bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect Of Maternal Cigarette Smoking On Newborn Iron Stores
Authors: Pateva Irina , Kerling Elisabeth , Carlson Susan , Reddy Manju , Chen Dan , Tancabelic Jakica
Published: October 2019

Objective

Previous small-scale studies suggest that maternal smoking lowers neonatal body iron. Our objective was to study and compare the relationship between maternal and infants’ body iron in smokers and non-smokers in a large matched-pair cohort.

Method

This was a prospective cohort study involving 144 mothers – 72 smokers and 72 non-smokers and their respective infants. Samples were obtained from maternal blood and infants’ cord blood at delivery for serum transferrin receptor (sTfR) and ferritin levels. Serum TfR and ferritin levels were measured by RAMCO ELISA and RIA assays. The total body iron (TBI) was calculated using the sTfR/ferritin ratio.

Results

Maternal total body iron and smoking status

Women who smoked had lower sTfR, higher ferritin and higher body iron compared to nonsmoking women.

Infant’s total body iron, measurements at birth and smoking status

In contrast to their respective mothers, we found a small but statistically significant negative correlation between smoking and infants’ total body iron. The number of PPD smoked was negatively correlated with infants’ ferritin and total body iron. The number of days smoked during pregnancy was also negatively correlated with infants’ ferritin and total body iron and positively correlated with infants' sTfR.

Birth weight was lower in babies of smokers compared to nonsmokers (mean /- SD =3270 +/-475 vs. 3393 g +/- 475 g, p=0.03). Correlation studies revealed that birth weight in infants of smokers was negatively correlated with PPD smoked and number of days smoked. Birth length in the same infants was also negatively correlated with PPD smoked and number of days smoked.

Conclusion

Mothers who smoked during pregnancy had higher iron stores but their newborn infants had lower iron stores than those of non-smoking mothers. There may be a negative dose-dependent response between fetal smoke exposure and infant iron stores.

Disclosures:

No relevant conflicts of interest to declare.
Microparticle Profile During Painful Crisis and Steady State Period In Sickle Cell Anemia
Authors: Atmis Anil , Sasmaz Ilgen , Antmen Bulent Ali ,   Yurdanur - Kilinc ASH Member ID425205
Published: October 2019

Introduction

Sickle cell anemia is a disease which is characterized with hemolytic anemia, hypercoagulopathy and painful crisis. Microparticles are 0,1-1 µm sized little membrane particles which are derived during activation or apoptotic phase of cell cycle. It is reported that microparticles are increased in many systemic disease including sickle cell anemia.

Aim

In this study we aimed to investigate the role of microparticles on during crisis and non-crisis periods in sickle cell anemia patients.

Materials and method

Twenty nine patients, following by Cukurova University, Department of Pediatric Hematology, are included in this study. Blood samples were collected in 26 of these patients in non-crisis period. Control group formed with 18 healthy children without any systemic disease. Complete blood count, hemoglobin electrophoresis and biochemical parameters were studied in both groups. Also patients’ total microparticle levels, erythrocyte (CD235a), endothelial (CD106), monocyte (CD14) particle levels and tissue factor expressing (CD142) microparticle levels were studied by flow cytometry and whole data was statistically analyzed.

Findings

Hemoglobin and hematocrit levels were significantly low in sickle cell anemia patients (p<0,001). Levels of HbS were significantly high during crisis period comparing with mean HbS levels during non-crisis period (p<0,001). Total microparticle levels were significantly high in sickle cell anemia patients with painful crisis comparing with control group (p<0,05). Erythrocyte and monocyte microparticle levels were significantly high in patients with painful crisis comparing with non-crisis periods (p<0,05). Endothelial and tissue factor expressing microparticle levels were high in patiens with crisis comparing to non-crisis period but this was not statistically significant (p>0,05). There was not any significant relation with frequency of crisis and microparticle levels (p>0,05). Microparticle levels were low in patients whose were taking hydroxiurea treatment comparing with non-hydroxiurea treatment but this data was not statistically significant (p>0,05).

Result

As a result we found high levels of total microparticle, erythrocyte and monocyte microparticles in sickle cell anemia patients during painful crisis period. This important clue of crises was need further studies in order to understand the effect of microparticles on pathophysiology of sickle cell anemia.

Disclosures:

No relevant conflicts of interest to declare.
The Cerebral Vasculopathy In Malian Sickle Cell Anemia Children: Lesson From Routine Transcranial Doppler Screening
Authors: Diallo Dapa Aly , Guindo Aldiouma , Dorie Alain , Touré Boubacari Ali , Fané Baba , Sarro Yaya , Tchernia Gil
Published: October 2019

