bluuebunny/crossref_metadata_2025_split huggingface dataset:
Erythropoietin Signaling Inhibits the Short-Term Marrow Reconstitution.
Authors: Chang Ko-Tung , Pastore Yves D. , Nussenzveig Roberto H. , Prchal Josef T. 
Published: April 2020

Abstract
Murine hematopoietic stem cells (HSCs) transfected with gain-of-function human erythropoietin receptor (EPOR) transgene were reported to have a competitive advantage over wild type mouse HSCs in a bone marrow transplantation (BMT) model (Kirby, Blood; 95(12):3710, 2000). However, EpoRs may not be normally expressed in early progenitor/stem cells, moreover, whether the Epo/EpoR signaling plays a role on homing and repopulating of hematopoietic progenitor/stem cells is also unknown. Our lab has previously created a mouse model harboring either a knocked-in human wild type (wthEPOR) hypomorphic gene or a mutant human gain-of-function EPOR (mthEPOR) gene into the mouse EPOR locus (Divoky, PNAS; 98(3):986, 2001). The wthEPOR mice are anemic, while mthEpoR mice are polycythemic. We tested the possible advantage of mthEPOR HSCs in the competitive bone marrow (BM) transplantation assay using C57/Bl6 congenic mice. BM from wthEPOR (CD45.1/45.2 heterozygous or CD45.1) and mthEPOR (CD45.2) mice were co-transplanted (1:1 ratio) into lethally irradiated (137Cs &gt;11Gy split) normal recipients (CD45.1 or CD45.1/45.2). Unexpectedly, in three independent groups the peripheral blood chimerism derived from wthEPOR-bearing cells significantly out-competed the mthEPOR-bearing cells two weeks after BMT (52% vs. 40%, 52% vs. 45% and 50% vs. 42%, n=11, 8 and 6, p&lt;0.01). Furthermore, administration of exogenous Epo (10U/day/mouse) further impaired peripheral blood engraftment (including Ter119+ erythroid lineages) of mthEPOR cells (45% vs. 39%, n=8, p&lt;0.05) though the relative proportion of erythroid cells within the engraftment was increased. Thus, an enhanced Epo/EpoR signaling interferes with the short-term repopulation of hematopoietic progenitors resulting in a decreased engraftment, possibly by interfering with homing. A possible long-term advantage of mthEPOR HSCs is being evaluated in ongoing studies; however, these short term data suggest that Epo administration to BM transplant patients may impede engraftment.
Figure Figure
Human Mesenchymal Stem Cells Inhibit In Vitro Antibody Production Against Alloantigens.
Authors: Comoli Patrizia , Maccario Rita , Avanzini Maria Antonietta , Ginevri Fabrizio , Moretta Antonia , Podestà Marina , Marconi Massimo , Cometa Angela , Bernardo Maria Ester , Frassoni Francesco , Locatelli Franco 
Published: April 2020

Abstract
Antibodies directed against alloantigens are implicated in the pathogenesis of several immune reactions complicating transplantation. In particular, this humoral response unfavorably affects the outcome of solid organ transplantation, and it has been hypothesized to be responsible for some of the clinical manifestations related to graft-versus-host disease (GVHD). In detail, the presence of antibodies against donor cells is a contraindication to kidney transplantation because of the risk of hyperacute rejection. In the effort to expand the donor pool, trials of allograft transplantation across HLA-sensitization have been conducted by means of strategies including pre-transplant plasmapheresis, intravenous immunoglobulins (Ig), anti-B cell monoclonal antibodies and splenectomy, associated with high-intensity immunosuppressive regimens. These measures have proved only partially successful in preventing humoral rejection in high-risk patients. Thus, the development of new therapeutic tools able to blunt alloantibody production could be a welcomed implementation to existing protocols. Mesenchymal stem cells (MSC) have been demonstrated to possess immunomodulatory capacity, since they induce T-cell hyporesponsiveness in vitro, prolong survival of skin graft in a primate model, and seem to decrease GVHD incidence and severity in humans given hematopoietic stem cell transplantation.
To verify whether MSC may exert an inhibitory effect on antibody production, we stimulated B-cell-enriched peripheral blood mononuclear cells (PBMC) obtained from healthy controls (n=9) or sensitized prospective kidney recipients (n=5) in a mixed lymphocyte culture (MLC) against irradiated HLA-disparate stimulator PBMC (controls) or stimulators cells bearing HLA antigens matched with the positive cross-match (patients). Antibody production in the absence or in the presence of third-party allogeneic MSC (responder:MSC ratio:4:1) was then evaluated by ELISA. We found that the addition of MSC at the beginning of MLC considerably inhibited IgG and IgM production (median fold-decrease of IgG production: controls, 7; patients, 5; median fold-decrease of IgM production: controls, 17; patients, 4). Our preliminary findings indicate that third-party MSC are able to suppress antibody production in vitro, and may therefore help to overcome a positive cross-match in sensitized transplant recipients. These results may also contribute to partly explain the mechanism at the basis of the favourable effect played by MSC in patients with GVHD.
Gene Transfer to Macrophages with Nanoparticle-Loaded Platelets.
Authors: Scull Christopher M. , Kuruvilla Ruben , Fischer Thomas H. , Nichols Timothy C. 
Published: April 2020

