Comprehensive polymorphism analysis of ABO using allele-specific separation by bead technology and subsequent sequencing.

The ABO system is the most important blood group system in transfusion and transplantation medicine. Over the last decade ABO genotyping was introduced to complement serological analysis but because of the enormous diversity at the ABO locus, DNA-based typing is highly sophisticated. In some genotype combinations and especially when dealing with hybrid genes, cis/trans linkage analysis is necessary. Therefore, we developed a simple and reliable approach to isolate ABO haplotypes. The described method physically separates diploid genomic DNA using probe-dependent allele hybridization followed by magnetic bead separation. After this haplotype-specific extraction (HSE), sequencing based typing (SBT) was performed to analyze coding and non-coding respectively regulatory regions of ABO.

GFI1 as a novel prognostic and therapeutic factor for AML/MDS.

Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting histone deacetylases to target genes. We present data that low expression of GFI1 is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1 expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1 expression. Leukemic cells from animals that express low levels of GFI1 show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1 expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KD mice and from AML patients with low GFI1 levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1 expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.

Determinants of carcinogen-induced malignant conversion in rat brain: proliferative properties of neural precursor cell subpopulations analyzed by multi-parameter flow cytometry.

The induction of neuroectodermal tumors in BDIX rats by N-ethyl-N-nitrosourea (EtNU) is a model system for the analysis of transformation risk as a function of target cell properties. The yield of neural tumors induced by EtNU varies with the developmental window chosen for the carcinogen pulse; i.e. with the relative proportions of different neural precursor cells exposed to EtNU at distinct developmental stages. Different subsets of fetal brain cells have been characterized previously with respect to their relative risk of malignant transformation using monoclonal antibodies. As DNA replication of target cells is considered to be a prerequisite for malignant conversion, we analyzed the cell cycle distributions, using flow-cytometry, of 4 subsets of neural precursor cells considered to be at high or low risk, respectively, of malignant conversion by EtNU in vivo. Cell populations associated with an elevated risk of transformation exhibited higher proportions of cells in S-phase. One of the 2 putative low-risk populations exhibited a significantly lower fraction of S-phase cells, while the value of the second one exceeded those obtained for the 2 high-risk subpopulations. Therefore, a higher than average fraction of cells in S-phase appears to be positively correlated with the cellular risk of malignant transformation by EtNU, but does not represent a dominant risk determinant per se.

Determination of immunoreactive fraction and kinetic parameters of a radiolabeled monoclonal antibody in the absence of antigen excess.

Monoclonal antibodies (Mabs) directed against cell surface determinants and conjugated to fluochromes, radionuclids or drugs are of increasing importance in cell and tumor biology as well as in clinical oncology. Many of the applications of Mab require precise and quantitative information regarding the molecular interactions of labeled antibody with the respective antigen expressed on the cell surface. These interactions are characterized by the association rate constant (ka), the dissociation rate constant (kd) and the antibody affinity constant (K). The immunoreactive fraction (IRF) of the labeled antibody molecules directly influences these parameters. IRF is usually reduced below 100% by antibody purification and labeling procedures and, in case of radiolabeled antibodies, by radiation damage during antibody storage. Besides the calculation of kinetic parameters, IRF should, therefore, be determined for the quality control of any antibody preparation before experimental or clinical application. Commonly used methods for measuring IRF are based on radioimmunoassays (RIA) on intact cells performed under antigen excess. However, especially with Mabs directed against cell surface antigens expressed in small numbers per cell and for displaying low affinity constants, these assays often give unsatisfactory results. We have, therefore, established a method which permits us to determine IRF, ka, kd and K for an 125I-labeled Mab with precision even in the absence of antigen excess.

The effect of H-2 compatibility on pancreatic beta cell survival in the nonobese diabetic mouse.

