Human lymphocytic B-leukemia cell line treatment with the bacterial toxin listeriolysin O and rituximab (anti-CD20 antibody): Effects of similar localization of their receptors.

Small B-cell lymphocytic lymphoma/chronic lymphocytic leukemia, which typically affects elderly people, is a group of conditions that are not clinically uniform. It has been suggested that using the combined activity of the monoclonal antibody anti-CD20 (rituximab) and Listeria monocytogenes toxin listeriolysin O (LLO) for this condition could produce an enhanced treatment effect. Here, we tested the effect of the joint activity of rituximab and LLO, which is a cell membrane toxin, in human leukemia cell lines. The human B-leukemia Raji cell line, which expresses CD20, and the T-cell Jurkat cell line, which does not express CD20, for comparison were used in model tests. Cell cytotoxicity of rituximab or LLO and both applied jointly to the cell lines was compared in the presence of human plasma complement. Optimal cytotoxic effects dependent on rituximab or LLO concentration were tested separately. LD50 values were determined and used for optimal application of a mixture of the two factors. The cytotoxic effect on Raji cells of both rituximab and LLO was more than 2.5 times that of LLO alone and 1.5 times that of rituximab alone. At the highest tested concentrations, a mixture of the tested factors had a non-specific cytotoxic effect on the Jurkat cell line, as well. The rituximab and LLO binding sites appear to be in a similar region of the Raji leukemia cell membrane, suggesting an effective interaction of both factors. The joint interaction of these compounds in cell membrane pore formation suggests an explanation for the more effective cytotoxic activity that their combination was observed in this experiment.

Cytotoxicity of bacterial metabolic products, including listeriolysin O, on leukocyte targets.

Bacterial toxins can exhibit anticancer activities. Here we investigated the anticancer effects of the listeriolysin O toxin produced by Listeria monocytogenes. We found that supernatants of Listeria monocytogenes strains (wild type, 1189, and 1190) were cytotoxic to the Jurkat cell line and human peripheral blood mononuclear cells (PBMC) in a concentration-dependent manner. The supernatant of strain 1044, not producing listeriolysin O, was inactive. The supernatants of Listeria strains were also cytotoxic toward B cells of chronic leukemia patients, with no significant differences in activities between strains. We also tested supernatants of Bacillus subtilis strains BR1-90, BR1-S, and BR1-89 producing listeriolysin O. BR1-S and BR1-89 were cytotoxic to PBMC and to Jurkat cells, the latter being more sensitive to the supernatants. BR1-90 was not hemolytic or cytotoxic to PBMC, but was cytotoxic to Jurkat cells in the concentration range of 10-30%, suggesting that listeriolysin O is selectively effective against T cells. Overall, the response of human peripheral blood mononuclear and human leukemia cell lines to bacteria supernatants containing listeriolysin O depended on the bacteria strain, target cell type, and supernatant concentration.

CD4+/CD25+ cells in systemic inflammation in COPD.

The autoimmune reaction is recently suspected to play a role in the pathogenesis of chronic obstructive lung disease (COPD). As COPD is a systemic disease, the elements of an autoimmune response in circulatory system is of interest. It has been shown that regulatory T cells are important in the control of autoimmunity. There are some data on a role of adiponectin in the regulation of immune reactions. The objective of this study was to assess the elements of autoimmune reaction in the peripheral blood (PB) of patients with COPD. Twenty-eight patients with mild/moderate COPD and 20 healthy volunteers were investigated. Flow cytometry method with mixtures of monoclonal antibodies anti: CD14/CD45, CD3/CD19, CD4/CD25/CTLA4 and CD8/CD25 were used. Concentration of adiponectin was measured using ELISA method. We observed significantly lower proportion of CD4+/CD25+ as well as CD4+/CD25+ (high) cells in COPD patients than in healthy controls (15.3 versus 17.8% and 0.79 versus 1.54%, respectively, P < 0.05). The proportion of CTLA4+ cells in CD25+ cells and the mean fluorescence of CTLA4 on CD4+ cells were higher in patients than in healthy controls (10.4 versus 4.7%, P < 0.05, 189% versus 149%, non significant, respectively). We found significantly elevated concentration of adiponectin in patients when compared to healthy subjects (15.4 versus 8.5 µl/ml, P < 0.05). We found that the adiponectin/BMI ratio correlated with the decrease of FEV(1) %. The results of this study support the possible role of CD4/CD25/CTLA4 cells and adiponectin in the systemic inflammation in COPD.

