Interleukin-18 regulates acute graft-versus-host disease by enhancing Fas-mediated donor T cell apoptosis.

Interleukin (IL)-18 is a recently discovered cytokine that modulates both T helper type 1 (Th1) and Th2 responses. IL-18 is elevated during acute graft-versus-host disease (GVHD). We investigated the role of IL-18 in this disorder using a well characterized murine bone marrow transplantation (BMT) model (B6 --> B6D2F1). Surprisingly, blockade of IL-18 accelerated acute GVHD-related mortality. In contrast, administration of IL-18 reduced serum tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) levels, decreased intestinal histopathology, and resulted in significantly improved survival (75 vs. 15%, P < 0.001). Administration of IL-18 attenuated early donor T cell expansion and was associated with increased Fas expression and greater apoptosis of donor T cells. The administration of IL-18 no longer protected BMT recipients from GVHD when Fas deficient (lpr) mice were used as donors. IL-18 also lost its ability to protect against acute GVHD when interferon (IFN)-gamma knockout mice were used as donors. Together, these results demonstrate that IL-18 regulates acute GVHD by inducing enhanced Fas-mediated apoptosis of donor T cells early after BMT, and donor IFN-gamma is critical for this protective effect.

Oncoprotein 18 levels and phosphorylation mediate megakaryocyte polyploidization in human erythroleukemia cells.

Megakaryocytes undergo an unusual cell cycle during differentiation that results in polyploidy through largely unknown mechanism(s). It has been shown that serine phosphorylation of oncoprotein 18 (Op18) is required for cell cycle progression specifically at the G2/M transition. Moreover, mutant forms of Op18 that are defective in one or more of the four serine residues induce G2/M arrest and subsequent polyploidization. Op18 phosphorylation is rapidly induced with phorbol myristate acetate (PMA) treatment in a wide range of human cells. In this study, we investigated the role of Op18 in PMA induced polyploidization during megakaryocyte differentiation of the human erythroleukemia cell line. Crucial to the molecular analysis of megakaryocyte differentiation, is the ability to fractionate cell populations with different ploidy levels. We have utilized cell elutriation as a fractionation strategy to analyze Op18 expression in synchronized cell subpopulations in different phases of the cell cycle or with progressive megakaryocyte polyploidization. In the absence of PMA, increased phosphorylation of Op18 was observed in HEL cells during cell cycle progression, as for other proliferating cells. However, in contrast to Jurkat leukemia cells chosen as control, HEL cells exhibited a lack of Op18 phosphorylation in response to PMA, which was accompanied by polyploidization and differentiation along the megakaryocytic lineage. To further determine the role of Op18 in polyploidization, HEL cells were transfected with different Op18 expression constructs. Differences in cell survival and polyploidization were observed between high and low Op18 expressors. An increased Op18 level reduced cell survival during the early stage of PMA induced megakaryocyte differentiation, but enhanced polyploidization efficiency. Our findings suggest that maintenance of a high level of unphosphorylated Op18 is required for efficient polyploidization during the differentiation program of megakaryocytes.

Cytotoxicity and accumulation of ganciclovir triphosphate in bystander cells cocultured with herpes simplex virus type 1 thymidine kinase-expressing human glioblastoma cells.

