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Biomedical subjects

C B Lozzio

Publications and source records attributed to C B Lozzio.

At least 19 recordsLinked to original sources
PubMed2001

Simultaneous flow cytometric measurement of K-562 megakaryocytic differentiation and CD56+ large granular lymphocyte cytotoxicity.

K-562 cells have the capacity to undergo multi-lineage differentiation, which may be crucial to their ability to serve as target reservoirs for CD56+ large granular lymphocytes (LGL). Conventional techniques using chromium release assays to measure lymphocyte-mediated cytotoxicity suffer from disadvantages, including radioactive contamination and the inability to simultaneously determine K-562 and/or CD56+ lymphocyte phenotypes. We illustrate here a three-color flow cytometric method providing for the simultaneous evaluation of K-562-CD56+ LGL binding, K-562 cell viability, and the status of K-562 cell differentiation. Phorbol 12-myristate 13-acetate (PMA) engenders megakaryocytic differentiation in K-562 cell populations, as measured by presentation of the beta(3) integrin (gpIIIa, CD61), while maintaining a negative expression of MHC-I and MHC-II molecules. Using the auto-fluorescence of K-562 cells, flow cytometry can be used to demonstrate a significant decrease in CD56+ LGL activity against K-562 cells in populations pre-incubated with PMA. The capacity of three-color flow cytometry to measure lymphocyte-target cell binding and cell death kinetics, while simultaneously determining target cell phenotype, permits the specific localization of CD61-expressing K-562 cells to areas inconsistent with CD56+ LGL-mediated patterns of lysis.

Antigens, CD↗
PubMed2000

PMA-treated K-562 leukemia cells mediate a TH2-specific expansion of CD4+ T cells in vitro.

Highly enriched preparations of human CD3+CD4+ T-lymphocytes were stimulated with mitogen or OKT3 to determine the capacity of K-562 cells to function as accessory cells. Phorbol 12-myristate 13-acetate (PMA)-treated K-562 cells were induced to differentiate along the megakaryocytic lineage and could supplant monocyte-accessory cell function. Intracytoplasmic analysis of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) established that IL-4, and not IFN-gamma, was preferentially produced by the activated lymphocytes. This polarized stimulation is compatible with a type 2 or humoral immune response of purified T cells co-cultured with differentiated K-562 cells in vitro, and may have implications in immunoregulation due to disease progression.

CD3 Complex↗
PubMed1998

Differential regulation of interleukin-1alpha and interleukin-1beta in K-562 cells.

Interleukin (IL)-1alpha and IL-1beta are encoded by two separate genes, but both function as comitogens for lymphocyte activation. In this study, we observed K-562 cells to express constitutively mRNA for IL-1alpha, although IL-1alpha was not detected in the growth-conditioned medium (GCM). However, IL-1beta mRNA was not expressed unless the cells had been treated with phorbol myristate acetate (PMA). Both IL-1alpha and IL-1beta were detected in the GCM after the cells had been cultured with PMA, suggesting that IL-1 elaboration required PMA treatment. The K-562 cells treated with PMA differentiated to the myeloblastic stage, as observed by nuclear morphologic properties by electron microscopy. PMA treatment induced de novo expression of CD61 or gpIIIa, a marker associated with megakaryoblasts. These results showed that although K-562 cells constitutively expressed IL-1alpha mRNA, PMA treatment was required for secretion. On the other hand, both the expression and secretion of IL-1beta required treatment with PMA. This study showed that K-562 cells treated with PMA differentiated to the myeloblastic stage and expressed and secreted IL-1alpha and IL-1beta.

Cell Division↗
PubMed1992

Use of a molecular genetic approach to diagnosing the fragile X genotype.

We report the direct molecular detection of the fragile X genotype in 111 individuals from 17 families with a total of 31 cases of fragile X syndrome. Comparison of our molecular data with our previous cytogenetic and linkage data from these same families indicates the effectiveness of the direct molecular analysis. We have been able to assign a genotype unambiguously in 100% of the persons tested, and in all cases the molecular data correlated with the cytogenetic or linkage findings or both. Two of the three families presented in this study represent inheritance of this gene through normal transmitting males, and the third is strongly suggestive of this mode of inheritance. Our data show that the direct molecular approach will be of great utility for confirmation of the diagnosis and for the detection of female carriers and normal transmitting males who are at high risk for having affected children or grandchildren.

Blotting, Southern↗
PubMed1991

Cell death in the human leukemia cell line, K-562, induced by antiserum and monoclonal antibodies.

