bluuebunny/crossref_metadata_2025 huggingface dataset:
Health Consequences Of Benzene Exposure Among Children Following a Flaring Incident At Petroleum Refinery In Texas City
Authors: D'Andrea Mark , Mitter Ajay , Reddy G. Kesava
Published: October 2019

Objective

Human exposure to benzene is associated with multiple adverse health effects leading to hematological malignancies. The objective of this retrospective study was to evaluate the health consequences of benzene and other toxic chemical exposure in children following a flaring incident at the British petroleum refinery in the Texas City, Texas.

Methods

The study included children aged < 17 years who had been exposed and unexposed to benzene due to a flaring incident at the British petroleum refinery facility. Using medical charts, clinical data including white blood cell (WBC) counts, platelets counts, hemoglobin, hematocrit, blood urea nitrogen (BUN,) creatinine, alkaline phosphatase (ALP), aspartate amino transferase (AST), alanine amino transferase (ALT), and somatic symptom complaints by the children exposed to benzene were reviewed and analyzed.

Results

A total of 312 subjects (benzene exposed, n=157 and unexposed, n=155) were included. Hematologic analysis showed that WBC counts were significantly decreased in benzene exposed children compared with the unexposed children (6.8 ± 2.1 versus 7.3 ± 1.7, P=0.022). Conversely, platelet (X 103 per µL) counts were increased significantly in the benzene exposed group compared with the unexposed group (278.4 ± 59.9 versus 261.6 ± 51.7, P=0.005). Similarly, benzene exposed children had significantly higher levels of ALP (183.7± 95.6 versus 165 ± 70.3 IU/L, P=0.04), AST (23.6 ± 15.3versus 20.5 ± 5.5 IU/L, P = 0.015), and ALT (19.2 ± 7.8 versus 16.9 ± 6.9 IU/L, P=0.005) compared with the unexposed children.

Conclusion

Together, the results of the study reveal that children exposed to benzene experienced significantly altered blood profiles, liver enzymes and somatic symptoms indicating that children exposed to benzene are at a higher risk of developing hepatic or blood related disorders.

Disclosures:

No relevant conflicts of interest to declare.
Citrate Synthase Activity Is Increased In Children With Sickle Cell Disease (SCD) On Hydroxyurea (HU) Therapy
Authors: Morris Claudia R. , Latronica Cornelia , Kuypers Frans A , Rosenfeld Howard , Larkin Sandra K , Doniger Stephanie , Vichinsky Elliott , Shiva Sruti
Published: October 2019

Background

Mitochondrial dysfunction is associated with abnormal cell function, oxidative stress and cell death. A progressive impairment of mitochondrial function and/or increased oxidative damage likely contribute to the pathophysiology of SCD, however little is known about bioenergetics in SCD. While previous work demonstrated mitochondrial dysfunction in the platelets of adult patients with SCD characterized by deficient complex IV and V activity which was associated with hemolysis, no data has yet been described in children with SCD.

Objective

To initiate investigation into mitochondrial function in children with SCD.

Methods

Children with a diagnosis of SCD requiring parenteral opioids for vaso-occlusive pain episodes (VOE) were prospectively enrolled within 24 hours of presentation to an urban emergency department (ED). Blood was obtained for biomarkers of hemolytic rate, and estimated pulmonary pressures were noninvasively measured by a point-of-care Doppler echocardiography (echo). Platelet rich plasma was isolated from patient whole blood by differential centrifugation and flash frozen for mitochondrial studies. The activities of Complexes V and IV were measured since these enzymes are the sites of oxygen consumption (Cx IV) and ATP production (CxV). The activity of citrate synthase, a mitochondrial matrix protein, was also determined as an indicator of mitochondrial number.

Results

11 children with SCD seen in the ED or admitted for VOE were included in this pilot study. Mean age was 17±3 years (range: 9-21 years), 64% were male, and 45.5% had HbSS, 45.5% HbSC and 9% HbSbThal. Mean Hb was 9.9±2 g/dL, reticulocyte (retic) count was 7.6±5%, and 45.5% were on HU. Mean tricuspid regurgitant jet velocity (TRV) = 2.0±0.4 m/s. Of interest patients on HU had significantly higher citrate synthase activity (Figure 1), although Complex IV and V activities were similar in both groups. Citrate synthase activity inversely correlated with TRV (r=-0.63, p=0.04). Similar to the previously reported adult data, complex IV activity inversely correlated with retic count (R2=0.27), although not reaching statistical significance, likely due to small sample size. No difference in mitochondrial studies were found when HbSS patients were compared to those with HbSC.

Conclusions

These data suggest mitochondrial dysfunction and potentially decreased mitochondrial number in SCD children with increased hemolytic rate (TRV). Further, the greater citrate synthase activity in platelets of patients with SCD on HU compared to those not treated with HU may be indicative of increased mitochondrial number with HU therapy. These observations have never been previously described, and may suggest a novel mechanism of action for HU warranting further study.

Disclosures:

No relevant conflicts of interest to declare.
Toll Like Receptors Have mRNA Differentiated Expression In Dendritic Cells In Crisis-State Sickle Cell Anemia Patients, Suggesting a Pivotal Role Of These Molecules and Cell Type In The Maintenance Of Inflammatory Response
Authors: Cajado Cyntia , Fukutani Kyioshi Ferreira , Boas Wendell Vilas , Veloso Cerqueira Bruno Antônio , Tavares Natalia Machado , Leite Ivana , Siqueira Isadora , Lyra Isa , Adorno Elisangela , Goncalves Marilda Souza
Published: October 2019

Introduction

The vascular alterations play an important role in the SCA chronic inflammatory state with the participation of different cell types, including macrophages and dendritic cells (DCs).

