Extracellular double-stranded DNA participates in various processes in an organism. Here we report the suppressive effects of fragmented human double-stranded DNA along or in combination with cyclophosphamide on solid and ascites grafts of mouse Krebs-2 tumor cells and DNA preparation on human breast adenocarcinoma cell line MCF-7. Apoptosis and necrosis were assayed by electrophoretic analysis (DNA nucleosomal fragmentation) and by measurements of LDH levels in ascitic fluid, respectively. DNA internalization into MCF-7 was analyzed by flow cytometry and fluorescence microscopy. Direct cytotoxic activity of double-stranded DNA (along or in combination with cyclophosphamide) on a solid transplant was demonstrated. This resulted in delayed solid tumor proliferation and partial tumor lysis due to necrosis of the tumor and adjacent tissues. In the case of ascites form of tumor, extensive apoptosis and secondary necrosis were observed. Similarly, MCF-7 cells showed induction of massive apoptosis (up to 45%) as a result of treatments with double-stranded DNA preparation. Double-stranded DNA (along or in combination with cyclophosphamide) induces massive apoptosis of Krebs-2 ascite cells and MCF-7 cell line (DNA only). In treated mice it reduces the integrity of gut wall cells and contributes to the development of systemic inflammatory reaction.

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-1β, TNF-α, IL-10, TGF-β, 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.

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-β1 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.

We have characterized the human cell line arised from the Epstein–Barr virus (EBV) positive multiple myeloma aspirate subjected to the long-term cultivation. This cell line has acquired the ability to form free-floating spheres and to produce a xenograft upon transplantation into NOD/SCID mice. Cells from both in vitro culture and developed xenografts were investigated with a number of analytical approaches, including pathomorphological analysis, FISH analysis, and analysis of the surface antigens and of the VDJ locus rearrangement. The obtained results, as well as the confirmed presence of EBV, testify that both biological systems are derived from B-cells, which, in turn, is a progeny of the EBV-transformed B-cellular clone that supplanted the primordial multiple myeloma cells. Next we assessed whether cells that (i) were constantly present in vitro in the investigated cell line, (ii) were among the sphere-forming cells, and (iii) were capable of internalizing a fluorescent TAMRA-labeled DNA probe (TAMRA+ cells) belonged to one of the three types of undifferentiated bone marrow cells of a multiple myeloma patient: CD34+ hematopoietic stem cells, CD90+ mesenchymal stem cells, and clonotypic multiple myeloma cell. TAMRA+ cells were shown to constitute the fourth independent subpopulation of undifferentiated bone marrow cells of the multiple myeloma patient. We have demonstrated the formation of ectopic contacts between TAMRA+ cells and cells of other types in culture, in particular with CD90+ mesenchymal stem cells, followed by the transfer of some TAMRA+ cell material into the contacted cell.

Objective: Cell-based technologies are considered to be a new approach for the treatment of cerebral palsy (CP). Given the potent anti-inflammatory activity and high regenerative potential of M2 macrophages, these cells may be a promising source for cell transplantation. To evaluate the safety and efficacy of autologous M2 macrophages, we performed an open-label, Phase I/II, non-controlled clinical trial in children with severe CP. Patients and methods: Fifty-seven children with severe CP, including 33 boys and 24 girls, with a median age of 4 years were enrolled in the study. The patients were treated with intrathecal administration of autologous M2 macrophages. The primary outcome measure was safety, and the secondary outcome measure was functional improvement in neurologic scales, including the 66-item Gross Motor Function Measure test, Peabody Developmental Motor Scale-Fine Motor test, Ashworth scale, Medical Resarch Council scale, and an easy-to-understand unified questionnaire for evaluation of cognitive functions in our modification. Results: Intrathecal introduction of M2 cells in a median dose of 11.2×10⁶ did not induce any serious adverse events either related with cell injection or during 5 years of follow-up. After 3 months, the Gross Motor Function Measure score increased from 19±4.5 to 77±7.8, the Peabody Developmental Motor Scale-Fine Motor test score improved from 0.9±0.23 to 4.4±0.51, and the Ashworth score decreased from 3.5±0.11 to 2.5±0.16. The assessment of cognitive functions revealed an increase from 1.22±0.24 to 3.98±0.95, and a reduction of seizure syndrome was registered. In addition, M2 injection was accompanied by an increased production of brain-derived neurotrophic factor (p_U=0.015). Conclusion: The data obtained suggest that cell therapy based on M2 macrophages is safe, does not induce any severe cell-related reactions or long-term side effects and comorbidities, and is accompanied by significant neurologic improvements in severe CP patients.