bluuebunny/crossref_metadata_2025 huggingface dataset:
Iron Does Not Cause Arrhythmias in the Guinea Pig Model of Transfusional Iron Overload.
Authors: Kaiser Lana , Davis John , Patterson Jon , Olivier N. Bari , Bohart George , Schwartz Kenneth
Published: October 2019

Cardiac events, including heart failure and arrhythmias, are the major cause of death in patients with b-thalassemia. Although cardiac arrhythmias in humans are believed to result from iron overload, excluding confounding factors in the human population is difficult. The purpose of these studies was to evaluate the development of cardiac arrhythmias using the guinea pig model of secondary iron overload. Electrocardiograms were recorded via surgically implanted telemetry devices in guinea pigs loaded intraperitoneally with iron dextran and controls. Cardiac and liver iron concentrations were significantly elevated in the iron loaded animals when compared to control, and were in the range of those reported for humans with thalassemia. Arrhythmias were noted infrequently in both iron loaded and control guinea pigs. No life threatening arrhythmias were detected in either group. These data suggest that iron alone may be insufficient to cause cardiac arrhythmias, and that arrhythmias detected in individuals with iron overload may be the result of a complex interplay of factors.
A Novel Parameter for the Diagnosis of Iron Deficiency: The Transferrin/Albumin Ratio.
Authors: Pieroni Laurence , Jardel Claude , Merle-Beral Helene , Azgui Zahia , Baudet Sylvie , Tostivint Isabelle , Hainque Bernard , Maloum Karim
Published: October 2019

Iron deficiency (ID) is generally easily diagnosed by a serum ferritin concentration less than 12 μg/L. However, in patients with concomitant pathologies, sensitivity of ferritin for diagnosing ID is low, since inflammation or liver disease can lead to normal or increased ferritin values, even when iron deficiency. In this study, we propose a new marker for diagnosing ID in patients with chronic disease: the Transferrin/Albumin ratio (Tf/A ratio). Indeed, the synthesis and the elimination of albumin and transferrin are regulated by similar processes, excepted in ID. In the latter, the synthesis of transferrin is stimulated, whereas that of albumin is not. Therefore, the Tranferrin/Albumin ratio increases in case of ID, even when transferrin values are within the normal range. To determine the accuracy of this novel parameter, we studied 75 patients with chronic disease who were submitted to bone marrow aspirates and iron staining. Iron stores depletion was defined by less than 10% sideroblasts, without extra-cellular iron nor siderocytes. Blood samples were routinely undertaken at the time of the medullar sampling for determination of hematological and biochemical parameters. Iron status including ferritin, Tranferrin Saturation (TfSat), soluble Transferrin Receptor (sTfR), sTfR/log ferritin and Tf/A ratio, was determined. The diagnostic accuracy of the Tf/A ratio was compared to previously described parameters of iron status that we cited above. Receiver Operating Characteristics (ROC) curves were built to determine the best cut-off values for the prediction of iron deficiency. According to the Perls’ reaction, 25 of the 75 patients (33%) had depleted iron stores and 50 had normal or increased iron stores. Sixteen iron-depleted patients (67%) had anemia. Mann and Whitney U test showed that parameters significantly associated with ID were: Tf, Tf/A ratio, ferritin, TfSat, sTfR, sTfR/log ferritin, mean corpuscular volume, mean corpuscular hemoglobin, red blood cell and reticulocyte counts. In a multivariate analysis, the only significant, independent predictor of iron depletion was the Tf/A ratio (r = 0.637, p < 0.005). The sensitivity/specificity of Tf/A ratio at a cut-off point of 6.4% as given by ROC curve were 80%/88%. In conclusion, the Tf/A ratio is useful in the detection of iron depletion in patients with chronic disease and could dispense with bone marrow aspirate and Perls’ reaction in more than 80% of cases.
Liver Status in a Cohort of Polytransfused B-Thalassemia Major (BTM) on Long Term Desferrioxamine (DfO) or Deferiprone (L1).
Authors: Elalfy Mohsen Saleh , Abdin Iman
Published: October 2019

The liver is the main body site for iron storage. Iron chelators as DFo and L1 had problems of compliance and intolerance respectively. This prospective study enrolled 76 BTM patients aged from 9–33 years, mean of 14±4.4 to evaluate the effectiveness of both chelators judged by serum ferritin (sf) and liver iron content (LIC) using repeated liver biopsies. Fifty patients were on DFo 35 mg/kg/day 5 days per week and twenty-six were on L1 75 mg/kg/day on daily bases. Mean baseline sf values were 3678±1922 and 3573±1879 ng/ml while mean baseline LIC was 19.2±7.9 and 18.9±8.1 mg fe/g dw, while 12% and 14% showed evidence of liver fibrosis (score ≥ 3) respectively. Twenty-four months later; a decline in sf by 287±837 in Dfo (In Dfo compliant group [number = 30]who had received ≥ 75% of planned dose; sf dropped by 890±437 while increased in DFo non compliant by 621±513). While falling by 729±633 in L1 group. A decrease in LIC by 3.9±2.8 mg fe/g dw (−5.8±3.1 in Dfo compliant,+2.6±3.3 in Dfo non compliant) and falling by 2.2±3.4 mg fe/g dw in L1 group, the difference was statistically significant P< 0.05 between compliant and non compliant DFo and insignificant difference between Dfo and L1.The mean iron excretion: intake was 1.61,0.86 and 1.48 in Dfo compliant, non compliant and L1 respectively. A positive correlation was found between sf and LIC in both groups. Fibrosis was improved in Dfo compliant group better than L1 but no statistically significant difference was observed, while progressive fibrosis in 10% of Dfo non compliant group. All Dfo compliant group completed study, 18 out of 20 Dfo non compliant while six L1 patients discontinued the drug prematurely (3 severe gastrointestinal disturbances, 2 severe arthralgia and one repeated neutropenia). Conclusion: Both Dfo and L1 are effective iron chelators evident by drop in sf, LIC and improved fibrosis, however compliance and intolerance are still troublesome.
A Novel C to A Substitution in the CCAAT Box of beta Globin Gene.
Authors: Refaldi Chiara , Di Pierro Elena , Mocellini Maria C. , Cappellini Maria D.
Published: October 2019

