Biosonda Corporation

Santiago, Chile

Biosonda Corporation

Santiago, Chile
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Pizarro-Bauerle J.,University of Chile | Maldonado I.,University of Chile | Sosoniuk-Roche E.,University of Chile | Vallejos G.,University of Chile | And 6 more authors.
Frontiers in Immunology | Year: 2017

Molluskan hemocyanins are enormous oxygen-carrier glycoproteins that show remarkable immunostimulatory properties when inoculated in mammals, such as the generation of high levels of antibodies, a strong cellular reaction, and generation of non-specific antitumor immune responses in some types of cancer, particularly for superficial bladder cancer. These proteins have the ability to bias the immune response toward a Th1 phenotype. However, despite all their current uses with beneficial clinical outcomes, a clear mechanism explaining these properties is not available. Taking into account reports of natural antibodies against the hemocyanin of the gastropod Megathura crenulata [keyhole limpet hemocyanin (KLH)] in humans as well as other vertebrate species, we report here for the first time, the presence, in sera from unimmunized healthy donors, of antibodies recognizing, in addition to KLH, two other hemocyanins from gastropods with documented immunomodulatory capacities: Fisurella latimarginata hemocyanin (FLH) and Concholepas concholepas hemocyanin (CCH). Through an ELISA screening, we found IgM and IgG antibodies reactive with these hemocyanins. When the capacity of these antibodies to bind deglycosylated hemocyanins was studied, no decreased interaction was detected. Moreover, in the case of FLH, deglycosylation increased antibody binding. We evaluated through an in vitro complement deposition assay whether these antibodies activated the classical pathway of the human complement system. The results showed that all three hemocyanins and their deglycosylated counterparts elicited this activation, mediated by C1 binding to immunoglobulins. Thus, this work contributes to the understanding on how the complement system could participate in the immunostimulatory properties of hemocyanins, through natural, complement-activating antibodies reacting with these proteins. Although a role for carbohydrates cannot be completely ruled out, in our experimental setting, glycosylation status had a limited effect. Finally, our data open possibilities for further studies leading to the design of improved hemocyanin-based research tools for diagnosis and immunotherapy. © 2017 Pizarro-Bauerle, Maldonado, Sosoniuk-Roche, Vallejos, López, Salazar-Onfray, Aguilar-Guzmán, Valck, Ferreira and Becker.

Manubens A.,Fundacion Ciencia y Tecnologia para el Desarrollo | Manubens A.,BIOSONDA Corporation | Salazar F.,Fundacion Ciencia y Tecnologia para el Desarrollo | Haussmann D.,Austral University of Chile | And 7 more authors.
Cell and Tissue Research | Year: 2010

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their bestknown function is O2 transport. We studied the site of their biosynthesis in the gastropod Concholepas concholepas by using immunological and molecular genetic approaches. We performed immunohistochemical staining of various organs, including the mantle, branchia, and hepatopancreas, and detected C. concholepas hemocyanin (CCH) molecules in circulating and tissue-associated hemocytes by electron microscopy. To characterize the hemocytes, we purified them from hemolymph. We identified three types of granular cells. The most abundant type was a phagocytelike cell with small cytoplasmic granules. The second type contained large electron-dense granules. The third type had vacuoles containing hemocyanin molecules suggesting that synthesis or catabolism occurred inside these cells. Our failure to detect cch-mRNA in hemocytes by reverse transcription with the polymerase chain reaction (RT-PCR) led us to propose that hemocytes instead played a role in CCH metabolism. This hypothesis was supported by colloidal gold staining showing hemocyanin molecules in electron-dense granules inside hemocytes. RT-PCR analysis, complemented by in situ hybridization analyses with single-stranded antisense RNAs as specific probes, demonstrated the presence of cch-mRNA in the hepatopancreas; this was consistent with the specific hybridization signal and confirmed the hepatopancreas as the site of CCH synthesis. Finally, we investigated the possibility that CCH catabolism in hemocytes was involved in the host immune response and in the generation of secondary metabolites such as antimicrobial peptides and phenoloxidase. © Springer-Verlag 2010.

