bluuebunny/crossref_metadata_2025 huggingface dataset:
Pre-Infusion Characteristics of the Predominant Cord Blood Unit Correlate with Hematopoietic Engraftment in the Setting of Non-Myeloablative Double Cord Blood Transplant (DCBT).
Authors: Haspel Richard L. , Kao Grace S. , Yeap Beow , Spitzer Thomas R. , Ritz Jerome , Antin Joseph H. , Ballen Karen
Published: April 2020

It is well established that delayed engraftment following transplant of single cord blood unit is mainly due to low CD34 cell dose. Protocols utilizing sequential infusion of two cord blood units increase the total dose of CD34 cells and lead to more rapid hematopoietic engraftment. One of the two cord bloods usually predominates. We assessed the effect of the pre-infusion variables of each cord blood unit on engraftment. Twenty-one patients with hematologic malignancies underwent non-myeloablative conditioning with fludarabine, mephalan and antithymocyte globulin. Two cord bloods, each a minimum 4/6 HLA match with the recipient and each other, were then infused sequentially 1 to 6 hours apart. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. Chimerism analysis of peripheral blood leukocytes was performed by PCR amplification of short tandem repeat loci. Four patients with less than 4 weeks of chimerism data due to death or failed engraftment were excluded. The logrank test, using median values as the cutoff, was used to determine the relationship between pre-infusion variables and time to engraftment. All exact p-values are based on two-sided tests. In all patients, a single cord blood predominated by 3 months post-infusion. In 13 out of 17 transplants (76%), the first cord blood infused predominated (p=.05). The median times for engraftment were 41 days (range: 21–55 days) for platelets >20000 (P20), 65 days (range: 34–91 days) for platelets > 100000 (P100) and 18 days (range: 15–34 days) for ANC>500. Total (cord1 + cord2) CD34 cells/kg (median: 1.9 x 105; range: 6.3 x 104 to 1.0 x 106) and total nucleated cells/kg (median: 4.02 x 107; range 2.98 x 107 to 5.27 x 107) infused did not affect engraftment. However, patients receiving greater than 1.1x 105 CD34/kg in the predominant unit (median: 1.1 x 105; range: 3.0x104 to 8.7x105) but not losing unit (median: 6.92 x 104; range: 8.3.0x103 to 7.4x105) had faster P100 (p=.042 versus p=0.54). The median storage time of the cord blood units infused was 1374 days (range: 272–3047 days). Faster P100 was observed when the predominant unit had a shorter storage time relative to the losing unit (median difference between units: 392 days, p=.03). Our study demonstrated that in the setting of non-myeloablative sequential DCBT, the majority of predominant units engrafted is the first one infused. The CD34 dose and storage time of the predominant unit are factors that can affect engraftment. Although only a small number of patients were studied, these results suggest that the capacity of stem cell niche may be limited and the quality of the cord blood unit occupying this niche can affect the speed of hematopoietic engraftment.
Erythropoietin Signaling Inhibits the Short-Term Marrow Reconstitution.
Authors: Chang Ko-Tung , Pastore Yves D. , Nussenzveig Roberto H. , Prchal Josef T.
Published: April 2020

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 >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<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<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

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

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

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

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

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

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

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

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

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 (< vs > 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
Citation Impact of Breakthrough Interventions for Malignant Blood Disorders.
Authors: Djulbegovic Benjamin , Soares Heloisa P. , Kumar Ambuj
Published: April 2020

Background: Citation count is a widely accepted measure of how much a study has drawn attention of other scientists and clinicians. The studies that have generated new, breakthrough interventions that potentially can affect the lives of many patients are expected to be widely disseminated. In this work, we test hypothesis if the breakthrough interventions as discovered in the NCI sponsored phase III hematological malignancies trials received high citation count. NCI trials account for almost 100% of publicly sponsored phase III trials in the US.

