bluuebunny/crossref_metadata_2025 huggingface dataset:
Correlation of JAK2 V617F Mutation Status and Cytogenetic Findings in Myeloproliferative Disorders.
Authors: Sun Guoxian , Buccini Frank , Fuentes Elizabeth , Weisberger James
Published: October 2019

Detection of JAK2 V617F mutation is quickly becoming a front-line screening test for suspected myeloproliferative disorders (MPDs), as the mutation shows high frequency and specificity in non-CML MPDs, PV, ET or CIMF. Routine cytogenetics can detect chromosome abnormalities in approximately 20% of MPDs and is very helpful in establishing or confirming the presence of aberrant clonality, although chromosome changes are often numerical gains and losses, deemed non-specific. To see if there is correlation between JAK2 mutation and karyotypes, we studied 57 consecutive patients with clinically and morphologically confirmed diagnosis of non-CML MPDs. JAK2 V617F mutation performed using allele-specific PCR (sensitive to 10% using pyrosequencing) was found in 72% of patients (41/57), whereas clonal chromosome abnormalities were observed in 15.8% (9/57). There was no correlation between JAK2 mutational status and karyotypes. In 41 patients positive for the JAK2 mutation, 6 were cytogenetically abnormal and 35 normal. In 16 patients negative for the mutation, 3 showed abnormal karyotypes and 13 had normal karyotypes (X2 test, p>0.5). Among 6 patients with both JAK2 mutation and an abnormal karyotype, JAK2 mutation was seen in >50% of each sample in 4 patients, consistent with a homozygous mutation. Interestingly, in two cases, one with PV and trisomy 9 and another with MPD unclassifiable and trisomy 9p resulting from an unbalanced translocation between chromosomes 9p and 13, JAK2 mutation was present in >65% of each sample. Trisomy 9 and trisomy 9p are common abnormalities in MPDs, particularly in PV, seen in over 20% of cytogenetically abnormal cases. JAK2 gene is located on 9p24. Mitotic recombination is considered the most likely cause of loss of heterozygosity (LOH) and thus mutant homozygosity which is undetectable at the cytogenetic level. However, in cases with trisomy 9 or 9p, the JAK2 allele genotypes may be G/T/T and/or T/T/T as well as the usual G/T and/or T/T. Our observations suggest that trisomy 9 or 9p should be taken into consideration when interpreting JAK2 mutation status and that further molecular studies are needed to delineate the implication of trisomy 9 or 9p in potential mutant allele selective advantage and clonal evolution in JAK2 mutation positive MPDs.
Outcome of Hematopoietic Stem Cell Transplantation for Pediatric Acute Lymphoblastic Leukemia in Third Complete Remission (CR3): A Vital Role for Graft-Versus-Host-Disease/Graft-Versus-Leukemia Effect in Survival.
Authors: Gassas Adam , Ishaqi Kashif , Doyle John
Published: October 2019

Children with acute lymphoblastic leukemia (ALL) who suffer 2 relapses could be salvaged by hematopoietic stem cell transplantation (HSCT) when a suitable stem cell source is available provided they respond to the pre HSCT chemotherapy and at least enter morphological remission. However, these patients are at very high risk for post HSCT relapse and also at a high risk for transplant related mortality (TRM). Our objective, herein, was to review the outcome of children (0–18years) with ALL who received allogeneic HSCT in third complete remission (CR3) at our institution. Between January 1994 – August 2005, twenty-two consecutive children in CR3 received HSCT in the Hospital for Sick Children, Toronto, Canada. Conditioning regimens included single dose of VP16 (60mg/kg infused over 4 hours) and fractionated total body irradiation (TBI; 1200cGy) in six fractions over 3 days (VP16/TBI) in 10 patients (1994–1998) and cyclophosphamide 50mg/kg infused over 1 hour daily for 4 days followed by the same dose of fractionated TBI (CY/TBI) in 12 patients (1999–2005). Graft-versus-host disease (GVHD) prophylaxis included cyclosporine A and a short course of methotrexate for the majority of patients, and all patients were in complete morphological remission prior to HSCT. Median age was 8.4 years (range 3–15.4). Donor source was as follows: matched sibling donor (MSD), 8; matched unrelated donor (MUD) 6; one antigen mismatch related donor (MMRD) 4; one antigen mismatched unrelated donor (MMUD) 3; and one patient received 1 antigen mismatched cord progenitor stem cells. White cell engraftment was successful in all patients at a median of 18 days (range 9–29). Ten patients died of TRM, seven relapsed, one died from other causes and four patients are long term survivors at a median follow up of 3.7 years (range 1–10.2). All patients who did not develop clinical acute or chronic GVHD relapsed and died. Event free survival was (EFS 19% ± 4%). Three out of the 4 survivors received MMUD and all 4 survivors had moderate to severe acute GVHD and three had chronic GVHD, limited in two and extensive in one.

Conclusion: Children with ALL in CR3 receiving HSCT are extremely high risk for relapse and transplant related mortality. These children have already relapsed twice and demonstrated chemotherapy resistance and GVL/GVHD plays a key role in leukemia eradication. Although, TRM is high in such patients and GVHD could potentially increase TRM, there are no survivors without GVHD and exploring means of inducing GVHD by reduction of immunosuppressive medications or other means of immunotherapy should seriously be considered in these patients.
Gleevec as Therapy for CML Relapsed after Allogeneic Stem Cell Transplantation: High Rate of BCR-ABL PCR Negativity and Donor Graft Reconstitution.
Authors: Funke Vaneuza A.M. , Nabhan Samir K. , Oliveira Michel M. , Ruiz Jefferson , Setubal Daniela C. , Azambuja Ana Paula , Lima Denise H. , Kojo Terezinha K. , Bitencourt Henrique , Hamerschlak Nelson , Clementino Nelma , Neves Heliz Regina A. , Pasquini Ricardo
Published: October 2019

INTRODUCTION: CML relapsed after HSCT has been treated with donor lymphocyte infusions (DLI), with responses achieving 60–70%, and even better results in earlier relapses. However, complications as chronic GVHD and myelossupression can be a problem, especially on those who receive larger DLI doses. The present study has the objective of evaluate quality of response and chimerism in patients who received Gleevec as therapy for relapsed CML after HSCT.

