Regenerative Potential of Granulocyte Colony-Stimulating Factor Immobilized by Using Electron-Beam Synthesis Nanotechnology in an Experimental Model of Ovarian Reserve Depletion.

The pharmacological activity of granulocyte CSF (G-CSF) immobilized using electron-beam synthesis nanotechnology (imG-CSF) was evaluated in an experimental model of ovarian reserve depletion. The effectiveness of the drug was compared with that of its unmodified form. Depletion of the ovarian follicular pool in female Sprague-Dawley rats was caused by a single intravenous injection of the antitumor drug etoposide in the maximum tolerated dose. The effectiveness of the studied drugs was assessed by serum concentration of anti-Mullerian hormone (AMH) measured by ELISA and by the number of primordial, two-layer, multilayer, and atretic follicles counted on serial sections of the ovaries (5-µm thick; through the entire organ) stained with hematoxylin and eosin. It was found that imG-CSF prevents depletion of the ovarian reserve in the model used, which was confirmed by high AMH concentration and higher numbers of primordial, two- and multilayer follicles in comparison with the corresponding parameters in the control (etoposide), and by a decrease in the severity of atretic processes. Unmodified form of the drug demonstrated lower efficiency.

Assessment of Ante- and Postnatal Development of the Offspring of Male Rats Crossed in Delayed Periods after Treatment with Methotrexate in Low Doses.

We studied ante- and postnatal development of the offspring of intact female rats crossed with males injected with low doses of methotrexate 3 and 6 months before mating. The time of crossing corresponded to the manifestation of the cytostatic effect on spermatogonial stem cells. The offspring of methotrexate-treated males was characterized by increased preimplantation losses and fetal growth restriction in the antenatal period and inhibition of physical development, delayed formation of sensory-motor reflexes, and impaired learning abilities in the postnatal period.

Delayed Consequences of the Toxic Effect of Paclitaxel on the Testes of Prepubertal Rats and Their Correction with p-Tyrosol.

Delayed gonadotoxic effects were revealed in outbred male sexually mature rats (SD) after exposure to paclitaxel in the prepubertal period, and the possibility of their correction with p-tyrosol was shown. It was found, that administration of paclitaxel does not inhibit the ability of animals to conceive, but impairs the reserve capacity of the testicular tissue. In intact female rats crossed with male rats receiving paclitaxel, increased post-implantation fetal death was observed. Combined administration of paclitaxel and p-tyrosol alleviated the delayed effects of the cytostatic treatment on the prepubertal testis.

Estimation of the Regenerative Potential of para-Tyrosol in the Model of Testicular Insufficiency Caused by Damage to Spermatogonial Stem Cells.

The regenerative properties of p-tyrosol were investigated in a model of testicular insufficiency caused by a toxic effect on spermatogonial stem cells (single administration of paclitaxel in the maximum tolerable dose). Against the background of p-tyrosol administration, we observed an increase in the number of normal spermatogonia and Sertoli cells, stimulation of spermatogenesis, and renewal of the spermatogenic tissue. The treatment with p-tyrosol also led to a decrease in DNA damage in cells of the testicular tissue. These changes were accompanied by a decrease in the level of free radicals, an increase in antioxidant protection, and normalization of the redox potential.

Experimental Evaluation of Long-Term Toxic Effects of Methotrexate on Male Reproductive System.

The morphological and functional state of the reproductive system was studied in male outbred rats (SD stock) and male F1(CBA×C57BL/6) mice after long-term (3 months) methotrexate administration. The drug was administered subcutaneously once a week for 4 weeks, the dose for male rats was 1 mg/kg, for male mice 2.2 mg/kg. It was found that male rats retained the ability to conceive, their reproductive potential was not limited by increased risk of embryo death. At the same time, signs of astheno- and pathospermia were revealed. The testicular tissue was characterized by reduced content of the sources of the proliferative pool of spermatogenesis. In mice treated with methotrexate, increased content of DNA breaks was detected in the testicular cells.

Evaluation of the Effect of p-Tyrosol on the Level of DNA Damage in the DNA Comet Assay In Vivo.

The effect of p-tyrosol on the spontaneous level of DNA damage in the cells of the bone marrow, liver, kidney, and rectum of mice (series I) and on the genotoxic effects of cytostatic drugs with different mechanisms of action in rat testicular cells (series II) was studied by DNA comet assay on C57BL/6 mice. p-Tyrosol was administered in a dose of 40 mg/kg once (series I) or for 5 days before and 5 days after cytostatic exposure (busulfan, paclitaxel, methotrexate; series II). It was found that p-tyrosol reduced spontaneous level of DNA damage in all studied organs. p-Tyrosol exhibited an antigenotoxic effect with respect to the DNA-damaging action of methotrexate and produced no genoprotective effect in case of busulfan and paclitaxel.