The ability of herpes simplex virus type 1 thymidine kinase (HSV-TK)-expressing cells incubated with ganciclovir (GCV) to induce cytotoxicity in neighboring HSV-TK-negative (bystander) cells has been well documented. Although it has been suggested that this bystander cell killing occurs through the transfer of phosphorylated GCV, there is little direct proof that bystander cells can accumulate GCV nucleotides. We have studied the ability of U251 human glioblastoma cells expressing HSV-TK (U251tk cells) to induce cytotoxicity in neighboring U251 bystander cells that lack the viral kinase (U251beta gal cells) and evaluated whether this bystander cell killing is mediated by GCV nucleotides. The cytotoxicity studies demonstrated that the ratio of HSV-TK-expressing cells:bystander cells was important in determining the sensitivity of both cell types to GCV. U251tk cells cocultured with an equal number of U251beta gal cells (a 50:50 ratio) exhibited a sensitivity to GCV similar to that observed in the absence of bystander cells, with >99.8% cell kill at 1 microm GCV. However, in cultures with 10% U251tk cells and 90% bystander cells (a 10:90 ratio), 1 microM GCV decreased the survival of U251tk cells by only 54%. Strong bystander cell killing was observed at both ratios. In a 50:50 coculture of U251tk and U251beta gal cells, the survival of bystander cells was decreased by >99.5% with 3 microM GCV, whereas 30 microM GCV was required to effect a similar decrease in bystander cell survival when 90% of the culture consisted of U251beta gal cells. To determine whether this bystander cell killing may be mediated by GCV nucleotides, we developed a technique to separate the two cell populations after coculture. A U251 bystander cell line was developed from the parental cell line by transfection with the cDNA coding for green fluorescent protein (U251gfp cells), which permitted the separation of U251gfp cells from nonfluorescing U251tk cells by flow cytometry with cell sorting. With this technique, bystander cells were isolated in a viable state with >97% purity within 1 h after harvest, permitting analysis of the nucleotide pools for the presence of phosphorylated GCV. The results demonstrated that significant levels of the triphosphate of GCV (GCVTP) accumulated in bystander cells within 4 h of coculture, and this accumulation was dependent upon the percentage of HSV-TK-expressing cells as well as the concentration of GCV and the length of incubation. The proportion of GCVTP in bystander cells was consistently 50-80% of that in HSV-TK-expressing cells in the 50:50 or 10:90 cocultures, suggesting a facile transfer of phosphorylated GCV. However, the actual amount of GCVTP was as much as 8-fold lower in both the U251tk and U251beta gal cells cocultured at a ratio of 10:90 compared to those cocultured at a ratio of 50:50, which is consistent with the lesser effect on cell survival. When U251tk and U251gfp cells were cultured with 1-beta-D-arabinofuranosylthymine (araT), the 5'-triphosphate of araT accumulated in the bystander cells, demonstrating that the transfer of phosphorylated compounds between these cell types is not restricted to GCV nucleotides. However, the proportion of araT-5'-triphosphate in bystander cells compared to that in HSV-TK-expressing cells was lower than that for GCVTP, and the amount was not sufficient to decrease survival in the bystander population.

Flow cytometric analysis of DNA damage in nucleoids from cultured human breast epithelial cells treated with hydrogen peroxide.

Flow cytometric analysis of nucleoids is useful to investigate nuclear matrix-DNA associations in cells. We have now applied this technique to examine whether differences in nucleoid structure can be detected between tumor and normal-like human breast epithelial cells. We have previously shown that MCF-7 tumor and MCF-10A normal-like human breast cells exhibit different levels of endogenous oxidative DNA damage as well as differences in response to hydrogen peroxide. We therefore examined whether flow cytometric analysis of nucleoids can be used to detect both endogenous DNA damage and DNA damage induced by hydrogen peroxide in these cells. Nucleoids were prepared by lysis of MCF-7 and MCF-10A cells with a high-salt buffer. The size of the DNA loops around the nuclear matrix core was detected by measuring the forward light-scatter signal in the presence of ethidium bromide. The relaxation and supercoiling of DNA in the presence of low and high concentrations of ethidium bromide, respectively, was similar in MCF-7 tumor and MCF-10A normal-like cells. After treatment of cells with 100 microM and higher of hydrogen peroxide, there was a statistically significant increase in the forward light scatter from nucleoids prepared in the presence of high concentrations of ethidium bromide, and this increase was similar in both cell lines. The changes in the forward light scatter signal with increasing hydrogen peroxide concentration did not mimic the patterns of increases in single strand breaks or formation of 5-hydroxymethyl-2 '-deoxyuridine that we reported previously. This indicates that the flow cytometric technique probably detects other types of changes induced by hydrogen peroxide.

