bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelets Provide Signals for CD34+ Progenitor Cells to Home to Sites of Vascular Injury.
Authors: Van Zonneveld Anton Jan , Verseyden Caroline , Loomans Cindy J.M. , Rabelink Ton J. , De Boer Hetty C.
Published: October 2019

Introduction Accumulating evidence indicates that vascular function is modulated by circulating progenitor cells (PCs). Circulating PCs have been shown to enhance angiogenesis, promote vascular repair, improve endothelial function, inhibit atherosclerosis and increase ventricular function after myocardial infarction. How circulating PCs home to their target sites is not known.

Hypothesis Since vascular damage, ischemia and atherosclerosis coincides with the activation of coagulation and platelets we hypothesized that fibrin, activated platelets and platelet products might trigger circulating PCs to home to sites of injury. Homing involves (1) escape of PCs from flowing blood, (2) migration towards an injury and (3) differentiation into an endothelial cell phenotype.

Methods & Results (1) Escape from flowing blood was tested in a flow model using tissue factor expressing endothelial cells (ECs) on which a fibrin network containing thrombi was formed under flow. The platelet thrombi offered flowing CD34+ cells a landing site leading to deceleration and firm attachment to the surrounding ECs. Tethering of the CD34+ was dependent on P-Selectin expressed by the platelets, since blokkage of P-Selectin or P-Selectin glycoprotein ligand-1, expressed by the CD34+, prevented deceleration of the CD34+ cells. (2) Migration of PCs was tested in a Boyden chamber in which fibrin, fibrin+platelets or platelets alone was used to attract CD34+ cells. Fibrin+platelets induced migration of CD34+ cells, while fibrin or platelets had no effect. (3) Differentiation of PCs was tested in a culture system using a fibrin clot containing platelets and a culture medium lacking serum and additional growth factors. In this way, secreted platelet products like VEGF, transforming growth factor, platelet activating factor or lysophosphatidic acid determine the culture conditions. After 10 days of culture on fibrin+platelets, CD34+ PCs showed uptake of acetylated LDL, expression of eNOS, VEGF receptor 2 and CD31, indicating that the cells exhibited endothelial-like features.

Conclusion Our results indicate that activation of the coagulation system and consequently activation of platelets, may offer circulating PCs a way to efficiently home to sites of vascular injury and thus contribute to neovascularization.
Stem Cell Factor (SCF) for Hematopoietic Stem Cell (HSC) Mobilization: Results of the Randomized IFM 99-01 Trial.
Authors: Bourin Philippe , Huynh Anne , Recher Christian , Berthou Christian , Garderet Laurent , Benbouker Lofti , Perry Anne-Marie , Facon Thierry , Hulin Cyrille , Sotto Jean-Jacques , Voillat Laurent , Michallet Mauricette , Attal Michel
Published: October 2019

Introduction : In multiple myeloma, the usual mobilization protocol is toxic because of the use of cyclophosphamide. Several studies showed the interest of SCF.

Objective : To compare two mobilization protocols: endoxan 4g/m2 + G-CSF 5μg/kg/j (arm A) versus G-CSF 10μg/kg/j + SCF 25μg/kg/j (arm B) in a prospective, open and, randomized trial.

Patients and methods : the studied criteria were the quality of the cell collections (objective > 5.106 CD34+ cells into 2 cytapheresis), the toxicity of the mobilization and graft phases, as well as the post-graft hematopoietic reconstitution. Multiple myeloma patients, less than 65 years old, with 0 or 1 risk factor (ß2microglobulin > 3 mg/L or chromosome 13 deletion) and who have a response ≥ 50% after 3 cures of VAD were included. After a fourth cure of VAD each patient was planed to receive a tandem transplant (IFM 99-02 trial).

Results : 150 patients (pts) were included and 138 were eligible (arm A = 67 pts, arm B = 71 pts). Pts and disease characteristics were similar in each arm. The objective of HSC collection was obtained with 92% pts in arm A and 81% pts in arm B (non significant). The total number of CD34+ cells collected were similar: 16.106 CD34+/kg (arm A) versus 15.106 CD34+/kg (arm B). Toxicity of HSC mobilization procedure was significantly different: duration of neutropenia < 500/mm3 (8 days in arm A versus 0 days in arm B, p<0.00001), duration of thrombopenia < 50 000/mm3 (5 days in arm A versus 0 days in arm B, p<0.0001), use of antibiotherapy (43% pts in arm A versus 9% pts in arm B, p<0.00001). 31% pts receiving SCF had local erythema at injection point. One pts experienced a grade 3 allergy in arm B. Hematopoietic reconstitution after first graft (high dose Melphalan 140 mg/m2, G-CSF at day 7) was not significantly different in either arm: duration of neutropenia < 500/mm3 (8 days in arm A versus 10 days in arm B), duration of thrombopenia < 50 000/mm3 (7.5 days in arm A versus 9 days in arm B), number of red blood cells units transfusions (1.5 versus 1.5). The 36 months overall survival probability was not significantly different with HDC mobilization (64%) versus SCF + G-CSF mobilization (87%).

