Epstein-Barr virus (EBV) is a ubiquitous B-cell trophic herpesvirus associated with a variety of histologically diverse B-cell lymphomas, each associated with specific viral-latency gene expression programs. Initial infection drives resting B-cells to differentiate via an atypical germinal centre reaction into memory B-cells, where the virus resides in a latent state. The mechanisms that underpin this process have yet to be fully elucidated. EBV expresses more than 40 microRNAs (miRNAs). The alternatively spliced BamHI A rightward transcripts (BARTs) are the template for two large miRNA clusters (BARTs A and B), that comprise the majority of all known EBV-miRNAs. Although BART-miRNAs are abundantly expressed in all latency programs, few BART-miRNA targets have been identified and their function is poorly understood. The early B-cell factor 1 (EBF1) was identified using bioinformaticss analysis as a novel target of EBV-miRNA BART11-5p, encoded by BART cluster B. EBF1 is an important B-cell transcription factor that regulates many B-cell specific genes including Pax5, BCR and CD40 and is critical for germinal centre formation. Using luciferase reporter assays and a series of BART-constructs, we confirmed silencing via the EBF1 3’ untranslated region (UTR) and identified the target site as 2137-2159 bp after the stop codon. Results were confirmed following transfection of a BART11-5p mimic, which was able to silence via the predicted target site. Our findings highlight a potential role of BART-miRNAs in the regulation of B-cell differentiation.
EBV; microRNA; BamHI A rightward transcripts; early B-cell transcription factor; B-cell
Background: Several studies revealed that MSC from human bone marrow can downregulate graft-versus-host disease (GVHD) after allogeneic HSCT. Methods: Herein we present 50 patients with acute GVHD who got 74 (1-4) MSC infusions for 54 separate episodes of aGVHD. Results: aGVHD was defined as steroid resistant grade IV aGVHD in 42 cases. The major presentation was gastrointestinal GVHD; two (n=18) or more (n=21) systems were involved in the majority of cases. The 1st infusion with MSC was given on day +27 (range, 1 to 136); d+45 (range, +11 to +150) post diagnosis of aGVHD and HSCT, respectively. In 2/3 of the cases treatment was performed with frozen stocked MSCs; in 62 cases early passages (1-3) were used. The median number of infused cells was 1.14±0.47 million per kg in the first injection and up to 4.27 (1.70±1.10) millions in total. The two patients with aggressive liver GVHD received MSCs injections intra hepatic arteries without changes of blood flow or evidence cytolysis, but also without a visible effect. Disease free survival at 3.6 years was 56%. We observed better overall survival in patients with GVHD grade < 4, in responders to the 1st treatment with MSC, and in pediatric group. The multivariate analysis demonstrated independent influence on survival of initial response and younger age. There were no immediate or late toxicity or side effects. Conclusion: Injection of MSCs seems to be a promising and safe treatment of GVHD. The encouraging results obviously should be confirmed in a randomized prospective study.
Mesenchymal stromal cells (MSC); mesenchymal stem cells; hematopoietic stem cell transplantation; graft versus host disease; steroid resistance
Myelodysplastic syndromes (MDS) encompass a range of myeloid neoplasms characterised by a defect in haematopoietic stem cell maturation, resulting in peripheral cytopenias. As a major consequence, most MDS patients become anaemic, so as to require red blood cell transfusions. To investigate the costs and the impact on quality of life (QOL) of MDS-separately in transfusion-independent (TI) and -dependent (TD) patients-a literature search was conducted. From Medline and Embase, 742 studies were identified, of which 17 were considered eligible. Total medical costs per patient/year range from $ 9,840 to $ 19,811 for the TI condition and from $ 29,608 to $ 51,066 in the TD condition, more than doubling when moving from the former condition to the latter. With regard to QOL, in the transition from TI to TD, QOL could be reduced by half depending on the studies. The TD condition negatively impacts on costs and the QOL of patients with MDS. Therapeutic strategies that reduce transfusion dependence may lead to broad benefits for patients and the community.
Myelodysplastic syndromes; anemia; transfusion dependence; quality of life; medical costs
Neutrophils provide first-line defense against infections and are potent effectors in innate and adaptive immunity. Recently neutrophils have been shown to play important roles in multiple antitumor reactions. A subset of mature neutrophils in human systemic inflammation has been identified as a unique circulating population of myeloid cells, which is capable of inhibiting T cell responses. These neutrophils show unique immunophenotype (CD11c bright/CD62L dim/CD11b bright/CD16 bright). This study reports detection of mature neutrophils with similar immunophenotype in the peripheral blood samples of cancer patients using flow cytometry analysis. This population of neutrophils is not detected in peripheral blood samples of normal controls. Thus this finding suggests the involvement of mature neutrophils in antitumor immunity.
