bluuebunny/crossref_metadata_2025 huggingface dataset:
Prostaglandin E2 (PGE2) Regulates Osteoblastic Jagged1 and Expands Primitive Hematopoietic Cells In Vivo.
Authors: Calvi Laura M. , Frisch Benjamin J. , Gigliotti Benjamin J. , Christianson Christina A. , Weber Jonathan M. , Jordan Craig T. , O’Keefe Regis J.
Published: April 2020

Parathyroid Hormone (PTH) targets osteoblastic cells (OBs) in the bone marrow microenvironment and expands hematopoietic stem cells (HSC) through Notch activation. Since PTH stimulates the Notch ligand Jagged1 (J1) in OBs, we have focused on the signaling pathways involved in this PTH effect in order to identify novel activators of the HSC niche. Osteoblastic Protein Kinase A (PKA) activation is required for the PTH-dependent J1 increase in OBs. Therefore, we hypothesized that alternative PKA activators could also regulate osteoblastic J1, alter the HSC niche, and provide additional pharmacologic tools to expand HSC in vivo. Consistent with this hypothesis, direct PKA agonists 8-bromo-cAMP and dibutyryl-cAMP stimulated J1 in osteoblastic UMR106 cells. In addition, PGE2, a member of the prostaglandin family known to stimulate PKA in OBs, was studied in vivo and in vitro. By real-time RT-PCR analysis, J1 mRNA was increased up to 5 fold at 2 hours in UMR106 cells when treated with PGE2 (10−7 M) compared to vehicle. J1 protein was also increased after treatment with PGE2. The PGE2-dependent J1 increase was blocked in the presence of the specific PKA inhibitors H89 and myristoylated PKA Inhibitory Peptide (14–22)(PKI) (200ug/ml), demonstrating that PKA is necessary for osteoblastic J1 stimulation by PGE2. Since systemic PGE2 is known to have bone anabolic effects in both humans and animal models, adult wild-type FVB/N male mice were treated with PGE2 (6mg/kg/day i.p.) for 12 days. This regimen has previously been shown to have bone anabolic effects in rats. At day 12, histologic analysis demonstrated an anabolic effect mainly on cortical bone, as was evident in the femurs and tibiae of PGE2-treated mice compared to control. This histologic finding was confirmed by histomorphometry (trabecular bone area means 41% vs 12%,p=0.0916, n=3 in both groups; cortical thickness means 138 vs 85 μm, p=0.0071, n=3 in both groups). Frequency of hematopoietic stem cells (c-Kit+, Sca1+, lin−) was increased in bone marrow from PGE2-treated vs control mice by over 20% (p=0.0018, n=8 in both groups). In summary, PGE2 stimulates J1 in osteoblastic cells through PKA activation and increases mainly cortical bone in vivo. Ongoing studies will confirm whether in vivo PGE2 treatment expands HSC, and whether osteoblastic J1 regulates this process. This study identifies PGE2 as a novel regulator of osteoblastic J1, and as a potential new microenvironmental modulator of HSC, which could be used for in vivo therapeutic HSC niche manipulation.
Increased Levels of Inflammatory Mediators in Lung Cancer and Their Modulation by Oral Anticoagulant Treatment.
Authors: DeMir Muzaffer , Hoppensteadt Debra A. , Cunanan Josephine , Iqbal Omer , Fareed Jawed
Published: April 2020

Venous thromboembolic events (VTE) are associated with lung cancer and significantly contributes to the increased mortality in these patients. While the increased prevalence of VTE is fully recognized in lung cancer, its pathogenesis is not fully understood. Even the patients undergoing therapeutic interventions are at high risk to develop VTE. These patients provide a unique clinical setting to investigate the pathogenesis of lung cancer associated thrombosis. In a prospective, randomized, controlled study patients with inoperable lung cancer (n=100) were randomized to receive chemotherapy, radiation and warfarin (INR 1.5–2.5) or chemotherapy, radiation without warfarin (n=50). Blood samples were drawn prior to and after the second treatment cycle with warfarin. All samples were analyzed for tumor necrosis factor alpha (TNF a), CD 40 ligand (CD 40L), C-reactive protein (CRP), interleukin 1 beta (IL-1b), asymmetric dimethylarginine (ADMA) and nitric oxide (NO) as summarized in Table 1. All of the surrogate markers of inflammation showed a decreased trend (13–50%) in the warfarin treated group, whereas the non-warfarin treated group exhibited an increase (18–46%) in all markers except CRP and ADMA. The levels of various inflammatory markers are upregulated in lung cancer suggesting a pathogenic role of this process in lung cancer. Warfarin down regulated the inflammatory process in contrast to the non-warfarin treated group. The clinical relevance of these observations require additional analysis.