Methods: We evaluated all randomized controlled trials (RCTs) that have been conducted by six NCI sponsored cooperative oncology groups (COGs). We included all trials that were completed by 2002. Here, we focus on hematological malignancies trials. We arbitrarily defined as “breakthrough interventions” those interventions that were judged by the investigators highly preferred so they should become standard of care and/or had an effect size was so large that their log hazard ratio for survival or event-free survival was −1 or less. The study which received more than 1,000 citations is considered as highly-cited clinical research paper. (Ioannidis JPA, JAMA 2005)

Results: We evaluated 133 hematological-malignancy trials/comparisons. Ninety-two trials studied leukemias, 38 lymphomas and 3 either multiple myeloma or myelodysplastic syndrome. 26 (20%) interventions were found to meet the criteria for the “breaktrough interventions”. None of the papers met criteria for highly-cited research. Citation counts ranged from 4–311 (median: 62). The highest-cited paper reported the use of All-trans-retinoic acid for induction and maintanence of acute promyelocytic leukemia. This paper is currently cited in NCCN guidelines and the NCI PQD® treatment website.

Conclusions: The best clinical research in hematological malignancies has received relatively little scientific attention. Elucidating the factors that could explain these findings may help identify the barriers that prevent generation and dissemination of evidence of vital importance for improving outcomes of patients with malignant blood disorders.
Evaluation of Trends in Imatinib Dosing over Time: A Retrospective Cohort Study.
Authors: Hamdan Muhammad , Yood Marianne Ulcickas , Sanders Lynn M. , Hirji Ishan , Hoy Tracey , Oliveria Susan A.
Published: April 2020

Background: The recommended imatinib starting dose is 400mg/day for chronic myelogenous leukemia (CML) patients in chronic phase and 600mg/day for patients in accelerated phase or blast crisis. Some studies have reported improved response rates and progression-free survival in chronic phase patients receiving higher imatinib doses. However, little information is available regarding tolerability of higher doses and the actual initial dosing of imatinib dispensed in the clinical setting. To investigate initial imatinib dosing over time, we conducted a retrospective cohort study using the HealthCore Integrated Database. The data used for this analysis captured pharmacy and medical encounters for a base population of approximately 13 million covered lives.

Methods: We identified all patients with an ICD-9 coded diagnosis of CML (205.1) and at least one prescription for imatinib from January 1, 2001 through December 31, 2004. Using the pharmacy database, we identified the first imatinib prescription and calculated the daily dose of that first prescription (based on number of pills dispensed, dose per pill and prescription days supply).

Results: A total of 486 patients were exposed to imatinib (N=40 patients with starting dose <400 mg/day; N=356 patients with starting dose 400 mg/day; N=5 patients with starting dose 500mg/day, N=52 patients with starting dose 600 mg/day; and N=33 patients with starting dose >600 mg/day). Figure 1 presents initial dosing by calendar year for patients starting at 400mg/day and >600 mg/day.

Imatinib Starting Dose by Year 2001–2004 Imatinib Starting Dose by Year 2001–2004

Among patients receiving imatinib >600mg/day, 15% (95% confidence interval (CI) 5.8–30.4%) had a subsequent dose decrease (defined as a decrease of at least 100 mg/day). Among patients starting imatinib 600mg/day, 22.6% had a dose decrease (95% CI 12.9–35.3%), and 5.8% of the 361 patients receiving imatinib 400mg/day had a dose decrease (95% CI 3.7–8.6%).

