bluuebunny/crossref_metadata_2025 huggingface dataset:
Hodgkin’s Disease
Authors: SHIMKIN MICHAEL B.
Published: October 2019

Mortality from Hodgkin’s disease in the United States during the period 1921 through 1951 was analyzed with respect to race, sex and age incidence and distribution. The findings were compared with those reported for leukemia.

The recorded death rate from Hodgkin’s disease rose from 6.9 in 1921 to 17.0 per million in 1951. During this period, the death rate from leukemia rose from 14 to 61 per million.

The death rate among males is higher than among females for both diseases; the male predominance is more marked in Hodgkin’s disease than in leukemia. The rate is higher among whites than non-whites for both diseases; the white predominance is more marked in leukemia than in Hodgkin’s disease. There is no peak in rate during childhood for Hodgkin’s disease as there is for leukemia, and the increase in rate with age is much less steep for Hodgkin’s disease than for leukemia.

The mean age at death of adults dying from Hodgkin’s disease and from leukemia increased by 3.5 and 8.0 years, respectively, between 1925 and 1950. The male-female sex ratio for Hodgkin’s disease decreased slightly, and increased slightly for leukemia between 1925 and 1950.
Hypoproaccelerinemia
Authors: LEWIS JESSICA H. , FERGUSON JOHN H.
Published: October 2019

This communication is concerned with the case of a 3-year old girl who had shown a moderately severe bleeding tendency since the age of 6 months. The cause of the bleeding could be clearly attributed to a marked deficiency in plasma proaccelerin and platelet accelerator factor. Abnormalities were also found in the clotting time, prothrombin consumption and Quick test. That this was probably familial in nature was suggested by moderately reduced plasma proaccelerin levels in the patient’s parents and in one sibling.*
Studies of Coagulation and Fibrinolysis by New Technic of Continuous Recording
Authors: VON KAULLA KURT N. , WEINER MURRAY
Published: October 2019

1. The Hartert instrument is a useful tool for recording continuously changes in a physical property of the clot during its formation and dissolution.

2. Alterations in fibrinogen content, platelets, and plasma clotting factors influence the pattern of the coagulogram. These patterns do not necessarily parallel the results of classical clotting time studies based on observation of the time necessary to form a visible clot. Clearly visible clots may not be sufficiently firm to be recorded by the coagulograph.

3. Coagulograph studies require careful handling of blood specimens to avoid artefacts of extrinsic origin.

4. The lysis of clots by different enzymes follows significantly different patterns which are not detectable by the standard methods for observing the dissolution of a clot.
Absorption Microspectroscopy of Bone Marrow Cells
Authors: DE CARVALHO SÉRGIO
Published: October 2019

Using a microspectrographic technic, reflecting optics and narrow bands of the continuum (5500, 4150 and 2650 Å), the author has obtained pictures of human and rat bone marrow cells in unfixed dry smears as well as in suspensions in vivo; erythroblasts showed different absorption patterns. Violet light microspectrography, specific for heme absorption, allowed us to demonstrate the presence of intranuclear heme granules through various stages of maturation of these cells. This confirmed previous and simultaneous results arrived at by cytochemical and other methods.

The presence of nucleic acids has been demonstrated in neutrophil granulations.
Megaloblastic Anemia of Pregnancy in Two Sisters
Authors: HOLLINGSWORTH JAMES W. , ADAMS FAE M.
Published: October 2019

1. Two sisters with megaloblastic anemia of pregnancy were reported.

2. Erythrocytes from the patients, both before and after therapy with folic acid, were tagged with radioactive sodium chromate and injected into normal subjects. The results of these studies were discussed.
Marrow Fibrosis in Myelodysplastic Syndromes - Its Significance in the Context of the WHO Classification of Disease and the International Prognostic Scoring System.
Authors: Buesche Guntram , Ganser Arnold , Wilkens Ludwig , Schlegelberger Brigitte , Hecker Hartmut , Georgii Axel , Kreipe Hans H.
Published: October 2019

Marrow fibrosis (MF) is rarely considered in myelodysplastic syndromes (MDS) although the frequency of this complication ranges from 10 to 50 % in the few reports on this issue, and there are no data on occurrence and significance of this complication in the context of the International Prognostic Scoring System (IPSS) and the World Health Organization (WHO) classification of disease. In a retrospective study, diagnostic bone marrow biopsies from a total of 936 patients with MDS were examined for MF and its relevance to the course of disease. Frequency of MF varied markedly between different types of MDS ranging from 3 % (RARS) to 37 % (MDS, therapy-related; WHO classification, P < 0.000005). Risk of MF furthermore correlated with multilineage dysplasia (P < 0.000005). However, there was no obvious correlation to the IPSS or to karyotype abnormalities. The survival time of patients was significantly reduced by about 50 % from 11 (RAEB-1/-2) - 55 (RARS, RCMD-RS) down to 6 (RAEB-1/-2) - 33 months (RARS, RCMD-RS) in median when MF was detected independently of the IPSS and the classification of disease (FAB, WHO; P = 0.0001). We conclude that MF is an unfavorable complication of MDS significantly shortening the survival time of patients independently of the IPSS and the classification of disease.
HIV-1 Nef Interferes with M-CSF Receptor Signalling through Hck Activation and Inhibits M-CSF Bioactivities.
Authors: Okada Seiji , Suzu Shinya
Published: October 2019

