bluuebunny/crossref_metadata_2025 huggingface dataset:
Clonal Gammopathies, Immune Cytopenias and Autoimmune Thyroid Dysfunction Following Alemtuzumab Containing Reduced-Intensity Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Akhtari Mojtaba , Raj Kavita , Ingram Wendy , Milojkovic Dragana , El-Hemaidi Ihab , Devereux Stephen , Pagliuca Antonio , Ho Aloysius , Mufti Ghulam
Published: October 2019

Reduced-intensity or “nonmyeloablative” conditioning (RIC) regimens, which include Alemtuzumab, results in stable donor hematopoietic and lymphoid engraftment with minimal graft versus host disease. At our institution 182 patients with myeloid and lymphoid malignancies have undergone RIC transplantation between March 1998 and March 2004. In order to ascertain whether the inclusion of Alemtuzumab affects post transplant immune reconstitution, we have assessed the incidence of clonal gammopathies and organ specific autoantibodies in all patients in whom information was available. We used 44 patients who had conventional nommyeloablative HSCT as our control group. In the RIC HSCT group 40(22%) developed clonal gammopathies. The median age at RIC HSCT was 55 years (range 24–66); the underlying dignosis was MDS(n=15), AML(n=15), NHL(n=8) and CML(n=2). A gammopathy was detected at a median of 19 weeks post RIC HSCT (range 8–44). Bone marrow aspiration and biopsy did not show increased numbers of plasma cells. Chimerism studies (unfractionated bone marrow and CD3+, CD15+, CD19+ and CD56+ peripheral blood fractions) showed that 18(45%) were fully donor, 18(45%) had mixed chimerism and it was not available for 4(10 %). By contrast clonal gammopathies were detected in only 4 of 44 patients who had conventional nonmyeloablative conditioning. There was a significant difference between two groups (P=0.05). 9 of 182 (5%) patients (AML=6, MDS=1, MCL=1) developed immune cytopenias at a median of 149 days(range 24–238 days). Those comprised of ITP(n=4), AIHA(n=3), Evans’ syndrome(n=2), red cell and megakaryocytic aplasia(n=1). Chimerism studies (unfractionated bone marrow and CD3+, CD15+, CD19+ and CD56+ peripheral blood fractions) showed that 6(66%) patients were fully donor, 1(11%) was mixed chimeric and for 2(23%) patients chimerism studies were not available. One patient did not need any treatment; one patient with AIHA had a complete response to prednisolone. 2 patients (1 ITP and 1 Evans’ syndrome) responded to IVIG. The five non-responders to IVIG (2 AIHA, 2 ITP, and 1 red cell aplasia and megakaryocytic aplasia) resonded to treatment with Rituximab 375mg/m2 weekly for 4 weeks with a maximal response at a mean of 30 days from commencing the treatment. 3 of 182(1.6%) patients developed autoimmune thyroid dysfunction. All patients had normal thyroid function tests and no antibodies to thyrotropin receptor before RIC HSCT. None of the patients, who had clonal gammopathy, immune cytopenia and autoimmune thyroid dysfunction, developed PTLD. We hypothesize that the RIC regimens incorporating monoclonal antibody Alemtuzumab causes clonal gammopathies, immune cytopenias and autoimmune thyroid dysfunction due to dysregulated B cell proliferation secondary to prolonged T cell recovery time.
Effect of Wine Phenolics and Their Analogs on Suppression of Tissue Factor Induction in Mononuclear Cells.
Authors: Kaur Gurjeet , Pendurthi Usha
Published: October 2019

Several epidemiologic studies suggest that moderate consumption of alcohol, particularly red wine, lowers mortality rates from coronary heart diseases. Aberrant expression of tissue factor (TF), a cellular receptor of coagulation factor VIIa, is thought to contribute to the pathogenesis of coronary heart diseases (CHD) and acute coronary events associated with atherosclerosis. Therefore we hypothesized earlier that consumption of red wine provides protection against CHD by suppressing the aberrant expression of TF in vascular cells, and showed two red wine phenolics, resveratrol and quercetin, suppressed TF induction in endothelial cells and mononuclear cells. However these studies revealed that relatively high concentrations of these compounds (10 to 100 μM), which were unlikely to be achieved in vivo after moderate consumption of red wine, were needed to suppress TF induction. The present study was undertaken to investigate whether other red wine phenolics potentiate the inhibitory effects of resveratrol or quercetin and evaluate the effect of various resveratrol analogs, which were shown to be 10 to 1000 times more effective than resveratrol in modulating other cellular effects, on TF induction in mononuclear cells. Mononuclear cells isolated from healthy human blood donors were treated with various wine phenolics alone or in a combination for 1 h and the cells were stimulated with LPS (10 ng/ml for 6 h) to induce TF expression. TF activity in cell lysates was measured as its ability to support factor VIIa catalyzed activation of factor X. Studies of treating mononuclear cells with varying concentrations of resveratrol and quercetin alone or in a combination revealed that their inhibitory effects were additive and not synergistic. Treatment of cells with a cocktail of red wine phenolics (in concentrations that present in red wine) completely suppressed LPS-induced TF activity. Exclusion resveratrol, epicatechin, catechin or rutin from the cocktail did not diminish the inhibitory effect. In contrast, exclusion of quercetin from the cocktail fully attenuated the inhibitory effect of the cocktail. Further experiments showed that quercetin (55 μM) alone fully suppressed TF induction. Among various resveratrol analogs tested, the cis-form methylated derivative of resveratrol (3,5,4′-trimethoxystilbene, TMS) was 10-times more effective than resveratrol in inhibiting TF induction. A 2 μM of TMS inhibited the induction of TF by more than 75%. None of the wine phenolics or resveratrol analogs had a significant effect on the cell viability. Overall these data suggest that quercetin is the principal inhibitor of TF induction among red wine phenolics and no synergism exists among various wine phenolics in inhibiting TF induction. Comparison of our data on the effect of resveratrol analogs on TF induction with previously published studies on other cellular functions suggests that resveratrol analogs exhibit varying potencies in modulating different cellular activities.
ATP-Induced Modulation of Norepinephrine Exocytosis in Human and Porcine Heart: Role of E-NTPDase1/CD39.
Authors: Machida Takuji , Heerdt Paul M. , Reid Alicia C. , Schaefer Ulrich , Silver Randi B. , Broekman Marinus Johan , Marcus Aaron J. , Levi Roberto
Published: October 2019

