bluuebunny/crossref_metadata_2025 huggingface dataset:
Systematic Review of Randomized Controlled Trials of Prophylactic Clotting Factor Concentrate in Hemophilia.
Authors: Stobart Kent , Iorio Alfonso
Published: October 2019

Introduction: One of the current approaches to managing persons with hemophilia entails the use of prophylactic treatment with clotting factor concentrates (CFC) to prevent bleeds and bleeding related complications. However, this process is not practiced in all countries, or is based on the synthesis of evidence from data of all trials.

Objective: To conduct a systematic review/meta-analysis (SR/MA) regarding the effectiveness of clotting factor concentrate prophylaxis in the management of people with hemophilia A or B.

Methods: We conducted a SR of all randomized controlled trials (RCTs) that studied the effect of prophylactic CFCs in people with hemophilia A or B. We searched all major electronic databases (MEDLINE, LILACS, EMBASE, and the Cochrane Cystic Fibrosis and Genetic Disorders Group’s Controlled Trials Register) as well as performing handsearches of journals and relevant meeting abstract books. We included RCTs that that compared the use of prophylactic CFCs to a control group.

Results: We identified 29 studies, of which four (including 37 participants) were eligible for inclusion. Three studies evaluated hemophilia A; one showed a decrease in frequency of joint bleeds with prophylaxis compared to placebo (non-physiological dose), with a rate difference (RD) −10.80 (95% confidence interval (CI) −16.33 to −5.27) bleeds per year. The remaining two studies evaluating hemophilia A compared two prophylaxis regimens, one study showed no difference in joint bleed frequency, RD −5.04 (95%CI −17.02 to 6.94) bleeds per year and another failed to demonstrate an advantage of factor VIII dosing based on individual pharmacokinetic data over the standard prophylaxis regimen with RD −0.14 (95% CI −1.34 to 1.05) bleeds per year. The fourth study evaluated hemophilia B and showed fewer joint bleeds with weekly (15 IU/kg) versus bi-weekly (7.5 IU/kg) prophylaxis, RD −3.30 (95% CI −5.50 to − 1.10) bleeds per year.

Conclusion: There is insufficient evidence to determine whether prophylactic clotting factor concentrates decrease bleeding and bleeding-related complications in hemophilia A or B, compared to placebo, on-demand treatment, or prophylaxis based on pharmacokinetic data from individuals. Well-designed RCTs are needed to assess the effectiveness of prophylactic clotting factor concentrates.
Interobserver Agreement of Centrally Adjudicated Venous Ultrasound Video Documents in Asymptomatic Patients after Hip and Knee Replacement.
Authors: Schmidt Benjamin A. , Guenther Matthias , Halbritter Kai , Beyer Jan , Schellong Sebastian M.
Published: October 2019

Feasibility of venous ultrasound as an endpoint measure in multicenter trials critically depends on the type and quality of documents which can be assessed by central adjudication. We evaluated the offline assessability of standardized video documents of complete compression ultrasound (CCUS) in asymptomatic patients 7 days after hip or knee replacement. The study was performed at a single study center. Consecutive asymptomatic patients one week after hip or knee replacement were screened for deep vein thrombosis by complete compression ultrasound of proximal and distal veins including muscle veins by one trained sonographer. Examinations were recorded on video tape in a standardized format. Video sequences with an approximate duration of 60 seconds demonstrated femoral veins, popliteal veins, peroneal veins and tibial posterior veins, respectively. The video documents were digitized and electronically stored in a central adjudication unit. All videos were read by two independent readers, and veins were scored as normal, thrombotic or nonevaluable. All thrombotic findings were re-assessed by two senior readers according to the same protocol. Analysis of inter-observer agreement was performed by Cohen’s Kappa coefficient. Video documents of 300 legs were recorded in 150 patients. Documents of 10 patients (6.7%) or 13 legs (4.3%) were scored as non-evaluable by at least one reader. Reading A revealed 5.5% proximal DVT and 18.7% distal DVT including muscle vein thrombosis. Reading B revealed 4.1% and 23.6%, respectively. Kappa values for different types of DVT are given in the table.

Conclusion: In a single center setting, interobserver agreement of centrally adjudicated standardized CCUS documents in the diagnosis of asymptomatic postoperative DVT is strong for all categories of DVT. It remains to be established how these figures will be affected by a multicenter setting.

Interobserver Agreement Vein segment Kappa (95% CI) 1) Interobserver agreement according to number of legs (n=300) All vein segments 0.92 (0.84–0.99) Proximal deep vein segments 0.87 (0.70–1.00) Distal deep vein segments 0.84 (0.73–0.94) Femoral veins - Popliteal and confluent veins 0.87 (0.70–1.00) Peroneal veins 0.79 (0.60–0.99) Posterior tibial veins 0.83 (0.60–1.00) Calf muscle veins 0.77 (0.64–0.91) 2) Interobserver agreement according to number of patients (n=150) All vein segments 0.90 (0.81–0.99) Proximal deep vein segments 0.85 (0.65–1.00) Distal deep vein segments 0.81 (0.65–0.97)
Immune Tolerance Resulting in a Dramatic Clinical Improvement in a High-Responding Inhibitor Hemophilia A Patient Using Immunosuppression with Mycofenolate Mofetil and a Factor VIII Concentrate Containing Von Willebrand Factor.
Authors: Lambert Thierry , Goujard Cécile , Rafowicz Anne , Guillet Benoît , Taoufik Yacine , Dreyfus Marie
Published: October 2019