Responses of Blood System to Doxorubicin/Docetaxel Chemotherapy in Patients with Breast Cancer.

We studied the effects of combined chemotherapy with doxorubicin/docetaxel on erythroid and granulocytic hematopoietic lineages with particular attention focused on their recovery in patients with stages III-IV breast cancer. Intensification of differentiation of erythroid and granulocytic CFU (even under conditions of their suppressed proliferation) provided the increase in the content of mature and morphologically differentiated elements in the bone marrow and peripheral blood. High proliferative activity of erythroid and granulomonocytic precursors resulted from enhanced production of hematopoiesis-stimulating activities by microenvironment elements.

Effectiveness of Phenolic Antioxidants in Experimental Model of Benign Prostatic Hyperplasia.

Experimental model of sulpiride-provoked benign prostatic hyperplasia was employed to comparatively assess the effect of phenolic antioxidants (dihydroquercetin, p-thyrozol, dibornol, and prostagenin) on prostate morphology. All examined agents decreased the degree of hyperplasia in acinar epithelium; the greatest efficacy was demonstrated by prostagenin. Moreover, dihydroquercetin and p-thyrozol increased the cross-section area of acinar lumina and prostate volume, which is inadmissible in this pathology. These results suggest that the use of phenolic antioxidants in the therapy of benign prostatic hyperplasia should be strictly controlled.

Endothelial Progenitor Cells and Notch-1 Signaling as Markers of Alveolar Endothelium Regeneration in Pulmonary Emphysema.

We studied the effects of elastase, cigarette smoke extract, D-galactosamine hydrochloride, and tyrosine kinase inhibitor SU5416 on endothelial progenitor cells and angiogenesis precursors, as well as on Notch-1 expression by immature endothelial cells. Simultaneously with pulmonary emphysema, different damaging factors with diverse mechanisms of action caused pathological changes in the microvascular network of the lungs and destroyed the alveolar endothelium in female C57Bl/6 mice. D-galactosamine hydrochloride disturbed mobilization of endothelial progenitor cells expressing VEGFR (CD45-CD309+) and angiogenesis progenitors (CD45-CD309+CD117+) and their migration into emphysema expanded lungs. Elastase inhibited VEGFR-expressing endothelial progenitor cells, while cigarette smoke extract inhibited cells with CD45-CD31+CD34+ phenotype. In pulmonary emphysema provoked by elastase or D-galactosamine hydrochloride, angiogenesis was provided by endothelial cells with CD45-CD31+CD34+ phenotype, whereas in emphysema modeled with SU5416 or cigarette smoke extract, it was provided by the endothelial VEGFR-expressing cells and mature CD31+ endothelial cells, respectively. Replenishment of immature endothelial cells damaged by elastase and SU5416 involved Notch-1+ angiogenesis precursors and Notch-1+ endothelial progenitor cells with VEGFR.

Experimental Study of the Effectiveness of Phenolic Antioxidants in Male Infertility Caused by Pathospermia.

The effect of phenolic antioxidants (dihydroquercetin, p-tyrosol, dibornol) on the morphology, functions, and redox processes in the reproductive cells of male rats was studied on the model of experimental pathospermia. All antioxidants reduced the percentage of degenerative forms of spermatozoa. Dibornol was most effective. Dihydroquercetin and p-tyrosol did not increase the total number of spermatozoa and the percentage of their mobile forms. These indicators were improved only by dibornol. After administration of all test drugs, the antioxidant potential of spermatozoa increased and did not significantly differ from the baseline values.

Mechanisms of Granulocytopoiesis Recovery Stimulation with Filgrastim in Breast Cancer Patients Receiving Chemotherapy.

We studied myelotoxicity of modern schemes of chemotherapy for breast cancer (docetaxel/doxorubicin and cyclophosphamide/doxorubicin/5-fluorouracil) towards granulocytopoiesis, the mechanisms determining the differences of hematological effects of these schemes, and the efficiency of correction of the observed changes with granulocyte CSF (filgrastim). Granulocytopoiesis stimulation with filgrastim during the treatment with docetaxel/doxorubicin combination was more pronounced than during cyclophosphamide/doxorubicin/5-fluorouracil therapy. The observed differences were found at all levels of granulocyte lineage organization (central and peripheral), which is related to different effects of the cytostatic substances used in the proposed protocols on the structures controlling hemopoiesis.

Experimental Analysis of the Efficacy of Dihydroquercetin on the Model of Chronic Nonbacterial Inflammation of the Prostatic Gland.