The promoter of the human beta-globin gene contains three positive cis-acting elements required for maximal transcription: the CACCC box located between −86 and −90, the CCAAT box located between −72 and − 76 and the TATA box located between −28 and −31 relative to the start site of transcription. Naturally occurring mutations within the TATA and the CACCC box regions have been recorded in patients with beta+ thalassemia. Mutations within the TATA box disrupt assembly of the basal transcription complex, while mutations at the CACCC box prevent binding of an erythroid-specific transcription factor EKLF. Surprisingly, no mutations have so far been identified in the highly conserved element CCAAT box and the transcription factors responsible for the regulatory activity of the CCAAT site in vivo have been less intensively studied. We report a novel mutation −76 C>A (HBB c. −126) detected by sequencing analysis of beta globin gene in a Italian beta+ thalassemic patient. The transversion C>A hits the first nucleotide in the CCAAT box of the beta globin gene. The carrier, a male 44 years old, shows a mild hypochromic and microcytic anaemia with reduced mean corpuscular volume and mean corpuscular haemoglobin (MCV 75 fl, MCH 25 pg) and Hb A2 level slightly increased (3.9%). Recently, studies in vitro in gel-shift and reporter assays, investigating the transcriptional activity of human beta globin CCAAT box, have identified five factors: NF-Y (CP1) a ubiquitous CCAAT box binding complex, GATA-1 an erythroid-specific transcription factor, C/EBPbeta, C/EBPgamma and C/EBPdelta members of CCAAT/enhancer-binding protein family involved in hemapoietic regulation. This represents the first report of a natural mutation of the human beta-globin CCAAT box and confirms its functional significance for in vivo transcription.
Contribution of Platelets and Erythrocytes to Thrombin Generation in Patients with β-Thalassemia.
Authors: Valles Juana , Piñon Marta , Dasi Maria A. , Moscardo Antonio , Fuset Maria P. , Latorre Ana , Santos Maria-Teresa
Published: October 2019

Patients with β-thalassemia show an abnormally high proportion of red blood cells (RBCs) with exposed phosphatidyl serine (PS) molecules on the RBC membrane, which may contribute to thrombophilia in these patients (1). We have reported previously (2) that thromboxane A2 and free-arachidonate—substances released from activated platelets—induce PS exposure in a subpopulation of normal RBCs. The aims of the present study were first, to evaluate the presence of activated circulating platelets in patients with β-thalassemia, and second, to determine tissue factor (TF)-initiated thrombin generation in plasma and the influence of RBCs, platelets and their cooperation on this process in normal subjects and patients with β-thalassemia. Thrombin generation was assessed according to Hemker et al. (3) in 12 patients with β-thalassemia diagnosed by molecular biology, age 5–33 years and in 18 normal subjects. Two patients had thalassemia major (T-M) requiring blood transfusion every 3 weeks and the analysis were performed just prior transfusion. Two had thalassemia intermedia (T-I) with esplenectomy without transfusion requirements and eight had thalassemia minor (T-m). Thrombin generation was evaluated in platelet-poor plasma (PP), platelet-rich plasma (PRP), RBCs in the plasma (P-RBC) and in the presence of both cell types (PRP+RBC). The presence of activated platelets in circulation was evaluated in whole blood by flow cytometry. We monitored P-selectin (CD-62) and the activated αIIbβ3 receptor, defined as PAC-1 binding. The percentage of platelets exposing activation markers CD-62 (mean± SEM, 0.72± 0.17 vs. 4.03± 1.39) and PAC-1(0.75± 0.14 vs. 12.26± 6.47) were significantly higher in patients than in control subjects (P < 0.05). In normal subjects, the presence of erythrocytes in plasma (P-RBC) triggered a quicker thrombin generation and a significant elevation in the thrombin peak (32 ± 5 nM in PP vs. 86 ± 16 nM in P-RBC; P < 0.05), in the absence of cell lysis. These effects of RBCs were markedly higher in the patients with β-thalassemia, who had 2.5-fold higher thrombin generation at 3 min and higher peak thrombin levels (86 ± 16 nM in controls vs. 130 ± 15 nM in patients; P < 0.05). Thrombin generation in PRP was also higher in the patients with thalassemia at early times (3 min; 19 ± 3 nM in controls vs. 43 ± 8 nM in patients; P < 0.05). Importantly, the rate and peak of thrombin generation were significantly amplified by the presence of both cell types (PRP+RBCs), suggesting a biochemical cooperation between activated platelets and RBCs for thrombin generation. This was enhanced in patients with β-thalassemia, particularly those with T-I. In conclusion, chronic platelet activation in patients with β-thalassemia may influence TF-induced thrombin generation in platelets and erythrocytes. Thrombin generation appears to be a novel mode of platelet-erythrocyte cooperation in thrombogenesis.
Liver Iron Measurement by SQUID Compared to Liver Biopsy.
Authors: Pakbaz Zahra , Fischer Roland , Butz John , Foote Drucilla , Hackney-Stephens Ekua , Fung Ellen , Fagaly Bob , Harmatz Paul , Vichinsky Elliott
Published: October 2019

