bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigation of TCR-Vβ CDR3 Spectrotypes in CD4 and CD8 Lymphocytes from Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients.
Authors: Nakamura Akiko , Shichishima Tsutomu , Noji Hideyoshi , Ikeda Kazuhiko , Maruyama Yukio
Published: April 2020

PNH is one disorder of bone marrow failure syndromes, including aplastic anemia and myelodysplastic syndrome. It is considered that immunologic mechanisms by cytotoxic T lymphocytes (CTLs) and interferon-γ (IFN-γ) contribute to hypoplastic bone marrow of these disorders. In addition, PNH is an acquired clonal disorder of the hematopoietic stem cell. Recently, it has been reported that analysis of T cell-antigen receptor (TCR)-Vβ repertoires, especially TCR-Vβ CDR3 (complementarity- determining region 3) spectrotypes, is an effective tool to study immunologic mechanisms by CTLs in pathophysiology of PNH (Karadimitris et al, Blood, 2000; Kook et al, Blood, 2002; Risitano et al, Blood, 2002). In the present study, we investigated 21 kinds of TCR-Vβ repertoires by flow cytometry in CD4 and CD8 lymphocytes from 5 PNH patients and a healthy volunteer and the TCR-Vβ CDR3 spectrotypes using polymerase chain reaction assay in CD4 and CD8 lymphocytes from 3 of 5 PNH patients and the control. We also quantitated intracellular IFN-γ in CD4 and CD8 lymphocytes from 5 PNH patients and the control according to the method by Sloand et al (Blood, 2002). We found no specific TCR-Vβ repertoires in CD4 and CD8 lymphocytes from PNH patients compared with the control. The TCR-Vβ repertoires with relative increase of CD4 or CD8 lymphocytes (over 10 of ratio of the proportion of each TCR-Vβ repertoire in a PNH patient/the proportion of the same TCR-Vβ repertoire in a healthy volunteer) were 13.6 or 4 and 22 in Case 1, 3 and 11 or 1 in Case 2, 3 and 13.6 or 3 in Case 3, 5.3 and 7.2 or 2, 3, 7, and 18 in Case 4, and 4, 5.2, 13.6, 16, and 23 or 1 and 14 in Case 5, respectively. TCR-Vβ CDR3 spectrotyping showed that in CD4 lymphocytes most CDR3 patterns were chiefly polyclonal, except for one oligoclonal (Case 1) and one monoclonal (Case 3) patterns of TCR-Vβ25; in CD8 lymphocytes most CDR3 consisted of polyclonal, oligoclonal, and/or monoclonal patterns, suggesting the possibility that CD8 lymphocytes recognize much more antigens of abnormal cells, probably including PNH clones, than CD4 lymphocytes. Unfortunately, we found the same patterns as described above in CD8 lymphocytes from the control, although CD4 lymphocytes from the control presented only polyclonal pattern of CDR3. Quantitative analyses of IFN-γ showed that index values of IFN-γ in CD4 and CD8 lymphocytes from PNH patients were higher than those from the control. However, we did not find any significant correlations between the spectrotypes of TCR-Vβ CDR3 and the index values of IFN-γ in PNH patients, suggesting that TCR-Vβ repertoires with monoclonal and oligoclonal CDR3 patterns do not necessarily produce much IFN-γ. In conclusion, our findings suggest that TCR-Vβ CDR3 spectrotyping is more effective tool to resolve some immune mechanisms of pathophysiology in PNH, especially by auto-reactive CTLs.
Sustained Polyclonal Hematopoietic Repopulation after Successful SCID-X1 Gene Therapy by Means of a Non Random Integrating Pseudotyped Gammaretrovector.
Authors: Schwarzwaelder Kerstin , Schmidt Manfred , Howe Steven , Prinz Claudia , Wissler Manuela , Schmidt Sonja , Deichmann Annette , Glimm Hanno , Gaspar Bobby , Trasher Adrian , von Kalle Christof
Published: April 2020

The integration of a retroviral vector as used in hematopoietic gene therapy trials produces a transition sequence from the vector DNA into the genomic DNA and may thus serve as a stable molecular marker unique for each cell clone. High sensitive linear amplification mediated PCR (LAM-PCR) allows the detection of specific retroviral integration sites. Thus it is possible to determine the clonal composition of the hematopoietic system in vivo (1). We could show that the hematopoietic repopulation in human SCID-X1 patients was derived from various, long-lived progenitor cell clones indicating retroviral transduction into pluripotent cells (manuscript submitted). In two cases of lymphoproliferative disorder after successful SCID-X1 gene therapy integration of the retroviral vector into the LMO-2 oncogene was probable the main reason for malignancy (2). The distribution of integration sites over the whole genome, the potential preference for integration at certain loci and which cells receive genetic correction and engraftment are therefore of considerable interest. Recently, another gene therapy trial has successfully corrected 4 infants suffering from SCID-X1. A GALV-pseudotyped MLV-based vector carrying the therapeutic common gamma chain gene was used for transduction of autologous CD34+ cells. The patients did not receive any conditioning therapy before transplantation. We analyzed lymphoid and myeloid DNA from all patients. The transduction efficiency of T lymphocytes and myeloid cells was up to 100 and 1 %, respectively. In vivo clonality analysis of CD3+ cells showed a polyclonal composition 1 to 2 years after transplantation. The myeloid repopulation also consisted of various different clones. These data may indicate transduction of pluripotent and long term active stem or progenitor cells. We here report on more than 300 sequenced integration sites of the patients, whereas 250 sequences could be assigned unequivocally to a unique locus. So far, no vector integration in the LMO-2 oncogene could be detected in this trial, and the patients do not reveal any other evidence for malignancy or clonal deformation of their stem cell compartment. We could show that integration of the mammalian gammaretroviral vector in this gene therapy trial is not random. Integration of the vector happens generally within or close to specific regions of genes. We found common integration sites (CIS) in RefSeq gene regions. The targeted RefSeq genes were classified according to the Gene Ontology database. Our data strongly support the presumption that curative gene therapeutic treatment requires a sustained polyclonal contribution of ex vivo manipulated stem and progenitor cells and provide an important insight into the integration manner of GALV-pseudotyped MLV-based vectors.
Imatinib Mesylate Disrupts Cell Cycle Progression, Modifies the Nucleoskeleton and Suppresses Activation-Induced Transcription in Human T Cells.
Authors: Dietz Allan , Johnson William B. , Knutson Gaylord J. , Bulur Peggy A. , Schulenberg Bertie , Beechem Joe M. , Prendergast Franklyn G. , Vuk-Pavlovic Stanimir
Published: April 2020