Conclusion : In myeloma patients, HSC mobilization with SCF + G-CSF is as effective as HDC + G-CSF, and gives very significant lower toxicities.
Relation of CD4+CD25+ Regulatory T Cells to Immune Status in Patients with HIV Infection.
Authors: Tsunemi Sachi , Iwasaki Tsuyoshi , Imado Takehito , Higasa Satoshi , Kakishita Eizo , Okamoto Takahiro , Shirasaka Takuma , Sano Hajime
Published: October 2019

Human immunodeficiency virus (HIV) infection is characterized by marked defects in CD4+ helper T cell (Th) functions that commonly progress to a substantial decline in peripheral CD4+ T cell counts. However, the mechanisms responsible for the loss of Th functions in HIV-infected patients independent of CD4+ T cell counts remains unclear. CD4+CD25+ regulatory T cells (T Reg) are essential for down-regulation of both autoreactive and alloreactive T cells. Therefore, we decided to investigate the role of T Reg in immune status of HIV-infected patients. We examined the expression of cell surface CD25, cytoplasmic IL-4 and cytoplasmic IFN-gamma in peripheral blood CD4+ T cells from both healthy controls (n=9) and HIV-infected patients (n=43). We also compared T Reg functions between the 2 groups. CD4+CD25+ T Reg isolated from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3, and suppressed the proliferation of CD4+CD25− T cells, suggesting that CD4+CD25+ T cells from both healthy controls and HIV-infected patients possess phenotypic and functional characteristics of Treg. CD4+CD25high T cells are a subset of circulating CD4+CD25+ T cells in normal humans and exhibit strong in vitro regulatory functions similar to those reported for murine CD4+CD25+ T Reg. We measured the frequency of CD4+CD25high T Reg by analysis of surface CD25 on CD4+ T cells in peripheral blood samples. We also examined Th1 and Th2 frequencies by analysis of cytoplasmic IFN-gamma and IL-4 levels in CD4+ T cells. T Reg from HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high cell frequency (p<0.05) compared to healthy controls, with T Reg frequencies inversely proportional to CD4+ T cell numbers (p<0.01). However, in HIV-infected patients with undetectable plasma HIV-RNA, frequencies of CD4+CD25high T Reg were not increased and not related to CD4+ T cell numbers. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable: p<0.05, undetectable: p<0.001), but positively related to Th2 frequency (detectable: p<0.01, undetectable: p<0.001). Our results indicate that increased frequencies of peripheral blood T Reg were related to disease progression as measured by detectable plasma HIV-1 RNA, decreased peripheral blood CD4+ T cell counts, and polarization toward Th2 immune responses in HIV-infected patients. HIV infection may lead to induction of T reg that inhibit antiviral immune responses, resulting in the progression of the disease. Manipulation of T Reg could help restore antiviral immune responses in HIV infection, and prevent the progression of HIV infection.
CalDAG-GEFI Is a Key Signal Integrator in Platelet Activation and Thrombus Formation.
Authors: Bergmeier Wolfgang , Crittenden Jill R. , Piffath Crystal L. , Wagner Denisa D. , Housman David E. , Graybiel Ann M.
Published: October 2019

Inside-out activation of platelet integrin αIIbβ3 is a key step in agonist-induced platelet aggregation. Recent studies suggested the involvement of the small GTPase Rap1b in this process as it is highly expressed in platelets and becomes activated during platelet activation. In cell lines, overexpression of the Rap activator CalDAG-GEFI increased αIIbβ3-dependent adhesion, while overexpression of RapGAP, which inactivates Rap1, reduced αIIbβ3 activity. Here we provide evidence that CalDAG-GEFI is an essential component of this pathway in vivo. To generate CalDAG-GEFI knockout mice, we engineered mouse embryonic stem (ES) cells with a deletion that results in a frameshift mutation and a premature stop codon at the position encoding the 37th amino acid of CalDAG-GEFI. These ES cells were then used to derive chimeric mice that yielded germline transmission of the CalDAG-GEFI mutation. Deficiency of CalDAG-GEFI in mutant mice was confirmed by immunohistochemistry and western blot analysis. CalDAG-GEFI−/− platelets showed impaired Rap1b activation and aggregation in response to various agonists, with aggregation being completely blocked when platelets were activated with ADP, thromboxaneA2 analog, or calcium ionophore. Under physiological flow conditions in vitro and in vivo, CalDAG-GEFI-deficient platelets showed normal tethering to basement membrane components but failed to form thrombi. Mice deficient in CalDAG-GEFI were further characterized by a greatly increased bleeding time as well as by a strong protection against collagen-induced pulmonary thrombosis. In summary, we identified CalDAG-GEFI as a key signal integrator in the cascade leading through Rap1 and integrin αIIbβ3 to platelet aggregation and thrombus formation. The fact that CalDAG-GEFI knockout mice are resistant to collagen-induced thrombosis, and do not undergo spontaneous hemorrhaging, suggests that CalDAG-GEFI may be a promising new target for antithrombotic therapy.
Role for Interleukin-6 and Insulin like Growth Factor-I Via PI3-K/Akt Pathway in the Proliferation of CD56-Negative and Positive Multiple Myeloma Cells.
Authors: Sahara Naohi , Takeshita Akihiro , Kobayashi Miki , Shigeno Kazuyuki , Nakamura Satoki , Shinjo Kaori , Naito Kensuke , Otsuki Takemi , Ohnishi Kazunori , Ohno Ryuzo
Published: October 2019

