bluuebunny/crossref_metadata_2025 huggingface dataset:
Nodular Lymphocyte Predominant Hodgkin Lymphoma. Experience in a Single Institution in Argentina
Authors: Alfaro Elizabeth M , Sanchez La Rosa Cristian G , Guitter Myriam , Millan Natalia , Montanari Andrea , Galluzzo Laura , Felice Maria Sara , Zubizarreta Pedro
Published: October 2019

Background: Nodular Lymphocyte Predominant Hodgkin Lymphoma (NLPHL) is a rare subtype of Hodgkin Lymphoma (HL) that represents approximately 5% of HL and discloses a distinct clinical-pathological features with an overall good prognosis. Treatment strategies have been based on protocols for HL. Selected patients can be treated with surgery alone.

Objective: Our aim was to describe the clinical characteristics, management and outcome of patients with NLPH.

Methods: Between March 2000 and June 2016, 16 patients (14 M/2 F), median age: 9.8 (range 4.9-14.5) years, were consecutively diagnosed in our institution. Initial chemotherapy schedules were ABVD (Doxorubicin, Bleomycin, Vinblastine and Dacarbazine), COPP/ABVD or AV-PC (Adriamycin/Vincristine/Prednisone/Cyclophosphamide) were administered to 14 patients. When response was partial or null, additional chemotherapy was administered, IF-RT, Anti CD20 and/or HDC/Autologous SCT. A watch-and-wait strategy after lymph node complete surgery was used in two cases when this strategy became trustworthy.

Results: Fourteen patients (87.5%) showed nodal compromise being cervical localization the most frequent localization. Thirteen patients (81.2%) presented localized disease (stage IA: 8, stage IB: 1, stage IIA:4) and 3 (18.7%) advanced disease (stage IIIA:1, stage IVA:1, IVB:1). Two patients had B symptoms. Two IA stage cases (12.5%) had complete node resection. Five patients (31.2%) received 4 cycles of ABVD. One patient (6.2%) received COPP/ABVD. Five patients (31,2%) received 3 cycles of AV-PC. One patient (6.2%) received 6 cycles of AV-PC plus 4 doses of Rituximab. One patient (6.2%) presented progressive disease after 3 cycles of ABVD, additional chemotherapy was administered and HDC/Autologous SCT. Three (18%) patients received IF-RT plus chemotherapy. One patient is still on treatment. Response to treatment was: CR in 12 pts (75%), Partial-response: 2 pts (12.5%), NR: 1 pts (6.2%). Median follow up: 58.4 (range 2-89) months. All patients remain in CR. Conclusion: Although the management of NLPHL was not uniform in our retrospective analysis, the results were promising. Watch and wait strategy plays an important role to avoid adverse effects associated with therapy. Tailored therapy according to staging and disease response, seemed to be a good strategy in our setting.

Disclosures

No relevant conflicts of interest to declare.
Pharmacogenetic Markers of Treatment Response of Imatinib Mesylate in Chronic Myeloid Leukemia Brazilian Patients
Authors: Vieira Natália M , Leite Renata , Reginato Fabíola , Zandoná Marília , Vanelli Costa Tito , Fogliatto Laura , Linden Rafael , Venzon Antunes Marina , Capron Arnaud , Wallemacq Pierre , Wagner Sandrine , Mattevi Vanessa , Capra Marcelo
Published: October 2019

The treatment of chronic myeloid leukemia (CML) was revolutionized by the introduction of imatinib mesylate (IM). However, approximately 20% of patients are non-responsive and interpatient variability in response to IM is still a phenomenon lacking explanation. Single nucleotide polymorphisms (SNPs) located in genes encoding proteins involved in IM pharmacokinetics are potentially involved in the causes of this variation. In this study, we investigated the association of SNPs in the genes encoding IM metabolizing enzymes CYP3A4 (rs35599367 and rs2740574) and CYP3A5 (rs776746) and efflux transporter proteins ABCB1 (rs3213619, rs1128503, rs2032582 and rs1045642), ABCG2 (rs2231142) and ABCC4 (rs9561765) with response to treatment and with IM plasma through levels and hair concentrations. The analyzed sample was constituted of 182 CML patients on IM treatment. DNA samples were genotyped for the nine SNPs using real-time polymerase chain reaction. Hair and plasma trough IM concentrations were measured through liquid chromatography coupled to mass spectrometry methods. Clinical response was defined according to the European Leukemia Net guidelines. The number of responders to standard IM therapy was 104. A trend to a higher frequency of CYP3A4 *22 (rs35599367) allele carriers was observed among responders to IM (12.5 vs. 3.4% of non-responders, P = 0.087), although no significant differences in plasma or hair concentrations between genotypes were found. Pharmacogenetics may become a valuable approach to optimize therapy with IM in CML, but many factors still need to be clarified to make possible its application in clinical practice.

Disclosures

No relevant conflicts of interest to declare.
Germline Calr Mutation and Thrombocytosis Presenting with Concomitant BCR-ABL1+ CML
Authors: Baughn Linda B. , Gilles Scott , Courville Elizabeth L. , Nelson Andrew C. , Sachs Zohar
Published: October 2019

CALR mutations are present in 70-84% of JAK2 wild-type myeloproliferative neoplasms (MPN) and 67% and 88% of essential thrombocytopenia (ET) and primary myelofibrosis (PMF) respectively. Most cases of MPN are apparently sporadic, but 7-11% have evidence of familial predisposition. While germline mutations in ET-associated genes, MPL and JAK2, have been described in hereditary thrombocytosis, germline mutations in CALR have not been described in any setting. Two types of CALR mutations are common in MPN: a 52-base pair deletion (bp) and a 5 bp insertion, both in exon 9. With rare exceptions, CALR mutations are generally mutually exclusive with JAK2 or MPL mutations and have very rarely been reported in conjunction with the BCR-ABL1translocation.

