bluuebunny/crossref_metadata_2025 huggingface dataset:
Imatinib Mesylate Has a Stage-Specific Inhibitory Role in Generation of CD26highCD8+ Central Memory T Cells in Vitro and in Vivo.
Authors: Yokoyama Kazuaki , Nagamura-Inoue Tokiko , Nakayama Shin , Ishige Ikuo , Ogami Kazuo , Futami Munenori , Kobayashi Seiichirou , Tojo Arinobu
Published: October 2019

CD26 is a transmembrane glycoprotein with intrinsic dipeptidyl peptidase IV (DPPIV) activity as well as costimulatory activity of mitotic signals triggered by the CD3/TCR complex. Based on the expression level of CD26, CD4+ and CD8+ T cells can be divided into 3 (high/intermediate/low or negative) subsets. The significance of CD26 has been studied mainly on CD4+ T cells, and CD26highCD4+ T cells are considered to represent effector memory T cells of a typical Th1 phenotype producing IL2 and IFNg. Furthermore, we reported a significant decrease of this subset in CML patients under imatinib therapy in comparison to those under IFNa therapy and normal volunteers. In contrast, the role of each subset of CD8+ T cells has not yet been clarified.

Multi-parameter flow cytometry analysis was performed to characterize CD8+ T cells differentially expressing CD26 in combination with intracellular detection of effector molecules such as perforin (P) and granzyme B (Gr). The capacity to secrete effector cytokines such as IFNg following short-term stimulation was also assessed. As a result, according to the expression level of CD26, we could clearly categorize CD8+ T cells as follows: CD26highCD8+ T cells are defined as central memory T cells which has a phenotype of CD45RO+CD28+CD27+ IFNg+Gr−P+/−, CD26intCD8+ T cells as naïve T cells of CD45ROCD28+ CD27+ IFNg−Gr−P−, and CD26lowCD8+ T cells as effector memory/effector T cells of CD45RO−/+ CD28−CD27−IFNg++Gr++P++, respectively. We next investigated the effects of imatinib on 3 distinct subsets during CD8+ T cell differentiation program. Peripheral blood mononuclear cells were primed with anti-CD3/CD28 MAb and subjected to the grading doses of imatinib for short term culture, followed by flow cytometory. CFSE labeling was used for monitoring cell proliferation. Intriguingly, we found that imatinib dose-dependently inhibits activation, cytokine production and proliferation of CD26highCD8+ central memory T cell subsets in a differentiation stage-specific manner. Finally, we compared the absolute number of peripheral blood CD26highCD8+ T cell subsets between 20 patients with CML in imatinib-induced CCR and 20 normal volunteers, clearly indicating a significant decrease of this subset in CML patients (22.30/ml vs 45.60/ml, p<0.01).

The present study offers another evidence for immunomodulatory effects of imatinib or the critical role of Abl (-related) kinase in T cell development, and draws special attention to susceptibility to viral infection of CML patients under long-term imatinib therapy.

Figure Figure
Leukemia Relapse after Allogeneic Transplants in AML: Predictive Role of WT1 Expression and Protective Effect of DLI.
Authors: Bacigalupo Andrea , Dominietto A. , Di Grazia C. , Pozzi S. , Bertolotti F. , Podesta’ M. , Lamparelli T. , Gualandi F. , Ibatici I. , Bregante S. , Pinto V. , Frassoni F.
Published: October 2019

Background: relapse remains a significant problem in patients with acute leukemia undergoing an allogeneic hemopoietic transplant (HSCT) and a predictive molecular marker of disease would be useful.

Aim of the study. Assess the role of WT1 expression in AML patients undergoing an allogeneic HSCT, to

predict relapse and drive immune intervention

Methods. We studied 81 patients with AML who received an allogeneic HSCT between 30/1/04 and 11/2/08 in our Unit: 52 patients were in first remission, and 29 patients had more advanced disease. Median age 45 (16–66), prepared with CY-TBI (n=52) or RIC regimen (n=29). WT1 mRNA levels of expression were normalized with respect to the number of ABL transcripts and expressed as WT1 copy numbers every 104 copies of ABL. Patients bone marrow cells were monitored for WT1 expression, and cytology, pretransplant, at day +30 and every month untill day +180, then at scheduled follow up. Patients were considered WT1 positive, when WT1 copy numbers were greater than 180. Escalating doses of donor lymphocyte infusions (DLI) were given, when possible, in WT1+ patients.

WT1 pre-transplant. WT1 pre-HSCT was available in 63 patients, 39 were WT1- and 24 WT1+: their relapse risk was 10% vs 62% (p=0.00002) and their crude survival 79% vs 29% (p=0.0001). When CR1 patients were selected, relapse was 10% vs 50% for WT1- vs WT1+ patients (p=0.02), and survival 81% vs 50% (p=0.9).

WT1 post-transplant. Fifty patients were always WT1- after HSCT, and 31 were found WT1+ at a median interval from HSCT of 80 days (range 24–696): relapse was 8% vs 68% for WT1- vs WT1+ patients (p=<0.0001), and survival 78% vs 26% (p=0.0001).

Cox analysis: in multivariate analysis on relapse WT1 pre-transplant (p<0.0001) and post-transplant (p<0.0001) were independent predictors, whereas disease phase (CR 1/>1) was not (p=0.39). Survival was predicted by all 3 variables, WT1+ pre (p=0.001), WT1+ post (p=0.03) and phase of disease (p=0.002).

Immune intervention. Of the 31 WT1+ patients, 13 had BM blasts on cytology (6– 80%) at the time of first WT1 positivity, and were treated with chemotherapy. 18 patients were WT1+ and had marrow in CR on cytology: 9 of these received DLI and 9 did not; hematologic relapses were seen in 2/9 and 7/9 respectively (p=0.02) and crude survival is 6/9 vs 2/9 (p=0.07). The median interval between WT1+ and relapse was 96 days (39– 246).

