p16(INK4a) induces differentiation and apoptosis in erythroid lineage cells

OBJECTIVE: Hematopoiesis is regulated by proliferation, differentiation, and death. p16(INK4a) has been reported to regulate apoptosis and differentiation of diverse cells, as well as arresting the cell cycle at G1 phase. The aim of this study is to explore the properties of p16 in apoptosis and differentiation of erythroid cells. METHODS: We transfected the INK4a gene to K562 cells, which defect the INK4a gene, and compared the effect of enforced expression of p16(INK4a) with that of various additives, topoisomerase I inhibitor (SN 38), interferon-alpha, phosphatidyl-inositol-3 kinase inhibitor (LY294002), and serum deprivation, which arrest cell cycle at different phases. We also investigated the role of p16(INK4a) in normal day-6 human erythroid colony-forming cells by transfecting the INK4a gene. RESULTS: p16(INK4a) induced cell cycle arrest at the G0/G1 phase, and promoted erythroid differentiation in viable K562 cells, but induced apoptosis in K562 cells with incomplete differentiation. The apoptosis induced by p16 was accompanied with downregulation of bcl-x and nuclear NF-kappaB. These findings were not observed in K562 cells treated with various additives. p16(INK4a) decreased the cell viability and promoted apoptosis in day-9 ECFC. CONCLUSION: We propose that p16(INK4a) plays a role in maintaining homeostasis during erythroid differentiation, and that the mechanisms for this effect are not confined to those inducing cell cycle arrest.     

A monoclonal antibody recognizing CD43 (leukosialin) initiates apoptosis of human hematopoietic progenitor cells but not stem cells

CD43 (the major sialoglycoprotein of leukocytes) is an adhesion molecule broadly expressed on hematopoietic cells. A monoclonal antibody recognizing this molecule induces apoptosis of lineage marker- negative bone marrow hematopoietic progenitor cells (HPCs) that express CD34 at a high density (CD34hiLIN-). However, not all cells within this population undergo apoptosis on stimulation via CD43. Dividing progenitor cells are most highly affected, whereas more primitive quiescent cells survive anti-CD43 monoclonal antibody treatment. These surviving cells (1) are enriched for cobblestone area-forming cells, (2) repopulate fragments for human fetal bone implanted into C.B-17 scid/scid mice, (3) have a potential to differentiate in vivo to myeloid and lymphoid cells, and (4) have a high proliferative potential in long-term stromal cell-free liquid culture. These data indicate that cells with hematopoietic stem cell characteristics are relatively resistant to CD43-mediated apoptosis as compared with HPCs. Thus, CD43 may be specifically involved in the regulation of HPC proliferation.     

The monoclonal antibody TER-119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage

The antigen specificity of a rat monoclonal antibody TER-119 was investigated. In adult mice, TER-119 reacted with mature erythrocytes, 20-25% of bone marrow cells and 2-3% of spleen cells but not with thymocytes nor lymph node cells. In fetal haematopoietic tissues, 30-40% of d 10 yolk sac cells, 80-90% of d 14 fetal liver cells and 40-50% of newborn liver cells were reactive with TER-119. TER-119+ cells in adult bone marrow expressed significant levels of CD45 but not myeloid (Mac-1, Gr-1) or B-cell (B220) markers. Morphological examination and haematopoietic colony-forming assays for isolated TER-119+ cells revealed that TER-119 reacts with erythroid cells at differentiation stages from early proerythroblast to mature erythrocyte, but not with cells showing typical erythroid blast-forming unit (BFU-E) and erythroid colony-forming unit (CFU-E) activities. Erythroleukaemia cell lines do not express the TER-119 antigen even after stimulation with dimethylsulphoxide. TER-119 immunoprecipitated protein bands with molecular masses of 110 kDa, 60 kDa, 52 kDa and 32 kDa from erythrocyte membrane, whereas only a 52-kDa band was detected by TER-119 in Western blot analysis. Further molecular and cellular analyses indicated that the TER-119 antigen is a molecule associated with cell-surface glycophorin A but not with glycophorin A itself.     

