bluuebunny/crossref_metadata_2025 huggingface dataset:
Reduced-Intensity Allogeneic Peripheral Blood Stem Cell Transplantation for High-Risk Acute Lymphoblastic Leukemia: A Potential Therapeutic Approach for High Risk ALL Patients Not Eligible for Full Intensity Transplantation
Authors: Stein A. , Palmer J , O’Donnell M , Kogut N. , Spielberger R , Slovak ML , Tsai NC , Forman S. J
Published: April 2020

Acute lymphoblastic leukemia has a poor prognosis in adults, with a two-year survival rate of 35–40% despite aggressive therapy (American Cancer Society: Cancer Facts and Figures 2008 Atlanta, GA: American Cancer Society, 2008). Reduced-intensity allogeneic stem cell transplantation (SCT) relies mainly on graft versus leukemia (GVL) for its efficacy; however, the role of GVL in ALL is less well-defined. Between 5/7/02 and 6/15/07, 25 adult ALL patients were uniformly treated with fludarabine 25mg/m2 daily for 5 days and melphalan 140mg/m2 followed by ALLO-SCT using peripheral blood stem cells. The indications for the reduced intensity regimen were:

≥ 50 years (10 pts) (40%), decreased organ function (9 pts) (36%) and previous ALLO-SCT (6 pts) (24%).

Patient demographics include: median age, 47 years (range, 23–68 yrs.), remission status at ALLO-SCT: first complete remission (CR) (12 pts) (48%), first relapse (3 pts) (12%), second CR (5 pts) (20%), >second CR (4 pts) (16%), induction failure (one pt) (4%), t(9;22) or Philadelphia positive (Ph+) ALL(9 pts) (36%). Donor source was matched sibling in 9 pts (36%), and matched unrelated in 16 pts (64%). Graft versus host disease (GVHD) prophylaxis: was cyclosporine (CSA)/+ mycophenylate (MMF) (3 pts) (12%), CSA/MMF/methotrexate (MTX) (8 pts) (32%), CSA/MMF/ATG (one pt) (4%), tacrolimus/sirolimus (7 pts) (28%) and tacrolimus/sirolimus/MTX (6 pts) (24%). With a median follow-up for surviving patients of 28.5 months (range: 12.8–72.5 months), the two year cumulative probability of overall survival and disease-free survival were both 59% (95% CI: 46–69.2%). The relapse rate was 16% (95% CI: 6.4,–37.4%),. Incidence of acute GVHD was 60% for grade II–IV and 20% for grade III–IV. Of the patients at risk for chronic GVHD, 8% developed limited chronic GVHD and 60% developed extensive chronic GVHD. The 100-day, one year and two year non-relapse mortality rates were 12% (CI: 4.6–29.3%), 24.2% (CI: 14–39.9%) and 29.3% (CI: 18.4–44.6%), respectively. There was no difference in survival outcomes based on donor source. The results of this study show that reduced intensity ALLO-SCT using fludarabine/melphalan based conditioning with primed peripheral stem cells from a matched related or unrelated donor provides a curative option for patients with high risk ALL who are not eligible for full intensity transplant.

Figure Figure
Adoptive Immune Cell Therapy for the Control of Fungal Infections in Hematopoietic Stem Cell Transplant Patients
Authors: Chang Lung-Ji , Liang Yin , Lien Lily , Tong Chun- Rong , Dong Lu-Jia , Wu Tong , Fu Yinchieh , Yang Fu-Hung , Tang Jih-Luh , Lu Dao-Pei
Published: April 2020

Similar to virus infections, fungal infections are commonly seen in immunosuppressed transplant patients and can be life-threatening. Invasive Aspergillosis and Candidiasis are principal fungal infections among hematopoietic stem cell transplant (HSCT) patients, but Aspergillosis and other molds are the leading cause of deaths by fungal infections in immunocompromised allogeneic HSCT patients. The most effective treatment for fungal infections is preemptive and empirical anti-fungal therapies using agents such as fluconazole and amphotericin B deoxycholate (AmB-D). However, the success rate of antifungal therapy is generally low (in the 30–40% range) and associated with high toxicity. Both diagnosis and treatment for fungal infections are expensive and often ineffective. While improved formulations of AmB-D, second-generation triazoles, and echinocandins may be tolerable, newer generations of anti-fungal agents are very expensive. In animal studies, it has been shown that Aspergillosis can be successfully treated using Aspergillus-specific cytotoxic T cells (CTLs). Therefore, it is conceivable that CTLs specific to fungal antigens are effective in controlling fungal infections in allogeneic HSCT patients. To explore anti-fungal immune cell therapy, we used two different approaches to generate fungus-specific immune cells:

Trichoderma and Rhizopus fungal lysates as antigen source to pulse dendritic cells (DCs), and pooled antigenic peptides to pulse DCs.

The antigen-primed DCs were then co-cultured with lymphocytes to generate antigen-specific immune effector cells. The ex vivo generated anti-fungal immune cells displayed antigen-specific effector functions as illustrated by intracellular IFN-γ and CD107a staining. Interestingly, the fungus-specific immune effector cells are mostly CD4 T cells for all three species of fungal antigens. In a pilot clinical study, patients were selected when diagnosed with invasive aspergillosis based on galactomannan and beta-glucan assays, radiographs, CT scans, and/or blood cultures, or after an extended unsuccessful anti-fungal treatment with non-tolerable organ toxicity. Early indications suggest that the infusion of anti-fungal immune cells is safe, with therapeutic efficacy based on objective clinical evidence and importantly, is cost-effective. Nevertheless, more effective diagnosis and surveillance tools are needed to document the effectiveness of our anti-fungal immune cell treatment.
Respiratory Tract Lophonomas Blattarum Infection of Allograft Hemopoietic Stem Cell Transplantation: A Case Report and Review of the Literature
Authors: Miao Miao , Depei Wu , Sun Aining , Yan Lingzhi , Wu Xiaojing , Tie Tiemei
Published: April 2020

Objective To explore the features of respiratory tract lophonomas blattarum infection in allograft hemopoietic stem cell transplantation.

