Mononuclear Fraction Research Articles

Transplantation of Cultured Mononuclear Fraction of Bone Marrow Cells in the Treatment of Heymann Nephritis in Rats.

In an experiment on Wistar rats, a Heymann's active nephritis model was reproduced. After the chronic course of the disease was confirmed, we compared the effectiveness of single systemic and local transplantation of allogeneic cultured stromal cells of the mononuclear fraction of the bone marrow. Both methods of cell therapy reduced clinical manifestations of active Heymann's nephritis: proteinuria decreased and glomerular filtration rate increased 30 days after cell administration. At the histological level, signs of the inflammatory process manifested in an increase in the number of CD68+ cells. Thus, in the case of autoimmune glomerulonephritis, two transplantation methods provide a comparable level of correction of functional disorders in Heymann's active nephritis model.

The Role of Decreased Expression of Glucocorticoid Receptors in the Development of Itsenko-Cushing Syndrome

Background: Itsenko-Cushing syndrome is caused by unregulated excessive cortisol incretion by the adrenal cortex. The causes of increased stimulation of cortisol incretion by the adrenal cortex, leading to hyperplasia and tumors (adenoma and cancer) of the adrenal cortex, as well as to adenoma of the pituitary gland itself, remain unclear to date. Materials and Methods: 6 patients with adrenal Itsenko-Cushing syndrome with adrenal cortex adenoma were under observation. The age of the patients ranged from 24 to 87 years. The patients underwent removal of the adrenal gland on the affected side. The control group consisted of 6 individuals who underwent surgical treatment of varicocele and hernias of various localization. In patients of the main group and control group, with their consent, a small fragment of skin from the edge of the surgical wound was taken for examination, which were fixed in a 10% formalin solution. Results: The presence of adrenal Itsenko-Cushing syndrome in patients of the main group was confirmed by laboratory and instrumental studies, as well as characteristic clinical manifestations. The average Histochemical score of the expression of glucocorticoid receptors (GR) of the skin in patients of the main group with Itsenko-Cushing adrenal syndrome was 2.7 times less than the same value in the control group. Conclusions: A decrease in the formation or absence of glucocorticoid receptors (congenital or acquired) blocks the negative feedback of cortisol suppression of corticoliberin production by the hypothalamus and ACTH-pituitary gland. The resulting excessive incretion of corticoliberin becomes the main link in pathogenesis of pituitary adenoma formation, and an excessive incretion of ACTH leads to the formation of cortical hyperplasia and tumors (adenomas, cancers) of the adrenal cortex. Unregulated excessive cortisol production by hyperplastic adrenal tissues or benign/malignant adrenal tumors leads to overcoming resistance to glucocorticoids and, according to the principle of negative feedback, suppresses the production of corticoliberin by the hypothalamus and ACTH-pituitary gland. As a result, the pituitary form of Itsenko-Cushing syndrome turns into an adrenal form with an unregulated increase in cortisol levels and a decrease in ACTH levels. Transfusion of the mononuclear fraction of peripheral blood obtained from healthy donors, which allows the pluripotent stem cells contained in it to partially replace the altered cells of all tissues of patients, is a promising method of treating patients with Itsenko-Cushing syndrome due to a hereditary decrease in the expression of glucocorticoid receptors.

In vitro results with minimal blood toxicity of a combretastatin A4 analogue.

Cancer is a disease caused by uncontrolled cell growth that is responsible for several deaths worldwide. Breast cancer is the most common type of cancer among women and is the leading cause of death. Chemotherapy is the most commonly used treatment for cancer; however, it often causes various side effects in patients. In this study, we evaluate the antineoplastic activity of a parent compound based on a combretastatin A4 analogue. We test the compound at 0.01mg mL- 1, 0.1mg mL- 1, 1.0mg mL- 1, 10.0mg mL- 1, 100.0mg mL- 1, and 1,000.0mg mL- 1. To assess molecular antineoplastic activity, we conduct in vitro tests to determine the viability of Ehrlich cells and the blood mononuclear fraction. We also analyze the cytotoxic behavior of the compound in the blood and blood smear. The results show that the molecule has a promising antineoplastic effect and crucial anticarcinogenic action. The toxicity of blood cells does not show statistically significant changes.

