological molecules with antimicrobial properties represent a promising alternative to combat pathogens. Bovine lactoferrin (bLF) is a key component of the innate host defense system, and its peptides have exhibited strong antimicrobial activity. Based on these properties, we evaluated the parasiticidal activity of these peptides on G. intestinalis. Trophozoites were incubated with different peptide concentrations for different periods of time, and the growth or viability was determined by carboxyfluorescein-succinimidyl-diacetate-ester (CFDA) and propidium iodide (PI) staining. Endocytosis of peptides was investigated by confocal microscopy, damage was analyzed by transmission and scanning electron microscopy, and the type of programmed cell death was analyzed by flow cytometry. Our results showed that the LF peptides had giardicidal activity. The LF peptides interacted with G. intestinalis and exposure to LF peptides correlated with an increase in the granularity and vacuolization of the cytoplasm. Additionally, the formation of pores, extensive membrane disruption, and programmed cell death was observed in trophozoites treated with LF peptides. Our results demonstrate that LF peptides exhibit potent in vitro antigiardial activity.

Protein profiles of Taenia solium cysts obtained from skeletal muscles and the central nervous system of pigs: Search for tissue-specific proteins.

Taeniasis/cysticercosis caused by the tapeworm Taenia solium is a parasite disease transmitted among humans and pigs, the main intermediate host. The larvae/cysts can lodge in several tissues of the pig, i.e. skeletal muscles and different locations of the central nervous system. The molecular mechanisms associated to tissue preferences of the cysts remain poorly understood. The major public health concern about this zoonosis is due to the human infections by the larval form in the central nervous system, causing a highly pleomorphic and debilitating disease known as neurocysticercosis. This study was aimed to explore the 2DE protein maps of T. solium cysts obtained from skeletal muscles and central nervous system of naturally infected pigs. The gel images were analyzed through a combination of PDQuest™ and multivariate analysis. Results showed that differences in the protein patterns of cysts obtained from both tissues were remarkably discrete. Only 7 protein spots were found specifically associated to the skeletal muscle localization of the cysts; none was found significantly associated to the central nervous system. The use of distinct protein fractions of cysts allowed preliminary identification of several tissue-specific antigenic bands. The implications of these findings are discussed, as well as several strategies directed to achieve the complete characterization of this parasite's proteome, in order to extend our understanding of the molecular mechanisms underlying tissue localization of the cysts and to open avenues for the development of immunological tissue-specific diagnosis of the disease.

The endocrine-immune network during taeniosis by Taenia solium: The role of the pituitary gland.

It is well known that sex hormones play an important role during Taenia solium infection; however, to our knowledge no studies exist concerning the immune response following complete or lobe-specific removal of the pituitary gland during T. solium infection. Thus, the aim of this work was to analyze in hamsters, the effects of lack of pituitary hormones on the duodenal immune response, and their impact on T. solium establishment and development. Thus, in order to achieve this goal, we perform anterior pituitary lobectomy (AL, n = 9), neurointermediate pituitary lobectomy (NIL, n = 9) and total hypophysectomy (HYPOX, n = 8), and related to the gut establishment and growth of T. solium, hematoxylin-eosin staining of duodenal tissue and immunofluorescence of duodenal cytokine expression and compared these results to the control intact (n = 8) and control infected group (n = 8). Our results indicate that 15 days post-infection, HYPOX reduces the number and size of intestinally recovered T. solium adults. Using semiquantitative immunofluorescent laser confocal microscopy, we observed that the mean intensity of duodenal IFN-γ and IL-12 Th1 cytokines was mildly expressed in the infected controls, in contrast with the high level of expression of these cytokines in the NIL infected hamsters. Likewise, the duodenum of HYPOX animals showed an increase in the expression of Th2 cytokines IL-5 and IL-6, when compared to control hamsters. Histological analysis of duodenal mucosa from HYPOX hamsters revealed an exacerbated inflammatory infiltrate located along the lamina propria and related to the presence of the parasite. We conclude that lobe-specific pituitary hormones affect differentially the T. solium development and the gut immune response.