HIV-1 Nef protein is a major determinant of the pathogenicity of the virus. It has been shown that Nef activates Hck, a member of Src family kinase, in monocytes/macrophages and that the interaction is critical for AIDS-like disease progression in a mouse model. However, it was unclear how the molecular interaction in monocytes/macrophages leads to the disease progression. Here, we show for the first time that Nef interferes with the M-CSF/M-CSF receptor pathway. In this study, we introduced a conditionally active Nef into myeloid leukemia TF-1-fms cells and analyzed their responsiveness to M-CSF. We found that Nef-activated Hck constitutively associated with M-CSF receptor complex. The formation of the molecular complex should occur under physiological conditions, i.e., upon M-CSF stimulation. Due to the aberrant molecular association, the tyrosine-phosphorylation/activation of the receptor in response to M-CSF was markedly diminished in Nef-active cells. Consequently, Nef activation caused the inhibition of M-CSF-mediated proliferation of TF-1-fms cells and macrophage differentiation of the cells induced by M-CSF and 12-O-tetradecanoylphorbol 13-acetate. These results indicate that HIV-1 Nef interferes with M-CSF receptor signalling through Hck activation and thereby inhibits M-CSF functions in monocytes/macrophages.
Gene Conversion of Sialic Acid Binding Domains in CD33-Related Siglecs by Adjacent Pseudogenes: A Novel Mechanism To Change Sialic Acid Binding Specificity.
Authors: Hayakawa Toshiyuki , Angata Takashi , Margulies Elliott H. , Mikkelsen Tarjei , Green Eric D. , Varki Ajit
Published: October 2019

Siglecs (sialic acid-binding immunoglobulin superfamily lectins) are a family of cell surface receptors involved in regulating the immune response. The CD33-Related Siglecs (CD33rSiglecs, namely Siglec-3, -5 through -11 and -XII in humans) are a subgroup of these molecules found primarily on cells of the innate immune system. All are type-1 transmembrane proteins with an N-terminal sialic acid-recognizing V-set domain followed by a variable number of C-2 set domains, a transmembrane region and a cytosolic C-terminal domain that includes two tyrosine-based signaling motifs. Available data suggest an inhibitory signaling role in the innate immune response, mediated by recognition of host sialic acids as “self”. Nine of the 13 known primate Siglec genes along with 14 Siglec pseudogenes comprise the CD33-related Siglec gene cluster on human chromosome 19. Gene conversion is a mechanism for copying part of a genomic sequence into another, contributing to genetic diversity. Pseudogenes are known to play role in generating functional diversity of related genes (e.g., antibody diversity via gene conversion in chickens). We recently analyzed genomic sequences of the CD33-related Siglec gene cluster in three primates (human, chimpanzee and baboon) and found evidence for rapid evolution in this gene family (Angata et al., PNAS, in press). Additional evolutionary studies using distance-based phylogenetic trees shows evidence for three partial gene conversions between Siglec genes and adjacent Siglec pseudogenes. All three involve the coding regions for extracellular domains that mediate sialic acid recognition, and two involve a pseudogene converting a known Siglec gene. Functional analyses using recombinant proteins show marked differences in sialic acid-binding properties between the converted Siglec and its non-converted ortholog. These findings suggest that gene conversion with pseudogenes has contributed to the rapid functional evolution of the Siglecs, and provides a novel mechanism for changing sialic acid binding specificity. We hypothesize that this mechanism allows for rapid evolutionary adjustments in the recognition of endogenous sialic acids as “self”, a potential factor in controlling the innate immune response.
Randomized Multicenter Phase 3 Trial of High-Dose Dexamethasone (dex) with or without Oblimersen Sodium (G3139; Bcl-2 antisense; Genasense) for Patients with Advanced Multiple Myeloma (MM).
Authors: Chanan-Khan Asher Alban , Niesvizky Ruben , Hohl Raymond J. , Zimmerman Todd M. , Christiansen Neal P. , Schiller Gary J. , Smith Sandra , Frankel Stanley
Published: October 2019

Over-expression of Bcl-2 has been linked to acquired dex resistance in MM cells. Conversely, reduction of Bcl-2 using oblimersen has been shown to enhance dex-induced apoptosis in MM cell lines and in fresh MM cells in culture (Liu et al., Blood101:4105, 2003). Preliminary non-randomized clinical studies have suggested that oblimersen may potentiate the activity of VAD and dex/thalidomide in patients (pts) with resistant MM. We have conducted a randomized, multinational, Phase 3 trial of pts with advanced MM using dex given with or without oblimersen. Eligibility requirements included: relapsed or refractory MM; measurable M-protein in serum or urine; ≤ 6 prior regimens; ECOG PS ≤ 3; serum Cr ≤ 1.5 mg/dL; ANC > 1,000/ml; plts ≥ 50,000/ml. Exclusions: previous allogeneic transplant; other significant medical disease. Pts were stratified according to number of prior treatments (1 or 2 vs. ≥ 3 regimens), prior autologous stem cell transplant (yes/no), and primary resistance to vs. relapse from primary treatment. All pts received dex 40 mg p.o. x 4 days during weeks 1, 2 and 3. Pts randomized to oblimersen received 7 mg/kg/d x 7 days by IV infusion, beginning 3 days before dex treatment during the 1st and 3rd weeks, and during all subsequent dex cycles. Beginning on week 5, additional 4-day dex cycles were repeated in stable or responding pts every 3 weeks, for a maximum of 12 months. All pts received prophylactic H-2 antagonists, TMP/SMZ, and a bisphosphonate. Response assessments were conducted prior to each treatment cycle. The primary endpoint of the study was to compare time-to-disease progression between the 2 treatment groups. Secondary endpoint comparisons included overall survival, event-free survival at 6 months, overall response rates, response duration, clinical benefit, and safety. A total of 224 pts were randomized into the study. All pts have completed > 12 mos follow-up, and final results of this trial will be presented.
Phase II Study of the Combination of Low-Dose Thalidomide, Prednisone, and Etanercept (“PET regimen”) in the Treatment of Anemia, Splenomegaly, and Constitutional Symptoms Associated with Myelofibrosis with Myeloid Metaplasia (MMM).
Authors: Mesa Ruben A. , Steensma David P. , Li Chin Y. , Hoering Antje , Powell Heather , Tefferi Ayalew
Published: October 2019

