Evolution of Mechanical Stress (In-vitro) on Properties of Granulocytes in Acute Lymphoblastic Leukemia.

Acute lymphoid leukemia (ALL) affects the lymph cells or lymphocytes that make up the lymph tissue and prevents the proper maturation of the bone marrow cells. The processes through which cells convert mechanical stimuli into biochemical signals are called mechanical transitions and result in the sensation of specific cellular responses. In the present study, the functional properties of granulocytes of the patients with ALL were investigated using the in vitro mechanical stress model. The experimental part of the work was executed using blood from patients with ALL (n=30) being treated in the Hematological Department of Belgorod Region Hospital, Belgorod, Russia. The patients were in the age range of 18-45 years. Sample blood was obtained from all the patients who underwent a standard course of chemotherapy. Blood sampling was performed using a venepuncture and collected into the vacuum tubes Vacuette K3E. Blood samples from each experimental group were divided into two groups of control and experiment. The injection model of mechanical stress was used for the experiment group in vitro. Subsequently, the adenosine triphosphate (ATP) concentration increased by 1.8 times in this group, compared with the controls. Young's module, which numerically characterizes the rigidity of the granulocytes' plasmalemma, decreased by 54.4% (P<0.05) under the influence of mechanical stress. The surface potential of plasmalemma was not significantly different between samples in the group of control and experiment in patients with ALL. However, the adhesive force between erythrocyte and granulocyte increased by 30.7% (P<0.05). The osmotic load test showed an increase in the cell's volume during incubation. The use of membrane reserve by granulocytes increased by 47% (P<0.05) at the initial seconds of incubation. The obtained results pointed to the regulatory role of ATP molecules in intercellular signaling and add to the present literature regarding the mechanisms of intercellular interaction in the microvasculature on the development of leukemia. Moreover, the obtained results can be taken into account for the development of new pharmacological immune correctors.

[Activity of cAMP System in Sheep Lymphocytes and Platelets Exposed to External γ-Radiation in vivo].

The activity of the cyclic adenosine-3,5-monophosphate (cAMP) in lymphocytes and platelets of sheep has been studied for 15 days after animal exposure to total external γ-radiation at a dose of 4 Gy (LD50/30). The basal and E1 stimulated activity of adenylate cyclase and phosphodiesterase activity were determined by the thinlayer chromatography technique, and the cAMP content was determined using RIA-kits with 125J. Modification was revealed in the cAMP system functioning in the cell populations studied from the first day following radiation exposure. In particular, the basal adenylate cyclase activity in lymphocytes increased 1.7-4.3 times at all study dates, and stimulated enzyme activity increased on days 1-7, 21-8.8-fold compared to the initial data. Platelets showed increase in the basal adenylate cyclase activity on days I and 7, 2.7 and 35 times, and stimulated activity at the same times--9 and 5.7 times, respectively. The adenylate cyclase stimulation coefficient in lymphocytes raised 1.8 and 2.3 times on days 1 and 5, respectively, on day 7 it corresponded to the initial data and on days 10-15 it dropped below the control. However in platelets, this parameter did not have significant differences from the initial data. The phosphodiesterase activity in lymphocytes increased 3-fold on day 3, 2.4-fold on day 7, and in platelets it practically remained at the control level. The cAMP content in lymphocytes dropped within the first 12 hours and on day 3, 2.71 and 2.38 times, respectively, whereas in platelets it decreased 3.08 times within 12 hours and increased 1.91 times on day.3. At other times the cAMP content in lymphocytes and platelets did not differ from the initial data. The results suggest that the identified modification of cAMP system activity in lymphocytes and platelets in various periods after exposure of animals is based on different mechanisms. In the early period, alteration in the enzyme activity of cAMP system and cAMP content is caused by radiation effects on cytoplasmic membrane of cells circulating in the peripheral blood of animals. In the later periods, the recorded variations in the cAMP system activity are defined by the prevalence in the blood of more resistant to radiation damage cell subpopulation with an enhanced activity of cAMP enzymes and cAMP concentration corresponding to unexposed animals.