PATIENTS AND METHODS: We report 32 CML patients who received Gleevec as therapy for relapse after HSCT. Age: 13 – 56 (Median: 38 y); sex M/F: 17/15; Allogeneic HSCT: 31 pts, syngeneic: 1 pt. Time from HSCT to relapse: 0–122 m (M 16m). Relapse was hematologic in 29 and cytogenetic in 3 patients. Phase at hematologic relapse: CP-14 pts, AP-11 pts and BC-4 pts; clonal evolution: 7 patients. Previous VNTR was available in 16 pts: 10–35% donor in 11, 0% donor in 3 and 95% donor in 2 pts. Previous DLI: 14 pts. Dose: 600mg QD in 15 pts, 400 mg QD - 17 pts.

RESULTS: Therapy duration: 7–1795 d (M 365 d). 27 (84%) pts reached complete hematologic response (CHR). Time to CHR: 7–60 d (M 28 d). Cytogenetic response was available for 25 pts, being complete in 16 (48%), partial in 2 and absent in 7 pts. Competitive Q-PCR was available in 21 patients, of whom 10 had major molecular responses (3-log reduction at the BCR-ABL/ABL ratio). In 6 (18%) patients, BCR-ABL was negative by nested PCR. Four (13%) pts relapsed after a initial response (1 BC, 3 AP). Nine from 10 pts with sequential VNTR, improved chimerism to more than 95% donor cells after Gleevec therapy. One patient had autologous recovery of Ph negative cells. Hematologic toxicity grade II–IV was observed in 21 pts (63%). Eleven pts developed grade IV neutropenia with a duration of 0–8m (M 2m). Five pts developed non-hematologic toxicity grade III–IV. Dose was held in 4 pts, increased in 6 and reduced in 11 pts. Only two pts developed skin and liver chronic GVHD, and two patients who had c-GVHD before therapy with imatinib did not flare. Estimated 5y OS is 67%.

CONCLUSIONS:Gleevec can be used safely as therapy for BMT relapse, with durable cytogenetic and molecular responses in chronic phase;Complete chimerism is often reestablished by Gleevec therapy after allogeneic BMT;Increased incidence of GVHD was not observed in this group of patients.
Poor Prognosis for Patients after Myelo- and Non-Myeloablative Conditioning Therapy Followed by Allogeneic Peripheral Blood Stem Cell Transplantation Admitted to Intensive Care Unit.
Authors: Neumann Frank , Lobitz Oliver , Fenk Roland , Kondakci Mustafa , Graef Thorsten , Bruns Ingmar , Kelm Malte , Huenerlituerkoglu Ali-Nuri , Haas Rainer , Kobbe Guido
Published: October 2019

Background and Aims: The role of intensive care unit (ICU) support for patients following allogeneic peripheral blood stem cell transplantation (PBSCT) is controversial. In an era of constrained resources, we assessed prognostic factors predictive for survival in patients after myeloablative (MAC) and non-myeloablative allogeneic (non-MAC) PBSCT over a period of seven years.

Patients: Between January 1999 and February 2006 three-hundred and one patients with various hematological malignancies underwent allogeneic stem cell tranplantation in our institution (MAC 196, non-MAC 105). Of these, sixty-four patients (21,3%) with a median age of 47 years (range 18–64 years; female 24, male 40; MAC 49, non-MAC 15) were admitted to the ICU during the first two years following PBSCT (median 55 days, range 1–631 days). We looked for variables defining the SOFA (Sequential Organ Failure Assessment) and the SAPS (Simplified Acute Physiology Score) score on the day of ICU admission and five days later to discriminate patients with poor and good prognosis with regard to survival. We also looked for variables such as age of patients, diagnosis, disease status, donor type, time between transplantation and ICU admission, reason for ICU admission and occurrence of veno-occlusive disease and GVHD.

Results: Mechanical ventilation was required by all patients admitted to the ICU. Median survival following referral to ICU was 22 days (range 0 – 959 days). The main reason for death was sepsis (56%). Among 49 patients who had received MAC 19 (39%) survived the ICU stay with a median survival time of 11 months (range 0–29 months). In the group of patients who had received non-MAC 3 out of 15 patients (20%) could be discharged from the ICU with a median survival time of 5 months (range 4–12 months). Looking at the twenty-two ICU survivors there were seven patients who survived the following year resulting in an overall survival of 11% one year after ICU admission. Only the SOFA score (p = 0.002) on the day of ICU admission was of prognostic relevance for survival.

Conclusion: ICU admission and respiratory failure are associated with poor prognosis after allogeneic stem cell transplantation The probability of survival is independent from the type of conditioning therapy. The SOFA score is a predictor for short term survival but fails to identify long term survivors.
“Designer” Cells for Cellular Immunotherapy: Zinc Finger Nuclease (ZFN) Stimulated Targeted Recombination for the Simultaneous Disruption of the Endogenous Glucocorticoid Receptor and Site-Specific Addition of the IL13-Zetakine.
Authors: Reik Andreas , Holmes Michael C. , Urnov Fyodor D. , Zhou Yuanyue , Mendel Matthew , Liu Pei-Qi , Moehle Erica , Rebar Edward J. , Miller Jeffrey C. , Ando Dale , Gregory Philip D. , Jensen Michael
Published: October 2019

Immunotherapy utilizing genetically-modified cytolytic T-lymphocytes (CTL) provides a promising therapeutic approach for treating a variety of diseases, including cancer.

