bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary Hypertension in Adults and Children with Sickle Cell Disease in Nigeria: Prevalence, Clinical Characteristics and Role of Endemic Tropical Infections.
Authors: Aliyu Zakari Y. , Vandana Sachdev , Mamman Aisha I. , Babadoko Aliyu , Akpanpe Peter , Attah Ester , Suleiman Yusuf , Aliyu Nurudeen , Yusuf Jamilu , Mendelsohn Laurel , Coles Wynona , Danbauchi Solomon , Taylor James , Castro Oswaldo , Gordeuk Victor R. , Blackwelder William C. , Kato Gregory J. , Gladwin Mark T.
Published: April 2020

Pulmonary hypertension has a prevalence of 30% in patients with sickle cell disease (SCD) in the United States with mortality rates of 40% at 40 months after diagnosis. The global burden of SCD is highest in sub-Saharan Africa where more than 200,000 children are born with the disease annually. The prevalence of pulmonary hypertension among individuals with SCD in Africa has not been previously reported. We performed Doppler echocardiographic assessments of pulmonary-artery systolic pressure in 206 consecutive hydroxyurea sickle cell patients at steady state in Nigeria, West Africa (101 males and 105 females; age range 10–52, mean [+/−SD] age, 21.5 +/− 7.7 years; 196 homozygous sickle cell and 10 compound heterozygotes SC). A control group consisted of 93 healthy Nigerians. Hemoglobin gentotype was determined by electrophoresis and DNA sequencing. Pulmonary hypertension was defined prospectively as a tricuspid regurgitant jet velocity (TRV) of at least 2.5 m per second. We collected clinical data on the patients and controls, and blood specimens for clinical laboratory measurements. Doppler-defined pulmonary hypertension occurred in 25% of sickle cell patients (21% with TRV 2.5 – 2.9 m/sec, 4% with TRV ≥ 3 m/sec). The presence of pulmonary hypertension was inversly associated with age (p=0.04) and hemoglobin (p=0.0016), and positively associated with reticulocyte count, serum levels of lactose dehydrogenase (p=0.03), creatine kinase, and blood urea nitrogen and systolic (p=0.03) and diastolic blood pressure (p=0.002) in bivariate analyses. In a multivariate linear regression model age, diastolic blood pressure and blood urea nitrogen had significant independent associations with pulmonary hypertension. There were no significant associations of HIV/AIDS, hepatitis B and C co infections and malarial parasitemia rate with pulmonary hypertension. Our findings suggest that pulmonary hypertension is common among sickle cell patients in Africa and it appears to be a complication of chronic hemolysis and vasculopathy. The prevalence of pulmonary hypertension decreases with age in Nigerian SCD patients, in sharp contrast to U.S. SCD patients, who demonstrate increasing prevalence with age. The public health implications of this finding are significant considering the potential number of individuals at risk for this complication. Large prospective cohort studies to determine the outcome of pulmonary hypertension in sickle cell patients in Africa are needed.
Puerto Rican Child with Combined Heterozygocity for Hemoglobin S and Porto Alegre.
Authors: Lojo Liliana , Santiago Pedro J. , Rivera Enid , Rivera Doris E. , Renta Jessica , Cadilla Carmen L.
Published: April 2020

Hemoglobin S is the most common abnormal hemoglobin detected in newborn screening in Puerto Rico. The purpose of this report is to present laboratory and clinical data from a newborn proven to have combined heterozygocity for hemoglobin S and a very rare hemoglobin. Abnormal newborn screening results using isoelectric focusing prompted us to further study an infant with an unknown hemoglobin. Testing with cellulose acetate and citrate agar electrophoresis revealed that this patient had, besides hemoglobin S, a rare fast moving band. We consulted the Haemoglobinopathy Screening Service at St. James’s University Hospital in Leeds, UK where our patient’s hemoglobin was identified as hemoglobin Porto Alegre by means of mass spectrometry. DNA sequencing at the molecular genetics laboratory in the Medical Sciences Campus, University of Puerto Rico confirmed our patient’s hemoglobin to be Hb Porto Alegre. The patient’s father was found to be heterozygous for hemoglobin S and her mother a carrier of hemoglobin Porto Alegre. Our patient has been followed-up for the last two years. She has remained asymptomatic, except for usual mild childhood diseases. Laboratory results up to 18 months of age have revealed Hgb 11.4–11.6 g/dl, MCV 87.5 fl, MCH 28.7 pg, reticulocyte count 2%, normal total bilirubin and LDH levels. Hemoglobin Porto Alegre involves a mutation in ß9(A6)Ser→Cys which promotes hemoglobin polymerization via disulfide bonds (Baudin-Creuza 2002). This mutation does not seem to alter hemoglobin function and apparently does not increase tendency for sickling. Tondo (1963) has reported that in vivo hemoglobin Porto Alegre exists in its normal tetrameric form, with no associated hemolysis. Martinez (1977) has suggested that hemoglobin Porto Alegre can exist in its tetrameric functional form by means of high levels of glutathione in red blood cells. Therefore, patients with combined hemoglobin S and Porto Alegre would be expected to be asymptomatic. Hemoglobin Porto Alegre has been documented in a few patients from Brazil, Portugal, Argentina, Cuba, and the Canary Islands. To our knowledge this is the first reported double heterozygote for hemoglobin S and Porto Alegre. We are following our patient closely for the development of any significant hematologic or clinical symptoms.
The Prevalence of Renal Dysfunction in Young Adults with Sickle Cell Disease.
Authors: Osarogiagbon Raymond U. , McHugh Laura M. , Kronish Lori E. , Chismark Elisabeth A.
Published: April 2020

