Automatic feedback control by image processing for mixing solutions in a microfluidic device.

Two approaches of an automatic control were studied through mathematical fitting obtained from color mixing saturation curves in polydimethylsiloxane microfluidic devices: The integrative control with variable integral gain and integrative control with constant integral gain. The aim of this work is to control the color percentage decrement when dye is injected. The results indicate that microfluidic systems are very sensitive to changes in flow and the control variable needs to change slowly; that is, it must be small (at least 100 times less than the theoretically calculated values). The control and stabilization of the microfluidic system were achieved for dye percentages above 60%. The controlling color percentage could provide a tool to regulate other parameters' concentration applied to cell culture and alkalinity control (pH) of solutions in microfluidic devices.

Purification of human interleukin 2 to apparent homogeneity and its molecular heterogeneity.

Interleukin 2 (IL-2), produced with and without co-stimulation by the Burkitt's lymphoma line Daudi, was purified 37,000-fold to apparent homogeneity from lymphocyte conditioned medium by (NH4)2SO4 precipitation, DEAE-cellulose ion-exchange chromatography, gel filtration, and chromatography on blue agarose and on Procion-red agarose. The purified IL-2 showed a 10(6) U/mg protein sp act. IL-2 produced in the absence of Daudi cells had a mol wt of 26,000 as measured by gel filtration and an isoelectric point of 6.7. This IL-2 showed a 16,000 and 17,000 mol wt in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). IL-2, produced in the presence of Daudi cells (10(6)/ml), showed a mol wt of approximately 14,000, as measured by both gel filtration and SDS-PAGE, and an isoelectric point of 8.1. The purified IL-2 lacked detectable interferon (alpha and gamma), granulocyte-macrophage colony-stimulating factor, B cell growth factor, T cell-replacing factor, and thymocyte-differentiating activity and was free of any contaminating proteins as judged by silver staining in SDS-PAGE. All three molecular forms of IL-2 were biologically active at concentrations of 10(-11) - 10(-10) M, supporting the growth of human and murine cytotoxic T cell lines.

Interleukin 2 regulates the expression of Tac antigen on peripheral blood T lymphocytes.

We investigated the effect of OKT3 antibody and interleukin 2 (IL-2) on Tac antigen expression and the proliferation of human peripheral blood mononuclear leukocytes. OKT3 monoclonal antibody at low, nonmitogenic concentrations (25 pg/ml) or IL-2 alone at optimal concentrations (20 U/ml) did not induce IL-2 receptor expression, as measured by Tac antibody or by T cell proliferation. However, costimulation with these concentrations of OKT3 antibody and IL-2 led to Tac antigen expression and T cell proliferation. These data suggest that the T cells are activated in two steps: OKT3 antibody at 25 pg/ml does not induce Tac antigen expression, but preactivates T cells to become responsive to IL-2. The addition of exogenous IL-2 then leads to expression of the IL-2 receptor, as recognized by Tac antibody, and to subsequent proliferation.

Biological activities of a human pluripotent hemopoietic colony stimulating factor on normal and leukemic cells.

We studied the biological effects of pluripoietin, a human pluripotent hemopoietic colony-stimulating factor (CSF) purified from the 5637 bladder carcinoma cell line. We found that this human CSF appears to be a unique hemopoietic growth factor, differing from interleukin 3 (IL-3) by virtue of its leukemia differentiating activity in mouse and man, and from mouse granulocyte CSF, which does have differentiation-inducing activity, but lacks pluripoietic activity. In addition, differences from IL-3 were observed in cross-species activity on normal and leukemic cells.

Synthesis of granulocyte colony-stimulating factor and its requirement for terminal divisions in chronic myelogenous leukemia.

In this paper we demonstrate that maturing neoplastic cells from patients with chronic myelogenous leukemia (CML) constitutively produce G-CSF and are also receptive for this molecule. G-CSF functions as an autocrine growth factor in stable phase CML, and thus is responsible for divisions of maturing leukemic cells leading to an expansion of the compartment of mature cells. This observation is well in line with in vivo features of CML in stable phase, i.e., the hyperplasia of the mature granulocyte compartment. In acute blastic phase of CML expression of the G-CSF gene seems to be less common and not related to autonomous blast growth.

Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells.

Experiments were conducted to isolate and characterize the gene and gene product of a human hematopoietic colony-stimulating factor with pluripotent biological activities. This factor has the ability to induce differentiation of a murine myelomonocytic leukemia cell line WEHI-3B(D+) and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). A complementary DNA copy of the gene encoding a pluripotent human granulocyte colony-stimulating factor (hG-CSF) was cloned and expressed in Escherichia coli. The recombinant form of hG-CSF is capable of supporting neutrophil proliferation in a CFU-GM assay. In addition, recombinant hG-CSF can support early erythroid colonies and mixed colony formation. Competitive binding studies done with 125I-labeled hG-CSF and cell samples from two patients with newly diagnosed human leukemias as well as WEHI-3B(D+) cells showed that one of the human leukemias (ANLL, classified as M4) and the WEHI-3B(D+) cells have receptors for hG-CSF. Furthermore, the murine WEHI-3B(D+) cells and human leukemic cells classified as M2, M3, and M4 were induced by recombinant hG-CSF to undergo terminal differentiation to macrophages and granulocytes. The secreted form of the protein produced by the bladder carcinoma cell line 5637 was found to be O-glycosylated and to have a molecular weight of 19,600.

Interleukin 1 stimulates T lymphocytes to produce granulocyte-monocyte colony-stimulating factor.

T lymphocytes are thought to cooperatively interact with monocytes to produce colony-stimulating factors (CSF). However, little is known about monocyte-mediated signals leading to CSF-secretion by T lymphocytes, although soluble monocyte products have been implicated. We have employed monoclonal antibody anti-T3B covalently coupled to CnBr-activated Sepharose 4B beads, to show that multimeric ligation of T cell antigen receptor leads to T cell receptiveness to interleukin 1 (IL-1), as indicated by T cell production of CSF, which induces growth of myeloid progenitor cells into neutrophil, eosinophil, and monocyte colonies. To investigate the molecular basis of these findings, total RNA was extracted from T3B Sepharose-primed and IL-1-stimulated T lymphocytes and probed for granulocyte-monocyte-CSF (GM-CSF), granulocyte-CSF (G-CSF), and monocyte-CSF (M-CSF) mRNA. GM-CSF, but not G-CSF or M-CSF, messages were detected. Nuclear "run on" assays revealed that IL-1 action is effective primarily at the level of GM-CSF gene transcription. These results suggest a previously unrecognized role of IL-1 in the regulation of GM-CSF secretion by T cells.

Granulocyte-macrophage colony-stimulating factor induces cytokine secretion by human polymorphonuclear leukocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effects of GM-CSF on polymorphonuclear leukocytes (PMN) more extensively. Using Northern blot analysis, we show that PMN are able to accumulate mRNAs for different cytokines, including tumor necrosis factor-alpha (TNF-alpha); G-CSF, and M-CSF, all of which are involved in inflammation and hematopoiesis. Biological assays and immunoassays demonstrate that PMN translate these mRNAs, except TNF-alpha, into secretory proteins. However, the expression of these cytokines is dependent on stimulation by exogenous signals, preferentially provided by the T cell-derived lymphokine GM-CSF. Stimulation of hematopoiesis and amplification of defense mechanisms after T cell activation thus might involve not only monocytes but also PMN, a cell type previously believed to be biosynthetically inactive.

Mithramycin selectively inhibits collagen-alpha 1(I) gene expression in human fibroblast.

