bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical Implications of Anemia in Heart Failure (HF) Patients (pts).
Authors: Puente Dolores , Colorio Cecilia , Ratto Roxana , Descalzo Martin , Rossi Andrea , Perea Francisco , Diez Mirta , Forastiero Ricardo , Tabares Maria , Favaloro Liliana , Pombo Gonzalo , Feldman Leonardo
Published: April 2020

Previous studies have shown that anemia is frequently associated with higher morbidity and mortality in HF pts. Our aim was to determine the impact of anemia in pts hospitalized with congestive HF at our hospital. We analyzed data from 277 pts with diagnosis of HF admitted between 1 June 2004 and 31 December 2005, with a follow up of at least 6 months. Anemia was defined as hemoglobin (Hb) <11,5 g/dl. HF was classified according to Framingham criteria. Previous history of arterial hypertension, diabetes, dislipemia, chronic renal insufficiency and ACE inhibitors treatment was recorded. Ischaemic and non- ischaemic etiology of cardiopathy was established. Renal disfunction was defined as creatinin concentraction >1.9 mg/dl. Pts with HF were assigned to group A (with anemia) or B (control). Statistical analysis was performed using Pearson’s Chi square, Spearman’s rho, Fisher test and Kaplan Meyer survival function.

Results: We evaluated 229 (82,7%) pts with a median follow up of 594 days (range: 1–1129 days). Mean age was 68,02 years (median 71, range: 17–91). 143/229 (62,4%) were male. In 75 /229 (32.75%) cases a Hb < 11,5 g/dl was measured at admission. The mean Hb was 13,1 g/dl for the entire group. Anemia pts showed a mean Hb of 10.6 g/dl. Demographic, clinical and outcome features are shown in table 1.Group A showed a higher number of readmissions because of HF and other cardiac.

Conclussions: In our analysis the prevalence of anemia was 33% in pts hospitalized for HF. There was no significant differences among clinical variables between anemic and non-anemic pts. Anemia was associated with worse clinical outcome (Group A pts required more readmissions for HF and other cardiological causes)and all cause mortality. Mean survival was longer in Group B pts.according to Kaplan Meyer analysis.

TABLE 1: RESULTS GROUP A (n=75) GROUP B (n= 154) p Male 42 (56%) 101 (65.5%) Mean age (years) 70.38 (R:20–90) 67.1 (R:17–91) Mean Hb (g/dl) 10.6 14.3 Ischaemic cardiopathy 38(50,6%) 78(50.6%) Non ischaemic cardiopathy 41(54,6%) 78 Arterial hypertension 54(72%) 106(68.8%) Diabetes 24(32%) 33(21.4%) Dislipaemia 35(46.6%) 64(41.5%) Chronic renal insufficiency 18(24%) 19(12.3%) 0.03 Previous ACE inhibitors treatment 25(33%) 71(46%) 0.08 Acute pulmonary edema 12(16%) 15(9.7%) Creatinin concentration > 1.9 mg/dl 16(21.3%) 18 (11.6%) Hospitalization days (mean) 9.6 (R: 1–51) 8.1(R:1–59) Readmission for HF 37 190 0.001 Readmission for other cardiologic causes 20 109 0.017 Readmission for non cardiologic diseases 18 58 Mean survival (days) 499 (R:1–1091) 658 (r=1–1129) 0.166 Global mortality 25 (33%) 32(21%) 0.05 HF related mortality 13 (20.9%) 24 (14.3%) Figure Figure
L-Glutamine Therapy Improves Minute Ventilation of Sickle Cell Anemia Patients.
Authors: Macan Henry R. , Porszasz Janos , Tsurugaya Hideki , Razon Rafael L. , Koh Han , Casaburi Richard , Tanaka Kouichi R. , Niihara Yutaka
Published: April 2020

We have demonstrated previously that oral L-glutamine administration can improve NAD redox potential in sickle red blood cells (RBC) and decrease the endothelial adhesion rate of sickle RBC. Additionally, preliminary results showed that L-glutamine therapy improved the exercise endurance of sickle cell anemia patients. Following up on this study, minute ventilation and work rate were evaluated. After proper consent, 6 homozygous sickle cell anemia volunteers participated in this two-part study. The first part was incremental work rate testing where the work rate was increased incrementally. The second part was the constant work rate test, in which 80% of the maximum work rate of the incremental test was utilized. Patients were observed for changes in ventilation. These tests were conducted at baseline and after 8 to 12 weeks of therapy with oral L-glutamine at 30 grams a day. There were 6 patients who completed the incremental work rate test and 4 patients who completed the constant work rate test, respectively, at baseline and after treatment. Two of the patients withdrew prior to the completion of the follow-up testing. The results were as follows: On the incremental test, there was an upward trend in peak work rate with an average of 3.5 ± 8.7 watts/min (88.7 ± 25.6 vs. 91.5 ± 21.7; ρ> 0.05). Minute ventilation (VE) showed an average improvement of −4.8 ± 3.9 liters/min (61.2 ± 9.8 vs. 56.4 ± 9.9; ρ value of 0.05). The improved ventilation may be secondary to increased pulmonary blood flow inferring an increased cardiovascular oxygen delivery. On the constant work rate test, there was significant increase in exercise duration with an average of 1.4 ± 0.8 minutes (5.9 ± 1.6 vs. 7.3 ± 1.1; ρ< 0.05). The minute ventilation (VE) showed an average decrease of −11.0 ± 11.6 liters/min (66.5 ± 24.9 vs. 55.5 ± 14.5; ρ> 0.05). This could be indicative of improved oxygen delivery to the exercising musculature despite the absence of a preparatory exercise training program. The data confirm our preliminary report suggesting that L-glutamine therapy improves the exercise endurance of sickle cell anemia patients.

