Biomedical subjects

G Konwalinka

Publications and source records attributed to G Konwalinka.

At least 73 records · Page 4Linked to original sources

PubMed1986

Effect of human plasmalipoproteins on erythropoietic progenitor cells in serum-free cultures.

The purpose of the present study was to investigate the influence of human lipoproteins on CFU-e and BFU-e proliferation from human bone marrow in a serum-free system. In our previously described miniaturized agar system the main lipoprotein-density-classes from human plasma, namely very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), low density lipoproteins (LDL), high density lipoproteins2 (HDL2) and HDL3 and a mixture of all the five lipoproteins were added in rising concentrations (from 1/10 to normal human plasma concentration) to serum-free medium containing delipidated and deionized bovine serum albumin (BSA), iron saturated transferrin and erythropoietin. The results demonstrate that all lipoproteins markedly increased the CFU-e and BFU-e proliferation after 7 and 14 days of incubation, respectively. Moreover, the lipoproteins induced a shift towards a lower threshold concentration of erythropoietin. Serumlike conditions were obtained if LDL and the mixture of lipoproteins were added to serum-free medium. Furthermore, in the serum-free cultures a maturation to the mature erythrocyte could be found.

Agar↗

PubMed1986

Human erythropoiesis in vitro and the source of burst-promoting activity in a serum-free system.

Bovine serum albumin (BSA) can markedly increase the number of size of erythropoietic bursts produced by mononuclear cells from human bone marrow and peripheral blood, and reduce the threshold amount of erythropoietin (Epo) required for initial burst formation. The purpose of this study was to determine a possible burst-promoting activity (BPA) of BSA. The experiments were performed in a miniaturized agar system, in which the addition of sheep Epo to cultures with or without BSA was delayed for five days. The results obtained have shown that, with or without BSA, Epo deprivation of up to five days (an epoprival state) did not markedly decrease the number of bursts produced by unfractionated peripheral mononuclear cells compared to the number produced in the presence of Epo from the beginning of culture. Similar results were found whether the fetal calf serum (FCS) concentration was 15% or 2%. The preservation of potential BFU-e formation during the epoprival state has therefore been attributed to the ability of T-lymphocytes and/or monocytes to supply BPA. In order to reduce the endogenous amount of BPA, a nonadherent, E-rosette-negative cell fraction was cultured in the presence of Epo, with or without BSA, in serum-free medium containing transferrin (TF). Under these conditions, an equal number of bursts was obtained in FCS and in serum-free medium containing Epo, BSA and TF, whereas no BFU-e growth was found in the presence of Epo and TF, but without BSA. If Epo was withheld for up to five days, the capacity to form erythroid colonies was still retained by the monocyte- and T-lymphocyte-depleted cell fraction in the continuous presence of BSA. However, BPA could not be detected in the BSA. This observation was further supported by experiments in serum-free medium using human recombinant Epo, in which no BFU-e colony formation could be detected in the presence of BSA. From our investigations carried out at limited cell density and in serum-free medium, it could be concluded that the crude Epo preparation was the source of BPA.

Animals↗

PubMed1985

CFU-gm assay, cytochemical and electron microscopic studies in agar in patients with preleukemic syndrome and aplastic anemia.

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.

Aged↗

PubMed1985

A regulatory role of activated T-lymphocytes on human megakaryocytopoiesis in vitro.

Cellular interactions responsible for regulating in vitro megakaryocytopoiesis were studied using a microagar culture system which permits the simultaneous proliferation of human megakaryocytic progenitor cells (CFU-M) and T-lymphocytic colonies (CFU-TL). The proliferation of these colony types depends mainly on two factors: phytohaemagglutinin (PHA) and erythropoietin (EP). The direct addition of increasing PHA concentrations to the liquid overlayer resulted in a parallel increase of CFU-M and CFU-TL. If the T-lymphocytes were removed by an E-rosetting technique a marked diminution of CFU-M and CFU-TL numbers was observed. However, monocyte depletion resulted in a marked augmentation of CFU-M proliferation compared to unfractionated mononuclear cells. In order to confirm that the reduction of CFU-M proliferation observed after T-depletion was primarily mediated by the absence of T-lymphocytes, we have co-cultured different concentrations of previously removed autologous T-lymphocytes with a constant number of T-depleted bone marrow cells. A parallel increase of CFU-TL and CFU-M was found if 0.75 - 7.5 X 10(4) T-lymphocytes were added to the culture. In conclusion, our results indicate that activated T-lymphocytes augment proliferation of human bone marrow CFU-M and that monocytes are less important for the growth of megakaryocytic colonies.

Bone Marrow Cells↗

PubMed1985

Studies on differentiation of committed hemopoietic progenitor cells with monoclonal antibodies directed against myeloid differentiation antigens.