A 40 year-old Caucasian patient with severe familial hemophilia A (FVIII <1%) related to an intron 22 inversion developed at the age of 2 a high-responding inhibitor. His elder brother also suffers from hemophilia A with high-responding inhibitor. Until 2000, the patient had been treated either using activated prothrombin complex concentrates (Autoplex® and Feiba®) or factor VIII (FVIII) concentrates of human (1983) and porcine origin (1992), resulting respectively in an inhibitor level rise at 360 Bethesda Units (BU) and 1800 BU. Between 2000 and 2003, the patient received exclusively recombinant activated Factor VII (NovoSeven®), and his inhibitor levels stabilized at a plateau of 15–20 BU. Between 2000 and 2003, several life or function threatening bleeding episodes occurred, such as hematomas of the iliopsoas muscles, spinal cord hematoma with transient paraplegia. Furthermore, due to hemarthroses the patient was confined to a wheelchair. Given the major impact of the inhibitor on the patient’s functional prognosis, life expectancy and quality of life, immune tolerance (IT) treatment was initiated, despite the high risk of failure (initiation 36 years after inhibitor onset, historical peak titer at 1800 BU, persistence of a plateau of 15–20 BU despite the absence of any stimulation with FVIII within the 3 last years). It started with an immunosuppressive drug, mycofenolate mofetil (Cellcept®) first given in may 2003 (no effect alone on inhibitor titer) and then in November 2003, infusions of a FVIII concentrate rich in von Willebrand factor, Factane® (LFB, Les Ulis, France) using 12,000 IU/day (150 IU/kg) of FVIII. The inhibitor peaked at 520 BU on D19 and was 0.5 BU by May 2004. Thus, the FVIII dosage was progressively reduced to 7000 IU/day. In July 2005, 24 hours after a 7000 IU FVIII infusion, inhibitor level was 0.7 BU, the residual FVIII level was 0.04IU/ml with a recovery of FVIIIc of 1.37%/IU/kg infused and a half-life of 4.9 hours. No significant change in the immunophenotype of peripheral blood lymphocytes was observed during this course. The patient’s quality of life was dramatically improved, with no hospitalization required and no bleeding episode observed within the 16 last months. The patient can now stand and has returned to his previous social activities.

These preliminary results show that this IT treatment, performed despite a high theoretical risk of failure, resulted in this patient in a dramatic clinical improvement, even though biological criteria of success have not yet been achieved. The respective roles of the type of FVIII concentrate, and of immunosuppression remain to be assessed, as well as the cost/benefit analysis on a longer follow-up period.
Intratumoral Treg Cells Completely Inhibit the Induction and Function of Tumor-Infiltrating CD8+ T-Cells in B-Cell NHL.
Authors: Yang Zhi-Zhang , Novak Anne J. , Stenson Mary J. , Witzig Thomas E. , Ansell Stephen M.
Published: October 2019

Background: Numerous studies have shown that lymphoma B-cells are resistant to CTL-mediated death, however the underlying mechanism for this resistance is not clear. In previous work, we have identified a subset of CD4+CD25+ T-cells overrepresented in the tumor sites of B-cell NHL that display a phenotype compatible with regulatory T cells (Treg cells). These cells express high levels of Foxp3 and CTLA-4, and are capable of suppressing the proliferation and cytokine production of autologous infiltrating CD4+CD25- T cells in B-cell NHL.

Goal: To explore whether Treg cells exert a suppressive effect on the induction and function of autologous tumor-infiltrating CD8+ T-cells in B-cell NHL.

Results: Using fresh specimens obtained from patients with B-cell lymphoma, we found that intratumoral Treg cells had a significantly suppressive effect on autologous tumor-infiltrating CD8+ T-cells in B-cell NHL. When activated CD8+ T-cells were cocultured with infiltrating CD4+CD25- T-cells, they displayed less proliferation than CD8+ T-cells cultured alone. However, we found that with a 1:4 ratio of stimulating cells (Treg cells) to responding cells (CD8+ T-cells), intratumoral Treg cells completely inhibited the proliferation of autologous tumor-infiltrating CD8+ T-cells activated with PHA. Furthermore, we have observed that 20% of infiltrating CD8+ T-cells in fresh isolated samples from B-cell NHL coexpress perforin and granzyme B. When intratumoral Treg cells were cocultured with CD8+ T-cells, the Treg cells inhibited the activation-induced production of perforin and granzyme B of autologous tumor-infiltrating CD8+ T-cells in a dose-dependent fashion. We also found that cytokine treatment (IL-2 and IL-12) or PHA activation induced the capability of tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells, which was accompanied by upregulation of expression of CD107a on the surface of cytotoxic CD8+ T-cells. Intratumoral Treg cells significantly inhibit cytotoxicity of activated infiltrating CD8+ T-cells to lymphoma B-cells. Finally, we found that there was an inverse correlation of cell frequencies between intratumoral Treg cells and tumor-infiltrating CD8+ T cells in patients with B-cell NHL, suggesting that Treg cells may inhibit the migration of cytotoxic T-cells to the sites of B-cell lymphoma.

Conclusion: Intratumoral Treg cells dramatically suppress the proliferation and activation-induced production of granules in infiltrating CD8+ T-cells in B-cell NHL. These Treg cells also inhibit the ability of activated tumor-infiltrating CD8+ T-cells to kill lymphoma B-cells in vitro, and may prevent the migration of CD8+ cells to the sites of lymphoma. Taken together, these data indicate an important role for intratumoral Treg cells in CTL-mediated anti-tumor immunity in B-cell NHL.
Incidence and Risk Factors for Central Nervous System Manifestation in Elderly Patients with Aggressive Non-Hodgkin’s Lymphoma.
Authors: Ösby E. , Hagberg H. , Kvalöy S. , Teerenhovi L. , Anderson H. , Cavallin-Ståhl E. , Holte H. , Myhre J. , Öst Å. , Pertovaara H. , Björkholm M.
Published: October 2019

Introduction: Manifestation of central nervous system (CNS) disease in patients with aggressive non-Hodgkin’s lymphoma (NHL) during or after first line treatment is often a devastating condition, which is associated with a poor prognosis. There is today no general consensus regarding prophylactic treatment in this patient category, particularly in the elderly. The aim of this study was to define the incidence and risk factors for CNS manifestation in a large cohort of elderly (>60 years) patients with aggressive NHL and no CNS disease at diagnosis.