Aim

Here, we evaluated the mRNA expression of TLRs in DCs cells pre and post LPS stimulus and also measured IL-1β, TNF-α and IL-10 cytokines in plasma and supernatant of DCs pre and post LPS challenge, in order to indentify a possible role of this cell and receptor in the inflammatory state described in sickle cell anemia.

Methods

In vitro generation of immature DCs (imDC) monocyte-derived was obtained of monocytes from peripheral blood mononuclear cells (PBMC) of seven hospitalized HbSS patients. Relative mRNA abundance of each transcript was investigated by quantitative real time PCR and cytokines levels were measured by ELISA.

Results

We found that different HbSS patients showed specific DCs TLR expression and a differential levels of IL-1β among HbSS patients with and without pneumonia history (p=0.0253) and with hemoglobin concentration (p=0.0288) (Fig. 1). There was difference of TLR4 mRNA expression between HbSS patients groups without and with leg ulcers history (0.320+0.302 and 1.60+0.014) respectively (p=0.0016); with platelets count between HbSS patients groups with TLR2 expression < 50th (569.7+3.512) and > 50th (304.5+111.7) (p=0.0102) and reticulocytes count between HbSS patients groups with TLR2 expression < 50th (15.97+2.450) and > 50th (2.025+2.417) (p=0.0007) and of TNF-alpha concentration in DCs supernatant culture among HbSS patients with TLR5 expression < 50th (30.57+11.92) and > 50th (7.053+6.918) (p=0.0496) and of IL-10 concentration in DCs supernatant culture among HbSS patients with TLR5 expression < 50th (3.525+0.921) and > 50th (5.790+1.226) (p=0.0374) (Fig.2). It was also found statistic difference of IL-1 beta concentration in DCs supernatant culture post LPS stimulus between HbSS patients without and with pneumonia history (P=0.0253); analysis of TLR4 mRNA expression between HbSS patients without and with leg ulcer history (P=0.0016); analysis of hemoglobin concentration between HbSS patients with different plasma IL-1 beta concentration (P=0.0288)

Conclusion

Ours results show that these molecules and cell type possibly have an important role in maintain the inflammatory state described in this disease.

Disclosures:

No relevant conflicts of interest to declare.
Pyknotic Lymphocytes In Infectious Mononucleosis
Authors: Dittman William A.
Published: October 2019

Context

Pyknotic lymphocytes were infrequently observed in routine blood film evaluation of patients with clinical infectious mononucleosis. A marked increase in frequency was observed in 1995-1997.

Objective

To characterize the cause of these changes.

Design

Collection of samples from patients (25) with clinical infectious mononucleosis to ascertain the effect of anticoagulant and/or time lapse on lymphocytes and to correlate with monospot or Epstein-Barr virus antibody testing. Control samples (33), mixed hematopoietic disease (29) and lymphoproliferative disorders (36) were obtained also. Additional patients (53), identified by variant lymphocyte morphology during routine CBC’s, were verified by monospot or EBV testing and slides were made at 24 and 48 hours for differentials. Finally, single specimens (86) were obtained to establish predictability or correlation between pyknotic cells and monospot testing

Setting

Inpatient and outpatient hematology laboratory.

Results

Patients with clinical infectious mononucleosis demonstrated 8-10% pyknotic lymphocytes in 24 and 48 hour samples in all three anticoagulants (see Figure 1) compared with controls, “other diseases” (Table 2) and lymphoproliferative disorders (Table 3). An unexpected predictive correlation of pyknotic cells with positive monospot or Epstein-Barr Virus antibodies was demonstrated when pyknotic cells were seen with or without significant atypical lymphocytes during daily routine differential counting.

Conclusion

Observation of pyknotic lymphocytes adds a significant and inexpensive element to the diagnosis of infectious mononucleosis and consideration of Cytomegalic Virus infection in older patient

Disclosures:

No relevant conflicts of interest to declare.
The Effect Of Thrombotic Markers On The Severity Of Bleeding Symptoms In Women With Inherited Bleeding Disorders
Authors: Halimeh Susan , Davies Joanna , Kadir Rezan A
Published: October 2019

Background

Women with inherited bleeding disorders demonstrate heterogenicity in bleeding symptoms that do not always correlate to underlying clotting factor levels. This is problematic when predicting the bleeding risk, especially when haemostatic challenge is anticipated. The effect of thrombotic variables in reducing the overall bleeding tendency, have demonstrated a protective effect in cases of severe haemophilia.

Aims

To assess the correlation between bleeding tendency, haemostatic and thrombotic variables in women with inherited bleeding disorders.

Method

Quantitative measurement of the bleeding tendency was assessed using the standardized ISTH Bleeding Assessment Tool. Pictorial Blood Assessment Chart (PBAC) was used to quantify the subjective assessment of menstrual blood loss. 78 women with inherited bleeding disorders (75 with von Willebrand disease and 3 carriers of haemophilia A) were included. Blood samples were then obtained from individuals for assessment of clotting factors (factor VIII and von Willebrand factor) and thrombophilia screen (lupus anticoagulant, antithrombin, protein C and protein S, factor V Leiden mutation and prothrombin mutation).

