bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional Vanilloid Receptor VR1 Is Expressed on Megakaryocytes and Platelets, and Acts as a Sensor for Hypertheimia Generating Endocannabinoids.
Authors: Maruyama Ikuro , Soejima Kentaro , Shimi Toshiaki , Abeyama Kazuhiro , Hashiguchi Teruto
Published: October 2019

Recent investigations have been revealing that platelet can sense shear stress and cold stimulation. These mechanical and physical stimuli alter the platelet functions, including induction of activation and irreversible dysfunction. These data may suggest that platelets, most small-sized cells, can efficiently circulate in the microcirculatory vasculature, and can sense the peripheral physical conditions including temperature.

Here we show that human megakaryocytic cell line, MEG-01 and platelets express vanilloid receptor VR1, a receptor for capsaicin. It has been identified that VR1 is linked to thermal sensor, and this can sense hyperthermia around 43 °. Therefore we examined the physiological significance of VR1 expressed on MEG-01 and platelets.

First we identified the expression of VR1 on human platelets and MEG-01 by immunoblotting, flowcytometer and RT-PCR, and identified the expression of VR1 on these cells. Hyperthermia stimulation of MEG-01 at 40–43 ° generated a endocannabinoids, 2-arachidonylglycerol(2-AG) and anandamide. Since this effect of hyperthermia stimulation was abolished by the incubation with capsazepine, an antagonistic analogue of capsaicin, it was suggested that the effect of hyperthermia might through VR1. It is well known that 2-AG and anandamide act on their specific receptors, CB1, CB2 and VR1 expressed on various cell-types, and act as an endocannabinoids, and accomplish diverse physiological functions, including vessel dilatation. Hyperthermia stimulation also affected the cell-cycle and induced G1-phase arrest in MEG-01 cells, partially through autocrine manner of generated endocannabinoids by acting of CB1 and CB2 receptors. Moreover prolonged hyperthermia stimulation up to 6 hours induced cellular apoptosis in MEG-01 by releasing cytocrome C and resulting caspase cascade activation.

Based on these results, we hypothesize that VR1 on platelets may act as a hyperthermal sensor and generate endocannabinoid, and this may regulate the diverse systems including vessel functions.
Clinical Outcomes of Recombinant Activated Factor VII (rVIIa) Use in a Tertiary Care Hospital: 70 Treatment Courses in 63 Patients.
Authors: DeLoughery Thomas G. , Boshkov Lynn K. , Schreiber Martin A. , Thorburg Per
Published: October 2019

rVIIa was originally developed and released for use in hemophilia patients with inhibitors. Recently the suggested uses for rVIIa have expanded dramatically. However, most of the data on the novel uses of rVIIa have been published as case reports or limited series. We reviewed our inpatient experience with rVIIa over the past 4 years. Diagnoses included CNS bleeding (12 of 14 on warfarin), inherited coagulation defects (2 patients with Glanzmann’s thrombasthenia (GT), one with factor VII deficiency and one with XI deficiency), inhibitors (1 hemophilia A, 2 hemophilia B, and 8 acquired factor VIII). “Last ditch” included 4 patients bleeding from severe GVHD after stem cell transplant, 3 cases of refractory sepsis, bleeding due to pulmonary hypertension, and CNS bleed due to tumor), surgery (4 of 7 cardiac) and trauma. 6 patients (2 inhibitor, 1 surgery, and 3 inherited) received more than one course of rVIIa. The average dose was 90 ug/kg. Overall 42 patients (60%) were alive at discharge with the congenital coagulation deficient and acquired inhibitor patients having 100% survival. Patients with liver disease or those patients given rVIIa as a “last resort” had the worst survivals despite being effective for controlling bleeding in most patients. rVIIa is effective for patients with GT, factor deficiencies, or inhibitors and its use is associated with good outcomes. In patients with bleeding due to their underlying medical problems, rVIIa was modestly effective at controlling bleeding. However, their outcomes were determined by the survivability of their disease process. For effective use of rVIIa patient selection appears to be crucial.

Diagnosis Number % Alive at D/C Effective% Avg # Doses Given CNS Bleed 14 57% 85% 1.2 Inherited Defects 7 100% 100% 10 Inhibitor 16 100% 93% 19 “Last Ditch” 9 0 66% 1.3 Liver Disease 6 16% 50% 1.6 Surgery 7 85% 100% 1.5 Trauma 13 46% 92% 1.3
Recurrent Vascular Access Site Thrombosis in Patients on Hemodialysis - a Problem of Thrombophilia?.
Authors: Klamroth Robert , Seibt Frank , Rimpler Hartmut , Landgraf Helmut
Published: October 2019

Introduction: Vascular access site thrombosis in patients receiving hemodialysis is a major cause of hospital admission and recurrent surgery. The underlying pathologic cause is often stenosis of the venous vessel due to fibromuscular hyperplasia. But in the case of early failure occasional studies have investigated that hypercoagulability could play an important role in this context.

Aim of the study: Is there a higher prevalence of hereditary and acquired thrombophilic risk factors in patients with vascular access thrombosis in comparison to patients without?

Patients: In 2002 and 2003 we examined 52 consecutive patients (mean age 66,1 years) receiving hemodialyisis. 27 patients (pts) in group 1 had a history of vascular access site thrombosis and 25 pts in group 2 had not and an open vascular access for longer than at least six months. All pts in group 1 had a history of at least two occlusions of vascular access. 10/27 pts in group 1 with prosthetic grafts had a history of thrombosis of arteriovenous fistula before implantation of PFTE graft.

