bluuebunny/crossref_metadata_2025 huggingface dataset:
THE DIAGNOSIS OF HODGKIN'S DISEASE BY ASPIRATION BIOPSY
Authors: LOSEKE LUCILE , CRAVER LLOYD F.
Published: October 2019

1. Among 242 cases of histologically proved Hodgkin's disease treated at Memorial Hospital for the most part within the past five years, the diagnosis was established by open biopsy in 228, while in 14 cases aspiration biopsy yielded a reliable diagnosis.

2. In the 14 cases of Hodgkin's disease in which the diagnosis was made by examination of the sectioned clot obtained by aspiration biopsy, the material was from lymph nodes in 9 cases, from a presternal nodule in 1 case, from the lung in 3 cases, and from the spleen in 1 case.

3. Among the 228 cases in which formal biopsy was used to prove the diagnosis, aspiration biopsy had been previously attempted but had been unsuccessful in 11 cases.

Thus in 25 cases of the total number of 242, aspiration biopsy had been selected for good reason as the first method to try, and was diagnostically successful in 56 per cent of the group.
THE CARDIOVASCULAR SYSTEM IN ANEMIA
Authors: WINTROBE MAXWELL M.
Published: October 2019

The symptoms and signs referable to the cardiovascular system that are associated with anemia are discussed, and the physiologic adjustments to anemia that take place in the cardiovascular system are considered. The capacity for adjustment when anemia develops gradually, appears to be very great. The remarkable changes found in the cardiovascular system in cases of sickle cell anemia may be the result of adjustments to severe anemia of exceptional chronicity.
PLATELET COUNTS AND PLATELET FUNCTION
Authors: AGGELER PAUL M. , HOWARD JOAN , LUCIA S. P. , MILLS EDITH
Published: October 2019

Determinations of the platelet count, clot retraction, bleeding time, capillary fragility, and coagulation time were made in 64 normal subjects and in 404 patients suffering from various diseases.

The normal values for the platelet count done by an experienced technician using the Rees and Ecker method were: mean, 409,000 per cu. mm.; standard deviation, 68,000 per cu. mm.; normal range (M ± 2.σ), 273,000 per cu. mm. to 545,000 per cu. mm.

There was a statistically significant relationship between the platelet count and the results of tests of clot retraction, bleeding time, and capillary fragility, but there was no significant relationship between the platelet count and the coagulation time. Factors other than platelet count or platelet function which may influence the results of these tests are discussed.

The critical level of the platelet count, below which abnormal bleeding is likely to occur, was found to be approximately 190,000 per cu. mm. in primary thrombocytopenic purpura and 230,000 per cu. mm. in secondary thrombocytopenic purpura. However, platelet counts as low as 100,000 per cu. mm. were found in one patient without abnormal bleeding, and counts as high as 280,000 per cu. mm. were found in another patient with classical primary thrombocytopenic purpura.

In all patients in the active phase of bleeding in primary thrombocytopenic purpura and in most with secondary thrombocytopenic purpura, the bleeding time was markedly prolonged and clot retraction was definitely diminished. In approximately one half of the patients suffering from thrombocytopenia without bleeding or from thrombocytopenia complicating other hemorrhagic states, the results of these tests were abnormal. Capillary fragility was increased in approximately three fourths of the patients with primary thrombocytopenic purpura, one half with secondary thrombocytopenic purpura, and less than one half with thrombocytopenia without bleeding or with thrombocytopenia complicating other hemorrhagic states.

In the stage of recovery from thrombocytopenic purpura, dissociation of the results of the various tests was sometimes found. In some patients the platelet counts returned to normal hut abnormalities persisted in the tests of the bleeding time, clot retraction, or capillary fragility. In other patients the results of one or all of these tests returned to normal before the platelet count had reached the normal range. These results have been interpreted as evidence of variability in the functional capacity of the platelets.
SPECIFICITY OF ISOAGGLUTININ RESPONSE FOLLOWING INJECTION OF GROUP SUBSTANCES INTO GROUP O INDIVIDUALS
Authors: BOYD WILLIAM C. , LOWELL FRANCIS C.
Published: October 2019

The anti-A and anti-B titers in group O individuals may vary independently following injection of group-specific substances, although such independent variation was not always seen. The rise in anti-B which followed the injection of A sub stance in some instances may have been due to traces of B activity in the A substance or to nonspecific stimulation of antibody which may follow the injection of any antigen. The part played by such factors was not determined.

It is concluded that the presence of A and B agglutinins in O individuals is not dependent on a single cellular characteristic responsible for the simultaneous formation of the isoagglutinins.
Gene Interaction Resulting in Suppression of Blood Group Substance B
Authors: LEVINE PHILIP , ROBINSON ELIZABETH , CELANO MARINO , BRIGGS OLIVE , FALKINBURG LEROY
Published: October 2019

A family is presented in which the propositus and 2 siblings, apparently in group O, have in their sera anti-H in addition to anti-A and anti-B. Inspection of the family tree reveals apparent violations of Mendelian heredity which can be explained on the basis of a rare suppressor gene which in the homozygote state suppresses in the propositus the actions of gene B and secretion gene Se. The genes B and Se are fully expressed when transmitted to her offspring who are heterozygous for the suppression gene.

The same mechanism may be assumed to operate in the 9 other so-called group O bloods containing anti-H.
Observations on the Mechanism of Intranasal Absorption of Vitamin B12 in Pernicious Anemia
Authors: MONTO RAYMOND W. , REBUCK JOHN W.
Published: October 2019

Satisfactory clinical and hematologic response was obtained from the single application to the nasal mucosa of 200 and 150 micrograms of crystalline vitamin B12 in two patients with pernicious anemia.