Here, we report a patient with a germline CALR mutation, thrombocytosis, and subsequent development of BCR-ABL+ CML. A 67-year-old female with no significant medical history presented with severe abdominal pain and nausea. Peripheral blood analysis revealed a marked leukocytosis composed of 66% neutrophils, 16% myelocytes, 6.5% monocytes, 3.5% basophils, 2.5% promyelocytes, 2.5% metamyelocytes, 1.5% lymphocytes, 1.5% blasts, and no eosinophils. The patient was non-anemic and had a normal platelet count (340,000/mm3). Bone marrow biopsy revealed a hypercellular marrow with myeloid predominant trilineage hematopoiesis and 1-2% blasts with morphology consistent with chronic myelogenous leukemia (CML). Fluorescence in-situ hybridization analysis of peripheral blood identified a BCR-ABL1fusion in 98.5% of interphase cells.

After 3 months of standard imatinib therapy, quantitative RT-PCR showed a reduction of BCR-ABL1/ABL1 in the peripheral blood, however platelet count was elevated at 539,000/mm3. Thrombocytosis persisted over 2 years with a maximal platelet count of 584,000/mm3. Given the patient's thrombocytosis, her peripheral blood was subjected to a next generation sequencing of JAK2, MPL, and CALR genes. A 52-bp out-of-frame deletion in exon 9 of the CALR gene was detected (52% allele frequency) in peripheral blood. In addition, the same 52-bp CALR deletion (63% allele frequency) was present at the time of diagnosis and within a buccal specimen (47% allele frequency) when the BCR-ABL1 transcript was 1% in the peripheral blood. Immunostain of the buccal sample was strongly positive for cytokeratin (CK) AE1/AE3 but CD45 was not detected indicating no leukocyte contamination. This case reports the first instance of a germline CALR mutation associated with thrombocytosis and is the fourth report of the co-occurrence of BCR-ABL1 and CALR mutation in a single patient. Evolution to BCR-ABL1+ CML suppressed the CALR-mutant thrombocytosis phenotype, emphasizing the effect of these genes on lineage determination in abnormal myeloid proliferation.

Disclosures

No relevant conflicts of interest to declare.
Luspatercept Response in ESA-NaïVe/RS+ Patients and RS- Patients with Low-Intermediate Risk Myelodysplastic Syndromes (MDS)
Authors: Platzbecker Uwe , Germing Ulrich , Götze Katharina , Kiewe Philipp , Wolff Thomas , Mayer Karin , Chromik Joerg , Radsak Markus , Donovan Eileen , Wilson Dawn M. , Zhang Xiaosha , Laadem Abderrahmane , Sherman Matthew L. , Attie Kenneth M. , Giagounidis Aristoteles
Published: October 2019

Background: Management of anemia is a common therapeutic challenge in patients with myelodysplastic syndromes (MDS). Luspatercept (ACE-536), a fusion protein containing modified activin receptor type IIB, is being developed for treatment of anemia in lower-risk MDS. Luspatercept binds GDF11 and other TGF-β superfamily ligands to promote late-stage erythroid differentiation and increase hemoglobin (Hgb) levels (Suragani R, Nat Med, 2014 and Attie K, Am J Hematol, 2014).

Aims: This is an ongoing, phase 2, multicenter, open-label study to evaluate the effects of luspatercept in patient (pts) with low-intermediate risk MDS. Endpoints included erythroid response (IWG HI-E), RBC transfusion independence (RBC-TI, ≥ 8 weeks), duration of HI-E, pharmacodynamic and iron metabolism biomarkers, safety, and pt-reported QoL.

Methods: Inclusion criteria included age ≥ 18 yr, Hgb < 10 g/dL (if < 4U RBC/8 weeks), no prior HMA, and no current lenalidomide or erythropoiesis-stimulating agent (ESA). An expansion cohort of up to 56 patients was added to this phase 2 study to evaluate response to luspatercept in pts who do not qualify for the phase 3 MEDALIST trial (for RS+ positive patients with baseline EPO ≥ 200 U/L and ≥ 2U RBC/8 weeks). These include pts with low transfusion burden (< 4U RBC/8 weeks) who are either 1) ring sideroblast (RS)+ (≥ 15% RS in bone marrow) with baseline EPO ≤ 200 U/L and no prior ESA use, or 2) RS- with any baseline EPO level and any prior ESA use. Patients are treated with 1.0 mg/kg of luspatercept every 3 weeks for up to 5 doses, with titration up to 1.75 mg/kg. Patients may rollover to an open-label extension study for up to an additional 2 years of treatment.

Results: Results for the initial patient cohorts have demonstrated a high proportion of HI-E and RBC-TI responses in RS+ patients. Data for the additional ESA-naïve RS+ patients with low EPO levels and RS- patients with 3 months of treatment will be presented at the meeting.

Conclusions: Erythroid response to luspatercept has been demonstrated in RS+ patients with lower-risk MDS and is being explored in ESA-naïve RS+ patients with low EPO levels and RS- patients. A Phase 3 study of luspatercept in regularly-transfused RS+ patients with lower-risk MDS according to IPSS-R is ongoing (MEDALIST study; NCT02631070).

