[Clinical effectiveness of combination treatment with α1-adrenoblokator and type 5 phosphodiesterase inhibitor for patients of advanced and senile age suffering from lower urinary tract symptoms.]

The results of the pilot study are presented, the main purpose of which was to assess the effectiveness and safety of the combined application of α-adrenoblocator and type 5 phosphodiesterase inhibitor for the treatment of patients with symptoms of the lower urinary tract of old and old age. The study involved 60 patients over 50 years of age having middle-severe lower urinary tract symptoms, erectile dysfunction, moderately pronounced infravesical obstruction, prostate volume greater than 30 cc and prostate-specific antigen levels less than 4 ng/ml. The duration of treatment in all groups was 6 months. The study participants were divided into three groups, comparable in age, clinical manifestations and laboratory-instrumental indicators: Group 1 patients received type 5 phosphodiesterase inhibitor daily, group 2 patients - α1-adrenoblocator, group 3 - α1-adrenoblocator and type 5 phosphodiesterase inhibitor. A clinical study showed that long-term combination treatment of lower urinary tract symptoms α1-adrenoblocator and type 5 phosphodiesterase inhibitor in elderly and senile patients proved to be effective, well tolerated and hardly caused side reactions, significantly improving quality of life.

[Features of urolithiasis in patients of advanced and senile age.]

There were 105 patients of different gender of elderly and senile age with urolithiasis. The entire contingent of patients with ICD was divided into 2 groups: the main group (55 people aged 61-85 years) and the comparison group (50 people aged 40-60). All patients went throught a general clinical examination: a detailed history of the anamnesis, an objective examination, laboratory and instrumental methods of investigation. It was revealed that in patients of elderly and senile age the clinical picture is more worn out, the renal colic is less often registered; more significant metabolic disorders that can participate in stone formation. Among the concomitant pathology in patients of this age category, a special role is assigned to: hypertension, diabetes, gout, chronic pyelonephritis and benign prostatic hyperplasia. According to the data of echographic and X-ray studies of these patient groups, it is noted that in patients aged 61-85 years, the calculi of the kidneys reach large sizes in comparison with patients 40-60 years old, and the main localization of stones in the ureter is its lower third.

Inactivation of Fac in mice produces inducible chromosomal instability and reduced fertility reminiscent of Fanconi anaemia.

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.

A newly discovered class of human hematopoietic cells with SCID-repopulating activity.

The detection of primitive hematopoietic cells based on repopulation of immune-deficient mice is a powerful tool to characterize the human stem-cell compartment. Here, we identify a newly discovered human repopulating cell, distinct from previously identified repopulating cells, that initiates multilineage hematopoiesis in NOD/SCID mice. We call such cells CD34neg-SCID repopulating cells, or CD34neg-SRC. CD34neg-SRC are restricted to a Lin-CD34-CD38- population without detectable surface markers for multiple lineages and CD38 or those previously associated with stem cells (HLA-DR, Thy-1 and CD34). In contrast to CD34+ subfractions, Lin-CD34-CD38- cells have low clonogenicity in short-and long-term in vitro assays. The number of CD34neg-SRC increased in short-term suspension cultures in conditions that did not maintain SRC derived from CD34+ populations, providing independent biological evidence of their distinctiveness. The identification of this newly discovered cell demonstrates complexity of the organization of the human stem-cell compartment and has important implications for clinical applications involving stem-cell transplantation.

Gene therapy model for stromal precursor cells of hematopoietic microenvironment.

Marker bacterial Neor gene was transduced by retroviral gene transfer into stromal precursor cells making up the hematopoietic microenvironment in murine long-term bone marrow cultures (LTBMC). Cultures were infected six times during the first 3 weeks of cultivation. At 4 weeks, the adherent cell layers (ACLs) were implanted under the renal capsule of syngeneic unirradiated and irradiated mice. Cells from newly formed ectopic foci were explanted into secondary LTBMC. ACLs containing the marker gene were detected by polymerase chain reaction. About 74% of stromal cells in ACLs contained Neor gene. The possibility of stable gene transduction into stromal precursor cells competent to transfer the hematopoietic microenvironment was established.