ATP co-released with norepinephrine (NE) from cardiac sympathetic nerve terminals increases NE exocytosis via a positive feedback mechanism. Since excessive NE release is an established cause of dysfunction in ischemic heart disease, we studied the role of endogenous and exogenous nucleotidases as part of the development of a novel therapeutic approach to myocardial ischemia and its consequences. Using sympathetic nerve terminals isolated from guinea pig heart (cardiac synaptosomes), we previously established that activation of ATP-gated ionotropic presynaptic P2X purinoceptors (P2XR) promotes exocytosis of NE. This effect was increased by inhibition of the endogenous ecto-nucleotidase (E-NTPDase1/CD39) and decreased by solCD39, a recombinant form of human E-NTPDasel/CD39. Furthermore, depolarization of synaptosomes with K+ also evoked NE exocytosis. This was potentiated by inhibition of E-NTPDase1 with ARL67156 (a selective E-NTPDase inhibitor), and attenuated by addition of solCD39. These findings indicated that ATP released by depolarization of sympathetic terminals enhances NE exocytosis, and that E-NTPDase1 plays an important modulatory role in adrenergic neurotransmission in the heart. In the present study, we investigated the role of E-NTPDasel in exocytosis of NE and ATP in porcine as well as human heart. Cardiac E-NTPDase1 is selectively localized in human and porcine cardiac neurons, and depolarization of cardiac tissue elicits an ω-conotoxin-inhibitable release of both NE and ATP. Inhibition of E-NTPDase1 with ARL67156 markedly potentiated release of ATP, demonstrating that E-NTPDase1 is a major control system for ATP availability at sympathetic nerve endings. Importantly, when ATP exocytosis was increased via inhibition of E-NTPDase1, NE exocytosis increased in parallel. Moreover, when ATP release was reduced by administration of solCD39, NE exocytosis was markedly diminished. Thus, ATP regulates NE exocytosis by activating presynaptic P2XR. This concept is further validated by our finding that the strong correlation between ATP and NE release was abolished by the P2XR antagonist PPADS. We conclude that released ATP governs NE exocytosis by activating presynaptic P2XR and that this is controlled by E-NTPDase1/CD39. Clinically, excessive NE release is a major cause of arrhythmic and coronary vascular dysfunction during myocardial ischemia. By curtailing NE release, solCD39 may offer a novel therapeutic approach to ischemic complications in the myocardium in addition to its role as an anti-thrombotic agent.
A Pilot Study Comparing Megestrol Acetate Concentrated Suspension (MA-CS) to Megestrol Acetate Oral Suspension (MA-OS) on Weight Gain and Body Composition in Patients with HIV-Associated Unintended Weight Loss (UWL).
Authors: Cilla Donald D. , Gutierrez Jodi L. , Femia Robert A. , Kramer Lynn D. , Wanke Christine
Published: October 2019

BACKGROUND: MA-CS is a new NanoCrystal® technology formulation of megestrol acetate that is bioavailable in the fasted state, which may provide benefit in the treatment of UWL in HIV patients.

METHODS: 63 patients with HIV-associated UWL (weight loss to 90% of the lower limit of ideal body weight) were recruited from South Africa, India and the US and then randomized to receive MA-CS (575 mg/5 mL) or MA-OS (800 mg/20 mL) once-daily for 12 weeks in a randomized, open-labeled, multi-center, pilot proof-of-principle study (sample size determined empirically). Patients had weight, body composition (bioimpedance analysis - BIA), and anthropometric measurements obtained at baseline and weekly thereafter, except BIA which was measured at 6 and 12 weeks. Weight changes were compared using the Wilcoxon Rank Sum test.

RESULTS: The demographics were comparable between the two groups. The mean weight change from baseline to Week 12 for MA-CS was 5.4 kg (55.6 kg to 61 kg, 10% of baseline body weight). The mean weight change from baseline to Week 12 for MA-OS was 3.5 kg (54.4 kg to 57.9 kg, 6% of baseline body weight). Differences from baseline, in the mean changes in weight, were observed as early as Day 3 (p=0.024) for MA-CS, however no increase was noted until the second week for MA-OS. Similar between group differences were noted consistently at the weekly assessment intervals until week 12 (p= 0.024). Of these weight changes, lean body mass accounted for ~40% of the increase in both treatment groups (MA-CS was ~5% greater than MA-OS). Of the anthropometric measures, the mean triceps skin fold increased by ~36% at Week 12, relative to baseline, in both MA-CS and MA-OS treatment groups. Other anthropometric changes were all smaller (less than 7%) and comparable between treatments. The types and incidence rates of adverse events were similar between MA-CS and MA-OS.