We have demonstrated that CTLs encoding a chimeric T-cell receptor (IL13-zetakine), consisting of an extracellular IL-13 domain and a cytoplasmic CD3 domain, can be re-directed to target malignant glioma both in vitro and in animal models. This chimera re-targets the antigen-specific effector functions of modified CTLs to recognize glioblastomas due to the high expression of IL13R in these tumors. However, practical application of this approach is limited by the fact that patients undergoing surgical resection of the tumor often require treatment with glucocorticoids to control the resulting inflammation. Such treatment blocks the activity of the re-directed CTL clones and thus inhibits their therapeutic action.

To overcome this limitation and render these tumor-specific CTLs resistant to glucocorticoids we have chosen to employ engineered ZFNs to specifically disrupt the endogenous glucocorticoid receptor (GR) gene. Heterodimeric ZFNs, consisting of the cleavage domain of the restriction enzyme FokI linked to engineered zinc finger DNA-binding domains, can be designed to specifically cleave a predetermined site in the genome. We have shown that these ZFN-induced double strand breaks can promote homologous recombination with high efficiency. In the present study we have investigated the use of ZFNs to simultaneously effect functional inactivation of human GR via specifically targeting the integration of the IL13-zetakine expression cassette into the GR locus itself. We can show that GR-specific ZFNs cleave their intended target sequences with high specificity and efficiency - resulting in the disruption of GR and the creation of glucocorticoid resistant cells. Moreover, we can demonstrate that these ZFNs coupled with an appropriate IL13-zetakine containing donor-DNA molecule can stimulate the integration of this chimeric T-cell receptor directly into the GR locus. Thus, this procedure results in the simultaneous knockout of GR and addition of the IL13-zetakine in a genetically defined manner.

These data support the notion that ZFN-modified cells can be engineered to express chimeric antigen receptors from a predetermined genomic locus and may provide a general approach to generating effective cellular immunotherapy strategies.
Tumor-Activated Human NK Cells Specificall Ylyse Autologous and Allogeneic NK-Resistant Tumor Targets.
Authors: Lowdell Mark W. , Bakhsh Ismail , North Janet , Marden Chloe , Addison Elena , Mackinnon Stephen
Published: October 2019

Pre-incubation of resting human NK cells with the leukemia cell CTV-1 primes NK cells to lyse NK-resistant cell lines, primary leukemias and solid tumors, even when HLA-matched; allogeneic or autologous. The primed NK cells remained non-responsive to HLA-C matched and mismatched normal peripheral blood or bone marrow mononuclear cells from multiple donors and did not affect in vitro colony forming unit assays. NK cells isolated from normal healthy donors and co-incubated with irradiated CTV-1 tumor cells synthesized CD69 within 60 min with maximal expression achieved within 6 hr. The tumor-activated NK cells (T-ANKs) lysed NK resistant RAJI and Daudi cells in a 4-hour assay (Fig 1). The degree of lysis was equivalent to that of matched lymphokine activated killer cells (LAK) after non-specific activation with IL-2. CTV-1 cells are HLA-C type 2 homozygous and express HLA-Bw4 alleles. They thus can ligate KIR2DL1 (CD158a) and KIR3DL1 (CD158e1) on NK cells. Co-cultures of HLA-matched or mis-matched NK with CTV-1 showed there was no significant difference in the generation of T-ANK activity in the presence or absence of KIR ligation. T-ANK cells from allogeneic donors were capable of lysis of primary AML cells of all FAB types, the relatively NK-resistant breast cancer cell line, MCF-7, and primary tumor cells isolated from resected tissue from patients with breast cancer and ascities from patients with ovarian cancer. Autologous T-ANK cells were generated from five AML patients in clinical remission; all of whom had low level or undetectable NK activity to their presentation disease. T-ANK cells from all five donors consistently induced significantly greater lysis of autologous AML blasts than the matched NK cells (p<0.05) (Fig 2). To investigate the tumor-restriction of allogeneic T-ANK cells we isolated NK cells from normal donors and either activated them with CTV-1 cells overnight or maintained them in media. These T-ANK cells were then compared with NK cells from the same donor with respect to lysis of normal autologous and allogeneic PBMC. Neither NK nor T-ANK cells lysed autologous PBMC nor did they lyse PBMC from HLA-C mismatched normal donors. To determine the likelihood of bone marrow suppression by T-ANK cells we established hematopoietic colony forming assays with bone marrow from 5 normal donors and added T-ANK from HLA-C mismatched donors at increasing ratios. CFU-GM, BFU-E and CFU-GEMM were not affected by co-incubation with HLA-mismatched T-ANK even at effector:target ratios of 20:1. We believe that this demonstrates a two stage process for NK activation; priming and triggering, the ligands for which are distinct. Tumor priming of NK primes cells capable of lysing a broad spectrum of NK-resistant tumors which may irrespective of KIR ligation which may represent an “off-the-shelf” immunotherapy product.