Anti-adhesive Properties of Epoxy-Treated Xenopericardium Modified with Polyvinyl Alcohol: in vitro Study of Leukocyte Adhesion in the Pulsatile Flow Model.

Fragments of unmodified (control) and modified with polyvinyl alcohol epoxy-treated bovine pericardium were incubated in the dedicated chambers connected to a pulsatile flow system (Ibidi GmbH, Germany). During 48 h incubation was conducted in whole donor plasma containing 3·106 of mononuclear fraction cells. To simulate plasma flow, the shear stress on the inflow and outflow sides of of bioprosthetic heart valve in the aortic position was set to 50 and 20 dynes/cm2, respectively. After the experiment was completed, the surface of the studied samples was subjected to scanning electron microscopy and immunofluorescence using antibodies to the pan-leukocyte marker CD45. Adhesion of leukocytes (CD45+) was seen for both the serous (outflow side) and fibrous (inflow side) surfaces of the control epoxy-treated bovine pericardium, whereas both surfaces of the material modified with polyvinyl alcohol were clear of immune cells. Scanning electron microscopy confirmed the adhesion of leukocytes to intact biological tissue: the cells on the surface of the control xenopericardium were of an irregular shape and formed numerous pseudopodia. The suggested modification of epoxy-treated bovine pericardium with polyvinyl alcohol prevents the adhesion of immune cells to the implant surface and can potentially protect bioprosthetic heart valves from immune rejection.

Классические и активирующие химерные антигенные рецепторы PD-1 как элемент мультитаргетного подхода в лечении гематологических и солидных новообразований

Aim. To generate anti-PD-L1 CAR-T effectors carrying extracellular domain PD-1 as antigen-recognizing site and to study their cytolytic activity as well as to functionally assess the anti-PD-L1 CAR-T effectors in vitro with a view to apply them in multi-targeted tumor therapy.
 Materials & Methods. Chimeric antigen receptor PD-1 was constructed using molecular cloning of PD-1 antigen-recognizing region (12–170 amino acids) into mammalian expression plasmid vector adding activation and co-stimulatory domains. Primary Т-lymphocytes of healthy donor peripheral blood mononuclear fraction were derived by expanding monoclonal antibody combination on surface markers CD3/CD28. Anti-PD-L1 CAR-T effectors were obtained by lentiviral transduction of primary T-lymphocyte genome of a healthy donor. Chimeric antigen receptor PD-1 expression and transduction efficiency were assessed by flow cytofluorometry. Specific cytotoxicity of the anti-PD-L1 CAR-T effectors was analyzed in vitro by means of real-time cytotoxicity assay (RTCA) with HeLa_PD-L1 target cell line co-cultivation. The level of cytokines IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A was assessed by flow cytofluorometry using Human Th1/Th2/Th17 CBA Kit (BD, USA).
 Results. The efficiency of lentiviral transduction and the proportion of the anti-PD-L1 CAR-T effectors were 42 %. The specificity of cytotoxic response of the anti-PD-L1 CAR-T effectors with a low effector/tumor ratio (1:20) was verified during HeLa_PD-L1 co-cultivation by a 1.5-fold decrease in the cell index (CI = 0.738) versus control (CI = 1.0645). The increase in synthesis of cytokines IL-2 (1000 pg/mL), IL-6 (438.5 pg/mL), TNF-α (44 pg/mL), and IFN-γ (1034 pg/mL) during HeLa_PD-L1 target cell line co-cultivation confirms the functionality of the analyzed effector cells.
 Conclusion. Anti-PD-L1 chimeric antigen receptor was constructed and tested in vitro. Anti-PD-L1 CAR-T lymphocytes specifically recognize and promote the cytolysis of tumor target cells by increased secretion of pro-inflammatory cytokines IFN-γ, TNF-α, IL-6, and IL-2. Chimeric antigen receptor PD-1 can be modified into chimeric switch receptor (CSR) by deleting CD3ζ-domain and can be used together with other CARs without predicted non-specific toxicity.

Synthetic 1,2,4-triazole-3-carboxamides Induce Cell Cycle Arrest and Apoptosis in Leukemia Cells.