[View on the problem of Klatskin tumor].

Klatskin tumor arises from the common hepatic duct and its bifurcation the bile ducts and is the most common primary malignancy of the biliary tree. the location of the tumor and its close relationship with vascular structures at the hepatic hilum have resulted in a low resectability and high morbidity and mortality. Improvement of instrumental diagnostics and operative techniques allows to perform extended resection and complex interventions on the liver, bile ducts and vascular structures at the hepatic hilum. The role of chemoratiotherapy and photodynamic therapy is not fully understood. thus, questions of treatment and prognosis of the disease are remain relevant and require further study.

[BILIARY DECOMPRESSION ASSOCIATED WITH TUMOURS OF PROXIMAL BILE DUCTS].

The treatment of tumours of proximal hepatic ducts (Klatskin tumours) remains a serious challenge because most patients are referred to the clinic at later stages of the disease. Its most common symptom is obstructive jaundice syndrome. Various methods of bile tract decompression have been proposed to resolve manifestations of this syndrome in the course of preoperative preparation for surgery of potentially resectable tumours and palliative treatment of incurable neoplasms. However analysis of the literature shows that the choice of an adequate method for the purpose remains a matter of debate.

[Methods of mathematical modeling in morphological diagnostics of Chernobyl factor influence on the testes of coal miners of Donbas--the Chernobyl disaster fighters].

The morphometric estimation of parenchyma and stroma condition included the determination of 29 parameters in testicles at 27 persons. The mathematical model of morphogenesis of testicles was created by Bayes' method. The method of differential diagnosis of testicles tissues' changes conditioned by the influence of the Chernobyl factor and/or unfavorable terms of the work in underground coal mines have been worked out. Its practical use provides exactness and reliability of the diagnosis (not less than 95%), independence from the level of the qualification and personal experience of the doctor, allows us to unify, optimize and individualize the diagnostic algorithms, answer the requirements of evidential medicine.

[Endogenous content of the nitric oxide in the cell layers of the eye retina].

Nitric oxide is a universal molecule that regulates many different functions in an organism. In the eye retina nitric oxide plays both a regulatory role by modulation of the synaptic transmission between photoreceptors and bipolar cells and a toxic role in apoptosis induction in the outer nuclear layer and in the layer of ganglion cells. In this paper there has been made the first attempt to estimate the endogenous NO concentration in retina layers in vivo. The concentration of the nitric oxide was determined by two indepen- dent techniques: ESR spectrometry using spin trap for in vivo determination and NO-sensitive microelectrode for in situ determination in the survival isolated frog retina. The distinct NO con- centration was detected only in the ganglion cells layer (~0.25 µM) and in the inner segments layer of the photoreceptors (~0.6 µM). The activity and the kinetic characteristic of the NO-synthase localized in the same layers were also determined. Key words: retina cells layers, nitric oxide, ESR, NO-sensitive microelectrodes.

[METHODS OF MATHEMATICAL MODELING IN MORPHOLOGICAL DIAGNOSTICS OF CHORNOBYL FACTOR INFLUENCE ON PROSTATE GLAND OF COAL MINERS-- THE CHERNOBYL DISASTER FIGHTERS].

The morphometric estimation of parenchyma and stroma condition included the determination of 25 parameters in a prostate gland at 27 persons. The mathematical model of morphogenesis of prostate gland was created by Bayes' method. The method of differential diagnosis of a prostate gland tissues' changes conditioned by the influence of the Chernobyl factor and/or unfavorable terms of the work in underground coal mines have been worked out. Its practical use provides exactness and reliability of the diagnosis (not less than 95%), independence from the level of the qualification and personal experience of the doctor, allows us to unify, optimize and individualize the diagnostic algorithms, answer the requirements of evidential medicine.

[Morphology and structural peculiarities of Basidiomycetes pathogens].

