Tarin-Loaded Nanoliposomes Activate Apoptosis and Autophagy and Inhibit the Migration of Human Mammary Adenocarcinoma Cells.

Background: Tarin, a lectin purified from Colocasia esculenta, promotes in vitro and in vivo immunomodulatory effects allied to promising anticancer and antimetastatic effects against human adenocarcinoma mammary cells. This makes this 47 kDa-protein a natural candidate against human breast cancer, a leading cause of death among women. Tarin encapsulated in pegylated nanoliposomes displays increased effectiveness in controlling the proliferation of a mammary adenocarcinoma lineage comprising MDA-MB-231 cells. Methods: The mechanisms enrolled in anticancer and antimetastatic responses were investigated by treating MDA-MB-231 cells with nano-encapsulated tarin at 72 µg/mL for up to 48h through flow cytometry and transmission electron microscopy (TEM). The safety of nano-encapsulated tarin towards healthy tissue was also assessed by the resazurin viability assay, and the effect of nanoencapsulated tarin on cell migration was evaluated by scratch assays. Results: Ultrastructural analyses of MDA-MB-231 cells exposed to nanoencapsulated tarin revealed the accumulation of autophagosomes and damaged organelles, compatible with autophagy-dependent cell death. On the other hand, the flow cytometry investigation detected the increased occurrence of acidic vacuolar organelles, a late autophagosome trait, along with the enhanced presence of apoptotic cells, activated caspase-3/7, and cell cycle arrest at G0/G1. No deleterious effects were observed in healthy fibroblast cells following tarin nanoencapsulated exposition, in contrast to reduced viability in cells exposed to free tarin. The migration of MDA-MB-231 cells was inhibited by nano-encapsulated tarin, with delayed movement by 24 h compared to free tarin. Conclusion: The nanoliposome formulation delivers tarin in a delayed and sustained manner, as evidenced by the belated and potent antitumoral and anti-migration effects on adenocarcinoma cells, with no toxicity to healthy cells. Although further investigations are required to fully understand antitumorigenic tarin mechanisms, the activation of both apoptotic and autophagic machineries along with the caspase-3/7 pathway, and cell cycle arrest may comprise a part of these mechanisms.

Do Fetal Microchimeric Cells Influence Experimental Autoimmune Myocarditis?

Objective: We investigated the presence and influence of fetal microchimerism in the cardiac tissue of mated female mice submitted to experimental autoimmune myocarditis. Materials and methods: Nulliparous BALB/c females and BALB/c females mated with either BALB/c males (syngeneic mating) or C57BL/6 males (allogeneic mating) were immunized with cardiac myosin peptide MyHC-α614-629 or kept as non-immunized controls. Immunization occurred 6-8 weeks after delivery and mice were assessed after 21 days. Results: Immunized mice of allogeneic mating had a lower production of anti-MyHC-α614-629 antibodies compared to immunized nulliparous mice. Immunized nulliparous females had an intense mononuclear inflammatory infiltrate in cardiac tissue, associated with fibroplasia, while mated females had a lower inflammatory reaction. An increase in the frequency of microchimeric fetal cells was observed in mice submitted to allogeneic mating following immunization. Conclusion: Allogeneic cells of fetal origin could contribute to mitigating the inflammatory response in experimental myocarditis.

Taro Lectin Can Act as a Cytokine-Mimetic Compound, Stimulating Myeloid and T Lymphocyte Lineages and Protecting Progenitors in Murine Bone Marrow.

Taro (Colocasia esculenta) corm is traditionally consumed as a medicinal plant to stimulate immune responses and restore a health status. Tarin, a taro lectin, is considered responsible for the immunomodulatory effects of taro. In the present study, in order to investigate the effects of tarin on bone marrow hematopoietic population, murine cells were stimulated with tarin combined with a highly enriched conditioned medium containing either IL-3 or GM-CSF. Cells challenged with tarin proliferated in a dose-dependent manner, evidenced by the increase in cell density and number of clusters and colonies. Tarin exhibited a cytokine-mimetic effect similar to IL-3 and GM-CSF, increasing granulocytic cell lineage percentages, demonstrated by an increase in the relative percentage of Gr-1+ cells. Tarin does not increase lymphocytic lineages, but phenotyping revealed that the relative percentage of CD3+ cells was increased with a concomitant decrease in CD19+ and IL-7Rα+ cells. Most bone marrow cells were stained with tarin-FITC, indicating non-selective tarin binding, a phenomenon that must still be elucidated. In conclusion, taro corms contain an immunomodulatory lectin able to boost the immune system by promoting myeloid and lymphoid hematopoietic progenitor cell proliferation and differentiation.