Disclosures

Donovan: Acceleron Pharma: Employment. Wilson:Acceleron Pharma: Employment, Equity Ownership. Zhang:Acceleron Pharma: Employment. Laadem:Celgene Corporation: Employment, Equity Ownership. Sherman:Acceleron Pharma: Employment, Equity Ownership, Patents & Royalties. Attie:Acceleron Pharma: Employment, Equity Ownership. Giagounidis:Celgene Corporation: Consultancy.
Long-Term Use of Azacitidine in Private Practice in Brazil: An Analysis of Patients Profiles
Authors: Centrone Renato , Garcia Laura Yolanda Chialanza , Santucci Rodrigo , Bellesso Marcelo
Published: October 2019

The AZA-001 study showed that azacitidine has a significant role in prolonging overall survival (OS) in High-Risk Myelodysplastic Syndromes (HR-MDS), particularly in patients with unfavorable cytogenetics, even in the subset of patients without any recorded response except progression. Otherwise, the benefit of azacitidine treatment in OS of HRMDS shows a lack of improvement in survival over the years in most of the patients in a Multicentric Spanish Study Group.

Since in our institution we had 34 patients who received Azacitidine for HR-MDS and 20-30% blast Acute Myelogenous Leukemia (AML) during last 36 months, we performed a retrospective analysis of our current long-term treatment patients currently taking Azacitidine, to understand their characteristics. We used WHO classification, IPSS- R score, bone marrow (aspirate) blast count, conventional cytogenetics-based risk, performance status (ECOG) and transfusional dependence.

We studied 11 patients with MDS who received at least 06 cycles of Azacitidine,(75 mg/ m2 D1 to D7) until data cut on 1st August 2016. The median age was 71 (range 46-84 years) and 72 % of the patients are male. WHO 2008 classification before beginning of treatment was: 3 AML (2 former RAEB-2 with recent progression), 03 Refractory Anemia with Excess Blasts-2 (RAEB-2), 02 Refractory Anemia with Excess Blasts-1 (RAEB-1), one Refractory Cytopenia with Multilineage Dysplasia (RCMLD) and 02 Refractory Anemia (RA). All but one patient had "good" cytogenetic risk and IPSS-R ranged from 2 to 8,5 (low to high risk), median 4,7 points.

Regarding treatment, patients received from 6 to 31 cycles of Azacitidine, with responses according to IWG including one complete response (CR), 03 partial responses (PR), 06 stable diseases (SD) and one Hematological improvement (HI). Transfusion dependence was present at the beginning of treatment in 07 cases, and no change in this parameter happened until those patient's last visit. Performance Status was very good (ECOG=0) in 06 patients before the first dose of azacitidine and good (ECOG-1) in the remaining subjects. At data cut, we had 09 patients with ECOG=0 in the group.

This analysis showed that patients had no real improvement of disease parameters since 07 of 11 did not achieve even a partial response, and the same number of patients stay as transfusion-dependents. However, even the worse IPSS-R classified patients had at least a better quality of life and they survived at least one year with treatment. The main finding of this evaluation is that patients who achieve a response other than progression or refractoriness have a good cytogenetic risk, most of them with normal Karyotype, confirming literature.

Table Table.

Disclosures

No relevant conflicts of interest to declare.
Functional Consequences of Spliceosome Mutations
Authors: Halene Stephanie
Published: October 2019

Mutations in key factors of the spliceosome occur in over 50% of patients with myelodysplastic syndromes (MDS) and thus splicing represents the most frequently affected pathway in MDS. The vast majority of mutations cluster in four genes: SF3B1, SRSF2, U2AF1, and ZRSR2. Mutations occur in a mutually exclusive manner suggesting a common mechanism by which they cause disease, and yet they segregate to specific subtypes of myeloid malignancies suggesting unique, mutation-specific functions. With the exclusion of ZRSR2, mutations occur at highly restricted residues and are strictly heterozygous, suggestive of gain of function mutations. We and others have shown that mutations affect RNA binding affinity and specificity, and can lead to a variety of outcomes including exon inclusion or skipping, alternative splice site selection, intron retention, and most recently alternative polyadenylation. Overall, these splice changes are subtle: only a small subset of all splice events is affected by these mutations and changes in isoform usage are < 10% for most alternative splice events identified. Furthermore, a direct connection between alternatively spliced transcripts and differentially expressed genes is often not seen. However, these alternative splice events affect genes critical to cellular function and hematopoiesis. Examples include EZH2, ABCB7, macroH2A1, and ATG7. Mutations in SRSF2 result in inclusion of a poison exon in EZH2 resulting in reduced protein levels of this critical epigenetic regulator. Mutations in SF3B1 cause alternative splicing of ABCB7 slating its RNA for non-sense mediated decay. Intriguingly, mutations in U2AF1 promote usage of a distal polyadenylation site in an essential autophagy factor, ATG7, resulting in inefficient translation and decreased protein levels. As a consequence, autophagy is impaired, which can promote cellular transformation, providing a possible explanation for the clonal advantage of cells with splicing factor mutations. Additional research is critical to further determine how alternative splicing contributes to the MDS phenotype with impaired hematopoietic differentiation and cellular function and eventual malignant transformation. Latest findings will be presented here with a focus on mechanistic, functional, and biologic effects of splicing factor mutations.