Test Mean + SD ρ value Pre Post Paired Δ (two-tailed t-test) Incremental Work Rate Peak Work Rate (watts/min) 88.7 ± 25.6 91.5 ± 21.7 3.5 ± 8.7 0.37 VE (liters/minute) 61.2 ± 9.8 56.4 ± 9.9 −c4.8 ± 3.9 0.05 Constant Work Rate −Duration (minutes) 5.9 ± 1.6 7.3 ± 1.1 1.4 ± 0.8 0.04 −VE (liters/minute) 66.5 ± 24.9 55.5 ± 14.5 −11.0 ± 11.6 0.15
Pipkiia and Beta-Globin: Transcripts Differentially Expressed in Reticulocytes and Associated with High Levels of Hb H in Two Siblings with Hb H Disease.
Authors: Sonati Maria F. , Wenning Marcia R. , Mello Maricilda P. , Andrade Tiago G. , Lanaro Carolina , Albuquerque Dulcineia M. , Titular Sara Saad , Titular Fernando F. Costa
Published: April 2020

Heterogeneous amounts of Hb H have been detected in peripheral blood of alpha-thalassemic patients even when they carry the same mutation association. This suggests the involvement of other modulating factors besides the thalassemia determinants. To address this issue, we investigated differential gene expression in reticulocytes from two Brazilian siblings of mixed ethnical origin (Chinese and African) in whom Hb H disease is caused by the -a3.7/--SEA genotype. One is a 21-year-old male, and the other a 19-year-old female. Their hemoglobin H levels are 18.7 and 5.0%, respectively. By using the mRNA differential-display reverse-transcription polymerase chain reaction (DDRT-PCR) and suppression subtractive hybridization (SSH) techniques, we identified two main transcripts in both procedures, one corresponding to the phosphatidylinositol-4-phosphate-5-kinase type II-alpha (PIP5KIIA) gene and the other corresponding to the beta-globin gene, both overexpressed in the patient with the higher percentage of Hb H. This result was validated by quantitative RT-PCR using two endogenous genes (GAPDH and BAC). PIP5KIIA expression, in arbitrary units (AU), was 0.108701 in the patient with the higher Hb H, 0.031646 in the other and 0.01561 in the normal control, while beta-globin gene expression was 1.13882, 0.71080 and 0.37631 AU, respectively. Reticulocyte RNA samples obtained from three beta-thalassemia intermedia patients showed a very low level of PIP5KIIA expression. Type II PIP kinases produce phosphatidylinositol 4.5 biphosphate (PI4.5P2) by phosphorylating phosphatidylinositol-5-phosphate. PI4.5P2 is a pleiotropic regulatory molecule involved in diverse cellular activities, including modulation of actin cytoskeleton, protein localization and gene expression. Our results suggest that PIP5KIIA may be one of the factors related to the beta-globin gene expression and to the different levels of Hb H in alpha-thalassemic patients. Its exact mechanism of action, however, remains to be determined.
Neurological Manifestations in Gaucher Disease Patients and Relatives.
Authors: Giraldo Pilar , Alfonso Pilar , Latre Paz , Pocovi Miguel ,   Spanish Group Gaucher Disease
Published: April 2020

Aims: Gaucher disease (GD) is characterized by a wide spectrum of manifestations. Several reports have indicated that GD relatives could developed neurological abnormalities. In order to detect neurological symptoms in GD patients and their relatives we have designed a specific prospective inquiry in Spanish GD families.

Patients and Methods: the Spanish GD Registry is an independent registry working since 1993, sponsored by the Spanish Gaucher Foundation (FEETEG). From January to December 2006 we have perform a postal survey contacting 42 physicians and 92 families to evaluate neurological symptoms and correlated with genetic characteristics. The data were included in a SPSS 12.0 data base to perform statistical analysis using descriptive parameters, ANOVA, t-test and correlation study including Pearson coefficient.

Results: We have obtained information from 72 families (78.3% responses) that included 92 type 1 and 7 type 3 GD patients and 266 relatives. Thirty (32.6%) of type 1 GD reported any neurological problem: tremor 8 (8.7%), uncordinated movements 9 (13.8%), concentration defects 11 (11.9%), strabismus 7 (7.6%), deafness 8 (8.7%), Parkinson disease (PD) 7 (7.6%), peripheral neuropathy 10 (10.9%). Thirty six (13.5%) first or second degrees relatives from 72 families presented neurological manifestations: PD 14 (5.3%), epilepsia 8 (3.0%), tremor 7 (2.6%), deafness 2 (0.7%), others 5 (1.9%). Patients with PD had mutations: S364R, D409H, L444P, [IVS2-2A>G]+ [c.(− 203)A>G], c.500insT, L336P. In relatives with PD a wide spectrum of mutations were observed: L444P, N370S, V398I, G202R, c.1439–1445del7, [E326K]+[N188S] and c.953delT, in others neurological manifestations as epilepsia, tremor or peripheral neuropathy predominant mutations were D409H, G195W, R120W; R147X, L336P and G377S.

Conclusions: We have found a higher incidence than expected of PD and other neurological symptoms in GD1 patients and relatives. These manifestations appear frequently in carriers of L444P or rare mutations. It is important to perform a systematic neurological exam in GD1 patients and carriers with risk mutations. This work is partially supported by the grant FIS: 06/1253.
Clinical Significance of Cytocentrifuge Preparation Analysis of Low-Cell-Count Cerebrospinal Fluid Specimens.
Authors: Lajeunesse-Viens Guylaine , Dufault Bernadette , Weng Xiaoduan , Lemieux Bernard , Florescu Marie
Published: April 2020

Introduction: The usefulness of performing a cytocentrifuge preparation (CCP) analysis on cerebrospinal fluid (CSF) containing less than 5 cells has never been studied. To determine if the CCP analysis has a clinical impact on central nervous system (CNS) management of patients with CSF samples containing less than five cells, we correlated the result of CCP to cytomorphology and flow cytometry.

Methods: From November 20th 2005 to July 18th 2007, CCP analysis were performed on 105 CSF consecutive samples containing less than 5 cells from patients with various cancers. For each of these samples, a research via computerized data was conducted to obtain cytomorphology and flow cytometry results. If results were positive or inconclusive, the clinical history and treatment were reviewed.