Thirty-two patients with pernicious anemia in relapse and 55 in remission have been controlled satisfactorily by intranasal B12 either in saline solution, lactose powder, or as crystals. Absorption of the vitamin was probably direct and did not require binding with "intrinsic substance." Chronic disease of the nose did not contraindicate this modality of therapy; local irritation of the mucous membranes by the crystalline B12 or the vehicles employed has not been observed.
The Application of the Prussian Blue Stain to Previously Stained Films of Blood and Bone Marrow
Authors: SUNDBERG R. DOROTHY , BROMAN HARRIETTE
Published: October 2019

A simple method for staining non-hemoglobin iron in erythrocytes, normoblasts, macrophages, and other cells containing particulate iron in new or old films of cellular fluids or imprints of tissues and organs is presented. This method consists in using the prussian blue reagent as a type of counterstain; no separate decolorization is necessary. The preparations obtained resemble the original preparations except that iron stands out as a vivid blue-green material.

The method is particularly useful in studying conditions accompanied by varying degrees of iron excess or hemosiderosis of the marrow.

The stainable iron is all virtually the same color. The diffuse blue-green color of the cytoplasm of macrophages might be attributed to the more soluble form of iron, ferritin.

Stainable iron is visible in normoblasts and erythrocytes as well as in macrophages in sections subjected to the prussian blue reaction.

A prussian blue method using formalin fixation on fresh films of marrow7 has also been shown to be useful in the demonstration of particulate iron in previously stained films of marrow and blood. The formalin fixation appears to bring about a higher percentage of siderocytes.
Hemolytic Disease of the Newborn Due to Anti-A1
Authors: HUBINONT P. O. , LATIERS P. , MASSART-GUIOT T.
Published: October 2019

1. The authors report clinical, hematological and serological data in a case of A1 iso-sensitization of pregnancy.

2. The mother’s serum displayed immune characters specific to A1 cells immediately after delivery. On the 24th day post partum the specificity extended to A2 cells.

3. The disease exhibited by the infant was very mildly hemolytic. It was marked by a deep jaundice, repeated alimentary vomiting and a progressive state of drowsiness. There was no anemia. The direct anti-globulin test was negative.

4. It is shown that the mildness of the hemolytic process in cases of placental transfer of immune anti-A or anti-B into the incompatible A or B fetus is probably dependent upon a peculiar "resistance" of the fetal cells. This may be demonstrated in vivo and in vitro. In the present case replacement transfusion with A1adult blood resulted in its in vivo sensitization, detectable by the antiglobulin test and eventually leading to hemolytic anemia.
Multiple Myelomatosis Treated with a Combination of Urethane and an Oral Nitrogen Mustard
Authors: INNES JAMES , RIDER W. D.
Published: October 2019

1. 17 Cases of advanced multiple myelomatosis have been treated with combined chemotherapy using small doses of urethane and an oral preparation of nitrogen mustard.

2. The object of such therapy was to test the hypothesis that a synergistic action existed between these compounds.

3. The results indicate that significant palliation was obtained in many patients treated and that in some the progress of the disease was modified.

4. In the dosage employed such combined therapy appears to be free from undesirable toxic manifestations and suitable for out-patient administration over long periods.
Refractory Anemia Occurring in Survivors of the Atomic Bombing in Nagasaki, Japan
Authors: LANGE ROBERT D. , WRIGHT STANLEY W. , TOMONAGA MASANOBU , KURASAKI HIROTAMI , MATSUOKE SHIGERU , MATSUNAGA HARUJI
Published: October 2019

In this paper the case histories of six Nagasaki atomic bomb survivors who developed refractory anemia are presented. Four of these individuals received undoubted radiation injury. The fact that refractory anemia may occur as a late manifestation of exposure to atomic radiation is pointed out.
Clinical Manifestations of Inherited Abnormal Hemoglobins
Authors: STURGEON PHILLIP , ITANO HARVEY A. , BERGREN WILLIAM R.
Published: October 2019

A clinical and hematologic description is presented of the family demonstrating the existence of the genetically determined hemoglobin-D originally described by Itano.

The interaction of hemoglobin-D with hemoglobin-S in two members of this family has resulted in a hemolytic process and a mild anemia. Clinically, one of the patients is asymptomatic. The other has repeated painful episodes characteristic of those seen in sickle cell crises.

Clinical and hematologic evaluations of two members of the family inheriting normal hemoglobin and hemoglobin-D revealed no abnormalities.

The only feature of hemoglobin-D which distinguishes it from normal hemoglobin is its electrophoretic mobility which is similar to that of hemoglobin-S. Hemoglobin-D is distinguished from hemoglobin-S by a higher solubility and by a failure to sickle and from hemoglobin-C by a lack of target cell formation and a different electrophoretic mobility.

Clinical, hematologic and special hemoglobin studies are presented of a family exhibiting a new abnormal hemoglobin (hemoglobin-E). Its interaction with thalassemia trait in one of its members also is described.

Hemoglobin-E does not sickle; under alkaline conditions it migrates slowly at a rate comparable to hemoglobin-C, while at an acid pH it migrates at a rate similar to hemoglobin-S.

