Immunotherapeutic Potential of Mollusk Hemocyanins in Murine Model of Melanoma.

The development of antitumor drugs and therapy requires new approaches and molecules, and products of natural origin provide intriguing alternatives for antitumor research. Gastropodan hemocyanins-multimeric copper-containing glycoproteins have been used in therapeutic vaccines and antitumor agents in many cancer models. MATERIALS AND METHODS: We established a murine model of melanoma by challenging C57BL/6 mice with a B16F10 cell line for solid tumor formation in experimental animals. The anticancer properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) were evaluated in this melanoma model using various schemes of therapy. Flow cytometry, ELISA, proliferation, and cytotoxicity assays, as well as histology investigations, were also performed. RESULTS: Beneficial effects on tumor growth, tumor incidence, and survival of tumor-bearing C57BL/6 mice after administration of the RtH or HaH were observed. The generation of high titers of melanoma-specific IgM antibodies, pro-inflammatory cytokines, and tumor-specific CTLs, and high levels of tumor-infiltrated M1 macrophages enhanced the immune reaction and tumor suppression. DISCUSSION: Both RtH and HaH exhibited promising properties for applications as antitumor therapeutic agents and future experiments with humans.

Penaeid shrimp counteract high ammonia stress by generating and using functional peptides from hemocyanin, such as HMCs27.

Agricultural and anthropogenic activities release high ammonia levels into aquatic ecosystems, severely affecting aquatic organisms. Penaeid shrimp can survive high ammonia stress conditions, but the underlying molecular mechanisms are unknown. Here, total hemocyanin and oxyhemocyanin levels decreased in Penaeus vannamei plasma under high ammonia stress. When shrimp were subjected to high ammonia stress for 12 h, 24 hemocyanin (HMC) derived peptides were identified in shrimp plasma, among which one peptide, designated as HMCs27, was chosen for further analysis. Shrimp survival was significantly enhanced after treatment with the recombinant protein of HMCs27 (rHMCs27), followed by high ammonia stress. Transcriptome analysis of shrimp hepatopancreas after treatment with or without rHMCs27 followed by high ammonia stress revealed 973 significantly dysregulated genes, notable among which were genes involved in oxidation and metabolism, such as cytochrome C, catalase (CAT), isocitrate dehydrogenase, superoxide dismutase (SOD), trypsin, chymotrypsin, glutathione peroxidase, glutathione s-transferase (GST), and alanine aminotransferase (ALT). In addition, levels of key biochemical indicators, such as SOD, CAT, and total antioxidant capacity (T-AOC), were significantly enhanced, whereas hepatopancreas malondialdehyde levels and plasma pH, NH3, GST, and ALT levels were significantly decreased after rHMCs27 treatment followed by high ammonia stress. Moreover, high ammonia stress induced hepatopancreas tissue injury and apoptosis, but rHMCs27 treatment ameliorated these effects. Collectively, the current study revealed that in response to high ammonia stress, shrimp generate functional peptides, such as peptide HMCs27 from hemocyanin, which helps to attenuate the ammonia toxicity by enhancing the antioxidant system and the tricarboxylic acid cycle to decrease plasma NH3 levels and pH.

Exploring the Antimicrobial Potential and Biofilm Inhibitory Properties of Hemocyanin from Hemifusus pugilinus (Born, 1778).

The seafood industry plays a huge role in the blue economy, exploiting the advantage of the enriched protein content of marine organisms such as shrimps and molluscs, which are cultured in aquafarms. Diseases greatly affect these aquatic organisms in culture and, hence, there is need to study, in detail, their innate immune mechanisms. Hemocyanin is a non-specific innate defense molecule present in the blood cells of several invertebrates, especially molluscs, arthropods, and annelids. It is concerned with oxygen transport, blood clotting, and immune enhancement. In the present study, this macromolecular metalloprotein was isolated from the hemolymph of the marine snail Hemifusus pugilinus (Born, 1778) using Sephadex G-100 gel filtration column chromatography. It occurred as a single band (MW 80 kDa) on SDS-PAGE. High-performance liquid chromatography (HPLC) of the purified hemocyanin showed a single peak with a retention time of 4.3 min. The secondary structure and stability of the protein were detected using circular dichroism (CD), and the spectra demonstrated negative ellipticity bands close to 208 nm and 225 nm, indicating ß-sheets. Further exploration of the purified hemocyanin revealed remarkable antimicrobial and antibiofilm activities against Gram-positive (Enterococcus faecalis and Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa and Proteus vulgaris) at a concentration of 1-5 µg/mL. Spectrophotometric and in situ microscopic analyses (CLSM) unveiled the potential of the purified hemocyanin to inhibit biofilm formation in these bacteria with a minimal inhibitory concentration of 40 µg/mL. Furthermore, H. pugilinus hemocyanin (10 µg/mL concentration) displayed antifungal activity against Aspergillus niger. The purified hemocyanin was also assessed for cytotoxicity against human cancer cells using cell viability assays. Altogether, the present study shows that molluscan hemocyanin is a potential antimicrobial, antibiofilm, antifungal, anticancer, and immunomodulatory agent, with great scope for application in the enhancement of the immune system of molluscs, thereby facilitating their aquaculture.