Identification and purification of human erythroid progenitor cells by monoclonal antibody to the transferrin receptor (TÜ 67)

Summary Anti-TÜ 67 is a murine monoclonal antibody that recognizes the transferrin receptor. With respect to hematopoietic cells TÜ 67 is expressed by human multipotent colony-forming cells (CFU-Mix), erythroid progenitor cells (BFU-E and CFU-E) and a fraction of granulocyte/monocyte colony forming cells, but is not expressed by mature hematopoietic cells including erythrocytes, platelets, lymphocytes, and peripheral blood myeloid cells. The TÜ 67-positive fraction of normal bone marrow, separated by fluorescence-activated cell sorting (FACS) or immune rosettes, contained 87% of the erythroid progenitor cells. Erythroid progenitor cells were enriched up to 50-fold by using a combination of monoclonal antibodies to deplete mature hematopoietic cells, followed by positive selection of BFU-E and CFU-E by TÜ 67 antibody.Supported by the Deutsche Forschungsgemeinschaft (He 1380-2/1)     

Cell cycle exit during terminal erythroid differentiation is associated with accumulation of p27(Kip1) and inactivation of cdk2 kinase

Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21(Cip1) and p27(Kip1) during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21(Cip1) and p27(Kip1) proteins were induced during erythroid differentiation, but only p27(Kip1) associated with the principal G(1) CDKs-cdk4, cdk6, and cdk2. The kinetics of binding of p27(Kip1) to CDK complexes was distinct in that p27(Kip1) associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27(Kip1) to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27(Kip1) binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G(1) growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27(Kip1) binding or loss of cyclin D from the complex. The data demonstrate that p27(Kip1) differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27(Kip1) until a specified time in differentiation when cdk2 kinase activity is targeted by p27(Kip1) to elicit G(1) growth arrest. Further, the data imply that p21(Cip1) may have a function independent of growth arrest during erythroid differentiation. (Blood. 2000;96:2746-2754)     

Unimpaired terminal erythroid differentiation and preserved enucleation capacity in myelodysplastic 5q(del) clones: a single cell study

Background

Anemia is a characteristic of myelodysplastic syndromes, such as the rare 5q- syndrome, but its mechanism remains unclear. In particular, data are lacking on the terminal phase of differentiation of erythroid cells (enucleation) in myelodysplastic syndromes.

Design and Methods

We used a previously published culture model to generate mature red blood cells in vitro from human hematopoietic progenitor cells in order to study the pathophysiology of the 5q- syndrome. Our model enables analysis of cell proliferation and differentiation at a single cell level and determination of the enucleation capacity of erythroid precursors.

Results

The erythroid commitment of 5q(del) clones was not altered and their terminal differentiation capacity was preserved since they achieved final erythroid maturation (enucleation stage). The drop in red blood cell production was secondary to the decrease in the erythroid progenitor cell pool and to impaired proliferative capacity. RPS14 gene haploinsufficiency was related to defective erythroid proliferation but not to differentiation capacity.

Conclusions

The 5q- syndrome should be considered a quantitative rather than qualitative bone marrow defect. This observation might open the way to new therapeutic concepts.     

Cross-reactivity of a monoclonal antibody to the amino terminal region of thyrotropin receptor with the serum protein alpha(1)-antitrypsin.

In a study designed to detect the presence of soluble, secreted A subunit of thyrotropin hormone receptor (TSHR) in serum, using anti-TSHR murine antibodies (mAbs) and peptide specific antiserum for Western blotting of human serum proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) it was consistently observed that only one mAb, termed A10, reacted strongly with the 53 kd serum protein. The reaction was specific with the mAb A10 only, but not with another mAb or polyclonal antiserum. Furthermore, A10 immunoreactivity was documented in a variety of sera from healthy donors and patients, including patients whose thyroid gland was ablated during treatment for thyroid cancer. This suggests that the A10 cross-reactive protein was not derived from thyroid cells. The A10 cross-reactive protein was purified from normal serum and subjected to N-terminal sequence analysis, which identified the protein as alpha(1)-antitrypsin. Further experiments by enzyme-linked immunosorbent assay (ELISA) and the binding of antibody with deglycosylated or elastase-treated purified serum protein confirmed the cross-reactivity of mAb A10 with alpha(1)-antitrypsin. Alignment of the TSHR amino acid sequence with that of alpha(1)-antitrypsin identified five identical amino acids in a short stretch of residues 34-39 (EEDFRV) in TSHR and residues 205-210 (EEDFHV) in alpha(1)-antitrypsin. Analysis of the structural model of alpha(1)-antitrypsin revealed that these residues were exposed on the surface of alpha(1)-antitrypsin and were accessible for antibodies. Autoantibodies in patients with Graves' disease do not appear to recognize this region of the receptor and hence do not react with serum alpha(1)-antitrypsin.     

