Intranasal Immunotherapy with M2 Macrophage Secretome Ameliorates Language Impairments and Autistic-like Behavior in Children.

Background/Objectives: The intranasal delivery of various neurotropic substances is considered a new attractive therapeutic approach for treating neuropathologies associated with neuroinflammation and altered regeneration. Specific language impairment (SLI) that arises as a result of damage to the cortical speech zones during the developmental period is one of the most common problems in preschool children, and it is characterized by persistent difficulties in the acquisition, understanding, and use of language. This study's objective is to evaluate the efficacy and safety of intranasal immunotherapy using the M2 macrophage secretome as a rich source of immunoregulatory and neurotrophic factors for the treatment of severe language impairment in children. Methods: Seventy-one children (54 boys and 17 girls, aged 3 to 13 years) were recruited to participate in a clinical trial (NCT04689282) in two medical centers. The children were examined before, 1 month after, and 6 months after the start of therapy. In the vast majority of children (55/71), language impairment was associated with autistic-like symptoms and attention deficit hyperactivity disorder (ADHD). Results: Daily intranasal inhalations of M2 macrophage-conditioned medium (for 30 days) were well tolerated and led to a decrease in the severity of language impairments, autistic-like behavior, and ADHD symptoms. The clinical effect appeared within a month after the first procedure and persisted or intensified during a 6-month follow-up. Two-thirds of the children showed a clear clinical improvement, while the rest had less pronounced improvement. Conclusions: Thus, the use of the M2 macrophage secretome and its intranasal delivery is safe, well tolerated, and clinically effective in children with severe language impairments.

Decreased circulating myeloid-derived suppressor cell count at the engraftment is one of the risk factors for multiple myeloma relapse after autologous hematopoietic stem cell transplantation.

Recent studies demonstrated that myeloid-derived suppressor cells (MDSCs) are involved in the pathogenesis and progression of multiple myeloma (MM). Nevertheless, data on the quantitative and functional changes in MDSCs during standard MM treatment remain poorly understood. Here, we determined that monocytic MDSCs (M-MDSC; CD14+HLA-DRlow/-) and granulocytic MDSCs (PMN-MDSC; Lin-HLA-DR-CD33+CD66b+) in MM patients in remission following induction therapy (IT) were significantly increased, while early MDSCs (E-MDSCs; Lin-HLA-DR-CD33+CD66b-) were decreased compared to the donor group. In progression, MM patients had the most pronounced decrease in E-MDSCs and enhanced levels of PMN-MDSCs. IT was accompanied with a decrease in the expression of arginase-1 (Arg-1). In MM patients with relapse or resistance to IT, Arg-1+ cell frequency in M-MDSCs and E-MDSCs, as well as PD-L1+ M-MDSCs, was increased, which may facilitate tumor immunosuppression. G-CSF administration led to a significant increment in the MDSC subsets. At the engraftment, circulating M-MDSC and PMN-MDSCs were temporarily increased, with a gradual decline to the pre-transplant levels in 12 months. The percentage of E-MDSCs was decreased at the leukocyte recovery. Patients with a higher (>Me) M-MDSC count at the engraftment had a shorter post-transplant leukopenia duration (Me 11 vs. 13 days; pU = 0.0086). The advanced MM stage, depth of response, and lower relative count of circulating E-MDSCs at the engraftment were independent risk factors associated with a lower progression-free survival. The obtained data allow us to hypothesize that MDSCs may play a positive role at the stage of leukocyte recovery by ameliorating the long-term anti-tumor response in MM.

The Macrophage Activator GcMAF-RF Enhances the Antitumor Effect of Karanahan Technology through Induction of M2-M1 Macrophage Reprogramming.