Liver biopsy, which is the gold standard for liver iron assessment in patients with hemosiderosis, is painful, associated with risk of infection or bleeding, and may be affected by uneven iron distribution and variation in analytical methods. Monitoring iron overload by serum ferritin is still a routine practice although its limitations are well known. Therefore, using non-invasive liver iron assessment by quantitative MRI or by liver susceptometry with a SQUID biomagnetometer (BLS) would be the better alternatives. This report explores the accuracy of liver iron concentration (LIC) measured by BLS at CHRCO compared to liver biopsy, in patients with thalassemia and sickle cell disease. A total of 33 patients with chronically transfused thalassemia (n=16) or sickle cell disease (n= 17) were prospectively assessed for LIC measured by BLS and liver biopsy within 2 month of each other. LIC was measured by a low temperature SQUID biosusceptometer system (Ferritometer®) under the standardized Hamburg-Torino-Oakland protocol. Iron in fresh tissue liver biopsy samples (with a dry weight larger than 1 mg) was measured at Mayo clinics by Inductively Coupled Plasma Mass Spectrometer (ICP-MS). Median value for the wet to dry weight of the tissue samples was 3.8 (3.1–6.2). Subjects ranged in age from 7 to 40 years (median: 18 y). Median LIC (wet weight) measured by SQUID was 1915 (415–4826) [mg/g wet weight ], median LIC by biopsy (dry weight) was 12681(1854–32864) [mg/g dry weight]. Serum ferritin ranged from 405 to 9843 (median: 1831). The Spearman rank correlation between wet weight LIC assessed by BLS and dry weight LIC from fresh tissue sample measured by ICP-MS was highly significant (RS=0.93, p<0.0001). The conversion factor between the two methods obtained from a weighted linear regression with zero intercept was 6.1 ± 0.3 (R2 = 0.92, cn2 = 0.5), while the median value for wet-to-dry weight ratio of the actual samples is 3.8. This conversion factor agrees with the value reported earlier by Piga et al (Blood2005; 106(11): 755a). The role of the wet-to-dry weight ratio needs to be explored further as the conversion factor generated from the linear regression is significantly larger.

Fig1. LInear regression LIC (BLS)=(0.165 ± 0.008) *LIC (biopsy) weighted by the uncertainties to the two methods (R2 = 0.92, reduced chi-square χ2= 0.5). Fig1. LInear regression LIC (BLS)=(0.165 ± 0.008) *LIC (biopsy) weighted by the uncertainties to the two methods (R2 = 0.92, reduced chi-square χ2= 0.5).
Bcl11a Causes Leukemia/Lymphoma in Cooperation with Nf1 Inactivation.
Authors: Yin Bin , Jeske-Geurts Jennifer L. , Diers Miechaleen D. , Shannon Kevin M. , Largaespada David A.
Published: October 2019

Loss of the NF1 (Neurofibromatosis Type 1) tumor suppressor gene is associated with chronic and acute forms of myeloid diseases including juvenile myelomonocytic leukemia (JMML). In the BXH-2 strain of mice, acute myeloid leukemia (AML) is caused by chronic infection by a murine leukemia virus (MuLV). We have reported the identification of 37 common proviral insertion sites (CIS), loci containing candidate genes whose mutation could cooperate with inactivation of Nf1 to result in BXH-2 AML. Among these candidate genes, Bcl11a, was shown to be expressed in JMML and CMML leukemia samples, and to confer a growth advantage when expressed in Nf1-deficient myeloblast cells. In this report, we have first tried to determine whether dysregulation of Bcl11a affects cellular behavior other than promoting cell growth. Using a granulocyte-colony stimulating factor induced myeloid terminal differentiation of 32D cell culture model, we found no difference in the percentage of differentiated cells between Bcl11a-transduced cells and empty vector-transduced cells, indicating overexpression of Bcl11a may have no influence on myeloid differentiation. Moreover, no effect of Bcl11a on 32D survival after IL-3 withdrawal was detected. To investigate the potential role of Bcl11a in the development of leukemia with Nf1 loss, a bone marrow transplantation assay was then performed. Bone marrow cells were collected from the Nf1(+/+) or Nf1(Flox/Fcr) donor mice carrying pIpC-induced expression of Cre, and transduced with Bcl11a-expressing retroviral construct or empty vector, before transplantation via the tail vein into the lethally irradiated recipient BoyJ mice. Successful bone marrow engraftment, pIpC-induced deletion of Nf1 and delivery of Bcl11a were confirmed by PCR on peripheral blood cells. Interestingly, 5 months after pIpC induction, 10 out of 11 Nf1(Flox/Fcr);Cre+;Bcl11a-transduced bone marrow recipient mice became moribund, whereas no or only 1 out of 10 or 11 recipient mice in each control groups died for unknown reason. The Nf1(Flox/Fcr);Cre+;Bcl11a-transduced mice display myeloid blast-like cells in their peripheral blood, enlarged lymph node and liver, and splenomegaly. Spleen touchpreps also showed excessively infiltration of blast-like cells in their spleens. Unexpectedly, two out of ten moribund recipient mice also developed markedly enlarged thymi. These tumors were demonstrated to be derived from transplanted donor cells by immunophenotyping for the presence of the differential cell surface marker CD45.2, expressed on the donor cells. The nature and cell lineage origin of these tumors are under study. These data show that BXH-2 mouse is a powerful genetic system to dissect Nf1-cooperating genetic events in leukemogenesis, and suggest that overexpression of Bcl11a can induce leukemia/lymphoma with Nf1 gene inactivation.
Interleukin 21-Based Therapy Can Induce Human B Cells To Produce Granzyme B and Express Other Features of Cytotoxic Lymphocytes.
Authors: Jahrsdoerfer Bernd , Blackwell Sue M. , Weiner George J.
Published: October 2019

B cells are not currently known to be capable of producing granzyme B or being cytotoxic. We recently found that human B cells activated with Interleukin 21 (IL-21) and antibodies to the B cell receptor (BCR) or immunostimulatory oligonucleotides (CpG ODN), can produce granzyme B. Further studies were done to assess the biological and potential therapeutic significance of this finding. Granzyme B ELISpot, intracellular staining for granzyme-B, quantitative real time RT-PCR for granzyme B messenger RNA and gene expression array confirmed B cells obtained from the peripheral blood of normal individuals (Fig. 1) and many B cell lines including Namalwa, Daudi, Ramos and EBV-transformed lymphoblasts, can be induced to produce granzyme B. This granzyme B is functional as demonstrated by cleavage of a granzyme B-sensitive colorimetric substrate. IL-21 based treatment also increased the transcription of the gene for perforin and the production of Interferon-γ in select B cell populations. We conclude that IL-21 based therapy can induce B cells to produce functional granzyme B and other components known to be present in the cytotoxic granules of CTL and NK cells. These unexpected findings could have significant implications on our understanding of the role of B cells in immune regulation and for a variety of immune phenomena including auto-, cancer and infectious immunity.