Cerebral vasculopathy is one of the major complications of Sickle Cell Disease (SCD). 11% of the homozygous SCD patients experience stroke by age of 20 years. The use of Transcranial Doppler (TCD) allows identification of patients at risk for stroke and leads to implementation of a preventive treatment that contributes to limit the burden of SCD particularly during childhood. Using TCD screening, we evaluated the prevalence and incidence of cerebral vasculopathy in Malian SCD children. During the years 2008 to 2013, 572 children, 249 girls and 323 boys, age range (2-17yrs) were routinely screened by TCD at our Sickle Cell Disease Research and Control Center of Bamako, Mali. The overall prevalence of cerebral vasculpathy defined by conditional (1.5-1.79 cm/sec) and abnormal TCD (≥ 1.80 cm/sec) in this population is 17.1%. The highest proportion (92.9%) was observed in homozygous SCD patients while the percentage of affected patients was much lower in S/β0thalassemia (4.1%) and in SC (3.1%) patients. No cases were observed in S/β+thalassemia patients. SCD children <9 years old were more susceptible to cerebral vasculopathy complications than those above this age threshold (P<0.001). Low hemoglobin levels and low fetal hemoglobin were associated with an increased risk of cerebral vasculopathy. During the study, 4 of 444 children with normal TCD converted to conditional TCD, while 5 of 68 children with conditional TCD converted to abnormal TCD. This conversion from conditional to abnormal TCD was associated with a mean decrease in Hb value of 0.37g/dL (P=0.002). In conclusion this study shows high prevalence and incidence of cerebral vasculopathy in Malian SCD children. While chronic transfusion programs significantly reduces the risk of stroke in SCD patients with abnormal TCD, at present there are no well articulated strategies to prevent conversion from conditional to abnormal TCD. A more comprehensive approach would hopefully reduce the morbidity and mortality due to cerebral vasculopathy in SCD affected children.

Disclosures:

No relevant conflicts of interest to declare.
Thalassemia and Hemoglobinopathy Screening By HPLC Method and Comparison With Conventional Methods
Authors: Hosseeini Soudabeh , Kalantari Ebrahim , Dorgalaleh Akbar , Bamedi Taregh , Farzi Massomeh , Saeed Dorgalele
Published: October 2019

Background

Thalassemia and hemoglobinopathies are heterogeneous group of inherited disorders that affects men and women equally. HPLC is a valuable method for hemoglobinopathy and/or thalassemia carrier screening. This study evaluate the role of cation exchange HPLC along with adjunctive tests as needed in the diagnosis of thalassaemias/haemoglobinopathies and to see the frequency of these disorders in the Iranian population.

Methods

This five-year study was conducted on 3780 patients. Initially complete blood count was done by autoanalyzer and then for detection of abnormal hemoglobins HPLC and HB electrophoresis methods was used. In cases with low MCV and MCH indices (MCV<80 fl,MCH<27 pg) and Hb-A2< 3.5% and normal Hb-electrophoresis, α-thalssemia trait(αα/--)was considered in the list of differential diagnosis. In cases with low MCV for exclusion of iron deficiency serum ferritin was meseared.

Results

Our rerults revealed that 1932 (51.11%) had normal electrophoretic pattern, 781 (20.66%) had β-thallasemia trait and 487(12.84%) had β-thallasemia major or intermedia,328( 8.67% ) had normal electrophoresis along with iron deficiency and 142 ( 3.75%) had normal Hb -electrophoresis and normal iron status but low MCV and MCH indices.We also identified 11(0.29%) with Alpha thalasemia variants Hb-H disease/alpha trait and 22(o.58%)with sickle trait and 18(o.47%)with sickle disease and 9(0.23%) S-Thal double heterozygote and 5(0.13%) with E- trait and 32(0.84%) with Hb-D variant and 1(0.026%) with heterozygote Hb-C variant and 5(0.13%) with Hb-D Iran and 1(0.026%) with Hb-J trait and 1(0.026%) Hb-S/D double heterozygote, and 1(0.026%) with Hb-D/J double heterozygote and 1(0,026%) with Hb-constant spring/ HB-H double heterozygote.

Conclusion

HPLC is a fast and reliable method in clinical laboratories specially in premarital, and neonatal screening laboratories.

Disclosures:

No relevant conflicts of interest to declare.
Substrate Reduction Therapy With Miglustat In Type 1 Gaucher Disease In Spain. Nine Years Outcomes Update On ZAGAL Study
Authors: Giraldo Pilar , Andrade Marcio M , Medrano Blanca , Alfonso Pilar , Irún Pilar , Atutxa Koldo , Fernandez-Galan Angeles , Barez Abelardo , Franco Rafael , Roig Inmaculada , Giner Vicente , Villalon Lucia , Martinez-Estefano Elisa , Luño Elisa , Loyola Ines , Salamero Olga , la Serna Javier de , Pocovi Miguel
Published: October 2019

Substrate reduction therapy (SRT) has showed as an useful therapy in type 1 GD patients with mild or moderate disease. The main limitation to use miglustat has been the gastrointestinal adverse events. Since 2004 we have conducted the ZAGAL (Zavesca en Gaucher Leve) study for monitoring the real-life use of miglustat in Spanish adult patients with mild-to moderate disease. The study included GD1 patient’s naïve to therapy as well as patients who have previously been treated with ERT. For follow-up, the therapeutic goals for GD therapy by Pastores GM et al were applied.

To date a total of 351 GD1 patients have been diagnosed in Spain (FEETEG unpublished. 2013); from 2004 until 2013, 53 of them have been exposed of miglustat (15.1%). In 16 patients (30.2%) miglustat was the first line of therapy and the remaining 38 switched from ERT.

Currently 37 patients (mean age 43.6 y, range 22-83) 50.9% females, are on miglustat therapy (20 at least for 5 years and 13 during more than 8 years). Related to efficacy, the therapy permit to achieve a stable hemoglobin concentration level and spleen reduced volume, but we recorded a decrease of absolute platelet count in 15 patients (28.3%) (mean: 35x109/L, range: 10-86x109/L). Related to biomarkers changes an increase in CT activity was observed in 56.6% of patients (mean: 2,448 nmol/mL.h, range 135-13,687) and 43.4 % for CCL18/PARC (mean: 250 ng/mL, range: 19-1016). Seventeen patients (32.1%) had transitory gastrointestinal disturbances. Sixteen patients (30.2%) discontinued therapy: one of them for pregnancy, two by bone crisis, two by weight loss, one for bad compliance and ten by gastrointestinal discomfort or intolerance (18.8%). 60% of patients has a fine tremor in first months on therapy. 4 died by non-related causes (2 cancer, 1 hearth attack, 1 liver failure),

Conclusion

The long term follow-up of GD patients treated with Miglustat shows that more than 69% had achieved and maintains the therapeutic goals. A high individual variability had been observed related to miglustat gastrointestinal intolerance apparently no related with GBA genotype, gender and age, but possibly associated with disacaridases inhibition and food habits.

