Purification and biologic characterization of plasma-derived megakaryocyte growth and development factor

The isolation and cloning of the ligand for the cytokine receptor, Mpl, have been recently described. In this report we present details of the purification of this novel cytokine (megakaryocyte growth and development factor [MGDF]) from aplastic canine plasma. Two forms of canine MGDF, with apparent molecular weights of 25 kD and 31 kD and sharing a common N-terminal amino acid sequence, were isolated. The sole contaminant detected in purified 25-kD or 31-kD MGDF was canine Ig. Canine MGDF is characterized as a human megakaryocyte colony- stimulating factor that acts synergistically with human recombinant stem cell factor but not interleukin-3. MGDF also appears to be physiologically regulated in response to platelet demand. In canine and murine models, serum levels of MGDF activity peak during the thrombocytopenic periods after irradiation, 5-fluorouracil, or antiplatelet antisera injections. These data indicate that the megakaryocyte-stimulating activity that accumulates in plasma in response to platelet losses is a novel cytokine that functions through an interaction with the Mpl cytokine receptor.     

Acute lymphoblastic leukemias with deletion of 11q23 or a novel inversion (11)(p13q23) lack MLL gene rearrangements and have favorable clinical features

Balanced translocations affecting the 11q23 region are among the most frequent chromosomal abnormalities in childhood acute lymphoblastic leukemia (ALL), comprising 5% to 6%. These cases consistently have a rearranged MLL gene and are associated with high-risk presenting features, hyperleukocytosis and younger age, and a poor treatment outcome. To assess the clinical and biologic significance of 11q23- associated structural chromosomal abnormalities other than translocations, we studied 17 cases of childhood ALL [14 with del(11)(q23) and 3 with inv(11)(p12q23)] that were identified among 785 cases with successful chromosome analysis. In contrast to reported cases with 11q23 and MLL gene rearrangement, our series was characterized by relatively low leukocyte counts (median, 15.1 x 10(9)/L), expression of CD10 antigen but not myeloid-associated CD15 and CDw65 antigens, a relatively high frequency of T-cell immunophenotypes, and a generally favorable prognosis. All 13 cases with interpretable molecular analysis lacked MLL gene rearrangements. We suggest that most cases with deletions or inversions affecting the 11q23 region represent clinically and biologically different entities as compared with those defined by 11q23 translocation.     

The expression of p53 protein in non-Hodgkin's lymphomas is not always dependent on p53 gene mutations

p53 overexpression has been found to be a fairly common feature in high grade lymphomas in the majority of tumoral cells. The results vary from series to series, from 25% to 33% of cases. To assess whether immunohistochemical positivity for p53 correlated with the presence of structural gene abnormalities, DNA from 16 non-Hodgkin's lymphomas with high and low p53 values was amplified and sequenced to determine the existence of point mutations in the highly conserved regions of the p53 gene. In the group of 8 cases containing high levels of protein, 3 cases showed missense point mutations at the codons mapping between exons 5 through 8. Of the 8 cases of tumors containing undetectable or low levels of p53 protein, 1 case presented a nonsense point mutation giving a stop codon. No missense mutations were detected in this group. The finding of p53 mutations in 4 of 16 cases confirms the presence of p53 gene mutations in high grade lymphomas distributed over different histologic groups. These include Burkitt's lymphoma, together with centroblastic, immunoblastic, and large cell lymphoma of mucosa origin. Nevertheless, the absence of mutations in 5 of the 8 cases that overexpressed p53 suggests that the nuclear or cytoplasmic stabilization of p53 protein could also depend on other factors. The absence of detectable levels of p53 protein cannot discount the existence of p53 mutations, as is shown by a case of Burkitt's lymphoma in which a nonsense mutation was detected. The impact of this range of p53 alterations on clinical course and treatment response of the patients deserves to be explored, in an attempt to differentiate the specific consequences of each one.     

Basic fibroblast growth factor promotes the proliferation of human megakaryocyte progenitor cells