Several studies including ours have suggested that lack of CD56 expression in multiple myeloma (MM) defines a unique patient subset with poorer prognosis. However, the mechanism underlying this aggressive behavior of CD56− MM has not been well elucidated. In this study, we sorted out both CD56− and CD56+ fractions from MM cell lines or patients with MM, and investigated their different responsiveness to interleukin-6 (IL-6) or insulin like growth factor-I (IGF-I), and tried to clarify the course of action in cell cycle distribution. After stained with PE-CD56, CD56− and CD56+ fractions in KMS-21-BM and U-266 cell lines were isolated by the cell sorter, and cultured separately either in the presence or absence of IL-6 (2 ng/ml and 10 ng/ml, respectively). Although CD56− cells in both KMS-21-BM and U-266 cell lines responded significantly to IL-6 (P=0.001 and 0.009, respectively), CD56+ cells did not. Ki-67+ cells in CD56− KMS-21-BM cells, that were significantly fewer than that in CD56+ ones (P=0.0003), increased significantly upon 24-hour incubation with IL-6 (P<0.0001). Western blotting analysis showed that the level of cyclin D1 and p27 protein in CD56− KMS-21-BM cells were up- and down-regulated by IL-6 in a time dependent manner, respectively. IL-6 also brought phosphorylation of Akt (ser473) in the CD56− cells. LY-294002 completely blocked these effects of IL-6. On the other hand, Ki-67+ cells in the CD56+ cells did not respond to IL-6. Although IGF-I did not increase Ki-67+ cells either in the CD56− and CD56+ cells from KMS-21-BM, anti-IGF-I mAb significantly reduced Ki-67+ cells only in the CD56+ cells (P=0.006). IGF-I up-regulated the level of cyclin D1 and phosphorylated Akt in CD56+ KMS-21-BM cells. LY294002 completely blocked these effects of IGF-I. Same results were obtained in the analysis of U-266 cell lines. The MM cells sorted from 17 patients with MM were also examined for CD56 and Ki-67 expression. Four and 13 patients were distributed to the CD56− and CD56+ group, respectively. These MM cells from the patients were cultured with or without IL-6 (10 ng/ml) or IGF-I (500ng/ml) for 24 hours. IL-6 increased the percentage of Ki-67+ cells in the CD56− group more than those in the CD56+ group (P=0.007). Although MM cells did not respond to IL-6, IGF-I significantly increased Ki-67+ cells in the CD56+ group (P=0.005). These results suggest that CD56− and CD56+ MM cells could be stimulated by different cytokines. We here found that CD56− MM cells were proliferated more than CD56+ MM cells in the presence of IL-6, and that this effect of IL-6 was mainly mediated by the activation of PI3-K/Akt pathway. In addition, our results suggest that IGF-I play an important role in the proliferation of CD56+ MM cells via PI3-K/Akt pathway.
Cladribine (CdA) or Fludarabine (F) or High-Dose Intermittent Chlorambucil (Chl) as First-Line Treatment of Symptomatic Chronic Lymphocytic Leukemia? First Interim Analysis of Data from the International Randomized Phase III Trial.
Authors: Karlsson Karin , Strömberg Mats , Jönsson Viggo , Gill Devinder , Hammerström Jens , Wallvik Jonas , Mulligan Stephen , Bradstock Kenneth , Liliemark Jan , Juliusson Gunnar
Published: October 2019

Purine analogues are effective treatment for CLL, but despite improved response rates and durations for purine analogues as compared to chlorambucil no survival advantage was documented. Our trial is the first comparing CdA and fludarabine (F) as first-line treatment of symptomatic B-CLL with high dose intermittent chlorambucil (Chl) as control. Patients aged 18–75 years from Scandinavia, Australia and UK were since November 1997 randomised between monthly courses of Chl 10mg/sqm orally day 1–10, F 25mg/sqm iv day 1–5, or CdA 5mg/sqm iv/sc day 1–5. Protocol is seen at www.cck.ki.se/CLL-study/index.html. Randomisation was stratified according to age and stage. Up to six courses were given according to response and toxicity. We now report a scheduled interim analysis of the first included 150 patients; 139 were evaluable and 5 excluded for not fulfilling eligibility criteria and 6 for incomplete data reporting. There was no significant difference in age, Binet stage or time from diagnosis to inclusion between the groups. Median age was 63 years and median time from diagnosis to inclusion was 16 months. Overall responses according to NCI criteria and intention to treat were documented in 57%/67%/74% of patients in the Chl/F/CdA arms, respectively (Chl vs CdA, p=0.06, Chi-test). The number of CR/nPR/PR in the different treatment groups were for Chl 2/3/22 (n=47), for F 0/2/28 (n=45), and for CdA 2/3/30 (n=47). NCI grade III and IV platelet toxicity was seen in 25% and 20% for Chl, 24% and 5% for F, and 36% and 11% for CdA. NCI grade III–V infections were seen in 25%/28%/30%, respectively. One F patient died in septic shock from E coli, and two CdA patients died from RS virus infection and aspergillosis, respectively; none of them responded to CLL-treatment. Autoimmune hemolysis was seen only with purine analogues, 4 during F and 3 during CdA treatment. We conclude that toxicity was feasible, and infections did not differ between arms. So far there is no significant difference in response rates between the therapies, but CdA seems at least as good as F. Inclusion is currently ongoing but will close during 2004, and a full evaluation is scheduled thereafter.
Evaluation of ADAMTS-13 Activity and VWF:Ag Levels in Patients with Ischemic Stroke.
Authors: Krause Manuela , Boehm Martina , Stier-Brueck Ingrid , Scharrer Inge
Published: October 2019

Background: The role of von Willebrand factor cleaving protease (ADAMTS-13) in the pathogenesis of ischemic stroke is still undefined. ADAMTS-13 cleaves ultra-large multimers of von Willebrand factor (ULVWF), and is believed to regulate the size thus the activity of VWF, especially on sites of high shear stress. By assuming the hypothesis, that a deficiency of ADAMTS-13 influences the thrombotic tendency such as intravascular aggregation and platelet thrombus formation, we analysed adult patients with onset of cerebral ischemia.

Methods and Patients: 158 unrelated patients (female:89 /male:69) with an objectively confirmed ischemic stroke (n=126) or TIA (n=32), median age at first onset:42 years (range:17–71 years), and 82 healthy subjects, median age:28 years (range:16–52 years), were studied. In our study group we analysed on the one hand 95 patients (pts) with early onset of stroke/TIA <45 years (median age:32 years), and on the other hand 63 elderly pts >45 years (median age:54 years). None of the pts enrolled had overt evidence of autoimmune or malignancy disease. ADAMTS13 was measured with an assay based on the positive correlation between multimeric size and Ristocetin Cofactor activity of the VWF.

