bluuebunny/crossref_metadata_2025_split huggingface dataset:
Angiopoietin-1 Supports SRC Activity Acquisition of Human CD34-Bone Marrow Cells.
Authors: Ando Kiyoshi , Nakamura Yoshihiko , Yahata Takashi , Muguruma Yukari , Sato Tadayuki , Matsuzawa Hideyuki , Uno Tomoko , Kato Shunichi , Hotta Tomomitsu 
Published: October 2019

Abstract
CD34 negative hematopoietic stem cells (CD34− HSCs) were identified in mice and humans. Human HSCs are evaluated as severe combined immunodeficient mouse (SCID)-repopulating cells (SRCs), originally identified by the ability to reconstitute hematopoiesis in nonobese diabetic (NOD)/SCID mouse. CD34− cord blood (CB) cells have been hard to engraft in NOD/SCID mice until recent report of successlul engraftment by intra-bone marrow transplantation (iBMT). However, CD34− bone marrow (BM) cells have not been analyzed precisely. We prepared lineage negative CD34 negative (Lin-CD34−) cells by negative selection using CD2,3,7,14,16,19,20,33,34,36,41,56,127, and GlyA antibody. Lin-CD34− BM cells did not engraft in NOD/SCID mice even by using iBMT (0/6). In the previous study, we reported that Lin-CD34− BM cells were able to differentiate into CD34+ cells accompanied by the emergence of colony forming activity after 7 days of stroma-dependent culture, while SRC activity was not detected. (BMT 28, 587–595, 2001) Here we cultured Lin-CD34− BM cells on stroma cells transfected with human angiopoietin-1 cDNA (AHESS-5), since we detected Tie-2 expression on Lin-CD34− BM cells. AHESS-5 supported induction of CD34 much better than HESS-5 cells or empty vector transfected control cells (EVHESS-5), and the effect was blocked by anti-Tie-2 antibody (Fig.1). Furtheremore, CD34+ cells produced from CD34− BM cells engrafted in NOD/SCID mice (11/12). As previously reported, CD34− CB cells differentiate CD34+ cells and acquire SRC activity by stroma-dependent culture without angiopoietin-1. These results highlighted the characteristic differences of CD34− HSCs of BM from CB and the unique role of BM niche for CD34− HSCs.
Fig. 1 CD34 expression on Lin − CD34 − BM cells after 7 days of culture Fig. 1. CD34 expression on Lin−CD34− BM cells after 7 days of culture
Hematopoietic Origin of Hepatic Stellate Cell.
Authors: Miyata Eri , Masuya Masahiro , Ishikawa Fumihiko , Yoshida Shuro , Kato Keizo , Ohishi Kohshi , Katayama Naoyuki 
Published: October 2019

Abstract
Hepatic stellate cells are believed to play a key role in the development of liver fibrosis. They undergo a gradual transition from a quiescent, fat-storing phenotype to an activated myofibroblast-like phenotype and then produce high amount of extracellular matrix such as collagen by liver injury. They express mesenchymal markers such as vimentin and desmin, or neural/neuroectodermal markers such as glial fibrillary acidic protein (GFAP). Based on the characteristic phenotype, the embryonic origin of stellate cells is thought to be the septum transversum mesenchyme or neural crest. However, their origin in the adult liver is still unknown. Recently, several studies have reported that crude bone marrow (BM) cells can give rise to hepatic stellate cells. However, since adult BM cells are thought to contain hematopoietic stem cells and mesenchymal stem cells, it is important to clarify which type of stem cells is the true source of hepatic stellate cells. We hypothesized that hepatic stellate cells are derived from hematopoietic stem cells. To test this hypothesis, we generated chimeric mice by transplantation of singe enhanced green fluorescent protein (EGFP)-marked hematopoietic stem cells (Lin− Sca-1+ c-kit+ CD34− cells) into lethally irradiated nontransgenic mice and examined the histology of liver tissues obtained from chimeric mice with carbon tetrahydrochloride (CCl4)-induced injury. Following 12 weeks treatment of CCl4, hepatic nodules and bridging fibrosis developed in all livers. We detected EGFP+ cells in the liver and some of them contained intracytoplasmic lipid droplets, which were proved by oil red O staining. Immunohistochemical analysis demonstrated that 60% of EGFP+ cells were negative for leukocyte common antigen (CD45); however, they expressed vimentin, GFAP and ADAMTS-13, which is a circulating zinc metalloproteinase synthesized in hepatic stellate cells. Moreover, nonparenchymal cell populations were isolated from the livers of chimeric mice with CCl4 treatment and were incubated on noncoated glass slides for 3 days. EGFP+ cells were also positive for type I collagen. These phenotypes are consistent with those of hepatic stellate cells. Our findings suggest that hematopoietic stem cells contribute to the generation of hepatic stellate cells upon liver injury.
An Investigation of Factor XIII Binding to Recombinant γ′/γ′ and γ/γ′ Fibrinogen.
Authors: Gersh Kathryn C. , Lord Susan T. 
Published: October 2019

