Selection of CD34-Positive Blood Cells for Allogeneic Transplantation: Approaches to Optimize D34-Cell Recovery, Purity, Viability, and T-cell Depletion.

BACKGROUND: Methods for clinical-scale selection of CD34-positive hematopoietic stem and progenitor cells have facilitated allogeneic transplants using HLA-mismatched healthy donors. We examined different approaches to purify mobilized CD34+ cells, focusing on yield, purity, and viability of the selected cells and T-cell depletion levels. METHODS: Sixty-seven CD34-positive selections were performed for a total of 37 allogeneic transplantations, 23 of which from HLA-haploidentical donors. The selection devices were the Isolex((R)) 300i (v. 1.12) used alone (n =13) or with the SuperMACS (n = 29); the CliniMACS (n = 3); and the Isolex 300i (v. 2.0b1). The latter was used for CD34-positive selection (n = 7) and combined CD34+/CD4 8 19-negative selections (n =15). DNAse was included to reduce cell clumping. RESULTS: With the Isolex 300i (v. 1.12), the median CD34+-cell recovery increased from 51% (without DNAse) to 61% (15 mg DNAse) and 70% (7.5 mg). DNAse (5 mg) was used for 22 selections with the Isolex (v. 2.0b1) without cell clumping. CD34-positive cell purity, yield, and viability, as well as the degree of CD3 depletion varied with the selection device and procedure used. CONCLUSION: With regard to all of the above-mentioned parameters, the best results were obtained with the Isolex 300i (v. 2.0b1). Values achieved for CD34-positive cells were 98% for purity, 50-60% for yield, and > 96% for cell viability; T-cell depletion was 4.5 to > 5 log. The automated and closed system provides target cells that are free of both magnetic particles and murine monoclonal antibody. Copyright 2000 S. Karger GmbH, Freiburg

Induction of the bradykinin B2-receptor, but not of the bradykinin B1-receptor, by interleukin-1beta in cultivated human decidua-derived cells.

Aspects of the pathophysiological steps leading to preterm labor after intrauterine infection can be studied in a cell culture model of decidua-derived cells. In this model, bradykinin (BK) increases the release of arachidonic acid (AA), the precursor of labor-promoting prostaglandins. The release is more than additively increased when cells are pretreated with interleukin-1beta (IL-1beta). Binding studies indicate that the expression of the bradykinin B2-receptor (B2R) protein rises to up to 300% of control after incubation with IL-1beta for 24 h. Thus, there is an increased capacity of mediating the response to BK. These findings suggest a pivotal role for the kallikrein-kinin system (KKS) during labor caused by intrauterine infection. However, there was no binding to the bradykinin B1-receptor (B1R), and it could not be induced by IL-1R, which is a unique finding compared with other cell systems.

CD99 (MIC2) expression in paediatric B-lineage leukaemia/lymphoma reflects maturation-associated patterns of normal B-lymphopoiesis.

We have recently shown that CD99 (MIC2) is differentially expressed during normal early B-cell development in the bone marrow (BM). Since immature B-cell precursors (BCP) are assumed to correspond to some extent to acute lymphoblastic leukaemia (ALL) and non-Hodgkin's lymphoma (NHL) cells with respect to patterns of phenotypic differentiation, we wondered whether the particular maturation-associated expression patterns of CD99 in the normal BCP stages were conserved also in malignant cells. Therefore we compared malignant and physiological B cells from paediatric ALL/NHL and normal BM samples with respect to CD99 expression using selective gating and semi-quantitative flow cytometry. Common-ALLs (n = 45) were similar to their corresponding, very immature BCPs (stage 1) in expressing very high levels of CD99. Most pre-B ALLs (n = 16) were also CD99hi and thus differed from the patterns found in normal cytoplasmic mu-chain+ (cmu+) pre-B cells (stage 2, CD99lo). In particular, we found that those pre-B-ALL cases which were CD34+ also showed higher CD99 expression than the CD34- cases. This prompted us to investigate the levels of CD99 in those rare normal BCPs which also coexpress CD34 and cmu; these cells, which are transitory from stage 1 to stage 2, were found also CD99hi, thus precisely reflecting the patterns of CD34+ pre-B ALLs. The blasts of Burkitt-type B-cell ALL/NHL samples (n = 13) expressed considerably less CD99, similarly to the more differentiated BCP stages 2 (cmu+) and 3 (surface mu-chain+). In summary, we found that paediatric B-lineage malignancies display remarkable synchrony regarding the levels of CD99 expression compared to their putative normal counterparts.

