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Transferrin receptor 1 (TfR1) is a type II membrane protein that mediates cellular iron uptake. Transferrin receptor 2(TfR2), another receptor for transferrin (Tf), has recently been cloned. We examined expression levels of TfR1, TfR2-α (membrane form) and TfR2-β (non-membrane form) transcripts in cells from 67 patients with de novo acute myeloid leukaemia (AML) using reverse transcription-polymerase chain reaction (RT-PCR), and correlated the results with a variety of clinical features and disease outcomes of these patients. Significant correlations were noted between the levels of both TfR1 and TfR2-α ( r = 0·771, P < 0·001) and TfR1 and TfR2-β ( r = 0·534, P < 0·001). Unexpectedly, initial white blood cell (WBC) counts were inversely correlated with levels of expression of either TfR1( r = −0·357, P = 0·003), TfR2-α ( r = −0·486, P < 0·0001), or TfR2-β ( r = −0·435, P = 0·0003). Only TfR2 expression was significantly associated with either serum iron ( r = −0·270, P = 0·045) or serum ferritin ( r = −0·364, P = 0·008). Multivariate analyses using Cox's proportional hazard model showed that elevated TfR2-α, but not TfR1 or TfR2-β mRNA levels significantly contributed to a better prognosis for AML patients. Furthermore, a group with high expression levels of both TfR2-α and TfR2-β survived significantly longer than a group without high expression of both of them ( P < 0·01 by log-rank). The present study suggests that (i) TfRs-independent iron uptake might have an important role in in vivo proliferation of AML cells; (ii) expression of TfR2 (especially the α form) is a novel prognostic factor for patients with AML. [ABSTRACT FROM AUTHOR]

The pathogenesis of B-cell lymphoproliferative disorders in general and B-cell chronic lymphocytic leukaemia in particular appears to involve dysfunctional regulation of humoral and cellular immunity with the subsequent development of genetic aberrations in B cells. In theory, either component may arise de novo or may be influenced by environmental exposures including infectious agents, antigens, genotoxic chemicals, or radiation. As an intermediary within the exposure-disease continuum, monoclonal B-cell lymphocytosis may be a helpful biomarker for teasing out these various contributions to risk. This article introduces a series of papers that resulted from an International Workshop held in May 2007 entitled ‘Monoclonal B-cell Lymphocytosis and Chronic Lymphocytic Leukemia: Environmental and Genetic Risk Factors’. Research efforts, such as those described in this issue, should lead to improved interventions, more predictive biomarkers, more effective treatments, and a greater appreciation of how the immune system functions over the entire human lifespan. [ABSTRACT FROM AUTHOR]

ImMucin, a 21-mer cancer vaccine encoding the signal peptide domain of the MUC1 tumour-associated antigen, possesses a high density of T- and B-cell epitopes but preserves MUC1 specificity. This phase I/ II study assessed the safety, immunity and clinical response to 6 or 12 bi-weekly intradermal ImMucin vaccines, co-administered with human granulocyte-macrophage colony-stimulating factor to 15 MUC1-positive multiple myeloma ( MM) patients, with residual or biochemically progressive disease following autologous stem cell transplantation. Vaccination was well tolerated; all adverse events were temporal grade 1 2 and spontaneously resolved. ImMucin vaccination induced a robust increase in γ-interferon ( IFN-γ-producing CD4+ and CD8+ T-cells (≤80-fold), a pronounced population of ImMucin multimer CD8+ T-cells (>2%), a 9·4-fold increase in peripheral blood mononuclear cells proliferation and 6·8-fold increase in anti-ImMucin antibodies, accompanied with T-cell and antibody-dependent cell-mediated cytotoxicity. A significant decrease in soluble MUC1 levels was observed in 9/10 patients. Stable disease or improvement, persisting for 17·5-41·3 months (ongoing) was achieved in 11/15 patients and appeared to be associated with low-intermediate PDL1 ( CD274) bone marrow levels pre- and post-vaccination. In summary, ImMucin, a highly tolerable cancerous vaccine, induces robust, diversified T- and B-cell ImMucin-specific immunity in MM patients, across major histocompatibility complex-barrier, resulting in at least disease stabilization in most patients. [ABSTRACT FROM AUTHOR]