BACKGROUND: Disease manifestations in myelofibrosis with myeloid metaplasia (MMM) that require therapy include anemia, thrombocytopenia, hepatosplenomegaly, and constitutional symptoms. We have previously reported on the therapeutic value of low-dose thalidomide in combination with prednisone for the treatment of both anemia and thrombocytopenia in MMM (Blood2003;101:2534) and of etanercept (tumor necrosis factor-alpha antagonist) for alleviation of constitutional symptoms (Blood2002;99(6):2252). In the current study, we investigated the value of combining all three drugs (PET).

METHODS: Study eligibility criteria included a histologically confirmed diagnosis of MMM and either severe anemia (hemoglobin < 10 g/dL) or symptomatic splenomegaly. Protocol treatment was initiated with 50 mg of oral thalidomide daily, etanercept 25 mg subcutaneously twice-a-week, and a three month prednisone taper starting at 0.5 mg/kg/day for the first month followed by a 50% reduction for the second months and a further 50% reduction for the third and final month of treatment with prednisone. Patients with a demonstrable clinical response after three months were able to remain on thalidomide and etanercept.

RESULTS: To date 12 patients have been enrolled to the trial (median age 67 years, range, 54–77; 11 males). At enrollment, all patients were severely anemic (6 were red cell transfusion dependent), 6 were at least moderately thrombocytopenic (platelet count < 100 x 109//L), and 4 had severe constitutional symptoms. The responses to date included resolution of constitutional symptoms in all 4 pertinent patients, a 12–326% improvement in thrombocytopenia in 4 of 6 pertinent patients, and anemia response in 4 patients including one patient who became red cell transfusion independent. A more than 50% decrease in splenomegaly was documented in 3 patients. Only one patient experienced greater or equal to grade 3 toxicity (i.e. elevated bilirubin and infection). Follow-up bone marrow examination, when available, did not show post-treatment histological changes.

CONCLUSIONS: The preliminary findings from the current ongoing phase II study of combination therapy with thalidomide-prednisone-etanercept in patients with MMM confirms the previously recognized value of etanercept in alleviating constitutional symptoms. However, at least preliminarily the PET regimen does not appear to be superior to thalidomide-prednisone combination in terms of therapeutic value for anemia, thrombocytopenia, or splenomegaly.
Imatinib Mesylate Therapy for Patients (pts) with Polycythemia Vera (PV).
Authors: Borthakur Gautam , Kantarjian Hagop , Verstovsek Srdan , O’Brien Susan , Giles Francis , Koller Charles , Rios Mary Beth , Cortes Jorge E.
Published: October 2019

Imatinib is a potent inhibitor of tyrosine kinases, including abl, c-kit, and platelet-derived growth factor receptor (PDGFR). A role for PDGF in PV has been suspected, and direct inhibition of PV colonies with imatinib has been reported. We initiated a phase II trial of imatinib in pts with PV diagnosed by the Polycythemia Vera Study Group criteria. Pts were treated with imatinib at 400 mg daily; if no significant response after 1 month (mo) of therapy, the dose could be escalated to 800 mg daily. Hydroxyurea could be used only during the first mo of therapy, but anagrelide was allowed at any time. 9 patients have been treated (6 male, 3 female). Median age was 51 years (yrs) (range 28 to 72 yrs); median duration of PV was 8 years (range 1/4–35 years). ECOG performance status was 0 for 2 pts and 1 for 7 pts. Most common symptoms included fatigue (n=7), intractable pruritus (n=5) and headache (n=4). 4 pts had splenomegaly, 1 had hepatomegaly, and 1 had prior splenectomy. All patients were dependent on repeated phlebotomies. All pts had diploid cytogenetics except one who had 20q− (prior therapy with 32 P). FISH and PCR confirmed absence of Bcr-Abl translocation.

After a median follow-up of 4 mo (range, 2 to 37 mo), 3 pts have ≥50% reduction in phlebotomy requirements. These include one pt with complete remission defined as hemoglobin < 14 g/dl, no phlebotomies and complete resolution of splenomegaly. Pruritus improved significantly in 3 pts; fatigue improved in 3 pts, headache and visual symptoms improved in one pt each. The dose of imatinib was increased to 800 mg in 3 pts. Grade 3 diarrhea and liver dysfunction occurred in 1 pt requiring dose reduction to 300 mg daily. Grade 2 toxicities included edema (n=2), rash (n=1), bone pain (n=1) and diarrhea (n=1). We conclude that imatinib has activity in PV, leading to improved symptoms and decreased phlebotomy requirements.
Aberrant Stabilization of c-Myc Protein in Lymphoblastic and Myelogenous Leukemia Cell Lines.
Authors: Malempati Suman , Sears Rosalie C.
Published: October 2019