Table 1 Test Warfarin Treated Warfarin Treated Warfarin Treated Non-Warfarin Treated Non-Warfarin Treated Non-Warfarin Treated Pre Post % Change Pre Post % Change TNF α (pg/ml) 63±22 44.4±18.2 ↓30 50.7±18 62.9±19.2 ↑24 CD 40L (ng/ml) 671±212 470±123 ↓29 563±176 712±234 ↑26 CRP (μg/ml) 19.1±8.5 9.6±2.8 ↓50 18.2±9.2 9.9±3.1 ↓46 NO (μM) 33.4±9.8 29±8.5 ↓13 29.9±8.8 35.2±8.2 ↑18 IL-1β (pg/ml) 14.6±5.7 11.1±5.3 ↓24 15.3±6.2 15.2±5.8 NC ADMA (μ/ml) 2.0±0.8 1.53±0.8 ↓24 2.0±0.8 1.9±0.7 ↓18
The Tumor Suppressor Gene SLC5A8 Is Selectively Silenced in MLL Partial Tandem Duplication-Positive Acute Myeloid Leukemia with Normal Cytogenetics.
Authors: Whitman Susan P. , Wen Jing , Muto Gina , Hackanson Bjoern , Yu Li , Liu Chunhui , Sanfilippo Joseph L. , Marcucci Guido , Rush Laura J. , Plass Christoph , Caligiuri Michael A.
Published: April 2020

The Mixed Lineage Leukemia Partial Tandem Duplication (MLL PTD) defect, present in 4–10% of adults with acute myeloid leukemia (AML) and normal cytogenetics (NC), predicts for shorter complete remission duration. Functionally, the MLL PTD is associated with aberrant histone methylation. Since posttranslational histone modifications are also frequently associated with changes in the methylation status of gene promoters, we evaluated whether AML with the MLL PTD is associated with a pattern of DNA methylation distinct from AML with NC and wild type MLL (MLL WT). Significant differences in DNA methylation patterns of over 2,000 CpG island fragments, most of them located in the 5′-region of genes, were observed between NC AML with the MLL PTD (n=9) and NC AML with MLL WT (n=23) using Restriction Landmark Genomic Scanning (RLGS) (P<0.001). This analysis revealed 201 RLGS spots were lost, corresponding to DNA methylation events, at a higher frequency in MLL PTD+ AML compared with MLL WT AML. Among the non-polymorphic, methylated spots, spot 3D41 corresponding to the SLC5A8 gene was found methylated in 78% of MLL PTD+ AMLs compared with only 17% of MLL WT AMLs (P<0.003). The SMCT1 protein, encoded by SLC5A8, is a known tumor suppressor reported to be silenced by DNA methylation in colorectal, glioma, thyroid and gastric cancers. This plasma membrane protein mediates intracellular transport of substrates such as butyrate, a histone deacetylase inhibitor (HDACi), and when SLC5A8 is underexpressed, this predicts worse prognosis in colon cancer patients. In MLL PTD+ AML samples exhibiting 3D41 loss by RLGS, bisulfite-PCR sequencing confirmed CpG hypermethylation within the SLC5A8 proximal promoter (range: 24–78% promoter methylation) compared with those AML samples (0%–12% promoter methylation) exhibiting no 3D41 loss. Furthermore, SLC5A8 transcript levels were significantly reduced in MLL PTD+ AML patient samples compared to MLL WT+ AML patient samples (P<0.002). The demethylating agent, decitabine (Dacogen®, MGI Pharma), alone or in sequential combination with one of the following HDACis- depsipeptide, valproic acid or MS275, induced expression of SLC5A8 and cell death in the MLL PTD+ EOL−1 cell line. These results suggest that aberrant promoter DNA methylation and the reduced expression of the tumor suppressor gene SLC5A8 may be associated with the short remission duration observed in MLL PTD+ AML patients. These data also support further investigation of DNA demethylating agents, such as recently FDA-approved Dacogen® and the appropriate timing of administration of HDACi, such as butyrate analogs, as a therapeutic approach for this poor prognostic AML subgroup.
Preclinical Pharmacokinetic and Metabolism of Apixaban, a Potent and Selective Factor Xa Inhibitor.
Authors: He Kan , He Bing , Grace James E. , Xin Baomin , Zhang Donglu , Pinto Donald J. , Luettgen Joseph M. , Knabb Robert M. , Lam Patrick Y.S. , Wexler Ruth R. , Humphreys William G. , Shyu Wen-Chyi , Grossman Scott J.
Published: April 2020

Apixaban is a potent, orally available, highly selective, and reversible factor Xa inhibitor, and currently under development for prevention and treatment of thrombosis. The preclinical pharmacokinetic and metabolism attributes of apixaban feature a small volume of distribution, a low systemic clearance, good oral bioavailability, multiple elimination pathways and minimal potential for drug-drug interactions. Apixaban is well absorbed in chimpanzees, dogs and rats with a mean oral bioavailability of 51, 88 and 34%, respectively. The mean volume of distribution of apixaban is 0.17, 0.29 and 0.31 L/kg in chimpanzees, dogs and rats, respectively, suggesting apixaban is primarily distributed (30–50%) to blood where the therapeutic action resides. The small volume is not due to extensive plasma protein binding, but possibly attributed to limited extravascular tissue distribution, given that the unbound fraction is approximately 13, 5, 8 and 4% in human, chimpanzee, dog and rat serum, respectively. The systemic clearance is <3% of hepatic blood flow in chimpanzees (0.018 L/h/kg) and dogs (0.052 L/h/kg), and <10% in rats (0.26 L/h/kg). Consistent with this low clearance, the in vitro metabolic clearance of apixaban is low, as indicated by the lack of significant metabolism in human liver microsomes and hepatocytes, and in chimpanzee and dog liver microsomes. The primary metabolite identified in vitro is the O-demethylated product which is formed mainly by CYP3A4 in human liver microsomes. The elimination of apixaban involves multiple pathways including renal and intestinal excretion of the parent and metabolism. The biliary clearance is low in dogs, accounting for approximately 2% of the systemic clearance. Apixaban did not inhibit CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 activities in cDNA-expressed enzyme systems and human liver microsomes, nor induced 1A2, 2B6 and CYP3A4 in human hepatocytes. No glutathione adduct with apixaban was formed in dog and rat, and in human hepatocytes and liver microsomal incubation in the presence of glutathione, suggesting low potential for the formation of reactive metabolites. In conclusion, apixaban shows excellent pharmacokinetic and metabolic properties for clinical development.
Role of 4-1BB as a Regulator for NKG2D Costimulation in Human CD8+ T Cells.
Authors: Kim Young-June , Han Myung-Kwan , Broxmeyer Hal E.
Published: April 2020