Patients with multiple myeloma ( MM) undergoing high dose therapy and autologous stem cell transplantation ( SCT) remain at risk for disease progression. Induction of the expression of highly immunogenic cancer testis antigens ( CTA) in malignant plasma cells in MM patients may trigger a protective immune response following SCT. We initiated a phase II clinical trial of the DNA hypomethylating agent, azacitidine ( Aza) administered sequentially with lenalidomide (Rev) in patients with MM. Three cycles of Aza and Rev were administered and autologous lymphocytes were collected following the 2nd and 3rd cycles of Aza- Rev and cryopreserved. Subsequent stem cell mobilization was followed by high-dose melphalan and SCT. Autologous lymphocyte infusion ( ALI) was performed in the second month following transplantation. Fourteen patients have completed the investigational therapy; autologous lymphocytes were collected from all of the patients. Thirteen patients have successfully completed SCT and 11 have undergone ALI. Six patients tested have demonstrated CTA up-regulation in either unfractionated bone marrow ( n = 4) or CD138+ cells ( n = 2). CTA ( CTAG1 B)-specific T cell response has been observed in all three patients tested and persists following SCT. Epigenetic induction of an adaptive immune response to cancer testis antigens is safe and feasible in MM patients undergoing SCT. [ABSTRACT FROM AUTHOR]

Rituximab is widely used in autoimmune diseases including immune thrombocytopenia ( ITP), although the mechanism of effect remains unclear. This study describes the effects of rituximab on platelet-associated antibodies ( PA- APAs), B and T cell counts and clonality ( IGHV and TRG@ gene rearrangements), FCGR3A ( Fcγ RIIIa) and FCGR2A ( Fcγ RIIa) polymorphisms and correlation to anti- CD40 ligand (CD40L) response. PA- APA levels fell more frequently in responders (6/8) than in non-responders (2/10: P = 0·08-0·15). Two responders had no PA- APAs. Two non-responders with a fall in PA- APAs had very high CD8 levels. One non-responder had a B cell clone, one responder and one non-responder had a T cell clone. 15/16 patients had the same responses to rituximab and anti CD40 L. Patients with FCGR3A V/ V polymorphisms were more likely to respond to rituximab ( P = 0·03). In summary, the fall in PA- APAs in responders confirms the humoural effect of rituximab. Failure to respond in patients with very high CD8 levels, despite PA- APA fall indicates a role for T cell-mediated platelet/megakaryocyte destruction. Concordance of response to anti- CD40 L suggests autoantibody-producing cells are under T cell control. Finally, the effect of FCGR polymorphisms on response confirms the importance of FCGR-mediated depletion of B cells in autoimmunity. This has implications on the pathology of ITP as well as the immunological effect of B cell depletion. [ABSTRACT FROM AUTHOR]

Summary Lenalidomide has demonstrated impressive antileukaemic effects in patients with chronic lymphocytic leukaemia (CLL). The mechanism(s) by which it mediates these effects remain unclear. Clinically, CLL patients treated with lenalidomide demonstrate an acute inflammatory reaction, the tumour flare reaction that is suggestive of an immune activation phenomenon. Samples from CLL patients treated with lenalidomide were used to evaluate its effect on the tumour cell and components of its microenvironment (immune cellular and cytokine). Lenalidomide was unable to directly induce apoptosis in CLL cells in vitro, however it modulated costimulatory (CD80, CD83, CD86) surface molecules on CLL cells in vitro and in vivo. Concurrently, we demonstrated that NK cell proliferation was induced by lenalidomide treatment in patients and correlated with clinical response. Cytokine analysis showed increase in levels of TNF-α post-lenalidomide treatment, consistent with acute inflammatory reaction. Furthermore, the basal cytokine profile (high IL-8, MIG, IP-10 and IL-4 levels and low IL-5, MIP1a, MIP1b, IL12/p70) was predictive of clinical response to lenalidomide. Collectively, our correlative studies provide further evidence that the antileukaemic effect of lenalidomide in CLL is mediated not only through modulation of the leukaemic clone but also through elements of the tumour microenvironment. [ABSTRACT FROM AUTHOR]

Serum levels of pro-[vascular endothelial growth factor (VEGF)] and anti-[thrombospondin-1 (TSP)] angiogenic cytokines were prospectively measured in a phase II trial of chemoimmunotherapy (CIT) for chronic lymphocytic leukaemia (CLL) patients ( n = 56). Pretreatment VEGF levels were lower among patients who achieved complete remission (CR) or nodular partial remission (nPR) relative to those with partial remission (PR) or stable/progressive disease (median 122·0 pg/ml vs. 246·8 pg/ml; P = 0·03). VEGF:TSP ratio was lower (anti-angiogenic phenotype) among patients who achieved CR/nPR. The pretreatment VEGF:TSP ratio also correlated with overall survival ( P = 0·008). A pro-angiogenic profile appears associated with diminished response and inferior survival in CLL patients receiving CIT. [ABSTRACT FROM AUTHOR]