Macrophages are the immune cells of high-immunological plasticity, which can exert both pro- and anti-inflammatory activity, as well as repolarize their phenotype to the opposite or neutral one. In this regard, M2 macrophages of the tumor-associated stroma (TAS) are a promising therapeutic target in treating malignant neoplasms. Using FACS assay, we have estimated the CD11b+/Ly-6G+/Ly-6C+ fraction of macrophages from the peritoneum and TAS in intact healthy mice and those with developed Lewis carcinoma, both untreated and treated according to Karanahan technology in combination with group-specific macrophage activator (GcMAF-RF). As well, the pattern of pro- and anti-inflammatory cytokines mRNA expression in different groups of experimental and tumor-bearing animals was assessed. It was found that: (i) exposure of intact mice to GcMAF-RF results in the increased number of CD11b+/Ly-6C+ peritoneal macrophages and, at the same time, the expression pattern of cytokines in peritoneal macrophages switches from that characteristic of the mixed M1/M2 phenotype to that characteristic of the neutral M0 one; (ii) combination of Karanahan technology and GcMAF-RF treatment results in M0/M1 repolarization of TAS macrophages; (iii) in tumor-bearing mice, the response of peritoneal macrophages to such a treatment is associated with the induction of anti-inflammatory reaction, which is opposite to that in TAS macrophages.

The Molecular Aspects of Functional Activity of Macrophage-Activating Factor GcMAF.

Group-specific component macrophage-activating factor (GcMAF) is the vitamin D3-binding protein (DBP) deglycosylated at Thr420. The protein is believed to exhibit a wide range of therapeutic properties associated with the activation of macrophagal immunity. An original method for GcMAF production, DBP conversion to GcMAF, and the analysis of the activating potency of GcMAF was developed in this study. Data unveiling the molecular causes of macrophage activation were obtained. GcMAF was found to interact with three CLEC10A derivatives having molecular weights of 29 kDa, 63 kDa, and 65 kDa. GcMAF interacts with high-molecular-weight derivatives via Ca2+-dependent receptor engagement. Binding to the 65 kDa or 63 kDa derivative determines the pro- and anti-inflammatory direction of cytokine mRNA expression: 65 kDa-pro-inflammatory (TNF-α, IL-1ß) and 63 kDa-anti-inflammatory (TGF-ß, IL-10). No Ca2+ ions are required for the interaction with the canonical 29 kDa CLEC10A. Both forms, DBP protein and GcMAF, bind to the 29 kDa CLEC10A. This interaction is characterized by the stochastic mRNA synthesis of the analyzed cytokines. Ex vivo experiments have demonstrated that when there is an excess of GcMAF ligand, CLEC10A forms aggregate, and the mRNA synthesis of analyzed cytokines is inhibited. A schematic diagram of the presumable mechanism of interaction between the CLEC10A derivatives and GcMAF is provided. The principles and elements of standardizing the GcMAF preparation are elaborated.

Effect of Differently Polarized Human Macrophages on the SH-SY5Y Cells Damaged by Ischemia and Hypoxia In Vitro.

Macrophages are the major cells of innate immunity with a wide range of biological effects due to their great plasticity and heterogeneity. Macrophages play a key role in neuroregeneration following nervous tissue injury. However, the neuroregenerative potential of various macrophage phenotypes, including those polarized by efferocytosis, remains unexplored. The aim of this study was to compare the neuroregenerative and neuroprotective activity of soluble factors secreted by variously activated human macrophages on the functions of neural progenitors in an in vitro model of ischemia or ischemia/hypoxia. Macrophages were polarized by interferon-γ (M1), IL-4 (M2a), or interaction with apoptotic cells (M2(LS)). The effect of macrophages conditioned media on the proliferation, differentiation, and survival of SH-SY5Y cells damaged by serum deprivation alone (ischemic conditions) or in combination with CoCl2 (ischemic/hypoxic conditions) was assessed. All studied macrophages stimulated the proliferation and differentiation of SH-SY5Y cells. On day 3, the pro-proliferating effect of M1 and M2 was similar and did not depend on the severity of the damaging effect (ischemia or ischemia/hypoxia), while on day 7 and under ischemic/hypoxic conditions, the effects of M2(LS) exceeded those of M1 and M2a cells. The prodifferentiation effects of macrophages were manifested in both short- and long-term cultures, mainly under ischemic/hypoxic conditions, and were most characteristic of M2(LS) cells. Importantly, the ischemia/hypoxia model was accompanied by the pronounced death of SH-SY5Y cells. Only macrophages with the M2 phenotype demonstrated antiapoptotic activity, and the effect of M2(LS) was higher than that of M2a. The results obtained indicate that human macrophages have neuroprotective and neuroregenerative activity, which is mediated by soluble factors, is most characteristic for macrophages activated by efferocytosis (M2(LS)), and is most prominent under in vitro conditions simulating the combined effect of ischemia/hypoxia.