Disclosures

No relevant conflicts of interest to declare.
Ferroportin Mediated Control of Iron Metabolism and Disease
Authors: Lakhal-Littleton Samira
Published: October 2019

Ferroportin, the only known mammalian iron export protein, releases iron from the duodenum, reticuloendothelial system and liver, the sites of iron absorption, recycling and storage respectively. By downregulating ferroportin, the liver-derived hormone hepcidin controls systemic iron availability in response to erythroid demand and inflammation. This ferroportin/hepcidin axis has long been recognized as essential for systemic iron homeostasis. However, both ferroportin and hepcidin are found in tissues not recognized for their role in systemic iron control, such as the heart, the kidney, the brain and the placenta. Co-existence within the same tissue suggests a possible function for hepcidin and ferroportin in local iron homeostasis. However, this hypothesis has not been formally explored. Using mouse models with cardiac-specific manipulation of hepcidin and ferroportin, we have uncovered a role for the cardiac hepcidin/ferroportin axis in cell-autonomous iron homeostasis within cardiomyocytes. Disruption of this cardiac pathway leads to fatal cardiac dysfunction, even against a background of normal systemic iron homeostasis. One the one hand, loss of cardiac ferroportin causes by fatal cardiac iron overload that is preventable by dietary iron restriction 1. On the other hand, loss of cardiac hepcidin or of cardiac hepcidin responsiveness causes fatal cardiomyocyte iron deficiency that is preventable by intravenous iron administration. Comparative study of cardiac iron homeostasis and function in cardiac versus systemic models of ferroportin/hepcidin disruption provides insight into the interplay between systemic and cellular iron homeostasis. A role for the hepcidin/ferroportin axis in cell-autonomous iron control, demonstrated here in the context of the heart, has not previously been described in any other tissue. A pertinent question is whether our findings in the heart extend to other tissues that express both hepcidin and ferroportin, such as the kidney, brain and placenta. Disturbances in iron homeostasis are of clinical importance in cardiovascular disease, renal failure, neurodegeneration and developmental defects. Our findings have two clinically relevant implications: a) that disruption of the local hepcidin/ferroportin axis may in itself have a disease-modifying effect, and b) that therapeutic strategies developed to target the systemic hepcidin/ferroportin axis may have off-target effects relating to local iron control within some tissues.

Reference

1.Lakhal-Littleton S, Wolna M, Carr C, et al. Cardiac ferroportin regulates cellular iron homeostasis and is important for cardiac function. PNAS. 2015; 10;112(10):3164-3169.

Disclosures

No relevant conflicts of interest to declare.
Long-term Live Single Cell Quantification of Transcription Factor Dynamics
Authors: Schroeder Timm
Published: October 2019

Hematopoiesis is highly complex and dynamic, and consist of large numbers of different cells expressing many molecules. Despite intensive research, many long-standing questions in hematopoiesis research remain disputed. One major reason is the fact that we usually only analyze populations of cells - rather than individual cells - at very few time points of an experiment. Tracking of individual cells would be an extremely powerful approach to improve our understanding of molecular cell fate control. We are therefore developing imaging systems to follow the fate of single cells over many generations. We program new software to help recording and displaying the divisional history, position, properties, interaction, etc. of all individual cells over many generations. In addition, novel microfluidics devices are designed and produced to allow improved observation and manipulation of cells. Our technologies allow continuous long-term quantification of protein expression or activity in living cells. Among other approaches, we generate knock in models expressing transcription factor to fluorescent protein fusions from endogenous gene loci. This enables non-invasive long-term live quantification of transcription factor protein dynamics in single stem and progenitor cells throughout their differentiation. The resulting novel kind of continuous quantitative single cell data is used for the generation and falsification of models describing the molecular control of hematopoietic cell fates.

Disclosures

No relevant conflicts of interest to declare.
Toll-Like Receptors: Mechanism and Relation to Human Populations
Authors: Freedman Jane
Published: October 2019

Inflammation and infection are known to alter platelet count, production, and function and major studies have demonstrated that acute infection is associated with a transient 5-fold increased risk of thrombotic vascular syndromes including stroke, acute myocardial infarction, and peripheral vascular occlusion. Platelets play an intricate role in thrombosis, myocardial infarction, and thrombotic stroke and are now being appreciated for their functional innate immune receptors. More recently, platelets have been connected to the host's immune system via their ability to trap bacteria and present them to leukocytes for destruction. This interaction appears crucial to the host's immune defense. Platelets are known to express functional interleukin 1 receptor (IL1R), toll like receptor (TLR)2, TLR3, TLR4, TLR7, and TLR9 with other immune receptors under investigation. It is known that activation of TLR2, TLR7, or TLR4 leads to platelet-neutrophil interaction.Platelet-leukocyte communication has also been observed during different bacterial or viral infections.

We have used animal models and large-scale human clinical data to examine if platelets demonstrate specific immunity or broadly express TLRs and characterize how this expression is causative in immune responses. Our studies have included thousands of participants of the Framingham Heart Study in addition to investigations of active infection and inflammatory diseases. We have also examined if presence of immune receptors in platelets is associated with vascular inflammation and cardiovascular disease or risk factors. We have found widespread and varied immune and inflammatory platelet receptor and gene expression that varies remarkably between individuals and diseases.

Additionally, anucleate platelets contain transcripts that might relate to other physiological or pathophysiological conditions and are known to be released into the circulation, participate in protein formation, and engage in horizontal RNA transfer to other vascular cells. These platelet transcripts include microRNAs (miRNAs), which are small noncoding RNAs involved in many molecular processes, most notably regulation of gene expression and release of these noncoding RNAs also may be regulated by innate immune receptors. In platelets and their released exosomes, these noncoding RNAs seem to participate in vascular homeostasis, inflammation, and platelet immune function. In addition, they are associated with the presence or extent of cardiovascular diseases, such as atrial fibrillation and peripheral vascular disease. Thus, platelets are known to contain a myriad of receptors that regulate hemostasis and thrombosis; however, there is growing appreciation of the diverse expression of receptors traditionally associated with immune and inflammatory cells.

