Gaucher disease: Genetics, diagnosis and management.

Gaucher disease is a storage disorder with varied manifestations. As the first storage disorder for which treatment is available, it is of considerable clinical and scientific importance. We review the history, clinical syndromes, molecular genetics and manifestations as well as modern diagnosis and therapy of the adult non-neuronopathic variant of Gaucher disease. (c) 2001 Prous Science. All rights reserved.

Human monoblastoid cell line U-937 cultured in protein-free medium: immunophenotype, cytochemical and biochemical markers.

Human monoblastoid cell line U-937 was adapted to grow in protein-free (protein-free hybridoma--PFH) medium and cloned by limiting dilution. Resulting cell subline (U-937/PF) cultured in protein-free medium was characterized by immunological, cytochemical and biochemical techniques. There were no major differences in immunophenotype (determined by FACS analysis with monoclonal antibodies directed to HLA and CD antigens) and cytochemical markers between the U-937/PF cells cultured in protein-free cell culture medium and parental U-937 cell cultured in serum-supplemented medium. Maximal cell density was slightly decreased in protein-free culture as compared to the parental cell line in FCS-supplemented medium. Cell viability and cell DNA histograms (determined by propidium iodide cytofluorimetry) showed no major differences between parental U-937 and U-937/PF cells. Phorbol ester (TPA)-induction of differentiation-associated cell markers resulted in a proliferation arrest and accumulation of G0/G1 cells in both sublines. All-trans retinoic acid and, to a lesser extent, TPA-stimulated NBT reduction was higher in parental U-937 cells cultured in serum-supplemented medium as compared to U-937/PF cells. Quantitative differences in the expression and inducibility of some cytochemical markers (beta-glucuronidase, chloroacetate esterase) were found between both examined sublines. Described U-937/PF subline cultured in a protein-free cell culture medium (PFH) appeared as a potential tool for studies of in vitro inducing agents and serum components with differentiation promoting (or inhibiting) activities.

Differential modulation of leucocyte surface antigens by various inducers on the T-cell line MOLT-4.

Pretreatment of MOLT-4 T-cell line with 12-0-tetradecanoylphorbol 13-acetate (TPA) potentiated the interferon alpha-induced cell surface expression of class I HLA. Simultaneous combined action of TPA and rIFN-alpha resulted in a synergistic effect upon the up-regulation of class I HLA. Neither TPA nor rIFN modulated the cell surface expression of HLA class II (DR and DP) antigens. Leucocyte common antigen (CD45) cell surface expression was quantitatively increased on MOLT-4 cells by TPA induction, unaltered by rIFN and up-regulated by combined action of TPA and rIFN. CD8 antigen was down-regulated by both TPA and rIFN.

Biochemical and immunological phenotype alterations in human monoblastoid cell line U-937 induced by physiological (interferon-alpha, retinoic acid) and nonphysiological (phorbol ester) inducers.

Human monoblastoid cell line U-937 was induced by recombinant or leukocyte human interferon-alpha, retinoic acid, by their combination, or by 12-0-tetradecanoyl-phorbol-13-acetate (TPA), to express some differentiation-associated markers and characteristics. Induced biochemical alterations were studied with the aid of two-dimensional electrophoretic analysis (isoelectrofocusing/SDS-PAGE) of 125I-lactoperoxidase radioiodinated cell surface proteins, 35S-methionine metabolically radiolabeled cell proteins and 32P orthophosphate radiolabeled cell phosphoproteins. Alteration of immunophenotype markers was studied by continuous flow immunocytofluorometry with a panel of monoclonal antibodies directed to leukocyte antigens, cell proliferation, DNA, RNA, and protein synthesis by radioactive precursor incorporation techniques. Furthermore, cytochemical markers, cell adherence and nitroblue tetrazolium (NBT) reduction were utilized to follow the induction of differentiation-associated characteristics. Differential alterations of some immunophenotype markers induced by diverse inducers were observed. Major induced alterations included down-regulation of CD4 (induced by TPA, and to a lesser extent by IFN-alpha), TPA-induced decrease of cell surface expression of transferrin receptor (unmodified by IFN-alpha) and IFN-alpha induced increase of antigen density (fluorescence intensity) of MHC class I antigen. Marked retinoic acid and interferon-alpha induced increase in membrane expression of antigen(s) detected by monoclonal antibodies BraC6 and BraC8, elicited with healthy donor's granulocytes, was also observed.

