bluuebunny/crossref_metadata_2025 huggingface dataset:
Anti-Leukemic CTL Generated by RNA Loaded Cord Blood-Derived Dendritic Cells Control Leukaemia in a NOD-SCID Mouse Model.
Authors: Hsu Andy K.W. , Kerr Beverley M. , Lock Richard B. , Hart Derek N.J. , Rice Alison M.
Published: October 2019

Acute Lymphocytic Leukaemia (ALL) patients who relapse after transplantation have few therapeutic options. An immunotherapeutic approach that enhances the graft versus leukemia effect may improve their survival. We have developed a method for generating cord blood derived dendritic cells (CBDC) and transfecting them with high efficiency (>90% eGFP positivity) and viability (>90%) (BBMT 2006 12:855–867). We postulate that CTL generated from total ALL RNA-loaded CBDC may be exploited in therapy and therefore tested their ability to control the kinetics of leukaemic growth in a NOD-SCID mouse model of human ALL.

CTL induced using total RNA from the pre-B ALL Nalm-6 cell line lysed 49±11% of Nalm-6 cells compared to 7%±1% of autologous and 2.5%±1.3% of allogeneic mononuclear cells (p=0.03 and p=0.022 respectively). Using total RNA from patient derived ALL xenograft, we generated anti-leukemic CTL cultures comprising a mixed population of 69.9±2% CD4, 12.9±2% CD8 and 17.6±1% NK cells. These CTL lysed 63±6% of ALL xenograft targets and 22±7% of autologous MNC (p=0.04) and also lysed autologous survivin mRNA-transfected DC (16±5.4%). Preliminary results suggest that some of the ALL xenograft CTL also recognize the cell surface protein, HM1.24, which is specifically up-regulated in metastatic tumor cells and chemoresistant cancer cells. This suggests that the total ALL RNA generated CTL comprise a polyclonal population, which may prove efficacious in vivo. To test the in vivo efficacy of these anti-leukemic CTL, we established a NOD-SCID mouse model of ALL using patient samples. By varying the inocula of ALL cells, we established a predictable and reproducible model of emerging relapse.

Administration of anti-leukaemic CTL 2, 3, 4 and 5 weeks post ALL inoculation, in increasing doses (low - 2.5×106 cells/mouse, medium - 10×106/mouse and high - 20×106/mouse), altered the kinetics of ALL growth in vivo (p=0.00032, see figure). By 8 weeks post transplant, mice given ALL alone had 3.6% human CD45+CD19+ leukemic cells in the blood, whereas recipients of the highest CTL dose had only 0.66% CD45+CD19+ leukemic cells in the blood (p=0.05). After 11 weeks the disease load was the same in mice who received ALL alone and those who received ALL and 4 doses of 20 × 106 CTL. These results suggest that anti-leukemic CTL generated by RNA-loaded CBDC can control leukaemia in a NOD-SCID mouse model and that disease control may be enhanced by antigen-specific restimulation of the anti-leukaemic CTL and/or more refined administration. These results represent an important step forward in the development of clinical immunotherapy protocols for the treatment of ALL and its potential application after cord blood transplantation.

Anti-leukaemic CTL infusions alter the kinetics of ALL engraftment Anti-leukaemic CTL infusions alter the kinetics of ALL engraftment
Targeted Immuotherapy of Muc1 Positive Carcinomas with Genetically Engineered Human T Cells.
Authors: Chinnasamy Nachimuthu , Shaffer James , Chinnasamy Dhanalakshmi
Published: October 2019

Mucin1 (Muc1), a tumor-associated antigen, is overexpressed in many adenocarcinomas including breast, pancreas, ovary, prostate, colon, and in multiple myeloma. Therefore, tumor associated Muc1 is an attractive target for immunotherapy. Several types of immunotherapeutic approaches targeting Muc1 are currently being tested in preclinical and clinical settings. A major limitation of vaccination strategies involving tumor antigens is the lack of induction of sufficient number of antigen-specific effector T cells systemically to achieve objective clinical response. In an effort to redirect activated T cells towards tumor cells, we constructed lentiviral vectors expressing chimeric receptors consisting of a novel single chain variable domain (scFv) of a monoclonal antibody against Muc1 combined with various cytoplasmic signaling domains such as CD3 Zeta, CD28 and OX40. Human peripheral blood T cells were lentivirally transduced with chimeric immune receptors in order to redirect them towards Muc1 positive tumor cells. A sufficiently high level of transduction (>80) was achieved without any positive selection. Transduced T cells were stimulated and expanded over a period of 10–15 days in the presence of IL-2. Gene-modified T cells efficiently recognized Muc1 expressing tumor target cells in an MHC-independent manner as demonstrated by antigen-specific tumor cell cytolysis. Furthermore, transduced T cells showed antigen-specific proliferation and secreted interferon gamma upon ligation with Muc1 on tumor cells. These results indicate that generation of large numbers of antigen specific T cells for clinical application is possible through the use of this chimeric immune receptor gene transfer strategy. This approach might be an attractive strategy in a minimal residual disease setting complementing traditional therapies based on surgery, radiation and/or chemotherapy. Therefore, present study suggests a promising new strategy for cancer immunotherapy using gene-modified T cells.

Figure Figure
Successful Reversion of Refractory Paroxysmal Nocturnal Hemoglobinuria (PNH) by Allogeneic Peripheral Blood Stem Cell Transplantation.
Authors: Pang Ying , Feng Ying , Chen Ruizhen , Ye Xu
Published: October 2019

Objective: To observe the safety and effect of allogeneic peripheral blood stem cell transplantation (allo-PBSCT) on hematopoietic reconstitution of refractory paroxysmal nocturnal hemoglobinuria(PNH)and reversion of hemolysis.