Sickle cell disease is typified by dramatic acute painful episodes, which consume the majority of patients’ and healthcare providers’ attention. However, the cumulative damage to vital organs that occurs in this disease is often the determinant of early death. The kidneys are a major target organ in sickle cell disease and renal failure is a highly morbid event. It is also strongly associated with early death. Glomerular hyperfiltration is often the earliest sign of renal injury. This is followed by return to apparently normal filtration rates, then successively lower rates, culminating in end-stage renal disease. Microalbuminuria follows hyperfiltration and progresses to frank proteinuria. We evaluated the prevalence of renal dysfunction in a cohort of patients with sickle cell disease managed at the University of Tennessee Cancer Institute’s adult sickle cell program. At entry into the program, patients underwent a routine battery of tests, including 24 hour urine collection for measurement of creatinine clearance and urine protein. Our findings are summarized in the table below.

By the age of 25 years, almost 80% of patients with sickle cell disease showed significant abnormality of glomerular filtration and 40% had significant proteinuria. This worsens with age. By a median of 37 years, 2 in 3 patients have developed significant proteinuria and hypofiltration has become the predominant pattern of glomerular filtration abnormality. Since renal injury is reversible in the early stages, more emphasis needs to be placed on aggressive early screening, surveillance and intervention. Intervention at the stage of hyperfiltration and microalbuminuria is easier and much more likely to be successful. Patients and their healthcare providers need to be educated on the high prevalence of renal damage. Efforts at education, screening, surveillance and, where necessary, treatment should begin well before adolescence.
Expression of High Levels of Human γ-Globin in Adult Mice Carrying a Transgene of the Brazilian Type of Hereditary Persistence of Fetal Hemoglobin.
Authors: Cunha Anderson F. , Brugnerotto Ana F. , Melo Monica B. , Corat Marcus A.F. , Gimenes Ana P. , Passos Luiz A.C. , Devlin Emily E. , Bodine David M. , Saad Sara T.O. , Costa Fernando F.
Published: April 2020

The term, hereditary persistence of fetal hemoglobin (HPFH), describes a hereditary benign disease, characterized by an increase in fetal hemoglobin (HbF) during adult life. Non-deletional forms of HPFH are characterized by single-base mutations in the promoter region (most of them between −114 and −202 from the cap site) of either the Gγor Aγ-globin gene, resulting in an increase of HbF ranging from 3 to 20% in heterozygotes. Many point mutations in this region have been described, including the Aγ-195 C →G mutation that causes the Brazilian type of HPFH (HPFH-B). A previous study showed that the -195 mutations alone was not able to increase gene expression in vitro and a modest increase was observed when a LCR element fragment (HS2) was introduced in the construction. This study showed too, that the the mechanism of HbF elevation by the -195 mutation was neither mediated by the Sp-1 transcription factor nor by the creation of a CACCC box, as described for the -198 mutation. Thus, other proteins may be involved in the over-expression of the γ-globin chain and/or may depend on DNA structure and these mechanisms remain to be clarified. To better understand this mechanism we have developed HPFH-B transgenic mice. The promoter with mutation was amplified using the genomic DNA of a HPFH-B patient and cloned in a μLCRAγψβδβ cosmid. This construct, containing the micro-LCR and other essential elements of human beta-globin gene cluster was microinjected into single cell mouse embryos. To detect the differences in developmental regulation of the human gamma-globin gene expression in the transgenic mice, we analyzed the yolk sac derived embryonic blood at embryonic day 10.5 (E10.5) and the fetal liver of mouse embryos at E13.5 of both mutated and non-mutated transgenic mice by RNAse Protection Assay (RPA) and Real time PCR (RT-PCR). Levels of expression of murine alpha-globin mRNA were used as internal controls in the RPA experiment and levels of murine-GAPDH were used in RT-PCR. mRNA levels of human gamma-globin in fetal liver of transgenic mice containing mutation were clearly higher, as compared with control transgenic mice bearing cosmid construct with wild a type sequence gamma promoter. Additionally a nearly 10-fold increase in human gamma-globin expression was detected in fetal livers of HPFH transgenic mice at E13.5 compared to the yolk sac. Thus, our data indicate that the - 195 mutation is the unique cause of elevation of HbF in Brazilian HPFH, but the exact mechanism needs to be clarified.
Automated Sorting of Monocytes from Whole Blood for Reproducible Gene Expression Profiling.
Authors: Poggel Carsten , Adams Timo , Martin Sabine , Pickel Carola , Prahl Nicole , Schmitz Jurgen , Bosio Andreas
Published: April 2020