Neutrophils; immunosuppressive; cancer; CD62L
Delayed recovery of platelet count post allogeneic hematopoietic stem cell transplantation (allo-HSCT) has been associated with worse transplant outcomes. Thrombopoietic agents have been successfully used in immune mediated thrombocytopenia or thrombocytopenia from bone marrow failure syndromes; however, the experience regarding their use after allo-HSCT is limited. Here we report on the safety and efficacy of romiplostim used in 3 consecutive patients with thrombocytopenia post allogeneic transplantation. Two patients had prolonged platelet recovery due to poor graft function while one had secondary failure of platelet recovery, likely immune mediated, post transplantation. Successful use of such agents post-transplant may improve platelet recovery, decrease rates of complications and potentially improve outcomes.
Post-transplant thrombocytopenia; romiplostim; allogeneic hematopoietic stem cell transplantation
Background: Platelet microparticles (PM) are the most abundant cell-derived microparticles in the blood, and accumulate in thrombo-inflammatory diseases. Platelets produce PM upon aging via an apoptosis-like process and by activation with strong agonists. We previously showed that long-term treatment of monocytic cells with apoptosis-induced PM (PMap) promotes their differentiation into resident macrophages. Here we investigated shorter term effects of various types of PM on monocyte signalling and function. Methods and results: Flow cytometry and scanning electron microscopy revealed that PM formed upon platelet aging (PMap) or ultra-sonication (PMsonic) expressed activated αIIbβ3 integrins and tended to assemble into aggregates. In contrast, PM formed upon platelet activation with thrombin (PMthr) or Ca2+ ionophore (PMiono) had mostly non-activated αIIbβ3 and little aggregate formation, but had increased CD63 expression. PM from activated and sonicated platelets expressed phosphatidylserine at their surface, while only the latter were enriched in the receptors CD40L and CX3CR1. All PM types expressed P-selectin, interacted with monocytic cells via this receptor, and were internalised into these cells. The various PM types promoted actin cytoskeletal rearrangements and hydrogen peroxide production by monocytic cells. Markedly, both aging- and activation-induced PM types stimulated the phosphoinositide 3-kinase/Akt pathway, suppressing apoptosis induced by several agonists, in a P-selectin-dependent manner. On the other hand, the PM types differentially influenced monocyte signalling in eliciting Ca2+ fluxes (particularly PMap) and in releasing secondary mediators (complement factor C5a with PMap, and pro-inflammatory tumour necrosis factor-α with PMthr). Conclusions: In spite of their common anti-apoptotic potential via Akt activation, aging- and activation-induced PM cause different Ca2+ signalling events and mediator release in monocytic cells. By implication, aging and activated platelets may modulate monocyte function in different way by the shedding of different PM types.
Aging; apoptosis; microparticles; monocytes; platelet activation; tumour necrosis factor
Systemic mastocytosis (SM) is a hematopoietic neoplasm characterized by pathologic expansion of tissue mast cells in one or more extracutaneous organs. In most children and most adult patients, skin involvement is found. Childhood patients frequently suffer from cutaneous mastocytosis without systemic involvement, whereas most adult patients are diagnosed as suffering from SM. In a smaller subset of patients, SM without skin lesions develops which is a diagnostic challenge. In the current article, a diagnostic algorithm for patients with suspected SM is proposed. In adult patients with skin lesions and histologically confirmed mastocytosis in the skin (MIS), a bone marrow biopsy is recommended regardless of the serum tryptase level. In adult patients without skin lesions who are suffering from typical mediator-related symptoms, the basal serum tryptase level is an important diagnostic parameter. In those with slightly elevated tryptase (15-30 ng/ml), additional non-invasive investigations, including a KIT mutation analysis of peripheral blood cells and sonographic analysis, is performed. In adult patients in whom i) KIT D816V is detected or/and ii) the basal serum tryptase level is clearly elevated (> 30 ng/ml) or/and iii) other clinical or laboratory features are suggesting the presence of occult mastocytosis, a bone marrow biopsy should be performed. In the absence of KIT D816V and other indications of mastocytosis, no bone marrow investigation is required, but the patient’s course and the serum tryptase levels are examined in the follow-up.
