In Vitro and In Vivo Cysticidal Effects of Carica Papaya Cell Suspensions.

Parasitic diseases are a major public health problem worldwide. Plant-derived products appear to be ideal candidates from a biotechnological perspective, being sustainable and environmentally friendly. The antiparasitic properties of Carica papaya have been attributed to some of its components, including papain and other compounds that are concentrated in the latex and seeds. This study demonstrated in vitro a high and insignificantly different cysticidal activity of soluble extract that was obtained after the disruption of nontransformed wild-type (WT) cells as well as transformed papaya calluses (PC-9, PC-12, and PC-23) and papaya cell suspensions (CS-9, CS-12, and CS-23). In vivo, cell suspensions of CS-WT and CS-23 that had been previously lyophilized were tested with respect to their cysticidal effects, compared with those of three commercial antiparasitic drugs. CS-WT and CS-23 together reduced the number of cysticerci, the number of buds, and the percentage of calcified cysticerci in a similar extent to albendazole and niclosamide, whereas ivermectin was less effective. Mice were then orally immunized with CS-23 that expressed the anti-cysticercal KETc7 antigen (10 µg/mouse), CS-WT (10 mg/mouse), or both together to evaluate their preventive properties. CS-23 and CS-WT significantly reduced the expected parasite and increased the percentage of calcified cysticerci as well as recovery, being more effective when employed together. The results reported in this study support the feasibility of the development of an anti-cysticercosis vaccine from cells of C. papaya in in vitro cultures, as they are a source of an anthelmintic, natural, and reproducible product.

Immunogenic and antigenic analysis of recombinant NSP1 and NSP11 of PRRS virus.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus in the order Nidovirales, family Arteriviridae, genus Betaarterivirus. Antibodies against nonstructural proteins (NSPs) from this virus can be found in pigs starting 4 days postinfection and they remain detectable for several months. OBJECTIVE: The goal of this study was to evaluate the immunogenicity and antigenic properties of recombinant proteins NSP1 and NSP11 expressed in Escherichia coli cells, as well as to assess the neutralization activity that they elicit. METHODS: We obtained the complete ORF-1 genes coding for NSP1 and NSP11 from PRRSV using the VR-2332 strain. Cloning was performed with the pET23a(+) vector with a histidine tag (His6), linearized by restriction enzyme digestion; the expression of the NSP1 and NSP11 clones was induced in OverExpress C41(DE3) chemically competent cells. Recombinant proteins were used to generate hyperimmune sera and we perform serological assays to confirm neutralizing antibodies. RESULTS: The expressed recombinant NSP1 and NSP11 were found to be immunogenic when injected in pigs, as well as demonstrated higher specificity in recognition of antigen in field sera from pigs positive infected with PRRSV. Furthermore, both NSP1 and NSP11 recombinant proteins elicited PRRSV neutralizing antibodies. CONCLUSIONS: In this study, we demonstrated the immune humoral response to NSP 1 and NSP11, and neutralizing-antibody response to PRRSV VR2332 strain in sera from hyperimmunized pigs.

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.

Role of porcine serum haptoglobin in the host-parasite relationship of Taenia solium cysticercosis.

Human and porcine cysticercosis is a parasitic disease caused by the larval stage (cysts) of the tapeworm Taenia solium. Cysts may live in several host tissues such as skeletal muscle or brain. We have previously described the presence of host haptoglobin (Hp) and hemoglobin (Hb) in different protein extracts of the T. solium cysts. Here, we report the binding of host Hp and Hb to a number of cyst proteins, evaluated through measuring electrophoretic and light absorbance changes. In the sera obtained from 18 cysticercotic pigs, Hp-Hb complexes were abundant, whereas free Hp was undetectable. In contrast, in the sera from non 18 cysticercotic pigs, Hp-Hb and free Hp were found. In the soluble protein fraction of cysts tissue, free Hp was detected showing a considerable Hb-binding ability, whereas in the vesicular fluid, Hp is mainly bound to Hb. Interestingly, assays carried out with the insoluble fraction of T. solium cysts tissue, showed binding of Hp and Hp-Hb in a saturable way, suggesting the existence of specific interactions. Our results suggested that the parasite can take advantage of the uptaken host Hp and Hb, either free or in complexes, as a source of iron or as a way to modulate the inflammatory response surrounding the T. solium cysts.

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.

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis.

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity.

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity. .

Isolation and characterization of goat ovarian aromatase cDNA: assessment of the activity using an intact cell system and placental expression.

Goat ovarian follicles produce estrone and estradiol from androgens. The synthesis of C18 estrogens from C19 androgens requires cytochrome P450 aromatase, but little information about this key enzyme is available in the goat. We report here for the first time the cDNA sequence of the goat ovarian aromatase, the activity of the enzyme in a cell system, and its expression in the term goat placenta. A cDNA library from goat ovarian poly(A)+ RNA was constructed. Human aromatase cDNA was selected as probe to screen the library; several clones were isolated, but none was complete. The longest clone was 3.1 kb long, but it lacked the sequence coding for a few amino acids in the NH(2)-terminal. To obtain the missing sequence, we performed reverse amplification of the cDNA end (RACE). Sequence analysis indicated that goat aromatase possessed a very long 3'-untranslated region ( approximately 1790 bp), and a polyadenylation signal (AATAAA) located at position 3320 downstream from the ATG start codon. The coding region of goat cDNA was inserted in an expression vector and transfected into HEK-293 cells that were cultured in presence of [14C]-androstenedione, steroids extracted and further separated by TLC. The transfected cells efficiently transformed [14C]-androstenedione into estrone. This activity was inhibited by 4-hydroxyandrostenedione. We also investigated the presence of mRNA for P450 aromatase in the goat placenta, using reverse transcription-polymerase chain reaction (RT-PCR) and primers derived from the cDNA ovarian sequence and confirmed the expression of the mRNA in term placenta.