The effect of major histocompatibility (MHC) antigens on the survival of newborn pancreatic iso and allografts was assessed in the nonobese diabetic (NOD) mouse, which develops a spontaneous autoimmune diabetes. The NOD mouse (H-2Kd,Db) rejects skin allografts from CBA (H-2k) within a 12-day period, indicating normal immune function toward alloantigens. A pancreatic allograft into the NOD mouse represents a presumed first-set allogeneic response, as well as a possible second-set immune response to islets. To assess the effect of donor H-2 antigen and the influence of autoimmune disease on pancreatic graft survival, newborn pancreata from various strains of mice were transplanted into diabetic NOD mice treated with 40 mg/kg/day cyclosporine (CsA) that prevented skin allograft rejection. The grafts were then harvested at day 10 to histologically assess the graft viability. CBA pancreatic grafts, incompatible at all MHC loci, showed the least lymphocytic infiltration, and good donor beta cell survival. Furthermore, CBA newborn pancreata under appropriate conditions were able to cure or improve the diabetic condition in 3/6 NOD mice. In the graft sharing class I MHC antigens, lymphocytic infiltration was significantly increased, while the donor beta cell number clearly decreased. The intensity of the graft destruction was intermediate in C57BL/6 allografts sharing H-2Db antigen, and strongest in BALB/c allografts sharing H-2Kd and in NOD isografts. The results indicate that in diabetic NOD mice the CsA dose controlling allograft rejection is incapable of controlling antiislet immunity. This antiislet immunity appears to exert its effect in an H-2-restricted manner. These findings may have important implications for the transplantation of pancreatic tissue in treating type I diabetes in humans.

Age-dependent change of the effect of a cytostatic drug on the proliferation kinetics of a solid tumor of the mouse.

The aim of the present investigations was to study the effectiveness of a cytostatic drug (VCR) during different phases of tumor growth (1st, 7th, 14th days a.t.) and at the periphery and at the centre of the tumor (on the 14th day a.t.). Furthermore the inducement of a partial synchronization in the proliferation of tumor cells was attempted. It was found that the intensity of the cytostatic effect significantly decreased both with the passage of time after transplantation and within the tumor from the periphery to the centre. The changing cytostatic sensitivity probably is due to the diminishing vascular supply and the decreased rate of cell proliferation; especially the decline of the growth fraction. A partial synchronization of the proliferation of tumor cells could not be demonstrated.

Experimental investigations on a sequential combination chemotherapy protocol.

This paper deals with experimental investigations concerning the composition of a cytostatic three-drug-protocol in diploid Ehrlich-Ascites-Tumor (EAT) cells in vivo at a far advanced stage of the disease. Hydroxyurea (HU) and vincristine (VCR) were used in very low doses to induce a modification of the growth pattern of tumor cells alike partial synchronization. Adriamycin (ADM) was selected as cytocidal drug during DNA synthesis of the partially synchronized cells. It was found that the sequential combination of HU and VCR (first HU and 12 h thereafter VCR) caused the greatest alteration of growth pattern compared with other combination protocols. A further statistically significant increase of the degree of synchrony was observed after a second VCR administration -- 22 h after HU. By means of this protocol the EAT was subdivided into two proliferating subpopulations, a diploid and tetraploid one. The tetraploid population resulted from surviving cells being not able to perform dytokinesis correctly, so that polynuclear cells and cells with a large single nucleus containing tetraploid DNA values were created. With respect to therapy, the administation of ADM at the time of DNA synthesis of the partially synchronized cells resulted in a statistically significant prolongation of the mean survival time and in 30% of cures of the animals. The dosage of ADM was 2.6 mg/kg, i.e., a nonlethal dose (50% of the LD10). Other combinations, i.e., simultaneous or reversed sequential combinations, did not show any therapeutic improvement compared to single drug therapy of ADM.

The presence and significance of the Thomsen-Friedenreich antigen in mammary gland. II. Its topochemistry in normal, hyperplastic and carcinoma tissue of the breast.