Diagnostic differentiation of chronic B-cell malignancies using monoclonal antibody L161 (CD1c)

The expression of membrane CD1c, as defined by monoclonal antibody L161, was examined on malignant lymphoid cells from 191 cases of chronic lymphoproliferative disease and on eight 'normal' enriched tonsil B-cell extracts. Of 79 cases of chronic lymphocytic leukaemia (CLL) studied, 77 showed low (less than 20% positive cells) CD1c expression whereas 63/71 (89%) cases of B-PLL, HCL and B-NHL showed increased CD1c+ (but not CD1a or CD1b) components. In contrast, malignancies corresponding to terminal stages of B-cell differentiation (immunocytoma and myeloma) generally showed low CD1c expression as did lymphoid cells from 10 cases of post-thymic malignancy. Although there was some correlation between the expression of membrane CD1c and immunoglobulin (SIg) light chain densities (P less than 0.001), it is relevant in diagnostic terms that seven cases of B-NHL with low SIg staining intensities more typically associated with CLL were CD1c+. CD1c expression was not, however, correlated with the presence of CD23 or FMC7 determinants but did show a similar pattern of expression to that previously reported for beta-2 microglobulin. Determination of cellular CD1c by APAAP immunocytochemistry confirmed the presence of higher antigen densities in malignant B-cells at intermediate/late stages of differentiation and this interpretation was further supported by the finding that the majority of phenotypically mature tonsil B-cells were also CD1c+. The determination of CD1c expression by malignant B-cells may therefore be of particular value in the diagnostic differentiation of chronic lymphoproliferative disorders.     

Inhibition of differentiation and affinity purification with a monoclonal antibody to a myeloid cell differentiation-inducing protein

The normal myeloid hematopoietic regulatory proteins include four growth-inducing proteins called colony-stimulating factors (CSF), including interleukin-3 (IL-3), or macrophage and granulocyte inducers, type 1 (MGI-1), and another type of protein (MGI-2) with no myeloid cell growth-inducing activity that induces differentiation of normal myeloid precursor cells and certain clones of myeloid leukemic cells. An IgG2a monoclonal antibody was prepared and it neutralized two forms of MGI-2 (MGI-2A and MGI-2B) produced by mouse Krebs ascites tumor cells. The monoclonal antibody was used for affinity purification of MGI-2. This antibody also neutralized MGI-2 produced by normal mouse macrophages, normal myeloblasts incubated with IL-3, and MGI-2 produced by the lungs and found in the serum of mice injected with lipopolysaccharide (LPS). The anti-MGI-2 antibody did not inhibit the activity of any one of the four myeloid growth-inducing proteins (CSF or IL-3 = MGI-1), IL-1, tumor necrosis factor, or lymphotoxin. This antibody also inhibited induction of differentiation of myeloid leukemic cells by LPS, which is mediated by the endogenous production of MGI-2, but did not inhibit induction of differentiation in these leukemic cells by dexamethasone or cytosine arabinoside, which is not mediated by MGI-2. Anti-MGI-2 antibody thus inhibited differentiation when MGI-2 was added externally to cells or when it was mediated by endogenously produced MGI-2.     

Effects of erythroid differentiation factor (EDF) on proliferation and differentiation of human hematopoietic progenitors.

The effects of erythroid differentiation factor (EDF) on normal human hematopoietic progenitor cells were examined by bone marrow colony assay. Addition of EDF to the erythroid colony assay system enhanced erythroid burst-forming unit (BFU-E)-derived colony formation, and this effect disappeared on removal of adherent cells. Conditioned medium of EDF-treated monocytes also enhanced BFU-E colony formation, whereas conditioned medium of EDF-treated T cells did not. In contrast, EDF inhibited erythroid colony-forming unit (CFU-E) colony formation dose-dependently, although it had no effect on colony formation by myeloid cells. These data show that EDF has a specific effect on human hematopoietic progenitors of the erythroid lineage. The results also indicate that EDF enhanced BFU-E colony formation by stimulating adherent cells to produce factors with burst-promoting activity (BPA), but suppressed CFU-E colony formation by promoting differentiation of these cells.     