Methods A 16 year-old male, was diagnosed of chronic myelolytic leukemia by MICM. On 2007-10-23, he underwent HLA-matched sibling donor allograft hemopoietic stem cell transplantation(Allo-HSCT). The preconditioning of transplantation is BU/CY(CCNU 250mg/m2*1d, HU40mg/Kg*2, Ara-C 2g/m2*1d, Bu 0.8mg/Kg/6h*3d, CTX 1.8g/m2*2d). CSA and MTX were used to prevent GVHD. Chest CT scan showed left inferior pneumonia on 18 days after transplantation. The patient underwent anti-bacteria, fungi and virus treatments, but his symptoms didn’t improve. Fiberoptic bronchoscopy(FOB) and bronchial alveolar lavage(BAL) was performed. It was confirmed that the pathogen was lophonomas blattarum We reported the clinical manifestation and management of respiratory tract lophonomas blattarum infection after Allo-HSCT and reviewed the clinical features of another eleven cases from the literature.

Results Infection with lophonomas blattarum mostly occurs in patients of hypoimmunity, its living conditions many in south hot and damp area. The clinical symptoms are severe and difference. Lophonomas blattarum can be observed under microscope. Metroindazole is always effctive.

Conclusion Lophonomas blattarum is a recessive infection in patients with Allo-HSCT, and co-infection of different pathogen may be involved. In Allo-HSCT patients with unexplained respiratory tract infection which do not respond to ordinary therapy, rare pathogens such as lophonomas blattarum need to be considered. Bronchoalar lavage and fiberoptic bronchoscopy examination are safe and fiberoptic bronchoscopy examination are safe and effective method in the early pathogenic diagnosis of respiratory tract infection after Allo-HSCT.
Long-Term Outcomes of Myeloablative Conditioning and Matched-Related Donor Hematopoietic Cell Transplantation for Patients with High-Risk and Advanced-Stage Hematolymphoid Malignancies
Authors: Benjamin Jonathan E. , Laport Ginna G. , Johnston Laura J. , Arai Sally , Weng Wen-Kai , Shizuru Judith A. , Miklos David B. , Wong Ruby M. , Elder Linda V. , Blume Karl G. , Negrin Robert S. , Lowsky Robert
Published: April 2020

Patients with high-risk hematolymphoid malignancies who relapse or who do not achieve a complete remission to induction chemotherapy generally do not achieve long-term survival when treated with the best available non-transplant therapies. The benefit of allogeneic hematopoietic cell transplantation has been well described for patients in first complete remission, but less so for patients with advanced disease. We report the long-term follow-up of 131 patients with leukemia or lymphoma who received an HLA-matched related donor transplant following myeloablative conditioning with fractionated total body irradiation (1320cGy), etoposide (60mg/kg), and cyclophosphamide (60mg/kg). Eligibility for transplantation under this protocol included induction failure or high-risk disease that was beyond first remission. All patients were treated at a single institution. Diagnosis at the time of transplantation included ALL (n=57), AML (n=38), NHL (n=20), CML (n=10), MDS (4), JMML (n=2). Of the 95 patients with acute leukemia, 62 (65%) were not in remission at the commencement of the conditioning regimen. The median age at transplantation was 29 years (range 2–55). Seventy-four (56%) patients received unmanipulated bone marrow and the remainder received filgrastim-mobilized peripheral blood. Median follow-up of surviving patients was 8 years (range 0.3–17). The estimated five-year overall survival and event-free survival were 34% (95% confidence interval: 22–42%) and 32% (95% confidence interval: 24–39%), respectively. Leading causes of death included relapse (n=43), infection (n=11), acute graft-versus-host disease (n=8), respiratory failure (n=5) and hepatic veno-occlusive disease (n=4). Grade II–IV acute graft-versus-host disease occurred in 26% of patients. The cumulative incidence of extensive chronic graft-versus-host disease among those patients who survived beyond day 100 was 32%. These results indicate that patients with high-risk or advanced disease can experience long-term disease-free survival following an aggressive conditioning regimen that combines radiotherapy, etoposide, and cyclophosphamide. Relapse remains the most significant cause of mortality, and future efforts should focus on augmenting the graft-versus-malignancy effect.
Factors Affecting the Results of Allogeneic Hematopoietic Stem Cell Transplantation in Hematologic Malignancies
Authors: Mehta J. , Frankfurt O. , Altman J. , Evens Andrew M , Gordon L. , Tallman M. , Williams S. , Winter J. , Krishnamurthy J. , Grinblatt D. , Kaminer L. , Singhal S.
Published: April 2020

130 patients who were unsuitable for conventional-intensity conditioning underwent RI-HSCT after 100 mg/m2 melphalan (+ 50 mg/kg cyclophosphamide if no prior autograft). GVHD prophylaxis comprised cyclosporine (HLA-matched siblings) or tacrolimus (all others) and MMF.