Microarray-based gene expression profiling has been used to develop clinically relevant molecular classifiers for many different diseases. Furthermore, it has been shown for various chronic diseases that specific gene expression patterns are reflected at the level of blood cells. However, blood is a complex tissue comprising numerous cell types. Therefore, the contribution of rare cell types to a whole blood expression profile might not be detected and a substantial proportion of what is usually reported as “up-regulation” or “down-regulation” might actually be the result of a shift in cell populations and not of a true regulatory process. In order to circumvent these problems, several techniques have been established to analyze purified subpopulations rather than whole blood samples. Previously, it has been shown, for example, that reproducible gene expression profiles can be generated by positive selection of blood cell subsets from PBMCs1. As the preparation of PBMCs by, for example, Ficoll is time-consuming, inconvenient, and not amenable to automation, we have set up a combined direct whole blood cell separation and gene expression profiling protocol. By using Whole Blood CD14 MicroBeads in combination with the autoMACS Pro™ Separator, the separation protocol generally allowed enrichment of monocytes from whole blood within 30 min with purities higher than 90%. In combination with the depletion of neutrophils, the major source of contaminating RNA, purities increased to over 95% for all tested blood donors. Monocytes included the CD14bright/CD16− as well as the CD14dim/CD16+ populations. To assess the reproducibility of gene expression profiles and the influence of several experimental parameters, monocytes were sorted from 5 ml whole blood. RNA was extracted and hybridized to microarrays and the Pearson correlation coefficients of pairwise comparisons were calculated. Technical repeats of monocyte analysis from blood donated at different days showed a higher correlation coefficient than whole blood RNA. Blood storage at room temperature resulted in a strong deregulation of many genes, whereas blood stored at 4°C showed minimal changes, which is in agreement with previous studies. Skipping the centrifugation step, which is used to remove unbound MicroBeads did not alter the gene expression profiles. Incubation of sorted cells in PrepProtect™ Stabilization Buffer showed no alteration of gene expression thus enabling the shipping of cells without liquid nitrogen. Monocytes play a crucial role in diseases like atherosclerosis. Our rapid and simple protocol for combined direct cell sorting from whole blood and gene expression profiling of monocytes might help to ease the discovery of new biomarkers and to screen and monitor patients. 1 Lyons et al., BMC Genomics (2007), 8:64.
Mycophenolic Acid Causes Dysfunction of Human Polymorphonuclear Neutrophils and Dendritic Cells during the Interaction with Aspergillus fumigatus.
Authors: Loeffler Juergen , Mezger Markus , Einsele Hermann
Published: April 2020

Immunosuppressive drugs are a variety of substances to prevent transplant rejection. There is increasing data that immunosuppressants not only modulate lymphocyte function but also interfere with other immune effector cells. In this study, we analyzed the influence of Mycophenolic acid (MPA) on the activity of human polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs) during the interaction with the mold Aspergillus fumigatus. PMNs were obtained from blood using Bicoll separation. Release of reactive oxygen species (ROS) was detected photometrically by using dichlorofluorescein. Monocytes were isolated by magnetic-associated cell sorting followed by differentiation into DCs using GM-CSF and IL-4. DCs were either co-cultivated with MPA (10 μg/ml), with A. fumigatus germ tubes (MOI=1) or both stimuli. Whole-genome microarray analyses (Affymetrix U133A) or quantitative real-time PCR assays were performed to quantify differentially regulated genes. MPA could be identified as an enhancer of the A. fumigatus induced oxidative burst of PMNs while fungal killing efficiency was not affected. Incubation of fully differentiated Mo-derived DCs in the presence of MPA did not alter gene expression patterns. However, if DCs were treated during early differentiation processes, DC development was influenced by MPA resulting in increased apoptosis. In addition, we revealed impaired expression of a variety of pro-inflammatory immune response genes (TNF-α, CXCL10, IL-12). In conclusion, MPA influences PMN and DC function during immune defense against fungi. MPA triggers an acute inflammatory syndrome by enhancing the A. fumigatus induced oxidative burst. Furthermore, innate and adaptive immune responses might be impaired because of a reduced number of DCs and lower levels of pro-inflammatory cytokines and chemokines.
The Role of Nramp1 in Erythrophagocytosis.
Authors: Soe-Lin Shan , Sheftel Alex D. , Ponka Prem
Published: April 2020

Erythrocytes contain the majority of the body’s iron in the form of heme, a component of the red blood cell pigment hemoglobin. Erythrocytes transport oxygen bound to hemoglobin from the lungs to the tissues, and become progressively more damaged as they age. Macrophages are responsible for the detection and degradation of these senescent red blood cells, and play a central role in the recycling of hemoglobin-derived iron via transferrin to the bone marrow in order for erythropoiesis to occur. This recycling process, and the influence of inflammatory mediators on it, are poorly understood. Nramp1 is a protein expressed within macrophages, and is an iron transporter discovered to be mutated in mice susceptible to tuberculosis and leprosy infections. As Nramp1 localizes to phagosomal membranes following phagocytosis, we speculate that Nramp1 may play a role in the recycling of hemoglobin-derived iron following the internalization of effete erythrocytes. To test this possibility, we have utilized a model of erythrophagocytosis using both Nramp1 replete and deficient RAW264.7 murine macrophages, and macrophages elicited from wild type and Nramp1-knockout 129SV mice. We have found that Nramp1 protein is increased in primary macrophages following internalization of opsonized murine erythrocytes. This increase peaks at 6 hours of erythrocyte exposure, and decreases to baseline levels overnight. Our model of erythrophagocytosis did not induce macrophage activation as no nitric oxide production was observed. This therefore discounts the possibility that inflammatory factors are the cause of the increase in Nramp1 protein levels. Using the fluorescent iron-binding probe, calcein, we monitored changes in the macrophage labile iron pool (LIP) following erythrocyte loading. This transit pool of loosely bound, chelator-accessible iron is thought to reflect the overall iron status of the cell. We have found that, following erythrophagocytosis of opsonized murine erythrocytes, Nramp1-replete RAW264.7 macrophages experience a greater-fold increase in labile iron as compared to Nramp1-deficient macrophages. Furthermore, we have also found that primary peritoneal macrophages elicited from wild type 129SV mice phagocytose more erythrocytes when compared to macrophages elicited from their Nramp1-knockout counterparts. Interestingly, we have also observed that treatment with erythropoietin, the hormone responsible for the promotion of erythrocyte precursor survival and ultimately the increase in red blood cell production, resulted in a modest increase in Nramp1 protein levels. Our observations show distinct differences in iron metabolism between Nramp1 replete and deficient macrophages undergoing erythrophagocytosis. We speculate that Nramp1 may serve to promote a more efficient intracellular transport of iron following red blood cell engulfment.
Multi-Scale Analysis of Granulopoiesis: A Bottoms-Up Approach.
Authors: Lee Daniel W. , Thompson Christopher , Obeyesekere Mandri N. , Corey Seth J.
Published: April 2020

