Clinical-grade quality platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelet concentrates promoted expansion of mesenchymal stromal cells.

BACKGROUND AND OBJECTIVES: The aim of our study was to test a platelet-rich plasma releasate (PRP-R/SRGF) from CaCl2 -activated platelets as a source of growth factors for the expansion of mesenchymal stromal cells (MSCs). PRP-R/SRGF, obtained with a low-cost procedure, is characterized by a reduced variability of growth factor release. MATERIALS AND METHODS: PRP-R/SRGF is a clinical-grade quality solution obtained from CaCl2 -activated platelets. Its activity was evaluated by measuring the proliferation, the phenotype, the differentiation potential and the immunosuppressive properties of MSCs derived from bone marrow (BM) and adipose tissue (AT). RESULTS: PRP-R/SRGF was more active than FBS to expand BM- and AT-derived MSCs. PRP-R/SRGF treatment did not affect the expression of typical MSCs surface markers, neither MSCs differentiation potential nor their capability to inhibit activated T-cell proliferation. CONCLUSIONS: The clinical-grade PRP-R/SRGF may be used in the clinical setting for the expansion of MSCs.

Gefitinib inhibits the ability of human bone marrow stromal cells to induce osteoclast differentiation: implications for the pathogenesis and treatment of bone metastasis.

Significant relief of bone pain in patients with bone metastases was observed in a clinical trial of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor gefitinib in breast cancer. Osteoclast activation and differentiation are regulated by bone marrow stromal cells (BMSC), a heterogeneous cell compartment that comprehends undifferentiated mesenchymal stem cells (MSC) and their specialized progeny. In this regard, we found that human primary BMSCs express immunoreactive EGFR. Expression of EGFR mRNA and protein was also demonstrated in two human, continuous MSC-like cell lines, HDS-1 and HDS-2 cells. Treatment of HDS cells with EGF produced a significant increase in the levels of activated EGFR which was not observed in the presence of gefitinib. A significant reduction in the basal levels of activation of the EGFR and of Akt was observed in HDS cells following treatment with gefitinib. Treatment of HDS cells with gefitinib produced a significant reduction in the levels of secreted macrophage colony-stimulating factor (M-CSF) and cell-associated receptor activator of NF-kappaB ligand (RANKL) in both cell lines, as assessed by using specific ELISA and Western blotting techniques. Finally, the ability to sustain the differentiation of pre-osteoclasts of conditioned medium from gefitinib-treated HDS cells was reduced by approximately 45% as compared with untreated HDS cells. These data have demonstrated for the first time that the EGFR regulates the ability of BMSCs to induce osteoclast differentiation and strongly support clinical trials of gefitinib in breast cancer patients with bone disease.

CD30L up-regulates CD30 and IL-4 expression by T cells.

CD30L is frequently expressed on acute myeloid leukemia (AML) blasts. Its presence is associated with the co-expression of interleukin-4 (IL-4) receptor and with the expansion of specific T-helper 2 (Th2) cell subsets producing IL-4 and expressing CD30. Recombinant CD30L-bearing cells up-regulated the expression of surface CD30 and increased the production of IL-4 and soluble (s) CD30 by co-cultured T cells. These findings were confirmed with AML blasts expressing surface CD30L, where blocking anti-CD30 antibodies completely abolished the release of sCD30 and reduced the production of IL-4. Our data indicates a direct role of CD30L(+) neoplastic cells in driving the immune response toward a Th2-polarized non-protective state.

In vitro and in vivo effects of 2'-deoxycoformycin (Pentostatin) on tumour cells from human gammadelta+ T-cell malignancies.