Abstract
Increased levels of the alternatively spliced γ′ chain of fibrinogen have been measured in patients suffering from coronary artery disease (Lovely et al. Thromb. Haemost.2002;88:26–31). Others have suggested that the γ′ chain serves as a binding site for factor XIII (Siebenlist et al. Biochemistry1996;35:10448–10453), thus increasing the amount of cross-linking and therefore the stability of the γ′-containing clots (Falls and Farrell JBC1997;272:14251–14256). We have expressed recombinant γ′/γ′ homodimer and γ/γ′ heterodimer fibrinogen variants in CHO cells and characterized their polymerization by turbidity. Compared to normal recombinant γ/γ fibrinogen, γ′/γ′ fibrinogen displayed a slower rate of polymerization and a lower final absorbance. γ/γ′ Heterodimer fibrinogen had both a polymerization rate and final absorbance that was intermediate between γ/γ and γ′/γ′. These observations suggest that the presence of the γ′ chain slows lateral aggregation and leads to the formation of thinner fibers. These variants were then used to investigate the binding of factor XIII to fibrinogen. We designed an ELISA assay to measure this binding by immobilizing fibrinogen on a 96-well plate, incubating with factor XIII, and measuring bound factor XIII with an anti-factor XIII A-subunit antibody. Normal recombinant γ/γ fibrinogen bound factor XIII zymogen (A2B2) with a KD of 4.1 x 10−8 M +/− 1.3 x 10−8 M, similar to previous data for plasma fibrinogen. Surprisingly, both γ′/γ′ and γ/γ′ fibrinogen variants bound factor XIII zymogen with the same KD as did γ/γ fibrinogen. As shown by ELISAs with antibodies specific for either γ or γ′, the C-termini of both the γ and the γ′ chains are accessible on this immobilized fibrinogen. These findings suggest that the C-terminus of the γ′ chain is not a critical element in the binding of factor XIII zymogen. Our findings differ from previously reported findings. Further studies are in progress to reconcile these differences.
Factor VIII’s C Domains Bind to Platelets.
Authors: Hsu Ting-Chang , Pratt Kathleen P. , Thompson Arthur R. 
Published: October 2019

Abstract
The C domains of factor VIII contain the primary binding site for the cofactor, activated factor VIII, to interact with the phospholipid membranes, including those on the platelet surface. Isolated C2 domain has been shown to bind to phosphotidyl-L-serine-rich lipids and platelets; under flow cytometry, binding to activated platelets was confirmed. For comparison, C1C2, expressed in E.coli, was prepared with up to mg quantities isolated. Fresh, gel-filtered platelets were then studied in a flow cytometer either with or without activation by the thrombin receptor peptide, SFLLRN-amide. Depending upon the conditions, up to 80% of the platelets could be stained with a monoclonal antibody to C2 (ESH8) that is known not to compete with lipid or von Willebrand factor binding. The results were confirmed using a S2296C mutant C1C2 where the free suflhydryl group was either biotinylated and detected by fluorescein labeled streptavidin or directly labeled with fluorescein. As shown in the figure, essentially all platelets bound directly fluorescein labeled C1C2. Using standardized, labeled microbeads, it was estimated that there are 7000–10,000 binding sites per platelet. After platelet activation, the number of platelets binding C1C2 increased with all three detecting systems but only by 15–30%. In contrast, binding of isolated C2, as determined either by ESH8 or as a C2296 biotinylated species, was much lower when the same molar amounts were added, and was primarily detectable following platelet activation. C1C2 binding appeared independent of von Willebrand factor as platelets from two unrelated subjects with severe, type 3 von Willebrand disease gave the same patterns on flow cytometry as seen in platelets from normal subjects. ESH4, a monoclonal antibody known to inhibit binding of C2 to lipid membranes effectively competed C1C2 binding to platelets. Although an indirect alteration the C2 domain conformation cannot be excluded, results support a direct role of C1 in enhancing platelet binding.
Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets Binding of direct florescein-labeled C1C2 to SFLLRN-amide-activated platelets
The Neutrophil Proteases, Elastase and Cathepsin G, May Modulate the Activity of Factor VIII.
Authors: Gale Andrew J. , Rozenshteyn Diana , Riceberg Justin 
Published: October 2019

