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PubMed2000

Estimation of stem cell fractions in peripheral blood stem cell harvest by using an SE-9000 hematology analyzer.

We inquired whether stem cell fractions in peripheral blood stem cell harvest could be precisely detected by using a hematopoietic progenitor cell (HPC) counting system applied to an automated hematology analyzer, SE-9000. Although there was an apparent increase in the HPCs 20 h after storage in nondiluted conditions, samples diluted with RPMI-1640 containing 1.0 mg/ml EDTA-2K showed a relatively stable number of HPCs. There was a significant relationship between HPCs and CD34 cells (n = 75, r = 0.769). This method may represent the least expensive and most time-effective way for stem cell estimation in harvest products.

Anticoagulants↗
PubMed1988

Effect of lymphokine-activated killer cell fraction on the development of human hematopoietic progenitor cells.

Lymphokine-activated killer (LAK) cells from cultures of human peripheral blood mononuclear cells with recombinant interleukin-2 (rIL-2) have been clinically used in adoptive immunotherapy for cancer patients. To study their influence on human hematopoiesis, the LAK cell fraction was cocultured with marrow nonphagocytic cells from normal subjects in an assay system of hematopoietic progenitors. The fraction suppressed colony growth from relatively mature erythroid progenitors in a dose-dependent manner. Although unactivated cells, which were produced without IL-2, augmented the growth of early erythroid progenitors, the LAK cell fraction did not. This fraction suppressed colony growth from mature granulocyte-macrophage progenitors (day 7 CFU-GM) especially with an 18-h preincubation prior to coculture. It also suppressed both immature granulocyte-macrophage progenitors (day 14 CFU-GM) and multipotential hematopoietic progenitors. The suppressive effects were observed on colony growth from autologous marrow cells as well as allogeneic marrow cells. The suppression of day 7 CFU-GM colony growth by supernatants due to preincubation with marrow cells and the LAK cell fraction suggested that the humoral factor contributes to the suppression by the LAK cell fraction. These data suggest that the LAK cell fraction suppresses the development of human hematopoietic progenitor cells.

Cells, Cultured↗
PubMed1981

Regulation of antigen-induced lymphoproliferation by BSA gradient-separated T cell fractions.

Human T cells from 6 volunteers immunized with keyhole limpet hemocyanin (KLH) or tetanus toxoid were fractionated on bovine serum albumin (BSA) gradients. These T cell fractions were then recombined with autologous unseparated mononuclear cells or unseparated T cells to determine the effect of each fraction on the proliferative (DNA synthetic) response to the immunizing antigen. Using this separation technique, we were able to define 2 fractions of T cells of widely different densities that had a suppressive effect on the proliferative response to KLH of unfractionated T cells. The demonstration of suppressive activity was possible only if cells were tested within 8 wk of immunization with KLH. These suppressive T cell fractions differed not only in density but also in surface characteristics. Fraction 1 cells had a high proportion of Ia+ cells but no Fc receptors for IgG (FcRG-) were seen, whereas cells from fraction 4 (of higher density) were Ia- and were composed of 12.1 +/- 1.2% FcRG+ cells. this suppression was shown to be specific for the immunizing antigen. One individual who had been immunized with KLH for more than 1 yr in which suppression of KLH-induced proliferation was no longer demonstrable was given primary immunization to tetanus toxoid. Suppression could be demonstrated in fractions 1 and 4 specific for tetanus toxoid; there was no demonstrable effect of these T cell fractions on the response to KLH. We postulate that these are different functional populations of suppressor T cells that regulate antigen specific lymphoproliferation.

Animals↗
PubMed1984

Monoamine oxidase activities of dissociated cell fractions from rat ventricular muscle.