Mastocytosis; tryptase; KIT D816V; diagnostic algorithm; staging
MyD88 was originally described as a primary response gene up-regulated during myeloid differentiation after IL-6 induction. Later, MyD88 was shown to be a key molecule necessary for IL1, IL18 and Toll-like receptor signaling. Since these receptors recognize abundantly produced cytokines during infection or molecular patterns of pathogens, MyD88 itself was suggested to be an important regulator of the first line of defense against invading pathogens, including the differentiation and maturation of myeloid cells. Here we describe that MyD88 is important for early and late hematopoietic events that occur independently of antigen under steady-state conditions. In MyD88-deficient mice the earliest alteration in hematopoiesis was found at the level of long-term hematopoietic stem cells. Moreover, we found that MyD88 influences not only the development of the myeloid lineage but also the differentiation of B cells. The B cell defect observed in Btk-deficient mice is further enhanced when both molecules, Btk and MyD88, are not expressed. Therefore, MyD88 affects myeloid as well as lymphoid hematopoiesis. Since Btk and MyD88 deficiencies influence differentially myeloid and lymphoid development, both molecules seem to act in different signaling pathways important for appropriate developmental events during myelo- and lymphopoiesis.
MyD88; Btk; hematopoiesis; myelopoiesis; lymphopoiesis
Patients over age 60 comprise the majority of those diagnosed with acute myeloid leukemia (AML), but treatment approaches in this population are variable, with many uncertainties and controversies. Our group conducted a literature review to summarize the latest information and to develop a consensus document with practical treatment recommendations. We addressed five key questions: selection criteria for patients to receive intensive induction chemotherapy; optimal induction and post-remission regimens; allogeneic hematopoietic stem cell transplantation (HSCT); treatment of patients not suitable for induction chemotherapy; and treatment of patients with prior hematological disorders or therapy-related AML. Relevant literature was identified through a PubMed search of publications from 1991 to 2012. Key findings included the recognition that cytogenetics and molecular markers are major biologic determinants of treatment outcomes in the older population, both during induction therapy and following HSCT. Although disease-specific and patient-specific risk factors for poor outcomes are more common in the older population, age is not in itself sufficient grounds for withholding established treatments, including induction and consolidation chemotherapy. The role of HSCT and use of hypomethylating agents are discussed. Finally, suggested treatment algorithms are outlined, based on these recommendations.
Acute myeloid leukemia; chemotherapy; cytogenetics; prognosis; hematopoietic stem cell transplantation; hypomethylating agent
Purpose: Recent whole genome and/or exome sequencing in a cohort of 32 Multiple Myeloma (MΜ) patients reported the incidence of BRAF mutations at 4%, while in another exome sequencing study, BRAF mutations were reported in up to 13% of cases tested. We ran a confirmatory study by using High Resolution Melting Analysis (HRMA), which is a low-cost, straightforward and sensitive screening test for detection of BRAF exon 15 mutations in MM and Waldenström’s macroglobulinemia (WM) patients, in order to investigate their incidence in every day clinical practice. We considered this investigation to be of clinical relevance following the recent emergence of potent anti-BRAF compounds. Patients and Methods: We used genomic DNA isolated from 31 bone marrow aspirates obtained from 25 MM patients and 3 patients with WM (14 female; 14 male) who signed an informed consent. Patients’ median age was 69 years (range 43-86) and median follow-up time was 45 months. Myeloma subtypes were as follows: 7 IgGκ, 6 IgGλ, 7 IgAλ, 4 IgAλ and 1 non-secretory. The bone marrow plasma cells ranged from 12 to 100% (mean/median value 45%). By International Staging System (ISS) 9/25 patients were stage Ι, 6/25 stage ΙΙ, 7/25 stage ΙΙΙ, while in 3 cases staging information was missing. In 3 MM cases matched paired samples at diagnosis and at relapse were also available. DNA samples were screened using HRMA. HRMA results were confirmed by subsequent ds-bi-directional sequencing (Sanger method) for somatic mutations in exon 15 of BRAF. Results: At a limit of detection ≥2.5% mutant allelic content by HRMA, we did not detect any BRAF mutations in exon 15 in any of our 31 samples. Conclusions: By using HRMA we do not confirm previously reported results. Lack of detection of BRAF exon 15 mutations in our MM and WM series may be related to different sensitivity of the assays used and/or the relatively small sample size. In any case, we consider that existing data should be taken into account when considering the clinical development of BRAF inhibitors in plasma cell neoplasms.