Modulation of CALLA (CD10) antigen on cultured ALL (REH) cells: effect of various modulators.

Phorbol ester (TPA)-induced down-regulation of the common ALL (CALLA) antigen was studied by continuous flow immunocytometry with the aid of several CD10 monoclonal antibodies, including a new CD10 monoclonal antibody (DGH-10-1-A9), shown to be of IgG1 isotype, recognizing a 100 kDa cell surface protein and effectively inhibited by a series of reference CD10 monoclonal antibodies. The TPA-induced down-regulation of CALLA on REH cells was demonstrated with the aid of the following CD10 monoclonal antibodies: J-5, VIL-A1 and DGH-10-1-A9. No major modulations in cell surface expression of CALLA on REH cells were observed after induction with 1,25-(OH)2 vitamin D3, retinoic acid, recombinant interferon (IFN) alpha 2a and recombinant interleukin 2.

Isoforms of leukocyte common antigen (CD45) on human neoplastic hematopoietic cell lines.

Leukocyte common antigen (LCA) isoform expression patterns were examined on different human hematopoietic cells and cell lines with the aid of the immunoblotting technique utilizing two anti-LCA monoclonal antibodies elicited by an ALL cell line and able to detect four isoforms (180, 195, 205 and 220 kDa) of LCA on peripheral blood lymphocytes from healthy donors and some neoplastic hematopoietic cell lines. Fourteen leukemia/lymphoma cell lines of different origin and immunophenotype, as well as three nonhematopoietic human tumor cell lines were examined by this technique. Human hematopoietic cell lines expressed two to four different polypeptide chains (180, 195, 205 and 220 kDa). Non-T, non-B cell lines, some B-cell lines and monocyte lymphoma cell line U 937 predominantly expressed higher molecular weight isoforms. In myeloid leukemia cell lines examined the expression of lower molecular weight isoform(s) predominated. Individual variability in LCA isoform patterns on cell lines precluded the strict correlation between these patterns and immunophenotype of the examined cell lines.

Monoclonal antibodies (series Bra-) of broad (non-lineage) or restricted (myelomonocytic) reactivities elicited with granulocytes or K 562 cells.

A series of monoclonal antibodies was obtained by hybridoma technology after immunization with granulocytes from healthy donors or K 562 immature erythroid-myeloid leukemia cells. Three different types of reactivities with examined hematopoietic-, nonhematopoietic cells and cell lines were observed by microscopic immunofluorescence, immunocytofluorometry and enzyme-linked immunoassay (ELISA), as follows: (i) broad, non-lineage type of two monoclonal antibodies (Bra10G and Bra7F1) with examined hematopoietic and nonhematopoietic human neoplastic cell lines, (ii) non-lineage type of reactivity restricted to hematopoietic cell lines, and (iii) restricted (myelomonocytic) pattern of binding to myeloid cell lines and healthy donors' granulocytes (monoclonal antibodies Bra4F1, BraC8 and Bra1F2). Monoclonal antibodies Bra10G and BraC6 were shown to immunoprecipitate specifically a heterodimeric two-chain cell surface protein p200,95 from cell lysates of lactoperoxidase radioiodinated U 937 cells with recognized epitope localized on the heavy chain (as shown by immunoblotting experiments). Antibodies with restricted myelomonocytic type of reactivity exhibited minor quantitative differences in their microscopic immunofluorescence and immunocytofluorometric patterns of reactivities with examined myeloid leukemia cell lines (K 562, HL 60, U 937) and healthy donors' granulocytes. The monoclonal antibody Bra4F1 was defined by the 4th International Workshop on Leukocyte Differentiation Antigens as CD15 (with typical selective reactivity towards myelomonocytic leukemia cells and cell lines as well as healthy donors' granulocytes) recognizing the X-hapten carbohydrate antigenic determinant.

Monoclonal antibody directed to MHC class I antigen (Bra23/9): characterization and utilization for study of antigen expression in differentiation of U 937 cell line.