Results: In our population the ADAMTS-13 activity was in median 95% (range= 40–185%) significantly higher than 91% (range=52–150%) among controls (p=0.002; OR 0.54). In the subgroup analysis we found no statistical differences in the mean ADAMTS-13 activities among pts with early onser (99% vs 91%; p=0.76, OR 0.93), and among pts with onset at age 45 years as compared to healthy controls (88% vs 91%; p=0.30, OR 1.25). Furthermore, we found statistical differences in the mean VWF:Ag levels among all pts as compared to healthy controls (135% vs 110%; p=0.01, OR 0.63), and in the elderly pts (140% vs 110%; p=0.01, OR 1.63). The young pts showed no statistical differences in the mean VWF:Ag levels compared with the control group (127% vs 110%; p=0.98, OR 0.99).

Conclusion: The results from this study show that higher levels of ADAMTS-13 activity and VWF:Ag as compared to healthy controls are associated with ischemic stroke/TIA. Only in the elderly pts we observed a tendency to a negative association between ADAMTS-13 and VWF (lower ADAMTS-13 levels and higher levels of VWF:Ag). It may be, that higher ADAMTS-13 levels have a relevance to prevent thrombotic events.

Further studies are needed to confirm whether the relationship between VWF and ADAMTS-13 plays an additional role in the pathogenesis on the onset of ischemic stroke.
Routine Prophylactic Platelet Transfusions Are Not Necessary for Patients in Clinically Stable Condition after Autologous Peripheral Stem Cell Transplantation(ASCT): Updated Results in 106 Patients.
Authors: Schaefer-Eckart Kerstin , Frank Markus , Wilhelm Martin , Wandt Hannes
Published: October 2019

We present our extended experience with an only therapeutic platelet transfusion strategy in patients after autologous peripheral stem cell transplantation(ASCT). Clinically stable patients(fever < 38,5°Celsius, no local infections, no sepsis syndrome) received single donor apheresis platelet transfusions only in the case of bleeding WHO ≥ II°, while prophylactic platelet transfusions were given to clinically instable patients if the morning platelet count was < 10/nl. In a first analysis after 50 patients we have shown that this strategy was safe, with no bleeding greater than WHO II°. In a retrospective matched-pair analysis the total number of platelet units transfused was reduced to 50% compared to our former strategy with routine platelet transfusions given when the morning platelet count was below 10/nl. (ASH 2002).

We now analysed 106 patients with a total number of 140 ASCTs. Median age was 54 years(19–70): The diagnoses were acute leukemia(17), lymphoma(34), solid tumors(9) and multiple myeloma(46). The conditioning regimens corresponded to standard protocols.

Median days of thrombocytopenia < 20/nl and 10/nl were 6(0–92) and 3(0–62) respectively, with a total number of days with thrombocytopenia <20/nl and <10/nl of 989 and 508. Hemorrhages WHO I° and II° was observed in only 49 out of 140(35%) ASCTs. We observed no bleeding greater than WHO II°. The median number of platelet units was 1(0–18). 48 out of 140(34%) transplantations could be performed without platelet transfusions. In multiple myeloma this percentage was even higher: 32/68(47%). The indications for prophylactic transfusions were mainly FUO(21/61 – 34%) and mucositis with or without fever(19/61–31%). Considering age below or above the median age of 54 years or different diagnoses, there was no difference in days with platelets <10/nl, <20/nl, bleeding complications or median number of platelet units transfused. The total number of 234 transfusions in these 140 transplantations could have been even further reduced, because 15%(36/234) of the transfusions were given without a clear indication regarding the study regimen, because of a learning effect with this new strategy. This new strategy has shown to be very safe and prophylactic platetelet tansfusions are probably not necessary in clinically stable patients with fever as the only sign of an infection.

We are just starting a multicenter randomised study comparing this new strategy with the former strategy of routine prophylactic platelet transfusion.
Accessing the Erythrocyte Cytoplasm: A Method for Manipulating the Intracellular Environment of Erythrocytes for the Study of Malaria Invasion, Trafficking and Growth.
Authors: Murphy Sean C. , Bhattacharjee Souvik , Harrison Travis , Haldar Kasturi
Published: October 2019

Invasion of erythrocytes by malaria parasites requires participation of both parasite ligands and host determinants. Further recent studies show that erythrocyte G protein signaling regulates malarial infection. Many of the Gs-associated signaling components reside on the cytoplasmic leaflet of the erythrocyte plasma membrane, rendering them inaccessible to most extracellular probes. Since erythrocytes are enucleated and terminally differentiated, they cannot be transfected to express exogenous transgenes. We have modified methods of hypotonic lysis and isotonic resealing to generate loaded erythrocyte ghosts that can be efficiently infected by Plasmodium falciparum and sustain normal levels of intraerythrocytic parasite growth and replication. Further, we show that these ghosts can be filled with various membrane-impermeable peptides or other proteinaceous cargoes for studying signaling and transport events inside the erythrocyte. Resealed ghosts morphologically resemble normal erythrocytes, albeit with reduced hemoglobin content. Other measures of erythrocyte function and malarial infection are being investigated. Studies will be presented on the use of ‘reconstituted’ erythrocytes in elucidating mechanisms parasite invasion as well as parasite protein trafficking in erythrocytes infected by P. falciparum.
The Venezuelan Type of Nondeletional Hereditary Persistence of Fetal Hemoglobin Is Due C →T Mutation at Nucleotide -211 of the Aγ Globin Gene.
Authors: Arends Anabel , Urquiola Martha Bravo , Montilla Silvia , Lic Gloria García , De L. Dalia Velásquez , Alvarez Maritza , Gerard Natalie , Krishnamoorthy Rajagapol
Published: October 2019

Hereditary Persistence of Fetal Hemoglobin (HPFH) is a clinically benign condition characterized by continuous synthesis of fetal hemoglobin (Hb F) in adult life without major related hematological changes. The nondeletional HPFH is characterized by hemoglobin F level between 5% and 30%, in heterozygotes state. This condition has been observed among Blacks, Italians, Greeks, English and Chinese. Two different forms are known, characterized by the type of chain involve, either Gγ or Aγ. Several point mutations in the promoter region of the Aγ globin gene have been identified in ndHPFH: −202, −198, −196, −195, −117, −114, deletion 4pb (−222 −225) and −158.

