M. A. Tikhonova

Application of autologous bone marrow stem cells in the therapy of spinal cord injury patients.

We studied the safety and efficiency of transplantation of autologous bone marrow cells in complex therapy of patients with spinal cord injury in the late period of the disease. In control group patients, meningomyeloradiculolis was performed, while in the main group surgical treatment was supplemented by transplantation of autologous bone marrow cells. Transplantation of BM stem cells into the cyst cavity and intravenously was well tolerated, did not cause allergic or inflammatory reactions in the early and delayed periods after surgery, and did not induce the formation of ossification foci in the nervous tissue. Analysis of the neurological status by ASIA, Bartel, and Ashworth scales showed that in the main group the positive clinical dynamics was more often observed than in the control. The decrease in neurological deficit included improvement of sensory and motor activity and conducting sensory function. Thus, transplantation of autologous bone marrow cells can be a novel safe strategy for the treatment of patients in the late period after spinal trauma.

Formation of different abzymes in autoimmune‐prone MRL‐lpr/lpr mice is associated with changes in colony formation of haematopoietic progenitors

It was shown that IgGs from the sera of 2–7‐month‐old control non‐autoimmune (CBA x C57BL)F1 and BALB/c mice and 2–3‐month‐old autoimmune prone MRL‐lpr/lpr mice (conditionally healthy mice) are catalytically inactive. During spontaneous development of deep systemic lupus erythematosus (SLE)‐like pathology a specific reorganization of immune system of these mice leads to conditions associated with a production of IgGs hydrolyzing DNA, ATP and polysaccharides with low catalytic activities (conditionally pre‐diseased mice).A significant increase in DNase, ATPase and amylase IgG relative activities associated with a transition from pre‐diseased to deep diseased mice is correlated with additional changes in differentiation and proliferation of mice bone marrow haematopoietic stem cells (HSCs) and lymphocyte proliferation in different organs.The highest increase in all abzyme activities was found in mice immunized with DNA, which in comparison with pre‐diseased and diseased mice are characterized by a different profile of HSC differentiation and by a suppression of cell apoptosis. Abzyme activities in the serum of pregnant females were comparable with those for pre‐diseased mice, but the profile of HSC differentiation and cell apoptosis levels in pregnant and pre‐diseased mice were quite different. Right after the beginning of lactation (4 days after delivery) and in a late time of lactation (14 days after delivery) there was an observed increase in cell apoptosis and two different stages of significant change in the HSC differentiation profiles; the first stage was accompanied with a significant increase and the second with a remarkable decrease in abzyme activities. Overall, all mouse groups investigated are characterized by a specific relationship between abzyme activities, HSC differentiation profiles, levels of lymphocyte proliferation, and cell apoptosis in different organs. From our point of view, the appearance of ATPase, DNase activities may be considered the earliest statistically significant marker of mouse spontaneous SLE and a further significant increase in their activities correlates with the appearance of SLE visible markers and with an increase in concentrations of anti‐DNA Abs and urine protein. However, development of autoimmune (AI)‐reactions and the increase in the sera anti‐DNA antibodies (Abs) and in the abzyme activities in pregnant and lactating mice do not associate with SLE visible markers and proteinuria. The possible differences in immune system reorganizations during pre‐disease, disease, pregnancy and lactation leading to production of different auto‐antibodies and abzymes are discussed.

Characteristics of bone marrow cells under conditions of impaired innervation in patients with spinal trauma.

We studied quantitative and functional parameters of bone marrow stem cells and mature lymphocyte population under conditions of impaired innervation in patients with injuries to the cervical and thoracic portions of the spinal cord. Our findings indicated the absence of deficiency of quantitative and proliferative potentials of stem cells and demonstrated intact subpopulation structure of mature lymphocytes and T-cell proliferative activity similar to that in donors. The content of CD34+ cells in patients did not differ from that in donors. The percentage of CD34+CD38− hemopoietic stem cells was elevated in patients, presumably due to increased proliferative activity of hemopoietic stem cells. The possibility of derivation and in vitro culturing of fibroblast-like cells with mesenchymal stem cell phenotype was demonstrated.