G-CSF versus GM-CSF for stimulation of peripheral blood progenitor cells (PBPC) and leukocytes in healthy volunteers: comparison of efficacy and tolerability.

This study compared two recombinant human (rh) hematopoietic growth factors in healthy volunteers for stem cell stimulation. Granulocyte colony-stimulating factor (G-CSF, n=9) or granulocyte-macrophage colony-stimulating factor (GM-CSF, n=8) was given subcutaneously for 5 days (5 microg/kg/day). Controls (n=5) received no growth factor. Laboratory parameters and side effects were monitored for 8 days. Within 24 h, both cytokines led to a rapid increase of leukocytes, the majority of which were granulocytes. Compared with the controls (n=5), the increase on day 5 in the G-CSF/GM-CSF groups was 37-/10-fold (CD34+ cells), 5.2-/2.4-fold (leukocytes), 7.2-/3.0-fold (granulocytes), 7.4-/4.4-fold (monocytes), 1.7-/1.1-fold (lymphocytes), 9.8-/2.7-fold (basophils), 2.3-/9.6-fold (eosinophils), and 1.9-/1.6-fold (reticulocytes). The mobilization of myeloblasts, promyelocytes, myelocytes, and metamyelocytes coincided with the pronounced increase of CD34 + PBPC observed on day 4. Serum levels of uric acid (UA) and lactic dehydrogenase (LDH) increased under G-CSF, and platelets decreased after G-CSF discontinuation. Rash at the injection site occurred in 50% of the GM-CSF-treated volunteers. Seven volunteers in the GM-CSF group and six in the G-CSF cohort complained of flu-like symptoms, including musculoskeletal pain. We conclude that, in terms of tolerance and mobilization of CD34+ cells and leukocytes, G-CSF is superior to GM-CSF, but higher levels of UA and LDH and late decrease in platelets make monitoring of these parameters necessary.

The bradykinin B2-receptor in human decidua.

Bacterial infection of the amniotic cavity is one of the most frequent causes of preterm delivery. Bacterial products activate a network of autocrine and paracrine mediators in fetal membranes and decidua, with prostaglandins finally inducing contractions of the myometrium. Bradykinin and its B2-receptor (B2R) seem to be part of this network. In cultured decidua-derived cells, bradykinin stimulates the release of arachidonic acid, interleukin-6 (IL-6), and interleukin-8 (IL-8). These effects are prevented by the specific B2R antagonist Hoe 140. Using a pooled antiserum against peptide sequences of the B2R protein, the receptor can be visualized immunocytochemically. The cells contain mRNA for the B2R, as shown by reverse transcriptase polymerase chain reaction (RT-PCR). Binding studies reveal specific and saturable binding sites for bradykinin with characteristics of the B2R. Binding of bradykinin to the cells is enhanced by the inflammatory mediator interleukin-1beta. In summary, human decidua-derived cells express the B2R, its expression is upregulated in response to interleukin-1beta, and bradykinin stimulates the secretion of further mediators by these cells. Thus, bradykinin and the B2R could play a central role in decidual activation. If so, B2R antagonists would add to established tocolytic therapies that are applied together with antibiotics in cases of chorioamnionitis at low gestational age.

Flow cytometric monitoring of hematopoietic reconstitution in myeloablated patients following allogeneic transplantation.