Disclosures

No relevant conflicts of interest to declare.
The Diagnosis of Thalassemia Trait by Starch Block Electrophoresis of the Hemoglobin
Authors: GERALD PARK S. , DIAMOND LOUIS K.
Published: October 2019

Starch block electrophoresis of the hemoglobin has been performed for a group of adults who are the parents of children with thalassemia major. The hemoglobin electrophoretic pattern was found to be constantly abnormal in this group, in that the minor component with E-like mobility (designsated the A2 component) constituted a greater than normal proportion of the total hemoglobin. Reduced mean cell (erythrocyte) volume was likewise found to be present in all members of the patient group.

If it is assumed that typical childhood thalassemia major represents the homozygous state for the thalassemia gene, then the patient group studied constitutes a populations of adults heterozygous for thalassemia. Since microcytosis and an increase in the A2 content were constantly present in this group, they are suggested as suitable minimum diagnostic criteria for thalassemia trait.

The degree of elevation of the A2 fraction was noted to have a discontinuous distribution. Preliminary studies have demonstrated that the degree of elevation appears to be identical in affected members of single pedigrees. The discontinuity in distribution observed is thus apparently under genetic control.

The rather small difference in A2 content between some normal and some thalassemia trait adults implies that the alteration of hemoglobin synthesis is a secondary phenomenon, since such "low-valued" thalassemia traits were not different in their clinical expression. Despite the smallness of this difference, quantitation of the A2 fraction sufficed to distinguish these "low-valued" thalassemia heterozygotes from normal individuals.
The Detroit Strains of Human Epithelial-like Cells from Nonleukemic Peripheral Blood
Authors: BERMAN LAWRENCE , STULBERG CYRIL S.
Published: October 2019

1. The development of 6 strains of Ep-L cells from human blood was reported. In each instance the Ep-L cells appeared in cultures which had some connection with the administration of estrogenic materials, either to the donor of the blood or to the primary explant of blood in vitro. The possible role of estrogens was discussed, but no final conclusions were reached.

2. The development of stable human Ep-L cells did not require the participation of cells from leukemic blood.

3. Morphologic, karyotypic, and mitotic data on the Detroit blood cell strains were presented.

4. The morphogenesis of the Ep-L cells was described. The most likely source of the Ep-L cells in blood cultures appeared to be lymphocytes, but the role of monocytes could not be ruled out.

5. The significance of our findings with respect to transplantation studies was indicated.
A Method for Obtaining Polymorphonuclear Leukocytes from Intraperitoneal Exudates
Authors: ESTES FRANCES L. , SMITH SHIRLEY , GAST JOSEPH H.
Published: October 2019

By modification of existing methods, technics have been developed for obtaining polymorphonuclear leukocytes in good yield from intraperitoneal exudates of guinea pigs. The cells were 80-98 per cent viable at the time of harvest and they maintain this viability for at least 24 hours if stored under refrigeration. Good yield, viability and metabolic activity are dependent on careful handling of the cells and the exclusion of extraneous material from the suspending medium. Since the cells are maintained without extraneous material, they are especially suitable for metabolic studies.
Role of the Spleen and Effect of Splenectomy in Sickle Cell Disease
Authors: SPRAGUE C. C. , PATERSON J. C. S.
Published: October 2019

The survival of sickle cell anemia and sickle-cell/Hb-C erythrocytes was determined by the radioactive chromium method, both in their parent circulations and in the circulations of compatible normal recipients.

In sickle cell anemia patients with splenomegaly the average erythrocyte survival time (T½) was found to be 3.7 days. After splenectomy T½ increased to an average of 11.4 days.

In sickle cell anemia patients without splenomegaly the average T½ was found to be 10 days. In five instances an average T½ of 9.2 days was found, whereas the average T½ for the same cells in the circulations of normal recipients was 4.4 days.

In sickle-cell/Hb-C disease the average T½ was 15.7 days. Following splenectomy in two patients, T½ was unchanged in one and increased in the second. T½ was shortened in two of three instances when these cells were transfused into normal recipients, but the data are insufficient to permit conclusions to be drawn.

The role of the spleen in hemolysis is discussed briefly and it is concluded that the hemolytic process in sickle cell anemia is accelerated in the presence of a spleen. This finding is compatible with the diminution in the severity of sickle cell anemia frequently recognized in the adult patient whose spleen has atrophied, and in the child following splenectomy. The validity of the chromate-tagging method for determining erythrocyte survival is discussed. Splenectomy was performed in children in whom the erythrocyte survival was shortened (T½ less than 6 days).
The Serum Lactobacillus casei Folate Clearance Test in Various Hematologic Disorders
Authors: HOGAN JAMES A. , MANIATIS ALICE , MOLONEY WILLIAM C.
Published: October 2019

Serum folic acid clearance curves using Lactobacillus casei were determined on 10 normal individuals, 5 patients with hemolytic anemias and active hemolysis, 15 patients with myeloid metaplasia, and 12 patients with miscellaneous hematologic disorders.

In general, there was a correlation between the initial serum folate value and the subsequent serum folate clearance curve. Both values were determined by L. casei. In many instances of initial serum folate values classified as indeterminate, the serum folate clearance clarified the initial impression.