CONCLUSIONS: Both products were successful at increasing body weight. There was a greater and more rapid mean change from baseline in the weight gain for MA-CS than MA-OS. The observed weight changes reflected increases in both lean body mass and body fat. MA-CS had a greater change in lean body mass. This trial supports the principle that improved bioavailability of megestrol acetate in the fasted state with MA-CS may be associated with faster time to onset of changes in body weight.
Molecular Karyotyping by High Resolution Arrray CGH Uncovers New Deleted and Amplified Genomic Regions That Contain Genes of Biological Interest in Multiple Myeloma Cells.
Authors: Largo Cristina , Alvarez Sara , Blesa David , Prosper Felipe , Calasanz M. Jose , Cigudosa Juan
Published: October 2019

Multiple Myeloma (MM) is a malignancy characterized by clonal expansion of plasma cells. In 50% of the cases, the neoplastic transformation begins with a chromosomal translocation that juxtaposes the IGH gene locus to an oncogene. After defining the IGH-translocations present in a panel of MM cell lines, we conducted expression-profiling analysis. Supervised analysis identified 166 genes significantly differentially expressed among the cell lines harboring MMSET/FGFR3 (4p16), MAF (16q) and CCND1 (11q13) rearrangements. Besides translocations, gene copy number changes are also frequent in MM but far less characterized than in other neoplasias. We conducted array Comparative Genomic Hybridization (arrayCGH) with the same cDNA platform that was used for the expression analysis with the double purpose of characterizing the amplified genome regions and identifying the amplified and overexpressed (A/O) genes within these regions. We focused in five chromosomes recurrently affected by gains/amplifications in primary samples and cell lines where we found 60 A/O genes. Among them, twenty-five (42%) were only overexpressed when amplified, and six, CHI3L1, ELMO1, BNIP3L, PLAG1, LOC157567, and VPS28, showed a significant association between overexpression and gain/amplification.

In a second step, we are conducting a high resolution analysis of copy number changes in flow sorted CD138 myeloma cells from patients with normal karyotype, as stated by conventional cytogenetics. We are using the Human Genome CGH Microarray 44A platform from Agilent Technologies (Palo Alto, CA), which contains 44.000 60-mer oligonucleotides covering the human genome with an average resolution of 40 Kb. As expected, this genomic approach, performed on selected cells, allowed the identification of a high number of deletions and gains. We have found small regions (below 500 Kb of size) that were rearranged as follows:

Copy number changes on selected CD138+ MM cells Chromosome Rearrangement Some genes of biological interest loss 1p12-p21.3 STXBP3, SORT1, HRMT1L6, PSMAS loss 6q24.1–q24.2 STXBP5, IL20RA, MYB loss 11q14.1–q22.3 MMP1, 3, 7, 8, 10, 12, 13, 20, BIRC2 & 3 loss 14q12–q21.3 FOXA, FBXO33, SNX6 loss 16p12.1 TP53TG3, ZNF267, CREBPB gain 1q21 CKS1B, AIM2, SELL, CHI3L1 gain 8q24.12–q24.23 C-MYC, PTK2
Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis: A Report of the European Blood and Marrow Transplantation Group (EBMT).
Authors: Saccardi Riccardo , Mancardi Gianluigi , Tyndall Alan
Published: October 2019

Haematopoietic Stem Cells Transplantation (HSCT) has been successfully used in the last decade for the treatment of severe Autoimmune Diseases (ADs), including Multiple Sclerosis (MS). A retrospective analysis of 183 Multiple Sclerosis patients recorded in the database of the EBMT is reported. The patients received an autologous HSCT in the period 1995–2004. The disability at the time of HSCT was severe, with a median EDSS of 6.5 (range 3.5–9). Cumulative survival is 86% at 9 years with median follow-up time of 3.5 years. Overall Transplant-Related Mortality (TRM) is 5.3%; no toxic deaths were reported in the 62 patients transplanted after the year 2000. Conditioning regimens including Busulphan were statistically associated to TRM, both in uni- and in multivariate analysis. No toxic deaths were reported among the 53 patients treated with the BEAM/ATG regimen without graft manipulation, irrespective of their clinical condition at the time of the transplant. Sixty-three percent of the 145 patients evaluable for the neurological outcome showed a sustained improvement or stabilization of their disability. A trend of a better clinical outcome in patients transplanted within 5 years of diagnosis was shown. HSCT was shown as an effective procedure to stop disease progression in patients affected by severe, progressive MS; safety and feasibility of the procedure can be significantly improved by appropriate patient selection and choice of transplant regimen.
SCIO-469, a Potent and Selective Inhibitor of the p38a MAPK, Inhibits TNFa-Induced Adhesion of Multiple Myeloma Cells to Bone Marrow Stromal Cells Via Downregulation of Chemokines CXCL10/IP-10 and CCL8.
Authors: Nguyen Aaron N. , Young Gilbert O’ , Quon Diana , Damm Debby , Kapoun Ann M. , Higgins Linda S. , Navas Tony A.
Published: October 2019

Multiple myeloma (MM) is a plasma cell cancer characterized by the accumulation and clonal expansion of malignant cells in the bone marrow (BM). Recent findings indicate that adhesion of MM cells to BM stromal cells (BMSCs) protects MM cells from drug-induced apoptosis and leads to upregulation of interleukin-6 (IL-6), a cytokine that promotes MM cell growth and survival. However, the molecular mechanism that determines the adhesion of MM cells to BMSCs is relatively unclear. Here we show that SCIO-469, a potent and selective inhibitor of the p38a mitogen-activated protein kinase (MAPK), prevents TNFa-induced adhesion of MM cells to BMSCs. Interestingly, TNFa-induced expression of ICAM-1 and VCAM-1, molecules that have been reported to mediate cell-cell adhesion, is not affected by SCIO-469 treatment. In an effort to identify additional factors that may play a role in the adhesion of MM cells to BMSCs, we performed a DNA microarray experiment on BMSCs. Of the BMSC genes that were strongly upregulated by TNFa exposure and reversed by SCIO-469 treatment, a set of chemokines was most prominent. To determine whether these chemokines are important for MM adhesion to BMSCs, various chemokines (CCL2, CCL7, CCL8, CXCL1, CXCL3, CXCL6, CXCL10/IP-10, and CXCL11) were added together with SCIO-469 in the adhesion assays. We found that reintroduction of the chemokines CXCL10 and CCL8 reversed the inhibition of adhesion by SCIO-469. These results suggest that SCIO-469 inhibits TNFa-induced adhesion of MM cells to BMSCs by downregulating the chemokines CXCL10 and CCL8. Intriguingly, these two chemokines were also recently demonstrated to be involved in leukocyte adhesion to endothelial cells. Thus, chemokines, especially CXCL10 and CCL8, appear to have a general function of localizing blood cells to various sites within the body. Together, our findings demonstrate another potential therapeutic role for SCIO-469 in MM in addition to its role of inhibiting the production of growth factors such as IL-6 and VEGF in the MM bone marrow microenvironment.
XBP-1 a Selective and Specific Target for Immunotherapy in Myeloma.
Authors: Bae Jooeun , Carrasco Ruben , Sukhdeo Kumar , Tassone Pierfrancesco , Neri Paola , Yu-Tzu Tai , Li Xianfeng , Coffey Rory , Prabhala Rao , Anderson Kenneth , Munshi Nikhil
Published: October 2019