Methods: In every patient hereditary and acquired thrombophilic risk factors were determined including antithrombin (AT), protein C (PC), protein S (PS), factor V-G1691A-mutation (FVM), prothrombin-G20210A-mutation (FIIM), homocysteine, lipoproteine (a) (Lpa), lupus anticoagulant (LA), cardiolipin antibodies IgG and IgM (ACA), fibrinogen and factor VIII. Platelet hyperreactivity was studied by light transmittance aggregometry in platelet rich plasma (Aggregometer PAP 4, moelab inc.). Aggregation was recorded as the maximum percentage change in light transmittance from baseline using platelet poor plasma as a reference. We defined sticky platelets as platelet aggregation > 30% after induction with different concentrations of ADP (10, 1 and 0,5 μmol) in platelet rich plasma.

Results: We found in 14/27 pts with vascular access site thrombosis antiphospholipd antibodies (LA and/or ACA) in comparison to only 2/25 in pts without thrombosis. Activated platelets like the sticky platelets syndrome was shown in 11/27 pts in group 1 and 4/25 pts in group 2. In both groups hyperhomocysteinaemia (23/27 pts and 21/25 resp.), factor VIII elevation (21/27 pts and 22/25 resp.), fibrinogen elevation (22/27 pts and 21/25 resp.) and high levels of Lpa (7/27 pts and 6/25 resp.) were quite similar. There were no significant differences in the number of hereditary risk factors like AT, PC, PS, FVM and FIIM in both groups.

Conclusions: In patients receiving hemodialysis we found a high prevalence of acquired thrombophlic risk factors like elevation of factor VIII, homocysteine and fibrinogen. There seems to be causal relation between vascular access site thrombosis and espacially antiphospholpid antibodies and activated platelets (sticky platelets syndrome). The evaluation of these thrombophilic risk factors in patients with recurrent vascular access site thrombosis could lead to an improved antithrombotic therapy.
Manifestations of the Antiphospholipid Syndrome in Patients with Malignancies.
Authors: Miesbach Wolfgang A. , Asmelash Geneth , Puetz Birgit , Boehm Martina , Scharrer Inge
Published: October 2019

The presence of antiphospholipid antibodies has been reported in a large variety of malignancies. It is not clear, however, if the antiphospholipid antibodies are related to thrombotic associations of the antiphospholipid syndrome (APS) in these patients. We investigated the frequency of thrombotic manifestations in 58 patients with the presence of antiphospholipid antibodies and a history of neoplasia, including haematologic and lymphoproliferative malignancies.

Methods Antiphospholipid antibodies were detected by clotting assay (lupus anticoagulant, LA) or by enzyme-linked immunosorbent assay (anticardiolipin antibodies). LA were tested by more than 2 different methods according to the proposed criteria of the SSC of the ISTH.

Results 39/58 patients suffered from solid tumours mostly from carcinoma of the breast, prostate, and colon and 19/58 patients from malignant haematologic or lymphoproliferative diseases mostly from Non-Hodgkin lymphoma. One patient was suffering simultaneously from two carcinomas of the prostate and the testicle and a Non-Hodgkin’s lymphoma.

Among the patients with solid tumours 18/39 (46 %) patients had thromboembolic complications of the antiphospholipid syndrome. Among the patients with haematologic and lymphoproliferative malignancies only 6/19 (32 %) suffered from thromboembolic complications. Thrombotic manifestations were more common on the arterial than the venous site. There was no relation between the titres of aCL antibodies and the rate of clinical manifestations. In two patients aPL disappeared after the effective treatment of the tumor. Especially patients with very high titres did not present any thromboembolic manifestation.

Conclusion The presence of antiphospholipid antibodies may identify a subset of cancer patients with high risk of developing thrombotic complications but the frequency of thrombosis is lower in aPL positive patients with lymphoproliferative and haematological malignancies. Especially in these patients very high titres of aCL antibodies do not seem to be associated with clinical manifestations of the APS.
Age May Modify the Relationship between Factor V Leiden Mutation - but Not G20210A Prothrombin Gene Variation - and Idiopathic Venous Thromboembolism: An Hospital-Based Case-Control Study.
Authors: Le Gal Gregoire , Lacut Karine , Couturaud Francis , Oger Emmanuel , Mottier Dominique
Published: October 2019

Introduction:

Factor V Leiden is the most common inherited risk factor for venous thromboembolism (VTE). A four- to sevenfold increased risk of VTE for the heterozygous state has been reported by numerous epidemiological studies but most of them did not include patients over 70 years. Surprisingly, we found in a previous study no association between Factor V Leiden and VTE in patients over 70 years.

Methods

Therefore we conducted a large hospital-based matched case-control study to test the hypothesis of an interaction between age and the factor V mutation, as well as G20210A prothrombin gene variation.

Results:

We analysed 392 patients experiencing VTE not related to a major acquired risk factor and their matched controls. Factor V Leiden was not associated with VTE in patients aged 80 years and over: odds ratio 0.8 (95%CI 0.2-3.4). There was a significant interaction between age and the mutation for VTE risk (p=0.03). Conversely, the association between the G20210A variant and VTE was consistent across age-groups: odds ratio 2.8 (95%CI 1.4–5.8).

In conclusion, age may modify the relation between factor V Leiden and VTE. The prevalence of the factor V mutation decreased with increasing age among patients with VTE but not among controls.
Rapid Detection of 871G>A Mutation by Sequence Specific PCR in ABO*A301 Blood Donors.
Authors: Novaretti Marcia C.Z. , Domingues Alexandre E. , Pares Madalena M.N.S. , Dorlhiac-Llacer Pedro E. , Chamone Dalton A.F.
Published: October 2019

Background: A3 blood subgroup is heterogeneous at molecular level. To date only two cases of ABO*A301 allele containing a single substitution 871G>A (Asp291Asn) were found in A3B individuals exclusively. The purpose of this study was to use a sequence specific PCR (PCR-SSP) to detect 871G>A mutation in A3 and A3B samples.