Effect of recombinant human granulocyte colony-stimulating factor treatment of mice on spleen colony-forming unit number and self-renewal capacity.

The aim of this investigation was to find out whether the self-renewal capacity of spleen colony-forming units (CFU-S) changed after treatment of CBF1 (CBA-LacxC57B1/6) F1 female mice with granulocyte colony-stimulating factor (G-CSF). In addition, the possibility of synergism between erythropoietin (Epo) and G-CSF in blood stem cell mobilization was studied. Twenty-five and 250 micrograms/kg/d injections of recombinant human (rh) G-CSF expanded the number of CFU-S in peripheral blood four- and 32-fold. The self-maintenance potential of CFU-S did not change significantly. The treatment of mice with 250 micrograms/kg/d rhG-CSF resulted in a two-fold increase of spleen cellularity and a 15-fold augmentation of CFU-S number, without noticeable changes in their self-renewal capacity. Moderate changes in CFU-S number were observed after Epo administration in spleen and peripheral blood; however, no significant synergistic effect of Epo was detected with either dose of rhG-CSF. The multifold increase of CFU-S number in peripheral blood following rhG-CSF administration, with no reduction in their generating potential, suggests that rhG-CSF-mobilized blood stem cells provide an appropriate source for reconstitution of the hematopoietic system.

Hematopoietic compartment of Fanconi anemia group C null mice contains fewer lineage-negative CD34+ primitive hematopoietic cells and shows reduced reconstruction ability.

Fanconi anemia (FA) is a complex recessive genetic disease that causes bone marrow failure in children. The mechanism by which the gene for FA group C (Fancc) impinges on the normal hematopoietic program is unknown. Here we demonstrate that the bone marrow from Fancc-/- mice have reduced ability for primary and secondary long-term reconstitution of myeloablated recipients compared to wild-type or heterozygous mice, indicating that the Fancc gene product is required for the maintenance of normal numbers of hematopoietic stem cells. Long-term and secondary transplant studies suggested that there also were qualitative changes in their developmental potential. Consistent with the reduction in reconstitution, flow cytometric analysis of the primitive subfractions of hematopoietic cells obtained from the bone marrow of Fancc -/- mice demonstrated that they contained 40 to 70% fewer lineage-negative (Lin-)Thy1.2-/lowScal(+) c-Kit(+)CD34+ cells compared to controls. In contrast, the number of Lin Thy1.2-/ lowScal(+)c-Kit CD34(-)cells was comparable to that of wild-type mice. The differential behavior of Lin(-)Thy1.2-/lowScal+c-Kit+CD34+ and Lin(-)Thy1.2-/lowScal(+)c-Kit CD34 subfractions also was observed in mice treated with the DNA cross-linking agent mitomycin C(MMC). Fancc-/- mice treated with MMC had an 92% reduction of CD34 cells as compared to Fancc+/+ mice. The number of CD34 cells only was reduced about 20%. These results suggest that the Fancc gene may act at a stage of primitive hematopoietic cell development identified by CD34 expression.

Characterization and retroviral transduction of an early human lymphomyeloid precursor assayed in nonswitched long-term culture on murine stroma.

