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PubMed1993

Human primitive hematopoietic progenitor cells are more enriched in KITlow cells than in KIThigh cells.

To clarify the phenotypes of various classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with CD34 and a newly developed mouse antihuman c-kit proto-oncogene product (KIT) monoclonal antibody (MoAb). We characterized three cell fractions in CD34+ cells that express KITlow and KIThigh cells in addition to KIT- cells. A clonogenic assay showed that most granulocyte-macrophage colony-forming cells (GM-CFC) were present in CD34+KIThigh populations, whereas erythroid burst-forming cells (BFU-E) were detected mainly in the CD34+KITlow population. CD34(+)-KIT- fraction contained a small number of BFU-E. Morphologic analysis showed that blast-like cells were more enriched in the CD34+KITlow fraction. KITlow cells contained CD34+CD38- cells that were considered to be very primitive progenitor cells, as determined by a replating assay. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell functions by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At week 2, more CFC recovered from the culture in the fraction initiated with a CD34+KIThigh population. However, more LTC-IC were present during weeks 5 to 9 in the CD34+KITlow population. These results indicate that primitive progenitors are more enriched in the KITlow population and that the KIThigh population contains many GM-committed progenitor cells. We also showed that anti-KIT MoAb inhibited the ability of CD34+ cells to generate CFC on the stromal layer in the LTC system. This suppressive effect was more evident in the generation of BFU-E by CD34+KITlow cells. Moreover, we confirmed that CD34+KIThigh cells emerged from CD34+KITlow cells during coculture with allogeneic stromal cells or from liquid culture in the presence of stem cell factor (SCF), interleukin-6, and erythropoietin. These results emphasize the pivotal role of the KIT and SCF interaction in hematopoiesis and indicate that KITlow cells are more primitive than KIThigh cells.

Animals↗

PubMed1994

Flow cytometric terminal deoxynucleotidyltransferase analysis. Evaluation of Triton X-100 and methanol permeabilization methods compared with immunofluorescence microscopy.

We evaluated the detection of terminal deoxynucleotidyltransferase by flow cytometry using two commonly reported methods of permeabilization and compared them with immunofluorescence microscopy. Thirty-four consecutive cases referred for leukemia workup were studied prospectively. We found decreased viability of cells with both flow cytometry methods in association with shifts in forward and side scatter patterns. Both procedures demanded careful manipulation of cells to avoid total lysis. The Triton X-100 method had a sensitivity of 100% and a specificity of 93%, while the methanol method had a sensitivity of 54% and a specificity of 100%. Therefore, the Triton X-100 method correlated better with immunofluorescence microscopy than did the methanol method. Despite technical difficulties, the advantages of increased objectivity, increased diagnostic capability associated with multicolor analysis, and the increased sensitivity in detecting minimal residual disease make flow cytometry a more attractive and advantageous procedure.

Adolescent↗

PubMed1993

A genetic map of chromosome 20q12-q13.1: multiple highly polymorphic microsatellite and RFLP markers linked to the maturity-onset diabetes of the young (MODY) locus.

Multiple highly polymorphic markers have been used to construct a genetic map of the q12-q13.1 region of chromosome 20 and to map the location of the maturity-onset diabetes of the young (MODY) locus. The genetic map encompasses 23 cM and includes 11 loci with PIC values > .50, seven of which have PICs > .70. New dinucleotide repeat polymorphisms associated with the D20S17, PPGB, and ADA loci have been identified and mapped. The dinucleotide repeat polymorphisms have increased the PIC of the ADA locus to .89 and, with an additional RFLP at the D20S17 locus, the PIC of the D20S17 locus to .88. The order of the D20S17 and ADA loci determined genetically (cen-ADA-D20S17-qter) was confirmed by multicolor fluorescence in situ hybridization. The previously unmapped PPGB marker is closely linked to D20S17, with a two-point lod score of 50.53 at theta = .005. These markers and dinucleotide repeat markers associated with the D20S43, D20S46, D20S55, D20S75, and PLC1 loci and RFLPs at the D20S16, D20S17, D20S22, and D20S33 have been used to map the MODY locus on chromosome 20 to a 13-cM (sex averaged) interval encompassing ADA, D20S17, PPGB, D20S16, and D20S75 on the long arm of chromosome 20 and to create a genetic framework for additional genetic and physical mapping studies of the region. With these multiple highly polymorphic loci, any MODY family of appropriate size can be tested for the chromosome 20 linkage.