Material and methods: Twenty blood samples from unrelated donors were classified serologically as A3 and A3B using tube test technique and gel-test simultaneously. Agglutination of red cells by lectins was performed according to manufacturer’s instructions using anti-A1 lectin from Dolichos biflorus and anti-H from Ulex europaeus. Genomic DNA was prepared from peripheral blood cells by a salting-out method. ABO genotyping was performed using a polymerase chain reaction method with sequence specific primers: 95s (5′-CAC CAG GCC ATG ATG GTC A-3′) for consense G detection, 96s (5′-CAC CAG GCC ATG ATG GTC A-3′) for mutation A detection and 244as (5′-CGC AGT GAA CCT CAG CTT C-3′) for both detections on gene ABO exon VII (Seltsam, Transfusion 2003, 43(4): 428-39). PCR amplification was carried out under the following conditions: 5 initial cycles at 94°C for 10 minutes and 65°C for 1 minute; 20 cycles of 94°C for 10 seconds and 65°C for 1 minute; 20 final cycles of 94°C for 20 seconds, 61.5°C for 50 seconds and 72°C for 30 seconds. PCR amplification of 0.1 μg of genomic DNA was performed in a total volume of 25 μL containing 0.2 mM of each dNTP, 5 units Taq DNA polymerase in the buffer supplied, 2.5 mM MgCl2 and 12.5 pmoles of each primer. Fragment of 177 bp derived from each mutation or consense assay was separated for 90 minutes at 102V using 0.5 μg.mL−1 ethidium bromide-stained 2.5% agarose gels and visualized in a UV light apparatus (Eagle Eye II).

Results: We found 871G>A mutation in heterozygosis in three blood donors (15%), two of them typed as A3B and one as A3.

Conclusion: The 871G>A mutation is relatively commom in A3B and A3 individuals and can be easily detect by PCR-SSP.
Autoantibody Formation and Alloimunization in Sickle Cell Disease Patients.
Authors: Novaretti Marcia C.Z. , Jens Eduardo , Pagliarini Thiago , Rodrigues Andreia L. , Dorlhiac-Llacer Pedro E. , Chamone Dalton A.F.
Published: October 2019

Background: Alloantibody and autoantibody formation to red blood cell (RBC) antigens is one of the observed complications in sickle cell disease patients (SCD). The incidence of alloimmunization and autoantibodies in this selected group of patients is particularly high, although the clinical implication of autoantibodies in sickle cell disease patients is not clear. The purpose of this study is to evaluate the rate of alloantibody and autoantibody formation in SCD patients.

Study design and methods: A retrospective analysis of transfused sickle cell disease patients followed at Fundacao Pro-Sangue Hemocentro de Sao Paulo between 1988 and 2004 were retrieved. Data on transfusion history, were correlated with development of alloantibodies and autoantibodies.

Results: The study group was composed by 43 sickle cell disease patients followed for a mean of 89 months (22–116). The number of RBC units transfused (mean) was 64 (4–208). The development of the first alloantibody was detected after a mean of 40 months (1–107) after the first transfusion in our institution. Out of these patients, 31 (72.1%) were identified with RBC alloantibodies; 9 of these patients (21%) had both allo and autoantibodies to RBC antigens, whereas 5 (55.6%) developed autoantibodies after alloimmunization. The one remainder had only autoantibodies.

Conclusion: The alloimmunization rate was extremely high (72.1%) and can be partially explained because of the extended time of follow-up (mean of 89 months). Different from the literature the development of autoantibodies preceeded alloantibodies in 44.4%. The impact of this observation in clinical practice warrants further investigation.
High Frequency of HPA-2B Allele in Brazilian Apheresis Blood Donors Detected by Sequence Specific PCR.
Authors: Novaretti Marcia C.Z. , Bonifacio Silvia L. , Manhani Reinaldo , Dorlhiac-Llacer Pedro E. , Mendrone Alfredo , Chamone Dalton A.F.
Published: October 2019

Background: Human platelet antigens (HPA), mainly HPA-1 and HPA-2, play a significant role in alloimmune thrombocytopenic syndromes. Accurate HPA typing is important for the diagnosis and therapy of these patients. HPA gene frequencies vary in different populations. Thus, it is relevant to determine HPA genotype in distinct populations. The purpose of this study was to perform HPA-1,-2 genotyping in apheresis Brazilian blood donors.

Material and Methods: Genomic DNA was prepared from whole blood of 57 unrelated repeated apheresis Brazilian blood donors using a commercial DNA isolation kit (Qiagen,. GmbH, Germany). Sequency specific PCR (SSP-PCR) was performed amplifying HPA-1A/HPA-1B and HPA-2A/2B genes using a pair of primers for each allele as described elsewhere (HPA-1A ACT TAC AGG CCC TGC CTC T; HPA-1B ACT TAC AGG CCC TGC CTC C; HPA-1AS AGC CGG AGT GCA ATC CTC TG; HPA-2A CCC CCA GGG CTC CTG AC; HPA-2B CCC CCA GGG CTC CTG AT and HPA-2AS GCC AGC GAC GAA AAT AGA GG). A fragment of the human growth hormone gene (HGH) served as internal positive control. PCR was carried out in a final volume of 20 μL, containing 0.2 mg of genomic DNA, 0.2 mM of each dNTP, 5% of glycerol, 1.5 units of platinum Taq polymerase (Invitrogen, Brazil) in the buffer supllied, 5.0 mM MgCl2 and 10.0 pmoles of each primer. Fragments of 196 bp derived from HPA-1A/1B mutation and of 241 bp for HPA-2A/2B were separated for 90 minutes at 102V using 0.5 μg.mL−1 ethidium bromide-stained 2.0% agarose gels and visualized in a UV light apparatus (Eagle Eye II, EUA).