NKG2D is a potent costimulatory receptor which is involved in activating various CD8+ T cell responses. Cells undergoing various stresses including tumor formation and virus infection express MHC class I related chain (MIC), a ligand for NKG2D. Thus, MIC acts as a signature molecule of the target cells for CD8+ T cells to recognize in killing. In contrast, MIC also plays a role in impairing effector CD8+ T cell responses because it rapidly internalizes NKG2D from the cell surface. It is unclear how these opposing effects of NKG2D ligation on CD8+ T cell fate are regulated. In this study, we demonstrated that 4-1BB, another costimulatory receptor, might be an important regulator for induction and maintenance of NKG2D expression after its internalization, thereby enhancing effector CD8+ T cell responses. Whereas NKG2D is constitutively expressed on naïve human CD8+ T cells, 4-1BB is induced only by activation. Costimulation of NKG2D with anti-NKG2D and suboptimal anti-CD3 eliminated NKG2D but efficiently induced 4-1BB on naïve CD8+ T cells isolated from human umbilical cord blood, even in the presence of TGF-ß1 which inhibited 4-1BB expression in the absence of NKG2D costimulation. In vitro chromium release assays indicated that the CD8+ T cells costimulated with NKG2D remained low in their cytotoxic activity against C1R B lymphoma cell line expressing MIC which was used as target cells. We found these results despite the fact that the CD8+ T cells were fully activated and expressed 4-1BB and granzyme B at high levels. This is correlated with a lack of NKG2D on the cell surface. Subsequent 4-1BB costimulation restored NKG2D to the cell surface, even in the presence of TGF-ß1. The resulting CD8+ T cells expressing both NKG2D and 4-1BB showed extremely potent cytotoxic activity. Importantly, the NKG2D co-expressed with 4-1BB was unique for being highly refractory to the downregulation by TGF-ß1 and internalization from the cell surface. IL-4 markedly promoted downregulation of NKG2D and 4-1BB expression induced by TGF-ß1, generating a CD8+ T cell type lacking both NKG2D and 4-1BB on the cell surface. These cells were inert and unable to gain cytotoxic as well as proliferative activity, suggesting that CD8+ T cells lacking both NKG2D and 4-1BB may represent terminally incapacitated cells. In summary, NKG2D may be involved in inducing 4-1BB expression and in turn 4-1BB may play a critical role for stabilizing surface NKG2D expression to overcome tumor immune evasion especially in tumor microenvironments enriched with soluble MIC and TGF-ß1. Due to such coupled supports, co-expression of NKG2D and 4-1BB may be a key biomarker for defining effective tumor infiltrating CD8+ T cells that can be used in a clinical setting of immune therapeutic strategies.
Interleukin-10 Severely Impairs TLR Mediated Activation of Antigen Presenting Cells.
Authors: Knödler Anja , Bringmann Anita , Weck Markus M. , Brauer Katharina M. , Kanz Lothar , Grünebach Frank , Brossart Peter
Published: April 2020

Danger signals provided by pathogens are sensed by specialized receptors, Toll-like receptors (TLR), on the cell surface of antigen presenting cells (APC) leading to activation of dendritic cells (DC) accompanied by their phenotypic and functional changes. Activation of DC via the TLR is believed to be a critical event regulating the strength and outcome of an immunological response. IL-10 is an immunosuppressive cytokine shown to have inhibitory effects on function and differentiation of APC by interfering with signaling pathways involved in DC activation like the PI3-kinase or NFκB pathway.

In our study we analyzed the function of human dendritic cells generated in the presence of IL-10 upon activation by Toll-like receptor ligands (TLR2, TLR3, TLR4, TLR7/8). Exposure of DCs to IL-10 resulted in a skewed phenotypic maturation in response to stimuli provided by TLR ligands, a reduced production of cytokines like IL-12, IL-6 or TNF-α and an impaired capacity to stimulate T cell activation. Furthermore, CCR7 upregulation in DC exposed to TLR stimulation as well as migration toward CCL19/MIP-3β were strongly reduced. As a suitable mechanism for these effects, IL-10 was found to downregulate MyD88, an essential adaptor molecule for TLR signaling, and to decrease TLR-induced nuclear expression of the NFκB transcription factor members c-Rel and Rel-B as well as IRF-3 and IRF-8. This was not due to the inhibition of the MAP kinase pathway as phosphorylation of p38 and ERK was not affected but was rather mediated by inhibitory effects on the PI3-kinase pathway. IL-10 treatment of APC resulted in a reduced TLR induced mTOR phosphorylation. In summary, our results demonstrate that IL-10 can inhibit TLR-mediated activation of APC via a MyD88-dependent and independent pathway.
Transcriptional Activation of Mdm2 in Germinal-Center B Cells Is Induced by IRF8.
Authors: Zhou Jeff X. , Lee Chang-Hoon , Qi Chen-Feng , Maghashfar Zohreh , Zhao Ming , Morse Herbert C.
Published: April 2020