Basic fibroblast growth factor (bFGF), a multifunctional growth factor produced by bone marrow stromal cells, is known to be a potent modulator of hematopoiesis. Because bFGF is present in both human megakaryocytes (MKs) and platelets, we have hypothesized that this growth factor might affect human megakaryocytopoiesis. To test this hypothesis, either low density bone marrow (BM) cells (LDBM), a human BM subpopulation (CD34+ DR+) enriched for the colony-forming unit megakaryocyte (CFU-MK) or a BM subpopulation (CD34+ DR-) enriched for the more primitive burst-forming unit megakaryocyte (BFU-MK) were assayed in the presence of this growth factor. The effect of bFGF on MK colony formation differed according to the cell population assayed. bFGF alone had on MK colony-stimulating activity (MK-CSA) when either CD34+ DR+ or CD34+ DR- BM cells were cloned, but exhibited MK-CSA equivalent to that of interleukin-3 (IL-3) when LDBM cells were used as the target cell population. The MK-CSA of bFGF was inhibited by the addition of neutralizing antisera to either IL-3 and/or granulocyte- macrophage colony-stimulating factor (GM-CSF) but not IL-6. The addition of excess amounts of either IL-3 or GM-CSF to cultures containing bFGF plus anti-IL-3 or anti-GM-CSF reversed the inhibition by the corresponding antisera. The addition of bFGF and IL-3 to assays containing CD34+ DR+ or CD34+ DR- cells increased the size of both CFU- MK- and BFU-MK-derived colonies, respectively, when compared with assays containing IL-3 alone. This increase in MK colony size mediated by bFGF was not affected by addition of either an anti-GM-CSF or anti- IL-6 neutralizing antisera. When LDBM cells were assayed, bFGF alone increased CFU-MK-derived colony size when compared with control values. However, this potentiation of MK colony size by bFGF could be reversed by the addition of either anti-IL-3 or anti-GM-CSF but not anti-IL-6 antisera. In addition, the effects of bFGF and IL-3 on the size of MK colonies cloned from LDBM were not additive. These results suggest that bFGF affects human megakaryocytopoiesis by directly promoting MK progenitor cell proliferation and stimulating BM accessory cells to release growth factor(s) with MK-CSA, such as IL-3 and GM-CSF. We conclude that bFGF, likely produced by cellular components of the BM microenvironment, plays an important role in the control of human megakaryocytopoiesis.     

The B7/BB1 antigen is expressed by Reed-Sternberg cells of Hodgkin's disease and contributes to the stimulating capacity of Hodgkin's disease-derived cell lines

The B7/BB1 molecule has recently been found to be expressed on professional antigen-presenting cells and to be the natural ligand for CD28 and CTLA-4 on T cells. On binding of B7/BB1, CD28 transduces a signal that synergizes with triggering of the T-cell antigen receptor, resulting in enhanced cytokine secretion. In view of the data supporting an antigen-presenting function of Reed-Sternberg cells, we evaluated the expression of B7/BB1 in lymph nodes affected by Hodgkin's disease. B7/BB1 was found to be strongly expressed by the Reed- Sternberg cells in all 47 cases of Hodgkin's disease studied. Moreover, Reed-Sternberg cells were frequently surrounded by CD28-expressing T cells. Evidence for a functional role of B7/BB1 on Reed-Sternberg cells was obtained by our findings that T-cell proliferation and interleukin- 2 (IL-2) production in the primary allogenic mixed lymphocyte reaction (MLR), using the B7/BB1-expressing Hodgkin's disease-derived cell lines L428 and KM-H2 as stimulators, could be partially blocked by adding anti-B7 monoclonal antibody. B7/BB1 expression was also evaluated in a group of non-Hodgkin's lymphomas (n = 46). Whereas B7/BB1 was not expressed by the neoplastic cells of most non-Hodgkin's lymphomas, including T-cell-rich B-cell lymphoma (n = 11), it was present on the neoplastic cells of anaplastic large-cell lymphoma (Ki-1 lymphoma) (n = 5) and follicular lymphoma (n = 4). Our data provide further evidence for an accessory cell function of Reed-Sternberg cells. The accessory cell function of Reed-Sternberg cells might lead to pronounced T-cell activation in vivo, which might contribute to the Hodgkin's syndrome. In addition, our study indicates that B7/BB1 may be a useful marker for differentiating Hodgkin's disease from morphologically similar conditions such as T-cell-rich B-cell lymphoma.     

Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils

The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.     

Amylose ostéoarticulaire et dysglobulinémie: à propos de deux cas

Amyloidosis of bone and joint related to plasma cell dyscrasia.We report two cases of bone and joint amyloidosis involvement related to plasma cell dyscrasia. The radiographie appearances mimic numerous benign or malignant diseases. MR imaging shows a diffuse low signal in TI and an heterogenous low or mild low signal in T2 weighted spin-echo sequence.     