Increased levels of naturally occurring regulatory T cells (TReg cells) have been found in a variety of solid tumours and haematological malignancies. In multiple myeloma (MM), evidence suggests that TReg cells are increased though controversy exists with regards to their function and no relationship to disease stage and treatment has been demonstrated. Here, we demonstrate significantly elevated levels of functional CD4+CD25+FoxP3+ TReg cells in a large cohort of patients with MM as well as monoclonal gammopathy of uncertain significance (MGUS) in comparison to age-matched, healthy controls. The frequency of Double Negative TReg cells was also evaluated, demonstrating that these cells were reduced in patients with MM. Furthermore, a characteristic profile of immunomodulatory cytokines in the peripheral blood and bone marrow of patients with MM and MGUS was demonstrated, compared with healthy controls. This data adds further evidence to the understanding of the role of TReg cell subsets in tumour immunology and the fundamentals of the host/tumour immune conflict. [ABSTRACT FROM AUTHOR]

The disturbed erythropoiesis in patients with refractory anaemia with ring-sideroblasts (RARS) is characterized by intramedullary apoptosis of erythroid precursors and increased iron accumulation in mitochondria. To gain insight into these pathophysiological mechanisms we compared the gene expression profile (GEP) of erythroid precursors from RARS patients to the GEP of normal erythroid precursors. Three hundred sixty four probe sets were up-, and 253 probe sets downregulated in RARS cells. Interestingly, Growth Differentiation factor 15 (GDF15), a cytokine from the TGFβ family, was dramatically upregulated in all RARS patients. Measurement of GDF15 in the sera from twenty RARS patients confirmed this finding by showing significantly, 7·2-fold, increased protein levels (3254 ± 1400 ng/ml vs. 451 ± 87 ng/ml in normals). In vitro studies demonstrated erythroid-specific production of GDF15 and dependence on erythropoietin. Induction of apoptosis by arsenic trioxide, a drug which acts via reduction of the mitochondrial membrane potential, also stimulated GDF15 production. Downregulation of endogenous GDF15 production in erythoblasts by specific siRNA led to diminished erythroid differentiation. Taken together, our findings demonstrate a new role for GDF15 in normal erythropoiesis as well as in the ineffective erythropoiesis of RARS patients. [ABSTRACT FROM AUTHOR]

Bone marrow angiogenesis is suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML) and endothelial cells may mediate chemosensitivity. This study investigated in vitro endothelial effects of coculture of microvascular endothelial cells (MVEC) with AML cells derived from 33 consecutive AML patients. A proliferation assay showed that (i) AML cells from the majority of patients examined increased endothelial cell proliferation, while cytokine neutralizing experiments had divergent effects on proliferation and (ii) the angiopoietin/Tie2 system was important for growth of AML cells, and angiopoietin-1 induced phosphorylation of signal transducers and activators of transcription (STAT) proteins in AML cells. Finally, gene expression profiling of MVEC cocultured with AML cells was conducted in non-contact cultures. Microarray analysis revealed that the majority of significantly expressed genes could be categorized into gene ontology terms involved in transcription, cellular organization and intracellular signalling. Our study indicates a role for the leukaemic-endothelium crosstalk in leukaemogenesis with enhancement of endothelial cell growth and increased AML cell proliferation possibly mediated by angiopoietin-1 and the STAT signalling pathway. [ABSTRACT FROM AUTHOR]

The haemophagocytic syndrome (HPS) is a rare but frequently fatal disorder of immune regulation caused by hypercytokinemia. Using cytometric bead array technique, the serum T-helper cell type 1 (Th1) and 2 (Th2) cytokines including interferon-γ (IFN-γ), tumour necrosis factor (TNF), interleukin (IL)-10, IL-6, IL-4 and IL-2 were determined in 24 children with de novo HPS and 87 children as control. The median levels of serum IFN-γ, IL-10 and IL-6 in the acute phase of HPS were 901·7, 879·0 and 63·8 pg/ml, respectively, significantly higher than those after remission, and in the healthy volunteers and patients with viral infection. IL-4 was slightly elevated while IL-2 and TNF were within normal range in acute phase. Patients with bacterial sepsis showed an extremely high level of IL-6 and moderate level of IL-10, whereas IFN-γ was only slightly elevated. Five patients were diagnosed with HPS according to the Th1/Th2 cytokine pattern 3–13 d earlier than they fulfilled the relevant diagnostic criteria. IL-10 level >2000 pg/ml was an unfavorable prognostic factor for HPS treatment response ( P = 0·033) and outcome ( P = 0·009). We conclude that the significant increase of IFN-γ and IL-10 and a slightly increased level of IL-6 is an early, specific and prognostic cytokine pattern for childhood HPS. [ABSTRACT FROM AUTHOR]