The c-Myc oncoprotein is a key regulator of cell fate decisions including proliferation, differentiation, and apoptosis. Orderly control of c-Myc protein levels is important in maintaining regulated cell proliferation in normal cells. While c-Myc overexpression is seen in many hematopoietic malignancies, the reason for high protein levels in most cases is unknown and, in general, is not the result of translocations or gene amplification. C-Myc levels vary with cell cycle and are kept very low in quiescent cells. Protein half-life is controlled by phosphorylation at two specific N-terminal sites, Serine 62 and Threonine 58, which regulate c-Myc degradation by the ubiquitin proteasome pathway. Two Ras-dependent signaling pathways (Raf/MEK/ERK and PI(3)K/Akt) modulate phosphorylation at Serine 62, which stabilizes the protein, and Threonine 58, which targets Myc for ubiquitination and subsequent degradation. We recently reported that a stabilized form of c-Myc (c-Myc T58A) contributes to oncogenic transformation of human cells in culture (Yeh et al, Nat. Cell Bio.6:308–318, 2004). Here we describe the role of c-Myc protein stabilization in 2 pediatric ALL cell lines (REH and Sup-B15), 1 AML cell line (HL-60), and 1 CML cell line (K562). Markedly higher expression of c-Myc protein was seen in all 4 cell lines as compared to normal peripheral blood mononuclear cells (PBMCs). FISH analysis demonstrated amplification of the c-myc gene in HL-60 cells as has been previously reported, but not in REH, Sup-B15, or K562 cells. Using [35S]methionine pulse-chase analysis we demonstrate that the half-life of c-Myc in REH (55 minutes), Sup-B15 (47 minutes), and K562 (40 minutes) cells is longer than in normal PBMCs (9 to 15 minutes), but is not significantly prolonged in HL60 cells. We provide additional functional evidence for aberrant protein stabilization based on greater elevation of c-Myc protein after proteasome inhibition in PBMCs and HL-60 cells than in REH, Sup-B15, or K562 cells. These results suggest that that abnormalities in c-Myc degradation exist upstream of ubiquitination in the ALL and CML cell lines. Consistent with this hypothesis, experimental inhibition of the PI(3)K pathway knocked down c-Myc levels in REH and Sup-B15 cells, an effect that was abrogated by concomitant proteasome inhibition. This result suggests that abnormal activation of the PI(3)K pathway could participate in c-Myc stabilization in these cells. In addition, destabilization of c-Myc by PI(3)K inhibition correlated with a significant decrease in cell proliferation. In conclusion, we demonstrate that aberrant stabilization of c-Myc protein occurs in human leukemia cell lines. Affecting the c-Myc degradation pathway in hematopoietic malignancies that have stabilized c-Myc may constitute a novel therapeutic target. Additional experiments are ongoing to assess c-Myc stability in primary cells from leukemic bone marrow samples.
MYC Inactivation Induces Tumor Regression through the Recovery of a Functional DNA Damage Response.
Authors: Rabin Karen , Giuriato Sylvie , Ray Suma , Felsher Dean W.
Published: October 2019

Cancer is caused by genetic events that result in the activation of oncogenes or the inactivation of tumor suppressor genes. Using the Tetracycline system, our laboratory has generated a transgenic mouse model in which MYC is conditionally overexpressed in hematopoietic cells, allowing us to turn MYC expression on and off at will. We have previously demonstrated that the inactivation of this single oncogene in an established and even highly invasive and metastatic lymphoma is sufficient to reverse cancer, suggesting that MYC may be an effective therapeutic target. In a variety of conditional oncogene models, we and others have found that tumor regression following oncogene inactivation involves similar phenomena, including cell cycle arrest, apoptosis, and differentiation of the tumor cells. The similarity in the regression process across a variety of different oncogenes and types of cancer strongly suggests the existence of a common signaling pathway following oncogene inactivation, which culminates in the cessation of cell proliferation and the induction of apoptosis and differentiation. Recently, we have demonstrated that MYC activation disrupts the repair of DNA breaks and results in genomic instability. We speculated that MYC inactivation may cause tumor regression by restoring the ability of tumor cells to recognize that they are genomically damaged, which could subsequently lead to the cell cycle arrest, differentiation and apoptosis that is generally observed. Indeed, we now report that upon MYC inactivation, tumor cells activate DNA damage signaling pathways and begin to repair their DNA breaks. We have found evidence for activation of a functional DNA damage response both by immunofluorescent staining for phosphorylated ATM and Mre11, which demonstrates foci formation following MYC inactivation; and by the Comet assay, which shows a quantitative decrease in severity of DNA breaks following MYC inactivation. Our results suggest that MYC inactivation may induce tumor regression at least in part through the restoration of a DNA damage checkpoint response.
Cyclin D1 Deregulation in Mantle Cell Lymphoma: Tethering of the Cyclin D1 Promoter and 3′ Immunoglobin Enhancers by CTCF and Nucleophosmin.
Authors: Epner Elliot M. , Liu Hui , Jin Wang , Huang Jing , Nichols Craig R. , Maziarz Richard T.
Published: October 2019

Translocations involving the immunoglobulin heavy chain gene locus (IgH) are common in B cell malignancies. One common target gene is cyclin D1, which is deregulated in most patients with mantle cell lymphoma (MCL) and 20–30% of patients with multiple myeloma (MM). Cyclin D1 is known not to be expressed in normal lymphocytes, yet is deregulated by IgH translocations and insertions. The molecular mechanisms of long distance cyclin D1 activation by IgH regulatory elements in B cell malignancies have been obscure. Chromatin immunoprecipitation (ChIP) assays demonstrated the presence of the CCCTC-binding factor (CTCF) at both the cyclin D1 promoter and 3′ IgH regulatory regions only in cyclin D1 expressing malignant B cell lines containing IgH translocations or insertions. The nucleolar protein nucleophosmin (B23), a newly identified CTCF interaction partner, exhibited a similar distribution in ChIP assays. Combined FISH/immunofluorescence labelling studies have shown the presence of the cyclin D1/IgH translocated locus at the outer nucleolar membrane colocalized with CTCF and nucleophosmin. Thus, tethering of the cyclin D1 promoter to the 3′ IgH regulatory regions at the nucleolar membrane by CTCF/nucleophosmin provides a mechanism for spatial juxtaposition of the cyclin D1 promoter and 3′ IgH regulatory elements. We postulate RNA polymerase II then transfers from the 3′ IgH enhancers to the cyclin D1 promoter and initiates cyclin D1 transcription.
Profiling of Caspase Signaling Pathways Predicts Clinical Response to Chemotherapy in Diffuse Large B-Cell Lymphomas.
Authors: Oudejans Joost J. , Muris Jettie J.F. , Ossenkoppele Gert J. , Cillessen Saskia A.G.M. , van Houdt Inge , Kummer Alain J. , van Krieken Han , Kluin-Nelemans Hanneke C. , Meijer Chris J.L.M.
Published: October 2019