Results: The genotype frequencies presented 77.2% of HPA-1A/1A, 19.3% of HPA-1A/1B and 3.5% of HPA1B/1B; for HPA-2 the frequencies were 56.1% of HPA-2A/2A, 35.1% of HPA-2A/2B and 8.8% HPA-2B/2B. The gene frequencies in apheresis blood donors are 0.87 for HPA-1A, 0.13 for HPA-1B, 0.74 for HPA-2A and 0.26 for HPA-2B.

Conclusion: In this studied population we found high gene frequency homozygous HPA-2B when compared with other data already published. (Castro et al, European Journal of Immunogenetics,1999;26:355–60).
Mobilisation and Harvest of Peripheral Blood Stem Cells from 187 Healthy Donors. A Comparison of Two Different Apheresis Machines and Three Apheresis Programs.
Authors: Kumlien Gunilla , Bergstrom Gunilla , Ayoub Amal , Kanogo Liisa , Hagglund Hans
Published: October 2019

Background:

Between January 1995 and July 2004, mobilisation and harvest of peripheral blood stem cells for allogeneic transplantation was performed in 136 related and 51 unrelated healthy donors. Four related and one unrelated donor were harvested on two separate occasions after renewed G-CSF mobilisation. For logistic reasons, automated harvest programs were used until recently. Automated apheresis programs for peripheral blood stem cell harvest are less labour intensive but unfortunately also less efficient. One of the potential health hazards for donors is the depletion of platelets, thus it is essential to minimize platelet depletion.

Method:

Between 1995 and 1999 CS3000 (Baxter) cell separator with an automated stem cell harvest program was used. Since 1995 Cobe Spectra (Gambro) cell separator is used, initially with the automated program AutoPBSC and since March 2004 with the “manual” program MNC. The 187 medical reports were analysed retrospectively.

Results:

Median age was 40 years (range 14–71), median weight was 75 kg (range 42–130) and the male/female ratio was 1.3 (107/80). In a majority of donors (97%) antecubital veins were used as access to the circulation. In 4% of donors antecubital veins were unsuitable, and a femoral catheter was therefore employed.

Donors were mobilised with G-CSF 10 ug/kg and harvested on days five and six. Transplantation dose was median 6,3 x 10(6) CD34+ cells/kg recipient (range 1,2–98,6). Harvest yield was median 5,8 x 10(6) CD34+ cells/kg donor (range 1,4–16,3). Donor platelet count after second apheresis was median 129 x 10(9)/L (range 57–268) (normal range 150–400).

There were no significant differences regarding yield of CD34+ cells between male and female donors or between the two automated apheresis programs CS3000 and CobeSpectra AutoPBSC but the shift to CobeSpectra MNC-program led to a significantly increased yield only after six (three male and three female) donors (Mann-Whitney Test p<0,005) and median yield doubled to 10 x 10(6) CD34+ cells/kg donor.

There was no significant difference between CS3000 and Cobe Spectra MNC regarding platelet depletion but Cobe Spectra AutoPBSC was significantly less platelet depleting compared both to CS3000 (Mann-Whitney Test p<0,001) and Cobe Spectra MNC (Mann-Witney Test p<0,001).

Conclusion:

No serious side effects were reported during G-CSF mobilisation or apheresis. Cobe Spectra MNC program is significantly more efficient regarding yield of CD34+ cells than the two automated programs. Cobe Spectra AutoPBSC is significantly less platelet depleting than both CS3000 and Cobe Spectra MNC.
Gene Expression Profiles, Differentiation and Lineage Commitment of Human Myeloid Progenitors.
Authors: Edvardsson Louise , Dykes Josefina , Olofsson Tor
Published: October 2019

With the objective to further elucidate the mechanism behind commitment to erythroid and neutrophil lineages, we isolated by cell sorting common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs) and megakaryocyte/erythrocyte progenitors (MEPs), based on the surface expression of CD123 (IL3R-α) and CD45RA on human CD34+ bone marrow cells (first described by Manz et al. PNAS, 2002;99:11872). Methylcellulose cultures supporting the growth of myeloid and erythroid progenitors, and real-time RT-PCR mapping the gene expression of Flt3, c-kit, TpoR, GATA-2, GATA-1, SCL, NF-E2, EpoR, ABO, β-globin, GPA, PU.1, C/EBPα, C/EBPε, G-CSFR, proteinase 3 (PR3) and lactoferrin, were used to validate and characterize the progenitors and their progeny. Cell sorted progenitors were labeled with CFDA, SE to track cell division and cultured in suspension to induce neutrophil or erythroid differentiation (SCF+G-CSF for neutrophil and Epo+IL–3+GM–CSF for erythroid culture). After 3–5 days, cells that had gone through 1–8 divisions were sorted and changes in clonogenicity and gene expression were studied. The CMP-population retained some clonogenicity after as many divisions as were tested (at the most six divisions in erythroid and eight in neutrophil culture) and the CMPs differentiated along the lineage defined by the culture system, as evidenced both by the methylcellulose cultures and an increasing expression of GATA-1 and EpoR in erythroid and PU.1, G-CSFR and PR3 in neutrophil cultures, respectively. On the other hand, the GMP-population displayed granulocyte/monocyte (G/M)-differentiation irrespective of the culture system used, although it divided fewer times and lost its clonogenic capacity faster in erythroid culture. Little or no clonogenicity remained after 4–5 divisions in erythroid culture, while some colony-forming capacity remained even after seven divisions in neutrophil culture (maximum number tested). The increased expression of the granulopoiesis-associated genes was also less pronounced in the erythroid culture. The MEP-population dominated by erythroid differentiation capacity retained colony-forming capacity for at least 6–7 divisions in both erythroid and neutrophil cultures, although with a higher overall clonogenicity in erythroid culture. Unexpectedly, however, MEPs were restricted to G/M-differentiation when cultured in neutrophil culture. In cells from erythroid culture the expression of GATA-1, EpoR and β-globin increased, while a corresponding pattern was seen for PU.1, G-CSFR and PR3 in neutrophil culture. Overall, our data support the progenitor classification, based on the surface expression of CD123 and CD45RA, with regard to CMP and GMP populations but question it with regard to the MEP-population. The change in differentiation course for the MEPs in neutrophil culture could be a result of an initially present G/M-potential or a less strict commitment susceptible to cytokine-induced redifferentiation.
In Vitro Hematopoietic Lineage Interconversion from Human Bone Marrow Stem and Progenitor Cells.
Authors: Chen Ling , Jean-Noel Stephanie , Hall Kevin , Shi Ying , Rodgers Griffin P.
Published: October 2019

