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Biomedical subjects

F R Seiler

Publications and source records attributed to F R Seiler.

At least 37 records · Page 2Linked to original sources
PubMed1991

Effect of granulocyte-macrophage colony-stimulating factor on the induction of unresponsiveness by lymphoid cells.

Skin grafts can be significantly prolonged in ALS-treated mice by the injection of 25 x 10(6) donor bone marrow cells or 50 x 10(6) spleen cells. Lymph node cells and thymocytes are only minimally effective in prolonging grafts. The effect of a hematopoietic growth factor, granulocyte-macrophage colony stimulating factor (GM-CSF) was studied in this model of unresponsiveness. C3H/He lymphoid cell donors were treated with GM-CSF. Either normal or GM-CSF-treated cells were injected into ALS-treated B6AF1 mice grafted with C3H/He skin. GM-CSF treatment significantly augmented the effect of marrow in prolonging graft survival at doses of 1 to 25 x 10(6) cells. In contrast, GM-CSF had no effect on the graft-prolonging effect of spleen cells when 50 x 10(6) cells were given. When the dose of cells was reduced to 25 x 10(6), graft survival in the group given GM-CSF-treated cells was prolonged compared with survival in the group given normal cells. Grafts in the group given GM-CSF-treated lymph node cells were rejected in sensitized fashion. When marrow and spleen are separated on a Percoll gradient, the cell active in promoting graft survival is recovered primarily in the 52.5% fraction. The graft-prolonging effect of the 52.5% marrow fraction was not affected by GM-CSF treatment. In contrast, GM-CSF-treated marrow cells in the 60% fraction significantly prolonged graft survival, while normal marrow cells in this fraction had no effect on graft survival. GM-CSF-treated spleen cells in the 52.5% and 60% fractions significantly decreased graft survival compared with normal cells when given at a dose equal to the number of cells recovered from 50 x 10(6) cells. When the dose of fractionated spleen cells was reduced, GM-CSF-treated spleen cells were more effective than normal cells in prolonging graft survival. These results indicate that GM-CSF activates a cell in marrow that promotes graft survival. This cell is recovered in the 60% Percoll fraction. In contrast, GM-CSF appears to affect two cell populations in spleen, one beneficial and one detrimental to graft survival. The predominant effect depends on the dose of spleen cells that is given.

Animals↗
PubMed1991

Studies on the efficacy of mast cell growth factor (c-kit ligand) in vitro as well as in vivo.

We tested the ability of recombinant human (rhu) mast cell growth factor (MGF), also known as c-kit ligand, to stimulate the colony formation of human bone marrow cells in semisolid medium alone and in combination with rhu erythropoietin (EPO), rhu Interleukin 3 (IL-3), rhu granulocyte colony stimulating factor (G-CSF) and rhu granulocyte-macrophage colony stimulating factor (GM-CSF). The addition of MGF to cultures containing EPO or EPO + IL-3, GM-CSF and G-CSF, resp., resulted in macroscopic erythroid burst-forming units (BFU-E). Multipotential (colony-forming unit granulocyte, erythroid, monocyte, megakaryocyte [CFU-GEMM]) progenitors were stimulated by MGF in the presence of EPO. Colony-forming unit granulocyte-macrophage (CFU-GM) were activated by MGF only in combination with GM-CSF. The combination of MGF with EPO was used for synergism studies in healthy cynomolgus monkeys. In the chosen concentration MGF alone had no effect on white blood cell (WBC) counts and on platelets, but a slight effect on reticulocytes. EPO by itself increased reticulocyte counts with no effects on WBC or platelets. The combination of both factors resulted in a significant increase of reticulocytes. No other effects were seen. These studies demonstrate the potent synergistic interaction of MGF and other hematopoietic growth factors.

Animals↗
PubMed1991

An enzyme linked immunosorbent assay for the detection of human interleukin 3 in serum.

A sandwich enzyme immunoassay was developed for measuring human Interleukin 3 (IL-3) in human and animal sera. Polyclonal and monoclonal antibodies were raised against the recombinant human protein. These have been used to develop an immunoassay which can detect down to 10 pg/ml of human IL-3. The assay involves a polyclonal rabbit antibody coupled to a solid phase and a mouse monoclonal antibody-horseradish peroxidase conjugate as the detection antibody. Unlike the classical bone marrow assay and other cell line based bioassays for IL-3, the immunoassay was specific for the cytokine showing no or only negligible cross-reactivity with IL-1, IL-2, IL-6, erythropoietin, granulocyte colony-stimulating factor (G-CSF) and GM-CSF. The assay does not exhibit interfering matrix effects when used for the estimation of human IL-3 in serum samples.