A number of studies demonstrate the efficacy of ribavirin against various cancer types in in vitro and in vivo models. However, ribavirin induces the development of multiple side effects, suggesting a high demand for ribavirin analogues with improved therapeutic indexes. This study was focused on the analysis of ribavirin, its aglycon 1,2,4-triazole-3-carboxamide, and several of its derivatives activities in blood cancer cells in vitro. Four 1,2,4-triazole-3-carboxamide derivatives were designed and synthesized. Antiproliferative effects were evaluated in chronic myeloid leukemia cells К562 and acute lymphoblastic leukemia cells CCRF-SB as well as in the cells of whole blood mononuclear fraction of healthy volunteers by cell counting using the trypan blue exclusion method. Cell cycle distribution and apoptosis under the influence of the compounds were analyzed by flow cytometry with PI staining, and then apoptosis data were confirmed by Western blot analysis for PARP1 and caspase-3 cleavage. We demonstrated the significant antiproliferative effect of 5-(tetrahydropyran-2-yl)-1,2,4-triazole-3- carboxamide and 1-(tetrahydropyran-2-yl)-1,2,4-triazol-3-carboxamide in leukemia cell lines in vitro in comparison to non-transformed monocytes, providing the rationale for further studies of 1,2,4-triazole-3-carboxamide derivatives as anti-leukemia drugs. These results implied that the 1,2,4-triazole-3-carboxamide derivatives exhibited their antiproliferative activities by induction of cell cycle arrest. Consequently, 5-(tetrahydropyran-2-yl)-1,2,4-triazole-3-carboxamide and 1-(tetrahydrofuran-2-yl)-1,2,4-triazol-3-carboxamide may present antimetabolites with potential anticancer efficacy.

Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

BACKGROUND AND AIMS - Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. METHODS - Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins. RESULTS - WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq. In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a >50% reduction in clonogenic potential was observed after nucleofection. CONCLUSIONS - Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade. Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

Stem Cell Therapy in Stargardt Disease: A Systematic Review.

This article aimed to review current literature on the safety and efficacy of stem cell therapy in Stargardt disease. A comprehensive literature search was performed, and two animal and eleven human clinical trials were retrieved. These studies utilized different kinds of stem cells, including human or mouse embryonic stem cells, mesenchymal stem cells, bone marrow mononuclear fraction, and autologous bone marrow-derived stem cells. In addition, different injection techniques including subretinal, intravitreal, and suprachoroidal space injections have been evaluated. Although stem cell therapy holds promise in improving visual function in patients with Stargardt disease, further investigation is needed to determine the long-term benefits, safety, and efficacy in determining the best delivery method and selecting the most appropriate stem cell type.

Quantitative ratio of mRNA expression of IGF/INS system receptors in multiple myeloma

Background. Individuals with increased expression of components of the IGF/INS system, are more likely to develop various malignancies. And in the case when the components of the IGF/INS system are overexpressed in tumors, this adversely affects the prognosis of the disease, including leading to a decrease in relapse-free survival. A characteristic feature of the IGF/INS system is the ability of the same ligands to bind to different receptors and vice versa (cross interactions) and activate different signaling pathways in the cell. This feature of the system requires an integrated approach to the study of the expression of its components, namely, the study of the quantitative ratio of the expression of individual components. The result obtained will make it possible to determine possible combinations of ligand-receptor bonds and, ultimately, will have both prognostic and evaluative value: in terms of a therapeutic target.Aim. To establish the quantitative ratio of mRNA expression of the IGF/INS system receptors: IR-A, IR-B, IGF-1R, and IGF-2R in the IM9 lymphoblastoid cell line and in three myeloma cell lines: RPMI1640, RPMI8226, H929, and to identify frequency of expression of these receptors in the mononuclear fraction of bone marrow aspirates obtained from treated patients with multiple myeloma.Materials and methods. We used human lymphoblastoid cells and 3 types of human myeloma cells, differing in the degree of differentiation and, as well as bone marrow aspirates obtained from 19 treated patients with stage III multiple myeloma. Expression of mRNA in cells was studied by quantitative real-time reverse transcription polymerase chain reaction and in bone marrow aspirate samples by semi-quantitative reverse transcription polymerase chain reaction.Results. During the study, we found that within each cell line, the receptor IR-A is predominant compared to the receptor IR-B. Patients with MM have a high frequency of IR-A expression compared to IR-B. The minimum ratio of IGF-1R:IR-A and IGF-1R:IR-B mRNA is in IM9 lymphoblastoid cells, and for myeloma cells these ratios are high. The ratio of IGF-2R:IR-A is maximum for IM9 lymphoblastoid cells, and for myeloma cells this ratio is three or more times less.Conclusion. Based on the study of the quantitative ratio of receptor mRNA, we state that in myeloma cells there is a high probability of the presence of IGF-1R/IGF-1R and IR-A/IR-A homodimers, and an IGF-1R/IR-A heterodimer. These data have both prognostic and evaluative value, since these combinations of receptors suggest a significant increase in the mitogenic effect due to activation by three ligands: IGF-1, IGF-2 and INS, which is an unfavorable factor, especially when a patient with multiple myeloma with concomitant Diabetes mellitus was prescribed insulin therapy along with chemotherapy. Based on our findings, we recommend simultaneously inhibiting both the IGF-1R receptor and the IR-A receptor as a therapeutic target.

