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G Fritsch

Publications and source records attributed to G Fritsch.

At least 73 records · Page 4Linked to original sources
PubMed1994

Flow cytometric assessment of human MIC2 expression in bone marrow, thymus, and peripheral blood.

The cell-surface expression of the MIC2 antigen defined by the monoclonal antibody 12E7 was investigated on human leukocytes in bone marrow (BM), thymus, and peripheral blood (PB) using multiparameter flow cytometry and cell sorting. In contrast to preceding reports, we found that the MIC2 antigen is not restricted to T cells and monocytes. We show that it is also expressed in the B cell and in the granulocytic lineage, the levels of expression being related to distinct maturational stages. CD34+ cells of BM were found to express the antigen at high levels. Along the granulocytic maturation pathway from CD34+CD33+ blasts to mature granulocytes, MIC2 densities appeared progressively reduced with a considerable decline at the myelocyte stage. In B lymphopoiesis, the earliest CD34+ CD10+ B-cell precursor (BCP) cells, further subdivided by expression of CD19, displayed the highest MIC2 density of BM leukocytes. All later BCP stages showed lower MIC2 expression levels, with a remarkable reduction concomitant with loss of the CD34 antigen at the CD10+CD20- surface mu-chain- stage, and a subsequent slight upregulation along with maturation to CD10-CD20high surface mu-chain+ BCPs. The brightest MIC2 expression of all cells tested was displayed by the most immature thymic T-lineage cells characterized by the antigenic profile CD34weakor- CD7++ surface CD3-CD1a(weak) CD4weak CD8-or weak. Common thymocytes stained slightly less intense with 12E7, whereas all subsequent stages of T-lineage cells in thymus, PB, or BM showed markedly reduced MIC2 levels. Mature peripheral CD4+ as well as CD8+ T cells displayed a bimodal distribution of MIC2. In the CD4+ population, the distinct MIC2 levels were related to the well-studied functional subdivision by differential expression of CD45 isoforms, the helper-inducer/memory subset showing higher MIC2 expression than helper-suppressor/naive CD4+ T cells. Similarly high MIC2 densities were found on CD16+ natural killer cells and on CD14+ monocytes, whereas mature peripheral B cells exhibited low or intermediate expression, and granulocytes exhibited no or only dim expression. These results document that the MIC2 antigen (1) is expressed on all leukocyte lineages; (2) is differentially expressed during T- and B-lymphoid, as well as granulocytic maturation; (3) shows highest expression in the most immature lymphocytic and granulocytic developmental stages; and (4) is also differentially expressed on functional T-cell subsets. We speculate that these observations imply a functional significance of MIC2 in the network of hematopoietic adhesion pathways.

12E7 Antigen↗
PubMed1994

Does cord blood contain enough progenitor cells for transplantation?

We analyzed 125 blood samples obtained from umbilical cord immediately after delivery of full-term neonates. Between 0.1 and 10.4% (mean 1.13%, SD 1.34) of the density-separated glycophorin A (GPA)-negative mononuclear cells (MNC) expressed CD34 as analyzed by flow cytometry. These hematopoietic progenitor cells did not coexpress CD19, and the majority were negative for CD45RA. The number of MNC determined per ml cord blood ranged from 1 x 10(5) to 200 x 10(5) (mean 20.2 x 10(5), SD 24.7). Regression analysis revealed that a mean of 56% (n = 26, R = 0.8) and 120% (n = 35, R = 0.94) of the analyzed CD34+ MNC gave rise to day 14 colonies in the clonogenic assay when cultured without or with stem cell factor (SCF). The number and the exact phenotype of progenitor cells required for successful transplantation are not known. If the transplantation of 5 x 10(5) CD34% cells/kg body weight is required for engraftment and one-third of the progenitor cells are lost to cell processing, and if 180 ml blood can be collected from a single umbilical cord (and placenta), our data suggest that 90% of the collections do not contain enough precursors to transplant a 25 kg recipient. To meet these conditions, an average of 1439 ml cord blood would be necessary for transplantation.

Colony-Forming Units Assay↗
PubMed1994

[Intracerebral hematoma as an acute manifestation of intracranial tumors].