Granulopoiesis constitutes a dynamical system, able to respond acutely to internal or external stimuli, with production of neutrophils. Because neutrophils are short-lived (<24 hr) in the peripheral circulation, a major component of bone marrow activity is devoted to their generation. The hematopoietic growth factor, granulocyte colony-stimulating factor (G-CSF), is the most important growth factor for the production of granulocytes. When it or its cognate receptor, the G-CSF Receptor, is genetically ablated in mice, severe neutropenia results. The two most commonly expressed isoforms are the normal full-length G-CSF Receptor (class I) and a truncated, differentiation-defective receptor (class IV). We hypothesized that expression of these two receptor isoforms critically control granulopoiesis. We have developed a quantitative PCR assay and have found that class IV expression is variably upregulated in primary AML and leukemic cell lines. Receptor endocytosis rates differ between class I and class IV, which contributes to proximal phosphoprotein signaling profile (e.g. Lyn, Erk1/2, Akt). Aberrant expression of class IV leads to profound changes in intracellular signaling of both positive and negative effectors, resulting in proliferation without differentiation. Using a multi-scale approach, we have developed a mathematical model containing ordinary differential equations (ODE) to explain the kinetic properties of this dynamical system. This model involves three major steps: probability of receptor dimerization, single cell fate decision making, and population distribution of differentiated and non-differentiated cells. Computer simulation of receptor isoform expression and endocytosis revealed a decreased ratio of class I to class IV over time until steady state (∼120 min). Further validation is underway. This model can be applied to better understand disease states such as congenital neutropenia and acute myeloid leukemia, where aberrant G-CSF Receptor signaling can occur.
Human Meprin Alfa Metalloprotease Is Differentially Expressed in Peripheral Blood Cells.
Authors: Dunham Kimberly , Yura Renee , Bao Lei , Bond Judith , Sun Qi
Published: April 2020

Meprin metaloproteases are highly expressed in the apical brush-border of the intestinal and renal proximal tubule epithelia. However, the expression and function of these metaloproteinease in other tissues have not been well established. Using a specific antisera raised against rat but cross-reactive with human Meprin Alfa, we examined the expression of this protein in human peripheral blood cells. Flow cytometry analysis showed that the post-, but not the pre-, immune sera stained strongly for the CD14+ monocytes and moderately for CD56+ NK cells, but not for CD3+ T and CD19+ B cells. To confirm Meprin Alfa expression at the RNA level, peripheral blood cells were fractionated based on the expression of the above surface markers, followed by detection of Meprin Alfa message RNA by semi-quantitative RT-PCR. Consistent with the flow cytometry data, Meprin Alfa messages were detected only in cell fractions containing monocytes and NK cells but not T and B cells. Subsequent sequencing of the PCR amplified fragments confirmed the specificity of the PCR amplification. Interestingly, flow cytometry analysis further revealed that while the expression of Meprin Alfa was maintained in the macrophages differentiated in vitro from isolated monocytes, its expression was reduced in monocytes-derived immature dendritic cells (DC) and diminished in mature DC. The differential expression of Meprin Alfa in the peripheral blood cells suggests important physiological function of this protein in the immune system.
Plasma Cell Dyscrasia in Human Immunodefficiency Disease.
Authors: Jajeh Ahmad , Nukala Suresh
Published: April 2020

Plasmacytosis is commonly seen in the bone marrow biopsies of patients with acquired immunodefficiency disease AIDS and human HIV virus infection. Polyclonal increase of immunoglobulin is very common. However few cases of plasma cell dyscrasia are reported. The purpose of this abstract is to show our institution experience with plasma cell dyscrasia and HIV infection. For the last ten years ten patients were identified based on bone marrow biopsy, immunoelectophoresis and immunofixation, twenty four hours urine collection and urine electrophoresis and immunofixation. Immuno staining for kappa and lambda light chain to check for clonality were done on four cases. Mean age of patients is 49 years (range 33 to 59 years). Seven were black, two hispanic and one white. Nine were males and one female. All patients had plasmacytosis on bone marrow more than 30% and protiurea of more than one gram. five patients had nephrotic range protien urea. one patient had IgA lambda, one patient had Ig G kappa All patients had low CD4 counts except two patients with CD4 above 200 and have long standing HIV infection. One patient presented with hepatosplenomegally and increased IgM level and waldnestrome looking plasmacytoid lymphocytes. One patient had plasmablast and plasma cells syncitia who had rapidly fatal disease. one patient expired of respiratory failure before treatment. One patient, a female presented with plasma cell leukemia and did respond to Doxil, vincristine and dexamethasone therapy. She is alife. Two other patients with long standing HIV infection and CD4 count above 200 are still alife both patients were treated initially with Thalidomide and dexamethasone with excellent response. one of them failed Thalidomide after more than one year response and currently on his fourth cycle of velcaid (proteosome inhibitor). All patients were not on HIV therapy at time of diagnosis. In conclusion plasma cell dyscrasia with HIV infection do respond to treatment with durable response. Repetetive antigenic stimulation with HIV-1 p24 antigen are the proposed mechnism of oncogenesis.
Emerging Epidemics of Babesiosis in Hematology Consultation at a University Hospital.
Authors: Hosur Srilatha , Liu Delong , Seiter Karen , Nelson John , Aguero-Rosenfeld Maria , Brodsky Sergey , Ahmed Tauseef , Wormser Gary
Published: April 2020

