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.

Lupus progression deteriorates oogenesis quality in MRL/lpr mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the activation of the immune response against self antigens. Numerous reproductive complications, including reduced birth rate and complications for the mother and the fetus during pregnancy, have been observed in women with SLE. In the present study, we aimed to investigate the effect of SLE development on oocyte meiosis in lupus-prone mice. Lupus-prone MRL/lpr mice were used for the experiments: disease-free (4 weeks of age) and sick (20 weeks of age, virgin and postpartum). The immune response was monitored by flow cytometry, ELISpot, ELISA, and histology. Oocytes were analyzed by fluorescence microscopy based on chromatin, tubulin, and actin structures. The lupus-prone MRL/lpr mice developed age-dependent symptoms of SLE with increased levels of various autoantibodies, proteinuria, and renal infiltrates and a tendency for the immune response to worsen with changes in cell populations and the cytokine profile. The number and quality of oocytes were also affected, and the successful pregnancy rate of MRL/lpr mice was limited to only 60%. Isolated oocytes showed severe structural changes in all studied groups. Systemic alterations in immune homeostasis in SLE affect the quality of developing oocytes, which is evident from a young age. The data obtained is in line with the trend of reduced fertility in lupus-prone MRL/lpr mice. The phenomenon can be explained by changes in the microenvironment of the relevant organs and close connection between ovulation and inflammatory processes.

DNA and protein-generated chimeric molecules for delivery of influenza viral epitopes in mouse and humanized NSG transfer models.

Purified subunit viral antigens are weakly immunogenic and stimulate only the antibody but not the T cell-mediated immune response. An alternative approach to inducing protective immunity with small viral peptides may be the targeting of viral epitopes to immunocompetent cells by DNA and protein-engineered vaccines. This review will focus on DNA and protein-generated chimeric molecules carrying engineered fragments specific for activating cell surface co-receptors for inducing protective antiviral immunity. Adjuvanted protein-based vaccine or DNA constructs encoding simultaneously T- and B-cell peptide epitopes from influenza viral hemagglutinin, and scFvs specific for costimulatory immune cell receptors may induce a significant increase of anti-influenza antibody levels and strong CTL activity against virus-infected cells in a manner that mimics the natural infection. Here we summarize the development of several DNA and protein chimeric constructs carrying influenza virus HA317-41 fragment. The generated engineered molecules were used for immunization in intact murine and experimentally humanized NSG mouse models.

Exploring the Role of CD74 and D-Dopachrome Tautomerase in COVID-19: Insights from Transcriptomic and Serum Analyses.

The COVID-19 pandemic has posed a significant threat to public health worldwide. While some patients experience only mild symptoms or no symptoms at all, others develop severe illness, which can lead to death. The host immune response is believed to play a crucial role in determining disease severity. In this study, we investigated the involvement of CD74 and D-DT in COVID-19 patients with different disease severities, by employing an in silico analysis of a publicly available transcriptomic dataset and by measuring their serum levels by ELISA. Our results showed a significant increase in MIF levels in PBMCs from COVID-19 patients, as well as a significant increase in the D-DT levels in PBMCs. However, we observed no modulation in the serum levels of D-DT. We also observed a concordant reduction in the serum levels and PBMCs expression levels of CD74. Furthermore, we found a negative correlation between CD74 serum levels and IL-13. In conclusion, our study sheds light on the involvement of CD74 and D-DT in COVID-19, with potential implications for disease severity and treatment. Further studies are needed to fully elucidate the mechanisms underlying these observations and to explore the potential therapeutic value of targeting CD74 and IL-13 in COVID-19.

Crocus sativus Extract as a Biological Agent for Disease-Modifying Therapy of Collagenase-Induced Mouse Model of Osteoarthritis.