Human Macrophages Polarized by Interaction with Apoptotic Cells Produce Fibrosis-Associated Mediators and Enhance Pro-Fibrotic Activity of Dermal Fibroblasts In Vitro.

Apoptosis and subsequent removal of dead cells are an essential part of wound healing. Macrophages phagocytize apoptotic cells (efferocytosis) and contribute to the resolution of inflammation. However, their participation in fibrogenesis and the mechanisms of influence on this process remain unclear. In the present study, we focused on the fibrogenic properties of human monocyte-derived macrophages polarized in the M2 direction by interaction with apoptotic cells. We studied their influence on the proliferation ([3H]-thymidine incorporation), differentiation (by the expression of α-SMA, a myofibroblast marker) and collagen-producing activity (ELISA) of dermal fibroblasts compared to classically (LPS) and alternatively (IL-4) activated macrophages. Macrophages polarized by the interaction with apoptotic cells had a unique phenotype and profile of produced factors and differed from the compared macrophage subtypes. Their conditioned media promoted the proliferation of dermal fibroblasts and the expression of α-SMA in them at the level of macrophages stimulated by IL-4, while the stimulating effect on the collagen-producing activity was more pronounced compared to that of the other macrophage subtypes. Moreover, they are characterized by the high level of production of pro-fibrotic factors such as TIMP-1, TGF-ß1 and angiogenin. Taken together, M2-like macrophages polarized by efferocytosis demonstrate in vitro pro-fibrotic activity by promoting the functional activity of dermal fibroblasts and producing pro-fibrotic and pro-angiogenic factors.

Comparing the Biological Properties of Double-Stranded DNA Extracted from Human and Porcine Placenta and Salmon Sperm.

Background: Double-stranded fragmented extracellular DNA is a participant, inducer, and indicator of various processes occurring in the organism. When investigating the properties of extracellular DNA, the question regarding the specificity of exposure to DNA from different sources has always been raised. The aim of this study was to perform comparative assessment of biological properties of double-stranded DNA obtained from the human placenta, porcine placenta and salmon sperm. Methods: The intensity of leukocyte-stimulating effect of different dsDNA was assessed in mice after cyclophosphamide-induced cytoreduction. The stimulatory effect of different dsDNA on maturation and functions of human dendritic cells and the intensity of cytokine production by human whole blood cells was analyzed ex vivo. The oxidation level of the dsDNA was also compared. Results: Human placental DNA exhibited the strongest leukocyte-stimulating effect. DNA extracted from human and porcine placenta exhibited similar stimulatory action on maturation of dendritic cells, allostimulatory capacity, and ability of dendritic cells to induce generation of cytotoxic CD8+CD107a+ T cells in the mixed leukocyte reaction. DNA extracted from salmon sperm stimulated the maturation of dendritic cells, while having no effect on their allostimulatory capacity. DNA extracted from human and porcine placenta was shown to exhibit a stimulatory effect on cytokine secretion by human whole blood cells. The observed differences between the DNA preparations can be caused by the total methylation level and are not related to differences in oxidation level of DNA molecules. Conclusions: Human placental DNA exhibited the maximum combination of all biological effects.

Impact of Double-Stranded RNA Internalization on Hematopoietic Progenitors and Krebs-2 Cells and Mechanism.