Base Sequence↗

PubMed1993

[Characterization of human immature T cell precursors in fetal liver and postnatal thymus].

The sequence of events that accompany development of immature T cell precursors in man is not precisely known. Recently, we identified subpopulations of thymocytes that may represent intermediate stages between CD3-CD4-CD8- (TN) and CD3+CD4+CD8+ (DP) thymocytes. Multicolor analysis provided evidence for the existence of CD3-CD4+CD8- and CD3-CD4+CD8 alpha+beta- populations. In vitro culture of TN thymocytes with IL-7 gave rise to cells of these phenotypes, suggesting that the novel subpopulations we found are the progeny of TN thymocytes. Expression of CD34 was detected on most of TN thymocytes and progressively reduced as cells differentiate. The novel subpopulations were shown to express CD34. 6-22-week-old human fetal liver cells were also analyzed in search for the possible prethymic T cell precursors. It was revealed that most of CD3-CD7+ cells which had been considered as T cell precursors were CD34-CD56+ and exhibited non-MHC-restricted cytotoxicity. Thus, although they expressed cytoplasmic CD3 antigens, these cells seemed to represent NK cells rather than T cell precursors. Existence of the latter is still to be demonstrated.

Cell Differentiation↗

PubMed1993

Morphologic characteristics and chemical composition of Christmas tree cataract.

PURPOSE: Christmas tree cataract consists of highly refractile multicolored "needles" crisscrossing the lens fibers of the deep cortex. The fact that the colors vary according to the angle of the incident light, and that in retroillumination only a dim outline of the cataract is seen, would suggest that Christmas tree cataract is a diffractive phenomenon. This study was performed to unravel the ultrastructure and chemical composition of the Christmas tree needles. METHODS: Eight lenses from donor eyes and four extracapsularly extracted lenses with Christmas tree cataract were investigated by scanning and transmission electron microscopy. The chemical composition was studied with energy-dispersive x-ray microanalysis and Raman microspectroscopy. RESULTS: Scanning electron microscope examination showed that the needles are smooth, rectangular, plate-like elements bordered by membranes and amorphous material and running crisscross through the lens. In the specimens for transmission electron microscopic examination, the needles proved to be largely dissolved, but the remains showed regular spacings of approximately 5 nm. Material identical in spacing and electron density was found in neighboring cells bound to a reticular membranous network originating from the fiber-limiting membranes. Energy-dispersive x-ray and Raman microanalysis showed that the needles have a high sulfur content and pronounced S-S, CS-SC, and C-S vibrations. The cytoplasm adjacent to the needles and reticular meshwork had an elevated Ca++ content. CONCLUSIONS: It is concluded that cystine is the most likely candidate for the Christmas tree needles and that the needles probably are formed as the result of an age-related aberrant breakdown of crystallins induced by elevated Ca++ levels.

Aged↗

PubMed1993

Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death.

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2-lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.

Adolescent↗

PubMed1993

The Q-switched Nd:YAG laser effectively treats tattoos. A controlled, dose-response study.

BACKGROUND AND DESIGN: The Q-switched ruby laser was recently shown to remove tattoos without scarring. The Q-switched neodymium: yttrium aluminum garnet (Nd:YAG) laser, which targets black ink with a longer wavelength that has less absorption by melanin, should effectively treat tattoos with fewer pigmentary alterations. A prospective, blinded, controlled study was conducted to assess the ability of the Q-switched Nd:YAG laser (1064 nm, 10 ns, 5 Hz) to remove tattoos. Twenty-five patients with 39 blue-black or multicolored tattoos (14 previously untreated, 25 Q-switched ruby laser-resistant) were exposed in randomly derived quadrants with 6, 8, 10, or 12 J/cm2 at 3 to 4 week intervals for a total of four treatment sessions. RESULTS: An excellent response (> 75% ink removal) was seen in 77% of the black tattoos and more than 95% of the black ink cleared in 11 (28%) of 39 tattoos at 10 to 12 J/cm2 after four treatment sessions. Colored inks were not as effectively removed. Response was related to fluence with greatest improvement noted in the quadrants treated with 10 and 12 J/cm2. No significant side effects, including pigmentary changes or scarring, were noted. Histopathologic examination demonstrated persistence of tattoo ink in clinically clear areas and confirmed the absence of fibrosis and granulomatous changes. CONCLUSION: The Q-switched Nd:YAG laser (1064 nm) effectively treats black tattoos with an excellent cosmetic outcome. Bright colors were minimally responsive to treatment. Higher doses were more effective and equally well tolerated.