The monoclonal antibody Bra23/9 (IgG2a) elicited by a non-T, non-B ALL cell line (REH), reacted in microscopic immunofluorescence, enzyme-linked immunoassay (ELISA), complement-dependent cytotoxicity tests and immunocytofluorometric measurements with hemopoietic cell lines and peripheral blood lymphocytes, monocytes and granulocytes from healthy donors in a pattern characteristic for MHC class I antigens. Immunoprecipitation of lactoperoxidase radioiodinated cell surface proteins and surface sialoglycoproteins radiolabeled by sodium metaperiodate/tritiated sodium borohydride technique confirmed the structure gp44,p12 (consisting of a 44 kDa glycopeptide linked with a nonglycosylated 12 kDa peptide) typical for MHC class I antigen(s) as a structure recognized by the Bra23/9 monoclonal antibody. The increase of MHC class I antigen density (immunofluorescence intensity) induced by a phorbol ester (TPA) in monoblast U 937 lymphoma cell line was observed by immunocytofluorometry as a predominant tendency in several TPA-induction experiments, where a certain variability among individual experiments in TPA-induced MHC class I antigen alterations was observed.

Cytogenetic changes of human peripheral blood lymphocytes in vitro after exposure to cis-DDP (cis-diamminedichloroplatinum II) and oxo-Pt (cis-diamminedichloro-trans-dihydroxyplatinum IV).

Human peripheral blood lymphocytes were tested for induction of micronuclei, chromosome aberrations and sister chromatid exchanges (SCEs) frequency after exposure to two platinum complexes (cis-DDP Platidiam and oxo-Pt) and a comparison was made with the inhibitory effect of these drugs on mitotic activity. When the cis-DDP cell samples were compared with the untreated controls, there was a distinct increase in the frequency of micronuclei and chromosome aberrations, and the statistically significant increase in SCE frequency was accompanied by a significant decrease in mitotic activity. In oxo-Pt cell samples, using an identical concentration of the drug, only a slight increase in micronuclei and chromosome aberration frequency was observed. However, the increase in the SCE frequency was not significant and neither was the decrease of mitotic activity when compared with the controls.

Human neoplastic cell line distribution, immunoprecipitation and immunohistopathological study of a gp200 cell surface glycoprotein (LCA) detected by a monoclonal antibody elicited with an ALL cell line.

The antigen recognized by a newly produced monoclonal antibody (bra55; IgG1) elicited by the non-T, non-B acute lymphoblastic leukemia cell line REH 6, was expressed on all examined hemopoietic neoplastic cell lines (including non-T, non-B, T, B and myeloid leukemia cell lines), but not on examined nonhemopoietic human tumor cell lines (such as carcinoma, sarcoma, melanoma and neuroblastoma cell lines), as demonstrated by indirect immunofluorescence and enzyme-linked immunoassay. Specific immunoprecipitation of 125I-lacto-peroxidase radioiodinated cell surface proteins and sodium metaperiodate/tritiated sodium borohydride 3H-radiolabeled cell surface sialoglycoproteins followed by electrophoretic analysis (SDS-PAGE) demonstrated that the immunoprecipitated antigen is a cell surface 200 kDa sialoglycoprotein (on the non-T, non-B ALL cell line REH 6), with variation in its electrophoretic mobility (in the Mr range of 170,000-210,000) on different examined cell lines. These properties are characteristic for the leukocyte common antigen (LCA, T200). Immunoperoxidase staining of several normal and malignant tissues, as well as some nonhemopoietic tumor tissues confirmed the type of antigen tissue distribution pattern characteristic for LCA.

Supplementary characteristics of anti-MHC class II monoclonal antibodies elicited by an ALL cell line: immunofluorescence cytofluorometry, C-dependent cytotoxicity, two-dimensional analysis of antigen.