Patients and methods: This Nordic study included a total of 455 previously untreated patients with advanced (stage II–IV) aggressive NHL without CNS involvement at diagnosis (Blood2003;101:3840). The vast majority had diffuse large B cell lymphoma. Patients (median age, 71 years; range, 60–86 years) were randomised to receive CHOP (doxorubicin 50 mg/m2) or CNOP (mitoxantrone 10 mg/m2) with or without G-CSF (5 microg/kg from day 2 until day 10–14 of each cycle every 3 weeks; 8 cycles). Intrathecal methotrexate was given as prophylaxis to patients with lymphoma bone marrow involvement and in patients with testicular, orbital, sinus and epidural sites of presentation.

Results: At this time point information is available for 441/455 patients. After a median observation time of 113 months in surviving patients 30/441 (6.8%) developed CNS disease. Advanced stage (p=0.004) and a high age-adjusted International Prognostic Index (IPI; p=0.034) were associated with an increased risk of CNS disease. Age, sex, performance status, bone marrow involvement/extranodal disease, serum lactate dehydrogenase level, or treatment received were not related to the risk of CNS manifestation. All patients with CNS disease were dead at follow-up.

Conclusion: A significant proportion of elderly patients with advanced aggressive NHL and no CNS manifestation at diagnosis develop CNS disease despite prophylactic measures in clinically defined high-risk patients. CNS occurrence is related to clinical stage and age-adjusted IPI. The prognosis of elderly aggressive NHL patients with CNS manifestation is poor.
Selective Induction of the p53-Pathway as Treatment for Multiple Myeloma.
Authors: Stühmer Thorsten , Zöllinger Angela , Chatterjee Manik , Bommert Kurt , Bargou Ralf C.
Published: October 2019

The p53 pathway is central to cellular defences against neoplastic transformation, and mutations that impair p53 function are prominent oncogenic events. However, certain malignancies, such as multiple myeloma (MM), rarely present with p53 defects except in late stages. MM might therefore be vulnerable to p53 induction therapy, but it is unknown if the p53 pathway remains actually functional in this disease, and if it can be sufficiently well triggered to induce tumor cell death. We have used nutlin-3, a newly developed small-molecule antagonist against the p53 suppressor murine double minute 2 (MDM2), to analyze the effect on myeloma cell lines and a large panel of patient tumor samples. Nutlin-3 specifically and exclusively induced p53 downstream targets in myeloma cells with wild-type p53, and a large majority of primary medullary myeloma samples was susceptible to nutlin-3-mediated apoptosis. Comparison with the clinically relevant genotoxic drugs melphalan and etoposide showed that nutlin-3 was as effective or better at inducing apoptosis of primary tumor cells, and that combinations of nutlin-3 with genotoxic cytostatics at low doses produced better than additive anti-tumor effects. Importantly, broad anti-tumor activity of nutlin-3 persisted even when primary tumor cells were kept in coculture with bone marrow stromal cells (BMSCs). However, primary tumor isolates often contain a fraction of cells apparently unaffected by drug treatment, and some primary samples were conspicuously less sensitive to nutlin exposure when the cells were kept in coculture with BMSCs, as compared to culture in medium supplemented with interleukin-6. We have begun to analyze to what extent heterogeneity for p53 induction within the tumor cell population may underlie these differences, and if the capacity to activate intracellular growth and survival pathways has an influence on the result of p53 pathway activation. Nutlin-3, as a non-genotoxic agent to specifically induce or strengthen p53-mediated apoptotic responses may be a promising agent to complement standard genotoxic regimens for the treatment of MM.
Hsp90 Is Critical for the Regulation of Human T Lymphocytes and NK Cell Phenotype and Function.
Authors: Bae Jooeun , Mitsiades Constantine , Prabhala Rao Prabhala , Yu-Tzu Tai , Martinson Jeff , Hartson Steven , Hideshima Teru , Catley Lawrence , Li Xianfeng , Batchu Ramesh Babu , Bertheau Robert , Masood Shammas , Samala Sushima , Miller Jeff , Anderson Kenneth , Munshi Nikhil
Published: October 2019