The result of its interaction with thalassemia is a microcytic, hypochromic anemia clinically resembling thalassemia major of an intermediate degree of severity. The major defect appears to be in hemoglobin synthesis, although the slight persistent reticulocytosis suggests a mild hemolytic process.
Comparative Incorporation of S35 L-Cystine and S35 Sodium Sulfate by Normal and Leukemic Leukocytes
Authors: WEISBERGER AUSTIN S. , SUHRLAND LEIF G.
Published: October 2019

1. Certain differences are demonstrated in the metabolic turnover of l-cystine sulfur and inorganic sulfur by normal and leukemic leukocytes. These differences are apparent both in vivo and in vitro and may be associated with factors peculiar to the metabolism of leukemic leukocytes.

2. The incorporation of S35 l-cystine or S35 sodium sulfate by leukocytes appears to be associated primarily with the presence of immature granulocytes.

3. Both l-cystine (or l-cysteine) and sulfate may be of importance in the metabolism of leukocytes.
Action of Disodium Ethylenediamine Tetracetate on Blood Coagulation
Authors: TRIANTAPHYLLOPOULOS DEMETRIOS C. , QUICK ARMAND J. , GREENWALT TIBOR J.
Published: October 2019

A. The action of EDTA on the coagulation mechanism was studied. EDTA was found:

1. To exert no direct effect on prothrombin or thrombin.

2. To have an inhibitory action on labile factor.

3. To increase the speed of conversion of prothrombinogen to prothrombin.

B. The effect of incubation or aeration of plasma on thrombin time was studied. It was found that aeration or incubation increases the thrombin time of plasma. The following observations lead one to attribute this prolongation to a heparinoid activity:

1. The property of incubated plasma to prolong the thrombin time of fresh plasma when added to it.

2. The ability of protamine to shorten the thrombin time of incubated or aerated plasma.

3. The sensitivity of the thrombin time of incubated or aerated plasma to pH changes and calcium concentration.

C. The incidental observation was made that cooling reverses the prolonged thrombin time produced by incubation.
Autoerythrocyte Sensitization A Form of Purpura Producing Painful Bruising Following Autosensitization to Red Blood Cells in Certain Women
Authors: GARDNER FRANK H. , DIAMOND LOUIS K.
Published: October 2019

Four patients with purpura who manifested an unusual response to bruising were studied. This response was characterized by the development of an area of painful ecchymosis at the site of trauma followed by progressive erythema and edema. This unusual tissue response was seen only in women. The various features of the cases suggested an autosensitization by the patients to their own blood.

Special studies utilizing skin testing procedures indicated an abnormal tissue response of sensitivity to red blood cells. The factor responsible was present in the red cell stroma and was not associated with the hemoglobin.

The clinical manifestations and possible therapy are discussed.

This syndrome may represent another example of autosensitization such as has been speculated for lupus erythematosus, some forms of acquired hemolytic anemia and of thrombocytopenic purpura, and for an increasing number of disease states.
The Effect of Various Metabolites on the Growth of Marrow Cells in Vitro
Authors: SWAN HAROLD T. , REISNER EDWARD H. , SILVERMAN MORRIS
Published: October 2019

Marrow was cultured on chick plasma clots, glass surfaces and in suspension and the influence of various metabolites and antimetabolites on the growth and differentiation of red cell precursors was studied.

Vitamin B12 appeared to stimulate cell growth in cultures grown on clots and to convert megaloblastic hematopoiesis to normoblastic in cultures grown on glass.

In suspension cultures of megaloblastic marrows in pernicious anemia serum significant cell increases were obtained with the addition of folinic acid, but not with vitamin B12. Both vitamin B12 and folinic acid appeared to accelerate the conversion of megaloblasts to normoblasts in differential counts of stained smears of such cultures. No enhancing effect of intrinsic factor upon the activity of B12 in suspension cultures of megaloblasts was observed. The effect of folinic acid could be blocked by colchicine and folinic acid could reverse the inhibitory effects of Amethopterin. These facts indicated that folinic acid acted in suspension cultures by stimulating mitosis.

Megaloblasts could be produced from normoblastic marrows by culturing them in pernicious anemia serum, or in normal serum in the presence of Amethopterin.
Susceptibility to Thrombosis in Normal Young, Aging, Cortisone-treated, Heparinized and X-irradiated Hamsters as Tested by Topical Application of Thrombin
Authors: BERMAN HERBERT J. , FULTON GEORGE P. , LUTZ BRENTON H. , PIERCE DAVID L.
Published: October 2019

1. Thrombin applied topically to the everted cheek pouch of the hamster produced platelet and not red thrombi in exposed, uninjured blood vessels with circulating blood. Red thrombi were produced in stagnant blood. Thrombus formation occurred in the venules for the most part and seldom in arterioles or capillaries.

2. An in vivo test for platelet thrombus susceptibility, based on the thrombin reaction and the resistance of the hamster to thrombosis, has been described.

3. Thrombus susceptibility, measured by the thrombin test, increased with age and during cortisone treatment, and decreased after heparin injection and following large doses of whole body x-irradiation.

4. The thrombin susceptibility test could be correlated with the platelet count in x-irradiated hamsters, showing a relatively critical minimum concentration of blood platelets (100,000/cu.mm.) required for platelet thrombosis.