Conclusions. This study suggests that WT1 positivity before and after HSCT are independent predictors of relapse and survival in patients with AML. WT1 expression predicts relapse better than disease phase. The interval between WT1 positivity and relapse is approximately 3 months: the use of DLI in this time interval appears to protect against hematologic relapse.
Late Mortality and Relapse Following HLA-Identical Sibling Donor Marrow Transplantation for Chronic Myeloid Leukemia.
Authors: Copelan Edward , Crilley Pamela , Szer Jeffrey , Dodds Anthony J. , Stevenson Dustin E. , Phillips Gary , Elder Patrick , Nivison-Smith Ian , Avalos Belinda , Penza Sam , Topolsky David , Sobecks R. , Kalaycio Matt , Bolwell Brian J.
Published: October 2019

We studied the incidence and reasons for late failure following allogeneic transplantation for chronic myeloid leukemia (CML). Three-hundred thirty-five adult patients (median age 37, 229 chronic, 62 accelerated, 44 blastic phase) who underwent allogeneic marrow transplantation from HLA identical siblings at 7 institutions from 1984 – 1995 following preparation with busulfan and cyclophosphamide (BuCy2), the most commonly used regimen in this circumstance. Cyclosporine or tacrolimus-based regimens were used to prevent graft-versus-host-disease (GVHD). Median follow-up of surviving patients exceeded 14 years. Of 182 patients alive and free of leukemia at 3 years, 77.0% (95% CI: 65 to 83.6%) survive at 18 years and 61.9% (95% CI: 52.1 – 70.3%) survive without relapse. Twenty-seven (42.2%) of the 64 total relapses occurred beyond 3 years. Death occurred less frequently (P<.001) and the interval from relapse to death was longer (P=.01) in patients who relapsed late compared to those who relapsed early. The hazard rate for relapse fell rapidly from year 1 through 5, then remained low but constant through year 15. The cumulative incidence of late (beyond 3 years) relapse for patients alive and free of disease at 3 years was 22.8%.. The cumulative incidence of non-relapse mortality was 18.1% for those alive and leukemia-free at 3 years and in the 36 patients who died beyond 3 years, the primary cause of death was chronic GVHD in 11, relapse in 7, new malignancy in 6, organ failure in 6 (pulmonary in 4) and infection in 2. Multivariate analysis demonstrated that advanced disease stage (HR 2.75, P=.001) and older age (HR 1.39, P = .032) were associated with diminished LFS of patients alive and free of disease at 3 years. Overall survival was also adversely influenced by older age (HR 3.86, P < .001) and advanced stage (HR 1.54, P=.024). Advanced disease phase was associated with a higher incidence of late relapse (HR ≥ 2.50, P=.039) and late NRM (HR 3.18, P=.008). Acute GVHD and short interval from diagnosis to transplant influenced early, but not late failure. Chronic GVHD was associated with lower incidence of late relapse (HR=.30, P=.002) but did not significantly influence survival or LFS. Overall, nearly 2 of every 5 patients alive and free of leukemia at 3 years died or relapsed over the subsequent 15 years. No plateau in LFS was seen.

Figure Figure
Pre-Transplant Conditioning Using Intravenous Busulfan Is a Feasible and Effective Option in Reduced-Intensity Cord Blood Transplantation.
Authors: Uchida Naoyuki , Yamamoto Hisashi , Matsuno Naofumi , Ishiwata Kazuya , Tsuji Masanori , Takagi Shinsuke , Araoka Hideki , Kato Daisuke , Yoshimi Mayumi , Seo Sachiko , Masuoka Kazuhiro , Wake Atsushi , Narita Madoka , Sagawa Kimiko , Yoneyama Akiko , Makino Shigeyoshi , Taniguchi Shuichi
Published: October 2019

Although cord blood (CB) transplantation with reduced-intensity conditioning (RICBT) has been widely applied to those who lack available related or unrelated donors and are not eligible for conventional conditioning, the optimal pre-transplant conditioning has not been established yet. Intravenous busulfan (ivBu) has been incorporated in conditioning regimens before bone marrow or peripheral blood stem cell transplantation, but the feasibility in cord blood transplantation has been controversial. In order to address this issue, we retrospectively reviewed patients who underwent RICBT including ivBu. Twenty-one patients, who were at ECOG performance status of 2 or less and received RICBT at our institute from June 2007 to April 2008 consecutively, were included in this study. Median age was 56 years (range, 21–71). Seventeen were AML, 3 were ALL, and 1 was MDS RA. All but 2 (MDS RA and ALL in CR2) were not in remission, 5 had CNS involvement, and 9 had history of prior transplantation. Seventeen had HCT-specific comorbidity index score ≥1, including 8 ≥2, and 6 ≥3. Fludarabine phosphate 125–180mg/m2 and ivBu 6.4–12.8mg/kg were used in all patients. All but 2 received additional melphalan (Mel) or total body irradiation (TBI); Mel 80–140mg/m2 for 10, 2–8Gy of TBI for 8, and Mel 40mg/m2 plus 4Gy of TBI for 1. Graft-versus-host disease prophylaxis consisted of tacrolimus plus mycophenolate mofetil in 11, tacrolimus alone in 8, and cyclosporine alone in 1. Single cord blood units, matched at 4/6 in 20 and 5/6 in 1, of 2 x 107/kg of recipient body weight at minimum were infused. Seventeen achieved neutrophil recovery ≥500/μl on day 19 post-transplant at medium (range, 12–30). Among 4 who failed to achieve neutrophil recovery, 2 experienced early disease progression, 1 showed rejection, and 1 died before engraftment due to interstitial pneumonia. All 17 patients who achieved neutrophil engraftment and 1 who died before engraftment showed complete donor-type chimerism on 13 days post-transplant at medium (range, 8–35). Median follow-up time of survivors was 398 days (range, 162–532) post-transplant. Cumulative incidence of non-relapse mortality at day 100 and day 365 post-transplant were both 19%. No VOD/SOS was observed. Disease-free survival and overall survival (OS) at 1 year post-transplant were 40±11% and 61±9%, respectively. Thus, ivBu can be safely administered, and have potential to achieve successful engraftment as well as sufficient disease control, indicating possible benefit of incorporating this drug in pre-transplant conditioning of RICBT. The optimal dosing schedule or combination with other therapeutic modalities needs further investigation in prospective clinical trials.
Genetic Variations in CYP2B6 and Risk of Cyclophosphamide Toxicity in Patients Undergoing Myeloablative Stem Cell Transplantation.
Authors: Melanson Stacy E , Antin Joseph H. , Court Michael H. , Ho Vincent , Ritz Jerome , Kim Haesook T. , Kuo Frank C , Longtine Janina A , Jarolim Petr
Published: October 2019