Hepatosplenic gammadelta+ T-cell lymphoma represents a rare neoplasm of post-thymic phenotype, characterized by an aggressive clinical course and a poor response to conventional chemotherapy. In the present study, we have examined the cytotoxic effects of the purine analogue 2'-deoxycoformycin (dCF) on cultured mononuclear cells and purified gammadelta+ tumour cells from bone marrow or peripheral blood of four patients with hepatosplenic gammadelta+ T-cell lymphoma. At a concentration of 10 microM, dCF, in the presence of 2'-deoxyadenosine (dAdo), displayed an early and selective cytotoxic effect on gammadelta+ tumour T cells. After 48 h of in vitro exposure to dCF, the absolute number of viable CD3+/gammadelta+ tumour T cells was reduced by more than 90% in all samples with respect to control cultures, with absolute counts of viable CD3+/alphabeta+ lymphocytes being reduced only by 6-40% of the initial cell input. Analysis of cultures after 5 d of exposure to dCF plus dAdo revealed the persistence of normal CD3+/alphabeta+ T cells, which accounted, however, for only 20-25% of the initial cell input. Accordingly, the combination of dCF (10-100 microM) plus dAdo was able to induce a dose-dependent inhibition of clonogenic growth and [3H]-thymidine incorporation in purified CD3+/CD4-/CD8- gammadelta+ tumour cells. We also report that one patient with hepatosplenic gammadelta+ T-cell lymphoma in terminal leukaemic phase showed a striking haematological response to single-agent dCF given as fourth-line treatment. In particular, the selective clearance of gammadelta+ tumour T cells in peripheral blood and bone marrow was observed starting after the second course of treatment. Our results suggest that dCF may represent a potentially active drug for the management of this aggressive form of T-cell lymphoma.

CD30 ligand (CD30L)-expressing acute myeloid leukemias: a new model of paracrine interactions for the regulation of blast cells proliferation.

CD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals through its specific counterstructure CD30. Even though there are indications that CD30L plays a key role as a paracrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known about its biological functions in other human hemopoietic malignancies, despite the demonstration of the frequent expression of CD30L in hemopoietic neoplasms of both myeloid and lymphoid origin. The present review summarises structural and biological properties of CD30L, and focuses on CD30L+ acute myeloid leukemias (AMLs) by recapitulating some phenotypic and clinical features of this subset of acute leukemias. We also discuss some mechanisms by which CD30L-expressing leukemic blasts may gain a proliferative advantage through direct interaction with specific cells, in turn expressing its specific counterreceptor CD30. In particular, data has been provided suggesting that CD30L+ AMLs may evoke a sort of polarized T-cell response with the preferential production of Th2-like cytokines, mainly IL-4, by specific CD30-expressing T cell subsets. On the other hand, leukemic blasts presenting surface CD30L, have been shown to express a peculiar cytokine-receptors pattern that makes them an ideal target for T cells-produced Th2-like cytokines. Furthermore, some Th2-like cytokines, such as IL-4, are able to enhance blast cells proliferation, as well as to up-regulate the surface expression of specific adhesion molecules that have been shown to be associated with the presence of CD30L on AML blasts.

The RET receptor tyrosine kinase, but not its specific ligand, GDNF, is preferentially expressed by acute leukaemias of monocytic phenotype and is up-regulated upon differentiation.

The RET gene product represents the signal-transducing molecule of a surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF), which includes GDNFR-alpha as a ligand-binding component. By a semi-quantitative competitive RT-PCR approach, we have analysed the relative abundances of RET transcripts in blasts purified from 40 acute myeloid leukaemia (AML) cases, revealing significant amounts of RET transcripts in 60% of AML cases (24/40). RT-PCR data was confirmed by immunocytochemical detection of RET protein in leukaemic blasts. The highest RET mRNA levels, almost exclusively confined to FAB M4/M5 AMLs, directly correlated with the presence on leukaemic cells of adhesion molecules and surface structures typically expressed by blasts of monocytic lineage and were inversely associated with the expression of the stem cell antigen CD34. Consistently, differentiation of the monoblastic cell line U937 resulted in an up-regulated expression of RET proto-oncogene, which was maximal upon exposure to agents inducing a more complete monocytic differentiation. Finally, while transcripts specific for GDNF and GDNFR-alpha were never found in leukaemic blasts, stromal cells of the haemopoietic microenvironment expressed, in the absence of RET, significant amounts of both GDNF and GDNFR-alpha. Our results suggest a role for RET in the functional regulation of AMLs through interactions with GDNF- and GDNFR-alpha-producing stromal cells.

Characterization of anti-CD138 monoclonal antibodies as tools for investigating the molecular polymorphism of syndecan-1 in human lymphoma cells.