Abstract
Hemorrhagic fever, traumatic brain injury, and inflammatory bowel syndrome are among a wide range of pathologies involving inflammation that results in bleeding. The vascular injury that occurs in these conditions is closely associated with macrophage activation. An inherent function of macrophages is to phagocytose platelets that have adhered to sites of vascular injury, and thus platelets are an ideal vehicle for site-specific delivery of anti-inflammatory genes to macrophages at wound sites. Here we report the preparation of reporter gene nanoparticle-platelet conjugates and gene transfer to macrophages. Plasmid-containing nanoparticles were prepared by condensing cDNA for β-galactosidase, green fluorescent protein, or luciferase with polyethyleneimine. Scanning electron microscopic analysis showed that this anion/cation agglomeration reaction yields spherical particles with diameters of 100 to 300 nm. Plasmid-containing nanoparticles were then incubated with platelets that were isolated from human blood by differential centrifugation. Flow cytometric and transmission electron microscopic analysis demonstrated that the nanoparticles associated with the surface-connected canalicular system with most platelets containing one or more particles. The ability of the nanoparticle-loaded platelets to transfer the reporter genes to macrophages was tested in tissue culture using monocyte-derived macrophages incubated with the nanoparticle-loaded platelets. Subsequent fluorescent microscopic, luminescent and histological analysis demonstrated that the macrophages readily phagocytosed the nanoparticle-loaded platelets and expressed the reporter genes. This result provides the basis for studies to deliver anti-inflammatory genes to macrophages in animal model systems.
Expansion of Genetically Modified Primary Human Hematopoietic Cells Using a Cell Growth Switch.
Authors: Wang Linlin , Wood Brent , Levin Greg , Blau C. Anthony 
Published: April 2020

Abstract
Methods for regulating the growth of transplanted cells have many applications in gene and cell therapy. One such method uses conditional signaling molecules that are activated by artificial ligands called chemical inducers of dimerization (CIDs). Here we examine the response of human cord blood cells to a CID-triggered Fibroblast Growth Factor Receptor 1 (F36VFGFR1) signal in vitro and in vivo. In vitro, CD34+ cord blood cells were transduced with a lentivirus vector incorporating a chicken b-actin promoter/cytomegalovirus enhancer (CAG) that also allows for expression in human embryonic stem cells, but which is not expressed in human erythroid cells. Cells cultured in the presence of the CID expanded 40-fold relative to cells cultured in the absence of CID. The cell types evident in culture were predominantly granulocytes/monocytes but B-lymphocytes were also observed. The absence of an effect on erythroid cells is most likely attributable to the lack of transgene expression by the CAG element in erythroid cells. Contrary to our results in murine system using F36VFGFR1, cell expansion was transient, peaking at week 3 of culture. Activation of F36VFGFR1 was associated with a transient increase in CFU-GM. Ex vivo studies using a lentivirus vector in which F36VFGFR1 expression is regulated by an MSCV promoter, allowing for expression in all hematopoietic lineages, have been initiated. Preliminary results from immune deficient mice transplanted with cord blood CD34+ cells transduced with the MSCV based vector lentivirus indicate that F36VFGFR1 activation can support the CID-dependent expansion of a much wider repertoire of hematopoietic lineages, including granulocytes/monocytes, erythroid cells and lymphocytes, compared to our previous studies using a conditional derivative of the thrombopoietin receptor (F36VMpl). These findings establish CID-activated receptors as growth factors for genetically modified human hematopoietic cells.
Insulin Activation of PI3K Reduces Inflammation in a Mouse Model of Endotoxemia.
Authors: Mackman Nigel , Schabbauer Gernot , Tencati Michael , Holscher Todd , Luyendyk James P. 
Published: April 2020