The cell surface antigen, CD133, marks a fraction of hematopoietic stem and progenitor cells and has been successfully used to study their differential biology. To evaluate the differentiating capacity of stem/progenitor cells, we cultivated purified normal human bone marrow CD133 selected cells for 2 weeks with erythropoietin (EPO) or granulocyte colony-stimulating factor (G-CSF) to induce erythroid or myeloid differentiation, respectively. After the second week of cultivation, we reversed the seeding environment of the two populations by placing EPO treated cells into a G-CSF environment and G-CSF treated cells into an EPO environment for an additional 2-week culture. The cells produced in the culture were phenotypically defined by morphology and flow cytometry, and genotypically by RNA and proteomic analyses. Three-color flow cytometry was used for identifying CD133+ progenitors, CD36+ erythroid and CD13+ myeloid cells, as shown in Table 1. The morphology of the cultured cells, assessed by Wright-Giemsa staining, is consistent with the conversion of cellular specific markers. Rapid analysis of gene expression demonstrated co-expression of 76% of 266 genes analyzed among the erythroid and myeloid lineages. Furthermore, proteomic analysis exhibited the sharing of 33% of 9518 expressed protein spots assayed in the two populations after the first 2-week culture, and 32% after 2 weeks of the switch culture. Our data clearly demonstrate that the committed erythroid and myeloid precursors are able to change their fate and can switch into the opposite cell type by a conversion pathway under a specifically defined condition. We termed this switch as interconversion, considering conversion of hematopoietic cells to non-hematopoietic cells. Furthermore, the observations presented in this study show that cytokines used can improve the conversion. We are developing a mathematical model describing the kinetics of hematopoietic stem/progenitor cell transitions into specific lineages, along with the conversion of committed cells based on multiple potential energy wells corresponding to different cell states and cytokines.

Table 1. Expression of cell surface markers after 4-week culture

D0 1 week 2 weeks 4 weeks CD expression (%) E G E G E2w →G2w → G2w E2w Data are presented as a mean of at least 2 experiments. E: EPO; G: G-CSF; E2w or G2w: EPO or G-CSF treatment for two weeks. CD133+ 96.19 15.74 13.6 0.24 0.36 0.01 0.63 CD36+ 0 60.37 27.39 96.37 25.87 45.41 68.54 CD13+ 0.43 35.41 57.29 24.41 92.1 85.87 37.76 CD133+ / CD36+ 0.44 22.24 15.97 0.12 0.18 1.55 7.65 CD133+ / CD13+ 1.24 19.43 13.36 0.36 1.09 13.31 14.92 CD36+ / CD13+ 0.09 41.25 17.80 23.69 54.1 46.60 79.41
Autologous Bone Marrow Cells for Treatment of Myocardial Infarction in Colombia.
Authors: Cuéllar-Ambrosi Francisco , Manuel Juán , Velásquez Oscar , Velásquez-Lopera Margarita , Navas Claudia , García Luis Fernando
Published: October 2019

Experimental studies in animals demonstrate the ability of the bone marrow stem cells to differentiate in cardiomyocytes, vascular endothelium cells and smooth muscle cells. There is evidence that these cells can regenerate infarcted myocardium and induce myogenesis and angiogenesis. Clinical studies in humans suggest the feasibility and safety of the utilization of the stem cells to recovery the ventricular function in patients with acute myocardial infarction. We report the first experience in Colombia using autologous bone marrow cells for cardiomyoplasty in isquemic heart disease.

This report shows the two months follow-up of four patients, three with acute myocardial infarction of the anterior myocardial wall and one patient with severe chronic isquemic heart disease. Extensive myocardial damage demonstrated by absence of viability in scintigraphic images and ejection fraction less than 40%. The patients received an optimum postinfarction medical treatment, successful coronary percutaneous intervention (three patients) or direct intramyocardial injection (one patient) to transfer of autologous bone marrow cells mobilised with granulocyte-colony stimulating factor during five days.

Demographics and results

The exercise capacity improve importantly, evidenced by increase in contractility, the six minutes test, the treadmill exercise time and the functional capacity in METS. There were not changes in the myocardial perfusion at two months follow-up, there were not complications related to the cellular transplant or the utilization of the granulocyte-colony stimulating factor. This is the first experience in Colombia with the bone marrow cells and selective intracoronary transplantation for myocardial regeneration and angiogenesis. We observed functional recovery of the left ventricle, improvement in the exercise capacity without adverse effects or complications related to the therapy.

