Changes In Cell Surface Antigens Research Articles

Prolonged Azacitidine Exposure Alters Immunophenotype in Myeloid Leukemia Cell Lines

Hypomethylating agents (HMA), namely azacitidine and decitabine, are used for treatment of MDS and AML. Although HMAs have demonstrated efficacy in a subset of patients, their mechanism of action is not well established. Some studies indicate that HMAs induce expression of specific target proteins, thus enhancing the efficacy of targeted therapies. For example, studies of HMAs used in combination with gemtuzumab ozogamicin (GO) suggest that they can apply additional epigenetic stress to AML cells and increase surface CD33 expression, thereby increasing the efficacy of GO. Despite suggestions of epigenetic modulation of target antigens, data on comprehensive response to HMAs is lacking. We proposed to evaluate the full extent of surface antigen expression in different AML cell lines after prolonged exposure to azacitidine.The dose and duration of azacitidine therapy used for prolonged exposure was established in Kasumi-1 cells (t(8;21), N822K KIT mutation). A daily dose of 250 nM allowed continued cellular proliferation for 18 days. We then treated two cell lines, Kasumi-1 and MV4-11 (t(4;11), FLT3 -ITD+) as above for 14-18 days. Cells were submitted for multi-dimensional flow cytometry (MDF) every 2-4 days.Prolonged azacitidine exposure resulted in broad changes in hematopoietic cell surface antigens, as demonstrated by MDF analysis. CD33, the premier cell surface target for AML immunotherapy, increased significantly in Kasumi-1 cells, approximately doubling by day 14 of treatment (p=0.003, Fig. 1). C -kit, or CD117, which is overexpressed in Kasumi-1 cells and is a potential target of tyrosine-kinase inhibitor (TKI) therapy in t(8;21) AML, was significantly decreased with azacitidine exposure (p <0.0001, Fig. 1). In contrast, treatment of MV4-11 cells resulted in significant decrease in CD33 with a corresponding increase in CD117 (Fig. 1).Kasumi-1 and MV4-11 both exhibited significant increases in surface CD38, CD13, and CD36, and significant decreases in CD64, CD123, and CD11b. Cell marker changes observed in only one cell line include significantly increased CD34 in Kasumi-1 and significantly increased CD56 in MV4-11. Many of the observed changes peaked after day 10 of treatment. No significant changes were seen in either line in CD7, CD14, CD16, CD19, CD45, and HLA-DR. Notably, the shared pattern of surface antigen changes in both Kasumi-1 and MV4-11 indicates that prolonged azacitidine exposure may induce a less mature myeloid phenotype, as typified by increased CD38 and decreased CD11b and CD123 (Fig. 1).The results above were validated in an experiment in which 4 leukemia cell lines were exposed to azacitidine as above for 10 days, with MDF analyses on days 5 and 10. Cell lines included Kasumi-1, MV4-11, K562 (t(9;22)), and the ALL cell line REH (t(12;21)). Similar trends in surface antigens for Kasumi-1 and MV4-11 were again observed. K562 cells exhibited increased CD38, CD36, CD7, CD14, and CD19, and decreased CD33, CD56, CD64, and CD45 (Fig. 1). No change was seen in K562 in CD117, CD13, CD11b, CD16, CD34, CD123, and HLA-DR (Fig. 1). Of these myeloid markers tested, REH cells had only a significant increase in CD13, a marker also seen in very early B-lymphocytes. Thus, HMAs do not appear to promote lymphoid to myeloid phenotype shift in REH.We observed broad changes in cell surface antigen expression in myeloid leukemia cell lines treated with prolonged azacitidine, which may signify changes in gene expression induced by hypomethylation. Among AML cell lines, prolonged azacitidine exposure resulted in a less mature immunophenotype. We observed significant changes in CD33 and CD117, both of which are considered potential therapeutic targets in AML. CD33 expression was increased by azacitidine exposure in Kasumi-1 cells, and reduced in MV4-11 and K562. C -kit was also noted to variably change in response to azacitidine. Downregulation of cell surface antigens is a known resistance mechanism to immunotherapeutic agents, including antibody-drug conjugates such as GO. Resistance to c -kit-targeted agents has been proposed to occur through a variety of mechanisms, including cell surface expression, therefore changes to c -kit levels induced by HMAs may be an important avenue of investigation in averting TKI resistance. The use of prolonged, low-dose hypomethylating agents warrants further investigation as a potential means to modulate surface protein levels for therapeutic benefit. [Display omitted] DisclosuresPardo:Hematologics Inc: Employment. Loken:Hematologics Inc: Employment, Equity Ownership. Brodersen:Hematologics Inc: Employment.