Figure 1. Interleukin 21 (IL-21) induces de-novo synthesis of Granzyme B by activated B cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the above depicted agents for 18 hours. After further incubtion with Brefeldin A for 4 hours cells were harvested, fixed, permeabilized, stained with PE- and PE-Cy5-labeled antibodies against Granzyme B and CD19, and analyzed flowcytometrically. Figure 1. Interleukin 21 (IL-21) induces de-novo synthesis of Granzyme B by activated B cells. Peripheral blood mononuclear cells (PBMC) were stimulated with the above depicted agents for 18 hours. After further incubtion with Brefeldin A for 4 hours cells were harvested, fixed, permeabilized, stained with PE- and PE-Cy5-labeled antibodies against Granzyme B and CD19, and analyzed flowcytometrically.
Effects of Niflumic Acid on Thrombocytic Cytoplasmic Free Calcium and Platelet Aggregation in Patients with Diabetes Mellitus.
Authors: NIE Danian , Yin Songmei , Xie Shuangfeng , Wang Xiaogang , Li Yiqing , Ma Liping , Wang Xiuju , Wu Yudan , Feng Jianhong
Published: October 2019

Background: Platelet activation plays an important role in cardiovascular complications which are frequently seen in patients with diabetes mellitus. It has been demonstrated that platelets are hyperactive in diabetes patients and this is supposed to be due to high platelet cytoplasmic free calcium ion concentration ([Ca2+]i). There are chloride channels on the membranes of platelets and the activation of chloride channels may participate in the transportation of calcium. Our previous study also showed that niflumic acid (NFA), a chloride channel blocker, can inhibit the elevation of platelet [Ca2+]i in healthy volunteers. The goal of this study was to investigate effects of NFA on platelet [Ca2+]i and platelet aggregation rate (PAG) in diabetic patients.

Methods: We prospectively enrolled 17 consecutive patients with diabetes mellitus at diagnosis. The control group was formed from 17 healthy volunteers. Platelet [Ca2+]i was detected by Fura-2 fluorescent technique and PAG was detected by platelet aggregometer. We studied the effects of NFA, nifedipine, a calcium channel blocker, and their combining effects on platelet [Ca2+]i and PAG in diabetic patients.

Results: The PAG, platelet resting [Ca2+]i, Ca2+ release and Ca2+ influx in diabetic patients were significantly higher than control group(P<0.05). NFA and nifedipine reduced the platelet Ca2+ influx which induced by thrombin in a dose-dependent manner in diabetic patients. 50% inhibition concentration (IC50) of NFA was 50μmol/L. At this concentration, NFA reduced platelet [Ca2+]i by (54.7±14.5)% and decreased PAG by (32.3±21.4)%. The IC50 of Nifedipine was 7.5μmol/L. At this concentration, nifedipine reduced platelet [Ca2+]i by (17.9±11.9)% and decreased PAG by (32.3±20.4)%. NFA(50μmol/L) combining with nifedipine (7.5μmol/L) inhibited platelet Ca2+ influx induced by thrombin in diabetes patients and their effects were independent.

Conclusions: Platelets in patients with diabetes mellitus are hyperactive. NFA reduces the PAG and platelet [Ca2+]i influx possibly by blocking the chloride channels on platelet membrane in diabetic patients. There are no interactions between NFA and nifedipine on the movement of platelet [Ca2+]i.
CIB1 Positively Regulates Outside-In Signaling through αIIbβ3 by Enhancing FAK and Src Activity.
Authors: Naik Meghna U. , Naik Ulhas P.
Published: October 2019

Integrin αIIbβ3, the platelet fibrinogen receptor, mediates outside-in signaling upon fibrinogen binding. Calcium- and integrin-binding protein 1 (CIB1) specifically interacts with the cytoplasmic domain of αIIb and is involved in platelet spreading. Here we show that CIB1 binding to αIIb is an essential step in the propagation of outside-in signaling through integrin αIIbβ3. Incorporation of BAPTA-AM completely blocked outside-in signaling and CIB-1 binding to aIIb, suggesting that an intracellular Ca2+ rise induced by fibrinogen binding is required for CIB1 interaction with αIIb. When CIB1-binding to αIIb was inhibited by introduction of a function blocking antibody or a synthetic peptide corresponding to αIIb tail, CIB1 localization at the tip of the filopodia, but filopodia formation was not blocked. This result suggests that interaction of CIB1 with αIIb is not required for filopodia formation, but is needed for CIB1 accumulation at the filopodia, as well as platelet spreading. Immunoprecipitation experiments showed that during outside-in signaling, CIB1 associates with FAK. Although association of FAK and CIB1 does not require dynamic rearrangement of the cytoskeleton, their accumulation at the filopodia and the activation of FAK is dependent on cytoskeletal rearrangement, as treatment with cytochalasin D after the platelets form filopodia affects CIB1 localization. Interestingly, the interaction of CIB1 with αIIb upon fibrinogen binding is also necessary for FAK and Src activation. However, Src activity is not required for CIB1 accumulation at the filopodia since this accumulation was not stopped by the Src inhibitor PP2, despite blocking platelet spreading. Our results suggest that during outside-in signaling an intracellular Ca2+ rise occurs that facilitates CIB1 interaction with αIIb. CIB1 recruits FAK to this complex and is localized to the filopodia dependent upon dynamic cytoskeletal rearrangement. Furthermore, activation of Src and FAK requires interaction of CIB1 with αIIb. Although Src activity is not required for the accumulation of CIB1 at the filopodia, it is required for platelet spreading on immobilized fibrinogen. Thus we provide evidence for initial sequence of outside-in signaling in platelets.
Analysis of Immunoglobulin Gene Mutation Status in Lymphoplasmacytic and Splenic Marginal Zone Lymphomas with Bone Marrow Infiltration.
Authors: Nathalie Gachard , Marie Parrens , Barbara Petit , Isabelle Soubeyran , Valerie Costes , Pierre Brousset , Michel Cogne , Dominique Bordessoule , Jean Feuillard
Published: October 2019

Splenic Marginal-Zone lymphomas (SMZL) and Lymphoplasmacytic lymphomas (LPL) correspond to entities that shared clinical, morphological and immunohistochemical features and are not always easy to discriminate. Immunoglobulin variable heavy chain gene (VH) usage and somatic mutation pattern were analyzed to clarify the relationship and the pathogenesis of these 2 entities.