Patient # 1 2 3 4 MI= myocardial infarction, IHD= isquemic heart disease, B/A= before/after 2 months, EF= eyection fraction, ESV=end systolic volume Age/sex 51/M 23/M 59/M 53/M Diagnosis acute MI acute MI acute MI chronic IHD Diagnosis to transplant 3 weeks 2 weeks 4 weeks > 1 year CD34 dose 19.7x10(6) 16.8x10(6) 19.5x10(6) 21.7x10(6) EF% B/A 36/43 26/40 40/41 45/55 ESV (cc) B/A 80/60 116/103 65/70 101/84 METS B/A 4/14 4/17 5/12 5/14 6-min test (mts) B/A 420/540 216/462 260/450 414/727
Characterization and Neural Differentiation of Fetal Lung Mesenchymal Stem Cells.
Authors: Han Zhong Chao , Gang Cun , Wu Feng , Jun Qing , Hong Shi , Ying Hai , Mao Zong , Liu Bin , Han Zhi Bo
Published: October 2019

Mesenchymal stem cells (MSCs) have been successfully isolated from a broad range of adult, fetal and other non-embryonic tissues. Fetal lung has been identified as a rich source of MSCs capable of differentiating into multilineage cells of mesenchymal origin. However, the biological characteristics and differentiation potential of fetal lung MSCs remain to be explored. In this study, we have established a series of methods for isolation and expansion of fetal lung MSCs. These MSCs could withstand 40 passages without obvious decline in proliferation ability, significant changes in morphology and expression of cell markers. Cell cycle analysis revealed that when the MSCs reached their log phase of growth, more than 90% of the cells were in G0-G1 phase while the proportion of cells in S phase and G2-M phase were about 5.56% and 2.08% cells individually. Flow cytometric analysis showed that fetal lung MSCs expressed CD13, CD29, CD44, CD90, CD105, D117, CD166 and HLA-ABC, but not CD14, CD31, CD34, CD38, CD41a, CD42b, CD45, CD49d, CD61, CD106, CD133 and HLA-DR. These MSCs could differentiate into neural cells in addition to their mesenchymal differentiation potential. Our data suggest that the fetal lung MSC population is an alternative source of stem cells for cell-based therapy of neurological defects or mesenchymal originated diseases.
Lysosomal Glycohydrolase Activities in Dendritic Cells: Is It a Function of Hematopoietic Stem Cells Differentiation Process?.
Authors: Berardi Anna C. , Manieri Pamela , Ciraci Elisa , Tribuzi Roberto , Di Girolamo Ilaria , Cavalieri Cristina , Isacchi Giancarlo , Emiliani Carla , Bottazzo Gianfranco , Orlacchio Aldo , Martino Sabata
Published: October 2019

A key mechanism responsible for processing of peptide-MHC class II complexes in mature Dendritic Cells (DCs) is the generalized activation of lysosomal function. Mechanisms underlie these developmental changes are controversial. Thus, it is unclear whether immature DCs can present self antigens, and which are the checkpoints that regulate antigen presentation in immature and mature DCs. Here we generated in-vitro human DCs from peripheral blood CD34+ hematopoietic stem cells (HSCs), by adding to the medium culture Flt-3, GM-CSF, IL-4, and TNF-a (cytokine cocktail, CC) at 37°C for 14 days, and analysed the lysosomal glycohydrolases production and function. Lysosomal enzymes, b-N-Acetyl-Hexosaminidase, a-Mannosidase, b-Galactosidase and b-Glucoronidase are highly increased in a wide range in DCs (14 days of culture) with respect to the CD34+HSCs. All the glycohydrolases activities measured at 3 and 7 days in-vitro culture, were similar and four times more than CD34+HSCs (day 0) respectively. Interestingly, no activities increase were observed, even when SCF, an early acting cytokine, promoting cellular proliferation, were added to the CC medium, indicating that this phenomenon is independent from the proliferation process. Moreover, LPS treatment, to induce DCs maturation, slightly enhance the specific activities of all enzymes that we tested as respect to the untreated cells. and support the evidence that the lysosomal glycohydrolases activation is up-stream to DCs maturation process. Furthermore, for the first time, this date indicated that lysosomal glycohydrolases are regulated during the stem cell differentiation process. Understanding the key mechanism leading this phenomenon is critical for therapeutic application in immunologic or neoplastic disease.
Rapid In Vivo Mapping of Hematopoietic Cis-Regulatory Elements.
Authors: Stamatoyannopouylos John A. , Hawrylycz Michael , Humbert Richard , Wallace James C. , Yu Man , McArthur Michael , Dorschner Michael O.
Published: October 2019

Cis-acting sequences play defining roles in the control of genes involved in hematopoiesis. However, for the vast majority of such genes, regulatory sequences remain to be defined. We describe a powerful, generic approach to identification of cis-regulatory sequences that may be applied to any gene locus in the context of any cell type. We used densely tiled primers and a novel real-time PCR-based assay to create continuous, high-resolution quantitative profiles of in vivo chromatin structure across entire gene domains. Such profiles can be analyzed using robust statistical algorithms to pinpoint disruptions in chromatin structure that are characteristic of cis-regulatory elements. We analyzed >1Mb of human genomic terrain from diverse gene loci in the context of several hematopoietic cell lines and cleanly delineated a spectrum of classical cis-regulatory activities including enhancers, promoters, insulators, and locus control regions. The approach displayed outstanding sensitivity (100%) and specificity (>99.6%) for known elements and was successful in defining novel elements even in heavily-explored terrain such as the alpha- and beta-globin loci. Since only small quantities of cells are required, the approach can be used readily in the context of hematopoietic progenitors. Systematic application of quantitative chromatin profiling to relevant genes promises to expand dramatically our understanding of the regulation of hematopoiesis.
Myelosuppresive Effects of Interferon [slpha] and Transforming Growth Factor β Can Be Reversed by Novel p38 MAPK Inhibitor SD-282 through Inhibition of Cell Cycle Arrest.
Authors: Mohindru Mani , Pahanish Perry , Collins Robert , Navas Tony , Higgins Linda , Verma Amit
Published: October 2019

Cytokines play important roles in the regulation of normal hematopoiesis and a balance between the actions of hematopoietic growth factors and myelosuppressive factors is required for optimal production of cells of different hematopoietic lineages. Even though the effects of Type I Interferons (IFNs α,β) and Transforming Growth Factor βs (TGF βs) as negative regulators of hematopoiesis are well documented, the exact molecular mechanisms by which such effects occur remain unknown. Our previous studies had shown that pharmacological inhibition of the p38 MAPK with commercially available inhibitors SB203580 and SB 202190 was able to reverse the myelosuppresion caused by IFN and TGF β. These inhibitors cannot be used in human studies due to toxicity and are also questioned for their selectivity in inhibiting the p38 MAPK. Thus, to confirm the role of p38 MAPK in regulating hematopoeisis, we conducted experiments with SD-282, a more potent and selective inhibitor of p38 α.