Detection of Blood Cell Surface Biomarkers in Septic Mice.

Sepsis arises when an infection induces a dysregulated immune response, resulting in organ damage. New methods are urgently needed to diagnose patients in the early stages of sepsis, and identify patients with a poor disease prognosis. One promising approach is to identify the rapid changes in cell surface antigens (biomarkers) that occur during sepsis, as a consequence of leukocyte mobilization and activation. This chapter describes the method for staining whole blood with fluorescently conjugated antibodies that detect cell surface biomarkers, and performing flow cytometry analysis to quantify biomarker-positive cells. Our protocol is designed to detect blood cell surface biomarkers in septic mice, but could also be applied to study potential biomarkers in blood obtained from human patients with sepsis and other medical conditions.

Investigation of Macrophage Polarization Using Bone Marrow Derived Macrophages

The article describes a readily easy adaptive in vitro model to investigate macrophage polarization. In the presence of GM-CSF/M-CSF, hematopoietic stem/progenitor cells from the bone marrow are directed into monocytic differentiation, followed by M1 or M2 stimulation. The activation status can be tracked by changes in cell surface antigens, gene expression and cell signaling pathways.

CD5-Positive Diffuse Large B Cell Lymphoma Infiltrating the Central Nervous System Presenting Guillain-Barr^|^eacute;-Like Syndrome after Chemotherapy

An 83-year-old woman was admitted to our hospital with abdominal pain. Examination revealed mediastinal lymphoadenopathy, hepatosplenomegaly, and infiltration of abnormal cells into the bone marrow with hemophagocytosis, and CD5-positive diffuse large B cell lymphoma was diagnosed. Chemotherapy was administered and progressive weakness of the limbs, resembling a Guillain-Barré-like syndrome, subsequently appeared. Cerebrospinal fluid examination indicated lymphoma cell infiltration. Although immune globulin and steroid therapies were not effective, intrathecal injection of methotrexate, predonisolone, and cytarabine improved these symptoms. Subsequent to chemotherapy, cell surface antigen changes were observed in the cerebrospinal fluid relative to those in bone marrow.

Genetic diversity in Campylobacter jejuni is associated with differential colonization of broiler chickens and C57BL/6J IL10-deficient mice

Previous studies have demonstrated that Campylobacter jejuni, the leading causative agent of bacterial food-borne disease in the USA, exhibits high-frequency genetic variation that is associated with changes in cell-surface antigens and ability to colonize chickens. To expand our understanding of the role of genetic diversity in the disease process, we analysed the ability of three C. jejuni human disease isolates (strains 11168, 33292 and 81-176) and genetically marked derivatives to colonize Ross 308 broilers and C57BL/6J IL10-deficient mice. C. jejuni colonized broilers at much higher efficiency (all three strains, 23 of 24 broilers) than mice (11168 only, 8 of 24 mice). C. jejuni 11168 genetically marked strains colonized mice at very low efficiency (2 of 42 mice); however, C. jejuni reisolated from mice colonized both mice and broilers at high efficiency, suggesting that this pathogen can adapt genetically in the mouse. We compared the genome composition in the three wild-type C. jejuni strains and derivatives by microarray DNA/DNA hybridization analysis; the data demonstrated a high degree of genetic diversity in three gene clusters associated with synthesis and modification of the cell-surface structures capsule, flagella and lipo-oligosaccharide. Finally, we analysed the frequency of mutation in homopolymeric tracts associated with the contingency genes wlaN (GC tract) and flgR (AT tracts) in culture and after passage through broilers and mice. C. jejuni adapted genetically in culture at high frequency and the degree of genetic diversity was increased by passage through broilers but was nearly eliminated in the gastrointestinal tract of mice. The data suggest that the broiler gastrointestinal tract provides an environment which promotes outgrowth and genetic variation in C. jejuni; the enhancement of genetic diversity at this location may contribute to its importance as a human disease reservoir.