X-box binding protein 1 (XBP-1) is a transcription factor is essential for the differentiation of plasma cells and the unfolded protein response. XBP-1 is significantly up-regulated in myeloma cells compared to both normal plasma cells and B-cells. Selective and specific requirement of XBP-1 for differentiation of B cells to plasma cells and its further up-regulation in multiple myeloma (MM) cells makes it a promising target for immunotherapy directed at MM. We have evaluated XBP-1 as a target antigen to develop MM-specific immunotherapy. In order to generate XBP-1 antigen-specific cytotoxic T lymphocytes (CTLs), we have identified HLA-A2-specific peptides derived from non-spliced (short form) and spliced (long form) XBP-1 proteins. We have further modified the peptide by altering one amino acid, which is critical for HLA-A2 affinity, to obtain epitope, which has higher stability to HLA-A2 clefts. The modified XBP-1 peptides were able to evoke higher levels of IFN-g release compared to native peptides and were able to induce CTLs highly specific to MM, demonstrated by Calcein-released cytotoxicity assay. Cytotoxic activity of these XBP1 peptide-specific-CTLs against U266, a HLA-A2+/XBP-1+ MM cell line was 60% and 79% by CTLs stimulated by non-spliced peptide and 69% and 88% by CTLs stimulated by spliced peptide at Effector:Target ratios of 20:1 and 60:1, respectively. The CTLs did not lyse HLA-A2− or XBP-1− cells. These results were further confirmed by the novel CD107 cytotoxicity assay, which detects MM-specific cytolytic CD8+ T cells by flow-cytometric analyses. The CTLs generated with XBP-1 peptide displayed distinct phenotypes, showing high CD69+CD45RO+ cell population (62% in non-spliced peptide-CTLs, 67% in spliced peptide-CTLs, 4% in unstimulated control-CTLs) and CD45RA+CCR7+ cell population (1% in non-spliced peptide-CTLs, 1% in spliced peptide-CTLs, 24% in unstimulated control-CTLs). In conclusion, we report the identification of highly immunogenic heteroclitic XBP-1 epitopes that have ability to generate MM-specific CTLs. Confirmation of in vivo activity of these CTLs in SCID mouse model is currently underway prior to its evaluation in clinical studies.
Autocrine Signaling by Osteopontin Regulates Actin Remodeling in Human Primary Erythroid Progenitors.
Authors: Wickrema Amittha , Zhou Ying , Reynolds Jessica , Ulaszek Jodie , Satgurunathan Nilesh , van Besien Koen , Kang Jeong-Ah
Published: October 2019

Osteopontin (OPN) is a secreted protein shown to have multiple functions depending on cell type and context. The cell types known to express OPN are typically assocoated with immune and /or bone remodeling functions. Here we demonstrate that OPN is also expressed and secreted by primary human erythroid progenitors and that it is involved in actin remodeling in erythroid cells. We initially identified OPN as an important cytokine in erythroid differentiation by screening a cytokine/receptor cDNA microarray. Subsequently using a culture system of highly purified primary erythroid progenitors (sorted for glycophorin A) we examined OPN mRNA and protein expression during terminal erythroid differentiation. We demonstrated that OPN mRNA is expressed throughout differentiation and that OPN protein is also detectable at basophilic, polychromatic and orthochromatic stages during the maturation program. Furthermore by ELISA we were able to detect secretion of OPN at each of the major stages of differentiation. In addition we show that CD44 and integrin alpha 4, two of the receptors for OPN are highly expressed in these cells. Stimulation of these cells with rOPN resulted in phosphorylation of several proteins one of which was identified as the heat shock protein-27 (Hsp27). In subsequent studies we were able to demonstrate that OPN regulates phosphorylation of Hsp27 at serine-78 site through the p38 MAP kinase pathway. We then effectively knocked down the expression of OPN by siRNA methodology, which resulted in formation of distinct F-actin bundles in the perimembrane region of the cell. By contrast in cells transfected with control siRNA maintained F-actin in a punctate appearance. Altogether our studies suggest a critical signaling function for OPN in regulation of atleast one of the erythrocyte cytoskeletal elements during erythroid differentiation.
Mapping of the Glycolytic Enzyme Binding Sites on Human Erythrocyte Membrane Band 3.
Authors: Chu Haiyan , Low Philip S.
Published: October 2019