Disruption of the apoptosis signaling cascade is a probable cause for resistance to chemotherapy in patients with diffuse large B-cell lymphomas (DLBCL). We investigated whether inhibition of either or both caspase 8 and 9 apoptosis signaling pathways in biopsy specimens of primary nodal (DLBCL) predicts clinical outcome. Expression levels of c-Flip and numbers of active caspase 3 positive lymphoma cells were used to determine the status of the caspase 8 mediated pathway. Expression levels of Bcl-2 and XIAP were used to determine the status of the caspase 9 mediated pathway. Expression of c-FLIP, XIAP and Bcl-2 and caspase 3 activity all provided prognostic information. According to these immuno-histochemical parameters, inhibition of either or both caspase signaling pathways was detected in all cases. Three groups were identified: one with a caspase 8 inhibition profile, one with a caspase 8 and 9 inhibition profile and one with a caspase 9 inhibition profile. The caspase 9 inhibition profile was strongly associated with a poor response to chemotherapy and a usually fatal outcome, whereas the caspase 8 inhibition profile was associated with excellent clinical outcome. Functional analysis of apoptosis sensitivity confirmed that apoptosis profile was directly related to chemotherapy sensitivity. Thus, our data strongly suggest that inhibition of the caspase 9 and not the caspase 8 mediated pathway is a major cause for therapy resistance in patients with nodal DLBCL.
Negative Regulation of Platelet Function by a Secreted Cell Repulsive Protein, Semaphorin 3A.
Authors: Kashiwagi Hirokazu , Shiraga Masamichi , Kato Hisashi , Kamae Tsuyoshi , Yamamoto Naoko , Tadokoro Seiji , Kurata Yoshiyuki , Tomiyama Yoshiaki , Kanakura Yuzuru
Published: October 2019

Semaphorin (Sema) 3A is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and it regulates vascular morphogenesis by inhibiting integrin function in an autocrine fashion. Since platelet interaction with endothelial cells is critical for thrombus formation as well as hemostasis, we investigated effects of Sema3A on platelet function.

We used two distinct Sema3A fusion proteins in this study: human Sema3A fused to Fc (Sema3A/Fc) and human Sema3A fused to placental alkaline phosphatase (Sema3A/AP). We detected dose-dependent and saturable binding of Sema3A fusion proteins to platelets. Flow cytometric analysis with PAC1 or soluble fibrinogen showed that Sema3A/Fc and Sema3A/AP inhibited activation of αIIbβ3 by all agonists examined, including ADP, thrombin, U46619, convulxin, and PMA. Sema3A also inhibited aggregation induced by thrombin or collagen. Moreover, Sema3A inhibited CD62P and CD63 expression after agonist stimulation, indicating that Sema3A inhibits granular secretion as well as activation of αIIbβ3. However, Sema3A did not inhibited thrombin-induced rapid intracellular calcium increase. Sema3A inhibited platelet adhesion to immobilized fibrinogen partially, and it severely impaired spreading of adhered platelets and even shrinking of spread platelets was observed after addition of Sema3A, suggesting that Sema3A impairs rearrangement of platelet cytoskeleton profoundly.

Activation of platelets by thrombin or PAR1 peptide increases F-actin contents, and Sema3A inhibited the agonist-induced elevation of F-actin contents. Sema3A treatment also decreased phosphorylation of cofilin in both resting and thrombin-stimulated platelets, suggesting that Sema3A may keep cofilin in the activated state leading to increase severing of F-actin. Since phosphorylation of cofilin was regulated by LIM-kinase, an effecter of Rac-PAK signaling pathway, we next examined effects of Sema3A on Rac1 activation after thrombin stimulation. PAR1 peptide induced rapid activation of Rac1 in platelets, and Sema3A almost completely inhibited the activation of Rac1. These results suggest that Sema3A inhibits agonist-induced actin rearrangement via Rac1-dependent pathway including phosphorylation of cofilin.

In conclusion, we demonstrated that Sema3A binds to platelets and inhibits platelet functions extensively. The inhibition of platelet functions by Sema3A appeared to be mediated at least in part through impairment of agonist-induced Rac1-dependent actin rearrangement.
Dynamic Patterns of Migration and Expansion of Hematopoiesis during MGMT Mediated Drug Selection.
Authors: Lin Yuan , Cheung Perrin , Wilson David L. , Gerson Stanton L.
Published: October 2019