The antiapoptotic gene A1/BFL1 is a WT1 target gene that mediates granulocytic differentiation and resistance to chemotherapy

AbstractPrevious work has demonstrated that WT1 (–Ex5/–KTS) potentiates granulocyte colony-stimulating factor (G-CSF)–mediated granulocytic differentiation. This WT1 isoform suppresses cyclin E, which may contribute to the prodifferentiation effect by slowing proliferation, but WT1 target genes that affect survival might also be involved. We screened a cDNA array and identified the bCL2 family member A1/BFL1 as a new WT1 target gene in 32D cl3 murine myeloblast cells. Induction of WT1 (–Ex5/–KTS) expression is accompanied by up-regulation of A1 on the cDNA array, and this up-regulation was confirmed by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). Moreover, both promoter-reporter assays and chromatin immunoprecipitation assays suggest that this isoform of WT1 activates the promoter directly. Constitutive expression of A1 in 32D cl3 cells induces spontaneous granulocytic differentiation, with both morphologic and cell-surface antigen changes, as well as resistance both to chemotherapy and to withdrawal of interleukin-3 (IL-3). Finally, we note an association between WT1 expression and A1 expression in primary acute myeloid leukemia samples. Taken together, these results demonstrate that A1 is a new WT1 target gene involved in both granulocytic differentiation and resistance to cell death, and suggests that these genes might play an important role in the biology of high-risk leukemias.

Postadaptational resistance to benzalkonium chloride and subsequent physicochemical modifications of Listeria monocytogenes.

Many studies have demonstrated that bacteria, including Listeria monocytogenes, are capable of adapting to disinfectants used in industrial settings after prolonged exposure to sublethal concentrations. However, the consequent alterations of the cell surface due to sanitizer adaptation of this pathogen are not fully understood. Two resistant and four sensitive L. monocytogenes strains from different sources were progressively subcultured with increasing sublethal concentrations of a surfactant, benzalkonium chloride (BC). To evaluate the effects of acquired tolerance to BC, parent and adapted strains were compared by using several morphological and physiological tests. Sensitive strains showed at least a fivefold increase in the MIC, while the MIC doubled for resistant strains after the adaptation period. The hydrophobicities of cells of parent and adapted strains were similar. Serological testing indicated that antigen types 1 and 4 were both present on the cell surface of adapted cells. The data suggest that efflux pumps are the major mechanism of adaptation in sensitive strains and are less important in originally resistant isolates. A different, unknown mechanism was responsible for the original tolerance of resistant isolates. In an originally resistant strain, there was a slight shift in the fatty acid profile after adaptation, whereas sensitive strains had similar profiles. Electron micrographs revealed morphological differences after adaptation. The changes in cell surface antigens, efflux pump utilization, and fatty acid profiles suggest that different mechanisms are used by resistant and sensitive strains for adaptation to BC.

Enhancement of Metastatic Activity of Colon Cancer as Influenced by Expression of Cell Surface Antigens

Background.Cell surface antigens are contributory factors toward metastatic activity. There have been no detailed studies on changes in cell surface antigens of colon cancer cell lines. To control life-threatening metastasis, it is necessary to evaluate what types of changes in cell surface antigens exert an influence on metastatic activity.Materials and methods. In vivoselection was performed using the human colon cancer-derived cell line KM12SM to obtain variants of metastatic activity. A murine spleen injection-liver metastasis procedure reflecting the latter half of the metastatic process was adopted and repeated four times. Flow cytometric analyses were carried out to detect expression of antigens: Lewis a (Lea), Lewis x (Lex), sialyl Lewis a (sLea), sialyl Lewis x (sLex), E-cadherin, CD44v6, integrin α2 (CD49b), integrin α3 (CD49c), integrin α4 (CD49d), integrin α5 (CD49e), and integrin β1 (CD29).Results. In vivoselection produced variants with higher metastatic activity. In the original line KM12SM, sLea, E-cadherin, CD49b, CD49c, or CD29 were positive in more than 40% of the cells. After selection, the percentage of cells positive for Lea, sLea, and all examined integrins significantly increased. Lex, sLex, and CD44v6 increased slightly, while E-cadherin decreased slightly.Conclusions. In vivoselection and flow cytometric analysis revealed that Lea, sLea, CD49b, CD49c, and CD29 appear to be involved in the increase of metastatic activity. The changes of integrin expression in this study suggest that integrins collaborate in the promotion of adhesion to an extracellular matrix.