The experimental study of polyelectrolyte coatings suitability for encapsulation of cells.

Living cells encapsulated in polymeric shells are receiving increasing attention because of their possible biotechnological and biomedical applications. The aim of this work is to evaluate how different polyelectrolyte coatings, characterized by different numbers of polyelectrolyte layers and by different polyelectrolyte conformations, affect the viability of encapsulated biological material. We demonstrate the ability to individually encapsulate HL-60 cells as well as rat pancreatic islets within polymeric shells consisting of different PE layers using the layer-by-layer process. Coating of HL-60 cells allows for surviving and functioning of cells for all applied PE as well as for different numbers of layers. The islets encapsulated in applied polyelectrolytes exhibited the lower level of mitochondrial activity as compared to non-encapsulated islets. Nevertheless, encapsulated islets exhibited comparable absorbance values during the whole period of culture. Polyelectrolyte coating seems to be a promising way of allowing capsule void volume minimization in a model of encapsulated biological material for local production of biologically active substances.

Effect of IFN-gamma stimulation on expression of intercellular adhesion molecule-1 (ICAM-1) on alveolar macrophages in patients with non-small cell lung cancer.

An impairment of in vitro cytotoxicity and tumoricidal function of alveolar macrophages (AMs) in patients with lung cancer was reported in a number of studies. The aim of our study was to evaluate the expression of intercellular adhesion molecule-1 (ICAM-1) on AMs after stimulation with interferon-gamma (IFN-gamma) in patients with non-small cell lung cancer (NSCLC). The study was performed in 13 patients with NSCLC, 6 patients with various nonmalignant pulmonary diseases, and 6 healthy volunteers. AMs were isolated from bronchoalveolar lavage fluid (BALF) by adherence and then cultured with or without IFN-gamma for 24 h. The expression of ICAM-1 on AMs was analyzed by flow cytometry. Stimulation with IFN-gamma caused increased expression of ICAM-1 on AMs in all studied groups (p < 0.05). The degree of the increase in ICAM-1 expression on AMs after IFN-gamma stimulation was significantly lower in patients with NSCLC compared with healthy volunteers (p = 0.002) or the other patients (p = 0.022). The results suggest impaired reactivity of ICAM-1 expression on AMs after stimulation with IFN-gamma in patients with NSCLC, which might be involved in functional defects of AMs in patients with NSCLC.

Treatment of Philadelphia leukemia in severe combined immunodeficient mice by combination of cyclophosphamide and bcr/abl antisense oligodeoxynucleotides.

BACKGROUND: Philadelphia cells are human chronic myelogenous leukemia (CML) cells that contain the BCR/ABL oncogene (a fusion of the BCR and ABL genes). Selective eradication of these cells in vitro can be achieved by combined treatment with antisense phosphorothioate oligodeoxynucleotides ([S]ODNs) specifically targeted to this oncogene (bcr/abl [S]ODNs) and a suboptimal (for use as a single agent) dose of mafosfamide (the in vitro active form of cyclophosphamide). PURPOSE: We evaluated the ability of bcr/abl antisense [S]ODNs, alone or subsequent to treatment with a single injection of cyclophosphamide, to suppress the leukemic process induced in severe combined immunodeficient (SCID) mice by Philadelphia cells (i.e., primary CML-blast crisis [CML-BC] cells). In addition, we studied potential mechanisms that might explain the efficacy of the bcr/abl antisense [S]ODN-mafosfamide combination against Philadelphia cells in vitro. METHODS: The effects of treating leukemic mice with cyclophosphamide (25 mg/kg body weight; 25% of the dose required to eradicate evidence of leukemia in SCID mice) and/or bcr/abl antisense [S]ODNs were assessed by analysis of survival, by examination of bone marrow for the presence of leukemia cells (using a colony formation assay or using coupled reverse transcription and the polymerase chain reaction to screen for bcr/abl messenger RNA), and by examination of a variety of tissues for the presence of infiltrating leukemia cells. The induction of apoptosis (a cell death program) in vitro in primary CML-BC cells following treatment with bcr/abl antisense [S]ODNs plus or minus prior treatment with mafosfamide was monitored by use of a commercial assay. Relative cellular uptake of [S]ODNs by CML-BC cells treated in vitro with or without prior treatment with mafosfamide was determined by use of confocal microscopy and flow cytometry (for fluorescent [S]ODNs) or by use of blotting techniques that employed radioactively labeled probes (for extracted, unlabeled [S]ODNs). Levels of specific proteins in treated and untreated cells were determined by use of western blotting methods. Reported P values are two-sided. RESULTS: The disease process in leukemic mice was retarded substantially by combination treatment with cyclophosphamide and specific bcr/abl antisense [S]ODNs (P < .001, relative to treatment with specific antisense [S]ODNs alone, cyclophosphamide alone, or cyclophosphamide plus nonspecific [i.e., control] antisense [S]ODNs); 50% of the mice treated with cyclophosphamide and specific antisense [S]ODNs appeared to be cured of leukemia. The combination treatment was associated with increased induction of apoptosis. In addition, cellular uptake of bcr/abl antisense [S]ODNs appeared to be increased twofold to sixfold by prior treatment with mafosfamide. This increased uptake of [S]ODNs was associated with enhanced suppression of p210bcr/abl protein levels. CONCLUSIONS AND IMPLICATIONS: Combination therapy with antisense [S]ODNs targeted to specific oncogenes and less toxic doses of anticancer drugs may represent a rational strategy to purpose for the treatment of human leukemias.