In myeloablative stem cell transplantation (SCT), most patients receive cyclophosphamide (CPA) conditioning based on body weight or body surface area, under the assumption that each individual will metabolize the drug with the same efficiency. However, CPA requires biological activation, primarily by cytochrome P450 2B6 (CYP2B6), to exert its effects; therefore, patients with increased protein activity, as reported for *4 and *6 allelic variants, may have an increased risk for CPA-associated toxicity compared to patients with decreased protein activity, as reported for *5 and *7 allelic variants. In this study, we determine the relationship between allelic variants of CYP2B6 and toxicity outcomes and attempt to clarify the influence of CYP2B6 on the development of severe cardiac toxicity, veno-occlusive disease (VOD), and idiopathic pneumonia syndrome (IPS) early after SCT. A total of 259 DNA samples were obtained from patients who received 3.6 g/m2 or 120 mg/kg of CPA as part of a myeloablative allogeneic SCT between 2002 and 2006. Genotyping was performed using pyrosequencing to detect three SNPs, 516A>G, 785A>G and 1459C>T, associated with four allelic variants: *4 (785A>G), *5 (1459C>T), *6 (785A>G, 516G>T) and *7 (785A>G, 516G>T, 1459C>T). The patients’ clinical history was reviewed and any instance of hepatic VOD, severe cardiac toxicity (i.e. myopericarditis ≥ grade 3) or IPS from 0 to 100 days post-transplant was recorded. Overall, 60 (24%) patients experienced toxicity (cardiac = 20; VOD = 26; IPS = 14). The prevalence of the *4, *5, *6 and *7 alleles was 3%, 8%, 23% and 2%; respectively. Patients heterozygous for *4, *5, *6 and *7 allelic variants and homozygous for the *6 allelic variant had similar rates of toxicity to those patients without allelic variants of CYP2B6, even when comparing each toxicity separately (Table 1). However, patients with decreased protein activity, *1/*5 and *1/*7, had a lower rate of toxicity compared to those with wild-type or increased protein activity, *1/*1, *1/*4, *1/*6 and *6/*6 (14.6% (7/48) vs. 26.1% (53/203)). In conclusion, variant alleles of CYP2B6 may predict a decreased risk of VOD, IPS, or cardiac toxicity within the first 100 days of SCT, but do not appear to predict an increased risk of toxicity.

Table 1. Association of toxicities with genotypes

Genotype Patients All Toxicities Cardiac Grade ≥3 VOD IPS *One patient had both cardiac and VOD toxicity * 1/*1 97 27 (28%) 7 (7%) 12 (12%) 8 (8%) * 1/*4 8 3 (38%) 0 (0%) 2 (25%) 1 (13%) * 1/*5 38 5 (14%) 2 (6%) 1 (3%) 1 (3%) * 1/*6 80 18 (23%) 7 (9%) 8 (10%) 3 (4%) * 1/*7 10 2 (20%) 1 (10%) 1 (10%) 0 (0%) * 6/*6 18 5 (28%)* 3 (17%) 2 (11%) 1 (6%)
Allogeneic Stem Cell Transplantation for Patients with Acute Leukemia Transformed from Myelofibrosis
Authors: Ciurea Stefan O , De Lima Marcos , Giralt Sergio , Qazilbash Muzaffar H. , Alousi Amin , Kebriaei Partow , Khouri Issa F. , Anderlini Paolo , Andersson Borje , Hosing Chitra , Verstovsek Srdan , Kantarjian Hagop , Champlin Richard , Popat Uday
Published: October 2019

Background and Rationale: Transformation of myelofibrosis (MF) to acute leukemia (>20% blasts) portends a grave prognosis. Allogeneic stem cell transplantation (ASCT) is a curative approach for patients with acute leukemia; however, the outcomes of ASCT in patients with MF transformed to acute leukemia are currently unknown.

Methods: Fifty-one consecutive patients with either primary (PMF) or secondary (SMF) were transplanted at UTMDACC after 1994. Thirteen patients who developed AML (25%), 10 arising from PMF and 3 with SMF, received an ASCT from a sibling or matched unrelated donor. Median age was 59 years. Five patients (38%) had prior splenectomy. JAK2V617F mutation analysis was performed in 7, and was present in 5 patients. Cytogenetics were intermediate in 10 and poor-risk in 3 patients. Seven patients (54%) were not in remission at the time of transplant. Eleven of 13 patients received induction chemotherapy; 6 achieved remission, while 7 had persistent disease at the time of transplant. One patient had a prior autologous transplant and 2 patients had prior allogeneic transplant for myelofibrosis. The donors were matched siblings (7 patients), matched unrelated (4 patients) and 1 antigen mismatched relatives (2 patients). The stem cell source was peripheral blood in 9 and bone marrow in 4 patients. Nine patients received a reduced-intensity conditioning regimen with a fludrabine-melphalan-based regimen, and 4 myeloablative conditioning (3 fludarabine-busulfan, 1 busulfan-cyclophosphamide).