Methods: A 20 years old female patient diagnosed with PNH complicated with cerebral infarction underwent allo-PBSCT. The donor was the patient’s 18 years old biological brother. Six loci on HLA-A, B, and DRB1 were completely matched with those of the recipient. Four days after the administration of G-CSF (250 μg/day) to the recipient, peripheral blood stem cells (PBSCs) were collected from the donor for 2 consecutive days. A total volume of 112 ml was harvested. A median nucleated cell (MNC) count of 6.2×108/kg with 2.4×106 /kg of CD34+ cells were actually administered to the recipient. The standard conditioning regimen was busulfan/cyclophosphamide (BU/CY). The recipient was started on intravenous infusion (IV) of cyclosporine A (CSA) on Day -1 and a plasma concentration of 150–250 ng/L was maintained. IV methotrexate (MTX) 10mg/m2 was given on Days +1, +3, +6, and +11. Oral mycophenolate mofetil(MMF)was administrated from Days +1 to +28 for prophylaxis of acute graft-versus-host disease (GVHD). If the concentration of hemoglobin (Hb) decreased below 70 g/L and/or platelet count (BPC) below 15×109 /L, the recipient was transfused with 60Co 25cGy irradiated and leuko-depleted red blood cells (RBCs) and/or single-donor platelets. Starting from Day +3, G-CSF was given to the recipient until white blood cells (WBC) increased to above 3.0× 109 /L. EPO and Interleukin-11 were also administrated to stimulate the proliferation of the progenitors of erythrocytes and megakaryocytes.

Results: DNA-STR on Day +33 indicated a complete chimerism in the recipient. Chromatin bodies were observed as 46XY on Day +45. CD55/CD59 expression on the membranes of leucocytes and erythrocytes, hemolysis index, and coagulation parameters returned to normal. The patient has been relapse-free for one year since the time of discharge.

Conclusion: 1. When conventional therapy with hormone and immunosuppressant becomes unsuccessful to control PNH, allo-PBSCT may be an alternative measure with promising effect. 2. In comparison to the reduced dose regimen, the standard dose regimen of BU/CY shown lower rate of relapse. 3. To decrease the mortality rate related to transplantation, allo-PBSCT needs to be considered as early as possible for refractory paroxysmal nocturnal hemoglobinuria.
A Case of SAA with Secondary Hemochromatosis: Can Continuous Iron Chelation Reverse Both Bone Marrow and Multiple Organ Failures?.
Authors: Park Soo-Jeong , Han Chi-Wha
Published: October 2019

A 24-year-old male patient with hemochromatosis due to multiple packed red blood cell transfusions, was referred to our emergeny center for a treatment of severe aplastic anemia and dyspnea. He was diagnosed with aplastic anemia at the age of 11 and had developed a transfusional hemochromatosis after 5 years of multiple blood transfusions. He had dilated cardiomyopathy with ejection fraction of 4 % requiring diuretics and digitalis, multiple endocrine dysfunctions (hypothyroidism, hypoparathyroidism with hypocalcemia, cataract, and intracranial calcifications, diabetes, and gonadal dysfunction), liver dysfunction, generalized bleeding, and skin pigmentation (Fig.1-left). A total volume of packed red blood cell transfusion before deferoxamine therapy was about 96,000 ml and the number of transfused units of platelet concentrates were innumerable. He had received regular iron chelation therapy (continous intravenous infusion of deferoxamine, 50 mg/kg/day for 5 days q 3–4 weeks) for 7 years after multiple organ failures. His cytopenias and organ dysfunctions (heart, liver and skin) began to recover progressively in 2002, after four years of deferoxamine therapy. He had had complete normal ranges of his peripheral blood cell counts, heart size, and liver function two years ago (Fig.1-right & Fig.2). He has not received any transfusions for the last four years. This finding suggests that continuous deferoxamine infusion may play a role in immune regulation in addition to iron chelation effect.

Figure 1. Posteroanterior chest radiographs reveal a marked cardiomegaly with right pleural effusion before the treatment (left) and a normalized heart after the deferoxamine therapy (right). Figure 1. Posteroanterior chest radiographs reveal a marked cardiomegaly with right pleural effusion before the treatment (left) and a normalized heart after the deferoxamine therapy (right). Figure 2. Hemoglobin changes and clinical course. Figure 2. Hemoglobin changes and clinical course.
Retrospective Review of Patients of Southeast Asian Descent with Persistent Hyperferritinemia Who Are Negative for Hereditary Hemochromatosis.
Authors: Yenson Paul R. , Tsang Peter W.K. , Li Charles , Chung Henry , Yoshida Eric M.
Published: October 2019

Background and Methods. Isolated hyperferritinemia is a common clinical finding. However, known HFE mutations C282Y and H63D have not been well-described in the Asian population. As well, evaluation of hyperferritinemia in the Asian population is confounded by chronic hepatitis B infection. We performed a retrospective case review of 79 patients of Southeast Asian ethnicity referred to three subspecialists in a tertiary care teaching hospital from January 1997 until July 2006 for assessment of hyperferritinemia. A subset of patients may represent a novel syndrome of isolated hereditary hyperferritinemia.

Results. 35 patients had identifiable secondary causes for their hyperferritinemia. 4 had clinical or biopsy proven iron overload consistent with hemochromatosis. 40 had isolated hyperferritinemia. Of the 35 patients with secondary causes, 26 had liver disease; 16 with viral hepatitis. Other causes included hematologic disorders (10), malignancy (2) and inflammatory arthritis (2). On liver biopsy, 8 patients showed no increase in stainable iron while 10 showed mild-moderate increase. 7 patients were negative for C282Y and H63D mutations. Of the 40 cases of isolated hyperferritinemia, 38 had normal liver enzymes. 2 had liver biopsies showing mild increase in stainable iron. 9 patients underwent C282Y and H63D genotyping with 1 patient being H63D heterozygous. 8 index patients had first-degree relatives with hyperferritinemia. 3 families were identified with more than 2 members affected, suggestive of a possible hereditary hyperferritinemia syndrome.