BACKGROUND: We report a routine flow cytometric (FACS) approach to quantify circulating leukocytes (NC) in myeloablated patients before and during regeneration after allogeneic transplantation of either whole bone marrow (BM) or of highly purified (> 99%) blood-derived CD34(+) cells (PBSC). METHODS: Blood samples were analyzed daily between infusion of the transplant and hematopoietic reconstitution. Significant differences in the composition of NC types and CD34(+) cells were observed between the two CD34 sources. The detection threshold for NC was roughly 1 cell per w L blood. RESULTS: The cell nadir of < 100 NC/ microL was reached on Day +4 (BM) and on day 0 (PBSC), when unusual CD34(+) cells of recipient genotype were detected in all patients. They were not clonogenic, showed high CD34 expression, but were negative for CD45, CD38, CD33, CD50, HLA-DR and Stro-1. Between Days +5 and +16, the onset of hematopoietic reconstitution was clearly detectable in multi-parameter evaluation of the FACS data. This was a median of 3.5 days before NC increased above 200/ w L blood and 4-10 days before granulocyte counts were > 500/ microL. It was marked by the appearance of monocytes, immature (CD38(+)) granulocytes, and clonogenic donor CD34(+) cells exhibited normal size and phenotype. DISCUSSION: We conclude that dynamic FACS analyses can reliably detect hematopoietic reconstitution, but also graft rejection, before a visible increase NC numbers. This may have considerable impact on clinical management strategies.

Comparative phenotype mapping of normal vs. malignant pediatric B-lymphopoiesis unveils leukemia-associated aberrations.

Leukemic cells of B-lineage acute lymphoblastic leukemia (ALL) are regarded as the malignant counterparts of immature, physiologic B cell precursors (BCPs). To determine whether phenotypic differences exist between these corresponding cell types, we investigated samples of normal pediatric bone marrow (n=30) as well as of B-precursor ALL at diagnosis (n=53; common and pre-B subtype). Using three-color multiparameter flow cytometric analysis, we compared the leukemic populations with the physiologic BCPs of corresponding maturity with respect to the intensity with which they expressed a series of antigens. In some of these antigens, leukemia-associated aberrations were frequently observed. In particular, overexpression of CD10 was displayed by 65% of ALL samples, whereas 58% of leukemic cases aberrantly exhibited very low or no CD45RA expression. Regarding CD11a and CD44, 47% and 35% of ALL populations were aberrant as defined by either the absence or significant overexpression of the antigen. In contrast, antigen densities of CD49d, CD49e, and CD99 on leukemic cells were in the normal range of values for BCPs. Combining the patterns of frequently aberrant markers in a comprehensive analysis, we were able to identify individual phenotypic leukemic cell aberrations in up to 98% of investigated cases. CD10 and/or CD45RA were aberrant in 86% of cases overall, emphasizing the high discriminative potential of these two markers. Using comparative phenotype mapping based on quantitatively aberrant, leukemia-associated antigenic patterns, we were able to detect leukemic blasts among normal bone marrow cells at frequencies as low as 10(-5). We speculate that our approach may have a profound impact on the development of new strategies for minimal residual disease investigations in patients with BCP-ALL.

Quantification of CD34+ cells: comparison of methods.

BACKGROUND: Quantification of CD34+ stem and progenitor cells is predominantly performed by flow cytometric analysis of cells prepared by whole blood staining and red cell lysis. This method also includes cell washing, which is thought to cause the destruction and loss of some of the nucleated cells (NCs). To address this cell loss and its influence on the outcome of enumeration, three techniques for preparing cells for quantification of CD34+ cells were compared. STUDY DESIGN AND METHODS: Blood (n = 179), bone marrow (n = 60), and leukapheresis components (n = 64) were examined by the use of density separation of mononuclear cells (MNCs) and two red cell-lysis procedures (wash and no-wash). Cell counts were determined in the original materials and after cell preparation. Absolute CD34+ cell counts were calculated using the flow cytometry-analyzed proportions of CD34+ cells and the various white cell counts. RESULTS: Depending on the cell source and the cell preparation chosen, the loss of NCs ranged between 12 percent and 89 percent of the original white cell number. This loss of NCs was exclusively due to cell washing and predominantly affected granulocytic cells. Analysis of the flow cytometry data revealed that the relative CD34+ values in blood and bone marrow were roughly threefold higher in density separated MNCs than in those that underwent the lyse-and-wash procedure. Calculation of absolute CD34+ cell counts confirmed that the MNC procedure underestimated the CD34+ cell content by a median of 26 percent (blood), 21 percent (bone marrow), and 5 percent (leukapheresis component) when compared with the median yield from analysis and cell counting performed after the lyse-and-wash procedure. On the other hand, the conventional lysis procedure, which applies the original white cell counts for CD34+ quantification, was shown to overestimate the CD34+ cell content by a median of 1.2-fold, 1.33-fold, and 1.13-fold, respectively. CONCLUSION: Neither density separation nor the whole-blood lysis procedure seems appropriate for optimal CD34+ quantification.