Results: All patients engrafted, 75% achieved full donor chimerism, on day 30. Neutrophil and platelet engraftment occurred after a median of 13 and 21.5 days. Twelve evaluable patients achieved remission; 3 subsequently relapsed. JAK2V617F mutation became negative after transplant in all tested patients and reappeared in 1 patient who later relapsed. Grade 2–4 aGVHD developed in 3 patients (grade 3–4 in one) and cGVHD in 4/11 evaluable patients (extensive in two). After a median follow-up of 17.2 months (range 7.2–128.6 mo), OS and EFS were 49% (SE 15%) and 44% (SE 14%), respectively (Figure1). Six patients died, related to disease relapse (2), pneumonia (2), GVHD (1), and hepatic failure (1).

Conclusion: Patients with acute leukemia transformed from myelofibrosis with good performance status can achieve durable complete remissions with ASCT.

Figure 1. Overall survival (continuous line) and disease-free survival (interrupted line) in 13 patients with acute leukemia transformed from MF post ASCT after a median follow-up of 17.2 months. Figure 1. Overall survival (continuous line) and disease-free survival (interrupted line) in 13 patients with acute leukemia transformed from MF post ASCT after a median follow-up of 17.2 months.
Evaluation of Serum Free Light Chains and Outcome in Multiple Myeloma Patients with an Intact Monoclonal Immunoglobulin Treated with Autologous Stem Cell Transplantation.
Authors: Trieu Young , Xu Wei , Anglin Peter , Chen Christine , Kukreti Vishal , Trudel Suzanne , Reece Donna E.
Published: October 2019

Introduction: The serum free light chain (FLC) assay is a useful tool in diagnosing and monitoring multiple myeloma (MM) patients (pts) with non-secretory and light chain only disease. In addition, the detection of an abnormal serum FLC ratio is an adverse prognostic factor in pts with monoclonal gammopathy of undetermined significance. However, the relationship of the FLC assay to the outcome of patients with an intact monoclonal immunoglobulin following a single autologous stem cell transplantation (ASCT) has not been studied. Thus, the objective of this single centre, retrospective review study was to evaluate the usefulness of the FLC assay as a predictor for response rate and progression free survival (PFS) in this category of pts.

Patients & Methods: We identified in our Princess Margaret Hospital MM database a total of 290 pts who underwent a single ASCT between June 2003 and May 2006. Of these, 65 had an intact monoclonal immunoglobulin (IgG in 47, IgA in 16 and IgD in 2) detected at diagnosis plus FLCs measured at referral. Normal range for FLC measurements is as follows: kappa 3.3–13.1 mg/L, lambda 5.7–26.3 mg/L, and kappa/lambda ratio of 0.26–1.65.

Results: The median age at diagnosis was 59 years (range, 34–73); 33 (51%) were male. The median time from diagnosis to ASCT was 9.0 months (range, 5.0–29), with a median follow-up time of 27 months (range, 1.0–58.0). Assessment of best response following ASCT revealed that 20 (31%) pts achieved CR/nCR, 21 (32%) VGPR, 21 (32%) PR, 2 (3%) MR, and 1 (2%) was not evaluable for response. No prognostic factors for response were identified. To date, only 9 pts have died and the median overall survival is not yet reached. The median PFS is 25.4 months, with 36 patients progressing after ASCT. An elevated kappa and lambda light chain was detected in 30 (46%) and 22 (34%) of the 65 pts, respectively. Additionally, 52 (82%) of the 65 pts were found to have an abnormal kappa/lambda ratio. There was no significant difference in the PFS of patients with abnormal vs. normal free kappa light chains or FLC ratio. However, a decreased PFS was associated with elevated levels of serum free lambda light chains (p=0.01), β-2 microglobulin (p=0.007) and LDH (p=0.01).

Conclusions:

The majority of pts with an intact monoclonal immunoglobulin also have an abnormally high level of the corresponding serum FLC and an abnormal FLC ratio; an elevated serum free lambda level as well as increased β-2 microglobulin and LDH levels, as previously described, were identified as adverse prognostic factors for PFS in this population; we continue to routinely assess serum FLC for all pts at referral; however, longer follow-up is needed to further evaluate the prognostic significance of this parameter on the clinical outcome of pts.
Molecular Signatures to Improve Diagnosis, Prognostication and Identification of Oncogenic Pathways in Peripheral T and NK Cell Lymphoma.
Authors: Iqbal Javeed , Weisenburger Dennis , Greiner Timothy C. , Nakamura Shigeo , Vose Julie M. , Seto Masao , Delabie Jan , Berger Francoise , Loong Florence , Au Wing-Yan , Ko Young-Hyeh , Sng Ivy , Smith Lynette M , Kucuk Can , Geng Huimin , Armitage James , Chan Wing C.
Published: October 2019

Background: Peripheral T-cell lymphoma (PTCL) consists of an uncommon and heterogeneous group of lymphomas that are often challenging to diagnose and classify. Since most patients also have a poor survival with standard multiagent chemotherapy, more effective therapeutic approaches are needed to improve patient outcome.

Table1:

Pathological diagnosis Number of cases profiled AITL 36 ALK(+)ALCL 19 ALK (−)ALCL 08 ATLL 12 T/NK 14 PTCLU 44 Other rare entities 10

Methods: A mRNA profiling study using Affymetrix HGU133+2 arrays on 143 cases of PTCL and NK-cell lymphoma (NKCL) from the International Peripheral T-cell Lymphoma Project, was conducted on pre-treatment biopsies. These included the following pathologically classified cases (Table 1). In addition, we also profiled nine NK cell lines, seven T cell lines, normal resting and activated CD4+ and CD8+ T cells and resting and IL2- activated NK cells from healthy individuals. BRB-ArrayTools was used to develop gene classifiers for the major PTCL entities and survival predictors for AITL based on gene expression data.

