The First Experience of Application the Umbilical Cord-Derived Human Autologous Mesenchymal Stromal Cells for the Rehabilitation Therapy of Premature Infants with Extremely Low Body Weight and Hypoxic-Ischemic Encephalopathy.

Two clinical cases of the use of cell therapy with umbilical cord-derived autologous mesenchymal stromal cells in the rehabilitation therapy of extremely premature newborns (27-28 weeks gestation, body weights 900 and 870 g, respectively) with hypoxic-ischemic encephalopathy are described. The girls were born by caesarean section; the 1-min Apgar score was 6 points. After resuscitation including artificial ventilation, stabilization of the condition was achieved against the background of the development of hypoxic-ischemic encephalopathy. Rehabilitation therapy included administration of umbilical cord-derived mesenchymal stromal cells harvested at birth. The cells were injected in a dose of 1.6-7 million/kg body weight at the age of 3, 6, 12 months (the first patient) and 3, 6, 9, 15 months (the second patient). Psychoneurological developmental delay was scored using the Hellbrügge scale. Cell therapy induced no significant adverse reactions and improved the psychomotor development of children.

Dichotomy of Local Th1- and Systemic Th2/Th3-Dependent Types of Immune Response in Rheumatoid Arthritis.

Biomarkers of activity of rheumatoid arthritis were analyzed in the blood serum and synovial fluid of affected joints in 84 patients. Significant differences between the serum and synovial fluid levels were revealed for 10 of 17 analyzed biomarkers; the levels of IgM and proinflammatory cytokines TNFα, IL-1ß, IFNγ, and IL-6 were higher in the synovial fluid. The concentration of IL-10 in the synovial fluid was also elevated. In the peripheral blood, the content of antinuclear antibodies, circulating immune complexes, and cytokines IL-4 and TGF-ß was elevated. These findings attest to the development of local Th1 type immune response in affected joints paralleled by compensatory elevation of immunosuppressive cytokine IL-10 and systemic Th2/Th3 type immune response (judging from peripheral blood parameters) in patients with rheumatoid arthritis.

The Use of Autologous Mesenchymal Stem Cells for Cell Therapy of Patients with Amyotrophic Lateral Sclerosis in Belarus.

We studied a new method of treatment of amyotrophic lateral sclerosis with autologous mesenchymal stem cells. Autologous mesenchymal stem cells were injected intravenously (intact cells) or via lumbar puncture (cells committed to neuronal differentiation). Evaluation of the results of cell therapy after 12-month follow-up revealed slowing down of the disease progression in 10 patients in comparison with the control group consisting of 15 patients. The cell therapy was safe for the patients.

Effect of platelet releasate on osteogenic differentiation of human mesenchymal bone marrow stem cells.

We studied the effect of platelet releasate on osteogenic differentiation of human mesenchymal BM stem cells. Histological staining and reverse transcription PCR analysis showed that addition of platelet releasate to osteogenic differentiation medium stimulated the formation of calcium ossificates and alkaline phosphatase and increased expression of osteopontin and alkaline phosphatase genes.

[Highly efficient transfer and stable expression of two genes upon lentivirus transduction of mesenchymal stem cells from human bone marrow].

The efficiency of human bone marrow (BM) mesenchymal stem cell (MSC) transduction with a bicistronic lentivirus vector was estimated, and the stability of transgene expression in genetically modified MSCs was determined. First-passage BM MSCs were capable of efficient transduction with the bicistronic lentivirus vector. The transduction efficiency depended on the multiplicity of infection (MOI), being 64 +/- 6.5 and 88.6 +/- 2.9% at MOI 10 and 20, respectively. The lentivirus transduction efficiency proved independent on the number of passages of a BM MSC culture, and expression of the egfp and dsRed1 transgenes in genetically modified MSCs remained stable for one month of culturing. A comparison showed that the level of egfp and dsRed1 transgene expression was preserved upon hepatogenic differentiation in vitro. The results provide a basis for further development of multigenic modification of human BM MSCs for research and/or therapeutic purposes.

Neurogenic induction of human mesenchymal stem cells in fibrin 3D matrix.