Differential expression of amino acid transport systems A and ASC during erythroleukemia cell differentiation

The human erythroleukemic cell K-562 serves as an in vitro model to study changes in cell surface antigens and mechanisms regulating globin gene expression associated with in vivo erythropoiesis. In this report we have examined the regulation of amino acid transport systems, in particular, systems A and ASC, during differentiation of erythroleukemic cells. For additional comparison we examined the uptake of leucine, 3-aminoendobicyclo-(3,2,1)-octane-3-carboxylic acid (BCO), arginine, and glutamate. Hexamethylene-bis-acetamide (HMBA), dimethyl sulfoxide, and butyrate induce cell differentiation with a block in G1-G0 phase of the cell cycle. These agents caused a significant downregulation of 2-(methylamino)isobutyric acid uptake by system A. In contrast, the Na(+)-dependent threonine uptake by system ASC remained unaltered. The uptake of leucine, BCO, arginine, and glutamate by as yet unidentified systems was, however, stimulated after HMBA treatment. Hemin, a potent inducer of hemoglobin synthesis in K-562 cells, does not block cell cycle events and, interestingly, had no significant effect on both systems A and ASC. These differences in inducer actions suggest that system A activity may be related to specific stages of cell differentiation and perhaps to other cellular signals.

The paradoxical effects of diethyldithiocarbamate: Comparisons between New Zealand Black/White F 1 hybrid and [formula omitted] mice

Diethyldithiocarbamate (DTC), an immunomodulative agent suggested to enhance T cells, has been shown to prolong life in autoimmune MRL- lpr lpr mice. In addition to increased survival, MRL- lpr lpr mice treated with DTC displayed a number of changes in expression of cell surface antigens as well as decreased serum autoantibody levels. To determine if DTC treatment would have similar positive effects on another murine model of autoimmune disease, we studied the New Zealand Black/White F 1 hybrid ( NZB W ). In addition, the effects of DTC treatment on cell surface antigen expression were compared between the NZB W and a normal murine strain, the Balb c . DTC treatment increased the density of cell surface antigens in the NZB W , but decreased the density of these antigens in the Balb c . Treatment with DTC induced distinct changes in the percentage of cells expressing specific surface antigens that differed between the NZB W and the Balb c . There was no affect on serum anti-DNA and anti-histone antibody levels or on survival in NZB W mice treated with DTC. Therefore, while DTC treatment did not successfully influence the disease course in the NZB W , it did result in specific changes in cell surface antigens. These data demonstrate that DTC is capable of inducing a variety of immunologic changes depending upon the strain treated.

Up-regulation of gamma interferon receptors on the human monocytic cell line U937 by 1,25-dihydroxyvitamin D3 and granulocyte-macrophage colony stimulating factor.

The human monoblast-like cell line U937 can be induced to differentiate by a variety of agents including phorbol esters, retinoic acid, gamma interferon (IFN gamma), and 1,25-dihydroxyvitamin D3 (VD3). Increased expression of OKM1 antigen, Fc receptors, and other cell surface antigens occur with the differentiation of this cell line along the macrophage lineage. Whereas 10(-8) M VD3 alone induces changes in cell surface antigens, there were no changes in the number or affinity of IFN gamma receptors. Incubation of U937 with VD3 and 100 U/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in further increases in OKM1 antigen expression and an up-regulation of IFN gamma receptors. The number of IFN gamma receptors increased between two- and fourfold and was maximal after 48 h incubation with VD3 and GM-CSF. Scatchard analysis revealed a single class of receptors before or after differentiation, although the increase in receptor number was associated with an overall decrease in receptor-binding affinity. Incubation of U937 with VD3 plus GM-CSF and IFN gamma resulted in further increases in the density of OKM1 antigen expressed per cell. This increase in OKM1 expression was greater than that observed for U937 incubated with VD3 and GM-CSF or VD3 and IFN gamma alone. These results suggest that GM-CSF up-regulates IFN gamma receptors on VD3-stimulated U937 and enables these cells to be induced further along the pathway of macrophage differentiation, possibly by subsequent interaction with additional cytokines such as IFN gamma.