Multiparameter phenotype mapping of normal and post-chemotherapy B lymphopoiesis in pediatric bone marrow.

We studied the differentiation profiles of B cell precursors (BCP) in normal and post-chemotherapy pediatric bone marrow (BM) using multiparameter flow cytometry. The goal of our study was to draw a comprehensive phenotypic map of the three major maturational BCP stages in BM. By correlating lineage-associated markers, CD45RA, and several adhesion molecules, the stage-specific patterns were found to differ in certain details from previously published concepts. Among the earliest BCP, a subset of CD34+ CD10(lo) precursors was repeatedly observed in addition to the well characterized CD34+ CD10(hi) CD19+ majority of cells. Only two-thirds of these CD34+ CD10(lo) cells expressed CD19. However, uniformity of phenotypic features, absence of T lineage markers, and the regeneration kinetics after chemotherapy suggest the B lineage affiliation of the CD34+ CD10(lo) precursors in general. In the more mature BCP, expression of CD10, CD20, cytoplasmic and surface mu chains (c mu and s mu) was observed to overlap more than previously recognized. We found that CD20 and c mu appear early during B cell ontogeny (already on CD34+ BCP), and that CD10 is lost late, following the onset of s mu expression. Differences between normal and post-chemotherapy BM specimens regarding the phenotypic appearance of BCP were exclusively due to differences in the subset composition, as post-chemotherapy samples showed a preponderance of immature stages. Our observations may build a framework for comparing leukemic cells with their normal counterparts to define possible leukemia-associated aberrations useful for residual disease studies.

Evidence for bradykinin B2-receptors on cultured human decidua cells.

Bradykinin is known to be present at sites of acute inflammation and to exert its potent inflammatory effects mainly via the bradykinin B2-receptor. Recently, bradykinin dependent processes have been described in cultured human decidual cells, so that bradykinin may expand the list of paracrine factors involved in labour induction. In this paper we present the results of in vitro studies giving evidence that these cells carry the bradykinin B2-receptor. By immunocytochemical methods the receptor protein was localized on decidual cells. Analysis of cellular extracts of cultured decidual cells by RT-PCR showed the presence of the specific mRNA coding for the bradykinin B2-receptor. Binding studies revealed a single, saturable and specific binding site for bradykinin of high affinity (Kd = 0.85 nM, Bmax = 436 fmol/mg protein). Competitive binding studies showed displacement of [3H]-bradykinin by HOE 140, but not by the ligands for the bradykinin B1-receptor, des-Arg10-kallidin and [Leu8]-des-Arg9-bradykinin. The results are consistent with the presence of the bradykinin B2-receptors.

Bradykinin stimulates interleukin-6 and interleukin-8 secretion of human decidua derived cells.

Cytokines are known to participate in the process of parturition within a paracrine network in fetal membranes. Bradykinin can also contribute to this process, increasing the release of eicosanoids from decidua cells. In this study, we present evidence for a cross-link between bradykinin and the cytokines. Short and long term cultures of human decidua cells were incubated for 4 h and 24 h with and without bradykinin. IL-6 and IL-8 were determined in the supernatants by ELISA. Results show a large interindividual variability in the basal secretion of IL-6 and IL-8 and a clear increase in the secretion of both ILs in response to bradykinin. By this amplifying effect on cytokine secretion, bradykinin can initiate local and systemic effects of amniochorionitis.

The B2 receptor on cultured human decidua cells: release of arachidonic acid by bradykinin.

In vitro studies on cultured human decidual cells present evidence for the expression of the bradykinin B2 receptor. This protein was localized by immunocytochemical methods at the cellular plasma membrane and the mRNA was found in cellular extracts by RT-PCR. As a biological effect the release of 14C-AA was observed. Cells that had been incubated for 24 h with 14C-AA showed a bradykinin (BK)-induced release of radioactivity into the culture medium. This response was prevented by the specific bradykinin B2 receptor antagonist HOE 140. Pretreatment with LPS resulted in an increase of the spontaneous release of radioactivity. Adding BK, a further release of about 40-50% was to be shown. We conclude that BK can play a role in the case of bacterial infection of the fetal membranes AND may stimulate labour by an augmented production of prostaglandins.