Arancibia S.,Fundacion Ciencia Y Tecnologia para el Desarrollo FUCITED | Espinoza C.,Fundacion Ciencia Y Tecnologia para el Desarrollo FUCITED | Salazar F.,Fundacion Ciencia Y Tecnologia para el Desarrollo FUCITED | Del Campo M.,Fundacion Ciencia Y Tecnologia para el Desarrollo FUCITED | And 10 more authors.
PLoS ONE | Year: 2014

Hemocyanins, the huge oxygen-transporting glycoproteins of some mollusks, are used as immunomodulatory proteins with proven anti-cancer properties. The biodiversity of hemocyanins has promoted interest in identifying new anti-cancer candidates with improved immunological properties. Hemocyanins promote Th1 responses without known side effects, which make them ideal for long-term sustained treatment of cancer. In this study, we evaluated a novel hemocyanin from the limpet/gastropod Fissurella latimarginata (FLH). This protein has the typical hollow, cylindrical structure of other known hemocyanins, such as the keyhole limpet hemocyanin (KLH) and the Concholepas hemocyanin (CCH). FLH, like the KLH isoforms, is composed of a single type of polypeptide with exposed N- and O-linked oligosaccharides. However, its immunogenicity was significantly greater than that of KLH and CCH, as FLH induced a stronger humoral immune response and had more potent anti-tumor activity, delaying tumor growth and increasing the survival of mice challenged with B16F10 melanoma cells, in prophylactic and therapeutic settings. Additionally, FLH-treated mice demonstrated increased IFN-γ production and higher numbers of tumor-infiltrating CD4+ lymphocytes. Furthermore, in vitro assays demonstrated that FLH, but not CCH or KLH, stimulated the rapid production of pro-inflammatory cytokines (IL-6, IL-12, IL-23 and TNF-α) by dendritic cells, triggering a pro-inflammatory milieu that may explain its enhanced immunological activity. Moreover, this effect was abolished when deglycosylated FLH was used, suggesting that carbohydrates play a crucial role in the innate immune recognition of this protein. Altogether, our data demonstrate that FLH possesses increased anti-tumor activity in part because it activates a more potent innate immune response in comparison to other known hemocyanins. In conclusion, FLH is a potential new marine adjuvant for immunization and possible cancer immunotherapy. © 2014 Arancibia et al.

Vargas F.,University of Santiago de Chile | Vargas F.,Biosonda Corporation | Becker M.I.,Biosonda Corporation | Friguet B.,University Pierre and Marie Curie | And 2 more authors.
Journal of the Brazilian Chemical Society | Year: 2016

Deleterious effect mediated by glucose degradation, as a parallel pathway to Maillard reaction, was analyzed in terms of the feasibility of inducing photo-crosslinking in isolated bovine crystalline proteins when exposed to ultraviolet A (UVA)-visible light. These experiments showed evidence supporting the generation of a glucose-derived chromophore (GDC). The ability of this chromophore to induce oxidative damage in lens protein and bovine lens epithelial cells (BLEC) was further assessed. The analysis of dityrosine and carbonyl levels in lens proteins irradiated at 5 and 20% O2 indicates the occurrence of mixed type I/type II photosensitizing mechanisms. When BLEC were exposed to photosensitized reactions induced by GDC a decrease in cellular viability and intracellular reduced (GSH) and oxidized (GSSG) glutathione ratio was observed, as well as an increase in the amount of intracellular reactive oxygen species. Our data suggest a major effect of type I photosensitizing mechanism in both lens proteins photo-oxidation and oxidative stress induced in BLEC. © 2016 Sociedade Brasileira de Química.