Babesiosis was first reported in NY in 1975. 560 cases were reported from 1986–2001 in NY, mostly from Long island. Human babesiosis is a tick- borne disease caused by B.microti in the US. Most of the cases occur between May and August in the north eastern states. The risk factors for severe disease include older age, asplenia, immunocompromised state. As the same tick is the vector for lyme disease and ehrlichiosis, co-infection can occur. In this abstract we report ten cases of babesiosis encountered in hematology consultation since 2005, with 5 cases occurring this year. The patients’ ages ranged between 36–84 yrs. Three patients had neither travel history nor tick bite /outdoor activity. Three patients were immunocompromised (AML, CLL, HIV). Most common presentations were malaise, nausea, and high fever. Two patients had splenomegaly. Six patients had hyperbilirubinemia, anemia and thrombocytopenia. Three patients were also positive for lyme disease. One patient developed splenic rupture. All the patients recovered after treatment with atovoquone and azithromycin. This series of case reports emphasizes the varied atypical presentations of babesioisis including uncommon season of occurrence, absence of a rash or tickbite history, co-existing infection with Lyme disease and complications including splenic rupture.
Effect of Rituximab and/or High-Dose Therapy with Autotransplant at Time of Relapse in Patients with Follicular Lymphoma.
Authors: Coiffier Bertrand , Brice Pauline , Delarue Richard , Haioun Corinne , Souleau Bertrand , Mounier Nicolas , Feugier Pierre , Tilly Hervé , Sebban Catherine
Published: April 2020

Follicular lymphoma (FL) is considered as an incurable disease but treatments have changed with the introduction of high-dose therapy with autologous stem cell transplant (HDT) then of rituximab (RTX). The effect of these 2 strategies on the outcome of FL patients is well described in first line but not for relapsing patients. We analyzed 2 cohorts of patients treated in 2 successive randomized studies with the same treatment, CHVP plus interferon, to evaluate the role of RTX and HDT in first disease progression or relapse. Of the 364 patients <61 years old included in these 2 studies, 255 progressed or relapsed and constitute the analyzed population. Among them, 93 had been treated with HDT, 63 with RTX alone or combined with chemotherapy, and 30 with RTX followed by HDT. Characteristics of the patients were similar in all subgroups. If event-free survival (EFS) was similar in patients treated within GELF-86 or GELF-94 studies(10-year EFS of 20% and 23%), overall survival (OS) was longer in GELF-94 study (10-year OS of 66% compared to 48%, p=0.014) witnessing a change in the management of relapsing patients. HDT was associated with a statistically significant benefit in terms of event-free survival from relapse (EFSR) and survival after relapse (SAR), p=0.004 and p=0.01, respectively (Table). Rituximab during salvage therapy was associated with a larger benefit than HDT for these 2 endpoints: p<0.0001 and p<0.0001, respectively. When both treatments were combined, 5-year SAR is over 90% (Table). In follicular lymphoma patients treated with chemotherapy in first line, the combination of salvage regimen containing rituximab followed by HDT seems to be superior to rituximab-containing chemotherapy alone. These retrospective results must be confirmed within a prospective study.

Salvage without rituximab without HDT (A) Salvage without rituximab with HDT (B) Salvage with rituximab without HDT (C) Salvage with rituximab * p values between A and C =0.0002 and <0.0001 for EFSR and SAR, respectively; p values between B and D <0.05 and =0.01 for EFSR and SAR, respectively Patients 87 59 29 30 5-year  EFSR 24% (15–34) 44% (31–56) 42% (21–62) 67% (42–86) 5-year  SAR 32% (22–42) 62% (45–73) 70% (42–86) 92% (71–98) Figure Figure
Study on Dendritic Cells in Peripheral Blood of the Patients with Autoimmune Hemolytic Anemia/Evans Syndromes.
Authors: Shao Zonghong , Xing Limin , Wang Huaquan , Tu Meifeng , Fu Rong
Published: April 2020

Objective To study the alteration of the numbers and the subsets of DCs in perpheral blood of the patients with AIHA/Evans syndromes and also the costimulation molecules on the surface of DCs.

Methods Labeled with three-color immune monoclonal antibodies, total DC, pDC and mDC in the peripheral blood (PB) of 14 cases with AIHA and 21 normal controls were detected by Facs. The expressions of CD80, CD86 andCD40 on DCs in the PB of 14 cases with AIHA/Evans syndomes and 6 normal controls were detected.