Imatinib mesylate (imatinib, Gleevec®, Novartis, Basel, Switzerland) inhibits T cells in vitro and in vivo (Dietz et al., Blood104: 1094–1099, 2004; Cwynarski et al., Leukemia18: 1332–1339, 2004). The drug blocks T cell cycle progression rather uniquely as it neither inhibits expression of CD69, an early marker of T cell activation, nor induces apoptosis. To characterize the molecular effects of imatinib leading to this mode of T-cell inhibition, we measured the changes in transcriptome (by Affymetrix U133 chips), proteome and phosphoproteome (by Western blotting, differential phosphoprotein expression and mass spectrometry). We found that phytohemagglutinin activated T cells pre-treated with imatinib had reduced expression of 983 transcripts and increased expression of 271 transcripts when compared to untreated PHA activated T cells by the factor of 1.5 or more (p<0.05). Among the prominently down-regulated transcripts were granzyme B, CTLA-4 and IL-2-receptor α-chain (CD25), all characteristic of activated T cells, as well as cyclins D2 and D3 and cyclin-dependent kinases 3, 4 and 7, the molecules regulating cell cycle progression. Among the up-regulated transcripts were Kruppel-like transcription factors 2 and 7, and p27, a finding compatible with the observed cell cycle inhibition. Furthermore, we selected and identified 30 proteins from 2-D gels that were up-regulated and/or hyperphosphorylated in imatinib treated activated T cells. Among these were four heterogeneous ribonucleoproteins, three lamins and γ-actin, all components of the nucleoskeleton at the interface of chromatin and inner nuclear membrane and involved in replication and transcription (Herrmann and Foisner, Cell. Mol. Life Sci.60: 1607–1612, 2003; Shumaker et al. Curr. Opinion Cell Biol.15: 358–366, 2003). Thus, imatinib-borne interference with T cell signal transduction affects the nuclear structure indicating for the first time that nucleoskeleton structural changes are associated with T cell activation status.
Sudden Blastic Transformation (SBT) in Patients (pts) with Chronic Myeloid Leukemia (CML) Treated with Imatinib Mesylate.
Authors: Vidal Marieberta , Kantarjian Hagop , O’Brien Susan , Rios Mary Beth , Verstovsek Srdan , Cortes Jorge
Published: April 2020

Imatinib has become standard therapy for pts with CML in chronic phase (CP). SBT has been reported in pts receiving IFN-α, at a rate of 0.5% to 2.5% during the first 3 years of therapy. There is little information on the occurrence of SBT among pts treated with imatinib. Here we report 3 pts who developed SBT, defined as occurring after being found in complete cytogenetic remission (CG CR). These 3 pts represent 0.5% of 557 pts who received imatinib at MD Anderson for chronic phase CML. The total population has been followed for a median of 33 months (range, 1 to 56). Pt #1 was a 54 yr old female with a variant Ph translocation, t(9;22;19;10) (q34;q11.2;p13.1;q22). Sokal risk was low. She started imatinib 400mg/day within 4 mos from diagnosis, achieved a CG CR within 3 mos and was still in CG CR after 9 mos with BCR-ABL/ABL 0.66%. On routine follow-up at 12 mos she was found in myeloid blast phase with 48,XX,+8,t(8;21)(q22;q22),t(9;22;19;10) (q34;q11.2;p13.1;q22), +der(22)t(9;22;19;10) in all 20 metaphases. She received standard induction chemotherapy followed by bone marrow transplantation and is alive and in CR after 24+ months. Pt #2 was a 51 yr old female initially treated with IFN-α. She achieved a CG CR but discontinued therapy because of toxicity after 18 mos. Fourteen mos after stopping IFN-α she had a CG recurrence (35% Ph-positive) and was started on imatinib 400mg/day. Three mos later she was in CG CR and BCR-ABL was undetectable by nested PCR; a similar result was found after 9 mos of therapy. 9 mos later (18 mos after start of imatinib) she had a lymphoid SBT (precursor B-cell). CG showed 45,XX,-8, t(9;22) (q34;q11.2), −20,+mar in one metaphase, and 11 metaphases were diploid. No ABL mutations were identified. She achieved CR with HCVAD + imatinib and is currently receiving a BMT. Patient #3 was a 27 yr old pt who received imatinib 800 mg/d as first line of therapy for CML in CP with intermediate Sokal risk score. Three mos after start of therapy he was in CG CR and repeated analyses showed continued CG CR up to mo 15, with the lowest BCR-ABL/ABL 0.12 at mo 6. On mo 18 he had a lymphoid SBT (precursor B phenotype) with 43–46,XY, −7, −8,i(9)(q10),t(9;22)(q34;q11.2), −13, −17, −21,+der(22)t(9;22),+mar in 13 metaphases and one diploid metaphase. No ABL mutations were identified. He was treated with HCVAD + imatinib and achieve a CR, becoming undetectable for BCR-ABL by nested PCR. He is undergoing a BMT. In pts 2 and 3, transformation was associated with a sudden drop in platelet counts. These isolated events should alert the physicians to continued monitoring of pts on imatinib and consider marrow studies in the event of late, unexpected peripheral blood count changes. Still, SBT is rare, probably less common that seen with IFN-α therapy.
Identification of BCR-ABL Dependent Gene Regulation by Using a Phosphoproteomics Approach.
Authors: Ziegler Patrick , Balabanov Stefan , Hartmann Ulrike , Kammer Winfried Kammer , Nordheim Alfred , Brummendorf Tim H.
Published: April 2020