Results

Bleeding score was inversely related to von Willebrand factor antigen (vWF:Ag), ristocetin cofactor (vWF:RCo) levels. A positive correlation was demonstrated with antithrombin III activity. Ten women tested positive for thrombotic markers. Multiple regression analysis did not reveal a significant correlation with presence of thrombotic marker on bleeding score.

Conclusion

Bleeding score is a useful assessment for bleeding phenotype and seems to correlate with vWF:Ag and vWF:RCo, which may by useful for assessment of the bleeding risk. Although there was no significant correlation demonstrated by the presence of a thrombotic marker, the levels of antithrombin III appears to affect the bleeding phenotype and this requires further consideration.

Disclosures:

Halimeh: Octapharma AG: Investigator Other, Research Funding.
Changes Of Coagulation Parameters During The Menstrual Cycle
Authors: Halimeh Susan , Rott Hannelore , Kappert Guenther , Siebert Manuela
Published: October 2019

Introduction

Von Willebrand disease is the most common bleeding disorder with a prevalence of 1-2% of the population. Nevertheless diagnosis of a von Willebrand Syndrom Typ 1 is still challenging. In a newer publication (1), 30 studies about the haemostatic variables during the menstrual cycle were compared. 11 studies were focused on the von Willebrand parameters but only in one study these parameters in patients with von Willebrand disease were observed. We investigated possible cyclic variations in women with menorrhagia, which can lead to a diagnosis of a coagulation disorder.

Samples and Methods

We conducted a laboratory workup in 122 women sent to our lab for menorrhagia. The following tests were conducted: Blood count, VWF:RCo, VWF:Ag, VWF:CB, Fibrinogen (Clauss), activities of FII, FV, FVII, FVIII (clotting and chromogenic), FIX, FX, FXI, FXII, FXIII during the menstrual cycle on predefined time points (day 1-6, day 7-11, day 12-18, day 19-23, day 24-28).

Results

In 51 (40%) patients a von Willebrand disease could be detected, 37% hat other coagulation disorders like p. e. factor-VII-deficieny and factor-XIII-deficiency. 11.5% had an iron deficiency. In 11.5% no coagulation disorder could be found.

In patients with von Willebrand disease we found cyclic variations especially in the VWF:Ag (p = 0.02). They showed the lowest level during the ovulation. For other coagulation parameters no significance for variations during the menstrual cycle were found.

Conclusion

There are cyclic variations in von Willebrand antigen. To investigate women on predefined time points during the menstrual cycle can be useful to diagnose a von Willebrand disease particularly in mild cases in which no other suspicious bleeding symptoms exists.

Literature

Knol H.M., Kemperman R.F.J., Kluin-Nelemans C., Mulder A.B., Meijer K., Haemostatic variables during normal menstrual cycle, Thrombosis and Haemostasis 107.1/2012

Disclosures:

Halimeh: Octapharma AG: Investigator Other, Research Funding.
Paroxysmal Nocturnal Hemoglobinuria With Budd-Chiari Syndrome Treated With Complement Inhibitor Eculizumab; A Case Report
Authors: Valeri Federica , Borchiellini Alessandra , Schinco Piercarla , Boccadoro Mario
Published: October 2019

Introduction

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired haemolytic anaemia caused by somatic mutation in the phosphatidylinositol glycan-complementation class A gene, resulting in absence of a key complement regulatory protein, CD59.

Thrombosis occurs in up to 40% of PNH patients; it usually involves abdominal and cerebral veins and it is the leading cause of death disease related.

Methods

We describe the response to Eculizumab (Soliris, Alexion) in 28 years old male with PNH diagnosed as a consequence of Budd Chiari Syndrome, acute liver dysfunction, mild haemolytic anaemia and thrombocytopenia.

Results

The patient was admitted to the gastroenterology department with acute abdominal pain, fatigue, hemolytic anaemia, thrombocytopenia and transaminitis. Abdominal doppler ultrasonography (US) was immediately performed with detection of Budd Chiari Syndrome, portal vein thrombosis, initial portal hypertension and ascites.

He was started on low dose low molecular weight heparin (platelets < 40x10^9/L), but despite anticoagulation progressive liver damage occurred, with poor pain control and worsening ascites. At the same time, we observed rapid exacerbation of thrombocytopenia and increasing in hemolysis tests with lactate dehydrogenase (LDH) reaching 1766 U/L, unresponsive to steroids administration. Bone marrow biopsy was negative but peripheral blood flow cytometry characterized a large PNH clone (85% total red blood cells). Furthermore, liver biopsy identified advanced stage of idiopathic cirrosis.

Eculizumab therapy was then initiated at a dose of 600 mg weekly for 4 weeks and then 900 mg every 14 days. During the first month, transaminases progressively normalized and platelets settled permanently above 40x10^9/L, allowing therapeutic dose of anticoagulation. LDH dropped from basal value of >1000U/L to 600U/L and progressive reduction in abdominal pain was observed.

Recanalization of portal vein thrombosis was found out at the US doppler after 6 weeks of anticoagulation, but recanalization of sovraepatic veins was not yet detectable.

Conclusions

Currently, after 17 Eculizumab administrations, platelets are 44 x 10^9/L, Hb 11.9 g/dl, AST 26 mg/dl, ALT 55 mg/dl, GGT 123 mg/dl, LDH 518 U/L. No further thrombotic episodes occurred, no ascites was detected as well as portal hypertension signs, performing ultrasonography monitoring.

This case shows that Eculizumab can block intravascular haemolysis and platelet consumption and can improve hepatic failure, allowing full dose of anticoagulant as therapy for current thrombosis or as prophylaxis for future events.