Fig 1 Fig 1. Fig 2 Fig 2.
Playing the Game Together: Coexpression of a Single Chain T Cell Receptor and a T Cell Receptor Constant-Alpha Domain Triggers Tumor Reactivity.
Authors: Thomas Simone , Voss Ralf H. , Intan Ratna , Engel Renate , Kuball Juergen , Huber Christoph , Theobald Matthias
Published: October 2019

Grafting T cells by tumor-antigen specific T cell receptors (TCR) could trigger the initiation of effector function and redirect T cell cytotoxicity towards tumors. We utilized various HLA-A2.1 transgenic mice to bypass human MDM2- and p53-specific self-tolerance. In contrast to the use of HuCD8×A2Kb transgenic mice to generate an MDM2-specific CD8-dependent TCR, we generated a high-affinity, CD8-independent p53-specific TCR in single human A2.1 transgenic mice. The efficiency of double chain (dc) TCR modified T cells could be affected by the incorrect TCR α/β chain pairing between endogenous and transgenic TCR constructs to form hybrid TCR potentially leading to autoimmunity. To address this concern, chimeric A2.1-restricted peptide-specific murine single chain (sc) TCRs were constructed (Vα-Li-VβCβ) and retrovirally transduced into human T cells. Despite detectable surface expression, these chimeric receptors were not able to convey any MDM2- or p53-specific cytolytic activity. Therefore we developed a truncated TCR-alpha domain (Cα) comprising solely the TCRα signal peptide, the ecto-domain, the transmembrane region as well as the cytoplasmic tail and cotransduced these construct with the scTCRs. We anticipated that Cα would stabilize scTCR expression by interacting with the single chain beta chain. Indeed, this approach not only led to increased expression levels of the chimeric scTCRs, but also induced specific lysis of A2.1 positive MDM2 or p53 peptide-pulsed target cells as well as solid tumor cell lines. Recognition of malignant targets by p53 specific scTCR transduced CD4 and CD8-positive T cells was equivalent to that observed with double-chain p53 TCR gene modified effector cells. To test whether this concept is applicable to human TCRs as well, we constructed a human gp100-specific scTCR and a human Cα domain. In contrast to the gp100-specific double chain TCR, only a marginal expression pattern was observed for the human scTCR / Cα constructs. Introduction of an additional disulfide bond within the constant domains in order to stabilize TCR surface expression showed no effect. Since murine TCR are expressed on human T cells to a much higher extent, the human constant β-domain of the scTCR was replaced by murine Cβ. Comparable to the murine scTCR concept, the chimerized scTCR coexpressed with murine Cα demonstrated high cell surface expression and triggered cytotoxicity of malignant A2.1/gp100-positive targets.

In summary, our results lay a commonly applicable conceptual basis for the construction of therapeutic scTCR to prevent recombination of natural and transgenic dcTCR alpha and beta chains.
Effect of Cytosolic Hereditary Spherocytosis Mutations of Human Erythrocyte Anion Exchanger 1 on Its Interaction with Cytoskeletal Protein 4.2.
Authors: Bustos Susan P. , Reithmeier Reinhart A.F.
Published: October 2019

Anion exchanger 1 (AE1, Band 3) is the predominant membrane protein of erythrocytes. Human AE1 has two functionally independent domains: its 52 kDa C-terminal membrane domain catalyzes the exchange of chloride for bicarbonate across the membrane while its 43 kDa N-terminal cytosolic domain (cdb3) anchors the membrane to the cytoskeleton, giving the red cell its stability and flexibility. Several proteins bind to cdb3 including cytoskeletal protein 4.2, ankyrin, glycolytic enzymes and deoxyhemoglobin. Three mutations in cdb3 (E40K, G130R and P327R) are associated with the hemolytic anemia hereditary spherocytosis (HS) and decreased levels of erythrocyte protein 4.2 while maintaining a normal amount of AE1 at the red cell membrane. Wild-type and mutant cdb3 proteins were expressed in E. coli and purified and it was shown through a variety of biophysical methods that these three HS mutations do not cause major structural changes in this domain. Each of these mutations introduces a positive charge at the surface of the protein that is predominantly negatively charged, which may have an effect on protein interactions while maintaining its native folded structure. Full-length wild-type AE1 or HS mutants were co-expressed with protein 4.2 in HEK-293 cells in order to study their interaction in a mammalian cell line. All three HS mutant proteins were expressed at similar levels to wild-type in these cells and were shown to be present at the membrane using immunofluorescence and confocal microscopy. These proteins were also co-expressed in LLCPK-1 cells for the purpose of co-localization studies using immunofluorescence. A series of GST fusion proteins of protein 4.2 domains were designed based on a homology model of protein 4.2 and regions of the protein known to interact with AE1. These fusion proteins were expressed in E. coli and purified on glutathione-Sepharose resin and were used to study their interaction with purified wild-type and HS mutant cdb3 proteins in vitro. Blot overlay analysis showed that a protein 4.2 GST fusion protein containing a putative β-hairpin region (Asp145 to Glu203) binds specifically to wild-type cdb3 while GST does not. It is hypothesized that since these three HS mutations do not cause major structural changes in cdb3, the decreased level of protein 4.2 in the red cells of these patients is a result of impaired binding that occurs due to these mutation sites.
Gfi1b Association to Human Hematological Abnormalities.
Authors: Villegas Ana , Gonzalez Fernando A. , Anguita Eduardo
Published: October 2019

Lineage specific transcription factors play essential roles in regulation of hematopoietic development. Transcription factor abnormalities have been frequently described in acute leukemia, mostly through cytogenetic changes. Nevertheless, point mutations can be easily missed. Recently, mutations in the erythroid and megakaryocyte specific transcription factor GATA1 have been discovered in patients with dyserythropoietic anemia and acute megakaryoblastic leukemia (AML-M7) with Down syndrome. Besides GATA-1, located on the X-chromosome, point mutations have been described in biallelic genes. This is the case of AML1 (RUNX1). PU1 and C/EBPalpha also represent examples of transcription factors in which point mutations are found in leukemia.

A new zinc finger transcription factor involved in erythropoiesis is Gfi1b. Gfi1b was recently identified by sequence homology with oncogene Gfi1. Gfi1b knockout has demonstrated that this gene is essential for development of both erythroid and megakaryocytic lineages, and in its absence no enucleated erythrocytes are produced.

Several Gfi1b DNA and protein targets (GATA1, Gfi1, AML1, p21WAF1, IL-6 Socs1 and Socs2) have been described that might be involved in malignancy. These findings indicate that Gfi1b is at the centre of hematopoiesis and may be a good candidate gene to be involved in hematological abnormalities.