We studied the potential of neurogenic induction of human bone marrow mesenchymal stem cells in fibrin-based 3D matrix. The best results were obtained after incubation of mesenchymal stem cells in fibrin 3D matrix in the presence of neurogenic medium containing EGF and bFGF growth factors. Under these conditions, most cells formed numerous branching processes and expressed neural cell marker ßIII-tubulin. Optimal combination of 3D matrix and neurogenic factors of the medium can provide new insight into neurogenic potential of mesenchymal stem cells, which can be used for the therapy of neural traumas and neurodegenerative diseases.

Hepatogenic potential of human bone marrow and umbilical cord blood mesenchymal stem cells.

Conditions of human BM and umbilical cord blood MSC in vitro differentiation in the hepatogenic direction were studied. Changes in cell morphology, phenotype, acquisition of the capacity to produce albumin and accumulate glycogen, express cytokeratin, alkaline phosphatase, and albumin mRNA indicated that BM and umbilical cord blood MSC differentiated in vitro into immature hepatocyte-like cells.

Neurons and stromal stem cells as targets for polycation-mediated transfection.

Expression of transgenes in neurons and stromal/mesenchymal stem cells (MSC) can greatly enhance their therapeutic potential. In transfection experiments, we studied properties of linear and branched (dendrimers) polycations as transgene delivery vehicles. Linear polyethyleneimine transfected neurons, but was ineffective in MSC. Polyamidoamine dendrimers showed greater transfection efficiency and mean GFP fluorescence intensity compared to phosphorus dendrimers of the same (4th) generation. Expression of neurotrophic factor BDNF in MSC transfected with polyamidoamine dendrimers was also by more than 10 times higher.

[Neurogenic differentiation of mesenchymal stem cells: a transgenic approach].

Mesenchymal stem cells (MSC), alongside with "traditional" osteogenic, chondrogenic and adipogenic differentiation potentials, are considered by many researches as capable of giving rise to neurogenic lineage as well. We overview transgenic approaches to the study of neurogenic differentiation of MSC, including expression of neurotrophic factors, signalling molecules and other transgenes with neurogenic properties.

[Use of LAK-cells and systemic chemotherapy with hyperthermia in the management of chemo-resistant tumors].

Tentative results of LAK-cell and whole-body hyperthermia (WBH) were evaluated in 19 children with advanced chemorefractory tumors. LAK-cells were obtained by extracorporeal incubation of peripheral blood lymphocytes: a germ-cell rhabdomyosarcoma was detected in 4, Askin's tumor--2--2, renal cell carcinoma--2 and miscellaneous--7. Autologous LAK-cells were infused twice: on completion of WBH as body temperature fell to as low as (+) 40 deg. C and on day after WBH. The latter was well tolerated. Complete or partial response to thermochemobiotherapy was reported in 8 patients. Overall 5-year survival was 43% (median follow-up--12.6 months).

[Gene therapy based on human mesenchymal stem cells: strategies and methods].

Major technologies of transient and stable transgene expression in hMSC are reviewed. Properties and efficiencies of recombinant lentiviruses, adenoviruses, AAV and baculoviruses used for hMSC transduction are compared. The aims oftransgenesis include directed differentiation of hMSC, function improvement, correction of pathology factors and proliferatrion control, as well as many basic research challenges.

Plasticity of human mesenchymal stem cell phenotype and expression profile under neurogenic conditions.

Human bone marrow MSC cultured in neurogenic medium containing EGF and FGFbeta demonstrated alteration of the phenotype and expression of neuronal precursor/early neuron markers nestin and NSE. Signals of expression of neuronal and oligodendroglial markers MAP-2, dm-20, and MBP were detected after prolongation of incubation in neurogenic medium to 2 weeks. Cells with neuronal morphology were immunopositive to early neuronal marker beta-III-tubulin. Replacement of neurogenic medium for alpha-MEM with 10% fetal calf serum induced reversion of the phenotype to that typical for human MSC. This indicates high plasticity of the phenotype and expression profile of neuronal markers in MSC cultured under neurogenic conditions or possibility of dedifferentiation of MSC reaching the stage of neuronal precursors/early neurons.