Antibodies, Monoclonal↗
PubMed1991

Highly specific and highly sensitive enzyme immunoassays for antibodies to human interleukin 3 (IL-3) and human erythropoietin (EPO) in serum.

Two versatile, rapid, easy-to-perform and highly sensitive enzyme immunoassays (ELISAs) were developed for the detection and quantification of antibodies to human IL-3 and human EPO in serum samples from both man and laboratory animals. These one-step assays are based on the "inverse sandwich" principle, where antibodies present in the sample linked both the solid phase antigen bound to a microtiter plate and the free antigen, which had been covalently coupled to horseradish peroxidase. The limits of detection are lower than those of the neutralization bioassays; antibodies to IL-3 and to EPO were detected at concentrations as low as 2 ng/ml and 5 ng/ml, respectively. No cross-reactivity with related proteins and no interfering matrix effects were observed when used for the estimation of antibodies against rhu IL-3 and rhu EPO in serum samples of various species. Thus both assays were used without modification for the screening of antibodies in serum samples from both man and animal during treatment with these hematopoietic hormones.

Animals↗
PubMed1991

Optimizing autologous blood donation by recombinant human erythropoietin (rhu-EPO) and interleukin 3 (IL-3).

The transfusion of autologous blood protects surgical patients from both the transfusion transmitted diseases (AIDS, posttransfusion hepatitis) and the immunosuppressive effects of homologous blood. Nevertheless, the use of autologous blood is still unsatisfactory, mainly because of the elaborated logistics, organization and technique required and the often insufficient amounts of autologous blood gained. Today, the major growth-factors of erythropoiesis are available as recombinant analogues. In the studies reviewed here, we investigated the effects of rhu-EPO and IL-3 on perioperative erythropoiesis in two canine models of acute isovolemic hemodilution. Different therapeutic concepts are compared with respect to preoperative changes in hematocrit, the volume of autologous blood gained and the duration of postdilutional anemia.

Animals↗
PubMed1991

Bone marrow obtained during hip surgery: a novel source for studies of hemopoiesis in human long-term bone marrow culture (LTBMC).

Studies with human bone marrow cells are often impaired by the poor quality of sternal aspirates due to varying numbers of contaminating blood cells before enrichment procedures and insufficient progenitor cell yields. In this study we report on experiments performed with human bone marrow cells isolated from a) spongiose bone fragments collected during hip surgery in patients with osteoarthritis or b) sternal aspirates. After Ficoll-Histopaque density gradient centrifugation absolute cell numbers were always lower in the samples obtained from sternal aspirates. However, both sources proved to yield the same proportions of the respective myeloid cell populations. Human long-term bone marrow cultures (LTBMC), semi-solid agar assays (CFU-GM day 14), and 3H-thymidine incorporation assays proved the comparability of the two bone marrow sources.

Adult↗
PubMed1991

Development of a rapid, highly sensitive, non-radioactive assay system for hematopoietic growth factors.

The aim of this study was to develop non-radioactive cell line proliferation assays. The human leukemic cell line TF1 (Kitamura et al., 1989) was used for the determination of the specific biological activity of recombinant human (rhu) granulocyte-macrophage colony-stimulating factor (GM-CSF) and rhu Interleukin 3 (IL-3) by a simple and economical fluorometric assay with a sensitivity similar to the measurement of 3H-thymidine uptake. The TF1 cell line responds to rhu IL-3, rhu GM-CSF and to a lesser extent to rhu Erythropoietin (EPO) and mast cell growth factor (MGF), but not to rhu G-CSF. It is dependent upon rhu GM-CSF for survival in culture. For the proliferation assay 1 x 10(4) TF1 cells were incubated with 20 ng - 0.256 pg rhu GM-CSF or rhu IL-3 at 37 degrees C and 5% CO2 in humidified atmosphere. After 48 h the cells were washed twice with PBS and were incubated with 4-Methylumbelliferyl-heptanoate for 60 min. Fluorescence was determined on a Titertek Fluoroskan II (Flow Lab.), and results were given as fluorescence units using a 355 nm excitation filter and a 480 nm emission filter. The developed assay showed an interassay variability lower than 15%. The sensitivity of the proliferation assays in the same range as the thymidine incorporation assays.

Biological Assay↗
PubMed1991

Therapeutic effects of 15-deoxyspergualin in acute and chronic relapsing experimental allergic encephalomyelitis (EAE) as models for multiple sclerosis (MS).