It is well-established that double-stranded RNA (dsRNA) exhibits noticeable radioprotective and radiotherapeutic effects. The experiments conducted in this study directly demonstrated that dsRNA was delivered into the cell in its native form and that it induced hematopoietic progenitor proliferation. The 68 bp synthetic dsRNA labeled with 6-carboxyfluorescein (FAM) was internalized into mouse hematopoietic progenitors, c-Kit+ (a marker of long-term hematopoietic stem cells) cells and CD34+ (a marker of short-term hematopoietic stem cells and multipotent progenitors) cells. Treating bone marrow cells with dsRNA stimulated the growth of colonies, mainly cells of the granulocyte-macrophage lineage. A total of 0.8% of Krebs-2 cells internalized FAM-dsRNA and were simultaneously CD34+ cells. dsRNA in its native state was delivered into the cell, where it was present without any signs of processing. dsRNA binding to a cell was independent of cell charge. dsRNA internalization was related to the receptor-mediated process that requires energy from ATP. Synthetic dsRNA did not degrade in the bloodstream for at least 2 h. Hematopoietic precursors that had captured dsRNA reinfused into the bloodstream and populated the bone marrow and spleen. This study, for the first time, directly proved that synthetic dsRNA is internalized into a eukaryotic cell via a natural mechanism.

The New General Biological Property of Stem-like Tumor Cells (Part II: Surface Molecules, Which Belongs to Distinctive Groups with Particular Functions, Form a Unique Pattern Characteristic of a Certain Type of Tumor Stem-like Cells).

An ability of poorly differentiated cells of different genesis, including tumor stem-like cells (TSCs), to internalize extracellular double-stranded DNA (dsDNA) fragments was revealed in our studies. Using the models of Krebs-2 murine ascites carcinoma and EBV-induced human B-cell lymphoma culture, we demonstrated that dsDNA internalization into the cell consists of several mechanistically distinct phases. The primary contact with cell membrane factors is determined by electrostatic interactions. Firm contacts with cell envelope proteins are then formed, followed by internalization into the cell of the complex formed between the factor and the dsDNA probe bound to it. The key binding sites were found to be the heparin-binding domains, which are constituents of various cell surface proteins of TSCs-either the C1q domain, the collagen-binding domain, or domains of positively charged amino acids. These results imply that the interaction between extracellular dsDNA fragments and the cell, as well as their internalization, took place with the involvement of glycocalyx components (proteoglycans/glycoproteins (PGs/GPs) and glycosylphosphatidylinositol-anchored proteins (GPI-APs)) and the system of scavenger receptors (SRs), which are characteristic of TSCs and form functional clusters of cell surface proteins in TSCs. The key provisions of the concept characterizing the principle of organization of the "group-specific" cell surface factors of TSCs of various geneses were formulated. These factors belong to three protein clusters: GPs/PGs, GIP-APs, and SRs. For TSCs of different tumors, these clusters were found to be represented by different members with homotypic functions corresponding to the general function of the cluster to which they belong.

The new general biological property of stem-like tumor cells Part I. Peculiarities of the process of the double-stranded DNA fragments internalization into stem-like tumor cells.

Stem-like tumor cells of ascites carcinoma Krebs-2 and Epstein-Barr virus-induced B-lymphoma were shown to possess the innate capability of binding and internalizing the TAMRA-labeled double-stranded DNA (dsDNA) probe. The process of binding and internalizing is rather complicated and composed of the following successive stages: 1) initiating electrostatic interaction and contact of a negatively charged dsDNA molecule with a positively charged molecule(s) on the surface of a stem-like tumor cell; 2) binding of the dsDNA probe to a tumor stem cell surface protein(s) via the formation of a strong chemical/molecular bond; and 3) the very internalization of dsDNA into the cell. Binding of DNA to cell surface proteins is determined by the presence of heparin/polyanion-binding sites within the protein structure, which can be competitively blocked by heparin and/or dextran sulfate, wherein heparin blocks only the binding, while dextran sulfate abrogates both binding and internalization. The abrogation of internalization by dextran sulfate implies the role of scavenger receptors in this process. Cells were shown to uptake DNA in amounts constituting ∼0.008% of the haploid genome. Inhibitors of caveolae-dependent internalization abrogate the DNA uptake in Krebs-2 cells, and inhibitors of the clathrin/caveolar mechanism block the internalization in B-lymphoma cells. In the present report, it is shown for the first time that in contrast to the majority of committed tumor cells, stem-like tumor cells of Krebs-2 and B-lymphoma carry a general positive charge on their surface.

