Morphofunctional Characteristics of Lung Macrophages in Rats with Acute Respiratory Distress Syndrome.

A comprehensive morphofunctional study of the lungs and alveolar macrophages was carried out in Sprague-Dawley rats with acute respiratory distress syndrome (n=10) induced by intratracheal administration of E. coli LPS 0111:B4 in a dose of 15 mg/kg. On the first day after LPS administration, bronchopneumonia was observed in the lungs, the number of macrophages of the bone marrow origin and the number of M1 macrophages with the proinflammatory phenotype in the bronchoalveolar lavage increased, the expression of proinflammatory cytokines increased and the expression of anti-inflammatory cytokines decreased, which was accompanied by an increase in LPS and C-reactive protein in the blood serum. The revealed changes correspond to the development of acute respiratory distress syndrome in humans, and the decrease in the number of macrophages in the lungs and their predominant polarization to the M1-proinflammatory phenotype substantiate the use of cell therapy with reprogrammed M2 macrophages.

Differential Markers of Subpopulations of Epithelial Cells of the Larynx in Squamous Cell Carcinoma.

In squamous cell carcinoma of the larynx, the population of epithelial cells in the tumor tissue is initially heterogeneous and, in addition to tumor cells invading the organ mucosa, includes normal epithelial cells of protein-mucous glands and cells of the stratified epithelium covering the mucous membrane. A search for differential markers to separate these subpopulations was carried out. The surface marker CD44 and cytokeratins 5 and 17 that are often used to verify carcinoma cells, are common markers for all epithelial cells of the larynx. In highly differentiated carcinoma, subpopulations of normal and tumor epithelial cells can be separated by the level of expression of cytokeratins 10 and 18 and nuclear markers Ki-67 and p63. However, in moderately differentiated carcinoma, tumor cells and normal cells of the basal layer of the stratified epithelium covering the mucous membrane of the larynx have similar phenotypes, which should be taken into account when conducting experimental studies.

Expression of Metalloproteinases and Type I and III Collagens during Healing of Excisional Skin Wound on the Abdomen and Back in Rats.

The study was carried out using a novel rat model developed in our laboratory, namely16 mm diameter circular excisional wounds were generated on the abdomen which resulted in minimal scarring. Restoration of the skin integrity was completed by day 60 after the wounding surgery. By this time, regenerates on the abdomen were stronger than on the back (at, respectively, 58 and 17.4 % of the tensile strength of the intact skin at corresponding location) and the ratio of type I and type III collagens in regenerates on the abdomen reached the level of intact skin at the same location. On days 3 to 14, the ratio of Mmp9/Timp1 expression levels on the abdomen was higher than on the back. On days 20 and 30, the Mmp9/Timp1 ratio on the abdomen was identical to the level of intact skin, whereas the increased MMPs expression levels on the back were maintained until day 30. It has been shown for the first time that according to functional and molecular characteristics, wound healing on the abdomen of an adult rat is more similar to complete regeneration than scarring repair of the back skin.

Analysis of the Expression of Regulator Genes in Kupffer Cells and Monocytes.

Differences in the gene expression profiles in resident macrophages (in particular, Kupffer cells) and monocytes were revealed. However, these differences in gene expression profiles do not allow considering resident liver macrophages as purely M2 macrophages and monocytes as purely M1 macrophages. At the same time, a significant number of the genes upregulated in Kupffer cells are associated with normal regulation of liver functions (Arg 1, Flt, iNOs, and Kng). In monocytes, the expression of genes Alox15, Alox12, Tlr2, Tlr4, Tlr7, and Tlr8 (typical functional genes of macrophages) was also upregulated in comparison with Kupffer cells.

Quantitative and Qualitative Characterization of Phagocytic Activity of Macrophages of Bone Marrow and Fetal Origin.

We compared phagocytic activity of macrophages of monocyte origin and Kupffer cells under the influence of M1 and M2 inducers and without activation. Cultures of monocyte-derived macrophages and Kupffer cells were characterized by intensive expression of CD68 that was not affected by activation factors. At the same time, these cultures demonstrated different dynamics of phagocytic activity. Monocyte-derived macrophages initially had more pronounced absorption capacity that gradually increased during the experiment. Kupffer cells were characterized by abrupt fluctuations of phagocytic activity: sharp growth and rapid saturation. Despite these differences, the endosomes produced by monocyte-derived macrophages and Kupffer cells had similar degrees of maturity.

Dynamics of Expression of Cytokine Genes and Macrophage Content in the Lungs and Kidneys after Subtotal Hepatectomy in Rats.