Cell fractions enriched in cardiac muscle cells (myocytes), on the one hand, and in non-myocytes, on the other, were prepared by dissociation of rat ventricular tissue with collagenase. Amine oxidase activities in homogenates of these cell fractions and also in homogenates of the corresponding undissociated ventricular tissue were compared. In addition, the activity of alkaline phosphatase (AP), an enzyme found predominantly associated in the heart with non-myocytes, particularly capillary endothelial cells, was also measured. No significant difference in the activity of MAO-A (assayed with 1 mM 5-hydroxytryptamine) was found between myocyte and non-myocyte fractions. In contrast, the activities of alkaline phosphatase (AP) and also the semicarbazide-sensitive amine oxidase (SSAO), assayed with 1 microM benzylamine (BZ), were both significantly higher in non-myocytes, by several-fold, than in myocyte fractions. Studies of the inhibition by clorgyline of 1 mM BZ metabolism confirmed that both MAO-A and MAO-B can also contribute to BZ oxidation in the rat heart. These experiments indicated different ratios of MAO-A: MAO-B in the various cell fractions. The ratios of the percentage contributions of MAO-A and MAO-B, respectively, to the total metabolism of 1 mM BZ were 78:20 (myocytes), 43:52 (non-myocytes) and 57:32 (undissociated tissue). These results suggest that MAO-B, in addition to AP and SSAO, may be associated preferentially with non-myocyte constituents of the rat heart. Although cardiac myocytes appear to contain predominantly MAO-A, this enzyme form is also localized, with high activity, to the non-myocyte fraction. However, since the non-myocyte fraction is heterogeneous in its cell content, containing vascular components of the coronary microcirculation, as well as other cells of connective tissue origin, the exact cellular localization of the enzyme activities within this fraction has not yet been defined.

Alkaline Phosphatase↗
PubMed1980

Skin test and lymphocyte stimulation in delayed hypersensitivity against staphylococcal antigens. Relation to bacterial cell fractions.

Staphylococcal homogenate was fractionated into cell walls (CW), cell membranes (CM) (insoluble part left after removal of the cell wall fraction) and a soluble fraction. The capacity of these fractions to evoke delayed skin reactions and to stimulate lymph node and peripheral blood lymphocytes from guinea pigs sensitized with staphylococcal homogenate in Freund's complete adjuvant was tested 21 days after sensitization. Highest skin reactivity was observed with the cell wall fraction. In the lymphocyte stimulation test similar results were obtained with all three fractions. With peripheral blood lymphocytes higher stimulation indices were observed than with lymph node lymphocytes.

Animals↗
PubMed1983

[Separation of natural ribonucleoside triphosphates and their determination in an acid-soluble cell fraction by anion exchange high performance liquid chromatography].

An anion-exchange HPLC procedure for CTP, UTP, ATP and GTP determination in the acid-soluble fraction of cells is described. Ribonucleoside triphosphates are separated on LiChrosorb AN isocratically with NH4H2PO4-acetonitrile. The dependence of the separation efficiency on the salt and acetonitrile concentrations and pH was analyzed and the optimal conditions were chosen. The range, wherein the linearity between the ribonucleoside triphosphate amount and the area of the corresponding peak is observed, was defined and the regression equations were derived. The CTP, UTP, ATP and GTP content in the ovarian cancer cells CaOv in culture was found to be 418 +/- 32, 1122 +/- 21, 9262 +/- 442 and 1036 +/- 49 pmole/10(6) cells, respectively. After 2 hr incubation with 6-mercaptopurine (10(-4) M) the level of ATP and GTP is reduced by 55%, and after 24 hr incubation--by 73% for ATP and 85% for GTP. At the same time the UTP and CTP content is decreased by 12-31%.

Adenosine Triphosphate↗
PubMed1982

Purification of human monocytes by adherence to polymeric fluorocarbon. Characterization of the monocyte-enriched cell fraction.