Disclosures:

No relevant conflicts of interest to declare.
The Profile Of The T Helper, Cytotoxic T and Natural Killer Cells In Sickle Cell Anemia Patients With Ischemic Attack and The Relation With Clinical Prognosis
Authors: Kilinç Yurdanur , Atmis Bahriye - , Atmis Anil , Yilmaz Mustafa , Sasmaz Ilgen , Karagün Barbaros Sahin
Published: October 2019

Purpose

The study is conducted in 29 sickle cell anemia (SCA) patients, who are in ischemic attacks (in vasoocclusive crises period) and in steady state; the level of T helper cells, cytotoxic T cells and natural killer cells were determined and the effect of ischemic attack on clinical prognosis and the immune functions.

Materials and Methods

In the study, 29 patients with sickle cell anemia in ischemic attacks with painful crises and in steady state and 24 healthy children were chosen in the same age group for control group. These groups were examined with complete blood cells count, Hb electrophoreses and the blood biochemistry. The study was performed by flowcytometric method to find the level of CD3 monoclonal antibody for total T lymphocyte, CD4 monoclonal antibody for T helper cells, CD8 monoclonal antibody for cytotoxic T cells and CD16+56 monoclonal antibodies for natural killer cells in the mentioned groups to compare the statistical data.

Findings

The average of HbS in ischemic periods in SCA patients are to be found 83±6,6 %. There were decrease in hemoglobin and hematocrit levels in SCA versus control group (p<0,001). The levels of CD3 are lower in SCA patients with crises period (62,31±7,79 %) than in steady state (65,53±5,72 %) and in control group (69,09±9,18 %) (p=0,007). The natural killer cells in crisis period (13,07±7,67 %) and in steady state (12,71±5,62 %) were lower than in control group (8,11±4,67 %) (p=0,009). In SCA patients, the level of CD3,CD4, CD8 and CD16+56(+) T cells are not changed with the frequency of crisis (p>0,05).

Results

As a result of the study, in the patients with sickle cell anemia whom associated with chronic hemolysis and tissue hypoxia during the ischemic attack, the levels of total T lymphocyte (CD3) was found significantly lower than the control groupp<0.001. The levels of T helper cells (CD4) and cytotoxic T cells (CD8) did not differ between SCA patients and control group. The levels of natural killer cells (CD16+56) was found higher in crises period and steady state than control group. To find the T cell subset levels in clinical and prognostic effects in SCA, it is necessary to do study with bigger groups of patients and wide range of study.

Disclosures:

No relevant conflicts of interest to declare.
Self- and Parent/Proxies-Reported Health-Related Quality Of Life In Children and Adolescents With Bleeding Disorders: Cross Sectional Investigation and Comparison With Healthy Siblings and Healthy Peers
Authors: Bruno Neuner , von Mackensen Sylvia , Halimeh Susan , Holzhauer Susanne , Klamroth Robert , Kurnik Karin , Kruempel Anne , Fruewald Michael , Krampe Henning , Nowak-Goettl Ulrike
Published: October 2019

Background

Hereditary bleeding disorders (HBD) affect children from birth during their life course. Medical treatment and the overall prognosis of children with HBD have significantly progressed. But even in patients cared for in specialized treatment centers it remains unclear whether survival till adulthood goes along with health-related quality of life [Hr-QoL] comparable to other patients groups respectively comparable to population children. Therefore aim of the present study was to assess self- as well as parents/proxies reported Hr-QoL in children and adolescents with HBD and to compare the results with findings from children affected by another chronic medical condition respectively with healthy siblings and peers.

Methods

91 patients with HBD (thrombosis / deep venous thrombosis, DVT, respectively Haemophilia A and B) aged 8 to 16 years from 6 Hemophilia study centers were investigated regarding Hr-QoL. Hr-QoL was assessed in patients and parents/proxies with the generic KINDL-R questionnaire exploring overall well-being and 6 sub-dimensions (physical well-being, psychological well-being, self-esteem, family-related well-being, friend-related well-being, and school-related well-being). Findings were compared with 70 children with stroke / TIA respectively with healthy controls (45 healthy siblings and 106 healthy peers).

Results

Overall well-being in children with thrombosis / DVT (77.6 ± 9.7 points) was comparable to healthy controls but in children with Haemophilia A or B (76.0 ± 9.6) lower compared with healthy peers (80.2 ± 9.7, p = 0.005). No differences occurred between healthy controls and children with stroke/TIA (74.2 ± 10.3). In children with HBD none of the KINDL-R sub-dimensions showed values below healthy siblings. Two KINDL-R sub-dimensions showed lower values compared with healthy peers. Parents/proxies rated the Hr-QoL of their children with HBD similar to their healthy children. The internal consistency of overall well-being in all study subgroups was acceptable (> 0.7) but showed poor results (< 0.5) in two KINDL-R sub-dimensions.