Syndecan-1 (CD138) is a surface proteoglycan consisting of long unbranched glycosaminoglycan (GAG) chains covalently attached to a protein backbone. High levels of a putatively syndecan-1 isoform have recently been found on neoplastic cells of primary effusion lymphoma (PEL). As opposed to murine systems, studies on syndecan-1 isoforms in humans have been hampered by the lack of a precise characterization of anti-CD138 monoclonal antibodies (mAbs). We have therefore investigated the reactivity of anti-CD138 mAbs (B-B4, B-B2, 1D4, MI15 and 104-9) with either intact native proteoglycans or a recombinant unglycosylated form of syndecan-1 core protein, and utilized these reagents to dissect the molecular heterogeneity of syndecan-1 in human lymphoma cells. Our results indicated that: (a) mAb B-B2 recognized only nondenatured syndecan-1, being poorly reactive by immunoblotting with both intact and recombinant syndecan-1 protein; (b) mAb 104-9 was unable to recognize native syndecan-1, but showed a significant reactivity with intact and unglycosylated syndecan-1 protein upon immunoblotting; (c) mAbs B-B4, 1D4 and MI15 recognized both the intact molecule and the core protein of syndecan-1, and showed a comparable reactivity in flow cytometry and immunoblotting. Cross-blocking experiments indicated these latter mAbs recognizing the same or closely related epitopes of syndecan-1. Using these mAbs, we have demonstrated that: (a) tumour cells from PEL expressed a syndecan-1 isoform with a higher molecular weight than that present on malignant plasma cells; (b) syndecan-1 expressed by PEL cells had a core protein identical in size to that expressed by plasma cells, suggesting that differences in syndecan-1 size were due to different GAG chains attached to an identical protein backbone; (c) the PEL-specific isoform of syndecan-1, which probably represented the major proteoglycan expressed by these cells, was effective in mediating cell adhesion to type I collagen substrates. This data represents the first evidence describing the existence of a molecular polymorphism, of syndecan-1 in human lymphomas.

Differential expression of the RET gene in human acute myeloid leukemia.

The RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multi-subunit membrane receptor complex for at least two different types of transforming growth factor (TGF)-beta-related neurotrophic factors. We have previously shown that RET gene expression in acute myeloid leukemia (AML) occurs more frequently in AMLs displaying either a monocytic (FAB M4/M5) or intermediate-mature myeloid phenotype (FAB M2/M3) than in leukemias reflecting an earlier stage of myeloid differentiation (FAB M0/M1). To further verify the association between RET expression and the relative maturation stage of AML cells, we have performed a quantitative estimation of relative abundances of RET transcripts among various FAB subtypes of AMLs. By analyzing 13 AML samples and normal hematopoietic cells through a competitive-quantitative RT-PCR approach, we were able to show that the relative levels of RET-specific mRNAs continuously increase with blast cell maturation in human AML, i.e., the amounts of RET gene-specific transcripts differ among RET-expressing AMLs, being higher in the more differentiated FAB phenotypes. In addition, we provide evidence that the relative amounts of RET transcripts increase upon in vitro and in vivo differentiation of leukemic promyelocytes from FAB M3 AML patients, becoming overall comparable to those found in normal granulocytes. These results indicate that RET expression in human AMLs is maturation-associated, probably mirroring the developmental regulation of this gene during differentiation of normal hematopoietic cells.

Hodgkin's disease: a disorder of dysregulated cellular cross-talk.

Hodgkin's disease (HD) is a peculiar type of human malignant lymphoma characterized by a very low frequency of tumor cells, the so called Hodgkin and Reed-Sternberg (H-RS) cells, embedded in a hyperplastic background of non-neoplastic (reactive) cells recruited and activated by H-RS cells-derived cytokines. H-RS cells can be functionally regarded as antigen-presenting cells (APC) able to elicit an intense, but anergic and ineffective, T-cell mediated immune response along with a hyperplastic inflammatory reaction which involves several cell types including T- and B-cells, neutrophils, eosinophils, plasma cells, fibroblasts and stromal cells. In tissues involved by HD, malignant H-RS cells and their reactive neighboring cells are able to cross-talk via a complex network of cytokine- and cell contact-dependent interactions. As a result of such interactions, mediated by specific surface receptors and adhesion molecules on both tumor and non-neoplastic cells, H-RS cells may receive several proliferative and anti-apoptotic signals favoring the cellular expansion and tumor cell survival in HD. The ineffective T-cell immune response elicited by the abnormal APC function of H-RS cells may further contribute to the biologic and clinical progression of HD. Innovative therapeutic strategies aimed at blocking the pathways of dysregulated cellular cross-talk among H-RS cells and bystander reactive cell populations might be beneficial in the treatment of HD patients.