Cytotoxic Activity of Dendritic Cells as a Possible Mechanism of Negative Regulation of T Lymphocytes in Pulmonary Tuberculosis

The PD-1/B7-H1-mediated induction of T cell apoptosis/anergy as a possible mechanism of immune response failure was studied in 76 patients with pulmonary tuberculosis (TB) with normal and low-proliferative response to antigens of M. tuberculosis (purified protein derivative (PPD)). It was revealed that dendritic cells (DCs), generated in vitro from patient blood monocytes with GM-CSF + IFN-α, were characterized by increased B7-H1 expression, upproduction of IL-10, and reducing of allostimulatory activity in mixed lymphocyte culture (MLC). Moreover, DCs of patients with TB were able to enhance T cell apoptosis and to block T-cell division in MLC. It was shown that neutralizing anti-PD1 antibodies significantly decreased the proapoptogenic/tolerogenic effect of DCs. Correlation analysis revealed a direct relationship between IL-10 production and level of B7-H1 expression in the general group of investigated patients. It was demonstrated that generation of healthy donor DCs in the presence of IL-10 led to an increase in the number of DCs-expressed B7-H1 molecule, DC proapoptogenic activity, and a decrease in their allostimulatory activity. Obviously, the revealed phenomenon of the PD-1/B7-H1-mediated pro-apoptogenic activity of DCs is clinically significant since the cytotoxic/tolerogenic potential of DCs is more pronounced in patients with PPD anergy.

Interferon alpha induces generation of semi-mature dendritic cells with high pro-inflammatory and cytotoxic potential

Dendritic cell-based vaccines are considered as a new and promising immunotherapeutic approach for cancer treatment. However, the choice of optimal protocol of dendritic cell generation in vitro represents the major challenge. Here, we compared phenotype and functional characteristics of human monocyte-derived dendritic cells (DCs) generated in the presence of IL-4/GM-CSF (IL4-DCs) and IFNα/GM-CSF (IFN-DCs). We showed that IFN-DCs displayed semi-mature phenotype and expressed higher level of CD123, TNF-related apoptosis-inducing ligand (TRAIL) and B7-H1 molecules in comparison with IL4-DCs. LPS-stimulated IFN-DCs were characterized by greater production of Th1/pro-inflammatory (IFN-γ, IL-2, IL-1β, TNF-α, IL-17), Тh2/anti-inflammatory cytokines (IL-10, IL-5), hematopoietic growth factors (G-CSF) and chemokines (MCP-1). These data indicated more pronounced ability of IFN-DCs to induce cellular immune response as well as humoral immune response compared to IL4-DCs. LPS-stimulated IFN-DCs possessed higher direct cytotoxic activity against TRAIL-sensitive tumor cell line Jurkat and similar cytotoxicity against TRAIL-resistant tumor HEp-2 cells. Besides, IFN-DCs and IL4-DCs equally induced apoptosis of activated CD4(+) and CD8(+) T cells. These results suggest that IFN-DCs can be used as potent cell-based curative therapies for individuals with cancer.

Cytotoxic activity of ex-vivo generated IFNα-induced monocyte-derived dendritic cells in brain glioma patients.

Recent studies have revealed that besides the important role in triggering the adoptive antitumor immunity, dendritic cells (DCs) possess direct cytotoxic antitumor activity. Here, we investigated brain glioma patient monocyte-derived DCs generated in the presence of IFNα and GM-CSF (IFN-DCs). These DCs were characterized by reduced cytotoxic activity against TRAIL-resistant HEp-2 cells. The impairment of DC cytotoxic function was observed mainly in high-grade glioma patients and associated with poor survival. The dysfunction of patient DC cytotoxicity was partially restored under in vitro pretreatment of DCs with double-stranded human DNA as well as rIL-2. In contrast to healthy donors, IFN-DCs in a part of high-grade glioma patients also failed to lyse primary autologous or allogeneic glioma cells. Our findings point to possible contribution of DC impairment in tumor pathogenesis in brain glioma and justify the necessity to evaluate and correct DC cytotoxic function when exploring DCs as cancer vaccines in glioma.

Use of interferon-α-induced dendritic cells in the therapy of patients with malignant brain gliomas

Clinical and immunological analysis of the efficiency of combined immunotherapy with the use dendritic cells for the treatment of malignant glioma of the brain was carried out. Dendritic cells generated in the presence of granulocyte-macrophage CSF and IFN-α retain their functional characteristics in patients with gliomas, which suggests the possibility of their use for the treatment of malignant tumors (glioma) of the brain. Combined therapy using interferon-induced dendritic cells was associated with generation of antigen-specific immune response during vaccinations. The results indicate satisfactory tolerance of combined immunotherapy using dendritic cells and the absence of toxic side effects at the stage of adoptive immunotherapy and at the stage of vaccinations with dendritic cells. Clinical trials showed that vaccinations with dendritic cells included into combined immunotherapy improved the quality of life and survival of patients with malignant gliomas.