We have searched for Gfi1b point mutations in 122 patients with acute leukemia of all FAB types at diagnosis or relapse and 9 cases of congenital dyserythropoietic anemia. We have amplified Gfi1b promoter, coding and non-coding exons (Nucleic Acids Res2004;32:3935–46, MN 004188) by high fidelity PCR and screen for point mutations through dHPLC (Wave, Transgenomic) followed by sequencing of the cases with abnormal pattern. SNIPs in the promoter and exons were further confirmed in at least another PCR, cloned in pGEM-T easy vector system (Promega) and sequenced. Alleles with promoter SNIPs were cloned in pGL3-Enhancer vector (Promega), and transiently cotransfected with pEGFP-C2 (Clontech) to K562 cells. Luciferase activity was determined with Dual-Luciferase Reporter Assay (Promega).

We found two promoter SNIPs in sequences conserved from chicken to human, one of them affecting a GATA-1 site, reducing promoter in vitro activity by 60 and 50% respectively. We also discovered a congenital exonic SNIP causing a mammalian conserved serine change to leucine. We excluded these to be frequent polymorphisms by dHPLC analysis of 96 blood donors. Although we cannot at present establish a clear relation between the former SNIPS and leukemia, we will discuss the presence of other milder hematological abnormalities. So far this is the first report of Gfi1b mutations that can be related to human pathology.
Paroxysmal Nocturnal Hemoglobinuria (PNH): Long Term Follow up of 48 Patients in a Single Institution.
Authors: Ruiz Jefferson , Medeiros Larissa A. , Funke Vaneuza A.M. , Bonfim Carmem M.S. , Bitencourt Marco A. , Setubal Daniela C. , Pettengil Claudia , Nunes Elenaide C. , Oliveira Michel M. , Neto Jose Zanis , Medeiros Carlos R. , Malvezzi Mariester , Pasquini Ricardo
Published: October 2019

Background: PNH is a clonal acquired disease characterized by poor expression of CD55 and CD59, adding higher lytic activity to the complement pathway, ending up in hemolysis, pancytopenia and thrombosis. The clinical variability and the lack of effective therapeutics make the treatment of PNH a real challenge.

Material and methods: A retrospective analysis of 48 patients diagnosed with PNH between feb/1997 and march/2006 admitted to our center was performed. Before 1999, diagnosis was made by HAM and SACAROSIS tests. From this point immunophenotyping analysis was used to establish diagnosis. Bone marrow aspirate and biopsy, cytogenetic and biochemistry analysis were performed for exclusion of comorbities. Patients were divided in two groups: 1) de novo PNH associated with marrow failure (MF) and 2) PNH as an evolution of severe aplastic anemia (SAA) or mielodysplastic syndrome (MDS). Response was defined as reduction at hemolytic episodes as well as improvement of CBC and transfusion requirements.

Results: Median age was 24 years (range 9–75); male patients constituted 58% of the sample (male/female: 1,4:1). De novo PNH with associated marrow failure occurred in 22 patients (group 1) and evolutive PNH in 15, being 13 from previous SAA and 2 from previous hypoplastic MDS (group 2). Thirty seven patients were followed at our clinic during this time: 25 (67,5%) received immunesupression with cyclosporine and steroids. Overall response (partial or complete) after one year of therapy was 84% (31/37). The remaining 11 patients (32,5%) received steroids alone, androgens or only observation, four of them remained with stable disease. HLA match hematopoietic stem cell transplantation (HSCT) was performed in 18/48 (38%), being 11 from group 1 and 7 from group 2. Main indications were: pancytopenia (77%), thrombosis (17%) and AML (6%).

Only nine patients died (19%): five after HSCT and 4 of those followed at our clinic (massive thrombosis and hemolysis). Overall survival after HSCT was 72% with median follow-up of 890 days. Overall survival among outpatient clinic patients was 86% with a median follow-up of 2340 days. Most frequent outpatient events were: hemolysis (48%), pancytopenia (13%), thrombosis (13 %), renal failure (9%) and infection (8%).Fourteen patients (37%) remained without symptoms.

Conclusions:Immunosuppressive therapy with cyclosporine and corticosteroids is an excellent strategy in patients with PNH and associated marrow failure, leading to durable clinical responses.The present data supports previous published indications for HSCT: ASS and thrombosis.Overall survival of PNH patients who received HSCT achieved 72%.
HbA Is More Effective Than HbF for Improving the Clinical Course in Sickle Cell Disease.
Authors: Bezerra Marcos A.C. , Fattori Andre , Santos Magnum N. , Albuquerque Dulcineia M. , Sonati Maria F. , Saad Sara T.O. , Araujo Aderson S. , Costa Fernando F.
Published: October 2019

It is well known that high levels of HbF exert a protective effect on the clinical outcome of sickle cell disease. For this reason hydroxyurea, a drug that increases levels of HbF, is standard treatment for sickle cell disease patients with severe clinical courses. We present, herein, two groups of patients with sickle cell disease (HbS / β thalassemia) with differing genotypes who present similar quantities of HbS, but who clearly have differences in the amounts of HbF and HbA. The table below presents the genotypes, and the hematological and clinical data of the two groups of patients. Data presented in the table demonstrate that the clinical course of the patients with a high percentage of HbA is very mild, whilst the clinical course of the patients with high levels of HbF is severe. It is important to note that the amount of HbA + HbF is not statistically different in the two groups (22.8±8.3 e 28.1±1.8). There were no other factors, such as haplotypes or α genotypes that could have interfered with the clinical presentation of the patients. Taken together, the data presented suggest that a protection against hemoglobin S polymerization in patients may be more effective in the presence of HbA compared to similar amounts of HbF. In this case, it would be possible to speculate that a therapy aiming to induce the production of HbA, even in small amounts, may be more effective in the treatment of sickle cell disease than the increase in the production of HbF.