Disclosures:

No relevant conflicts of interest to declare.
Modified Heparin Assay For The Quantitation Of Rivaroxaban
Authors: Sanfelippo Michael , Tillema Veronica
Published: October 2019

The anticoagulant rivaroxaban is not routinely monitored due to its predictable anticoagulant action. However, drug levels are clinically useful in patients requiring surgery as well as in assessing compliance. We have modified a commercial heparin assay based on inhibition of activated factor X (Xa) that was previously modified for the assay of fondaparinux (Am J Clin Pathol 2009, 132:608-612) by replacing the fondaparinux calibrators with commercial rivaroxaban calibration plasmas. Technoview rivaroxaban calibrator plasmas were purchased from Dia Pharma (West Chester, Ohio). These calibrators were substituted for the fondaparinux calibrators. The assay was run on a Siemens BCS XP, as previously described. The assay was linear in the range of 0 to 650 ng/ml. The limit of detection was 14 ng/ml, and carryover at 285 ng/ml was < 1%. The intra-assay precision at 100 ng/ml was standard deviation of 1.04 ng/ml, coefficient of variation of 1.03%. The inter-assay precision at 105 ng/ml was standard deviation 3.5 ng/ml, coefficient of variation of 3.3%. The assay is unaffected by warfarin, but can be affected by other Xa inhibitors such as heparin, low molecular weight heparin, fondaparinux, and apixaban.

Disclosures:

No relevant conflicts of interest to declare.
Specific recognition of human leukemic cells by allogeneic T cells: II. Evidence for HLA-D restricted determinants on leukemic cells that are crossreactive with determinants present on unrelated nonleukemic cells
Authors: Sosman JA , Oettel KR , Smith SD , Hank JA , Fisch P , Sondel PM
Published: October 2019

Transplantation of immunocompetent cells present within allogeneic bone marrow has been associated with the elimination of residual host leukemia, both in animal tumor models and in patients receiving marrow transplants for leukemia. This observation has been called the “graft- versus-leukemia effect.” We have attempted to study this phenomenon in vitro by characterizing the cytolytic response of T cells from normal donors after in vitro activation with allogeneic leukemic cells. As expected, most T cells that react against an allogeneic patient's leukemic cells recognize their foreign HLA antigens and lyse the patient's nonleukemic remission lymphoid cells. In addition, we have shown that a small fraction of the T cells recognize and lyse foreign leukemic targets without lysis of nonmalignant remission targets from the same leukemic patient. These T cells have been isolated and characterized as CD3+, CD4+ cells expressing the alpha/beta T cell receptor (TCR). Their lysis appears to reflect specific antigen recognition mediated via the CD3-TCR complex and interactions involving the CD4 receptor. Some of these “leukemic specific” T cell lines, which are restricted by HLA class II molecules, can also lyse occasional nonleukemic cells from certain unrelated donors. This recognition appears to involve crossreactive determinants shared by the leukemic cells and the unrelated allogeneic nonleukemic cells. These specific interactions may represent an in vitro model of the graft-versus- leukemia effect.
Synergistic effect of recombinant interferon-gamma and interleukin-3 on the growth of immature human hematopoietic progenitors
Authors: Kawano Y , Takaue Y , Hirao A , Abe T , Saito S , Matsunaga K , Watanabe T , Hirose M , Ninomiya T , Kuroda Y
Published: October 2019

Purified peripheral blood hematopoietic progenitors from children in early remission from cancer respond to recombinant human interleukin-3 (IL-3), but not to granulocyte colony-stimulating factor (G-CSF). With these purified cells as a target, we studied the effect of recombinant human interferon-gamma (IFN-gamma) on progenitor growth, using both liquid-suspension limiting dilution assay (LDA) and regular methylcellulose culture of progenitors. We found that in LDA with IL-3, IFN-gamma directly stimulated the growth of blood progenitors in a dose- dependent manner with single-hit kinetics, whereas IFN-gamma suppressed the growth of G-CSF-supported progenitors obtained from bone marrow. The stimulatory effect was also observed in methylcellulose culture, but the addition of antibodies for G-CSF, granulocyte-macrophage CSF, IL-1 alpha, IL-1 beta, IL-6, or tumor necrosis factor did not result in a decrease of the colony number, supporting further the possible direct effect of IFN-gamma on progenitor growth. These results suggest that the inhibitory effect of IFN-gamma on hematopoietic progenitors is limited to those in an advanced stage of maturation. IFN-gamma may be one of the essential lymphokines upregulating the growth of human hematopoietic progenitor cells.
Consistent involvement of the bcr gene by 9;22 breakpoints in pediatric acute leukemias
Authors: Suryanarayan K , Hunger SP , Kohler S , Carroll AJ , Crist W , Link MP , Cleary ML
Published: October 2019