Introduction: The selective tyrosine kinase inhibitor imatinib (formerly STI571, Glivecâ) has been shown to block phosphorylation of tyrosine residues by occupying the ATP binding site of the Abl tyrosine kinases Bcr-Abl, c-Abl, v-Abl and Abl-related gene (ARG), as well as platelet-derived growth factor receptor (PDGF) alpha and beta and of the receptor for human stem cell factor (SCF) c-kit. We chose a large scale phospho-proteomics approach to identify novel downstream targets of imatinib which could possibly be utilized for combined treatment strategies.

Material and Methods: Phospho-proteomics was performed by comparison of large scale phosphotyrosine-immunoprecipitation of imatinib (10 mM/2 hours) versus DMSO treated K562 cells separated by one-dimensional polyacrylamide gelelectrophoresis. In addition enriched CD34+ cells (>70%) of a newly diagnosed Bcr-Abl positive CML patient were used immediately after purification and treated in the same way as described above. Resulting differentially immuno-precipitated proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Protein identification via peptide mass-fingerprinting and peptide sequencing was performed using Mascot search tool and NCBI nr database. Differential phosphorylation was confirmed by combined immunoprecipitation and western blot analysis of selected candidate proteins.

Results: Phospho-proteomics of K562 cells revealed 8 differentially phosphorylated proteins after a two hour treatment with imatinib including the recently identified c-cbl, and Bcr-Abl itself, the latter confirming autophosphorylation. Ship2 which was originally identified as beeing constitutively phosphorylated in chronic myelogenous leukemia progenitor cells showed reduced, imatinib sensitive phosphorylation. Remaining candidates could be classified as being involved in protein folding or in ATPase activities associated with a variety of functions (type II AAA). The analysis of primary CD34+ cells from a CML patient showed a predominance of the nonmuscular myosin heavy chain protein in different molecular weight forms.

Discussion: We detected significant imatinib-dependent differences in protein phosphotyrosine-immunoreactivity of the Bcr-Abl -dependent cell line K562. Previously identified down-stream targets of Bcr-Abl could be confirmed and novel candidate proteins were identified. Phosphorylation of Ship2, a previously identified down-stream target of Bcr-Abl, was found to be inhibited by imatinib treatment. Ongoing studies are aimed at the characterization of the role of the identified phospho-proteins, particularly type II AAAs for Bcr-Abl induced signal transduction as well as for the development of resistance to imatinib.
Critical Role of Dok-1 and Dok-2 in Leukemia Suppression.
Authors: Niki Masaru , Di Cristofano Antonio , Zhao Mingming , Hirai Hisamaru , Van Aelst Linda , Cordon-Cardo Carlos , Pandolfi Pier Paolo
Published: April 2020

Chronic myelogenous leukemia (CML) is characterized by the presence of the chimeric p210bcr/abl oncoprotein which shows elevated and constitutive protein tyrosine kinase activity relative to the normal c-abl tyrosine kinase. While several p210bcr/abl substrates have been identified, their relevance in the pathogenesis of the disease is unclear. We have identified a family of proteins, Dok (downstream of tyrosine kinase), coexpressed in hematopoietic progenitor cells. Members of this family such as p62dok (Dok-1) and p56dok-2 (Dok-2) associate with the p120 rasGTPase-activating protein (rasGAP) upon phosphorylation by p210bcr/abl as well as receptor and non-receptor tyrosine kinases. Here we report the generation and characterization of single and double Dok-2 or Dok-1/Dok-2 KO mutants. Single KO mice displayed normal steady state hematopoiesis. By contrast, concomitant Dok-1 and Dok-2 inactivation resulted in aberrant hemopoiesis and Ras/MAP kinase activation. Strikingly, all Dok-1/Dok-2 double KO mutants spontaneously developed transplantable CML-like leukemia due to increased cellular proliferation and reduced apoptosis. Furthermore, Dok-1 or Dok-2 inactivation markedly accelerated leukemia and blastic crisis onset in Tec-p210bcr/abl transgenic mice known to develop, after long latency, a myeloproliferative disorder resembling human CML. These findings unravel the critical and unexpected role of Dok-1 and 2 in tumor suppression and control of the hematopoietic compartment homeostasis.
Evaluation of the Profile of Thrombin Generation during the Process of Whole Blood Clotting as Assessed by Thromboelastography.
Authors: Rivard Georges E. , Kathleen Brummel , Li Fan , Angelique Hofer , Eli Cohen , Kenneth Mann G.
Published: April 2020