Conclusion. Hyperferritinemia appears to be relatively common in Asians but classic hemochromatosis is not. Liver disease is a frequent cause of hyperferritinemia in the Asian population. We have, however, discovered a small group of healthy patients with no evidence of liver disease and isolated hyperferritinemia that in some cases appears to be familial. This has not been previously reported.
Re-Organization of Cardiac Band 3 (cAE1) in Neonatal Cardiomyocytes Submitted to Alkali Loading.
Authors: Baratti Mariana O. , Lima Paulo R.M. , Alberto Daniella C. , Borges Luciene , Franchini Kleber G. , Costa Fernando F. , Saad Sara T.O.
Published: October 2019

Band 3 was initially described as the first member of the anion exchanger family (AE1). It is an integral membrane protein, initially characterized in erythrocytes. The AE family is encoded by four genes, AE1, AE2, AE3 and AE4. Three AE1 isoforms have been described up to now: erythroid (eAE1), kidney (kAE1), and cardiac (cAE1). Increased expression and activity of AE1 and AE3, as well as of the Na+/H+ exchanger have been demonstrated in hypertrophic myocardium of rats. A physical association of band 3 with cardiac actin has been previously described by our group, using yeast two hybrid screening. It has also been suggested that this binding occurs in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane. In the present study, immunolocalization analysis by confocal laser scanning microscopy was used to investigate the band 3 re-organization in neonatal cardiomyocytes (Wistar rats 1 to 2-day-old) submitted to different stimuli, such as pH changing or mechanical stretch (a model leading to cardiac hypertrophy). In the untreated cells, band 3 was dispersedly distributed in the cytoplasm, preferentially in the perinuclear region. On the other hand, when these cells were submitted to cyclic stretch (2 hours), band 3 appears between actin filaments, in the sarcomeric units. Similar distribution was showed in cardiac cells submitted to alkali loading (pH 8.9 for 2 hours). However, in experiments where the culture medium was pH 6.8, band 3 was dispersed in the cytoplasm, but was not concentrated in the perinuclear region. Intracellular pH (pHi) is a major regulator of diverse cellular processes including metabolic pathways, Ca2+ homeostasis, cell contractility, cell excitability and cell death; a rise in pHi activates Cl−/HCO3− exchanger. On the other hand, the mechanical stretch stimulates a rapid secretion of Angiotensin II, rise of pHi and activation of intracellular signaling molecules such as protein kinase C and tyrosine kinases. These processes induce cell proliferation, cardiac hypertrophy and reorganization of actin cytoskeleton. The demonstration of reorganization of band 3 and its localization in the sarcomeric units after these stimuli suggest that band 3 is involved in the cardiac hypertrophy physiopathology. Band 3 could function as an anchorage for the contractile apparatus, as well as in the transport of anions in the myocardium, which are well described processes in normal erythrocytes.
CobaSorb, a New Non-Radioactive Vitamin B12 Absorption Test.
Authors: Hvas Anne-Mette , Morkbak Louise , Nexo Ebba
Published: October 2019

After diagnosing vitamin B12 deficiency, the cause of the deficiency has to be clarified. For many years Schillings test was used for this purpose. However, Schillings test has almost disappeared from the marked. Recently, a new vitamin B12 absorption test, CobaSorb, was introduced. CobaSorb is based on oral intake of non-radioactive vitamin B12 followed by measurement of an increase in holotranscobalamin (holoTC) 24 hours later. In the present study, we further explored the design of CobaSorb to identify the best marker for reflection of vitamin B12 absorption and to determine the duration of the test. Seventy-eight healthy individuals (age 21–81 years, 40 males) were included and three oral doses of 9 microgram vitamin B12 per day were administered for 5 successive days. Non-fasting blood samples were collected on days 1 to 5 before administration of vitamin B12 and in the morning day 8. Plasma cobalamins were assayed on the Advia Centaur (Bayer A/S, Germany), and holoTC was measured by an in-house sandwish ELISA after removal of the apoTC with vitamin B12 coated beads. Baseline levels were 151–528 pmol/L for plasma cobalamins and 19–33 pmol/L for holoTC. We found a significant positive association between age and baseline holoTC (p=0.03), whereas no association was found between baseline plasma cobalamins and age (p=0.55). Baseline levels were not associated with sex. Because a vitamin B12 absorption test is relevant only in individuals with borderline or low levels of holoTC or plasma cobalamins, we analysed in detail the pattern of absorption in those having baseline levels below the 75% percentiles. We used a change greater than 2×CVday-to-day in holoTC (22%) and plasma cobalamins (12%) to indicate a change caused by absorption of vitamin B12. Among individuals with a baseline holoTC below the 75% percentile (<75 pmol/L, n=57), the increase in holoTC from baseline (day 1) to day 3 was > 22% for 56 (98%). Only 41 (72%) of the individuals with baseline plasma cobalamins below the 75% percentile (<335 pmol/L, n=57) had an increase in plasma cobalamins >12% from day 1 to day 3. We concluded that in healthy individuals with baseline holoTC <75 pmol/L, vitamin B12 absorption was well reflected by an increase in holoTC-but not plasma cobalamins-after 2 days administration of oral vitamin B12. We suggest that future use of CobaSorb is done based on this design.
Clinical Observation of Combination Therapy of Anti-Thymocyte Globulin Cyclosporine A, Hemopoietic Growth Factor and Androgen for 47 Severe Aplastic Anaemia Cases.
Authors: Shen JianPing , Zhou YuHong , Zheng Zhiying , Chen Junfa , Lin Shengyun , Shen YiPing , Hu ZhiPing , Luo Xiusu , Ye BaoDong , Yu LongXi
Published: October 2019

