bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Helper T Cell Response to CMV Predicts Probability of CMV Vireamia Following Allogeneic Stem Cell Transplantation.
Authors: Buyck Hubertus C. , Paston Samantha J. , Lowdell Mark W. , Mackinnon Stephen , Emery Vincent C.
Published: October 2019

CMV reactivation after allogeneic stem cell transplantation remains problematic and is increasing in frequency with the use of reduced intensity conditioning regimens. A functional immune response is critical to control viral replication and hence disease. In this study, 19 allogeneic stem cell transplant recipients at risk for CMV reactivation were followed for a median of 185 days. Six patients received myeloablative conditioning and thirteen patients received reduced intensity conditioning therapy. Functional CMV specific immune responses were monitored by flow cytometry, measuring CD4 and CD8 specific intracellular interferon gamma production in response to stimulation with peptide pools of 15 mer overlapping peptides spanning the entire length of the immunodominant PP65 and IE1 proteins. In addition the CD4 response to whole CMV antigen, which is capable of presenting multiple viral antigens, was also determined. 53% of patients experienced a CMV reactivation with a median time to 1st reactivation of 53.5 days (range 20–134). In comparison with the CMV reactivation group, the mean day 50 CD4 whole CMV antigen response was approximately 2 logs lower in the reactivated group (0.065X106/L) than the non-reactivated group (3.620X106/L) suggesting that CMV specific CD4 function is an important predictor of patients at risk of CMV reactivation. The incidence of CMV reactivation in patients receiving Campath-1H was 71% (10 out of 14 patients) while none of the 5 patients in the non-Campath-1H group reactivated. The use of Campath-1H as part of the conditioning regimen was associated with a significantly lower day 50 CD4 whole CMV antigen response (0.06X106/L) vs (4.53X106/L) for the non-Campath-1H group, suggesting a mechanism for the observed increased frequency of CMV reactivation with the use of this agent. The relative contribution of the CD4 and CD8 responses to IE1 and PP65 has also been determined in this patient group. In summary, the absolute functional helper T cell response to CMV was predictive of viral reactivation following transplant.
Biallelic Mutation of SOCS-1 Impairs JAK2 Degradation and Sustains Phospho-JAK2 Action in MedB-1 Mediastinal Lymphoma Line.
Authors: Moller Peter
Published: October 2019

Mediastinal B-cell lymphoma is a well-defined subtype of diffuse large B-cell lymphoma. Molecular cytogenetics revealed frequent gains of 9p24. JAK2, mapping in this region, is presently regarded as a candidate oncogene since expression profiling showed high JAK2 transcript levels and JAK2 was found to be constitutive phosphorylated in mediastinal B-cell lymphomas. We confirm that in MedB-1 mediastinal cell line, harbouring a trisomy 9, JAK2 transcription is elevated and the product is highly phosphorylated. However, JAK2 is not over-expressed at the protein level. On top, JAK2 protein turnover is even delayed. This unexpected finding coincides with a biallelic mutation of the SOCS-1 gene in this cell, which perpetuates SOCS box function of the protein. Ectopic expression of wt-SOCS-1 in MedB-1 leads to growth arrest, dramatic reduction of phospho-JAK2 and its downstream partner phospho-STAT5. Ultimately, the target gene cyclin D1 is repressed in transfectants while Rb1, which is silenced in MedB-1, is induced. We conclude that, at least in MedB-1, JAK2 is not a constitutive oncogene but secondarily accumulates in its active form due to defective SOCS-1. Hence, SOCS-1 qualifies as a novel tumor suppressor. Of note, the biallelic SOCS-1 mutation is also present in the parental tumor of MedB-1.
Apoptotic Microparticles Derived from Endothelial Cells, Smooth Muscle Cells and Monocytes Induce Thrombin Generation Via Different Pathways.
Authors: Isobe Hirotaka , Kaufmann Viktoria , Mager Jutta , Bochkov Valery , Binder Bernd
Published: October 2019

Apoptotic microparticles (MPs) have been shown to be produced in atherosclerotic plaques and could be involved in the development of atherothrombosis. In the present study, we compared the procoagulant activities of apoptotic MPs derived from cultured endothelial cells (ECs), smooth muscle cells (SMCs), and monocytes, the major constituents of the plaque formation. Apoptotic MPs were generated from ECs, SMCs, monocytes and monocytic cell line, U937 cells, collected and purified by differential centrifugation. MPs were analyzed for the expression of phosphatidylserine and tissue factor (TF). Procoagulant activities were measured using a novel thrombin generation assay (TECHNOTHROMBIN® TGA) with normal and factor deficient plasmas. All MPs derived from ECs, SMCs, and U937 cells bound with annexin V, expressed TF, and induced thrombin generation (TG) dose dependently, indicating the thormbogenic properties. Among these MPs, SMC-MPs and EC-MPs showed the higher levels of TG activities than those of monocytic MPs. When antibodies against TF were used to inhibit TG generated by MPs, maximum level of TG of monocytic MPs was quenched dose dependently, and that of SMC-MPs was partially inhibited, but that of EC-MPs was not inhibited. To further investigate this phenomenon, factor VII, VIII, IX, XI and XII deficient plasmas were used. Although the initiation of TG induced by all MPs was dependent on factor VII, the amplification mechanisms of TG were different among these MPs. EC-MP induced amplification of TG was dependent on factors VIII, IX, XI and XII, and not dependent on factor VII. SMC-MPs and monocytic MPs induced TG dependently on an amplification loop including factor VII, and independently of factors VIII, IX, XI and XII. From these data we conclude that, apoptotic MPs derived from cultured ECs, SMCs and monocytes could induce TG via different coagulant pathways and could critically contribute to the process leading to thrombus formation after plaque rapture.
Transcriptional Repressor Gfi-1 Integrates Cytokine-Receptor Signals Controlling B Cell Differentiation.
Authors: Rathinam Chozhavendan , Klein Christoph
Published: October 2019