Adult↗

PubMedDate not supplied

[Microdochectomy with perioperative dye localization of ducts in patients with nipple secretion].

A series of 1321 women having a discharge from the nipple were examined at the outpatient clinic. Of seven basic types of discharge: milky, multicolored, purulent, watery, yellow (serous), serosanguineous and bloody, only the last four represent and indication for surgery. Galactography was performed in 55 patients as a necessary element in the surgical plan of management. In 36 women, the operation of microdochectomy was indicated, but only 24 accepted to be subjected to that procedure. The authors describe a new technique of colour mammary ductal localization which identifies the duct from which the discharge is emerging and assures its complete excision. The procedure combines preoperative methylen blue dye injection followed by periareolar incision of the involved duct using a pair of binocular loops for magnification of the nipple. In all tissue specimens, the cause of a pathological discharge was found: 15 (62.5%) cases presented with fibrocystic disease, and in 6 carcinoma was present. This technique provides a safe and effective method of complete excision of the pathologically involved mammary duct with good cosmetic results.

Adult↗

PubMed1995

Distribution and ecology of the mosquito fauna in the southwestern Saudi Arabia.

A mosquito Larval survey was carried out in the Southwestern region of Saudi Arabia for a two subsequent years period. Nine mosquito species were identified; 4 Anopheline species; Anopheles arabiensis, An. sergentii, An. multicolor and An. tenebrosus and 3 Culicine species, Culex pipiens, Cx. quinquefaciatus and Cx. theileri as well as one Aedine species, Aedes caspius and Culiseta subochrea. A map was made showing the distribution of the encountered species and a full description of all breeding places was made also. The association of different species in each breeding site was also recorded.

Aedes↗

PubMed1996

Gene transfer into human bone marrow hematopoietic cells mediated by adenovirus vectors.

Human bone marrow mononuclear cells (BMMNCs) and enriched CD34 positive (CD34+) cells were transduced with adenovirus vectors encoding Escherichia coli beta-galactosidase gene. Tranductions were carried out by 24-hour coincubation with adenovirus vectors at different multiplicities of infections (moi). Efficacy of gene transfer into BM cells and expression of the gene product (ie, beta-galactosidase) were studied using X-Gal histochemical staining and flow cytometric analysis. X-Gal staining demonstrated that the percentage of positive cells at mois of 5 to 500 was 3.4% to 34.5% for BMMNCs and 6.0% to 20.0% for enriched CD34+ cells. Similar results (1.5% to 35.7% for BMMNCs and 5.4% to 24.2% for enriched CD34+ cells) were obtained with flow cytometric analysis using fluorescein di-beta-D-galactopyranoside (FDG). Multicolor flow cytometry analysis, which included FDG, demonstrated that BM progenitors (CD34+ or CD34+CD38-), T cells (CD2+), B cells (CD19+), natural killer cells (CD56+), granulocytes, and monocytes all expressed the adenovirus transgene. To ascertain the effects of adenovirus vectors on normal BM progenitors, the numbers of colony forming unit-granulocyte/macrophage (CFU-GM), burst-forming unit-erythrocyte (BFU-E), and high-proliferative potential-colony-forming cells (HPP-CFC) after 24-hour coincubation with adenovirus vectors were determined. When BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at mois of 5 and 50, no significant differences in the numbers of CFU-GM, BFU-E, and HPP-CFC were observed compared with the uninfected control cells. However, the numbers of CFU-GM were significantly (P < .01) decreased when BMMNCs or enriched CD34+ cells were incubated with adenovirus vectors at a moi of 500, compared with the uninfected control cells. The adenovirus infected cells, purified by cell sorting for FDG expression, were capable of growing in culture and gave rise to various colonies (ie, CFU-GM, BFU-E, and HPP-CFC). These data indicate that recombinant adenovirus vectors can be used to transfer genes to human BM hematopoietic cells with expression of the exogenous gene at a high transduction efficiency.

Adenoviridae↗

PubMed1996

Ultrasoft 1.5 keV aluminum K X rays are efficient producers of complex chromosome exchange aberrations as revealed by fluorescence in situ hybridization.