High and low molecular weight kininogen and plasma prekallikrein/plasma kallikrein in villous capillaries of human term placenta.

This study examined the expression and presence of components of the kallikrein-kinin system in human term placenta. Immunohistochemical studies localized H-kininogen and plasma prekallikrein/plasma kallikrein to endothelial cells of placental villous capillaries. In larger placental blood vessels and umbilical cord, neither kininogens nor kallikreins were detected. High (H) and low (L) molecular weight kininogen, plasma prekallikrein and plasma kallikrein were detected by Western blot analysis in human term placenta and in maternal and fetal blood, whereas tissue kallikrein was not. Furthermore, mRNA of plasma prekallikrein was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in placental homogenates, while mRNA of H-kininogen, L-kininogen and tissue kallikrein was not. Because H-kininogen and plasma prekallikrein circulate in a complexed form, we suggest that endothelial cells bind kininogen and plasma prekallikrein in which they are secreted by the fetal liver from fetal blood. The co-localization of kininogen and plasma prekallikrein/plasma kallikrein suggests that kinins could be generated locally in placental capillaries. When released, they may play a role in regulating placental blood flow and transplacental transport of substrates and metabolites.

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood.

Flow cytometric DNA analysis was performed in combination with three-colour immunological staining of cell surface antigens on density-separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell-cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5/micrograms per kg body weight of either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-simulating factor (GM-CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34- MNC, the proportion of cells in S/G2M phase increased from < 0.1% to 0.75 +/- 0.4% (GM-CSF) and to 1.34 +/- 0.75% (G-CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non-cycling status of PB and progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cells sources. However, in vitro stimulation of these progenitor cells using IL3, GM-CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2m phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized 'stem cells' which may require the BM micro-environment for adequate proliferation in vivo.

The composition of CD34 subpopulations differs between bone marrow, blood and cord blood.

Our previous data obtained by flow cytometry and by clonogenic assay had consistently shown a lower cloning efficiency of hematopoietic progenitor cells in bone marrow (BM) compared to those in blood (PB) or in cord blood (CB). Also, recent clinical reports have described more rapid reconstitution after PB than after BM transplantation. We have applied two- or three-color flow cytometric analysis using monoclonal antibodies directed against the stem- and progenitor cell antigen CD34, in combination with other cell surface markers. We report significant differences in the composition of progenitor cells contained in BM (238 specimens from 53 healthy donors and from patients in remission), PB (301 samples from 92 patients with or without cytokine support) and CB (n = 37). Leukapheresis products (Pher, n = 69) were included in the study as well as positively selected CD34+ cells obtained from BM (BMsel, n = 2), PB (PBsel, n = 28) and CB (CBsel, n = 5). We used monoclonal antibodies directed against CD7, CD19, CD34, CD38, CD45RA and glycophorin A. The highest proportion of CD34+ cells (in % of the MNC) was found in BM (mean 5.37% +/- 4.5). In the other sources, the mean values were 1.79% +/- 2.46 (PB), 1.48% +/- 1.81 (Pher) and 1.1% +/- 1.69 (CB). However, BM was the only source in which a considerable proportion of the CD34+ cells coexpressed the B cell antigen CD19 (mean 30.1%, median 28, range 0 to 84%). The amount of earlier myeloid progenitors as determined by their non-expression of the CD45RA antigen was lowest among BM CD34+ cells (26.7% +/- 16.6). In the other sources, the respective values were 57.5% +/- 17.9 (PB), 63.6% +/- 13.9 (Pher) and 70.4% +/- 16.1 (CB). These results were confirmed by subtype analyses of the CD34+ cells positively selected from the three sources. Enrichment showed minor CD34+ subpopulations to be identified. The mean proportions of B cell progenitors were 0.11% +/- 0.24 (PBsel) and 1.3% +/- 1.42 (CBsel) of the CD34+ cells. The CD34+ cells from all cell sources coexpressed GPA (median 0.15%, range 0 to 1.8%) and CD7 (median 0.25%, range 0 to 1.2%). The proportion of CD38- cells ranged from 0.7 to 4% of the CD34+ MNC. Thus, despite higher CD34 counts in BM, the relative proportions of myeloid progenitors are higher in PB and in CB. This suggests that, if timely reconstitution depends on the number of CD34+ cells transplanted, the mean number of "stem cells' (SC) required is 1.4-fold (for myeloid cells) or 2.2-fold (for earlier myeloid cells) higher for BM than for PB.