Role of phosphatidylinositol 3-kinase-Akt pathway in nucleophosmin/anaplastic lymphoma kinase-mediated lymphomagenesis.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in a subset of anaplastic large cell lymphomas, encodes a Mr 75,000 hybrid protein that contains the NH2-terminal portion of the nucleolar phosphoprotein nucleophosmin (NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocations. Our studies showed that NPM/ALK, similar to other members of this family, activates phosphatidylinositol 3-kinase (PI3K) and its downstream effector, serine/threonine kinase (Akt). PI3K was found in complex with NPM/ALK. Both PI3K and Akt kinase were permanently activated in NPM/ALK-transfected BaF3 murine hematopoietic cells and in NPM/ALK-positive, but not in NPM/ALK-negative, patient-derived anaplastic large cell lymphoma cell lines. In addition, Akt was phosphorylated/activated in protein samples isolated from four patients diagnosed with ALK-positive T/null-cell lymphomas. The PI3K inhibitors wortmannin and LY294002 induced apoptosis in NPM/ALK+ cells but exerted only minor effects on the control BaF3 parental cells and peripheral blood mononuclear cells stimulated by growth factors. Furthermore, retroviral infection of NPM/ALK+ BaF3 cells with a dominant-negative PI3K mutant (delta p85) or a dominant-negative Akt mutant (K179M) inhibited proliferation and clonogenic properties of the infected cells. Finally, the Akt mutant (K179M) suppressed the tumorigenicity of NPM/ALK-transfected BaF3 cells injected into syngeneic mice. In conclusion, our data indicate that NPM/ALK constitutively activates the PI3K-Akt pathway and that this pathway plays an important role in the NPM/ALK-mediated malignant transformation.

Role of signal transducer and activator of transcription 5 in nucleophosmin/ anaplastic lymphoma kinase-mediated malignant transformation of lymphoid cells.

The NPM/ALK fusion gene, formed by the t(2;5) translocation in anaplastic large-cell lymphoma, encodes a M(r) 75,000 hybrid protein that containsthe amino-terminal portion of the nucleolar phosphoprotein nucleophosmin(NPM) joined to the entire cytoplasmic portion of the receptor tyrosine kinase anaplastic lymphoma kinase (ALK). NPM/ALK encodes a constitutively activated tyrosine kinase that belongs to the family of tyrosine kinases activated by chromosomal translocation. Our studies show that NPM/ALK, similar to other members of this family, activates signal transducer and activator of transcription 5 (STAT5) and that this activation is essential for lymphomagenesis. NPM/ALK-mediated activation of STAT5 was demonstrated by detection of: (a) constitutive tyrosine phosphorylation and enhanced DNA binding ability of STAT5 in NPM/ALK-transformed cells; and (b) NPM/ALK-dependent stimulation of STAT5-mediated transactivation of the beta-casein promoter. Retroviral infection of NPM/ALK+ cells with a dominant-negative STAT5B mutant (STAT5-DNM) inhibited the antiapoptotic activity of NPM/ALK in growth factor and serum-free medium. In addition, STAT5-DNM inhibited proliferation and diminished the clonogenic properties of NPM/ALK-positive cells. Finally, SCID mice injected with NPM/ALK+ cells infected with a virus carrying STAT5-DNM survived significantly longer than mice inoculated with NPM/ALK+ cells infected with the empty virus. Necropsy identified a widespread ALK+ lymphoma in lymph nodes and liver of the affected animals. Together, our data indicate that NPM/ALK-induced activation of STAT5 may play an important role in NPM/ALK-mediated lymphomagenesis.

