Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate

Background: Phosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments. Methods: Ovarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5–3.0 × 1.5–3.0 × 0.5–0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5–3.0 × 1.5–3.0 × 1.5–2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). Results: For Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative). Conclusion: The presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue

Selective Silencing of DNA-Specific B Lymphocytes Delays Lupus Activity in Mrl/Lpr Mice

The pathological DNA-specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross- links their surface immunoglobulins with the inhibitory Fc gamma IIb surface receptors. A hybrid molecule was constructed by coupling the DNA- mimicking DWEYSVWLSN peptide to a monoclonal anti-mouse Fc gamma RIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/1pr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti-DNA but not of anti-diphtheria IgG antibody-producing cells. Intravenous infusions with the DNA-peptide antibody chimera to 7-wk-old animals prevented the appearance of IgG anti- DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA-peptide chimeric antibody to 18 wk-old mice with full-blown disease resulted in the maintenance of a flat level of IgG anti-DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis . The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease-associated B cells in autoimmune diseases

Efficacy and safety of Aviron Rapid&amp;reg; in 18-60-year-old patients with clinical diagnosis of acute respiratory viral infection: a multicenter, randomized, double-blind, placebo-controlled clinical trial

Aim: Prevention and treatment of upper respiratory tract infections are given the highest priority because of the tremendous health and economic impact of these diseases. Development of novel effective and safe options for treatment can contribute considerably to decrease the burden of disease.Materials and methods: We designed a multicenter, randomized, double-blind, placebo-controlled study in ambulatory-treated adult patients with a clinical diagnosis of acute upper respiratory tract viral infection. The patients (18-60 years old) were randomized into two groups and followed-up for 5 days. Group 1 received the standard symptomatic therapy + Aviron Rapid&amp;reg;, and Group 2 received the standard symptomatic therapy + placebo. The primary endpoint of the study was defined as the duration of disease measured by the percentages of disease-free patients for every 12-hour period of the study.&amp;nbsp;Results: Treating clinically relevant patients with the natural product Aviron Rapid&amp;reg; for 5 days decreases the duration of disease, the intake of antipyretics and the severity of symptoms. Significant difference between the tested groups for most of studied parameters was found as soon as 12 or 24 hours after initiation of administration in favour of active arm and was the most prominent on days 3 and 4. Significant decrease in the total score of symptoms severity was achieved on day 4 and extended to the end of study. There were no dif-ferences in the adverse events between the groups and the tested product demonstrated excellent safety profile.&amp;nbsp;Conclusions: This study is a clinical confirmation of well documented antiviral activity of the product targeting multiple points in viral replication and covering broad spectrum viral pathogens

<i>Crocus sativus</i> 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

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

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

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

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&ndash;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