OBJECTIVES: Osteoarthritis (OA) is an age-related joint disease that involves the degeneration of cartilage and is the most prevalent form of arthritis, affecting a large part of the population. OA is a multifactorial disorder, and no single etiological mechanism has been found to be common to all forms of the disease. Currently used therapies for control of the disease are mainly nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroid medications. The aim of this study was to investigate the extract from Crocus sativus as a biological disease-suppressing therapy agent. METHODS: Balb/c mice were injected intra-articularly with Clostridium histolyticum type IA for induction of osteoarthritis. The mice were randomized to five groups: control group, I group (CIOA untreated), II group (CIOA + 100 mg/kg/daily saffron), III group (CIOA + 50 mg/kg/daily saffron), IV group (CIOA + 25 mg/kg/daily saffron). Flow-cytometry analysis was used to study the splenocytes' phenotype isolated from the treated animals. The serum levels of inflammatory and anti-inflammatory cytokines were analyzed with ELISA. The histological assessment was used to analyze the saffron extract effect on histopathological alterations. RESULTS: Saffron treatment significantly decreased osteoarthritis-associated joint histological manifestations and decreased serum TNFα levels. The flow-cytometry analysis showed a decrease in pro-inflammatory immune cell subtypes in the spleen. CONCLUSIONS: The results obtained suggest that saffron affected the disease progression and could be a potential therapeutic approach in osteoarthritic patients' therapy.

Targeted suppression of Dpt-specific B cells in humanized Rag2- γc- mouse model of HDM allergy.

Der p 1 is one of the major allergenic molecules of Dermatophagoides pteronyssinus, causing house dust mite (HDM) allergy. The pathological B cells produce allergen-specific IgE antibodies that mediate the hypersensitivity reaction, therefore the selective elimination of these B cells is a legitimate therapeutic goal in allergy. Chimeric molecule Dp51-72 able to cross-link B cell inhibitory complement receptor type 1 and BCR on Der p 1-specific B cells was constructed. The signalling capabilities of this molecule have been tested on human B cells. A humanized mouse model of HDM allergy has been used to test the in vivo effects of the chimeric molecule administration. Administering the chimeric molecule to immunodeficient Rag2- γc- mice transferred with PBMCs from allergic patients resulted in reduction of allergen-specific IgE antibodies in the sera, and reduced infiltration of immune cells in lung histology preparations. Reduced numbers of human CD45+ and CD4+ cells in the lungs as well as inhibition of mast cell degranulation were also observed. The treatment with Dp51-72 chimera significantly decreased the local levels of anti-Dpt IgE antibodies in the bronchoalveolar lavage fluid (BALF). The binding of the chimeric molecule to tonsillar B cells triggers the tyrosine phosphorylation of 30-32 kDa protein, which is most likely involved in the inhibitory process. Administration of constructed chimeric molecules to humanized mice with developed inflammation resulted in specific suppression of disease-associated IgE antibody-producing cells and preserved lung histology. This effective approach could be further developed into a therapeutic agent for treatment of patients with HDM allergy.

Selective Silencing of Disease-Associated B Lymphocytes from Hashimoto's Thyroiditis Patients by Chimeric Protein Molecules.

Hashimoto's thyroiditis is one of the most common endocrine disorders, affecting up to 20% of the adult population. No treatment or prevention exists except hormonal substitution for hypothyroidism. We hypothesize that it may be possible to selectively suppress anti-thyroglobulin (Tg) IgG antibody-producing B lymphocytes from HT patients by a chimeric protein molecule containing a monoclonal antibody specific for the human inhibitory receptor CR1, coupled to peptide epitopes derived from Tg protein. We expect that this treatment will down-regulate B-cell autoreactivity by delivering a strong inhibitory signal. Three peptides-two epitope-predicted ones derived from Tg and another irrelevant peptide-were synthesized and then coupled with monoclonal anti-human CR1 antibody to construct three chimeric molecules. The binding to CD35 on human B cells and the effects of the chimeric constructs on PBMC and TMC from patients with HT were tested using flow cytometry, ELISpot assay, and immunoenzyme methods. We found that after the chemical conjugation, all chimeras retained their receptor-binding capacity, and the Tg epitopes could be recognized by anti-Tg autoantibodies in the patients' sera. This treatment downregulated B-cell autoreactivity and cell proliferation, inhibited Tg-specific B-cell differentiation to plasmablasts and promoted apoptosis to the targeted cells. The treatment of PBMCs from HT patients with Tg-epitope-carrying chimeric molecules affects the activity of Tg-specific autoreactive B lymphocytes, delivering to them a strong suppressive signal.

