[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.

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.

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.

Distinct classes of human stem cells that differ in proliferative and self-renewal potential.

The composition of the human hematopoietic stem cell compartment is poorly understood due to the absence of experimental tools with which to characterize the developmental program of individual stem cells. We report here that human stem cells differ markedly in their repopulation capacity and self-renewal potential, as determined using nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice transplanted with retrovirally transduced cord blood stem cells, called SCID-repopulating cells (SRCs). Clonal stem cell analysis based on the identification of unique retroviral integration sites within serial bone marrow aspirates showed that repopulation was generally oligoclonal with extensive variability in the lifespan and proliferative capacity of individual SRCs. Most clones contributed to human cell engraftment for several weeks after transplantation and then disappeared but others appeared later and persisted. Further evidence for stem cell heterogeneity was found in the secondary transplantation capacity of SRCs. These data point to the existence of different classes of human stem cells with variable self-renewal potential and short- or long-term repopulating capacity.

Transduction of human CD34+ CD38- bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors.

The major limitations of Moloney murine leukemia virus (MoMLV)-based vectors for human stem cell applications, particularly those requiring bone marrow (BM) stem cells, include their requirement for mitosis and retroviral receptor expression. New vectors based upon lentiviruses such as HIV-1 exhibit properties that may circumvent these problems. We report that novel third-generation, self-inactivating lentiviral vectors, expressing enhanced green fluorescent protein (EGFP) and pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G), can efficiently transduce primitive human repopulating cells derived from human BM and cord blood (CB) tested by the SCID-repopulating cell (SRC) assay. Highly purified CD34+ CD38- CB or BM cells were efficiently transduced (4-69%) and stably expressed in EGFP for 40 days in culture following infection for only 24 h without fibronectin, polybrene, or cytokines. Nonobese diabetic/severe combined immune-deficient (NOD/SCID) mice transplanted with transduced cells from either CB or BM donors were well engrafted, demonstrating maintenance of SRC during the infection procedure. Serially obtained femoral BM samples indicated that the proportion of EGFP+ cells within both myeloid and lymphoid lineages was maintained or even increased over time, averaging 42.3 +/- 6.6% for BM donors and 23.3 +/- 7.2% for CB at 12 weeks. Thus, the third-generation lentivectors readily transduce human CB and BM stem cells, under minimal conditions of ex vivo culture, where MoMLV-based vectors are ineffective. Since CB is inappropriate for most therapeutic applications, the efficient maintenance and transduction of BM-derived SRC during the short infection procedure are notable advantages of lentivectors.

Expansion of human cord blood CD34(+)CD38(-) cells in ex vivo culture during retroviral transduction without a corresponding increase in SCID repopulating cell (SRC) frequency: dissociation of SRC phenotype and function.

Current procedures for the genetic manipulation of hematopoietic stem cells are relatively inefficient due, in part, to a poor understanding of the conditions for ex vivo maintenance or expansion of stem cells. We report improvements in the retroviral transduction of human stem cells based on the SCID-repopulating cell (SRC) assay and analysis of Lin(-) CD34(+)CD38(-) cells as a surrogate measure of stem cell function. Based on our earlier study of the conditions required for ex vivo expansion of Lin(-)CD34(+) CD38(-) cells and SRC, CD34(+)-enriched lineage-depleted umbilical cord blood cells were cultured for 2 to 6 days on fibronectin fragment in MGIN (MSCV-EGFP-Neo) retroviral supernatant (containing 1.5% fetal bovine serum) and IL-6, SCF, Flt-3 ligand, and G-CSF. Both CD34(+)CD38(-) cells (20.8%) and CFC (26.3%) were efficiently marked. When the bone marrow of engrafted NOD/SCID mice was examined, 75% (12/16) contained multilineage (myeloid and B lymphoid) EGFP(+) human cells composing as much as 59% of the graft. Half of these mice received a limiting dose of SRC, suggesting that the marked cells were derived from a single transduced SRC. Surprisingly, these culture conditions produced a large expansion (166-fold) of cells with the CD34(+)CD38(-) phenotype (n = 20). However, there was no increase in SRC numbers, indicating dissociation between the CD34(+)CD38(-) phenotype and SRC function. The underlying mechanism involved apparent downregulation of CD38 expression within a population of cultured CD34(+)CD38(+) cells that no longer contained any SRC function. These results suggest that the relationship between stem cell function and cell surface phenotype may not be reliable for cultured cells. (Blood. 2000;95:102-110)

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.