Comparison between S/ beta thal IVSI-5 and S / beta thal IVSI-6 patients: Genotypes (S / beta thal) Hb (g/dL) HbS (%) HbA (%) HbF (%) S (n) T (%) HAI / HAC (%) S: Splenectomy (no of patients); T: Transfusion (more than 3 transfusion events); HAI: Hospital Admission for Infection events; HAC: Hospital Admission for pain Crisis (frequency: more than 3 vaso-occlusives episodes). All values (with the exception of HbS) were statistically different between the 2 groups (p<0.001, Wilcoxon rank sum test). S / IVSI-5(G-> C) n = 18 7.4±1.0 72.9±8.2 5.2±1.3 17.6±9.1 6 72 89(16) S / IVSI-6(T -> C) n = 20 11.6±0.9 67.4±1.7 26.3±1.4 1.8±1.2 0 0 5 (1)
Hematological and Clinical Findings Predictive of Mortality in Italian Patients with Sickle-Cell Disease: A Case-Control Study.
Authors: Maggio Aurelio , Di Salvo Veronica , Rigano Paolo , Giangreco Antonino , Renda Maria Concetta , Renda Disma
Published: October 2019

Background: the number of cases with Sickle-Cell Disease (SCD) registered in Italy at 2000 was 928. Six-hundred-twenty-two (67%) of these were with Sickle-Cell/βthalassemia. Moreover, while the main causes of death in Afro-American patients with SCD is well known, we do not have many informations in Italian subjects. For this reason, we carried ahead a case-control study in this group of patients.

Aim of the study: the evaluation of the main hematological and clinical parameters predictive of mortality.

Materials and Methods: clinical and hematological findings of 14 SCD died patients were compared with a SCD alive control population homogeneous for age, sex, phenotype and number of painful crises per year. Two alive control subjects were studied for each died patient.

Results: the main causes of death were: Acute Chest Syndromes (ACS) (n°6), Cardiovascular Diseases (n°3),Hepatic Failure (n°1),Hodgkin Lymphoma (n°1), Stroke (n°1), Lung Cancer (n°1), Surgery Complication (n°1). The main correlated hematological findings with mortality were Hb (gr/dl) levels (p=<.05), WBC count (103 / ml) (p= <.05), ferritin levels (ng/ml) (p=<.01). The main correlated clinical features were: the number of crises >3/year (Odd Ratio= 1.64; p= <.01), previous stroke (Odd Ratio= 1.53; p= <.01), HU treatment (Odd Ratio=<.01; p=<.01), leg ulcers (Odd Ratio=1.46;p=<.01).

Conclusions: these data suggest that the main causes of death in Italian patients with SCD are similar to those reported for Afro-American subjects (M.H. Steinberg, JAMA, April 2, Vol. 289, N°13, 1645–1651). Particularly, the lack of HU treatment, anemia, high ferritin levels, number of crises per year and previous stroke are the most important predictive factors of mortality.
HbQ-Iran [α75 (EF4) Asp→His]/−α3.7/β° IVSII.1 G→A in an Iranian Child: First Reported Case.
Authors: Muniz Adriana , Nagel Ronald L. , Rahimi Zhoreh , Raygani Asad V. , Akramipour Reza , Bahremand Fariborz
Published: October 2019

HbQ-Iran [α75 (EF4) Asp→His] is an α-chain variant first described in 1970, which substitution is not involved in the interchain contacts of hemoglobin. It has a slow-moving migration pattern on cellulose acetate at alkaline pH, that resembles the Hb S, but with normal solubility. The quantity of this variant in the heterozygous state has been reported to be 17–19%. Heterozygotes individuals with HbQ-Iran are hematologically asymptomatic and the combination with α+-thalassemia and β0- thalassemia has not been reported up to now.

We here described the hematological characteristics of a 5 years old child referred to the clinic of Kermanshah University. Physical examination revealed no enlargement of the spleen, and the initial blood count indicated a mild microcytic anemia with low levels of Hb (10g/dL), a hematocrit of 30.5% and low MCV and MCH (59.3fL and 19.6pg) respectively. DNA analysis revealed the presence of HbQ-Iran, −α3.7 kb deletion of the alpha globin genes and also the presence of the mutation (IVSII.1.G→A) β0-thalassemia in the child. The study of the family revealed the presence of a minor β-thalassemia in the father and the HbQ-Iran in the mother. Both parents were heterozygous for the −α3.7 deletion (−α3.7/α α) in the α-globin genes. The coinheritance of HbQ-Iran with −α3.7 deletion and β-thalassemia resulted in an elevation of the levels of HbQ-Iran to 22.4%. However, the remainder hematological values of the young patient indicated a significant reduction in MCV (59.3 fL) and MCH (19.6 pg), an elevation of HbF (6.3%) and of HbA2 (3.7%) according to a picture of a minor β-thalassemia. This result demonstrated that HbQ-Iran is a benign structural variant, that in combination with β0-thalassemia and in the presence of a α+-thalassemia, produce a minor β-thalassemia picture with moderate anemia and elevation of HbF. The application of DNA technologies allowed the identification for the first time of the presence in one individual of three already known mutations, emphasizing the unique genetic heterogeneity of this population.
Beneficial Effect of Hydroxyurea on Avascular Necrosis of Femoral Head in Adult Sickle Cell Disease (Homozygous) - One Year Prospective Study in Orissa, India.
Authors: Jena Rabindra , Swain Trupti R.
Published: October 2019

Avascular nacrosis (AVN) of femoral head in Sickle cell Disease, (Homozygous) (SCD) is a progressive and crippling complication seen commonly in India. AVN evolves slowly from cumulative effect of recurrent small episodes of ischemia or infarction within the spongiosa of bone. This complication can occur in the absence of painful crisis for which a satisfactory treatment is yet to be found. Though Hydroxyurea inhibits polymerization of HbS by raising HbF level, its role on AVN of femoral head is not known. Forty six adult SCD with AVN of Femoral head with or without painful crisis received hydroxyurea at a dose of 20 mg/ kg/ day orally while 18 adult SCD with AVN of femoral head, without painful crisis were taken as control. Both the groups received folic acid (1 mg/ day), red cell transfusion and ibuprofen as required. All patients were advised adequate rest and to avoid prolong standing and walking. Clinical, hematological and radiological evaluation were done once in three month for one year.