Characterization of a platelet membrane protein of low molecular weight associated with platelet activation following binding by monoclonal antibody AG-1

With the exception of the major platelet glycoproteins IIb/IIIa and Ib, which function as receptors for fibrinogen and von Willebrand factor, little is presently known regarding the possible role of other platelet surface proteins in mediating platelet aggregation. We report the production of a murine monoclonal antibody (AG-1) recognizing human platelet membrane surface protein of relatively low molecular weight (mol wt) that may be involved in this process. AG-1 added to human platelet-rich plasma induces dense granule secretion and aggregation, with lag phase and maximal extent of aggregation dependent on antibody concentration. Aggregation induced by AG-1 is inhibited by AG-1 Fab fragments, indicating that the response is not Fc receptor-mediated. Although AG-1 continues to produce platelet shape change in the presence of EDTA, aggregation is fully inhibited and appears to be mediated by fibrinogen binding to glycoproteins IIb/IIIa. AG-1 is a potent stimulus of thromboxane formation, but full inhibition of thromboxane production by 30 mumol/L indomethacin does not significantly inhibit platelet aggregation induced by 25 micrograms/mL AG-1, indicating that aggregation induced by AG-1 may proceed by way of an endoperoxide-independent pathway. Quantitation of AG-1 Fab binding to platelets reveals approximately 65,000 binding sites per platelet. When intact platelets are radioiodinated, immunoprecipitation of NP-40 lysates by AG-1 reveals an intensely labeled protein with an apparent mol wt of approximately 21,000 daltons, and several additional bands in the mol wt range of 22,000 to 28,000 daltons, all sharing the AG-1 epitope. These bands appear to be distinct from glycoprotein IX or from the beta-chains of glycoprotein Ib or IIb. Finally, studies with platelets labeled by the periodate-[3H]borohydride procedure suggest the possibility of complex formation between subpopulations of glycoprotein Ib and the low-mol-wt glycoproteins recognized by AG-1.     

Phenotype of early erythroblastic leukemias

Nine cases of early erythroblastic leukemia, unidentified by usual criteria, have been diagnosed using a panel of antibodies. Three cases arose in patients with Down's syndrome, one in a patient with therapy- related leukemia, and four patients were in blast crisis of chronic myeloid leukemia; only one case arose de novo. Blast cells could be assigned to two main stages of erythroid differentiation: presence of all erythroid-specific proteins in two patients, a phenotype corresponding to an immature erythroblast; absence of the erythroid markers such as glycophorin A and spectrin in the presence of carbonic anhydrase isoenzyme I, ABH group antigens, and the antigen defined by FA6 152 monoclonal antibody in six patients, a phenotype related to a late erythroid progenitor (CFU-E). One patient had an intermediate phenotype. All patients except one demonstrated a megakaryocytic component. In three patients, chromosomal abnormalities were present, detected both in blasts and in erythroid colonies. In conclusion, these findings indicate that most "cryptic erythroleukemias" are blocked at a "CFU-E-like" stage of differentiation, it may be a frequent event in Down's syndrome and chronic myeloid leukemia, and these erythroleukemias are phenotypically heterogeneous.     

Isolation of the full-length murine erythropoietin receptor using a baculovirus expression system

The full-length murine erythropoietin receptor was expressed in Spodoptera frugiperda (Sf9) cells using a recombinant baculovirus vector. Erythropoietin receptor protein production was maximal 48 hours after infection, as determined by metabolic labeling and immunoblotting; receptor protein varied in molecular mass from 62 to 76 kD. Erythropoietin receptors produced in Sf9 cells could be solubilized using CHAPS in a form capable of binding erythropoietin, and the solubilized receptor bound to immobilized Concanavalin A (Con A) and wheat germ agglutinin, as well as to immobilized recombinant human erythropoietin. Analysis of the distribution of erythropoietin receptors in Sf9 plasma membrane and cytosol fractions using lectin affinity chromatography revealed that membrane-bound receptor had a higher apparent molecular mass and contained the bulk of receptors that bound to wheat germ agglutinin. The receptor was purified by sequential affinity chromatography on Con A-Sepharose and immobilized erythropoietin. Erythropoietin receptors expressed in Sf9 cells were inserted into the plasma membrane in the correct orientation, bound 125I-erythropoietin with a single affinity (kD, 330 pmol/L), and were internalized after ligand binding. However, kD varied inversely with the number of cell surface receptors. Solubilized erythropoietin receptors in whole-cell lysates and isolated plasma membranes exhibited high-affinity binding, with kD values of 92 and 57 pmol/L, respectively. Erythropoietin bound to the surface of infected Sf9 cells could be cross-linked to two proteins with molecular masses of 90 and 65 kD using the homobifunctional cross-linker, disuccinimidyl suberate (DSS). Similar results were obtained with solubilized receptors in whole-cell lysates, and both proteins could be immunoprecipitated by an antiserum to the erythropoietin receptor carboxyl-terminal domain.