The materials of studies of morphology and structural peculiarities of viruses, fungi and bacteria, which affect Basidiomycetes under biotechnology process and nature biocenosis conditions are given. The analysis of infection development in button mushroom (Agaricus bisporus) (J.Lge) Imbach and in oyster mushroom (Pleurotus ostreatus Kumm.), which served as model objects in the experiments of various levels of complexity has been carried out. Other kinds of edible and medicinal mushrooms, which were a source of biochemical fractions to form biologicals were investigated.

[Photoinduced production of reactive oxygen species by retinal derivatives and conjugates].

The formation of reactive oxygen species (ROS) during all trans-retinal illumination was studied by ESR. The quantity of ROS was estimated by the ESR signal of a spin adduct of DEPMPO (5-dietoxyphosphoryl-5 methyl-1-pirroline-N-oxide). The ESR signal was found to be a superposition of the adducts: 80%--superoxide anion radical, 15%--hydroxyl radical and 5%--an unknown radical. It was shown that the addition of an equimolar albumin concentration resulted in the 5-fold decrease of the relative quantum yield for ROS generation, and a 4-fold albumin excess led to decreased ROS production by 30-fold. It is supposed that ATR and A2E in the photoreceptor membrane are the sources of photodamage induced by ROS. ATR in the cell is known to be transported by interphotoreceptor retinal binding proteins that belong to the albumin family. The ATR binding with proteins is supposed to prevent the ROS production in the photoreceptor cell and thus produces the light damage protection action.

[Morphological verification problems of Chernobyl factor influence on the testis of coal miners of Donbas-liquidators of Chernobyl accident].

Problem of a diagnostic of Chernobyl factor influences on different organs and systems of Chernobyl accident liquidators are remain actually until now. Though morbidly background which development at unfavorable work conditions in underground coalminers prevents from objective identification features of Chernobyl factor influences. The qualitative and quantitative histological and immunohistochemical law of morphogenesis changes in testis of Donbas's coalminer - non-liquidators Chernobyl accident in comparison with the group of Donbas's coalminers-liquidators Chernobyl accident, which we were stationed non determined problem. This reason stipulates to development and practical use of mathematical model of morphogenesis of a testis changes.

[Morphological verification problems of Chernobyl factor influence on the prostate of coalminers of Donbas--liquidators of Chernobyl accident].

Problem of a diagnostic of Chernobyl factor influences on different organs and systems of Chernobyl accident liquidators are remain actually until now. Though morbidly background which development at unfavorable work conditions in underground coalminers prevents from objective identification features of Chernobyl factor influences. The qualitative and quantitative histological and immunohistochemical law of morphogenesis changes in prostate of Donbas's coalminer-non-liquidators Chernobyl accident in comparison with the group of Donbas's coalminers-liquidators Chernobyl accident which we were stationed non determined problem. This reason stipulates to development and practical use of mathematical model of morphogenesis of a prostatic gland changes.

DNA-damaging effects of dental bleaching agents.

We studied DNA-damaging effects of dental bleaching systems containing hydrogen peroxide and/or carbamide peroxide by the "comet assay" (alkaline version). Dental bleaching systems in a hydrogen peroxide concentration range from 0.03 to 30 mM produced a genotoxic effect on isolated HeLa cells in vitro comparable with the effects of pharmacopoeial hydrogen peroxide or urea peroxide. Catalase protected the cells against products containing hydrogen peroxide and had no effect on the genotoxicity of samples containing carbamide peroxide.

[Modification of the nitric oxide concentration regulates the development of the apoptosis in the eye retina].

Using model elaborated it was shown that the retinal ischemia initiated the development of the apoptosis in the inner layers of the retina. Administration of NOS inhibitor prevented the development of the apoptosis in the retina. To ascertain if nitric oxide could induce the retinal apoptosis by itself the nontoxic donor of nitric oxide (dinitrosyl iron complex) was injected intravitreally. Administration of DNIC in low concentrations induced the development of the apoptosis in the same retinal layers as in ischemia. The injection of dinitrosyl iron complex at the higher concentration resulted in the decrease of the apoptosis level. Administration of dinitrosyl iron complex with excess of glutathione didn't lead to the development of the retinal apoptosis. The obtained data demonstrates the neurotoxic properties of the excess of nitric oxide in the retina.