Sixteen patients with spontaneous intracerebral haematoma due to intracranial tumours are discussed. The total number of patients with intracranial tumour treated in the period concerned was 594, so the incidence of tumour haemorrhage was 2.7%. In most of the patients we found metastatic neoplasm (n = 6) and glioblastoma (n = 3). All patients were acute admissions. Seven were comatose, seven were somnolent, and two patients were alert. In ten patients the haemorrhage represented the first reliable clinical sign. In six patients a tumour disease was known. Fourteen patients were operated on. Six patients died. Extensive neuro-radiological examination is very important, particularly since 30% of these acute intracerebral haemorrhages occur in patients with benign intracranial tumours.

Acute Disease↗
PubMed1993

Myeloperoxidase expression in CD34+ normal human hematopoietic cells.

Bone marrow (BM), adult peripheral blood (aPB), and umbilical cord blood (CB) samples contain small proportions of CD34+ cells that include virtually all hematopoietic progenitor cells. Myeloperoxidase (MPO) is considered to be selectively expressed in cells committed to granulomonocytic differentiation. Using flow cytometry and an antibody against MPO, we studied at which stage of normal hematopoietic differentiation CD34+ cells being to express MPO. We consistently observed a characteristic MPO/CD34 staining pattern and found that 35% +/- 9% of CD34+ BM cells express MPO. The MPO+ CD34+ subset and the CD33+ CD34+ subset were of similar size and overlapped considerably. MPO+ CD34+ cells expressed high levels of HLA-D molecules, were weakly CD71/transferrin receptor positive to negative, were CD45RA+ and lacked the CD45RO isoform of the leukocyte common antigen. Additionally, MPO+ CD34+ cells were on average larger in size than MPO- CD34+ cells. Virtually identical phenotypic features have previously been described for in vitro colony-forming granulomonocytic progenitor cells. In vitro clonogenic assays performed with MPO-enriched and MPO-depleted fractions of CD34+ BM cells performed by us also suggest, but do not formally prove, that at least a portion of MPO+ CD34+ cells have in vitro cluster (10 to 50 cells/colony) or colony-forming unit granulocyte-macrophage (> or = 50 cells/colony) forming capacity. CD34+ cells from CB and aPB resembled CD34+ BM cells in that considerable proportions of them coexpressed CD33. However, in contrast to BM, CD34+ cells from CB and aPB samples lacked significant MPO expression and, in line with this, the majority of them (CB, 59% +/- 7%; aPB, 66% +/- 5%) coexpressed CD45RO.

Adult↗
PubMed1993

Rapid discrimination of early CD34+ myeloid progenitors using CD45-RA analysis.

Mononuclear cells (MNC) isolated by density centrifugation of cord blood and healthy bone marrow, and of peripheral blood (PB) from patients treated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF after chemotherapy, were double-stained with anti CD34 monoclonal antibody (MoAb) (8G12) versus anti CD45, CD45-RB, CD45-RO, and CD45-RA, respectively, and analyzed by flow cytometry. In all specimens, CD34+ MNC co-expressed CD45 at a low level and the expression of CD45-RB was similar or slightly higher. Most CD34+ MNC were negative for CD45-RO, a weak coexpression was only seen in some bone marrow (BM) and blood samples. In contrast, CD45-RA could subdivide the CD34+ population into fractions negative, dim (+), and normal positive (++) for these subgroups, and typical staining patterns were observed for the different sources of hematopoietic cells: in BM, most CD34+ MNC were RA++. In PB, their majority was RA++ after G-CSF but RA+ or RA- after GM-CSF. In cord blood, the hematopoietic progenitors were mainly RA-/RO-. Semisolid culture of sorted CD34+ MNC showed that clusters and dispersed (late) colony-forming unit-GM (CFU-GM) originated from 34+/RA++ cells, while the 34+/RA- MNC formed compact and multicentric, both white and red colonies derived from early progenitors. Addition of 20 ng stem cell factor per milliliter of medium containing 34+/RA- cord blood MNC led to a change of many burst-forming unit-erythrocyte (BFU-E) to CFU-mix which was not, at least to this extent, seen in blood and BM. We conclude that early myeloid CD34+ cells are 45+/RA-. Because this population excludes 34+/19+ B cells and 33+ myeloid cells, both of which are RA++, two-color flow cytometric analysis using CD34 and CD45-RA facilitates the characterization and quantification of early myeloid progenitor cells.

Antibodies, Monoclonal↗
PubMed1993

Condensation of the chromatin at the membrane of an apoptotic nucleus is not associated with activation of an endonuclease.