Table 1: Patient characteristics

Age (y) 19–71 (median 55) Male 55% Refractory disease 57% ECOG performance status 0 34% 1 48% 2 14% 3 4% Diagnosis Lymphoma 42% Leukemia 36% Myeloma 22% Donor Matched sibling 49% Mismatched sibling 2% Matched unrelated 33% Mismatched unrelated 15% Donor age (y) 21–71 (median 44; 45% >45) Male donor 65% Prior autograft 45%

Refractory disease, HLA mismatch, donor age >45, elevated creatinine, low DLCO, thrombocytopenia, male donor, poor performance status, and low albumin affected OS adversely in univariate analysis. The following factors affected outcome independently:

Table 2: Cox analysis

Adverse variable Relative risk P TRM HLA mismatch 4.78 0.0003 Performance status 2–3 3.07 0.044 Relapse Donor age >45 y 2.0 0.012 Male donor 1.98 0.026 Refractory disease 1.84 0.05 EFS Performance status 2–3 0.48 0.022 HLA mismatch 0.53 0.023 Refractory disease 0.55 0.016 Male donor 0.61 0.039 Donor age >45 y 0.63 0.034 OS Performance status 2–3 0.44 0.025 HLA mismatch 0.31 0.0001 Refractory disease 0.49 0.007 Donor age >45 y 0.55 0.012 High LDH 0.53 0.007 Platelets <100 0.58 0.021

These prognostic factors were similar to those found on studying the first 63 patients in this series (Mehta et al. Bone Marrow Transplant 2006) –with the additional emergence of HLA mismatch and thrombocytopenia which were significant in univariate but not multivariate analysis then.

These data suggest that RI-HSCT is useful for some patients with hematologic malignancies who are ineligible for conventional-intensity HSCT. However, alternative HSCT techniques or non-HSCT options are needed for high-risk patients.

Figure 1 shows the effect of the number of adverse prognostic factors on the cumulative incidence of relapse and transplant-related mortality. Figure 1. shows the effect of the number of adverse prognostic factors on the cumulative incidence of relapse and transplant-related mortality. Figure 2 shows the effect of the number of adverse prognostic factors on survival. Figure 2. shows the effect of the number of adverse prognostic factors on survival.
Study of the Relationship of Product Infused with Survival in Patients with Hodgkin’s Disease and No Hodgkin’s Lymphoma Undergoing Autologus Stem Cell Transplantation
Authors: Salido Eduardo , Majado Maria Juliana , Minguela Alfredo , Moraleda Jose Maria , Gonzalez Consuelo , Parrado Antonio , Funes Consuelo , Martinez-Sanchez Maria Victoria , Morales Alfonso
Published: April 2020

Absolute lymphocyte count (ALC) at diagnosis (ALC-D), as well as ALC recovery, have been described as prognostic factors in overall survival (OS) and progression free survival (PFS) in patients with non-Hodgkin’s and Hodgkin’s lymphoma(NHL and HL) who underwent autologous stem cell transplantation (ASCT). The aim of this study is to verify those findings, as well as the influence of infused lymphocyte, MNC, CD19 and CD3 in OS and PFS in our patients, in order to give some support to the importance of the immune system for disease control.

A total of 55 patients were reviewed, 35 males and 20 females, 44 had NHL and 12 HL. Variables analyzed with regard to PFS and OS were: ALC-D, ALC at day 15 post-transplant (ALC15), infused lymphocyte ×106/Kg, MNC X108 / Kg, CD19 X106/ Kg and CD3×106/ Kg. Variables were dichotomized at the median, and PFS and OS estimates were calculated with the Kaplan-Meier method and compared using log-rank test; regression Cox test was used for multivariate study.

Results: The median follow-up was 71 months (14–180). Relapse or progression occurred in 20 patients (36%) between 1 and 148 months (median 32). Eighteen patients (33%) died at a median of 28 months (14–110). Median ALC-D was 1.5×109/L (0.2–5.2 ×109/L). ALC15 count was 0.6 ×109/L (0.1–1.9). Infused MNC were 5.0×108/Kg (0.04– 22.2). Infused lymphocytes were 241.6 ×106/Kg (0.1–1222). Median CD19 cells infused were 1.12 ×106/Kg (0.0–79.7). Infused CD3 cells were 106.8 ×106/Kg (0.02–678.2). No difference was found in OS or PFS in patients in groups above and below median in ALC-D, ALC15 counts, neither in MNC, lymphocytes, CD19 nor CD3 cells infused in Kaplan-Meier study. MNC infused have statistical significance in OS (p=0.042) and PFS (p=0.023) in Cox regression analysis. In our series we do not find a clear predictor in relation to the immune reconstitution in NHL and HL patients receiving an ASCT; althoug it is possible that among MNC should be immune progenitors able to offer a better outcome to these patients, that could be studied in future.
Promoter Methylation of Multiple Genes in Germinal Center Hyperplasia and Lymphomas of Germinal Center Origin
Authors: Paz-Carreira Jose M , Losada Raquel , Garcia-Rivero Arantxa , Alvarez Augusto , Bal Fernando , Alba Jesus
Published: April 2020

INTRODUCTION. Germinal centers (GC) are unique sites in peripheral lymphoid tissue where clonal selection of B cells takes place. This occurs as a response to stimulation by various antigens originating, sometimes, follicular hyperplasia (FH). GC have been known as a major source of B-cell lymphomas including follicular (FL) and diffuse large cell (DLCL). DNA methylation of tumor-suppressor genes is a mechanism of gene silencing involved in the pathogenesis of FL and DLCL. Much less is known about the role of methylation in FH. We determined the methylation status of 6 tumor-suppressor genes in 43 patients with FH, 18 patients with FL and 49 patients with DLCL in order to see the differential implication of this epigenetic mechanism in the pathological and the physiological development of GC.

MATERIAL AND METHODS. Genomic DNA extracted from paraffin-embedded samples of 43 FH, 18 FL and 49 DLCL after being treated with EZ DNA-Methylation Kit (Zymo Research) with the manufacturer’s instructions, were analyzed by methylation-specific polymerase chain-reaction to determine promoter hypermethylation of DAP-k, SHP1, Rarβ, p14, SHP1, MGMT and PDRM1. All samples were obtained mostly from lymph nodes and tonsils. Diagnosis was based on morphology and immunohistochemistry analysis. All cases were matched for age, sex and ethnic origin.