Jewel Orchid's Biology and Physiological Response to Aquaponic Water as a Potential Fertilizer.

Ludisia discolor is commonly known as a jewel orchid due to its variegated leaves. Easy maintenance of the orchid allows it to be used as a test system for various fertilizers and nutrient sources, including aquaponic water (AW). First, we applied DNA barcoding to assess the taxonomic identity of this terrestrial orchid and to construct phylogenetic trees. Next, the vegetative organs (leaf, stem, and root) were compared in terms of the level of metabolites (reducing sugars, proteins, anthocyanins, plastid pigments, phenolics, and antioxidant activity) and nutrient elements (carbon, nitrogen, sodium, and potassium), which highlighted the leaves as most functionally active organ. Subsequently, AW was used as a natural source of fish-derived nutrients, and the orchid growth was tested in hydroponics, in irrigated soil, and in an aquaponic system. Plant physiological status was evaluated by analyzing leaf anatomy and measuring chlorophyll content and chlorophyll fluorescence parameters. These results provided evidence of the beneficial effects of AW on the jewel orchid, including increased leaf formation, enhanced chlorophyll content and photosystems' productivity, and stimulated and prolonged flowering. The information acquired in the present study could be used in addressing additional aspects of the growth and development of the jewel orchid, which is also known for its medicinal value.

Collagenase-Induced Mouse Model of Osteoarthritis-A Thorough Flow Cytometry Analysis.

OBJECTIVES: Osteoarthritis (OA) is a chronic degenerative disorder of the joint characterized by cartilage breakdown and synovial inflammation. A number of different cells of innate and adaptive immunity contribute to joint pathology during OA inflammation. The interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is still unknown. The objective of this study was to investigate the role of the different types of immune cells in the development of OA. METHODS: Collagenase-induced osteoarthritis was induced in Balb/c mice; flow cytometry analysis; and histopathological damages were assessed in histological sections stained with H&E, Toluidine blue, and Safranin O. RESULTS: Flow cytometry analysis showed B lymphocyte infiltration in the active phase of inflammation and an increase in the effector T cell population into the synovium. An increased activation state of cytotoxic T cells and of NK cell populations in the spleen and synovium was also found. The differentiation of NK cells from a cytotoxic phenotype in early OA to cells with an effector phenotype in the chronic phase of the disease followed. CONCLUSIONS: A number of different cells contribute to inflammatory processes in OA. The correlation between their phenotype and the inflammatory pathophysiology could result in the development of novel approaches to suppress destructive changes in the joint.

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.

Morphometric and Nanomechanical Features of Platelets from Women with Early Pregnancy Loss Provide New Evidence of the Impact of Inherited Thrombophilia.

Pregnancy is associated with hypercoagulation states and increased thrombotic risk, especially in women with thrombophilia. We combine atomic force microscopy (AFM) and flow cytometry to examine the morphology and nanomechanics of platelets derived from women with early pregnancy loss (EPL) and control pregnant (CP) and non-pregnant (CNP) women. Both control groups exhibit similar morphometric parameters (height and surface roughness) and membrane stiffness of platelets. EPL patients' platelets, on the other hand, are more activated than the control groups, with prominent cytoskeletal rearrangement. In particular, reduced membrane roughness (22.9 ± 6 nm vs. 39.1 ± 8 nm) (p < 0.05) and height (692 ± 128 nm vs. 1090 ± 131 nm) (p < 0.05), strong alteration in the membrane Young modulus, increased production of platelets' microparticles, and higher expression of procoagulant surface markers, as well as increased occurrence of thrombophilia (FVL, FII20210A, PLA1/A2, MTHFR C677T or 4G/5G PAI-1) polymorphisms were found. We suggest that the carriage of thrombophilic mutations triggers structural and nanomechanical abnormalities in platelets, resulting in their increased activation. The activation state of platelets can be well characterized by AFM, and the morphometric and nanomechanical characteristics might serve as a new criterion for evaluation of the cause of miscarriage and offer the prospect of an innovative approach serving for diagnostic purposes.