Multiple myeloma; BRAF; mutation; WaldenstrÖm’s macroglobulinemia; high resolution melting analysis
Chromosomal translocations resulting in chimeric fusion genes are prototypic for pediatric leukemia patients. The most known fusions are ETV6-RUNX1 or BCR-ABL1 in B-cell progenitor (BCP)-ALL, and rearrangements of MLL in pediatric ALL and AML. Genome-wide sequencing projects have revealed additional, recurrent gene mutations in B cell malignancies. One of these mutations comprises the IKZF1 gene, encoding the IKAROS transcription factor which is one of the essential transcription factors driving lymphoid development. IKZF1 deletions were first identified by SNP arrays in ALL patients, and later identified with a high prevalence in BCR-ABL1+ patients. IKZF1 deletions turned out to be an independent prognostic marker associated with a poor outcome. Here, we characterized IKZF1 deletions in pediatric BCP-ALL patients by combining MLPA mapping experiments with long distance inverse PCR. The aim of our study was also to compare existing methods with our approach. Our attempt confirmed many of the existing data but revealed a more complex pattern of recombination sites, including a total of 4 recombination hotspots. This extended knowledge was translated into a novel, multiplex PCR assay that allows to perform IKZF1 deletion analyses by using a 2-tube PCR approach.
Childhood leukemia; cancer genetics; gene deletion; IKAROS; IKZF1; leukemia markers
High dose cyclophosphamide (HiCY) without stem cell rescue has been shown to induce remissions in patients with severe autoimmune disorders (SADS). However, up to 80% of these patients ultimately relapse. Here we review the outcomes of seven patients treated with two cycles and one patient treated with three cycles of HiCY. The diseases re-treated were scleroderma, multiple sclerosis, three patients with severe aplastic anemia (SAA), and three patients with myasthenia gravis (MG). All but two patients with SAA had received standard immunomodulatory therapy for their disease up front and had been refractory. All patients had complete hematologic recovery. Overall survival in this cohort was 100%. All patients relapsed after the initial cycle but event free survival thereafter was 93.3%. All are still in remission except two MG patients, one of whom relapsed after a severe GI infection requiring hospitalization, and the other relapsed 3 years after the second treatment and she did not respond to the third treatment. This shows that HiCY can be safely re-administered in patients with SAA and refractory SADS. The quality and duration of second remissions appears to be equal or superior to the first remission.
Autoimmunity; cyclophosphamide; severe autoimmune diseases
Signal transducers and activators of transcription (STAT) proteins function in the JAK/STAT signaling pathway and are activated by phosphorylation. As a result of this signaling event, they affect many cellular processes including cell growth, proliferation, differentiation, and survival. Increases in the expressions of STAT5A and STAT5B play a remarkable role in the development of leukemia in which leukemic cells gain uncontrolled proliferation and angiogenesis ability. At the same time, these cells acquire ability to escape from apoptosis and host immune system. In this study, we aimed to suppress STAT-5A and -5B genes in K562 CML cells by siRNA transfection and antisense oligonucleotides (ODN) targeting and then to evaluate apoptosis rate. Finally, we compared the transfection efficiencies of these approaches. Quantitative RT-PCR and Western blot results indicated that STAT expressions were downregulated at both mRNA and protein levels following siRNA transfection. However, electroporation mediated ODN transfection could only provide limited suppression rates at mRNA and protein levels. Moreover, it was displayed that apoptosis were significantly induced in siRNA treated leukemic cells as compared to ODN treated cells. As a conclusion, siRNA applications were found to be more effective in terms of gene silencing when compared to ODN treatment based on the higher apoptosis and mRNA suppression rates. siRNA application could be a new and alternative curative method as a supporting therapy in CML patients.