5. The relationship of platelet concentration to platelet thrombus formation and predisposition to hemorrhage has been discussed.
Multilineage Dysplasia Is Associated with Worse Clinical Outcome Independent of the Diagnosis of Acute Myeloid Leukemia or Myelodysplastic Syndrome.
Authors: Amin Hesham M. , Pierce Sherry A. , Estey Elihu H. , Kantarjian Hagop M. , O’Brien Susan M. , Giles Francis J. , Albitar Maher
Published: October 2019

Acute myeloid leukemia (AML) and the myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematopoietic neoplasms. The World Health Organization (WHO) classification includes the two new categories: AML with dysplasia in more than one lineage (multilineage dysplasia; MLD) and refractory cytopenia with MLD. However, the relevance of dysplasia in one lineage versus more than one lineage and the importance of separating MDS with MLD from AML with MLD are not known. We studied 1110 patients with AML and 171 patients with MDS classified according to the WHO criteria and treated at MD Anderson Cancer Center from 1995 to 2003. All MDS patients had advanced disease requiring treatment and included 19 patients with refractory anemia (RA), 7 with RA with ringed sideroblasts (RARS), and 145 with RA with excess blasts (RAEB). Survival analysis showed no statistical significance in overall survival between MDS patients and AML patients when classified according to the WHO classification (P=0.196). When AML and MDS patients were considered as one group, but divided based on the presence or absence of MLD, patients with MLD had significantly shorter survival (P=.004; Figure 1). More importantly, multivariate Cox proportional hazard model showed that the shorter survival in the combined AML and MDS patients with MLD was independent of cytogenetic abnormalities, antecedent hematologic disease, performance status, and the original diagnosis of AML or MDS. As expected, shorter survival was found in AML patients with MLD as compared with those with no dysplasia or dysplasia of one lineage (P=.003). The presence of dysplasia in one lineage was irrelevant for the clinical course. Apoptosis detection using annexin V staining or mitochondrial membrane potential analysis showed significantly higher apoptosis in AML patients with MLD as compared with those patients without MLD and the presence of increased apoptosis correlated with shorter survival (P=.04). The present study demonstrates that the criteria established by the WHO classification for the diagnosis of AML and MDS can be significantly enhanced if the presence or absence of MLD is considered irrespective of the percentage of blasts. Furthermore, apoptosis can be detected in AML and MDS patients using annexin V staining or mitochondrial membrane potential analysis and it can be utilized to indirectly reflect the level of dysplasia in these patients. With the progress in targeted therapy, the classification of leukemia and myelodysplasia based on biological abnormalities may provide more helpful clues for therapy and clinical management.

Figure Figure
The Severe Chronic Neutropenia International Registry - 10 Years of Follow-Up.
Authors: Dale David C. , Bolyard Audrey Anna , Schwinzer Beate , Pracht Gusal , Bonilla Mary Ann , Boxer Laurence , Freedman Melvin , Donadieu Jean , Kannourakis George , Alter Blanche P. , Cham Bonnie , Winkelstein Jerry , Kinsey Sally E. , Fier Carol , Zeidler Connie , Welte Karl
Published: October 2019

2004 marks the 10th anniversary of the Severe Chronic Neutropenia International Registry (SCNIR), a registry organized to improve understanding and treatment of the hematological disorders causing severe chronic neutropenia. The SCNIR enrolls patients with blood neutrophil counts intermittently or continuously less than 0.5x109/L whose neutropenia is not attributed to cancer, cancer chemotherapy, or systemic autoimmune diseases. Longitudinal data has now been collected on 1163 patients with a range of follow-up of 0.01 to 15.66 years. By diagnostic category the patients include severe congenital neutropenia (422), cyclic neutropenia (205), idiopathic neutropenia (349), autoimmune neutropenia (68), glycogen storage disease (42), Barth syndrome (10), myelokathexis (8), Shwachman-Diamond syndrome (37), immune deficiency syndromes (7), and others (15). Overall, 1053 (90.5%) of these patients have been treated longitudinally with G-CSF (dose range 0.02 to 300 mcg/kg/day, median 3.33 mcg/kg/day) or received G-CSF transiently. Among other treatments, 76 patients were treated with allogeneic bone marrow or stem cell transplants (SCT). The reasons for SCT were myelodysplasia (MDS) or acute myeloid leukemia (AML) (43), chromosomal aberrations or G-CSF receptor mutation (9), partial or non-response to G-CSF (21), or other reasons (3). G-CSF has dramatically changed the natural history of these disorders reducing the occurrence of infection, hospitalization, and antibiotics, and improving patients’ quality of life. A small percentage of patients, 14% (163/1163), show evidence of osteoporosis/osteopenia; the predisposing factors for this complication remain unclear. MDS and AML has occurred in 63 patients: severe congenital neutropenia (13.7%, 58/422), Shwachman-Diamond syndrome (8.1%, 3/37) and 2 others with the clinical diagnoses of cyclic neutropenia (1) and idiopathic neutropenia (1). The SCNIR has also provided a rich resource for studies on the genetic and molecular basis for these disorders. These include the finding of mutations in the gene for neutrophil elastase (ELA2) in causing cyclic and congenital neutropenia, the role of mutations in the gene for the G-CSF receptor in the evolution of severe congenital neutropenia to AML and the importance of apoptosis as the cellular mechanism for several diseases causing severe chronic neutropenia. The SCNIR illustrates the value of a patient registry to improve our understanding of rare hematological diseases.
Idiopathic Hypereosinophilic Syndrome (HES) with FIP1L1-PDGFRA Rearrangement Can Be Effectively Treated with Imatinib.
Authors: Martinelli Giovanni , Cilloni Daniela , Ottaviani Emanuela , Malagola Michele , Messa Francesca , Rondoni Michela , Bosi Costanza , Gottardi Enrico , Rosti Gianantonio , Ricci Paolo , Gaitani Stavroula , Pane Fabrizio , Testoni Nicoletta , Mecucci Cristina , Soverini Simona , Piccaluga Pier Paolo , Amabile Marilina , Tiribelli Mario , Zaccaria Alfonso , Grafone Tiziana , De Vivo Antonello , Pileri Stefano , Fattori Pierpaolo , Bocchia Monica , Serra Anna , Maurillo Luca , Fanin Renato , Cross Nicholas C.P. , Merante Serena , Cazzola Mario , Alimena Giuliana , Frassoni Francesco , Galieni Piero , Russo Domenico , Gobbi Marco , Gugliotta Luigi , Lauria Francesco , Mazza Patrizio , Ferrara Felicetto , Gherlinzoni Filippo , Leoni Pietro , Musto Pellegrino , Baccarani Michele , Saglio Giuseppe
Published: October 2019