Reed-Sternberg cells of classical Hodgkin's disease react with the plasma cell-specific monoclonal antibody B-B4 and express human syndecan-1.

Although the cellular origin of Reed-Sternberg (RS) cells of classical Hodgkin's disease (HD) has been a controversial issue for many years, recent immunophenotypic and molecular studies have suggested that RS cells of a subset of classical HD cases may be related to B cells. To further define the immunophenotypic features and the differentiation stage of RS cells, a series of 56 HD samples, including both nodular lymphocyte predominance (LP) (eight cases) and classical HD (nodular sclerosis [NS], 32 cases; mixed cellularity [MC], 16 cases) with a non-T-cell phenotype, were evaluated for the immunohistochemical expression of the B-B4 antigen, a specific marker for terminally differentiated B cells. Because the cDNA of the B-B4 antigen encodes syndecan-1, a member of a family of transmembrane heparan sulfate proteoglycans thought to be involved in binding cells of the B lineage to the interstitial matrix, the B-B4 immunoreactivity was correlated with the expression of syndecan-1 in HD-derived cell lines (L428, KM-H2), as detected by both reverse transcriptase polymerase chain reaction (RT-PCR) studies and Western blotting. Our results show that B-B4 reacts with RS cells and their morphological variants of all cases of classical HD, irrespective of their antigenic phenotype (B, undetermined), albeit at a varying degree of cellular expression. Notably, a high reactivity and staining intensity for the B-B4 monoclonal antibody (MoAb) was restricted to tumor cells from NS HD. In cases of the latter subtype, B-B4 positivity was also found in sclerosis-trapped spindle cells (fibrocytes/fibroblasts). Conversely, the putative tumor cells of nodular LP HD were consistently unreactive with the B-B4 MoAb. Finally, we have demonstrated by RT-PCR, flow cytometry, and Western blotting that cultured RS cells, of B and undetermined phenotype, express syndecan-1 mRNA and produce a form of syndecan-1, recognized by the B-B4 MoAb, which is predominantly associated with glycosaminoglycans and is present at the cell surface. Our detection of the plasma cell-specific antigen B-B4 (syndecan-1) on tumor cells of classical HD further supports that RS cell progenitors may be related to germinal/postgerminal center mature B cells and suggests that expression of syndecan-1 may contribute to some of the typical biologic and histopathologic features of classical HD, with a special regard to the NS subtype.

Expression of the RET receptor tyrosine kinase and GDNFR-alpha in normal and leukemic human hematopoietic cells and stromal cells of the bone marrow microenvironment.

The RET proto-oncogene product is a receptor tyrosine kinase representing the signal-transducing molecule of a multisubunit surface receptor complex for the glial cell line-derived neurotrophic factor (GDNF), in which a novel glycosyl-phosphatidylinositol (PI)-linked protein (termed GDNFR-alpha) acts as the ligand-binding component. We have analyzed expression of RET and GDNFR-alpha in purified normal hematolymphopoietic cells, leukemia/lymphoma cell lines, and 154 primary samples from patients with hematopoietic malignancies encompassing different lineages and differentiation stages. Relatively low amounts of RET mRNA were found in early CD34+ hematopoietic progenitors, but RET transcripts appeared to increase after myelomonocytic maturation. No expression of RET was found in peripheral blood and tissue B and T lymphocytes. Analysis of human myelomonocytic cell lines was overall consistent with results obtained on purified normal cells. Accordingly, RET expression was mainly confined to acute myeloid leukemias (AMLs) displaying either monocytic (French-American-British M4 and M5) or intermediate-mature myeloid (M2 and M3) phenotypes, being less frequently detected in early myeloid (M0 and M1) AMLs. In contrast, RET mRNA was sporadically detected in B-cell tumors, whereas, among T-cell malignancies, RET transcripts were mainly detected in cells of postthymic and mature T-cell phenotype. RET broad detection in primary tumors was not paralleled by the mutual expression of GDNFR-alpha, which was detected only in 2 isolated primary samples and in 3 leukemia/lymphoma cell lines. However, GDNFR-alpha transcripts, in the absence of RET mRNA, were found in normal bone marrow stromal cells (BMSC), in BM fibroblasts, and in two osteoblast cell lines previously described to support normal hematopoiesis. In the presence of GDNF-receptors derived from BMSC by PI-specific phospholipase C cleavage, GDNF efficiently bound RET-expressing AML blasts and was functionally active by reducing their clonogenic growth and triggering the monocytic maturation of leukemic cells.