Experimental allergic encephalomyelitis (EAE) is an inflammatory and paralytic auto-immune disease of the central nervous system (CNS) resulting from the sensitization of susceptible laboratory animals with whole brain homogenate, lyophilized spinal cord or heterologous myelin basic protein (MBP) in complete Freunds adjuvant (cFA). Acute EAE generally presents as a monophasic disease that produces perivascular inflammation and sometimes small areas of demyelination. The chronic relapsing form of EAE in particular offers important similarities to the human disease multiple sclerosis (MS). Due to its immunosuppressive mode of action, the authors wished to examine the therapeutic effects of 15-deoxyspergualin (15-DOS) in the two models of acute and chronic relapsing EAE in Lewis rats. In the first model, sensitization of adult rats (8-12 weeks of age) with guinea pig spinal cord in cFA results in an acute clinical episode of severe EAE, and by day 17 all animals died. Lewis rats treated with 15-DOS showed a delayed and reduced onset of clinical symptoms and even low amounts of the drug prevented mortality. The protection afforded by 15-DOS was long-lasting and no subsequent relapse has been observed. On rechallenging, such convalescent rats were totally resistant to re-induction of the disease. Passive transfer of EAE to naive Lewis rats was possible with encephalitogenic (antigen-specific primed) spleen cells from diseased control animals but could be prevented by fresh 15-DOS treatment. Passive transfer of acute EAE was suppressed when spleen cells from diseased Lewis rats but 15-DOS-treated animals were used.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1991

Immunosuppressive activity of 15-deoxyspergualin (15-DOS) on various models of rheumatoid arthritis.

Based on the promising results obtained thus far in various auto-immune disease models, the authors have further elucidated the disease-modifying potential of the new immunosuppressive drug 15-deoxyspergualin (15-DOS) in models of inflammatory and chronic degenerative joint disease of adjuvant arthritis (AA) and collagen type-II induced arthritis (CIA) in Lewis rats and spontaneously developing polyarthritis in MRL/1 mice. Treatment of AA animals with 15-DOS starting with the day of adjuvant injection prevented the disorder from spreading to the non-injected extremity. The drug also reduced the arthritis index (47% inhibition). The adjuvant disease can thus be prevented nearly totally, even after discontinuation of drug application. Even when starting the therapy during the established disease, 15-DOS still exerts a protective action on the development of the adjuvant disease. In the present model of CIA, the animals showed the same progression as reported by others. Approximately 80% of the animals had bilateral hind paw swelling. The production of autoreactive anti-type-II collagen antibodies and also high levels of circulating rheumatoid factor (RF) can be demonstrated in the serum of CIA rats. Treatment of these animals with 15-DOS was very effective in inhibiting swelling of the joints. Average antibody titres were also depressed and circulating RF was reduced. Even when treatment with 15-DOS started on day 15 after CIA induction on established lesions of polyarthritis, not only a significant suppression of the paw diameters occurred, but also the humoral response (antibody formation) could be inhibited in animals with established CIA disease. The disorder of MRL/1 mice is characterized by the development of auto-antibodies most prevalently directed against double-stranded DNA (dsDNA) and type-II collagen. These mice also have circulating rheumatoid factor (RF) and develop histological changes in their joints characterized by pannus formation, cartilage and bone erosions. Treating MRL/1 mice with 15-DOS resulted in a decrease in the amount of auto-antibodies and inhibited developing polyarthritis. Even in an established disease, 15-DOS reduced circulating RF and increase in paw volume (signs of polyarthritis) was inhibited. These results suggest that 15-DOS might be used as a therapeutic agent for human RA and that this new immunosuppressive drug could be an effective nonsteroidal antirheumatic agent.

Animals↗
PubMed1990

Cytofluorometric and cytomorphologic analysis of human bone marrow cells derived from stromal cultures stimulated by granulocyte-macrophage colony-stimulating factor, interferon-gamma and splenopentin pentapeptide.

We studied the influence of human recombinant granulocyte-macrophage colony-stimulating factor (hrGM-CSF), human recombinant interferon-gamma (hrIFN-gamma) and splenopentin pentapeptide (Sp-5), either alone or in combination, on the proliferation and differentiation of human bone marrow cells in modified Dexter's cultures. After 10, 14 and 21 days cells were analyzed by classical staining according to Pappenheim and by cytofluorometry with a set of different monoclonal antibodies. IFN-gamma inhibited the proliferation of progenitor cells and provided signals promoting monocytic differentiation, whereas GM-CSF induced the proliferation of blastoid elements which expressed HLA-DR and M2 (VIM-2 monoclonal antibody), but progressively lost surface CD34. Furthermore, an increase of CD15+ cells was also observed. When GM-CSF was tested in combination with IFN-gamma, it abolished the inhibitory effect of IFN-gamma and both cytokines synergized to promote the expression of CD11c, CD14 and M2 surface antigens. Sp-5 alone had only a marginal activity, but it potentiated the effects of GM-CSF. These findings suggest that GM-CSF may induce the transition from stem cells to committed myeloid progenitors. In contrast to IFN-gamma, Sp-5 can serve as an additional proliferative signal with negligible effects on cell maturation.