The transcription factor, interferon regulatory factor 8 (IRF8), is expressed at low levels in both naïve and terminally differentiated B cells but at high levels in germinal center (GC) B cells where it contributes to transcriptional activation of two genes critically involved in the GC reaction: BCL6, a BTB/POZ-zinc finger transcriptional repressor; and AICDA (also known as AID) a single stranded DNA deaminase (Lee et al. J. Exp. Med.201(1):63–72, 2006). AID is responsible for the generation of double stranded DNA breaks (DSDB) that occur physiologically in B cells during the processes of somatic hypermutation (SHM) of immunoglobulin (Ig) gene variable region sequences and Ig class switch recombination (CSR). The response of most cell types to DSDB is marked by activation of p53 (also termed TP53) that induces cell cycle arrest or apoptosis. p53 also functions at a critical checkpoint to prevent aberrant repair of DSDB in Ig genes that can result in chromosomal translocations that activate proto-oncogenes. GC B cells, however, must be able to tolerate these breaks without experiencing p53-dependent apoptosis. Recent studies showed that functional inactivation of p53 in GC cells is governed in part by transcriptional repression of the p53 gene by BCL6 (Phan and Dalla-Favera Nature432(7017):635–639, 2004). Here we report that IRF8 also suppresses p53 function via transcriptional activation of MDM2. MDM2 is a p53-binding protein and E3 ubiquitin ligase that blocks the transcriptional activity of p53 and stimulates p53 degradation. The levels of both Mdm2 mRNA and its encoded protein were decreased in GC B cells of Irf8 knockout mice as well as in a B lymphoma cell line with siRNA-induced knockdowns of IRF8 expression. Conversely, MDM2 protein levels were increased in cells induced to overexpress IRF8. Transfection of a B lymphoma cell line with an IRF8 expression vector resulted in marked activation of a Mdm2 promoter reporter construct. Oligonucleotide pull-down assays using sequences from the Mdm2 promoter similar to the interferon stimulated response element, a known IRF8 target sequence, were found to bring down IRF8. IL-21, produced by GC T helper cells, promotes the differentiation of B cells activated through the BCR, while inducing apoptosis of unengaged cells. Studies of a B lymphoma cell line treated with IL21 showed that the rate of apoptosis was significantly increased when IRF8 expression was suppressed by siRNAs. The same pattern was true for cells treated with etoposide, a drug that induces DSDB. These results indicate that IRF8 shepherds B cells through the GC reaction by stimulating expression of BCL6 and MDM2 thereby suppressing p53-mediated cell cycle arrest or death in response to DNA breaks.
Prevention of Alloimmunization to Platelets and Cardiac Transplants by Mirasol‚ Pathogen Reduction Technology (PRT).
Authors: Asano Hiroshi , Lee Chih-Yuan , Baldwin William M. , Marschner Susanne , Goodrich Raymond
Published: April 2020

Functional WBCs in blood components may be responsible for a number of adverse transfusion effects, including alloimmunization and alloimmune platelet refractoriness. Attempts to reduce these undesired effects have used WBCs reduction filters and γ-irradiation. Previous studies have shown that exposure of platelet concentrates to riboflavin and light (Mirasol PRT treatment) causes irreparable modification of nucleic acids that results in inactivation of a wide range of pathogens as well as inhibition of the immunological responses mediated by the WBCs present in platelet concentrates. This study evaluated the ability of Mirasol PRT treatment to prevent alloimmunization and to modify the immune response to transplanted tissue in rats. Over a 10 week period, test animals (Lewis rats) received 8 transfusions of untreated or Mirasol treated platelet products containing leukocytes from DA rats. A third group of animals received saline injections. Antibody levels (IgG, IgM) in the recipient animals were monitored weekly. Animals were subsequently challenged with heart transplant under cyclosporine treatment to assess the effect of Mirasol PRT treatment on presensitization and transplant rejection. The IgM and IgG response in rats that received Mirasol PRT treated platelets was almost completely abolished compared to animals that received untreated platelets (see Figure below). In preliminary experiments, animals that mounted an IgG response also rejected the subsequent heart transplant. In summary, Mirasol PRT treatment prevented the development of an Ig response in transfused animals. This inhibition of an Ig response, in particular IgG, shows that Mirasol PRT treatment prevents alloimmunization. The inability to reject the heart transplant in the absence of an IgG response indicates that Mirasol PRT treatment may be effective in preventing alloimmune refractoriness to platelets and presensitization to transplants.

Figure Figure
Carbon Monoxide Atmosphere Allows Platelets To Retain Function and Hinder Pathogen Growth.
Authors: Shaklai Nurith , Amit Yonit , Tsemakhovitch Vladimir , Samuelov-Zubare Meirav , Shaklai Matityahu
Published: April 2020

Platelets are the sole blood fraction which is currently preserved at room temperature and thus exposed to microorganisms, specifically bacterial, growth. Bacterial contamination in platelet concentrate (PC), in a range of up to 0.4%, is the major cause of morbidity and mortality in transfusion medicine. To extend the current limited shelf life of 5 days for platelets, fast screening methods have been developed. Alternatively, methods for inactivation of bacterial growth by DNA crosslinking are provided. The latter include treatment by psoralen or photodynamic inactivation such as riboflavin activated by irradiation. The drawback of these efficient methods which relates to large investment and unknown future side effects, calls for alternative preservation methods. In the current study we demonstrate an option to store PC under sealed anaerobic, carbon monoxide atmosphere, in which neutral pH is kept by bicarbonate (Cob). The procedure is simple and cost effective.