Role of NO-synthase in regulation of protein metabolism of stretched rat m. soleus muscle during functional unloading.

Gravitational unloading causes atrophy of muscle fibers and can lead to destruction of cytoskeletal and contractile proteins. Along with the atrophic changes, unloaded muscle frequently demonstrates significant shifts in the ratio of muscle fibers expressing fast and slow myosin heavy chain isoforms. Stretching of the m. soleus during hindlimb suspension prevents its atrophy. We supposed that neuronal NO-synthase (NOS) (which is attached to membrane dystrophin-sarcoglycan complex) can contribute to maintenance of protein metabolism in the muscle and prevent its atrophy when m. soleus is stretched. To test this hypothesis, we used Wistar rats (56 animals) in experiments with hindlimb suspension during 14 days. The group of hindlimb suspended rats with stretched m. soleus was injected with L-NAME to block NOS activity. We found that m. soleus mass and its protein content in hindlimb-suspended rats with stretched m. soleus were preserved due to prevention of protein degradation. NOS is involved in maintenance of expression of some muscle proteins. Proliferation of satellite cells in stretched m. soleus may be due to nNOS activity, but maintenance of muscle mass upon stretching is regulated not by NOS alone.

Protective effect of L-Arginine administration on proteins of unloaded m. soleus.

Cytoskeletal and contractile proteins degenerate during functional unloading of muscle. The ratio of myosin heavy chain (MHC) expression changes simultaneously. We have supposed that NO can be a signal molecule related to the regulation of protein metabolism upon muscle unloading. To test this hypothesis, Wistar rats underwent functional unloading for 14 days without and with peroral administration of L-arginine (500 mg/kg) as NO precursor. Significant decreases in m. soleus mass, NO, nNOS, dystrophin, Hsp90, p-S6K, and type I MHC mRNA contents were found in the group of animals with unloading without preparation compared to those in control and in the group with unloading and administration of L-arginine; at the same time, increased contents of atrogin-1/MAFbx and MuRF-1 (p < 0.05) were found. No difference in the IGF-1 mRNA content between all three groups was found. Atrophy was significantly less pronounced in the group with unloading and L-arginine administration compared to that without the amino acid, and no destruction of cytoskeletal proteins was observed. We conclude that administration of L-arginine upon functional unloading decreases the extent of m. soleus atrophy, prevents the decrease in it of type I MHC mRNA, and blocks destructive changes in some cytoskeletal proteins. Such effect can be due to the absence of increase in this group of the content of some ubiquitin ligases and decreased intensity of the p70S6 kinase synthesis marker.

[Carbohydrate metabolism after pancreatoduodenal resections in patients with chronic pancreatitis].

The carbohydrate metabolism was analyzed in 70 patients with the complicated chronic pancreatitis before and after pylorus-preserving pancreatoduodenal resection (PPDR). Patients were aged 42±1,15 years, of them 64 were men and 6 - women. Bodymass index was 2,2±0,54kg/m2. Patients were divided in groups according to pre- and postoperative glucose metabolism disorders to define the diabetes morbidity by chronic pancreatitis and the influence of surgery on the first. According to the preoperative data, diabetes mellitus of mild severity had 9 of 70 operated patients, 18 patients had medium diabetes. After the operation only one patient demonstrated the aggravation of diabetes. However, the onset of diabetes mellitus was registered in 8 patients postoperatively. The increased glucose blood level in early postoperative period proved to have a high prognostic significance. Therefore, all patients of that category must be strongly recommended to be under the endocrinologist's observation after hospital discharge. That measure would provide a timely diagnose and treatment of the diabetes mellitus. All patients with chronic pancreatitis should be thoroughly investigated concerning glucose metabolism disorders before the operation.

[The distal pancreatic resection with spleen preservation: which collaterals can we rely on?].