Flow cytometric analysis of P-glycoprotein expression and drug efflux in human soft tissue and bone sarcomas.

Twenty-two fresh surgical specimens of human sarcomas (soft tissue and bone) from 20 patients were analyzed by flow cytometry for the expression of drug resistance-related P-glycoprotein (P-gp) and cellular daunorubicin (DNR) accumulation with or without the presence of DNR efflux blockers. Single-cell suspensions prepared from the tumor specimens were analyzed by dual-color flow cytometry after reaction with MRK-16 (anti-P-gp) and anti-CD45 (pan-leukocyte) antibodies. MRK-16 reactivity of tumor cells was evaluated after exclusion of CD45-positive cells by electronic gates. Parallel samples were incubated with DNR alone or in combination with DNR efflux blockers, verapamil (VPL), or dipyridamole (DPD) for determination of cellular DNR accumulation and the effect of the efflux blockers. Extensive heterogeneity was observed in both P-gp expression and DNR accumulation of the tumor specimens examined. Eight of the 22 tumor specimens had significant numbers of P-gp-positive cells. In three of the eight P-gp-positive tumors, cellular DNR accumulation was significantly increased by co-incubation with the efflux blockers VPL or DPD. These results indicate that both quantitative and functional analysis of P-gp expression may be essential in determining the cellular drug resistance phenotype of tumor cells and its correlation with therapeutic outcome.

A sensitive flow cytometric method for measuring the oxidative burst.

We report a novel method of flow cytometric detection of the oxidative burst in human neutrophils. The cells were covalently labeled on the plasma membrane by incubation with 4-carboxydihydrotetramethylrosamine succinimidyl ester on ice. Activation of neutrophils resulted in an increase in red fluorescence at 575 nm using 514 nm excitation. The sensitivity of this assay in detecting the response to chemotactic peptide N-formyl-met-leu-phe (FMLP) and phorbol 12-myristate 13-acetate (PMA) was comparable to that observed with the cytochrome c oxidation test. We compared the new method to previously described methods using intracellular fluorescent stains. Our method showed the lowest nonspecific oxidation and the best sensitivity to FMLP-induced activation. It was equally or slightly less efficient than the green fluorescent stain dihydrorhodamine 123 in detection of neutrophil activation by immune complexes and PMA but much more sensitive than red fluorescent stains hydroethidine and dihydrotetramethylrosamine. It can be successfully used in kinetic experiments and yields a fluorescence signal that remains stable for at least 1.5 h after formaldehyde fixation with antioxidant added.

Quantitative measurements of the efficacy of new anti-cancer agents on fresh human AML cells by using multivariate flow analysis.

The testing of new human leukemia-specific drugs for activity against primary acute myeloid leukemia (AML) blasts is severely limited by the low and variable clonogenic potential of primary human leukemias in culture. To circumvent this problem, we have modified a previously described flow cytometric approach to permit the simultaneous determination of live/dead cells, and the quantitation of the surviving cell fraction as a ratio of viable cells in treatment and control groups. The method utilizes the combination of calceinAM as a probe for intracellular esterase activity (green fluorescence,) which has the advantage over carboxyFDA of pH insensitivity and superior signal-to-noise ratio, and propidium iodide (red fluorescence) as an indicator of plasma membrane integrity. Suspension cultures of AML blood and marrow samples from patients were treated with known active agents as well as several new agents arising from a clonogenic disk assay screen. Quantitative dose-response values obtained from surviving cell fractions assayed by flow cytometry at 24 h following drug exposure demonstrated the utility of this assay for quantitating drug-induced cytotoxic effects on primary human AML cells in short-term culture.

T lymphocytes in the murine vaginal mucosa are phenotypically distinct from those in the periphery.