Glycolytic enzymes (GEs) including aldolase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH) are known to associate with the inner surface of the human erythrocyte membrane. Previous studies have suggested that the N-terminus of the cytoplasmic domain of band 3 (cdb3) constitutes the likely binding site for aldolase, GAPDH and PFK, however, the membrane docking sites for PK and LDH have not been elucidated. In this study, we demonstrate that PK and LDH exhibit no affinity for band 3, regardless of whether the association is measured by co-immunoprecipitation assay, binding competition studies, or catalytic inhibition analyses. We further find that the binding sites for GAPDH, aldolase and PFK on band 3 are distinct but partially overlapping, as evidenced by the fact that: 1) deletion of residues 1–11 of cdb3 eliminates the binding of aldolase, but not PFK or GAPDH, 2) fusion of thioredoxin (Trx) to the N-terminus of residues cdb3 blocks aldolase binding, but not the association of GAPDH or PFK, 3) deletion of sequences 1–50, 1–40, 1–31, or 1–23 of cdb3 blocks cdb3 association with all three GEs, whereas deletion of residues 12-23 only abrogates aldolase binding (while reducing the affinity of PFK and GAPDH), 4) the presence of both sequences, 6–DDYED-10 and 19-EEYED-23, are necessary for cdb3 association with aldolase, whereas the presence of either sequence alone (especially 19-EEYED-23) is sufficient to maintain association with GAPDH and PFK, 5) mutation of all of the acidic residues in the above two sequences to their corresponding amides (E→Q and D→N) results in loss of affinity for all GEs. Because i) kidney cdb3 (which lacks residues 1-65 of cdb3) shows no affinity for any of the GEs, ii) residues 1–55 of cdb3 show near normal affinity for aldolase, GAPDH and PFK, and iii) GAPDH, aldolase, and PFK all compete with each other for cdb3, we conclude that the binding sites for aldolase, GAPDH and PFK are all located within the first 23 residues of cdb3, with the docking site for aldolase likely residing somewhat more N-terminal than the binding sites for GAPDH and PFK. Finally, because each band 3 monomer contains three homologous sequences (6-DDYED-10, 19-EEYED-23 and 902-DEYDE-906) that are found in other proteins that bind GEs (e.g. actin, β-tubulin, troponin T), and since the first two of these sequences were shown to be required for cdb3 binding, the question naturally arose whether different GEs might also associate with residues 902-DEYDE-906 at the extreme C-terminus of band 3. Similar binding studies demonstrate that such an interaction does not occur. Thus, our data show that GAPDH, aldolase, and PFK all bind near the N-terminus of band 3 and that PK and LDH must dock somewhere else on the red cell membrane.
Myocardial T2* Is Not Affected by the Structural Effects of Myocardial Fibrosis, Ageing or Impaired Left Ventricular Function.
Authors: Kirk Paul , Pennell Dudley J.
Published: October 2019

Background

The myocardial T2* technique has been validated as a reproducible non-invasive measurement of myocardial iron load and is now widely used for measurement of myocardial iron in iron overload diseases such as thalassaemia. The reduction in myocardial T2* seen in iron overload conditions is substantially greater than is seen in any other clinical circumstance, but there has been no direct comparison of myocardial T2* in normals and other conditions such as increasing age, myocardial infarction or impairment in left ventricular function. We aimed therefore to compare the findings in patients affected by these conditions with normals.

Method

A total of 38 patients in total were scanned using the myocardial T2* technique. Fifteen patients had normal hearts, 18 had impaired LV function and 6 had chronic myocardial infarction affecting the anteroseptal wall, where myocardial T2* measurements are normally made.

Results

The mean myocardial T2* in normals was 36.0 +/− 6.4 ms, yielding a lower limit of normal of 23ms. In patients with impaired LV function, the mean myocardial T2* was 39.0 +/− 11.7ms (p= 0.37 vs normals). In patients with anteroseptal myocardial infarction, the mean myocardial T2* was 34.7ms +/− 3.9ms (p= 0.64 vs normals). The frequency distribution of the myocardial T2* values are shown in figure 1. These approximate to normal, and are very similar in distribution. In addition, the age distribution of myocardial T2* in the 15 normals is shown in figure 2. There was no significant relation between myocardial T2* and age (r2 = 0.066, p=0.82).

Conclusion

There is no significant reduction in myocardial T2* associated with fibrosis from chronic myocardial infarction, impairment of left ventricular function, or increasing age. This suggests that structural changes associated with remodelling, infarction and fibrosis, and ageing do not have significant effects on the absolute measure of myocardial T2*, and in particular do not cause a reduction below 20ms as is seen in myocardial overload conditions. Thus these date suggest that myocardial T2* is robust to these structural alterations, and that myocardial iron overload can be ascertained from reduced myocardial T2* values, in a similar manner to that which can be achieved in normals.

Figure 1 Figure 1. Figure 2 Figure 2.
Assessment of Serum Free Light Chain Assay for Screening for Plasma Cell Disorders.
Authors: Smith David E. , Abadie Jude , Bankson Daniel , Mead Graham
Published: October 2019

Introduction and Methods: The purpose of this study was to evaluate the serum free light chain (FLC) assay in its ability to improve performance of protocols designed to screen for plasma cell disorders. We measured M-protein levels using serum protein electrophoresis (SPEP) in 312 consecutive patients being screened for plasma cell disorders at the Veterans Administration Medical Center - Puget Sound. The serum kappa and lambda free light chain levels were quantitated using the serum FLC assay in these same patients. The kappa/lambda ratio was calculated using the free kappa and free lambda results from the serum FLC assay.

Results: SPEP results indicated the presence of a possible monoclonal gammopathy in 77 of the 312 patients in this study. In this group of 77 patients, a plasma cell disorder was diagnosed in 27 of them. The serum FLC assay showed an abnormal kappa/lambda ratio in 20 of these 77 patients, all 20 of whom were diagnosed with multiple myeloma. In the group of 235 patients with normal SPEP results, 17 were found to have an abnormal kappa/lambda ratio. Of these 17 patients, 15 were diagnosed with multiple myeloma, one with lymphoma, and one with bladder cancer.

Conclusions: Because a number of disorders and diseases can increase production of immunoglobulins, there were a significant number of false positives in the SPEP results. At the same time, there were also several false negative SPEP results. The number of both false positives and false negatives was smaller for the serum FLC assay. Further, use of SPEP and the serum FLC assay together resulted in significantly improved sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). (See Table 1.) These results indicate an important role for the serum FLC assay in screening for monoclonal gammopathies.

