ALBERT

Description: Four distinct intragenic polymorphisms in the coagulation factor IX gene which have been reported to be important for family diagnosis of Caucasian hemophilia B were studied in 51 normal Japanese subjects (21 males and 30 females). High-molecular-weight DNA prepared from peripheral blood lymphocytes were digested with endonuclease, Ddel, Mspl, Taql or Xmnl, and were studied by Southern blot analysis with factor IX complementary DNA as a probe. None of the minor fragments produced by these enzymes was found in the normal Japanese DNA samples tested, although the probe detects minor allelic forms in control Caucasian DNA samples. Our data suggest that the frequent polymorphic sites found in Caucasians are possibly absent in the Japanese population.

Description: Although it is well established that the addition of 1,25- dihydroxyvitamin D3 (D3) to the culture of normal human granulocyte/macrophage progenitors induces monocyte/macrophage (Mo/M phi) colonies, the target cells of D3 in the Mo/M phi differentiation have not been identified. We examined whether neutrophilic promyelocytes are the target cells. As a source of the promyelocyte fraction, we used colonies after 5 days of culture (5-day colonies) of colony-forming unit-granulocyte. The culture contained granulocyte colony-stimulating factor (G-CSF) as the growth factor and generated only neutrophilic colonies. The promyelocytic nature of the 5-day colonies was confirmed morphologically, cytochemically, and ultrastructurally. After morphological evaluation on part of the individual colonies, they were transferred into new semisolid cultures with or without D3 (10(-7) mol/L) in the presence of G-CSF, then incubated for the subsequent 7 days. With D3, the colonies were loose, and all the constituent cells were morphologically small macrophages, which were positive for alpha-naphthyl butyrate (alpha NB) esterase, strongly positive for CD14 antigen, and plastic-adherent. While without D3, the colonies were rather compact, and all the constituent cells were morphologically mature neutrophils, which were positive for naphthol ASD-chloroacetate esterase and weakly positive for CD14 antigen. Secondary culture of the 8- or 10-day colonies with D3 induced a lower number of alpha NB-positive cells, in proportion to the percentage of promyelocytes at the time of transfer in each colony. Four days of secondary culture with D3 was sufficient to induce alpha NB-positive cells. G-CSF was not an essential factor to induce alpha NB- positive cells. These findings indicate that D3 differentiates normal human neutrophilic promyelocytes into the Mo/M phi lineage in vitro.

Description: Recently, it has been shown that the homozygous deletion of the cyclin- dependent kinase-4 inhibitor (CDK4I;p16) gene, which is mapped to chromosome 9p21, is frequently observed in a wide spectrum of human cancers, including leukemias. Therefore, the CDK4I gene is thought to be a putative tumor-suppressor gene. We report here that both alleles of the CDK4I gene were completely or partially deleted in human leukemia cells derived from both patients and established cell lines. Thirty-seven hematopoietic cell lines and samples from 72 patients with leukemias were examined for homozygous loss of the CDK4I gene locus by Southern blot analysis. We found that a part or the whole of the CDK4I gene was homozygously deleted in 14 of the 37 (38%) cell lines and 4 of 72 (6%) samples from leukemia patients, including 45 with acute myelocytic leukemia, 14 with acute lymphocytic leukemia (ALL), and 13 with chronic myelocytic leukemia in blastic crisis. In the cell lines, the homozygous deletion of the CDK4I gene was detected in a variety of cell lineages, whereas all 4 cases showing the homozygous deletion were confined to ALL. It should be noted that 2 of them had no cytogenetic abnormalities of chromosome 9. Our results suggest that loss of the CDK4I function may contribute to immortalization of human leukemia cells and play a causative role at least in development of human lymphocytic leukemias.

Description: The expression of adhesion molecules on human tonsillar follicular dendritic cells (FDCs) in the secondary lymphoid follicle (LF) in vivo and in vitro was investigated using cryostat sections and cytospin preparations of FDCs isolated with a magnetic cell sorter, respectively. FDCs were immunochemically positive for Mac-1 (CD11b), sialyl-Lex (CD15s), CD22, integrin beta 1 (CD29), CD40, very late activation antigen (VLA)-alpha 3 (CD49c), VLA-alpha 5 (CD49e), VLA-alpha 6 (CD49f), intercellular adhesion molecule (ICAM)-3 (CD50), ICAM-1 (CD54), B7 (CD80), and vascular cell adhesion molecule (VCAM)-1 (CD106). With respect to ligands on B cells for these adhesion molecules, the CD11b-CD54, CD50-leukocyte function-associated molecule (LFA)-1 (CD11a/18), and CD106-VLA-4 (CD49d/29) interactions in the apical light (ALZ) and basal light (BLZ) zones; the CD15s-L-selectin (CD62L) and CD106-CD49d/29 interactions in the mantle zone; and the CD54-CD11a/18 interaction in the entire LF may participate in FDC-B cell adhesion. Namely, the adhesion molecules participating in FDC-B cell interactions may differ in each of the five zones. Furthermore, the immunochemical evidence that FDCs were fibronectin (VLA-5, CD49e/29) and laminin (VLA-6, CD49l/29) receptor-positive discussed above was confirmed by immunoelectron microscopy and binding assays. Immunoelectron microscopy revealed fibers surrounded by cytoplasmic FDC extensions that were CD29-, CD49e-, and CD49f-positive. In the binding assays, the numbers of FDCs bound to fibronectin- and laminin-coated dishes and LFs of cryostat sections of human tonsils were reduced markedly by pretreatment with monoclonal antibodies against CD29, CD49e, and CD49f. These data indicate clearly that FDCs bind to reticulin and laminin fibers in LFs via their respective receptors.