RESULTS: DAP-k promoter methylation occurred with higher frequency in FL(89%) than in DLCL(78%) and FH(40%). SHP1 was methylated in 61% of FL, 58% of FH and 23% of DLCL. RARb was methylated in 67% of FL patients, 30% of DLCL and only 12% of FH. Eight (44%) FL, seventeen (35%) DLCL and four (10%) BFH patients showed MGMT methylation. Promoter hypermethylation of p14 was detected only in 5 (12%) FH, 2 (4%) DLCL and none FL patients. Methylation of PRMD1 was present only in 1 (6%) FL, 2 (6%) DLCL and 1 (4%) FH samples.

CONCLUSIONS. Inactivacion of DAP-K and SHP1 is present in B-cell malignancies, DLCL and FL, and BFH. Therefore, it may represent a physiologic event conferring a temporal survival advantage necessary for a GC hyperplastic response. Inactivation of the retinoic acid response through the methylation of Rarâ is significantly more frequent in lymphomas than in FH. As reported in other tumors methylation of MGMT is more frequent in lymphomas than in FH. With our data methylation of Cyclin dependent kinase inhibitors p14 is not a differential pathogenic event in lymphomas of GC origin, in fact it is more frequent in FH. Promoter Methylation of PDRM1 is not the mechanism involved in lymphomagenesis in FL and DLCL, the two FH positive deserve further follow-up to determine its significance.
Expression of NFATc1 in Diffuse Large B Cell Lymphomas (DLBCL) Is Decreased in HIV Positive Patients
Authors: Homan S. M , Patel R. A , Sheehan C. E , Hayner-Buchan A. , Ross J. S , Nazeer T.
Published: April 2020

Background: DLBCL is the most common non-Hodgkin lymphoma (NHL), comprising 30 to 40% of adult NHLs. It may present with nodal or extranodal involvement or it may be primary to the central nervous system (CNS). HIV positive patients comprise a significant percentage of patients diagnosed with DLBCL, however, differences in molecular characteristics based on HIV status have not been elucidated. Understanding the molecular basis for lymphomas is integral to the development of targeted therapy. The NFAT (nuclear factor of activated T cells) family is a group of cytoplasmic transcription factors that undergoes nuclear translocation when activated and plays an important role in intracellular signaling. This study evaluated the differential expression of the NFATc1 protein in HIV positive and negative patients with DLBCL (nodal, extranodal and primary CNS).

Design: Formalin-fixed, paraffin-embedded archival tissue from 34 nodal/extranodal DLBCLs and 41 primary CNS DLBCLs were immunostained with antibodies against NFATc1 by automated methods (NFATc1 (7A6):sc-7294, Santa Cruz Biotechnology, Inc, Santa Cruz, CA;Ventana Medical Systems, Inc, Tucson, AZ). Nuclear immunoreactivity in neoplastic tissue was semiquantitatively assessed and the results were correlated with HIV status and overall patient survival.

Results: Nuclear NFATc1 immunoreactivity was noted in both nodal/extranodal and primary CNS DLBCLs with 13/34 (38%) nodal/extranodal DLBCLs and 13/41 (32%) primary CNS DLBCLs showing nuclear NFAT1c positivity. Of the 59 total HIV negative cases, 42% (25/59) showed nuclear NFAT1c immunoreactivity. In contrast, less than 1% (1/16) of HIV positive cases demonstrated nuclear NFAT1c immunoreactivity (p=0.007). Within the nodal/extranodal DLBCL subgroup, nuclear NFATc1 positivity was observed in 12/23 (52%) HIV negative cases versus 1/11 (9%) HIV positive cases; while within the primary CNS subgroup nuclear positivity was noted in 13/36(36%) HIV negative cases versus 0/5(0%) HIV positive cases. There was no correlation with overall survival in either subgroup.

Conclusions: Similar to previous studies, nuclear NFAT1c immunoreactivity was demonstrated in DLBCLs and primary CNS lymphomas. Interestingly, this nuclear NFAT1c immunoreactivity was significantly decreased among patients with HIV positive status suggesting activation of alternative intracellular signaling pathways. These findings highlight potential variations in gene expression among DLBCLs based on HIV status. This decreased expression of NFAT1c amongst HIV positive individuals may be of particular importance when developing targeted therapy for aggressive B-cell malignancies.
Phasee I Biological Study of IV and SQ Azacitidine (Vidaza®) to Determine the Optimal Biological Dose in Hematologic Malignancies
Authors: Yang Allen S. , Mohrbacher Ann , Douer Dan , Gorospe Gerry , Byun Hyang-Min
Published: April 2020

Azacitidine (5-azacytidine, Vidaza) is a DNA methylation inhibitor that has been shown to have a survival benefit in patients with high-risk myelodysplastic syndrome and is under investigation in other cancers. The purpose of this study was to determine the optimal dose and route of administration for azacitidine to inhibit DNA methylation in patients with hematologic malignancies. Eligibility included patients with hematologic malignancy who provided informed consent. Enrollment criteria varied depending on the type of cancer. Patients were enrolled into 5 escalating dose levels for the first course of therapy (25mg, 50mg, 75mg, 100mg or 150mg IV per m2 per day for 5 days). On day 28 all patients received 75mg/m2/day IV for 5 days. For course 3 patients received 75mg/m2/day SQ × 5 days. Beyond course 3 patients received 75mg/m2/day either SQ or IV × 5 days every 4 weeks. Peripheral blood was collected on days 1, 3 and 5 for each course and LINE1 DNA methylation was measured using bisulfite-PCR Pyrosequencing to measure global DNA methylation changes induced by azacitidine. To date, 17 patients have been treated (3 at 25mg, 4 at 50mg, 4 at 75mg, 3 at 100mg, and 3 at 150mg/m2). Diagnosis included 6 patients with MDS, 9 patients with AML (3 untreated older patients, 6 relapsed or refractory patients), 1 patient with CML (imatinib refactory), and 1 patient with non-Hodgkin’s lymphoma (relapsed disease). At the time of submission (August 2008) 12 patients were evaluable for response with 1 CR, 3 PR, 7 SD and 1 PD reported. The median number of cycles given was 2 (range 0–12+). LINE1 DNA methylation decreased by 1.4, 2.3, 4.8, 1.9 and 4.0% on day 5 for the 25mg, 50mg, 75mg, 100mg, and 150mg/m2 dose levels respectively. Mean decrease in LINE1 DNA methylation with 75mg/m2 IV was 3.7% and only 2.6% by 75mg/m2 of azacitidine SQ. There was a large amount of inter-patient variability but less intra-patient variability in DNA methylation response to azacitidine. DNA methylation and data analysis is ongoing. Azacitidine is effective at inhibiting DNA methylation at multiple dose levels for both IV and SQ routes of administration. There is a high degree of patient-to-patient variability in DNA methylation changes, but 75mg/m2 lead to the greatest mean decrease in DNA methylation by a 5 day IV regimen. Updated analysis for this study will be presented.
Acquired Multiple Coagulation Factor Inhibitors Associated Bleeding Disorder
Authors: Chen Qin-fen , Li Pei , Xie Yi , Chen Bao-An
Published: April 2020