ABL-fusion oncoproteins activate multi-pathway of DNA repair: role in drug resistance?

Chromosomal translocations of tyrosine kinase c-ABL gene from chromosome 9 may generate oncogenic kinases exhibiting constitutive tyrosine kinase activity. Recently, we have shown that ABL-fusion oncogenic tyrosine kinases, BCR/ABL and TEL/ABL, specific to hematopoietic malignances, induced resistance to DNA-damaging agents. To elucidate the role of DNA repair in this phenomenon we examined the capacity of murine BaF3 lymphoid cells and their TEL/ABL-transformed counterparts to repair DNA lesions caused by gamma- and UV-radiations and the anti-cancer drug, idarubicin. TEL/ABL-transformed cells displayed resistance to these DNA damaging agents as evaluated by MTT assay and the survival advantage was associated with an accelerated kinetics of DNA repair as measured by the alkaline comet assay. Deoxyribonucleosides (dNTPs) supplementation of the repair medium further stimulated DNA repair and the effect was specific to the DNA damage agent used in the experiment but only the transformed cells displayed this feature. A variety of damages induced imply the multi-pathway of DNA repair involved. We also examined the capability of BCR/ABL-fusion to modulate the repair of oxidative lesions, considered as a major side effect of various anti-cancer drugs including idarubicin and radiation. Employing the free radical scavenger alpha-phenyl-N-tert-butyl nitrone (PBN, a spin trap) and DNA repair enzymes: endonuclease III (EndoIII) that nicks DNA at sites of oxidized bases, we found that BCR/ABL-transformed cells repaired oxidative DNA lesions more effectively than control cells. Our results suggest, that oncogenic ABL-dependent stimulation of DNA repair may contribute to the cell resistance to genotoxic treatment.

Antitumor activity of tributyrin in murine melanoma model.

Butyric acid has been known to inhibit growth and to induce differentiation of a variety of tumor cells. Butyrate-treated tumor cells have also been observed to undergo apoptosis. Although butyrate compounds have demonstrated antitumor activity in murine tumor models and have already been admitted to clinical trials in tumor patients, the exact mechanism of their antitumor effects has not been elucidated. The results of our study showed antitumor activity of tributyrin, a butyric acid prodrug, in murine melanoma model and are strongly suggestive that antiangiogenic effects could participate in antitumor effects of butyrate compounds in vivo.

The kinetics of immunologic and hematologic recovery in mice after lethal total body irradiation and reconstitution with syngeneic bone marrow cells treated or untreated with mafosfamide (ASTA Z 7654).

The immunohematopoietic reconstitution of mice lethally irradiated (TBI) and reconstituted with syngeneic bone marrow cells untreated or treated with mafosfamide (ASTA Z 7654) [TBI + SBMT or TBI + SBMT-Maf mice, respectively] was examined. The number of CFU-S was greatly reduced in TBI + SBMT-Maf mice compared with those in TBI + SBMT mice. The recovery of blood parameters (hematocrit, reticulocytes, erythrocytes, white blood cells, granulocytes, platelets) and of bone marrow and spleen cells, but not of peritoneal exudate cells, was slightly delayed in TBI + SBMT-Maf mice compared with those in TBI + SBMT mice. The time for immune system regeneration was, however, considerably longer in TBI + SBMT-Maf than in TBI + SBMT mice, as measured by the incidence of Ig+, Thy-1.2+, L3T4+, Lyt-2+, and IL-2R+ cells in the spleens. The appearance of Mac-1+ and asialo-GM 1+ cells was only slightly prolonged or unchanged, respectively.

Hematological effects of intermittent 2-hour infusions of cladribine in multiple sclerosis patients: a comparison of 2 dosage patterns.