Conclusions: These data indicate that a greater proportion of CML patients now receive high starting imatinib doses, potentially due to the fact that more aggressive dosing is used in chronic phase patients. Although phase of disease has not been verified at this point, medical record review is underway to determine imatinib indication and reasons for dose changes. Between 15–20% of patients receiving high starting doses of ≥ 600 mg/day underwent dose reduction of at least 100 mg/day.
Mortalin-2 (mthsp70/PBP74/Grp75), a Novel Mediator of Erythropoietin Signal.
Authors: Ohtsuka Rie , Abe Yasunobu , Fujii Tomomi , Nagasawa Eriko , Shimada Hirotoshi , Tachikawa Yoshimichi , Muta Koichiro
Published: April 2020

Erythropoietin (EPO) stimulates erythroid growth by enhancing the proliferation, maturation and survival of late stage erythroid progenitor cells. Intracellular signaling molecules such as Janus kinase 2 (JAK2) and phosphoinositide-3 kinase (PI-3K)/Akt are considered mediators of the EPO signal, however the entire process of EPO stimulation remains undetermined. To further clarify the intracellular mechanism by which EPO affects the growth of erythroid progenitor cells, we analyzed protein obtained from purified human erythroid colony forming cells (ECFCs) cultured with, or without EPO, by 2-dimensional gel electrophoresis. Five protein spots were apparently related to EPO stimuli, and these proteins were then examined by peptide mass fingerprinting. One of the 5 was identified as mortalin-2, which is a member of the heat shock protein 70 family of chaperones. Mortalin-2 is thought to perform multiple functions relevant to stress response, intracellular trafficking, antigen processing, control of cell proliferation and differentiation. When the expression of mortalin-2 was confirmed by Western blotting, the amount of mortalin-2 was found to be greater in ECFCs cultured with EPO. The amount of mortalin-2 mRNA was then analyzed by quantitative real time PCR. In the presence of EPO, the amount of mortalin-2 mRNA gradually increased during erythroid maturation, peaked on day 6, and then decreased in the terminal stage of maturation. When day 6 ECFCs were incubated with different concentrations of EPO, the amount of mortalin-2 mRNA increased in a dose-dependent manner. Next, we designed a small interfering RNA (siRNA) to RNA encoding mortalin-2, and used this in a transient transfection assay. When the siRNA was transfected into day 6 ECFCs, the expression of mortalin-2 mRNA was suppressed to 40–60% of that seen in the control culture. In the presence of different concentrations of EPO (0–1.0 u/ml), the effect of the siRNA on ECFC growth was determined by MTT assay. The siRNA significantly suppressed the growth of ECFCs in the presence of relatively low concentrations of EPO (0.5 u/ml). These data suggest that mortalin-2 mediates the EPO signal to stimulate the growth of erythroid progenitor cells.
Regulation of the Anti-Apoptotic Protein, Bcl-xL, during Differentiation of Megakaryocytes from Stem Cells.
Authors: Kojima Hiroshi , Kozuma Yukinori , Nagasawa Toshiro
Published: April 2020

This study was conducted to elucidate the anti-apoptotic mechanism operating during differentiation of megakaryocytes (Mgks) from hematopoietic stem cells. As previous reports have indicated the relevance of Bcl-xL for maintaining primitive hematopoietic cells, we focused on Bcl-xL among various anti-apoptotic proteins. Expression of Bcl-xL, which is relatively low in stem cells, increased gradually within 2–3 days of the culture of c-kit+/Lineage- stem cells in the presence of TPO. Expression of Bcl-xL temporally correlated well with that of GATA-1. Inhibition of GATA-1 expression in TPO-independent Meg-O1 megakaryocytic cells by using siRNA resulted in the decrease in Bcl-xL expression, suggesting that expression of Bcl-xL is directly or indirectly regulated by the GATA-1 transcription factor. On the other hand, when TPO was withdrawn from TPO-dependent UT-7 megakaryocytic cells, expression of Bcl-xL protein decreased dramatically within 48 hours despite the stable expression of Bcl-xL mRNA, indicating the post-transcriptional regulation of Bcl-xL after TPO depletion. As a broad caspase inhibitor z-VAD-fmk significantly reduced the decrease in Bcl-xL expression, we considered that proteolysis of Bcl-xL by a caspase might be responsible for down regulation of Bcl-xL. Indeed, corresponding with the time course of decrease in Bcl-xL protein after TPO depletion, activation of caspase-3 was recognized by immunostaining. As these results suggest that TPO-mediated signaling maintains caspase-3 inactive, we cultured UT-7 cells in the presence of a PI3K inhibitor or a MAPK inhibitor in search of the signaling molecule involved in this process. As PI3K inhibitor, wortmannin, induced the activation of caspase-3 even in the presence of TPO, we concluded that PI3K, in the downstream signaling pathway of c-Mpl, maintains caspase-3 inactive, which prevents cleavage of Bcl-xL by caspase-3. Withdrawal of TPO from primary culture of megakaryocytes similarly resulted in the activation of caspase-3 and decreased expression of Bcl-xL protein. Taken together, we conclude that expression of Bcl-xL protein is regulated transcriptionally by GATA-1 and post-transcriptionally by caspase-3 whose activation is prevented by TPO-mediated PI3K activation.
Cytokine Exposure Changes the Hierarchy of Molecular Pathway Usage in Bone Marrow Homing.
Authors: Boenig Halvard , Priestley Gergory V. , Papayannopoulou Thalia
Published: April 2020