Hsp90 inhibitor has shown promising anti-tumor activity through the destabilization and eventual degradation of Hsp90 client proteins critical for cell survival. In this study, we examined the in vitro effects of Hsp90 inhibitor on the phenotype and function of human T lymphocytes and NK cells. We observed no significant effects of Hsp90 inhibitor treatment on cell survivals. However, Hsp90 inhibitor treatment for 24 hours led to irreversible down-regulation of expression of critical T-cell surface antigens including CD3, CD4, CD8, CD28, CD154 (CD40L) and TCRab. Among the antigens evaluated, expression of CD4 antigen was most significantly downregulated (untrt vs. trt = 326 vs. 88 in Mean Fluorescence Intensity) following Hsp90 inhibitor treatment. Decreased CD3+ T lymphocytes proliferation (untrt vs. trt = 222839 cpm vs. 111102 cpm, 3[H]-thymidine incorporation) and reduced IFN-g secretion (untrt vs. trt = 77 vs. 48 pg/ml) was observed upon stimulation with allogeneic dendritic cells following 24 hrs treatments of T cells with Hsp90 inhibitor. Furthermore, CD3+ T-cell proliferation in response to mitogen stimulation, as measured by flow cytometry using CFSE was decreased following Hsp90 inhibitor treatment (untrt vs. trt = 41% vs. 3%, CFSE). Specifically, the CD4+CD28+ (untrt vs. trt = 32% vs. 1%) and CD8+CD28+ (untrt vs. trt = 27% vs. 17%) activated T-cell subpopulations displayed a significant decrease in proliferation in response to mitogen. Similarly, NK cells displayed decreased activation receptor expression including CD2, CD11a, CD94, NKp30, NKp44, NKp46, and KARp50.3 and reduced cytotoxic activity against multiple myeloma cells (untrt vs. trt = 49% vs. 11% against MM1S cells, 65% vs. 8% against ARP cells) following Hsp90 inhibitor treatment. These studies demonstrate that Hsp90 inhibitor treatment significantly affects phenotype and function of human T-lymphocytes as well as NK cells, and suggest the need to monitor immune functions in patients being treated with Hsp90 inhibitor in our future studies.
Low Dose Radiation Induces a Highly Effective p53 Response and Rapid Tumor Regression in Follicular Lymphoma.
Authors: Knoops Laurent , Haas Rick L. , de Kemp Sanne , Broeks Annegien , van ‘t Veer Laura J. , de Jong Daphne
Published: October 2019

Involved field radiation therapy with 30 to 40 Gy is a valuable local treatment for follicular lymphoma (FL) that is routinely used in clinical practice. We previously showed that very low dose radiation (2x2 Gy, days 1 and 3) is also effective, with rapid and often long lasting remissions in up to 90% of FL patients (Haas et al, JCO, 2003). However, the biological mechanism of this extremely effective response is not known. To study the molecular response to low dose radiation therapy in FL, gene-expression profiling using 35K spotted 60-mer oligo-arrays was performed from lymph node biopsy samples taken before treatment and 24 hours after the second dose of 2 Gy irradiation, in 15 patients. The clinical response was excellent (10 CR, 5 PR). In all patients, a major and consistent induction of p53 and p53 target genes was seen, reflecting both proliferation arrest (e.g., P21, repression of cell-cycle regulated genes) and apoptosis induction (NOXA, PUMA, BAX, TRAIL/DR5 and FAS). P53 upregulation, p53-mediated proliferation arrest and apoptosis were substantiated using immunohistochemistry with dramatic increase of p53 protein levels in B-cells, T-cells and accessory cells. There was also a significant increase in the numbers of cleaved-caspase 8 positive cells (death receptor pathway apoptosis) with a minor increase of cleaved-caspase 3 positive cells and morphological features of apoptosis, suggesting a relatively early stage in the apoptotic process.

The other induced genes revealed an ‘immune signature’, with a whole set of biologically meaningful genes related to macrophages (e.g., CD68, TLR4), TH1 immune response (e.g., IL18, CXCL9, 10, 11), clearance of apoptotic cells (e.g., C1Q, lysosomal enzymes), tolerance (ILT-3, IL-4, IDO) and death receptor ligands (FASL, TRAIL). Immunohistochemical analysis did not show an increase in T-cell subsets and macrophages density, rather suggesting an activation or differentiation of resident macrophages by radiation and/or apoptotic cells than recruitment of novel cell populations.

This is the first global analysis of the direct molecular effect of radiotherapy and p53 related apoptosis in vivo in human lymphoma. Moreover, the ‘immune signature’ suggests that radiation-induced apoptosis in FL is not an immunologically silent process, but rather an early event that could contribute to the death and clearance of tumor cells. These insights may have important implications for modulation of the cancer-related immune response and for immunotherapeutical approaches in FL.
Altered Hepcidin Regulation in Hereditary Hemochromatosis and Dysmetabolic Hyperferritinemia.
Authors: Nemeth Elizabeta , Loreal Olivier , Le Lan Caroline , Ropert Martine , Troadec Marie-Bérengère , Detivaud Lénaïck , Jard Christine , Pouchard Michele , Le Treut André , Deugnier Yves , Ganz Tomas , Brissot Pierre
Published: October 2019

Hepcidin, the key iron-regulatory hormone synthesized by the liver, blocks iron absorption in duodenum and iron recycling from macrophages. Hepcidin dysregulation is implicated in the pathogenesis of several iron disorders. Hepcidin deficiency was observed in most types of hereditary hemochromatosis, including the HFE-related hemochromatosis, where decreased levels of hepcidin mRNA were found in patients with HFE mutations and in mice lacking HFE. However, levels of the bioactive hepcidin peptide in this disease have not been evaluated. We analyzed urinary hepcidin concentrations in a large cohort of patients with hepatic diseases associated with iron dysregulation, including untreated and treated HFE hemochromatosis (HH), dysmetabolic hyperferritinemia (DYSH) and alcoholic cirrhosis (AC). In untreated HH patients (n=33), hepcidin levels were marginally increased in comparison to controls (n=71). However, the hepcidin/ferritin ratio, an index of appropriateness of hepcidin response to iron load, was decreased in untreated HH group compared to controls, suggesting that hepcidin levels were inappropriately low for the degree of iron loading.

In treated HH patients (n=41), hepcidin levels were decreased when compared to either controls or untreated HH group, but the hepcidin/ferritin ratio was not statistically different from the control group ratio. Since hemoglobin levels in the iron-depleted HH group remained unchanged, hepcidin decrease is likely not related to hypoxia, but rather demonstrates partial responsiveness to changes in iron stores.