Bone marrow frozen samples from 27 patients, 16 LPL and 11 SMZL, were studied. B-cell clonality was first analysed using the standard PCR method with the Biomed 2 primers. Informative PCR products were then purified, sequenced and analyzed on IMGT and Ig-Blast websites.

Overall, median rate of somatic mutations was 9% in LPL and 4% in SMZL (p=0.013).

All cases of LPL were mutated. Mutation rate was always higher than 5%. A preferential usage of VH3 gene was observed in 13/16 cases (80%), with a predominance of VH3–23 and VH3–74, both representing 60% of cases. VH4 gene was used only in 2/16 cases (13%). Regarding JH usage, a predominance of JH4 gene rearrangement was observed in 11/16 cases (69%). JH5 and JH6 were used in 20% and 13% of cases respectively.

In the group of SMZL, mutation rates and usage of VH and JH genes were more heterogeneous. Thirty six percent of SMZL were unmutated and 19% had a mutation rate lower than 5%. VH3 was used in 4/11 cases (36%), VH4 in 3/11 cases (27%) and VH1 in 4/11 cases (36%). JH4 and JH6 were used in 4/11 cases (36%) and the JH5 in 1/11 cases (9%).

Therefore, the profile of somatic mutations and the VH and JH segment preferentially used are different in SMZL and LPL, suggesting a different process of antigen-driven selection and of transformation of the initial B-cell clone. These findings consolidate the existence of LPL as a genuine entity, different of SMZL despite their histopathological overlaps.
Expression and Mutation Analysis of the Myc Antagonist Gene Mad1 in Acute Leukemia.
Authors: Pan Ling , Zhang Xuejun , Suo Xiaohui , Wang Fuxu , Niu Zhiyun , Dong Zuoren
Published: October 2019

Objectives: Overexpression of Myc genes has been observed in many solid tumors and hematological malignancies, and Myc proteins are believed to act as transcription factors. In the past decade, several Myc-related genes have been identified, and the interaction mechanisms between these genes have been clarified. Members of Myc superfamily proteins belong to four different families: the Max family of common dimerization partners (including Max and MLx), the Myc family of proto-oncoproteins (including c-, N-, and L-Myc), the Myc antagonists including Mad family (including Mad1 and Mxi1) and Rox (Mnt). Mad1 gene maps to human chromosome 2p13 and encodes a 221-amino acid protein. Mad1 protein was shown to counteract the effects of Myc proteins and to inhibit the proliferation and tumorigenicity of glioblastoma and astrocytoma cells, and therefore was considered to be a kind of tumor suppressor. All Myc super-family members share a highly conserved structural domain, basic helix-loop-helix leucine zipper domain (bHLHZip). This bHLHzip domain is essential for these proteins to function during molecular switching from proliferation to differentiation. To function in vivo, Myc and Mad1 must heterodimerize with Max through the HLHZip motif. Then through the b (basic) motif, the heterodimers bind with CACGTG (E-box) containing DNA to activate or inactivate transcription. The present study is thus designed to examine the expression of Mad1 gene and to search for mutations in bHLHzip encoding domain.

Methods: we screened bone marrow mononuclear cells (BMMNC) from 26 patients with AL and peripheral blood mononuclear cells (PBMNC) from 30 healthy volunteers, using reverse transcription- polymerase chain reaction, single strand conformation polymorphism analysis and sequencing.

Results: Mad1 gene was expressed in all samples. Two polymorphisms of Mad1 were found in BMMNC from AL patients and PBMNC from health volunteers. A point mutation was detected at a very important position, a substitution of A397G inducing Lys78Glu in two patients. This might have significant implication for the function of Mad1, as Mad1 will heterodimerize with Max at this very point. Mutation at this point could thus disrupt proper intermolecular alignment between Mad1 and Max. The two patients with Mad1 gene mutation had poor clinical outcomes, patient No.9 never achieved CR and died 2 months after diagnosis, while patient No.11 got CR, after two courses chemotherapy, but relapsed quickly and died 3 months after diagnosis.

Conclusion: Mad1 gene expressed and displayed mutations in acute leukemia patients. It would be interesting to know whether these mutations influence either the heterodimerizing or DNA binding potentials of Mad1 protein, by performing functional analysis of the mutant protein.
Increased Midkine Gene Expression in Childhood Acute Leukemia.
Authors: Hidaka Hirokazu , Yagasaki Hiroshi , Takahashi Yoshiyuki , Horibe Keizo , Kadomatsu Kenji , Kojima Seiji
Published: October 2019

Midkine (MK) is a heparin-binding growth factor and overexpressed in a number of solid tumors, contributing to their growth. Its expression in acute leukemia, however, has not been clarified. We examined relative levels of MK gene expression defined in K562 cells as 1.00 using real-time quantitative polymerase chain reaction (PCR) in 94 children with acute leukemia and compared with those in normal bone marrow (BM; n=18) and peripheral blood (PB; n=10). Median age was 6 years (range, 0–15 years). Diagnosis included B-precursor ALL (n=41), T-ALL (n=14), B-ALL (n=4), Ph1-ALL (n=6), infant ALL (n=5) and AML (n=24). 8 normal BM samples were separated into CD34+ and CD34− fractions. MK gene expression was detected in normal control samples. Median MK gene expression level was significantly lower in normal PB (2.9×10e-3) than in normal BM (8.0×10e-3) (p<0.01). Expression of MK gene was higher in normal CD34+ BM cells (0.78×10e-3) than in normal unfractionated BM cells (p<0.001). Overexpression of MK was difined as expression at least twice as high as the maximal MK gene expression in normal PB and BM. In 30 of the 41 patients with B-precursor ALL (73.2%), MK gene is overexpressed. We classified B-cell lineage ALL into 3 stage:

DR+,CD19+,CD10+,CD20−,slg−, DR+,CD19+,CD10+,CD20+,slg−, DR+,CD19+,CD20+,slg+.