Results: All 105 samples had negative CCP results. Of the 105 samples, 74 (70,5%) had available cytomorphologies, with 5 (6,8%) being positive, confirming central nervous system infiltration. All 5 patients received treatment for their CNS involvement. The sensitivity of cytomorphology was 83,3%(5/6) and its specificity 100% (68/68).Of the 105 samples, 23 (21,9%) had available flow cytometries: 4 of the 23 (17,4%) cytometries were positive, 2 correlated by positive cytomorphology and treated. 2 had negative cytomorphology with one patient receiving treatment. The sensitivity of cytometry was 75% (3/4) and the specificity 94,7% (19/20).

Conclusion: We found no advantage to performing CCP analysis on CSF samples of less than 5 cells. However, this suggests that performing both cytomorphology and flow cytometry allows a better detection and management of CNS infiltration.
Sequential Serum Cytokine Levels after Autologous, Related and Unrelated Allogeneic Stem Cell Transplantation: Reflection of Immune Reactivity and Reconstitution.
Authors: Fagoaga Omar R. , Nehlsen-Cannarella Sandra L. , Ratanatharatorn Voravit , Martinelli Christine M. , Lum Lawrence G. , Uberti Joseph P.
Published: April 2020

Conditioning regimen in HSCT induces production of inflammatory, regulatory and growth-promoting cytokines. Cytokines are known to mediate alloresponses and heighten GVHD. We prospectively studied sequential cytokine levels in 38 recipients of HSCT (autologous 10, allogeneic related 10 and unrelated 18). Serum TNFα, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IFNγ, G-CSF, GM-CSF, MIP1α, MIP1β levels were measured. Also, Th1 and Th2 cytokines were evaluated as a ratio: Th1/Th2 = IL-2 + IFNγ /IL-4 + IL-10 and Type 1/Type 2 = IL-2 + IFNγ / IL-4 + IL-5 + IL-10. Samples were collected at baseline prior to conditioning regimen and at 4–6 day intervals up to day 95 postHSCT. We report only the significant findings here. In autologous HSCT, IL-5 peaked 192-fold (288.5pg/mL, p=0.0005) at 7d postHSCT over preHSCT levels (1.5pg/mL). At day 15d postHSCT, IL-1α levels decreased 1.9-fold to 0.2 pg/mL under preHSCT levels of 2.3pg/mL and increased 1.9-fold at 40d postHSCT (p=0.0301). In related alloHSCT, the levels of IL-1β increased 3.1-fold to a peak of 16.2 pg/ml at 51d postHSCT (p=0.001) compared to preHSCT level of 5.2pg/mL. IL-2 levels increased 2.2-fold (p=0.001) during the 2nd month postHSCT compared to preHSCT (1.5pg/mL) levels. IL-4 decreased 8.1-fold (p=0.001) at 27d postHSCT compared to preHSCT levels (4.9 pg/mL). IFNγ levels increased 1.5-fold to a plateau after 51d postHSCT compared to preHSCT levels (5.8pg/mL, p=0.017). In unrelated alloHSCT, the levels of TNFα increased 1.7-fold at 60d and 68d postHSCT compared to preHSCT levels of (8.94 pg/mL). IL-10 levels increased 5-fold (p=0.028) to peak at 27d postHSCT compared to preHSCT levels of 2.5pg/mL and to levels after the 1st month postHSCT. The Type1/Type2 ratio decreased 2.5-fold (p=0.023) to a nadir at 27d postHSCT from baseline and returned to preHSCT values by approximately 3 months postHSCT. In unrelated HSCT, Th2 polarization increased in the first month postHSCT due to higher levels of IL-5 and IL-10. After the first month, Th2 dominance subsided, probably due to T-cell recovery and its response to histoincompatibility. The related HSCT showed marked Th1 polarization after the first month of HSCT during which high levels of IL-4 declined and levels of IL-2 and IFNγ increased (Figure 1). Increased IL-5 levels in autologous HSCT is consistent with reconstitution of humoral response in the absence of allostimulation. In summary, we demonstrate some of the dynamic cytokine interactions occurring in the clinical course of recipients undergoing autologous, allogeneic related or unrelated HSCT. The kinetics of Th1 and Th2 levels may reflect the cellular immune reconstitution that could help us design immunotherapeutic interventions.

Figure Figure
ETP in the Discrimination of Liver Disease from DIC.
Authors: Tran Minh H. , Hassett Andrea L. Cortese
Published: April 2020

Introduction Endogenous Thrombin Potential (ETP) is felt to offer a global assessment of coagulation status using continuous photometric measurement of fluorescence emitted, followed by cleavage of a synthetic thrombin substrate. In this study we sought to explore differences in the ETP profile of patients diagnosed with Dissemintated Intravascular Coagulation (DIC) and to examine whether ETP can aid in the discrimination of patients with DIC or End Stage Liver Disease, who can often display similar testing profiles.

Methods Patients with test results interpreted as diagnostic for DIC, Liver Disease (LD), and neither DIC nor Liver Disease (NCD) were selected for evaluation. A total of 55 potential patients were identified and, of these, 45 (82%) were suitable for testing. Samples for testing had been collected in trisodium citrate, double centrifuged, and stored at −70° C for a mean of 15 months. The ETP test (Dade Behring, Marburg, Germany) was performed utilizing the BCS instrument according to the manufacturer’s guidelines. Results for T Lag, T Max, C Max, and AUC were compared between patient groups using two tailed T-test with a P value of <0.05, considered significant.

Results Tests were uninterpretable in 9/45 samples (20%) and therefore the total patient data set included 36 samples (10 in the LD group, 14 in the DIC group, and 12 in the NCD group). There were 7 female and 13 male controls (NC). Liver disease patients express lower values for T Lag and T Max than do DIC patients and lower C Max and AUC than either NCD or NC groups. All ETP variables are significantly lower in the DIC group vs either NCD or NC. NCD and NC groups exhibit minor, but significant differences in all variables, the greatest differences being lower C Max and AUC in NCD vs NC (see Figure 1). Similar differences were observed in gender specific analysis.