Anti-ANX A1 Antibody Therapy in MRL/lpr Murine Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease characterized by dysfunction of immune regulation, overproduction of inflammatory cytokines and attack on normal tissues by self-reactive cells and antibodies. The main role in the pathogenesis plays the autoreactive tandem of B-T cells, responsible for lupus progression and acceleration. Both activated B and T cells express a phospholipid binding protein Annexin A1 and abnormal levels of the protein were found in murine and human autoimmune syndromes, potentiating its role as a therapeutic target. Here, using anti-annexin A1 antibody we explore its property to modulate the autoimmune response in MRL/lpr mouse model of lupus. Anti-ANX A1 antibody was tested in vitro using spleen cells from MRL/lpr mice to determine the effect on lymphocyte activation, plasma cells differentiation, apoptosis and proliferation by flow cytometry and ELISpot assays. Subsequently, several groups of young (disease-free) and old (sick) MRL/lpr mice were treated with the antibody to determine the levels of panel auto-antibodies and cytokines, T cell arrest and migration. Treatment of splenocytes with anti-ANX A1 antibody inhibited T-cell activation and proliferation, suppressed anti-dsDNA antibody-producing plasma cells and affected B cell apoptosis. Administration of the antibody to MRL/lpr mice resulted to decreased autoantibody levels to various lupus antigens, suppressed T cell migration from lymph nodes and increased the levels of IL4 mRNA compared to the control group. Anti-ANX A1 antibody therapy suppresses B and T cell over-activation and down- modulates disease activity.

Monoclonal antibody therapy that targets phospholipid-binding protein delays lupus activity in MRL/lpr mice.

Systemic lupus erythematosus is an autoimmune syndrome characterized by the development of autoantibodies to a wide range of antigens. Together with B cells, respective self-reactive T cells have an important contribution in disease progression as being responsible for inflammatory cytokines secretion, B cell activation and promoting amplification of the autoimmune response. Annexin A1 is expressed by many cell types and binds to phospholipids in a Ca2+ -dependent manner. Abnormal expression of annexin A1 was found on activated B and T cells in both murine and human autoimmunity suggesting its potential role as a therapeutic target. In the present study, we have investigated the possibility to suppress autoimmune manifestation in spontaneous mouse model of lupus using anti-annexin A1 antibody. Groups of lupus-prone MRL/lpr mice were treated with the anti-annexin A1 monoclonal antibody, and the disease activity and survival of the animals were following up. Flow cytometry, ELISA assays, and histological and immunofluorescence kidney analyses were used to determine the levels of Annexin A1 expression, cytokines, anti-dsDNA antibodies and kidney injuries. The administration of this monoclonal antibody to MRL/lpr mice resulted in suppression of IgG anti-dsDNA antibody production, modulated IL-10 secretion, decreased disease activity and prolonged survival compared with the control group.

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.

Suppression of Disease-Associated B Lymphocytes by GAD65 Epitope-Carrying Protein-Engineered Molecules in a Streptozotocin-Induced Mouse Model of Diabetes.