Characterization of a monoclonal antibody against a differentiation antigen of human natural killer cells (NK cells)]

The new murine monoclonal antibody BL-(H5) reacts with a novel surface molecule which is mainly expressed on human NK and B cells. The antigen is not expressed on peripheral T lymphocytes, thymocytes and different human T-cell lines. BL-(H5) does not bind to erythrocytes and platelets. The monoclonal antibody reacts in western blotting experiments with an antigen of 78kDa.     

Immunoelectron microscopic demonstration of antigenic sites on lymphoid cells using a human monoclonal antibody (Ha6D3)

The reactivity of a human monoclonal antibody directed against human B and T lymphocytes was tested for the first time at the ultrastructural level. The antigenic sites detected by this antibody were localized on the surface membrane of lymphocytes and, to a lesser extent, in the cytoplasm on membranes of the endoplasmic reticulum and perinuclear envelop of some centroblasts and immunoblasts. Ultrastructural demonstration of target antigen detected by human monoclonal antibodies may be important prior to therapeutic application of these antibodies.     

Diagnostic utility of a mouse monoclonal antibody (5-21-3) employed as a competitive probe in an immunoassay to detect antibody to HIV-1 gp41.

A mouse monoclonal antibody, designated 5-21-3, was raised against HIV-1 gp41 using detergent-disrupted virus as the immunogen. Antibody 5-21-3 was conjugated to horseradish peroxidase (HRP) and employed as a competitive probe against normal and HIV-1 antibody-positive sera in an immunoassay to detect the presence of antibody to HIV-1 gp41. The diagnostic utility of the competitive monoclonal immunoassay was assessed by correlation to a similar assay which employed HRP-labeled polyclonal IgG from a gp41-seropositive donor as the competitive probe. The monoclonal immunoassay was greater than 98% as sensitive and 99% as specific as the polyclonal immunoassay, regardless of the geographic source or disease state of the donor. The monoclonal immunoassay also was nearly as effective as the polyclonal immunoassay in detecting points of seroconversion in individuals enrolled in longitudinal studies. Of particular interest was the finding that the epitope recognized by monoclonal antibody 5-21-3 did not map to the well-characterized gp41 immunodominant region.     

Phase 1 study results of the type II glycoengineered humanized anti-CD20 monoclonal antibody obinutuzumab (GA101) in B-cell lymphoma patients

Whereas the chimeric type I anti-CD20 Ab rituximab has improved outcomes for patients with B-cell malignancies significantly, many patients with non-Hodgkin lymphoma (NHL) remain incurable. Obinutuzumab (GA101) is a glycoengineered, humanized anti-CD20 type II Ab that has demonstrated superior activity against type I Abs in vitro and in preclinical studies. In the present study, we evaluated the safety, efficacy, and pharmacokinetics of GA101 in a phase 1 study of 21 patients with heavily pretreated, relapsed, or refractory CD20(+) indolent NHL. Patients received GA101 in a dose-escalating fashion (3 per cohort, range 50/100-1200/2000 mg) for 8 × 21-day cycles. The majority of adverse events (AEs) were grades 1 and 2 (114 of 132 total AEs). Seven patients reported a total of 18 grade 3 or 4 AEs. Infusion-related reactions were the most common AE, with most occurring during the first infusion and resolving with appropriate management. Three patients experienced grade 3 or 4 drug-related infusion-related reactions. The best overall response was 43%, with 5 complete responses and 4 partial responses. Data from this study suggest that GA101 was well tolerated and demonstrated encouraging activity in patients with previously treated NHL up to doses of 2000 mg. This trial is registered at www.clinicaltrials.gov as NCT00517530.     

抗MTB FurB单克隆抗体制备及特性分析

目的制备针对结核分支杆菌FurB蛋白的单克隆抗体(mAb)并分析其特性。方法利用纯化的FurB蛋白,采用腹股沟皮下包埋硝酸纤维素膜和腹腔内注射FurB的方法免疫小鼠,然后进行细胞融合、克隆化制备抗FurB mAb,用Western-blot鉴定其特异性,用ELISA法初步分析其特异性位点和相对亲和力。结果获得3株抗FurB mAb,分别为DD12、BH1和DH8,经Western-blot分析都可与FurB蛋白特异性结合。三株mAb中DH8效价最高达到10-10,相对亲和力,BH1>DH8>DD12,3株mAb的抗原表位相近。结论获得的抗FurB mAb效价高、特异性强,可以作为进一步研究FurB功能和作用的有效工具。     

Reaction of rheumatoid factors with IgG3 monoclonal anti-Rh(D) antibodies: more frequent reactivity to a monoclonal antibody of the Gm allotype G3m(5) in rheumatoid patients negative for G3m(5).