The Effects of Immunosuppressive Drugs on the Characteristics and Functional Properties of Bone Marrow-Derived Stem Cells Isolated from Patients with Diabetes Mellitus and Peripheral Arterial Disease.

Diabetic patients (DPs) with foot ulcers can receive autologous cell therapy (ACT) as a last therapeutic option. Even DPs who have undergone organ transplantation and are using immunosuppressive (IS) drugs can be treated by ACT. The aim of our study was to analyze the effects of IS drugs on the characteristics of bone marrow-derived stem cells (BM-MSCs). The cells were isolated from the bone marrow of DPs, cultivated for 14-18 days, and phenotypically characterized using flow cytometry. These precursor cells were cultured in the presence of various IS drugs. The impact of IS drugs on metabolic activity was measured using a WST-1 assay, and the expression of genes for immunoregulatory molecules was detected through RT-PCR. Cell death was analyzed through the use of flow cytometry, and the production of cytokines was determined by ELISA. The mononuclear fraction of cultured cells contained mesenchymal stem cells (CD45-CD73+CD90+CD105+), myeloid angiogenic cells (CD45+CD146-), and endothelial colony-forming cells (CD45-CD146+). IS drugs inhibited metabolic activity, the expression of genes for immunoregulatory molecules, the production of cytokines, and the viability of the cells. The results indicate that IS drugs in a dose-dependent manner had a negative impact on the properties of BM-MSCs used to treat ischemic diabetic foot ulcers, and that these drugs could affect the therapeutic potential of BM-MSCs.

Human NK cells internalize recombinant major stress protein HSP70

Heat shock proteins 70 kDa (HSP70) protect intracellular proteins from the damaging effects of stress factors of various natures. Moreover, HSP70 play an important role in the vital activity of cells under normal physiological conditions, performing chaperone functions. These functions are realized in the intracellular space; however, in some cases, these proteins are also found on the cell surface and in the extracellular environment. The causes and mechanisms of HSP70 translocation to the cell surface and secretion into the extracellular space have not yet been well understood, but such an unusual localization of HSP70 activates the immune system. The surface HSP70 and their extracellular pool stimulate the cytotoxic activity of NK cells. However, direct experimental evidence for the internalization of HSP70 molecules by NK cells has not yet been demonstrated. This paper presents the results of the interaction of the extracellular HSP70 pool with NK cells from the peripheral blood. The results demonstrated the confirmation of the internalization of exogenous HSP70 molecules by NK cells. To this end, fluorescently labeled recombinant stress-inducible human HSP70 were obtained. The electrophoretic data indicated the absence of protein degradation during the labeling process, the purity and stability of the modified protein. To assess the interaction of HSP70 with NK cells, the fluorescently labeled HSP70 was added to an in vitro culture of NK cells isolated by magnetic separation from the peripheral blood mononuclear fraction and analyzed by confocal microscopy. This analysis indicated that living NK cells internalize extracellular HSP70 with localization both in lysosomes and in phagosomes. Our experiments illustrated for the first time the process of penetration of the extracellular form of HSP70 into these cells. The results suggest that the activation of NK cells under the action of exogenous HSP70 could be associated with the internalization of these protein molecules.