Rabbit polyclonal antiserum, or derived gamma globulin, to K-562 cells induces decreased TdR uptake within hours and cell death without cytolysis in 2-4 days. A panel of nine mAb, reactive with K-562 cells, was grouped on the basis of no effect on growth or TdR uptake, increased uptake, or decreased uptake. Treatment of cells with antiserum, gamma globulin, or mAb of the last group caused single-strand, but not double-strand, DNA fragmentation at a time when the cells were still viable. Cycloheximide did not inhibit the antibody effect suggesting that protein synthesis was not required. Aurintricarboxylic acid at certain concentrations markedly enhanced TdR uptake and protected the cells when antiserum was used but did not protect from mAb treatment.

Animals↗
PubMed1989

Interaction between selenium and zinc in the pathogenesis of anencephaly and spina bifida.

We present preliminary evidence that Selenium (Se) enhances zinc (Zn) accumulation in cultured skin fibroblasts from fetuses with NTD. Zn accumulation was decreased in NTD cells compared to controls and was increased but remained below the basal levels of controls when a 3-fold dose of Se was added to the culture media for 48 hours prior to the addition of Zn. These studies at the cellular level are in agreement with the epidemiologic evidence suggesting a correlation between low levels of Se in the diet and higher incidence of NTD.

Anencephaly↗
PubMed1989

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus.

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.

Cell Line↗
PubMed1988

Detection of surface differences between closely related cell populations by partitioning. Cultured K-562 cell sublines.

The K-562 cell line is a culture of human leukemia stem cells originally derived from a patient with chronic myelogenous leukemia in blast crisis. We have applied a sensitive method capable of detecting subtle differences in charge-associated and noncharge-related cell surface properties between closely related cell populations to K-562 cells from different sources and having different histories. The method consists of isotopically labeling aliquots of each of two cell populations to be compared with 51Cr-chromate and mixing the labeled cells with an excess of unlabeled cells with which they are to be compared. The mixtures are subjected to countercurrent distribution in either a charge-sensitive or a noncharge-sensitive dextran-poly(ethylene glycol) aqueous two-phase system. The distribution curves are analyzed for total cells (in terms of electronic counts) and labeled cells (in terms of cpm). Alterations in relative specific activities through the distribution curves are indicative of differences in surface properties between such cell populations. Using this method we have found surface differences, both charge-associated and noncharge-related, between any two K-562 cell sublines examined. Interestingly, whereas we observed differences among K-562 sublines, we never witnessed a change in surface properties of the respective sublines. The differences among the sublines examined remained unaltered for more than 40 passages in our hands. It thus appears likely that the event(s) leading to an altered K-562 cell surface, detectable by partitioning, does not occur gradually.

Cell Line↗
PubMed1988

Lowered susceptibility of K-562 cells treated with gamma interferon in serum-free medium to natural killer cell-mediated cytolysis.

K-562 cells grown in serum-free medium were treated with gamma interferon (IFN-gamma) and they became significantly less susceptible to natural killer (NK) cell-mediated cytolysis. To examine if this loss in susceptibility was related to induced differentiation events, the presence of various antigens was determined after induction. There was a coincident expression of class I HLA common antigen, although it is not clear if this is a direct causal relationship. The level of the constitutively expressed myelomonocytic antigen, reactive with anti-Leu-M1, was not affected by IFN-gamma induction and three normally nonexpressed monocytic antigens, defined by monoclonal antibodies, remained unexpressed. IFN-gamma did induce an enhanced expression of IL-2 receptors on K-562 cells after 2 days of treatment but, thereafter, the expression appeared to be suppressed. Electron microscopy of IFN-gamma-treated cells revealed the development of increased surface blebbing and electron-dense cytoplasmic inclusions. These ultramicroscopic changes could not be correlated with definitive differentiation events. We suggest that IFN-gamma treatment of K-562 cells induces class I HLA expression and morphological changes that may be important to differentiation events that render the cells less susceptible ot NK-mediated cytolysis.

Cell Differentiation↗
PubMed1987

Do some patients with Seckel syndrome have hematological problems and/or chromosome breakage?

We report on a 12-yr-old female and a 14-yr-old male with Seckel syndrome. The 12-yr-old female had pancytopenia, which is seen occasionally in patients with Seckel syndrome and is also a feature of Fanconi anemia, a well-recognized autosomal recessive dwarfism syndrome with chromosome instability. Chromosome breakage analysis of both of our patients also indicated chromosome instability. We suggest that there may be a subgroup of Seckel syndrome patients with chromosome instability and/or hematological problems.

Abnormalities, Multiple↗
PubMed1987

Continuous or modulation expression of hematopoietic cell antigens in sublines of the leukemia cell line, K-562.