Type 1 diabetes mellitus is an autoimmune syndrome defined by the presence of autoreactive T and B cells, which results in destruction of insulin-producing beta cells. Autoantibodies against GAD65 (glutamic acid decarboxylase 65)-a membrane-bound enzyme on pancreatic beta cells, contribute to beta cells' destruction and the loss of pancreatic functions. Mouse FcγRIIb on B lymphocytes possesses an inhibitory effect on the activity of these cells. We hypothesized that it may be possible to suppress GAD65-specific B cells in mice with diabetes using chimeric molecules, containing an anti-FcγRIIb antibody, coupled to peptide B/T epitopes derived from the GAD65 protein. With these engineered chimeras, we expect to selectively co-cross-link the anti-GAD65-specific B cell receptor (BCR) and FcγRIIb, thus delivering a suppressive signal to the targeted B cells. An anti-FcγRIIb monoclonal antibody and two synthetic peptide epitopes derived from the GAD65 molecule were used for chimeras' construction. The suppressive activity of the engineered molecules was tested in vivo in mice with streptozotocin (STZ)-induced type 1 diabetes. These chimeric molecules exclusively bind disease-associated B cells by recognizing their GAD65-specific BCR and selectively deliver a strong inhibitory signal through their surface FcγRIIb receptors. A reduction in the number of anti-GAD65 IgG antibody-secreting plasmocytes and an increased percentage of apoptotic B lymphocytes were observed after treatment with protein-engineered antibodies of mice with STZ-induced type 1 diabetes.

Correction: Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

[This corrects the article DOI: 10.1371/journal.pone.0129108.].

Targeting of Influenza Viral Epitopes to Antigen-Presenting Cells by Genetically Engineered Chimeric Molecules in a Humanized NOD SCID Gamma Transfer Model.

Antiviral DNA vaccines are a novel strategy in the vaccine development field, which basically consists of the administration of expression vectors coding viral antigen sequences into the host's cells. Targeting of conserved viral epitopes by antibody fragments specific to activating cell surface co-receptor molecules on antigen-presenting cells could be an alternative approach for inducing protective immunity. It has been shown that FcγRI on human monocytes enhances antigen presentation in vivo. Various DNA constructs, encoding a Single-chain variable antibodies (scFv) from mouse anti-human FcγRI monoclonal antibody, coupled to a sequence encoding a T- and B-cell epitope-containing influenza A virus hemagglutinin inter-subunit peptide were inserted into the eukaryotic expression vector system pTriEx-3 Neo. The constructed chimeric DNA molecules were expressed by transfected Chinese hamster ovary cells and the ability of the engineered proteins to interact with FcγRI-expressing cells was confirmed by flow cytometry. The fusion protein induced a strong signal transduction on human monocytes via FcγRI. The expression vector pTriEx-3 Neo containing the described construct was used as a naked DNA vaccine and introduced directly to experimental humanized NOD SCID gamma mice with or without boosting with the expressed fusion protein. Immunization with the generated DNA chimeric molecules and prime-boost with the expressed recombinant proteins induced significant serum levels of anti-influenza immunoglobulin G antibodies and strong cytotoxic T lymphocyte activity against influenza virus-infected cells in humanized animals.

Annexin A1 as a target for managing murine pristane-induced systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a polygenic pathological disorder which involves multiple organs. Self-specific B cells play a main role in the lupus pathogenesis by generating autoantibodies as well as by serving as important autoantigen-presenting cells. Autoreactive T lymphocytes, on the other hand, are responsible for B cell activation and proliferation, and cytokine production. Therefore, both factors promote the idea that a down-modulation of activated self-reactive T and B cells involved in the pathogenic immune response is a reasonable approach for SLE therapy. Annexin A1 (ANX A1) is expressed by many cell types and binds to phospholipids in a Ca2+ dependent manner. Abnormal expression of ANX A1 was found on activated B and T cells in both murine and human autoimmunity, suggesting its potential role as a therapeutic target. While its role on T lymphocytes is through formyl peptide receptor-like molecules (FPRL), and the formed ANX A1/FPRL pathway modulates T cell receptor signalling, there is still no fool-proof data available for the role of ANX A1 in B cells. We employed a lupus model of Balb/c mice with pristane-induced SLE which very closely resembles human lupus. In the present study, we investigated the possibility to modulate the autoimmune response in a pristane-induced mouse model of SLE using an anti- ANX A1 antibody. Administration of this monoclonal antibody resulted in the inhibition of T-cell activation and proliferation, suppression of IgG anti-dsDNA antibody-secreting plasma cells and of proteinuria, decreased disease activity and prolonged survival compared to control group.