Efficient bone marrow (BM) homing is a prerequisite for successful engraftment of transplanted hematopoietic progenitor cells (HPC). Contradictory conclusions about the dependence of homing on SDF-1/CXCR4 have clouded our understanding of its participation in homing and cooperation with other defined molecular pathways. Based on the principle that homing is the coordinated outcome of several pathways, we sought to unravel cooperative and compensatory molecular mechanisms guiding BM homing of BM-HPC. BM homing of fresh BM-HPC does not require SDF-1/CXCR4 or other Gi-protein signals, since fresh BM-HPC rendered either SDF-1 unresponsive (by AMD3100, evaluated 3h after transplantation) or Gi-signaling refractory (by Pertussis toxin (PTX), evaluated 18h after transplantation) homed similarly to untreated control BM-HPC (0.56±0.03% vs. 0.60±0.01% of injected CFU-C recovered per femur (−/+AMD3100 @3h); 0.89±0.08% vs. 0.80±0.07% (−/+PTX @18h)). This was likely due to compensation by α4-integrin interacting with VCAM-1: a contribution of SDF-1/CXCR4- or Gi-protein-mediated signals to BM homing became apparent when inhibition was exerted on α4−/− HPC, significantly augmenting the intrinsic homing defect of α4−/− HPC (0.31±0.02% vs. 0.11±0.03% (−/+AMD3100 @3h); 0.52±0.08% vs. 0.02±0.01% (−/+PTX @18h) (p<0.005)). Similar conclusions were revealed when VCAM-1 deficient hosts were used as recipients of PTX-treated BM-HPC (p<0.005 compared to VCAM-1−/− recipients of untreated control BM-HPC), but not when E/P-selectin−/− recipients were used. Thus Gi-signals do not synergistically interact with E/P-selectins, although α4 integrins do so for the homing of fresh BM-HPC (p<0.005 compared with α4−/− to WT or WT to E/P−/− transplantation). When cytokine incubated BM cells were used for transplantation (SCF, 100 ng/mL for 16–18 h) the hierarchy of molecular pathway usage in homing was shifted towards a dominance of SDF-1/CXCR4 and other Gi-signals (0.60±0.05% vs. 0.37±0.08% (−/+AMD3100 @3h, p<0.05); 0.74±0.05% vs. 0.16±0.02% (−/+PTX @18h, p<0.005)), with only partial compensation through α4/VCAM-1 and endothelial selectins. Of note, expression of α4-integrin on short-term SCF-incubated HPC was not appreciably different from untreated controls. Moreover, SCF+PTX-treated α4−/− HPC homing was virtually abrogated (p<0.05 compared to similarly treated WT HPC). E/P−/− recipients supported normal homing of SCF-incubated HPC, but homing of SCF+PTX-incubated HPC in E/P-selectin−/− recipients was further decreased (0.02±0.00%, p<0.005). Additional homing studies confirmed that these findings likewise apply to SCF-incubated c-kit+lin− cells, a population enriched for stem cells, and that this functional phenotype is not specific to SCF, since it was observed for TPO±PTX incubated BM-HPC. These studies suggest a flexible hierarchy of cooperating homing pathways, in which dominant players are repositioned with changing cytokine milieu, and possibly source of HPC.
Receptor Specificity in the Self-Renewal, Survival and Differentiation of Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.
Authors: Lintmaer Ingrid , Weinreich Michael A. , Wang Linlin , Liggitt Denny , Harkey Michael , Blau C. Anthony
Published: April 2020