Laboratory safety evaluation of lokivetmab, a canine anti-interleukin-31 monoclonal antibody, in dogs.

Lokivetmab (Cytopoint®, Zoetis) is a canine monoclonal antibody that specifically binds and neutralizes interleukin (IL)-31. Lokivetmab is approved for use in dogs for the treatment of atopic dermatitis (AD) and allergic dermatitis. The laboratory safety of lokivetmab was evaluated in 2 studies by adapting the science-based, case-by-case approach used for preclinical and early clinical safety evaluation of human biopharmaceuticals. The main objectives were to demonstrate the safety of lokivetmab in healthy laboratory Beagle dogs by using integrated clinical, morphologic, and functional evaluations. In Study 1, dogs were treated s.c. with saline or lokivetmab at 3.3 mg/kg (1X, label dose) or 10 mg/kg (3X intended dose) for 7 consecutive monthly doses, with terminal pathology and histology assessments. In Study 2, the functional immune response was demonstrated in naïve dogs using the T-cell dependent antibody response (TDAR) test with 2 different dose levels of unadjuvanted keyhole limpet hemocyanin (KLH) as the model immunogen. The primary endpoint was anti-KLH IgG antibody titer, and secondary endpoints were ex vivo IL-2 enzyme-linked immunospot (ELISpot) and peripheral blood mononuclear cell lymphoproliferation assays. Both studies included monitoring general health, periodic veterinary clinical evaluations, serial clinical pathology and toxicokinetics, and monitoring for anti-drug antibodies. In both studies, the health of dogs receiving lokivetmab was similar to controls, with no treatment-related changes uncovered. Extensive pathology evaluations of immune tissues (Study 1) revealed no lokivetmab-related morphologic changes, and in dogs treated at 10 mg/kg lokivetmab, immunization with the model antigen KLH did not impair the functional antibody or T-cell recall responses. There were no immunogenicity-related or hypersensitivity-related responses observed in either study. These studies in healthy laboratory dogs showed that lokivetmab was well-tolerated, did not produce any treatment-related effects, and had no effect on immune system morphology or its functional response. These studies also demonstrated the utility of a science-based case-by-case approach to the safety evaluation of a veterinary biopharmaceutical product.

Organic copper promoted copper accumulation and transport, enhanced low temperature tolerance and physiological health of white shrimp (Litopenaeus vannamei Boone, 1931).

This study was conducted to assess the effects of dietary copper source and level on hematological parameters, copper accumulation and transport, resistance to low temperature, antioxidant capacity and immune response of white shrimp (Litopenaeus vannamei Boone, 1931). Seven experimental diets with different copper sources and levels were formulated: C, no copper supplementation; S, 30 mg/kg copper in the form of CuSO4·5H2O; SO, 15 mg/kg copper in CuSO4·5H2O + 7.5 mg/kg copper in Cu-proteinate; O1, O2, O3 and O4, 10, 20, 30 and 40 mg/kg copper in the form of Cu-proteinate, respectively. A total of 840 shrimp (5.30 ± 0.04 g) were randomly distributed to 21 tanks (3 tanks/diet, 40 shrimp/tank). An 8-week feeding trial was conducted. The results showed that there was no significant difference in growth performance and whole shrimp chemical compositions among all groups. Compared with inorganic copper, dietary organic copper (O2 and O3) increased total protein, albumin, and glucose content of plasma, while decreased triglyceride and total cholesterol of plasma. Copper concentration in plasma and muscle and gene expression of metallothionein and copper-transporting ATPase 2 like in hepatopancreas were higher in shrimp fed organic copper (SO, O2, O3 and O4). The lowest mortality after low temperature (10 °C) challenge test was observed in the O2 and O3 groups. Organic copper (SO, O2, O3 and O4) significantly enhanced the antioxidant capacity (in terms of higher activities of total superoxide dismutase, copper zinc superoxide dismutase, catalase, glutathione peroxidase and total antioxidant capacity, lower malondialdehyde concentration of plasma, and up-regulated gene expression of superoxide dismutase, copper zinc superoxide dismutase, catalase and glutathione peroxidase of hepatopancreas). Organic copper (SO, O2, O3 and O4) enhanced the immune response (in terms of higher number of total hemocytes, higher activities of acid phosphatase, alkaline phosphatase, phenoloxidase, hemocyanin and lysozyme in plasma, and higher gene expressions of alkaline phosphatase, lysozyme and hemocyanin in hepatopancreas). Inorganic copper (Diet S) also had positive effects on white shrimp compared with the C diet, but the SO, O2, O3 and O4 diets resulted in better results, among which the O2 diet appeared to be the best one. In conclusion, organic copper was more beneficial to shrimp health than copper sulfate.