Use of Blood Mononuclear Cells Autologous Fraction for Treatment of Menstrual Disorders (Case Report)

The aim of our work was to investigate the effectiveness of circulating SC in the blood mononuclear fraction to restore normal functioning of the human ovaries. AH270 patient complained to the clinic about an irregular menstrual cycle in the last 6 months (duration of the menstrual cycle from 38 to 60 days). On day 10 of the menstrual cycle, under general anesthesia, 2 ml of a solution containing a fraction of native nucleus-containing cells isolated from a pool of peripheral blood was administered transvaginally to the right and left ovaries. Microscopic examination of these cells showed that the obtained fraction contains polymorphic nucleus-containing cells of different sizes and high viability, which was 95-98%. After 2 months, the patient came for a follow-up examination, during which an ultrasound of the pelvic organs was performed, as well as laboratory diagnostics of blood tests for AMH and FSH levels. Were taken according to the results of ultrasound of the pelvic organs, the following changes were observed - in the right and left ovaries there were 2 antral follicles in each, up to 3 mm in diameter. The duration of the menstrual cycle after the introduction of stem cells has decreased to 30-35 days. Thus, it is shown that the pool of nuclear-containing peripheral blood cells, which contains circulating progenitors and stem cells, has a powerful regenerative effect with concentrated topical administration, which in this case led to a full functional recovery of the ovaries and normalization of the menstrual cycle.

Peripheral blood monocytes as a therapeutic target for marrow stromal cells in stroke patients.

BackgroundSystemic administration of marrow stromal cells (MSCs) leads to the release of a broad range of factors mediating recovery in rodent stroke models. The release of these factors could depend on the various cell types within the peripheral blood as they contact systemically administered MSCs. In this study, we assessed the immunomodulatory interactions of MSCs with peripheral blood derived monocytes (Mϕ) collected from acute stroke patients.MethodsPeripheral blood from stroke patients was collected at 5–7 days (N = 5) after symptom onset and from age-matched healthy controls (N = 5) using mononuclear cell preparation (CPT) tubes. After processing, plasma and other cellular fractions were removed, and Mϕ were isolated from the mononuclear fraction using CD14 microbeads. Mϕ were then either cultured alone or co-cultured with MSCs in a trans-well cell-culture system. Secretomes were analyzed after 24 h of co-cultures using a MAGPIX reader.ResultsOur results show that there is a higher release of IFN-γ and IL-10 from monocytes isolated from peripheral blood at day 5–7 after stroke compared with monocytes from healthy controls. In trans-well co-cultures of MSCs and monocytes isolated from stroke patients, we found statistically significant increased levels of IL-4 and MCP-1, and decreased levels of IL-6, IL-1β, and TNF-α. Addition of MSCs to monocytes increased the secretions of Fractalkine, IL-6, and MCP-1, while the secretions of TNF-α decreased, as compared to the secretions from monocytes alone. When MSCs were added to monocytes from stroke patients, they decreased the levels of IL-1β, and increased the levels of IL-10 significantly more as compared to when they were added to monocytes from control patients.ConclusionThe systemic circulation of stroke patients may differentially interact with MSCs to release soluble factors integral to their paracrine mechanisms of benefit. Our study finds that the effect of MSCs on Mϕ is different on those derived from stroke patients blood as compared to healthy controls. These findings suggest immunomodulation of peripheral immune cells as a therapeutic target for MSCs in patients with acute stroke.

FUNCTIONAL AND PHENOTYPIC CHARACTERISTICS OF CIRCULATING PHAGOCYTES IN RATS WITH DIFFERENT MODELS OF ALZHEIMER’S DISEASE

Aim. The purpose of the study was to evaluate manifestations of systemic inflammation in rats with Alzheimer disease (AD) induced by injections of Aβ1-40 and Aβ25-35 by the assessment of functional polarization of circulating phagocytes. Methods. AD was induced by intracerebral injections of Aβ1–40 and Aβ25–35 Wistar male rats. Intact and sham-operated animals were used as a control. AD development was affirmed by the assessment of cognitive impairment in behavioral tests (‘Open field’ test, apomorphine test, Barnes maze test), as well as by the level of death of dopaminergic neurons. The functional polarization of circulating phagocytes was designated by phagocytic activity, oxidative metabolism, and the expression of phenotypic markers CD80 and CD206, which were examined by flow cytometry. Results. Circulating phagocytes from rats with Aβ1-40-induced AD were characterized by increased fraction of phagocytizing monocytes with decreased endocytic activity, moderately up-regulated granulocyte ROS generation along with temperate increase of CD86+ mononuclear phagocyte fraction and high level of CD206 expression. Two widely accepted indices of systemic inflammation: NLR and SII were higher in these animals than those in control rats by 6,5 and 7,5 times respectively. In rats with Aβ25-35-induced disease, significantly increased granulocyte ROS generation was registered. NLR and SII values in these animals were slightly higher than those in control rats. Conclusion. Therefore, Aβ1-40 AD model reproduces disease-associated systemic inflammation at the greater extent than Aβ25-35-induced pathology, and is more appropriate for the study of inflammation in the disease pathophysiology.