While hematopoietic engraftment kinetics are well appreciated after lethal irradiation in the mouse, most observations have been limited to blood samples or terminal examination of marrow or spleen. The development of non-invasive bioluminescence in vivo imaging technology allows a dynamic picture of engraftment and clonal expansion to be defined. We have extended this technology to the process of drug resistance gene therapy. We hypothesized that drug selection would profoundly affect the extent and dynamics of hematopoietic stem cells (HSC) engraftment and clonal expansion after lentiviral mediated gene transfer of the P140KMGMT gene into murine HSC. In previous studies, we have shown that P140KMGMT gene containing retroviral and lentiviral transduced bone marrow cells provided significant protection against chemotherapeutic drugs BCNU and TMZ given with BG (O6-Benzylguanine), in vitro and in vivo. We generated a bicistronic lentiviral vector containing P140KMGMT gene and firefly luciferase gene linked by 2A sequence of FMDV(Foot-and-Mouth Disease Virus), which will cleave itself during ribosomal translation. Whole bone marrow cells was collected from BALB/c mice 4 days after 5-FU treatment and transduced with P140KMGMT-luc lentiviruses at MOI of 1.4. Transduced bone marrow cells were transplanted into lethally irradiated or non-myeloablated syngeneic recipient mice at different cell numbers. Initial bioluminescent signal emerged 6–8 days after transplantation in both lethally irradiated and non-myeloablated recipients. The onset of bioluminescent foci after transplantation occurred in a cell dose dependent manner. The initial signal emitted predominantly from bone marrow, especially femurs, humeri and vertebrae during the early stage of clonal expansion. Intense signal appeared in spleen at days 12–14 and became weaker or even disappeared by days 20–28. Clonal expansion and engraftment greatly increased after a single course of BG+TMZ treatment and initiated strong hematopoiesis in non-myeloablated recipients. Total body bioluminescence intensity of drug treated mice increased 24 fold and 7 fold compared to non-treated mice in both non-myeloablated and lethally irradiated recipients, respectively. A transient phase suggesting migration through the lymphatic system and in the spleen occurred in most mice and was exacerbated by drug selection, but this was less clear in lethally irradiated mice, where engraftment was more confined to the marrow spaces. Bioluminescence in vivo imaging reveals active migration between the bone marrow and the spleen during hematopoiesis. Drug selection has a significant impact on the patterns of engraftment and clonal expansion of HSC and progenitor cells after transplantation.
Role of G-Protein Coupled Inwardly Rectifying Potassium (GIRK) Channels in ADP-Mediated Thromboxane A2 Generation.
Authors: Shankar Haripriya , Prabhakar Janani , Lakhani Parth , Kunapuli Satya P.
Published: October 2019

Platelet activation is associated with platelet shape change, fibrinogen receptor activation, platelet aggregation, secretion of granule contents and phospholipase A2 (PLA2) activation. Agonist-induced PLA2 activation is important for liberation of arachidonic acid from membrane phospholipids, which is then subsequently converted to thromboxane A2 (TXA2) via the sequential effects of cyclo-oxygenase and thromboxane synthase respectively. Thus the generated TXA2 plays a very predominant role in potentiating the effects of other physiological platelet agonists like collagen, thrombin and ADP. ADP-induced TXA2 generation requires co-stimulation of the P2Y1 and P2Y12 receptors, fibrinogen receptor activation and subsequent outside-in signaling. We recently demonstrated that G-protein gated inwardly rectifying potassium channels (GIRKs) are important for P2Y12 receptor-mediated platelet functional responses, namely platelet aggregation, dense granule secretion and Akt phosphorylation. In this study, we evaluated the role of GIRK channels in ADP- mediated TXA2 production in human platelets. ADP-mediated TXA2 generation was inhibited in the presence of SCH23390 and U50488H in a concentration-dependent manner, with maximal inhibition occurring at 10 μM. At these concentrations of GIRK blockers, none of the other P2Y12 receptor-dependent platelet functional responses including aggregation, Akt phosphorylations were affected. We confirmed our findings by performing similar experiments with 2-MeSADP, a potent and selective P2Y1 and P2Y12 agonist. We observed that GIRK channel blockers inhibit 2-MeSADP-mediated TXA2 generation. These results suggest the existence of two different populations of GIRK channels;-one that plays an important role in TXA2 formation (sensitive to low concentrations of the GIRK blockers) and the other population that plays an important role in platelet aggregation (sensitive to higher concentrations of GIRK blocker). Experiments done in the presence of SKF38393, a structurally similar but inactive analog of SCH23390 did not have any effect on ADP or 2-MeSADP-induced TXA2 production in the non-aspirinated platelets. Finally, we also observed that arachidonic acid-induced TXA2 production is not affected by 10 μM concentrations of SCH23390 and U50488H. These data suggest that GIRK channel blockers inhibit TXA2 formation by interfering with agonist-induced PLA2 activation. Based on these findings we conclude that there exist different populations of GIRK channels, one that contributes to ADP- and 2-MeSADP-induced TXA2 and the other that contributes to P2Y12 receptor-dependent platelet functional responses. Also, each of the two populations of GIRK channels has varying susceptibilities to the GIRK channel blockers.
Arg333 and Arg334 in the C-Terminus of the Human Platelet P2Y1 Receptor Are Crucial for Gq Coupling.
Authors: Ding Zhongren , Tuluc Florin , Bandivadekar Kavita R. , Zhang Lili , Jin Jianguo , Kunapuli Satya P.
Published: October 2019