The products of the collagen-alpha 1(I) and -alpha 2(I) genes form the triple helical molecule collagen type I, which constitutes the major ECM protein in tissue fibrosis. The collagen-alpha 1(I) gene is mainly transcriptionally regulated, and its promoter activity depends on the interaction of the transcription factors NF-I and Sp1 with a tandem repeat of evolutionary conserved NF-I/Sp1 switch elements. An increased affinity of Sp1 to these elements has been observed in experimental liver fibrosis. Here, we demonstrate that the DNA binding drug mithramycin displays a high affinity binding to the GC-rich elements in the collagen-alpha 1(I) promoter as measured by DNAse I protection and gel retardation assays. Mithramycin interferes with Sp1 but not with NF-I binding to these sites. At a concentration of 100 nM, mithramycin efficiently reduces basal and TGF-beta-stimulated alpha 1(I) gene expression in human primary fibroblasts. The transcriptional activity and mRNA steady state levels of other genes, including the collagenase gene, as well as the growth rate of fibroblasts remained unchanged on exposure to this drug. Taken together, our results indicate that the transcriptional activity of the type I collagen gene highly depends on its GC-rich regulatory elements, and further, that these elements can be differentially blocked, thereby changing the balance between ECM structural and degrading gene activities in human fibroblasts.

Participation of the cytokines interleukin 6, tumor necrosis factor-alpha, and interleukin 1-beta secreted by acute myelogenous leukemia blasts in autocrine and paracrine leukemia growth control.

Autonomous in vitro growth of myeloid leukemic colony-forming cells may in part result from autocrine production of colony-stimulating factors (CSF). Some acute myeloid leukemia (AML) samples, however, fail to synthesize CSF despite growing autonomously in agar, and are therefore believed to bypass CSF requirements. Cytokines such as IL-6, tumor necrosis factor (TNF)-alpha, and IL-1, products of cells of the myeloid lineage, are known to be involved in growth control of myeloid progenitor cells. Since these molecules may also contribute to autocrine and paracrine growth regulation of myeloid leukemias, we screened a series of AML for cytokine production. In addition, possible roles of IL-6, TNF-alpha, and IL-1 in growth control of AML were investigated in vitro. We show that a substantial proportion of AML cells produce IL-6, TNF-alpha, and IL-1-beta and use these mediators to stimulate their growth by disparate mechanisms: IL-6 acts as a costimulator to enhance CSF-induced clonogenicity of AML blasts. TNF-alpha induces CSF production by endothelial cells and may therefore provide a paracrine loop to support leukemia growth.

Production of and in vitro response to interleukin 2 in the acquired immunodeficiency syndrome.

To test the hypothesis that deficient interleukin 2 (IL-2) secretion may underlie the impaired capacity of T cells from patients with Acquired Immunodeficiency Syndrome (AIDS) and the AIDS-related complex (ARC) to generate the macrophage-activating lymphokine, gamma interferon (IFN-gamma), we used five specific microbial antigens to examine IL-2 production. Mononuclear cells from only one of 32 (3%) AIDS patients secreted normal levels of IL-2, and 21 (66%) failed to produce any detectable IL-2. For 36 ARC patients, IL-2 generation was normal in nine (25%) and absent in 11 (31%). Given these results, recombinant (r) IL-2 was tested for its capacity to stimulate or enhance IFN-gamma production. rIL-2 (10 U/ml) alone stimulated cells from controls, ARC, and AIDS patients to secrete 93 +/- 25, 99 +/- 33, and 7 +/- 3 U/ml of IFN-gamma, respectively. rIL 2 (10 U/ml) plus antigen induced no change in mean IFN-gamma levels for controls, a 4.4-fold increase for 17 AIDS patients (16 +/- 16 vs. 71 +/- 21 U/ml), and a 7.2-fold increase (18 +/- 5 vs. 130 +/- 27 U/ml) for 19 ARC patients with abnormal IFN-gamma generation to antigen alone. Individual responses indicated that six of the 17 (35%) AIDS patients with opportunistic infections and 12 of the 19 (63%) with ARC were apparent responders to 10-100 U/ml of rIL-2. These results (a) document profound impairment in antigen-induced IL-2 secretion by AIDS and ARC T cells, (b) indicate that, in vitro, mononuclear cells from certain patients can respond to rIL-2 with enhanced IFN-gamma production, and thus (c) suggest that in selected patients rIL-2 might have a potentially beneficial therapeutic (AIDS) or prophylactic (ARC) effect against opportunistic infections.