We studied the efficiency of dihydroquercetin on the model of chronic nonbacterial inflammation of the prostatic gland in rats. It was found that administration of dihydroquercetin was followed by a significant decrease in the area of the connective tissue in the prostatic gland to initial levels, which attested to antifibrotic properties of this oxidant. Additionally, the substance prevented the development of atrophy of acinus epithelium. After administration of reference drug Prostamol Uno, only moderate antifibrotic effects were observed.

Role of Sertoli and Leydig Cells in the Regulation of Spermatogonial Stem Cell and Development of Reproductive Disorders in Male C57Bl/6 Mice with Type 1 Diabetes Mellitus.

Course administration streptozotocin to male C57Bl/6 mice induces a complex of symptoms typical of type 1 diabetes mellitus: hyperglycemia and insulin deficiency, focal inflammatory infiltration of the pancreas, destructive changes in the Langerhans islets, damage to the insular apparatus (reduced number of PDX1+ cells and insulin expression by the secreting cells). Male reproductive disorder are serious complications of type 1 diabetes mellitus. In "diabetic" mice, interstitial edema with inflammatory infiltration and microvascular disorders in the testicular tissue are observed, the number of endothelial precursors (CD45-/CD31+) and the total number and percentage of motile spermatozoa decreased, immature spermatogenic epithelium cells are desquamated of into the lumen of the tubules. Disturbances in the proliferation and differentiation of various spermatogonial stem cell populations (c-kit-/CD90+, c-kit+/CD90+, and CD51-/CD24+/CD52+) in diabetes can be explained by the inhibitory influence of inflammatory factors on testosterone-producing Leydig cells.

Assessment of Erythroid and Granulocytic Hematopoietic Lineages in Patients with Non-Small-Cell Lung Carcinoma.

The toxic effects of combined cisplatin/docetaxel therapy cycles on erythroid and granulocytic hematopoietic lineages as well as their intercycle recovery were examined in patients with stage III-IV non-small-cell lung carcinoma. Responsiveness of the blood system to this therapy remained at a high level. Combined therapy pronouncedly activated the key elements of the erythroid and granulocytic hematopoietic lineages leading to accumulation of immature and mature myelokaryocytes in the bone marrow, enlargement of the medullary pool of mature neutrophils, and increase in the count of medullary erythroid and granulocytic precursor cells under conditions of their accelerated maturation.

Regenerative Potential of Spermatogonial Stem Cells, Endothelial Progenitor Cells, and Epithelial Progenitor Cells of C57Bl/6 Male Mice with Metabolic Disorders.

The properties of spermatogonial stem cells, endothelial progenitor cells, and the epithelial progenitors of C57Bl/6 mice under conditions of metabolic disorders were studied using the model of busulfan-induced suppression of spermatogenesis and in vitro culture technique. Spermatogonial stem cells CD117-CD90+ and epithelial progenitors CD45-CD31-Sca-1+CD49f+ derived from the testes of mice with metabolic disturbances demonstrated 17- and 28-fold increase in the respective cell mass and generated cell colonies in vitro. In contrast, spermatogonial stem cells with immune phenotype CD51-CD24+CD52+ had reduced selfrenewal capacity. Spermatogonial stem cells CD117-CD90+ and CD117+CD90+ as well as endothelial progenitors CD45-CD31+ derived from the testes of donor mice with metabolic disorders demonstrated high transplantation capacity in C57Bl/6 mouse testes damaged by cytostatic busulfan.

Regenerative Potential of Stem and Progenitor Cells from Ischemic Testes of C57Bl/6 Mice in Culture and in the Model of Spermatogenesis Suppression Caused by Busulfan.

The regenerative potential of stem and progenitor cells from ischemic testes of C57Bl/6 mice was studied in vitro (cell culture) and in vivo (mouse model of busulfan-induced suppression of spermatogenesis). Spermatogonial stem cells with phenotypes CD117-CD90+ and CD51-CD24+CD52+ from ischemic testes demonstrated 33-fold and 7-fold increments of cell mass and generated colonies in vitro. Epithelial (CD45-CD31-Sca-1+CD49f+) and endothelial (CD45-CD31+) precursors exhibited lower self-renewal capacity. On day 30 after injection of stem and progenitor cells from ischemic testes to the rete testis zone of the testes of busulfantreated animals, an increase in the count of CD117-CD90+ spermatogonial stem cells, total count, and mobile sperm count in the testes of recipient mice was observed. In addition, we observed an increase in Sca-1+ cell count, recovery of the spermatogenic epithelium in the seminiferous tubules, and appearance of immature Leydig cells in "busulfan" testes; the level of tissue testosterone and fertility index also increased.