SD-282 also performs very similarly in animal and cell models to a p38 inhibitor now in the clinic. Our results show that SD-282 is able to inhibit p38 MAPK selectively in primary human erythroid progenitors (at CFU-E stage of maturation) and suppress activation of downstream kinase MapKapK-2 after IFN α stimulation. In methycellulose clonogenic assays with mobilized CD34+ cells, IFN α treatment resulted in marked suppression of both erythroid (BFU-E) and myeloid (CFU-GM) colonies, which could be reversed in the presence of p38 inhibitor SD-282. In a similar manner TGF-β2 was not able to effectively inhibit both erythroid and myeloid colonies in the presence of p38 blockade by SD-282. In further studies, we demonstrate that the primary mechanism by which the p38 MAPK pathway mediates IFN mediated hematopoietic suppression is by regulation of cell cycle progression and is unrelated to induction of apoptosis. Treatment with p38 inhibitors led to significantly lesser numbers of cells in G0/G1 phase of cell cycle arrest induced by exposure to IFN α. Altogether, these findings confirm that the p38 MAPK signalling pathway is a common effector for type I IFN and TGF beta signaling in human hematopoietic progenitors and plays a critical role in the induction of the suppressive effects of these cytokines on normal hematopoiesis. Our studies also provide a rationale for the use of SD-282 and other p38 inhibitors in cytokine mediated hematological diseases.
Berbamine-Induced Apoptosis in Human Leukemia K562 Cells.
Authors: Zhao Xiaoying , Xu Lei , Wu Dong , Xu Rongzhen
Published: October 2019

Purpose: To investigate apoptosis-inducing effects of Berbamine on human leukemia cells and to explore the underlying mechanism.

Materials and methods: Berbamine was dissolved in 0.9% sodium chloride to an initial concentration of 1mg/ml and subsequently diluted to desired concentrations with cell culture medium. MTT was used to examine the effect of Berbamine on cell proliferation of K562 cells. Characteristic cellular morphological changes were used as indicators of apoptosis in K562 cells while the rate of apoptosis was measured by flow cytometry assay. Expression levels of apoptosis related genes bcl-2 and bax were determined by RT-PCR and the levels of bcr/abl were evaluated by nested-PCR. Levels of Caspase 3 were measured by flow cytometry assay.

Results: Berbamine inhibited the cell proliferation significantly and in a dose-dependent manner in tested K562 cells. Its IC50 value was 5.23ug/ml. As determined by morphological observations and flow cytometry assay, Berbamine was able to induce apoptosis of K562 cells within 6 hours. The apoptosis rate of K562 was also dose-dependent. Steady-state transcript levels of bcr/abl decreased dramatically (half-quantity ratio from 1.284 to 0.506 within 72 hours following 8mg/ml Berbamine treatment. On the other hand, the protein levels of Caspase 3 surged from 18.36% to 38.25% (p<0.001) within 24 hours after treatment of 12mg/ml Berbamine. During the same period, no changes of bcl-2 or bax transcript levels were detected in the cells that were treated with 8mg/ml Berbamine.

Conclusions: Our results suggest that Berbamine is a potent inhibitor of cell proliferation and a strong inducer of apoptosis in human K562 cells. The Berbamine-induced apoptosis pathway involves down regulation of bcr/abl and up regulation of Caspase 3 expressions. Neither bcl-2 nor bax plays substantial roles in Berbamine-induced K562 cell apoptosis.
Cyclin D1 Alternative Spliced Forms Expressed in B-Cells Display Different Effects on Apoptosis and Cell Cycle.
Authors: Sola Brigitte , Gauduchon Juliette , Krieger Sophie , Roussel Mikel , Lesage Denis , Troussard Xavier
Published: October 2019

Cyclin D1 is an essential regulator of cell cycle progression. Over-expression of cyclin D1 is associated with tumorigenesis both in solid cancers and in hematological malignancies such as mantle cell lymphoma (MCL), hairy cell leukemia and multiple myeloma (MM). The CCND1 gene coding for cyclin D1 exhibit an A/G polymorphism at the nucleotide 870 which is responsible for the generation of an alternative splice variant called form b. The cyclin D1b is identical to the typical form a, except in the C-terminal part. The cyclin D1b has lost the PEST domain responsible for protein degradation and the threonine 286, the site of phosphorylation by the glycogen synthase kinase-3 essential for the nuclear export. Cyclin D1b has been recently described as a nuclear oncogene (Lu et al., Cancer Res63, 7056, 2003; Solomon et al., J Biol Chem278, 30339, 2003). Using real time quantitative RT-PCR, we have detected cyclin D1b mRNA in all cyclin D1a-expressing MM and MCL cell lines and in MCL patients. The cyclin D1b protein, detected with an antibody specific of the form b (a generous gift of Dr Alan Diehl) is present in a limited number of samples (1/6 MM and 1/1 MCL cell lines) and localized both in the nucleus and in the cytoplasm. We have generated B-cells conditionally (ecdysone system) expressing cyclin D1a or cyclin D1b proteins. B-cells were stimulated with ponasterone A to induce cyclin D1 synthesis and then with cycloheximide. The two alternative proteins forms displayed similar stability: calculated half-lives are 35 min and 50 min for a and b respectively. By co-immunoprecipitation and western blots experiments, we determined that, although cyclin D1b was able to bind efficiently CDK4 (to the same extent than cyclin D1a), cyclin D1b/CDK4 complexes weakly catalyzed the phosphorylation of pRB. We further showed by cytofluorometric analysis of propidium iodide-stained cells, that cyclin D1b did not allow the entry of quiescent cells within the cell cycle and did not allow the progression through the G1 phase. This implies that cyclin D1b plays a minor role in cell cycle regulation. However, cyclin D1b, such as cyclin D1a (Duquesne et al., Cell Death Different8, 51, 2001) potentiated the apoptosis induced by growth factor deprivation. It us tempting to speculate that the contribution of cyclin D1b for B-cell hemopathies does not occur through deregulation of the pRb pathway but more likely through deregulation of apoptotic pathways. We will study these aspects in the future.
Ex Vivo Expansion of Human Marrow Mesenchymal Stem Cells: A Serum-Free Medium Compared to Classical α-MEM Medium.
Authors: Meuleman Nathalie , Tondreau Tatiana , Delforge Alain , Dejeneffe Marielle , Massy Martine , Bron Dominique , Lagneaux Laurence
Published: October 2019