Hematopoietic growth factors have had many applications in clinical medicine. However the entry of new growth factors into the clinic has been hampered by the ability of many ligands to affect non-targeted tissues, or to activate more than one receptor type in the targeted tissue. Here we test whether receptors, rather than ligands, can be used to differentially regulate hematopoiesis in vitro and in vivo. Using modified receptors whose activity is solely dependent on a synthetic ligand, called a chemical inducer of dimerization (CID), we compared a conditional derivative of fibroblast growth factor receptor -1 (F36VFGFR1) with an analogous derivative of the thrombopoietin receptor (F36VMpl) in the transduced bone marrow cells of mice. While both receptors allowed for the sustained expansion of transduced marrow cells in culture, only F36VFGFR1 allowed for the expansion of short term repopulating HSCs, and the survival of long term repopulating HSCs, in culture. Both receptors supported the CID dependent expansion of erythroid cells in vivo, however F36VFGFR1 was much more potent, and also induced a marked CID-dependent expansion of platelets and granulocytes, lineages in which F36VMpl had little effect. Cultures in the presence of inhibitors of cell signaling showed a surprising amount of overlap, with both receptors exhibiting equal susceptibility to Jak2 inhibition, whereas F36VFGFR1 was relatively more sensitive to inhibitors of src kinases and phospholipase Cgamma.
Prevalence of Pulmonary Hypertension and Renal Dysfunction by Systemic Blood Pressure Categories in Sickle Cell Disease.
Authors: Gordeuk Victor R. , Gladwin Mark T. , Kato Gregory , Castro Oswaldo
Published: April 2020

In an emerging paradigm of sickle cell disease (SCD) vasculopathy, the risk for pulmonary and systemic hypertension, renal dysfunction, proteinuria, stroke and early death may be related to the degree of hemolysis, nitric oxide scavenging and resultant damage to the vasculature (JAMA2005;293:1653). The systemic blood pressure (BP) in sickle cell anemia (SS) is lower than in normal subjects. Yet, the concept of relative systemic hypertension has been proposed because SS pts. Have higher BP than subjects with other forms of congenital anemias (Am J Med Sci 1993). Furthermore, apparently minor BP increases in SS are associated with stroke risk (Am J Med Sci1993;305:150, Am J Med1997;102:171).

We hypothesized that in contrast to otherwise healthy individuals without SCD, systolic blood pressures (sBP) of 120 to 139 mm Hg define relative hypertension in SCD and identify patients at increased risk for vasculopathy and its complications. We analyzed entry data from 195 adult patients enrolled in the prospective Sickle Cell Pulmonary Hypertension Screening Study (NEJM2004;350:886), and stratified their ECHO-determined tricuspid regurgitant jet velocity (TRV) and serum creatinine concentration according to sBP categories.