Hematopoietic stem cell differentiation is specified by cytokines and transcription factors but the mechanisms controlling instructive and permissive signalling networks are poorly understood. We provide evidence that common lymphoid progenitor (CLP)-1-dependent IL7-receptor mediated B cell differentiation is critically controlled by the transcriptional repressor Gfi1. Gfi1 is expressed selectively in CLP1 but not in CLP2 cells. Furthermore, in Gfi1-deficient hematopoiesis, CLP1 cells are significantly reduced, while CLP2 cells are present in normal numbers. Hardy fractions B/C, proposed to represent IL7-sensitive stages of early B cell development, are also reduced in Gfi1-deficient bone marrow. In contrast to bone marrow B cells, the phenotype of Gfi1-deficient thymic B cells was comparable to wildtype mice, suggesting that IL7-mediated signalling is perturbed. This hypothesis was further substantiated by a side-by-side comparison of Gfi1- and IL7-deficient mice; both knockout mice revealed a similar B cell phenotype in bone marrow and thymus. In vitro, Gfi1-deficient Sca1+ckit+lin- hematopoietic stem cells did not differentiate into B220+IgM+ B cells in the presence of SCF and IL7, suggesting that instructive IL7-mediated signals are not operative in the absence of Gfi1. Upon retroviral Gfi1 gene transfer, B differentiation was re-established Furthermore, whereas a combination of SCF and IL7 maintained the viability and induced proliferation of Gfi1-deficient HSC, their viability and proliferative capacity was significantly reduced in the presence of IL7 only, suggesting that both trophic and proliferative responses to IL7 depend on Gfi1. In addition, RT-PCR analysis revealed that in early B cell development the coordinated upregulation of the B cell specific transcription factors E2A, EBF, and Pax5 was significantly reduced in sorted Hardy fractions obtained from Gfi1-deficient mice. We reasoned that defective IL7-mediated responses might be due to defective Jak/Stat signalling. Indeed, phosphorylation of STAT5 was almost undectable in Gfi1-deficient B-progenitor cells. This may be due to enhanced expression levels of SOCS3 as determined by RT-PCR and Western Blot analysis. Thus, Gfi1 selectively specifies IL7-dependent development of B cells from CLP1 progenitors, providing clues to the transcriptional networks integrating cytokine signals and lymphoid differentiation.
Allogeneic Stem Cell Transplantation for High-Risk Acute Myeloid Leukemia: No Increase in Relapse with Alemtuzumab-Depleted Grafts.
Authors: Thomson Kirsty J. , Peggs Karl S. , Morris Emma C. , Khwaja Asim , Linch David C. , Goldstone Anthony H. , Mackinnon Stephen
Published: October 2019

The role of T cell depletion in allogeneic stem cell transplantation for acute myeloid leukemia (AML) remains controversial. This study describes the results of an alemtuzumab-containing myeloablative regimen used to transplant 51 consecutive patients with hematological malignancy. Diagnoses were AML (n=48) of which 8 were secondary (6 transformed MDS; 2 therapy-related AML) or MDS (n= 3). Median age at transplant was 38yrs (range 13–57), and 29 patients had unrelated donors. Sixteen patients were HLA-mismatched at up to 3/10 loci (8x1; 7x2; 1x3). The majority of patients (39/51; 76%) were high-risk for relapse, as defined by induction failure (>15% blasts after one course of induction chemotherapy or >5% blasts after 2 courses), adverse cytogenetics, >CR1, or secondary disease. Six patients (12%) had residual detectable disease at the time of transplant despite at least 3 courses of induction therapy. Patients received cyclophosphamide 120 mg/kg and fractionated total body irradiation (14.4 Gy). Those with unrelated donors also received fludarabine 90 mg/m2. Stem cell source was bone marrow in 11 patients and PBSC in the remaining 40. Stem cells underwent in vitro T cell depletion using 20mg alemtuzumab added to the bag. All patients also received cyclosporin A for GvHD prophylaxis. Median follow-up for the surviving patients is 29 months (range 3–49). The estimated event-free survival is 70% at 1yr and 58% at 3yrs. Acute GvHD grade II occurred in 8% (no grade III–IV) and extensive chronic GvHD in 22%. Non-relapse mortality was 17% at 1yr and 25% at 3yrs, and relapse has occurred in 9 patients, giving an estimated relapse risk of 21% at 3yrs (24% in high-risk patients, n=39). For EFS and NRM, the only significant variable is age >45yrs at transplant, with no significance for donor type or presence of HLA mismatch. For patients aged ≤ 45yrs at transplant (n=42), of whom 81% were high risk for relapse, the outlook is very good with estimated NRM of 15% at 3yrs, and EFS of 69%. Use of this regimen therefore permits the successful transplantation of younger patients with high-risk disease and HLA-matched or mismatched unrelated donors, with minimal acute GvHD, low non-relapse mortality and no evidence of an excessive relapse rate, when compared to regimens without T cell depletion.
Long Term Remission in Follicular Lymphoma (FL) and Mantle Cell Lymphoma (MCL) with Rituximab Consolidation after High-Dose Chemotherapy and Autologous Stem Cell Transplantation: Extended Follow-Up of a Multicenter Phase II Study.
Authors: Brugger Wolfram , Hirsch Judith , Repp Roland , Frank Gruenebach , Guenther Schlimok , Wichard Vogel , Markus Manz , Peter Brossart , Kaufmann Martin , Ganser Arnold , Fehnle Karl , Gramatzki Martin , Kanz Lothar
Published: October 2019