MK gene expression decreased with B-cell lineage differentiation of leulkemic blast. It was also overexpressed in more than half of the patients with FAB M1 and M2 types of AML. In contrast, overexpression of MK gene was observed only in one of the 13 patients with T-ALL and none of the 6 patients with AML-M5. Supporting the possible involvement of MK in carcinogenesis, overexpression of MK may be not only merely assoziated with total development but also related to leukemic transformation of hematopoietic progenitors. Quantification of MK gene by real-time PCR is relatively simple and widely applicable in patients with acute leukemia. This technique offers particular promise as a prognostic marker and a marker for minimal residual disease in children with B-precursor ALL.
Aberrantly Expressed Neutrophil Elastase (ELA2) Cleaves Cyclin E (CCNE) in the Nucleus and Cytoplasm of Acute Lymphocytic Leukemia Yielding Novel Leukemia-Associated Antigens.
Authors: Ishiyama Ken , Kondo Yukio , Wieder Eric D. , Lu Sijie , Molldrem Jeffrey J.
Published: October 2019

We have shown that donor-derived cytotoxic T lymphocytes (CTL) are specific for two HLA-A2-restricted peptides derived from CCNE (CCNE1144–152, ILLDWLMEV and CCNE2144–152, ILLDWLLEV) specifically lyse lymphoid and myeloid leukemia cells that aberrantly express CCNE. The CCNE protein is overexpressed in AML, ALL, and CML, and in solid tumors such as breast, lung, and gastric cancers. Furthermore, five low molecular weight forms (LMWFs) of CCNE, which are constitutively active to promote cell division, are found only in malignant cells, though it is not known how LMWFs are formed in leukemia. We hypothesized that the cleavage of CCNE into LMWFs occurs by enzymatic cleavage from aberrantly expressed ELA2 in leukemia, which renders the leukemia susceptible to killing by ELA2- and CCNE-specific CTL. Whole cell lysates from U937, HL60, and bone marrow from patients with B-ALL, but not from PBMC or bone marrow cells from healthy donors or ALL patients in remission, showed high expression of CCNE and LMWF by western blot (WB). Recombinant ELA2 added to B cell-derived whole cell lysates increased LMWFs, and the leukocyte elastase inhibitor Elafin prevented this cleavage. Subcellular fractions studied by coimmunoprecipitation showed that ELA2 was bound to CCNE in the nucleus, cytoplasm, and membrane-bound organelles of B-ALL, but not in normal cells. Because nuclear expression of CCNE increases during normal cell division, we studied healthy donor PBMC stimulated with anti-CD3 and anti-CD28 and found no expression of ELA2 or CCNE LMWFs by WB. We conclude that ELA2, normally expressed only in myeloid cells, is also expressed in some ALL blasts, and this data explains how CCNE LMWFs are formed in leukemia. Our findings also suggest how ELA2-mediated cleavage of the PML-RARα fusion product, required for leukemic transformation, could occur when ELA2 is aberrantly expressed in the nucleus. Finally, this work implies that overexpression of CCNE and the LMWFs that contain the immunogenic peptides could increase susceptibility of leukemia cells to CCNE-CTL lysis.
Selection of Human Anti-CD22 scFvs from an Acute Lymphocytic Leukemia Related scFv Library with Ribosome Display.
Authors: Rothe Achim , Hosse Ralf J. , Nathanielsz Anne , Oberhaeuser Frank , von Strandmann Elke Pogge , Engert Andreas , Hudson Peter J. , Power Barbara E.
Published: October 2019

Novel in vitro methods for screening of antibody libraries against disease-related antigens have led to the development of powerful protein-based biotherapeutics. Ribosome display represents an emerging and innovative in vitro technology for the rapid isolation and evolution of high-affinity peptides or proteins. Eukaryotic ternary ribosome complexes can be used to display human single chain antibodies (scFvs) in order to isolate specific binding reagents. Here we present the isolation of human scFv against the validated immunotherapeutic target antigen CD22 from a leukaemia patient-derived human scFv library using ribosome display technology. The ribosome-displayed scFv were enriched against the human CD22 conjugated to magnetic beads. Isolated constructs were further affinity-matured and specific target recognition was demonstrated by surface plasmon resonance and validated using in vitro cell assays.
Oral Etoposide Is Equivalent to Intravenous Administration within the CHOEP-Regimen - Results of a Comparative Pharmacokinetic Study.
Authors: Kroschinsky Frank P. , Friedrichsen Kai , Mueller Juliane , Haenel Mathias , Ehninger Gerhard , Schleyer Eberhard
Published: October 2019

The addition of etoposide to the CHOP protocol (CHOEP) has been shown to improve outcome particularly in younger patients (<60 years) with aggressive non-Hodgkin’s lymphoma [Pfreundschuh, Blood 2004]. In the previous trials a dose of 100 mg/m2 etoposide was given intravenously on three consecutive days representing a disadvantage of CHOEP compared to standard CHOP in terms of patients’ convenience. Therefore we investigated the pharmacokinetic equivalency of etoposide as an oral preparation on days 2 and 3 compared to the intravenous route. Ten patients (male, n=7; female, n=3; median age 56 years) with aggressive B-cell- (n=9) or T-cell- (n=1) lymphoma were included. The CHOEP regimen consisted of cyclophosphamide 750 mg/m2, doxorubicin 50 mg/m2, vincristine 2 mg/m2 and prednisone 100 mg orally days 1 to 5. Etoposide 100 mg/m2 was given intravenously on day 1, and 200 mg/m2 orally on days 3 and 4, respectively. EDTA blood for pharmacokinetic study were taken on days 1 (i.v. study) and 3 (p.o. study) before etoposide and after administration at 30, 60 and 90 min, every hour until 8 hours, followed by samplings at 16, 20, 24 and 48 hours. The samples were centrifuged immediately at 5°C for 15 min, and the plasma was aliquoted into cryo vials and stored at −20°C until assayed. Etoposide levels in plasma were determined by high-performance liquid chromatography (HPLC). The area under the plasma etoposide concentration versus time curve (AUC), plasma etoposide clearance (CL), volume of distribution in steady state (VDss) and terminal etoposide plasma half-life (t½) for the intravenous and oral administered drug were calculated based on the TOPFIT computer program. The median bioavailability after oral etoposide was 58 % with an interpatient variation (coefficient of variation, CV) of 26 %. AUC was very similar after 200 mg/m2 oral etoposide and intravenous administration of 100 mg/m2 of the drug, however the pharmacokinetic variation was higher after oral uptake compared to the parenteral route (35 % vs. 23 %). Data are presented in detail (medians and ranges) in Table #1. We conclude that, within the CHOEP regimen, the intravenous administration of 100 mg/m2 etoposide on days 2 and 3 can be replaced by 200 mg/m2 of the oral preparation, which simplifies the treatment in the outpatient setting.