In the hierarchy of human hematopoietic progenitors, long-term culture-initiating cells (LTC-IC) and extended LTC-IC belong to the earliest cell populations that can be assayed in vitro. We report the identification of a multipotential lymphomyeloid progenitor detected in a nonswitch culture system. We observed the emergence of CD33+ myeloid and CD19+ B-lymphoid cells following plating of lineage-depleted (Lin-) CD34 -enriched or purified CD34+ CD38- cord blood cells on MS-5 stroma in the absence of exogenous cytokines. Both CD19+ CD20- pro-B and CD19+ CD20+ pre-B lymphocytes coexist with myeloid cells in long-term culture. A limiting dilution approach was used to show that a single CD34+ CD38- cell can generate lymphomyeloid progeny in conventional (5-week) and extended (10-week) cultures. Most of the clones in long-term culture or extended long-term culture contained not only lymphoid and myeloid cells, but also myeloid clonogenic progenitors. A high proportion of CD34+ CD38- cells gave rise to lymphomyeloid clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively), which distinguishes the assay reported here from those using switch culture conditions. We performed retroviral gene transfer experiments involving 1-3 days of exposure of Lin CD34+ -enriched cells to virus encoding enhanced green fluorescent protein. Monitoring of gene transfer efficiency into LTC-IC by enhanced green fluorescent protein fluorescence showed that it is possible to achieve marking of lymphomyeloid LTC-IC, albeit to a lesser extent than myeloid-restricted LTC-IC.

Biodegradation of synthetic biphasic calcium phosphate by human monocytes in vitro: a morphological study.

Biodegradation processes (both intra- and extracellular) occur immediately after implantation of calcium phosphate (CaP) ceramics. Monocytes and macrophages, among the first cells to appear in wound healing, are largely implicated in phagocytosis and may be involved in CaP degradation because of their sensitivity to secreted cytokines. We tested the behaviour of human monocytes placed on the surface of hydroxyapatite (HA) and biphasic calcium phosphate (BCP) tablets in the presence of vitamin D3 (VD3) and interferon gamma (INF gamma). After short-term culture (6 days), morphological events were observed in histological and scanning electron microscopy studies, and degradation lacunae were characterized. There were cell prints but no pits on the HA surface, but pits appeared near cells on the BCP surface. Preincubation of biomaterial in culture medium was essential. Variations in cell morphology were observed in different culture types. In the presence of VD3, degradation was greater than in the control, and cells were more polarized and rounded. With INF gamma, cells were extensively spread out on the sample surface, and the biomaterial seemed to be extracted from the surface by cells. Thus, monocytes are clearly influenced by soluble factors (vitamins, cytokines) and could be key cells in initiating the degradation of biomaterial.

Culturing of cells from giant cell tumour of bone on natural and synthetic calcified substrata: the effect of leukaemia inhibitory factor and vitamin D3 on the resorbing activity of osteoclast-like cells.

Osteoclastic cells from giant cell tumour of bone (GCT) of bone provide a rich source for investigation of cellular mechanisms leading to formation of multinucleated cells, the resorption process and involvement of hormones and cytokines in these events. In the present study we investigated the effect of 1,25-dihydroxyvitamin D3 (VD3) and leukaemia inhibitory factor (LIF) on the resorbing potential of osteoclast of GCT origin using quantitative image-analysis of resorption lacunae in an in vitro dentine model. While VD3 unsignificantly increased the number of resorption pits and implicated surface after 7 days of GCT cell culturing, the stimulative effect of LIF was statistically significant. In cultures supplemented with LIF (5000 U/ml) the number of lacunae and resorption surface increased by 38% and 55%, respectively, when compared with control cultures. We suggest that both osteotropic agents increased osteoclastic activity, as the number of multinucleated cells was similar in control and experimental cultures. Seeding of GCT cells on biphasic calcium phosphate substratum revealed the relative inability of osteoclastic cells to resorb this synthetic material.

In vivo dynamics of human stem cell repopulation in NOD/SCID mice.

Primitive human hematopoietic cells can be assayed on the basis of their ability to repopulate immune-deficient NOD/SCID mice and have been termed SCID repopulating cells (SRCs). The in vivo biological fate of individual SRCs can be tracked by following the unique retroviral insertion site in the progency of transduced SRCs. Distinct human SRCs were identified that differ in the proliferative and self-renewal capacity indicating that the primitive cell compartment is functionally heterogeneous.

High-pressure liquid chromatographic--mass spectrometric determination of delta9-tetrahydrocannabinol in human plasma following marijuana smoking.