Objective: To inquire into the clinical features of acquired multiple coagulation factors inhibitors associated bleeding disorder.

Methods: A case of acquired multiple coagulation factors inhibitors in clinical manifestations, diagnosis, treatment and result was described and related literatures were reviewed.

Results: A 74-year-old man developed sustained wound bleeding after implantation of heart pacemaker. Prothrombin time (PT), activated partial thromboplastin time (APTT) were prolonged significantly. But thrombin time (TT), fibrinogen (Fg), platelet count(PLT) and liver function were normal. 3P test was negative. Further tests revealed that the activities of factor 2, 5, 7,8,9,10,11,12 were all less than 10%, and the inhibitors of these factors could be detected, with the titers ranging from 8~64 Bu. So acquired multiple coagulation factor inhibitors was diagnosed. Transfusion with frozen plasma and cryoprecipitate was ineffective, whereas the combination therapy with glucocorticoid plus plasma exchange seemed to be successful. The patient was cured.

Conclusion: Acquired multiple coagulation factors inhibitors is a rare bleeding disorder. In fact, this is the first case, as far as we are aware. It may develop serious bleeding. Immunosuppressive agents, such as corticosteroids, used for suppression of autoantibodies formation and plasma exchange, used for eradication of inhibitors may have beneficial effects.
Radiosynovectomy in Children with Congenital Haemostatic Defects
Authors: Laguna Pawel PL , Zbikowski Piotr , Cwikla Jaroslaw , Klukowska Anna , Walecki Jan , Matysiak Michal
Published: April 2020

Aims: To evaluate the clinical benefits of radiosynovectomy (RS) in children with congenital coagulative disorders, reduce bleeds and improve progression free survival (PFS).

Methods: 35 patients aged 7 to 17 yrs, (mean age 12.6 yrs) were included in the study. 29 had haemophilia A, (4 with inhibitor factor VIII), 3 had haemophilia B (1 with inhibitor factor IX) and 3 had von Willebrand’s disease. There were 43 therapies including injections into the followed joints: knee 21, elbow 13, ankle 6 and shoulder 3. In 30 cases this was initial therapy and in 5 cases repeat therapy due to recurrence or deterioration. Injected activity of 186Re colloid was dependent on type and size of joints, range: 60–180 MBq. Post therapy imaging was performed in each case 1 h after injection of radioisotopes, and then on day 2 and 3. The number of bleedings into joints per month, improvement of joint movements, and other clinical changes were evaluated before and after therapy in 3 month intervals. US images were used to assess changes in joint effusion and synovial overgrowth. PFS in terms of significant clinical improvement within joints was assessed using standard Kaplan Meier methods. The prognostic significance of selected parameters was tested using discriminate function analysis.

Results: After injection of 186Re into joints, post-therapeutic images showed intraarticular distribution of tracer and there was no leakage. A significant reduction in bleeds into joints was noted (before therapy mean 1.8 per month and after treatment mean 0.3 per month, p<0.001).

Conclusions: This study suggests that use of 186Re in early stages of arthropatic haemophilia reduces the number of bleeds into joints and improves PFS.
Increased Plasminogen Activity Is An Independent Predictor of Bleeding in Hemophiliacs
Authors: Gerhardt Andrea , Pesch Melanie , Scharf Rudiger E. , Zotz Rainer B.
Published: April 2020

Rationale and Objectives: Apart from the differences in factor VIII:C levels, it is difficult to explain the significant variation in bleeding events observed among individual patients with similar levels of FVIII:C and standard laboratory profiles. We hypothesize that additional dispositional (genetic) and expositional (non-genetic) factors of hemostasis may augment or attenuate the likelihood of bleeding symptoms in hemophiliacs.

Patients and Methods: To test this hypothesis, we studied 61 patients diagnosed with hemophilia A. The following laboratory parameters were examined in multivariate analysis using a case-only analysis: genotyping for a2C807T, HPA-1 of aIIbb3, factor V G1691A, prothrombin G20210A; coagulation factor activity/antigen of fibrinogen, factor II, V, VII, VIII, IX, X, XI, XII, XIII, vWF; in-vitro bleeding time (PFA-100 closure times), and plasminogen.

Results: After adjustment for age, low levels of factor VIII (p=0.0296) and increased levels of plasminogen (p=0.0415), and collagen/ADP closure times (PFA-100) (p=0.04) could be identified as independent predictors of mucosal bleeding. 10% higher activity of plasminogen increased the bleeding risk 10.4 fold. For joint bleeding low levels of fibrinogen (p=0.003) and longer collagen/epinephrine closure times (PFA-100) could be identified as bleeding predictors.