Freshly isolated platelets (PC) were prepared by the Israel Central Blood Bank and stored in plastic flasks either allowing air transfer (control) or in gas non-transparent, Cob, at 22°C. Growth of inoculated bacteria in Cob stored PC was arrested immediately or after one log. Integrity of Cob platelets was better than control as assessed by leak of lactic dehydrogenase (LDH). Leak of LDH was lower as compared to control cells. P-selectin exposure on long term preserved platelets (days 6–9) was lower in Cob preserved platelets as compared to identical control PC. Phosophatydil serine (PS) measurement using flow cytometry indicated a large exposure in Cob platelets stored longer than 4 days, concomitant with consumption of all glucose, the energy source for anaerobic metabolism, in plasma. However, introduction of air resulted in a reversible reduction of the exposed PS. To better mimic in vivo viability of stored platelets, additional experiments were carried out using reconstituted blood (R-blood) prepared as follows: Platelets were removed from freshly drawn blood and stored PC were added to yield a concentration of ~ 350,000/μl. Aggregability of control and Cob R-blood were compared using a “paleletworks” kit (Helena company). Aggregability of stored PC was reduced with time. Nevertheless, aggregability of Cob platelets stored in R-blood was always significantly higher than its twin, air stored, sample. Adhesion of the stored platelets was estimated using Cone and Plate Analyzer (Diamed). No differences were found, within error, between air and Cob stored R-blood. In vivo survival of the stored platelets was assessed using a previously described rabbit model. Survival time declined with prolonged preservation in both control and COb stored platelets. No differences within error were observed between control and COb preserved platelets. These results indicate the option to use COb method for storage of platelets in a viable, pathogen reduced manner.
Generation of Kinins during Preparation and Storage of Leukoreduced Platelet Concentrates.
Authors: Moreau Marie Eve , Thibault Louis , Désormeaux Anik , Marceau François , de Grandmont Marie-Joëlle , Lemieux Réal , Robillard Pierre , Rivard Georges E. , Lepage Yves , Adam Albert
Published: April 2020

BACKGROUND. Severe hypotensive reactions can occur following transfusion of blood components, particularly with platelets concentrates (PCs) leukoreduced with negatively- charged filters. Bradykinin (BK)-related peptides were proposed as a possible cause of this side-effect. This study evaluated the effects of leukoreduction and storage conditions on the levels of two kinins (BK and des-Arg9-BK) in PCs.

METHODS. Whole blood donations (n=35) were processed using current PRP (platelet-rich plasma) procedure to prepare leukoreduced and unfiltered components by Leukotrap® RC-PL Whole Blood Collection, Filtration and Storage System. PCs and plasma were stored for 7 days at 20–24°C with agitation. Levels of BK and des-Arg9-BK were measured by specific enzyme immunoassays and HPLC at day 0, 2, 5 and 7 of storage. Mechanisms potentially responsible for accumulation of BK and des-Arg9-BK were studied.

RESULTS. On day 0, kinins were measured in significantly higher concentrations in leukoreduced (BK: 101 ± 157 pg/mL; des-Arg9-BK: 194 ± 191 pg/mL) vs unfiltered PCs (BK: 71 ± 121 pg/mL; des-Arg9-BK: 98 ± 114 pg/mL). During storage, both kinins peaked on day 5, with concentrations higher than 1 ng/mL in 22% of leukoreduced as well as unfiltered PCs. Physicochemical and pharmacological characterization of immunoreactive kinins confirmed their nature. In vitro activation of the contact system of the corresponding platelet-poor plasma (PPP) showed that a high kinin concentration on day 5 of the storage corresponded to a low kinin-forming capacity of plasma. On day 7, BK was no longer elevated presumably due to its degradation and the depletion of kinin-forming capacity of the plasma in stored PCs. The activity of metallopeptidases that metabolize BK-related peptides in plasma from PCs were at levels similar to those recorded in the plasma of a normal reference population and were unaffected by storage.

CONCLUSIONS. Our results indicate that filtration and storage conditions of PCs contribute to generation of pharmacologically relevant bradykinin levels that might pose a risk in susceptible patients. The clinical relevance of such high concentrations of kinins in PCs remains to be established but could potentially be significant especially in patients treated with angiotensin I-converting enzyme-inhibitors that affect the pathway of BK degradation.
Signaling Pathways in HIT Immune Complex Stimulation of Monocyte Tissue Factor.
Authors: McKenzie Steven E. , Cassel Diana L. , Casasanto Peter , Taylor Scott M. , Reilly Michael P.
Published: April 2020