Characterization of hematopoietic stem cells.

On the basis of density-separated mononuclear cells isolated from bone marrow, peripheral blood, and cord blood, we have repeatedly shown good correlation between two-color flow cytometric (FACS) CD34 analysis and colony formation in the clonogenic assay. We have analyzed the distributions of CD34 subpopulations in these three stem cell sources using patients' and donors' bone marrow biopsies (n = 196), cord blood samples from full-term deliveries (n = 14), and peripheral blood from patients mobilized by chemotherapy and/or cytokine treatment (n = 258). Irrespective of absolute cell counts, the mean (+/- SD) proportions of CD34+ cells were clearly higher in bone marrow (5.6 +/- 4.6% of mononuclear cells) than in peripheral blood (1.9 +/- 2.6) and cord blood (1.7 +/- 2.6). However, two-color FACS analyses revealed significant differences among these cell sources with regard to their distribution of CD34 subpopulations: B-cell progenitors coexpressing CD34 and CD19, at considerable concentrations of > 0.5%, were only found in bone marrow (mean 30 +/- 24.3% of CD34+ mononuclear cells, median 28.7%, minimum 0%, maximum 83.3%). In addition, CD34+ cells in S/G2M phase were never detected in peripheral blood or cord blood, but only in bone marrow at a concentration of 10-15% of CD34+ mononuclear cells. On the other hand, the proportions of relatively immature myeloid progenitors, as characterized by not expressing CD45RA and by higher clonogenic capacity, were significantly higher in cord blood (76.7 +/- 17.2) and peripheral blood (58.2 +/- 17.5) than in bone marrow (26.4 +/- 16.7). These data were confirmed by analysis of apheresis products and of progenitors positively selected from different cell sources, and they may explain why, in autologous transplantations of analogous amounts of CD34+ cells, peripheral blood is superior to bone marrow. We conclude from our results that if successful transplantation and timely recovery depend on the number of CD34+ cells transplanted, the mean amount of stem cells required is 1.4- (for myeloid cells) or 2.2-fold (for early myeloid cells) higher for bone marrow than for peripheral blood.

[Phenotypic differences between CD34 positive cells in various transplantation tissues]. / Phänotypische Unterschiede zwischen CD34 positiven Zellen in verschiedenen Transplantations-Materialien.

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)

Visualization of tissue kallikrein in human breast carcinoma by two-dimensional western blotting and immunohistochemistry.

Tissue kallikrein is well known to liberate the vasoactive peptide kallidin from L-kininogen. Recently it was reported to activate matrix degrading metalloproteinases in vitro and to be present in gastric carcinoma cells. By immunohistochemistry we localized tissue kallikrein in the cytoplasm of ductal breast cancer cells. In addition, two-dimensional Western blotting was used to further characterize its biochemical properties. By this method immunoreactive tissue kallikrein was found to have a molecular mass of 25 kDa and an isoelectric point close to pH 6. Furthermore its presence in human milk could be demonstrated.

Identification of immunoreactive tissue kallikrein in human ductal breast carcinomas.

Various proteases have been shown to be present in malignant breast tissue. Although the question of the involvement of tissue kallikrein, a serine protease, in the pathophysiology of tumours has been raised, the presence of this enzyme in human breast carcinoma has so far not been examined. In the present study, both neoplastic and normal human breast are scanned by immunocytochemistry for the presence and cellular localization of tissue kallikrein. In the healthy breast, tissue kallikrein was observed as a deposit of immunoreactive material that localized in the apical portion of duct cells. In the malignant breast tumours surveyed, the enzyme was observed only in ductal carcinomas, whereas lobular carcinomas were devoid of immunostaining. In ductal carcinomas, the immunoreactivity for tissue kallikrein appeared to be associated with gradations of malignancy, being absent in dedifferentiated tumours. The presence of tissue kallikrein in malignant breast tumours poses the question of the role of this enzyme in malignant breast tissue. The enzyme may participate within the tissue either in proteolytic processes (it has been shown to activate procollagenase) or by enhancing vascularity or mitogenicity by the generation of kinins.