The propositus is a 36 year old black female with a moderate increased level of Hb F, also her sister and her sibling had the same condition. She was discovered during studies due to the presence of splenomegaly and gastroesophageal reflux. The Hbs A, A2 and F were eluted and quantified by cation exchange high-performance liquid chromatography (HPLC-CE), using Beta Tal short program, Variant BioRad (Hercules, California, USA). DNA was isolated from peripheral blood leukocytes by a salting-out extraction procedure. The Aγ and Gγ promoters globins gene were amplified independently by PCR and the presence of the HPFH mutation was determined by denaturing gradient gel electrophoresis technique (DGGE) as previously described Gottardi et al., (1992). The Aγ promoter globin gene was sequenced in three members of the family using the BigDyeTM Terminator Cycler Sequencing Kit and the ABI PRISMTM 310 Genetic Analyzer (PE Applied Biosystems, Foster City, CQ, USA).

Her hematological studies revealed Hb: 14.2 g/dl, Hcto: 41.1 %, MCV: 94.4 fl, MCHC: 34.5 fl, Hb A: 81.9 %, Hb A2: 2.1 % and Hb F: 6.3 %. The sibling hematological finding was Hb: 13.1 g/dl, Hcto: 41.8 %, MCV: 83.7 fl, MCHC: 31.3 fl, Hb A: 83.2 %, Hb A2: 3.2 % and Hb F: 5.1. The sister was Hb: 14.3 g/dl, Hcto: 46.4 %, MCV: 105.9 fl, MCHC: 30.8 fl, Hb A: 83.7 %, Hb A2: 3.3 % and Hb F: 2.7 %. No spleen enlargement was seen in the other two affected members of the family. The DGGE results in the Aγ promoter, suggested the presence of a non-deletion form of HPFH in heterozygotes state. The Aγ promoter region revealed the presence of a substitution C→T at position −211. This mutation does not modify any restriction site.

To our knowledge the −211 (C→T) mutation in the Aγ promoter have not been reported previously. Since modulation of g-globin gene expression in adults could provide a therapeutic intervention in hemoglobinopathy, the study of the molecular mechanisms generating HPFH is medically relevant.
Erythropoietin (EPO) Plasma Levels Reflect the Course of Clinical Vaso-Occlusive Complications in Sickle Cell Disease.
Authors: Duits Ashley J. , Akin Sakir , Ozveren Erkan , Mac Gillavry Melvin R. , Brandjes Dees P. , Schnog John-John B.
Published: October 2019

Erythropoietin (EPO) has an important role in both erythropoieses and angiogenesis. EPO levels are elevated in sickle cell patients due to severe anemia, but tissue hypoxia, due to for example vaso-occlusion, may also contribute to EPO elevation. In this study we sequentially determined EPO plasma levels in HbSS sickle cell patients (n=14, 39 samples) presenting with vaso-occlusive complications at our emergency ward and the period thereafter. Patients were also requested to fill out a pain-intensity score during these events. Our results show a striking correlation between the course of vaso-occlusive events and plasma EPO levels. Vaso-occlusive crises were accompanied by impressive increments of plasma EPO levels with quick reduction of plasma EPO levels upon crisis amelioration. Changes in plasma EPO levels also closely reflected changes in pain intensity. The results of this study warrant further analysis of the potential role of EPO in the pathophysiology of sickle cell disease related complications, as well as the applicability of employing EPO levels as a marker of vaso-occlusive disease activity.
Prevalence of Pulmonary Hypertension (PHTN) in Sickle Cell Disease (SCD) Adolescents with Pulmonary Complications.
Authors: Onyekwere Onyinye C. , Campbell Andrew , Williams James , Gaskin Peter , Rana Sohail , Gordeuk Victor R. , Castro Oswaldo L.
Published: October 2019

Despite the high prevalence of PHTN in adults with SCD, the prevalence in the pediatric population with SCD is not known. We hypothesized that elevated pulmonary artery pressures may be found in SCD adolescents with history of pulmonary complications, such as acute chest syndrome (ACS), obstructive sleep apnea (OSA), asthma, and reactive airway disease. Thirty such sickle cell disease adolescents were screened at Howard University or University of Michigan for PHTN with Doppler echocardiography. We defined PHTN as a tricuspid regurgitant jet velocity (TRV) of at least 2.5 m/sec (corresponding to a pulmonary artery systolic pressure greater than 35 mm Hg). PHTN was found in 16 SCD patients (53.3%) and 5 (16.7%) had TRV > 3.0 m/sec. Clinical findings according to the presences or absence of PHTN are shown in the table.

Potential factors contributing to PHTN in patients with SCD include chronic hemolysis and chronic hypoxia. Our results suggest that PHTN is common among SCD adolescents with a history of pulmonary complications. Consideration should be given to screening such patients for PHTN and exploring treatment options. Further studies are urgently needed to clarify the prevalence and mechanisms of PHTN in adolescents with SCD.