Effect of Granulocyte Colony-Stimulating Factor Immobilized by the Electron-Beam Synthesis Nanotechnology on Reparative Regeneration of Spermatogenous Tissue.

Effectiveness of the granulocyte colony-stimulating factor immobilized by using electronbeam synthesis nanotechnology was investigated on the model of experimental testicular failure caused by the toxic effect on stem spermatogonia. Administration of the drug to experimental paclitaxel-treated animals increased the number of sources of the proliferative pool of spermatogenesis and its productivity. The effectiveness of immobilized granulocyte colony-stimulating factor was based on its ability to stimulate reparative regeneration of the spermatogenic tissue, which manifested in a decrease in spermatogenic layer maturity and increase in the number of microenvironment cells. Effectiveness of the immobilized form of the drug was superior to that of non-immobilized form.

Role of Hematopoietic Stem Cells in Inflammation of the Pancreas during Diabetes Mellitus.

The model of streptozotocin-induced diabetes mellitus in C57Bl/6 mice was employed to study the role of precursors of insulin-producing ß-cells, hematopoietic stem cells, and progenitor hematopoietic cells in inflammation. In addition to provoking hyperglycemia, streptozotocin elevated serum levels of IL-1ß and hyaluronic acid, induced edema in the pancreatic insular tissue and its infiltration by inflammatory cells (neutrophils, lymphocytes, and macrophages) and fibroblasts. Inflammation in pancreatic islets was accompanied by necrotic processes and decreasing counts of multipotent progenitor ß-cells (CD45(-), TER119(-), c-kit-1(-), and Flk-1(-)), oligopotent progenitor ß-cells (CD45(-), TER119(-), CD133(+), and CD49f(low)), and insulinproducing ß-cells (Pdx1(+)). Pancreatic infl ammation was preceded by elevation of the number of short-term hematopoietic stem cells (Lin-Sca-1(+)c-kit(+)CD34(+)) relative to long-term cells (Lin(-)Sca-1(+)c-kit(+)CD34(-)) in the bone marrow as well as recruitment of hematopoietic stem and progenitor cells into circulation. Transplantation of bone marrow hematopoietic stem and progenitor cells from diabetic C57Bl/6 donor mice to recipient CBA mice with 5-fluorouracilinduced leukopenia accelerated regeneration of granulocytopoiesis in recipient mice.

[Radionuclide Methods in the Assessment of Anthracycline-Induced Cardiotoxicity].

PURPOSE: To compare diagnostic efficacy of multigated acquisition (MUGA) scan and Tc-99m MIBI gated SPECT for evaluation and forecast of anthracycline-induced cardiotoxicity. MATERIAL AND METHODS: We included into this study 80 patients (72 women and 8 men, mean age 43 ± 4.2 years) with malignant tumors without overt pathology of the cardiovascular system. These patients received doxorubicin (50 mg/m2 per course) based cytostatic therapy. All patients were studied either by MUGA (n = 40) or 99MTc MIBI gated SPECT (n = 40) before initiation of chemotherapy 1 hour after first administration of doxorubicin and after the 4th course. RESULTS: After administration of 50 mg/m2 of doxorubicin 14 patients according to MUGA scan and 16 patients according to 99mTc MIBI gated SPECT had significant (≥ 10%) reduction of left ventricular ejection fraction (LVEF). Significant inhibition of systolic function in these patients remained after the 4th course of treatment. Individual analysis of the MUGA and 99mTc MIBI gated SPECT data registered after the fourth course (achievement of total doxorubicin dose of 200 mg/m2) showed that ≥ 10% LVEF reduction in response to the first dose of doxorubicin could predict the development of cumulative cardiotoxicity. CONCLUSION: MUGA scan and 99mTc MIBI gated SPECT can be applied with equal effectiveness for assessment of acute and chronic anthracycline-induced cardiotoxicity in patients with malignant tumors. LVEF reduction ≥ 10% in response to the first dose of doxorubicin appeared to be a predictor of development of chronic cardiotoxicity.

Role of PI3K, MAPK/ERK1/2, and p38 in implementation of the proliferative and differentiation potential of erythroid progenitors after blood loss.

The involvement of PI3K, ERK and p38-dependent signaling system in the regulation of functional activity of erythroid precursors after blood loss (30% of circulating volume) was studied. We demonstrated the important role of PI3K and p38 in the suppression of differentiation of erythroid precursors the contribution of p38 to stimulation of mitotic activity of erythroid CFU, which maintains the growth potential of the precursors at the optimal physiological level. The classical MAPK/ERK-kinase pathway does not determine the proliferative and differentiation status of erythroid CFU.