Normal tissue as well as various benign and malignant lesions of the breast were histochemically examined for the presence of the Thomsen-Friedenreich (TF)-antigen. Fluorescein- or 3H-labelled peanut agglutinin was used for this purpose, a lectin that is known to have a high affinity for the TF-antigen. The occurrence of this TF-antigen seemed in all cases, even in the carcinoma lobulare in situ that is regarded as being derived from myoepithelial cells by some authors, to be associated with a secretory condition. Its presence (free and neuraminic acid covered) in normal, hyperplastic and malignant breast tissue, however, cannot be considered a specific tumour associated antigen as has been previously assumed. Furthermore the investigations have shown that the intensity of fluorescence for peanut agglutinin (PNA)-receptors was generally stronger in differentiated carcinomas than in undifferentiated carcinomas of the breast. The histochemical findings are discussed with regard to diagnostical and immunotherapeutical aspects.

Frequency of clonal B lymphocytes in chronic myelogenous leukemia evaluated by fluorescence in situ hybridization.

The demonstration of the Philadelphia (Ph) chromosome in B lymphocytes from patients with chronic myelogenous leukemia (CML) has provided evidence that the disorder originates in a pluripotent progenitor cell. Divergent results, however, exist as to the degree of contribution of clonally derived cells to the B-cell compartment. To address this issue, B lymphocytes were selected from the blood of seven patients in the chronic phase of Ph-positive CML and were examined with dual-color fluoresence in situ hybridization for the presence of the Ph translocation. The purity of the B-cell preparations ranged from 88% to 97% (mean 93%). The Ph translocation was detected in 22-34% (mean, 27%) of the sorted B cells. There was no evidence that the duration of the disease affects the ratio of Ph-positive and -negative B cells. In summary, clonally derived circulating B lymphocytes were present in all patients studied but made only minor contribution to this compartment.

Effect of H-2 compatibility in autoimmune destruction of islet allografts from B10 congenic lines to nonobese diabetic mice.

Autoimmune diabetes involves multiple antigens, and both cellular and humoral immune responses. Using CBA (H-2k) C57BL/6 (H-2b), and BALB/c (H-2d) newborn mouse pancreata, we previously demonstrated that acute and strong destruction of islet allografts by anti-islet autoimmunity in the nonobese diabetic (NOD) mouse H-2Kd, Db) is under the influence of major histocompatibility complex (MHC) antigens. In the current study, we have attempted to confirm these results in the absence of minor alloantigenic differences using B10 congenic strains as pancreatic donors. Pancreata from B10.BR (H-2k), C57BL/10SnJ (H-2b), and B10.D2 (H-2d) were transplanted under the kidney capsule of NOD mice within 1 month of diabetes onset. These recipients were immunosuppressed with cyclosporine (CsA) in a dosage that effectively prevents rejection of skin allograft, but not islet isograft destruction that is mediated by anti-islet autoimmunity. On day 10, the grafts were harvested and examined histologically to assess viability. Pancreatic allografts from B10.D2, sharing the H-2Kd with the NOD mouse, showed the strongest lymphocytic infiltration, and neither islets nor beta cells were found in all seven grafts. C57BL/10SnJ grafts, sharing the same H-2Db, also showed severe lymphocytic infiltration, and no intact islets, and only a few beta cells were found, as single cells, in three of eight grafts. In contrast, B10.BR grafts, completely incompatible at the H-2, showed the least infiltration, and normal islets containing many beta cells were found in 10 of 11 grafts. These results again suggested the hypothesis that islet allograft destruction by diabetic NOD mice is MHC restricted.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation potential of a monoclonal antibody-defined neural progenitor cell population isolated from prenatal rat brain by fluorescence-activated cell sorting.