[Effect of interleukin-2 on apoptosis and function of human lymphocytes in in-vitro cytostatics culture].

In vitro experiments were conducted to determine the impact of interleukin-2 (IL-2) on apoptosis and function of cytostatic-cultured lymphoid (mononuclear) cells (MNC) of peripheral blood from healthy subjects and children with cancer. Neither slight or any effect on vepesid-16 or carboplatin--cultured MNC apoptosis, nor any phytohemagglutinin--induced proliferation was found. By contrast, in carboplatin- cultured MNC from healthy subjects, IL-2 significantly potentiated their toxicity for tumor cells by producing interferon-gamma. It was concluded that IL-2 predominantly supported MNC functional activity rather than inhibited lymphoid MNC apoptosis in in vitro culture with cytostatic drugs.

Discrepancy between direct and antibody-dependent cytotoxic activities of human LAK cells.

Human lymphokine-activated killer (LAK) cells display cytotoxic activity against natural killer (NK)-resistant tumor cells in an antibody-independent and -dependent manner. We compared LAK cell-mediated antibody-independent cytotoxicity (LAK activity) and antibody-dependent cellular cytotoxicity (ADCC) against untreated and antibody-coated Raji cells, respectively. Human lymphocytes showed drastically increased LAK activity after stimulation with interleukin-2 (IL-2) for 3 or 7 days when compared to non-activated cells. The level of ADCC was reduced for 3-day-generated LAK cells and augmented for 7-day-generated LAK cells as compared to non-activated cultured lymphocytes. Phenotypical analysis revealed IL-2-induced up-regulation of the proportion of CD11b+ (but not CD16+) lymphocyte subpopulation in 7-day-generated LAK cells. The data imply that human LAK cells exhibit antibody-dependent and -independent cytotoxic activities via distinct effector pathways at different stages of generation. These stages may be associated with changes in adhesion molecule (CD11b/CD18) expression on the surface of IL-2-activated lymphocytes.

Plasma level of tumor necrosis factor-alpha (TNF-alpha) correlates with leukocytosis and biological features of leukemic cells, but not treatment response of children with acute lymphoblastic leukemia.

We have analyzed the clinical significance of the plasma level of tumor necrosis-alpha (TNF-alpha) detection in childhood acute lymphoblastic leukemia (ALL) at diagnosis. 49 patients (pts) with B-lineage ALL (3-17 years of age) and 30 healthy children (age-related control) were enrolled in study. The mean value of the level of TNF-alpha was 43.4 +/- 8.1 pg/ml in ALL pts and 30.7 +/- 3 pg/ml in control. In ALL pts the level of TNF-alpha positively correlated with blast cell count in peripheral blood (R = +0.432; P = 0.008); negatively correlated with percentages of S-phase leukemic cells (R = -0.446; P = 0.042) and culture-induced apoptotic cells (R = -0.411; P = 0.057). Pts with TNF-alpha level above the median value were characterized by higher WBC count (P = 0.025), lower percentage of S-phase leukemic cells (P = 0.02) and tendency to lower percentage of apoptotic cells (P = 0.07) vs pts with TNF-alpha level below the median value. No significant association of the level of TNF-alpha with treatment response at days 8 and 15 or 3-year overall survival was defined. We conclude that the elevated plasma level of TNF-alpha is a useful marker to assess disease activity/progression, but not prognosis of childhood B-lineage ALL.

Different patterns of cytokine regulation of phagocytosis and bacterial killing by human neutrophils.

We describe here the capacity of human recombinant interleukin-1-beta (IL-1beta), interferon-gamma (INF-gamma), interferon-alpha (INF-alpha), tumor necrosis factor-alpha (INF-alpha), interleukin-6 (IL-6) and interleukin-2 (IL-2) to modulate phagocytosis and bacterial killing by human neutrophils. IL-1beta, INF-gamma and TNF-alpha enhance phagocytosis of bacteria but do not have consistent effect on bacterial killing. In contrast, IL-6 augments bacterial killing but not phagocytosis of bacteria by neutrophils. INF-alpha augments and IL-2 depresses both antibacterial reactions of neutrophils. A direct effect of TNF-alpha (inhibition) and IL-2 (promotion) upon bacterial growth has been observed. It is concluded that (a) phagocytosis and intracellular bacterial killing are independent reactions of neutrophil-mediated antibacterial defence on the basis of different patterns of up- and down-regulation by cytokines, and (b) cytokines can exhibit direct effects on bacterial growth.