Clinical and demographic data of 30 SCD adolescents with pulmonary findings who underwent echocardiography at Howard University Hospital or University of Michigan

PHTN (N = 16) No PHTN (N = 14) P Age in years (mean +/− SD) 15.9 +/− 3.2 17.4 +/− 2.3 0.17 Females (no. and %) 5 (31.3) 7 (50) 0.5 Hemoglobin SS Phenotype (no and %) 14 (87.5 11 (78.6) 0.5 Hemoglobin concentration (mean +/− SD) 8.0 +/− 2.1 9.3 +/−1.9 0.11 White blood cells (mean +/− SD) 10.9 +/− 2.9 9.7 +/− 3.7 0.4 Platelet (mean +/− SD) 475 +/− 172 364 +/− 240 0.17 Hemoglobin F percent (mean +/− SD) 5.1 +/− 3.5 6.4 +/− 5.5 0.6 Lactate dehydrogenase (mean +/− SD) 505 +/− 162 264 +/− 50 0.002 Total bilirubin (mean +/− SD) 4.1 +/− 2.6 3.4 +/− 2.6 0.5 Creatinine concentration (mean +/− SD) 0.6 +/− 0.2 0.7 +/− 0.2 0.18 Aspartate transaminase (mean +/− SD) 48 +/− 27 36 +/− 16 0.18 Alanine transaminase (mean +/− SD) 51 +/− 37 39 +/− 20 0.3
Detailed Analysis of Neutropenic Risk Factors in Patients with Prostate Cancer Treated with Chemotherapy.
Authors: Laber Elizabeth R. , Laber Damian A.
Published: October 2019

Background: Although thousands of patients (Pts) receive chemotherapy every year in the United States, only few retrospective studies have been done to address the risk factors for chemotherapy (CT) induced neutropenia in patients with cancer. The data in androgen-independent prostate cancer (AIPC) patients is extremely scarce. We decided to perform a retrospective analysis of patients with metastatic AIPC treated with chemotherapy in an open phase II clinical trial looking for predictive factors for neutropenia.

Methods: We reviewed the records of all patients with AIPC who completed at least 2 cycles of CT with cyclophosphamide 50 mg/m2, etoposide 50 mg/m2 and estramustine 280 mg orally every night for 14 days out of a 28 day-cycle. We divided the patients into 2 groups according to neutropenia grade 3–4 (Group 1) versus 0–2 (Group 2). Risk factors reviewed included age, number of organs involved with metastases, number of bone metastasis, prior radiotherapy (RT) to the prostate/pelvis, prior number of areas treated with palliative RT, prior CT regimens, baseline PSA, absolute neutrophil count (ANC) and ECOG performance status (PS).

Results: See table and figures.

Group 1 2 Neutropenia Grade 3–4 0–2 # of Pts 7 11 Age 68 (58–86) 63 (52–74) # organs involved 2.1 1.6 # bone metastases 10.9 9.5 RT prostate/pelvis 0.1 0.4 # palliative RT courses 1 0.6 # prior CT regimens 0.4 0.2 Baseline ANC 3.6 3.8 Baseline PS ECOG 1.3 1.4 Baseline PSA 584 (26–2268) 157 (37–65)

Figure Figure

Conclusions: Average age and PSA were the only risk factors that were different between the patients who developed severe neutropenia and the ones who did not, albeit not statistically significant. Studies with a larger sample size may detect clinically and statistically significant differences.
The Effect of Adenosine on TLR Signaling Pathways in Human Monocytic THP-1 Cells.
Authors: Chen Chin-Fu , Cheng Chun-Huai , Vos Seychelle
Published: October 2019

Adenosine is an important metabolite that serves as a potent regulator of inflammation and mediates various biological functions in different cell types. Adenosine inhibits the proinflammatory actions of inflammatory and immune cells via interaction with its receptors, particularly A2A receptors. Adenosine receptors belong to the seven-transmembrane G-protein coupled receptors and via the different G proteins transfer signals through different effectors including adenyl cyclase, PKA, PKC, PI3K, and MAP kinases. The mechanisms by which the adenosine regulates immune responses and how adenosine receptor pathways interact with other signaling pathways are currently unknown. Toll-like receptors (TLRs) of the innate immune cells recognize conserved microbial structures, such as bacterial lipopolysaccharide and viral double-stranded RNA, and activate signaling pathways that result in innate immune responses against microbial infections. Fcγ receptors of the innate cells play a critical role in the clearance of pathogens, regulation of inflammation and co-ordination of the immune response. We seek to understand the interaction between adenosine (via adenosine receptors) and TLR- and Fcγ R- mediated signaling pathways. We have initiated study on the effect of adenosine and lipopolysaccharide (LPS) on expression of TLR2, TLR4, FcγRI and Fcγ RII receptors in the human monocytic cell line THP-1. We incubated cells with 100μM adenosine for three hours at 37°C and assayed the expression of receptors using flow cytometry. Our results suggest that adenosine increases the expression of TLR2 and TLR4 in both undifferentiated cells and the cells induced to become macrophages by phorbol ester. Incubation with adenosine for 24 hours further increases the expression of TLR2 and TLR4 in both undifferentiated and differentiated THP-1 cells. Similarly, incubation with LPS for three hours increases the expression TLR2 and TLR4 in both undifferentiated and differentiated THP-1 cells. In contrast, the expression level of FcγRI and FcγRII receptors do not change in the presence of either adenosine or LPS. These observations suggest that adenosine specifically enhances expression of TLRs but not Fcγ receptors. To further understand the interaction between adenosine and TLR pathways, we are continuously investigating the effect of adenosine on expression and the protein modification (e.g. phosphorylation) of TLR2, TLR4, and the molecules in the TLR signaling cascades including MyD88, IRAK and MAP kinases using real-time RT-PCR and western blotting.
Recurrent Histiocytosis Evolving to Myelodendritic Leukemia.
Authors: Rubio Araceli , Recasens Valle , Perez-Barrachina Carmen , Orfao Alberto , Giraldo Pilar
Published: October 2019