Chronic myeloid leukemia; K562; STAT5; siRNA knockdown; antisense oligonucleotides; apoptosis
In chronic lymphocytic leukemia (CLL) signals from the B cell receptor (BCR) play a major role in disease development and progression. In this light, new therapies that specifically target signaling molecules downstream of the BCR continue to be developed. While first studies on the selective small molecule inhibitor of Bruton’s tyrosine kinase (Btk), Ibrutinib (PCI-32765), demonstrated that Btk inhibition sensitizes CLL cells to apoptosis and alters their migratory behavior, these studies however did not address whether Btk-mediated signaling is involved in the process of CLL leukemogenesis. To investigate the requirement of Btk signaling for CLL development, we modulated Btk expression in the IgH.ETμ CLL mouse model, which is based on sporadic expression of the simian oncovirus SV40 T-antigen in mature B cells. To this end, we crossed IgH.ETμ mice on a Btk-deficient background or introduced a human Btk transgene (CD19-hBtk). Here we show that Btk deficiency fully abrogates CLL formation in IgH.ETμ mice, and that leukemias formed in Btk haplo-insufficient mice selectively expressed the wild-type Btk allele on their active X chromosome. Conversely, Btk overexpression accelerated CLL onset, increased mortality, and was associated with selection of non-stereotypical BCRs into CLL clones. Taken together, these data show that Btk expression represents an absolute prerequisite for CLL development and that Btk mediated signaling enhances leukemogenesis in mice. We therefore conclude that in CLL Btk expression levels set the threshold for malignant transformation.
Chronic lymphocytic leukemia (CLL); bruton’s tyrosine kinase (Btk); B cell receptor signaling
Excess body iron could persist for years after allogeneic hematopoietic cell transplantation (HCT) with possible deleterious sequels. An iron depletive therapy with phlebotomy seems rational. Kinetics of iron removal by phlebotomy without erythropoietin support in non-thalassemic adult patients with iron overload after HCT and the impact of pre- and post-HCT hemochromatosis (HFE) genotype on iron mobilization were investigated. Patients and methods: Phlebotomy was initiated in 61 recipients of allografts due to hematologic malignancies (median age 48 years) after a median of 18 months. The prephlebotomy median serum ferritin (SF) was 1697ng/ml and the median number of blood transfusions 28 units. Alanine aminotransferase (ALT)/aspartate aminotransferase (AST), alkaline phosphates (AP), and bilirubin were elevated in 55.7%, 64% and 11.5% patients respectively. HFE-genotype was elucidated by polymerase chain reaction using hybridization probes and melting curve analysis. Results: Phlebotomy was well-tolerated irrespective of age or conditioning. A negative iron balance in 80% of patients (median SF 1086 ng/ml) and a rise in hemoglobin were observed (p<0.0001). Higher transfusional burden and SF were associated with a greater iron mobilization per session (p=0.02). In 58% of patients, a plateau after an initial steady decline in SF was followed by a second decline under further phlebotomy. The improvement in ALT (p=0.002), AST (p=0.03), AP (p=0.01), and bilirubin (p<0.0001) did not correlate with the decline in SF. Mutant HFE-gene variants were detected in 14/55 (25%) pre-HCT and 22/55 (40%) patients post-HCT. Overall, dissimilar pre- and posttransplantational HFE-genotypes were detected in 20/55 (40%) patients. Posttransplantational mutant HFE variants correlated with a slower decline in SF (p=0.007). Conclusions: Phlebotomy is a convenient therapy of iron overload in survivors of HCT. A negative iron balance and a rise in hemoglobin were observed in the majority of patients. Liver dysfunction improved irrespective of SF reduction suggesting a probable rapid decline of the deleterious labile plasma iron. In recipients of grafts with mutant HFE variants a “mixed chimerism” of HFE in body tissues might be created with a change in the set point for iron regulation. The transient plateau in SF after an initial decline might reflect iron mobilization from various tissues.
Iron overload; ferritin; phlebotomy; allogeneic HCT
The transcriptional repressor Gfi1 regulates the expression of genes important for survival, proliferation and differentiation of hematopoietic cells. Gfi1 deficient mice are severely neutropenic and accumulate ill-defined CD11b+GR1int myeloid cells. Here we show that Gfi1 expression levels determine mono- or granulocytic lineage choice in precursor cells. In addition, we identify CD48 as a cell surface marker which enables a better definition of monocytes and granulocytes in mouse bone marrow. Using the CD48/Gr1/Gfi1 marker combination we can show that the CD11b+GR1int cells accumulating in Gfi1 deficient mice are monocytes and not granulocyte precursors. Expression of CD48, Gr1 and Gfi1 define different bone marrow subpopulations that are either committed to the granulocytic lineage, or bipotential precursors of granulocytes or monocytes. Finally, a comparison of genes differentially expressed between murine Gfi1 high granulocytic precursors and mature granulocytes with gene expression changes from human myeloblasts versus neutrophils show a strong resemblance of human and mouse differentiation pathways. This underlines the value of the markers CD48 and Gfi1 identified here to study human and murine granulo-monocytic differentiation.