Discussion

The application of a generic Hr-QoL questionnaire in a sample of children and adults with HBD seems feasible. Their Hr-QoL seems comparable to their siblings but below their healthy peers.

Disclosures:

Halimeh: Octapharma AG: Investigator Other, Research Funding.
High-dose melphalan and granulocyte-macrophage colony-stimulating factor for refractory multiple myeloma
Authors: Barlogie B , Jagannath S , Dixon DO , Cheson B , Smallwood L , Hendrickson A , Purvis JD , Bonnem E , Alexanian R
Published: October 2019

High-dose melphalan has induced remissions in about 40% of patients with refractory myeloma, but the mortality has been high, at about 20%, due to complications of prolonged granulocytopenia. In an attempt to stimulate earlier granulocyte recovery, recombinant human granulocyte- macrophage colony-stimulating factor (GM-CSF) was administered subcutaneously to 23 patients with refractory myeloma who had been treated with melphalan at a high dose of 100 mg/m2. Thirty-nine percent of patients achieved marked tumor cytoreduction by at least 75%, 2 died within 2 months from infectious complications during severe neutropenia; and median durations of relapse-free and overall survival were 7 and 10+ months, respectively. The nine patients presenting with both advanced age over 50 years and a long history of prior therapy of over 1 year required significantly longer median times of 31 days for granulocytes and of 63 days for platelets to reach safe levels of at least 500/microL and 50,000/microL, respectively, than the 14 remaining patients who had none or only one of these adverse features (21 and 26 days, respectively). In a historic control of 43 patients treated previously with high-dose melphalan but without GM-CSF, hematologic recovery to the aforementioned levels of granulocytes and platelets proceeded over almost 5 weeks, regardless of age and prior treatment exposure. Thus GM-CSF seems to hasten marrow recovery, especially in patients with adequate normal marrow stem-cell reserve as defined by younger age or less prior therapy. While not shortening the duration of neutropenia, GM-CSF dose increments (from 0.25 to 0.5 to 0.75 mg/m2) increased the incidence of severe toxicity from 0% to almost 40%, especially among older patients. These results support the usefulness of low-dose GM-CSF (0.25 mg/m2) in stimulating marrow recovery in selected patients with adequate marrow reserve treated with high-dose melphalan for refractory multiple myeloma.
Quinine-induced immune thrombocytopenia associated with hemolytic uremic syndrome: a new clinical entity
Authors: Gottschall JL , Elliot W , Lianos E , McFarland JG , Wolfmeyer K , Aster RH
Published: October 2019

Three patients are described who developed severe thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure after ingestion of quinine. In one patient, the same clinical findings recurred several months later after another exposure to quinine. Serum from one patient contained quinine-dependent IgG antibodies reactive with the platelet glycoprotein (GP) Ib/IX complex. In the second and third cases, serum contained IgG and IgM antibodies reactive with both the GP Ib/IX and IIb/IIIa complexes in the presence of quinine. Quinine appears to have induced both immune thrombocytopenia and the hemolytic uremic syndrome (HUS) in these individuals. Findings made in these cases may have implications for the pathogenesis of some forms of HUS.
Mutations of the p53 gene in lymphoid leukemia
Authors: Sugimoto K , Toyoshima H , Sakai R , Miyagawa K , Hagiwara K , Hirai H , Ishikawa F , Takaku F
Published: October 2019

p53 is currently considered to be a tumor suppressor gene product, and its alterations are suggested to be involved in several human malignancies. Here we show evidence of the possible involvement of p53 gene mutations in lymphoid leukemias studied by reverse transcriptase- polymerase chain reaction, single strand conformation polymorphism analysis, and nucleotide sequencing. Fourteen patients with various leukemias were examined and two with acute lymphoblastic leukemia and one with Waldenstrom's macroglobulinemia were identified to have mutations in the coding region of the p53 gene. These mutations included point mutation, triplet deletion, and single nucleotide insertion. Furthermore, expression of the wild-type p53 mRNA was not detected in the samples from these three patients. In one of them, chromosome 17p was deleted, suggesting the absence of the nonmutated p53 gene, whereas in the other two patients, chromosome 17p seemed to be intact by cytogenetic analysis. Our results suggest that alterations of the p53 gene may have a role in the genesis of some leukemias.
Molecular biology of the Rh antigens
Authors: Agre P , Cartron JP
Published: October 2019

The RBC Rh antigens are of large clinical importance, but until recently have been poorly understood at a molecular level. The Rh polypeptides are a family of nonglycosylated Mr 30- to 32-Kd transmembrane proteins that are core structural components of the Rh antigens and have been purified and partially characterized biochemically. Rh polypeptides are present in RBCs from normal humans and other mammalian species and are probably required for normal membrane integrity, because they appear to be missing from the RBCs of the rare Rhnull individuals that express several membrane defects. The Rh polypeptides contain an exofacial free sulfhydryl that is important for Rh antigenic reactivity and several intracellular sulfhydryls that appear to be palmitylated, but most of the molecule appears to reside between the leaflets of the phospholipid bilayer. The cDNA coding for a 416-amino acid Rh polypeptide was recently isolated but was not found to share sequence homology with any known protein, and Northern analysis indicated that Rh is erythroid specific. The Rh antigens within the native membranes are thought to exist as a complex of Rh polypeptides and multiple other membrane components, including certain Rh-related glycoproteins. While it is thought that this assembly may be important for the Rh antigenic reactivity, the structural basis of this remains to be established. While the physiologic role of Rh is yet to be defined, several clues indicate that it may play a role in the organization of membrane phospholipids or synthesis or membrane expression of various glycoproteins. While our knowledge of Rh is still very incomplete, recent research has significantly advanced the molecular understanding of these important blood group antigens.
Detection of minimal residual disease in T-cell acute lymphoblastic leukemia using polymerase chain reaction predicts impending relapse
Authors: Neale GA , Menarguez J , Kitchingman GR , Fitzgerald TJ , Koehler M , Mirro J Jr , Goorha RM
Published: October 2019