Public Cord Blood Banks as a source of starting material for clinical grade HLA-homozygous induced pluripotent stem cells

BackgroundThe increasing number of clinical trials for induced pluripotent stem cell (iPSC)-derived cell therapy products makes the production on clinical grade iPSC more and more relevant and necessary. Cord blood banks are an ideal source of young, HLA-typed and virus screened starting material to produce HLA-homozygous iPSC lines for wide immune-compatibility allogenic cell therapy approaches. The production of such clinical grade iPSC lines (haplolines) involves particular attention to all steps since donor informed consent, cell procurement and a GMP-compliant cell isolation process.MethodsHomozygous cord blood units were identified and quality verified before recontacting donors for informed consent. CD34+ cells were purified from the mononuclear fraction isolated in a cell processor, by magnetic microbeads labelling and separation columns.ResultsWe obtained a median recovery of 20.0% of the collected pre-freezing CD34+, with a final product median viability of 99.1% and median purity of 83.5% of the post-thawed purified CD34+ population.ConclusionsHere we describe our own experience, from unit selection and donor reconsenting, in generating a CD34+ cell product as a starting material to produce HLA-homozygous iPSC following a cost-effective and clinical grade-compliant procedure. These CD34+ cells are the basis for the Spanish bank of haplolines envisioned to serve as a source of cell products for clinical research and therapy.

In-vivo reprogramming macrophages and dendritic cells with Allocetra-OTS: Successful mono- and combination-antitumor therapy.

2532 Background: Chimeric antigen receptor (CAR) T cells can activate an immune response to a cancer-specific antigen but is less effective in solid tumors. Immune check point inhibitors (ICI) revolutionized the treatment of solid tumors, however, in many tumors only partial response is achieved. Here we questioned the role of synergistic effect of Allocetra-OTS (cellular therapy for in-vivo reprogramming macrophages and dendritic cells, Enlivex Therap.) on solid tumor progression. Methods: To follow tumor growth in vivo, HeLa-CD19 cells were stably transduced with pLenti-PGK-V5-Luc-Neo. For CAR preparation, fresh mononuclear cells (MNC) were transfected with CD19-CAR plasmids. For the intraperitoneal solid tumor model, SCID-Bg mice were injected intraperitoneally (IP) with human HeLa-CD19 or HeLa-CD19-luciferase cells, 10×106 allocetra-OTS or vehicle, and 10×106 CD19-CAR T cells or mock T cells. In an immune-competent model, Balb/c mice were treated IP with AB12 (mesothelioma) with pLenti-PGK-V5-Luc-Neo and treated with anti-CTLA4 with or without Allocetra-OTS. Mice were monitored daily for clinical signs and peritoneal fluid accumulation and weekly for tumor growth. Kaplan-Meier log rank test was done for survival. Peritoneal cells were evaluated using single cell analysis and flow cytometry. Tumors were examined for bacterial presence by immunohistochemistry staining with antilipoteichoic acid (LTA) and antilipopolysaccharide (LPS). For allocetra-OTS preparation, enriched mononuclear fractions were collected by leukapheresis from healthy eligible human donors and induced to undergo early apoptosis. Results: SCID mice survived 30±5 days (range 27–37) and were sacrificed or died from solid tumor in the peritoneal cavity after accumulation of bloody peritoneal fluid and clinical deterioration. Results were verified using IVIS of intraperitoneal HeLaCD19-Luc cells. CAR T cell therapy significantly ameliorated survival to 55±11 days (p < 0.05 vs MOCK) but Alloctra-OTS further ameliorated survival to 75±10 (p < 0.001) with 20-40% complete remission. In AB12 model, anti CTLA4 therapy significantly ameliorated survival from 26±5 to 38 ±9 days (p < 0.05). However, Allocetra-OTS monotherapy ameliorated survival to 45 ±12 days (p < 0.02) and combinational therapy to 75±9 days (p < 0.0001) with complete remission in 60-75% of mice. Single cell analysis revealed that restoration of large peritoneal macrophages (LPM), were associated with antitumor activity. Conclusions: During intraperitoneal tumor progression, allocetra-OTS as monotherapy or combinational therapy with CAR or anti-CTLA4 significantly reduced tumor size and enable complete remission in up to 75% treated mice. Based on excellent safety profile in > 30 patients treated for sepsis and Covid19, human phase I/II of allocetra-OTS plus ICI, for peritoneal metastases, is planned for 2022.