We studied 141 patients with HES, of which 55 primary HES (39%) defined as a peripheral blood eosinophilia greater than 1,500 cells/μL for longer than 6 months, absence of other apparent aetiologies for eosinophilia and symptoms of organ involvement. All patients were studied by molecular analysis for PDGFRB-TEL, FGFR1-BCR and BCR-ABL transcripts, frequently associated with HES/CMML/MDS syndrome: all these transcripts were absent in our series. We also sought for the recently reported involvement of PDGFRα, cryptically translocated with FIP1L1 in some HES pts responsive to Imatinib therapy: 13 pts (23%) were positive for the FIP1L1-PDGFRA rearrangement and all of them showed previously unreported, abnormal-sized fusion transcripts. Curiously, all FIP1L1-PDGFRA positive pts were male. We enrolled in a national clinical trial 31 (55%) primary HES pts, including all 13 (23%) FIP1L1-PDGFRA positive, with imatinib mesylate (100 to 400 mg/day). Median follow up of treatment was 4,5 moths (range 2–28). Rapid and complete haematological responses to imatinib therapy were recorded only in all FIP1L1-PDGFRA positive pts (100%) after one months of therapy and partial response in only one cases with HES without FIP1L1-PDGFRA fusion transcript. Complete molecular response without evidence of FIP1L1-PDGFRA transcript by qualitative RT-PCR was also recorded in all responding pts after median 2 months of therapy. We conclude that FIP1L1-PDGFRA rearrangement may be useful molecular marker of myeloproliferative HES, a predictor of imatinib-responsiveness and as a means to follow therapy in this subgroup of pts.
European Multicenter Experience on Idiopathic Hypereosinophilic Syndrome (HES) with FIP1L1-PDGFRA Rearrangement treated with Imatinib.
Authors: Martinelli Giovanni , Apperley Jane , Reiter Andreas , Cross Nicholas P. , Cilloni Daniela , Grimwade David , Hochhaus Andreas , Ottaviani Emanuela , Cools Jan , Prin Lionel , Malagola Michele , Walz C , Preudhomme Claude , Messa Francesca , Rondoni Michela , Gottardi Enrico , Rosti Gianantonio , Mecucci Cristina , Pane Fabrizio , Testoni Nicoletta , Soverini Simona , Merante Serena , Alimena Giuliana , Baccarani Michele , Hehlmann Rudiger , Aventin Anna , Sureda Anna , Gomez Jose-Roman , Duyster Justus , Verhoef Gregor , Saglio Giuseppe
Published: October 2019

Idiopathic hypereosinophilic syndrome (HES) is an infrequent hematological disorder characterized by persistent eosinophilia with organ involvement, often presenting to other medical specialists. We studied 89 primary HES defined as a peripheral blood eosinophilia greater than 1,500 cells/μL for longer than 6 months, absence of other apparent aetiologies for eosinophilia and symptoms of organ involvement. All patients were negative by molecular analysis for TEL-PDGFRB, and/or BCR-ABL transcripts, frequently associated with HES/CMML/MDS syndromes. We also sought for the recently reported involvement of PDGFRalpha, cryptically fused with FIP1L1 in some HES patients responsive to Imatinib therapy: 34 patients were positive for the FIP1L1-PDGFRA rearrangement and all of them showed previously unreported, abnormal-sized fusion transcripts. A specific RT-PCR assay has been set up for this analysis and a quantitative Q-RT-PCR Taqman assay is in development. Of note, all FIP1L1-PDGFRA positive patients were male. 56 out of 89 (62%) primary HES patients, including 31/34 FIP1L1-PDGFRA positive, were sensitive to imatinib mesylate (100 to 400 mg/daily). Median follow up of treatment was 10 months (range 2–28). Rapid and complete haematological responses (CHR) to imatinib therapy were recorded in all FIP1L1-PDGFRA positive patients (91%) after one month of therapy and partial response in nine cases with HES but without FIP1L1-PDGFRA fusion transcript. Complete molecular response without evidence of FIP1L1-PDGFRA transcript by qualitative RT-PCR was also recorded in the majority of responding patients after median 2 months of therapy, however in few cases the complete molecular response could be recognised only after more than 18 months of imatinib therapy. We conclude that FIP1L1-PDGFRA rearrangement is a useful molecular marker of myeloproliferative HES, a predictor of imatinib-responsiveness and as a means to follow therapy in this subgroup of patients.
DLEU2, Located within the CLL 13q14 Deletion Locus, Encodes an Antisense RNA for the Putative Bicistronic RFP2/LEU5 Gene in Humans and Mouse.
Authors: Corcoran Martin M. , Hammarsund Marianne , Zhu Chaoyong , Lerner Mikael , Kapanadze Bagrat , Larsson Catharina , Forsberg Lars , Ibbotson Rachel E. , Einhorn Stefan , Oscier David G. , Grander Dan , Sangfelt Olle
Published: October 2019

Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies, including chronic lymphocytic leukemia and mantle cell lymphoma. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, strongly suggesting that a nonclassical tumor-suppressor mechanism may be involved. Our recent analysis demonstrates that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5. Further characterisation of the 13q14 deletion locus distal to the RFP2/DLEU2 region has resulted in the identification of two novel evolutionary conserved genes DLEU7 and DLEU8, the latter of which may function as both a protein encoding and an cis-antisense gene. The combined data indicates that the CLL 13q14 deletion locus encompasses a highly complex gene regulatory system, the partial deletion of which may affect the post-transcriptional regulation of non-deleted genes in the vicinity.
Glycoprotein (GP) VI Is Associated with GPIb-IX-V on the Membrane of Resting and Activated Platelets.
Authors: Berndt Michael C. , Arthur Jane F. , Gardiner Elizabeth E. , Ozaki Yukio , Kahn Mark L. , Andrews Robert K.
Published: October 2019

The platelet collagen receptor, glycoprotein (GP)VI, initiates platelet aggregation at low shear stress while GPIb-IX-V, which binds von Willebrand factor, elicits platelet aggregation under high shear conditions. To investigate the possibility that GPIb-IX-V and GPVI are associated on the platelet surface, we first ascertained that aggregation induced by a GPVI-specific agonist, collagen-related peptide, like collagen, is markedly cross-blocked by a GPIbα-specific monoclonal antibody, SZ2. Immunoprecipitation of GPIb-IX with anti-GPIbα from the 1% (v/v) Triton-soluble fraction of unstimulated platelets and immunoblotting with anti-GPVI demonstrated association between GPIb-IX and GPVI. This association was maintained when platelets were activated by thrombin. Pre-treatment of platelets with methyl-β-cyclodextrin to disrupt lipid rafts did not affect association in resting or activated platelets under these conditions of detergent lysis. The association is also independent of cytoskeletal attachment, since it was unaffected by treatment with N-ethylmaleimide or DNaseI, which dissociate GPIb-IX from filamin and the actin-containing cytoskeleton, respectively. Finally, the association involves an interaction between the ectodomains of GPIbα and GPVI, since soluble fragments of GPIbα (glycocalicin) and GPVI are co-precipitated from the platelet supernatant under conditions where GPVI is shed. A contribution of GPIb-IX-V to GPVI-induced platelet responses, and vice versa, therefore warrants further investigation.
CD63 Modulates Spreading and Tyrosine Phosphorylation of Platelets on Immobilized Fibrinogen.
Authors: Israels Sara J. , McMillan-Ward Eileen
Published: October 2019

CD63 is a member of the tetraspanin superfamily of integral membrane proteins. Present on a variety of cells, tetraspanins form lateral associations with integrins and may act as ‘organizers’ of multimolecular networks that modulate integrin-mediated signaling, cell morphology, motility and migration. In resting platelets CD63 is present on the membranes of dense granules and lysosomes. Following platelet activation and exocytosis, CD63 relocates to the plasma membrane where it associates with the platelet integrin αIIbβ3-CD9 complex, and with the actin cytoskeleton in an αIIbβ3-dependent manner. In the current study, D545, a monoclonal antibody directed at the second extracellular loop of CD63, was used to investigate the role of CD63 in platelet adhesion, spreading and tyrosine phosphorylation, as determined by immunofluorescence microscopy, confocal imaging and immunoblotting. As previously demonstrated, D545 did not alter adhesion of platelets to fibrinogen, but did affect platelet spreading. Thrombin-activated platelets showed marked spreading, F-actin reorganization, redistribution of vinculin and extensive tyrosine phosphorylation, all of which were inhibited by D545. Similarly D545 inhibited the phosphorylation of focal adhesion kinase in thrombin-activated adherent platelets. These results suggest that CD63 may modulate αIIbβ3-dependent cytoskeletal reorganization.

In some malignant cell lines CD63 has been shown to associate with phosphatidylinositol (PI) kinase. To address a similar association in platelets, lipid kinase assays were performed on D545 immunoprecipitates, using PI as substrate. CD63 co-immunoprecipitated with a lipid kinase that was stimulated by non-ionic detergents, inhibited by adenosine, but unaffected by wortmannin. These enzymatic properties are consistent with a PI 4-kinase type II. The CD63-PI 4-kinase complex was co-purified from both resting and activated platelets and therefore was not activation-dependent. The linkage of CD63 with PI 4-kinase may result in the recruitment of the signaling enzyme to specific membrane locations and influence phosphoinositide-dependent signaling.
Two Distinct Domains Mediate Self-Assembly of Knob Protein in Plasmodium Falciparum-Infected Human Erythrocytes.
Authors: Chen Huiqing , Li Xuerong , Oh Steven S. , Chishti Athar H.
Published: October 2019