Results: We have identified key molecular signatures for PTCL and NKCL that have allowed us to construct a robust classifier for AITL (207 transcripts), ALK+ ALCL (94), ATLL (225) and NKCL (127). PTCL-U group may have 3 or 4 molecular subgroups and additional studies with more cases, are necessary to further define this group. Misclassified cases were identified and re-assigned to the molecularly defined entities, including re-assigning of 9/44 PTCL-U to AITL. We have confirmed the enriched expression of genes identified in follicular helper T-cells in AITL, suggesting that AITL is derived from this T-cell subset. A number of oncogenic pathways (e.g. NF-κB, HIF-a,VEGF, IL6) and tumor/host interactions that contributed to local tumor-induced immunosuppression (e.g. TGF-b), were identified in AITL. A molecular predictor of outcome was developed for AITL and validated by leave one-out-cross validation. Since PTCL is an uncommon disease, future studies will require the collaboration of multiple large clinical groups with tissue resources for both discovery and validation.

Conclusion: This study has demonstrated that GEP will allow the construction of robust and biologically-meaningful classifiers for PTCL, and prognosticators can be derived for well-defined entities with a sufficient number of cases. GEP will also allow us to identify therapeutically-relevant oncogenic pathways and tumor/host interactions that may lead to improvement in the therapy and outcome of patients with PTCL and NKCL. (This study is a part of the International T-cell Lymphoma Project)
Limitations of Current Plasma VonWillebrand Factor (VWF) Activity Tests: A Comprehensive Comparison of Five VWF Activity Assays.
Authors: Chen Dong , Pruthi Rajiv , Nichols William L. , Heit John A.
Published: October 2019

Accurate measurement of plasma von Willebrand factor (VWF) activity is essential for the laboratory diagnosis and treatment monitoring of von Willebrand disease (VWD).

Currently available VWF activity assays include VWF ristocetin cofactor activity by manual light transmission platelet aggregometry (VWF:RCo–Agg) or flow cytometry (VWF:RCo–FL), collagen I and III binding activity (VWF:Co–I and –III) (Technozym), and platelet activity by latex agglutination (VWF:Lx) (Instrumental Laboratory). In this study we evaluated and compared the accuracy and precision of these 5 assay methods. Plasma samples from 11 normal donors and 41 patients categorized as type 1 (n=20) or type 2 (n=21) VWD based on clinical evaluation, fVIII:C activity, VWF:RCo–Agg, VWF antigen (VWF:Ag) level and plasma VWF multimer analysis by agarose gel electrophoresis were assayed for VWF activity by VWF:RCo–FL, VWF:Co–I, VWF:Co–III and VWF:Lx methods. The VWF:Ag/VWF activity ratio by VWF activity assay method was calculated for each sample. For normal donors and type I VWD patients, VWF:RCo–FL and VWF:Lx correlated well with VWF:RCo–Agg (R2=0.87, and 0.97, respectively), while VWF:Co–I and –III were lower compared to VWF:RCo-Agg. For type 2 VWD patients, different VWF:Ag/VWF activity ratio cutoffs (range 0.3–0.7) were used (Figure). Both VWF:RCo–Agg and –FL were sensitive (95%) and specific (97%) for type 2 VWD while the VWF:Lx was slightly less sensitive (81%) but was very specific (100%). VWF:Co–I and –III were the least sensitive (<90%) and specific (<90%); both methods had high false positive and negative rates for type 2 VWD. In Summary, for normals and type 1 VWD patients, VWF:RCo–FL and VWF:Lx correlate well with VWF:RCo–Agg and have similar sensitivities and specificities for type 2 VWD. VWF:Co–I and –III are unreliable for assessing plasma VWF activity.
Cell-Based Immunotherapy with Different Subsets of Tolerogenic Dendritic Cell in Idiopathic Thrombocytopenic Purpura.
Authors: Zhang Xiao-Lin , Peng Jun , Xu Shu-Qian , Liu Xin-Guang , Yu Yuan , Qin Ping , Shi Yan , Wang Lin , Hou Ming
Published: October 2019