Minimally Manipulated Bone Marrow-Derived Cells Can Be Used for Tissue Engineering In Situ and Simultaneous Formation of Personalized Tissue Models

Red bone marrow and autologous bone tissue (bone fragments and bone chips) of the donor were harvested intraoperatively during autoplasty of talus bone defect. Titanium chips were obtained by grinding a fragment of a microporous titanium-coated hip arthroplasty (Zimmer). Bone marrow mononuclear cells were isolated in the operating room, and bone and titanium fragments were incubated with a suspension of mononuclear cells. The quality of revitalization was assessed by fluorescence microscopy and histological examination after culturing of adherent cells on the bone and titanium fragments. During culturing on bone chips, bone marrow mononuclear fraction cells demonstrated significantly higher metabolic activity than bone marrow cells (p=0.04). Mononuclear fraction cells were also capable of stable colonization of titanium fragments with the formation of composite tissue model.

Therapeutic Angiogenesis in the Experimental Ulcerative-Necrotic Stage of Critical Lower Limb Ischemia

Introduction. The effectiveness of therapeutic angiogenesis in the ulcerative-necrotic stage is insufficient to preserve the limb.The aim of research was to study the effectiveness of therapeutic angiogenesis with "Udenafil", mononuclear fraction of autologous bone marrow and their combination in the experimental ulcerative-necrotic stage of critical lower limb ischemia.Materials and methods. Lower leg muscle ischemia was simulated on Wistar rats. All animals were divided into 4 groups, 20 animals in each group: with simulated critical ischemia without treatment (control group); with critical ischemia and monotherapy with udenafil (udenafil, orally, 8.6 mg/kg for 28 days, the first experimental group); with critical ischemia and monotherapy with mononuclear fraction of autologous bone marrow (a single paravasal injection of a mononuclear fraction of autologous bone marrow in 7 days after critical ischemia simulation, the second experimental group); with critical ischemia and combined therapy (udenafil orally, 0.86 mg/kg, once a day, for 28 days, and a single paravasal injection with a mononuclear fraction of autologous bone marrow, similar to the group with monotherapy, the third experimental group). The level of blood microcirculation in the lower leg muscles was measured in 21 and 28 days; after that, histological examination was performed.Results. In the control group, the regional blood flow rate in the ischemic shin muscle was 2.1 times less than in intact animals in 21 days and 1.8 times less in 28 days after simulated critical ischemia. In the first experimental group, the regional blood flow rate increased by 1.6 times in 21 days and 28 days after udenafil administration; in the second experimental group, the regional blood flow rate increased by 1.6 times and 1.7 times, respectively, after the mononuclear fraction of bone marrow was administered to animals; and in the third experimental group, the regional blood flow rate increased 1.8 times, compared with the animals of the control group after the combined use of bone marrow cells and udenafil. In 28 days after the start of the experiment, histological tests detected foci of necrosis with perifocal inflammation in the ischemic muscles of the lower leg in animals of the control group. In the first and second experimental groups, a decreased size and number of necrosis sites was registered due to formation of young connective tissue and new capillaries. None of newly formed microcirculatory bed was detected. In the third experimental group, the severity of necrotic changes was minimal, a large number of newly formed blood vessels (arterioles) were visualized in wide layers of the connective tissue (capillaries, venules); this was a new microcirculatory bed.Conclusion. Application of udenafil combined with the mononuclear fraction of the bone marrow results in an increased perfusion of ischemic tissues and development of a new microcirculatory bed.