Abstract
A recent clinical study showed that intensive insulin treatment in patients in intensive care units reduced morbidity and mortality. Other studies indicate that insulin has anti-inflammatory properties beyond its regulation of glucose metabolism. We determined the effect of insulin on LPS-induced inflammation in vivo. Insulin was administered via miniosmotic pump at a low dose (2.5 mU/h) that did not affect plasma glucose levels. Insulin treatment significantly reduced levels of plasma TNFα and IL-6 and prolonged the survival of endotoxemic mice. Next, we determined the role of the phosphatidyl-inositol-3 kinase (PI3K) pathway in insulin protection. Insulin is a potent activator of PI3K signaling. We and others have shown that inhibition of PI3K in monocytes, endothelial cells and in a model of endotoxemia enhances inflammation, suggesting that the PI3K pathway suppresses LPS signaling. We determined if insulin reduced inflammation in a PI3K-dependent manner by blocking PI3K in endotoxemic mice using wortmannin. We found that wortmannin abolished the protective effect of insulin. Finally, we examined the effect of insulin on p85β−/− mice, which have increased PI3K activity and are hyperresponsive to insulin. Endotoxemic p85β−/− mice exhibited reduced plasma TNFα and IL-6 and had a prolonged survival compared with endotoxemic wild-type littermate controls. Taken together, these results indicate that insulin reduces LPS-induced inflammation in a PI3K-dependent manner.
Vitamin D3-Mediated Regulation of the Antimicrobial Peptides CAMP and DEFB4 Is Evolutionarily Important for Innate Immunity in Humans and Primates.
Authors: Gombart Adrian F. , Chen Hong , Brandi Lisbet , Olgaard Klaus , Borregaard Niels , Adams John S. , Koeffler H. Phillip 
Published: April 2020

Abstract
The seco-steroid hormone 1,25(OH)2D3 (vitamin D3) and its analogs induce the expression of cathelicidin antimicrobial peptide (CAMP or CAP18) and human β-defensin 4 (DEFB4, formerly DEFB2) in human bone marrow cells, acute myeloid leukemia cells, monocytes/macrophages, neutrophils, skin, and cell lines from carcinomas of the lung and head and neck. The induction is mediated by a DR3-type vitamin D response element (VDRE) located in the promoter of each gene. We demonstrated the induction of CAMP by vitamin D3 does not occur in murine cells. The induction appears to be conserved in humans and chimpanzees, but not in other mammals as indicated by the absence of the VDRE in the murine, rat, and canine CAMP and DEFB4 promoters. The VDRE of the CAMP promoter is present in a short-interspersed nuclear element (SINE) of the Alu-Sx subfamily. This class of transposable elements is found throughout primate genomes. In contrast, the DEFB4 VDRE is not located within such an element. We hypothesized that this unique vitamin D3-regulated expression of antimicrobial peptides is evolutionarily conserved in other primates in addition to humans and chimpanzees. To test this, we designed oligonucleotides to highly conserved sequences on either side of the CAMP or DEFB4 VDREs and amplified by PCR the region from genomic DNA of either New World Monkeys (NWM; marmoset) or Old World Anthropoids (OWA; Rhesus macaque &Savanna monkey). Sequence for the orangutan CAMP promoter was obtained from the trace archive at NCBI. Sequencing and analysis of the PCR products revealed that the promoter and VDRE of the DEFB4 and CAMP genes were highly conserved among all the primates. The CAMP gene SINE from human, chimp, orangutan, and Savanna monkey was approximately 550 bp in size; however, the SINE from Rhesus macaque and the marmoset was about 850 bp in size. The size difference was due to an approximately 300 bp of additional sequence that was highly similar to the SINE element and contained the VDRE. The data suggest that the progenitor of both NWM and OWA possessed the 850 bp SINE and that approximately 300 bp was lost in some OWA. Sequencing of addtional NWM and OWA VDREs and functional studies of both the CAMP and DEFB4 VDREs by reporter gene and gel shifts assays are in progress. The CAMP protein hCAP18 is present at significant levels in the plasma. In preliminary studies, at 24 h post-treatment with vitamin D3 or an analog, hCAP18 levels in the plasma increased in one normal volunteer and in 4/10 patients on chronic hemodialysis (increase ranged from 25–200%). The levels dropped back down to pretreatment levels by 48 and 72 h post-treatment in the patients. The data suggest that vitamin D3-mediated regulation of the antimicrobial peptides CAMP and DEFB4 is evolutionarily important for innate immunity in humans and other primates as opposed to non-primate mammals.
Involvement of PKC Activation in Mer Tyrosine Kinase-Mediated Phagocytosis of Apoptotic Cells.
Authors: Wu Yi , Georgescu Maria-Magdalena , Birge Raymond B. 
Published: April 2020