Conclusion: Apart from low levels of factor VIII, increased activity of plasminogen is an independent predictor of mucosal bleeding in hemophiliacs. In consequence, it will be of importance to examine whether the critical subgroup of patients with increased plasminogen levels and mucosal bleeding can benefit from prevention with specific antifibrinolytic agents.
Interferon Gamma +874A/T Polymorphism in Children with Idiopathic Thrombocytopenic Purpura
Authors: Demircioglu Fatih , Ören Hale , Kizildag Sefa , Yilmaz Sebnem , Atabay Berna , Turker Meral , Vergin Canan , Irken Gulersu
Published: April 2020

A recent study showed that expression of Toll-like receptor and interferon-gamma associated genes is significantly increased in patients with chronic ITP. Interferon-gamma is an important protein which takes place in immunoregulation. +874A/T polymorphism in the first introne of interferon gamma gene is found to be associated with the development and clinical phenotype of some autoimmune diseases such as diabetes mellitus, thyroiditis, multiple sclerosis, and SLE. The aim of our study was to investigate whether interferon gamma +874A/T polymorphism is a risk factor for the development of ITP and whether it affects the clinical course and response to the treatment. Thirty five children with acute ITP and 40 children with chronic ITP who were followed for at least 6 months were included. Control group consisted 90 healthy children. Two millilitres of blood sample was taken into sterile tubes containing 0.1% EDTA from each child and all blood samples were stored at −20 until analysis. DNA was isolated from blood samples and interferon gamma +874A/T polymorphism was studied with real-time PCR and LightCycler TM. Twenty one patients had AA, 35 patients had AT, and 19 patients had TT genotype. In the control group, 47 children had AA, 36 children had AT, and 7 children had TT genotype. There was a statistical difference between ITP and control group regarding the genotype (p=0.001). The frequency of A and T alleles in ITP group was 52% and 48%, respectively. The frequency of A and T alleles in control group was 72.7% and 27.8%, respectively. The frequency of allele distribution was statistically different between the ITP and control groups (p<0.0001). There was a statistical significant difference between acute ITP and control group regarding the frequency of AA, AT, and TT gene polymorphisms and allele frequency (p=0.002, p=0.002). Similarly, there was a statistical significant difference between chronic ITP and control group regarding the frequency of AA, AT, and TT gene polymorphisms and allele frequency (p=0.008, p=0.002). The frequency of AA, AT, and TT gene polymorphisms and allele frequency showed no statistical difference between acute and chronic ITP groups (p=0.285, p=0.896). There was no correlation between interferon gamma +874A/T polymorphism and severity of bleeding (mild, moderate and severe) (p=0.09). There was no correlation between interferon gamma +874A/T polymorphism and response to long term treatment in patients with chronic ITP (p=0.568). In conclusion, there was a significant difference between patients with ITP and children in control group regarding interferon gamma +874A/T polymorphism and in the light of recent data involving other autoimmune disorders, we think that interferon gamma +874A/T polymorphism may be a risk factor for ITP.
Identification of Three Novel Substitutions in the Erythroid Promoter of the HMBS Gene: A New Erythroid Variant of Human Acute Intermittent Porphyria?
Authors: Di Pierro Elena , Besana Valeria , Brancaleoni Valentina , Tavazzi Dario , Cappellini Maria Domenica
Published: April 2020

Porphyrias are inherited disorders in the biosynthesis of heme. Acute intermittent porphyria (AIP), the most common form, is autosomal dominant and it is characterized by recurrent attacks of abdominal pain, gastrointestinal dysfunction, and neurologic disturbances. AIP is caused by molecular defects in the hydroxymethylbilane synthase gene (HMBS) causing a partial deficiency of the third enzyme of the heme biosynthetic pathway. This gene maps to chromosome 11q23.2 with a total of 15 exons. Two distinct promoters direct the synthesis of housekeeping and erythroid specific mRNAs by alternative splicing. The housekeeping promoter is located upstream the exon 1 while the erythroid promoter include a portion of intron 1, the exon 2 and the intron 2. The exon 1 and the exons 3 to 15 generate the housekeeping mRNA while the exons 2 to 15 generate the erythroid mRNA giving rise to the housekeeping HMBS isoform and to the erythroid HMBS isoform respectively. In the classic form of AIP, both the housekeeping and the erythroid HMBS isoforms are deficient since the must common molecular defects involve the common region of the gene from exon 3 to exon 15. Approximately 5% of AIP patients have normal levels of the erythroid HMBS isoform and defects in the housekeeping promoter or in the exon 1 which is specific for the housekeeping HMBS isoform, causing the ‘non-erythroid variant’ of AIP. So far no mutations are known in the erythroid promoter. In this study we searched for molecular defects in HMBS gene in three Italian patients with typical clinical and biochemical signs of AIP. The diagnosis was based on elevated urinary excretion of porphyrinic precursors and reduced erythrocyte HMBS activity. The entire HMBS gene has been amplified by PCR and submitted to direct automated sequencing: no mutations known as responsible for classic form of AIP have been identified. However, each of three patients had a substitutions in the erythroid promoter of HMBS gene that could justify the reduced erythrocyte HMBS activity: c.34–115 C>A, c.34–126 G>C and c.34–156G>A. In order to establish if these substitutions were polymorphisms, more than 200 alleles from Italian normal subjects were sequenced and none of them revealed the substitutions. These three mutations, located specifically in the intron 2, could affect the normal splicing of exon 3 causing an abnormal mRNA, to give rise to the classical form of AIP. The mRNA analysis, however, didn’t reveal any abnormal mRNA. Moreover real time experiments in the lymphocytes showed a 100% expression of housekeeping HMBS mRNA excluding the non-sense mediated decay mechanism. These data suggest that these mutations could affect the normal function of the erythroid promoter only, causing an erythroid variant of human acute intermittent porphyria. Further expression studies are in progress.
Effects of down-Regulating BCL11B Expression on the Proliferation and Apoptosis of Molt-4 Cells by RNA Interference
Authors: Chen Si , Li Yangqiu , Huang Xin , Yang Lijian , Chen Shaohua , Zhou Yubing
Published: April 2020