Gfi1; CD48; CD106; granulocyte; monocyte; myelopoiesis; neutropenia
Isocitrate dehydrogenase 1 (IDH1) gene aberrations have recently been reported in acute myeloid leukemia (AML). To evaluate the prognostic significance of IDH1 mutations in AML, we performed a meta-analysis. Fifteen studies covering a total of 8121 subjects were included in this analysis. The frequency of IDH1 R132 mutations were 4.4–9.3% for AML patients and 10.9–16.0% for cytogenetically normal (CN)-AML patients. The IDH1 mutations were associated with NPM1 mutations in 6 studies and normal cytogenetics in 5 studies. AML patients with IDH1 mutations had inferior overall survival compared to patients without the mutations (hazard ratio 1.17, 95% CI: 1.02–1.36). Additionally, in CN-AML patients, IDH1 mutations were associated with a lower complete remission rate (risk ratio 1.30, 95% CI: 1.04–1.63). Although the available literature is limited to observational studies, these results may justify the risk-adapted therapeutic strategies for AML according to the IDH1 status.
Acute myeloid leukemia; IDH1; mutation; prognosis; meta-analysis
The author reviewed pathologic features of 37 cases of malignant lymphoma in the gastrointestinal organs in the last 10 years in our pathology laboratory. The current WHO classification was adopted. The 37 cases consisted of 20 males and 17 female, and the age ranged from 46 to 89 years with a median of 69 years. Of the 37 cases, 25 cases (68%) were gastric lymphomas, 6 cases (16%) were small intestinal lymphomas, and 6 cases (16%) were colon lymphomas. Of the 37 cases, 35 cases (95%) were B-cell neoplasms and 2 cases (5%) were T-cell neoplasms. In the 25 gastric lymphomas (male:female=14:11, age range 46-84 years, median 70 years) 11 cases were diffuse large B-cell lymphomas, and 14 cases were extranodal marginal zone B-cell lymphomas (MALT lymphomas). The clinical endoscopic diagnosis was gastritis in 3, gastric ulcer in 3, gastric carcinoma in 7, carcinoid in 1, submucosal tumor in 1, malignant lymphoma in 2, and suspected MALT lymphoma in 8. In the 6 small intestinal lymphomas (male:female=2:4, age range 49-89 years, median 70 years), all cases were located in the ileum. Of the 6 cases, 4 were diffuse large B-cell lymphoma and 2 were peripheral T-cell lymphoma. One case showed multiple lymphomas, and one case was associated with rectal adenocarcinoma and one case with gastric MALT lymphoma. The clinical diagnosis was adenocarcinoma in 2, suspected lymphoma in 2, and ileal tumor in 2. In the 6 colon lymphomas (male:female=4:2, age range 69-86 years, median 74 years), 5 cases were diffuse large B-cell lymphomas and one case was follicular lymphoma. Clinical endoscopic diagnosis was GIST in 1, colon carcinoma in 4, and colon polyp in 1. Cases of Hodgkin’s disease, mantle cell lymphoma, Burkitt lymphoma were not recognized in the present series. In summary, the author reported pathologic features of 37 cases of gastrointestinal malignant lymphoma in our laboratory in the last 10 years.
Malignant lymphoma; gastrointestinal organs
The phagocyte NADPH oxidase (NOX2) is known to be expressed in Epstein-Barr virus (EBV)-transformed human B lymphocytes. Phosphorylation of the NOX2 cytosolic subunit p47phox is required for phorbol myristate acetate (PMA)-induced NOX2 activation in EBV-transformed B lymphocytes, however the role of this process in receptor-mediated NOX2 activation is not known. Here, we used pansorbin which acts by cross linking cell surface IgG and transfected cells with mutated p47phox to address if the phosphorylation of this subunit is required for receptor-mediated NOX2 activation. We show that pansorbin induced NOX2 activation in a time and concentration-dependent manner, albeit at levels only of 20% of those induced by PMA. GF109203X, a PKC selective inhibitor, inhibited pansorbin as well as PMA-induced NOX2 activation. Using specific anti-phospho serine antibodies we showed that pansorbin induced p47phox phosphorylation on Ser304, 315, 320, 328, and 345 and kinetics of these phosphorylations preceed NOX2 activation. To determine whether the phosphorylation of p47phox is required for pansorbin-induced NOX2 activation, we transfected EBV-transformed lymphocytes deficent in p47phox with a plasmid expressing wild type p47phox or p47phox with all the phosphorylated serines mutated to alanines, p47phoxS(303-379)A. Results show that pansorbin-induced NOX2 activation was greatly decreased in lymphocytes expressing the mutant as compared to the wild-type p47phox. These results show that pansorbin induced p47phox phosphorylation on multiple sites in EBV-transformed B lymphocytes and this process is required for pansorbin-induced NADPH oxidase activation in these cells.