We investigated the efficacy of rituximab given after high-dose therapy (HDT) and autologous peripheral blood stem cell transplantation (ASCT) in order to prevent relapse by clearance of minimal residual disease in patients with advanced FL and MCL. Eligibility included newly diagnosed stage III/IV t(14;18)+ FL or newly diagnosed or relapsed patients with stage III/IV t(11;14)+ MCL. 35% of patients had elevated LDH levels, 23% had B-symptoms. The age-adjusted IPI risk category was low-intermediate in 65% and high-intermediate in 35% of the patients. Initial cytoreduction consisted of VACOP-B (6 weeks) or CHOP chemotherapy, followed by G-CSF supported VIP-(E) or DexaBEAM therapy. PBSC were harvested, and positive selection of CD34+ cells was performed. HDT consisted of TBI (6x2 Gy) and cyclophosphamide (120 mg/kg), followed by CD34+ PBSCT (7.16x106 CD34+/kg; range 2.4–22.7). A median of 62 days (range 38–108) after transplantation, rituximab was given at a dose of 375 mg/m2 weekly for 4 weeks. No additional treatment was given thereafter. Between October 1997 and July 2001, 31 patients were included. Median age at transplantation was 49 (31–60) years. One patient died early after HDT due to neutropenic septicemia before rituximab administration. Therefore, only 30 patients were evaluable for safety and efficacy of rituximab after HDT and ASCT (FL n=20, MCL n=10). Twenty seven were evaluable for MRD. In the intent-to-treat (ITT) analysis, 7 treatment failures were observed: two FL patients relapsed at 25 and 33 months post transplant, four MCL patients relapsed at 24, 38, 47 and 48 months post transplant, and one MCL patient died unexpectedly in complete remission (CR) at 19 months post transplant. One of the MCL patients died at 37 months post transplant due to progressive disease. Currently, 28 of 30 patients are alive. With an extended median follow up of 61 months (18.7 – 87.5 months) after ASCT, the progression-free survival (PFS) for the ITT population is 76.7%, the median PFS has not yet been reached. In the FL population, the PFS at 5 years is 90% (95% CI: 65.6 – 97.4) whereas in the MCL population, the PFS is 46.2% (95% CI: 14.7 – 73.3) (log rank p=0.0168). The overall survival (OS) at 5 years for the ITT population is 93.3% (95% CI; 75.6 – 98.3) with 100% OS in the FL patients and 79.4% (95% CI: 39.6 – 94.4) (log rank p=0.0400) in MCL patients. In summary, at an extended follow-up of 5 years, we show that one 4-week course of Rituximab consolidation after HDT and ASCT induces prolonged remissions in both FL and MCL. Futher improvement in MCL therapy is warranted.
Reduction of Factor VIII Inhibitor Formation with F.VIII-Pulsed Tolerogenic Dendritic Cells in the Murine Hemophilia A Model.
Authors: Ragni Margaret V. , Wu Wenhu , Liang Xiaoyan , Lu Lina
Published: October 2019

Inhibitor formation is a severe complication of hemophilia, occurring in up to 25% and associated with poor response to factor replacement, uncontrolled bleeding, and high morbidity. Preventing inhibitor formation is, thus, a major goal of hemophilia management. The role of dendritic cells (DC) in regulating immune response has been increasingly recognized: immature DC (imDC) induce T regulatory cells in vitro and promote Ag-specific tolerance in vivo. We, therefore, studied the role of imDC propagated from bone marrow with GM-CSF + TGFβ to prevent inhibitor formation in the hemophilia A murine model. Following tail vein injection of recombinant F.VIII (Advate, Baxter) 2.5 U (0.2 μg) on days 0, 2, and 4 in hemophilia A exon 16 KO C57Bl/6 mice, anti-VIII antibodies were detected by semi-quantitative APTT (scored 1-4), peaking on day 6. On rechallenge with F.VIII 2.5 U on days 12, 14, and 16, anti-VIII was detected, peaking on day 17. Anti-VIII production was associated with high level splenic T cell proliferation in response to F.VIII stimulation in vitro, measured by 3H-thymidine incorporation in mixed lymphocyte reaction (MLR). By contrast, there was no antibody formation in F.VIII-treated Wt C57Bl/6 mice: the latter was associated with low T cell response to F.VIII in vitro. Functionally immature DC (imDC) were propagated from the bone marrow of hemophilia A mice with GM-CSF (4ng/ml) and TGFβ (0.2ng/ml). For comparison, functionally mature dendritic cells (mDC) were propagated with GM-CSF (4ng/ml) and IL-4 (1000U/ml).The former (imDC) demonstrated deficient NF-kB binding activity in nuclear protein as detected by gel shifting assay and expressed low level of costimulatory molecules CD80, CD86; by contrast, the latter (mDC) demonstrated enhanced NF-kB binding activity and high levels of co-stimulatory molecules. Administration of 2x106 F.VIII-pulsed imDC (20U/ml x 24h) 7 days before F.VIII dosing on days 0, 2, and 4, led to reduction in inhibitor formation on day 6 (score 1.6 vs. 2.3 in control group) which was further reduced on day 8 (score 1.0 vs. 2.0 in control group). The inhibitor could not be detected on day 8 in 2 of 4 mice pretreated with F.VIII-pulsed imDC. By contrast, high levels of inhibitor were detected in mice pretreated with F.VIII-pulsed mDC (score 3.3). Rechallenge with F.VIII on day 10 in imDC-treated mice resulted in no increase in the reduced or absent anti-VIII effect on day 12. Splenic T cells (CD3+) from the imDC-pretreated mice showed lower proliferative capacity when restimulated in vitro with F.VIII, suggesting that imDC induced F.VIII unresponsiveness. These studies show that FVIII-pulsed imDC reduce the intensity of inhibitor formation, and suggest the potential role of modified DC in preventing or reducing F.VIII inhibitor formation.
A Longitudinal Analysis of the T-Cell Receptor Vβ Repertoire in Aplastic Anemia Patients at Initial Presentation, Remission, and Relapse.
Authors: Cheng Yunfeng , Tang Yong , Green Spencer , Keyvanfar Keyvan , Bruno Tullia , Young Neal S.
Published: October 2019