Description: Multilineage and single-lineage hemopoietic precursors were studied in 102 bone marrow transplant recipients and their respective donors to determine their contribution to clinical outcome as measured by time to engraftment and survival. The patient population was heterogenous with respect to diagnosis and disease status. They included individuals with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), aplastic anemia, and a few other hematopoietic malignancies. The frequency of various clonogenic precursors in the normal donor population varied considerably. The data yielded a symmetrical distribution. In contrast, most bone marrow transplant recipients presented with significantly reduced numbers of clonogenic cells before transplantation, resulting in skewed distribution profiles. Serial studies of recipients demonstrated a significantly lower than normal level of clonogenic precursors even 3 and 4 years after transplantation. The median values and distribution profiles approximated those observed before transplantation but did not return to measurements obtained for normal donors. Patients with ALL deviated from this pattern. The median values and distribution profiles of clonogenic precursors before transplantation approximated the pattern of normal donors. The frequency of clonogenic progenitors after transplantation, however, remained significantly lower than that of their respective donor or pretransplant values. Cell cycle studies performed after normalization of peripheral blood hematopoietic parameters demonstrated for most recipients that a higher than normal proportion of multipotent cells was in S-phase (P = .011). By univariate and multivariate approaches, clonogenic precursors and clinical parameters were assessed for their contributions to clinical outcome as measured by time to engraftment and survival time. The number of nucleated cells in the transplant inoculum contributed to survival independent of other risk factors. Patients with a higher cell load had a higher probability of surviving than did patients with a lower cell concentration in the transplant inoculum (P = .042). The frequency of clonogenic precursors in the transplant inoculum altered neither survival nor time to engraftment. The time to engraftment was significantly influenced by the frequency of clonogenic megakaryocyte precursors (CFU-M) observed in recipients prior to transplantation (P = .003). Patients with high values engrafted faster than did patients with a low frequency of CFU-M. This was independent of both diagnosis and disease status of the patients at time of transplantation.

Description: Plasma fibronectin (Fn) induces phagocytosis of C3b-opsonized sheep erythrocytes (EC3b) by human peripheral blood monocytes. However, Fn does not induce erythrophagocytosis of EC3b by human polymorphonuclear leukocytes (PMN), unless the PMN have been exposed to C5a or N-formyl- methionyl-leucyl-phenylalanine. Because of this difference, it is of great interest to examine Fn binding to cells that possess the capacity to differentiate into either granulocytes or monocytes. Hence, we have examined the consequences of Fn binding to the human myelomonocytic cell line, HL-60, both before and after in vitro differentiation of the HL-60, along a monocytoid or a granulocytoid pathway. Fn receptors were not found on undifferentiated HL-60, but several differentiating agents promoted the HL-60 binding of Fn-coated microspheres (Fn-ms). The peak of Fn-ms binding occurred four to five days after the induction of differentiation with dimethylsulfoxide (DMSO), and two days after induction by PMA. In addition, cells that differentiated along either the monocytoid or the granulocytoid pathway showed a marked increase in the phagocytosis of both IgG-coated erythrocytes (EA) and EC3b when they were exposed to Fn. Comparison of the effects of anti-Fn monoclonals on the binding of Fn-ms to the monocytes, PMN, and HL-60 showed that the same monoclonals block Fn-ms-binding and Fn-induced EC3b phagocytosis by all three cell types. Two monoclonal antibodies, M1/70 and A6F10, directed against membrane antigens on PMN and monocytes, inhibited Fn-ms binding. Both also blocked Fn-induced EC3b ingestion by these cells. However, neither antibody blocked Fn-ms binding or EC3b ingestion by differentiated HL-60. We conclude that differentiated HL-60 cells express functionally active Fn receptors, similar to monocytes and activated PMN, which, nonetheless, differ from normal cells in their association with the antigens recognized by M1/70 and A6F10.

Description: Four distinct intragenic polymorphisms in the coagulation factor IX gene which have been reported to be important for family diagnosis of Caucasian hemophilia B were studied in 51 normal Japanese subjects (21 males and 30 females). High-molecular-weight DNA prepared from peripheral blood lymphocytes were digested with endonuclease, Ddel, Mspl, Taql or Xmnl, and were studied by Southern blot analysis with factor IX complementary DNA as a probe. None of the minor fragments produced by these enzymes was found in the normal Japanese DNA samples tested, although the probe detects minor allelic forms in control Caucasian DNA samples. Our data suggest that the frequent polymorphic sites found in Caucasians are possibly absent in the Japanese population.