As shown in Figure 1, among Hb SS and S beta thal patients, the prevalence of pulmonary hypertension (PHTN, TRV 2.5 m/sec or greater) was 26% with sBP <120 mm Hg, 36% with sBP 120–139 mm Hg and 89% with sBP 140 mm Hg or higher (P <0.0005 for trend). Similarly, the prevalence of a serum creatinine concentration of 1.0 mg/dL or higher was 7% with sBP <120 mm Hg, 13% with sBP 120–139 mm Hg and 30% with sBP 140 mm Hg or higher (P = 0.002 for trend, Figure 2). The increasing prevalences of PHTN and renal dysfunction with the three progressively higher sBP categories are consistent with our hypothesis that sBP 120–139 represents relative hypertension and increased risk for vascular complications in patients with SCD. Whether treatment of relative hypertension in sickle patients would improve vasculopathy and lower their risk for PHTN, renal impairment or other complications such as stroke, is unknown. Consideration should be given to clinical trials to answer this important question.

Figure Figure Figure Figure
Matched Sibling Donor Hematopoietic Cell Transplantation for Sickle Cell Disease Using a Reduced Intensity Conditioning Regimen Can Lead To Stable Long Term Engraftment.
Authors: Krishnamurti Lakshmanan , Wu Catherine , Baker K. Scott , Wagner John E.
Published: April 2020

Hematopoietic Cell Transplantation (HCT) from sibling donors has been demonstrated to cure sickle cell disease. Concerns about high regimen related toxicity particularly in older patients and in those with advanced organ damage and the potential for late sequelae such as chronic graft-versus-host disease (GVHD) and infertility have limited the applicability of HCT. HCT following a reduced intensity conditioning (RIC) regimen has the potential for reducing toxicity and making this curative therapy more acceptable and applicable to this group of patients. We report preliminary results on the first five patients enrolled on a pilot study to evaluate the safety and efficacy of HCT following a RIC regimen for patients with high risk sickle cell disease. All patients received bone marrow from a matched sibling donor. The conditioning regimen consisted of Busulfan 2 mg/kg orally q12 hr x 2 days (0.8mg/kg IV q 6 hr x 2 days for patient#3, 4, 5), Fludarabine 35 mg/m2/dose IV daily x 5 days, anti-thymocyte globulin 30 mg/kg/dose IV daily x 5 days and total lymphoid irradiation 500 cGy with shielding of the liver, lungs, heart, and gonads. GVHD prophylaxis consisted of cyclosporine A and Mycophenolate mofetil. Clinical characteristics, outcomes and donor chimerism in peripheral blood genomic DNA and of subjects are summarized in the Table.1 The preparative regimen was well tolerated with no serious infections or mucositis in any patient. No patient has had recurrence of previous sickle cell related symptoms. Lineage-specific chimerism analysis (patients # 3, 4, 5), reveal predominance of donor erythropoiesis (Table 2). These findings indicate that HCT for sickle cell disease following a RIC regimen is well tolerated and can lead to stable long term engraftment.

Clinical Characteristics of patients with sickle cell disease undergoing HCT from a matched sibling donor following a reduced intensity conditioning regimen PIN. Age, Indication Follow-up (days) Regimen Related Toxicity ANC<500(days) GVHD Acute or Chronic % Engraftment Day 100 % Engraftment Day180 % Engraftment Day 365 1. 8 yrs. Stroke, allosensitization 2100 None 7 None 89 100 100 2. 8 yrs. Repeated ACS 1800 None 8 Grad II Skin 75 81 81 3. 6yrs. Repeated ACS 750 None 9 None 75 85 81 4. 8 years Repeated ACS 295 None 14 None 79 71 - 5.18 yrs. Stroke, allosensitization 288 Mild 13 None 100 100 - Lineage Specific Chimerism PIN, Genotype Donor Genotype % donor erythroid day 100 % donor erythroid day 180 % Donor Lymphoid day 100 % Donor Lymphoid day 180 % Hemoglobin S Day 100 % Hemoglobin S Day 180 3. Hb SS Sickle trait 100 100 30 35 30 34 4. HbS/β Thalassemia β Thalassemia trait 100 81 33 58 4 3 5. HbSS Sickle trait 100 100 100 100 32 34