In alcoholic cirrhosis (n=43), hepcidin levels were decreased when compared to controls which may be due to decreased hemoglobin levels observed in these patients and to the replacement of hepcidin-producing hepatocytes by scar tissue. Patients with DYSH (n=40) had increased hepcidin levels in comparison to the controls. Although the underlying cause is yet unclear, the increased hepcidin levels in DYSH could be a contributing factor for the characteristic iron accumulation in both macrophages and parenchymal cells, similar to anemia of chronic diseases.

Our study suggests that dysregulation of hepcidin is a likely cause of iron metabolism abnormalities in HH and in DYSH.
Inherited Bone Marrow Failure Syndromes in Children: 32 Years Experience in a Single Institution.
Authors: Drelichman Guillermo I. , Basack Nora F. , Maro Alejandra , Schwalb Graciela , Freigeiro Daniel H. , Pennesi Sandra , Moran Lorena , Awdejczuk Ana , Aversa Luis
Published: October 2019

Inherited bone marrow failure syndromes (IBMFS) present chronic bone marrow failures, a familial incidence and high risk of malignancy.

Objective: we describe our experience with patients with IBMFS. Since 02/73 to 02/05, 60 patients (pts) with IBMSF were diagnosed.

Fanconi’s Anemia Blackfand-Diamond TAR-Shwachman-Diamond-Dyskeratosis Congenita Amegakaryocytic Thrombocytopenia.- Kostmann’s S. Familial AA-Pearson’s S N Pts 26 17 6 – 2 – 2 2 – 2 2 – 1 Median age at diagnosis 7.1 y 3 m 3m – 4 m – 6.4 y 2 m – 1.8 m 4 y – 3 y First hematologic sign Pancytopenia Anemia Thrombocytop. – Neutrop. – Pancytop. Thrombocytop. – Neutrop. Pancytop. – Anemia Bone Marrow Aplastic Erythroid Aplasia Megakar. Aplasia – Myeloid arrest– Aplastic Megakar. Aplasia– Myeloid arrest Aplastic – Vacuolated myeloid Physical Abnorm.(%) 88 47 100 – 50– 100 50 – 0 0 – 0 Aplastic Anemia (%) 100 0 0 – 0 – 100 50 – 0 100 – 100 Leukemia / MDS (%) 11.5 0 0 – 0 – 0 0 – 0 0 – 0 Mortality(%) 62 0 17 – 50 – 0 50 – 50 50 – 0

Conclusion: 68 % pts. presented with associated physical abnormalities, 53 % began the symptoms with bone marrow aplasia. With a median follow up: 16. 3 years (r: 1y – 32 y), 39 (65%) pts are alive (9 pts. post bone marrow transplantation) and 3 pts with Fanconi’s Anemia developed refractory Acute Myeloblastic Leukemia.
Comparison of Iron Load of the Liver and Gum of Patients with Thalassemia Major.
Authors: Caliskan Umran , Canatan Duran , Ciris Metin , Bozkurt Yesim , Caglayan Gurhan , Kuran Mine , Kosaci Sevgi , Oguz Nurgul , Karadogan Cavidan , Balta Nihal , Cosan Ruya
Published: October 2019

Various researchs have being conducted to replace liver biopsy which is accepted as a gold standart to evaluate prognosis and as the best indicator of total body iron storage in thalassemia major cases. Especially liver and heart iron storages determined by MR have been reported to correlate with tissue iron leves. However, these techniques have not been investigated sufficiently. We haven’t encountered any study comparing histopathological evaluation of gum and liver iron storage as an indicator of body iron storage. Therefore we have aimed to investigate and compare gum and liver iron storages by histological method which is techniqually easier and lesser noninvasive method. For this purpose, totally 22 beta thalassemia major, aged beetween 5–18 years, were investigated. As control group, 20 healthy person who experienced a tooth extraction, aged between 5–18 years, were included to study. Both gum and liver biopsies of the patients and only gum biopsies of controls were performed. Biopsy samples were stained with Prussia blue, hematoxyleneosin and Masson trichrome dyes. Collagen type-I and type-II were evaluated by immunohistochemical staining. Results; in all thalassemics, siderosis, fibrosis and hepatitis were found in hepatocellular, kupffer and portal areas. Iron in arteriolar wall and endotelium was seen in 22,7% and 50% of cases, respectively. Collagen type-I was found in 54,5% of cases. A statistically significant correlation was found between liver fibrosis, siderosis and iron overload but there was no significant correlation between liver and gum as fibrosis and siderosis in thalassemics. As a result, although periodontological and gingival problems in thalassemics were significantly important to the controls as clinically and histopatologically, however, there was no significant correlation between gum siderosis and hepatocellular siderosis as an indicator of body iron storage in thalassemic patients.
Pulmonary Dysfunction in Thalassemia Major Patients.
Authors: Gennari Alessandra , Bellagamba Maria Paola , di Stasio Alessandra , Ferraro Andrea , Cogo Annaluisa , Borgna-Pignatti Caterina , Capra Lucetta
Published: October 2019

The improved survival of patients with Thalassemia Major (TM) has allowed new complications to develop in the adult patients. These previously unknown clinical entities require a new approach to the management of thalassemia. Pulmonary dysfunction is one of the least understood, previously unrecognized, complications of TM.

Patients and Methods The aim of our study was to define the spirometric pattern and lung damage in 35 TM patients (18 M and 17 F, age range from 18 to 44 years). All were regularly transfused (pre transfusion hemoglobin level 9+/−0,4 g/dl) and chelated (25 with deferoxamine,5 with deferiprone and 5 with both chelators). Their serum ferritin level was 1071+/−628 ng/ml All patients had normal cardiac ejection fraction. Nine patients were active smokers (10+2 pack/years) while 9 had been smokers in the past (6+2 pack/years). Four had a mild allergic asthma under good clinical control, 1 had a mild pulmonary hypertension and 1 had mediastinal hematopoietic tissue. Pulmonary function tests were performed before transfusion and included pulmonary volumes and flows recorded by spirometry; residual volume (RV) and total lung capacity (TLC)measured by helium closed-circuit rebreathing technique; carbon monoxide diffusion capacity (TLCO)measured by single breath method.