Conclusion While ETP determination may be helpful as an additional piece of information in distinguishing between DIC and End Stage Liver Disease patients, differences between the two groups were minor and there was considerable overlap. Paradoxically, the T Max (time to peak thrombin generation) was longer, and both the C Max (which represents the slope of the rise in thrombin generation) and AUC (total thrombin generation) lower, in DIC than in all other groups compared. This is possibly the result of inhibition from significantly higher levels of fibrinogen degradation products and D-Dimers in the DIC vs Liver Disease and non-DIC groups (data not shown). Additional studies using ETP in DIC, Liver Disease, and other hyper- and hypo-coagulable states are necessary.

ETP Variables in Various Patient Groups ETP Variables in Various Patient Groups
Short Term Outcome after Allogeneic Stem Cell Transplantation in 70 Cases of Severe Aplastic Anaemia.
Authors: Ahmed Parvez , Ullah Khalil , Satti Tariq M. , Raza Shahid , Chaudhry Qamar-Un-Nisa , Kamal Khalid , Khan Badshah , Anwar Masood
Published: April 2020

Allogeneic stem cell transplantation (SCT) from HLA matched sibling donor is the standard treatment option in younger patients with severe aplastic anaemia (SAA). In the current study outcome of 70 patients with SAA undergoing allogeneic SCT at our institution from July 2001 to June 2007 is presented. Median follow up time was 727 days (range 100–2187). Three patients received 2nd SCT due to graft failure or rejection so actual number of SCT in the patients was 73. Median age of the patients was 16 years (range 5–38), 55 males, and 15 females. Seventeen had major ABO mismatch while sex mismatch in the form of female donor to male patient was present in 23 cases, 7 had both major ABO and sex mismatch. Sixty four patients were CMV positive while 6 had CMV negative status. Conditioning regimens included; cyclophosphamide (Cy) 200 mg/kg with either antilymphocyte globulin (ALG) 45 mg/kg (n=33) or antithymocyte globulin (rabbit ATG) 11.25 mg/kg (n=26); Cy plus Campath 100 mg (n=6), fludarabine 150 mg/m2 plus Cy 300 mg/m2 and ATG (n=8). All patient undergoing 2nd transplant received conditioning with Cy, fludarabine and ATG. GVHD prophylaxis was given with cyclosporin (CSA) plus prednisolone (41) with or without short course of methotrexate (29). Patients received PBSC (10) or bone marrow (12) alone or both (48). Mean mononuclear and CD34+ cell doses were 5.59 x 108/Kg and 4.8 x 106/Kg respectively. Median time to neutrophil recovery was 11 days (range 7–24). Neutropenic fever was seen in 60% cases, with mean duration of fever being 8 days. In majority (66%) no focus of infection could be found. Various isolates included gram negative rods (n=6), staphylococcus (n=2) and fungi (n=5). Other post-transplant infections were tuberculosis (n=2), herpes zoster (n=2) and transfusion associated falciparum malaria (n=2). Post-BMT non-infectious complications included acute GVHD (24%), chronic GVHD (08%), hemorrhagic cystitis (14%), seizures (5%), and VOD (3%). Graft rejection and primary graft failure was seen in 3 and 2 cases respectively. Three of them received 2nd transplant and had successful recovery while the other 2 died of septicemia. Six patients died during peri-transplant period, 3 at day 100, and 8 beyond day 100. One patient died of unrelated cause at 2 years post-transplant. Main causes of death were septicemia (n=4), conditioning regimen toxicity (n=3), VOD (n=2), GVHD (n=2) and disseminated aspergillosis (n=2). The overall and disease free survival was 76%. In univariate analysis using Logrank and Wilcoxon test factors correlated with better survival were patient’s age <15 years, disease duration <6 months, previous transfusions <20 events, conditioning with fludarabine/Cy/ATG, and absence of chronic GVHD. SCT from HLA matched sibling donor is effective treatment modality in majority of the young patients with SAA.
A Common Genetic Variant in the 3′UTR of IRF4/MUM1 Associates with Risk of Disease and Poor Prognosis in Chronic Lymphocytic Leukaemia.
Authors: Allan James M , Sunter Nicola , Hall Andrew , Mainou-Fowler Tryfonia , Jackson Graham , Summerfield Geoffrey , Harris Robert John , Pettitt Andrew , Houlston Richard , Bailey James , Pepper Chris , Fegan Chris , Pratt Guy , Allsup David J
Published: April 2020

High frequency low penetrance risk alleles for chronic lymphocytic leukaemia (CLL) have been identified at 6p25.3 (rs872071, IRF4/MUM1), 11q24.1 (rs735665, GRAMD1B), 15q23 (rs7176508), 2q37.1 (rs13397985, SP140), 2q13 (rs17483466, ACOXL) and 19q13.32 (rs11083846, PRKD2). Given a role in determining risk of disease it is plausible that these allelic variants also play a role in disease progression and overall survival. In order to test this hypothesis, polymorphic status was determined in a case series of 403 patients diagnosed with CLL recruited via 5 clinical centres in the United Kingdom. Mean follow-up time was 4447 days and 169 patients were deceased at the time of last follow-up. Kaplan-Meier survival analysis and the log-rank test were used to investigate the prognostic significance of constitutional genetic markers. Polymorphic variation at rs735665, rs7176508, rs13397985, rs17483466 and rs11083846 was not significantly association with time from diagnosis to first treatment (p>0.05, log-rank test). However, a genetic variant in the 3′UTR of the interferon regulatory factor 4 (IRF4)/multiple myeloma oncogene 1 (MUM1) gene was significantly associated with poor prognosis. Specifically, carriers of the disease-associated variant at rs872071 had a significantly shorter mean time from diagnosis to first treatment (3983 days), compared to non-carriers (7340 days)(p=0.001), although there was no significant difference in overall survival (p=0.29). The association with time to first treatment remained significant in multivariate Cox regression analysis that included age, gender, immunoglobulin heavy chain variable region mutational status and stage of disease at diagnosis in the model (p=0.021). IRF4 is expressed in germinal centre (GC) and post-GC B-cells, and is important for B-cell maturation and homeostasis. Taken together with the aetiological evidence, these data suggest that common allelic variation in IRF4 not only affects risk of CLL, but also predicts poor prognosis.
Junctional Adhesion Molecule a Helps Maintain Integrin αIIbβ3 in Resting State
Authors: Naik Meghna Ulhas , Stalker Timothy J. , Brass Lawrence F. , Naik Ulhas Pandurang
Published: April 2020