RESULT: Pain in the hip joints were completely absent both during rest and activity after a median period of 6 months in 11 patients (73.3%,out of 15 cases) in early stage of AVN (minimum necrosis, crescent sign).Radiological abnormalities disappeared completely in these patients after a median period of 9 months. Similar benefits were observed in 7 cases (35%, out of 20 cases) of SCD with segmental collapse of femoral head. Most advanced cases of AVN (complete degeneration and collapse of femoral head) were resistant with least benefits in 3 patients (25.4%, out of 11 cases) with only reduction of pain at rest without any improvement in radiological changes and quality of activity. In control groups though pain was relieved during rest in 4 cases (22.2%) no improvement in radiological picture and pain during activity was observed. Chi square analysis detected a significant rise in Hb F level and benefit in hydroxyurea treated group. Thus hydroxyurea administration in early stages of AVN due to SCD is beneficial.
Hb F Malta I in Association with Hb F Sardinia (AyT) and Hb Valletta in Heterozygotes: Quantification of the Six Globins Suggests Developmental Control of the XMN-I Site and Interplay with the (AT)xTy Sequence in Connection with Globin Gene Switching.
Authors: Felice Alexander , Borg Joseph , Cassar Wilma , Galdies Ruth , Pizzuto Monica , Caruana Maryrose , Scerri Christian
Published: October 2019

Although the precise biochemical mechanisms of globin gene switching remain elusive, considerable insight is gained by in vivo expression profiling through quantification of the hemoglobin / globin phenotype of informative heterozygosities and homozygosities / compound heterozygosities in the context of specific regulatory DNA sequence diversity such as the XMN-I or the [(AT)xTy] sequence polymorphisms.

The quantification of normal and abnormal globins of Hb F Malta-I (or a2b2, 117(G19)His>Arg) heterozygotes which are in tight linkage disequilibrium with Hb Valletta (or a2b2 287(f3)Thr>Pro) i.e. Gyo, GyFMalta-I, AyI, bV and bA together with extensive haplotyping of homozygotes and heterozygotes including the XMN-I dimorphism in the Gy promoter and the (AT)xTy polymorphism (BP1 binding site) 5′ to the b globin genes had suggested that the XMN-I dimorphism was largely inactive in the normal newborn. In contrast the Hb F levels and the proportion of Gy globin in anemic adult beta-thalassemia homozygotes and compound heterozygotes differed significantly, depending on the XMN-I genotype (TT, TC or CC) Here, we document the occurrence of seven newborn who were heterozygous at three globin loci permitting quantification by reverse phase liquid chromatography of the six globin products; Gyo, GyFMalta-I, AyI, AyT, bV and bA in the context of genotypic variation at the XMN-I and (AT)xTy sequences. The data were compared with those of newborn HbF-Malta-I-Hb-Valletta heterozygotes and anemic adult beta thalassemia homozygotes / compound heterozygotes. The globin quantification together with haplotype data were analysed using the general linear model (two-way ANOVA) by SPSS version 12. The data excluded significant effect of the XMN-I dimorphism alone on relative y/b globin gene expression in the newborn. On the other hand, the (AT)xTy polymorphism with BP1 binding sites of 21 [(AT)7T7], 23 [(AT)9T5], or 25 [(AT)11T3], nucleotides in trans over-ride XMN-I. In contrast, it is the XMN-I dimorphism that over-rides the (AT)xTy diversity in the anemic adult beta thalassemia homozygotes or compound heterozygotes. The GyFMalta-I/Gyo ratio of the newborn heterozygotes with Hb F Malta-I and the AyT/AyI ratio of the newborn heterozygotes with HbF-Malta-I and HbF-Sardinia suggested that the developmental regulation of the XMN-I site may be subject to cis/trans interplay with the (AT)xTy sequences.
Mechanism of Immune System Activation by (1,4)-α-D-glucan Isolated from Tinospora cordifolia in Macrophages.
Authors: Nair P.K. Raveendran , Melnick Steven J. , Khatib Ziad A. , Ramachandran Reshma , Escalon Enrique A. , Ramachandran Cheppail
Published: October 2019

We have characterized and reported the immunostimulating properties of a novel polysaccharide - (1,4)-α-D-glucan (RR1)- isolated from the medicinal plant Tinospora cordifolia [23]. This novel glucan is water soluble and having (1, 4)-α-D-glycosidic linkages in the main chain and (1, 6)-α-D-glycosidic linked side chains at an interval of 6, 7 glucose units. The signaling mechanism of the novel (1,4)-a-D-glucan (RR1) was investigated in macrophages to evaluate its immunostimulating properties. When RAW264.7 macrophages were incubated with RR1 at 4°C, the novel glucan inhibited the phagocytosis of unopsonized zymosan A bioparticles in a dose-dependent manner. RR1 also inhibited the binding and internalization of opsonized zymosan A bioparticles, although at a lower level than laminarin. Incubation of macrophages with anti-CD11b mAb followed by RR1 failed to show any inhibitory effect on RR1-induced TNF-α synthesis confirming that complement receptor 3 (CR3) is not involved in the opsonic binding and internalization of RR1 in macrophages unlike zymosan A. The anti-CD11b mAb has significant inhibitory effect on the zymosan A-induced tumor necrosis factor (TNF)-α synthesis. RR1 induced TNF-α synthesis in macrophages in a dose-dependent manner which can be completely inhibited by the NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or curcumin. RR1 activated NF-κB in a time- and dose-dependent manner and this modulation of nuclear NF-κB activity is associated with the degradation of I-κB a thus facilitating the translocation of NF-κB into the nucleus. RR1-induced NF-κB activity peaks at 8 h of RR1 stimulation while I-κB a degradation occurred within 1 h of stimulation. RR1-induced NF-κB activation occurred through TLR6 signaling as evidenced by the synthesis of IL-8 in TLR6-transfected HEK293 cells. These results show that the novel (1,4)-α-D glucan from Tinospora cordifolia activates the immune system through the activation of macrophages that occurs through TLR6 signaling, NF-κB translocation and cytokine production. (Supported by Miami Children’s Hospital Foundation research funds).
Haplotype Block for Inflammatory Cytokine Gene Polymorphisms in Fabry Disease.
Authors: Elstein Deborah , Safyan Rachael , Whybra Catharina , Beck Michael , Altarescu Gheona
Published: October 2019