There is growing evidence that tolerogenic dendritic cells (DCs) play an important role in maintaining peripheral tolerance through the induction of anergic or regulatory T cells. However, in humans, little is known about the ability of tolerogenic DCs to induce tolerance to autoantigens in autoimmune patients. Idiopathic thrombocytopenic purpura (ITP) is an immune-mediated disease in which platelets are destroyed by antiplatelet autoantibodies. Here, we explored in vitro the ability of four subsets of tolerogenic DCs (i.e., immature DCs (imDCs), IL-10-modulated DCs (IL-10-DCs), vasoactive intestinal peptide-modulated DCs (VIP-DCs) or plasmacytoid DCs (pDCs)) derived from patients with ITP, to induce an anergic state or regulatory T cells in autologous platelet glycoprotein (GP)-specific T cells. GPIIb/IIIa-reactive T cells were preincubated with GPIIb/IIIa-loaded imDCs, IL- 10-DCs, pDCs or VIP-DCs, and then rechallenged with autologous mature DCs (mDCs) in the presence of GPIIb/IIIa. Only when T cells were cultured with GPIIb/IIIa-loaded VIP-DCs in primary incubation, inhibited proliferation of GPIIb/IIIa-reactive T cells could be observed at rechallenge with GPIIb/IIIa-loaded mDCs. The anergic state of VIPDC- primed GPIIb/IIIa-reactive T cells could be reversed when rechallenged with GPIIb/ IIIa-loaded mDCs in the presence of a high concentration of exogenous IL-2. Meanwhile, GPIIb/IIIa-reactive T cells were also cultured with VIP-DCs loaded with tetanus toxoid (TT). In contrast to T cells pretreated with GPIIb/IIIa-loaded VIP-DCs, GPIIb/IIIareactive T cells pretreated with TT-loaded VIP-DCs proliferated when rechallenged with GPIIb/IIIa-loaded mDCs, which demonstrated that the induced anergy of autoreactive T cells is antigen specific. Additionally, functional analysis showed that VIP-DC-modulated T cells could not suppress the proliferation of newly induced GPIIb/IIIa-reactive T cells when cocultured with GPIIb/IIIa-loaded mDCs. These results indicated that VIP-DCs could induce autoreactive T cells anergic but not functionally suppressive. Moreover, we found that coculture of VIP-DCs with autologous PBMCs resulted in reduced production of anti-GPIIb/IIIa antibodies, suggesting that GPIIb/IIIa-reactive T cells lost their helper function for inducing autoantibody production by B cells. In contrast, reduced antibody production could not be found when autologous PBMCs were cocultured with imDCs, IL-10-DCs or pDCs. In conclusion, our studies revealed the therapeutic potential of VIPDCs, compared with imDCs, IL-10-DCs or pDCs, to induce autoreactive T-cell anergy to GP antigens, which would in turn facilitate the reestablishment of autoantigen-specific tolerance in patients with ITP.
Changing Trends in ITP Management from 1995 to 2007: An Interim Report from the UK Paediatric ITP Registry.
Authors: Grainger John D. , Rees Joanne L , Reeves Marie , Bolton-Maggs Paula HB
Published: October 2019

A UK national paediatric ITP registry (www.uk-itp.org) was set up in January 2007 designed to collect data from all newly diagnosed ITP over at least a subsequent 10 year period. By May 2008, 62 hospitals had recruited 114 children. Details about epidemiology, presentation and management were entered into the registry. The preliminary data presented was compared with results from two national UK audits in 1995 and 2000.

In this first analysis, 68% children had mild, 29% had moderate and 3% had severe disease as defined by a modified Buchanan bleeding score. The mean platelet counts at diagnosis for the groups were 18.3, 10.1 and 8.0 respectively. Bruising (mean 86.7%) and purpura (mean 73.0%) were the most common bleeding manifestations in ITP followed by epistaxis (mean 22.0%) and oral bleeding (mean 18.7%). Other bleeding sites such as bleeding from the gastrointestinal tract and menorrhagia are much less common and there were no intracranial bleeds. Children with mild disease have fewer bleeding sites (mean 1.3), compared to 2.3 with moderate and 3.0 for severe disease. These data are very similar to those reported in the previous national audits.

The proportion of children receiving platelet raising treatment was noted to decrease from 60.5% in 1995 to 37.8% in 2000. The current 2007 registry data shows a continued decrease in treatment to 20% of all the children. In the mild category 11.8% were treated, compared to 31% of the moderate and all of the severe. Of those receiving treatment 75% were treated with steroids alone, 15% IVIG alone and 10% received combination IVIG and steroids. Forty seven percent were admitted to hospital with a median duration of stay at 2 days. In summary the current UK Paediatric ITP Registry data shows similar epidemiological data to the previous UK audits. The previous observed decrease in treatment and adoption of a “watch and monitor” policy has increased to the extent that 80% of children do not receive platelet raising therapy. Continued recruitment and analysis is required to provide more data on which patients can safely avoid platelet raising therapy and which patients require prompt treatment to raise their platelet count.
PTPN22 Mutation Is Present in An Increased Proportion of ITP Patients.
Authors: D’Silva Karl , Zamora Mathew , Gerlach John , Schwartz Kenneth A.
Published: October 2019

Immune-mediated thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by low platelet count and antibody mediated platelet destruction. ITP may be due to a failure of T-cell tolerance. Protein tyrosine phosphatase non receptor type 22 (PTPN22) is an important negative regulator of signal transduction from the T cell receptor. A single nucleotide polymorphism 1858C>T in the PTPN22 gene has been associated with various autoimmune disorders. We hypothesize that the PTPN22 mutation 1858C>T would be present in a higher proportion of ITP patients and patients who carry the mutation would have an increased concentration of autologous antiplatelet IgG on their platelet surface. We genotyped 45 patients with ITP by a PCR based restriction length polymorphism(RFLP) to identify the mutation 1858C>T in the gene PTPN22. Ten patients (22%) were heterozygous for the PTPN22 mutation and 2 were homozygous for the mutation (4.4%). This compares with a frequency of 8.6% observed in a published population study of 960 controls who did not have autoimmune disease (p<0.0001) (Am. J. Hu. Genetics2004:75:330). All of the 12 ITP patients who carried the PTPN22 mutation had an increase in autologous platelet surface IgG (≥1250 molecules IgG/platelet)(Schwartz Am. J. Heme1990;33:167). We conclude that the PTPN22 1858C>T mutation is present in an increased proportion of ITP patients and that all 12 of the PTPN22 mutation positive patients were also positive for autologous antiplatelet IgG. Our results indicate that PTPN22 increases the probability of developing ITP.
Drug Dependent Immune Destruction of Human Platelets in the NOD/SCID Mouse.
Authors: Bougie Daniel W. , Boylan Brian , Nayak Dhirendra , Newman Peter J. , Aster Richard H.
Published: October 2019