Sensitization with Eustrongylides sp. (Nematoda: Dioctophymatidae) antigens induce production of specific IgG and IgE in murine model.

Fish consumption plays an important role in human diet. Hoplias malabaricus, commonly known as traíra, is a freshwater fish widely appreciated in several Brazilian states and frequently infected by Eustrongylides sp. fourth-instar larvae (L4). The aim of the present study was to evaluate allergenic potential of Eustrongylides sp. L4 crude extract (CEE). BALB/c mice were immunized intraperitoneally (IP) by 10 µg CEE with 2 mg of aluminum hydroxide on days 0 and 35. Specific IgG and IgE antibody levels were determined after immunization and cellular immunity was evaluated by assessing intradermal reaction in ear pavilion. Epicutaneous sensitization was performed in dorsal region by antigen exposure using a Finn-type chamber containing 50 µg of CEE or saline solution, followed by evaluation of specific antibody levels. IP immunization resulted in a gradual increase in IgG antibody levels and transitory IgE production. Significant increase in ear thickness was observed in cellular hypersensitivity reaction. In case of antigen exposure by epicutaneous route, CEE was able to induce meaningfully increased levels of specific IgG and IgE antibodies as well as heightened cellular immunity. Both intraperitoneal immunization and epicutaneous contact with Eustrongylides sp. larval antigens were observed for first time to be capable of inducing immunological sensitization in mice.

Short-Term Betanin Intake Reduces Oxidative Stress in Wistar Rats.

Oxidative stress is a common condition described in risk factors for cardiovascular disease. Betanin, a bioactive pigment from red beetroot demonstrates anti-inflammatory and antioxidant properties. The main aim of this study was to evaluate the short-term intake of betanin against oxidative stress in a rodent model, a common condition described in several risk factors for cardiovascular disease. Oxidative stress was induced in Wistar rats by a hyperlipidemic diet for 60 days, followed by betanin administration (20 mg·kg-1) through oral gavage for 20 days. Plasma biochemical parameters and antioxidant enzyme activities were evaluated. Lipid peroxidation and histopathological changes were determined in the liver. The hyperlipidemic diet caused hyperglycemia, hyperinsulinemia, insulin resistance, and increases in alanine transaminase and aspartate transaminase levels. Oxidative stress status was confirmed by reduction of antioxidant enzyme activities, increased lipid peroxidation, and liver damage. Purified betanin regulated glucose levels, insulin, and insulin resistance. Hepatic damage was reversed as evidenced by the reduction in alanine transaminase and aspartate transaminase levels and confirmed by histological analyses. Betanin reduced hepatic malondialdehyde and increased superoxide dismutase, catalase, and glutathione peroxidase activities. Short-term betanin intake modulated biochemical parameters, reversed hepatic tissue damage, and attenuated oxidative stress in Wistar rats.

Circulation of spotted fever group rickettsiae among dogs seropositive for Leishmania spp. in an urban area of Brazil.

INTRODUCTION: Dogs play an epidemiological role in several vector-borne diseases that affect human and animal health worldwide. We aimed to identify rickettsial circulation among dogs with canine visceral leishmaniasis (CVL) from a region endemic for both diseases. METHODS: CVL-seropositive dogs were screened for spotted fever group rickettsiae using an indirect immunofluorescence assay. RESULTS: Among the CVL-positive dogs, anti-Rickettsia rickettsii antibodies were identified in one asymptomatic and one oligosymptomatic dog. CONCLUSIONS: This study shows low circulation of antibodies to R. rickettsii in CVL-seropositive dogs. It is recommended that surveillance studies in dogs should continue in order to monitor this scenario.