A current hypothesis holds that chromatin fragmentation into oligonucleosomal patterns is an early event during apoptosis. In contrast, induction of apoptosis in cultured hepatocytes by TGF-beta 1 was not associated with DNA fragmentation into oligonucleosomes in hepatocyte monolayers and apoptotic fragments. For a more rigorous test of the hypothesis we performed a number of experiments. We compared nuclear changes resulting from TGF-beta 1 with those induced by Ca2+, a known activator of endonuclease. The morphology of apoptotic and Ca(2+)-treated nuclei was different as judged by DNA staining with Hoechst 33258. Likewise, electron microscopy of apoptotic nuclei showed characteristic condensation of the chromatin as well as dissolution of the nucleolar structure and nuclear fragmentation, changes not seen after Ca2+ treatment, after three hours of incubation. Analysis of DNA fluorescence of nuclei by FACS revealed that treatment with Ca2+ reduced the signal by 20%. In contrast, nuclei from TGF-beta 1-treated hepatocytes did not exhibit a reduced signal and after sorting by FACS, apoptotic nuclei remained in the 2N and 4N fractions. The absence of detectable DNA fragmentation in apoptotic nuclei was further verified by in situ nick translation, not only in hepatocytes but also in a mouse lymphoma cell line. From these findings we conclude that activation of an endonuclease is not an early event on the pathway to morphologically recognizable apoptosis.

Animals↗
PubMed1993

Use of flow cytometric CD34 analysis to quantify hematopoietic progenitor cells.

This review summarizes our experiments on flow cytometric analysis of CD34 positive mononuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only 1%-9% of the CD34+ MNC. Due to the variable numbers of CD34+ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Antigens, CD↗
PubMed1992

Recovery kinetics after chemotherapy and circulating mononuclear cells expressing the CD34 antigen in pediatric cancer patients.

Hematopoietic progenitor cells collected from the peripheral blood are capable of restoring hematopoiesis after myeloablative therapy. The numbers of circulating mononuclear cells expressing the CD34 antigen were calculated and the colony-forming capacity was determined in 26 blood samples, which were drawn during rapid rise of leukocytes after chemotherapy cycles that were followed by aplasia. Culture assay after 14 days revealed a median 507 (210-2029) myeloid progenitors (CFU-GEMM/GM) per 10(5) nucleated cells (NC) in 13 CD34-positive samples, and only a median 76 (9-224) in 13 CD34-negative ones (p less than 0.001). Median 343 (175-2450) erythroid burst-forming units (BFU-E) per 10(5) NC were detected in the CD34-positive samples, whereas only 72 (10-315) per 10(5) NC were found in the negative ones (p less than 0.01). The percentage of CD34-positive cells clearly correlated with the growth of CFU-GEMM/GM and BFU-E (p less than 0.01). The content of CD34-positive cells in circulation was determined within 120 min by FACS analysis and predicted colony-forming capacity of circulating mononuclear cells. These observations will help to select the optimal individual days for leukaphereses.

Adolescent↗
PubMed1992

Induction of apoptosis in cultured hepatocytes and in the regressing liver by transforming growth factor-beta 1 occurs without activation of an endonuclease.

In previous studies in vivo apoptotic liver cells were found to be positive for transforming growth factor-beta 1 (TGF-beta 1). In hepatocyte cultures TGF-beta 1 induced rounding up and fragmentation of the cells into multiple vesicles. As revealed by the DNA specific stain H33258 the chromatin of these cells condensed and segregated into masses at the nuclear membrane, followed by nuclear fragmentation. Ultrastructurally the cytoplasm was well preserved as demonstrated by the presence of intact cell organelles. These features strongly suggest that occurrence of apoptosis. Furthermore we administered TGF-beta 1 in vivo using an experimental model in which regression of the liver was initiated by a short preceding treatment with the hepatomitogen cyproterone acetate (CPA). Two doses of 1 nM TGF-beta 1/kg each augmented the incidence of apoptotic hepatocytes 5-fold. These studies strongly suggest that TGF-beta 1 is involved in the initiation of apoptosis in the liver In TGF-beta 1 treated hepatocytes both from the liver and monolayer culture no DNA fragmentation into oligosomes could be detected. Comparison of nuclei in which endonuclease was activated by Ca2+ with apoptotic nuclei revealed no obvious similarities, as demonstrated by FACS analysis, H33258 staining and electron microscopy. Thus, apoptosis induced by a growth inhibitor obviously occurs without activation of an endonuclease.