For therapy to severe aplastic anaemia (SAA), the effective and survival rate of combination regime using anti-thymocyte globulin (ATG), globulin, cyclosporine A (CsA), hemopoietic growth factor and androgen are similar to those of hemopoietic stem cell transplantation(HSCT). we have treated 236 cases of SAA from Oct. 1995 to Dec. 2005. 47of them took combination therapy mainly with ATG. The range of age was 10 to 56 (median age was 32). Of all the patients, totle effective rate was 84.5%, 41% achieved complete remission and 43.5% got partial remission. With the median follow-up time of 53 months, 41 patients are survival (87.2%),6 died, 3 relapsed, 4 patients stayed the same, 1 coincided with PNH. The combination therapy mainly with ATG began to work after 1 to 11months (median time was 2.5 months). If one can be treated in 6 months after diagnosis, the effective rate can be 95% after 3 months’ management, but if not, it was only 43%. For patients whose bone marrow hemopoietic cell >30% before treatment, the effective rate was 87.5%, but if it<5%, the effective rate was only 15%. No patients was severely affected by allergy or serum disease caused by ATG during treatment. By using infection preventing method of platelet transfusion and supporting treatment, most of the patients went through risk time and showed therapeutic reaction. We believe that the combination therapy mainly with ATG is safe and effective for SAA. It is considerable for SAA patients without HLA matched donors.
Uniform Approach Better Defines Natural History of Transient Myeloproliferative Disorder (TMD) in Down Syndrome (DS) Neonates: Outcomes from Children’ Oncology Group (COG) Study A2971.
Authors: Sharma Mukta , Alonzo Todd A. , Sorrell April , Gerbing Robert B. , Hilden Joanne M. , Arceci Robert J. , Massey Gita , Doyle John J. , Bayer Lisa , Perentesis John P. , Ross Julie , Smith Franklin O. , Brynes Russell , Hussong Jerry , Hathaway Lois , Gamis Alan
Published: October 2019

TMD is solely found in DS infants in the first few months of life & is distinguished from congenital AML primarily by its spontaneous resolution. A2971, the largest study of TMD conducted to date, defines the natural history of this disorder by utilizing uniform observation & treatment guidelines. A total of 136 DS patients (pts) <90 days (d) of age were enrolled from 1999–2004 with median follow-up of 745d(0–2187). Pts were assigned to observation(n=110) or intervention(n=26) arms based upon clinical severity. Median age at diagnosis was 5d(0–58) with median hematologic values of WBC 32,800(4,800 – 259,100), hemoglobin 15(5–22.5), platelets 125K(10–958K), peripheral blasts 25%(0–92) & bone marrow blasts 15%(0–95). Hepatomegaly (HM) was seen in 59%, splenomegaly in 39%, & pleural or pericardial effusion in 10% & 19%. Pts required intervention due to hyperviscosity(11%), blast count >100,000/μl(25%), organomegaly(OM) with respiratory(resp) compromise(43%), CHF(11%), hydrops fetalis(21%), liver dysfunction(43%), renal dysfunction(14%), or DIC(25%). For pts who required intervention(n=29, 21%), 9 pts(31%) received leukopheresis/exchange transfusion (Leuk/Exch). 26(90%) pts received low-dose AraC (3.33 mg/kg/24 hrs continuous infusion on days 0–4) for OM, organ dysfunction causing resp compromise, or continued symptoms after Leuk/Exch. Among all pts, 3-year overall survival(OS) was 77 ± 8% & event-free survival(EFS) was 73 ± 9%. 28 deaths were noted (15 related, 12 unrelated to TMD or therapy, 1 unknown). Median time to TMD resolution (defined as resolution of peripheral/ bone marrow blasts, OM, effusions & organ dysfunction) was 46d(7–757). Peripheral blast resolution was attained in a median of 35d (2–147). To date, 16 pts (12%), including 4 treated with AraC, developed AML/MDS at a median time of 355d(118–888). We identified 3 distinct groups of TMD pts: low risk (without palpable HM or hepatic dysfunction, n=52, 38%), intermediate risk (HM with non-life threatening hepatic dysfunction, n=55, 40%), & high risk (WBC>100K or life threatening cardio-resp compromise due to TMD, n=29, 21%). The OS for low, intermediate & high risk groups was 92 ± 8%, 82 ± 11% & 49 ± 20% and EFS was 83 ± 11%, 64 ± 15% & 36 ± 19%, respectively(p<0.001 for low vs high & intermediate vs high risk groups for OS & EFS). WBC >100K (Hazards Ratio-HR 3.57, p=0.004) & presence of hepatomegaly (HR 3.6, p=0.009) were significant adverse risk factors for OS in univariate analyses. This study confirms that most TMD pts undergo spontaneous resolution of disease without intervention, further clarifies the natural history of TMD, & provides baseline comparisons for upcoming COG trials.
Effect of the Terminal Complement Inhibitor Eculizumab on Patient Reported Outcomes in Paroxysmal Nocturnal Hemoglobinuria (PNH): Phase III Triumph Study Results.
Authors: Brodsky Robert A. , Muus Petra , Dührsen Ulrich , Hill Anita , Bessler Monica , Coutre Steven , De Paz Raquel , Moskovits Tibor , Nakamura Ryotaro , Van den Neste Eric , Zanella Alberto , Quinn-Senger Kerry , Kroon Henk-Andre , Cella David
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia in which complement-sensitive RBCs are destroyed leading to chronic intravascular hemolysis. PNH patients suffer from diverse and serious hemolysis-induced morbidities leading to a poor quality of life (QoL). Fatigue in PNH patients may be disabling and levels are similar to anemic cancer patients. Fatigue is multifactoral, related to both the underlying anemia and hemolysis. Patients suffer from reduced global health status, patient functioning, pain and dyspnea. Treatment with the terminal complement inhibitor eculizumab reduces intravascular hemolysis and improves anemia. The impact of eculizumab treatment on levels of fatigue and other patient reported outcomes was prospectively examined in a double-blind placebo controlled study (TRIUMPH) using two distinct instruments, the FACIT-Fatigue and the EORTC QLQ-C30. Improvements in QoL were quantified using standardized effect sizes (SES), a measure of the magnitude of the clinical benefit in various instruments. Eculizumab treatment, as compared to placebo, was associated with a very large and significant improvement in fatigue as measured by the FACIT-Fatigue scale (SES=1.13, P<0.001) as well as the EORTC QLQ-C30 fatigue subscale (SES=1.12, P<0.001). Similarly, the percentage of patients achieving a pre-specified minimally important difference (MID) was 53.7% versus 20.5% of eculizumab- and placebo-treated patients, respectively (P=0.003) using the FACIT-Fatigue; 67.7% versus 24.4%, respectively (P<0.001) with the EORTC QLQ-C30. Treatment independent univariate analyses showed that reduction in intravascular hemolysis (decreased LDH levels) and improvement in anemia (increased hemoglobin levels) were both significantly associated with an improvement in fatigue. Further, multivariate analyses indicated that reduction in hemolysis was more predictive than improvement in anemia of an improvement in fatigue. Eculizumab treatment was also associated with significant improvements with moderate to large SES in the following EORTC QLQ-C30 subscales: global health status (0.87, P<0.001); role functioning (0.93, P<0.001); social functioning (0.57, P=0.003); cognitive functioning (0.78, P=0.002); physical functioning (1.01, P<0.001); emotional functioning (0.51, P=0.008); pain (0.65, P=0.002); dyspnea (0.69, P<0.001); and appetite loss (0.50, P<0.001). These data demonstrate that resolution of intravascular hemolysis with eculizumab treatment results in large and clinically meaningful improvements in patient reported outcomes including fatigue, global health status, patient functioning, and disease-related symptoms in PNH.
Antithymocyte Globulin (ATG) Plus Cyclosporine (CSA) for Aplastic Anemia (AA) and Low-Risk Myelodysplastic Syndrome (MDS).
Authors: Lee Je-Hwan , Choi Seong-Jun , Lee Jung-Hee , Seol Miee , Kim Keun Hee , Park Jae-Hoo , Kim Hawk , Joo Young-Don , Lee Won-Sik , Zang Dae Young , Kim Hyo Jung , Lee Kyoo Hyung
Published: October 2019