NADPH oxidase; NOX2; p47phox; B lymphocytes; pansorbin; ROS; phosphorylation
In patients with myeloproliferative neoplasia (MPN) the development of fibrosis and increased vessel density correlate with poor prognosis. The JAK2V617F mutation constitutively activates JAK2, which phosphorylates signal transducer activator of transcription (STAT), up-regulating vascular endothelial growth factor (VEGF), which might be responsible for angiogenesis in MPN. Galectins are involved in the development of fibrosis and angiogenesis and might also be involved in activation of the JAK/STAT pathway in MPN.
106 MPN patients, 36 essential thrombocythemia (ET), 25 polycythemia vera (PV) and 45 primary myelofibrosis (PMF), were analyzed for the expression pattern of galectin-1, galectin-3, pSTAT3, pSTAT5 and MVD by immunostaining of bone marrow biopsy sections followed by automated image analysis. The JAK2 mutational status was analysed through real time PCR in blood samples.
The expression of galectin-1 was significantly higher in all MPN patients compared to normal controls. Galectin-3 was expressed more in PV patients. MVD was significantly higher in all MPN patients and correlated with galectin-1 and pSTAT5 expression. pSTAT5 expression showed a trend of higher expression in patients carrying the JAK2V617F mutation as well as in PV patients. PMF patients and all JAK2V617F positive patients showed a significantly higher pSTAT3 expression compared to control and ET patients.
The findings suggest the involvement of galectin-1 in MPN development, regardless of the subtype. Furthermore involvement of galectin-3 in PV development, pSTAT5 in that of PV and JAK2V617F positive patients and angiogenesis, as well as pSTAT3 is involved in the pathogenesis of PMF.
MPN; myeloproliferative neoplasia; galectin; JAK; STAT; angiogenesis; MVD
To detect factors associated with quality of life (QOL) of patients with myelodysplastic syndrome (MDS) and to compare the MDS patients’ self-assessed QOL with that perceived by their physicians. In an observational, non-interventional, prospective, multicentre study, QOL was evaluated in 148 patients with newly diagnosed low- and intermediate-risk IPSS MDS. QOL measures (QOL-E v.2, LASA and EQ-5D) and patient-related candidate determinants of QOL were assessed for up to 18 months. Patients' QOL scores were compared with those obtained by appointed hematologists’ assessment and with ECOG performance status (PS). Fatigue was not prevalent at diagnosis, though physical QOL and energy levels were low. Transfusion-dependent patients had worse QOL scores. In multivariate analysis, Hb levels and comorbidities were a major determinant of QOL. Physicians’ perception of patients’ well-being correlated with patients’ QOL. Physicians underestimated the impact of disturbances on patients’ QOL, mainly in the MDS-specific components. ECOG PS did not discriminate patients according to QOL status. In conclusion, the association of anemia with QOL is confirmed, while co-morbidities emerge as an independent predictor of QOL in MDS. Fatigue is not a major concern. ECOG PS is not a valuable surrogate of patient’s QOL, thus highlighting that physician’s judgment of patient’s well-being must not substitute patient-reported outcomes. Appropriate questionnaires should be used to assess MDS patients’ QOL in order to improve communication and therapeutic choice.
Myelodysplastic syndromes; quality of life; comorbidities; anemia; transfusion-dependence; patient-reported outcomes
T-cell subset enumeration in HIV patients is routinely performed for monitoring infection stage and response to antiretroviral therapy. Studies have examined the effect of specimen refrigeration and age for single-platform (SP) methods, but there is limited data for time and temperature requirements of dual-platform (DP) methods.
Using a DP method, we analyzed peripheral blood (PB) from 52 HIV patients at room temperature (RT) at 24, 72, and 96 hours. PBs from 34 HIV patients had baseline RT analysis within 24 hours, and then were refrigerated and analyzed at 24, 48, and 72 hours. The coefficient of variation (CV) and residuals (changes in lymphocyte subsets) were recorded at each time point and compared to assess the precision and bias under the various conditions. Testing performance under different conditions was compared by linear regression.