CD30 ligand is frequently expressed in human hematopoietic malignancies of myeloid and lymphoid origin.

CD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals leading to either cell death or proliferation through its specific counterstructure CD30. Although several lines of evidence indicate that CD30L plays a key role as a paracrine- or autocrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known regarding its distribution and biologic significance in other human hematopoietic malignancies. By analyzing tumor cells from 181 patients with RNA studies and immunostaining by the anti-CD30L monoclonal antibody M80, we were able to show that human hematopoietic malignancies of different lineage and maturation stage display a frequent and broad expression of the ligand. CD30L mRNA and surface protein were detected in 60% of acute myeloid leukemias (AMLs), 54% of B-lineage acute lymphoblastic leukemias (ALLs), and in a consistent fraction (68%) of B-cell lymphoproliferative disorders. In this latter group, hairy cell leukemia and high-grade B-cell non-Hodgkin's lymphoma (B-NHL) expressed a higher surface density of CD30L as compared with B-cell chronic lymphocytic leukemia and low-grade B-NHL. Purified plasmacells from a fraction of multiple myeloma patients also displayed CD30L mRNA and protein. A more restricted expression of CD30L was found in T-cell tumors that was mainly confined to neoplasms with an activated peripheral T-cell phenotype, such as T-cell prolymphocytic leukemia, peripheral T-NHL, and adult T-cell leukemia/lymphoma. In contrast, none of the T-lineage ALLs analyzed expressed the ligand. In AML, a high cellular density of CD30L was detected in French-American-British M3, M4, and M5 phenotypes, which are directly associated with the presence on tumor cells of certain surface structures, including the p55 interleukin-2 receptor alpha-chain, the alpha(M) (CD11b) chain of beta2 integrins, and the intercellular adhesion molecule-1 (CD54). Analysis of normal hematopoietic cells evidenced that, in addition to circulating and tonsil B cells, a fraction of bone marrow myeloid precursors, erythroblasts, and subsets of megakaryocytes also express CD30L. Finally, we have shown that native CD30L expressed on primary leukemic cells is functionally active by triggering both mitogenic and antiproliferative signals on CD30+ target cells. As opposed to CD30L, only 10 of 181 primary tumors expressed CD30 mRNA or protein, rendering therefore unlikely a CD30-CD30L autocrine loop in human hematopoietic neoplasms. Taken together, our data indicate that CD30L is widely expressed from early to late stages of human hematopoiesis and suggest a regulatory role for this molecule in the interactions of normal and malignant hematopoietic cells with CD30+ immune effectors and/or microenvironmental accessory cells.

The role of eosinophils in the pathobiology of Hodgkin's disease.

BACKGROUND: Even though the presence of a prominent tissue eosinophilia represents a common histopathologic feature of Hodgkin's disease (HD), eosinophils have been mainly regarded as 'innocent' bystanders recruited and activated during the cellular reaction typical of HD. To evaluate the putative role of eosinophils or eosinophil-derived cytokines on tumor-cell regulation in HD, we have analyzed these cells for the functional expression of surface ligands (L) of the tumor necrosis factor (TNF) superfamily, whose specific receptors are known to transduce proliferation signals at the surface of Hodgkin (H) and Reed-Sternberg (RS) cells. MATERIALS AND METHODS: Eosinophils from peripheral blood of healthy donors and patients with HD, primary hypereosinophilic syndrome (HES), or secondary hypereosinophilia (HE), were purified by density gradient centrifugation and immunomagnetic depletion of residual granulocytes. RESULTS: By immunostaining and mRNA analysis, we were able to show that eosinophils from normal donors and patients with HD, HES, and HE express a number of receptors and ligands of the TNF superfamily, including CD40, CD40L, CD30L, CD95/Fas, CD95/FasL and 4-1BB. In addition, we provide evidence that cytokines regulating eosinophil proliferation and activation, i.e., interleukin (IL)-5, IL-3, and granulocyte-macrophage colony-stimulating factor, are able to enhance the cellular density of several TNF superfamily ligands and/or receptors at the surface of cultured eosinophils. Finally, we have shown that native CD40L and CD30L at the surface of purified eosinophils are functionally active and able to transduce proliferative signals on CD40+ and CD30+ target cells, including cultured H-RS cells. CONCLUSIONS: Our data suggest that eosinophils may act as important elements in the pathology of HD by providing cellular ligands for TNF-superfamily receptors (CD40, CD30, CD95/Fas) able to transduce proliferation and antiapoptotic signals at the surface of H-RS cells. The presence on eosinophils of receptors for TNF ligands expressed by activated T cells (i.e., OX40L, FasL, CD40L, 4-1BBL), also suggest that eosinophils may contribute to the deregulated network of interactive signals between H-RS cells, T cells, and other surrounding reactive cells.