After achieving remission, approximately one-third of patients with T- cell acute lymphoblastic leukemia (T-ALL) relapse due to the resurgence of residual leukemic cells that cannot be detected in remission by morphologic methods. Thus, the early detection of residual disease is highly desirable to monitor the efficacy of therapy, or to institute an alternative mode of therapy. Toward this aim, we have examined the applicability of polymerase chain reaction (PCR) amplification in the detection of minimal residual disease (MRD) in bone marrow samples from patients with T-ALL in morphologic remission. Two different approaches were taken to identify leukemic clone-specific sequences that could be used as targets for PCR amplification. The first technique used T-cell receptor-delta (TCR-delta) gene rearrangements that were sequenced directly after PCR amplification of leukemic DNA. This method was successful in generating clone-specific probes for 76% of T-ALL patients screened. An alternative method was used to clone and sequence a TCR-beta chain gene from leukemic cells to generate a specific probe. The PCR assays that we used were specific for each patient's leukemic clone, and were capable of routinely detecting one leukemic cell in 10(4) normal cells. Using these sensitive PCR-based assays, we found no evidence for persistence of the leukemic clone in any of the bone marrow samples from four T-ALL patients who are in long-term (3.9 + to 8.1 + years) remission. In contrast, we detected residual disease in clinical remission samples from two patients who subsequently relapsed. In one patient, where we had appropriate samples, we observed a dramatic expansion of the leukemic clone 3 months before clinical relapse. These results suggest that PCR-based assays for detection of MRD in T-ALL patients have great potential in predicting impending relapse, and in determining the efficacy of the anti-leukemic therapy. These methods may also allow the identification of long-term survivors.
Retinoic acid-induced granulocytic differentiation of HL60 human promyelocytic leukemia cells is preceded by downregulation of autonomous generation of inositol lipid-derived second messengers
Authors: Porfiri E , Hoffbrand AV , Wickremasinghe RG
Published: October 2019

Inositol phosphates (InsPs) and diacyglycerol (DAG) are second messengers derived via the breakdown of inositol phospholipids, and which play important signalling roles in the regulation of proliferation of some cell types. We have studied the operation of this pathway during the early stages of retionic acid (RA)-induced granulocytic differentiation of HL60 myeloid leukemia cells. The autonomous breakdown of inositol lipids that occurred in HL60 cells labeled with [3H] inositol was completely abolished following 48 hours of RA treatment. The rate of influx of 45Ca2+ was also significantly decreased at 48 hours, consistent with the role of inositol lipid- derived second messengers in regulating Ca2+ entry into cells. The downregulation of inositol lipid metabolism clearly preceded the onset of reduced proliferation induced by RA treatment, and was therefore not a consequence of decreased cell growth. The generation of InsPs in RA- treated cells was reactivated by the fluoroaluminate ion, a direct activator of guanine nucleotide-binding protein(s) (G proteins) that regulate the inositol lipid signalling pathway. Subtle alterations to a regulatory mechanism may therefore mediate the RA-induced downregulation of this pathway. The data are consistent with the hypothesis that the autonomous generation of inositol lipid-derived second messengers may contribute to the continuous proliferation of HL60 cells, and that the RA-induced downregulation of this pathway may, in turn, play a role in signalling the cessation of proliferation that preceedes granulocytic differentiation.
Identification of autoantibodies specific for the neutrophil adhesion glycoproteins CD11b/CD18 in patients with autoimmune neutropenia
Authors: Hartman KR , Wright DG
Published: October 2019

Anti-neutrophil antibodies have been described in a variety of clinical conditions associated with neutropenia. However, relatively little is known about the antigenic specificities of naturally occurring anti- neutrophil autoantibodies. We investigated the possibility that anti- neutrophil antibodies specific for the neutrophil adhesion glycoprotein (GP) complex CD11b/CD18 might be present in the sera of some patients with autoimmune neutropenia. These membrane GPs have been shown to be highly immunogenic in the production of murine monoclonal antibodies against neutrophil antigens. Moreover, autoantibodies to the platelet membrane GP complex IIb/IIIa, another member of the integrin family of cell adhesion proteins, have been demonstrated in immune thrombocytopenic purpura. Sera from 50 patients known to have anti- neutrophil IgG antibodies were evaluated using an immunobead “antigen capture” assay, modeled after a method used to identify anti-platelet GPIIb/IIIa autoantibodies. This assay detected anti-CD11b/CD18 autoantibodies in seven of the 50 sera. Each of these seven sera demonstrated decreased IgG binding to the neutrophils of a patient with congenital deficiency of CD11b/CD18. The patient with the highest levels of anti-CD11b/CD18 suffered recurrent skin infections and cellulitis, and died of respiratory failure during one of multiple episodes of pneumonia. Purified IgGs from five of these patients demonstrated effects on adhesion and/or opsonin receptor-mediated functions when tested with intact neutrophils in vitro. Our findings indicate that some patients with autoimmune neutropenia have autoantibodies specific for the functionally important neutrophil adhesion proteins CD11b/CD18. Our findings also raise the possibility that these autoantibodies may, in some cases, interfere with neutrophil function, thereby amplifying the risk of infection associated with neutropenia.
Culture of phenotypically defined hematopoietic stem cells and other progenitors at limiting dilution on Dexter monolayers [see comments]
Authors: Weilbaecher K , Weissman I , Blume K , Heimfeld S
Published: October 2019