We have studied the differentiation potential in vitro of a subpopulation of neural progenitor cells from BDIX-rat brain. These cells transiently express a cell surface determinant (CSD) specified by monoclonal antibody (Mab) RB13-2 (Kindler-Röhrborn, A. et al., Differentiation 30 (1985) 53-60), and recently identified as a set of O-acetylated gangliosides (Reinhardt-Maelicke, S. et al., submitted) also recognized by Mab D1.1 (Levine, J.M. et al., J. Neurosci., 4 (1984) 826-831) and partly by Mab JONES (Schlosshauer, B. et al., J. Neurosci., 8 (1988) 580-592), respectively. As analyzed by immunofluorescence, Mab RB13-2 binding brain cells (prenatal days 11-22; postnatal days 7 and 89) were localized in different areas of the proliferative ventricular layer of the prenatal cerebrum and in the external granular layer of the early postnatal cerebellum. No Mab RB13-2 positive brain cells were found in adult brain. Following their isolation by fluorescence activated cell sorting on prenatal day 18, the differentiation potential of Mab RB13-2 binding brain cells was studied by double-immunofluorescence analysis under different conditions of monolayer culture. In the presence of 10% fetal calf serum (FCS), these cells differentiated into glial fibrillary acidic protein (GFAP) positive flat astrocytes, whereas neurons (neurofilament-positive) and a smaller number of stellate astrocytes (GFAP-positive) developed in a chemically defined medium containing 0.5% FCS. Neural progenitor cells binding Mab RB13-2 may thus either retain more than one option for differentiation into specific cell types, or the expression of the CSD specified by Mab RB13-2 may be common to more than one subset of neural progenitor cells (with or without predetermined unidirectional differentiation pathways) whose survival and/or proliferative behavior could be differentially affected by microenvironmental conditions.

A standardized protocol for flow cytometric analysis of cells isolated from cerebrospinal fluid.

Flow cytometry (FC) is an useful tool for the analysis of subpopulations in complex cell suspensions. When applying this method to the cerebrospinal fluid (CSF), some characteristic properties of this cell type must be taken into consideration: there are only few cells which decay rapidly in their native medium and during centrifugation. One aim of the immunostaining procedure preceding flow cytometric analysis must be to minimize cell loss in order to get an undistorted picture of 'true' CSF cell populations. Consequently, morphological flow cytometric plots of high resolution are an indispensable precondition for reliable determination of subpopulations defined by monoclonal antibody (Mab) binding. We describe a standardized protocol for the flow cytometric examination of CSF cells which minimizes undesired cell loss. By the use of a 'quality control' the extent of cell loss could be monitored. Examples of morphological flow cytometric plots are given. The subsequent determination of Mab binding subpopulations is critical when fluorescence intensities of antigen positive and negative cells are non-disjunct. A statistical test was developed for these cases often seen when cell surface determinants are expressed at low levels only.

Simultaneous measurement of cellular P-glycoprotein content and function by multiparametric flow-cytometry.

OBJECTIVE: A multiparametric approach was applied to simultaneously determine expression and function of the drug efflux pump P-glycoprotein (PGP) in multidrug-resistant (MDR) human leukemic lymphoblast cell lines and isolated leukemic blasts using flow-cytometry in a patient with acute myeloid leukemia (AML). METHODS: The antigen was measured by staining PGP using the monoclonal antibody 4e3 which does not inhibit the function of PGP. The 4e3 antibody binds to an external epitope of PGP and can therefore be used for staining living cells. Drug transport, mediated by PGP, was determined simultaneously by measuring rhodamine 123 (rho123) efflux. The MDR cell lines, CEM/VLB10-2 and CEM/VBL100 are 10-fold and 270-fold resistant to vinblastine (VBL), respectively, compared to the human PGP-negative parent cell line CEM/WT and they express different amounts of PGP. Initially, living cells were stained using the 4e3 antibody and a secondary antibody labeled with 7-amino-4-methylcoumarin-3-acetic acid (AMCA). Cells were then incubated for 60 min with rho123 (10 microM) and analyzed for rhodamine and AMCA-derived fluorescence. The decrease in rho123 fluorescence was determined after a further period of 30 min. RESULTS: CEM/VLB100 cells expressed larger amounts of PGP, and rho123 fluorescence after 30 min was 85% lower than the parent cell line. PGP expression and rho123 efflux were also detected in CEM/VLB10-2 cells which display a low degree of resistance, thus reflecting the high sensitivity of this method. PGP-expressing blasts and moderate rho123 efflux were also observed in a specimen derived from a patient with clinically resistant acute myeloid leukemia (AML). CONCLUSION: A multiparametric approach using flow-cytometry allows the reliable and sensitive measurement of both PGP expression and function simultaneously in single cells.