Abstract
Efficient phagocytosis of apoptotic cells is crucial for many cellular processes. One of earliest signals to the phagocyte is the expression of phosphatidylserine (PS) on the outer surface of the apoptotic cell that provides a potent ‘eat-me’ signal. Mer-family tyrosine kinases and αvβ5(3) integrin are major receptors recognizing PS through the opsonizing proteins MFG-E8, or Gas6, respectively. The previous studies have suggested Mer and integrin αvβ5 signaling pathways converge on tyrosine phosphorylation of FAK and p130Cas, as well as downstream CrkII/Dock180/Rac1 activation, evolutionally-conserved for engulfment. In this study, we aimed at investigating how Mer initiates early signaling events to mediate phaogcytosis and activation of integrin αvβ5, and found that Gas6-Mer-induced tyrosine phosphorylation of FAK and p130Cas was inhibited by calphostin C, and engulfment of apoptotic cells by 293T cells overexpressing wild-type Mer was also suppressed by membrane-permeable PKC inhibitory peptide, suggesting that PKC activation is an early signaling event in Mer-mediated phagocytosis. Consistently, we found that Mer activation stimulates tyrosine phosphorylation of PLCγ2, an upstream event of PKC activation. More interestingly, using constitutively active form for Mer (CDMer) and CDMer mutants, we further found that Tyr 867 of Mer is predominantly responsible for PLCγ2 phosphorylation. In addition, Mer activation stimulated complex formation between integrin αvβ5 and Rack1, a receptor for activated PKC. Taken together, PKC is required for Mer-mediated activation signals for phagocytosis, and might be involved in the crosstalk between Mer and integirn αvβ5 via interaction with Rack1.
Defective Regulated Exocytosis in Mast Cells from Synaptotagmin-2 Knockout Mice.
Authors: Sansores-Garcia Leticia , Melicoff Ernestina , Teich Abigail H. , Murthy Jayasimha , Thakurdas Shakeel M. , Adachi Roberto 
Published: April 2020

Abstract
Synaptotagmins (Syts) are a family of membrane proteins present on a variety of intracellular organelles. They contain a short N terminus, a single transmembrane domain and two calcium binding C2 domains. Twelve Syt isoforms have been identified, in vertebrates. Syt-1 is the postulated calcium sensor in neuronal regulated exocytosis. Syt2 is functionally and structurally closely related to Syt1, it is the major Syt isoform in mast cells (MC) and it localizes on the MC secretory granules. MC are central effectors of allergic reactions and enhancers of host immune responses against bacterial infections. MC respond to a variety of stimuli with three effector mechanisms: secretion of preformed inflammatory mediators stored in their granules via regulated exocytosis, release of newly synthesized cytokines and chemokynes by constitutive exocytosis, and secretion of prostaglandins and leukotrienes by an exocytosis-independent mechanism. Based on the role of Syt1 in neuronal exocytosis, we postulate that Syt2 is the calcium sensor in MC regulated exocytosis. To investigate this possibility we generated a Syt2 knockout mouse using homologous recombination. Disruption of Syt2 results in low viability (around 14 days survival), growth impairment (approximately half size and weight of the control littermates) and severe ataxia and muscular weakness. We derived MC from bone marrow and lungs of 2 weeks-old Syt2−/− mice and control littermates (Syt2+/−, Syt2+/+). We found no morphological or developmental difference in Syt2−/− MC when compared with controls, and after 6 weeks in culture Syt2−/− MC have normal surface expression of FcεRI and c-kit as measured by flow cytometry. However, we found a 50% inhibition in β-hexosaminidase and histamine release upon stimulation by IgE crosslinking, compatible with a defect in degranulation. Furthermore, other effector responses seem not to be compromised, as there was no difference in stimulated LTC4 release when compared with controls. Our data proves the essential role of Syt2 in MC regulated exocytosis. We are now performing additional functional assays and single-cell electrophysiology.
Incompatibilities of HA-1 and 3 Polymorphic Adhesion Molecules Including CD62L, CD31 codon563 and CD31 codon125 Induce Graft-Versus-Leukemia Effect Rather Than GVHD Resulting in Long-Term Survival in HLA Identical SCT.
Authors: Kuba Nahoko , Shiobara Shintaro , Hurukawa Tatsuo , Tsukada Jyunichi , Nakao Sinji , Harada Mine 
Published: April 2020