The evaluation of thrombin generation during blood coagulation has become the preferred clinically relevant approach to assess the global integrity of this complex process. In order to give the full picture of the physiology of this multifactorial reaction, all cellular and soluble plasma participants to this reaction have to be present in their natural whole blood environment. Whole blood thrombin generation tests are best suited for this purpose but are somewhat cumbersome for use in real time clinical practice. Thrombelastography has long been recognized as a clinically useful technology for assessment of whole blood coagulation. The purpose of this study was to evaluate the possibility of linking the tracing generated by clotting of whole blood in a Thrombelastograph® (TEG®) Hemostasis System with the generation of thrombin as assessed by thrombin/antithrombin complex (TAT). 320μL of citrated whole blood (3.2% buffered citrate and 1000μg/ml of corn trypsin inhibitor; 1 part to 9 parts of blood) from volunteers was clotted in the presence of 20μL CaCl2 (200mM) and 20μL of Innovin (1/100 diluted in PBS/albumin 4%, pH 7.4). Clotting was monitored with 8 channels of the TEG® system. At different time points (4 to 40 min) the whole blood TEG® reaction cups were put in 2640μL of cold quenching solution (EDTA 50mM, L-benzamidine 10mM, NaCl 150mM, HEPES 2mM, pH 7.4, and 10μL of 10mM D-Phe-Pro-Arg CH2Cl chloromethylketone diluted in 0.01M HCl), vortexed, and centrifuged, and the supernatants were kept at −80°C until assayed for TAT by ELISA. Total Thrombus Generation (TTG) was calculated from the first derivative of the TEG® waveform and was compared to the thrombin generation as measured by TAT as described above. The two vectors of values - the TAT thrombin generation data and the corresponding TEG® TTG - were analyzed using Pearson correlation coefficients and linear, non-linear, and natural log (Ln) transformation of TAT values for least-squares goodness-of-fit curves. The best least-squares fit is the exponential curve (Fig. 1) and its corresponding natural log transformation of TAT value as shown by Fig. 2, with R2 = 0.8787 (P < 0.0001). The prediction equation for TAT thrombin generation is y = 3122.7e0.0005x, where x is the TEG® TTG variable (P < 0.0001). Linearizing using the natural log of the TAT thrombin generation variable (Fig. 2), produces the same R2 as the exponential curve. The prediction equation is y = 8.0465 + 0.0005x, significant at P < 0.0001, where y is the TAT thrombin generation in the Ln transformation variable and x is the TEG® TTG variable. The high magnitude of R2 and the high significance of the prediction equation demonstrate the high efficacy of the prediction of TAT thrombin generation using the TEG® TTG.
Host γd T cells Exacerbate Experimental Acute Graft-Versus-Host Disease through Activation of Host Antigen Presenting Cells.
Authors: Maeda Yoshinobu , Reddy Pavan , Liu Chen , Keith Bishop D. , Ferrara James L.M.
Published: April 2020

Large numbers of T cells bearing γd T cell receptors are present in graft-versus-host disease (GVHD) target tissues. We investigated the potential role of host γd T cells during acute GVHD in a well-characterized GVHD model following full intensity conditioning (11 Gy TBI). BM and spleen T cells from BALB/c (H2d) donors were transplanted into wild type (wt) B6, aß T cell deficient B6 (aß −/−) or γd T cell deficient B6 (γd −/−) hosts. γd −/− hosts demonstrated significantly better day 35 survival (85%) than wt (40%) or aß−/− hosts (18%) (P<0.05). Reconstitution of γd −/− B6 hosts with B6 type γd T cells 24 hr prior to BMT restored lethal GVHD (50 % day 35 survival). In vivo, γd −/− B6 hosts demonstrated at least a five fold reduction in donor T cell expansion and cytokine production. In vitro, T cells proliferated less when co-cultured with allogeneic γd −/− dendritic cells (DCs) than with wt DCs (40,127 ± 1634 vs. 72,503 ± 1296, P<0.05). BM-derived DCs cultured with γd T cells caused greater proliferation of allogeneic T cells than DCs cultured with aß T cells (15.1 ± 21 x 104 vs. 5.1 ± 1.2 x 104, P<0.05). We next tested the effect of γd T cells on host DCs in vivo using a model system in which only the DCs injected prior to BMT expressed the alloantigen that stimulated the GVHD reaction. MHC Class II −/− B6 mice that had been depleted of γd T cells were given 11 Gy TBI and injected one day prior to BMT with B6 DCs that had been co-cultured either with γd T cells or with medium. On day 0 both groups of recipient mice were injected with BM plus splenic T cells from allogeneic bm12 donors. On day +5, CD4+ donor T cells expanded four times more in recipients of DCs co-cultured with γd T cells than in recipients of control DCs and serum levels of TNF-a were significantly higher (36.7 + 6.8 vs. 21.3 + 3.7 pg/ml, P<0.05). Together these data demonstrate that γd T cells amplify the stimulatory function of host DCs and increase the severity of GVHD, suggesting that a new therapeutic target for the prevention of the major BMT toxicity.
Keratinocyte Growth Factor Ameliorates Acute Graft-versus-Host Disease in a Novel Nonmyeloablative Haploidentical Transplantation Model.
Authors: Bos Gerard M.J. , Vanclee Ariane , Oving Ellis , Lutgens Ludy , Deutz Nicolaas , Schouten Harry
Published: April 2020

Allogeneic stem cell transplantations (SCT) are currently being used as a therapy for hematological malignancies, some solid tumors and non-malignant bone marrow deficiencies. Nevertheless, clinical applicability is limited due to toxicity of conditioning regimens, graft-versus-host disease (GVHD) and the scarcity of HLA-identical family donors. New concepts are based on nonmyeloablative conditioning to reduce toxicity, prevention or amelioration of GVHD and the use of haploidentical donors to increase donor availability.

To meet these requirements, we have developed a nonmyeloablative conditioning regimen in a haploidentical F1 → F1 mouse model. These mini-transplantations, consisting of low dose total body irradiation and cyclophosphamide-based chemotherapy, resulted in stable full donor chimerism, but also in the development of GVHD. Administration of keratinocyte growth factor (KGF) before and after SCT resulted in reduced GVHD, evident as reduced weight loss and a lesser degree of dermatitis than in saline-treated controls. KGF preserved plasma citrulline and TNF-a levels, both indicative for reduced radiation-induced injury to the gastrointestinal tract. Citruline has recently been described as a new marker for gastrointestinal toxicity. Six months after transplantation, survival rates were significantly higher in KGF-treated animals (57% as compared to PBS-treated controls (5.3%).