Long-Time Cooling before Cryopreservation Decreased Translocation of Phosphatidylserine (Ptd-L-Ser) in Human Ovarian Tissue.

OBJECTIVES: To translocation (externalization) of phosphatidylserine lead at least the five negative effects observed during cells cryopreservation: hypoxia, increasing of intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species (ROS) and lipid peroxidation. The aim of this study was to test the intensiveness of the phosphatidylserine translocation immediately after thawing and after 45 d xenografting of human ovarian tissue, which was either frozen just after operative removal from patient or cooled before cryopreservation to 5°C for 24 h and then frozen. MATERIALS AND METHODS: Ovarian fragments from twelve patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following four groups. Pieces of Group 1 (n=30) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n=30) after operation were cooled to 5°C for 24 h, then frozen after 24 h pre-cooling to 5°C, thawed and just after thawing their quality was analyzed. Group 3 pieces (n=30) were frozen immediately after operation without pre-cooling, thawed, transplanted to SCID mice and then, after 45 d of culture their quality was analyzed. Group 4 pieces (n=30) were frozen after 24 h pre-cooling to 5°C, thawed, transplanted to SCID mice and then, after 45 d their quality was analyzed. The effectiveness of the pre-freezing cooling of tissuewas evaluated by the development of follicles (histology) and by intensiveness of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). RESULTS: For groups 1, 2, 3 and 4 the mean densities of follicles per 1 mm3 was 19.0, 20.2, 12.9, and 12.2, respectively (P1-2, 3-4 >0.1). For these groups, 99%, 98%, 88% and 90% preantral follicles, respectively were morphologically normal (P1-2, 3-4 >0.1). The FACS analysis showed significantly decreased intensiveness of translocation of phosphatidylserine after pre-cooling of frozen tissue (46.3% and 33.6% in Groups 2 and 4, respectively), in contrast with tissue frozen without pre-cooling (77.1% and 60.2 % in Groups 1 and 3, respectively, P1, 3-2, 4 <0.05). CONCLUSIONS: Long time (24 h) cooling of ovarian tissue to 5°C before cryopreservation decreased translocation of phosphatidylserine that evidences about increases the viability of the cells in the tissue after thawing.

Helix pomatia hemocyanin - a novel bio-adjuvant for viral and bacterial antigens.

BACKGROUND: New generated subunit vaccines are characterized by increased safety and lack of side effects, however they suffer from weak immunogenicity. The adjuvants are substances that have the ability to enhance the magnitude and duration of the immune response and to increase vaccine efficacy, but the different vaccines may require diverse adjuvants. The urgent need of novel adjuvant formulations occurs, thus ensuring protective cellular and humoral responses against infectious pathogens. The hemocyanins, oxygen binding copper proteins in the hemolymph of molluscs and arthropods, are widely used as peptide carriers and vaccine adjuvants. RESULTS: In the present study we promote the hemocyanin isolated from the terrestrial gastropod Helix pomatia (HPH) as bio-adjuvant, combined with standard antigens. The purified HPH combined with influenza virus hemagglutinin intersubunit peptide (IP) or with tetanus toxoid (TT) were used for immunization. Administration of tetanus toxoid combined with HPH in mice resulted in an increased number of anti-TT IgG producing plasmocytes and induced a significant increase of B and T cell proliferation. The level of the anti-TT IgG antibodies in mice sera was comparable to the group administered with TT+Al(OH)3. An immunization of experimental animals with IP combined with H. pomatia hemocyanin led to generation of strong anti-influenza cytotoxic response. CONCLUSION: The vaccination of mice demonstrates that the HPH is acceptable as a potential bio-adjuvant for subunit vaccines and it could be used as a natural adjuvant or protein carrier.