Designing two-dimensional temperature profiles using tunable thermoplasmonics.

Heat flow generation and manipulation in nanometer-sized solids using light represents one of the up-and-coming tasks in thermonanophotonics. Enhanced light-matter interaction due to plasmon resonance permits metallic nanostructures to absorb light energy efficiently, and it results in extra optical heating. The net temperature increment of nanostructures is directly dependent on heat exchange with a thermostat. However, to the best of our knowledge, precise tailoring of optical heating at a fixed pump power is still of no practical implementation. In this paper, we focus on the tunable optical heating of a plasmonic nanostructure exposed to moderate light intensity (MW cm-2) based on slowing down heat exchange through a 1D waveguide heatsink bridging the nanostructure and the highly thermal conducting thermostat. The rationale for this concept is evidenced through optical heating of a 2D array of stacked titanium nitride (TiN) (plasmonic refractory nanoheater) and height-controlled silicon (Si) (1D waveguide heatsink) cylinders. Depending on the Si pillar height, the temperature rise of a TiN : Si voxel ranges from a few up to thousands of degrees at a fixed pump power. The temperature of the TiN : Si voxel is remotely measured from the Raman shift of the Si pillar. Using ellipsometry, we find a temperature threshold of 400 °C, above which the thin TiN film is chemically degraded due to oxidation. The latter enables fine tailoring of thermal gradients using TiN : Si voxels of equal size but different permittivity. These findings contribute towards the development of tunable thermoplasmonics by demonstrating programmable non-uniform temperature profiles in the steady-state regime under continuous-wave laser illumination for a variety of thermo-optical applications.

Analysis of the Biological Properties of Blood Plasma Protein with GcMAF Functional Activity.

The main problem related to the studies focusing on group-specific component protein-derived macrophage-activating factor (GcMAF) is the lack of clarity about changes occurring in different types of macrophages and related changes in their properties under the effect of GcMAF in various clinical conditions. We analyzed the antitumor therapeutic properties of GcMAF in a Lewis carcinoma model in two clinical conditions: untreated tumor lesion and tumor resorption after exposure to Karanahan therapy. GcMAF is formed during site-specific deglycosylation of vitamin D3 binding protein (DBP). DBP was obtained from the blood of healthy donors using affinity chromatography on a column with covalently bound actin. GcMAF-related factor (GcMAF-RF) was converted in a mixture with induced lymphocytes through the cellular enzymatic pathway. The obtained GcMAF-RF activates murine peritoneal macrophages (p < 0.05), induces functional properties of dendritic cells (p < 0.05) and promotes in vitro polarization of human M0 macrophages to M1 macrophages (p < 0.01). Treatment of whole blood cells with GcMAF-RF results in active production of both pro- and anti-inflammatory cytokines. It is shown that macrophage activation by GcMAF-RF is inhibited by tumor-secreted factors. In order to identify the specific antitumor effect of GcMAF-RF-activated macrophages, an approach to primary reduction of humoral suppressor activity of the tumor using the Karanahan therapy followed by macrophage activation in the tumor-associated stroma (TAS) was proposed. A prominent additive effect of GcMAF-RF, which enhances the primary immune response activation by the Karanahan therapy, was shown in the model of murine Lewis carcinoma. Inhibition of the suppressive effect of TAS is the main condition required for the manifestation of the antitumor effect of GcMAF-RF. When properly applied in combination with any chemotherapy, significantly reducing the humoral immune response at the advanced tumor site, GcMAF-RF is a promising antitumor therapeutic agent that additively destroys the pro-tumor properties of macrophages of the tumor stroma.

Chronometric Administration of Cyclophosphamide and a Double-Stranded DNA-Mix at Interstrand Crosslinks Repair Timing, Called "Karanahan" Therapy, Is Highly Efficient in a Weakly Immunogenic Lewis Carcinoma Model.