Association of Kaposi's sarcoma-associated herpesvirus-positive primary effusion lymphoma with expression of the CD138/syndecan-1 antigen.

Primary effusion lymphoma (PEL) represents a novel B-cell non-Hodgkin's lymphoma (NHL) type associated with Kaposi's sarcoma-associated herpesvirus infection and typically growing as lymphomatous effusions in the body cavities. The precise B-cell subset from which PEL originates as well as the biologic mechanisms responsible for its peculiar growth pattern are unclear. In this study, we have analyzed PEL for the expression status of CD138/syndecan-1, a molecule selectively associated with late stages of B-cell differentiation and implicated in cell-to-cell and cell-to-extracellular matrix interactions. PEL patient samples (n = 7) and cell lines (n = 5) were investigated by multiple approaches, including immunocytochemistry, flow cytometry, RNA analysis, and Western blot studies. For comparison, lymphomatous effusions other than PEL (n = 13) and tissue-based NHL (n = 103) were also tested. Expression of CD138/syndecan-1 associates at high frequency with PEL (5 of 7 patient samples and 5 of 5 cell lines), whereas it is consistently absent among other lymphomatous effusions (n = 13). The CD138/syndecan-1 isoform expressed by PEL has an average molecular weight of 420 kD, which is substantially different from that of CD138/syndecan-1 molecules generally expressed by plasma cells. These data, along with previous immunophenotypic evidence, unequivocally define that PEL cells represent a preterminal stage of B-cell differentiation and may bear implications for the peculiar growth pattern of this lymphoma.

Human granulocyte-macrophage colony-stimulating factor supports the clonogenic growth of B-lineage acute lymphoblastic leukemias expressing myeloid antigens.

The effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3 and IL-6 on clonogenic growth of blast-cell progenitors from 19 immunologically defined CD10-positive B-lineage acute lymphoblastic leukemias (ALL) coexpressing (My+ALLs) or not (My-ALLs) myeloid antigens have been studied. Our results demonstrate that GM-CSF was able to support the clonogenic growth of blast cells from My+ALLs, being totally ineffective on My-All samples. Accordingly, both alpha and beta chains of GM-CSF receptor (R) were expressed by My+ALL blasts, as investigated by reverse-transcriptase polymerase chain reaction (RT-PCR). Colony cells from GM-CSF-stimulated My+ALL cultures displayed the same immunophenotype as primary leukemic cells at diagnosis (CD10+, CD19+, CD22+), and retained the expression of myeloid-associated antigens and of GM-CSF-R transcripts. Moreover, My+ALL blasts showed a preferential sensitivity to the growth-promoting activity of IL-3 and IL-6, as compared with My-ALL cells. In addition to rearrangements of the JH region of immunoglobulin genes, My+ALL cells showed aberrant rearrangements of gamma (three cases) and beta (two cases) T-cell receptor genes, as well as of bcr sequences (three cases). Our data, showing an unexpected cross-lineage response of My+ALLs to GM-CSF, and their preferential stimulation by IL-3 and IL-6, as compared with My-ALLs, further support the concept that My+ALLs represent a separate entity with unique biological features.

Human eosinophils express functional CD30 ligand and stimulate proliferation of a Hodgkin's disease cell line.