Animal models have been of limited value for studying survival and immune destruction of human blood cells because of xenoantibodies that mediate rapid elimination of the transfused cells. The NOD/SCID mouse is a genetic variant that lacks immunoglobulins, including xenoantibodies, and may therefore be useful for studying survival and immune destruction of human blood cells (Newman, PJ et al. J Thromb Haemost.2007; 5 Suppl 1:305–9). Human platelets, infused into the retroorbital plexus of NOD/SCID mice survive about three days, nearly as long as mouse platelets themselves. Injection of IgG from a patient with quinine-induced immune thrombocytopenia (TP) and a newly developed quinine-dependent murine monoclonal antibody (mAb) were without effect on platelet survival. However, both antibodies caused rapid platelet clearance after IP injection of quinine. 500 μg, but not 50 μg, of IgG from serum containing an anti-HPA-1a (PlA1) antibody also caused rapid clearance of HPA-1a positive platelets. These findings indicate that the NOD/SCID mouse can be valuable for the study of immune clearance of human platelets and perhaps other unresolved problems in transfusion medicine. However, it is not known whether mice recognize cells opsonized with human IgG or mouse IgG equally well. To examine this, we compared the survival of human platelets sensitized with 1) mAb 7E3 (IgG1) specific for an epitope in the beta A domain of GPIIIa or 2) c7E3 (from Centocor Inc), which recognizes the same epitope but possesses a human IgG1 Fc. In titration studies, we found that about four times as much c7E3 as 7E3 is needed to produce equivalent clearance of human platelets, indicating that mouse IgG is a more efficient opsonin than human IgG. However, this difference should not limit use of the model for studies of antibody-mediated blood cell destruction, since mg quantities of IgG can easily be isolated from small amounts (0.1– 0.2 ml) of human serum.
Rare SOX13 Sequence Variations in Pediatric Idiopathic Thrombocytopenic Purpura Patients.
Authors: Rischewski Johannes R , Wyss Johanna , Stocker Sylvie , Hergersberg Martin , Huber Andreas R , Kühne Thomas
Published: October 2019

Introduction: Idiopathic or immune-thrombocytopenic purpura (ITP) is defined as a bleeding disorder with the hallmark of autoimmune mediated thrombocytopenia. ITP is a diagnosis of exclusion. Primary biliary cirrhosis (PBC) is a presumed autoimmune disease of the liver. Many nonhepatic autoimmune diseases are found in association with PBC, and may prompt initial presentation. Several case reports described the co-occurrence of ITP and PBC. In some PBC patients, autoantibodies against SOX13 can be detected. SOX13 has been identified as a transcription factor promoting gamma-delta T-cell development while opposing alpha-beta T cell differentiation. As ITP has been linked to the autoimmune bicytopenic Evans syndrome, which may present as autoimmune lymphoproliferative syndrome (ALPS) with the hallmark of increased alpha-beta double negative T-cells, we are interested in a potentially etiological role of molecular SOX13 sequence variations in ITP patients.

Material and methods: After obtaining informed consent from ITP patients (n=34) and their legal guardians to participate in the IRB approved study genomic DNA was extracted from peripheral blood samples. The complete coding region of SOX13 including the classical non-coding splice sites was amplified by PCR. The amplicons were screened for sequence variations by Denaturing High Performance Liquid Chromatography (DHPLC) after heteroduplex induction. All samples with aberrant DHPLC retention patterns were directly sequenced.

Results: Two rare heterozygous sequence variations in the coding region of SOX13 were detected in a total of 4 patients (12%): c.1603C>T (Pro534Ser) and c.1836 C>T (synonymous). Discussion: In a healthy cohort with European ancestry the heterozygous c.1603C>T (Pro534Ser) genotype was detected in 1.7% of individuals. In our cohort 6% of the ITP patients were heterozygous. 2.6% of individuals in a cohort of African-Americans and Caucasians carried the heterozygous genotype c.1836C>T (synonymous). 6% of the investigated ITP patients were heterozygous. A surprising high proportion (12%) of pediatric ITP patients carries one of two rare SOX13 sequence variations. The rare heterozygous genotypes are 3.5 (c.1603C>T) respectively 2.3 times (c.1836C>T) more common than in published non-ITP cohorts.

Conclusion: It seems possible, that rare alleles of SOX13 accumulate in pediatric ITP cohorts. Larger case-control sample studies are needed to verify the results. The rare genotypes could influence the alpha-beta versus gamma-delta T-cell balance in the studied pediatric ITP patients. Immunological studies of our cohort are necessary to evaluate this suggested association.
A Transgenic Mouse Model for Congenital Dyserythropoietic Anemia Type I
Authors: Renella Raffaele , Roberts Nigel A , Sharpe Jacqueline A , Li Pik-Shan , Brown Jill M , Babbs Christian , Fugazza Cristina , Goardon Nicolas , Higgs Douglas R , Wood William G
Published: October 2019

The Congenital Dyseyrthropoetic Anemias (CDA) are a heterogeneous group of inborn defects characterized by anemia of varying severity and morphological abnormalities of bone marrow erythroblasts. Studying their pathology has the potential to contribute to the understanding of the normal process of erythropoiesis. In CDA type I, the phenotype is largely limited to the red cell lineage with erythroblasts showing characteristic spongy nuclear chromatin on electron microscopy. Its genetic basis is due to missense mutations in the CDAN1 gene (chromosome 15q15), but the biological function of the highly conserved and non-redundant codanin-1 protein remains entirely unknown.

We have produced a mouse model for CDA type I by generating transgenic mice from an ES cell line carrying a gene-trap (splice acceptor/β-galactosidase gene/neomycin resistance gene) in intron 25 of the murine CDAN1 gene. Blood and bone marrow from CDAN1gt/wt heterozygotes is morphologically and quantitatively indistinguishable from wild type animals. Northern blot analysis and RT-qPCR of codanin-1 RNA expression confirms a broad pattern of expression with the highest levels seen in erythroid tissue. Flow-cytometric analysis of β-galactosidase activity in erythroid cells from phenylhydrazine treated adult spleens shows the highest levels in the CD71high/Ter119low early erythroblasts, declining with further erythroid maturation. Furthermore, indirect immunofluorescence localizes the codanin-1/β-galactosidase fusion product in the nucleus of the CD71high/Ter119low early erythroblasts, particularly at the interface between euchromatin and heterochromatin. The presence of an in-frame β-gal in the gene trap insert, therefore, provides a method to analyze gene and protein expression from the CDAN1 promoter, and additional distribution data from heterozygous embryos and adults will be presented. Heterozygote crosses produced no homozygotes among liveborn progeny, suggesting embryonic lethality of this state. Analysis of embryos at different stages suggests development of homozygotes ceased at ~6.5d of gestation, prior to the onset of erythropoiesis.