Cladribine is a lymphocytotoxic purine nucleoside with potential for treatment of autoimmune diseases. However, optimal administration regimens remain to be established. Twenty multiple sclerosis patients enrolled into this study were given 30 intermittent 2-hour cladribine infusions (0.07 mg/kg per infusion) each. Ten patients received cycles of 5 consecutive daily infusions at 5-week intervals (clustered dosage) on an inpatient basis; the other 10 patients received 1 infusion weekly (nonclustered dosage) on an outpatient basis. Red blood cell (RBC), platelet, and total white blood cell (WBC) counts were assessed at 5-week intervals during the treatment and at 13-week intervals during a 26-week follow-up period. Major WBC and lymphocyte subsets were assessed cytometrically at 15-week intervals during the treatment and at 13-week intervals thereafter. The clustered dosage produced a lasting decline in granulocyte count, a delayed decrease in monocyte count, and a transient decrease in RBC count. The nonclustered dosage caused a larger and persistent decline in RBC count, a smaller (P = .051. compared over the study period) decrease in monocyte count, and no change in granulocyte count. Both regimens transiently reduced natural killer and B-cell subsets (by 40%-60% and >80%, respectively) and caused lasting declines in CD4+ T-cell subsets (by >50%). No significant change was found in CD8+ T-cell subsets. These results show similar potency of these regimens with respect to major lymphocyte subsets, while suggesting that the nonclustered dosage is less toxic to myeloid precursors and more toxic to erythroid lineage precursors.

Photochemical labeling of HL-60 cell membrane proteins with radioiodinated, 4-azidosalicylic acid acylated derivatives of gangliosides.

To detect HL-60 human promyelocytic leukemia cell proteins involved in the uptake of gangliosides from the culture medium we used photoreactive, 4-azidosalicylic acid (ASA) acylated and radioiodinated (200 Ci/mmole) derivatives of GM3, GD3, GM1, and FucGM1 gangliosides. Gangliosides-ASA, added to the medium at 15-20 nM concentration, followed a similar time course of uptake. After 1 min incubation cell bound gangliosides-ASA could not be removed with trypsin, but only 5-10% remained after incubation with BSA. The proportion of cell bound gangliosides-ASA resistant to BSA treatment increased with time of incubation up to 76% after 20 h. As shown on TLC, GM3- and GD3-ASA were catabolized to LacSph-ASA and ceramide-ASA, while GM1-ASA was hydrolyzed to GM2-ASA. FucGM1-ASA was converted to GM1-ASA very slowly. Upon irradiation with UV lamp, cell bound gangliosides-ASA crosslinked to and photolabeled many proteins but the distribution of radioactivity after SDS/PAGE was very uneven and did not correlate with Coomassie staining. In all experiments the 42 kDa protein bands were most intensely photolabeled. Photolabeling of 42 kDa proteins decreased with time of incubation as compared to lower molecular mass pro teins. With all gangliosides-ASA used similar but not identical protein photolabeling patterns were obtained. Photolabeling patterns with GM3- and GD3-ASA differed from those with GM1- and FucGM1-ASA.

Immunogenicity of cyclophosphamide-treated tumor cells.

The immunogenicity of cyclophosphamide-treated murine tumor cells was tested on syngeneic or semisyngeneic mice. The majority of mice immunized with modified Ehrlich carcinoma cells, L1210 leukemia cells or AA sarcoma cells survived after challenge with a lethal dose of unmodified tumor cells. Plasmacytomas RPC-5 and MOPC 104E were less immunogenic and L-1 sarcoma cells were not immunogenic.

Augmented pro-apoptotic effects of TRAIL and proteasome inhibitor in human promonocytic leukemic U937 cells.

TRAIL, Tumor necrosis factor-related apoptosis-inducing ligand), a member of the TNF family, is known to be cytotoxic for a high proportion of tumor cell lines. However, successful application of TRAIL in tumor therapy may depend on finding other agents that can potentiate its antitumor effects. The present study showed that the cytostatic/cytotoxic TRAIL activity against U937 cells could be significantly augmented by proteasome inhibitor PSI, as revealed by MTT assay. Increased cytostatic/cytotoxic effect on U937 cells by TRAIL/PSI combined treatment was caused by apoptosis, as shown by an increased PARP cleavage rate. TRAIL/PSI did not affect the level of mRNA expression for TRAIL receptors (DR4, DR5, DcR1) and other apoptosis signal transduction molecules (TRADD, caspase-8).

Interstitial lung disease in systemic sclerosis: comparison of BALF lymphocyte phenotype and DLCO impairment.