Table #1 100 mg/m2 Etoposide i.v. 200 mg/m2 Etoposide p.o. C max [μg/ml] 16.7 (10.1 – 21.1) 13.4 (5.8 – 16.8) V ss [L] 14.4 (10.7 – 18.3) 15.1 (10.6 – 25.4) CL total [ml/min] 45.9 (29.6 – 62.8) 82.5 (45.5 – 151.0) CL renal [ml/min] 17.2 (9.4 – 52.6) 16.3 (4.7 – 39.7) t 1/2 [h] 4.1 (3.8 – 5.4) 5.8 (3.2 – 8.3) AUC [μg × h/ml] 72.5 (44.3 – 93.4) 81.6 (44.0 – 147.0)
Re-Treatment with Rituximab Plus Chemotherapy in Patients with Aggressive Lymphoma Treated Previously with CHOP or CHOP-Like Combinations Plus Rituximab.
Authors: Palacios Andres , Navarrete Mayda , Gallur Laura , Zuazu Javier , Barrenetxea Cristina , Sanchez Eva , Lopez Andres
Published: October 2019

Introduction. CHOP plus Rituximab has become the standard treatment for patients with aggressive B-cell lymphoma - particularly DLBCL -, after having shown a better response rate and event free survival over CHOP. However, a number of patients are resistant or relapse (R/R) after a good response. The aim of this study is to assess the efficacy of different Rituximab-containing regimens as salvage therapy in refractory/relapsed (R/R) patients with aggressive lymphoma who had been treated with R-CHOP or R-CHOP-like regimens as first-line therapy.

Patients and Methods. Thirty-six consecutive patients (34 evaluable for response) from the the same institution were reviewed. Twenty-five patients had diffuse large B-cell lymphoma, 4 mantle-cell lymphoma, and 7 Grade 3b follicular lymphoma. Patients received R-CHOP (n=21), DA-R-EPOCH (n=12) orR-Hyper-CVAD (n=3) as first-line therapy. The following salvage regimens were used in this study: R-ESHAP (n=1), R-ICE (n=10), R-TT (n=4), R-GemOx (n=13), Rituximab monotherapy (n=7), and R-MC (n=1). The first three of these regimens were considered ‘aggressive’ (AG) and the three remaining regimens were considered ‘less aggressive’ (LAG).

Results. Median patient age was 59.6 years (range: 31–84 years). The overall response rate (ORR) was 53% (38% complete response [CR]; 15% partial response {PR]. CR directly correlated with IPI score at relapse or therapy failure. ORR was similar in both types of regimen, however the response quality (determined as % CR) was 45% with the LAG regimen and 22% with the AG regimen.With a median follow-up of 29 months the overall survival (OS) was 53% and the event free survival (EFS) was 22%. EFS correlated inversely with IPI at relapse or therapy failure (P=0.015) and directly with response to salvage therapy (P=0.0002). in addition, EFS was slightly higher in patients treated with LAG regimen (42% vs 20%); however this was not statistically significant.

Conclusion. Some authors have hypothesised that ORR appears to decrease when patients who had previously received Rituximab + chemotherapy for first-line therapy were treated with Rituximab + chemotherapy as salvage therapy at relapse. However, our data indicate that responses to Rituximab + chemotherapy continue to occur in these patients. In addition, ‘aggressive’ salvage regimens do not achieve better responses. Large, prospective studies are requires in order to verify the findings of this study.
Abnormal Polarization of T Lymphocyte in Myelodysplastic Syndrome Marrow and Its Negtative Effect on Hematopoiesis.
Authors: Wu Lingyun , Li Xiao , Chang Chunkang , Ying Shaoxu , Pu Quan
Published: October 2019

Objective: To explore polarization of T lymphocyte and its relationship with apoptosis of marrow cells in patients with myelodysplastic syndromes (MDS).

Methods: Measurement of Th1, Th2, Tc1, Tc2 subsets of marrows from 35 patients with MDS and 13 normal controls was performed by flow cytometry. INF-γ and TNF-α in the bone marrow serum were tested by ELISA (Enzyme-linked immunosorbent assay). Furthermore, apoptosis index of marrow cells was detected by TUNEL (TdT-mediated dUTP nick end labeling). Correlation among Th1, Th2, Tc1, Tc2 subsets and INF-γ, TNF-α level as well as apoptosis index were analyzed, and relationship between TNF-α, INF-γ level and apoptosis index was also investigated.