Aplastic anemia is a bone-marrow-failure syndrome characterized by low blood-cell counts and a fatty bone marrow. In most cases, no obvious etiological factor can be identified, but clinical responses to immunosuppressive treatment (IST) strongly suggest an immune pathophysiology. Our previous study of T-cell receptor (TCR) Vβ (variable region of β-chain) repertoire usage by flow cytometry suggested that aplastic anemia results from antigen-specific lymphocyte attack on hematopoietic progenitors (Risitano et al. Lancet2004; 364:355). In the current work, 7 patients were investigated for Vβ pattern expression before first immunosuppresive treatment, at the remission, and again on relapse. The TCR Vβ repertoire was analyzed for CD4+ and CD8+ subsets, separately, by flow cytometry, using a monoclonal antibody set of 22 different Vβ chains. Most patients had very different patterns of Vβ usage from healthy individuals, and all but one showed expansion of at least one Vβ family before immunosuppressive treatment (Vβ family expansions were defined as 2 standard deviations (SD) from the means in controls). The median number of expanded Vβ families was 4 per patient among CD8CD28dim effector cells. At remission, almost all the initially expanded Vβ subfamilies decreased to less than 2SD of controls. At relapse, most of the expanded Vβ subsets were increased again. However, 5/7 patients showed new expanded Vβ subsets at recurrence of cytopenias, suggesting antigenic spread of new epitopes recognized by immune systems. Although no common pattern of specific expanded Vβ subsets could be identified among different patients, some Vβ subfamilies appeared to be more frequently involved (Vβ 5.1 and Vβ 5.2 were expanded in 4 of 7 patients both at initial presentation and relapse ). These data suggest that monitoring Vβ subsets in aplastic anemia, and potentially in other immune-mediated human diseases of a similar pathophysiology could be used to guide individual therapeutic decisions and in the development of new treatments.
Zoledronic Acid Plus Thalidomide Is Safe and Well Tolerated When Used as Post-Autologous Stem Cell Transplant Maintenance Therapy in Patients with Nonprogressive Multiple Myeloma.
Authors: Spencer Andrew , Roberts A. , Neeman T. , Cremers S. , Schran H. , Lynch K.
Published: October 2019

Background: Thalidomide (THAL), an immunomodulatory agent with antiangiogenic properties, has become standard of care for patients with refractory or relapsed multiple myeloma (MM). Zoledronic acid (ZOL) is effective in preventing skeletal complications from MM, has cytotoxic effects, and enhances dexamethasone and THAL cytotoxicity in vitro (Ural A, et al. Int J Hematol.2003;78:443–449). However, reports have suggested that combining ZOL with THAL increases the risk of renal adverse events in MM patients. In the present study, the potential effect of this combination on renal function was investigated as part of a multicenter, randomized, phase III trial. The study design also allowed the investigation of a potential effect of THAL on serum PK of ZOL in a subgroup of patients.

Methods: Patients with nonprogressive MM who had previously undergone autologous stem cell transplantation were randomized to receive either THAL 200 mg od or no THAL. All patients received ZOL 4 mg via 15-minute infusion every 28 days and prednisolone 50 mg every other day. Total treatment and observation period was 12 months. Creatinine was determined in all pre-infusion serum samples. PK (Cmax and AUC) was determined from serum samples collected over 48 hours after the first 2 infusions of ZOL. The potential effect of THAL co-administration on serum creatinine and ZOL PK was investigated with a repeated measures analysis using a mixed model and paired t-tests. P < .05 was regarded as statistically significant.

Results: 243 patients have been randomized (114 to receive THAL, 129 without). Analysis has been completed on 171 patients (79/92) who have received a median of 11 and 9 doses of ZOL respectively. 6 patients had ZOL withheld because of a rise in serum creatinine (2 treated with THAL, 4 treated without THAL). There were no significant differences in creatinine levels between patients treated with or without THAL, over time (P = .58), although a small increase in mean creatinine levels was noted over time in both arms at 0.64 mmol/L per 4 week cycle. PK was investigated in a total of 24 patients equally distributed among the treatment arms. No statistically significant influence was found of THAL co-administration on the PK parameters of ZOL.

Conclusions: These results suggest that co-administration of THAL with ZOL does not result in an increased risk for nephrotoxicity. Serum PK of ZOL is not influenced by THAL. ZOL is safe and well tolerated in multiple myeloma patients when used in combination with THAL.
Genetic Rearrangements of FOXP1 Are Restricted to a Subset of Agressive B Cell Lymphoma with Extranodal Presentation.
Authors: Haralambieva Eugenia , Adam Patrick , Eichelbroenner Irina , Kalla Joerg , Rosenwald Andreas , Katzenberger Tiemo , Hoeller Sylvia , Banham Alison H. , Mueller-Hermelink Hans Konrad , Ott German
Published: October 2019

In most mature B-cell non-Hodgkin lymphoma (NHL) chromosomal translocations result in the transcriptional deregulation of an oncogene by juxtaposing it to regulatory sequences of genes that are constitutively expressed in mature B cells, most often the Ig genes. Recently, a novel recurrent translocation t(3;14)(p13;q32) that results in deregulation of the FOXP1 forkhead transcription factor has been reported in a variety of B cell lymphoma.