Plasmodium falciparum associated mortality is believed to originate from the adhesion of infected erythrocytes to the endothelial cells. The adhesion of mature parasite infected erythrocytes is mediated by the surface protrusions called knobs. A dominant constituent of knobs is the parasite derived knob-associated histidine-rich protein termed KAHRP or knob protein. Gene deletion experiments have shown that the knob protein is essential in malaria pathogenesis. Without the knob protein (KAHRP), adherence of malaria-infected erythrocytes is not established under physiological shear stress. The adhesive properties of infected erythrocytes are propagated by the direct interaction of KAHRP, a peripheral membrane protein, with the cytoplasmic domain of the P. falciparum erythrocyte membrane protein-1 (PfEMP1). Unlike the highly polymorphic extracellular domain, the cytoplasmic domain of PfEMP1 is conserved across species, hence the interest in delineating the mechanism of its interaction with KAHRP and components of the erythrocyte membrane. Our studies have observed recombinant KAHRP to self-associate into large aggregates in vitro. Under transmission electron microscopy, both knobs isolated from malaria-infected erythrocytes and self-assembled recombinant KAHRP in vitro have revealed a unique spiral shaped morphology. Based on these observations, we hypothesized that the unique knob spiral is formed by the self-assembly of KAHRP monomers and therefore must be encoded by distinct sequence elements within KAHRP. To investigate this speculation, we devised a solution binding assay and engineered a series of GST (glutathione S-transferase) and Trx (thioredoxin) fusion peptides to encompass the entire length of KAHRP. Binding measurements using heterologous combination of defined fusion peptides reveal a core histidine-rich amino terminal segment associating with a lysine-rich segment located within the carboxyl half of KAHRP. These findings suggest a novel head-to-tail interaction strategy employed by the intracellular parasite to assemble the spiral-shaped scaffold of KAHRP under the surface of electron dense knobs. Significantly, our results identify a novel self-interaction site within KAHRP that could be amenable to therapeutic intervention against cerebral malaria.
Red Cell Pyruvate Kinase Deficiency: Molecular Characterization of 10 New Variants.
Authors: Fermo Elisa , Bianchi Paola , Vercellati Cristina , Cotton Frederic , Zanella Alberto
Published: October 2019

PK deficiency is the most common glycolytic enzyme defect associated with chronic non-spherocytic hemolytic anemia. To date about 150 different mutations have been identified in the PK-LR gene. Among them only one large deletion has been described in Gipsy resulting in the loss of exon 11. We report 10 new variants of LR-PK gene in 8 families with pyruvate kinase deficiency. The entire coding region and intronic flanking regions were analyzed by direct sequencing. The results of the molecular analysis are reported in the table:

Pt Origin Hb (g/dL) Tx (n.) PK Activity (IU/gHb) Mutation Aminoacidic substitution SD Italy 15.1 0 10.6 107G / ? Ala36Gly/? TT Australia 8.9 0 nd 409A / del5006bp( IVS3-nt 1431) Ala137Thr / del ex 4-11 NR Italy 9 >50 5.5 661A /1209A Asp221Asn /Met403Ile NA Italy 9 >50 5.3 661A /1209A Asp221Asn /Met403Ile SA Italy 12 0 5.5 1456T/ 1209A Arg486Trp/ Met403Ile SC Italy nd nd 5.6 1529A/ 859C Arg510Gln/ Phe287Leu CM Italy 9.5 0 13.6 1456T/ 958A Arg486Trp/ Val320Met PS Italy 13.2 0 8 1094T /? Lys365Met /? VR Italy 10.5 0 10.7 1706A / ? Arg569Gln /? GR Guinea 14.4 0 7.6 1269A/ IVS9+43c Splice site/? Ref. Values 12.2–16 11.1–15.5

Mutations reported in bold are new. By comparing the amino acids sequences among several species (cat M1, chicken M, rat L, yeast and human), we found that mutations 661A, 859C, 958A, 1094T and 1209A involve highly conserved residues. Mutation 1209A when present in association with 661A (cases NA and NR) results in a severe clinical pattern with need of transfusion support, whereas in compound heterozygosity with 1456T (case SA, mother of NA and NR) is associated with a less severe clinical pattern. The variant 1706A was found in a patient carrying the polymorphism 1705C at the homozygous level; the mutation in association with the polymorphism determines the aminoacidic substitution Arg596Gln.

A deletion of 5006 nucleotides extending from intron 3 to the last 3 nucleotides of exon 10 has been found in an Australian baby dead at birth (case TT); the mutation results in a large cDNA deletion encompassing exon 4 and exon11 included. This is the largest abnormality so far detected in LR-PK gene.
Genome-Wide Identification of Cis-Regulatory Sequences Controlling Blood and Endothelial Development.
Authors: Göttgens Berthold , Donaldson Ian J. , Chapman Michael , Kinston Sarah , Landry Josette Renée , Knezevic Kathy , Piltz Sandie , Buckley Noel , Green Anthony
Published: October 2019

Haematopoiesis has long served as a paradigm for adult stem cell systems and studies over the last 20 years have established that transcriptional control is central to the specification and subsequent differentiation of haematopoietic stem cells (HSCs). With many of the key transcription factors known, haematopoiesis provides a powerful cellular system for the analysis of mammalian gene regulatory networks. The key missing ingredient, particularly for the stem and progenitor cell stages, is a set of experimentally validated gene regulatory regions together with a molecular understanding of their biological activity. Despite progress in lower organisms, genome-wide computational identification of mammalian cis-regulatory sequences has been hindered by increased genomic complexity and cumbersome transgenic assays. In order to identify transcriptional control regions active during early embryonic haematopoietic development, we have developed a strategy that is based upon a haematopoietic stem cell enhancer (+19 enhancer) identified downstream of the SCL transcription factor, a key regulator of haematopoietic stem cell formation. Starting with this well-characterised blood stem cell enhancer, we have developed computational tools which allow genome-wide identification of functionally related enhancers. This approach has been used to identify novel enhancers involved in the regulation of early blood and endothelial development, and which exhibit predicted biological activity in vitro and in transgenic mice. Transcription factors binding to these enhancers have been identified by chromatin immunoprecipitation. Our data allow the construction of an experimentally verified nascent transcriptional network, which controls the development of blood and endothelium, and which so far contains the SCL, GATA-2, Fli-1, Elf-1 and Hex transcription factors. Additional candidate members of the network have been identified and are being subjected to functional validation. This approach represents a widely applicable strategy for characterising the transcriptional networks controlling a broad range of mammalian developmental programmes.
Effects of Single Point Mutations within Proposed Copper Binding Sites on Specific Activity and Inter-Chain Affinity of Factor VIII.
Authors: Wakabayashi Hironao , Zhou Qian , Nogami Keiji , Fay Philip J.
Published: October 2019