Figure Figure

These data indicate that nonmyeloablative haploidentical transplantations might be feasible and that KGF might contribute substantially to reduction of lethal GVHD, also after nonmyeloablative procedures. For clinical transplantation such an effect would be significant if intensive T cell depletion might be prevented in haploidentical transplantation protocols and therefore reduced infection rates can be obtained.
Pathobiology of Hemophilic Synovitis II: Synovial Cell Proliferation.
Authors: Valentino Leonard A. , Hakobyan Narine , Kazarian Tamara , Jabbar Adnan A. , Jabbar Kausar J.
Published: April 2020

Hemophilia is a genetic disease due to the deficiency of blood coagulation factor VIII or IX. Bleeding is common, especially into joints where a proliferative synovitis develops resulting in a debilitating arthritis, hemophilic arthropathy. The pathogenesis of blood-induced hemophilic synovitis (HS) is poorly understood. Repeated episodes of bleeding induce synoviocyte hypertrophy and hyperplasia, an intense neovascular response and inflammation of the synovial membrane. The component(s) in blood that initiates these changes is (are) not known, although iron is often proposed as one possibility. Previously, we reported that iron promotes the proliferation of human synovial fibroblasts and increases c-myc expression in vitro (Blood2002; 100(3)912–6). Here, we describe the proliferative effects of joint bleeding following traumatic hemarthrosis in a murine model of severe, human hemophilia A. Controlled, blunt trauma to the right knee joint consistently resulted in joint swelling due to a combination of bleeding and inflammation. The left, control knee was sham injured and remained normal for the duration of the experiment. Hemarthrosis resulted in acute morbidity evidenced by inactivity, weight loss and immobility. Hemosiderin was found in the synovial membrane of the right knees only. The numbers of synovial cell layers present in the membrane of the right (injured) knee increased during the experiment (figure). Expression of Ki67 in the nucleus, a marker of cell proliferation, confirmed the increase in synovial cell proliferation in membranes from the right (injured) knees. Taken together these data support the notion that iron causes aberrant gene expression and is, at least in part, responsible for the pathological findings observed in the joints of patients following bleeding and contributes to the development of hemophilic synovits.

Figure Figure
Age and the Prevalence of Bleeding Disorders in Women Presenting with Menorrhagia.
Authors: Philipp Claire S. , Faiz Ambarina , Dowling Nicole , Dilley Anne , Michaels Lisa A. , Ayers Charletta , Miller Connie H. , Bachmann Gloria , Evatt Bruce , Saidi Parvin
Published: April 2020

Menorrhagia occurring during adolescence and perimenopause is presumed to be associated with anovulation, and during perimenopause, with uterine pathology, such as subserosal uterine myoma, as well. The frequency of bleeding disorders in women presenting with menorrhagia at the extremes of menstruating age, ie adolescence and perimenopause, are not known. We conducted a study to evaluate the frequency and types of hemostatic defects found in adolescent and perimenopausal age women diagnosed with menorrhagia. 115 women with a physician diagnosis of menorrhagia, including 25 adolescent women, 25 perimenopausal age women, and 65 women between the ages of 20 and 44, underwent comprehensive hemostatic testing for possible bleeding disorders. There were no significant differences between the three age groups in mean hemoglobin, percentage of women with anemia, duration of menses, mean pictorial blood assessment scores, or race. Overall, 48% of women were found to have hemostatic abnormalities.

Hemostatic abnormalities among women with menorrhagia

Overall ≤ 19 yrs 20–44 yrs ≥ 45 yrs p-value N=115 N=25 N=65 N=25 *Platelet aggregation, Von Willebrand factor, and/or coagulation factor Platelet Aggregation 44% 44% 48% 32% 0.48 Von Willebrand Factor 7% 4% 8% 8% 0.78 Coagulation Factor 5% 8% 6% 0% 0.34 Any Abnormality* 48% 48% 54% 32% 0.32

Adolescents and perimenopausal age women diagnosed with menorrhagia were as likely as women presenting between age 20 to 44 to have underlying hemostatic defects. Among the adolescents, neither age at presentation nor time from menarche were predictive of having a bleeding disorder. The platelet aggregation defects observed were similar among the three age groups with the exception of ADP induced platelet aggregation defects which were seen significantly more frequently in adolescents compared to older women (p≤ 0.01). Ristocetin and epinephrine induced platelet aggregation defects were the most common aggregation defects in women 20 years and older and ADP induced platelet aggregation defects were the most common among adolescents. There were no significant differences in platelet ATP release abnormalities between the three age groups. Our results demonstrate that a high proportion of adolescents and perimenopausal age women presenting with menorrhagia have bleeding disorders. These results suggest that menorrhagia in the adolescent, even if presenting soon after onset of menarche, and in perimenopausal age women, even if fibroids are present, may warrant hemostatic evaluation. Further age-specific studies evaluating treatment benefits and outcomes in women with menorrhagia diagnosed with bleeding disorders are warranted.
Survival of HIV Infected Individuals with Hemophilia after 20 Years: From the Canadian Hemophilia Registry.
Authors: Walker Irwin R. , Arnold Donald M. , Julian Jim A.
Published: April 2020

Background : Patients with hemophilia A and B have been tracked by the Canadian Hemophilia Registry since 1980. The total number on the Registry is 3307 (A=2721, B=586) of which 662 were/are HIV-positive. New HIV and HCV infections ceased after 1985 and 1988 respectively with the introduction of safer blood products; HIV-infected individuals with hemophilia therefore resemble a cohort with HIV infection that was acquired in a relatively narrow time period about 20 years ago. Current status of HIV-positive patients is Active 250 (37.8%), Deceased 406 (61.3%), Lost to Follow Up 9 (0.9%). Follow up data is available for 3135/3307 (95% of all patients), and for 656/662 (99% of HIV-positive patients). We previously reported (Haemophilia1998;4:714) a peak in the death rate of HIV-positive individuals during the years 1991 to 1993 with a subsequent decline.

Objective : To update the rate and causes of death among HIV positive hemophilia patients in Canada with current information.