Abstract
Neutrophils and monocytes express cathepsin G and elastase and also can bind to activated platelets, thus they can be localized to the site of active coagulation. Early studies suggested that cathepsin G and elastase inactivated factor VIII (FVIII) and were thus anticoagulant. But other studies have suggested procoagulant functions for cathepsin G and elastase in activation of factor V or activation of platelets among other possible mechanisms. Therefore, we investigated the effects of human neutrophil elastase and human neutrophil cathepsin G on FVIII/VIIIa. Elastase does inactivate both FVIII and FVIIIa but cathepsin G activates FVIII while having very little effect on FVIIIa. Cathepsin G activation of FVIII is enhanced by phospholipid vesicles, apparently due to enhanced rate of cleavage and stabilization of the resulting molecule. The maximum level of activation is less than that of thrombin, but it is still four-fold as measured in an APTT assay. Cleavage sites for both proteases in FVIII were identified by Edman degradation and gel analysis. FVIII cleavages are limited to a few specific sites that are mostly located near known activating and inactivating cleavage sites. A notable exception is a cleavage site for elastase after valine 26 in the A1 domain. Cathepsin G cleavage sites near to thrombin cleavage sites likely contribute to the partial activation of FVIII. The unique elastase cleavage site at valine 26 likely contributes to the inactivation of FVIII and FVIIIa. Therefore, it is possible that neutrophils and monocytes may provide some pro-coagulant effect by activating FVIII and may also provide negative feedback by inactivating FVIIIa as well.
dSLIM Immunomodulators Induce Anti-Tumor Responses Both In Vitro and In Vivo.
Authors: Schmidt Manuel , de Cristobal Javier , Sander Astrid , Brzezicha Bernadette , König Merediz Sven A. , Wittig Burghardt 
Published: October 2019

Abstract
Cytosine-guanine (CpG) motifs containing oligonucleotides (ODN) are commonly used for immunomodulatory purpose in cancer therapy and for the treatment of allergic diseases since they resemble bacterial DNA and serve as “danger signals”. These CpG-ODNs promote predominately a TH1-response with secretion of IL-12 and IFN-γ, In addition their broad potential includes activation of B-cell proliferation, monocyte stimulation and secretion of IgM and IL-6, and stimulation of plasmacytoid DC to produce IFN-α/-β and thus γδT-cells and NK-cells to express CD69 and secrete IFN-γ. Usually phosphorothioate (PS) modifications are to enhance the stability, but these are leading to several side-effects, like severe organ enlargements, morphological changes and immunosuppression in mice. We designed immunomodulatory molecules based on short covalently-closed dumbbell-like structures (dSLIM) to stabilize the DNA without the otherwise necessary PS-modification. To evaluate the anti-tumor effect of the dSLIM molecules we developed an in vitro anti-tumor assay. This assay uses supernatant from dSLIM-activated human PBMCs for incubation with tumor cells in vitro. We observed increased apoptosis and necrosis of the HT-29 tumor cell line after incubation with supernatant from dSLIM-treated PBMC which was significantly higher than the effect of supernatant from non-treated PBMC. In addition, supernatant from dSLIM-treated PBMC increased the expression of HLA-ABC on the tumor cells, a pre-requisite for tumor cell recognition by the immune system. These effects were confirmed with human HEK293 and murine Renca cell lines. Analyzing the effect with neutralizing antibodies to various apoptosis-related cytokines, we observed a crucial role of IFN-γ but not IFN-α or TNFα. To investigate the anti-tumor effects of dSLIM in vivo, we employed a SKH1 murine model which is prone to spontaneous development of papillomas. Using chemicals for initiation and weekly promotion of de novo papilloma development we compared groups of weekly s.c. or i.p. dSLIM injections, respectively, with the PBS control group. The number of papilloma developing mice was significantly lower in the dSLIM groups and the total number of papillomas on all mice was reduced by approximately 50%. In conclusion, we showed that dSLIM immunomodulators exhibit potent anti-tumor effects in vitro and in vivo.
Allogeneic Environment and Tissue Damage Provide Critical Stimuli for Regulatory T Cell Expansion and Survival In Vivo after Hematopoietic Cell Transplantation.
Authors: Nguyen Vu H. , Chang Daisy , Negrin Robert S. 
Published: October 2019