K-562 is described as a pluripotent leukemic cell line which expresses a diversity of hematopoietic cell differentiation antigens. With the use of monoclonal antibodies (MoAb), we show that the expression of these antigens is either "modulated", i.e. they are not expressed in early logarithmic growth but are expressed in late logarithmic growth, or "continuous", i.e. they are not affected by proliferation and phase of culture growth. Whether an antigen is modulated or continuously expressed appears to be an inherent property of the subline, since the expression of myeloid antigen binding the MoAb, PM81, was modulated on cells from a clone, whereas, the expression of this antigen was continuous by the parent subline and an independent subline F-1. Continuously expressed antigens appear to be an integral component of the K-562 cell surface matrix, while modulated antigen expression appears to be influenced by culture growth conditions.

Antibodies, Monoclonal↗
PubMed1987

Interferon-gamma-induced expression of class I HLA antigens on K-562 cells grown in serum-free medium.

Human interferon-gamma (IFN-gamma), encoded in Escherichia coli by recombinant human DNA, induces the expression of HLA antigens in the pluripotent hematopoietic stem cell line K-562 grown in serum-free growth medium. Expression was noted in 90% of the cells within 4 days and there was a high density of expression per cell, as determined by cytofluorography. Upon subculture, the cells rapidly lose their ability to express HLA, indicating that the continued presence of IFN-gamma is necessary for the expression. Reinduction of expression with IFN-gamma occurs in a high percentage of cells (up to 90%), which is comparable to the induction in freshly treated control cells.

Cell Line↗
PubMed1986

Diversity of cell surface hematopoietic antigens on K-562 sublines identified with monoclonal antibodies.

The surface antigen profile of 8 sublines of K-562 cells, the original line, and the clone RA6 was determined with a panel of 12 monoclonal antibodies reactive with hematopoietic cell differentiation antigens. Cells from all sublines expressed the precursor hematopoietic antigen reactive with RFB-1, the T-cell antigen reactive with OKT17, the B cell/granulocyte antigen reactive with BA-1, the My-1 antigen, and glycophorin A which reacted with R10. A low percentage of cells in some of the sublines expressed platelet/monocyte glycoprotein I binding AN51, monocyte antigen binding 63D3, and an erythroblast/monocyte/platelet antigen binding 5F1. The use of a panel of K-562 sublines demonstrates that K-562 cells do share several "common" antigens but express a marked diversity and variability of other hematopoietic antigens.

Antibodies, Monoclonal↗
PubMed1985

I-cell disease and pseudo-Hurler polydystrophy: heterozygote detection and characteristics of the altered N-acetyl-glucosamine-phosphotransferase in genetic variants.

The human disorders I-cell disease and pseudo-Hurler polydystrophy (also known as mucolipidosis II and III, respectively) are caused by an inherited deficiency of UDP-GlcNAc: lysosomal enzyme precursor GlcNAc-P transferase activity. The most common genetic variants of these diseases (complementation group A) can be identified in homozygotes and heterozygotes using a GlcNAc-P transferase assay with artificial acceptors and commercially available radiochemicals. The kinetic characteristics of the residual GlcNAc-P transferase activity in complementation group A fibroblasts indicates that the low activity is due to a low Vmax. The measured Michaelis-Menten constants for the substrates UDP-GlcNAc and alpha-methyl mannoside are in the normal range. Homozygotes and heterozygotes of another less common variant of pseudo-Hurler polydystrophy (complementation group C) have normal activity and normal kinetic characteristics with this assay using alpha-methyl mannoside as the acceptor substrate. Several PHP variants with unusual characteristics are discussed.

Cells, Cultured↗
PubMed1985

Natural killer cell resistance in K-562 cell sublines.

Sublines of the hematopoietic stem cell line K-562 were tested for their susceptibility to human natural killer (NK) cell activity. A correlation was found between the degree of NK-mediated lysis and the presence or absence of particular chromosomal markers. K-562 subline susceptible to lysis by NK were found to express karyotypically a deletion 9- and a marker 8(t1-18), whereas resistant sublines did not express these markers. A cloned K-562 subline B1V was chosen as representative of a resistant subline. This subline was resistant to lysis even after prolonged incubation and activation of the NK cells with interferon. However, it was found that B1V was lysed by both antibody-dependent, complement-mediated and antibody-dependent cellular mechanisms at levels comparable to those seen with NK-sensitive K-562 sublines. Subline B1V did compete poorly with NK-sensitive K-562 in cold-target inhibition; however, conjugate-formation assays demonstrated that the binding of NK cells to B1V cells is comparable to that of NK-sensitive K-562 cells. We suggest that cells of the cloned line B1V are recognized normally by NK cells but do not activate the lytic mechanism of the bound NK cell. Treatment of the resistant clone B1V with neuraminidase did not lead to enhanced levels of lysis. Protein extracts of NK-sensitive K-562 sublines efficiently inhibited lysis by NK cells but extracts of the resistant clone, B1V, did not inhibit lysis, suggesting that the clone lacks cell surface determinants involved in the post-recognitive activation of the NK cytolytic mechanism.

Cell Line↗