We investigated the effectiveness of immunosuppressive therapy using ATG plus CSA in adult patients with AA and low-risk MDS. Between APR 2000 and JAN 2005, 93 patients, 82 AA and 11 MDS, from 4 institutes in Korea were included in this prospective study. Patients received one of 3 types of ATG according to the availability in Korea: horse ATG (Atgam®, Upjohn, Kalamazoo, MI; 40 mg/kg/d × 4 days), rabbit ATG (Thymoglobuline®, IMTIX-SANGSTAT, Lyon, France; 2.5 mg/kg/d × 5 days), or horse ALG (Lymphoglobuline®, IMTIX-SANGSTAT; 10 mg/kg/d × 5 days). Methylprednisolone 2 mg/kg/d was given before each dose of ATG. Oral CSA 3 mg/kg twice a day was administered for 3–6 months. In 38 patients, the HLA phenotyping results were available. The median disease duration before ATG/CSA therapy was 31 days (range, 3–3126). Eight patients were not evaluable for response because of early deaths in 4, early follow-up loss in 3, and clonal evolution to AML in 1 within 60 days after treatment. Of 85 patients who were evaluable for response, 46 (54.1%) showed response to ATG/CSA therapy: partial response in 23 and complete response in 23. Responses were observed in 44 (56.7%) of 75 AA patients and in 3 (30.0%) of 10 low-risk MDS patients (P=0.103). Patients with HLA B40 phenotype showed higher response (66.7% vs. 32.1%, P=0.043), but other HLA phenotypes did not show any significant influence on response. Cumulative incidence of response was 55.5% at 2-year. Multivariate analysis indicated that disease duration before ATG/CSA therapy (≤ 90 days vs. > 90 days; OR 0.154; P=0.001) and type of ATG (horse ATG vs. rabbit ATG; OR 0.235; P=0.006) were independent predictive factors for response. Nine patients, 7 AA and 2 MDS, relapsed and cumulative incidence of relapse was 25.9% at 5-year. Clonal or disease evolution occurred in 3 patients with AA (PNH, MDS, and AML in 1 of each) and in 3 patients with MDS (RAEB-1 in 2 and RAEB-2 in 1). Eighteen patients died and actuarial survival was 69.8% at 5-year: 76.9% in AA and 36.0% in MDS. No patient who attained at least PR died. Our results demonstrated the effectiveness of ATG/CSA therapy in AA and low-risk MDS. The effects of rabbit ATG was significantly inferior in our study probably due to dose problem rather than ATG type itself. The influence of HLA phenotype on response to ATG/CSA should be further investigated.
Endothelial-Dependent Vasodilation Is Impaired in Children with Sickle Cell Disease (SCD).
Authors: De Montalembert Mariane , Aggoun Yacine , Niakate Assa , Szezepanski Isabelle , Bonnet Damien
Published: October 2019

Impairment of endothelium-dependent vasodilation has been reported in steady-state SCD adult patients, and could contribute to the occurrence of vaso-occlusive events. In order to study whether vascular tone abnormalities could be also observed in SCD children, we enrolled 21 SCD steady-state children (18 homozygous SS, 3 S-b0 thalassemics); 12 males, 9 females, mean aged 10.4 ± 3.3 yrs, mean Hb level: 7.6 ± 1.0 g/dL. None had had a transfusion in the 3 previous months, nor were treated with hydroxyurea. These SCD children were compared to a control group of 23 Afro-Caribbean AA and AS controls matched for age and gender. Blood pressure and vascular function parameters were measured after the child had been recumbent for at least 10 minutes. Flow-mediated (FMD) and nitroglycerine-mediated (GTNMD) dilation of the brachial artery were examined in cases and controls using echotracking techniques. Additionally, intima-media thickness (IMT) and mechanical properties of the common carotid artery were measured using previously described methods.