These results from the first transgenic mouse model for CDA type I, highlight a non-erythroid role for codanin-1 in early embryonic development, in addition to its role in adult human erythropoiesis. The embryonic lethality of the mouse model is consistent with the absence of homozygote null mutations in the CDAN1 genes analyzed so far in human patients. Creation of mice with the CDA type I phenotype may have to await a knock-in transgenic containing a human mutation, which is currently in preparation. The model described here should be valuable for further studies of the intracellular localization of codanin-1 and the identification of any relevant protein binding partners.
High Levels of Growth Differentiation Factor 15 in Patients with Congenital Dyserythtopoietic Anemia Type I
Authors: Tamary Hannah , Shalev Hanna , Avraham Galit , Zoldan Meira , Levi Itai , Swinkels Dorine W. , Tanno Toshihiko , Miller Jeffery L
Published: October 2019

Growth differentiation factor 15 (GDF15) is a member of the TGF-beta superfamily of cytokines previously found to suppress hepcidin in primary human hepatocytes. GDF15 is secreted from human erythroblasts, and extremely high serum levels are present in β-thalassemia patients (Tanno et al., Nat. Med. 2007, 13, 1096–1101). To determine if elevated GDF15 levels are unique for thalassemia or more generally associated with iron-loading related to ineffective erythropoiesis, we determined the GDF15 levels, as well as, serum hepcidin (Swinkels DW et al, PLoS ONE PLoS ONE. 2008;3:e2706), ferritin, erythropoietin (EPO) and soluble transferrin receptor (sTfR) in patients with the congenital dyserythropoietic anemia type I (CDA I). Seventeen Israeli Bedouins with CDA I were studied, all homozygous for the founder R1040W mutation in the CDAN1 gene. All of the patients studied were young adults with a mean age of 29 years. Two patients previously underwent splenectomy, and one patient is currently transfusion-dependent. For comparison, ten healthy volunteers (HV) were studied. The mean level of GDF15 in CDA I patients was significantly elevated [10,239 ± 3,049 pg/ml (range 5,530–17,008) compared to 269 ± 238 pg/ml in healthy controls; p = 1.5×10−10]. Consistent with a previous study of dyserythropoietic anemia patients, significantly higher levels of soluble transferrin receptor were detected among the CDA I population (sTfR; CDA I, 86.4 ± 14.0 nmol/L; HV, 21.4 ± 6.2 nmol/L, p = 7.4×10−15). Serum EPO levels were also elevated (EPO; CDA I, 118 ± 59 IU/dL; HV, 2.0 ± 1.5 IU/dL, p = 2.3×10−7). For iron analyses, three patients with extensive transfusion histories were excluded. Among the remaining 14 patients, iron overload was demonstrated by elevated serum ferritin (CDA I, 916 ± 507 ng/ml; HV, 72 ± 60 ng/ml, p = 1.4×10−5). Despite the significant elevation in iron stores, significantly elevated levels of hepcidin 25 (Hep25) were not detected in the CDA I patients. Instead, a minor decrease in serum Hep25 levels were detected (Hep25; CDA I, 3.3 ± 2.8 nM; HV, 4.1 ± 3.0 nM, p = 0.27). Correlation analyses were performed between the iron parameters (Ferritin and Hep25) and GDF15, sTfR, or EPO levels. Only GDF15 demonstrated a significant positive correlation with ferritin and significant inverse correlations with Hep25 and the Hep25/Ferritin ratio. Weaker correlations with EPO were identified. Unexpectedly, the correlation trends for sTfR were opposite those of GDF15 in this group. These results demonstrate that GDF15 is immensely over-expressed in CDA I, and further suggest this cytokine contributes to hepcidin dysregulation and secondary hemochromatosis in humans with ineffective erythropoiesis.
Hsp90 regulates Ulk1-mediated autophagic clearance of mitochondria
Authors: Kundu Mondira , Yang Chia-Ying , McCastlain Kelly , Wu Junmin , Zhang Ji , Lindsten Tullia , Ney Paul , Thompson Craig B.
Published: October 2019

Recent studies have suggested that the serine-threonine kinase Ulk1, a mammalian homologue of the yeast autophagy regulator, Atg1, plays an important role in mitochondrial clearance during reticulocyte maturation. In order to gain insight into the regulation and activity of Ulk1, we used an unbiased proteomics approach to identify Ulk1-interacting proteins. Here, we demonstrate that Ulk1 interacts with heat shock protein 90 (Hsp90) and Cdc37, and is among a growing list of proteins whose steady state levels are regulated by this kinase-specific chaperone complex. Treatment of murine embryonic fibroblasts (MEFs) with the HSP90 inhibitor, 17AAG, inhibits the AKT/Tor pathway and induces autophagy despite the decreased steady state levels of Ulk1. By contrast, Ulk1-deficient MEFs are defective in autophagy induction, as measured by LC3 puncta formation (but not LC3 conversion) and ultrastructural analysis. Exposure of terminally differentiating erythroid cells to HSP90 inhibitors prevents the normal accumulation of Ulk1 protein and results in impaired autophagic clearance of organelles, similar to the effect of Ulk1 gene deletion. These findings highlight the importance of Ulk1 protein levels in terminal erythroid maturation and induction of autophagy and its regulation by the Hsp90/Cdc37 chaperone complex.