Results The percentage of the total DCs in the PB (DC/PBMNC) of AIHA/Evans syndromes cases((11.41±3.91)%)was significantly higher than that of in the normal controls((1.95±0.66)%) (t=2.384, p=0.032).mDC in the PB of the AIHA/Evans syndromes patients ((10.29±3.90)%) was significantly higher than that of controls((1.26±0.46)%)(t=2.297, p=0.038). But pDC in the PB of AIHA/Evans cases((1.12±0.68)%)was not different from that of controls((0.68±0.23)%)(t=0.612, p=0.55). The proportion of pDC to DC in the PB of the patients((21.26±5.99)%)was significant lower than that of normal controls((40±3.14)%)(t=2.77, p=0.012). Whereas, the proportion of mDC to DC in the PB of the patients((75.93±6.79)%)was higher than that of normal controls((59.99±3.23)%)(t=2.77, p=0.012). mDC in the PB of normal controls((1.26±0.46)%) was not different from their pDC((0.68±0.23)%). mDC of the patient with AIHA/Evans cases((10.29±3.90)%) was higher than their pDC((1.12±0.68)%)(t=2.316, p=0.036). CD80, CD86 and CD40 expressions on DCs in PB of AIHA/Evans syndromes patients were (7.63±4.71)%, (13.55±4.57)% and (1.68±1.04)% respectively. Those of normal controls were (1.24±0.70)%, (3.21±1.55)% and (0.28±0.16)% respectively. There was a significant difference of the expression of CD86 between the patients and control groups (t=2.142, p=0.048).

Conclusions DCs, particularly mDCs, in the PB of AIHA/Evans syndromes patients significantly increased. The patients with AIHA/Evans syndromes have more activated DCs in their PB, on which costimulatory molecules, mainly CD86 are over expressed.
Generation of Influenza HA-Specific Lymphocytes from Autologous Umbilical Cord Blood-Derived Dendritic Cells.
Authors: Decker William K. , Li Sufang , Xing Dongxia , Robinson Simon N. , Yang Hong , Steiner David , Bollard Catherine M. , Shpall Elizabeth J. , Safdar Amar
Published: April 2020

INTRODUCTION: Umbilical cord blood transplantation (CBT) is being performed with increasing frequency for patients lacking HLA-matched donors. CBT is associated with delayed immune reconstitution and high rates of viral infections. Particularly problematic is the reactivation of latent viral infections such as CMV, EBV, and VZV, as is overt infection from common agents like adenovirus and influenza. To address these issues, we proposed to generate influenza-specific CB T-cells ex vivo for potential cell therapy applications, allowing for effective therapeutic interventions until such time that immune reconstitution has occurred.

METHODS: Dendritic cells (DC) were generated from both the adherent and the CD14+ CB fractions cultured in AIM-V medium supplemented with human serum, GM-CSF, and IL-4. Antigen loading was performed by incubation with recombinant, purified A/New Caledonia HA antigen. Maturation was performed with the ITIP inflammatory cocktail (IL-1β, TNF-α, IL-6, and PGE2). Autologous CB lymphocytes were co-cultured with mature DC at a ratio of 10:1 for 4 days in IL-12 after which they were expanded in a cytokine cocktail of IL-2, IL-7, IL-12, and IL-15. Lymphocytes were restimulated similarly every 7-10 days. Antigen specificity was demonstrated by IFN-γ ELISpot.

RESULTS: Mature DC expressed the appropriate markers including CD80, CD83, CD86, CD209, and HLA-DR. Some DC expressed CD14, others CD11c, an indication of a mixed interstitial/Langerhans myeloid phenotype. We demonstrated a 4–10 fold enhancement of IFN-γ ELISpots when primed lymphocytes were incubated with HA-loaded DC in comparison to autologous unloaded DC (p<0.003) or loaded allogeneic DC (p<0.0001). The CD8 content of the HA-specific lines was typically greater than 50% after two stimulations. At the time of phenotyping CD3+ cells were primarily CD62LposCD197(CCR7)neg and CD127(IL-7Rα)neg, an effector phenotype; however, 6–22% of cells expressed CD127 (effector or central memory), and up to 6% of cells were CD62LposCD197pos (central memory).

CONCLUSIONS: The results demonstrate that, despite the generally naïve nature of CB lymphocytes, influenza-specific responses can be generated ex vivo, and could potentially be used clinically in CBT patients who develop infectious complications prior to immune reconstitution.
European MCL Network: An Update on Current First Line Trials.
Authors: Dreyling Martin H. , Hoster Eva , Hermine Olivier , Kluin-Nelemans J.C. , Walewski Jan , Trneny Marek , Geisler Christian H. , Unterhalt Michael , Hiddemann Wolfgang
Published: April 2020

Background: Conventional chemotherapy achieves only short term remission despite high initial response rates of 70%–80%. In the current study generation, the European MCL Network investigates the impact of various combined immuno-chemotherapy regimens. Additionally, in elderly patients the role of rituximab maintenance is being evaluated, whereas in younger patients dose-intensified regimens with implementation of high dose cytarabine are investigated based on the excellent results of the HyperCVAD regimen.

Methods: In MCL elderly, patients are initially randomized between 8 cycles of R-CHOP or 6 cycles of R-FC (experimental arm). Patients who achieve either an PR or CR, receive subsequently either interferon maintenance (standard arm) or a single rituximab dose every 2 months. In MCL younger, the standard arm (R-CHOP induction followed by myeloablative consolidation: 12 Gray TBI, 2x 60mg/kg cyclophosphamide) is compared to the implementation of high dose cytarabine into induction (R-CHOP/R-DHAP) and consolidation (10 Gray TBI, 4x1,5 g/m2 Ara-C, 140mg/m2 melphalan).