Background and Aims: A new technology based on the chronometric administration of cyclophosphamide and complex composite double-stranded DNA-based compound, which is scheduled in strict dependence on interstrand crosslinks repair timing, and named "Karanahan", has been developed. Being applied, this technology results in the eradication of tumor-initiating stem cells and full-scale apoptosis of committed tumor cells. In the present study, the efficacy of this novel approach has been estimated in the model of Lewis carcinoma. Methods: To determine the basic indicative parameters for the approach, the duration of DNA repair in tumor cells, as well as their distribution along the cell cycle, have been assessed. Injections were done into one or both tumors in femoral region of the engrafted mice in accordance with the developed regimen. Four series of experiments were carried out at different periods of time. The content of poorly differentiated CD34+/TAMRA+ cells in the bone marrow and peripheral blood has been determined. Immunostaining followed by the flow cytometry was used to analyze the subpopulations of immune cells. Results: The high antitumor efficacy of the new technology against the developed experimental Lewis carcinoma was shown. It was found that the therapy efficacy depended on the number of tumor growth sites, seasonal and annual peculiarities. In some experiments, a long-term remission has been reached in 70% of animals with a single tumor and in 60% with two tumors. In mice with two developed grafts, mobilization capabilities of both poorly differentiated hematopoietic cells of the host and tumor stem-like cells decrease significantly. Being applied, this new technology was shown to activate a specific immune response. There is an increase in the number of NK cell populations in the blood, tumor, and spleen, killer T cells and T helper cells in the tumor and spleen, CD11b+Ly-6C+ and CD11b+Ly-6G+ cells in the tumor. A population of mature dendritic cells is found in the tumor. Conclusion: The performed experiments indicate the efficacy of the Karanahan approach against incurable Lewis carcinoma. Thus, the discussed therapy is a new approach for treating experimental neoplasms, which has a potential as a personalized anti-tumor therapeutic approach in humans.

Karanahan: A Potential New Treatment Option for Human Breast Cancer and Its Validation in a Clinical Setting.

INTRODUCTION: Karanahan, a cancer treatment technology aimed at eradicating tumor-initiating stem cells, has already proven effective in 7 tumor models. Karanahan comprises the following procedures: (1) collecting surgical specimens, (2) determining the duration of the DNA repair process in tumor cells exposed to a cross-linking cytostatic agent, and (3) determining the time point, when cells, including tumor-initiating stem cells, are synchronized in the certain phase of the cell cycle after triple exposure to the cytostatic, becoming vulnerable for the terminal treatment, which is supposed to completely eliminate the rest of survived tumor-initiating stem cells. Determining these basic tumor properties allows to design the schedule for the administration of a cross-linking cytostatic and a complex composite DNA preparation. Being conducted in accordance with the schedule designed, Karanahan results in the large-scale apoptosis of tumor cells with elimination of tumor-initiating stem cells. METHODS: Breast tumor specimens were obtained from patients, and basic tumor properties essential for conducting Karanahan therapy were determined. RESULTS: We report the first use of Karanahan in patients diagnosed with breast cancer. Technical details of handling surgical specimens for determining the essential Karanahan parameters (tumor volume, cell number, cell proliferation status, etc) have been worked out. The terminally ill patient, who was undergoing palliative treatment and whose tumor specimen matched the required criteria, received a complete course of Karanahan. CONCLUSIONS: The results of the treatment conducted indicate that Karanahan technology has a therapeutic potency and can be used as a breast cancer treatment option.

Antitumor efficacy of multi-target in situ vaccinations with CpG oligodeoxynucleotides, anti-OX40, anti-PD1 antibodies, and aptamers.