In heparin-induced thrombocytopenia (HIT), platelet-fibrin thrombi form in vivo due to generation of thrombin initiated by intravascular tissue factor (TF). Stimulation of monocytic cells in vitro by the HIT immune complex (IC), consisting of heparin/PF4 complexes and IgG anti-hep/PF4 antibodies, triggers TF expression. The HIT IC binds to Fcγ receptors on monocytic cells to initiate a signal cascade which results in new TF transcription. Though much is known about monocyte TF transcription as a consequence of LPS stimulation of TLR4, the mechanism by which HIT IC engagement of FcγR leads to TF transcription is unknown. Transcription of pro-inflammatory and anti-inflammatory cytokines as a consequence of IC engagement of FcγRs on monocytes has been reported to involve the ras/Erk and/or PI3K/Akt pathways downstream of syk. We tested the hypothesis that HIT IC- FcγR engagement signals via a PI3K/Akt pathway to trigger TF transcription. We created a model HIT IC consisting of an optimal ratio of heparin and recombinant human PF4, bound by well-characterized monoclonal anti-hep/hPF4 antibody KKO. Using human monocytic cell line THP-1, we observed that the HIT IC induction of new TF mRNA, assessed by qRT-PCR after treatment for 2 h at 37°C, was reduced approximately 80% by pretreatment of the cells with PI3K inhibitor wortmannin (100 nM; n = 3, p < 0.05). In addition, the same effect was observed with another IC, heat-aggregated human IgG, namely 80% inhibition of stimulated TF expression by wortmannin (pretreatment with 100 nM, 37°C, 30 min, n = 4, p< 0.05). Of note, the LPS-mediated increase in TF expression was enhanced over 2-fold (1 ug/ml, 2 h, p <0.05) by the same wortmannin pretreatment, in direct contrast to the result with IC stimulation. In preliminary studies with Akt inhibitor SH5, we have observed ~80% inhibition of IC-stimulated TF expression. We have verified the effects of PI3K inhibition of IC-stimulation of TF (75–80% inhibition by wortmannin pretreatment) by using primary peritoneal macrophages derived from FcγRIIa-transgenic mice, which express a similar FcγR pattern as human cells and our HIT mouse model. Immunoblot analysis of phospho-Akt using anti-Ser473 Akt antibody(generously provided by Donna Woulfe, PhD, Thomas Jefferson University) verified the phosphorylation of Akt by HIT IC treatment and marked reduction when PI3K was inhibited. In summary, HIT IC stimulation of monocytic cell TF involves a PI3K/Akt pathway in a way clearly distinct from LPS-stimulated TF expression. Further studies are in progress to elucidate the upstream activators and downstream effectors, the role of other signaling pathways and the identity of the responsible transcription factor complexes.
Comparative Proteome Analysis To Study Novel Markers of the Blood Platelet Lesion.
Authors: Schubert Peter , Thon Jonathan N. , Duguay Marie , Serrano Katherine , Devine Dana V. , Kast Juergen
Published: April 2020

Platelet transfusion is a very common live-saving medical procedure for patients with platelet-deficient diseases like leukemia. In contrast to other blood components, the availability of platelets is restricted since they have a limited shelf-life of 5 days for transfusion purposes. This is due to storage-related deterioration in product quality resulting in the clearance from circulation. To overcome this problem, it is important to understand the molecular mechanisms leading to blood platelet lesion during storage. We were using two different proteomic approaches combined with functional biochemistry to investigate time-dependent changes in the blood platelet proteome. One type of analysis consisted of the separation of the platelet proteome at two different time points of storage, day 1 and day 8, using 2-dimensional (2D) gel electrophoresis for qualitative and DIGE technology for quantitative analysis. The identification of proteins changing in spot intensity was carried out by liquid chromatography and tandem mass spectrometry. The second method was based on stable isotope labeling with ITRAQ/ICAT reagents in combination with protease treatment, equivalent mixing, separation of the resulting peptides and quantitative analysis by mass spectrometry. Taken together, for the 2D/DIGE approach we analyzed 977 spots corresponding to 103 different proteins and for the ITRAQ approach 1428 peptides corresponding to 355 proteins, resulting in 37 proteins significantly changing both quantitatively due to protein synthesis or degradation and qualitatively due to post-translational modification and enzymatic activity. The high degree of correlation between the two approaches validates the experimental set-up and confirmed the requirement for complementary tools to enhance proteome coverage. Among others, increased amounts of integrins and other proteins known to form receptor signaling complexes with these integrins as well as proteins observed in platelet activation were detected. This proves, for the first time, that there is an apparent link between blood platelet storage lesion and cell signaling.
Production of Megakaryocytes and Platelets from Common Marmoset (Callithrix jacchus) ES Cells by Constitutive Expression of tal1/scl Gene.
Authors: Kurita Ryo , Yokoo Tomoko , Sasaki Erika , Dong Yan , Suehiro Youko , Tanioka Yoshikuni , Soda Yasushi , Komatsu Norio , Tani Kenzaburo
Published: April 2020