The spleen preservation by distal pancreatic resection can be performed either with spleen vessels preservation or with the ligation of the least. The experiment evolved ligation of all gastric arteries but the short gastric arteries in 20 cadaveric organocomplexes, followed by ink perfusion through the left gastric and left gastro-epiploic arteries. The study was amplified with the intraoperative dopplerography of portal arteries of the spleen after crossclamping of the left gastro-epiploic and short gastric arteries. Ten patients after distal pancreatic resection with spleen preservation and splenic vessels ligation had the CT-angiography before and after the surgery. All the conducted studies demonstrated the incapability of short gastric arteries to supply the satisfactory spleen perfusion. The left gastro-epiploic artery proved to be the main source of splenic blood supply after splenic vessels ligation.

[Nitrites can be reduced in retinal vessels during hypoxia and protect the retina against ischemia and apoptosis].

The possibility and the mechanism of the reduction of nitrites in retinal vessels under the acute hypoxia in vivo have been investigated. An experimental model of the rat retina ischemia was elaborated using the laser coagulation of retinal vessels. It was demonstrated that the vessel thrombosis does not occur if the nitrite concentration in the vessels is increased. It was proposed that, under acute hypoxia, nitrites are reduced to NO, which results in a drastic vasodilatation. Considering that the effect takes less than a minute, this reduction cannot be due to hypoxic acidosis but is more likely associated with NO reduction by heme proteins. It was found that the increased concentration of nitrites protects the retina from the development of ischemia progress and that the preliminary administration of nitrites prevents apoptosis in the retina and a decrease in its photoelectric activity.

Focused ultrasound and microbubbles for enhanced extravasation.

The permeability of blood vessels for albumin can be altered by using ultrasound and polymer or lipid-shelled microbubbles. The region in which the microbubbles were destroyed with focused ultrasound was quantified in gel phantoms as a function of pressure, number of cycles and type of microbubble. At 2MPa the destruction took place in a fairly wide area for a lipid-shelled agent, while for polymer-shelled agents at this setting, distinct destruction spots with a radius of only 1mm were obtained. When microbubbles with a thicker shell were used, the pressure above which the bubbles were destroyed shifts to higher values. In vivo both lipid and polymer microbubbles increased the extravasation of the albumin binding dye Evans Blue, especially in muscle leading to about 6-8% of the injected dose to extravasate per gram muscle tissue 30 min after start of the treatment, while no Evans Blue could be detected in muscle in the absence of microbubbles. Variation in the time between ultrasound treatment and Evans Blue injection, demonstrated that the time window for promoting extravasation is at least an hour at the settings used. In MC38 tumors, extravasation already occurred without ultrasound and only a trend towards enhancement with about a factor of 2 could be established with a maximum percentage injected dose per gram of 3%. Ultrasound mediated microbubble destruction especially enhances the extravasation in the highly vascularized outer part of the MC38 tumor and adjacent muscle and would, therefore, be most useful for release of, for instance, anti-angiogenic drugs.

[Carbohydrate metabolism disturbances after pancreatoduodenal resection in patients with cancer of the head of pancreas].

Carbohydrate metabolism was explored in 52 patients with cancer of the head of pancreas before and after pylorus-preserving pancreatoduodenal resection. Glycemia, insulin dosage, glucosuria and acetonuria were assessed retrospectively to reveal correlation with postoperative carbohydrate metabolism disturbances. Preoperatively 46.2% of patients had no metabolic problems, whereas postoperatively only 38.5% of patients showed normal sugar metabolism. 9 (17.3%) patients developed disturbed glucose tolerance, 5 (9.6%) patients developed mild diabetes mellitus and 18 (34.6%) patients had diabetes of medium severity. Combination of first diagnosed diabetes mellitus, progressive weight loss and abdominal pain proved to be the negative prognostic set of symptoms and should urge on pancreatic tumor search. Glucose level in early postoperative period is a valuable prognostic criteria for the development of carbohydrate metabolism disturbances in long-term postoperative period.