Abstract
Mismatches of minor histocompatibility antigen (mHag) between HLA identical stem cell donor and host are known as a major risk factor for graft-versus-host disease (GVHD). We determined the alleles of 4 polymorphic molecules including HA-1 and 3 adhesion molecules; CD62L, CD31 codon563 and CD31 codon125 in 114 patients transplanted HLA identical stem cell grafts and investigated the association of mismatches as rates of relapse and GVHD. All 114 recipients underwent stem cell transplantation after myeloablative conditioning between 1985 and 2004. Risk ststus of the disease at SCT was standard (n=71) and high (n=43). After SCT, 36, 57 and 35 developed acute GVHD (≥2), chronic GVHD and relapsed, respectively. Our patients relapsed at rates of 14.3% and 38.0% with one or more and without incompatibilities (P=0.011). In standard risk group, the relapse rates of 20 and 51 patients with and without mHag incompatibilities were 5.0% and 39.2%, respectively (P=0.011). The relapse rates of patients with CD62L, CD31 codon563, HA-1, CD31 codon125 incompatibilities were 5.3%, 11.8%, 16.7%, 23.1% respectively. The frequency of acute GVHD (≥2) did not differ regardless of incompatibilities.
The probability of 10 year survival rates were 84.0% with incompatibility and 54.3% without incompatibility patients (P=0.009). Our data suggests that incompatibilities of at least one of 4 polymorphic molecules contribute to GVL effect rather than to GVHD, resulting in prolonged survival after HLA identical SCT.
Figure Figure
Rituximab May Allow To Improve Multi-Organ Involvement in Patients with cGVHD.
Authors: Zaja Francesco F. , Bacigalupo Andrea A. , Bosi Alberto A. , Scimè Rosanna R. , Milone Giovanna G. , Falda Michele M. , Vener C. , Laszlo D. , Alessandrino E. P. , Narni F. , Sica S. , Olivieri A. , Sperotto Alessandra A. , Fanin Renato R. 
Published: April 2020

Abstract
Background
Chronic graft versus host disease (cGVHD) resembles some clinical and biological features of autoimmune diseases. Only limited data are available concerning the use of rituximab in cGVHD. The preliminary reports in some autoantibodies-related immune disorders encouraged, recently, further application of this agent in the subset of cGVHD with interesting results, particularly in cases with skin involvement.
Objectives
The GITMO (Italian Group Bone Marrow Transplant) performed a retrospective multi-centre study on the use of rituximab in advanced and refractory cGHVD.
Methods
Twenty-eight patients, transplanted with a HLA-matched donor (sibling in 24 and unrelated in 4), 20 with a conventional and 8 with a reduced intensity conditioning, entered this study. The median age was 43 years (range 23–62) and the median interval elapsed from diagnosis of cGVHD to treatment with rituximab was 24 months. All patients had been treated with at least two lines of therapy including cyclosporine or tacrolimus and steroids. Rituximab was given at the conventional dose of 375 mg/m2 every week for a median of 4 administrations (range 1–9). Patients were continued on baseline immunosuppressive therapy. The evaluation of response was distinguished analyzing separately the specific organ site of involvement. Response criteria were the following: complete response (CR); partial response (PR); no response (NR).
Results
A better response rate (p=0.02) was evident for patients treated with rituximab earlier in the course of disease (&lt; vs &gt; 6 months). At a median follow up of 6 months from rituximab administration (range 1–72 months) 14 patients maintain a significant clinical response. During the period of rituximab administration 1 patient died after the third dose because of acute respiratory failure, 1 patient had renal failure and 1 patient developed Gram + septicaemia.
Conclusions
Rituximab can induce significant disease control in patients with cGVHD refractory to standard treatment. Its activity in patients with skin involvement, the subgroup higher represented in this survey, appears, at present, the better defined one. The probability to achieve a response seems related with an earlier timing of administration. Prospective studies in larger number of patients and with more homogeneous organ involvement are warranted
organ involvement and response to rituximab organ involvement patients CR PR NR not valuable skin 25 3 812%) 13 (52%) 7 (28%) 2 scleroderma 6/25 0 4 (66%) 2 (33%) liver 11 0 3 6 2 Gut 5 0 3 1 1 lung 6 0 2 3 1 eyes 9 0 5 3 1 mouth 10 1 4 4 1 thrombocytopenia 3 1 1 1 0 pur red cell aplasia 1 0 0 1 0 myasthenia gravis 1 1 0 0 0