Ph-negative chronic myeloproliferative disorders (CMPD) are characterized by constitutive Janus kinase-signal transducer and activator of transcription (JAK-STAT) activation. SOCS3, SOCS1 and PTPN6 ( SHP1) are negative regulators of the JAK-STAT pathway. We investigated epigenetic and genetic inactivation of SOCS3, SOCS1 and PTPN6 in 112 CMPD and 20 acute myeloid leukaemia (AML) post-CMPD. SOCS3 methylation occurred at high frequency in both CMPD (46/112; 41·1%) and AML post-CMPD (10/17; 58·8%) and was associated with transcriptional silencing. In contrast, methylation of SOCS1 and PTPN6 was observed in only a fraction of CMPD (15/112, 13·4% for SOCS1; and 8/112, 7·1% for PTPN6) and AML post-CMPD (3/20, 15% for SOCS1; and 1/20, 5% for PTPN6). No somatic mutations of SOCS1 were found in CMPD. SOCS3, SOCS1 and PTPN6 methylation occurred in both JAK2V617F-positive (35·1% for SOCS3; 14·9% for SOCS1; 8·1% for PTPN6) and JAK2V617F-negative (57·1% for SOCS3; 14·3% for SOCS1; and 9·5% for PTPN6) CMPD. These data indicate that methylation of SOCS3 and, to a lesser extent, SOCS1 and PTPN6 is a frequent event in both JAK2V617F-positive and -negative CMPD and may act as an alternative or complementary mechanism to JAK2 mutations, enhancing cytokine signal transduction. The frequent inactivation of SOCS3 is a novel finding in CMPD with potential implications for the molecular pathology of these disorders. [ABSTRACT FROM AUTHOR]

A human anti-RhD immunoglobulin G1 monoclonal antibody (mAb), R297, was tested in a phase I study to assess its ability to induce the clearance of antibody-coated autologous RhD+ red blood cells (RBCs) in healthy male volunteers. The clearance potency of R297 was compared with that of a marketed human polyclonal anti-D product (Rhophylac®). This mAb has been selected for its ability to strongly engage Fc-gamma receptor IIIA and to mediate a potent antibody-dependent cell cytotoxicity (ADCC) against RhD+ RBCs. Autologous RhD+ RBCs were sensitized with either Rhophylac® or R297 at three different coating percentages (25, 12·5 and 6·25%), before re-infusion. This phase I study showed that the human R297 mAb promoted rapid and complete clearance of RBCs, and showed activity that was at least as potent as the human polyclonal anti-D antibody preparation. Clearance of RBCs could still be observed when the percentage of R297 used to coat the RBCs was reduced to 6·25%. Finally, none of the adverse events was severe or considered to be related to R297. Thus, R297 is a promising candidate for the prevention of allo-immunization and represents a new generation of Fc-modified monoclonal antibodies with increased FcγRIII binding and increased ADCC. [ABSTRACT FROM AUTHOR]

Interleukin-1 alpha (IL-1 α) is a pro-inflammatory cytokine that is implicated in the initiation/maintenance of graft-versus-host disease (GVHD) and the immune response to infection. A cytosine (C) to thymine (T) transition at position −889 is believed to influence gene transcription. A previous single institution study showed that the presence of at least one IL1A T allele in the donor was associated with improved survival after unrelated donor haematopoietic stem cell transplant and lower transplant-related mortality if the donor and recipient each possessed the IL1A T allele. The present study sought to confirm these results in a larger homogeneous population. Thus the study population included 426 patients older than 18 years with chronic myeloid leukaemia (CML), transplanted in first chronic phase and receiving a total body irradiation and cyclophosphamide preparative regimen. Donor recipient pairs were categorised into four groups according to the presence or absence of an IL1A T allele in the donor and recipient. There were no significant differences in patient, donor and transplant characteristics between the groups. We did not observe an association with IL-1 α genotype in donor and/or recipient and transplant-outcome. These data suggest that the outcome of unrelated donor transplant for CML is not influenced by IL-1 α genotype. [ABSTRACT FROM AUTHOR]