Here we present the analysis of 275 B-cell NHL: 102 diffuse large B cell lymphoma (DLBCL) (49 nodal and 53 extranodal cases) 122 marginal zone lymphoma (MZL) from different localization (cutenous n= 9, salivary gland n= 13, pulmonary n=50, ocular adnexae n=36, thyroid n=8, gastric n=6), 30 follicular lymphoma (FL) and 21 mantle cell lymphoma (MCL). The presence of genetic aberrations involving the FOXP1 gene was studied by interphase fluorescence in situ hybridization (FISH) and FOXP1 protein expression - by immunohistochemistry using the JC12 monoclonal antibody.

In total, 5 of 275(2%) lymphoma were found to carry a chromosomal breakpoint within the FOXP1 gene. Further FISH studies indicated the presence of a FOXP1/IgH translocation in only 2 of these samples, suggesting the likely involvement of other translocation partner. All samples that harbored a FOXP1 breakpoint represented aggressive B cell lymphoma with proliferation fraction of 70–90% defined by the Ki 67% labeling pattern. Cytogenetic analysis and/or FISH suggested a presence of the complex chromosomal rearrangements. Four of 5 tumors presented at extranodal sites: gastro-intestinal tract (N=3); thyroid gland (N=1). All samples expressed B-cell markers and lacked CD5 and CD10 - a phenotype consistent with a marginal zone derivation. Interestingly, we failed to identify a FOXP1 breakpoint in any of the “true” low grade MZL in our series.

A gain of genomic material was found in 80 (29%) of these samples, indicated by detection of more than two FISH signals in a variable number of cells and/or cytogenetic data. In the vast majority of these cases trisomy 3 was further identified. All 5 samples that harbor FOXP1 chromosomal breakpoint showed strong nuclear labeling pattern when stained with JC12 monoclonal antibody. FOXP1 nuclear expression, in a variable number of the cells was detected in 95% of the cases with genomic gain/trisomy 3, however also in 40 % of the samples without evidence of any genetic aberrations in this locus, suggesting a range of mechanisms, regulating the FOXP1 expression.
Neutrophil Elastase Modulates Granulopoiesis through the Interaction of G-CSF and G-CSF Receptor.
Authors: Sato Takashi , Habara Masakazu , Kihara Hiroki , Kawaguchi Hiroshi , Nakamura Kazuhiro , Okada Satoshi , Ishikawa Nobutsune , Ohno Norioki , Kobayashi Masao
Published: October 2019

Primary granule proteases provide negative feedback on granulopoiesis. Recent study showed that neutrophil elastase potentially inhibits granulopoiesis by enzymatic digestion of G-CSF. We studied the effects of neutrophil elastase on granulopoiesis in the interaction of G-CSF and G-CSF receptor (G-CSFR). Primitive myeloid cells of the bone marrow in normal subjects were purified based on the expression of CD34 and G-CSFR by FACS Vantage. The cells were incubated with or without human neutrophil elastase for 2 hours in serum-deprived media. Cells were washed once and then cultured in various serum-deprived conditions. The proliferation and colony formation of CD34+/G-CSFR+ cells in the presence of G-CSF were significantly reduced by the pretreatment of elastase. Furthermore, those of CD34+/G-CSFR+ cells in response to hematopoietic factors without G-CSF were similarly inhibited by elastase. On the other hand, the responses of CD34+/G-CSFR− cells were not affected by the treatment of elastase in the stimulation of any hematopoietic factors. When CD34+ cells were incubated with or without elastase, further G-CSFR expression was analysed by flow cytometry. The treatment of elastase did not affect the expression of surface expression of G-CSFR on CD34+ cells. Next, the binding of G-CSF to G-CSFR was examined by flow cytometry using antibody to G-CSF. The mean channel of fluorescence of G-CSF binding to G-CSFR-positive cells was reduced by the treatment of elastase, suggesting the loss of the binding of G-CSF to G-CSFR. When CD34+/G-CSFR+ cells were treated with elastase, the observation by confocal immunofluorescence microscopy revealed neither accumulation of elastase on the surface of cell membrane nor in intracellular compartment. No evidences that cells treated with elastase directly cleave the G-CSF leading to inactivation of G-CSF. Collectively, these results suggest that G-CSFR-positive cells are selectively affected by neutrophil elastase, and that neutrophil elastase as negative regulator may play a role in modulating granulopoiesis through G-CSF-G-CSFR interaction.
Reversive Loss of Pulmonary Function Induced by Acute Chest Syndrome: A Report from the National Acute Chest Syndrome Study Group (NACSG).
Authors: Neumayr Lynne , Morris C. , Wen A. , Earles A. , Robertson S. , Abboud M. , Ballas S. , Browning I. , Bellevue R. , Daeschner C. , Groncy P. , Hume H.A. , Iyer R. , McMahon L. , Moncino M. , Orringer E. , Swerdlow P. , Waldron15 P. , Ware R. , Vichinsky E. ,   National Acute Chest Study Group (NACSG)
Published: October 2019

Acute Chest Syndrome (ACS) remains the leading cause of death and hospitalization in patients (pts) with sickle cell disease (SCD). There is limited data on the effects of ACS on lung function. From 1993 to 1997, 30 centers participated in the NACSG and prospectively analyzed 671 episodes of ACS in 538 pts. Pulmonary function tests (PFTs) included forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC), forced expiratory flow during 25% to 75% of FVC (FEF 25–75), peak expiratory flow rate (PEF) and the ratio of FEV1/FVC. Data is reported as percent-predicted of normal values based on age and height. 128 pts (mean 16yrs, 4 to 52 yr range) had PFTs during an ACS episode (within a mean of 2.5 days of diagnosis) and then 12 weeks later. 94% of pts had abnormal lung volumes, defined as either FEV1 or FVC < 80%. Mean lung volumes during ACS and at follow-up are shown below.