Results:

The percentage of Th1 cells (10.1±1.5%), Tc1 cells (24.2±3.5%) and Tc1/Tc2 ratio (49.7±10.8) was significantly increased in patients with MDS when compared to normal controls (4.0±0.5%, 5.8±0.6% and 13.4±2.7, respectively). Level of INF-γ (57.4±21.9pg/ml) and TNF-α (14.6±3.7pg/ml) in bone marrow serum of patients with MDS was markedly elevated compared to normal controls (0 and 0.3±0.2pg/ml, respectively). An increased apoptosis index of nucleated cells was also observed in patients with MDS (7.2±1.5%) when compared to controls (2.1±0.3%, P<0.05). The Th1 cells percentage showed a positive correlation with level of INF-γ and TNF-α (r=0.38, P<0.05 and r=0.39, P<0.05, respectively), and also a positive correlation with apoptotic index of nucleated marrow cells in patients with MDS (r=0.33, P<0.05). Furthermore, a positive correlation was observed when the relationship between INF-γ, TNF-α level and apoptotic index of marrow cells were analyzed (r=0.74, P<0.01 and r=0.73, P<0.01, respectively). Th1, Tc1 cells and Tc1/Tc2 ratio in patients with RCMD (according to WHO classification) was markedly elevated (P<0.01) while not significantly increased in patients with RCMD-RS, RAEB-1 and RAEB-2 when compared to normal controls. An elevation in the percentage of Th1 (11.1±1.9%), Tc1 (25.1±3.8%) and Tc1/Tc2 ratio (58.6±15.8%) was detected in patients with lower-risk (defined by IPSS), while there was no difference between higher-risk group and controls.

Conclusions: Th1/Th2, Tc1/Tc2 in bone marrow of patients with MDS were unbalanced, which were polarized to typeIreaction especially in patients with RCMD subtype and lower-risk group. The increased Th1 cells in marrow may account for apoptosis of nucleated marrow cells in MDS, which may act through proapoptotic cytokines such as INF-γ and TNF-α.
Comparative Pharmacokinetic/Pharmacodynamic Profiles in Japanese and Caucasian Patients with Transfusional Hemosiderosis Receiving Treatment with Deferasirox (Exjade®, ICL670).
Authors: Iki S. , Urabe A. , Hata T. , Ohyashiki K. , Nakao S. , Takatoku M. , Ishikawa T. , Kato J. , Tatsumi Y. , Mori H. , Tanii H. , Taniguchi J. , Kondo M. , Schran H. , Rabault B.
Published: October 2019

Introduction: In Japan, myelodysplastic syndromes (MDS) and aplastic anemia (AA) are the most common transfusion-dependent anemias. Deferasirox (Exjade®, ICL670) is a once-daily, oral iron chelator for the treatment of transfusional iron overload; its efficacy and tolerability have been established in adults and children with a range of transfusion-dependent anemias, including MDS. Here, deferasirox has been evaluated in Japanese patients and the pharmacokinetics (PK) and pharmacodynamics (PD) compared with those seen in a US study.

Methods: The tolerability, PK and PD of deferasirox have been assessed in a Phase I, open-label, unblinded, dose-escalation trial (1101) in Japanese patients with MDS and AA in 9 centers. Deferasirox was administered as a single dose of 5 (n=6), 10 (n=7), 20 (n=6) or 30 mg/kg (n=7). After a 7-day break, daily doses were administered (same doses/same patients) for 7 consecutive days. Data were compared with a study (0104) in 24 Caucasian β-thalassemia patients (age range 18–39 years) who were administered doses of deferasirox of 10, 20 or 40 mg/kg/day for 12 days. Throughout the study, iron intake and excretion were rigorously measured.

Results: A total of 26 Japanese patients (8 male, 18 female; mean age 65.6 years; 8 aged <65 years) completed single- and multiple-dosing periods (16 MDS; 6 AA; 4 other anemias). After a single dose and also at steady state, linear PK (Cmax and AUC) were observed at all doses. The figures show Cmax and AUC at steady state for both the Japanese patients (closed circles, solid lines) and the Caucasian patients (open squares, dotted lines), and demonstrate that PK were similar.

Figure Figure

Dose-dependent iron excretion (range 0.07–0.61 mg iron/kg/day) was observed, similar to that in the Caucasian patients (range 0.12–0.45 mg iron/kg/day). A linear relationship (PK/PD) was noted between AUC and iron excretion. Deferasirox was well tolerated, with generally infrequent and mild adverse events (AEs). The most common AEs related to deferasirox were diarrhea (n=2 each after single and multiple doses of 30 mg/kg/day) and nausea (n=1 each after multiple doses at 10 and 30 mg/kg/day). One patient had two serious AEs, pyrexia and duodenal ulcer, both suspected to be related to study drug, after multiple doses of 30 mg/kg/day.

Conclusions: Deferasirox is well tolerated both after single and multiple doses in Japanese MDS/AA patients, with similar PK/PD parameters to those in the Caucasian β-thalassemia cohort. Exposure to deferasirox in the Japanese patient cohort was dose dependent with a linear PK/PD relationship resulting in dose-dependent iron excretion. These data suggest that deferasirox is effective and well tolerated regardless of underlying disease, race or age.
Retrospective Analysis of Causes and Consequence of Delay in Diagnosis of Multiple Myeloma.
Authors: Kariyawasan Chitranga C. , Hughes Derralynn , Mehta Atul B.
Published: October 2019

We have conducted a retrospective study of 82 patients (49 - male, 33 - female, median age 67 years (Male), 63 years (Female) ) of whom 72 were de novo and 10 had a previous diagnosis of MGUS/Smouldering myeloma/Plasmacytoma. The duration of symptoms prior to diagnosis was used to categorise patients into 4 groups [A - 0 - 3months, B - 3 -6 months, C - 6 - 12 months, D > 12 months]. The incidence of complications of myeloma (infection, renal failure, bone disease, neurological disease and anaemia) were assessed in each group. 25 of 72 patients (35%) were in group A, 47 (65%) were in group B,C and D and 30 of these (64%) had presented initially to a general practitioner. The commonest presenting symptom was bone pain (60%) and skeletal disease was the commonest complication seeing in 43% of group A but in 57% of groups B,C and D. All 18 patients in group D had > 1 complication (median 2, range 1–4) while 9 of 25 patients (35%) in group A had no complication. Four of 25 (16%) patients in group A were in remission or first phase of treatment and 7 of 25 (28%) had survived 5 years or more. In contrast, 9 of 18 (50%) patients in group D had relapsed disease and only 2 had survived 5 years or more. Our findings indicate that a delay in diagnosing multiple myeloma has a significant impact on the course of the disease.