[Autoimmune disorders in liqvidators 11 years after the Chernobyl accident]. / Autoimmunye sdvigi u likvidatorov cherez 11 let posle avarii na ChAES.

We studied long-lasting consequences of the low-doses irradiation on the immune system of 71 clean-up workers who participated in the emergency work after the Chernobyl Plant accident in 1986 and 25 healthy donors from Belarus. In sera of the workers the level of autoantibodies to thyroid gland antigens (thyroglobulin and microsomal fraction of thyroid gland) was increased in 48% of cases, the level of autoantibodies to lens oculis antigen was increased in 44% of cases; the level of circulating immune complexes was elevated in 55%, and the serum level of thyroglobulin in 60% of people. Immunological disorders were found without any definite clinical evidences of diseases and this allows us to consider the examined contingent as a group of risk for the development of autoimmune pathology in the future.

[Changes in the functional activity of human neutrophils under the action of recombinant interleukin-2]. / Izmenenie funktsional'noi aktivnosti neitrofilov cheloveka pod deistviem rekombinantnogo interleikina 2.

The effect of recombinant interleukin 2 (rIL-2) on the functional activity of human polymorphonuclear cells (neutrophils) was studied. The study revealed that the preliminary incubation of neutrophils with rIL-2 at a concentration of 10 I.U./ml for 30 minutes significantly enhanced the chemotaxis of neutrophils. In the presence of rIL-2 the tendency towards an increase (p > 0.05) in the spontaneous migration of neutrophils and in the intensity of their respiratory burst was observed. When used at concentrations of 100-1000 I.U./ml, rIL-2 inhibited the phagocytosis of Staphylococcus aureus, and at concentrations of 10 I.U./ml and greater it inhibited the bactericidal activity of neutrophils. The conclusion on the direct action of rIL-2 on the functional activity of human neutrophils was made.

[Cytokines in vaccines and interferon preparations]. / Tsitokiny v vaktsinakh i preparatakh interferona.

Virus vaccines prepared on the basis of cells of mammals (rabies, poliomyelitis, measles and hepatitis A vaccines) contain cytokines (IL-1 beta, IL-6, TNF-alpha), whose concentration depends on the kind of the vaccine. Cell lines (green monkey kidney cells, VERO, 4647), used for the preparation of commercial and experimental vaccines, do not produce spontaneously any of the above cytokines. Cell line L-68, used for the manufacture of experimental measles vaccine, is capable of the spontaneous synthesis of IL-6. In Russian and foreign preparations of interferon the presence of IL-1 beta and TNF-alpha has been detected; the content of these cytokines is determined by the specific features of the methods used manufacturing these preparations.

[The neutrophil antibacterial function of patients with chronic bronchitis. Its immune correction by using inflammatory cytokines and allogeneic lymphocytes in vitro]. / Antibakterial'naia funktsiia neitrofilov u bol'nykh khronicheskim bronkhitom. Immunokorrektsiia s pomoshch'iu tsitokinov vospaleniia i allogennykh limfotsitov in vitro.

In patients with acute and chronic bronchitis decreased neutrophil phagocytic, induced O2-dependent metabolic, bactericidal activity and the content of lysosomal cationic proteins, increased spontaneous O2-dependent metabolic activity were revealed. The most pronounced neutrophil functional disorders were detected in patients with chronic obstructive bronchitis (COB). The use of human immunoregulatory recombinant cytokines (interleukin-1 beta, interferon-gamma, tumor necrosis factor alpha) failed to augment in vitro suppressed neutrophil antibacterial (phagocytic) activity in patients with COB. These patient's neutrophil antibacterial activity restored in vitro in the presence of allogeneic (nonactivated or interleukin-2-activated) lymphocytes. The data obtained provide evidence for healthy donor's lymphocytes use in immunocorrection of patients with chronic bronchitis.