Table 1: Decreased Lung Volumes During ACS PFT During ACS Follow-up p-value FEV1 52% 79% <.0001 FVC 55% 83% <.0001 FEF 25–75 50% 69% <.0001 PEF 61% 83% <.0001

Pts with abnormal lung volumes (FEV1 or FVC < 80%) and considered to have an obstructive pattern if the FEV1/FVC ratio was < to 85. Obstructive patterns in pts with abnormal lung volumes and the percent of pts who responded to bronchodilators (15% improvement in either FEV1 or FVC) are shown below.

Table 2: Patterns of Abnormal Lung Volumes in SCD SCD Patients During ACS Follow-up Abnormal PFTs 94% 45% Obstructive Pattern 48% 46% Respond to bronchodilators 25% 8%

In summary, ACS resulted in decreased pulmonary function in 94% of pts. It is striking that 49% pts had their PFTs reduced by half (FEV1 52% and FVC 55%). In pts with abnormal PFTs, 48% had evidence of obstruction and 25% of all pts tested improved with a bronchodilator. This is the first description of reversible abnormalities of pulmonary function occurring during ACS compared to baseline. An obstructive pattern is identified in a higher percentage of pts with SCD than in the local Oakland African American population (asthma prevalance 16%). While some pts PFTs improved with time, 45% remained abnormal at 12-week follow-up. ACS results in acute and chronic worsening of lung function. Future studies of ACS may reveal common pathogenic mechanisms with asthma, and lead to improved therapeutic interventions.
Posttranslational Control of Beta-Catenin Signaling Regulates Peripheral Blood Lymphocyte Activation.
Authors: Chung Eun Joo , Lee Min Jung , Lee Sunmin , Oh Ho Jung , Hwang Sang-Gu , Rubin Jeffrey S. , Trepel Jane B.
Published: October 2019

Lymphocyte proliferation is a critical step in the immune response to antigen. Previously we demonstrated that β-catenin is constitutively expressed in hematologic malignancies and that β-catenin regulates leukemia cell proliferation. Here we report that β-catenin is highly regulated in peripheral blood lymphocytes via posttranslational mechanisms, and that β-catenin signaling promotes lymphocyte proliferation. β-catenin protein was undetectable by western blot in resting PBL and readily detectable within 30 min after activation by TCR ligation, mitogen or PMA/ionomycin. Proteasome inhibitor alone induced β-catenin accumulation in resting PBL. Transfection of β-TrCP1, an adaptor protein that recruits serine-phosphorylated β-catenin to the Skp1-Cullin-F box ubiquitin ligase complex down-regulated β-catenin in activated cells and conversely, dominant-negative β-TrCP1 upregulated β-catenin in resting cells. β-catenin was serine/threonine phosphorylated in resting PBL and rapidly dephosphorylated after activation, consistent with regulation by an activation-induced phosphatase. In resting PBL, elevation of intracellular Ca2+ stabilized β-catenin protein. In activated PBL, β-catenin stabilization was inhibited either by blocking Ca2+ flux with EGTA and BAPTA/AM, or by incubation with cyclosporine A, an inhibitor of the calcium-activated phosphatase calcineurin. In activated PBL inhibition of β-catenin nuclear signaling with dominant-negative β-catenin, dominant-negative TCF or β-catenin antisense inhibited IL-2 expression and proliferation. Conversely, wild-type β-catenin increased IL-2 expression and stimulated proliferation. We conclude that

β-catenin protein is continuously synthesized and efficiently degraded in resting lymphocytes, β-catenin is rapidly stabilized in response to activation-induced Ca2+ flux, β-catenin nuclear signaling plays a critical role in peripheral blood lymphocyte activation.
Activity of the Histone Deacetylase (HDAC) Inhibitor PXD101 in Preclinical Studies and in a Phase I Study in Patients with Advanced Haematological Tumors.
Authors: Gimsing Peter , Wu Frank , Qian Xiaozhong , Jeffers Michael , Knudsen Lene , Sehested Maxwell , Lichenstein Henri S.
Published: October 2019