ADP-induced platelet aggregation plays an important role in hemostasis and thrombosis. Human platelets express two ADP receptors: the P2Y1 and P2Y12 receptors. The Gq-activating P2Y1 receptor plays an important role in ADP-induced platelet shape change, aggregation, and thromboxane A2 generation. In this study, we investigated the role of the carboxyl terminus of the human P2Y1 receptor in Gq activation. Human P2Y1 receptors, either wild type (P2Y1-WT) or a mutant in which the C-terminus was truncated (P2Y1-ΔT330-L373), were stably expressed with an HA-tag at the N-terminus in CHO-K1 cells. Stimulation of P2Y1-WT cells with 2-MeSADP caused inositol phosphate production and mobilization of calcium from intracellular stores. In contrast, P2Y1-ΔT330-L373 completely lost its response to 2-MeSADP, indicating that the C-terminus of the human P2Y1 receptor is essential for the activation of Gq. CHO-K1 cells expressing a chimeric P2Y12 receptor with the P2Y1 carboxyl terminus failed to elicit Gq functional responses, indicating that the P2Y1 carboxyl terminus is essential but not sufficient for Gq activation. Radioligand binding demonstrated that both the P2Y1-WT- and P2Y1-ΔT330-L373- expressing cells have almost equal binding of [3H]MRS2279, a P2Y1 receptor antagonist, indicating that C-terminus truncation did not drastically affect the conformation of the receptor. Two additional truncation mutants in the C-terminus, P2Y1-ΔR340-L373 and P2Y1-ΔD356-L373, were expressed in CHO-K1 cells, and responded to 2-MeSADP with functional responses. These results indicate that the 10 amino acids (330TFRRRLSRAT339) in the C-terminus of the human P2Y1 receptor are essential for Gq coupling. Finally, the cells expressing a double mutant P2Y1 receptor (R333A and R334A), in the conserved BBXXB region in the C-terminus of the Gq-activating P2Y receptors, completely lost its functional ability to activate Gq. We conclude that the two Arg residues (R333R334) in the C-terminus of the human platelet P2Y1 receptor are essential for Gq coupling and subsequent platelet activation.
Di-Annexin, a Novel Tool To Identify and Protect Phosphatidylserine Exposing Cells.
Authors: Kuypers Frans A. , Larkin Sandra K. , Emerson Renee K. , Allison Anthony C.
Published: October 2019

Phosphatidylserine (PS) is found exclusively in the inner monolayer of plasma membranes. During programmed cell death this well-preserved asymmetry of phopholipid distribution is lost, and PS is exposed on the outer surface. PS exposure is also found in subpopulations of red blood cells (RBC) in disease states such as sickle cell disease (SCD). PS exposure leads to increased cell-cell interactions as well as recognition and removal by macrophages. PS serves as a binding site for factors involved in blood coagulation and provides a target for secretory phospholipase A2 (sPLA2) mediated membrane degradation. Measurement of PS-exposing cells (PSRBC) relies on the use of proteins that bind to PS and can be fluorescently labeled and visualized by flow-cytometry. The human protein annexin V (AV) has a high affinity for PS and has been shown to interfere with the physiologic processes induced by the exposure of PS. To improve detection of PS exposing cells, and develop a probe that may be used in vivo, we generated plasmids encoding a dimer of human AV (DAV), and expressed the protein in E.coli. We show that DAV has a 5–8 fold higher sensitivity than AV in binding to PSRBC, and effectively identifies PSRBC in SCD blood samples. Moreover, DAV protects PSRBC from sPLA2-induced hemolysis. While normal RBC show only 2% hemolysis after incubation with 100 ng/ml sPLA2 for one hour, sPLA2 induces more than 90% hemolysis in PSRBC. DAV, when added to PSRBC before incubation with sPLA2, protects more than 90% of the PSRBC from hemolysis. Our data show that DAV effectively competes with factor Va for PS binding and inhibits thrombin formation in the presence of the pro-thrombinase complex. At low concentrations, DAV was significantly more efficient in inhibiting thrombin formation than AV (P<0.001). The increased size of DAV gives the molecule a significantly increased survival time in the murine circulation compared to AV, and allows visualization of PS exposing membranes in vivo. Together this data shows that DAV is a superior probe to identify PS-exposing membranes, protects PSRBC from sPLA2-induced hemolysis, and interferes with cellular hemostasis. The use of DAV will allow more precise determination of PS exposure by flow-cytometry as compared to currently available commercial probes. Moreover, it will allow the measurement of PS exposing membranes in vivo, and may ultimately be proven to be useful as a therapeutic agent in SCD pathology by masking PS exposing cell membranes.
Functional Analysis of Two Mutants of Pyrimidine 5′ Nucleotidase Causing Nonspherocytic Hemolytic Anemia.
Authors: Chiarelli Laurent , Mattevi Andrea , Galizzi Alessandro , Fermo Elisa , Bianchi Paola , Zanella Alberto , Valentini Giovanna
Published: October 2019

Pyrimidine 5′-nucleotidase type-I (P5′N-1) catalyzes the dephosphorylation of UMP and CMP to their respective nucleosides. In red blood cells, the enzyme has a major role in the catabolism of nucleotides formed from RNA degradation. P5′N-1 possesses also phospho-transferase activity suggesting an additional role of the enzyme in nucleotide metabolism. P5′N-1 deficiency is an autosomal recessive disorder characterized by hemolytic nonspherocytic anemia, heavy basophilic stippling in the peripheral blood smear, and accumulation of pyrimidine nucleotides within the erythrocytes. P5′N-1 deficiency is the third most common RBC enzymopathy causing hemolysis after G6PD and PK deficiency. Fourteen different mutations have been identified at the DNA level to date including four missense mutations. To increase our understanding on molecular basis of the P5′N-1 deficiency, after mutants N190S and G241R (Chiarelli et al, Blood 2003, abstract; Chiarelli et al, The Hematology Journal 2004, abstract), we have undertaken the biochemical characterization of D98V and L142P enzymes, identified respectively in a Norwegian family and in Japanese patients. The proteins were produced in E. coli cells as recombinant forms, and purified to homogeneity. The L142P protein showed a drastic reduction in the thermal stability (t1/2 at 37°C about 6 min compared to a fully stable wild-type), and kinetic properties slightly altered (kcat values nearly halved and Km 3–5 times higher). D98V exhibited reduced heat stability (t1/2 at 37° about 25 min) and catalytic efficiency turned especially versus UMP (about 25 times) owing the increased Km values. Thus, the decreased activity observed in Japanese patients homozygous for the L142P mutation is essentially due to lowered enzyme levels caused by protein instability, whereas the D98V mutation of Norwegian patients alters both stability and catalytic efficiency. We suggest that substitution D98V affects an amino acid residue involved in substrates binding site.
Efficient Inhibition of Notch3 and Notch4 Family Members In Vivo by a Dominant Negative Mutant of Mastermind.
Authors: Pross Seth , Millholland John M. , Sai Hong , Weng Andrew P. , Aster Jon C. , Pear Warren S. , Maillard Ivan
Published: October 2019