Results: Only the patient with mediastinal masses showed an obstructive pattern with the FEV1/FVC ratio of 53% of predicted value. Thirteen patients (37%)had a restrictive pattern (TLC<80%pred). No correlation was found with sex, age, smoking, asthma diagnosis, ferritin levels, chelation regimen. A decreased TLCO (<70%pred) that can be indicative of parenchymal disease was present in 29 patients (85,8%) even after correction for hemoglobin levels (Hb value equal to 12 g requires adjust of 8%, Hb value equal to 10 g requires adjust of 18%, Hb value equal to 7 g requires adjust of 45%). The restrictive lung function abnormality with reduced diffusion capacity found in our patients is suggestive of interstitial lung disease secondary to TM. We were unable to correlate the restrictive and interstitial lung disease to iron loading nor to chelation strategies. Further studies are needed to better understand these results and to determine whether different chelation regimens have different impact on pulmonary function. In order to minimize lung damage, however, every effort should be made to educate TM patients not to smoke, and to identify and properly treat TM patients with asthma.
Proteomic Detection of Beta Thalassaemia Trait Using Electrospray Mass Spectrometry-Mass Spectrometry.
Authors: Daniel Yvonne , Turner Charles , Dalton R. Neil
Published: October 2019

Introduction Mass spectrometry has proved a valuable tool in the unequivocal characterisation of haemoglobin variants detected during routine haemoglobinopathy screening using High Performance Liquid Chromatography (HPLC) and/or isoelectric focusing. We have recently described a rapid and specific electrospray mass spectrometry-mass spectrometry (MSMS) method, using proteomic mutation targeting, for simultaneous detection of the clinically significant haemoglobinopathies; haemoglobin (Hb)S, HbC, HbE, HbDPunjab and HbOArab. Here we report additional transitions within the 1min cycle time, using delta chains as a surrogate HbA2, with the objective of detecting beta thalassaemia trait.

Method Blood samples are digested using trypsin, which results in specific cleavage of globin chains at peptide bonds adjacent to lysine and arginine residues, to produce a series of well-characterised peptides. There is strong amino acid sequence homology between the beta and delta chains of haemoglobin, however tryptic digestion produces series of 15 (T1–T15) and 16 (T1–T16) peptides, for beta and delta chains respectively, with differences in T2, T3, T5, T10, T12, T13 and T14. In this report we have investigated the diagnostic utility of T2: beta sequence SAVTALWGK, average mass 932.1 daltons, delta sequence TAVNALWGK, average mass 959.1 daltons. In multiple reaction monitoring mode the respective T2 [M+2H]2+ ions, m/z 466.8 and 480.3, are selected, fragmented, and the singly charged ions of the informative peptide fragments, VTALWGK and VNALWGK, detected.

Results The percentage area ratio of delta/(beta+delta) was calculated and compared with the classical percentage HbA2 value obtained by HPLC. 66 samples with HbA2 values within our normal range (mean 2.7%, range 1.8–3.4), and 58 with HbA2 values indicative of beta thalassaemia trait (5.2%, 4.2–7.9) were analysed by MSMS and the corresponding delta/(beta+delta) ratios were 1.7%, 0.9–2.3 and 3.4%, 2.5–6.0. In addition, in 2 further HbS beta thalassaemia zero compound heterozygote samples confirmed by beta gene sequencing the ratios were 4.1% and 3.7%.

Conclusion This relatively small study provides evidence that beta thalassaemia trait may be diagnosed by MSMS and represents another step towards comprehensive MSMS population screening for clinically significant haemoglobinopathies.
Myocardial T2* in Patients with Cardiac Failure Secondary to Iron Overload.
Authors: Tanner Mark A. , Porter John B. , Westwood Mark A. , Nair Sunil V. , Anderson Lisa J. , Walker J. M. , Pennell Dudley J.
Published: October 2019

Introduction: Myocardial iron overload is a well established cause of heart failure in a number of haematological disorders and is the leading cause of death in β-thalassaemia major (TM). Once overt heart failure develops prognosis is very poor and it would therefore be desirable to identify patients at risk, prior to the development of symptomatic heart failure. Myocardial iron can now be rapidly and reproducibly assessed using a validated cardiac magnetic resonance (CMR) T2* technique. Left ventricular ejection fraction has been demonstrated to relate to myocardial T2* (normal >20ms), and accordingly iron overloaded patients with symptomatic heart failure are likely to have a low T2* but there are no reports of the myocardial T2* level in newly presenting patients with heart failure and the threshold for increased risk is also unknown.

Purpose: To establish the distribution of myocardial T2* values in patients presenting with symptomatic heart failure secondary to iron overload.

Methods: Database records of CMR T2* assessments over a 5 year period were reviewed to identify iron overloaded patients presenting with heart failure.

Results: 28 patients (median age 29y, 11–79) with iron overload and documented heart failure were identified. 22 patients had thalassaemia major, 3 hereditary haemochromatosis, and 3 had miscellaneous transfusion dependent anaemias. The mean myocardial T2* in all groups was 6.8+/− 2.2ms reflecting severe iron loading by clinical criteria. See figure 1. Myocardial T2* values were similar between sub-groups as follows: TM patients 6.7+/− 2.4ms, haemochromatosis 7.7ms (6.7–7.4ms), miscellaneous 6.8ms (4.8–9.1ms).

