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D Printz

Publications and source records attributed to D Printz.

51 records · Page 3Linked to original sources
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↗
PubMed1991

Isolation stress increases tyrosine hydroxylase mRNA in the locus coeruleus and midbrain and decreases proenkephalin mRNA in the striatum and nucleus accumbens.

Isolation of adult animals represents a form of psychological stress from which the animals cannot escape. In order to assess the effect of this stressor on neurochemical substrates in the brain, we assessed behavior and measured tyrosine hydroxylase and proenkephalin mRNA levels in selected brain areas by in situ hybridization histochemistry. Tyrosine hydroxylase (TH) mRNA levels in the locus coeruleus (LC) were significantly and progressively increased by 18, 42 and 68% after 7, 14 or 28 days of isolation, respectively. TH mRNA in the midbrain was transiently increased by isolation. Levels were significantly elevated by 34 and 48% above group-housed controls in the ventral tegmentum and the substantia nigra, respectively, after 14 days of isolation. In the forebrain, proenkephalin (PE) mRNA levels were found to be transiently decreased by 29% in the anterior and medial aspects of the caudate-putamen and the nucleus accumbens after 7 or 14 days of isolation stress, but the levels returned toward control levels after 28 days of isolation. Behavioral tests indicate that isolated animals progressively became more aggressive with duration of stress and showed a small but significant decrease in locomotor activity. The results demonstrate that a physically noninvasive stressor such as isolation of adult male rats can produce significant alterations in brain neurochemistry. The neurochemical responses observed may represent a brain mechanism designed to help the organism adapt to or protect from the deleterious effects of chronic psychological stress.

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↗
PubMedDate not supplied

[Phenotypic differences between CD34 positive cells in various transplantation tissues].

Transplantations to restore the hematopoietic system were originally performed with cells from the bone marrow (BM) (20) which was considered the only cell source comprising repopulating progenitor cells. The discovery that chemotherapy induced the mobilization of CD34+ cells into the peripheral blood (PB) (14) gave rise to the successful autologous transplantation of PBSC (1, 13). Also cord blood (CB) was found to contain considerable numbers of "stem cells", and to date at least 42 allogeneic transplantations have been performed with this cell source (22, J. Wagner, personal communication). Further investigations led to the successful autologous transplantation of positively selected CD34+ cells from BM and PB (18), and the latest results indicate that it is promising to transplant purified CD34+ cells obtained from cytokine-stimulated donors (4, 10, 15-16). Despite such achievements it remains unclear how many "stem cells" are required per kg of the recipient and how they are phenotypically characterized. In this communication we give examples of typical differences observed by flow cytometry and clonogenic assay between the CD34+ cells contained in the different cell sources. They may explain why it is not sufficient only to analyze the CD34+ cell populations which may represent progenitors of different lineages as well as of various states of differentiation. CB CD34+ cells are early myeloid progenitor cells with the highest incidence of CFU-mix among the three cell sources. They have a high proliferative potential in vitro. They hardly coexpress B cell antigens and they are partially negative for CD38.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMedDate not supplied

Kinetics of apoptosis and secondary necrosis in cultured rat thymocytes and S.49 mouse lymphoma and CEM human leukemia cells.

Cell culture systems are widely used to study metabolic changes during apoptosis. In cell culture, unlike in vivo, apoptotic cells are not phagocytosed and eventually lyse (secondary necrosis). This is of practical importance because metabolic changes seen in cultures may be due to the transition from apoptosis to necrosis, rather than to the induction of apoptosis itself. In the present study, we followed the kinetics of the occurrence of several indicators of cell death in rat thymocytes and mouse lymphoma (S.49), and human leukemia (CEM) cell cultures after dexamethasone treatment (10(-6) M). The presence of apoptosis and secondary necrosis was demonstrated by electron microscopy. Nuclear condensation and fragmentation, which are considered to reflect early stages of apoptosis, were visualized with Hoechst fluorescent dye H 33258 for quantitative determination by light microscopy. In S.49 and CEM cultures their incidence increased after glucocorticoid treatment, but remained at relatively low levels not exceeding 6-9% until 36 h (S.49) or 3-4% until 92 h (CEM). The trypan blue positive cells, however, increased steadily to about 60%. Furthermore, flow cytometry (single parameter DNA analysis after propidium iodide staining) revealed the occurrence of cells with reduced DNA fluorescence. Morphological and biochemical (internucleosomal DNA cleavage) analysis of FACS-sorted cells showed that early after dexamethasone the majority of them were apoptotic. In S.49 and CEM cell cultures no clear-cut time lag between increase in cells with reduced DNA fluorescence, chromatin condensation/fragmentation, and the uptake of trypan blue could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