Human mononuclear cells were obtained from peripheral blood by density gradients. Monocytes can be purified after cultivation of 2 hours by a modified adherence procedure on membranes of gas-permeable polymeric fluorocarbon (teflon). After further cultivation of 24-48 hours, monocyte-enriched cell fraction can be easily detached from the membranes with a viability greater than 98% and a final cell yield of approximately 50% of the peripheral monocyte count. The cells showed the morphological and cytochemical characteristics of human monocytes and differentiated into dense monolayers of macrophages within 10 days of cultivation. Immune-autoradiography with iodine-125-labelled xenogeneic antimonocytic antisera and staining with several monoclonal antisera in an indirect immunofluorescence technique revealed up to 92% of these cells to carry monocytic characteristics. To show their functional integrity, monocytes obtained by this procedure were activated by 48 hours' cultivation with synthetic alkyl-lysophospholipids to inhibit the proliferation of autologous tumor cells.

Autoradiography↗
PubMed1983

The distribution of fetal hemoglobin and the types of gamma chain in red cell fractions separated by gradient centrifugation from blood of patients with sickle cell anemia and other hemoglobinopathies.

Isopycnic separations of red cells from cord bloods, and from patients with sickle cell anemia, different forms of HPFH, S-beta O-thalassemia, and a beta +-thalassemia homozygosity were made in order to evaluate the distribution of Hb F and the relative levels of G gamma and A gamma chains over the cell fractions. As expected, the cord blood data showed decreased levels of both Hb-F and G gamma chains in the top cell fractions since the beta leads to gamma and high G gamma: A gamma low G gamma: A gamma switches are operative around the time of birth. Complete cell fractionations were made on the blood of three SS patients with low G gamma values (40%) and three SS patients with high G gamma values (60%). The proportion of G gamma chain was constant in all cell fractions, while the Hb-F level was higher in cells with higher densities. The difference in the quantities of the three types of gamma chain in the fetal hemoglobins of two SS patients with an A gamma T heterozygosity, one having a low G gamma value and the other a high G gamma level, can be explained by assuming an alteration in a regulatory mechanism. Considerable variation both in the level of Hb F and in the percentage of G gamma chain was observed in two G gamma A gamma-HPFH heterozygotes with a relatively low G gamma percentage of 30%; an inverse relationship was present between the two parameters. Such a phenomenon was not evident for the G gamma A gamma-HPFH homozygote, and also did not exist in two additional G gamma A gamma-HPFH heterozygotes with an associated alpha-thalassemia-2 heterozygosity who had a similar amount of Hb F but with higher G gamma values of about 50%. The difference in G gamma values between these two categories of G gamma A gamma-HPFH could be due to a higher affinity of the G gamma chains over A gamma chains for a slightly decreased amount of alpha chains as in an alpha-thalassemia-2 heterozygosity. 2 heterozygosity. Although an increased synthesis of Hb-F with G gamma chains was again observed after in vitro incubation of reticulocytes with [35S]methionine none of the isolated cell fractions contained a Hb F with the alpha 2 G gamma 2 composition.

Adolescent↗
PubMed1966

Application of cell fractionation techniques in the study of cells infected with polyoma virus and Newcastle disease virus.

Fisher, Harold W. (University of Rhode Island, Kingston), Hidemi Matsumiya, and Masanobu Azuma. Application of cell fractionation techniques in the study of cells infected with polyoma virus and Newcastle disease virus. J. Bacteriol. 91:1645-1651. 1966.-Techniques which permitted rigorous separation of nuclei and cytoplasm were applied to the study of the formation of Newcastle disease virus (NDV) in an established line of Chinese hamster cells and of polyoma virus (PYV) in mouse embryo fibroblasts. The results obtained for hemagglutinin and plaque-forming titers during virus growth were in agreement with those obtained by others, using different techniques. These indicated that NDV matures in the cytoplasm, and PYV in the nucleus, of host cells.

Animals↗
PubMed1981

Activation of the alternative pathway of complement in human serum by Propionibacterium acnes (Corynebacterium parvum) cell fractions.