Under physiological conditions, fibrinogen receptor integrin αIIbβ3 on the circulating platelets is in a low-affinity, or resting state, unable to bind soluble ligands. During platelet activation by agonists, a cascade of signaling events induces a conformational change in the extracellular domain of αIIbβ3, thereby converting it into a high-affinity state capable of binding ligands through a process known as “inside-out signaling”. What maintains this integrin in a low-affinity state is not well understood. We have previously identified JAM-A, junctional adhesion molecule A, on the platelet surface. We have shown that an antibody blockade of JAM-A dose-dependently activates platelets. To understand the molecular mechanism through which JAM-A regulates platelet aggregation, we used Jam-A null mice. Interestingly, the mouse bleeding times were significantly shortened in Jam-A null mice compared to wildtype littermates. Furthermore, the majority of these mice showed a rebleeding phenotype. This phenotype was further confirmed by FeCl3-induced carotid artery occlusion, a well-accepted in vivo model for thrombosis. Platelets derived from Jam-A-null mice were used to evaluate the role of JAM-A in agonist-induced platelet aggregation. We found that Jam-A null platelets showed enhanced aggregation in response to physiological agonists such as PAR4 peptide, collagen, and ADP as compared to platelets from wildtype littermates. JAM-A was found to associate with αIIbβ3 in unactivated human platelets, but this association was disrupted by both agonist-induced platelet aggregation and during outside-in signaling initiated upon platelet spreading on immobilized Fg. We also found that in resting platelets, JAM-A is phosphorylated on a conserved tyrosine 280 in its cytoplasmic domain, which was dephosphorylated upon platelet activation. Furthermore, JAM-A is rapidly and transiently phosphorylated on serine 284 residue during platelet activation by agonists. Interestingly, JAM-A also formed a complex with Csk, a tyrosine kinase known to be inhibitory to Src activation, in resting platelets. This complex was dissociated upon activation of platelets by agonists. These results suggest that tyrosine-phosphorylated JAM-A recruits Csk to αIIbβ3 in resting platelets, thus maintaining a low-affinity state of integrin αIIbβ3. Agonist–induced activation of platelets results in rapid dephosphorylation of JAM-A on Y280 and phosphorylation on S284 residues. This causes dissociation of JAM-A from integrin αIIbβ3 facilitating platelet aggregation.
Impact of Prior Invasive Pulmonary Aspergillosis (IPA) on Outcome in Patients Receiving Reduced Intensity Conditioning (RIC) Allogeneic Haematopoietic Stem Cell Transplantation (Allo-SCT).
Authors: El-Cheikh Jean , Wang Ling , Esterni Benjamin , Faucher Catherine , Furst Sabine , de Lavallade Hugues , Berger Pierre , Blaise Didier
Published: April 2020

Invasive Pulmonary Aspergillosis (IPA) is a major cause of morbidity in patients with hematological malignancies. The aim of this single centre retrospective study was to determine the impact of prior IPA on outcome after RIC Allo-SCT. All cases of proven or probable IPA diagnosed prior to performance of RIC Allo-SCT at the Paoli-Calmettes Institute Cancer Centre from 1 January 2000 through 31 December 2007 were included. 28 patients were identified among 434 patients undergoing Allo-SCT. Patients’ data were collected from a maintained database and by retrospective clinical chart review. Twenty eight cases were identified. Gender: M/F (16/12); median age at diagnosis was 53 years (18–65). 23 patients (82%) had acute myeloid leukemia. In all patients, IPA was diagnosed according to standard procedures. IPA therapy included: 20 patients (71%) were receiving Voriconazole; 4 patients Itraconazole, and 4 patients with an association of different antifungal drugs including Caspofungine, Liposomal Amphoteracine-B, or Posaconazole. The median duration of treatment prior to RIC was 8 months range (1–16). The median time between the diagnosis of IPA and transplantation was 6 months (1–27); 20 patients (71%) received a graft from a family donor, 4 patients had a MUD, and 4 patients received cord blood cells donors. 21 patients (75%) received a conditioning regimen with Fludarabine Busulfan and ATG, and 4 patient (14%) received a conditioning with Fludarabine Cyclophosphamide and TBI, 3 patients (11%) Fludarabine and TBI, Most patients (n=25; 89%) received or continued a secondary prophylaxis against aspergillosis at time of Allo-SCT: 17 (68%) had a prophylaxis with Voriconazole; 3 patients Itraconazole, 2 patients Posaconazole. After RIC transplantation only 4 patients (14%) had reactivation of their IPA. The latter 4 patients were experiencing severe acute GVHD treated with high dose corticosteroids. None of these patients died of IPA. Actually 18 patients (64%) are still alive with a median follow up of 23.5 months (12.6–48.5). Overall survival at 2 years was 59% [95%CI, 43–83]. In all, 3 patients died from other causes not directly related to IPA, 2 patients because of relapse or disease progression, and one due extensive chronic GVHD. In comparison to literature in standard myéloablative allo-SCT setting, these data suggest that adequately treated and controlled IPA prior to RIC Allo-SCT do not worsen outcome. The latter is likely due to multiple changes in transplantation practice, including the RIC nature of the conditioning regimens, the infusion of peripheral blood stem cells, rapid diagnosis of IPA, and use of modern efficient antifungal drugs.
Integrated Genomic Profiling Identifies Preferential Expression of the Insulin Receptor in CLL with Deletion 11q.
Authors: Malek Sami , Ouillette Peter , Shedden Kerby , Saiya-Cork Kamlai
Published: April 2020