To overcome immune tolerance to cancer, the immune system needs to be exposed to a multi-target action intervention. Here, we investigated the activating effect of CpG oligodeoxynucleotides (ODNs), mesyl phosphoramidate CpG ODNs, anti-OX40 antibodies, and OX40 RNA aptamers on major populations of immunocompetent cells ex vivo. Comparative analysis of the antitumor effects of in situ vaccination with CpG ODNs and anti-OX40 antibodies, as well as several other combinations, such as mesyl phosphoramidate CpG ODNs and OX40 RNA aptamers, was conducted. Antibodies against programmed death 1 (PD1) checkpoint inhibitors or their corresponding PD1 DNA aptamers were also added to vaccination regimens for analytical purposes. Four scenarios were considered: a weakly immunogenic Krebs-2 carcinoma grafted in CBA mice; a moderately immunogenic Lewis carcinoma grafted in C57Black/6 mice; and an immunogenic A20 B cell lymphoma or an Ehrlich carcinoma grafted in BALB/c mice. Adding anti-PD1 antibodies (CpG+αOX40+αPD1) to in situ vaccinations boosts the antitumor effect. When to be used instead of antibodies, aptamers also possess antitumor activity, although this effect was less pronounced. The strongest effect across all the tumors was observed in highly immunogenic A20 B cell lymphoma and Ehrlich carcinoma.

Highly proliferative and functional PD-1+ and TIM-3+ T cells are transiently increased in multiple myeloma following autologous hematopoietic stem cell transplantation.

The aim of our prospective study was to assess recovery dynamics and functional characteristics of PD-1+ and TIM-3+ T cells in multiple myeloma (MM) patients following high-dose chemotherapy (HDCT) with autologous hematopoietic stem cell transplantation (AHSCT). Peripheral blood, autograft and bone marrow samples were obtained from 46 MM patients before conditioning, at the engraftment, following six and 12 months post-transplant. Frequencies of CD4+ and CD8+ T cells expressing PD-1 and TIM-3 and intracellular expression of Ki-67 and Granzyme B were evaluated. Counts of PD-1+ and TIM-3+ T cells at the engraftment were significantly higher comparing with the levels before HDCT and 6-12 months following AHSCT. The post-transplant increase in the studied subsets was due to a temporary enhancement in proliferation activity. The cytotoxic potential of PD-1- and TIM-3-expressing CD8+ T cells was higher at the engraftment comparing with the pre-transplant and remained at the same level for at least 12 months. The increase in CD4+PD-1+ and CD8+TIM-3+ T cells at the engraftment was associated with higher absolute counts of their reinfused counterparts. Circulating PD-1+ CD8+ and TIM-3+ CD4+ T cells were increased in patients after post-transplant relapse comparing with the ones in remission. Homeostatic proliferation plays a key role in the upregulation of inhibitory checkpoint receptors on functional T cells under lymphopenic conditions. In this regard, it is difficult to predict both the efficacy and adverse reactions of therapy with checkpoint inhibitors on the course of MM after HDCT with AHSCT. Précis. Homeostatic proliferation plays apparently a key role in the upregulation of PD-1 and TIM-3 on functional T cells after AHSCT and appears to be a normal physiological process, contrary to relapse-associated increase in PD-1+ and TIM-3+ T cells.

Experimental Comparison of the In Vivo Efficacy of Two Novel Anticancer Therapies.

BACKGROUND/AIM: We compared the therapeutic efficacy of two recently developed experimental anticancer technologies: 1) in situ vaccination based on local immunotherapy with CpG oligonucleotides and anti-OX40 antibodies to activate antitumor immune response and 2) "Karanahan" technology [from the Sanskrit karana ('source') + han ('to kill')] based on the combined injection of cyclophosphamide and double-stranded DNA to eradicate cancer stem cells. MATERIALS AND METHODS: The anticancer approaches were compared on three types of mouse malignant tumors with different grades of immunogenicity: weakly immunogenic carcinoma Krebs-2, moderately immunogenic Lewis carcinoma, and highly immunogenic A20 В-cellular lymphoma. RESULTS: Our results indicated that in situ vaccination was the most effective against the highly immunogenic tumor А20. In addition, "Karanahan" demonstrated high efficiency in all types of tumors, regardless of their immunogenicity or size. CONCLUSION: "Karanahan" therapy showed higher efficacy relative to in situ vaccination with CpG oligonucleotides and anti-OX40 antibodies.

Efficacy of the new therapeutic approach in curing malignant neoplasms on the model of human glioblastoma.