Human monoclonal anti-Rh(D) antibodies of known IgG isotype and Gm allotype were bound to erythrocytes and then used as the target IgG antigens for rheumatoid factors (RFs) in a direct haemagglutination test. When serum samples from patients with rheumatoid arthritis (RA) were tested for RF specificity towards these IgG monoclonal anti-D antibodies the incidence and titre of reactivity towards an IgG3 monoclonal anti-D antibody was considerably greater than for a polyclonal anti-D antibody of the same Gm allotype, G3m(5). This difference was not explained by the amount of each anti-D antibody which bound to erythrocytes. Furthermore, when patients with RA were divided into groups according to their Gm phenotype, sera from a greater proportion of patients negative for the phenotype G3m(5) reacted to the G3m(5) monoclonal anti-D antibodies than sera from those patients positive for this allotype. Analysis of RF reactivities towards two IgG3 and three IgG1 monoclonal anti-D antibodies, each with different Gm allotypic epitopes, indicated, however, that individual serum samples contained RFs with a spectrum of specificities; some sera appeared to react to a single set of Gm alleles, whereas others also reacted to isotypic or iso-allotypic epitopes, or both. Our data suggest that RFs with specificity for Gm allotypes do not arise in patients who carry that particular allotype owing to tolerance induced in fetal-neonatal life. Conversely, RFs with apparent specificity for a Gm allotype formed in patients negative for that allotype may be reacting to a closely related but different epitope. Final proof requires precise specificities for each RF formed, and IgG3 monoclonal anti-D antibodies would be useful reagents for this purpose.     

In vitro studies of a bispecific monoclonal antibody antithrombotic conjugate [ATC(3)]

We assessed whether chemical conjugation of a monoclonal antibody (M735) against platelet glycoprotein IIb/IIIa receptors to urokinase and to a monoclonal antibody which binds to damaged endothelium (P14G11) resulted in enhanced inhibition of platelet aggregation with maintenance of localizing features (to damaged endothelium). Conjugation of M735 (an IgM monoclonal antibody) to urokinase and P14G11 (an IgG2a monoclonal antibody) was carried out using SPDP [N-Succinimidyl-3-(2-pyridyldithio)-propionate] as the cross-linking reagent. The resulting triple conjugate [ATC(3)] had fibrinolytic activity and retained platelet and damaged endothelium localizing features. Enhanced inhibition of platelet aggregation (induced by either adenosine diphosphate (ADP), collagen, or thrombin) was observed compared with both an unconjugated mixture of the three individual components and to the M735-urokinase conjugate. We conclude that it is possible to produce a bimonoclonal antibody-urokinase agent which has both potent antiplatelet aggregatory effects and additional targeting or localizing features.Presented at the 35th World Congress, International College of Angiology, Copenhagen, Denmark, July 1993R.S.M., M.J.B., M.U., and S.P. are supported by grants from the British Heart Foundation     

Use of anti-T-cell monoclonal antibody OKT3 to prevent acute graft-versus-host disease in allogeneic bone-marrow transplantation for acute leukaemia

Seventeen patients who received allogeneic bone-marrow transplants from matched or slightly mismatched (in four patients) siblings were observed for at least 60 days or until acute graft-versus-host disease (GvHD) developed. All donor marrows after preliminary manipulation were incubated with 1 mg of the murine monoclonal antibody OKT3 before infusion in an attempt to deplete them of immunocompetent T lymphocytes (opsonisation). In three of the seventeen patients acute GvHD of grade II or greater developed. Two of these patients died, but they had disseminated cytomegalovirus infection as well as GvHD. Eleven patients showed no evidence of acute GvHD, and four had transient limited skin rashes (grade I GvHD). Opsonisation of T lymphocytes has reduced the incidence of severe acute GvHD in this unit from 79% in an earlier group of 14 patients to 18% when added to prophylactic methotrexate.