Cadmium and copper mixture effects on immunological response and susceptibility to Vibrio harveyi in white shrimp Litopenaeus vannamei.

Cadmium (Cd2+) and copper (Cu2+) are considered immunotoxic metals and their presence in combination in the aquatic environment may cause effects on shrimp species as Litopenaeus vannamei. Thus, this research evaluates the combined effects of Cd2+ and Cu2+ on shrimp inoculated with Vibrio harveyi bacteria. The experiments were performed at 96-h of exposure to sublethal concentrations of both metals. No mortality was observed in organisms exposed to the sum of Criterion of Continuous Concentration (ΣCCC) in Cd + Cu mixture and those inoculated with V. harveyi. Higher clotting times were recorded in Cd + Cu + V. harveyi treatment at higher metal concentrations. No significant differences (P > 0.05) were recorded in hemocyanin content between shrimp exposed to metals and those experimentally infected. Significantly higher (P < 0.05) total hemocyte count (THC) was recorded at 96 h exposure in the ΣCCC and 10% treatments of Cd + Cu + V. harveyi experiment. Regarding Cd + Cu + V. harveyi bioassay, the highest phenoloxidase (PO) activity was recorded in shrimp inoculated with V. harveyi (0.326 ± 0.031 PO units/mg protein) at 96-h exposure. The lowest PO activity was observed in organisms exposed to Cd + Cu + V. harveyi. Regarding superoxide dismutase (SOD) activity, shrimp exposed to higher metal concentrations at 96 h showed the lowest hemolymph activity (6.03 ± 0.62 SOD units/mL). Protein decrease was observed in organisms exposed to metal mixture. The results showed that L. vannamei could be more susceptible to V. harveyi when exposed to Cd + Cu.

Functional Characterization, Antimicrobial Effects, and Potential Antibacterial Mechanisms of NpHM4, a Derived Peptide of Nautilus pompilius Hemocyanin.

Hemocyanins present in the hemolymph of invertebrates are multifunctional proteins that are responsible for oxygen transport and play crucial roles in the immune system. They have also been identified as a source of antimicrobial peptides during infection in mollusks. Hemocyanin has also been identified in the cephalopod ancestor Nautilus, but antimicrobial peptides derived from the hemocyanin of Nautilus pompilius have not been reported. Here, the bactericidal activity of six predicted peptides from N. pompilius hemocyanin and seven mutant peptides was analyzed. Among those peptides, a mutant peptide with 15 amino acids (1RVFAGFLRHGIKRSR15), NpHM4, showed relatively high antibacterial activity. NpHM4 was determined to have typical antimicrobial peptide characteristics, including a positive charge (+5.25) and a high hydrophobic residue ratio (40%), and it was predicted to form an alpha-helical structure. In addition, NpHM4 exhibited significant antibacterial activity against Gram-negative bacteria (MBC = 30 µM for Vibrio alginolyticus), with no cytotoxicity to mammalian cells even at a high concentration of 180 µM. Upon contact with V. alginolyticus cells, we confirmed that the bactericidal activity of NpHM4 was coupled with membrane permeabilization, which was further confirmed via ultrastructural images using a scanning electron microscope. Therefore, our study provides a rationalization for the development and optimization of antimicrobial peptide from the cephalopod ancestor Nautilus, paving the way for future novel AMP development with broad applications.