The role of the lungs and kidneys in liver regeneration after subtotal hepatectomy was studied on a rat model. It was found that production of hepatocyte growth factor (HGF) in the lungs and kidneys and expression of cytokine genes Il1b, Il6, Il10, and tnfa significantly increased. Analysis of the dynamics of lung macrophage population showed that accumulation of HGF and the increase in the expression of cytokine genes in the lungs were accompanied by simultaneous increase in the number of CD68+ cells, which attested to the leading role of macrophages in activation of HGF synthesis in the lungs. Macrophage content in the kidneys after subtotal hepatectomy did not increase.

[Cell therapy of critical lower limb ischemia (problems and prospects)].

Critical limb ischemia is a syndrome that combines several peripheral artery diseases with different ethiology and pathogenesis but with similar prognosis, high morbidity and mortality. Possibility of surgical and conservative treatment of critical limb ischemia almost completely exhausted. Some hopes have arisen due to progress in cell technology. The article provides a critical analysis of pathogenic prerequisites of stem/progenitor cells for the treatment of patients with a critical limb ischemia in detail the basic results of preclinical and clinical studies on the safety and efficacy of cell technology. Unsolved problems and prospects of practical application are also discussed.

Advantages and disadvantages of treatment of experimental ARDS by M2-polarized RAW 264.7 macrophages.

Innate immunity reactions are core to any immunological process, including systemic inflammation and such extremes as acute respiratory distress syndrome (ARDS) and cytokine storm. Macrophages, the key cells of innate immunity, show high phenotypic plasticity: depending on microenvironmental cues, they can polarize into M1 (classically activated, pro-inflammatory) or M2 (alternatively activated, anti-inflammatory). The anti-inflammatory M2 macrophage polarization-based cell therapies constitute a novel prospective modality. Systemic administration of 'educated' macrophages is intended at their homing in lungs in order to mitigate the pro-inflammatory cytokine production and reduce the risks of 'cytokine storm' and related severe complications. Acute respiratory distress syndrome (ARDS) is the main mortality factor in pneumonia including SARS-CoV-associated cases. This study aimed to evaluate the influence of infusions of RAW 264.7 murine macrophage cell line polarized towards M2 phenotype on the development of LPS-induced ARDS in mouse model. The results indicate that the M2-polarized RAW 264.7 macrophage infusions in the studied model of ARDS promote relocation of lymphocytes from their depots in immune organs to the lungs. In addition, the treatment facilitates expression of M2-polarization markers Arg1, Vegfa and Tgfb and decreases of M1-polarization marker Cd38 in lung tissues, which can indicate the anti-inflammatory response activation. However, treatment of ARDS with M2-polarized macrophages didn't change the neutrophil numbers in the lungs. Moreover, the level of the Arg1 protein in lungs decreased throughtout the treatment with M2 macrophages, which is probably because of the pro-inflammatory microenvironment influence on the polarization of macrophages towards M1. Thus, the chemical polarization of macrophages is unstable and depends on the microenvironment. This adverse effect can be reduced through the use of primary autologous macrophages or some alternative methods of M2 polarization, notably siRNA-mediated.

[The treatment by expanded ex vivo autologous regulatory T-cells CD4+CD25+FoxP3+CD127low restores the balance of immune system in patients with remitting-relapsing multiple sclerosis]. / Lechenie vyrashchennymi ex vivo autologichnymi regulyatornymi T-kletkami CD4+CD25+FOXP3+CD127low vosstanavlivaet balans immunnoi sistemy patsientov s remittiruyushchim rasseyannym sklerozom.

AIM: To assess clinical efficacy and safety of the autologous (own) regulatory T-cells (Tregs)CD4+CD25+Foxp3+CD127low isolated from the blood of patients with remitting-relapsing multiple sclerosis. Patients with autoimmune diseases have the decreased number of peripheral Tregs (pTreg) and impaired suppressive ability. In order to restore levels of pTreg, it is possible to isolate precursor cells, enter expanded ex vivo autologous Treg cells and introduce an expanded amount of autologous cells as Treg vaccine. MATERIAL AND METHODS: A method of ex vivo Tregs expansion by 30-40 times within 5-7 days has been developed. Expanded ex vivo Tregs are more than 90% CD4+CD25+Foxp3+CD127low and have high suppressor activity. Fourteen patients with remitting-relapsing multiple sclerosis were included in pilot studies.Ex vivoTregs were introduced subcutaneously in dosefrom 2.8 to 4.5 108 cell per injection. The duration of follow-up was 1 year. RESULTS AND CONCLUSION: The numbers of pTregs in the blood of these patients elevated by 1.5-2 times. No adverse-effects, a decrease of relapses and stabilization of disability index were observed. It has been suggested that ex vivo expanded Tregs can compensate the impaired function of pTregs and can be used for adoptive immunotherapyof multiple sclerosis.