Highly enriched, phenotypically defined hematopoietic stem, Thy-1loLin- Sca-1+, and progenitor cell populations from mouse bone marrow (BM) were tested at limiting dilution for their ability to reconstitute Dexter monolayers. Several classes of BM cells can reconstitute Dexter cultures, first forming discrete 'cobblestone” areas which then mature into colonies consisting primarily of maturing myeloid and erythroid cells. Most such colonies have a limited lifespan in culture. Only the Thy-1loLin-Sca-1+ cell fraction gives rise to colonies that survive longer than 3 weeks, which suggests that a limiting-dilution analysis for long-term reconstitution of Dexter cultures can serve as a quantitative measure of stem cell activity. Additional experiments were performed to assess the formation of new progenitor cells in reconstituted Dexter cultures. Again, only cultures seeded with the stem cell-enriched fraction contained expanded numbers of replatable WEHI-3 CM responsive colony-forming cells (CFU-GM). Quantitative analysis indicates that 97% of the replatable CFU-GM of whole BM is contributed by the Thy-1loLin-Sca-1+ cell fraction, again suggesting a potential stem cell-specific assay. Such quantitative in vitro assays might prove useful in characterization and isolation of human stem cells where in vivo assays are lacking.
Characterization of Ws mutant allele of rats: a 12-base deletion in tyrosine kinase domain of c-kit gene
Authors: Tsujimura T , Hirota S , Nomura S , Niwa Y , Yamazaki M , Tono T , Morii E , Kim HM , Kondo K , Nishimune Y
Published: October 2019

Homozygous mutant rats at the newly found white spotting (Ws) locus were anemic and deficient in mast cells and melanocytes. Because the phenotype of Ws/Ws rats resembled the phenotype of mice possessing a double-gene dose of mutant alleles at the W locus and because the c-kit gene was mapped at the W locus of mice, we characterized the c-kit gene of Ws/Ws rats. The authentic sequence of the rat c-kit cDNA was determined by using a cDNA library prepared from the hippocampus of Sprague-Dawley rats. The c-kit cDNA of Ws/Ws and normal (+/+) control rats was obtained by reverse transcriptase modification of the polymerase chain reaction. When compared with the authentic sequence, a deletion of 12 bases was found in the c-kit cDNA of Ws/Ws rats. This change was shown to be a result of the deletion of the genomic DNA. Four amino acids encoded by the deleted 12 bases (ie, Val-Lys-Gly-Asn) were located at two amino acids downstream from the tyrosine autophosphorylation site in the c-kit kinase and were conserved not only in mouse and human c-kit kinases but also in mouse and human c-fms kinases (ie, receptors of colony-stimulating factor-1). Taken together, the Ws/Ws rat is the first characterized mutant of the c-kit gene in an animal species other than the mouse.
Synergistic effects of interleukin-1 beta and interleukin-3 on the expansion of human hematopoietic progenitor cells in liquid cultures
Authors: Kobayashi M , Imamura M , Gotohda Y , Maeda S , Iwasaki H , Sakurada K , Kasai M , Hapel AJ , Miyazaki T
Published: October 2019

In the present study, we show that recombinant human interleukin-1 beta (rhIL-1 beta), which has no effect on the proliferation of human progenitor cells, has synergistic effects on the expansion of human progenitor cells induced by rhIL-3 in liquid cultures. The synergistic effects of rhIL-1 beta with rhIL-3 were observed in liquid cultures using not only fresh bone marrow mononuclear cells, but also selected populations of nonadherent cells, non-T nonadherent cells, and CD34- positive cells. Anti-granulocyte-macrophage colony-stimulating factor (anti-GM-CSF) antibody partially blocked the synergistic effects of rhIL-1 beta on the proliferation of colony-forming unit (CFU)-GM burst- forming unit-erythroid (BFU-E), and CFU-Mix in liquid cultures in the presence of rhIL-1 beta plus rhIL-3, suggesting that the synergistic effects of rhIL-1 beta plus rhIL-3 are explained in part by the secondary production of GM-CSF. Limiting dilution assays and liquid culture assays using CD34-positive cells indicate that rhIL-1 beta directly increases the numbers of colony-forming cells in liquid cultures. These results suggest that rhIL-1 beta has unique direct and indirect effects on the expansion of hematopoietic progenitor cells in liquid cultures.
Reassignment of the human CSF1 gene to chromosome 1p13-p21
Authors: Morris SW , Valentine MB , Shapiro DN , Sublett JE , Deaven LL , Foust JT , Roberts WM , Cerretti DP , Look AT
Published: October 2019