To investigate the relationship of bcr-abl fusion mRNAs with childhood acute lymphoblastic leukemias (ALL), we examined 27 pediatric Philadelphia chromosome (Ph1)-positive acute leukemias using a reverse polymerase chain reaction (PCR) procedure. In cells from 24 leukemias, single bcr-abl PCR products were detected that corresponded to breakpoints in the minor breakpoint cluster region (mbcr in intron 1 of the bcr gene) associated with production of the P190 fusion protein. Cells from the three remaining leukemias contained breakpoints in the major breakpoint cluster region (Mbcr) as shown by PCR and Southern blot analyses. These three leukemias also contained low levels of the mbcr PCR product that may have resulted from alternative splicing of the bcr-abl precursor RNA. A screen of 35 additional leukemias from patients who failed therapy before day 180 (induction failures or early relapses) found one case with unsuccessful cytogenetics to express Mbcr- abl RNA. All four children with Mbcr breakpoints had white blood cell levels in excess of 250,000 at presentation (compared with 2 of 24 with mbcr breakpoints) and two had hematologic and clinical features suggestive of chronic myelogenous leukemias (CML) in lymphoid blast crisis. Our results indicate that in Ph1-positive pediatric leukemias, all 9;22 breakpoints occur in one of the two known breakpoint cluster regions in the bcr gene on chromosome 22. The reverse PCR reliably detected all patients with cytogenetic t(9;22) and is capable of detecting additional Ph1-positive leukemias that are missed by standard cytogenetics. Furthermore, the Mbcr-type breakpoint, associated with production of p210, can be seen in childhood leukemias presenting either as clinical ALL or as apparent lymphoid blast crisis of CML, suggesting that t(9;22) breakpoint locations do not exclusively determine the biologic and clinical features of pediatric Ph1-positive ALL.
Use of polymerase chain reactions to monitor minimal residual disease in acute lymphoblastic leukemia patients
Authors: Yokota S , Hansen-Hagge TE , Ludwig WD , Reiter A , Raghavachar A , Kleihauer E , Bartram CR
Published: October 2019

T-cell receptor (TCR) delta gene rearrangements are observed in more than 80% of acute lymphoblastic leukemia (ALL) patients. Moreover, a preferential usage of specific genetic elements has been shown in different ALL subtypes: V delta 1 DJ delta 1 rearrangements predominate in T-ALL, while most B-precursor ALLs show a recombination of V delta 2 to D delta 3. Recently we have proposed a strategy for the detection of minimal residual disease (MRD) based on the isolation of clonospecific probes following the in vitro amplification of V delta 1 DJ delta 1 junctions by polymerase chain reaction (PCR) and now have adapted this method to the preparation of specific V delta 2 D delta 3 fragments. In the present study, clonospecific probes were generated from 11 T-ALL and 16 cALL patients (21 children, 6 adults). The sensitivity of these 27 probes in detecting residual leukemia cells varied between 10(-4) to 10(-6) as determined by semiquantitative evaluation of dilution experiments. PCR analysis of 55 bone marrow (BM) and peripheral blood (PB) samples obtained from the 27 patients during complete clinical remission showed the following results: (1) Evidence for MRD was obtained in the BM of all patients (eight of eight) investigated 2 to 6 months after remission induction and also in 6 of 11 cases on maintenance therapy 7 to 19 months after diagnosis. (2) In contrast, all patients but one (10 of 11) analyzed 6 to 41 months after the termination of treatment lacked apparent evidence for leukemia DNA; the exception was a girl exhibiting 10(-4) to 10(-5) residual cells in her PB 5.5 years after diagnosis. (3) Longitudinal analysis in nine patients disclosed marked individual differences in the intervals between achievement of clinical remission and complete eradication of the leukemia cell clone. (4) Differences in the duration of MRD were not associated with distinct clinical-hematologic features. (5) Detection of residual disease by PCR proceeded clinical relapse in two cases.
Identification and characterization of a low-affinity granulocyte- macrophage colony-stimulating factor receptor on primary and cultured human melanoma cells
Authors: Baldwin GC , Golde DW , Widhopf GF , Economou J , Gasson JC
Published: October 2019

Hematopoietic growth factor receptors are present on cells of normal nonhematopoietic tissues such as endothelium and placenta. We previously demonstrated functional human granulocyte-macrophage colony- stimulating factor (GM-CSF) receptors on small cell carcinoma of the lung cell lines, and others have reported that certain solid tumor cell lines respond to GM-CSF in clonogenic assays. In the current study, we examine human melanoma cell lines and fresh specimens of melanoma to determine whether they have functional GM-CSF receptors. Scatchard analyses of 125I-GM-CSF equilibrium binding to melanoma cell lines showed a mean of 542 +/- 67 sites per cell with a kd of 0.72 +/- 0.14 nmol/L. Cross-linking studies in the melanoma cell line, M14, showed a major GM-CSF receptor species of 84,000 daltons. Under the conditions tested, the M14 cells did not have a proliferative response to GM-CSF in vitro, nor was any induction of primary response genes detected by Northern analysis in response to GM-CSF. Studies to determine internal translocation of the receptor-ligand complex indicated less than 10% of the 125I-GM-CSF internalized was specifically bound to receptors. Primary melanoma cells from five surgical specimens had GM-CSF receptors; Scatchard analysis was performed on one sample, showing 555 sites/cell with a kd of 0.23 nmol/L. These results indicate that human tumor cells may express a low-affinity GM-CSF receptor protein that localizes to the cell surface and binds ligand, but lacks functional components or accessory factors needed to transduce a signal.
Examination of the roles of glycoprotein Ib and glycoprotein IIb/IIIa in platelet deposition on an artificial surface using clinical antiplatelet agents and monoclonal antibody blockade
Authors: Sheppeck RA , Bentz M , Dickson C , Hribar S , White J , Janosky J , Berceli SA , Borovetz HS , Johnson PC
Published: October 2019