Blood pressures in SCD and control children were comparable. IMT was not different in SCD children (0.42 ± 0.06 mm) vs controls (0.42 ± 0.03 mm). Systolic and diastolic diameters of the common carotid artery were significantly higher in SCD children than in controls (respectively, 6.8 ± 0.8 vs 5.7 ± 0.4, p<0.001, and 5.7 ±0.7 vs 5.0 ± 0.4 mm, p< 0.005) but the stiffness of the common carotid artery was not different in SCD children and in controls. Finally, FMD was significantly decreased in SCD children vs controls (5.6 ± 0.2 vs 8.0 ± 0.2 %, p: 0.008), while GTNMD was comparable in SCD patients and controls. We hypothesize that the increase of the carotid artery diameters, without modification of the distensibility, is related to the higher cardiac output secondary to anemia. Interestingly, we observed a significant decrease of the endothelial-dependent flow-mediated vasodilation in SCD children, which had never been reported before. This could result of impaired NO bioavailability, correlated with enhanced arginase activity and hemolysis rate. Oral arginine supplementation in SCD children could be an appropriate treatment.
Evaluation of the CapillaryS 2 CE System for Hemoglobinopathy Investigations.
Authors: McFarlane Andrew G. , Halchuk Linda M. , Eng Barry , Waye John , Crowther Mark A.
Published: October 2019

Traditional diagnosis of hemoglobinopathies rely on separation and accurate quantification of hemoglobin (Hb) fractions using alkaline and acid electrophoresis, isoelectric focusing (IEF) and/or High Performance Liquid Chromatography (HPLC). Recent reports have suggested capillary electrophoresis (CE) as an alternate method for screening. We evaluated a new CE system the Sebia CapillaryS 2 (Evry Cedex, France) and compared it to IEF and HPLC. For this evaluation samples referred to our laboratory for routine hemoglobinopathy screening or as cord bloods sent for confirmation of a variant detected as a result of newborn screening were analyzed using all three techniques. IEF was performed with the Resolve IEF kit (Perkin Elmer,Wallac Oy, Finland). HPLC was done on the BioRad Variant II (Munich, Germany) using the Hb A2/Hb A1c Dual program. The CE was performed using the “Capillarys Hemoglobin(E) kit”. Suspected rare variants were further investigated with DNA investigations, including PCR and direct nucleotide sequencing. Variant hemoglobins were detected in 156 of 764 samples (Table 1). The correlation between IEF, HPLC and CE was excellent. One variant (Hb Toulon) was not detected by the CE but ran with Hb F. The CE system did not report the Hb F value on samples with very low (< 1.0%) Hb F by HPLC. Two cases of Hb Constant Spring were identified by CE but not by either IEF or HPLC. The correlation of both Hb F and Hb A2 reported between the CE and HPLC systems were excellent (R > 0.99 and 0.98, Figure 1 and Figure 2 respectively). The positive bias of Hb A2 seen in HPLC when Hb S is present was lower in the CE. However, the CE system demonstrated a negative bias for Hb A2 if Hb C was present. CE was the only system capable of separating Hb A2 from Hb E. We conclude that CE is comparable to both IEF and HPLC for separation and quantification of hemoglobin fractions; this system has the added advantage of reporting Hb A2 in the presence of Hb E and consistently identifies Hb Constant Spring, which is not easily detected on IEF or HPLC. Further this CE system is user friendly and holds promise as a tool for newborn screening and routine laboratory hemoglobinopathy investigations.

Hemoglobinopathy Detected Hemoglobinopathy Number β Thalassemia 99 Hb S trait 55 Cord with Hb Barts 28 Hb C trait 13 Hb E trait 12 Hb D trait 8 α variant (not yet identified) 5 Hb Barts + Hb S trait 4 Hb Barts + Hb D trait 3 Hb S disease 3 Hb Barts + Hb E trait 2 Hb E +β Thalassemia 2 Hb H disease 2 Hb Constant Spring 2 Hb J Baltimore 2 Hb Toulon 2 δ variant (not identified) 2 Hb S + Hb C 1 Hb Barts + Hb C trait 1 Hb Q Thailand + --SEA/αα 1 Homozygous Hb E + Hb Constant Spring 1 Hb S + Hb Kenya 1 Hb J Roviga 1 Hb Beograd 1 Hb G Coushatta 1 Hb Sirian 1 Hb Titusville 1 B variant (not identified yet) 1 Total Abnormal 255 No hemoglobinopathy detected 509 Total 764 Hb F: HPLC vs CE Hb F: HPLC vs CE Hb A2: HPLC vs CE Hb A2: HPLC vs CE
High Calcium Requirement for Phosphatidylserine Exposure in Sickle Cell Disease.
Authors: DeJong Kitty , Miller Annabeth M. , Emerson Renee K. , Kuypers Frans A.
Published: October 2019

While the adverse effects of red blood cells (RBC) that expose phosphatidylserine (PS) in the sickle cell circulation are well recognized, the mechanism that underlies the formation of these cells is not well understood. Diminished flippase activity is found in a subset of sickle cells, and while this explains how PS persists in the outer RBC monolayer, it does not explain how the phospholipids are randomized across the membrane bilayer. Rapid scrambling of phospholipids, resulting in PS exposure, requires elevation of intracellular free Ca++ levels by Ca++ influx into the RBC. In this study we assessed how much Ca++ is required for PS exposure, and how this relates to previous findings of elevated Ca++ influx during deoxygenation of sickle cells. We loaded RBC for 30 minutes at 37°C with accurately established levels of intracellular Ca++ by balancing CaCl2 solutions with the chelator BAPTA, followed by Annexin V labeling under conditions that scrambling did not continue. In both normal and sickle RBC from either human or mouse, PS exposure was only found at or above 10 microM Ca++. Flippase inhibition with vanadate did not change this, indicating that phospholipid scrambling had not been initiated under these conditions. Scramblase sulfhydryl modification with NEM reduced the Ca++ requirement to lower than 1 microM, as did treatment with the scramblase stimulator oligomycin. Further experiments showed that Ca++ needs to be present during the entire duration of the scrambling process, and removal of Ca++ arrests the scrambling process. With 1 microM Ca++ in oligomycin-treated (scramblase stimulated) RBC and in absence of flippase activity (using vanadate), it took 5 minutes at 37°C to obtain a significant (>2%) population of PS-exposing cells. These conditions are unlikely met in all sickle cells. In addition, these experiments showed that the flippase is not inhibited by 1 or 100 microM Ca++, despite previous reports suggesting that Ca++ loading inhibits the flippase. Taken together, our data indicate that PS scrambling requires more Ca++ influx than has been reported for deoxygenated sickle cells (50–100 nM). We suggest that a small percentage of individual sickle cells contain Ca++ levels of 1 microM or above, which would lead to PS exposure in those RBC that have sustained flippase inhibition and scramblase modifications similar to those achieved with NEM or oligomycin. We have previously reported evidence for such scramblase modifications, as well as reduced flippase activity in a subset of sickle cells. Our data indicate that the degree of Ca++ influx may be the determining parameter for the extent of PS exposure in sickle cells.
Plasma Concentrations of Asymmetric Dimethylarginine, an Endogenous Nitric Oxide Synthetase Inhibitor, Are Elevated in Sickle Cell Patients but Do Not Increase during Painful Crises.
Authors: Landburg Pearl , Teerlink Tom , de Jong Sigrid , Muskiet Frits A. , Duits Ashley J. , Schnog John-John B.
Published: October 2019