Human macrophage colony-stimulating factor (CSF-1 or M-CSF) is encoded by a single gene that was previously assigned to the long arm of chromosome 5, band q33.1, in a region adjacent to the gene encoding its receptor (Pettenati MJ, et al, Proc Natl Acad Sci USA 84:2970, 1987). Using fluorescence in situ hybridization with genomic probes to examine normal metaphase chromosomes, we reassigned the human CSF1 gene to the short arm of chromosome 1, bands p13-p21. We confirmed this result by hybridizing a CSF1 cDNA probe to filters containing flow-sorted chromosomes and by identifying CSF1 sequences in DNAs extracted from human x rodent somatic cell hybrids that contained human chromosome 1 but not human chromosome 5. Our findings are consistent with studies that have shown tight linkage between the murine CSF1 and amylase genes, as part of a conserved linkage group between mouse chromosome 3 and the short arm of human chromosome 1, which also includes the genes encoding the beta subunits of thyrotropin and nerve growth factor. Assignment of the CSF1 gene to chromosome 1 at bands p13-p21 raises the possibility that it may be altered by certain nonrandom chromosomal abnormalities arising in human hematopoietic malignancies and solid tumors.
Expression of human recombination activating genes (RAG1 and RAG2) in neoplastic lymphoid cells: correlation with cell differentiation and antigen receptor expression
Authors: Bories JC , Cayuela JM , Loiseau P , Sigaux F
Published: October 2019

Regulation of V-(D)-J recombinations that occur in antigen receptor encoding genes remains poorly understood. Recently, two genes, RAG1 and RAG2, that are able to activate rearrangement of synthetic recombination substrates were cloned in mouse and a human gene homologous to RAG1 was described. To define the differentiation stages corresponding to RAG1 and RAG2 RNA expression, we have studied a large number of B- and T-lymphoid neoplasias. First, we show that a human gene homologous to the murine RAG2 is transcribed in humans. Moreover, using a polymerase chain reaction approach, we have shown that RAG are expressed not only in T-cell receptor (TCR)-negative T-cell acute lymphoblastic leukemias (T-ALLs), but also in some cases in which a significant percentage of cells expressed surface TCR. Absence of RAG expression was shown in certain T-ALLs at variable stages of thymic differentiation. Data obtained in B-lineage ALLs show that RAG RNAs are expressed in almost all slg- B-lineage ALLs but are not transcribed in the slg+ B-cell proliferations tested, including Burkitt's ALLs, follicular center cell lymphomas, and chronic leukemias. These findings are consistent with the involvement of RAG in the control of in vivo V- (D)-J recombinations. These findings are also of interest in the delineation of potential regulatory factors acting on RAG transcription and in the understanding of the mechanisms of specific chromosomal abnormalities occurring in immature lymphoid cells.
Differential usage of delta recombining element and V delta genes during T-cell ontogeny
Authors: Hara J , Takihara Y , Yumura-Yagi K , Ishihara S , Tawa A , Mak TW , Gelfand EW , Okada S , Kawa-Ha K
Published: October 2019

We analyzed the usage of the delta recombining element (delta Rec) and six V delta genes in cell samples from 15 patients with CD3- and 10 patients with CD3+ T-cell acute lymphoblastic leukemia in an attempt to define the hierarchy of genetic events that is associated with the T- cell receptor (TCR) alpha/delta gene complex during T-cell ontogeny. Based on the deletion patterns of these genes, we surmised their relative order on chromosome 14 to be as follows: 5′-V delta 4, V delta 6, V delta 1, V delta 5, delta Rec, V delta 2, D delta 1–3, J delta 1– 3, C delta, V delta 3–3′. In agreement with previous reports, V delta 1 was found to be preferentially rearranged in CD3+ samples. In CD3- samples, V delta 2 and V delta 3 rearrangements were observed at a high frequency. Incomplete V delta D delta rearrangements using V delta 2 or V delta 3, which are closest to C delta, were observed in three patients with CD3- and one patient with CD3+. These results suggest that V delta 2- and V delta 3-(Dn)D delta 3 recombinations are among the earliest recombinational events. Delta Rec was observed to be rearranged to phi J alpha on one allele. In addition, delta Rec rearrangements to J delta 1 and J alpha close to phi J alpha were also demonstrated on three alleles and one allele, respectively. Delta Rec rearrangements to J delta and J alpha other than phi J alpha also inhibit expression of the TCR delta locus. Approximately half of the alleles with J delta rearrangements showed no involvement of known V delta or delta Rec, indicating the existence of other, yet- uncharacterized V delta or delta Rec-like segments.
Isolation of cDNA coding for an ubiquitous membrane protein deficient in high Na+, low K+ stomatocytic erythrocytes
Authors: Stewart GW , Hepworth-Jones BE , Keen JN , Dash BC , Argent AC , Casimir CM
Published: October 2019

Human red blood cells (RBCs) that are deficient in an integral membrane- associated protein (“stomatin“) of apparent molecular mass 31 Kd show a catastrophic increase in passive membrane permeability to the univalent cations Na+ and K+ and are stomatocytic in shape. We have purified this protein from normal RBC membranes and isolated a cDNA clone coding for it. The deduced protein sequence is unrelated to that of any known ion- transport-related protein. Selective solubilization studies using detergents show that while the protein is strongly associated with the phospholipid bilayer, it also binds to the cytoskeleton. The predicted polypeptide has a single trans-membranous hydrophobic segment near the N-terminus, which would locate it in the membrane; the large C-terminal domain is hydrophilic and cytoplasmic in orientation and is presumed to be responsible for the attachment to the cytoskeleton. By inference, the protein has the function of closing a latent ion channel. The messenger RNA encoding this protein is ubiquitously distributed in different human cell types and tissues and is thus presumably a widely distributed regulator of transmembrane cation fluxes. As a membrane- bound inhibitor protein of Na+ and K+ transport, it is unique among the known components of membrane-transport proteins.
Clinical spectrum of clonal proliferations of T-large granular lymphocytes: a T-cell clonopathy of undetermined significance?
Authors: Dhodapkar MV , Li CY , Lust JA , Tefferi A , Phyliky RL
Published: October 2019