Results: In MCL elderly, 222 of 263 patients were evaluable based on the annual interim analysis. Median age was 70 years with 66% of patients displaying an intermediate high/high risk IPI. Induction was well tolerated with mainly hematological toxicity (grade III/IV in R-CHOP/R-FC): Leukocytopenia 62/72%, thrombocytopenia 13%/40%, but only rare febrile neutropenia (23%/7%) or infections (19%/23%). Despite the poor risk profile, combined immuno-chemotherapy (total group) achieved a remarkable 84% response rate (51% CR/CRu). Although the impact of maintenance is not yet evaluable, both progression-free and overall survival were encouraging with 77% and 86% at 12 months, respectively. In MCL younger, 247 of 271 patients were evaluable. Again, toxicity (grade III/IV in R-CHOP/alternating R-DHAP) was mainly hematological: leukocytopenia 58/77%, thrombocytopenia 14%/74%, but only rare febrile neutropenia (11%/22%) or infections (5%/7%). Combined immuno-chemotherapy achieved a 93% response rate (60% CR/CRu) before subsequent high dose consolidation. Again, both progression-free and overall survival are remarkable with both 90% at 12 months, respectively.

Discussion: Combined immuno-chemotherapy results in high response rates in two prospective international trials. Further recruitment and follow-up will determine the role of rituximab maintenance and high dose cytarabine in this distinct subtype of malignant lymphoma.
Identification of Transcriptional Targets of IRF8 in Germinal Center B Cells.
Authors: Shin Dong-Mi , Lee Chang Hoon , Abbasi Sadia , Morse Herbert
Published: April 2020

The development and function of cells of the innate and adaptive immune systems are governed by a series of hierarchical regulatory networks. The components of these networks are particularly well understood for the very early as well as the terminal stages of B cell differentiation; however, information regarding the set of regulatory factors governing B cell fate from the pro-B through the germinal center (GC) stage of differentiation is less well developed. A previous study identified IRF8 as an important component of the network as it contributes to the direct transcriptional activation of BCL6 and AID, key elements of the GC reaction. To identify additional targets of IRF8, we used the chromatin immunoprecipitation (ChIP)-on-Chip approach to study three human cell lines of GC origin - Ly1, Odh1 and Val - and the multiple myeloma cell line, MMS1. By coupling this study with ChIP-on-chip analysis for POLII using the same cell lines, we identified 248 transcriptionally active IRF8 targets. They comprised both known targets, such as B2M and ISGF3G, and a spectrum of novel targets that included important elements of innate immune responses such as RIG-1 and MDA-5. A functional classification of IRF8 targets by GO and a comparison of the GO term frequency with the global frequency in the human genome showed that IRF8 targets were significantly enriched in genes regulating the immune response, effecting DNA repair, and governing transcription. These results significantly enhance our understanding of the roles played by IRF8 in the transcriptional regulation of GC B cells, and identify innate immune mechanisms that contribute to their adaptive immune functions.
Characterization of Immune Reconstitution Following High Dose Chemotherapy in Acute Myeloid Leukemia.
Authors: Meyer Christian F. , Thoburn Christopher , Kos Ferdynand , Gocke Christopher , Levitsky Hyam I. , Briel Janet A. , Hess Allan , Karp Judith E.
Published: April 2020

Recovery of immune function after initial treatment of acute myeloid leukemia (AML) is critical, not only for protection against infections but also for surveillance against recurrent disease. A better understanding of the nature of lymphocyte recovery following induction and consolidation therapy for AML could guide the design of immunotherapy strategies aimed at boosting the anti-leukemia activity of a reconstituted immune system. Prior studies examining thymic T cell production following bone marrow transplantation (BMT) have found varying levels of thymic output post-transplant, as measured by T cell Receptor Excision Circle (TREC) levels in the peripheral blood of adult patients. Of note, relapse of chronic myeloid leukemia (CML) following BMT is correlated with decreased levels of TREC positive T cells. In order to characterize immune reconstitution in AML, we studied 26 patients after induction or consolidation time sequential chemotherapy. Their median age was 52 (range 23–69). Thirteen patients received cytarabine, daunorubicin, and etoposide (AcDVP-16) induction therapy, 3 patients received cytarabine, daunorubicin, and cytarabine (AcDAc) consolidation therapy and 10 patients received flavopiridol, cytarabine, and mitoxantrone (FLAM) either as induction or consolidation therapy. Peripheral blood samples were collected approximately every other day for 3–5 time points after each patient’s white blood cell count exceeded 200 cells/cubic mm on three consecutive days. Among the four patients evaluated to date, flow cytometry results show that a majority of cells seen early in immune reconstitution are CD3+ lymphocytes (range 69–97%). Subset analyses on 3 of these 4 patients have shown CD4:CD8 ratios ranging from 3:1 to 4:1, while the fourth patient exhibited an inverse of this ratio at 1:5. In addition, CD25+FOXP3+ T cells represented a median of 5.1% (range 2.5–12.3%) of the CD3+ T cells. Since T cells represented the abundance of cells in the peripheral blood during early bone marrow recovery, we then assessed whether these cells represented recent thymic emigrants or naïve T cells by examining TRECs using real time PCR (RT-PCR). TRECs were present in 24 of the 26 patients with levels ranging between 100 and 100,000 copies per 100,000 cells. Furthermore, 4 control samples from normal volunteers (ages 37–43) revealed the absence of TREC positive cells. Further analyses of these time points and correlations between TREC levels and clinical responses are ongoing.
Proteomics in the Study of Qualitative Platelet Defects: Validation of the Approach in the Gray Platelet Syndrome and Quebec Platelet Disorder.
Authors: Kasthuri Raj S. , Anderson Lorraine B. , Higgins LeeAnn , Di Paola Jorge , Rivard Georges E. , Hayward Catherine P.M. , Key Nigel S.
Published: April 2020