Previous investigations have shown that the novel hydroxamate PXD101 potently inhibits the enzymatic activity of histone deacetylase (HDAC) at nanomolar concentration, and the growth of cancer cell lines derived from solid malignancies at low micromolar potency. We have now examined and present results showing the activity of PXD101 on > 20 cell lines corresponding to a variety of haematological cancer types including acute T-cell leukaemia, cutaneous T-cell lymphoma, anaplastic large T-cell lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, diffuse large-cell lymphoma, plasma cell leukaemia and multiple myeloma (MM). Our data show that PXD101 strongly inhibits the growth of these cancer types at submicromolar potency. In addition, we will present in vitro data demonstrating that PXD101 efficiently inhibits the growth of doxorubicin-resistant leukemic cells, thereby supporting the possibility that this compound may be effective on drug-resistant haematological cancers. Finally, we will present results demonstrating that PXD101 used in combination with Velcade mediates greater growth-inhibition than does either drug used as monotherapy on a multiple myeloma cell line. These promising preclinical findings, taken together with previous results demonstrating the activity of PXD101 in a murine leukaemia animal model, support the evaluation of PXD101 for the treatment of haematological malignancies in a clinical setting. To this end, a Phase I trial using PXD101 in patients with advanced hematological tumors refractory to standard therapy was initiated. PXD101 is administered as a 30 minute i.v. infusion on days 1 to 5 of a 21 day cycle. Dose levels 600 mg/m2/d and 900 mg/m2/d have been examined (3 patients each) and recruitment to the dose level 1000 mg/m2/d is ongoing (5 patients to date). Patient characteristics are 8 males and 3 females with median age of 67 (range 58 – 73). Median number of prior therapies is 5 (range 4 – 7). Of the 11 patients enrolled to date, diagnoses were: MM (2), Non-Hodgkin’s lymphoma (5), CLL (4). Eleven patients have been treated with 1–8 cycles of therapy. Adverse events related to PXD101 treatment have mainly been grade 1–2. The most frequent adverse events were nausea (12/23 cycles), fatigue (9/23 treatment cycles), vomiting (8/23 cycles) and diarrhoea (7/23 cycles). Potential antineoplastic activity has been observed. One patient with MM had a possible tumor lysis syndrome with decrease in M-component, increasing urate and serum creatinine requiring dialysis. In addition, one patient with Richter transformation of CLL refractory to other treatments has now been in stable disease for > 6 months. We continue to accrue patients in this study, and updated data will be presented.
Genome-Wide Analysis of Copy Number Analysis of Myelodysplastic Syndromes Using High-Density SNP-Genotyping Microarrays.
Authors: Sanada Masashi , Nannya Yasuhito , Nakazaki Kumi , Yamamoto Go , Wang Lili , Hosoya Noriko , Hangaishi Akira , Kurokawa Mineo , Chiba Shigeru , Ogawa Seishi
Published: October 2019

Myelodysplastic syndromes (MDS) are clonal disorders of hematopoietic progenitors characterized by impaired blood cell production due to ineffective hematopoiesis and high propensity to acute myeloid leukemias. One of the prominent features of MDS is the high frequency of unbalanced chromosomal abnormalities that result in genetic imbalances and copy number alterations. Although the chromosomal segments involved in these abnormalities are thought to contain relevant genes to the pathogenesis of MDS, conventional analyses including FISH have failed to identify critical regions small enough to pinpoint their target genes.

Affymetrix® GeneChip® 100K/500K mapping arrays were originally developed for large-scale genotyping of more than 100,000/500,000 SNPs in two separate arrays, but the quantitative nature of the preparative whole-genome amplification and array hybridization thereafter also allows for accurate copy number estimate of the genome using these platforms at the resolutions of 21.3 kb and 5.4 kb with 116,204 and 520,000 oligonucleotide probes, respectively. Here we developed robust algorithms (CNAG) for copy number detection using 100K and/or 500K arrays and analyzed 88 MDS samples on these platforms in order to identify relevant genes for development of MDS.

With these huge numbers of uniformly distributed SNP probes, numerous copy number alterations were sensitively detected in cases with MDS with more numbers of abnormalities found in advanced diseases (RAEB and RAEB-t). In addition to large-scale alterations of various chromosomal segments previously reported in these syndromes, a number of small cryptic chromosomal abnormalities were identified that would escape conventional cytogenetic analysis or array CGH analysis. Minimum overlapping deletions in 5q, 7q, 12p, 13q, and 20q were precisely defined, although no pinpoint homozygous deletions were detected within these regions. A common 20q deletion spans a 400 kb segment harboring five transcriptomes and the common 12p deletion defines a 1.3 Mb region that contains the ETV6 gene. Other common overlapping abnormalities include deletions in 21q22, 17q13, and gains of 11q25. Genome-wide analysis of copy number changes using high-density oligonucleotide arrays provides valuable information about genetic abnormalities in MDS.
Antagonist Anti-CD40 Antibody, CHIR-12.12, Induces ADCC, Inhibits Tumor Growth, and Prolongs Survival in a Human Multiple Myeloma Xenograft Model.
Authors: Long Li , Tong Xia , Patawaran Montesa , Aukerman Sharon L. , Jallal Bahija , Luqman Mohammad
Published: October 2019

CD40 is expressed on all B-cell malignancies, including multiple myeloma, and represents an attractive target for antibody therapy. CHIR-12.12 is a fully human, highly potent, IgG1 antagonistic anti-CD40 monoclonal antibody generated using XenoMouse® mice (Abgenix, Inc). CHIR-12.12 can mediate antitumor activity by at least two mechanisms: blocking CD40-ligand-mediated survival signals and killing tumor cells by antibody-dependent cellular cytotoxicity (ADCC). We have previously reported that CHIR-12.12 mediates stronger in vitro killing of CD40+- and CD20+-expressing human non-Hodgkin’s lymphoma and lymphoblastoid B cells by ADCC than rituximab and significantly inhibits the growth of rituximab-responsive (Daudi) and rituximab-resistant (Namalwa) human lymphoma and lymphoblastoid B-cell (IM-9) xenografts in vivo. In this study, we examined the in vitro and in vivo efficacy of CHIR-12.12 against the human multiple myeloma cell line KMS-12-BM. CHIR-12.12 induced lysis of KMS-12-BM cells by ADCC in a dose-dependent manner, reaching maximum cell lysis at 0.1μg/ml with an EC50 of 17.5 pM. CHIR-12.12 efficacy in vivo was evaluated in orthotopic and subcutaneous KMS-12-BM xenograft models. In the staged orthotopic model, tumor cells were delivered intravenously and treatment was initiated 7 days post cell implantation. CHIR-12.12 significantly prolonged the median survival of tumor-bearing mice in a dose-dependent manner, with a median survival of 78 and 98 days in the groups treated with 1 mg/kg and 10 mg/kg CHIR-12.12 weekly, respectively, compared to a median survival time of 68 days in the control IgG1 group (P<0.0001). Bortezomib administered i.v. twice weekly at 0.5 or 1 mg/kg showed no survival benefit. In the staged subcutaneous model, CHIR-12.12 was administered weekly at 1 and 10 mg/kg after the mean tumor volume reached 100mm3. CHIR-12.12 significantly inhibited tumor growth, with a tumor volume reduction of 42% (P<0.05) and 63% (P<0.01), respectively. Bortezomib and melphalan/prednisone did not inhibit KMS-12-BM tumor growth at the doses and schedules reported for other human multiple myeloma xenograft models. Western blot analysis and immunohistochemical staining showed significantly increased levels of cleaved PARP in KMS-12-BM s.c. tumors 7 days after the initiation of CHIR-12.12 treatment, suggesting the induction of cell death by CHIR-12.12. Taken together, these data demonstrate that the anti-CD40 mAb CHIR-12.12 has potent activity against human multiple myeloma cells in vitro and in xenograft models in vivo. Currently CHIR-12.12 is in Phase I clinical trials for the treatment of B-cell malignancies.
The N-Terminus of Hepcidin Is Essential for Its Interaction with Ferroportin: Structure-Function Study.
Authors: Nemeth Elizabeta , Preza Gloria C. , Waring Alan J. , Ganz Tomas
Published: October 2019