We identified 68 patients with clonal T-large granular lymphocyte (T- LGL) proliferations who were seen at the Mayo Clinic between 1984 and 1992. Nineteen (28%) were asymptomatic at diagnosis, while the rest experienced fatigue (60%), B-symptoms (12%), and recurrent infections (15%). Associated comorbid conditions included rheumatoid arthritis (RA) in 26%. Severe anemia (hemoglobin [Hb] < 8g/dL) and neutopenia (absolute neutrophil count [ANC] < 500/microL) were seen in 19% and 40% of patients, respectively. Immunophenotypic studies showed CD3+, CD8+ phenotype in the majority (72%). Twenty-one patients (31%) have required no therapy, and remain relatively stable with a median follow- up period of 50 months. Treatment was required at either diagnosis (36 patients) or at subsequent follow-up (11 patients). Initial response rates were similar in patients treated with cyclophosphamide (CTX) with or without prednisone (69%), or prednisone alone (73%). Overall, 61 patients (90%) are alive with a median follow-up of 44 months. Actuarial median survival of this entire cohort is 161 months. The presence of anemia or symptoms does not appear to correlate with the tumor burden. In patients requiring therapy, a lower ANC and the presence of B-symptoms/infection were independently associated with a significantly lower probability of achieving a molecular or hematologic complete remission (H-CR). Intermittent immunosuppressive therapy is effective in achieving durable responses in a number of patients. T-LGL proliferations are associated with a favorable prognosis and response to therapy. However, significant heterogeneity exists in clinical presentation and associated comorbid conditions. These disorders should be included in the differential diagnosis of patients with unexplained cytopenias, particularly in the setting of RA and other autoimmune disorders. Analogous to the situation with monoclonal gammopathies, a term such as T-cell clonopathy of undetermined significance (TCUS) may be more appropriate to describe these patients.
Phlebotomy to Reduce Iron Overload in Patients Cured of Thalassemia by Bone Marrow Transplantation
Authors: Angelucci Emanuele , Muretto Pietro , Lucarelli Guido , Ripalti Marta , Baronciani Donatella , Erer Buket , Galimberti Maria , Giardini Claudio , Gaziev Djavid , Polchi Paola ,   the Italian Cooperative Group for Phlebotomy Treatment of Transplanted Thalassemia Patients
Published: October 2019

In thalassemia after successful bone marrow transplantation (BMT), iron overload remains an important cause of morbidity. After BMT, patients have normal erythropoiesis capable of producing a hyperplastic response to phlebotomy so that this procedure can be contemplated as a method of mobilizing iron from overloaded tissues. A phlebotomy program (6 mL/kg blood withdrawal at 14-day intervals) was proposed to 48 patients with prolonged follow-up (range, 2 to 7 years) after BMT. Seven patients were not submitted to the program (five because of refusal and two because of reversible side effects). The remaining 41 patients (mean age, 16 ± 2.9 years) were treated for a mean period of 35 ± 18 months. All were evaluated before and after 3 ± 0.6 years of followup. Values are expressed as mean ± standard deviation (SD) or as median with a range (25 to 75 percentile). Serum ferritin decreased from 2,587 (2,129 to 4,817) to 417 (210 to 982) μg/L (P < .0001), total transferrin increased from 2.34 ± 0.37 to 2.7 ± 0.58 g/L (P = .0001), transferrin saturation decreased from 90% ± 14% to 50% ± 29% (P < .0001). Liver iron concentration evaluated on liver biopsy specimens decreased from 20.8 (15.5 to 28.1) to 4.2 (1.6 to 14.6) mg/g dry weight (P < .0001). Aspartate transaminase decreased from 2.7 ± 2 to 1.1 ± 0.6 (P < .0001) and alanine transaminase from 5.2 ± 3.4 to 1.7 ± 1.2 (P < .0001) times the upper level of normality. The Knodell score for liver histological activity decreased from 6.9 ± 3 to 4.9 ± 2.8 (P < .0001). These data indicate that phlebotomy is safe, efficient, and widely applicable to ex-thalassemics after BMT.
CD4 Cross-Linking (CD4XL) Induces RAS Activation and Tumor Necrosis Factor-α Secretion in CD4+ T Cells
Authors: Tamma Seetha M. Lakshmi , Chirmule Narendra , Yagura Hirosuka , Oyaizu Naoki , Kalyanaraman Vaniambadi , Pahwa Savita
Published: October 2019

CD4 molecules are the primary receptors for human immunodeficiency virus (HIV) and bind the envelope glycoprotein gp120 of HIV with high-affinity. We have previously shown that cross-linking of CD4 molecules (CD4XL) in normal peripheral blood mononuclear cells (PBMC) results in secretion of cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), but not of interleukin-2 (IL-2) or IL-4. To investigate the intracellular signaling events associated with CD4-gp120 interaction, we incubated CD4+ T cells from peripheral blood of HIV-negative healthy donors with HIV envelope protein gp160 alone or performed CD4XL with gp160 and anti-gp160 antibody. This procedure resulted in tyrosine phosphorylation of intracellular substrates p59fyn, zap 70, and p95vav and also led to ras activation, as assessed by conversion of rasGDP to rasGTP. The role of ras in CD4 signaling was further investigated using CD4+ Jurkat cells transfected with a dominant negative ras mutant. CD4+ T cells expressing dn-ras secreted significantly reduced levels of TNF-α in response to CD4XL. These studies indicate that interaction of HIV gp160 with CD4 molecules activates the ras pathway in T cells, which may result in the cells becoming unresponsive to subsequent stimulation.