Patients with active hemolytic anemia showed rapid folate clearance curves following injection with a test dose of folic acid. In eight of 15 patients with myeloid metaplasia, the serum folate clearance was rapid; in 5 of these patients the serum folate clearance was consistent with marked folic acid deficiency. Several possible mechanisms for the folic acid deficiency in myeloid metaplasia are discussed, but the cause of the deficiency has not been established.
Cyclosporine as prophylaxis for graft-versus-host disease: a randomized study in patients undergoing marrow transplantation for acute nonlymphoblastic leukemia
Authors: Deeg HJ , Storb R , Thomas ED , Flournoy N , Kennedy MS , Banaji M , Appelbaum FR , Bensinger WI , Buckner CD , Clift RA
Published: October 2019

Seventy-five patients, 13 to 49 years of age, with acute nonlymphoblastic leukemia in first remission were treated with cyclophosphamide, fractionated total body irradiation, and marrow transplantation from an HLA-identical sibling and randomized to receive either cyclosporine (CSP) (n = 36) or methotrexate (MTX) (n = 39) as prophylaxis for graft-v-host disease (GVHD). All patients engrafted, and 22 who were given CSP and 21 who were given MTX, are alive at 20 to 47 (median, 35) months (P = .5). Engraftment as assessed by granulocyte recovery (P less than .0005) and platelet transfusion requirement (P = .01) was faster in patients on CSP. Twelve patients (33%) on CSP and 22 (56%) on MTX developed acute GVHD of grades II through IV (P = .07) and 15 of 30 on CSP and 14 of 32 on MTX that were at risk developed chronic GVHD. The most frequent causes of death were interstitial pneumonitis and marrow relapse of leukemia, which occurred with similar frequency in both groups. Beneficial effects observed in patients on CSP included less severe mucositis and shorter duration of hospitalization; adverse effects included renal function impairment and hypertension. These data confirm that CSP is a useful immunosuppressant in patients undergoing marrow transplantation but fail to show a significant improvement in survival as compared with the standard regimen of MTX.
Neonatal alloimmune thrombocytopenic purpura and congenital porencephaly in two siblings associated with a "new" maternal antiplatelet antibody
Authors: Friedman JM , Aster RH
Published: October 2019

We report a brother and sister, both of whom have porencephaly, hydrocephalus, optic atrophy, severe mental retardation, and spastic quadriplegia. In the younger child, abnormal intracranial structure was demonstrated by sonography at 32 weeks' gestation and was suspected earlier. Both children had transient severe thrombocytopenia as newborns. The mother is healthy and has never had purpura or other bleeding symptoms. However, her serum was found to react strongly with platelets from the father and from both children. The antibody in the mother's serum is platelet-specific but does not appear to be directed against any of the known antigens associated with neonatal alloimmune thrombocytopenic purpura (NATP) in other families, including PlA1, PlE2, or Baka. Although the mother's serum reacts with platelets from all 47 unrelated normal donors tested and from both the mothers and the fathers of 17 other children with suspected NATP, it does not react with her own platelets or with platelets from a patient with Glanzmann's thrombasthenia. These observations suggest that the serum from this woman identifies a previously undescribed high-frequency platelet-specific alloantigen and that sensitization to this determinant caused severe immune thrombocytopenia in both her children. It is likely that this led to intracranial hemorrhage in utero in these cases.
Mechanisms of corticosteroid action in immune thrombocytopenic purpura (ITP): experimental studies using ITP-prone mice, (NZW x BXSB) F1
Authors: Mizutani H , Furubayashi T , Imai Y , Kashiwagi H , Honda S , Take H , Kurata Y , Yonezawa T , Tarui S , Ikehara S
Published: October 2019

To determine the mechanism by which platelet counts increase after corticosteroid therapy for human immune thrombocytopenic purpura (ITP), we studied the platelet kinetics using prednisolone (PDN)-treated ITP- prone mice, (NZW x BXSB) F1 (W/B F1). An increase in platelet counts was observed in W/B F1 mice (n = 10, mean +/- SD, 1,202 +/- 202 x 10(3)/microL) 4 weeks after treatment with PDN (2 mg/kg/d); no increase occurred in nontreated W/B F1 mice (n = 5,651 +/- 126, P less than .005). Prolonged platelet life-spans (PLSs) were observed in treated W/B F1 mice (1.29 +/- 0.40 days), but not in nontreated controls (0.60 +/- 0.24 days, P less than .01). No increase in platelet production (platelet turnover) was found in PDN-treated W/B F1 mice, but significant decreases in platelet-associated antibodies (PAAs) and platelet-bindable serum antibodies (PBAs) were noted. Studies on organ localization of radiolabeled platelets showed that hepatic uptake significantly decreased in the treated W/B F1 mice, but not in nontreated W/B F1 mice. To elucidate the effect of PDN on the reticulo- endothelial phagocytic activity in W/B F1 mice, we studied in vivo clearance of IgG-sensitized, 51Cr-labeled autologous erythrocytes. W/B F1 mice treated with PDN showed a marked impairment of their ability to clear these cells, although PDN had little effect on the number of splenic or hepatic macrophage Fc gamma receptors. These results and our previous findings of splenectomy suggest that PDN improves platelet counts not only by suppressing systemic reticulo-endothelial phagocytic function but also by reducing antibody production.
Human recombination activating gene-1 in leukemia/lymphoma cells: expression depends on stage of lymphoid differentiation defined by phenotype and genotype
Authors: Yoneda N , Tatsumi E , Kawano S , Matsuo Y , Minowada J , Yamaguchi N
Published: October 2019