Animals↗
PubMed1991

Characterization of bovine haemopoietic progenitor cells using monoclonal antibodies and fluorocytometry.

Monoclonal antibodies against bovine leucocyte cell surface differentiation antigens were used in combination with a fluorescence activated cell sorter to enrich bovine haemopoietic progenitor cells present in bone marrow cell populations prior to in vitro culture. After two sequential centrifugations of the bone marrow cell suspension through Ficoll-Paque, the interface fraction was stained with a cocktail of monoclonal antibodies directed against mature monocytes/macrophages, granulocytes and lymphocytes. Using appropriate electronic window settings on a FACStar Plus, cells with a high 90 degrees light scattering property (granular cells), a low forward light scattering property (erythrocytes and reticulocytes) and cells positive for monoclonal antibodies specific for lineage-restricted leucocyte markers were removed and the negative cell fraction collected. These negatively-selected cells were stained with monoclonal antibodies specific for a pan-leucocyte or a MHC class II marker and the positive cell population was collected in a second sort and subsequently submitted to culture. All erythroid and granulocyte/macrophage colony forming cells expressed MHC class II antigens, as well as the pan-leucocyte antigen. These same progenitors did not bind any of a variety of monoclonal antibodies directed against lineage-specific antigens on lymphocytes, granulocytes or monocytes/macrophages, although they did bind monoclonal antibodies recognizing MHC class I antigens. Between 85% and 91% of the isolated cells seeded were capable of forming erythroid or granulocyte/macrophage colonies within 5 to 10 days, thus increasing the plating efficiency of these cell types in bone marrow populations by at least 60 fold.

Animals↗
PubMed1991

CD34-positive cell proportions in peripheral blood correlate with colony-forming capacity.

Blood samples were examined from 25 children with malignancies during hematopoietic recovery following chemotherapy-induced aplasia and from 9 children undergoing tonsillectomy. The proportion of CD34-positive peripheral blood mononuclear cells (PBMNC) evaluated by flow cytometry was compared with the number of colonies (granulocyte-macrophage colony-forming units, CFU-GM; mixed-lineage colony-forming units, CFU-GEMM; and erythroid burst-forming units, BFU-E) grown in methylcellulose medium within 2 weeks. A mean of 1387 myeloid colonies (495-4480) per 10(5) PBMNC seeded developed from 13 samples with detectable CD34 populations (between 0.9% and 5.6%), whereas only 152 (9-386, p = 0.002) and 65 (12-137, p = 0.005) colonies were formed from 12 patient and from 9 control samples in which the percentage of CD34-positive cells was too low for analysis. Linear regression analysis revealed that CD34 positivity correlates with colony-forming capacity (p = 0.0008, r = 0.782). Flow cytometric evaluation of the CD34 proportions can thus predict the in vitro colony-forming capacity of peripheral blood prior to leukapheresis.

Adolescent↗
PubMed1991

Relative enrichment of hematopoietic progenitor cells: efficiency of a repeated density centrifugation.

Low-density cells were prepared from 11 bone marrow samples by centrifugation on Ficoll-sodium diatrizoate. Repeated density gradient centrifugation of the cells collected from the interface of the first gradient removed most nonnucleated erythroid cells. A mean of 47.9% (19.4% to 76.0%) of the mononuclear cells collected after the initial centrifugation were recovered from the interface of the second gradient, whereas 13.3% (3.7% to 34.9%) of the MNCs were counted in the high-density pellet and 38.9% (3.8% to 65.7%) of the MNCs were lost unspecifically. In contrast, a mean of 71.7% (43.0% to 91.3%) of the colony-forming units were recovered from the interface after the second centrifugation (as determined by colony formation assays), whereas only 3.2% (0.5% to 7.0%) were found in the high-density pellet. The unspecific loss of colony-forming units was 25.1% (1.7% to 51.4%). The results demonstrate a relative enrichment of colony-forming units in the culture assay by 1.7 times (average). The method is recommended as an additional preparative step before fluorescence-activated sorting of viable cells, because removal of most erythrocytes and late normoblasts strongly reduces the time required for sorting.

Adolescent↗