Pro- and Anti-Inflammatory Cytokines in the Context of NK Cell-Trophoblast Interactions.

During pregnancy, uterine NK cells interact with trophoblast cells. In addition to contact interactions, uterine NK cells are influenced by cytokines, which are secreted by the cells of the decidua microenvironment. Cytokines can affect the phenotypic characteristics of NK cells and change their functional activity. An imbalance of pro- and anti-inflammatory signals can lead to the development of reproductive pathology. The aim of this study was to assess the effects of cytokines on NK cells in the presence of trophoblast cells in an in vitro model. We used TNFα, IFNγ, TGFβ and IL-10; the NK-92 cell line; and peripheral blood NK cells (pNKs) from healthy, non-pregnant women. For trophoblast cells, the JEG-3 cell line was used. In the monoculture of NK-92 cells, TNFα caused a decrease in CD56 expression. In the coculture of NK cells with JEG-3 cells, TNFα increased the expression of NKG2C and NKG2A by NK-92 cells. Under the influence of TGFβ, the expression of CD56 increased and the expression of NKp30 decreased in the monoculture. After the preliminary cultivation of NK-92 cells in the presence of TGFβ, their cytotoxicity increased. In the case of adding TGFβ to the PBMC culture, as well as coculturing PBMCs and JEG-3 cells, the expression of CD56 and NKp44 by pNK cells was reduced. The differences in the effects of TGFβ in the model using NK-92 cells and pNK cells may be associated with the possible influence of monocytes or other lymphoid cells from the mononuclear fraction.

Application of conditioned medium from mesenchymal stromal cells in the protocol for <em>ex vivo</em> production of megakaryocytes and platelets

Introduction. Of interest is the use of a conditioned medium from mesenchymal stromal cells in order to increase the expansion of CD34+ hematopoietic stem cells (HSCs).Aim — to analyze the effi cacy of two methods of ex vivo production of human megakaryocytes and platelets from CD34+ cord blood HSC using conditioned media from mesenchymal stromal cells and IMDM. Methods. Two cultivation methods that differ from each other by medium composition were compared. As a control of antigen expression of the donor, venous blood platelets were used. CD34+ HSCs were isolated from mononuclear fraction of cord blood using the immunomagnetic selection technique. The resulting cells were introduced at a concentration of 1 × 104 cells/mL into 24-well plates and cultured at 39 °C and 10 % CO2 for the first 7 days, after which the conditions were changed to 37 °C and 5 % CO2 and cultured for 14 days. In Group 1, up to day 7, the culture was performed using conditioned medium from mesenchymal stromal cell containing TPO (30 ng/mL), SCF (2 ng/mL), IL-6 (7.5 ng/mL), IL-9 (13.5 ng/mL), and in Group 2 a IMDM medium with the same cytokine cocktail was used. The cells were calculated using haemocytometer. CD34, CD41a, CD42b expression was evaluated using fl ow cytometry. Statistic data was processed with using R-language. The differences were evaluated as statistically signifi cant at signifi cance level p < 0.05.Results. Megakaryocyte production was observed starting from day 7 of culture. The expression level using conditioned medium from mesenchymal stromal cells (Group 1) according to CD41a was 5.84 ± 0.33 % versus 10.43 ± 1.08 % using IMDM medium (Group 2). On day 13 the ratio increased up to 42.05 ± 1.71 % in Group 1 and 61.78 ± 1.71 % in Group 2. CD41a+ megakaryocytes of Group 1 expressed the CD42b marker at the level of 96.85 ± 1.06 % versus 88.7 ± 0.56 % in Group 2. With the application of MSC conditioned medium the average number of nucleated cells was signifi cantly higher on the day 11 and it was equal 326.016 ± 1.86 × 104 cells/mL vs 197.26 ± 10.55 × 104 cells/mL in IMDM medium. Proplatelet formation was observed with microscopy staring from the day 12. The ratio of CD41a+ /CD42b+ platelets was 59.5 ± 3.85 % in conditioned medium, 65.9 ± 8.72 % in IMDM, and 96.11 ± 0.89 % in control platelets derived from venous blood.Conclusion. It was demonstrated that the use of MSC conditioned medium leads to an increase in the expansion of nucleated cells, however it decreases the rate of differentiation in megakaryocytes.