Abstract
CD4+CD25+ regulatory T cells (Treg) mediate alloresponses in murine models of bone marrow transplantation (BMT), leading to protection from graft-versus-host disease (GvHD). However, in vivo migration and tissue localization of Treg during this inflammatory response remain unclear. We previously demonstrated co-localization of Treg with effector T cells (Tcon) with initial expansion in secondary lymphoid organs prior to migration into inflamed tissues in a major MHC-mismatched BMT model. To explore the stimuli for Treg proliferation, we evaluated the role of the allogeneic environment by transferring FVB donor luciferase-expressing (luc+) Treg into lethally-irradiated syngeneic recipients. Unlike the allogeneic irradiated setting where Treg expand in the presence or absence of Tcon, adoptively transferred luc+ Treg were not detected in secondary lymphoid organs of syngeneic lethally-irradiated BMT recipients by in vivo bioluminescence imaging (BLI). Syngeneic luc+ Tcon also had significantly different in vivo dynamics, with a 4 day delay and only moderate expansion in lymph nodes. Proliferation was not detected in the spleen, unlike their allogeneic Tcon counterparts, nor in the bone marrow compartments, as seen in lymphopenic models. To assess whether irradiation induced the observed in vivo dynamics of Treg in the allogeneic setting, we transferred FVB luc+ Treg or luc+ Tcon into unirradiated Balb/c Rag2−/−gamma chain (γC) −/− recipients, which lack T, B, and NK cells. After adoptive transfer into Rag2−/−γC−/− recipients, robust Tcon proliferation was observed in secondary lymphoid organs and the bone marrow compartments; however, Treg expansion was weak, and specific localization to lymphoid or nonlymphoid tissues was not observed. Treg were stimulated to localize to and expand in secondary lymphoid organs by the co-transfer of Tcon in unirradiated Rag2−/− (γC) −/− or by conditioning Rag2−/− (γC) −/− recipients with irradiation. Exogenous IL2 administration two weeks following luc+ Treg transfer into unirradiated Rag2−/− (γC) −/− recipients similarly led to localization and expansion of Treg in secondary lymphoid organs. These studies indicate the critical role of proinflammatory cytokines, such as IL2, generated either by irradiation-induced tissue damage or donor Tcon, in the expansion and localization of Treg. Differences between Tcon and Treg expansion in syngeneic or unconditioned allogeneic Rag2−/− γC−/− hosts suggest an important role of conditioning with irradiation alone or in concert with the allogeneic environment, in providing distinct signals for Tcon versus Treg activation, proliferation, and localization.
Glucocorticoid Therapy Targets Proliferation, Differentiation and Bcl-2 Dependent Survival Signaling in ALL Blasts.
Authors: Rhein Peter , Scheid Stefanie , Ratei Richard , Hagemeier Christian , Seeger Karl , Kirschner-Schwabe Renate , Schrappe Martin , Spang Rainer , Ludwig Wolf-Dieter , Karawajew Leonid 
Published: October 2019