The mechanism of platelet thrombus growth on an artificial surface is incompletely understood. While glycoprotein (GP)Ib and GPIIb/IIIa are required for normal attachment and thrombus formation on subendothelium, their roles in platelet deposition to artificial surfaces remain unclear. Using selected platelet inhibitors (aspirin [ASA], low molecular weight dextran, monoclonal antibodies 10E5 [v GPIIb/IIIa], and 6D1 [GPIb]) we examined the mechanism of platelet deposition to polyethylene (PE) surfaces under steady laminar and oscillatory flow conditions. Polyethylene-100 (PE-100) tubes (0.86 mm internal diameter) were perfused under steady laminar flow with citrated human whole blood reconstituted with 111indium-labeled platelets at 312 seconds-1 shear rate in the presence and absence of platelet inhibitors. The effect of oscillatory flow on platelet deposition was examined in a microwell system using 3/16-inch diameter discs of National Heart, Lung, and Blood Institute primary reference PE as the test surface. ASA and dextran did not significantly (P greater than .05) inhibit platelet deposition in laminar flow (not tested in oscillatory). Antibody 10E5 was a potent inhibitor (laminar less than 1%, P less than .0001, oscillatory less than 1.6%, P less than .01) of platelet deposition in both systems, and in this case, true adhesion (first attached layer) was blocked. Antibody 6D1 unexpectedly inhibited 70% of platelet deposition (P less than .01) in steady laminar flow and 56.5% in oscillatory flow (P less than .01). Scanning electron microscopy demonstrated platelets atop platelets in the controls, rare platelets in the 10E5 group, and a patchy monolayer of platelets in the 6D1 group. Transmission electron microscopy of cross-sections confirmed these observations. We conclude that the adhesion of the first platelet layer to an artificial surface requires GPIIb/IIIa. The data also suggest that GPIb is required for the development of the second layer in vertical platelet thrombus growth.
Expression of interleukin-4 receptors on early human B-lineage cells
Authors: Law CL , Armitage RJ , Villablanca JG , LeBien TW
Published: October 2019

Interleukin-4 (IL-4) regulates multiple stages of the antigen-dependent phase of B-cell development. However, its precise role in regulating B lymphopoiesis in bone marrow is not as well defined. We examined whether surface IgM- normal and leukemic human B-cell precursors (BCP) expressed IL-4 receptors using biotinylated IL-4. Constitutive expression of IL-4 receptors was detected on both normal and leukemic BCP. A higher percentage of normal BCP (82% +/- 15%) expressed IL-4 receptors compared with leukemic BCP (44% +/- 8%). Using mean fluorescent intensity as an indicator of receptor level on the IL-4 receptor positive cells, normal (91 +/- 41) and leukemic (44 +/- 37) BCP expressed comparable numbers of receptors. IL-4 induced the expression of CD23 on 30% of the leukemic BCP cases examined. IL-4 induced CD23 on surface IgM+ fetal bone marrow lymphoid cells but not on the surface IgM- normal BCP, despite the presence of detectable receptors on the surface IgM- cells. IL-4 did not stimulate proliferation of normal BCP, nor could it enhance the effect of recombinant IL-7 or low molecular weight B-cell growth factor. However, IL-4 increased the expression of surface IgM and surface Ig kappa on in vitro differentiated pre-B cells. Our collective results identify no role for IL-4 in the proliferation of normal or leukemic BCP, but identify a role in the enhancement of surface Ig expression during pre- B to B-cell differentiation.
Molecular basis of the enhanced susceptibility of the erythrocytes of paroxysmal nocturnal hemoglobinuria to hemolysis in acidified serum
Authors: Wilcox LA , Ezzell JL , Bernshaw NJ , Parker CJ
Published: October 2019

When incubated in acidified serum, the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) are hemolyzed through activation of the alternative pathway of complement (APC), but normal erythrocytes are resistant to this process. PNH cells are deficient in decay- accelerating factor (DAF), a complement regulatory protein that inhibits the activity of both the classical and the alternative pathways. However, deficiency of DAF alone does not account entirely for the aberrant effects of acidified serum on PNH cells. Recently, we have shown that PNH erythrocytes are also deficient in another complement control protein called membrane inhibitor of reactive lysis (MIRL) that restricts complement-mediated lysis by blocking formation of the membrane attack complex (MAC). To determine the effects of the DAF and MIRL on susceptibility to acidified serum lysis, PNH cells were repleted with the purified proteins. DAF partially inhibited acidified serum lysis by blocking the activity of the amplification C3 convertase. MIRL inhibited acidified serum lysis both by blocking the activity of the MAC and by inhibiting the activity the C3 convertase. When DAF function was blocked with antibody, normal erythrocytes became partially susceptible to acidified serum lysis. By blocking MIRL, cells were made completely susceptible to lysis, and control of C3 convertase activity was partially lost. When both DAF and MIRL were blocked, the capacity of normal erythrocytes to control the activity of the APC and the MAC was destroyed, and the cells hemolyzed even in unacidified serum. These studies demonstrate that DAF and MIRL act in concert to control susceptibility to acidified serum lysis; of the two proteins, MIRL is the more important. In addition to its regulatory effects on the MAC, MIRL also influences the activity of the C3 convertase of the APC. Further, in the absence of DAF and MIRL, the plasma regulators (factor H and factor I) lack the capacity to control membrane- associated activation of the APC.
Fetal liver hematopoietic stem cells as a target for in utero retroviral gene transfer
Authors: Clapp DW , Dumenco LL , Hatzoglou M , Gerson SL
Published: October 2019