Immunostimulatory cytokines in somatic cells and gene therapy of cancer.

The use of immunostimulatory cytokines has become an increasingly promising approach in cancer immunotherapy. The major goal is the activation of tumour-specific T lymphocytes capable of rejecting tumour cells from patients with low tumour burden or to protect patients from a recurrence of the disease. Strategies that provide high levels of immunostimulatory cytokines locally at the site of antigen have demonstrated pre-clinical and occasional clinical efficacy. Animal models using poorly immunogenic tumours revealed that tumour cells genetically engineered to produce cytokines like IL-2, IL-4, IL-7, IL-12, IFNs, GM-CSF or TNF-alpha were found to be effective in eradicating disseminated tumours. Experimental data obtained from these different animal models are reviewed here to provide an overview of this rapidly evolving field. The data obtained so far from clinical trials involving cytokine gene-modified cells have provided important information regarding the feasibility, safety, immunological effects and occasional clinical responses.

Diagnostic and prognostic significance of the CFU-c assay in acute nonlymphoblastic leukemia.

A simplified system for classification of aggregate incidence and growth pattern in the CFU-c (colony-forming units in culture) assay, allowing simple and reproducible interpretation of test results, was developed and applied to 552 bone marrow samples from 202 patients with acute leukemia. Ninety-six consecutive patients with acute nonlymphoblastic leukemia were studied at diagnosis. The microcluster growth pattern ("acute myeloid leukemia-type") found in 57% of the patients was significantly associated with higher remission induction rates on both protocols (p = 0.004). No relationship between growth pattern at diagnosis and remission duration was observed. The acute myeloid leukemia-type growth pattern was found to be more frequent in leukemias exhibiting morphological evidence for partial myeloid or monocytic differentiation. The favorable prognostic significance of Auer rods previously described was recognized in two CFU-c growth pattern categories. Of patients exhibiting an acute myeloid leukemia-growth pattern and Auer rods, 89% obtained complete remissions compared to 38% in the Auer rod-negative group showing other growth pattern variants. The CFU-c assays performed during complete remission on 354 samples of 48 patients with acute nonlymphoblastic leukemia and, as a control, on 85 samples of 43 patients with acute lymphoblastic leukemia revealed marked spontaneous as well as chemotherapy-related fluctuations of aggregate incidence and growth pattern. These and similar observations obtained with other assay systems are probably of major pathophysiological significance but preclude clinical application of the CFU-c assay to the monitoring of remission status in patients with acute nonlymphoblastic leukemia.

Expression of cytokine genes, cytokine receptor genes, and transcription factors in cultured Hodgkin and Reed-Sternberg cells.

In the present study, we show by Northern blot analysis and enzyme linked immunosorbent assay that the Hodgkin's disease (HD)-derived cell lines HDLM-2 and KM-H2 express a variety of cytokine genes either constitutively or upon induction with phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Cytokine genes expressed by HD-derived lines include granulocyte-macrophage colony-stimulating factor (CSF), macrophage-CSF, interleukin (IL)-1-alpha, IL-3, IL-5, IL-6, IL-8, leukemia inhibitory factor, tumor necrosis factor-alpha, tumor necrosis factor-beta, and transforming growth factor-beta, while transcripts and the corresponding proteins for granulocyte-CSF, IL-1-beta, IL-2, IL-4, IL-7, IL-10, and the JE/macrophage chemoattractant and activating factor gene were not detectable in cytoplasmic RNA and culture supernatants obtained from both lines. In addition, IL-2 receptor (R) p55 and macrophage-CSF R (c-fms) genes were expressed by both lines. HDLM-2, but not KM-H2 cells, exhibited the IL-6 R p80 and the IL-2 R p75 chain. Analysis of nuclear proteins that bind to oligonucleotides containing the consensus sequences of the transcription factors activation protein 1, nuclear factor (NF) kappa B, and NFAT 1 revealed a pattern for HD lines resembling that of activated T-cells: HDLM-2 and KM-H2 cells constitutively expressed NF binding to the NF of activated T-cells (type 1), previously described to be T-cell specific. In addition, NF kappa B-binding proteins obtained from both lines showed, in electrophoretic mobility shift assays, the same migration pattern as T-cell-derived proteins but differed from monocyte- and B-cell-derived proteins. UV cross-linking experiments confirmed that NF kappa B-binding proteins of M(r) 85,000, 75,000, and 50,000/55,000 were detectable in nuclear extracts obtained from T-cells and both HD lines, while monocytes and B-cells displayed the M(r) 50,000/55,000 and 75,000 NF kappa B complex only. Both HD lines also constitutively expressed transcripts for c-fos and c-jun, which are involved in heterodimeric formation of the transcription factor activation protein 1, as well as for the NF kappa B/KBF1 gene.