A method was developed for analyzing delta9-tetrahydrocannabinol (I), a psychotomimetic constituent found in marijuana smoke. The developed method utilizes a high-pressure liquid chromatographic (HPLC) gradient elution program to separate I from the other major cannabinoids in marijuana smoke. To achieve the sensitivity required to detect I in human plasma following marijuana smoking, a mass spectrometric quantification method was developed to analyze the HPLC eluant. To 1 ml of human plasma was added a known amount of internal standard, trideuterated I. This stable isotope provided a check on extraction efficiency, a marker for UV monitoring of the HPLC effluent and subsequent collection, and a convenient mass for mass spectrometric quantification. An ion-counting technique was used in conjunction with the peak matching accessory of the mass spectrometer to provide for a rapid comparison between molecular ions of I and the internal standard. The method was linear, accurate, and reproducible over the concentration range expected for I in plasma following marijuana smoking; 2.5 ng/ml was the lower practical limit of detection. Plasma from 11 male subjects was analyzed by the method at appropriate intervals up to 24 hr after the smoking of a marijuana cigarette containing 10.8 mg of I. Results demonstrated that levels of I could be determined accurately in the plasma of marijuana smokers in the 1-hr period following smoking.

[Significance of the detection of fimbriae protein type adhesins on bacteria isolated in acute pyelonephritis in children]. / Intérêt de la recherche des adhésines de type protéines fimbriae sur les germes des pyélonéphrites aiguës chez l'enfant.

A prospective bacteriological study in 50 children with acute pyelonephritis (APN) (32 girls and 18 boys) and 132 children with lower urinary tract infections (LUTI) (89 girls and 43 boys) was conducted from May to December 1993. Infection was defined by Kass' criteria and APN was defined by the clinical findings. C-Reactive Protein (CRP) assay and postcontrast computed tomography in the presence of a doubt concerning the diagnosis. Escherichia coli (EC) was the bacterial species most frequently isolated (76%). A systematic search for fimbriae protein adhesins (group PAP: pyelonephritis associated pil) on the EC was performed by haemagglutination (human group A red blood cells). 64% of EC possessed fimbriae protein adhesions in the APN group versus only 20% in the LUTI group. In children in whom an organic abnormality was demonstrated, the incidence of fimbriae protein-positive EC was 33% while in children with no organic abnormality, particularly without reflux, 89% of EC presented fimbriae protein. A statistically significant difference was demonstrated between these two groups (p < 0.01). The results of this study illustrate the important role of these adhesins in the development of APN. These adhesins facilitate countercurrent ascension of bacteria in the ureter towards the upper urinary tract and can make the bacteria resistant to certain antibiotics. Testing for fimbriae protein can be useful in clinical practice when investigating the aetiology of APN in the absence of demonstrated reflux. A latex test should soon be available to facilitate the detection of fimbriae protein.

[The development of the hemopoietic system: colony-forming splenic units in mouse embryogenesis]. / Razvitie gemopoéticheskoi sistemy: kolonieobrazuiushchie selezenochnye edintsy v énmbriogeneze myshi.

Localization and time of appearance as well as dynamics of quantitative changes of splenic colony-forming units (CFU-S) in mouse (C57BL/6 X CBA)F1 embryos were studied. Cells taken from the whole embryo (day 8), yolk sac and embryo per se (day 9), and also liver (day 10) were injected into the lethally irradiated syngenic mice. 7-8 days after the injection the spleens were fixed and the number of macrocolonies was counted. Statistically significant number of CFU-S was detected starting from day 10 of development, first in the embryo (30-33 somites), then in yolk sac and blood (37-38 somites) and liver (after the 40 somites stage). Rapid increase of CFU-S number during days 11-12 (two-fold increase in about 4.6 hours) suggest that not only active proliferation of CFU-S but also maturation of CFU-S precursors take place.