A pharmacologic study of the relationship between lymphocyte function and surface antigen expression

The relationship between functional activity and distribution of lymphocyte surface markers has not been clearly defined. We have examined the relationship between cell surface markers and function under the influence of immunosuppressant therapy. We found that after immunization with EL4 cells, the development of the immune response in the BALB/c mouse was accompanied by a decrease in spleen cells which stained brightly with fluorescein-labeled monoclonal anti-Thy 1 and an increase in cells which stained with rabbit anti-mouse Ig as measured on the FACS. Low doses of azathioprine and cyclophosphamide, which affect functional activity of the cells, do not alter cell surface markers. However, at higher doses of the drugs normalization of immunization-induced marker changes were observed, and the Thy 1+ and Ig+ surface markers were maintained at levels seen in non-immunized mice. In spite of a nearly 3-fold increase in the total number of lymphocytes and an increase in the functional activity of cytotoxic T cells (Lyt 2+) after immunization, no alteration in the percentage of Lyt 2+T cells nor in the intensity of staining with FITC-labeled Lyt2 antibody was seen. Inhibition of the immune response with immunosuppressant also failed to change the Lyt2+-staining cell population. This study demonstrates that lymphocyte functional changes precede cell surface antigen changes, and that functional changes may occur without surface antigen changes. Thus cell surface markers are "late" indicators of functional changes.

Effect of retinoic acid on myeloid antigen expression and clonal growth of leukemic cells from children with acute non lymphocytic leukemia — A pediatric oncology group study

We have used a panel of 5 monoclonal antibodies against normal myeloid-differentiation antigens to determine retinoic acid-induced changes in cell surface antigens on ANLL bone marrow cells from 24 children at the time of diagnosis. Two of these antibodies (T5A7 and 5F1) detect antigens expressed on normal mature granulocytes and on all monocytes, respectively. The percentage of positive cells for each monoclonal antibody was determined by indirect immunofluorescence. After 5 days incubation with 1μM RA in liquid culture, cells from 11 of 24 patients showed substantially increased expression of one or both antigens detected by T5A7 and 5171. Leukemic bone marrow cells from these patients were also cultured in methylcellulose medium with and without 1 μ M RA for one week, and cells from 16 of 24 patients showed clonal growth. Cultures from 10 of these 16 patients showed RA-induced inhibition of colony growth; of these 10 patients, cultures from six patients showed RA-induced increases in antigens associated with maturing myeloid cells. This suggests that the RA-induced inhibition of clonal growth observed with leukemic cells from these patients may be accompanied by the increased expression of maturation-associated myeloid antigens by these cells in the presence of RA.

Changes in cell surface antigens during in vitro lizard myogenesis

Indirect immunofluorescence has been used to examine surface antigens of lizard myogenic cells during in vitro differentiation. At least two developmental stage-specific surface alterations have been identified. One of these is a compositional change and involves the appearance of a cell-surface antigen(s) as the cells differentiate. This antigen(s) (Ag1422) is muscle specific and is characteristic of some rounded-up G 0 myosin-positive myocytes, all stretched-back, G 0 myosin-positive myocytes, and all identifiable myotubes. The antigen is not found on proliferating myoblasts, extended G 1 (myosin-negative) cell-cycle-competent myoblasts or newly differentiated rounded-up, G 0 myosin-positive myocytes. Pretreatment of cells with neuraminidase, trypsin, or proteinase K indicates the antigen is not present in “masked” form on normally nonreactive cells. Proteinase K is effective in the removal or destruction of the antigen, indicating it is at least partially protein in nature. The antigen is expressed in a similar developmental stage-specific fashion on early-passage myogenic cells taken from both adult lizard tail regenerates and embryonic muscle. The antibodies identifying Ag1422 can be removed by adsorption with homogenates of mature skeletal muscle. Therefore, Ag1422 is not an artifact due to in vitro conditions or the expression of a transformation antigen unique to the continuous culture line. The second alteration is an apparent restriction in the mobility of surface components (antigens and lectin receptors). Upon treatment with multivalent ligands, undifferentiated myosin-negative myoblasts exhibit rapid patching and capping of cell surface components while well-differentiated myocytes and myotubes do not. This mobility restriction is evident after the appearance of Ag1422. Treatment with cytochalasin B (15 μg/ml) and/or colchcine (100 μ M) does not alter the restricted mobility of surface components seen on differentiated cells. Therefore, neither microfilaments nor microtubules seem to be involved in the mobility restriction. These observations are discussed in relation to current views of myogenesis.