Lysis of spontaneous murine breast tumors by human interleukin 2-stimulated syngeneic T-lymphocytes.

In vitro and in vivo cytotoxic assay systems show that primary explants of spontaneous mammary tumors from CD8F1 mice are susceptible to lysis by interleukin 2-stimulated, syngeneic T-lymphocytes.

Synergy of interleukin 3 and tumor necrosis factor alpha in stimulating clonal growth of acute myelogenous leukemia blasts is the result of induction of secondary hematopoietic cytokines by tumor necrosis factor alpha.

Colony growth of leukemic colony-forming units (L-CFU) obtained from patients with primary acute myelogenous leukemia stimulated with recombinant human interleukin 3 (rh IL-3) is significantly potentiated when recombinant human tumor necrosis factor alpha (rh TNF-alpha) is present in cultures. The costimulatory activity of tumor necrosis factor (TNF) alpha is dose dependent and maximum at TNF-alpha concentrations of 10 ng/ml. At high density, L-CFU proliferatively respond to TNF-alpha stimulation in the absence of exogenous rh IL-3. Studies of the mechanism of action of rh TNF-alpha on acute myelogenous leukemia L-CFU growth suggest that TNF-alpha acts by inducing release of growth stimulatory hematopoietic cytokines by the leukemic cells themselves, including IL-1 alpha, IL-1 beta, Granulocyte-macrophage colony-stimulating factor (CSF), granulocyte CSF, and IL-6. Treatment of L-CFU cultures, with neutralizing antibodies to IL-1 alpha, IL-1 beta, granulocyte-macrophage CSF, granulocyte CSF, and IL-6 to eliminate the endogenous source of these factors is associated with significant inhibition of the synergistic interplay of TNF-alpha and IL-3.

Pharmacokinetics of recombinant interleukin 2 in humans.

This report summarizes the pharmacokinetics in humans of recombinant interleukin 2 (IL-2) given as an i.v. bolus, i.v. or i.p. infusion, and i.m. or s.c. injection. Immediately after an i.v. bolus the serum IL-2 level equals the dose divided by the plasma volume, in a typical human 650 units/ml for a dose of 10(6) units/m2. The level initially decreases with a half-life of 12.9 min, followed by a slower phase with a half-life of 85 min out to 4 h after the bolus. The median steady state level during an i.v. infusion of 10(6) units/m2 over 6 h is 41 units/ml. A clearance rate of approximately 120 ml/min is obtained from either the i.v. bolus or infusion data and is consistent with the renal filtration being the major route of clearance. Serum levels remain fairly constant for about 8 h after s.c. or i.m. injection but are approximately 2% of the level seen immediately after an i.v. bolus. The area under the time-concentration curve suggests that about 30% of the IL-2 activity is transported from the site of an i.m. injection to the blood. After i.p. infusion IL-2 is only slowly transported to the blood. The median serum IL-2 levels are 430-fold lower than levels in the i.p. fluid and decrease with a median half-life of 6.3 h.

Phase I and II clinical and pharmacological study of 4-demethoxydaunorubicin (idarubicin) in adult patients with acute leukemia.