In the present study we evaluated the reactivity of monoclonal cytotoxic antibodies directed against myeloid differentiation antigens with hemopoietic precursor cells. VIM-D5 and VIM-2 inhibit the proliferation of clusters and colony formation after seven days of incubation. Day-14 CFU-GM are not affected by these antibodies. After complement-mediated cytolysis with VIM-2, the number of BFU-e was significantly reduced; however, this effect was largely abrogated by addition of leukocyte-conditioned medium to the cultures as an exogenous source of burst-promoting activity. Furthermore, the maturation of myeloid progenitor cells has been examined by delayed treatment with VIM-D5 and complement during the in vitro culture period. In these experiments a different maturation behavior of day-7 and day-14 CFU-GM was demonstrated. To study whether a cryptic carbohydrate structure is present on more immature CFU-GM, the effect of neuraminidase treatment of myeloid progenitor cells on reactivity with VIM-D5 was tested.

Antibodies, Monoclonal↗

PubMed1984

Subpopulations of colony-forming B lymphocytes exhibit distinct in vitro growth characteristics.

Human peripheral blood cells enriched for B lymphocytes were stimulated to focal proliferation in semisolid cultures with lymphocyte-conditioned media, Protein A (Prot A), lipopolysaccharide (LPS) and 2-mercaptoethanol (2-ME). After 6-8 days of incubation, two morphologically distinct colony types were observed. Type I colonies were diffusely proliferating aggregates within the agar layer, whereas another subset of B-cell-colony-forming cells (CFU-BL) formed round compact type II colonies which appeared to leave the agar layer and continued to proliferate in the liquid overlayer of our culture system. They reached maximum proliferation 2 days earlier than type I colonies. Cells derived from both colony types were positively identified as B lymphocytes by monoclonal antibodies using immunoperoxidase staining. In addition to this distinct growth pattern, both colony types exhibited different proliferative responses which were dependent on the kind of conditioned media used and the mitogen concentration. In secondary cultures both type I and type II colony-derived cells showed recloning capacity. However, after replating, both colony types gave rise to round compact type II colonies. These results demonstrate that there exist at least two subpopulations of colony-forming B lymphocytes, possibly one more primitive than the other, which can be distinguished by in vitro growth characteristics.

B-Lymphocytes↗

PubMed1984

[Proliferation of megakaryocytic precursor cells (CFU-M) in a micro-agar culture system].

The recently described micro-agar culture system for cloning erythropoietic progenitor cells was used to study the optimum conditions for the growth of CFU-M. In this system human mononuclear cells from normal human bone marrow were suspended in agar and incubated for 12 days. Various concentrations of phytohaemagglutinin lymphocyte conditioned medium (PHA-LCM) and prostaglandin E (PGE) were added to the liquid overlayer in the presence of 2-mercaptoethanol (2-ME) for the stimulation of CFU-M. Human AB serum was used instead of fetal calf serum (FCS) in all experiments. A sigmoidal dose-response curve, with a plateau at a concentration of 5 to 10%, was obtained by the addition of different concentrations of PHA-LCM in the presence of 10(-6)PG-E. Under optimal conditions (5% PHA-LCM, PGE 10(-6)M) a linear relation was obtained between the number of seeded cells and the megakaryocytic colonies formed. For routine morphological analysis the whole agar layer was stained using the Pappenheim method. For further characterization of CFU-M, an immunofluorescence test with rabbit antihuman factor VIII related antigen was performed on the whole agar layer.

Agar↗

PubMed1984

A miniaturized agar culture system for cloning human erythropoietic progenitor cells.

The micro-agar-culture technique for cloning early and late erythropoietic progenitor cells (BFU-E and CFU-E) was further modified and miniaturized in order to study the optimal growth conditions with a minimal consumption of erythropoietin (EP). Using microtiter plates, the total incubation volume was lowered from 0.5 to 0.1 ml and thus reduced the necessary amount of EP and cells by a factor of 5. The method consists of a 50 microliter agar layer, in which the mononuclear cells are suspended, and a 50 microliter liquid overlayer containing bovine serum albumin (BSA), transferrin (TF), and EP. After a seven- or 14-day incubation, the whole agar layers were fixed, transferred to microscopic slides, dried, stained using the Pappenheim method, and permanently preserved. The influences of FCS, BSA, and TF in the presence of EP were studied on the proliferation of human bone marrow CFU-E and BFU-E. The variation of FCS concentration showed an optimum at 10%. The addition of BSA in the presence of optimal concentrations of EP markedly increased the number of BFU-E, but not CFU-E. Furthermore, the threshold concentration of EP required for the initial burst formation could be reduced by half in the presence of BSA. By the addition of TF, a further increase in the number of BFU-E was obtained.

Agar↗

PubMed1983

[New aspects of megakaryopoiesis].