Since the successful establishment of human embryonic stem (ES) cell lines in 1998, transplantation of the differentiated ES cells to specific organ has been expected to cure its defective function. For the realistic medicine, the preclinical studies using animal model systems including non-human primates are essential. We have already demonstrated that 1) non-human primates of common marmosets (CM) are suitable for the laboratory animal models for preclinical studies of hematopoietic stem cell therapy and 2) tal1/scl gene transduction using a lentiviral vector is a very useful tool for the efficient hematopoietic cell differentiation from CM ES cells. In this study, we especially focused on the differentiation of CM ES cells to megakaryocytes and platelets in stroma cell-free conditions in order to investigate the feasibility of future new transfusion therapy. Firstly, we established CM ES cell line, which expressed tal1/scl gene constitutively (CM ES-tal1/scl). This ES cell line exhibited undifferentiated morphology and expressed stage-specific embryonic antigen (SSEA)-3 and SSEA-4, while SSEA-1 was not detected. The induction of hematopoietic cells from CM ES-tal1/scl was more efficient than that of original ES cell line. Next, by using this cell line, we planned to produce megakaryocytes and platelets efficiently. During semi-long culture (< 1 month) in the presence of thrombopoietin (10–100 ng/ml), we obtained some proplatelet-like structures, which showed CD41 expression by immunohistostaining, in the adherent cells. Furthermore, 2–4% of CD41 positive platelet-like fraction was observed in the culture supernatant by flow cytometry, although the induction efficiency was low. Now, we examine the efficient culture conditions including additional gene transduction such as gata1, runx1, and NF-E2 genes.
Prognostic Factors and Treatment Outcome in Lymphocyte Predominant and Classical Hodgkin’s Lymphoma: A Comprehensive Analysis of the German Hodgkin Study Group.
Authors: Nogova Lucia , Reineke Thorsten , Brillant Corinne , Ruediger Thomas , Mueller-Hermelink Hans K. , Bredenfeld Henning , Josting Andreas , Mueller Rolf P. , Diehl Volker , Engert Andreas
Published: April 2020

Introduction: Lymphocyte predominant Hodgkin lymphoma (LPHL) differs in histological and clinical presentation from classical Hodgkin lymphoma (cHL). Treatment of LPHL patients using standard approaches leads to complete remission (CR) in more than 95% of patients. However, differences in terms of relapse rates, overall survival (OS) and freedom from treatment failure (FFTF) between LPHL and cHL patients were suggested by a recent intergroup analysis. To obtain a more comprehensive picture, we reviewed all LPHL-cases registered in the GHSG database comparing patient characteristics and treatment outcome with cHL patients.

Patients and methods: We retrospectively analyzed 8298 HL patients treated within the GHSG trials (HD4 to HD12). 394 patients had LPHL and 7904 cHL. From 394 LPHL patients, 63% were in early favorable stage, 16% in early unfavorable and 21% in advanced stage of disease. Of the 7904 cHL patients analyzed, 22% were in early favorable, 39% in early unfavorable and 39% in advanced stages. 9% of LPHL patients had B symptoms compared to 40% in cHL patients.

Results: 91% LPHL vs. 86% cHL patients in early favorable stages, 86% vs. 83% in early unfavorable and 79% vs. 75% in advanced stages reached CR/CRu. 0.3% LPHL patients developed progressive disease (PD) compared to 3.7% cHL patients. The relapse rate of LPHL patients was very similar to cHL (8.1% vs. 7.9%). There were 2.5% secondary malignancies in LPHL and 3.7% in cHL patients. 4.3% LPHL patients and 8.8% cHL patients died. The FFTF rates for LPHL and cHL patients at a median observation of 41 or 48 months were 88% and 82% (p=0.0093), respectively. The OS for LPHL and cHL patients was 96% and 92%, respectively (p=0.0166). The analysis between LPHL stages showed significant differences in FFTF (p=0.0239). According to a multiple Cox-regression analysis, advanced stage (p=0.0089) and lymphocytes < 8% of white cell count was shown as negative prognostic factors for FFTF and age ≥ 45 years (p=0.008) as negative factor for OS in LPHL patients. Hb value < 10.5 g/dl was shown as negative factor for both, FFTF and OS (p=0.0125, p=0.0002).

Conclusion: This is the largest analysis comparing LPHL and cHL patients. We found differences in FFTF and OS rates between both groups and differences in FFTF between LPHL stages. New treatment protocols for LPHL patients with reduced intensity schedules are to be discussed.
Small Populations of PNH-Type Cells in Aplastic Anemia Patients Are Derived from PIG-A Mutant Stem Cell Clones without Proliferative Advantage.
Authors: Mochizuki Kanako , Sugimori Chiharu , Feng Xingmin , Lu Xuzhang , Reed Gudrun H. , Wittwer Carl T. , Nakao Shinji
Published: April 2020