The results from both clinical studies of women with recurrent vulvovaginal candidiasis and a murine model of experimental vaginitis indicate that systemic cell-mediated immunity may not represent a dominant host defense mechanism against vaginal infections by Candida albicans. Recent experimental evidence indicates the presence of local vaginal immune reactivity against C. albicans. The present study was designed to examine T-lymphocyte subpopulations in the vaginal mucosae of naive CBA/J mice. Vaginal lymphocytes (VL) were isolated by collagenase digestion of whole vaginal tissues. Cell populations were identified by flow cytometry, and the results were compared with those for both lymph node cells (LNC) and peripheral blood lymphocytes (PBL). The results of flow cytometry showed that 45% +/- 10% of lymphocytes in the vaginal mucosa are CD3+ compared with 75% +/- 5% in LNC and 50% +/- 5% in PBL. The majority (85%) of CD3+ VL are CD4+ and express the alpha/beta T-cell receptor (TCR), similar to the results for LNC and PBL. In contrast to LNC and PBL, VL contain a significantly higher percentage (15 to 20%) of gamma/delta TCR+ cells, 80% or more of which appear to express CD4. In addition, while CD4-CD8 cell ratios in LNC and PBL were 3:1 and 6:1, respectively, only 1% of VL expressed CD8, resulting in a CD4-CD8 cell ratio of > 100:1. Finally, while LNC and PBL recognized two epitope-distinct (GK 1.5 and 2B6) anti-CD4 antibodies, VL recognized only 2B6 anti-CD4 antibodies. Further analysis of VL showed that Thy-1 cells, but not CD4 cells, were reduced after intravaginal injection of complement-fixing anti-Thy-1.2 and GK 1.5 anti-CD4 antibodies, respectively. Taken together, these data suggest that T lymphocytes in the vaginal mucosae of mice are phenotypically distinct from those in the periphery and that CD4+ VL have an uncharacteristic or atypical expression of the CD4 receptor.

Zinc deficiency affects cell cycle and deoxythymidine kinase gene expression in HUT-78 cells.

Although zinc is known to be involved in cell proliferation and DNA synthesis, the mechanism by which zinc may regulate these processes is not understood. We have studied the role of zinc on cell proliferation and gene expression of a DNA synthesizing enzyme, deoxythymidine kinase (TK), in a T helper human malignant lymphoblastoid cell line (HUT-78). In zinc-deficient and zinc-sufficient media, the cell doubling time (mean +/- SD) of HUT-78 was 59 +/- 8 hours and 32.6 +/- 6 hours, respectively. The effect of zinc was T cell specific, inasmuch as the cell growth of another T malignant lymphoblastoid cell line, MOLT-3 (immature T cells), was not affected by zinc deficiency. Iron, copper, or manganese did not completely correct the cell growth of zinc-deficient HUT-78 cells. TK activity and the relative accumulation of TK-mRNA were significantly decreased in zinc-deficient cells during the G1 phase of cell cycle in comparison with zinc-sufficient cells. Nuclear run-on experiments and actinomycin-D studies showed that the transcription of TK-mRNA was affected adversely by zinc deficiency. Cell cycle studies showed that more zinc-deficient cells remained in S phase and did not undergo mitosis in comparison with zinc-sufficient cells. In conclusion, our data show that zinc is a T cell-specific growth factor and that a decreased gene expression of DNA-synthesizing enzyme TK in zinc-deficient HUT-78 cells in G1 phase affected adversely the DNA synthesis in S phase and delayed cell cycle.

In situ radiation sensitivity of recombinant human granulocyte colony-stimulating factor-recruited murine circulating blood and bone marrow progenitors (colony-forming unit [CFU]-granulocyte-macrophage and CFU-megakaryocyte): evidence for possible biologic differences between mobilized blood and bone marrow.