[Hematopoietic stem cells in mice during embryonic development preceding the establishment of liver hematopoiesis]. / Stvolovye krovetvornye kletki myshei v period émbrional'nogo razvitiia, predshestvuiushchii stanovleniiu pechenochnogo krovetvoreniia.

We studied the time course of appearance of CFUs (7-8 days old) in embryos of (C57B1/6 x CBA)F1 mice from the 8th day of embryonic development. Significant amounts of CFUs could be detected from the 10th day of development, initially in the body of the embryo from the stage of 30-33 pairs of somites, then in the yolk sac and still later, from the stage of about 40 pairs of somites, in liver anlage. CFUs could not be reliably detected until the 9th day of development either in the embryo itself or in the yolk sac. However, after incubation of nine day old embryos for four days in organ culture, such cultures contained CFUs. CFUs could be found in significant levels in embryos explanted from the embryos at the stage no earlier than 24 pairs of somites. When the yolk sac and the embryo were cultivated separately, CFUs could also be detected, however, the removal of liver primordium from the embryo did not influence the amount of CFUs in its body. CFUs were not found in cultures of liver primordium from nine day old embryos. Thus, we can detect pre-CFUs in 9 day old embryos at the stage 25-28 pairs of somites using the system of organ culture; at the same time CFUs cannot be found in intact embryos of the same age. These data provide evidence that before the establishment of liver hemopoiesis precursors of CFUs are located both in the yolk sac and in the embryo outside rudimentary liver. However, our results do not provide any data for the conclusion about the primary source of pre-CFUs in the mouse embryo.

[The introduction of the marker gene Neor into hematopoietic stem cells by electroporation]. / Vvedenie markernogo gena Neor v stvolovye krovetvornye kletki metodom élektroporatsii.

An electroporation method has been used to introduce marker gene Neor into mouse stem hemopoietic cells which are capable of long-term hemopoiesis maintenance in marrow long-term cultures. Integration of the gene was tested by polymerase chain reaction. The effect of the procedure averaged 50-80% of marked CFUc. Electroporation did little damage to hemopoietic cell precursors. Gene transfer can be made most effectively using bone marrow from mice injected 5-fluorouracil 4 days prior to the experiment.

[Induced hematopoietic foci in mice. I. Induction of extraskeletal hematopoietic areas using demineralized tooth matrix]. / Indutsirovannye ochagi krovetvoreniia u myshei. I. Induktsiia vneskeletnykh krovetvornykh territorii demineralizovannym zubnym matriksom.

Conditions of formation of induced osteo-hemopoietic foci in mice, in response to ectopic implantation to them of homologous demineralized tooth matrix, have been studied. It is shown that the hemopoietic tissue of the induced foci is similar to the bone marrow of the skeletal bones by the morphological parameters and cellular composition; differentiation of precursor cells takes place in all three directions of myelopoiesis. Induced foci formation under the capsule of the kidney occurs much later (after 5-8 months) than under the skin of muscular fascia (1-1.5 months). The induced hemopoietic foci functioning continues at least for one year.

Fuzzy regression modeling for tool performance prediction and degradation detection.

In this paper, the viability of using Fuzzy-Rule-Based Regression Modeling (FRM) algorithm for tool performance and degradation detection is investigated. The FRM is developed based on a multi-layered fuzzy-rule-based hybrid system with Multiple Regression Models (MRM) embedded into a fuzzy logic inference engine that employs Self Organizing Maps (SOM) for clustering. The FRM converts a complex nonlinear problem to a simplified linear format in order to further increase the accuracy in prediction and rate of convergence. The efficacy of the proposed FRM is tested through a case study - namely to predict the remaining useful life of a ball nose milling cutter during a dry machining process of hardened tool steel with a hardness of 52-54 HRc. A comparative study is further made between four predictive models using the same set of experimental data. It is shown that the FRM is superior as compared with conventional MRM, Back Propagation Neural Networks (BPNN) and Radial Basis Function Networks (RBFN) in terms of prediction accuracy and learning speed.