Background: Langerhans cells histiocytosis (LCH) is a rare disorder caused by proliferation of activated Langerhans cells. Although current therapies are very effective to induce a remission, multiple recurrences are common. Patients: In 1999 a 20-year-old female was admitted in the hospital to study by amenorreha for 5 months. In the exam a tumour located in the cranial base was identified and in lung a nodular infiltration was observed in the CT scan. The histological diagnosis was eosinophilic granuloma. The clinical manifestation was characterised by hypothalamic dysfunction, pituitary failure and diabetes insipidus (DI). Treatment: Telecobaltotherapy in CNS and chemotherapy (DAL HX-83/90). After therapy CNS MRI became normal, and body computed tomography (CT) scan showed minimal residual lung disease. The patient became asymptomatic, under hormonal substitutive therapy. Three years later, the patient was asymptomatic with ECOG 0, but in her peripheral blood a 3% of blast cells was detected (hemoglobin 8.2 g/dL, WBC 3.2x109/L neutrophils 3%, monocytes 8%, lymphocytes 86%, blasts 3%, platelets 213x109/L). Bone marrow: 0.5% blasts: CD45+, CD117+, CD33+, CD13+ weak, CD64++, CD15++, CD3−, CD2−, CD7−, CD79a−, CD19−, MPO−, CD34−, CD56++, HLADR+++, CD36+, CD11b−, CD7.1−/+lisozime+, CD1−/+, CD14 and 77.5% of cells with the same immunophenotype except CD117−, 7.1− and CD45−. One weak later we repeated the bone marrow exam and we found: 88.8% blasts cells with immunophemotype similar to blast as first exam. FISH: 88% translocation in 11q23. CT scan showed small and few nodules in lung and skin lesions were absent. She was diagnosed of leukemia involving progenitors of dendritic Langerhans’/monocytic differentiation cells: FAB AML-M0/M4. The patient has not sibling and she is receiving treatment with 2-Chlorodeoxyadenosine (0.12 mg/kg, days 1–5/28 x 6 courses) associated to epoetin-a. After two courses the patient had normalized her pheripheral blood (hemoglobin 12.5 g/dL, WBC 6.7x109/L with normal distribution and platelets 485 x109/L. The tolerance to therapy was excelent without adverse effects and she has a normal quality of life. Comments: The use of 2-Chlorodeoxyadenosine in LCH is based in its antiproliferative and immunomodulatory effects. CDA has been used in LCH with good results and minimal toxicity. Some cases published have showed a durable complete response in patients with LCH in skin, gastrointestinal, lungs, bone, diabetes insipidus and parenchymal CNS. In our knowledge, it has not been described previously a case of DMCL treated succesfully with CDA. Further studies are needed to determine the role of CDA in this disease, the optimal and schedule of therapy, but we have proved it may be effective even in high-risk patients with LCH. More therapies based on the pathogenesis of LCH are needed to investigate. This study has been supported by a grant from FEHHA
Clinical and Pathological Presentation of T-Cell Large Granular Lymphocyte Proliferations (T-LGL): A Single Institution Experience.
Authors: Battiwalla Minoo , Wallace Paul K. , Ford Laurie A. , Baer Maria R.
Published: October 2019

Proliferations of T-cell large granular lymphocytes (T-LGL) are a rare and heterogeneous disease entity characterized by a chronic course, cytopenias and circulating cytotoxic T lymphocytes. We present the results of a single institution retrospective review of the clinical presentation of T-LGL proliferations. A total of 23 patients evaluated for cytopenias were identified as having T-LGL by flow cytometry, ten male and thirteen female, with a median age of 67 years at diagnosis. Twelve patients had a prior malignancy (non-Hodgkins lymphoma=4, MDS/AML=4, breast cancer=2, squamous cell cancer (SCC) of the skin =2, lung SCC=1, vocal cord SCC=1, uterine cancer=1) and five patients had a history of autoimmune disease (rheumatoid arthritis=2, vasculitis=1, polyarthritis=1, Wegener’s granulomatosis=1), while seven patients had no previous history of autoimmune disease or malignancy. Four patients with prior malignancy had active disease and this led to death in three. Of the twelve patients informative for T-cell receptor gene rearrangements, clonal populations were found in eight. All patients who had cytopenias requiring treatment were initially treated with cyclosporine monotherapy, with the further addition of growth factors if necessary.

Multiparameter flow cytometric analysis of blood or bone marrow aspirates revealed that patients with a prior history of systemic malignancy had the common immunophenotype of CD3+(h), CD8+(h), CD2+, CD5+, CD7(h), CD56(h), CD57+, CD38+, CD16−. In contrast, patients who did not have a prior history of malignancy were CD3+, CD8+, CD2+, CD5+, CD7+, CD56+/−, CD57+, CD38+/(h), CD16+/− (h=heterogeneous). Thus patients with a prior malignancy had more heterogeneous expression of the T-cell markers CD3, CD8 and CD7 and were less likely to express CD16. We conclude that prior malignancy is more common in patients with T-LGL proliferations than previously recognized, may be twice as frequent as autoimmune disease in association with T-LGL and may have a distinct immunophenotype. Our data suggest that malignant cells may induce proliferations of T-LGL.
The Relationship between Systemic Anticoagulation during Cardiac Surgery and Heparin Platelet Factor 4 Antibody Formation.
Authors: Francis John L. , Drexler Alane , Walker Jamie , Rathmann Kristin
Published: October 2019