OBJECTIVE: Glioma is a highly invasive tumor, frequently disposed in essential areas of the brain, which makes its surgical excision extremely difficult; meanwhile adjuvant therapy remains quite ineffective. METHODS: In the current report, a new therapeutic approach in curing malignant neoplasms has been performed on the U87 human glioblastoma model. This approach, termed "Karanahan", is aimed at the eradication of cancer stem cells (CSCs), which were recently shown to be capable of internalizing fragments of extracellular double-stranded DNA. After being internalized, these fragments interfere in the process of repairing interstrand cross-links caused by exposure to appropriate cytostatics, and such an interference results either in elimination of CSCs or in the loss of their tumorigenic potency. Implementation of the approach requires a scheduled administration of cytostatic and complex composite double-stranded DNA preparation. RESULTS: U87 cells treated in vitro in accordance with the Karanahan approach completely lost their tumorigenicity and produced no grafts upon intracerebral transplantation into immunodeficient mice. In SCID mice with developed subcutaneous grafts, the treatment resulted in reliable slowing down of tumor growth rate (P < 0.05). In the experiment with intracerebral transplantation of U87 cells followed by surgical excision of the developed graft and subsequent therapeutic treatment, the Karanahan approach was shown to reliably slow down the tumor growth rate and increase the median survival of the mice twofold relative to the control. CONCLUSIONS: The effectiveness of the Karanahan approach has been demonstrated both in vitro and in vivo in treating developed subcutaneous grafts as well as orthotopic grafts after surgical excision of the tumor.

Epitope mapping of novel monoclonal antibodies to human angiotensin I-converting enzyme.

Angiotensin I-converting enzyme (ACE, CD143) plays a crucial role in blood pressure regulation, vascular remodeling, and immunity. A wide spectrum of mAbs to different epitopes on the N and C domains of human ACE have been generated and used to study different aspects of ACE biology, including establishing a novel approach-conformational fingerprinting. Here we characterized a novel set of 14 mAbs, developed against human seminal fluid ACE. The epitopes for these novel mAbs were defined using recombinant ACE constructs with truncated N and C domains, species cross-reactivity, ACE mutagenesis, and competition with the previously mapped anti-ACE mAbs. Nine mAbs recognized regions on the N domain, and 5 mAbs-on the C domain of ACE. The epitopes for most of these novel mAbs partially overlap with epitopes mapped onto ACE by the previously generated mAbs, whereas mAb 8H1 recognized yet unmapped region on the C domain where three ACE mutations associated with Alzheimer's disease are localized and is a marker for ACE mutation T877M. mAb 2H4 could be considered as a specific marker for ACE in dendritic cells. This novel set of mAbs can identify even subtle changes in human ACE conformation caused by tissue-specific glycosylation of ACE or mutations, and can detect human somatic and testicular ACE in biological fluids and tissues. Furthermore, the high reactivity of these novel mAbs provides an opportunity to study changes in the pattern of ACE expression or glycosylation in different tissues, cells, and diseases, such as sarcoidosis and Alzheimer's disease.

Effects of Recombinant Angiogenin on Collagen Fiber Formation and Angiogenesis in the Dermis of Wistar Rats.

PURPOSE: The purpose of this study was to assess the capability of recombinant angiogenin isolated from Pichia pastoris yeasts to stimulate regenerative processes in the dermis of experimental animals. PATIENTS AND METHODS: Wistar rats were administered with recombinant angiogenin intracutaneously. Morphological examination of the skin and the assessment of the proliferative activity of the epidermal cells were carried out. Additionally, cytokine production by human whole blood cells exposed to angiogenin was analyzed ex vivo. RESULTS: Administration of angiogenin stimulates collagen fiber formation and angiogenesis. This stimulation is tightly associated with an increase in the number of fibroblasts, an increased numerical density of dermal blood vessels and an increased density of collagen fibers; also, it activates the proliferation of basal cells. Angiogenin induces the production of MCP, IL-8, IL-6, IL-1ß, TNF-α, IL-10, TGF-ß, and VEGF by blood cells. CONCLUSION: The results obtained indicate a broad spectrum of actions of recombinant angiogenin during regenerative processes in the basal layer of the dermis.