Antitumor Properties of Epitope-Specific Engineered Vaccine in Murine Model of Melanoma.

Finding new effective compounds of natural origin for composing anti-tumor vaccines is one of the main goals of antitumor research. Promising anti-cancer agents are the gastropodan hemocyanins-multimeric copper-containing glycoproteins used so far for therapy of different tumors. The properties of hemocyanins isolated from the marine snail Rapana thomasiana (RtH) and the terrestrial snail Helix aspersa (HaH) upon their use as carrier-proteins in conjugated vaccines, containing ganglioside mimotope GD3P4 peptide, were studied in the developed murine melanoma model. Murine melanoma cell line B16F10 was used for solid tumor establishment in C57BL/6 mice using various schemes of therapy. Protein engineering, flow cytometry, and cytotoxicity assays were also performed. The administration of the protein-engineered vaccines RtH-GD3P4 or HaH-GD3P4 under the three different regimens of therapy in the B16F10 murine melanoma model suppressed tumor growth, decreased tumor incidence, and prolonged the survival of treated animals. The immunization of experimental mice induced an infiltration of immunocompetent cells into the tumors and generated cytotoxic tumor-specific T cells in the spleen. The treatment also generates significantly higher levels of tumor-infiltrated M1 macrophages, compared to untreated tumor-bearing control mice. This study demonstrated a promising approach for cancer therapy having potential applications for cancer vaccine research.

OX40L Inhibition Suppresses KLH-driven Immune Responses in Healthy Volunteers: A Randomized Controlled Trial Demonstrating Proof-of-Pharmacology for KY1005.

The safety, tolerability, immunogenicity, and pharmacokinetic (PK) profile of an anti-OX40L monoclonal antibody (KY1005, currently amlitelimab) were evaluated. Pharmacodynamic (PD) effects were explored using keyhole limpet hemocyanin (KLH) and tetanus toxoid (TT) immunizations. Sixty-four healthy male subjects (26.5 ± 6.0 years) were randomized to single doses of 0.006, 0.018, or 0.05 mg/kg, or multiple doses of 0.15, 0.45, 1.35, 4, or 12 mg/kg KY1005, or placebo (6:2). Serum KY1005 concentrations were measured. Antibody responses upon KLH and TT immunizations and skin response upon intradermal KLH administration were performed. PD data were analyzed using repeated measures analysis of covariances (ANCOVAs) and post hoc exposure-response modeling. No serious adverse events occurred and all adverse events were temporary and of mild or moderate severity. A nonlinear increase in mean serum KY1005 concentrations was observed (median time to maximum concentration (Tmax ) ~ 4 hours, geometric mean terminal half-life (t½) ~ 24 days). Cutaneous blood perfusion (estimated difference (ED) -13.4 arbitrary unit (AU), 95% confidence interval (CI) -23.0 AU to -3.8 AU) and erythema quantified as average redness (ED -0.23 AU, 95% CI -0.35 AU to -0.11 AU) decreased after KY1005 treatment at doses of 0.45 mg/kg and above. Exposure-response analysis displayed a statistically significant treatment effect on anti-KLH antibody titers (IgG maximum effect (Emax ) -0.58 AU, 95% CI -1.10 AU to -0.06 AU) and skin response (erythema Emax -0.20 AU, 95% CI -0.29 AU to -0.11 AU). Administration of KY1005 demonstrated an acceptable safety and tolerability profile and PK analyses displayed a nonlinear profile of KY1005. Despite the observed variability, skin challenge response after KY1005 treatment indicated pharmacological activity of KY1005. Therefore, KY1005 shows potential as a novel pharmacological treatment in immune-mediated disorders.

LvHemB1, a novel cationic antimicrobial peptide derived from the hemocyanin of Litopenaeus vannamei, induces cancer cell death by targeting mitochondrial voltage-dependent anion channel 1.