Patients with scleroderma (systemic sclerosis-SSc) frequently develop an interstitial lung disease. The role of lymphocytes in fibrosing alveolitis preceding lung fibrosis has been established. The purpose of this work was to evaluate cell profiles and lymphocyte phenotypes in the bronchoalveolar lavage (BAL) fluid and to correlate them with depression in lung function tests detected by depletion of diffusing capacity (DLCO). BAL was carried out in 25 untreated, non-smoking patients suffering from diffuse scleroderma and in 12 healthy non-smoking volunteers. For the analysis of lymphocyte sub-sets flow cytometry and monoclonal antibodies were used. The following cell sub-types were counted: T lymphocytes, B lymphocytes, helper lymphocytes, suppressor/cytotoxic lymphocytes, natural killer cells, cytotoxic T lymphocytes and activated T lymphocytes. The total cell count was higher in the group of patients with mild and moderate impairment in DLCO. The percentage of lymphocytes was greater in patients with DLCO lower than 65% of the predicted value since neutrophilia was found in patients with severe DLCO depletion, i.e. significant when compared with healthy subjects. The proportions of suppressor/cytotoxic lymphocytes and of activated T lymphocytes were higher in patients than in controls. The statistical analysis revealed significant differences between patients with moderate and mild changes in DLCO and the healthy volunteers. A decreased helper/suppressor ratio was noticed in these patients. We concluded that the BALF lymphocyte phenotype analysis may reflect the features of alveolitis in patients with SSc.

Serine proteinase inhibitor from lymphocytic leukemia cells. Properties and copurification with DNA.

A trypsin inhibitor was isolated from mouse lymphocytic leukemia L 1210 cells by ammonium sulphate precipitation and preparative isoelectric focusing. A 39-fold purification was attained. The inhibitor is a protein since its activity is destroyed by pronase and it binds to insolubilized trypsin. Two main forms of the inhibitor were found of pH 4.8 and 5.3. The inhibitor is copurified with DNA, although neither DNase II nor RNase A change its activity.

Apoptosis and some of its medical implications.

The following phases of apoptosis were proposed: decisive, executive and degradation. At least two checkpoints are recognized in the process of apoptosis: the p53 and Bcl-2/Bax protein family checkpoint and the protease-caspase and serine protease checkpoint. Signals to apoptosis from outside of the cell including Fas and Fas ligand interaction and sphingomyelin cycle were presented. Cellular morphological events in apoptosis: DNA chain breaks, changes in cell membrane and mitochondrial membranes were discussed. Some medical problems of cardiology in which apoptosis plays an important role were presented. The cancer treatment results depending on apoptosis of cancer cells were discussed as well.

Immunogenic and nondividing mafosfamide-treated L 1210 cells--comparison of lines resistant and nonresistant to cyclophosphamide.

Cyclophosphamide/mafosfamide-resistant L 1210 cell line [L 1210(Cy)R] was established from a sensitive parental line. The L 1210(Cy)R line was resistant to cyclophosphamide at the dose of 100 mg/kg. Cells of L 1210(Cy)R line were more immunogenic for semisyngeneic CD2F1, mice as compared with parental line. They grew slower in immunocompetent mice compared to immunosuppressed mice. It has been shown that L 1210(Cy)R cells treated with mafosfamide at high concentration not only retained immunogenicity but were even more immunogenic than parental L 1210 cells. In conclusion, it was possible to produce immunogenic, nondividing leukemia cells even when cells were resistant to the cytostatic used for cell modification.

Evaluation of a system producing the hemopoietic factor. WEHI-3B cell line function, when encapsulated in a polypropylene hollow fibre.

The purpose of our study was evaluation of functioning of WEHI-3B (an mouse cell line producing IL-3) cells encapsulated in hollow fibers (HF). In vitro: the WEHI-3B cells were encapsulated in HF of polypropylene K600 silikonized, and cultured over two weeks. In vivo: the encapsulated WEHI-3B after weeks culture, were implanted subcutaneously into mice for 1 week. After explantation encapsulated WEHI 3-B were cultured again in culture medium for one week. The production of IL-3 by encapsulated WEHI-3B cells was assessed by evaluation of IL-3 dependent, BaF3 cells viability. The percent number of one day survival of BaF3 cells in the culture medium supplemented with 15% of encapsulated WEHI-3B in vitro or encapsulated WEHI-3B after in vivo conditioned medium was comparable with positive control. Possible replacement of recombinant cytokines with HF encapsulated cytokine-producing cells may be a chance for continous supplementation of the factors for hematopoietic stem cells differentiation.