Adenoviral infections represent a major cause of morbidity and mortality following haematopoietic stem cell transplantation. Current anti-viral agents are virostatic and it is evident that elimination of adenovirus (ADV) infection is only achieved by recovery of cellular immunity. Using an interferongamma (IFN-γ) secretion and capture assay to isolate ADV-specific T cells, followed by a 2 week expansion and restimulation protocol, we generated ADV T cells that may be used for cellular immunotherapy. In contrast to virus-specific T cells for cytomegalovirus or Epstein-Barr virus, the ADV response was dominated by CD4+ T cells and the majority of captured cells exhibited an effector/memory immunophenotype. Highly specific antigen responses were demonstrated by intracellular IFN-c expression and cytotoxicity assays when the expanded cells underwent restimulation with ADV-pulsed target cells. Although T cells were initially generated in response to ADV species C, the expanded populations also showed strong activity against ADV species B, suggesting cross-reactivity across ADV species; a finding that has important clinical consequences in the paediatric setting, where the majority of infections are caused by ADV type B and C. The protocols can be readily translated to generate ADV-specific T cells suitable for clinical use and offer an effective immunotherapeutic strategy to control ADV infection. [ABSTRACT FROM AUTHOR]

The fetal sheep model was used to compare the in vivo haematopoietic potential of human neural stem cells (NSC) versus bone marrow (BM)-derived haematopoietic stem cells (HSC). To this end, sheep were transplanted with either 8 × 105 NSC ( n = 11) or HSC, CD34+Lin− ( n = 5), and subsequently analysed for haematopoietic chimaerism. While HSC-transplanted sheep displayed robust donor-derived haematopoiesis starting at less than 2 months post-transplant, NSC recipients exhibited haematopoietic engraftment at much later time points. Nevertheless, chimaerism persisted in both groups throughout the course of this study. Transplantation of secondary recipients with human CD45+/HLA-DR+ cells from the BM of NSC primary recipients at 14 and 16 months post-transplant demonstrated that long-term engrafting HSC were present in these animals. At 6 months post-transplant, both NSC- and HSC-transplanted sheep were mobilised with granulocyte colony-stimulating factor. In contrast to HSC-transplanted animals, levels of human blood cells in peripheral blood of NSC-transplanted sheep remained low throughout mobilisation. Our results show that, although human NSC were able to give rise to multilineage haematopoiesis in our model, the levels, timing of blood cell production and the ability to respond to cytokine mobilisation were different, suggesting that human NSCs latent haematopoietic potential is inherently different from that of true HSC. [ABSTRACT FROM AUTHOR]

Transcriptional silencing because of hypermethylation is now recognised to be a hallmark of human tumours. In contrast to acute myeloid leukaemia (AML), comparably little is known about aberrant methylation in myelodysplastic syndrome (MDS), a heterogeneous clonal stem cell disorder with a risk of transformation into secondary AML of up to 30%. Recent evidence demonstrates that suppressor of cytokine signalling SOCS-1, a negative regulator of cytokine pathways, may act as a tumour suppressor gene, and inactivation because of hypermethylation was shown in various malignancies. Employing a newly developed quantitative real-time polymerase chain reaction-based methylation assay we analysed, for the first time, SOCS-1 methylation in MDS and found disease-specific hypermethylation in 27 of 86 MDS patients (31%). Demethylation experiments provided direct evidence that aberrant methylation of SOCS-1 induces transcriptional silencing in myeloid cells. In addition, by analysing the expression of signal transducers and activators of transcription (STAT)-induced genes we provide for the first time evidence that the activity of the Janus kinase/STAT pathway is increased in primary patient samples showing SOCS-1 hypermethylation. [ABSTRACT FROM AUTHOR]

Adult T-cell leukaemia (ATL) is caused by human T-cell leukaemia virus type I (HTLV-I). It has been suggested that cytokines play a role in the development and in the neoplastic cell growth of ATL. However, the precise mechanism involved in this process still remains unclear. Interleukin-21 (IL-21) and its receptor (IL-21R) have been recently described. In this study, we examined the expression of IL-21R and the effect of IL-21 on ATL cells. Real-time reverse transcription polymerase chain reaction showed that HTLV-I-infected cell lines and primary ATL cells expressed IL-21R mRNA. Cell surface expression of IL-21R on these cells was confirmed by flow cytometric analysis using a newly developed monoclonal antibody against human IL-21R. In contrast to the expression of IL-21R, IL-21 mRNA was scarcely detectable in these cells. Notably, IL-21 induced the proliferation of ATL-43T and ED-40515(+) cells, both of which were derived from leukaemic cell clones of ATL. Concerning the intracellular signalling pathways, IL-21 activated the phosphorylation of the signal transducers and activators of transcription (STAT)3 and STAT5. Taken together, these findings provide the first evidence that ATL cells express functional IL-21R, suggesting that it may contribute to the pathophysiology of ATL. In addition, the IL-21/IL-21R system may represent a new target for the treatment of ATL. [ABSTRACT FROM AUTHOR]