Current cancer treatment regimens such as chemotherapy and traditional chemical drugs have adverse side effects including the appearance of drug-resistant tumor cells. For these reasons, it is imperative to find novel therapeutic agents that overcome these factors. To this end, we explored a cationic antimicrobial peptide derived from Litopenaeus vannamei hemocyanin (designated LvHemB1) that induces cancer cell death, but sparing normal cells. LvHemB1 inhibits the proliferation of human cervical (HeLa), esophageal (EC109), hepatocellular (HepG2), and bladder (EJ) cancer cell lines, but had no significant effect on normal liver cell lines (T-antigen-immortalized human liver epithelial (THLE-3) cells). In addition to its antiproliferative effects, LvHemB1 induced apoptosis, by permeating cells and targeting mitochondrial voltage-dependent anion channel 1 (VDAC1). Colocalization studies revealed the localization of LvHemB1 in mitochondria, while molecular docking and pull-down analyses confirmed LvHemB1-VDAC1 interaction. Moreover, LvHemB1 causes loss in mitochondrial membrane potential and increases levels of reactive oxygen species (ROS) and apoptotic proteins (caspase-9, caspase-3, and Bax (Bcl-2-associated X)), which results in mitochondrial-mediated apoptosis. Thus, peptide LvHemB1 has the potential of being used as an anticancer agent due to its antiproliferation effect and targeting to VDAC1 to cause mitochondrial dysfunction in cancer cells, as well as its ability to induce apoptosis by increasing ROS levels, and the expression of proapoptotic proteins.

Feeding treats containing cannabidiol (CBD) did not alter canine immune response to immunization with a novel antigen.

Due to the potential risk for cannabidiol (CBD) to negatively impact the immune system, the objective of the current study was to evaluate the effect of CBD on the canine immune response to immunization with a novel antigen, keyhole limpet hemocyanin (KLH). Thirty-two dogs (22.4 ± 6.3 kg BW) were utilized in a completely randomized design with treatments consisting of 5 mg CBD/kg BW/d and a control administered orally via treats. After a 7-d acclimation to treatments, dogs were immunized with 10 mg/dog of KLH via intramuscular injection into the semimembranosus muscle region, which was repeated in 14 d. Blood samples were collected at baseline and weekly for 28 d after initial KLH immunization for analysis of hematology, serum chemistry, and immunoglobulins. Data were analyzed using the MIXED procedure in SAS including the fixed effects of treatment, day, and the treatment by day interaction. Both primary and secondary KLH immunization produced robust immune responses. Most hematological and serum chemistry variables remained within normal reference ranges for dogs across both treatments throughout the study. Alkaline phosphatase, while within normal reference range and similar between treatments at baseline and on d 7 (P = 0.994 and 0.183, respectively), was elevated for CBD-treated dogs versus control on d 14, 21, and 28 (P = 0.006, 0.027, and 0.014, respectively). Both total and KLH-specific IgG and IgM were similar between treatments throughout the study (P > 0.05), although total IgM peaked earlier in control dogs compared to those receiving CBD. Despite the minor shift in the timing of the total IgM peak, CBD did not appear to exhibit humoral immunosuppressive effects when supplemented at 5 mg/kg BW/d. However, this work does highlight the potential for CBD to alter liver function and the need for further safety evaluations of CBD use in dogs utilizing longer-term studies and multiple CBD doses.

Hemocyanin Modification of Chitosan Scaffolds with Calcium Phosphate Phases Increase the Osteoblast/Osteoclast Activity Ratio-A Co-Culture Study.

The ongoing research on biomaterials that support bone regeneration led to the quest for materials or material modifications that can actively influence the activity or balance of bone tissue cells. The bone biocompatibility of porous chitosan scaffolds was modified in the present study by the addition of calcium phosphates or hemocyanin. The first strategy comprised the incorporation of calcium phosphates into chitosan to create a biomimetic chitosan-mineral phase composite. The second strategy comprised dip-coating of chitosan scaffolds with hemocyanin extracted from crayfish hemolymph. The cytocompatibility was assessed in a mono-culture of human bone marrow stromal cells (hBMSCs) and their differentiation to osteoblasts; in a mono-culture of human monocytes (hMs) and their maturation to osteoclasts; and in a co-culture of hBMSC/osteoblasts-hM/osteoclasts. Mineral incorporation caused an increase in scaffold bioactivity, as shown by reduced calcium concentration in the cell culture medium, delayed differentiation of hBMSCs, and reduced osteoclastic maturation of hMs in mono-culture. Dip-coating with hemocyanin led to increased proliferation of hBMSCs and equivalent osteoclast maturation in mono-culture, while in co-culture, both an inhibitory effect of mineral incorporation on osteoblastogenesis and stimulatory effects of hemocyanin were observed. It was concluded that highly bioactive scaffolds (containing mineral phases) restrain osteoblast and osteoclast development, while hemocyanin coating significantly supports osteoblastogenesis. These influences on the osteoblasts/osteoclasts activity ratio may support scaffold-driven bone healing in the future.