PubMed | Biosonda Corporation, Fundacion Ciencia y Tecnologia para el Desarrollo and Chile; and Biosonda Corporation
Type: Journal Article | Journal: Journal of immunology (Baltimore, Md. : 1950) | Year: 2016

Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks hemocyanins with varying structural and immunological properties, including hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1, IL-6, IL-12p40, and TNF-. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata hemocyanin in comparison with keyhole limpet hemocyanin and C. concholepas hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5 Collectively, our data demonstrate that hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.

Arancibia S.,Fundacion Ciencia y Tecnologia para el Desarrollo FUCITED | Campo M.D.,Fundacion Ciencia y Tecnologia para el Desarrollo FUCITED | Nova E.,Fundacion Ciencia y Tecnologia para el Desarrollo FUCITED | Salazar F.,Fundacion Ciencia y Tecnologia para el Desarrollo FUCITED | And 2 more authors.
European Journal of Immunology | Year: 2012

Hemocyanins, which boost the immune system of mammals, have been used as carrier-adjuvants to promote Ab production against haptens and peptides, as immunostimulants during therapy for bladder carcinoma and as a component in therapeutic vaccines for cancer. These biomedical applications have led to growing interest in obtaining hemocyanins with high immunogenicity. Here, we study the immunological properties of a modified oxidized Concholepas concholepas hemocyanin (Ox-CCH) obtained by the oxidation of its carbohydrates using sodium periodate. We assessed the internalization of Ox-CCH into DCs and its immunogenicity and antitumor effects. Transmission electron microscopy showed no changes in Ox-CCH quaternary structure with respect to native CCH, although proteolytic treatment followed by SDS-PAGE analysis demonstrated that Schiff bases were formed. Interestingly, DCs internalized Ox-CCH faster than CCH, mainly through macropinocytosis. During this process, Ox-CCH remained inside endosome-like structures for a longer period. Mouse immunization experiments demonstrated that Ox-CCH is more immunogenic and a better carrier than CCH. Moreover, Ox-CCH showed a significant antitumor effect in the B16F10 melanoma model similar to that produced by CCH, inducing IFN-γ secretion. Together, these data demonstrate that the aldehydes formed by the periodate oxidation of sugar moieties stabilizes the CCH structure, increasing its adjuvant/immunostimulatory carrier effects. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ebensperger L.A.,University of Santiago de Chile | Leon C.,University of Santiago de Chile | Ramirez-Estrada J.,University of Santiago de Chile | Abades S.,University of Santiago de Chile | And 7 more authors.
Physiology and Behavior | Year: 2015

One hypothesis largely examined in social insects is that cooperation in the context of breeding benefits individuals through decreasing the burden of immunocompetence and provide passive immunity through social contact. Similarly, communal rearing in social mammals may benefit adult female members of social groups by reducing the cost of immunocompetence, and through the transfer of immunological compounds during allonursing. Yet, these benefits may come at a cost to breeders in terms of a need to increase investment in individual immunocompetence. We examined how these potential immunocompetence costs and benefits relate to reproductive success and survival in a natural population of the communally rearing rodent, Octodon degus. We related immunocompetence (based on ratios of white blood cell counts, total and specific immunoglobulins of G isotype titers) and fecal glucocorticoid metabolite (FGC) levels of adults immunized with hemocyanin from the mollusk Concholepas concholepas to measures of sociality (group size) and communal rearing (number of breeding females). Offspring immunocompetence was quantified based on circulating levels of the same immune parameters. Neither female nor offspring immunocompetence was influenced by communal rearing or sociality. These findings did not support that communal rearing and sociality enhance the ability of females to respond to immunological challenges during lactation, or contribute to enhance offspring condition (based on immunocompetence) or early survival (i.e., to 3. months of age). Instead, levels of humoral and cellular components of immunocompetence were associated with variation in glucorcorticoid levels of females. We hypothesize that this covariation is driven by physiological (life-history) adjustments needed to sustain breeding. © 2014 Elsevier Inc.

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