Retroviral-mediated gene transfer into hematopoietic precursors often results in only short-term gene transduction in vivo. Loss of the transduced genetic material over time may be caused by the limited ability of retroviral infection to transduce genes into early, pluripotent hematopoietic stem cells. Because fetal liver contains actively proliferating multipotential stem cells that should be more susceptible to retroviral-mediated gene transfer than quiescent cells derived from adult bone marrow, these cells may be an ideal target for gene transduction. Furthermore, physiologic expansion of these cells during development obviates the need for marrow ablation during gene therapy in vivo. We performed in utero gene transfer by injecting high titer replication-defective retrovirus in vivo into the livers of 11, 14, 16, and 18 day gestation rats. After birth, the rats were analyzed for the presence of proviral integration and gene expression. The efficiency of gene transfer into bone marrow cells was greatest in rats infected at day 14 to 16 of gestation. In rats killed at 1 to 26 weeks of age, gene transfer was detected by Southern analysis in 48% and by polymerase chain reaction in 86% of bone marrow samples. The provirus was also detected in white blood cells, the granulocyte-macrophage colony-forming unit, thymus, spleen, liver, and lung. The presence of the transgene in bone marrow and other hematopoietic tissues at 26 weeks of age suggests that early hematopoietic precursors present in the fetal liver are susceptible targets for gene transfer and that these cells become resident in the bone marrow of the adult animal. This model is a new technique for gene transduction into proliferating hematopoietic cells in vivo that avoids bone marrow transplantation and has potential application in the correction of genetic defects in utero.
Successful allogeneic bone marrow transplantation in a 6.5-year-old male for severe aplastic anemia complicating orthotopic liver transplantation for fulminant non-A-non-B hepatitis
Authors: Kawahara K , Storb R , Sanders J , Petersen FB
Published: October 2019

We report the case of a 6.5-year-old male who received an unrelated orthotopic liver transplant for hepatic failure and encephalopathy following non-A-non-B hepatitis and subsequently developed severe aplastic anemia. For treatment of his aplastic anemia, he received a successful marrow transplant from his 9-year-old genotypically HLA- identical sister following conditioning with cyclophosphamide 200 mg/kg and anti-thymocyte globulin 90 mg/kg. Significant veno-occlusive disease of the liver and graft-versus-host disease did not occur. The patient remains alive without clinical chronic active hepatitis or need for blood product therapy. His hematocrit is 36%, white blood cell count 9.7 x 10(3)/mm3, and platelet count 1.7 x 10(5)/mm3 almost 2 years after marrow transplantation.
The effects on hematopoiesis of recombinant stem cell factor (ligand for c-kit) administered in vivo to mice either alone or in combination with granulocyte colony-stimulating factor
Authors: Molineux G , Migdalska A , Szmitkowski M , Zsebo K , Dexter TM
Published: October 2019

Stem cell factor (SCF) is the ligand for the receptor encoded by the c- kit proto-oncogene. Mutations of either c-kit or the SCF gene are responsible for the defects of W and SI mutant mice, which both suffer a macrocytic anemia, the former associated with defective stem cells and the latter with a defective hematopoietic microenvironment. PEGylated recombinant rat SCF was administered to normal or splenectomized mice for up to 21 days. SCF was found to be a modest stimulator of peripheral blood neutrophil numbers in both groups of animals. The peak in neutrophil numbers was higher and occurred earlier in splenectomized mice. Bone marrow and spleen cellularity changed little during treatment but the content of interleukin-3-responsive progenitor cells and spleen colony-forming cells (CFU-S) reached very high levels, particularly in the spleen. Using recombinant human granulocyte colony-stimulating factor (rhG-CSF), we have shown that SCF induces a greater than additive increase in both blood neutrophils and blood-borne CFU-S. This synergy was seen throughout the dose range and may indicate a clinical role for SCF either alone or in augmenting the activity of G-CSF upon blood neutrophils and transplantable stem cells.
Allogeneic bone marrow transplantation for high-risk acute lymphoblastic leukemia during first complete remission
Authors: Chao NJ , Forman SJ , Schmidt GM , Snyder DS , Amylon MD , Konrad PN , Nademanee AP , O'Donnell MR , Parker PM , Stein AS
Published: October 2019

Fifty-three patients with high-risk acute lymphoblastic leukemia (ALL) under age 50 with a histocompatible sibling donor received high-dose radiochemotherapy followed by allogeneic bone marrow transplantation (BMT). The high-risk factors used to identify the patients were: white blood cell count at initial presentation, cytogenetic abnormalities, age, extramedullary leukemic infiltration, and time from initial therapy to complete remission. Patients with one or more of the above risk factors who received BMT have a disease-free survival of 61% with a median follow-up of 66 months (range 11 months to 10.6 years), and an actuarial relapse rate of 10%. This study demonstrates that patients with high-risk ALL achieve a significant disease-free survival and cure rate with the use of allogeneic fully matched sibling BMT. However, a properly designed prospective study comparing the outcome of BMT with the best currently available chemotherapy data is required to define the ultimate role of BMT in this group of patients.
Involvement of a nuclear factor-kappa B-like protein in induction of the macrophage colony-stimulating factor gene by tumor necrosis factor
Authors: Yamada H , Iwase S , Mohri M , Kufe D
Published: October 2019