The presence of a prominent tissue eosinophilia represents a typical histopathologic hallmark of Hodgkin's disease (HD). To evaluate the putative role of eosinophils on tumor cell regulation in HD, we have analyzed these cells for the functional expression of CD30 ligand (CD30L), a surface molecule able to transduce CD30-mediated proliferation signals on Hodgkin's (H) and Reed-Sternberg (RS) cells. The results demonstrate that circulating and tissue eosinophils from normal donors and patients with HD or hypereosinophilic syndrome (HES), display CD30L mRNA and express CD30L protein, as shown by immunostaining with a specific monoclonal antibody (M80) and with a biotinylated soluble CD30-Fc fusion protein. The surface density of CD30L on eosinophils from HD and HES patients was remarkably higher compared with healthy donors, probably reflecting a cytokine-mediated upregulation in these pathologic conditions. Accordingly, we provide evidence that cytokines regulating eosinophils proliferation and activation, ie, interleukin-5 (IL-5), IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF), are able to enhance the cellular density of CD30L on purified eosinophils from normal subjects. Finally, we show that native CD30L on human eosinophils is a functionally active surface structure able to transduce proliferative signals on CD30+ target cells, including cultured H-RS cells. Our data suggest that eosinophils may not merely represent innocent bystanders, but rather act as important elements in the pathology of HD by contributing to the deregulated network of CD30/CD30L-mediated interactive signals between H-RS cells and surrounding reactive cells.

Effects on differentiation by the promyelocytic leukemia PML/RARalpha protein depend on the fusion of the PML protein dimerization and RARalpha DNA binding domains.

The block of terminal differentiation is a prominent feature of acute promyelocytic leukemia (APL) and its release by retinoic acid correlates with disease remission. Expression of the APL-specific PML/RARalpha fusion protein in hematopoietic precursor cell lines blocks terminal differentiation, suggesting that PML/ RARalpha may have the same activity in APL blasts. We expressed different PML/RARalpha mutants in U937 and TF-1 cells and demonstrated that the integrity of the PML protein dimerization and RARalpha DNA binding domains is crucial for the differentiation block induced by PML/RARalpha, and that these domains exert their functions only within the context of the fusion protein. Analysis of the in vivo dimerization and cell localization properties of the PML/RARalpha mutants revealed that PML/RARalpha--PML and PML/RARalpha--RXR heterodimers are not necessary for PML/RARalpha activity on differentiation. We propose that a crucial mechanism underlying PML/RARalpha oncogenic activity is the deregulation of a transcription factor, RARalpha, through its fusion with the dimerization interface of another nuclear protein, PML.

CD30 ligand expression in nonmalignant and Hodgkin's disease-involved lymphoid tissues.

The CD30 ligand (CD30L) is a type II transmembrane glycoprotein of the tumor necrosis factor ligand superfamily. Recent cloning of CD30L has enabled studies to explore its function and tissue distribution. For instance, recombinant CD30L has been shown to co-stimulate T cells and to act as mitogen for Hodgkin's disease (HD)-derived cell lines. The counter-receptor for CD30L, ie, CD30, is a type I cytokine receptor that is highly expressed by activated T cells, Hodgkin and Reed-Sternberg (H-RS) cells, and anaplastic large cell lymphoma cells. In the present study, recombinant membrane-bound and soluble human CD30L were instrumental to raise a monoclonal antibody (M80) recognizing membrane-bound CD30L on transfected and native cells. With this reagent, a panel of cultured lymphoma-derived cell lines as well as primary normal, reactive, and HD-involved lymphoid tissues were examined for expression of CD30L by immunostaining and flow cytometry. In reactive lymphnodes and tonsils, CD30L was expressed by a small subset of lymphoid cells, histiocytes, and granulocytes. Higher levels of CD30L expression were noted in HD lesions among bystander cells; ie, T cells and granulocytes that surrounded H-RS cells. Native CD30L displayed at the cell surface was functionally active as shown by the ability of fixed granulocytes to interact with CD30+ cell lines. Moreover, CD30L was detectable, although to a lower staining intensity, in primary H-RS cells of all HD tissues investigated regardless of the histological subtype and the phenotype of H-RS cells (ie, CD30+/CD40+ versus CD30-/CD40+). Co-expression of CD30 and CD30L that was seen on H-RS cells of all, except the CD30- nodular lymphocyte predominant, subtypes of HD may point to the use of this pair of molecules in paracrine and/or autocrine mitogenic cell interactions. Monoclonal antibody M80 may thus represent a useful tool for studying CD30L expression on cultured cell lines and primary cells from normal, reactive, and malignant tissues.