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.

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

Bone marrow failure in the Fanconi anemia group C mouse model after DNA damage.

Fanconi anemia (FA) is a pleiotropic inherited disease that causes bone marrow failure in children. However, the specific involvement of FA genes in hematopoiesis and their relation to bone marrow (BM) failure is still unclear. The increased sensitivity of FA cells to DNA cross-linking agents such as mitomycin C (MMC) and diepoxybutane (DEB), including the induction of chromosomal aberrations and delay in the G2 phase of the cell cycle, have suggested a role for the FA genes in DNA repair, cell cycle regulation, and apoptosis. We previously reported the cloning of the FA group C gene (FAC) and the generation of a Fac mouse model. Surprisingly, the Fac -/- mice did not show any of the hematologic defects found in FA patients. To better understand the relationship of FA gene functions to BM failure, we have analyzed the in vivo effect of an FA-specific DNA damaging agent in Fac -/- mice. The mice were found to be highly sensitive to DNA cross-linking agents; acute exposure to MMC produced a marked BM hypoplasia and degeneration of proliferative tissues and caused death within a few days of treatment. However, sequential, nonlethal doses of MMC caused a progressive decrease in all peripheral blood parameters of Fac -/- mice. This treatment targeted specifically the BM compartment, with no effect on other proliferative tissues. The progressive pancytopenia resulted from a reduction in the number of early and committed hematopoietic progenitors. These results indicate that the FA genes are involved in the physiologic response of hematopoietic progenitor cells to DNA damage.

Differential maintenance of primitive human SCID-repopulating cells, clonogenic progenitors, and long-term culture-initiating cells after incubation on human bone marrow stromal cells.

Many experimental and clinical protocols are being developed that involve ex vivo culture of human hematopoietic cells on stroma or in the presence of cytokines. However, the effect of these manipulations on primitive hematopoietic cells is not known. Our severe combined immune-deficient mouse (SCID)-repopulating cell (SRC) assay detects primitive human hematopoietic cells based on their ability to repopulate the bone marrow (BM) of immune-deficient non-obese diabetic/SCID (NOD/SCID) mice. We have examined here the maintenance of SRC, colony-forming cells (CFC), and long-term culture-initiating cells (LTC-IC) during coculture of adult human BM or umbilical cord blood (CB) cells with allogeneic human stroma. Transplantation of cultured cells in equivalent doses as fresh cells resulted in lower levels of human cell engraftment after 1 and 2 weeks of culture for BM and CB, respectively. Similar results were obtained using CD34+-enriched CB cells. By limiting dilution analysis, the frequency of SRC in BM declined sixfold after 1 week of culture. In contrast to the loss of SRC as measured by reduced repopulating capacity, the transplanted inocula of cultured cells frequently contained equal or higher numbers of CFC and LTC-IC compared with the inocula of fresh cells. The differential maintenance of CFC/LTC-IC and SRC suggests that SRC are biologically distinct from the majority of these in vitro progenitors. This report demonstrates the importance of the SRC assay in the development of ex vivo conditions that will allow maintenance of primitive human hematopoietic cells with repopulating capacity.

[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.

Assay of human stem cells by repopulation of NOD/SCID mice.

The only conclusive method to assay stem cells is to follow their ability to repopulate conditioned recipients, making it difficult to study human stem cells. The development of systems to transplant human hematopoietic cells into immune-deficient mice lays the foundation for such an experimental repopulation assay for primitive human cells. Cell purification and gene marking studies have shown that the repopulating cells, termed severe-combined immunodeficiency (SCID) mouse-repopulating cells (SRC), are primitive and distinct from most of the progenitors that are detected using short and long-term in vitro culture assays. The SRC are exclusively CD34+CD38- and poorly infected with retrovirus vectors. These gene marking data are reminiscent of the human clinical trials establishing that the SRC assay is a good surrogate to develop improved transduction methods. Limiting dilution analysis has been used to establish a quantitative assay for SRC that can be used to precisely determine the effect of various cytokine cocktails on the proliferation and differentiation of SRC during in vitro culture.

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.

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.