Plasma concentrations of asymmetric dimethylarginine (ADMA), an endogenously produced inhibitor of nitric oxide (NO) synthase, are elevated and related to endothelial activation in clinically asymptomatic patients with sickle cell disease (SCD). However, ADMA concentrations have not been defined during acute vaso-occlusive complications. We determined plasma ADMA and arginine concentrations in 96 samples (40 collected in the clinically asymptomatic state and 56 collected at presentation with a painful crisis) of 44 HbSS patients (mean age 32±13 years, m:f=23:21) and in 39 samples (22 collected in the clinically asymptomatic state and 17 collected at presentation with a painful crisis) of 22 HbSC patients (mean age 30±14 years, m:f=10:12). Thirty-five race, sex and age matched HbAA blood donors served as healthy controls. Plasma ADMA concentrations were elevated in asymptomatic sickle cell patients as compared to healthy controls (HbSS: mean 0.7±0.1 umol/L, HbSC mean 0.5±0.1 umol/L, HbAA mean 0.4±0.1 umol/L; p<0.001 one way ANOVA). Plasma arginine concentrations were lower in asymptomatic HbSS patients as compared to asymptomatic HbSC patients and healthy controls (HbSS: mean 59.6±25.8 umol/L, HbSC mean 75.5±25.8 umol/L, HbAA mean 74.0±27.1 umol/L; p=0.03 one way ANOVA). Ratio’s of ADMA to arginine (ADMA/Arginine × 1000) were highest in HbSS patients (HbSS: mean 16.2±11.6, HbSC mean 7.8±2.9, HbAA mean 6.3±3.2; p=0.001 one way ANOVA). In both HbSS and HbSC patients, neither ADMA (p=0.17/p=0.57), arginine (p=0.13/p=0.18) nor the ADMA/arginine ratio (p=0.88/p=0.08) differed between the asymptomatic phase and at presentation with a painful crisis. In conclusion, even though ADMA is elevated in SCD and therefore may well be involved in the development of chronic organ complications by limiting NO availability, ADMA concentrations are not affected during acute painful sickle cell crises.
Prediction of Adverse Outcomes in Children with Sickle Cell Anemia: Testing the CSSCD Early Prediction Model in the Dallas Newborn Cohort.
Authors: Quinn Charles T. , Lee Nancy J. , Shull Elizabeth P. , Buchanan George R.
Published: October 2019

The Cooperative Study of Sickle Cell Disease (CSSCD) reported that dactylitis, severe anemia, and leukocytosis in very young children with SCD increased the risk of later adverse outcomes, including death, stroke, recurrent acute chest syndrome (ACS), and frequent pain (NEJM2000;342:83). Specifically, in the CSSCD infant cohort, the occurrence of dactylitis in the first year of life (“early dactylitis”) and a steady-state hemoglobin (Hgb) concentration <7 g/dL in the second year of life (“severe anemia”) together predicted a high risk (0.36 probability) of a later adverse outcome. A steady-state leukocyte count of approximately 20,000/mm3 or higher in the second year of life (“leukocytosis”) in combination with either early dactylitis or severe anemia also predicted a high risk of an adverse outcome. The testing of this model in other cohorts has not been reported, so we evaluated its performance characteristics in the Dallas Newborn Cohort (Blood2004;103:4023). We studied all cohort members with sickle cell anemia (SS) or sickle-β0-thalassemia (Sβ0) who had 5 or more years of follow-up and complete laboratory data and medical records. We searched our electronic database and patients’ medical records to ascertain the occurrence of the three predictors (early dactylitis, severe anemia, and leukocytosis) and the four adverse events [death; clinically overt first stroke; frequent pain (average of 2 or more painful events/year for the entire follow-up period, with ≥2 events/year for 3 consecutive years); and recurrent ACS (average of 1 or more episode/year with ≥1 episode/year for 3 consecutive years)]. We only considered hospitalizations for pain in this analysis, unlike the CSSCD, because we do not systematically track episodes of pain treated only at home or in outpatient facilities. We studied a total of 119 children (68% male, 97% SS) who had a mean follow-up of 9.7 years (range 5 – 15.4). Twenty-four (20%) had early dactylitis, 5 (4%) had severe anemia, and 15 (12.6%) had leukocytosis. Eighteen subjects (15.1%) experienced adverse events: 2 (1.7%) died, 13 (10.9%) had a stroke, 3 (2.5%) had frequent pain, and 1 (0.8%) had recurrent ACS. Sensitivity and specificity of the model were 0 and 97%. Positive and negative predictive values were 0 and 84%. We used binary logistic regression to further evaluate the model. The whole model test was not significant (P=0.21), and R2 was 0.044. The area under the ROC curve was 0.64 (Figure), and better-performing cut-points for Hgb and leukocyte count could not be found. In summary, the CSSCD early prediction model had very low sensitivity and positive predictive value in the Dallas Newborn Cohort. Specificity and negative predictive value were high, however. This retrospective analysis has several limitations and biases, but it suggests that the CSSCD model does not well predict adverse outcomes in the Dallas Newborn Cohort.

