Hematopoietic differentiation activity of a recombinant human interleukin-6 (IL-6) isoform resulting from alternatively spliced deletion of the second exon.

We have previously identified and cloned an alternatively spliced form of human interleukin-6 mRNA lacking exon II, which encodes amino acid residues known to be important in gp130-mediated signal transduction pathways. We expressed and purified the recombinant protein (rIL6-alt) resulting from this alternatively spliced mRNA and now report the initial characterization of its biologic activities with comparison to full length IL6 (rIL6-full). rIL6-alt was found to have 10(4) to 10(5) fold less activity in proliferation assays with 7TD1 murine plasmacytoma cells and did not competitively inhibit the stimulatory activity of rIL6-full. In addition, like rIL6-full, rIL6-alt had antiproliferative activity toward M1 murine myeloblast cells and was 10-200-fold less active than rIL6-full. In contrast, in assays with human HL60 promyelocytic leukemia cells, rIL6-alt had greater antiproliferative activity than rIL6-full and more strongly upregulated phagocytosis as well as surface expression of the differentiation antigen CD11b. rIL6-full and rIL6-alt upregulated the level of lysozyme mRNA in HL60 cells approximately equally. These findings suggest that IL6-alt, which lacks amino acid residues encoded by the second exon of the gene, is not a natural inhibitor of IL6-full but may be relatively tissue specific and may play a role in modulation of hematopoietic cell growth and differentiation.

In vitro effects of boron-containing compounds upon glioblastoma cells.

Boron-neutron capture therapy (BNCT) is currently under investigation as a novel therapeutic modality for glioblastoma. This study was undertaken to determine whether boron-containing compounds 4-borono-2-fluoro-D,L-phenylalanine (FBPA) and FBPA-fructose have direct effects upon kinetics of A172, a glioblastoma cell line. Flow cytometry analyzed cell-cycle distribution and S-phase kinetics (bromo deoxyuridine [BUdR] incorporation). BUdR incorporation was increased during a 1-hr pulse after 24-hr or 72-hr exposure of cells to varying concentrations of FBPA or FBPA-fructose. Results suggest that boron-containing compounds may effect cell kinetics apart from neutron activation, and this effect should be further evaluated for potential impact upon tumor responsiveness to BNCT.

Flow cytometry of liver fine needle aspirates.

Fine needle aspiration analysis of intraabdominal masses is increasing in frequency. This study was designed to evaluate whether flow cytometry could be of value in the cytopathology interpretation of such specimens. Over a 4-year time span, 129 consecutive liver fine needle aspirates were evaluated by flow cytometry for DNA content, ploidy, and proliferation rate. Only excess cells remaining in the needle after cytology samples were prepared were included in this study. Overall sensitivity was 75% and specificity was 94%. In addition, flow cytometry results were pivotal for at least two specimens in achieving the appropriate diagnosis. For these reasons, it was concluded that flow cytometry could be a valuable adjunctive technology, to the cytopathologist in the interpretation of liver fine needle aspirates.

Flow cytometry as a prognostic indicator in women with borderline epithelial ovarian tumors.

Flow cytometry has been used to rapidly and reliably measure DNA content in malignant tumor cells. Although several studies have suggested that DNA ploidy is a powerful predictor of survival in women with epithelial ovarian cancer, few have determined the usefulness of this procedure in women with borderline tumors. Using data from a population-based tumor registry covering all of western Washington State, women who died prior to 1992 as a consequence of developing a borderline ovarian tumor between 1975 and 1986 were compared to an age, histology, and histologic stage-matched sample of women with the same diagnosis still living after at least 5 years of follow-up. Flow cytometry analysis was conducted using sections of tumor from the original paraffin blocks. Overall, 25% of the women who died and 24% of those still alive had aneuploid DNA tumors (odds ratio = 1.2, 95% CI = 0.3-4.9). This lack of association stands in contrast to the strong relationship of aneuploid status to mortality in an earlier, similarly designed, study of borderline ovarian tumors. We believe that additional studies are required prior to concluding that the clinical course of women with borderline tumors can be predicted by the ploidy status of their tumor's DNA.

Evaluation of in vivo breast fine needle aspirates by flow cytometry: an efficacy study.

BACKGROUND: Malignancy of the breast is frequently diagnosed through fine needle aspiration. In the hands of a skilled aspirator and cytopathologist, this can be a highly accurate procedure. PURPOSE: This study was undertaken to evaluate whether sufficient residual cells in the bore of the needle could be harvested and analyzed efficiently by flow cytometry analysis. The goal was then to determine the value of routine flow cytometry as an adjunctive technology in the interpretation of breast fine needle aspirations. METHODS: Cells were rinsed from the needles of 83 consecutive diagnostic fine needle aspirates after preliminary inspection had confirmed adequate material was obtained for cytopathology. Cells were washed, and nuclei prepared by detergent treatment. After ribonuclease treatment, DNA was stained with the fluorescent marker propidium iodide. DNA content per cell was determined by flow cytometry by measurement of right-angle fluorescence. RESULTS: Less than 4% of the samples were rejected for inadequate cell numbers. Flow cytometry criteria for evidence of malignancy included the presence of a DNA aneuploid population or an elevated rate of proliferation (13% or higher) of a diploid population. Accuracy of flow cytometry was based on cytopathologic interpretation in all cases except two which were based on results of excisional biopsy. The sensitivity of the flow cytometry analysis was 76%; the specificity was 100%, with results from flow cytometry pivotal in the correct diagnoses for two patients whose cytopathologic results were equivocal. Analysis of histograms indicated acceptable coefficients of variation for all populations. Gating analysis indicated the suitability of the material for this type of study, with an average of 85% of the events selected, or "gated in." Low recoveries were associated with the presence of necrotic debris in the sample. CONCLUSION: Flow cytometry can be a valuable adjunctive technology, capable of providing the cytopathologist with additional information regarding the character of cells analyzed.