Mesenchymal stromal cells improve early lymphocyte recovery and T cell reconstitution after autologous hematopoietic stem cell transplantation in patients with malignant lymphomas

Mesenchymal stromal cells (MSCs) possess a multi-lineage potential and immunoregulatory activities and provide a great potential in cell-based technologies. However, MSC suppressive activity raises concerns regarding the possible adverse effect of MSCs on the immune recovery. The influence of autologous MSC co-transplantation on recovery of T cell subsets in patients receiving autologous hematopoietic stem cell transplantation (AHSCT) for malignant lymphomas and multiple myeloma were characterized. Co-transplantation of MSCs improved lymphocyte recovery most effectively in patients with low input of hematopoietic stem cells or low absolute lymphocyte count in apheresis product. MSC co-transplantation improved early recovery of both memory and naive T cells with more prominent effect on naive CD4(+) T cells. Patients with MSC co-transplantation showed more effective reconstitution of recent thymic emigrants. These data indicate the positive impact of MSCs on immune reconstitution and note MSC co-transplantation is feasible to optimize the outcomes of AHSCT in malignant lymphoma patients.

Direct Antitumor Activity of Interferon-Induced Dendritic Cells of Healthy Donors and Patients with Primary Brain Tumors

Dendritic cells (DCs) are well known for their capacity to induce adaptive antitumor immune response through their unique ability to uptake, processing and presenting antigens (Ags), and tumor-specific T cell activation. In addition, cytokines produced by dendritic cells are able to regulate the direction and strength of immune response, activate the cytotoxic cells (NK-, NKT-cells) and participate in the coordination of the humoral immune response (Melief, 2008; Banchereau et al., 2000). An increasing number of reports evidenced that besides this role, DCs may display additional antitumor effects. Indeed, DCs in vitro can inhibit proliferation and provide a direct cytotoxic effect on tumor cells. In this, human monocyte-derived DCs might exert antitumor activity through multiple TNF family members (i.e. TNF-┙, lymphotoxin-┙1┚2, FasL, TRAIL), as well as perforin and/or granzyme (Wesa & Storkus, 2008; Chauvin & Josien, 2008). Direct tumor cell killing by DCs themselves appear to be highly important since involves immediate presentation of tumor-associated Ags in the context of MHC molecules for recognition by cognate T cells, inducing a specific immune response. Importantly, pleiotropy in DC mechanisms of cytotoxicity allows DCs to overcome the resistance of tumor cells that are heterogeneous with regard to their sensitivity to the various death pathways. A number of evidence suggests that the direct antitumor effect of DCs is not purely in vitro phenomenon, and is implemented in vivo. First, DCs are present in the tumors, and their higher content correlates with a more favorable prognosis (Becker 1999). Second, intra-tumoral injection of intact DC (not loaded with tumor antigen) has been shown to correlate with reduced tumor growth and even regression (Becker et al., 2001; Ehtesham et al., 2003). Finally, it is shown that the intra-tumoral introduction of DCs improves the effectiveness of chemotherapy, which may be due to synergistic effects of cytostatics and DCs (Vanderheyde et al., 2004).

Impairments of Antigen-Presenting Cells in Pulmonary Tuberculosis

The phenotype and functional properties of antigen-presenting cells (APC), that is, circulating monocytes and generated in vitro macrophages and dendritic cells, were investigated in the patients with pulmonary tuberculosis (TB) differing in lymphocyte reactivity to M. tuberculosis antigens (PPD-reactive versus PPD-anergic patients). We revealed the distinct impairments in patient APC functions. For example, the monocyte dysfunctions were displayed by low CD86 and HLA-DR expression, 2-fold increase in CD14+CD16+ expression, the high numbers of IL-10-producing cells, and enhanced IL-10 and IL-6 production upon LPS-stimulation. The macrophages which were in vitro generated from peripheral blood monocytes under GM-CSF were characterized by Th1/Th2-balance shifting (downproduction of IFN-γ coupled with upproduction of IL-10) and by reducing of allostimulatory activity in mixed lymphocyte culture. The dendritic cells (generated in vitro from peripheral blood monocytes upon GM-CSF + IFN-α) were characterized by impaired maturation/activation, a lower level of IFN-γ production in conjunction with an enhanced capacity to produce IL-10 and IL-6, and a profound reduction of allostimulatory activity. The APC dysfunctions were found to be most prominent in PPD-anergic patients. The possible role of APC impairments in reducing the antigen-specific T-cell response to M. tuberculosis was discussed.