Figure Figure
Anti-Inflammatory Effect of Hydroxyurea Therapy in Sickle Cell Disease.
Authors: Lanaro Carolina , Franco-Penteado Carla F. , Conran Nicola , Saad Sara T.O. , Costa Fernando F.
Published: October 2019

There is growing evidence that inflammatory stresses within the microvasculature may play a significant role in the vaso-occlusion that is characteristic of sickle cell disease. Inflammation, leukocyte adhesion to vascular endothelium, and subsequent endothelial injury appear to contribute to the pathogenesis of SCD. Whilst alterations in a number of inflammatory, anti-inflammatory cytokines and inflammatory biomarkers have been previously related, reports have been conflicting and a conclusive role for cytokines in SCD remains to be established. Furthermore, the effect of hydroxyurea therapy (HU) on the release of inflammatory mediators is not well understood. This study was undertaken to determine plasma levels of IL-8, IL-10, TNF-a and PGE2 in healthy controls, SCD patients in steady-state and in SCD patients on HU therapy (SCDHU; 20–30 mg/kg/day; 3-month minimum treatment duration) using specific ELISA assays (R&D Research). TNF-a levels were significantly higher in SCD individuals when compared to controls (2.95 ± 0.44 pg/ml, n=17; vs 1.43 ± 0.20 pg/ml, n=8, p=0.006). HU therapy, however, was observed to significantly reverse augmented TNF-a levels (SCDHU; 1.61 ± 0.18 pg/ml, n=15, p=0.007). Plasma IL-8 levels were significantly elevated in SCD individuals (16.58 ± 2.52 pg/ml, n=29, p<0.001) compared to healthy controls (5.67 ± 0.62 pg/ml, n=16); however HU therapy did not significantly alter augmented IL-8 in SCD (14.59 ± 1.57 pg/ml, n=22). Interestingly, detectable levels of the anti-inflammatory cytokine, IL-10 (>3.8 pg/ml), were found in 3 (15%) of 20 SCD patients, and 1 (11%) of 9 controls, whilst 7 (50%) of 14 HU-treated patients demonstrated detectable IL-10 levels. Levels of plasma PGE2, an inflammatory biomarker, were significantly increased in SCD patients compared to control individuals (128.35 ± 12.25 pg/ml, n=26; vs 88.45 ± 5.95 pg/ml, n=14, p=0.01) and similar levels were detected in patients treated with HU (143.6 ± 16.75 pg/ml, n=17, p=0.004, compared to control). Data demonstrate that the inflammatory cytokines, TNFa and IL-8, and the inflammatory biomarker, PGE2, are augmented in a population of SCD patients studied during steady-state, contributing to clarify conflicting data. Furthermore, HU therapy reversed augmented TNFa levels, and appears to have an augmenting effect on the anti-inflammatory cytokine, IL-10, suggesting an anti-inflammatory action of HU therapy that may be important for the prevention of vaso-occlusion.
Identification of a New Mutation in the 5′-UTR of Hepcidin Gene in beta-Thalassemia Major (TM) Patients.
Authors: Delbini Paola , Duca Lorena , Nava Isabella , Meo Anna , La Rosa Marina , Cappellini Maria D.
Published: October 2019

Hepcidin is a peptide hormone produced in the liver that acts as negative regulator of iron absorption from the enterocytes and of iron release from macrophages. Iron overload and inflammation up-regulate hepcidin synthesis, while anaemia and hypoxia suppress hepcidin expression. Thalassaemia Major (TM) is a hereditary haemolytic anaemia requiring long-life blood transfusions treatment with consequent iron overload. In β-thalassemias is a disorder in which hepcidin is regulated by opposing influences of ineffective erythropoiesis and concomitant iron overload. In order to get further insights on iron regulation in thalassemias, we screened hepcidin and HFE genes in fourty-three TM regularly transfused patients and sixty control subjects. Blood from TM was taken at least 48 hours after chelation therapy and just before blood transfusion. DNA was prepared from peripheral blood, according to standard protocols. Hepcidin and HFE sequences were amplified with PCR using specific primers and PCR products were sequenced, after purification, in a automatic sequencer. Moreover in all patients serum pro-hepcidin was evaluated by ELISA competitive binding assay (DRG,Germany); iron status was evaluated by serum ferritin (SF), percentage of transferrin saturation (TS) by standard procedures and non transferrin bound iron (NTBI) in serum by HPLC after nitrilotriacetic acid (NTA) chelation. Serum IL-6 as inflammation marker was measured by lateral flow immunoassay (Milenia QuickLine, Germany). Molecular analysis detected an undescribed G→T mutation at position +23 of the 5′-untranslated region in two unrelated TM patients; no mutations were found in control subjects. The probands have been regularly transfused since the age of 1 year, receiving 2–3 units of packed red cells and treated with Deferoxamine 40 mg/Kg/day 6 days/week. The first patient, wild type for HFE mutation, was a 29-years-old compound heterozygous IVS II-745/IVS I-110 man. The SF was 1052 ng/ml, TS 94% and NTBI 1.77 μM. Serum pro-hepcidin value was in normal range (213 ng/ml). The proband’s father was heterozygous for the same hepcidin mutation and showed signs of mild iron overload (SF 491 ng/ml and NTBI 0.50 μM). The second patient was a 26-years-old homozygous β039 man with high levels of SF (4346 ng/ml), TS (169%) and NTBI (2.10 μM), while serum pro-hepcidin was 269 ng/ml. HFE analysis revealed a homozygous genotype for H63D mutation. The patient’s mother was heterozygous for hepcidin and H63D mutation and showed mild iron overload (SF 500 ng/ml; NTBI 0.22 μM) whereas the father, heterozygous only for H63D, had normal iron status. According to recent findings (Bridle et al, 2003) our results indicate that hepcidin mutation in association with H63D synergizes the effect on iron homeostasis and it could be responsible for the development of marked iron overload poorly responsive to chelation therapy in β-thalassemia patients.