Chronic lymphocytic leukemia (CLL) is associated with characteristic genomic aberrations as defined by FISH and SNP arrays. CLL with del11q is characterized by a relatively aggressive clinical course, and about one third of cases carry mutations in ATM on the retained allele. Little information is available on additional molecular aberrations associated with del11q in CLL. We have analyzed del11q in a subset of 178 CLL cases using DNA from FACS-sorted CD19+ cells compared with buccal DNA using the Affymetrix 50k SNP array platform. Genomic copy number analysis using dChipSNP identified two non-overlapping minimal deleted regions on 11q, one of which spans ATM. We proceeded to use comparative expression array profiling of RNA derived from sorted CD19+ cells from 10 CLL cases with and nine cases without del11q using the Affymetrix U133 2.0 Plus platform. Analysis of normalized data identified ~85 probe sets with differential expression (del11q versus reference) that displayed a false discovery rate of <0.1. Approximately 70% of these genes were under-expressed and mapped to 11q22–23, thus providing confidence that genes lost through del11q displayed reduced expression under our assay conditions. Three probe sets identified the insulin receptor (INSR) to be expressed at substantially higher levels (log2 scale 2.5–3.1 fold) in del11q CLL as opposed to the reference cases that were used in the expression array-based analysis. Validation of differential INSR expression in CLL was accomplished by Q-PCR using cDNA synthesized from RNA purified from sorted CD19+ cells from 178 CLL cases. Using normalized Ct values for INSR expression (delta Ct INSR-GAPD) and a delta Ct cut-off of <10, 65% of CLL with del11q expressed the INSR as opposed to 15% (del13q14), 6% (del17p), 11% (normal FISH) and 23% (trisomy 12) and ~20% of all CLL cases, independent of genetic subtype. Finally, immunoblot analysis on cellular lysates derived from negatively enriched and >90% pure CLL cells from 10 cases with del11q and with high INSR expression versus cases without del11q and absent expression confirmed the accuracy of Q-PCR-based estimates of INSR expression. Work is in progress to measure effects of INSR expression on CLL survival and insulin-induced signaling in CLL in an effort to dissect functional consequences of INSR expression in CLL.
Patients with Severe Factor XI Deficiency Who Have An Inhibitor or IgA Deficiency Can Undergo Uneventful Major Surgery by a Single Infusion of Low Dose Recombinant Factor VIIa and Use of Tranexamic Acid.
Authors: Salomon Ophira , Tamarin Ilia , Livnat Tami , Horovitz Zeev , Kuriansky Joseph , Martinowitz Uriel , Mor Yoram , Seligsohn Uri
Published: April 2020

Inhibitors to factor XI (FXI) can develop following exposure to plasma products in 33% of patients with severe FXI deficiency who are homozygous for the type II nonsense mutation prevalent in Jews (Salomon et al. Blood2003;101:4783). Hemostasis in five such patients was previously achieved during surgery by infusion of recombinant factor VIIa (rFVIIa) and tranexamic acid, but two patients experienced arterial thrombosis following surgery (Schulman et al. Hemophilia2006; 12:223). Conceivably, the rather high dose of rFVIIa used was responsible for the thrombotic events in these patients. In a previous in vitro study, we showed that low concentrations of rFVIIa can correct thrombin generation in plasma of patients with severe FXI deficiency and an inhibitor to FXI (Livnat et al. J Thromb Hemost2006; 4:192). In the present study we addressed the question whether low doses of rFVIIa and use of tranexamic would be hemostatically effective and less thrombogenic during and after major surgery in patients with severe FXI deficiency in whom plasma derivatives could not be used. Three patients (two with FXI inhibitor and one with severe IgA deficiency) underwent major surgery managed by a single low dose infusion of rFVIIa and administration of tranexamic acid 1 G q.i.d for 7 days (Table). In patient 1 the infusion was given immediately prior to surgery and in patients 2 and 3 at the end of surgery. Hemostasis was achieved in all patients and no thrombosis occurred.

No. Sex/age FXI (U/dL) FXI inhibitor (B.U) Operation Single dose rVIIa (uG/Kg) Adverse events * IgA deficiency 1 M/62 < 0.01 5.0 Laparoscopic cholecystectomy 30 None 2 M/61 < 0.01 5.0 Transuretheral prostatectomy 16 None 3 M/63 0.02 0* Parathyroidectomy 15 None

These observations suggest that a single infusion of low dose rFVIIa and use of tranexamic acid can be a safe modality of treatment in patients with severe FXI deficiency who cannot receive blood products. Furthermore, the protocol outlined here may be used to avoid inhibitor formation in patients with extremely severe FXI deficiency who undergo surgery.
Glia Maturation Factor-Gamma Mediates Neutrophil Chemotaxis Via p38 MAPK and PTEN.
Authors: Aerbajinai Wulin , Chin Kyung , Zhu Jianqiong , Li Hongzhen , Rodgers Griffin P
Published: April 2020