The B-cell chronic lymphocytic leukemia (CLL)/lymphoma 11B (BCL11B /CTIP2/ RIT1/hRitα) gene, which encodes C2H2 zinc-finger transcription factors, plays critical roles during development of T lymphocytes and the central nervous system. Recently, BCL11B gene has been identified to be associated with hematopoietic malignancies, such as T-cell acute lymphoblastic leukemia (T-ALL). However, little is known regarding the molecular mechanism by which BCL11B may contribute as a tumor-suppressor gene. In order to clarify the role of BCL11B gene in T-cell malignancies, we analyzed the effects of BCL11B target-specific small interfering RNA (siRNA) on the proliferation and apoptosis of Molt-4 cells, which is the naturally BCL11B overexpressing cell line. BCL11B-specific siRNA and scrambled non-silencing siRNA control were designed and synthesized by Invitrogen. The siRNAs were transfected into MOLT-4 cells by nucleofector techique. The level of BCL11B mRNA expression was analyzed at 48 and 72 hours after nucleofection by Real-time quantitative PCR with Taqman technique. The cells proliferation rate was assayed by MTT method at 24 and 48 hours after transfection, respectively. Cell morphology was showed by Liu’s Stain. The changes of cell cycle and apoptosis rate were detected by flow cytometry. The results showed that the level of BCL11B mRNA expression was inhibited in siRNA transfected cells at 72 hours after transfection, as compared with scrambled controls. The survival rates of siRNA-transfected cells were significantly decreased at both 24 and 48 hours (P < 0.01). Apoptotic cells could be observed morphologically. The apoptosis rates of Molt-4 cells treated with BCL11BsiRNA were highly increased compared with scrambled control ( P < 0.05). However, the percentage of different phases did not show significantly difference between siRNAtransfected cells and control cells, which was suggested that the apoptosis could be induced all phases of cell cycle. In conclusion, our data indicated that, the survival of Molt-4 cells might be critically dependent on BCL11B gene. Downregulation of BCL11B expression by RNA interference selectively can inhibit the proliferation and induced the apoptosis effectively in transformed Molt-4 cells, farther investigation will be performed in primary T-ALL cells to confirm the effect of presented BCL11B-siRNA on T-ALL.
Method for Measuring Macrophage Iron Efflux in Vitro and in Vivo Using Magnetic Resonance Imaging
Authors: Andriopoulos Bill , Guimaraes Alexander R , Wojtkiewicz Gregory R , Chen Sandra , Xia Yin , Babitt Jodie L , Corradini Elena , Weissleder Ralph , Lin Herbert Y
Published: April 2020

Anemia of inflammation (AI) is the disturbance of iron homeostasis manifest by a decrease in absorption of dietary iron from the gastrointestinal tract and by iron sequestration by reticuloendothelial macrophages. It is widely recognized that elevated levels of proinflammatory cytokines and other mediators from activated immune cells are the primary causes of AI. More specifically, it is the abnormally decreased rate of iron efflux from macrophages and enterocytes that appear to be the crucial pathophysiology. Increasingly, the central role of the liver-derived factor hepcidin is recognized in mediating the disturbances of iron absorption, sequestration and utilization that characterize AI. Hepcidin decreases iron absorption from the duodenal enterocytes and promotes the sequestration of iron in the reticuloendothelial macrophages by down regulating the transmembrane iron export protein, ferroportin. Thus the regulation of hepatic hepcidin expression becomes of paramount interest in the study of AI. We have shown that bone morphogenetic protein is critical for the regulation of hepcidin expression. The aim of our study is to develop a model to study iron efflux both in vitro and in vivo using magnetic resonance imaging (MRI). This was done by experimentally measuring spin-spin relaxation times (T2 measured in sec-1) through multi-echo, spin-echo technique (16 echoes) on a 4.7 T Bruker pharmacan experimental MRI imager. Iron loaded cells exhibit lower T2 values than normal control cells. In vitro, hepatomas Hep3B and macrophages J774 and Raw 264.7 were loaded with magnetic iron oxide nanoparticles (MION) overnight. The next morning (baseline), the intracellular and extracellular iron content was serially measured for up to 24 hours using MRI. As expected, both J774 and Raw 264.7 cells lines displayed lower intracellular T2 values in comparison to baselines. In addition, both ferroportin and the iron storage protein ferritin were modulated following overnight MION treatment and 24 hours post baseline. In contrast, Hep3B cells did not take up any significant MION following overnight treatment. In vivo, normal mice on a normal diet were given an intravenous MION injection dosed at 10mg/kg iron. MRI was performed at baseline, 24, 48, 72 and 96 hrs post injection. 24 hours post injection, T2 increased in both liver and spleen (liver > spleen). Following the 24 hour spike, the T2 values in the mice decreased for up to 72 hours after injection with a rate in the liver mildly increased relative to the rate of decrease in the spleen.
Analysis of Mouse LMIR5/CLM-7 as An Activating Receptor: Differential Regulation of LMIR5/CLM-7 in Mouse Versus Human Cells
Authors: Yamanishi Yoshinori , Kitaura Jiro , Izawa Kumi , Oki Toshihiko , Takai Toshiyuki , Kitamura Toshio
Published: April 2020