Combining a Candidate Vaccine for Opioid Use Disorders with Extended-Release Naltrexone Increases Protection against Oxycodone-Induced Behavioral Effects and Toxicity.

Opioid use disorders (OUDs) and opioid-related fatal overdoses are a significant public health concern in the United States and worldwide. To offer more effective medical interventions to treat or prevent OUD, antiopioid vaccines are in development that reduce the distribution of the targeted opioids to brain and subsequently reduce the associated behavioral and toxic effects. It is of critical importance that antiopioid vaccines do not interfere with medications that treat OUD. Hence, this study tested the preclinical proof of concept of combining a candidate oxycodone vaccine [oxycodone-keyhole limpet hemocyanin (OXY-KLH)] with an FDA-approved extended-release naltrexone (XR-NTX) depot formulation in rats. The effects of XR-NTX on oxycodone-induced motor activity and antinociception were first assessed in nonvaccinated naïve rats to establish a baseline for subsequent studies. Next, OXY-KLH and XR-NTX were coadministered to determine whether the combination would affect the efficacy of each individual treatment, and it was found that the combination of OXY-KLH and XR-NTX offered greater efficacy in reducing oxycodone-induced motor activity, thigmotaxis, antinociception, and respiratory depression over a range of repeated or escalating oxycodone doses in rats. These data support the feasibility of combining antibody-based therapies with opioid receptor antagonists to provide greater or prolonged protection against opioid-related toxicity or overdose. Combining antiopioid vaccines with XR-NTX may provide prophylactic measures to subjects at risk of relapse and accidental or deliberate exposure. Combination therapy may extend to other biologics (e.g., monoclonal antibodies) and medications against substance use disorders. SIGNIFICANCE STATEMENT: Opioid use disorders (OUDs) remain a major problem worldwide, and new therapies are needed. This study reports on the combination of an oxycodone vaccine [oxycodone-keyhole limpet hemocyanin (OXY-KLH)] with a currently approved OUD therapy, extended-release naltrexone (XR-NTX). Results demonstrated that XR-NTX did not interfere with OXY-KLH efficacy, and combination of low doses of XR-NTX with vaccine was more effective than each individual treatment alone to reduce behavioral and toxic effects of oxycodone, suggesting that combining OXY-KLH with XR-NTX may improve OUD outcomes.

Antichagasic effect of hemocyanin derived from antimicrobial peptides of penaeus monodon shrimp.

Trypanosoma cruzi, the etiological agent of Chagas disease, is responsible for the infection of millions of people worldwide and it is a public health problem, without an effective cure. Four fragments with antimicrobial potential from the hemocyanin of Penaeus monodon shrimp were identified using a computer software AMPA. The present study aimed to evaluate the antichagasic effect of these four peptides (Hmc364-382, Hmc666-678, Hmc185-197 and Hmc476-498). The peptides were tested against the epimastigote, trypomastigote and amastigote forms of Trypanosoma cruzi Y strain (benznidazole-resistant strain) and cytotoxicity in mammalian cells was evaluated against LLC-MK2 lineage cells. Two fragments (Hmc364-382, Hmc666-678) showed activity against the epimastigote and trypomastigote forms and their selectivity index (SI) was calculated. The Hmc364-382 peptide was considered the most promising (SI > 50) one and it was used for further studies, using flow cytometry analyses with specific molecular probes and scanning electron microscopy (SEM). Hmc364-382 was able to induce cell death in T. cruzi through necrosis, observed by loss of membrane integrity in flow cytometry analyses and pore formation in SEM. Overall, Hmc364-382 open perspectives to the development of new antichagasic agents.

Intensive therapy with gastropodan hemocyanins increases their antitumor properties in murine model of colon carcinoma.