Cellular chemotaxis is a fundamental process in which cells migrate directionally when they are exposed to external chemical gradients. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity and chemotaxis, including the contribution of p38 MAPK and PTEN dependent migration of neutrophils toward their end target chemoattractants. However, it remains unclear how chemoattractant stimulation regulates F-actin dynamics and PTEN localization in migrating neutrophils. Glia maturation factor (GMFG) is a highly conserved protein expressed mainly in the blood and immune systems. Our lab previously reported that GMFG is upregulated in granulocyte-colony stimulating factor (G-CSF)-induced differentiated myeloid cells (Shi Y, et al, Genomics Proteomics Bioinformatics. 2006 Aug;4(3):145–55). Furthermore, while recent studies indicate that GMFG is involved in the regulation of actin cytoskeleton reorganization in microendothelial cells, little is known about the role of GMFG in hematopoietic myeloid cells. In this study, we investigated the role of GMFG in the chemotaxis of differentiated neutrophil-like HL-60 cells and how GMFG mediated neutrophils chemotaxis occurs in response to chemotactic signals. We have now shown that small interfering RNA-mediated depletion of GMFG expression in differentiated neutrophil-like HL-60 cells exhibit greater than 75% inhibition of basal cell migration, that could not be rescued in the presence of chemoattractant FMLP, but could rescued by IL-8. These data suggest that an endogenous expression of GMFG is required for chemotaxis toward end target chemoattractant FMLP, but not for toward intermediate chemokines IL-8. Moreover, immunofluorescence assay shows that gene silencing of GMFG abolished FMLP-induced the phosphorylation of p38 MAPK, localization of phosphatase PTEN, and F-actin reorganization. In contrast, transient overexpression of GMFG in K562 cells significantly enhanced basal cell migration by 2 fold (P=0.01) and chemotaxis toward FMLP by 2.2 fold (P=0.04), but there was no change with IL-8 stimulation. The overexpression of GMFG caused the activation of p38 MAP kinase and increased F-actin expression in the absence of fMLP stimulation. Pretreatment with p38 MAPK inhibitor SB203580 completely blocked the GMFG-dependent activation of p38 MAPK and enhanced expression of F-actin as well as cell migration. These results suggest that GMFG-induced cell migration in K562 cells is depending on p38 MAPK via F-actin reorganization. Taken together, our findings identify GMFG as a novel factor that mediates the FMLP-induced p38-dependent signal pathway leading to PTEN redistribution and F-actin reorganization during migrating neutrophils toward end target stimulation. These data, if confirmed, implicates GMFG as a potential target for therapeutic intervention in disorders associated with enhanced and pathological neutrophil infiltration.
Phase 1 Clinical Testing of HQK-1001, a Novel Oral Fetal Globin Gene Inducer
Authors: Perrine Susan , Welch William C , Keefer Jeffrey , Downey Robin L , Boosalis Michael S , Case Sally , Faller Douglas V , Berenson Ronald
Published: April 2020

Development of non-cytotoxic, orally-active therapeutics, which induce fetal globin production, would be beneficial for treatment of beta-thalassemias and hemoglobinopathies. The short-chain fatty acids (SCFA) butyrate and phenylbutyrate are active fetal globin inducers, but their clinical application has been limited by inhibition of erythropoiesis and unfavorable PK profiles. HQK-1001 is a SCFA which stimulates fetal globin gene expression in reporter gene assays, erythroid progenitors, transgenic mice, and anemic baboons. HQK-1001 also stimulates human erythroid progenitor (BFU-E) proliferation by a mean of 30% above controls, and increased red blood cell counts in phlebotomized baboons. HQK-1001 has demonstrated a wide safety margin in pre-clinical toxicology testing, with no significant adverse effects in sub-chronic (6-month) studies and negative mutagenicity testing. Based on these pre-clinical data, a placebo-controlled, randomized, blinded, single-dose escalating Phase 1 trial was conducted to evaluate HQK-1001 at 4 dose levels ranging from 2 to 20 mg/kg in 32 normal volunteers. The drug was welltolerated, with no significant adverse events. Pharmacokinetic analyses showed low intersubject variability, a linear increase in Cmax and AUC with increasing dose, and T½ of 11 hours, indicating suitability for once-daily dosing. Administration under fed conditions demonstrated equivalent AUC to fasting conditions. In a subsequent 14-day Phase 1 dosing study in 41 normal subjects, the drug was also well-tolerated, with no significant adverse effects at the studied dose levels (5, 10 and 15 mg/kg). F-reticulocytes increased in subjects treated with 5 to 10 mg/kg doses. Absolute reticulocytes increased significantly above baseline (p <0.02 – 0.002) following 14-day dosing at all dose levels, while no significant change occurred in placebo controls. These first-in-human clinical studies demonstrating safety of HQK-1001 in 73 normal volunteer subjects and an improved pharmacologic profile over prior SCFAs indicate that HQK-1001 merits further clinical investigation in anemic patients with beta-globin gene disorders.
Impact of Management of Iron Overload in Patients with Myelodsplastic Syndromes
Authors: Bozkaya Duygu , Migliaccio-Walle Kristen , Baladi Jean-Franòois
Published: April 2020

For many patients with myelodysplastic syndromes (MDS) chronic red blood cell transfusion is the standard of care to delay or prevent disease progression and to improve function. This is not without consequence, however, as transfusion-dependent patients are at risk for developing iron overload, potentially leading to cardiac disease, diabetes and hepatic complications. Iron chelators have been widely applied to remove excess iron from the body. Once-daily deferasirox given orally achieves iron reduction with easy administration. The purpose of the study was to develop a model to compare the impact of choosing deferasirox versus no chelation in managing transfusion-dependent patients with MDS. Mortality, progression to acute myeloid leukemia (AML), and development of complications related to iron overload were central elements of this model. Simulated patients were individually subject to these risks, dependent on use of chelation. The analysis was stratified by patients’ age group (≤77, >77) and by severity of MDS (Low, Int-1, Int-2 and High) as defined by the International Prognostic Scoring System (IPSS). Event risks, utilities associated with chelation and complication status were obtained from published or presented sources and deferasirox study data. The improved rates with deferasirox versus no chelation for mortality and progression to AML were taken from recent studies by Rose et al and Leitch, respectively. Complication rates for patients receiving transfusion, and the deferasirox-related reduction in these rates, were obtained from the literature. The cost of deferasirox and management costs were estimated from data obtained from literature and other US cost data sources. All costs are reported in 2008 US$. Utility values associated with deferasirox and no chelation were calculated as 0.74 and 0.67 using SF-36 measures from a substudy of a 1-year, open-label, single-arm, multi-center trial evaluating the efficacy and safety of oral chelation therapy. Costs and benefits were discounted at 3% per year. Univariate sensitivity analyses were implemented. Initial 10-year results estimated a cost per patient of $217,785 with deferasirox and $120,505 with no chelation. Deferasirox management resulted in 4.94 years survival (discounted) and 3.18 QALYs per patient. Compared with no chelation, deferasirox yielded an additional 1.63 years survival and 1.42 QALY. Resultant cost-effectiveness ratio was $68,699/QALY gained. Results of sensitivity analyses and other scenarios predicted a cost/QALY as low as $44,068. Chelation therapy with deferasirox is predicted to result in improved survival and substantially fewer complications due to excess iron accumulation in the range of accepted cost/QALY.
In Vivo Injection of Synthetic Small Interfering RNA to Lethally Irradiated Mouse as a New Model to Study Apoptotic Pathways.
Authors: Drouet Michel , Garrigou Philippe , Mayol Jean-François , Delaunay Christophe , Peinnequin Andre , Hérodin Francis
Published: April 2020