Increasing evidence especially stemming from peripheral blood progenitor transplantation studies points to a possible biologic difference between mobilized blood and bone marrow progenitor cells. The objective of this study was to compare the in situ radiation sensitivity of recombinant human granulocyte colony-stimulating factor (rhG-CSF)-recruited circulating granulopoietic (blood colony-forming unit-granulocyte-macrophage [CFU-GM(blood)]) and megakaryocytopoietic (blood CFU-megakaryocyte [CFU-Meg(blood)]) progenitors, with the nonmobilized fraction remaining in the bone marrow (CFU-GM(femur) and CFU-Meg(femur)). Splenectomized male B6D2F1 mice received 50 micrograms/kg/d rhG-CSF daily for 8 days to induce high levels of circulating progenitors, followed by either total body X-irradiation (TBI) or X-irradiation of the chest (CI) with 62.5, 125, 250, or 500 cGy. Progenitor cells were assayed 24 hours after irradiation. Circulating CFU-GM and CFU-Meg in the blood were decreased in a dose-dependent fashion by both TBI and CI, with TBI causing greater damage than CI. Average D0 values for TBI were 53 cGy for CFU-GM(blood) and 40 cGy for CFU-Meg(blood) D0 values for CI were 90 cGy for CFU-GM(blood) and 140 cGy for CFU-Meg(blood). As seen for blood progenitor cells, TBI caused a dose-dependent decrease of both CFU-GM(femur) (D0, 136 cGy) and CFU-Meg(femur) (D0, 148 cGy). However, radiation-induced bone marrow progenitor cell kill was significantly lower when compared with blood progenitors. Despite the fact that circulating blood elements only received a fraction of the total dose administered as Cl, the extent of blood progenitor kill caused by Cl was higher than the effects of identical TBI doses on bone marrow CFU. The results of this study showed that rhG-CSF-recruited CFU-Meg(blood) and CFU-GM(blood) were considerably more radiosensitive than femoral progenitors, thereby providing novel evidence for a biologic difference between rhG-CSF-recruited peripheral blood progenitors and the nonrecruited bone marrow CFU.

Prostate cancer: flow cytometric methods for detection of bone marrow micrometastases.

Up to 60% of patients with clinically localized prostate cancer will relapse despite potentially curative local treatment. Current staging tests have been limited in adequately identifying individual patients who are at a high risk for future relapse. Detection of bone marrow micrometastases may identify individuals destined to develop clinically detectable systemic metastases. Although immunohistochemistry and molecular approaches are being investigated, the most ideal test(s) are yet to be determined. In this report we describe methods for specific detection and isolation of prostate cancer micrometastases by multi-parameter rare event flow cytometric analysis. A model was developed and validated using three human prostate cancer cell lines, healthy donor marrow, dual marker labeling for cytokeratin (epithelial-specific marker) and CD45 (bone marrow-specific marker). The detection sensitivity of this model was at the level of one prostate cancer cell in 100,000 nucleated bone marrow cells. As a part of an ongoing clinical study, bone marrow aspirates from 15 patients with newly diagnosed prostate cancer have been analyzed. Six patients were found to have cytokeratin positive/CD45 negative cells in their bone marrow aspirates. We conclude that flow cytometric rare event analysis provides a sensitive and specific assay for detection of bone marrow micrometastases in patients with clinically localized prostate cancer.

Local tumor irradiation augments the response to IL-2 therapy in a murine renal adenocarcinoma.