We have analyzed leukocyte mono-Ig–like receptor 5 (LMIR5) as an activating receptor among paired LMIRs. Mouse LMIR5 (mLMIR5) is expressed in myeloid cells such as mast cells, granulocytes, macrophages, and dendritic cells. Cross-linking of transduced mLMIR5 in bone marrow–derived mast cells (BMMCs) caused activation events, including cytokine production, cell survival, degranulation, and adhesion to the extracellular matrix. mLMIR5 associated with DAP12 and to a lesser extent with DAP10, and mLMIR5-mediated functions of BMMCs were strongly inhibited by DAP12 deficiency. Importantly, cross-linking of endogenous mLMIR5 induced Syk-dependent activation of fetal liver–derived mast cells. Unlike mLMIR5, cross-linking of human LMIR5 (hLMIR5) induced cytokine production of BMMCs even in the absence of both DAP12 and DAP10, suggesting the existence of unidentified adaptors. Interestingly, hLMIR5 possessed a tyrosine residue (Y188) in the cytoplasmic region. Signaling via Y188 phosphorylation played a predominant role in hLMIR5-mediated cytokine production in DAP12-deficient, but not wild-type BMMCs. In addition, experiments using DAP10/DAP12 double-deficient BMMCs suggested the existence of Y188 phoshorylation-dependent and -independent signals from unidentified adaptors. Collectively, although both mouse and human LMIR5 play activatory roles in innate immunity cells, the functions of LMIR5 were differentially regulated in mouse versus human cells.
Diagnostic and Disease Management Algorithms for Gaucher Disease: A Guide for Haematologists
Authors: Mistry Pramod K , Cappellini Maria Domenica , Lukina Elena , Özsan Hayri , Pascual Sara Mach , Rosenbaum Hanna , Solano Maria Helena , Spigelman Zachary Simon , Villarrubia Jesús , Watman Nora Patricia , Massenkeil Gero
Published: April 2020

In Gaucher disease (GD), inherited deficiency in lysosomal acid beta-glucocerebrosidase leads to the widespread accumulation of glycolipid-laden macrophages in tissues and consequent multisystemic disease. The presenting symptoms of type 1 (non neuronopathic) Gaucher disease commonly reflect the haematologic components of the disease, i.e., splenomegaly, thrombocytopenia, anaemia, and bleeding tendency. Consequently the majority of patients are referred to haematologists for diagnosis and management. However, the remarkable heterogeneity of the disease compounded by its rarity presents a diagnostic challenge. Only around 20% of haematologists consider GD in differential diagnosis even though all classic manifestations of the disease may be present. Haematologic malignancy is the most often considered diagnosis in such patients. Lack of awareness of GD and its protean manifestations renders individuals with Gaucher disease vulnerable to misdiagnosis, diagnostic delays and risk of irreversible complications that are otherwise preventable by timely therapy. Missed diagnosis may place individuals at risk of bleeding even in the presence of mild thrombocytopenia, irreversible skeletal complications leading to disability and impaired growth, and an increased risk of malignancy, especially multiple myeloma. Imiglucerase (Cerezyme) enzyme replacement therapy is effective in reversing haematological, visceral and some skeletal manifestations of the disease. In a minority of patients in whom imiglucerase is not suitable, Miglustat substrate reduction therapy (Zavesca) is an alternative. An international group of physicians met to harness their respective experience of managing the diverse phenotypes of Gaucher disease in order to raise awareness of GD and to determine under which circumstances GD should be given greater priority in differential diagnosis. As a result of their discussions, simple diagnostic algorithms have been devised, which focus on splenomegaly as the most prevalent presenting manifestation of GD in individuals of both Ashkenazi Jewish and non Ashkenazi Jewish background. Given the prevalence of GD in Ashkenazim, a possible diagnosis of GD is prioritised in the presence of splenomegaly in this population. When challenged by differential diagnosis of splenomegaly, GD can be confirmed or eliminated by a straightforward assay for beta glucocerebrosidase activity. Diagnostic algorithms are accompanied by disease management algorithms, which reflect published therapeutic goals and monitoring guidelines, to simplify decision making in the evaluation, treatment and ongoing monitoring of GD in response to therapy.
CEACAM-1, -3, -6, and -8 Signaling in Human Neutrophils
Authors: Skubitz Keith M. , Skubitz Amy P.N.
Published: April 2020

Members of the carcinoembryonic antigen family (CEACAMs) are widely expressed, and, depending on the tissue, capable of regulating diverse functions including tumor promotion, tumor suppression, angiogenesis, and neutrophil activation. Four members of this family, CEACAM1, CEACAM8, CEACAM6, and CEACAM3 (recognized by CD66a, CD66b, CD66c, and CD66d monoclonal antibodies (mAbs), respectively), are expressed on human neutrophils. CD66a, CD66b, CD66c, and CD66d antibodies each increase neutrophil adhesion to human umbilical vein endothelial cell monolayers. This increase in neutrophil adhesion caused by CD66 antibodies is blocked by CD18 mAbs and is associated with upregulation of CD11/CD18 on the neutrophil surface. To examine potential interactions of CEACAMs in neutrophil signaling, the effects of a set of CD66 mAbs on neutrophil adhesion to human umbilical vein endothelial cells was tested following desensitization to stimulation by various combinations of these mAbs. The current data show that desensitization of neutrophils to any two CEACAMs results in selective desensitization to those two CEACAMs, while the cells remain responsive to the other two neutrophil CEACAMs. In addition, cells desensitized to CEACAM-3, -6, and -8 were still responsive to stimulation of CEACAM1 by CD66a mAbs. In contrast, desensitization of cells to CEACAM1 and any two of the other CEACAMs left the cells unresponsive to all CD66 mAbs. Cells desensitized to any combination of CEACAMs remained responsive to the unrelated control protein CD63. Thus, while there is significant independence of the four neutrophil CEACAMs in signaling, CEACAM1 appears to play a unique role among the neutrophil CEACAMs. A model in which CEACAMs dimerize to form signaling complexes could accommodate the observations. Similar interactions may occur in other cells expressing CEACAMs.