A simple method for counting small numbers of cells by flow cytometry.

We describe a flow cytometric method for the quantitation of small numbers of cells that facilitates proliferation studies in microcultures. A constant number of fluorescent latex microspheres/sample is added to single cell suspensions prepared from the cultures. By flow cytometric analysis, cells are easily distinguishable from the microspheres and can be quantitated on the basis of their light scattering and fluorescence properties is contour plots. As the number of microspheres/sample is known, the relative proportions of cells and microspheres, respectively, can be converted into absolute cell numbers. This quick method is useful for any type of studies where cell numbers less than 1 x 10(5) in a small volume are to be determined.

[In-vitro-technique for autoradiographic investigations of cell proliferation in benign and malignant alterations of the human urinary bladder mucosa from biopsy specimens (author' transl)]. / Zur Zellkinetik von Urotheltumoren. In-vitro-Verfahren zur autoradiographischen Untersuchung der Zellproliferation von gut- und bösartigen Veränderungen der menschlichen Harnblasenschleimhaut am Biopsiegewebe.

An in-vitro double labelling technique with 3H- AND 14C-Thymidine was applied to biopsy specimens of the human urinary bladder mucosa in order to determine proliferation kinetics of normal bladder epithelium as well as its benign and malignant alterations. Application of above mentioned technique led to evaluable autoradiographs. The mean 3H-Thymidine labelling-index in bladder cancer was remarkably higher than in normal bladder epithelium and in benign papillomas. There was a good correlation between histologic grading of malignancy and increase of the mean 3H-Thymidine labelling index. Addition of 5-Fluorodesoxyuridine to the incubation medium had no influence on the labelling of the incubated tissue.

[Nephroblastoma, partly appearing as a sarcoma botryoides of the renal pelvis (author's transl)]. / Nephroblastom, teils unter dem Bild eines Sarcoma botryoides des Nierenbeckens.

A renal tumour in a 4-year old girl morphologically appeared as both a nephroblastoma and, in parts, as a sarcoma botryoides of the renal pelvis. The histogenesis of this dysontogenetic tumour is most probably due to a disturbance of growth and differentiation both of the metanephrogenic blastema as well as partly of the urethral bud. This type of dysontogenetic tumour is extremely rare. Problems of diagnosis and operative therapy and the possibility of postoperative radiation therapy and cystostatic treatment are discussed.

[Myogenic differentiation in experimental malignant schwannomas (an immunohistochemical and molecular genetic study of the "triton" tumor)]. / Miogennaia differentsirovka v éksperimental'nykh zlokachestvennykh shvannomakh (immunogistokhimicheskoe i molekuliarno-geneticheskoe issledovanie opukholi "triton".

Spontaneous myogenic differentiation was observed in 2 out of 15 monolayer cultures from schwannomas induced in BD1X rats by transplacental exposure to N-enthyl-N-nitrosourea (ENU). Cells were sorted following fluorescence-activating method with monoclonal antibody (Mab) 217c. Myotubes and numerous mononucleated cells no longer expressed the Schwann cell antigens 217c and S-100 protein, but rather revealed the presence of desmin, the alpha-sarcomeric form (alpha-sr) of actin, and the cell surface antigen specified by Mab RB21-7, a glycoprotein sharing an epitope with the neural cell adhesion molecule (N-CAM). Subcutaneous reimplantation of such cells into syngeneic animals resulted in the appearance of tumours composed of both S-100 positive Schwann cells and desmin and alpha-sr-actin positive rhabdomyoblasts, thus closely resembling the human "triton" tumour. With the use of the polymerase chain reaction and allele-specific oligonucleotide hybridization, DNA isolated from individual myotubes was analysed for the presence of a T-->A transversion mutation at nucleotide 2007 of the neu gene, which is diagnostic of ENU-induced rat schwannomas. All of the amplified DNA isolated contained the mutant neu allele, thus providing direct genetic proof for the capacity of mammalian neuroectodermal cells for myogenic differentiation.