HIV-Related Mortality : The annual number of deaths in the HIV-positive group has been [year-#(deaths per 100 person-years)], 1980-0(0), 81-0(0),82-0(0),83-3(0.45),84-1(0.15),85-2(0.30),86-12(1.8),87-12(1.9),88-21(3.3),89-25(4.1),90-24(4.1),91-45(8.0),92-38(7.4),93-Currently, 256/662 (38.7%) are alive.

Causes of Death (HIV+) : The commonest primary causes of death in HIV-positive individuals have been: [cause #(%)] AIDS 284(70), liver failure 45(11), bleeding 18(4.4) and infections 15(3.7). Twenty-eight patients had liver disease listed as a secondary or contributing cause of death; thus liver disease was the primary or contributory cause of death in 45+28=73 individuals (18% of HIV-positive deaths).

HCV-Related Deaths : HCV status is positive in 1151 of the total population of 3307 (35%) individuals in the registry, negative in 1260 (38%) and unknown in 896 (27%). Of HIV-positive individuals, 448 (68%) are co-infected with HCV, 53 of whom have died with liver failure (12% overall, 18% of deaths). HIV/HCV co-infected individuals have higher overall mortality than those not co-infected (46% vs. 12%, p<0.0001). In addition, HCV singly-infected individuals have lower overall mortality (7%). 703 HIV-negative individuals are infected with HCV, of whom 7 have died with liver failure (1% overall, 13% of deaths).

Conclusions : The death rate of HIV-positive individuals peaked in the years 1991-3 and decreased thereafter with 38% of individuals still living at 20 years follow up. However deaths still occur at an annual rate of 3–5%. HIV/HCV co-infected individuals have the highest mortality and HCV single-infected individuals have the lowest mortality of those with transfusion-transmitted infection. Liver failure is a common mode of death in both HIV- and HCV-infected individuals.
Interferon-Gamma (IFN-γ) Secretion Defect in Peripheral Blood Mononuclear Cells (PBMCs) from Severe Hemophilia A (HA) Patients Prior to Therapeutic Exposure To Factor VIII (FVIII) Concentrates.
Authors: Gill Joan Cox , Stephany Karen , Helsell Craig
Published: April 2020

The recent availability of highly purified recombinant coagulation factor concentrates to treat hemorrhages in hemophilic patients is thought to circumvent perturbations in immune function by preventing chronic intravenous exposure to multiple foreign protein antigens. We previously reported a PHA-induced IFN-γ secretion defect in severe hemophilia A patients who had been exposed exclusively to highly purified coagulation factor VIII concentrates. We now report the results of cytokine secretion in response to PHA in cultured PBMCs from severe hemophilia A patients never exposed to FVIII concentrates. PBMCs were isolated from whole blood from three severe HA patients, 3 age-matched normal boys, 3 boys with severe hemophilia B and an adult control. Cells were stained with PKH26 (Sigma), a fluorescent dye with aliphatic tails that intercalate into membrane lipids; cells were than washed and cultured in complete media in wells of microtiter plates with and without PHA. Cells were harvested at 5 days and proliferation detected by loss of membrane PKH26 staining detected by flow cytometry. Cytokines secreted into the supernatants of 48 hour and 5 day cultures were measured by ELISA assays (BD Pharmingen). PBMCs from severe HA patients proliferated normally in response to PHA but failed to secrete IFN-γ when compared to normal boys and boys with severe HB:

PBMC Proliferation and Secretion of IFN-γ in Response to PHA

Subject Group HA HB Normal Boys Adult Control PHA Proliferation (% unstained cells) M± 1SD 79.3 ±3.7 78.2±13.3 95.4±1.5 89.3±5.3 INF-γ Secretion (pg/mL) M± 1 SD 6.7±4.7 429.0±464 69.1±32.4 770.1±148.7

The severe HA patients did not have improvement in INF-γ secretion following therapeutic exposure to FVIII concentrates. Addition of physiologic concentrations of FVIII or FIX to cultures with or without PHA did not alter the results of proliferation or cytokine secretion regardless of the inhibitor history of the patients. (1 HA and 1 HB patient developed high responder inhibitors.) Secretion of IL-4, IL-5 and IL-10 were moderately decreased in some HA patients as well. In summary, we have confirmed an INF-γ secretion defect in PBMCs from patients with HA in spite of normal proliferation in response to PHA. The defect is not present in HB or normal age-matched control subjects and is not corrected by addition of FVIII to cultures. We conclude that the defect is not due to exposure to intravenous foreign antigens. The precise clinical significance and any possible relation to the higher risk of inhibitor development in HA will require further investigation.

PBMC Proliferation and Secretion of IFN-γ in Response to PHA

Subject Group HA HB Normal Boys Adult Control PHA Proliferation (% unstained cells) M± 1SD 79.3 ±3.7 78.2±13.3 95.4±1.5 89.3±5.3 INF-γ Secretion (pg/mL) M± 1 SD 6.7±4.7 429.0±464 69.1±32.4 770.1±148.7
Persistence of High Viral Load Abrogates Long-Term HIV-Specific Memory CD4 T-Cells Producing Interleukin-2.
Authors: Boulassel Mohamed-Rachid , Younes Souheil-Antoine , Yacine-Diab Bader , Sekaly Rafick-Pierre , Routy Jean Pierre
Published: April 2020