The recombination activating gene, RAG-1, which is supposed to encode a molecule regulating V(D)J recombination, has been isolated. In the current study, the distribution of RAG-1 expression in human neoplastic hematopoietic cells was compared with the phenotypic and genotypic status of differentiation. Thirty-one hematopoietic cell lines (16 B- lineage, 9 T-lineage, 2 Hodgkin's disease, and 4 nonlymphoid cell lines) were investigated for the expression of human RAG-1 using the reverse-transcriptase polymerase chain reaction (RT-PCR). RAG-1 was not expressed in nonlymphoid, Hodgkin's disease, or mature-stage lymphoid cell lines, but was present in some acute lymphoblastic leukemia (ALL)/lymphoblastic lymphoma (LBL) cell lines. The investigation was extended to 45 cases of fresh ALL/LBL cells. The patterns of RAG-1 expression found in the cell lines and fresh ALL/LBL cells were similar. In B-lineage cells, the product of RAG-1 RT-PCR was detected in CD19+ CD10- CD20- CD5- stage (stage II, Nadler's classification) and was at the highest level in CD19+ CD10+ CD20- CD5- stage (stage III), but was absent or limited in CD19+ CD10+ CD20-+ CD5- (stage IV) or CD19+ CD10+ (or CD10-) CD5+. In stage II, monoclonal gene rearrangements of only the immunoglobulin heavy chain (IgH) were found, whereas monoclonal gene rearrangements of both IgH and T-cell receptor (TCR)-beta chain were frequently noted in stages III and IV. The expression of CD20 or CD5 antigen apparently correlated with the decline of RAG-1 expression. In T-lineage cells, RAG-1 was highly expressed in CD3- CD4+ CD8+/CD3+ CD4+ CD8+ thymic stages, but was negative or only weakly expressed in the CD3- CD4- CD8- prothymic or early thymic stage, in which the TCR-beta gene was often germline, or the CD3+ CD4+ CD8- mature thymic stage. The relative levels of RAG-1 mRNA give an additional delineating frame to the schemes of lymphoid differentiation based on phenotypic and genotypic status. RAG-1 is exhibited by cells of the thymic stage capable of synthesizing TCR or expressing it on the cell surface. The weak or absent expression of RAG- 1 in the prothymic or early thymic stage suggests that the contribution of RAG-1 to the gene rearrangement may differ quantitatively between TCR-delta/TCR-gamma and TCR-beta.(ABSTRACT TRUNCATED AT 400 WORDS)
Identification of the amino acid mutations associated with human erythrocyte spectrin alpha II domain polymorphisms
Authors: DiPaolo BR , Speicher KD , Speicher DW
Published: October 2019

Four distinct spectrin alpha II domain polymorphisms are known to occur in several nonwhite populations. Type 1 is essentially the only form found in whites and is also the most common form in nonwhites. In contrast to most other spectrin mutations that are single-base substitutions, two of the alpha II domain polymorphisms, types 2 and 3, are particularly unusual because they appear to involve 4-Kd insertions relative to type 1. We have identified the mutations responsible for these polymorphisms using biochemical approaches and a computer database of spectrin-domain peptides separated by two-dimensional gels. The type 3 mutation is characterized by an apparent 4-Kd increase in alpha II domain peptides with no change in pI. This apparent molecular weight increase is a sodium dodecyl sulfate (SDS) gel artifact resulting from an Arg-->His mutation at residue 22 of the domain. The type 4 polymorphism shows a basic charge shift with no apparent change in molecular weight on gels. This charge shift results from a mutation of Thr-->Arg at position 174 of the domain. This mutation appears to be linked to a “silent” mutation at position 130 from an Ile-->Val. Support for possible linkage was obtained from analysis of three unrelated donors with the type 2 polymorphism. The type 2 polymorphism shows both the charge shift characteristic of the type 4 mutation and the apparent size shift that defines the type 3 polymorphism. Analysis of type 2 peptides confirmed that the two mutations described above for type 4 as well as the mutation at residue 22 observed in type 3 occur simultaneously in type 2. The observation that the type 2 polymorphism is a composite of the type 3 and 4 mutations is especially surprising because the type 2 polymorphism occurs far more frequently than either the type 3 or 4 forms. The basis for apparent linkage between the mutations at residues 130 and 174, which are encoded by different exons, is also not clear. Identification of the mutations described here permits design of genetic screening analyses that can be applied to larger populations to evaluate this potential linkage.
Photochemical inactivation of cell-associated human immunodeficiency virus in platelet concentrates
Authors: Lin L , Londe H , Hanson CV , Wiesehahn G , Isaacs S , Cimino G , Corash L
Published: October 2019

Photochemical decontamination (PCD) of platelet concentrates, with adequate preservation of platelet function, has been shown using 8- methoxypsoralen (8-MOP) and long wavelength UV light (UVA). To further evaluate this technique, models for the inactivation of pathogenic human cell-associated viruses and integrated proviral sequences are required. We have assessed the ability of the PCD technique to inactivate cell-associated human immunodeficiency virus 1 (HIV-1) in platelet concentrates. We correlated PCD inhibition of HIV-1 infectivity with 8-MOP-DNA adduct formation in contaminating nucleated cells, and measured the inhibition of polymerase chain reaction (PCR)- mediated amplification of cellular DNA sequences as a surrogate for inactivation of integrated proviral nucleic acid sequences. After PCD treatment (8-MOP 300 micrograms/mL, UVA 17 mW/cm2) for 60 minutes, 0.5 x 10(6) plaque-forming units (PFU)/mL of cell-associated HIV-1 were inactivated and no virus was detectable by infectivity assay. After 60 minutes of PCD, 15 8-MOP-DNA adducts per 1,000 bp were formed, while in the absence of UVA, no adducts were formed. PCR-mediated amplification of a 242-bp cellular DNA sequence (HLA-DQ-alpha) was inhibited when greater than eight psoralen-DNA adducts per 1,000 bp were present. These studies indicate that high titers of cell-associated HIV-1 in platelet concentrates were inactivated by PCD, and the numbers of 8-MOP- DNA adducts in nucleated cells were sufficient to inhibit amplification of DNA segments that encode for as few as 80 amino acids. Based on the frequency of 8-MOP-DNA adducts, for the 10-kb HIV-1 genome, the probability of an integrated genome without at least one 8-MOP adduct after 60 minutes of PCD was 10(-33).
Prothrombinase assembly on an enveloped virus: evidence that the cytomegalovirus surface contains procoagulant phospholipid
Authors: Pryzdial EL , Wright JF
Published: October 2019