Hepcidin, a small peptide produced by the liver, is the principal iron-regulatory hormone. It acts by binding to the iron exporter ferroportin, inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive 25 amino acid peptide has a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing ferroportin-GFP fusion protein, by measuring the degradation of ferroportin-GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in vivo, by measuring hypoferremia in mice injected with hepcidin derivatives.

Serial deletion of N-terminal amino acids caused progressive decrease in activity which was completely lost when five N-terminal aa were deleted. Synthetic 3-aa and 6-aa N-terminal peptides alone, however, did not internalize ferroportin, and did not interfere with ferroportin internalization by native hepcidin. Deletion of two C-terminal amino acids did not affect peptide activity. Removal of individual disulfide bonds by pairwise substitution of cysteines with alanines also did not impact peptide activity in vitro. However, these peptides were significantly less active in vivo, likely due to their decreased stability in circulation. Peptides with a substitution G71D or K83R, previously described in human subjects, were fully active in vitro and in vivo. Zebrafish hepcidin, which is only 60% similar to human hepcidin, but with conservative substitutions at the N-terminus, was as active as its human counterpart.

Apart from the essential nature of the N-terminus, hepcidin structure appears permissive for mutations. Further studies of hepcidin structure in relation to its function are essential for the design of hepcidin antagonists and agonists which could be used for treatment of iron disorders.
Discordant Serum Folate Result Unique to the Beckman Access Assay.
Authors: Wakeman Lisa , Munro Roger , Lewis John , Beddall Andrew , Al-Ismail Saad
Published: October 2019

Most current commercial assays for serum folate use folate-binding protein with chemiluminescence detection. Historically serum folate methods have been calibrated using pteroylglutamic acid (PGA) as this is more stable than 5Methyltetrahydrofolate (5MTHF) which is the form present in patients not receiving supplementation. Poor specificity of these binding assays together with different detection technologies and different method designs have highlighted the need for greater standardisation to calibration regimes so that individual folate species can be better identified. We describe here discrepant serum folate results in a patient which appear to be unrelated to the issues described above.

Consequent to a procurement evaluation of multiple analytical systems, serum ferritin, vitamin B12 and folate assays were performed on the Beckman Access, Abbott Architect 12000SR, Bayer Centaur and Roche E170 analysers according to manufacturer’s instructions. Method comparison studies using serum samples from 500 individuals showed good agreement between all analytical systems for all three parameters. However, one serum sample from a 44 year old Caucasian male who had presented with weight loss, diarrhoea and a microcytic, hypochromic anaemia (Hb 10.7 g/L) gave discrepant serum folate results and was further investigated. He had no evidence of renal impairment. His only medication was a multivitamin tablet supplement which he had been taking for several weeks. IgA anti-TTG antibodies were 33 U/mL (NR 0 – 10) and IgA endomysial antibodies were weakly positive. There was a polyclonal increase in serum IgA = 4.62 g/L (NR 0.8 – 2.8). Otherwise routine chemistry, thyroid profile and a paraprotein screen were unremarkable. Parietal cell and intrinsic factor antibodies were negative. Serum ferritin = 16 ng/mL (NR 20 – 215); Vitamin B12 = 164 pg/mL (NR 150–600). The patient’s sample showed no discrepant inter-system results for ferritin or Vitamin B12. Serum folate assays (NR 3.0 – 15.0 ng/mL) were as follows:

Technique Serum Folate Calibration method Beckman Access >20.0 PGA Roche E170 2.2 Quantaphase II Abbott Architect 1.9 Quantaphase II Bayer Centaur 1.5 5MTHF

This pattern of results was confirmed on a second sample a month later. No discrepancies were found in twenty other patients with positive TTG and endomysial antibodies.

Current methods for folate assays give different results and cannot measure the various forms of folate due to lack of calibrant standardisation with different types of automated analysers. In this patient, it may be that the folate species in the Vitamin supplement are measurable by the Beckman Access but are undermeasured with the other assay techniques. This data suggests the need for a reference method capable of identifying individual folate species in all patients.

Technique Serum Folate Calibration method Beckman Access >20.0 PGA Roche E170 2.2 Quantaphase II Abbott Architect 1.9 Quantaphase II Bayer Centaur 1.5 5MTHF