Circulation of spotted fever group rickettsiae among dogs seropositive for Leishmania spp. in an urban area of Brazil

Abstract INTRODUCTION: Dogs play an epidemiological role in several vector-borne diseases that affect human and animal health worldwide. We aimed to identify rickettsial circulation among dogs with canine visceral leishmaniasis (CVL) from a region endemic for both diseases. METHODS: CVL-seropositive dogs were screened for spotted fever group rickettsiae using an indirect immunofluorescence assay. RESULTS: Among the CVL-positive dogs, anti-Rickettsia rickettsii antibodies were identified in one asymptomatic and one oligosymptomatic dog. CONCLUSIONS: This study shows low circulation of antibodies to R. rickettsii in CVL-seropositive dogs. It is recommended that surveillance studies in dogs should continue in order to monitor this scenario.

Tarin, a Potential Immunomodulator and COX-Inhibitor Lectin Found in Taro (Colocasia esculenta).

Taro (Colocasia esculenta) corm is a rustic staple food, rich in small starch granules, fibers, and bioactive phytoconstituents such as flavonoids, alkaloids, sterols, tannins, phytates, micronutrients, and proteins, including tarin, a GNA-related lectin. Tarin exhibits recognized biocide activities against viruses and insects, has antitumoral properties and is an immunomodulator molecule candidate. It has been isolated in highly purified form (>90%) from taro corms through low-cost and single-step affinity chromatography. It comprises 2-domain 27 to 28 kDa protomer, posttranslational cleaved into 2 nonidentical monomers, 11.9 and 12.6 kDa, held by noncovalent binding. At least 10 tarin isoforms sharing over 70% similarity have been described. The monomers assume the ß-prism II fold, consisting of 3 antiparallel ß-sheets formed by 4 ß-strands each. Tarin exhibits an expanded-binding site for complex and high-mannose N-glycan chains 49, 212, 213, 358, 465, and 477 found on cell surface antigens of viruses, insects, cancer, and hematopoietic cells, explaining its broad biological activities. Tarin may stimulate innate and adaptive immune responses, enabling hosts to recover from infections or immunosuppressed status inherent to several pathological conditions. In a murine model, tarin stimulates the in vitro and in vivo proliferation of total spleen and bone marrow cells, especially B lymphocytes. Granulocyte repopulation has also been demonstrated in long-term mice bone marrow cell cultures. As a potential immunomodulator, tarin, administered to immunosuppressed mice, attenuated cyclophosphamide-induced leukopenia. We propose a molecular model that unites the potential prophylactic and therapeutic action of tarin on hematopoietic and cancer cells, as a potential immunomodulator.

Principal component analysis of factors for sensitization to Anisakis spp. in postpartum women.

INTRODUCTION: Immunoreactivity to Anisakis spp. is believed to be associated with frequency of fish intake. The objective of this study was to evaluate, using principal component analysis, the main factors potentially involved in reactivity to these nematodes in postpartum women. METHODS: Retrospective study conducted on a database of 309 postpartum women. All completed a structured questionnaire and had blood samples collected for ELISA analysis of specific immunoglobulins against total Anisakis spp. antigens and assessment of reactivity. Parametric and nonparametric tests were used to assess factors for sensitization in the reactive and nonreactive groups, and a principal component analysis was performed. A Pearson correlation matrix with varimax rotation was used to assess the variables of interest (place of residence, age, number of prenatal visits, type of birth facility, fish intake and frequency, raw fish intake, fish handling, history of allergies). RESULTS: After exclusions, samples from 203 women were assessed. Of these, 52 (25.6%) were reactive for anti-Anisakis IgG. Most women claimed not to handle fish (n = 121) and eat fish only sporadically (n = 71). Significant differences in age were seen between the reactive and nonreactive groups (p = 0.001). The first two components explained 32.55% and 38.94% of variances in the nonreactive and reactive groups respectively. The adjusted matrix assigned greater probabilistic weight to weekly intake frequency (0.804), followed by raw fish intake (0.759), with differences in relation to the nonreactive group. CONCLUSION: Correlation matrices revealed a direct relationship between seroreactivity to Anisakis spp. and frequency of fish intake in a sample of postpartum women.