Table 1. Performance of SPEP, sFLC, and both assays in screening for plasma cell disorders SPEP Alone sFLC Alone Both SPEP and sFLC Sensitivity 64% 88% 100% Specificity 81% 98% 99% Positive Predictive Value 35% 88% 89% Negative Predictive Value 94% 98% 100%
Upshaw-Schulman Syndrome: A Masqueraded Thrombocytopenia during Pregnancy.
Authors: Matsuyama Tomomi , Matsumoto Masanori , Kato Seiji , Akiyama Nobu , Tomiyama Junji , Natori Kazuhiko , Kuraishi Yasunobu , Imamura Yutaka , Inoue Nobumasa , Kokame Koichi , Miyata Toshiyuki , Fujimura Yoshihiro
Published: October 2019

Upshaw-Schulman syndrome (USS) is a congenital thrombotic thrombocytopenic purpura (TTP) characterized by a deficiency of ADAMTS13 activity due to its mutations. More than half of USS patients have an episode of severe jaundice during the newborn period and require exchange blood transfusions. Beyond this period, however, the clinical manifestations of USS vary significantly from patient to patient, and do not usually include Moschowitz’s pentad, a hallmark of TTP. However, a consistent clinical sign in these patients is an occasional or chronic thrombocytopenia that is enhanced by precipitating factors such as viral infections. Therefore, USS patients are often incorrectly diagnosed with idiopathic thrombocytopenic purpura (ITP) or Evans syndrome during childhood, and the underlying ADAMTS13 gene defects are overlooked.

Here, we report on 7 female USS patients belonging to 4 different families, including 3 pairs of siblings who were uniformly diagnosed by episodes of thrombocytopenia or prevalence of TTP at 5~6 months of pregnancy. Plasma ADAMTS13 activity was measured with a highly sensitive ELISA using a monoclonal antibody directed against the VWF-A2 domain developed in our laboratory. The ADAMTS13 activity and gene mutations in these patients are described:

Family K: an elder sister K1 (activity 0.69%; gene mutation Y304C/G525D) and a younger sister K2 (activity <0.5%; gene mutation Y304C/G525D), Family L: an elder sister L1 (activity 1.3%; gene mutation 372insGT/Q1302X) and a younger sister L2 (activity 0.96%; gene mutation 372insGT/Q1302X), Family M: an elder sister M1 (activity <0.5%; gene mutation R193W/R349C) and a younger sister M2 (activity <0.5%; gene mutation R193W/R349C), and Family O: a patient (activity <0.5%;gene mutation I178T/Q929X). Figure indicates family pedigree of USS involved in this study.

Fig. Family pedigree of Upshaw-Schulman syndrome Fig. Family pedigree of Upshaw-Schulman syndrome
Interaction of beta2-Glycoprotein I with Members of the Low Density Lipoprotein Receptor Family.
Authors: van Lummel Menno , Pennings Maarten T.T. , Derksen Ronald H.W.M. , Westein Erik , Lenting Peter J. , de Groot Philip G.
Published: October 2019

The antiphospholipid syndrome (APS) is a non-inflammatory disease characterized by arterial and/or venous thrombosis and/or pregnancy morbidity in the presence of auto-antibodies that recognize beta2-glycoprotein I (beta2GPI) bound to cardiolipin. We have previously demonstrated that dimerization of beta2GPI by auto-antibodies induces platelet activation. This effect has been shown to be mediated by apoER2′. Here, we show that dimeric beta2GPI, and not plasma beta2GPI interacts with three different LDL-receptor family members; apolipoprotein E receptor 2′ (apoER2′), the low LDL-receptor related protein (LRP) and megalin. Interaction between dimeric beta2GPI and apoER2′ was best described with a one-site binding model (KD(app) 25 nM), whereas for LRP and megalin the interaction with dimeric beta2GPI was best described with a two-site binding model, representing a high- (3.1 nM) and a low-affinity site (192.1 and 241.2 nM, respectively). Binding kinetics of a dimeric beta2GPI mutant, that binds poorly to anionic phospholipids were similar to the binding kinetics of full length dimeric beta2GPI for all three LDL-receptor family members. Upon deletion of domain I or domain II of dimeric beta2GPI no changes in the binding kinetics were observed. Deletion of domain V however, significantly decreased the affinity for apoER2′, LRP and megalin. In conclusion, our data show that dimeric beta2GPI can interact with different LDL-receptor family members. This interaction is dependent on a recognition site in domain V of beta2GPI, which does not overlap with the phospholipid-binding site.
A Role of the Protein Tyrosine Kinase, Syk in Complement-Mediated Phagocytosis: A Mechanism on the Engulfment of Pathogen.
Authors: Tohyama Yumi , Shi Yuhong , Tohyama Kaoru , Yamamura Hirohei
Published: October 2019

A protein-tyrosine kinase, Syk plays a central role in Fc-gamma receptor-mediated phagocytosis in the adaptive immune system. Here we show that Syk also makes an essential role in compliment-mediated phagocytosis in innate immunity. For this purpose, HL60 cells induced to macrophage-like with vitamin D3 and TPA and C3bi-opsonized zymosan were used as the pathogen-phagocyte system. Time-lapse microscopic observation revealed that C3bi-opsonized zymosan particles were promptly attached to and sucked into the cells but non-opsonized zymosan was not, and therefore it was indicated that our phagocytosis system is mediated by the complement receptor 3 (CR3). Syk was tyrosine-phosphorylated by the treatment with C3bi-opsonized zymosan and marked accumulation of Syk was detected around the newly forming phagosomes. To further investigate the effect of Syk on complement-mediated phagocytosis, Syk-siRNA or dominant-negative Syk (DN-Syk) was introduced into HL60 cells. Quantitative analysis by flow cytometry showed that phagocytosis was severely impaired in DN-Syk or Syk-siRNA expressing HL60 cells. To determine whether the impaired capture of zymosan is due to reduced binding to CR3 or reduced engulfment after binding, the cells were treated with a quenching reagent which discriminates fluorescent zymosan particles between inside and outside the cells. In the parental HL60 cells most of the attached zymosan particles were inside the cells, while few particles were ingested in the case of DN-Syk or Syk-siRNA expressing HL60 cells. These data indicated that Syk is required for engulfment of C3bi-opsonized zymosan but not for attachment of zymosan to CR3. To clarify the molecular mechanism how Syk affects the phagosome formation and its engulfment, the behavior of F-actin was searched microscopically. At the early stage of complement-mediated phagocytosis, marked accumulation of F-actin occurred around each phagosome and was followed by rapid depolymerization in the parental HL60 cells. In contrast, phagosomes surrounded by the accumulated actin were significantly decreased in DN-Syk or Syk-siRNA expresssing cells. These results indicate that Syk promotes actin assembly around phagosomes prior to engulfment at the early stage of the phagocytosis. As for the analysis of the downstream pathway of Syk in our phagocytosis system, RhoA-signaling was investigated. Vav-RhoA pathway was activated in HL60 cells but transfer of DN-Syk or Syk-siRNA into HL60 cells reduced the activation of Vav-RhoA signaling. Therefore Syk may act as an activator of the RhoA-pathway in phagocytosis.