Virus-specific CD4 T-cells are believed to play a critical role in determining the persistence of memory and effector CD8 T-cell responses during viral infections. However, the reasons for which human immunodeficiency virus (HIV)-specific CD4 cells fail to generate effective CD8 cell responses remain incompletely understood. In this study, we analyzed the HIV-specific CD4 cells in 10 aviremic (viral load < 50 copies/ml) and 8 viremic (mean viral load 45,295 copies/ml) patients treated during primary HIV infection and followed for up to 6 years. At the time of apheresis, median CD4 T cell counts for aviremic and viremic patients were 671 cells/μl and 485 cells/μl respectively. Using Gag and Nef overlapping HIV peptides, the highly sensitive CFSE-based proliferation assay and intracellular staining techniques, we observed that proliferative Gag and Nef peptide-specific CD4 cell responses were 30-fold higher in aviremic patients compared to viremic. Several subsets of HIV-specific memory CD4 cells endowed with different proliferative and functional capacities were identified. We observed two subsets of HIV-specific memory CD4 cells in aviremic patients, CD45RA− CCR7+ central memory cells (Tcm) producing exclusively IL-2 and CD45RA− CCR7− effector memory cells (Tem) that produced both IL-2 and IFN-γ. In contrast, in viremic patients, Tem were found to unexpectedly produce IFN-γ exclusively. Longitudinal data showed that only cells, which were capable of producing IL-2, persisted as long-term memory cells. In viremic patients, HIV-specific CD4 cells that produce INF-γ were found only during periods of elevated viral loads. To test if the presence of IL-2 could restore the proliferation of these cells, we stimulated CD4 cells from viremic patients with a pool of peptides, which gave strong IFN-γ responses in these patients in the presence of exogenous IL-2. The addition of IL-2 during the in vitro peptide stimulation dramatically increased the fraction of proliferating cells. This experiment strongly suggests that the impaired proliferation of CD4 cells from viremic patients is not caused by a virus-mediated destruction of proliferating cells but a lack of producing IL-2. Altogether, these results indicate that long-term memory HIV-specific CD4 cell produce mainly IL-2, while those producing IFN-γ are short-lived. These findings favor a model in which Tcm are continuously produced in limited numbers but under continuous viral stimulation are rapidly induced to differentiate into IFN-γ only-producing cells that lack the capacity for self-renewal.These findings also indicate that treatment strategies aimed to increase the long-term memory CD4 cells are needed for future HIV vaccine development.
Zoledronic Acid (ZA)- Associated Nephrotoxicity: An Analysis of Adverse Drug Reaction (ADR) Reports by the Research on Adverse Drug Events and Reports (RADAR) Project.
Authors: McKoy June M. , Obadina Eniola , Yarnold Paul R. , Kut Victoria , Raisch Dennis W. , Bennett Charles L.
Published: April 2020

Background-The FDA originally approved zoledronic acid (ZA) in August 2001 for the treatment of hypercalcemia of malignancy and subsequently for prevention of skeletal related events among patients with multiple myeloma (MM), breast, and prostate cancer. Since its approval, the FDA has received several reports of nephrotoxicity. Many of these individuals have myeloma, a malignancy associated with renal damage. Herein, RADAR investigators reviewed the clinical characteristics of ZA-induced nephrotoxicity among cancer patients with multiple myeloma versus other malignancies. We investigated the clinical characteristics and reporting quality of all adverse event (AE) reports describing ZA-associated neprotoxicity.

Methods- We analyzed reports of ZA-associated nephrotoxicity from the FDA’s Adverse Event Reporting System, which included a total of 141 AE reports, with exclusion of cases/trials reported in duplicate.

Results- Overall, 141 cases of ZA-associated renal failure (RF) were identified: 92 case reports were from the United States and 44 were from non-United States countries. Reporting completeness was generally poor, with respect to serum creatinine levels and frequency of ZA administration, especially in reports from Canada and Great Britain respectively. Two thirds of the patients with scheduling information had received ZA every 4 weeks. Infusion rate was reported for 21%, and was usually 15 minutes or longer. NSAIDS and Cox-II inhibitor exposures were reported in 25%, and 20% of the patients who had prior diagnoses of renal insufficiency. Patients with MM (n=82) had similar mean age as patients with other cancers (n= 54) (72.05 versus 72.9 years). For patients for whom data was available (n=62), pamidronate was used by 97.8% in the MM group versus 86.9% in the non-myeloma group. The onset of RF occurred after a mean of 67.9 days in the MM group (range, 3–366) versus 73.7 days in the non-myeloma group (range, 0–546 days) post initiation of ZA; after a mean number of ZA doses (2.5 vs. 1.8 doses). Only one dose of ZA was received by 24.4% and 25.4% of MM versus non-myeloma patients respectively, which occurred after an average of 14.7 days and 30 days. RF manifestations included serum Cr > 2 for 59.7% of the MM and 83.1% of the other cancer patients. Outcomes of RF in MM and non-myeloma patients, respectively, included hospitalization (61% and 71%) and dialysis, (38% and 22%).

Conclusions- Close monitoring of serum creatinine prior to ZA, is important for both MM and other cancer patients; completeness of current case reporting efforts of ZA-associated renal insufficiency is poor in both US and non-US countries; and information about the potential occurrence of renal toxicity should be prominently described in the packet insert for ZA.
RNA Expression Levels of (Co)Chaperone Molecules of the Glucocorticoid Receptor Are Not Related to In Vitro Prednisolone Resistance in Childhood ALL.
Authors: Tissing Wim , Meijerink Jules P.P. , den Boer Monique L. , Brinkhof Bas , Pieters Rob
Published: April 2020

In vitro resistance to Glucocorticoids (GC) is an important adverse risk factor in the treatment of ALL. To induce apoptosis in ALL cells, GC have to bind to the GC receptor (hGR), which is tightly regulated by various (co)chaperone molecules. HSP70, ST13 and HSP40 facilitate hGR binding to HSP90 and HOP in an energy dependent fashion. This complex is stabilised by immunophillins FKBP 51, FKBP59, CYP40 and P23 and is necessary for the hGR to be able to bind GC. The ATPase BAG1 can function as negative regulator of HSP70, and may downregulate hGR activity. After the GC-hGR complex is formed, it is transported to the nucleus to regulate GC responsive genes. In this study, we tested the hypothesis that RNA expression levels of these (co) chaperone molecules are important determinants for in vitro prednisolone sensitivity in childhood ALL.