The four mammalian Notch receptors play multiple roles in the hematolymphoid system. Notch1 is critical for the generation of hematopoietic stem cells (HSCs) during embryonic development and for commitment to the T cell lineage, while Notch2 regulates marginal zone B cell development. In addition, important roles for Notch are starting to be identified in peripheral T cells, in which all four Notch family members are upregulated upon T cell activation. Canonical Notch signaling involves proteolytic cleavage of Notch receptors and translocation of intracellular Notch (ICN) to the nucleus where it interacts with the transcription factor CSL/RBP-J and recruits Mastermind-like proteins (MAMLs) for transcriptional activation. By inhibiting Notch1 and Notch2-dependent developmental decisions with a dominant negative mutant of MAML1 (DNMAML1), we have previously demonstrated that MAMLs critically regulate Notch signaling in vivo (Maillard et al., Blood 2004). However, it is unclear if Notch3 and Notch4 are equally dependent on the activity of MAMLs in vivo, and if DNMAML1 can thus be used as a true pan-Notch inhibitor.

To address this question, we used a modified fetal thymic organ culture (FTOC) system and Notch-mediated T lineage commitment as a readout for Notch function. Fetal liver cells (FLCs) were transduced with retroviruses expressing ICN1, ICN3 or ICN4 and a retrovirus expressing DNMAML1. Irradiated fetal thymic lobes were reconstituted with transduced FLCs and cultured in the presence of gamma secretase inhibitors to inhibit the activation of endogenous Notch receptors. In this manner the effect of a specific ICN could be evaluated in the absence of endogenous Notch signals. Gamma secretase inhibitors resulted in a profound block in T cell development and in the generation of intrathymic B cells, consistent with Notch1 inhibition. ICN1, ICN3 and ICN4 were all able to rescue commitment to the T cell lineage and to block B cell development, indicating that each ICN can deliver appropriate signals for T lineage commitment in the absence of endogenous Notch1 signaling. In cells doubly transduced with specific ICNs and DNMAML1, the rescuing effect of ICN1, ICN3 or ICN4 was blocked, leading to the generation of B cells. In this competitive situation, DNMAML1 only partially inhibited ICN1, the most potent of all four Notch family members, but it was able to completely inhibit ICN3 and ICN4. We conclude that DNMAML1 can efficiently inhibit Notch3 and Notch4 in vivo. The results validate the use of DNMAML1 as a bona fide pan-Notch inhibitor in multiple settings, including studying the role of Notch in HSCs and peripheral T cells.
Hemoglobin Levels and Bone Density in Older Persons: Results from the Inchianti Study.
Authors: Cesari Matteo , Pahor Marco , Lauretani Fulvio , Penninx Brenda W.H.J. , Bartali Benedetta , Russo Roberto , Cherubini Antonio , Woodman Richard C. , Bandinelli Stefania , Guralnik Jack M. , Ferrucci Luigi
Published: October 2019

Background: Hypoxemia has been recognized as a risk factor for bone loss. The aim of the present study is to investigate the relationship of anemia and hemoglobin levels with bone mass and density measures in a large sample of older community-dwelling persons.

Methods: The present study is based on data from 950 participants enrolled in the “Invecchiare in Chianti” (Aging in the Chianti area, InCHIANTI) study. All the analyses were performed considering continuous hemoglobin levels as well as the dichotomous anemia variable (defined according to WHO criteria as hemoglobin <12 g/dL in women and <13 g/dL in men). A peripheral quantitative computerized tomography (pQCT) scan of the right calf was performed in all participants to evaluate total bone density, trabecular bone density, cortical bone density, and the ratio between cortical and total bone area. Linear regression analyses were used to assess the multivariate relationship of pQCT bone measures with anemia and hemoglobin levels after adjustment for demographics, chronic conditions, muscle strength and biological variables.

Results: Participants were 75.0 (SD 6.9) years old. In our sample, 101 participants (10.6%) were anemic. In women, coefficients from adjusted linear regression analyses evaluating the association between pQCT bone measures (per SD increase) and hemoglobin levels/anemia showed significant associations of anemia with total bone density (ß=−0.335, SE=0.163; p=0.04), and cortical bone density (ß=−0.428, SE=0.160; p=0.008). Relationships with borderline significance were found for the associations of anemia with trabecular bone density and the ratio between cortical and total bone area. Significant associations were found between hemoglobin levels and trabecular bone density (ß=0.112, SE=0.049; p=0.02), total bone density (ß=0.101, SE=0.046; p=0.03), cortical bone density (ß=0.100, SE=0.046; p=0.03), and the ratio between cortical bone and total area (ß=0.092, SE=0.045; p=0.04). In men, significant associations were found for the associations of hemoglobin levels with total bone density (ß=0.076, SE=0.036; p=0.03) and cortical bone density (ß=0.095, SE= 0.41; p=0.02). A borderline significance was reported for the association between anemia and cortical bone density.

Conclusion: Anemia and low hemoglobin levels are negatively and independently associated with bone mass and density. The bone loss associated with hemoglobin levels mainly occurs in the cortical bone. Women with lower hemoglobin levels demonstrate a higher bone loss than men.