Effect of Clinoptilolite and Sepiolite Nanoclays on Human and Parasitic Highly Phagocytic Cells.

Nanoclays have potential applications in biomedicine raising the need to evaluate their toxicity in in vitro models as a first approach to its biocompatibility. In this study, in vitro toxicity of clinoptilolite and sepiolite nanoclays (NC) was analyzed in highly phagocytic cultures of amoebas and human and mice macrophages. While amebic viability was significantly affected only by sepiolite NC at concentrations higher than 0.1 mg/mL, the effect on macrophage cultures was dependent on the origin of the cells. Macrophages derived from human peripheral blood monocytes were less affected in viability (25% decrease at 48 h), followed by the RAW 264.7 cell line (40%), and finally, macrophages derived from mice bone marrow monocytes (98%). Moreover, the cell line and mice macrophages die mainly by necrosis, whereas human macrophages exhibit increased apoptosis. Cytokine expression analysis in media of sepiolite NC treated cultures showed a proinflammatory profile (INFγ, IL-1α, IL-8, and IL-6), in contrast with clinoptilolite NC that induced lees cytokines with concomitant production of IL-10. The results show that sepiolite NC is more toxic to amoebas and macrophages than clinoptilolite NC, mostly in a time and dose-dependent manner. However, the effect of sepiolite NC was comparable with talc powder suggesting that both NC have low cytotoxicity in vitro.

Protection against Amoebic Liver Abscess in Hamster by Intramuscular Immunization with an Autographa californica Baculovirus Driving the Expression of the Gal-Lectin LC3 Fragment.

In a previous study, we demonstrated that oral immunization using Autographa californica baculovirus driving the expression of the Gal-lectin LC3 fragment (AcNPV-LC3) of Entamoeba histolytica conferred protection against ALA development in hamsters. In this study, we determined the ability of AcNPV-LC3 to protect against ALA by the intramuscular route as well as the liver immune response associated with protection. Results showed that 55% of hamsters IM immunized with AcNPV-LC3 showed sterile protection against ALA, whereas other 20% showed reduction in the size and extent of abscesses, resulting in some protection in 75% of animals compared to the sham control group. Levels of protection showed a linear correlation with the development and intensity of specific antiamoeba cellular and humoral responses, evaluated in serum and spleen of hamsters, respectively. Evaluation of the Th1/Th2 cytokine patterns expressed in the liver of hamsters showed that sterile protection was associated with the production of high levels of IFNγ and IL-4. These results suggest that the baculovirus system is equally efficient by the intramuscular as well as the oral routes for ALA protection and that the Gal-lectin LC3 fragment is a highly protective antigen against hepatic amoebiasis through the local induction of IFNγ and IL-4.

Immunodiagnosis of porcine cysticercosis: identification of candidate antigens through immunoproteomics.

Cysticercosis, caused by the larval stage of Taenia solium, is a zoonotic disease affecting pigs and humans that is endemic to developing countries in Latin America, Africa and South East Asia. The prevalence of infection in pigs, the intermediate host for T. solium, has been used as an indicator for monitoring disease transmission in endemic areas. However, accurate and specific diagnostic tools for porcine cysticercosis remain to be established. Using proteomic approaches and the T. solium genome sequence, seven antigens were identified as specific for porcine cysticercosis, namely, tropomyosin 2, alpha-1 tubulin, beta-tubulin 2, annexin B1, small heat-shock protein, 14-3-3 protein, and cAMP-dependent protein kinase. None of these proteins were cross-reactive when tested with sera from pigs infected with Ascaris spp., Cysticercus tenuicollis and hydatid cysts of Echinococcus spp. or with serum from a Taenia saginata-infected cow. Comparison with orthologues, indicated that the amino acid sequences of annexin B1 and cAMP-dependent protein kinase possessed highly specific regions, which might make them suitable candidates for development of a specific diagnostic assay for porcine cysticercosis.