Qualitative platelet defects are a heterogeneous group of disorders characterized by abnormal platelet function. Currently available laboratory assays lack sensitivity and specificity for detecting Storage Pool and Platelet Secretion defects. The Gray Platelet Syndrome (GPS) is an autosomal recessive disorder characterized by a decrease or absence of alpha granule contents. The Quebec Platelet Disorder (QPD) results from proteolysis of alpha granule proteins due to increased amounts of platelet urokinase. A decrease or deficiency of multimerin is characteristic. Proteomics is a rapidly developing field with great promise in the study of pathophysiology and biomarker development. Recent advances have made measurement of relative protein abundance in multiple samples feasible. We used one such approach, “isotope Tagging for Relative and Absolute Quantitation” (iTRAQ) to characterize the platelet proteome of healthy volunteers and patients with GPS and QPD. iTRAQ allows analysis of up to 4 different samples simultaneously, and differences in protein abundance as low as 20% are reliably detected. The platelet proteomes of 4 healthy volunteers were analyzed. Platelet proteome analysis of study patients was as follows;

2 GPS patients compared to 2 controls; 2 heterozygous GPS patients compared to a homozygote and a control; and 3 QPD patients compared to a control.

For comparison, platelet proteins were grouped by location into 3 groups - receptor, alpha granule and cytoskeletal proteins. The results are shown in the figure. Protein abundance in healthy volunteers was very consistent, with inter-individual variability ≤ 20%. Distribution of receptor and cytoskeletal proteins in GPS patients mirrored the controls, but alpha granule proteins were significantly decreased as expected. Comparison of homozygous to heterozygous GPS platelets showed this to be true only in the homozygotes, again, as expected (figure inset). Variable decreases in alpha granule proteins were observed in QPD patients but this was less profound. Multimerin was detected in QPD patients, but at levels significantly lower than controls. While urokinase was not detected in QPD platelets in this study, it was detected in a parallel study evaluating platelet releasates. The iTRAQ technique was reproducible and yielded consistent results, but currently is a relatively expensive and time-consuming approach. In summary, our results suggest that the platelet proteome is tightly regulated and very stable in healthy individuals. A significant decrease in alpha granule proteins was noted in patients with GPS but this was less remarkable in the QPD group, probably because peptides resulting from urokinase-mediated proteolysis are still labeled and quantified.

Figure Figure
Platelet-Derived Microparticles Induce Angiogenesis In Vitro and In Vivo.
Authors: Chen Bao-An , Zhong Yue-Jiao , Huang Cheng-Yin , Gao Feng , Chen Jian , Gao Chong , Sun Yun-Yu , Wang Jun , Zhao Gang , Ding Jia-Hua , Chen Ning-Na , Pei Xiao-Ping
Published: April 2020

Objective: An attempt was made to investigate the effect of platelet-derived microparticles (PMPs) on the angiogenesis.

Methods: Thrombin was adopted to activate the platelets to release PMPs. Flow cytometry(FCM)was adopted to evaluate the efficiencies of different concentrations of thrombin to produce PMPs and BCA was adopted to evaluate the content of PMPs. By the carrier of human umbilical vein cell line ECV-304 cultivated in vitro, investigate the effect of PMPs on the proliferation and apoptosis of human umbilical vein endothelial cells using MTT and FCM. PMPs were put into CAM and observe the effects of PMPs on angiogenesis in Chick embryo chorioallantoic membrane (CAM).

Results: The efficiencies of PMPs activated by 1.0U/ml thrombin were 50.1%; PMPs induced proliferation of human umbilical vein cell line ECV-304 in a dose dependent manner. At the concentration of 40ug/ml PMPs, the proliferation rate of human umbilical vein cell line ECV-304 was as 1.8±0.3 times as control and there was no difference with the group of vascular endothelial growth factor (VEGF),which the proliferation rate was 1.9±0.5 times vs. control, p > 0.05;PMPs inhibited human umbilical vein cell line ECV-304 apoptosis. Compared with control group (apoptosis rate 9.4%±0.5%), apoptosis rate of PMPs (40μg/ml) is 3.9%±0.4%, which was significantly reduced, p<0.05. The addition of VEGF (10μl/ml) did not successfully prevented apoptosis of human umbilical vein cell line ECV-304 (apoptosis rate 8.0%±0.8%);After 72 h of incubation, showing an implant of PMPs by allantoic vessels developing radially towards it (implant) in a ’spoked-wheel’ pattern, at the concentration of 80μg/ml PMPs, number of vessel ramification is 112.5±11.31 and vessel area/CAM area is (6.19±1.29)%, compared with the VEGF(p>0.05). But there are not localized allantoic vessels developing in the NS control group(P<0.05).

Conclusion: 1.0U/ml thrombin activated platelet could get the best efficiency of PMPs, which could stimulate proliferation of human umbilical vein cell line ECV-304 and inhibit its apoptosis and PMPs have certain promotive effect on the formation of capillary in chick chorioallantoic membranes.