Copper ions appear important for the structural integrity and the function of factor VIII. Reconstitution studies have shown that Cu2+ increases specific activity of factor VIII as well as increases affinity between heavy chain (HC) and light chain (LC). Based on the ceruloplasmin homology, the existence of three Cu2+ binding sites has been proposed. These include a type 1 site in HC (A1 domain) coordinated by C310, H315, H267, and M320, a type 1 site in LC (A3 domain) coordinated by C2000, H1954, H2005, and M2010, and type 2 site spanning A1 and A3 domains coordinated by H99 and H1957 with possible other additional residues. We rationalized that point mutations within a putative Cu2+ coordination sites would diminish Cu2+ binding at that site, as detected by changes in factor VIII specific activity and/or inter-subunit affinity. We produced several mutant factor VIII proteins bearing point mutation of C310S, H315A, C2000S, H1954A, H99A, or H1957A using a B-domainless human factor VIII vector. Each mutant was stably expressed and purified by SP-sepharose, which bound factor VIII primarily though LC. Western blotting indicated a reduction in the relative amount of HC in C310S and H99A factor VIII forms, suggesting that inter-chain affinity was reduced by these mutations. EDTA-treated factor VIII forms in the presence of Ca2+ were titrated with Cu2+ and activity was monitored using a factor Xa generation assay. The concentration of free Cu2+ was controlled by the presence of EDTA and Ca2+ based on the known values for Cu2+-EDTA and Ca2+-EDTA affinities. The activity regain observed for wild type factor VIII following titration with Cu2+ yielded a Kd = 11.5 ± 2.2 fM. All of the mutants tested retained a high affinity response to Cu2+, suggesting that single point mutations were not sufficient to eliminate Cu2+ binding. However, while Kd values for H1957A (10.3 ± 2.2 fM), H99A (2.1 ± 1.9 fM), H315A (6.7 ± 3.1 fM), and C310S (12.7 ± 3.4 fM) retained similar or slightly reduced affinity for Cu2+, the Kd values for C2000S (304 ± 105 fM) and H1954A (365 ± 105 fM) showed a significant reduction in Cu2+ affinity. When factor VIIIa subunits were titrated with Cu2+ and monitored by intrinsic fluorescence, only purified A1 subunit showed a saturable effect of the signal (Kd = 11.9 ± 4.9 fM), indicating that this subunit contained a high affinity Cu2+ binding site. Taken together, these results suggest that occupancy of the type 1 Cu2+ binding site in A3 contributes to the Cu2+-dependent increase in specific activity of factor VIII and that the type 1 site in A1 and/or the type 2 site contributes to HC-LC association.
Safety and Efficacy of Low Molecular Weight Heparins for Hemodialysis in Patients with End-Stage Renal Disease: A Meta-Analysis of Randomized Trials.
Authors: Lim Wendy , Cook Deborah J. , Crowther Mark A.
Published: October 2019

Background: The anticoagulant management of patients with thromboembolic disease who also have end-stage renal disease is a frequently encountered and unresolved clinical problem. Low molecular weight heparins (LWMHs) are the preferred initial treatment for these disorders but are renally excreted, and generally avoided in patients with renal failure due to concerns of bioaccumulation and increased bleeding risks. Unfractionated heparin (UFH) remains the primary anticoagulant used in this patient population despite the efficacy and convenience of LMWHs. Although LMWHs are typically not used to treat thrombosis in patients with end-stage renal disease, they are frequently used during hemodialysis to prevent thrombosis of the extracorporeal dialysis circuit. While there are no randomized data evaluating the safety and efficacy of LMWHs in these patients, randomized trials have been performed using LMWHs in the setting of hemodialysis. We performed a systematic review and meta-analysis of these randomized trials summarizing this data.

Purpose: To evaluate the safety and efficacy of LMWHs compared to UFH for preventing thrombosis of the extracorporeal dialysis circuit.

Data sources: We identified studies using MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and FirstSearch, reviewed reference lists and contacted pharmaceutical companies.

Study selection: We included randomized, controlled trials that compared a LMWH with another anticoagulant during hemodialysis in patients with end-stage renal disease, and reported at least one of the following outcomes: bleeding, extracorporeal circuit thrombosis, or anti-Xa levels.

Data extraction: Two reviewers independently extracted data on methodologic quality, study design, clinical outcomes, and anti-Xa levels.

Data synthesis: We included 17 trials in this systematic review, 11 of which were included in the meta-analysis. We found that LMWH did not significantly affect the number of bleeding events (relative risk [RR] 0.96, 95% CI, 0.27 to 3.43), bleeding assessed by vascular access compression time (weighted mean difference −0.87, 95% CI, −2.75 to 1.02) or extracorporeal circuit thrombosis (RR 1.15, 95% CI, 0.70 to 1.91) as compared with UFH.

Conclusions: LMWHs appear to be as safe as UFH in terms of bleeding complications, and as effective as UFH in preventing extracorporeal circuit thrombosis. However, inferences from these trials assessing anticoagulation for patients undergoing hemodialysis will continue to be weak until larger, more rigorous randomized trials are conducted.