Various natural compounds have been tested as anticancer therapeutics in clinical trials. Most promising direction for antitumor therapy is the use of substances which enhance the immune system response stimulating tumor-specific lymphocytes. Hemocyanins are large extracellular oxygen transport glycoproteins isolated from different arthropod and mollusk species which exhibit strong anticancer properties. Immunized in mammals they trigger Th1 immune response that promotes unspecific stimulation and adjuvant activity in experimental therapeutic vaccines for cancer and antibody development. In the present study we used two hemocyanins - one isolated from marine snail Rapana thomasiana (RtH) and another one, from the terrestrial snail Helix pomatia (HpH) which have been investigated by using different administration schedules (intensive and mild) in murine model of colon carcinoma. The treatment with RtH and HpH generated high levels of antitumor IgG antibodies, antibody-producing plasma cells and tumor-specific CTLs, stimulated secretion of proinflammatory cytokines, suppressed the manifestation of carcinoma symptoms as tumor growth and size, and prolonged the life span of treated mice. Our results showed a significant anti-cancer effect of RtH and HpH hemocyanins on a murine model of colon carcinoma with promising potential for immunotherapy in various schemes of administration based on cross-reactive tumor-associated epitopes.

Folate-conjugated Helix lucorum hemocyanin - preparation, stability, and cytotoxicity.

This is the first report on the modification of a hemocyanin from Helix lucorum (HlH), a large molluscan respiratory protein, with folic acid (FA). In a two-step synthetic reaction, we prepared samples of HlH conjugated with 20 and 50 FA residues denoted as FA-HlH-1 and FA-HlH-2, respectively. Comparison of the attenuated total reflectance-Fourier transform infrared spectra in the amide I band region showed a structural rearrangement in the HlH that is due to FA conjugation. The changes in the secondary structure were more noticeable for FA-HlH-2. The thermal stability of HlH was not significantly affected by the FA modification, which is consistent with the observed structural similarities with the native protein. Preliminary cytotoxicity assays showed that FA-HlH-1 and FA-HlH-2 stimulate fibroblast proliferation when applied in concentrations of 50 and 100 µg/well. A negligible reduction of fibroblast growth was observed only for FA-HlH-1 and FA-HlH-2, exposed to 200 µg/well for 48 h. We found that FA-HlH-2 exhibits a low to moderate cytotoxic effect on two breast cancer cell lines, which express folate receptors, a hormone-dependent (MCF-7) and a hormone-independent (MDA-MB-231). FA-HlH-2 protects nontransformed cells and affects only neoplastic cells, which could be an advantage, and the protein could have potential in combination with other chemotherapeutics.

Biophysical Properties and Cytotoxicity of Feruloylated Helix Lucorum Hemocyanin.

For the first time Helix lucorum hemocyanin (HlH) has been feruloylated. Two HlH conjugates with 40- and 120- ferulic acid residues were prepared, denoted as FA-HlH-1 and FA-HlH-2. Expectedly, the feruloylation of HlH induced a rearrangement of the protein molecule, a decrease in the ?-helical structure at the expense of ß-structures was observed. Besides, the FA-HlH conjugates were more prone to aggregation, which is probably due to the stabilization of the partially unfolded protein molecules by non-covalent bonding. Interestingly, the thermal stability of HlH was not affected by the modification. The native and feruloylated HlH were not toxic to normal fibroblasts (BJ cells). We observed a decrease in cell viability of breast cancer MCF-7 cells to about 66% after a 48h exposure to 70 µg/well of FA-HlH-2.

Crayfish hemocyanin on chitin bone substitute scaffolds promotes the proliferation and osteogenic differentiation of human mesenchymal stem cells.

Crustacean chitin-hemocyanin-calcium mineral complexes were designed as bone biomimetics, with emphasis on their ability to bind or release calcium ions. Chitin scaffolds were prepared by dissolving chitin flakes in LiCl/dimethylacetamide, followed by gel formation and freeze-drying. Some of these scaffolds were modified by incorporation of CaCO3 . In some of the chitin-CaCO3 scaffolds, macroporosity was introduced by HCl treatment. Hemocyanin from the crayfish Cherax quadricarinatus was used to further modify the chitin scaffolds by dip coating. Cytocompatibility, cellular adherence and proliferation of human mesenchymal stem cells (hMSCs) were evaluated in terms of cell number as reflected in lactate dehydrogenase activity. The chitin, chitin-CaCO3 , and porous chitin-CaCO3 scaffolds were all found to facilitate cell attachment. Hemocyanin dip-coating of these scaffolds led to increased initial cell adhesion, enhanced proliferation, and osteogenic differentiation. Since the hemocyanin loading of the scaffolds was impaired by sterilization by gamma-irradiation (as required for biomedical applications), the hemocyanin loading was performed on previously sterilized scaffolds. All scaffolds facilitated osteogenic differentiation of osteoblasts, with the highest cell ALP-activity being found on hemocyanin-modified porous chitin-CaCO3 scaffolds. Thus, chitin-hemocyanin scaffolds enhanced the initial stages of bone cell development and could serve as promising biomaterials for bone regeneration.

