Fabián Salazar

A novel immunomodulatory hemocyanin from the limpet Fissurella latimarginata promotes potent anti-tumor activity in melanoma.

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.

Enhanced structural stability of Concholepas hemocyanin increases its immunogenicity and maintains its non-specific immunostimulatory effects

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.

Hemocianinas, una herramienta inmunológica de la biomedicina actual

Hemocyanins, the giant oxygen transporter glycoproteins of diverse mollusks, are xenogenic to the mammalian immune system and they display a remarkable immuno-genicity. Therefore they are ideal non-specific immunostimulants to treat some types of cancer. They are used as an alternative therapy for superficial urinary bladder cancer (SBC), that has been traditionally treated with Bacillus Calmette-Guerin (BCG). In contrast to BCG, hemocyanins do not cause side-effects, making them ideal for long-term repetitive treatments. Hemocyanins have also been exploited as carriers to develop antibodies against hapten molecules and peptides, as carrier-adjuvants for cutting-edge vaccines against cancer, drug addiction, and infectious diseases and in the diagnosis of parasitic diseases, such as Schistosomiasis. The hemocyanin from Megathura crenulata, also known as keyhole limpet hemocyanin (KLH), has been used for over thirty years for the purposes described above. More recently, hemoc yanin from the Chilean mollusk Concholepas concholepas (CCH) has proved to be a reliable alternative to KLH, either as carrier protein, and as a likely alternative for the immunotherapy of SBC. Despite KLH and CCH differ significantly in their origin and structure, we have demonstrated that both hemocyanins stimulate the immune system of mammals in a similar way by inducing a potent Thl-polarized cellular and humoral response.

Concholepas hemocyanin biosynthesis takes place in the hepatopancreas, with hemocytes being involved in its metabolism

Hemocyanins are copper-containing glycoproteins in some molluscs and arthropods, and their best-known 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 phagocyte-like 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.

Mollusk Hemocyanins as Natural Immunostimulants in Biomedical Applications

Hemocyanins, the massive oxygen-transporting glycoproteins found freely dissolved in the hemolymph of some mollusks, are potent natural immunostimulants when inoculated in mammals, enhance the innate and adaptive immune response with beneficial clinical outcomes. Hemocyanins are easily purified and molecularly correspond to large multisubunit structures, some over 107 Da (van Holde and Miller, 1995). Currently, hemocya‐ nins are commonly used as carriers/adjuvants for producing antibodies against different antigens. These antigens include tumor-associated antigens (TAAs), such as glycolipid and glycoprotein (mucin-like) antigens in cutting-edge therapeutic vaccines for cancer, along with idiotypes, the most commonly used tumor antigen to prepare vaccines for nearly all non-Hodgkin lymphomas. Other therapeutic strategies using hemocyanins include their use as adjuvants to disrupt self-tolerance to tumor antigens in the generation of ex vivo autologous tumor cell lysate-loaded dendritic cells (DCs) to induce T-cell responses in cancer patients (Del Campo et al., 2011). Furthermore, hemocyanins can be used as non-specific immunostimulants during therapy for recurrent superficial bladder cancer after transure‐ thral surgical resection with negligible toxic side effects, thus making them ideal for longterm on going treatments (Arancibia et al., 2012b).

Immunocompetence of breeding females is sensitive to cortisol levels but not to communal rearing in the degu (Octodon degus)

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 3months 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.

Household Transmission of Rotavirus in a Community with Rotavirus Vaccination in Quininde, Ecuador

Background We studied the transmission of rotavirus infection in households in peri-urban Ecuador in the vaccination era. Methods Stool samples were collected from household contacts of child rotavirus cases, diarrhea controls and healthy controls following presentation of the index child to health facilities. Rotavirus infection status of contacts was determined by RT-qPCR. We examined factors associated with transmissibility (index-case characteristics) and susceptibility (household-contact characteristics). Results Amongst cases, diarrhea controls and healthy control household contacts, infection attack rates (iAR) were 55%, 8% and 2%, (n = 137, 130, 137) respectively. iARs were higher from index cases with vomiting, and amongst siblings. Disease ARs were higher when the index child was <18 months and had vomiting, with household contact <10 years and those sharing a room with the index case being more susceptible. We found no evidence of asymptomatic infections leading to disease transmission. Conclusion Transmission rates of rotavirus are high in households with an infected child, while background infections are rare. We have identified factors associated with transmission (vomiting/young age of index case) and susceptibility (young age/sharing a room/being a sibling of the index case). Vaccination may lead to indirect benefits by averting episodes or reducing symptoms in vaccinees.

Hemocyanins in the Immunotherapy of Superficial Bladder Cancer

Chemoand immunotherapeutic approaches have been used to prevent recurrence of transitional cell carcinoma (TCC), the most common type of superficial bladder cancer (SBC). The bacillus Calmette-Guerin (BCG) vaccine for tuberculosis, which consists of an attenuated form of Mycobacterium bovis, is the most commonly used immunotherapeutic agent (Morales et al., 1976). Despite the successful results achieved with BCG, its serious side effects have led researchers to investigate other immunostimulatory substances. In the early 1970s, Olsson and collaborators reported that subcutaneous stimulation with keyhole limpet hemocyanin (KLH) from the Californian marine gastropod Megathura crenulata significantly reduced SBC recurrence frequency in TCC patients without any toxic side effects, making it ideal for long-term repetitive treatments (Olsson et al., 1974). These results provided promising support for the use of mollusk hemocyanins as alternative agents in SBC immunotherapy. Hemocyanins, blue respiratory glycoproteins that were discovered in 1878 by Leon Fredericq (Ghiretti-Magaldi & Ghiretti, 1992), are found freely dissolved in the blood of some mollusks and arthropods. These proteins are giant structures with molecular weights between 4 and 8 MDa, and they exhibit some of the most complex and sophisticated quaternary structures known. Hemocyanins are part of the type-3 group of copper proteins that includes phenoloxidases and tyrosinases (Decker & Tuczek, 2000). These proteins contain active copper-containing sites in which the Cu(I,I) state is oxidized to the Cu(II,II) state, thus accounting for their distinctive deep blue color. Because of these properties, the biochemistry of hemocyanins has been intensively studied (van Holde & Miller, 1995). The pioneering work of Weigle in the 1960s on the immunochemical properties of KLH demonstrated its remarkable immunostimulatory properties in an experimental animal model (Weigle, 1964). These results were quickly incorporated into clinical studies to evaluate its immunological effects. Because the primary amino acid sequences of mollusk hemocyanins are highly divergent from mammalian sequences, they are strongly recognized by the immune system, resulting in potent immunogenicity; these proteins can be used therapeutically as non-specific immunostimulants with beneficial clinical outcomes. Moreover, hemocyanins have been extensively used as carriers to generate antibodies against diverse hapten molecules and

TRANSMISSION OF NOROVIRUS WITHIN HOUSEHOLDS IN QUININDE, ECUADOR

We studied the transmission of norovirus infection in households in Quininde, Ecuador. Among household contacts of norovirus positive children with diarrhea, norovirus negative children with diarrhea and asymptomatic controls, infection attack rates were 33%, 8% and 18%, respectively (N = 45, 36, 83). Infection attack rates were higher when index children had a higher viral load.