Fifty-two adults treated previously with either acute leukemia (43 patients) or blastic-phase chronic myelogenous leukemia (nine patients) received 4-demethoxydaunorubicin (20 to 45 mg/sq m) i.v. over 2 to 3 days. Three of the ten patients with acute lymphocytic leukemia achieved a complete remission (CR) lasting 5 to 7 weeks. Five of the 28 patients with acute nonlymphocytic leukemia achieved a CR lasting 5 to 80 weeks. All remissions were induced with one course of treatment with a median time to CR of 28 days (range, 22 to 40 days). None of the patients with blastic chronic myelogenous leukemia or secondary leukemia achieved a CR. The drug was well tolerated; mucositis (36%), nausea and vomiting (35%), and hepatic dysfunction (26%) were the most common side effects. Pharmacokinetic observations on five patients demonstrated multiphasic clearance of 4-demethoxydaunorubicin and extensive formation and prolonged retention of 4-demethoxy-13-hydroxydaunorubicin; that metabolite accumulated in plasma on repeated daily dosing. 4-Demethoxydaunorubicin has sufficient antileukemic activity in both acute lymphocytic leukemia and acute nonlymphocytic leukemia to warrant a prospective comparison, in combination regimens, against the conventional anthracyclines, daunorubicin and/or doxorubicin.

Prevalence of N-ras mutations in children with myelodysplastic syndromes and acute myeloid leukemia.

The ras proto-oncogene family encodes a group of 21 kDa nucleotide-binding proteins. Activating mutations of ras genes are associated with certain types of malignancies, indicating that they are related in some way to the malignant process. We have examined bone marrow cells from nine children with myelodysplastic syndromes (MDS) and 35 with acute myeloid leukemia (AML) for activating point mutations of ras genes by in vitro amplification using polymerase chain reaction (PCR), oligonucleotide hybridization and sequencing of PCR products. We found N-ras mutations in cells from 3 of 9 children (33%) with MDS and only 2 of 35 children with AML (6%; 95% confidence interval is 0.7-19%). All mutations the second nucleotide of codon 12 or the first nucleotide of codon 61 of N-ras. There was no apparent correlation with clinical or laboratory characteristics, including karyotype; however, an association of N-ras activation with the most aggressive type of MDS was noted. Among the patients with MDS, 2 of 6 with monosomy 7 had N-ras mutations; however, three children with monosomy 7 which presented with AML lacked ras mutations. One patient was studied at time of diagnosis of MDS and again after progression to AML. At the preleukemic stage of disease, an N-ras mutation was identified; however, after development of AML this mutation was not present in the leukemic clone. In conclusion, these data show that ras mutations, while not necessary for leukemic transformation, may be important for the initiation of preleukemias evolving into overt AML.

Retrospective analysis of ras gene activation in myeloid leukemic cells.

Ras genes are activated by point mutations at critical sites of their coding regions. Activated N-ras genes with transforming ability have been detected in patients with myelodysplastic syndromes (MDS), acute myelogenous leukemia (AML) and in human myeloid cell lines. We used polymerase chain reaction (PCR), differential oligonucleotide hybridization and direct DNA sequencing to retrospectively analyze the N-ras gene of blast cells from the same patient (a) at time of diagnosis of MDS, (b) after the patient had developed AML. Two types of archival tissue samples served as a source of cells. Different passages of the KG-1 myeloid cell line which had been established from leukemic blasts of this patient were also analyzed. We found that native blast cells isolated at either of the two disease stages did not carry an N-ras mutation, and neither did early passage KG-1 cells. However, direct DNA sequencing of PCR-amplified DNA from nude mice transformants induced by DNA from late passage of the KG-1 cell line revealed two linked mutations involving both the second nucleotide of codon 12 and the third nucleotide of codon 15 of N-ras. The nucleotide substitution at codon 15 did not result in an amino acid substitution (silent mutation). The mutations probably occurred during prolonged passaging of the KG-1 cells and might have been overlooked by oligonucleotide hybridization assay.