Immunological characteristics of Interleukin-2 receptor subunit beta (IL-2Rß) in flounder (Paralichtlys olivaceus): Implication for IL-2R function.

Interleukin-2 receptor subunit beta of flounder (Paralichthys olivace, fIL-2Rß) was annotated on the NCBI, its gene was cloned and characterized functionally in this study. And then the amino acids sequences and tertiary structure of fIL-2Rß were analyzed, respectively. RT-PCR and ImageJ analyzed showed that fIL-2Rß mRNA were expressed in the gill, spleen, kidney, intestines, liver, blood, muscle and skin, which showed high signals in spleen and blood. And then the recombinant protein of fIL-2Rß extracellular region and its polyclonal antibodies were produced, native fIL-2Rß molecules in flounder peripheral blood leukocytes (PBLs) were identified at 60.7 kDa by Mass spectrometry, which were in accordance with the molecular mass of full fIL-2Rß protein calculated on the predicted protein sequence. Then the IL-2Rß+ cell in T/B lymphocytes were characterized by Flow cytometry and indirect immunofluorescence assay, respectively. The results showed that the percentages of IL-2Rß+ leukocytes, IL-2Rß+/CD4+, IL-2Rß+/IgM+ lymphocytes were 18.4 ± 2.7%, 4.5 ± 0.8%, 4.3% ± 0.5 in PBLs, and were 13.6 ± 0.9%, 4.6 ± 1.1%, 6.1% ± 0.4 in spleen, similarly, the percentages of IL-2Rß+ leukocytes, IL-2Rß+/CD4+, IL-2Rß+/IgM+ lymphocytes were 9.4 ± 0.3%, 4.0 ± 0.5%, 5.7 ± 0.1% in head kidney, respectively. After KLH injection, compared with control group, the gene expression of IL-2, IL-2Rß, CD3, TCR, CD79b and IgM in spleen of flounder were up-regulated, respectively (p < 0.05). And the FCM results showed that the percentages of IL-2Rß+ leukocytes in PBLs were significantly increased post Keyhole limpet hemocyanin (KLH) injection, which peaked 23.9 ± 0.9% at 9th day (p < 0.05). To our knowledge, those results first reported that the characteristics of IL-2R and IL-2R + molecules were expressed on both B and T lymphocytes in fish. At the same time, this study lays a foundation for further exploring the interaction between IL-2 and IL-2R to promote cell proliferation and carrying out biological functions.

Paraquat Preferentially Induces Apoptosis of Late Stage Effector Lymphocyte and Impairs Memory Immune Response in Mice.

Paraquat (PQ) is a toxic non-selective herbicide. To date, the effect of PQ on memory immune response is still unknown. We investigated the impact of PQ on memory immune response. Adult C57BL/6 mice were subcutaneously injected with 2 mg/kg PQ, 20 mg/kg PQ or vehicle control every three days for two weeks. A single injection of keyhole limpet hemocyanin (KLH) at day four after the initial PQ treatment was used to induce a primary immune response; a second KLH challenge was performed at three months post the first KLH immunization to induce a secondary immune response. In steady state, treatment with 20 mg/kg PQ reduced the level of serum total IgG, but not that of IgM; treatment with 20 mg/kg PQ decreased the number of effector and memory lymphocytes, but not naïve or inactivated lymphocytes. During the primary immune response to KLH, treatment with 20 mg/kg PQ did not influence the proliferation of lymphocytes or expression of co-stimulatory molecules. Instead, treatment with 20 mg/kg PQ increased the apoptosis of lymphocytes at late stage, but not early stage of the primary immune response. During the secondary immune response to KLH, treatment with 20 mg/kg PQ reduced the serum anti-KLH IgG and KLH-responsive CD4 T cells and B cells. Moreover, effector or activated lymphocytes were more sensitive to PQ-induced apoptosis in vitro. Treatment with 2 mg/kg PQ did not impact memory immune response to KLH. Thus, treatment with 20 mg/kg PQ increased apoptosis of late stage effector cells to yield less memory cells and thereafter impair memory immune response, providing a novel understanding of the immunotoxicity of PQ.