Flow cytometric analysis of pulmonary fluids and cells for the detection of malignancies.

Cells from a pulmonary or bronchial origin were analyzed with flow cytometry to assess the sensitivity and specificity of this method in diagnosing malignancy. In all instances, cells submitted for flow cytometry analysis were excess cells from specimens submitted for routine cytology. Less than 3% of all samples were rejected for insufficient material. Overall sensitivity from all sources was 86%, specificity 96%. Although cytology results were the standard for determining accuracy of flow cytometry, in a few patients cytology appeared normal and initial evidence for malignancy was obtained from flow cytometry. For this reason, flow cytometry may be a valuable adjunctive technology in the diagnosis of malignancy.

Flow cytometric determination of breast tumor heterogeneity.

Flow cytometric analysis was done on the DNA content of nuclei obtained from different sites of small breast tumors. Although specimens for analysis were obtained within a few millimeters of each other, dramatic differences were occasionally observed in the DNA histograms. In a limited study involving 141 consecutive breast specimens submitted for flow cytometry, 52% (74) were found to have at least one DNA aneuploid population. In 18% of DNA aneuploid tumors, one or more specimens from areas grossly identified as tumor had no DNA aneuploid population. Because of the proposed correlation of aneuploidy with a poorer prognosis and possible responsiveness to chemotherapy, multiple sites should be assayed when flow cytometric DNA analysis is done.

Vitamin B6 levels in patients with carpal tunnel syndrome.

Vitamin B6 levels were determined in patients with idiopathic carpal tunnel syndrome. Results from this limited study strongly suggest that vitamin B6 deficiency may accompany carpal tunnel syndrome. This study did not address the question of the causal relationship between vitamin B6 status and development of symptoms.

Colon cancer cells in peripheral blood of cancerous tamarins.

Diagnosing colon cancer in its early stages would lower the mortality rate. The cotton-top tamarin, Saguinus oedipus, serves as a model for the study of human colon cancer. This New World monkey has a high incidence of colitis and colon cancer. The mouse anti-human monoclonal antibody BR55.2, with specificity for human colon adenocarcinoma, was biotinylated. Peripheral blood mononuclear cells (PBMC) from animals with colon cancer were fluorescently stained with the biotinylated BR55.2. These results showed the cross-reactivity of mouse anti-human colon cancer monoclonal antibody to the PBMC of cancerous tamarins. Antibodies from either cancerous or chronic colitis tamarins were also biotinylated. Fluorescently labeled cells were detected when PBMC from cancerous tamarins were incubated with biotinylated antibodies from cancerous tamarins. Cytofluorographic analysis also showed a significant 4.5-fold difference in the percentage of fluorescently labeled PBMC between cancerous and chronic colitis tamarins when stained with biotinylated antibodies from cancerous tamarins. DNA flow cytometry analysis showed that PBMC from cancerous tamarins have a higher percentage of aneuploid cells than PBMC from chronic colitis tamarins.

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.

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.

Comparison of Clostridium difficile detection by monolayer and by inhibition of nucleoside uptake.

Detection and identification of Clostridium difficile toxin by traditional monolayer assay were compared with results obtained by a new procedure based on toxin-dependent inhibition of target cell uptake of a radioactive nucleoside. A high degree of correlation was noted between the two determinations. Although the new procedure was quantitative and objective, its value is seen at present as a rapid screen that may support results obtained in monolayers and as a potential assay for other, currently unidentified, toxins.

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.

Identification and quantitation of embryonic and three types of fetal hemoglobin produced on induction of the human pluripotent leukemia cell line K-562 with hemin.

The hemoglobins synthesized by the pluripotent K-562 leukemia cell line of human origin after induction with hemin have been isolated by DEAE-cellulose chromatography and characterized by electrophoresis, high pressure liquid chromatography, and a radioimmunological assay. Six hemoglobin zones have been observed with the following likely compositions. Zone 1: alpha 2 epsilon 2, or HB Gower-2; zone 2: zeta 2 epsilon 2, or HB Gower-1; zone 3: zeta 2 gamma 2, or HB Portland-I; zone 4: Hb F, or alpha 2 gamma 2; zone 5: a mixture of acetylated HB Portland-I and Hb F; zone 6: Hb Bart's, or gamma 4. The embryonic Hbs (zones 1, 2, and 3) constituted 50%-75% of the total Hb present; the quantities varied from one experiment to the other. Both Hb Gower-1 and Hb Gower-2 were present. The gamma chain was heterogeneous and contained the G gamma, A gamma I, and A gamma T types in a ratio of about 4:2:1, indicating a heterozygosity for the Ile leads to Thr substitution at position gamma 75. The methodology used can be applied for additional studies evaluating quantitative changes in Hb types due to in vitro manipulations.