We have previously demonstrated that local tumor irradiation effectively enhanced the therapeutic effect of IL-2 therapy on pulmonary metastases from a murine renal adenocarcinoma, Renca. Irradiation with 300 rad to the left lung only, followed by systemic IL-2 therapy, results in increased tumor reduction in both lungs, suggesting that radiation enhances the systemic effect of immunotherapy. In this study, we show that irradiation of the tumor-bearing organ is essential for the combined effect of both modalities. This effect is radiation dose-dependent as increases in the radiation dosage result in greater tumor reduction in the irradiated field as well as systemically in nonirradiated fields when combined with immunotherapy. We find that irradiation has a direct inhibitory effect on Renca cell growth in vitro. Irradiation of Renca cells also causes an upregulation in H-2Kd class I MHC antigen detectable at 300 rad and more pronounced with 800 rad. By in vivo selective depletion of lymphocyte subsets, we demonstrate the involvement of Lyt-2+ and L3T4+ T cell subsets and AsGM1+ cells, including NK cells, in the antitumor effect mediated by tumor irradiation and IL-2 therapy. Immunohistochemistry studies, performed on lung sections, showed a significant infiltration of CD3+ T cells and macrophages in the tumor nodules following treatment with tumor irradiation and IL-2 therapy. Our studies indicate that the mechanism of interaction between tumor irradiation and immunotherapy may include radiation-induced alterations in the tumor growth and antigenicity which may enhance or trigger an anti-tumor response elicited by IL-2 and mediated by T cells, AsGM1+ cells, and macrophages.

Growth and major histocompatibility antigen expression regulation by IL-4, interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha) on human renal cell carcinoma.

We have recently shown that human renal cell carcinoma (RCC) tumour lines express high-affinity IL-4 receptors. Binding of IL-4 to RCC cells induced a growth inhibition in the range of 20-68%. To enhance the growth inhibitory effect of IL-4, we have tested the effects of two additional cytokines capable of directly affecting tumour cell growth. IFN-gamma caused a significant inhibition of RCC tumour cell growth (up to 70%) in a dose-dependent manner, whereas the effect of TNF-alpha was more limited (0-20% inhibition). The addition of IL-4 to IFN-gamma on RCC cells sensitive to IL-4 induced a greater inhibition of cell growth than that seen with each cytokine alone. IL-4 and IFN-gamma rendered RCC cells more responsive to the inhibitory effect mediated by TNF-alpha. The combination of TNF-alpha with IL-4 and IFN-gamma induced an optimal growth inhibition (up to 90-98%) of RCC cells. In addition to a direct anti-proliferative effect, we have demonstrated that these cytokines can also enhance the expression of MHC antigens on the surface of RCC tumour cell lines which may render the cells more immunogenic. All RCC lines tested expressed class I antigens, but not class II antigens. IFN-gamma induced class II expression and up-regulated the expression of class I antigens on RCC cells. Class II antigen expression was detectable following 48 h incubation, and greater after 72 h with IFN-gamma. IL-4 minimally affected class I expression, whereas TNF-alpha up-regulated class I antigen expression. IL-4 or TNF-alpha did not induce class II expression. The combination of the three cytokines slightly augmented the up-regulation of class I and class II antigens observed with IFN-gamma alone. These observations confirm the direct interaction of IL-4, IFN-gamma and TNF-alpha with RCC tumour cells, both at the level of growth regulation and MHC antigen expression, and suggest a therapeutic potential of the combination of the three cytokines for renal cell carcinoma.

Flow cytometric DNA analysis of fresh prostatic resections. Correlation with conventional prognostic parameters in patients with prostate cancer.