Conclusion: In this database review, the patients developing heart failure secondary to iron overload all had abnormal myocardial T2*, and overall 89% of heart failure patients had values less than 10ms, which reflects severe myocardial iron loading. This data suggests a myocardial T2* <10ms should be considered a threshold for risk of heart failure (with its attendant high mortality) and such patients should be treated aggressively with increased iron chelation. It also suggests that clinical heart failure occurs nearly exclusively in patients with severe myocardial iron loading.

Distribution of myocardial T2+ values in symptomatic heart failure (n=28) Distribution of myocardial T2+ values in symptomatic heart failure (n=28)
Reduced Intensity Conditioning Matched Unrelated Donor Allograft for Kostmann Syndrome.
Authors: Thachil Jecko , Pizer Barry , Caswell Mark , Keenan Russell
Published: October 2019

Kostmann syndrome is a congenital disorder of myelopoeisis characterized by an absolute neutropaenia and severe bacterial infections. The introduction of G-CSF has dramatically changed the prognosis of these patients in recent years; though in G-CSF refractory patients, haemopoeitic stem cell transplant is still the only effective treatment. We describe the first case report of a patient who, unresponsive to escalating doses of G-CSF, underwent reduced intensity conditioning, Matched Unrelated Donor (MUD) allograft and has been cured from this condition. A male child presented shortly after birth with infection of the umbilicus. He developed repeated infections in the first six weeks of his life including pneumonia and scrotal abscess. Investigations at this stage included a bone marrow examination, which confirmed a diagnosis of severe congenital neutropaenia and he was started on treatment with G-CSF at 5μg/kg/day. Since diagnosis, he had numerous episodes of ear and skin infections and recurrent abscesses, most often with Methicillin resistant Staphylococcus aureus (MRSA) that required repeated hospitalisations. As the patient did not have a matched related donor, dose escalations were tried with G-CSF at 14-day intervals though he did not respond to doses up to 160μg/kg/day. At this stage, we had a child who was not responding to G-CSF injections, had several infections and was a carrier of MRSA and also Vancomycin Resistant Enterococci, who did not have a matched sibling donor. It was decided then to proceed with a MUD transplant with a non-myeloablative prepatory regimen. The pre transplant conditioning included fludarabine 30mg/m2 from day −8 to day −5, CAMPATH-1H 0.2 mg/kg from day -6 to day −2 and thiotepa 250 mg/m2 from day −4 to −2. Ciclosporin was started from day −1 and GCSF (Lenograstim) at 5μg/kg from day +7. A total of 10.4×106 CD 34+ cells/kg recipient’s weight of stem cells was infused. The patient successfully engrafted with a neutrophil count greater than 0.5×109/L on day 12 with an unsupported platelet count reaching above 50×109/L on day 33. The post transplant period was complicated with grade II skin and gut GVH that responded well to increased doses of ciclosporin alone. Slight falls in neutrophil count were noted on days 28 and 113 after transplant, which resolved by adjusting ciclosporin dosage. He is now ten months post transplant and has not had any infections needing hospitalization and maintains his neutrophil count.

Conclusion

We have therefore demonstrated for the first time that reduced intensity conditioning transplant from a matched unrelated donor is a safe and effective treatment for uncomplicated Kostmann syndrome.
Molecular Characterization of the Cannabinoid-Mediated Migration of Monocytes.
Authors: Park In-Woo , Anand Appakkudal R. , Groopman Jerome E.
Published: October 2019

2-Arachidonoylglycerol (2-AG), an endogenous ligand for the cannabinoid receptors CB1 and CB2, functions as a chemokine for monocyte migration. However, the molecular mechanism of its chemotactic effects is not clear. We found, consistent with previous data, that 2-AG induces the migration of differentiated but not undifferentiated monocytic cells in a dose-dependent manner. We first asked whether the expression of cannabinoid receptors changed during monocytic differentiation. Treatment with 1,25-(OH)2vitamin D3, a potent inducer of monocyte differentiation, or with 2-AG did not alter the surface expression of the CB1 and CB2 receptors, indicating that signaling downstream of receptor ligation accounted for the observed effect on monocyte migration. In addition, treatment of differentiated monocytic cells with inhibitors for adenyl cyclase and rho kinase blocked the 2-AG-mediated migration, directly implicating these signaling molecules in monocyte motility. Upon eliminating the concentration gradient of 2-AG, the motility of the cells from the upper to lower compartment was sharply reduced, but not completely abrogated. This suggested that the chemotactic effect may not fully explain the observed change in cell migration. Of note, treatment of monocytes with 2-AG resulted in an enhanced secretion of the chemokines MCP-1 and IL-8. Moreover, exposure of the cells to 2-AG inhibited their migration towards MCP-1, while exposure to MCP-1 did not alter migration toward 2-AG. Taken together, our findings demonstrate that intracellular signaling cascades as well as induction of chemokine secretion contribute to the cannabinoid-mediated migration of monocytes.
Protein-Biochemical Characterisation of Wilate®: Determination of the Upper Residual Moisture Limit during Terminal Dry-Heat Treatment (PermaHeat).
Authors: Kannicht Christoph , Stadler Monika , Neisser-Svae Andrea , Gruber Gerhard
Published: October 2019

Purpose: Wilate® is a novel, human plasma-derived, high purity, double virus inactivated coagulation factor concentrate. The safe and effective treatment of von Willebrand disease patients and haemophilia A has been demonstrated in clinical trials. The very high virus safety is achieved by an optimised solvent/detergent (S/D) method and terminal dry-heat treatment (PermaHeat) of the lyophilised product, not affecting the integrity of the proteins, which was investigated in this study.