Analysis of intrathymic differentiation patterns during the course of AKR leukemogenesis

Thymocytes from AKR mice in different stages of leukemia development were analyzed with the fluorescence-activated cell sorter (FACS) using monoclonal antisera to Lyt-1, Lyt-2, Thy-1.1, H-2K k, and Ia k. In addition, the number of cells bearing receptors for peanut agglutinin (PNA) was assessed. The results were correlated with the expression of murine leukemia virus (MuLV) antigen. Thymocytes from late preleukemic and leukemic stages were found to have a phenotype characteristic of a more mature cell population in that there was an increase in the expression of determinants encoded within the K end of H-2 k and Ia k. This was associated with a decrease in the number of thymocytes bearing receptors for PNA during the leukemic stage. Simultaneously, a shift from a Lyt-1 + 2 + thymocyte population to cells with varying expressions of Ly antigens was observed. Analysis of Lyt determinants on thymomas indicated that they could arise from cells bearing any of the different possible combinations of Ly phenotypes. The cell surface antigen changes occurred in temporal correlation with an increased expression of MuLV antigens.

Evidence for mutation in an I-A gene.

Mutant mouse strains are important tools for immunogenetic studies of the regulation, structure and function of major histocompatibility (H-2) antigens1-3. Several inbred strains have been established which carry H-2-linked mutations that cause changes in cell surface antigens as recognized by cytotoxic T cells and sometimes antibodies. Using intra-H-2 recombinant haplotypes in skin graft complementation studies, lesions in several of these mutant strains have been genetically mapped to genes encoded in the K- or D-end of the H-2 complex3. More precise mapping has often not been possible as many of the available mutant strains possessed lesions in the K-end of the H-2b haplotype and a recombinant separating the Kb and I-Ab regions was not previously available. The genetic locations of the lesions in some of these mutant strains could therefore only be inferred when a serological and/or structural alteration was detected (see refs 1, 2). We describe here the use of a newly established recombinant strain, B10.MBR (ref. 4), for skin graft complementation studies to map K-end mutations. These studies provide the first genetic evidence that the B6.C-H-2bm12 mutation involves an I-Ab gene and also confirm that the lesions in two other mutants can be mapped to a gene in the Kb region.

Chapter 27 The Use of Complement-Mediated Cytolysis to Analyze Specific Cell Populations of Eukaryote Cells in Cultures

The complement-mediated antibody cytotoxicity assay has been used in studies on the changes in cell surface antigens during neoplastic transformation, the differentiation of mammalian cells, the chromosomal localization of specific genes for cell surface antigens, the removal of specific types of cells from mixtures so that biochemical analysis can be performed, and the detection and isolation of specific populations of lymphocytes. The development of antibody cytolysis techniques allows preparing antibodies against cell surface antigens, the capability to sort cells from populations which are quantitatively or qualitatively heterogeneous for the antigen under study. This can be done without extremely complex and expensive equipment. The chapter reviews the mechanism by which activation of complement is thought to lyse cells, the techniques for the use of complement-mediated antibody cytotoxicity to analyze populations of neoplastically transformed and untransformed cells, and how other investigators have used these techniques.

Age-related changes in cell surface antigens of preleukemic AKR thymocytes.

Thymocytes from preleukemic AKR mice aged 5-6 mo have an altered pattern of cell surface antigens. The expression of four MuLV-related antigens on the cell surface (GIX, GCSA, gp70, p30) is markedly increased in comparison to 2-mo-old AKR mice and approximates the heightened levels of these antigens found on thymic leukemia cells. H-2 and Thy-1 alloantigens also show characteristic modifications in relation to age and leukemia development. In contrast to the high Thy-1/low H-2 levels on 2-mo-old AKR thymocytes, thymocytes from 6-mo-old mice and thymic leukemia cells frequently show a low Thy-1/high H-2 surface phenotype. As thymocytes from mouse strains with a low incidence of leukemia do not show these changes, they appear to represent a stage in the conversion of normal cells to leukemia cells.