The electron pairs generated by ultrasoft 1.5 keV aluminum K X-ray photons deposit their energy in tracks of length < 70 nm and provide an ideal tool for analyzing the spatial distribution of breaks and misrepair processes. We have undertaken the analysis of changes in chromosome structure produced by aluminum K X rays in untransformed HF12 human fibroblasts in G1 phase using fluorescence in situ hybridization (FISH). Multicolored chromosome-specific DNA probes for chromosomes 1 and 2 and an alpha-satellite pan-centromeric probe were used to examine in vitro radiation-induced chromosome-type exchange aberrations. After mean doses of 0.37-2.93 Gy the relative frequencies of complex exchanges, derived from three or more breaks in two or more chromosomes, ranged from 15-35%. For the classic break-age-and-rejoining theory to hold, very large interaction distances are needed to account for this high frequency of multibreak interactions, unless many sites pre-exist where several different chromosomes come very close together. Alternatively, damaged DNA may be able to interact with adjacent undamaged DNA, obviating the need for large rejoining distances.

Aluminum↗

PubMed1996

Cosmetic laser surgery.

The fact that multiple wavelengths of light (and, therefore, different lasers) are needed to treat multicolored tattoos and several laser sessions are required for tattoo eradication in most instances, elimination of tattoos remains an expensive endeavor in terms of money and time. The obvious benefit of lasers in the treatment of tattoos, however, lies in their ability to eliminate tattoos without scarring or dyspigmentation. While no single commercially available laser system currently exists that can treat all tattoos, combination laser systems may make treatment of a wide range of tattoo colors a reality in the near future.

Humans↗

PubMed1996

[Diagnostic value of color Doppler flow imaging in gestational trophoblastic diseases].

OBJECTIVE: To investigate the value of color doppler flow imaging (CDFI) and pulsed doppler (PD) determinations in early diagnosis, evaluation of therapeutic effects and prediction of prognosis of gestational trophoblastic diseases (GTD). METHOD: CDFI and PD were performed in 55 cases of GTD and 30 normal pregnant women as controls. RESULTS: The uterine muscular layer in GTD showed multicolored abundant blood flow and blood sinuses. Blood flow resistance was low with a resistance index (RI) less than 0.5. After chemotherapy the RI changed dramatically. Pathological evidences were found in surgical specimens of 8 patients. Findings of pelvic hysterography in 16 cases were in accordance with the results of CDFI and PD determinations. CONCLUSION: CDFI and PD as new methods for early diagnosis of GTD can also be used to evaluate the chemotherapeutic results as well as to determine the prognosis of the disease.

Adult↗

PubMed1996

Semiautomated assessment of loss of heterozygosity and replication error in tumors.

Loss of heterozygosity (LOH) and replication error (RER) are important phenomena in tumor development, with diagnostic and prognostic relevance. Therefore, screening for LOH and RER is a desirable first step in the molecular analysis of tumors. We used semiautomated procedures based on multicolor fluorescently labeled microsatellite markers and an automated sequencer for PCR amplification, electrophoresis of PCR products, and allele detection with a set of 16 microsatellites in 56 colorectal tumors. We improved existing software for computer-assisted assessment of LOH and RER. A comparison of these results with those of a conventional, radioactive technique and visual interpretation shows a high degree of correlation between the two methods. The detection rates of LOH and RER are similar to those reported previously. The main advantages of the semiautomated fluorescence-based typing are in the objective, observer-unrelated, easy, and rapid computer-based scoring, and the resulting quantitative assessment of RER.

Automation↗

PubMed1996

Multipurpose high sensitivity luminescence analyzer (LUANA): use in gel electrophoresis.

Many applications in molecular biology require the rapid and high-sensitivity detection of biological macromolecules. Here we describe a luminescence analyzer (LUANA), featuring xenon lamp-based illumination and a cooled CCD camera as detector. Luminescent samples (gels, membranes or microplates) are placed in a light-tight chamber, and computer software is used to control the camera and to display and analyze the images. LUANA can be used with a broad spectrum of excitation and emission wavelengths (200-1000 nm), allowing a choice of many fluorescent dyes and facilitating multicolor imaging. We show that LUANA is readily applicable to gel electrophoresis and highly sensitive; on gels we could detect < 100 fg of Cy5TM-labeled protein and 0.1 ng of plasmid DNA labeled with a fluorescent intercalator. We observed a linear response for sample concentrations differing by two orders of magnitude using a single acquisition time. This range could be further extended by using different acquisition times. We have also used LUANA in two-color imaging of DNA and for screening of antigen-antibody and peptide-protein complexes by gel bandshift.