Our results demonstrate that Syk is required for complement-mediated phagocytosis by regulating both actin accumulation around phagosomes and RhoA-signaling harmonically and finally leads to phagosome formation and its engulfment.
SFCE (Societe Francaise des Cancers de l’Enfant) Recommendations for the Management of Tumor Lysis Syndrome (TLS): A Validation Survey.
Authors: Bertrand Yves , Auvrignon Anne , Nelken Brigitte , Mialoux Valérie , Mechinaud Francoise , Patte Catherine , Plantaz Dominique , Le Guyader Nathalie , Urbieta Marisol , Perel Yves , Baruchel9 Andre , Leverger Guy
Published: October 2019

TLS is a metabolic complication of haematological malignancies, which can translate in renal impairment. As previously described (Goldman S.C. et al,Blood2001; 972998–3003 and Pui C.H.et al., J.Clin.Oncol.2001;19,697–704), some patients are at High Risk (HR) of developing TLS. Rasburicase (R) a recombinant urate oxidase which transforms uric acid into a highly soluble compound, allantoin, more easily excreted by the kidneys, is highly effective in the prevention and treatment of TLS. SFCE has made recommendations for the management of TLS in children and developed a prospective survey in order to validate these recommendations.

Patients and Methods: HR pts were defined as: Acute Lymphoblastic (ALL) or Myeloblastic (AML) Leukaemia with initial leucocytes counts of at least 50x109/L, B cell ALL, Stage III and IV T or B Non Hodgkin Lymphoma (NHL), any leukaemia or NHL with a plasma uric acid concentration (U) of at least 300mmoles/L if ≤ 10 years old or 350mmoles/L if ≥ 10 years old, or any disease with serum creatinine (C) or lactate deshydrogenase concentration (LDH) exceeding twice the upper limit of normal (N), hyperphosphatemia (P)≥ 2 mmoles/L. These pts received hydration (3L/m2) ± alkalinization and R 0,20 mg/kg/d x 5 days. If C >1,5 N or P> 3mmoles/L or U ≥ 200mmoles/l, R administration is prolonged until normalization. Low risk (LR) pts, defined as not HR, received hydration 3L/m2 and R 0,20 mg/kg/for only one day. R could be prolonged according to the same criteria as HR pts.

Results: Between May and December 2004, 174 patients including 91 boys and 83 girls (median age 5 years) were treated according to these recommendations, in 8 centers in France. Initial diagnosis was LA in141 and NHL in 33. 143 patients (82%) were classified HR and 31 LR (18%). In the HR group, the patients received a median of 5 days of treatment with R (1–12) and in the LR group a median of 1 day (0–5). 25% of HR patients required treatment prolongation, while the LR group treatment prolongation was needed for 1 patient (3%). No complication of TLS was observed.

Conclusions: these results show that the risk classification as established by the SFCE was accurate and that the treatment guidelines was effective for the control of hyperuricemia and TLS.
IL-21 Improves Rituximab-Mediated B Cell Depletion.
Authors: Hughes Steven D. , Ponce Rafael A. , Krejsa Cecile , Frings Werner , Bartsch Stephanie , Hill Julie P. , Rogge Mark C.
Published: October 2019

IL-21 is a cytokine produced by activated CD4+ T cells that augments the growth, survival, and function of NK, T and phagocytic cells. IL-21 also plays an important role in regulating B cell proliferation and differentiation. In a previous study of cynomolgus monkeys treated with recombinant human IL-21, we demonstrated increased numbers of NK cells and phagocytic cells expressing Fcγ RI and III, enhancement of ex vivo antibody dependent cellular cytotoxicity (ADCC) activity, altered lymphocyte trafficking, and improved B cell depletion with subclinical doses of rituximab. The current study aimed to extend these findings to a clinically relevant rituximab dose regimen, and to evaluate the safety and tolerability of IL-21 administration in this setting. Three groups of cynomolgus monkeys (n=8) were treated by IV injection once weekly for four weeks with rituximab (10 mg/kg), concurrently with vehicle or IL-21 (0.3 or 1.5 mg/kg). Standard toxicology data were collected. The number and activation state of peripheral blood leukocyte subsets were determined by flow cytometry. Effects on B cells within spleen and lymph nodes were evaluated by immunohistochemistry. Co-administration of rituximab and IL-21 was well tolerated at both dose levels of IL-21 tested. The primary toxicological responses to the combination treatment were moderate anemia and thrombocytopenia, which were IL-21 dose dependent and similar in magnitude to those observed in previous IL-21 toxicology studies. In animals treated with rituximab alone, circulating B cells were depleted to a nadir 0.3–3 % of baseline on Day 11 of the study (Figure 1). In contrast, significantly lower B cell nadirs were observed following the first treatment with rituximab and IL-21 combination therapy (p< 0.01, ANOVA). In animals receiving the 1.5 mg/kg dose of IL-21, circulating B cells were depleted to a nadir 0 −1 % of baseline on Day 4, followed by partial recovery and further depletion to 0.3 – 2% of baseline on Day 11. More importantly, B cell recovery was significantly delayed in the group treated with rituximab and 1.5 mg/kg IL-21, compared to rituximab treatment alone (p< 0.05, ANOVA/PLSD). Thirty days following the fourth and final treatment, B cell numbers had recovered to only 6% of baseline, compared to 22% in the rituximab monotherapy treated group. Additionally, coadministration of IL-21 with rituximab increased circulating activated monocytes, granulocytes and NK cells compared with rituximab monotherapy. In conclusion, addition of IL-21 to a standard weekly, 4-dose regimen of IV rituximab was well-tolerated in cynomolgus monkeys and resulted in more complete, rapid and durable depletion of circulating CD20-expressing target cells. As depletion of B cells in this model serves as a surrogate for clearance of CD20-expressing malignant cells, these data support the evaluation of IL-21 and rituximab combination therapy in patients with advanced CD20 positive malignancies.