BACKGROUND: Bone Marrow mesenchymal stem cells (MSC) are pluripotent cells that have the capacity to differentiate into several tissue lineages and also the ability to support hematopoiesis. Their use in gene and cell therapy requires their in vitro expansion. Maintenance and proliferation of MSC are strongly dependent on the culture conditions such as properties of the bovine serum added in the culture medium. However, duration and culture conditions are critical for the successful clinical use of MSC.

OBJECTIVE: We have evaluated the efficiency of a commercial serum-free medium (UltraCulture, Bio-Whittaker, Walkersville, MD) supplemented with a serum substitute (Ultroser, BioSepra, Cergy-Saint-Christophe, France) in order to work without FBS and to have a more constant composition. This medium (UC) was compared to the classical medium a-MEM containing 10% FBS (Invitrogen, Merelbeke, Belgium).

METHODS: BM-mononuclear cells collected from 11 healthy donors were plated in petri dishes at a concentration of 105/cm2. After 3 days, non adherent cells were removed and culture media were added to adherent cells which were maintained at 37°C until they reached confluence. MSC expansion was analysed after the primoculture (PM) and after the first passage (P1). CFU-F (colony forming units-fibroblastic) number, phenotypic analysis and differentiation potential were also evaluated.

RESULTS::The mean culture duration was 13±2 and 8±2 days respectively for PM and P1 but the confluence was reached more rapidly for cells cultured in UC. After PM, 0.35x106 and 1.21x106 cells were obtained for MEM and UC respectively (p<0.005). Moreover, around 20% of cells cultured in MEM were CD45+ while the level of CD45+ cells was frequently < 5% in UC indicating that UC medium favored the rapid elimination of hematopoietic cells. After P1, the expansion rate was significantly higher in UC than MEM: respectively 5.13 ± 1.2 and 22.6 ± 3 (p<0.0005). The numbers of CFU-F were always higher in UC demonstrating the enhanced proliferation in serum-free medium. The phenotype of MSC was similar in the both media: SH2+, SH3+, CD44+, CD45−, CD34−, HLA-DR−. More importantly, these cells remained able to differentiate into osteocytes, chondrocytes, adipocytes and neuron-like cells in the both media confirming their multipotentiality.

CONCLUSIONS: Our data strongly support UltraCulture medium supplemented with a serum substitute as an optimal medium for MSC culture. Indeed, it allows a better cell expansion, better proliferation and preserves multipotentiality. This medium, reducing culture period and containing low concentration of serum substitute, is of major interest for clinical production of MSC.
SHP-2 Tyrosine Phosphatase Directly Controls the Proliferative Potential of Neoplasm Cells of Human Leukemia.
Authors: Xu Rongzhen , Yu Yingzi , Zhao Xiaoying , He Zhiwen , Liang Yun , Zhang Xiaohong
Published: October 2019

Our previous studies have indicated a stringent requirement of SHP-2 tyrosine phosphatase for normal hematopoiesis, but little is known whether SHP-2 plays a role in human leukemogenesis. Here, we show that SHP-2 plays a pivotal role in regulating the proliferating potential of human leukemia cells. We identified three types of SHP-2 proteins: m-, n- and c-SHP-2 proteins from both leukemic and normal hematopoietic cells. The m- and n-SHP-2 proteins (termed as active SHP-2) are derived from the c-SHP-2 protein (inactive SHP-2), and highly expressed on the internal membrane of rapidly proliferating cells at S/G2 phase, and in nucleus of mitotic cells at prophase and prometaphase, respectively, whereas the c-SHP-2 protein is ubiquitously expressed in the cytoplasm of both proliferating and resting cells. Intriguingly, we found that both active SHP-2 protein and its mRNA were constitutively overexpressed in leukemic blasts from various human leukemia cell lines and nearly all cases of various subtypes of human leukemia tested, relative to normal hematopoietic progenitors. Moreover, the expression level of active SHP-2 protein is positively correlated with the hyperproliferative phenotype of leukemia patients, and inversely associated with differentiation degree of hematopoietic cells. Most importantly, block of SHP-2 expression induces apoptosis and growth inhibition of leukemic clonogenic cells both in vitro and in vivo. In addition, we found that both PI3k/Akt and Ras/Erk signaling pathways are constitutively activated in human leukemias. Finally, we identified no mutation in PTPN11 among all tested leukemia patients. Based on these findings, we propose that SHP-2 tyrosine phosphatase plays an essential role in human leukemogenesis in which it may directly controls the proliferative potential of neoplastic cells of human leukemia via regulating signaling pathways involved in survival, growth and apoptosis of leukemia cells such as PI3k/Akt and Ras/Erk signaling pathways.