BACKGROUND: DNA ploidy analysis has been investigated as a prognostic indicator in prostate cancer. Most of the data is derived from retrospective studies using paraffin-embedded tissue. This method has drawbacks related to the quality of DNA histograms and uncontrolled data collection. METHODS: DNA ploidy analysis of freshly resected prostatic tissue was prospectively compared with conventional prognostic variables in 97 men treated with radical prostatectomy for localized prostate cancer. RESULTS: Regarding the patients, 31.9% were African American and 66% had pathologic Stages C or D1 disease. Only 9.6% of patients with Stages A2 and B had a prostate-specific antigen (PSA) value greater than 10 ng/ml, whereas 97% of patients with PSA values greater than 20 ng/ml had pathologic Stages C and D1. PSA levels correlated with Gleason score (P = < 0.05); 51% and 100% of patients with Gleason score 5-7 and 8-10, respectively, had PSA values greater than 10 ng/ml. Twenty-two patients (23%) had DNA aneuploid tumors. Comparisons of mechanical to enzymatic cell suspensions indicated that DNA aneuploidy was better preserved in mechanical cell preparations. DNA ploidy correlated with pathologic stage (P = < 0.05) and Gleason score (P = < 0.05). Fifteen of 79 patients (18.9%) with Gleason score 5-7 had DNA aneuploid tumors versus 71.4% of patients with Gleason score 8-10. PSA groups correlated with ploidy status (P = 0.01). Although the majority of patients (19 of 22) with DNA aneuploid tumors had elevated preoperative PSA levels, none had a PSA value greater than 50 ng/ml. CONCLUSIONS: DNA ploidy analysis correlated with established prognostic indicators in prostate cancer; however, its independent correlation with natural history and treatment outcome must be established for it to have an effect on therapeutic decisions.

Development and optimization of tissue preparative methodology for DNA content analysis of soft tissue neoplasms.

Data regarding DNA content parameters in soft tissue sarcoma is limited. Development and optimization of tissue specific preparative techniques for DNA flow cytometry was undertaken prior to routine DNA content analysis of soft tissue neoplasms; 154 soft tissue tumors were studied. Dissociation dependent differences in cellular yields, viabilities, maintenance of DNA aneuploid populations, coefficients of variation, and DNA index supported the need for these developmental studies. Fifty-six of eighty-nine patients had DNA aneuploid soft tissue sarcomas. A relationship between DNA aneuploidy and grade was seen in this series with 38% with low grade, 59% with moderate grade, and 69% with high grade tumors demonstrating DNA aneuploid populations (P < 0.005). The mean S-phase fraction for DNA diploid and aneuploid sarcomas was 7.2% and 13.3%, respectively (P < 0.001). When classified by histologic grade of the primary tumor, a greater percentage of metastatic lesions were DNA aneuploid (4 of 7 grade 2 lesions, and 15 of 16 grade 3 lesions). Decreases in cellular yields and rate of DNA aneuploidy were observed in a subgroup of patients with localized high grade sarcoma treated preoperatively, as compared to patients treated with initial surgery. Prospective correlation of DNA content parameters to prognosis and response to cytotoxic therapy are now possible and are ongoing.

Non-specific binding of normal human IgG, including F(ab')2 and Fc fragments, to embryonic rat brain neurons and human cortex synaptosomes.

Binding of normal human IgG to embryonic rat brain neurons was quantitated by flow cytometry. IgG binding was linear between 0.05 and 1.5 mg/ml; slight binding was detectable even at normal cerebrospinal fluid concentrations. Similar binding curves were obtained for purified Fc and F(ab')2 fragments from normal human IgG. Normal human IgG also bound to synaptosomes (resealed nerve terminals) from human cerebral cortex. However, competition assays utilizing 125I-IgG showed no evidence for specific binding. This study indicates that the specificity of putative anti-neuronal antibodies should be confirmed by competition assays as for other receptor-ligand binding.

A human B-cell lymphoma line with a de novo multidrug resistance phenotype.

A human diffuse large cell lymphoma line (WSU-DLCL) expressing multidrug resistance (MDR) was established from a patient with primary chemotherapy-resistant disease. This cell line has the same phenotypic features as malignant cells from the patient. The established cell line has features of a mature B-cell neoplasm with no evidence for commitment to other lineages. WSU-DLCL grows in suspension forming relatively large clumps of cells with a doubling time of 20 hours. By light microscopic examination, the cells are very large with primitive lymphoid features, have a large amount of cytoplasm containing numerous vacuoles and an irregular outline. Immunophenotypic characterization by monoclonal antibodies and flow cytometric analysis showed a monoclonal IgM kappa B-cell phenotype with high expression of the multidrug-resistant P-glycoprotein compared with either normal peripheral blood lymphocytes or cells of the REH cell line. The cells were negative for T-cell and myeloid/monocyte antigens as well as Epstein-Barr virus nuclear antigen (EBNA). In addition, the cell line expressed high levels of MDR RNA. DNA histogram generated by flow cytometry indicated a DNA index of 1.83. Cytogenetic analysis confirmed hypertriploidy and showed complex chromosomal abnormalities including 14q+. This cell line should be a valuable tool to study the role of the MDR gene in the primary resistance of lymphomas to chemotherapy and to facilitate therapeutic investigations.