Figure Figure
Molecular Analysis of β-Thalassemia Cases in Poland.
Authors: Zdebska Ewa , Burzynska Beata , Mendek-Czajkowska Ewa , Maciag Malgorzata , Krawcewicz Angelika , Mokras Urszula , Adamowicz-Salach Anna , Koscielak Jerzy
Published: October 2019

β-thalassemia is a heterogenous, inherited disease resulting from reduced or absent synthesis of β-globin chain of haemoglobin. This disorder is very common in the Mediterranean, Middle Eastern, African and South-East Asian populations. The aim of the present investigation was to verify a common view that thalassemia in Poland is a very rare disease. 600 patients (270 men and 330 women), aged 2–85 years, with microcytosis and no evidence of iron deficiency, were examined for β-thalassemia. Hemoglobin A2 was increased in 106 patients. In 48 patients there was also an elevation of hemoglobulin F and in 42 patients of serum bilirubin.

All 106 patients were examined for 8 common Mediterranean mutations. 7 different mutations were detected in 46 heterozygous patients (number of patients wit a particular mutations are in square brackets): IVS1-6(T>C) [15]; IVS2-745(C>G)[14]; IVS2-1(G>A) [10]; IVS1-1(G>A) [2]; CD6-A [2]; CD39(C>T) [2]; IVS1-110(G>A) [1].

We also discuss the effect of these mutations on the expression level of the β-globin gene. Frequencies of individual mutations in Poland were different from those encountered in Mediterranean and some Centrak European countries. We also detect two novel mutations:

IVS-1-1(G>A) G^GTTGGT>AGATTGGT and 5′UTR; +33(C>T). The first novel mutation significantly decreased the mRNA level of the β-globin gene. The second mutation seems to be silent and does not affect β-globin synthesis. In this case, this particular mutation occurs with IVS-1-6(G>C) mutation in compound the heterozygotes stage.
Quality of Life in Survivors of Childhood and Adolescent Acute Myelogenous Leukemia (AML) Does Not Differ by Treatment (Bone Marrow Transplant (BMT) vs. Chemotherapy). A Report from the Children’s Oncology Group (COG).
Authors: Nicholson H. Stacy , Zhou Tianni , Chen Zhengjia , Moran Kellie , Sklar Charles , Zeltzer Lonnie , Neglia Joseph
Published: October 2019

Childhood and adolescent AML requires intensive therapy. Beginning in 1979, the Childrens Cancer Group (CCG) randomized patients with newly-diagnosed AML to allogeneic bone marrow transplantation (alloBMT) based solely on the availability of an HLA-identical sibling donor. Two decades of successive CCG trials using a similar design have shown a significant survival advantage for alloBMT. To determine whether BMT was associated with a diminished quality of life (QOL) in long-term survivors compared to those who had been treated with chemotherapy alone (chemo), we conducted a cross-sectional telephone survey study of AML survivors. To be eligible, survivors must have been enrolled on a CCG AML study (beginning in 1979), have survived at least five years, and reside in the US or Canada. QOL was measured using the Medical Outcomes Study Short Form 36 (SF-36) and the Child Health Questionnaire (CHQ). 206 survivors completed the study, including 124 treated with chemo, 54 treated with alloBMT and 28 treated with autologous BMT (autoBMT). Median age at diagnosis was 3 years (range, 0–20), median age at survey was 19 years (range 8–39) and median time from diagnosis was 13.4 years. 48% were male, and 87% were white. QOL summary scores for the 82 survivors treated with BMT did not differ from those of the 124 treated with chemo (see table). Also, the adjusted summary scores did not differ by treatment after adjustment for other factors, such as age at diagnosis, race, gender, relapse, etc. None of the physical or mental SF-36 or CHQ subscale domains significantly differed by treatment, although physical subscale domains trended to be better in the chemo group. Multivariate analyses found that females had significantly lower physical and mental scores, that relapse significantly diminished the physical summary score, and that age at diagnosis was positively and significantly associated with higher mental summary score. Our data suggest that survivors of childhood and adolescent AML have similar overall QOL scores, regardless of whether they were treated with BMT or chemo. In addition, the QOL scores in most physical and mental health domains were high, suggesting that AML survivors as a group enjoyed good QOL. These data suggest that QOL in long-term survivors is not diminished by BMT and that the primary concern in assigning treatment remains survival.

QOL Summary Scores Treatment Physical Score Mental Score Mean 95% CI p-value Mean 95% CI p-value All 49.3 (48.0,50,7) 49.9 (48.4,51.3) BMT (n=82) 49.0 (46.8,51.1) 0.68 51.3 (49.0,53.6) 0.11 Chemo (n=124) 49.6 (47.8,51.3) 48.9 (47.0,50.8)