Like erythro- and granulopoiesis megakaryocytopoiesis (MK poiesis) is controlled by specific feed-back mechanisms. Platelet production has been found to be regulated predominantly by the number or mass of circulating platelets. Our present understanding of MK poiesis is based on data from 3 major experimental areas, which are individually discussed in this paper. In vivo experiments on thrombocytotic or thrombocytopenic mice. Radioisotopic assays for thrombopoietin determination. Investigation by in vitro culture methods of megakaryocyte colony-forming precursor cells (CFU-M).

Animals↗

PubMed1983

[In vitro studies of clonable human B lymphocytes].

A recently developed micro agar culture system has been optimized for the in vitro growth of human B-lymphocytes. Enriched B-lymphocytes from the peripheral blood of normal individuals were suspended in an agar layer, above which a liquid overlayer containing 20% PHA-LCM, 2-ME and Prot A or LPS as stimulants was added. Two morphologically distinguishable colony types were observed using this culture technique: diffusely proliferating colonies (Type I) were found within the agar layer, and round, compact colonies (Type II) appeared to leave the agar layer and continue growth in the liquid overlayer. For both colony types a linear relationship was demonstrated between the number of seeded cells and the number of formed colonies. The appearance of the two colony types in vitro was not concurrent, and they exhibited differing sensitivity to mitogen concentration and to the type of serum used as additive to the culture medium. The implications of clonal in vitro cultivation of lymphocytes, both normal and pathological, are discussed.

Adult↗

PubMed1983

Clonal growth of human megakaryocytic progenitor cells in a micro-agar culture system: simultaneous proliferation of megakaryocytic, granulocytic, and erythroid progenitor cells (CFU-M, CFU-C, BFU-E) and T-lymphocytic colonies (CFU-TL).

A simple and reproducible micro-agar culture technique for cloning human CFU-M is described. Human bone marrow mononuclear cells were suspended in agar and incubated for 12 days. Stimulation was provided by the direct addition of phytohemagglutinin-P (PHA-P), erythropoietin (Epo) and 2-mercaptoethanol (2-ME) to the liquid overlayer. A shift from BFU-E and CFU-C proliferation to CFU-M and CFU-TL was observed with increasing PHA concentrations. Under optimal conditions (PHA 50 micrograms, Epo 1.2 IU, 2-ME 2 x 10(-4) M, 1% purified BSA, 0.04% human transferrin, saturated with Fe C13) a linear relationship between colonies formed and plated cell number were observed. For the routine morphological analysis, the whole agar layers were stained using the Pappenheim method. For further characterization of CFU-M, cytochemical stainings and immunofluorescence tests with rabbit-antihuman factor VIII-related antigen were performed on the whole agar layers.

Clone Cells↗

PubMed1983

Myelopoiesis of human bone marrow cells in a micro-agar culture system: comparison of two sources of colony stimulating activity (CSA).

Human bone marrow cells were grown in a micro-agar culture system in the presence of human placenta (HPCM) and giant cell tumor conditioned media (GCT). The effects of HPCM and GCT conditioned media on linearity, growth dynamics, and morphological composition of colonies were studied after 7 and 14 days of incubation. Under the described conditions the dose-response curves for HPCM and GCT were different: on day 7 maximal stimulation was obtained with 2.5% HPCM and 20% GCT; on day 14 a maximal response was reached with 1.25% HPCM and 5% GCT. Both stimuli produced maximal growth after 7 days of incubation, followed by a rapid decrease in the number of formed colonies up to day 14. The morphological study of aggregates showed that after 7 days of incubation 80% pure granulocyte and 20% mixed granulocyte-macrophage colonies were found in the presence of both stimuli. However, on day 14 the incidence of granulocyte-macrophage colonies increased to 60%, whereas the percentage of pure granulocyte colonies decreased to 20%. The frequency of eosinophil colonies was relatively low (median 15%) with both stimuli. The described system can be applied successfully for studies of myelopoiesis in vitro. Both sources of colony stimulating activitites (CSA) employed had no significant difference in their ability to stimulate myelopoiesis.

Cells, Cultured↗

PubMed1983

Studies of myelopoiesis in vitro on blood and bone marrow cells of patients with acute leukemia in long-term remission.