Mean CV was ≤7.3% and median residuals were <30/μl for absolute CD4 and CD8 determinations. There was good correlation between baseline analysis data at RT and at various time points, both at RT and 4°C.
Our results are similar to those published for SP methods for aging or refrigerated specimens. The high level of agreement between measurements supports the robustness of this DP methodology.
HIV; Absolute CD4 counts; flow cytometry; dual platform; specimen stability
Primary bone marrow presentation of diffuse large B-cell lymphoma (DLBCL) is unusual, and appreciation of the diffuse growth pattern may be difficult in cases with low-level involvement. In particular, subtle sinusoidal and interstitial bone marrow involvement and morphologic overlap of the tumor cells with pronormoblasts may result initially in a missed diagnosis. We describe the clinicopathologic features of 13 cases of morphologically subtle DLBCL involving the bone marrow, which were only identified with the aid of immunohistochemistry. The overwhelming majority of cases (12/13, 92%) presented with cytopenias, and 5 of 7 cases, with available information, had splenomegaly. The morphology of the tumor cells in the aspirate smears overlapped with pronormoblasts (immature erythroid precursors) in 12 of 13 cases. Similarly, in histologic sections, the tumor cells in virtually all cases (12/13) demonstrated round nuclear contours and oblong nucleoli, mimicking pronormoblasts. A CD20 immunohistochemical stain was essential in identifying the neoplastic infiltrate in all cases. The majority of cases (73%, 10/13) showed low-level bone marrow involvement by lymphoma, 10% or less. CD20 immunohistochemistry highlighted individually dispersed and small clusters of large lymphoid cells in a sinusoidal and/or interstitial growth pattern. Most of the cases that were assessed showed a non-germinal center phenotype (CD10-, BCL6-/+, IRF4/MUM1+). There was an aggressive disease course with a median overall survival of 6 months. We would recommend performing a CD20 immunostain in patients who present with unexplained cytopenias and/or splenomegaly. Further investigation is warranted to better describe the features of this unique and aggressive variant of DLBCL.
Diffuse large B cell lymphoma; bone marrow; immunohistochemistry; CD20; pronormoblast-like
Elimination of neoplastic cells from peripheral blood progenitor cells (PBPCs) is an important issue in transplantation-based high-dose chemotherapy in non Hodgkin’s lymphoma (NHL). The capacity to reliably assess the presence of residual lymphoma cells in PBPCs is mandatory in designing this type of protocols. Polymerase chain reaction (PCR) amplification of molecular rearrangements is widely used to detect minimal residual disease (MRD) in NHL patients. Although concordant data can be obtained in most of the cases from peripheral blood (PB) and bone marrow (BM) at diagnosis, the relationship between these two compartments and the role of their analysis in predicting the molecular status of PBPCs is still an open issue. Here we report data about MRD analysis in BM, PB and PBPCs in a series of mantle cell and indolent NHL patients who underwent high-dose chemotherapy: discordant results were obtained comparing PB, BM and PBPC molecular data. In addition, differences were noted among these results if molecular analysis was performed using well-known rearrangements (i.e., bcl-1/IgH and bcl-2/IgH) or patient specific oligonucleotides. We conclude that neither BM nor PB are reliable in predicting the molecular status of PBPCs and that caution must be adopted in interpreting molecular data obtained using patient specific oligonucleotides.
Minimal residual disease; peripheral blood; bone marrow; peripheral blood progenitor cells
Bim, a BH3-only Bcl-2-family protein, is essential for T-cell negative selection in the thymus as well as for the death of activated T cells in the periphery. The role of Bim has been extensively studied in T-cell responses to self-antigens and viral infections. Recent findings on Bim in autoimmunity triggered our interest in investigating whether Bim may play a role in another disease with inflammatory symptoms as graft-versus-host disease (GVHD). Here we report that Bim is required for optimal T-cell responses to alloantigens in vivo and for the development of GVHD. Using murine models of allogeneic bone marrow transplantation (BMT), we found that donor T cells deficient for Bim are impaired in the induction of GVHD primarily due to a significant defect in T cell activation and expansion in vivo. Upon TCR engagement, Bim-/- T cells exhibited selective defects in CD69 expression and phosphorylation of PLCγ1. Our studies uncover a novel aspect of Bim function in T-cell activation with important implications in understanding the mechanisms of T-cell activation and tolerance under allogeneic transplantation.
Bim; T cells; proliferation; apoptosis; alloantigen; GVHD; GVL; and BMT