The Fas/Fas-ligand system is a well known component of the extrinsic apoptotic pathway. Using a short term culture assay we have established that CD34+ hematopoietic stem and progenitor cells express Fas antigen within 10 hours following irradiation. Using the terminaldeoxynucleotidyl transferase test, we have also established that this expression was linked with apoptosis since only the Fas/Fas-ligand positive cells exhibited a high level of DNA fragmentation (Drouet et al, Stem cell 1999). However Fas is also involved in the CD34+ cells differentiation process as described in ex vivo expansion studies. Caspases are other important actors of radiation induced (RI) apoptosis process and our team has recently identified caspases one and six as key actors in RI apoptosis in CD34+ cells. The goal of the present study was to evaluate short term synthetic small interfering RNA (siRNA) as new tools to in vivo modulate apoptosis in order to allow pathophysiological studies at the hematopoietic niche level. Briefly, B6D2F1 mice were globally irradiated (9 Gy gamma, LD90% 30 days) and then injected at 2 hours following irradiation with siRNA (0.5 nmol/mice, Dharmacon). To ensure a proper delivery to the niche cell components, siRNA were intra-tibially injected under a volume of 30μl. The duration of gene inhibition is about 10 days long. Control mice were injected with non relevant mock siRNA (n=80). Treated animals were injected with Fas-siRNA (n=20) or Fas + a pool of siRNA against caspases 1+ 3 + 6 + 8)(n=20). All mice were given ciprofloxacin during a week and no early lethality was observed. The lethality curves show that animals treated with Fas-siRNA exhibited an accelerated death rate when compared with siRNA mice. These results are compatible with a janus model for Fas expression depending on the time following irradiation: initial proapoptotic role, then requirement for cell expansion. Globally this study suggests the feasibility of using synthetic siRNA in vivo to screen the role of apoptosis actors.
Long-Term Expansion and Self Renewal Is Contained in the CD34+, but Not the CD34- Subfraction of Nucleophosmin Mutated Acute Myeloid Leukemia Cells.
Authors: Woolthuis Carolien , Han Lina , van Gosliga Djoke , Kluin Philip , Vellenga Edo , Schuringa Jan J. , Huls Geert
Published: April 2020

Mutations in the nucleophosmin (NPM) gene are found in about 30% of cases of acute myeloid leukemia (AML) and lead to a dislocation of the nucleophosmin protein from the nucleus to the cytoplasm (NPMc+ AML). NPMc+ AML shows distinctive biological and clinical features, including a unique gene expression profile, a distinct microRNA signature, low percentage of CD34+ cells, increased incidence of Flt3-ITD (about 40% of cases), good response to induction chemotherapy and (in the absence of Flt3-ITD) a favourable prognosis. Despite significant progress in the characterization of the NPMc+ AML subgroup, questions remain about the leukemia-initiating cell. Distinct features of NPMc+ AML, including multilineage involvement and overexpression of HOX-genes, may point to an early progenitor as the leukemia-initiating cell, but the characteristic low percentage of CD34+ cells may point to a more differentiated leukemic stem cell in NPMc+ AML. To gain more insight in the leukemia-initiating cell in AML with mutated NPM, NPMc+ AML cells were sorted based on the expression of CD34 (n=8, the percentage of CD34+ in the total AML fraction varied between 0.06 and 37%). Western blotting, using an antibody that specifically recognizes the nucleophosmin mutant protein revealed that the NPM mutant protein is expressed in both CD34+ and CD34− cells, proving that the CD34+ NPMc+ AML cells belong to the leukemic clone. This was verified by sequencing the NPM gene in CD34+ and CD34− AML cells. Importantly, culture of sorted CD34+ and CD34− NPMc+ AML cells on a stromal layer revealed that the CD34+ but not the CD34− cells of NPMc+ AML were capable of expanding and initiating long-term growth. In the first 5 weeks of culture an at least 16 fold (range 16–208) expansion of CD34+ AML cells was seen in 5 out of 6 NPMc+ AML cases. This expansion was associated with the formation of cobblestone areas (CAs) under the stromal layer within 3 weeks after plating. The NPMc+ AML cells which expanded in culture were able to expand further after replating in 4 out of 5 investigated cases (fold expansion range 1.6–2.5), indicative of the self renewal capacity of these CD34+ NPMc+ AML cells. Gene expression analysis of CD34+ and CD34− NPMc+ AML cells of 4 cases analyzed thus far revealed the presence of the characteristic HOX-overexpression profile in both CD34+ and CD34− NPMc+ AML cells. In summary, this study shows that the NPM mutation is not only present in CD34−, but also in CD34+ cells of NPMc+ AML and that the properties of long-term expansion and self renewal belong exclusively to the CD34+ subfraction of NPMc+ AML.