Purification and characterization of the lectin from taro (Colocasia esculenta) and its effect on mouse splenocyte proliferation in vitro and in vivo.

Lectins are proteins found in a wide range of organisms, with the ability to bind reversibly to specific carbohydrates. They can display important biological activities, such as the activation of the cell cycle in lymphocytes. Storage proteins with lectin activity have been reported in tuberous plant species, such as Colocasia esculenta, popularly known as taro. A simple strategy based on Cibacron Blue chromatography was used to purify a 12 kDa polypeptide 1.3-fold, with a recovery of 30 %. The purified protein was identified as tarin by mass spectrometry, which indicated that it was present in G1a/G1d isoforms. Tarin exhibited both agglutinating activity against hamster erythrocytes and mitogenic activity in vitro and in vivo toward mouse splenocytes. Optimum cellular proliferation in vitro was achieved by 625 ng of the crude extract or 500 ng of the purified tarin. Total mouse splenocyte proliferation measured after 5 days of intraperitoneal inoculation of purified tarin was increased 3.3-fold in comparison to the control group. Half of the proliferating cells were identified as B lymphocytes by flow cytometry. These results show that this is an efficient and simple strategy to purify tarin and aid in establishing this protein as a new therapeutic drug, able to promote cell proliferation in a murine model.

Cross-sectional study of serum reactivity to Anisakis simplex in healthy adults in Niterói, Brazil.

Although the incidence of anisakiasis is rising worldwide, its frequency is still unknown in Brazil. The aim of this study was to verify immunoreactivity to Anisakis simplex antigens in healthy adults and determine its possible relationship with frequency of fish consumption and allergy symptoms. A prospective cross-sectional study was carried out with 67 volunteers recruited from a military facility in Niterói, Brazil. The subjects completed a structured questionnaire and serum titers of specific anti-Anisakis IgE and IgG antibodies were measured. The association between frequency of fish intake and IgE reactivity was evaluated by Fisher's exact test. Almost all subjects (97.0%, 65/67) that consumed seafood; 64.6% (42/65) ate fish at least once weekly. Of all seafood consumers, 56.9% (37/65) reported allergy symptoms, being gut allergies most often cited (35.5%). IgE seroreactivity to Anisakis simplex was found in 20.9% of subjects (14/67), with 13.4% (9/67) reacting exclusively to somatic antigen, 3.0% (2/67) exclusively to excretory/secretory antigens and 4.5% (3/67) to both antigens. There was a significant association between frequency of fish consumption and positive serology (p = 0.019). An immunoblot assay for Anisakis antigens showed different positive bands for IgG. The direct relationship between ELISA reactivity and frequency of fish intake and absence of association with allergy symptoms suggests previous contact with Anisakis simplex antigens.

A technique for the intra-gastric administration of live larvae of Anisakis simplex in mice.

To understand the mechanisms of infection and to attempt to simulate human infection by the Anisakidae family, many in vivo experimental approaches have been developed. The aim was to develop and present a technique for the induction of an oral infection through the use of an intra-gastric gavage of live Anisakis simplex in mice. A commercial pediatric gastric tube (No. 4) was cut longitudinally to produce a 3-cm slit at the distal end where the larva was placed to then be administered to the stomach of the mouse. There were no abnormal clinical complications before, during or after the procedure. In conclusion oral infection through the direct delivery of larvae in the stomach is simple and effective.

A new 2S albumin from Jatropha curcas L. seeds and assessment of its allergenic properties.

Significant effort has been made world-wide to boost biofuels with the expectation of a positive contribution to renewable fuel and greenhouse gas reduction. Jatropha curcas L. has proved to be an opportunistic crop in tropical areas, particularly in unfavorable environments. For this reason, analyses of toxicity and allergy caused by its seeds and pollen are necessary. A 12kDa, allergenic 2S albumin, denoted Jat c 1, was isolated from Physic nut (J. curcas) seeds. Jat c 1 binds IgE attached to rat mast cells, inducing histamine release. It also showed strong cross-reactivity with the major allergens from castor bean, Ric c 1 and Ric c 3.