The macrophage colony-stimulating factor (M-CSF) is required for the growth and differentiation of mononuclear phagocytes. Previous studies have demonstrated that tumor necrosis factor (TNF) induces transcription of the M-CSF gene in human myeloid cells. The present work examined the effects of TNF on cis-acting elements in the M-CSF promoter. Deleted forms of the M-CSF promoter were linked to the chloramphenicol acetyltransferase (CAT) gene and transfected by electroporation into HL-60 promyelocytic leukemia cells. The results demonstrate that an enhancer responsive to TNF stimulation is located between positions -406 and -344 upstream to the transcription start site. The fragment from positions -419 to -304 was cloned 5′ to the heterologous thymidine kinase (TK) promoter and linked to the CAT gene. Both orientations of this fragment enhanced TK-promoter activity in TNF- treated HL-60 cells. The results of gel mobility shift assays with the - 419 to -304 fragment demonstrate binding of a constitutive nuclear protein. A TNF-inducible protein also bound to this fragment and resulted in a different mobility pattern. Binding of the TNF-induced nuclear protein to the -419 to -304 fragment was inhibited by an oligonucleotide containing the nuclear factor-kappa B (NF-kappa B) consensus sequence. DNA footprinting demonstrated protection of an NF- kappa B binding site at positions -377 to -368. Methylation interference assays showed that the TNF-induced protein made contact points with guanine residues in the same NF-kappa B sequence. Taken together, the findings provide evidence for involvement of an NF-kappa B-like factor in transcriptional regulation of the M-CSF gene.
Intracellular heme coordinately modulates globin chain synthesis, transferrin receptor number, and ferritin content in differentiating Friend erythroleukemia cells
Authors: Battistini A , Coccia EM , Marziali G , Bulgarini D , Scalzo S , Fiorucci G , Romeo G , Affabris E , Testa U , Rossi GB
Published: October 2019

The effect of succinylacetone (SA), a highly specific inhibitor of ALA- dehydratase and heme synthesis, on hemoglobin (Hb) production, transferrin receptor (TfR), and ferritin expression was analyzed in differentiating Friend leukemia cells (FLC). This compound exerted a pronounced inhibitory effect not only on heme and Hb synthesis, but also on all the remaining above-mentioned parameters. In particular, SA induced: (1) a reduction of the level of alpha-globin mRNA; (2) a decreased number of exposed TfR molecules, without modification of their affinity for the ligand; (3) a reduced level of TfR RNA, without significant change of TfR gene transcription rate; and (4) a lower ferritin content. The addition of exogenous hemin to differentiating FLC exerted opposite effects, and particularly induced an increase of both the number of TfRs and ferritin content. These findings suggest that in erythroid cells optimal heme synthesis is required to coordinately sustain globin chains synthesis and TfR/ferritin production; thus, the intracellular heme level may represent a key regulatory factor in the Hb synthesis pathway.
Expression of human blood coagulation factor XI: characterization of the defect in factor XI type III deficiency
Authors: Meijers JC , Davie EW , Chung DW
Published: October 2019

Human factor XI (FXI) is a blood coagulation factor participating in the early phase of the intrinsic pathway of blood coagulation. It circulates in blood as a glycoprotein composed of two identical chains held together by a single disulfide bond between the fourth apple domains. FXI has been expressed in baby hamster kidney (BHK) cells, where it was synthesized as a single-chain molecule that was converted to the dimer before secretion. The recombinant protein was fully active in a clotting assay, indicating that it interacted readily with other components of the coagulation cascade. A mutant FXI in which Phe283 was converted to Leu (Phe283Leu) was also expressed in BHK cells. This amino acid change occurs in the fourth apple domain of FXI and corresponds to the type III deficiency in Ashkenazi Jews. The mutant protein was secreted at reduced levels (about 8%) compared with normal FXI. This was due to a defect in the dimerization of the molecule rather than a decrease in the transcription of type III messenger RNA. Once secreted, however, the mutant protein consisted of a dimer with full biologic activity. The in vitro expression of FXI indicated that the impaired dimerization and secretion of the Phe283Leu mutant can account for the defect found in patients who are homozygous for the type III FXI deficiency.
Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations
Authors: Bovill EG , Tomczak JA , Grant B , Bhushan F , Pillemer E , Rainville IR , Long GL
Published: October 2019

This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.
Internalization and intracellular fate of anti-CD5 monoclonal antibody and anti-CD5 ricin A-chain immunotoxin in human leukemic T cells
Authors: Ravel S , Colombatti M , Casellas P
Published: October 2019

We have investigated the entry and subsequent intracellular fate of T101 monoclonal antibody (MoAb) and T101-ricin A-chain (RTA) immunotoxin (IT) directed against the CD5 antigen (Ag) expressed on human leukemic CEM cells. We provide direct evidence for the internalization of T101 MoAb and the corresponding IT. Both the MoAb and IT were internalized at a relatively low rate. This slow internalization process could be related to the partial recycling of the MoAb/Ag or IT/Ag complexes. Analysis of the internalized molecules showed that their molecular weight was only partially altered after internalization and that no free A-chain could be found inside the cells, indicating that lysosomal degradation and cleavage of disulfide- linked conjugates is a quantitatively minor phenomenon compared with the localization of internalized anti-CD5 ITs in an endosomo-Golgi compartment, followed by their recycling to the cell surface. We believe that this is the major factor explaining the low efficacy of anti-CD5 IT when assayed in the absence of potentiating substances. Finally, we showed that the presence of ammonium chloride and monensin, which both dramatically enhance the kinetics of IT activity, did not affect the rate of internalization or the intracellular localization of the conjugate, suggesting that these activators could act at the postendocytotic level on a limited number of IT molecules.