Methods: Analyses of PermaHeat treated (+100°C for 120 min) Wilate® samples with different residual moisture (RM) contents were performed in order to evaluate the upper RM limit in lyophilised samples. Samples with RM up to 7.3% were subjected to SDS-PAGE, immunoblotting and peptide mapping. Further investigations were based on FVIII:C activity testing and phospholipid binding properties, VWF multimer analysis, Ristocetin cofactor (VWF:RCo) activity and collagen binding (VWF:CB).

Summary of results: The analyses confirmed that the structural and functional integrities of both VWF and FVIII were maintained during PermaHeat of Wilate® at +100°C for 120 minutes when the RM was kept at or below 1.8% during the heating procedure. Changes of protein-biochemical properties were detectable only for RM values exceeding 2%. Denaturation of samples occurred at RM of more than 3.5%.

Conclusion: Beside S/D treatment, optimised PermaHeat treatment of the lyophilised VWF/FVIII product ensures the high viral safety margin of Wilate®. According to the results, the RM content during PermaHeat treatment was specified with a great safety margin to a maximum of 1.6%. This limit is carefully controlled in each vial before heating by RM testing utilising near infrared (NIR). At or below this RM no structural or functional changes of VWF and FVIII were detected after PermaHeat treatment.
Undetected Factor VIII Level in a Type III von Willebrand Disease (vWD) Patient Masquerading as Severe Hemophilia A.
Authors: Mullah-Ali Ali M. , Chan Anthony , Pai Mohan K. , Decker Kay , Moffat Karen A. , Walker Irwin
Published: October 2019

Type III vWD is a rare disorder characterized by absent or <0.01u/ml of ristocetin cofactor (vW:RCoF), and a Factor (F) VIII:C of 0.03–0.10 u/ml. A total absence of FVIII:C has never been reported in type III vWD. We wish to report one such case where there was no measurable level of FVIII:C in a child leading to an initial diagnosis of severe haemophilia A. This patient presented at birth with post circumcision bleeding that was controlled with local measures. A packed RBC transfusion of 12 ml/kg body weight was given to correct acute blood loss. Coagulation studies performed before red cell transfusion revealed a FVIII:C of <0.01u/ml leading to diagnosis of severe haemophilia A. Patient required no further treatment to control bleeding. At 14 months of age the patient presented with an intracranial hemorrhage (ICH). rAHF (KogenateFS®) in doses of 50 to 400 u/kg resulted in poor recovery, half-life of 88 minutes and an in vivo survival of less than 4 hours. There was no FVIII:C inhibitor but vWF:RCoF, vWF:Ag and vWF multimers were totally absent. vWF concentrate (Humate-P®) in doses of 50–100/u/kg as vW:RCoF resulted in good clinical response, appropriate in vivo recovery and survival as shown in graphs below. This patient has made an excellent recovery and is fully functional 8 weeks after the ICH. Further enquiry into the family history revealed consanguinity with marriage between first cousins. A female cousin is also affected with easy bruising. Both parents have normal FVIII:C and borderline low vWF:RCoF. DNA analysis of the parents, propositus and unaffected siblings is in progress. Our case illustrates the importance of excluding Type III vWD at initial diagnosis even when the FVIII:C is <0.01u/ml instead of assuming it to be a severe haemophilia A.

Figure Figure Figure Figure
Development and Performance of the GTI ADAMTS-13 Activity Assay.
Authors: Pressman Melissa A. , Wuitschick Elizabeth , Chance Suzette C.
Published: October 2019

Thrombotic Thrombocytopenic Purpura (TTP) is a disease characterized by the sudden onset of a classical pentad of symptoms: thrombocytopenia, fever, renal insufficiency, neurologic deficit, and microangiopathic hemolytic anemia. It was recently discovered that ADAMTS-13 is the protease responsible for cleaving von Willebrand Factor; deficiency of ADAMTS-13 activity has been demonstrated in the plasma of TTP patients. The lack of ADAMTS-13 activity results in the accumulation of multimers of vWF in the plasma and ultimately intravascular platelet aggregation resulting in the clinical symptoms associated with TTP. Currently there are no commercially available diagnostic assays for TTP. Several laboratories have developed “in house” assays that measure the activity of the ADAMTS-13 protease. However, these assays are cumbersome, complex, difficult to perform, and there is a long turn around time, as they require long incubations. The results are difficult to interpret and are not easily quantitated. In addition, there is no standardization of results from laboratory to laboratory, mainly because the assays use various technologies. Determining the extent to which the ADAMTS-13 enzyme activity is decreased is believed to be important in distinguishing a patient with TTP from those presenting with similar clinical symptoms. Until ADAMTS-13 assays are more quantitative, there is not a good method for studying patients with severe decreases in ADAMTS-13 levels versus those who have some intermediate but less than normal level of enzyme activity. Furthermore, the lack of standardized results makes the assays less useful in monitoring the effectiveness of treatment of the TTP patients. GTI has developed a rapid, quantitative assay for ADAMTS-13 activity that is based on the cleavage of a substrate which consists of a peptide fragment of von Willebrand Factor and subsequent fluorescent detection. An initial study using plasma (obtained from the Blood Center of Wisconsin) from 5 TTP patients demonstrated good correlation between the GTI assay and the collagen binding assay, thus demonstrating feasibility of the assay. A more extensive evaluation using 40 normal samples and 55 samples from suspected TTP patients was initiated, the results of which will be reported. Assay sensitivity/limit of detection (based on dilution studies) was 3% normal ADAMTS-13 activity. Assay precision using 3 samples (mean values of 96%, 55%, and 9% normal ADAMTS-13 activity) run in duplicate over 8 days was evaluated. Assay imprecision was less than 10% cv for all samples tested.