Description: The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes by immunoaffinity chromatography using a monoclonal antibody specific for GP IIb-IIIa. GP IIb and IIIa were further separated in the presence of sodium dodecyl sulfate (SDS) by gel filtration high- performance liquid chromatography (HPLC). Two cycles of this procedure yielded almost complete separation of homogeneous preparations of GP IIb and IIIa. Each protein was then digested with lysyl endopeptidase (Achromobacter protease I), which cleaves at the carboxyl side of lysine residues, and the resulting oligopeptides from GP IIb and IIIa were fractionated with HPLC using a C18 reverse-phase column. Comparison of the elution profiles showed no obvious homology between the two proteins. Amino acid sequences of selected oligopeptides from each glycoprotein were determined using a gas-phase protein sequencer. Sixty amino acid residues (26 residues for IIb and 34 residues for IIIa) were identified.

Description: Plasma fibronectin (Fn) induces phagocytosis of C3b-opsonized sheep erythrocytes (EC3b) by human peripheral blood monocytes. However, Fn does not induce erythrophagocytosis of EC3b by human polymorphonuclear leukocytes (PMN), unless the PMN have been exposed to C5a or N-formyl- methionyl-leucyl-phenylalanine. Because of this difference, it is of great interest to examine Fn binding to cells that possess the capacity to differentiate into either granulocytes or monocytes. Hence, we have examined the consequences of Fn binding to the human myelomonocytic cell line, HL-60, both before and after in vitro differentiation of the HL-60, along a monocytoid or a granulocytoid pathway. Fn receptors were not found on undifferentiated HL-60, but several differentiating agents promoted the HL-60 binding of Fn-coated microspheres (Fn-ms). The peak of Fn-ms binding occurred four to five days after the induction of differentiation with dimethylsulfoxide (DMSO), and two days after induction by PMA. In addition, cells that differentiated along either the monocytoid or the granulocytoid pathway showed a marked increase in the phagocytosis of both IgG-coated erythrocytes (EA) and EC3b when they were exposed to Fn. Comparison of the effects of anti-Fn monoclonals on the binding of Fn-ms to the monocytes, PMN, and HL-60 showed that the same monoclonals block Fn-ms-binding and Fn-induced EC3b phagocytosis by all three cell types. Two monoclonal antibodies, M1/70 and A6F10, directed against membrane antigens on PMN and monocytes, inhibited Fn-ms binding. Both also blocked Fn-induced EC3b ingestion by these cells. However, neither antibody blocked Fn-ms binding or EC3b ingestion by differentiated HL-60. We conclude that differentiated HL-60 cells express functionally active Fn receptors, similar to monocytes and activated PMN, which, nonetheless, differ from normal cells in their association with the antigens recognized by M1/70 and A6F10.

Description: Multilineage and single-lineage hemopoietic precursors were studied in 102 bone marrow transplant recipients and their respective donors to determine their contribution to clinical outcome as measured by time to engraftment and survival. The patient population was heterogenous with respect to diagnosis and disease status. They included individuals with acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), aplastic anemia, and a few other hematopoietic malignancies. The frequency of various clonogenic precursors in the normal donor population varied considerably. The data yielded a symmetrical distribution. In contrast, most bone marrow transplant recipients presented with significantly reduced numbers of clonogenic cells before transplantation, resulting in skewed distribution profiles. Serial studies of recipients demonstrated a significantly lower than normal level of clonogenic precursors even 3 and 4 years after transplantation. The median values and distribution profiles approximated those observed before transplantation but did not return to measurements obtained for normal donors. Patients with ALL deviated from this pattern. The median values and distribution profiles of clonogenic precursors before transplantation approximated the pattern of normal donors. The frequency of clonogenic progenitors after transplantation, however, remained significantly lower than that of their respective donor or pretransplant values. Cell cycle studies performed after normalization of peripheral blood hematopoietic parameters demonstrated for most recipients that a higher than normal proportion of multipotent cells was in S-phase (P = .011). By univariate and multivariate approaches, clonogenic precursors and clinical parameters were assessed for their contributions to clinical outcome as measured by time to engraftment and survival time. The number of nucleated cells in the transplant inoculum contributed to survival independent of other risk factors. Patients with a higher cell load had a higher probability of surviving than did patients with a lower cell concentration in the transplant inoculum (P = .042). The frequency of clonogenic precursors in the transplant inoculum altered neither survival nor time to engraftment. The time to engraftment was significantly influenced by the frequency of clonogenic megakaryocyte precursors (CFU-M) observed in recipients prior to transplantation (P = .003). Patients with high values engrafted faster than did patients with a low frequency of CFU-M. This was independent of both diagnosis and disease status of the patients at time of transplantation.