Amino Acid Sequence↗
PubMed1990

Preclinical studies on synergistic effects of IL-1, IL-3, G-CSF and GM-CSF in cynomolgus monkeys.

The regulation of blood cell formation is mediated by a group of polypeptides classified as hematopoietic growth and differentiation factors. Overlapping as well as distinct functions have been described for three of these cytokines: interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Furthermore, interleukin 1 (IL-1) has been shown to promote hematopoietic regeneration after cytoreductive drug treatment. Evidence has been provided by in vitro studies that combinations of these factors exert a highly synergistic action on the proliferation and differentiation of committed hematopoietic progenitor cells. Additionally, these findings have been supported by studies of in vivo blood cell formation in nonhuman primates. We report here that IL-3 acts synergistically with GM-CSF or G-CSF on myelocytic cell development only if an administration time of eight days of IL-3 is followed by GM-CSF or G-CSF. Short-time IL-3 application of two and four days only resulted in platelet production. The reverse administration schedule did not show synergistically enhanced stimulation of myelocytic cells. However, G-CSF treatment followed by IL-3 did induce a two-fold increase in platelet numbers. This would appear to confirm previously reported in vitro findings that G-CSF shortens the G0 period of human hematopoietic stem cells, which subsequently proliferate in the presence of IL-3. The effects of IL-3 on myelocytic and megakaryocytic development seems to be differently regulated. Whereas, IL-1 failed to display synergistic activity with GM-CSF or G-CSF is sequentially applied. Only simultaneous application either in combination with GM-CSF or with G-CSF demonstrated enhanced efficacy.

Animals↗
PubMed1990

Erythropoietin accelerates the recovery from extreme hemodilution: a randomized, placebo-controlled study in dogs.

Six splenectomized beagles of either sex (13.8 +/- 2.2 kg) were randomly treated either with 500 U/kg recombinant human erythropoietin (rhu-EPO) (verum group, n = 3) or an equivalent volume of the vehicle (placebo group, n = 3). Both solutions were given intravenously for 3 days. At day 4 after onset of treatment, the dogs were anesthetized and subjected to isovolemic hemodilution using 6% Dextran 60 (MW 60,000) down to a hematocrit of 0.10. During the recovery period vehicle or rhu-EPO was given every other day until the hematocrit reached control values. Every day venous blood samples were withdrawn, and the hematocrit as well as the concentrations of hemoglobin and 2,3-diphosphoglycerate were determined. In addition, the platelets and reticulocytes were counted. Treatment with rhu-EPO shortened the time of hematocrit recovery from 20 (placebo) to 11 days (p less than 0.05). The reticulocyte count peaked at day 2 (verum) versus day 5 (placebo). These findings indicate a successful stimulation of red blood cell production after extreme hemodilution in animals treated with erythropoietin. Therefore, rhu-EPO may allow to optimize blood donation programs as well as preoperative hemodilution and yield both, higher amounts of autologous blood and an accelerated reversal of dilutional anemia.

Animals↗
PubMed1990

Physiochemical features of monoclonal idiotype-anti-idiotype complexes: importance of inter-chain disulfides for size distribution patterns and molecular geometries.

Cyclic tetramers represent the preferentially formed complexes of a murine monoclonal idiotype-anti-idiotype (Id-anti-Id) system consisting of IgG antibodies or (Fab')2 fragments at micromolar concentrations. The cleavage of inter-chain disulfides of both Id and anti-Id caused the predominant generation of cyclic dimers at the expense of larger aggregates, suggesting with regard to already published data that the hinge located interheavy-chain disulfides are essential for the strain.

Antibodies, Anti-Idiotypic↗
PubMed1990

Development of an ELISA for the detection and determination of contaminating proteins in recombinant DNA derived human erythropoietin.

An enzyme linked immunosorbent assay (ELISA) has been developed for the quantitative determination of the most probable contaminating proteins (MCP) of recombinant human Erythropoietin produced in the mouse fibroblast cell line C-127. The developed ELISA is a double polyclonal sandwich type immunoassay, which allows a quantitation of the MCPs in the range of parts per million. The polyclonal antibody, used for both coat and conjugate in this ELISA, was demonstrated to be reactive with the reference MCPs (a collection of the most probable protein contaminants) by immunoblot analysis and immunoabsorption of radiolabeled MCPs. Affinity purification of this antibody preparation using the immobilized MCPs resulted in an assay with higher signal-to-noise ratio. The assay was demonstrated to be very specific for the MCPs obtained from the rhu EPO purification process. Since the purification of each recombinant DNA derived protein expressed in mammalian cells requires its own unique process, no generic assay for contaminating proteins can be developed. There are only a few common criteria for the development of such multi-antigen ELISA, which will be discussed in this paper.

Animals↗