Methods: 20 children with leukemic cells in vitro sensitive to prednisolone (LC50<0.1μg/ml) were matched (according to age, immunophenotype and white blood cell count at diagnosis) each with an in vitro resistant patient (LC50 > 150 μg/ml). RNA expression levels of the different (co)chaperone molecules were measured by a quantitative real-time RT-PCR (Taqman) assay and standardised to endogenous GAPDH and RNAseP mRNA levels. In vitro resistance to prednisolone was measured by the MTT assay.

Results. The highest median expression levels, indicated as percentage of GAPDH levels were found for HSP90 (29%) and p23 (10%), whereas FKBP51 and ST13 were expressed at 2% and 1.6% respectively. HSP70 (0.1%), HSP40 (0.44%), HOP (0.2%), FKBP59 (0.4%), PPID (0.3%) and BAG-1 (0.4%) were expressed at relatively low levels. Using matched pair analysis, no significant differences were found for the expression levels of the various (co)chaperone molecules between in vitro sensitive and resistant patients. The ratio of different (co)chaperone molecules with opposing effect (HIP versus BAG-1, FKBP-51 versus FKBP-52, HSP-90 versus P23, and HSP-90 versus the functional GR-alpha receptor, positive and negative effect respectively) was not related to GC resistance as well.

Conclusion: Glucocorticoid resistance in childhood ALL can not be attributed to basal expression levels of the diverse (co)chaperone molecules involved in GC binding and transport of the GC-GR complex to the nucleus of the GR.
Immunophenotyping to Detect Minimal Residual Disease in Adult Acute Lymphoblastic Leukemia - Feasibility and Prognostic Significance.
Authors: Holowiecki Jerzy , Giebel Sebastian , Jagoda Krystyna , Krawczyk-Kulis Malgorzata , Stella-Holowiecka Beata , Kyrcz-Krzemien Slawomira , Paluszewska Monika , Kopera Malgorzata , Medrek Wlodzimierz , Bober Grazyna , Najda Jacek
Published: April 2020

Although well documented for pediatric patients, the prognostic value of minimal residual disease (MRD) detected with the use of immunophenotyping has not been established for adults with acute lymphoblastic leukemia (ALL) so far. With the use of standard “quadrans” analysis based on evaluation of the number of blasts bearing atypicial antigen combinations, the method may be applied to the majority of T-derived and only approximately half of B-lineage ALL. In this study we tested the feasibility and prognostic significance of a new “empty spaces” method taking into account the individual antigen expression pattern for each blast cell tested. The “forbidden” gates were established with the use of triple staining by comparison with the pattern obtained for healthy volunteer bone marrow donors. At least two antigen combinations were tested for each patient. MRD was evaluated after induction and after consolidation therapy. Only patients who achieved complete remission after a single course of induction were analysed with regard to the impact of MRD on long-term outcome.

Thirty adult ALL patients (B-lineage n=24, T-lineage n=6) were included in the study. For all cases it was possible to determine unique antigen expression pattern and to monitor MRD with the use of immunophenotyping at the level of 1/1000 cells. MRD was detected in 8/30 patients after induction and in 7/28 patients after consolidation tharapy (two patients relapsed during consolidation). For 11/30 patients the MRD resulted positive at least once.

At 20 months the relapse rate equaled 53% for patients with MRD detected after induction or consolidation and 28% for those with MRD always negative (p=0.08). The difference was more pronounced for patients with B-lineage ALL (67% vs. 26%, p=0.02). A single MRD evaluation after induction therapy had no significant prognostic value for the risk of relapse whereas there was a trend for higher relapse rate in B-lineage ALL patients with positive MRD after completion of consolidation therapy compared to those MRD-negative at that time-point (75% vs. 32%, p=0.06).

Results of the study prove the feasibility of immunophenotypic MRD evaluation in ALL. “Empty spaces” in addition to the standard “quadrans” analysis allows monitoring of the vast majority of patients regardless the immune subtype. Even with a small number of cases we were able to demonstrate its prognostic value for long-term outcome of adults with ALL.
Pharmacokinetics of Vincristine in Infants with Acute Lymphoblastic Leukemia.
Authors: Bomgaars Lisa , Hunter Chelsea , Huang Rong , Schmidt Mark , Dreyer ZoAnn , Blaney Susan , Berg Stacey , Murry D.J.
Published: April 2020

An understanding of vincristine (VCR) pharmacokinetics (PK) is important to achieve drug dosing that optimizes efficacy and minimizes toxicity. Limited information is available on the PK behavior of VCR in infants.

Objectives: To determine the PK of VCR in infants, and to compare the PK parameters of infants to those in older children.

Methods: A subset of 28 patients enrolled on the Pediatric Oncology Group 9407 infant leukemia study participated in this VCR PK study. PK samples were collected following the administration of VCR (0.05 mg/kg) i.v. push on week 1, day 1 of treatment. Twenty-eight patients, median age 7.1 months (range, 0.1–11.9 months) had blood samples collected for analysis of VCR concentrations at 5 minutes, and 0.5, 1, 4, and 24 h after drug administration. Samples were analyzed using a validated LC-MS method. A two-compartment model was fit to each patients data using Bayesian estimation (Adapt II software).

Conclusions: PK parameters in infants < and > 6 months of age were similar. CL in this population ranged from 7.8–31 ml/min/kg and was correlated with body weight, however only 25% of the variability in VCR CL could be accounted for by this variable. CL and t1/2 values are similar to those previously reported in older children. Evaluation of pharmacokinetic/pharmacodynamic correlation is ongoing.

Results:

VCR (mean ± SD) AUC (mcg•hr/L ) 41.7 ± 19 CL (ml/min/kg) 19.2 ± 6 Vc (L/kg) 1.3 ± 1 t 1/2 elimination (h) 17.1 ± 8.3 Kcp (hr-1) 5.2 ± 1.7 Kpc (hr-1) 0.3 ± 0.14