In vitro myelopoiesis in a group of ten patients with acute leukemia (6 AML, 1 APML, 3 ALL) in long-term remission has been studied. The patients remained in first stable remission for at least 4 years and maintenance therapy had been completed in all patients except one. In the patients, colony formation of bone marrow cells after 7 days of incubation was significantly increased compared to a representative control group. The proliferation of microclusters after 3 incubation days was also markedly enhanced, and accelerated proliferation of all aggregates (microclusters, macroclusters and colonies) could be demonstrated in culture with the patients' bone marrow cells. The use of autologous feeder layers and autologous serum showed no inhibitory effect on colony formation. The proportion of eosinophil colonies formed with the patients' bone marrow cells was in the normal range. In contrast to the high proliferation capacity of bone marrow precursor cells the CFU-c number of peripheral blood was significantly decreased in the patients' group. No significant correlation between CFU-c number of bone marrow and blood cells could be found. The colony stimulating activity of the patients' peripheral mononuclear cells was normal compared to healthy controls. We conclude from this study that even in long-term remission of acute leukemia certain in vitro abnormalities exist in myelopoietic proliferation and regulation.

Bone Marrow↗

PubMed1983

A case of acquired pure red cell anemia studied by cloning of erythroid progenitor cells in vitro.

A 70-year-old woman developed typical clinical symptoms of pure red cell anemia (PRCA) following a history of rheumatoid arthritis (RA). The patient's bone marrow erythropoietic progenitors cells were cloned in a micro agar culture system several times over a period of 11 months, revealing a diminished frequency of bone marrow erythroblasts paralleled by a markedly reduced number of CFU-e and BFU-e in vitro. No inhibitory activity in the patient's IgG fraction could be detected either by preincubation with IgG and/or rabbit complement, or in the continuous presence of IgG. Depletion of T lymphocytes from the patient's bone marrow cells led to an improved in vitro erythroid proliferation. Cytostatic therapy with cyclophosphamide clinically induced a marked increase in the bone marrow erythroblast and reticulocyte number, correlated in vitro by normalization of CFU-e levels and increase in the number of BFU-e. Nevertheless, BFU-e values never attained normal levels, which could be attributed to a reduced stem cell pool resulting from previous therapy with cyclophosphamide and/or antirheumatic drugs. Two independent factors, a reduced pool of committed stem cells as well as an autoimmune cell-mediated suppression, may both contribute to the pathomechanism of the disease in this patient.

Aged↗

PubMed1982

[Bone marrow culture, cytochemistry and electron microscopy in agar in patients with preleukemic syndrome and aplastic anemia].

Thirty-seven patients with chronic cytopenia were studied using a CFU-c assay in agar. On the basis of the growth pattern three types of preleukaemic syndrome (PL) and two types of aplastic anaemia were distinguished. Further evaluation of the bone marrow dysfunction was attempted with a combined application of cytochemistry and electron microscopy for the morphologic study of cells proliferating in vitro. Well-defined maturation defects in the growing cells from the bone marrow of patients with PL were demonstrated with cytochemical stainings performed in agar. These results were supported by electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukaemic cells. On the basis of our results a high risk group of PL for development of overt leukaemia and a group of patients with a grave prognosis in aplastic anaemia were distinguished. The data obtained are relevant for the clinical diagnosis and prognosis of patients with cytopenias.

Adult↗

PubMed1982

[A microagar culture system for clonal growth of human T-lymphocytes].

A simple and reproducible micro-agar culture system for cloning human T lymphocytes has been described. The system consists of an agar layer and a liquid phase, which enables the easy addition of test substances and a prolonged proliferation. Further advantages of this system are a low incubation volume and a better morphological, cytochemical, immunological and electron-microscopical study of colonies obtained from the liquid phase.

Culture Media↗

PubMed1982

A micro agar culture system for cloning human erythropoietic progenitors in vitro.

A simple and reproducible micro agar culture method for cloning erythropoietic progenitor cells from human bone marrow is described. Mononuclear cells (MNC) were immobilized in an agar layer and stimulated by erythropoietin (Ep), which was added to a liquid overlayer. The cultures were routinely incubated, fixed, transferred to microscopic slides, dried, and stained, and erythroid colonies were morphologically examined. The dynamics of growth observed from days 2 to 26 of incubation in the presence of 2.4 U Ep/ml showed basically three kinds of aggregates, which reached maximum growth on different days of incubation. A close Ep dose-response relationship was found for CFUE and BFUE at a concentration of 7.5 x 10(4) seeded cells. By varying the plated cell concentration between 2.5 and 10 x 10(4) cells a linear increase in the aggregates formed was found. On the basis of their growth dynamics and morphologic composition, the existence of three populations of erythropoietic progenitor cells in human bone marrow is tentatively proposed.

Agar↗

PubMed1982

[Proliferation of myeloid precursor cells in a microagar culture system].

A microagar-culture system for the in vitro study of myelopoiesis committed stem cells has been reported. Stimulation was induced by a commercially available conditioned medium obtained from culture of histiocytoma cells. The effect of different concentrations of conditioned medium and a linear relation between the seeded cells and the formed colonies has been established. The granulocytic-monocytic character of cell aggregates has been shown by morphologic studies. Further possibilities for the use of the described method have been briefly discussed.

Agar↗