Cloning, Molecular↗

PubMed1996

Quantitation of circulating hematopoietic progenitor cells early after transplantation.

By multicolor flow cytometry CD34 positive blood progenitor cells (BPC) were determined by standard procedures before and after reinfusion. Measurements of circulating progenitor cells after transplantation lead to cell numbers that are considered together with reconstitution potential, especially with the number of transfused apheresis platelet concentrates after transplantation. Decreased concentrations of CD34 positive cells are associated with an increased frequency of platelet transfusions after transplantation.

Antigens, CD34↗

PubMed1997

Topographic distribution of homing receptors on B and T cells in human gut-associated lymphoid tissue: relation of L-selectin and integrin alpha 4 beta 7 to naive and memory phenotypes.

In mice, integrin alpha 4 beta 7 is the main receptor used by lymphocytes that home to the Peyer's patches, although L-selectin contributes to the initial interaction with high endothelial venules. Less is known about the expression and function of these adhesion molecules in humans. The distribution of L-selectin and alpha 4 beta 7 on various B- and T-cell subsets was examined in human Peyer's patches (n = 8) and appendix (n = 4), collectively called gut-associated lymphoid tissue. Multicolor immunophenotyping was performed on cryosections, and dispersed cells were examined by flow cytometry. In cryosections, CD45RA+ T cells around and within interfollicular high endothelial venules, as well as surface (s)IgD+ B lymphocytes in the follicle mantles, often expressed abundant L-selectin but only intermediate levels of alpha 4 beta 7. CD45RO+ T cells and sIgD- B cells expressed higher levels of alpha 4 beta 7 and were often located near putative efferent lymphatics; only a small fraction (< 20%) of such memory cells expressed L-selectin. By flow cytometry, considerably more T than B lymphocytes co-expressed L-selectin and alpha 4 beta 7 (40% versus 25% and 67% versus 39%, respectively). In samples with many L-selectin+ cells (> 30%), more of these lymphocytes co-expressed alpha 4 beta 7 than in samples with few L-selectin+ cells. Because L-selectin and alpha 4 beta 7 were co-expressed on lymphocytes located near high endothelial venules, and because such co-expression was relatively common when many L-selectin+ cells were present, both of these molecules might participate in homing to human gut-associated lymphoid tissue. Such homing is probably most pronounced for T lymphocytes that were found to express L-selectin and alpha 4 beta 7 more often than B lymphocytes. The selective and relatively high expression of alpha 4 beta 7 on memory cells located near efferent lymphatics indicated a different migratory capacity; after exit from gut-associated lymphoid tissue, such stimulated cells might home mainly to mucosal effector sites.

Adolescent↗

PubMed1996

Echo Doppler color flow (EDCF) evaluation of vascular pathology in pediatric age groups.

Although rarely observed, vascular diseases in children constitute a complex clinical problem. Until recently the diagnostic approach to infant vascular diseases was based on invasive methods such as arteriography, but now the use of ultrasonographic methods such as Doppler c.w. and Echo Doppler Color Flow (EDCF) in angiology can offer new and interesting application even in the pediatric age range. In the present study 15 children affected by vascular diseases (eight with arteriovenous fistulas (AVF), two with pseudoaneurysms, and five with angiomas or cysts) either of iatrogenic or congenital etiology, were examined by Doppler c.w. and EDCF. In three additional cases a suspected vascular disease was not confirmed. The data obtained by Doppler c.w. and EDCF proved useful to perform a preoperative diagnosis; however some diagnostic criteria differed from those described in adulthood, as in the AVF cases, for instance, we observed the prevalence of indirect signs such as alterations of venous flow, and the presence of "multicolored speckled mass" and of "the spreading of the color in extravasal space" rather than a direct connection between an artery and a vein. Also pseudoaneurysms were easily detected by EDCF. In two cases of congenital AVF, the diagnosis was confirmed by arteriography. Surgery was performed in 9 children, 7 with AVF and 2 with pseudoaneurysm; in all cases, the vascular defect detected by ultrasonography was visualized and corrected. In 7 cases EDCF was also used in the follow-up. In conclusion this study suggests a possible more extensive use of ultrasonographic methods in the diagnostic approach to vascular diseases in pediatric age groups, and points out their validity in screening very young patients and in their surgical follow-up.

Arteriovenous Fistula↗