Analysis of distribution of tumor- and preneoplasia-infiltrating lymphocytes using simultaneous Hoechst 33342 labeling and immunophenotyping.

Hoechst 33342 in vivo staining was combined with immunofluorescent staining of cell surface antigens to quantify the distribution, relative to blood supply, of lymphocytes in a preneoplastic mammary lesion, the murine C4 hyperplastic alveolar nodule (HAN), and the C4 adenocarcinoma which develops from C4 HAN. The vascular supply to lymphocytes expressing Thy 1.2, L3T4, Ly2, and ASGM1 cell surface antigens was evaluated in both tissues. The distribution of ASGM1+ cells, which include natural killer cells, differed between the two tissues, being significantly increased in the 20% brightest Hoechst-stained lymphocyte fraction in HAN but not in C4 tumor. Distribution of T lymphocytes did not differ between the two tissues. The combination of in vivo Hoechst 33342 with in vitro immunofluorescence provides a simple method to evaluate the distribution with regard to blood supply of lymphocyte subsets in solid tumors and preneoplastic lesions.

Modulation of c-myc oncogene expression by phorbol ester and interferon-gamma: appraisal by flow cytometry.

A flow cytometric assay was developed to examine the expression of the cellular myc oncogene in relation to cell cycle in individual cells. C-myc-oncoprotein was detected by indirect immunofluorescence using a purified sheep polyclonal antibody, anti-human-myc. Specific binding of anti-human-myc was measured by flow cytometry. C-myc oncoprotein was detected in 90% of HL-60 and 75% of Daudi cells; human hematopoietic cell lines known to express high levels of c-myc oncogene. However, c-myc protein could not be detected in the REH cell line, normal human peripheral lymphocytes or thymocytes. Nuclear DNA content was measured simultaneously using propidium iodide staining. There was an equal level of c-myc protein in G0/G1, S and G2/M phases. The extent and kinetics of c-myc oncoprotein induction have been determined following phorbol ester, 12-O tetradecanoylphorbol 13 acetate (TPA) and interferon-gamma (IFN-gamma) exposure of both HL-60 and Daudi cells. TPA produced a gradual reduction in the level of c-myc protein and arrested the cells in G0/G1 phase in HL-60 cells. However, TPA failed to reduce c-myc protein or to change cell cycle distribution in Daudi cells. Interestingly, c-myc protein levels were stimulated by exposure of both HL-60 and Daudi cells to IFN-gamma. The results indicate that flow cytometric assay of oncogene expression is feasible, fast and requires relatively few cells. It also allows for the direct correlation of modulation of oncogene expression with cell kinetics.

Cell cycle distribution defect in PHA-stimulated T lymphocytes of sickle cell disease patients.

T lymphocytes from normal human controls and sickle cell disease (SCD) patients were isolated from peripheral blood and cultured for 72 hours following addition of phytohemagglutinin. The ratio for the fraction of cells in DNA synthesis (S phase) over the fraction in G2 phase (S/G2) was significantly higher in SCD patients in comparison to the controls (mean +/- SD) (4.01 +/- 0.78 vs. 2.78 +/- 0.76, P less than 0.02). Following in vivo zinc supplementation to two subjects, the S/G2 ratio was normalized. We conclude that the distribution of T lymphocytes in cell cycle is altered in SCD patients and that this effect may be zinc-dependent.