Fabry disease is an X-linked disorder associated with early-onset stroke, cardiomiopathy, and progression to end-stage renal failure. Correlations between IL-6 inflammatory cytokine gene polymorphisms and Mainz Severity Score Index (MSSI) scores have been shown. Therefore, polymorphisms of other key pro- and anti-inflammatory cytokines and correlation to clinical manifestations (MSSI scores) were attempted in order to build haplotype descriptions for Fabry disease. Genotyping for IL-10[819C/T;-592C/A]; IL-1b[+3954 C/T; -511C/T]; IL-1α[-889C/T]; and TNF-α[–308G/A] was performed in 76 patients and correlated with MSSI sub-scores and with enzyme (alpha-galactosidase A) levels. Fifty normal volunteers, age- and sex-matched, were also genotyped. Of 76 patients, 31(41%) were males and 45 (59%) were females. There was no correlation between enzyme levels and any cytokine levels. Statistically significant differences were found in prevalence of TNF-α [–308G/A] genotypes: 84%GG in patients versus 63%GG in controls (p=0.038) and for IL–1α [–889C/T] genotypes: 94%CC in patients versus 21%CC in controls (p<0.001). Statistically significant differences were found in the ratio between the two polymorphisms of IL-10 (p<0.0001), between the two polymorphisms of IL-1b (p=0.001); between IL-1α [–889C/T] and IL-1beta [3954C/T] (p=0.002); and between IL-10[–592C/T] and IL-1b [3954C/T] (p=0.041). Correlations between TNF-α [–308G/A] and both kidney and neurological MSSI sub-score (both: p=0.06) and between IL-10[-819C/T] and the MSSI neurological score (p=0.03) were noted. The relationship between inflammation and Fabry disease appears to exist on several levels. There was a multiplicity of associations among the two IL-10 polymorphisms and the two IL-1b polymorphisms and the one IL-1a polymorphism. These seem to indicate that the majority of patients have at least one C allele (but more often two C alleles) for each of the five polymorphic sites. On the other hand, the majority of patients had the GG genotype of the TNF-a[−308] polymorphism which is associated with decreased production of this pro-inflammatory cytokine. We speculate that sequence variations in regulatory DNA of genes coding for important members of the interleukin inflammatory family are associated with differential effects in Fabry disease and with increased sample size, haplotype blocks might be constructed.
The Spanish Gaucher Registry. An Observatoire To Detect Neurological Manifestations in Patients and Relatives.
Authors: Giraldo Pilar , Alfonso Pilar , Latre Paz , Fernandez-Galan Angeles M. , Alonso Dora , Ruiz Angeles M. , Vasco Ana , Masana Luis , Leon Pilar , Guzman Jose L. , Sanjurjo Pablo , Hernandez-Rivas Jesus M. , Albadalejo Jose , Loyola Ines , Martin-Hernandez Elena , Pocovi Miguel
Published: October 2019

Background: The Spanish Gaucher Foundation (FEETEG) is an independent and non-profit organization that keeps the Spanish Gaucher Registry (SGR) established in 1993. This SGR contains demographic, genetic, clinical, analytical and image data from a cohort of 306 Gaucher Disease (GD) patients (269 GD1, 22 GD2, 16 GD3), and 751 relatives. The SGR is a useful tool to perform epidemiological-genetic studies and analyse associated comorbilities. Patients and

Methods: From January to July 2006 an epidemiological survey to physicians and to the patients through GD Associations was conducted to ascertain the incidence of neurological symptoms in patients and their relatives as well as analyse genotype-phenotype relationship. The statistical analysis was performed in a SPSS 12.0 database using descriptive parameters, ANOVA, t-test and correlation study including Pearson coefficient.

Results: Twenty-four (38.7%) out of 62 GD1 patient respondents from 56 families have neurological symptoms, 8 (12.5%) tremor, 8 (12.5%) uncoordinated movements, 11 (17.7%) concentration defects, 3 (4.8%) strabismus and 8 (12.5%) deafness, 3 (4.8%) Parkinson Disease (PD). Twenty-one out of 56 families have one or more relatives with neurological manifestations. There were 29(8.6%) out of 336 relatives with neurological symptoms:13 (3.8%) PD, 5 (1.5%) epilepsy, 6 (1.8%) essential tremor, 5 (1.5%) others. Subjects with PD were carriers of mutations:: S364R, G202R, V398I, R47X, L336P, L444P, G195W, recNci and insertion alleles. In families with epilepsy the predominant carrier mutations were: L444P, G195W, R130W and in essential tremor: L444P.

Comments: There is a high incidence of neurological symptoms between GD1 patients and carriers. These manifestations appear frequently in carriers of rare mutations. It is very recommendable to perform a systematic neurological exam in GD1 patients and in carriers with risk mutations.