Heparin-induced thrombocytopenia occurs in approximately 1–3% of patients receiving unfractionated heparin. However, up to 50% of patients receiving heparin for systemic anticoagulation during cardiac surgery with cardiopulmonary bypass develop antibodies to the heparin-platelet factor 4 (HPF4) complex detectable by enzyme-linked immunoassays (ELISA). This high incidence of seroconversion is not diminished by off-pump cardiac surgery (OPCAB), despite significantly lower doses of heparin used for surgical anticoagulation. We tested the hypothesis that HPF4 antibodies develop following cardiac surgery even when heparin is not used for surgical anticoagulation. Patients (n=133) were randomized (2:1) to receive bivalirudin (The Medicines Company, Parsippany, NJ) or heparin for surgical anticoagulation during OPCAB. There were no restrictions on the pre-operative or post operative use of heparin. Blood samples were obtained pre-operatively (baseline), and 7 and 30 days after surgery. Serum samples were stored frozen until tested with a commercially available ELISA for HPF4 antibodies (GTI Inc., WI). An optical density (OD) ≥ 0.4 was considered positive, while an OD >1.0 was deemed strongly positive. The percentage of patients having a positive HPF4 antibody test (OD ≥ 0.4), was similar at baseline (6.7 vs. 6.8%) and at 7 days (27.1 vs. 29.3%) in bivalirudin and heparin-treated patients, respectively. At 30 days, the frequency of HPF4 antibodies was lower in the patients who received bivalirudin for surgical anticoagulation (32.5% vs. 40.9%). The percentage of patients having a strongly positive ELISA (OD > 1.0) was 0% at baseline in both groups, 4.3 vs. 7.3% at 7 days and 4.8 vs. 9.1% at 30 days for bivalirudin vs heparin, respectively. These data suggest that HPF4 antibody formation during hospitalization for cardiac surgery is not dependent only on the heparin used for anticoagulation during surgery. Antibody positivity in baseline samples presumably results from pre-operative heparin use and is consistent with previous reports. Since bivalirudin is not capable of eliciting an HPF4 antibody response, post-operative seroconversion presumably reflects a combination of both pre- and post-operative heparin use. However, when heparin was used for surgical anticoagulation, the number of patients subsequently developing strongly positive HPF4 antibodies was almost twice that seen in the bivalirudin group. This suggests that the use of bivalirudin as a surgical anticoagulant may reduce the incidence and risk of HIT following cardiac surgery.
Inhibitor Incidence in Previously Untreated Patients (PUP) with Hemophilia A and B - a 10-Year-Follow up of the Prospective Multicenter Study.
Authors: Kreuz Wolfhart , Auerswald Guenter K.H. , Budde Ulrich , Lenk Harald
Published: October 2019

The development of neutralizing FVIII-antibodies is one of the most serious complication in the replacement therapy with FVIII and IX concentrates in hemophilia A and B patients. In order to observe different parameters with possible impact on inhibitor development in PUPs with hemophilia A and B a prospective study was started in 1993 by the GTH (German, Swiss and Austrian Society on Thrombosis and Haemostasis Research).

The study protocol provides frequent inhibitor testing particularly during the initial 200 exposure days (ED) according to the modified Bethesda method (central lab Prof. Budde, Hamburg) and correlates inhibitor development with age at first exposure, therapy regimen, dosage, mutation type, type of concentrate and further variables which may have an impact on inhibitor development. Until September 2003, 293 patients with hemophilia A and B have been enrolled in this ongoing study. 206 have been treated (176 hemophilia A, 30 hemophilia B) at least once with plasma derived (pd) or recombinant (r) factor (F) VIII or IX concentrates.

Out of 176 hemophilia A patients 35 developed inhibitors (16 high titer >5 Bethesda Units/BU, 17 low titer >0.6–5 BU, 2 transient low titer inhibitors) after 12 exposure days (ED) in median (range 1–56) at the age of 0.9 years (median, range 0.25–10.8). Inhibitor development was predominantly observed in severe hemophilia A patients (31%) and to a lesser extent in moderate patients (7.7%). Inhibitor incidence was higher in patients receiving recombinant FVIII products than in those treated with plasma derived concentrates (p=0,14). No correlation between age at first exposure and inhibitor development was found. Early prophylaxis resulted in a lower inhibitor incidence than on-demand treatment (p=0.003).

Out of 16 severely affected hemophilia B patients 2 developed inhibitors.
Laboratory Analysis of Abnormal Closure Times with Relation to Low Ristocetin Cofactor Versus Other Causes.
Authors: Arthur Emma , Hassett Andrea Cortese , Bontempo Franklin A.
Published: October 2019

Closure times (CTs) have replaced the limitations of the previous testing methods. However, the clinical utility of detecting abnormal CTs remains poorly defined, with the differential diagnosis including von Willebrand’s disease (vWD), aspirin or NSAIDs, uremia, liver disease, or congenital defects. Ristocetin cofactor (RCoF) measures the functional activity of the vW molecule and when low, most often suggests vWD. CTs are frequently done in patients suspected by history of having vWD with some laboratories preferring to perform the collagen/epinephrine (Col/Epi) CT alone, while others additionally include the collagen/ADP (Col/ADP) CT.

METHODS: We analyzed 1480 consecutive CTs from a large reference laboratory done on a PFA-100 (Dade-Behring) and correlated the CTs with RCoF levels done on a BCS analyzer (Dade-Behring). Normal ranges were: Col/Epi CT 84-175 sec.; Col/ADP CT 65-117 sec.; RCoF 0.5-1.5 U/ml. Calculated means for CT of ≥ 300 sec. used a level of 300.

TABLE

Of the 260 studies with abnormal closure times, 131 (50%) had normal RCoF values, 47 (18%) were high and 18 (32%) were low. All studies with low RCoFs had at least one abnormal CT with 78/82 (95%) having both CTs prolonged, 2/82 (≫2%) with a long Col/EPI only, and 2/82 (≫2%) with a long Col/ADP only. Mean CTs in patients with low RCoFs and both CTs abnormal were 252 seconds for Col/EPI and 174 seconds for Col/ADP, both well above the upper limits of the normal range. Mean CTs in patients with low RCoFs and one abnormal CT were 180 seconds for Col/EPI and 123 seconds for Col/ADP, both just slightly above the upper limits of the normal range.

CONCLUSIONS: These findings suggest that when RCoF is low, both Col/EPI and Col/ADP CTs are prolonged, usually markedly, and the chances that a low RCoF will be missed is low, particularly if both CTs are used.

RESULTS: Total CTs analyzed: 1480 No.≥ 1 Abnormal CT 260/1480 (18%)

Number (%) Both CTs Abnormal Col/Epi ONLY Abnormal Col/ADP ONLY Abnormal Total Abnormal CTs 260 (100%) 203 (78%) 42 (16%) 15 (6%) Total Low RCoFs 82 (100%) 78 (95%) 2 (≈2%) 2 (≈2%)