Abstract
In the multicentric ALL-BFM (Berlin-Frankfurt-Munster) study, all patients are uniformly treated during the first week of induction therapy which uses glucocorticoids (GC) as the principal therapeutic agent. The GC response assessed at day 8 of therapy provides one of the basic parameters for further risk stratification. In spite of the clinical significance, molecular mechanisms of GC action in vivo are largely unknown. Our recent genome-wide analysis of gene expression in blasts persisting during induction therapy identified a common set of genes differentially expressed in blasts at day 8 (d8) and at diagnosis (d0) (n=457, false discovery rate <0.05). Expression changes indicated therapy-induced inhibition of cell cycling, expression shift towards normal mature B cells and downregulation of the apoptosis regulator Bcl-2. In the current study, we validated the key differences between d8 and d0 blasts at protein and cellular levels. DNA distribution and percentage of cycling blasts was determined by flow cytometry in a series of matched d8 and d0 samples (13 pts) and demonstrated the decreased proliferative activity of d8 cells (4.3-fold, p=0.014). Protein expression, investigated by flow cytometry in a total of 84 pts, demonstrated statistically significant expression decrease of the progenitor cell antigenes CD10, CD34 and TdT and expression increase of the B-cell antigene CD20 and the inflammatory response molecules CD11b and IFNGR1 (p<0.05). We were also able to confirm the lower expression values of the Bcl-2 protein in d8 blasts (p<0.05, n=57). Investigation of GC-sensitive B-lineage leukemia cell lines demonstrated that BCL-2 downregulation by GC was a pre-requisite of GC-induced apoptosis. Moreover, we found a considerable cross-correlation between viability, cell cycling and Bcl-2 expression levels. Upregulation of the Bcl-2 expression via IL-7 receptor signaling prevented GC-induced apoptosis in these cell lines. Collectively, GC therapy interferes with differentiation and proliferation programs in leukemic blasts which are closely related to the Bcl-2 specific apoptotic pathway.
Two Decades of Progresses in Adolescents with Acute Lymphoblastic Leukemia (ALL) Treated in the FRALLE Protocols: Adolescence Is No More a Bad Prognostic Feature If an Intensive Chemotherapy Is Applied.
Authors: Baruchel Andre , Auclerc Marie-Françoise , Perel Yves , Mechinaud Françoise , Auvrignon Anne , Schmitt Claudine , Piguet Christophe , Lejars Odile , Berthou Christian , Demeocq François , Schneider Pascale , Gandemer Virginie , Michel Gérard , Cayuela Jean-Michel , Leblanc Thierry , Leverger Guy ,   the FRALLE Group
Published: October 2019

Abstract
Adolescence has been claimed since the seventies to be associated to a a bad prognosis in childhood ALL. Out of 4658 patients with ALL, 258 adolescents (15–20 year old)(5.5%) were treated in the successive FRALLE 83, FRALLE 87–89, FRALLE 92 (pilot phase), FRALLE 93 and FRALLE 2000 protocols. The main characteristics were: a sex ratio of 1.8 (M/F), a B-lineage in 71% of the cases vs T lineage in 29%, and a median WBC of 12 G/L (9–1000). Translocation and fusion transcripts were searched for in 120 evaluable BCP-ALL: t(9;22)/BCR-ABL, 8 pts (6%); t(1;19)/E2A-PBX1, 12 pts (10%); t(4;11)/MLL-AF4, 4 pts (3%). Out of 75 evaluable pts t(12;21)/TEL-AML1 was found in only 4 pts (3%). 242 out of 258 adolescents were in CR at the end of induction therapy(EOI)(94%) without any significant difference according to the era. Nevertheless a major difference in the 3y and 5y EFS was found:
Number of pts CR at EOI (%) 3y EFS (%) 5y EFS (%) 10y EFS (%) *: p=.04; **: p=.04 Eighties (F83, F87–89) 100 93 42 +/− 5 35 +/− 5 35 +/− 5 Nineties (F92, F93) 84 93 71 +/−5* 67 +/− 5** 67 +/− 5 2000– (F2000) 74 96 86 +/−5* 86 +/− 10** NYA
The main modification introduced in the nineties was the adoption of a double delayed intensification for the good early responders. Autologous BMT or allogenic BMT were indicated in bad early responders (D8 poor prednisone response, D21 marrow M3 response) and/or unfavourable cytogenetics. The better results of the 2000 protocol can mainly be explained by the further intensification of chemotherapy between induction and delayed Intensification 1 and before delayed Intensification 2. These better results were obtained despite decreasing the indications of BMT (6 performed vs 20 in the nineties) and of CNS irradiation (100% in the nineties vs 35% in the current era, including the TBI for BMTs).
Conclusions:excellent results can now be achieved in adolescents with ALLthis study emphasizes again the need to treat adolescents with ALL according to pediatric intensive protocols and not “adult-type” protocols, as we recently suggested (Boissel et al, J Clin Oncol 2003).Whether this could also be applied to young adults remains to be demonstrated but seems appealing.