Monoclonal antibodies directed to MHC class II antigen(s), elicited by a non-T, non-B ALL cell line, were characterized by immunofluorescence flow cytofluorometry and ELISA immunofiltration measurements of their immunoreactivity with selected neoplastic hemopoietic cell lines, determination of their complement-dependent cytotoxic activity against isolated peripheral blood B and T lymphocytes and by two-dimensional electrophoretic analysis (isoelectric focusing, SDS-PAGE) of radiolabeled, immunoprecipitated by these antibodies cell surface antigens. Patterns of these immunological reactivities, as well as two-dimensional radioimmunoprecipitation patterns (acidic heavy chain p35 and basic light chain p30) of antigens recognized by these antibodies confirm their anti-MHC class II specificity. One of these antibodies (braFB6; IgG2b) displayed identical pattern of expression on cell lines and cell types as the typical anti-MHC class II antibodies, but immunoprecipitated only a single chain p30 radioiodinated cell surface protein (with two-dimensional pattern close to the beta-chain of MHC class II DR antigen). These properties indicate the ability of braFB6 monoclonal antibody to recognize a nonpolymorphic determinant of DP-MHC class II antigen.

Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. I. 35S-methionine labeled, lactoperoxidase radioiodinated and 3H-reductively methylated proteins.

Cell surface and cytoplasmic polypeptides of some human hemopoietic cell lines were radiolabeled by three different techniques: 35S-methionine metabolic radiolabeling, lactoperoxidase catalyzed radioiodination and reductive methylation with formaldehyde and tritiated sodium borohydride. Two-dimensional patterns of 35S-methionine labeled proteins revealed more than 500 separated protein spots with essentially similar general aspects of all examined cell lines, sharing major polypeptides such as those with electrophoretic mobilities of major cytoskeletal proteins. 125I-lactoperoxidase radio-iodinated cell surface protein patterns contained about 30 separated protein macromolecules with some marked differences between lymphoid versus myeloid lines and also between individual cell lines. Two-dimensional patterns of tritiated membrane polypeptides consisted of approximately 200 separated protein spots, some prominent of them appeared with electrophoretic mobilities of major cytoskeletal proteins being common to both 35S-methionine and reductive methylation patterns. Two-dimensional patterns of 35S-methionine labeled and cell surface radioiodinated proteins immunoprecipitated by monoclonal antibodies Bra30 and HL39, recognizing MHC class II antigens, appeared as bimolecular complex of larger acidic and smaller basic chains structurally corresponding to the expected two-dimensional patterns of MHC class II antigens.

Two-dimensional analysis of metabolically and cell surface radiolabeled proteins of some human lymphoid and myeloid leukemia cell lines. II. Glycosylated and phosphorylated proteins.

Cell surface glycoproteins, radiolabeled by sodium metaperiodate/tritiated borohydride technique and cell phosphoproteins, metabolically radiolabeled by 32P-orthophosphate were analyzed by two-dimensional electrophoretic analysis in some myeloid and lymphoid leukemia cell lines. Some markedly expressed major glycoproteins were predominant in some of cell lines (such as 95k and 100k glycoproteins with marked charge heterogeneity in non-T, non-B acute lymphoblastic leukemia cell lines NALM 6 and NALM 16), but markedly quantitatively reduced in other examined cell line, such as lymphoblastoid cell line UHKT 34/2. 32P-orthophosphate radiolabeled phosphoprotein two-dimensional patterns of examined lymphoid leukemia cell lines were essentially similar, with some minor differences, in examined lymphoid and myeloid leukemia cell lines, such as marked expression of a series of large phosphoproteins in the molecular weight range 80-100k in lymphoid cell lines and almost complete absence of these phosphoproteins on examined myeloid leukemia cell lines. Another configuration of acidic phosphoproteins (30-35k) exhibited individual cell line variability and differences between both individual myeloid leukemia cell lines and between lymphoid and myeloid cel lines examined.

Chromosomal aberrations, sister chromatid exchange (SCEs) and micronuclei induction with three platinum compounds (cis-DDP, CHIP, CBDCA) in V79 cells in vitro.

Three platinum compounds (cis-DDP, CHIP, CBDCA) were tested or induction of chromosome aberrations and micronuclei as well as for SCEs in V79 cells in vitro. There was a clear relationship between the degree of inhibitory activity of the tested drugs and the increase of studied markers of cytogenetic damage. Statistical significance was proved in increase of SCEs values in cis-DDP and CHIP-treated cells when compared with untreated controls.