Activation of the alternative pathway of complement is known to be initiated by bacterial structures. We have fractionated Propionibacterium acnes cells, purified various cell fractions, and tested their complement-activating ability in human serum chelated with ethyleneglycol bis-(beta-aminoethylether)-N,N1-tetraacetic acid. The majority of complement-activating activity was localized in the wall fraction. This activity was resistant to lipid extraction, protease, RNAse, DNAse and lysozyme treatment. NaIO4, formamide, and hot (but not cold) trichloroacetic acid (TCA) extraction ablated the complement-activating capacity of cell walls. Compounds removed by extraction failed to consume significant hemolytic activity against antibody-coated sheep erythrocytes (EA). Addition of TCA-extracted soluble material to cell wall suspensions resulted in an inhibition of hemolytic consumption by the cell wall. These results indicate that, in P. acnes, complement-activating molecules are located in the cell wall and are carbohydrate in nature. Peptidoglycan, lipid, protein, and nucleic acid do not appear to contribute to the cell wall's ability to activate complement.

Carbohydrates↗
PubMed2006

Robust computational reconstitution - a new method for the comparative analysis of gene expression in tissues and isolated cell fractions.

BACKGROUND: Biological tissues consist of various cell types that differentially contribute to physiological and pathophysiological processes. Determining and analyzing cell type-specific gene expression under diverse conditions is therefore a central aim of biomedical research. The present study compares gene expression profiles in whole tissues and isolated cell fractions purified from these tissues in patients with rheumatoid arthritis and osteoarthritis. RESULTS: The expression profiles of the whole tissues were compared to computationally reconstituted expression profiles that combine the expression profiles of the isolated cell fractions (macrophages, fibroblasts, and non-adherent cells) according to their relative mRNA proportions in the tissue. The mRNA proportions were determined by trimmed robust regression using only the most robustly-expressed genes (1/3 to 1/2 of all measured genes), i.e. those showing the most similar expression in tissue and isolated cell fractions. The relative mRNA proportions were determined using several different chip evaluation methods, among which the MAS 5.0 signal algorithm appeared to be most robust. The computed mRNA proportions agreed well with the cell proportions determined by immunohistochemistry except for a minor number of outliers. Genes that were either regulated (i.e. differentially-expressed in tissue and isolated cell fractions) or robustly-expressed in all patients were identified using different test statistics. CONCLUSION: Robust Computational Reconstitution uses an intermediate number of robustly-expressed genes to estimate the relative mRNA proportions. This avoids both the exclusive dependence on the robust expression of individual, highly cell type-specific marker genes and the bias towards an equal distribution upon inclusion of all genes for computation.

Arthritis, Rheumatoid↗
PubMed1970

Fowl cholera immunization in turkeys. I. Efficacy of various cell fractions of Pasteurella multocida as vaccines.

Cell fractions of Pastuerella multocida (P-1059) were tested as vaccines against fowl cholera in turkeys. These fractions were culture filtrate, cell wall, and cytoplasm. A second culture filtrate preparation made from cells grown on blood-agar rather than the standard medium was also tested along with a "combination" preparation made by recombination of the cell fractions. Each preparation was tested in three vehicles: saline, alum (0.5%), and Freund Incomplete Adjuvant (50%). The turkeys vaccinated with these preparations were challenged by exposure to an experimental epornitic of fowl cholera. The combination fraction appeared to be the most promising vaccine when compared to the protective action of the commercial bacterin included in the test as a positive control.

Agar↗
PubMed1961

Glycogen synthesis from uridine diphosphate glucose. The distribution of the enzyme in liver cell fractions.

The distribution in liver cell fractions of UDPG-glycogen transferase has been studied. In fasting animals which have been refed 6 hours before sacrifice, the distribution of the enzyme in the various cell fractions can be correlated with the glycogen content of each fraction. A purified glycogen fraction has been prepared by differential centrifugation in sucrose gradients. This glycogen fraction contains vesicular structures which resemble those seen in association with glycogen deposits in the intact liver cell. In addition, the glycogen pellet contains UDPG-glycogen transferase in high specific activity. Subfractionation of the glycogen pellet separates the majority of vesicular elements from the bulk of transferase activity and glycogen. The evidence presented suggests that the presence of UPDG-glycogen transferase in the glycogen pellet is to be attributed to its binding to glycogen rather than to its association with the structural elements found in the glycogen fraction.

Animals↗