In contrast to quiescent cells, we currently report that purified cytomegalovirus (CMV), strain AD169, constitutively expresses phosphatidylserine (PS)-like procoagulant activity. Initial evidence for this came from the observation of a CMV-dependent decrease in factor Xa clotting times. In a purified system, the assembly of a functional complex between factor Xa and the cofactor Va to form prothrombinase was found to be dependent on the addition of CMV. The corresponding dense bodies (DB) and noninfectious enveloped particles had similar activity. Quantification of the total virion and DB phospholipid, and comparison of prothrombin conversion rates to experiments conducted using known concentrations of PS-containing vesicles showed that 8.5% and 7.2% of the CMV and DB phospholipid, respectively is procoagulant. Direct binding studies of 125I-labeled factor Xa, active site-blocked factor Xa, or the zymogen factor X, with virions or DB showed a single class of Ca(2+)-dependent sites with dissociation constants in the order of 10(-7) MOL/l. Immunogold electron microscopy confirmed the specificity of the CMV/factor Xa association. Cumulatively, these data suggest that the CMV surface contains the necessary procoagulant phospholipid for coagulation enzyme complex assembly. This may enable CMV (and possibly other enveloped viruses) to bypass an important physiologic regulatory mechanism for the production of thrombin.
In vitro megakaryocytopoietic and thrombopoietic activity of c-mpl ligand (TPO) on purified murine hematopoietic stem cells
Authors: Zeigler FC , de Sauvage F , Widmer HR , Keller GA , Donahue C , Schreiber RD , Malloy B , Hass P , Eaton D , Matthews W
Published: October 2019

Recently, the ligand for c-mpl has been identified and cloned. Initial studies of this molecule indicate that it is the platelet regulatory factor, thrombopoietin (TPO). Previous work has indicated that c-mpl is expressed in very immature hematopoietic precursors and thus raised the possibility that TPO may act directly on the hematopoietic stem cell. Therefore, in these studies, we investigate the effects of TPO on hematopoietic stem cell populations isolated from the murine fetal liver and bone marrow. Cocultivation of stem cells with fetal liver stroma give rise to multilineage expansion of the stem cells but with little or no megakaryocytopoiesis. Addition of TPO to these cocultures gives significant megakaryocyte production. This production is enhanced in combination with Kit ligand or interleukin-3. The addition of TPO to stem cell suspension cultures produces a dynamic thrombopoietic system in which stem cells undergo differentiation to produce megakaryocytes and proplatelets. These experiments show that the megakaryocytopoietic and thrombopoietic activities of TPO are initiated at the level of an early progenitor cell or upon the hematopoietic stem cell.
Induction of anti-recombinant human granulocyte-macrophage colony- stimulating factor (Escherichia coli-derived) antibodies and clinical effects in nonimmunocompromised patients
Authors: Ragnhammar P , Friesen HJ , Frodin JE , Lefvert AK , Hassan M , Osterborg A , Mellstedt H
Published: October 2019

The pharmacokinetics of recombinant human granulocyte-macrophage colony- stimulating factor (rhGM-CSF), induction of anti-GM-CSF antibodies, and clinical effects related to the induction of the antibodies were analyzed in patients with metastatic colorectal carcinoma (CRC) who were not on chemotherapy (n = 20, nonimmunocompromised patients). rhGM- CSF (250 micrograms/m2/d; Escherichia coli-derived) was administered subcutaneously for 10 days every month for 4 months. Eight patients with multiple myeloma (MM) on intensive chemotherapy followed by rhGM- CSF treatment were also included (immunocompromised patients). After a single injection of GM-CSF at the first cycle in CRC patients, the maximum calculated concentration (Cmax) was 5.24 +/- 0.56 ng/mL; the half life (T1/2) was 2.91 +/- 0.8 hours; and the area under the concentration curve (AUC) was 30.86 +/- 6.03 hours x ng/mL (mean +/- SE). No anti-GM-CSF antibodies were detected. During the subsequent cycles, 95% of the CRC patients developed anti-GM-CSF IgG antibodies, which significantly altered the pharmacokinetics of rhGM-CSF at the third and fourth cycles with decreased Cmax (2.87 +/- 0.57 ng/mL; P < .05), T1/2 (1.57 +/- 0.2 hours; P < .05), and AUC (14.90 +/- 4.10 hours x ng/mL; P < .005). The presence of anti-GM-CSF antibodies significantly reduced the GM-CSF-induced enhancement of granulocytes, and there was a clear tendency for a decreased increment of monocytes. Antibodies diminished systemic side effects of rhGM-CSF. Only 1 of 8 MM patients showed a very low anti-GM-CSF antibody titer after GM-CSF therapy, as shown by enzyme-linked immunosorbent assay and Western blot. Therefore, in nonimmunocompromised patients, exogenous nonglycosylated GM-CSF induced an anti-GM-CSF IgG antibody response in practically all patients, which seemed to be of clinical significance. In immunocompromised patients, virtually no significant antibody response was shown.