Several studies revealed that a small number of glycosylphosphatidylinositol anchored protein-deficient cells are detectable in peripheral blood of healthy individuals but these PNH-type cells do not persist for a long time because they are originated from committed progenitor cells rather than primitive stem cells with PIG-A gene mutations. We have studied more than 700 patients with AA for the presence of CD55-CD59- granulocytes and red blood cells using highly sensitive flow cytometry and detected 0.003% or more PNH-type cells in about 50% of patients. In patients whose proportions of PNH-type are very low, the appearance of PNH-type cells may also be transient because they are not derived from true PIG-A mutant stem cell clones. In an attempt to characterize hematopoietic clones from which small populations of PNH-type cells are originated, we studied changes in the proportion of PNH-type granulocytes, clinical findings, and PIG-A mutations over 3–7 years on 52 AA patients (18 males and 33 females). The initial examination revealed less than 0.1% (0–0.075%) PNH-type granulocytes in 21 patients (Group A) and more than 0.1% (0.101–36%) PNH-type granulocytes in 31 patients (Group B). In three (14%) patients of Group A, PNH-type granulocytes became undetectable 2.9, 1.3 and 1.6 years after the initial examination. 0.007 % granulocytes became detectable 2.5 years after the first examination which produced a negative result in one patient. Among the other 17 (81%) patients of Group A, proportions of PNH-type granulocytes remained stable at 0.03–0.41% over 3–6 years. By contrast, 5 (16%) patients of Group B showed an apparent increase in the proportion of PNH-type cells (from 1.46%, 1.63%, 3.31%, 23%, and 36% to 79%, 21%, 83%, 55%, and 78%, respectively) 2 to 4 years after the initial examination. Four of the five developed clinical signs of hemolysis. The proportion of PNH-type cells in Group B patients remained stable in 33 (63%) patients and declined in 12 (23%) patients. When we sorted a small (0.04%) population of PNH-type granulocytes from a patients of Group A and analyzed PIG-A gene, only one point mutation at position 479, C to T was revealed in the exon 2. The finding that the proportion of PNH-type cells remained stable over 3 years in most Group A patients suggests that very low proportions of PNH-type cells are derived from primitive hematopoietic stem cell clones with PIG-A mutation. Among AA patients displaying small populations of PNH-type cells, only those whose proportion of PNH-type cells are more than 1% may be at a high risk of developing hemolytic PNH.
Roles of HLA-DR15 Alleles in the Pathogenesis of Acquired Aplastic Anemia (AA): DRB1*1502 Is in Linkages Disequilibrium with Unique Genes Which Determine Susceptibility to AA.
Authors: Sugimori Chiharu , Mochizuki Kanako , Feng Xingmin , Nakao Shinji
Published: April 2020

It is well known that HLA-DRB1 alleles corresponding to HLA-DR15 such as DRB1*1501 and DRB1*1502 are associated with susceptibility to acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH). However, little is known about how these DRB1 alleles contribute to the development of AA. We previously reported that DRB1*1501 is significantly associated with both an increase in the proportion of PNH-type cells and good response to a immunosuppressive therapy (IST) including antithymocyte globulin (ATG) plus cyslosporine (CsA) or CsA alone, while DRB1*1502, whose DR molecules differ from those derived from DRB1*1501 in only one amino acid, is not associated with either of the two factors. To gain insight into the reason for different contributions to the development of AA by the two DRB1 alleles, we determined DRB1 alleles and the proportion of PNH-type cells of 77 patients with recently diagnosed AA and analyzed the relationship of DRB1 alleles and the presence of increased PNH-type cells with response to ATG plus CsA therapy. Fifty-five of 77 patients (71%) improved with ATG plus CsA therapy. When the factors favorably affecting the response to IST in the AA patients were examined under a multivariate analysis, only the presence of PNH-type cells was significantly associated with the response to IST. Kaplan-Meier curves showed that there were significant differences in the probability of the response to IST between the DRB1*1501 + 1502 − patients and either the DRB1*1501 − 1502 + patients (P <0.01) or the DR15- patients (P =0.01). However, these differences in the probability of response to IST were no longer observed when the probability of response was compared in either the PNH + patients or the PNH − patients (Figure). Although DRB1*1502 was not associated with a good response to IST, the markedly high frequency of the allele in patients aged over 40 (52%) prompted us to study genes in linkage disequilibrium with DRB1*1502 which may determine susceptibility to AA. We compared the frequencies of 30 microsatellite marker alleles on chromosome 6 among three different groups of individuals possessing DRB1*1502 which include 18 PNH + AA patients, 13 PNH − AA patients, and 29 healthy individuals. There were no significant differences in the frequency of any of the polymorphic makers between PNH + AA patients and healthy individuals. On the other hand, one of the microsatellite maker alleles (C1-3-1) in close proximity to HLA-C locus was found at a significantly higher (Pc =0.01) frequency in PNH − AA patients than in PNH + AA patients and healthy individuals. These data suggest that DRB1*1501 contributes to development of AA via immune mechanisms leading to an increase in the proportion of PNH-type cells while DRB1*1502 is involved in the pathogenesis of AA by way of other genes in linkage disequilibrium with this DRB1 allele.

Figure Figure
Clinical Implications of Abnormal Cytogenetics at Diagnosis of Aplastic Anemia.
Authors: Kim Sung-Yong , Lee Jong-Wook , Cho Byung-Sik , Eom Ki-Seong , Kim Yoo-Jin , Lee Seok , Min Chang-Ki , Kim Hee-Je , Cho Seok-Goo , Kim Dong-Wook , Min Woo-Sung , Kim Chun-Choo
Published: April 2020

Because cytogenetic abnormalities of aplastic anemia at diagnosis have been reported fairly infrequently, their clinical implications have not known yet. A retrospective study was performed of the cytogenetics findings and clinical courses in patients with typical morphological and clinical features of aplastic anemia from a single institution for the years 1995 through 2005. The results of chromosome analysis of 610 patients were evaluable. Of the evaluable patients, 584 (95.7 %) had normal karyotypes and 26 (4.3 %) had abnormal karyotypes at diagnosis. The most frequent abnormality was trisomy 8 (n=13) followed by deletion 1q (n=5) and monosomy 7/deletion 7q (n=5). Other chromosome abnormalities were isochromosome 17q (n=1), trisomy 15 (n=1) and monosomy 21 (n=1). Among the 584 patients with typical aplastic anemia and no cytogenetic abnormalities, only two developed MDS/AML during the follow-up period, while 5 (19.2%) of 26 patients with typical aplastic anemia and abnormal cytogenetics subsequently developed MDS/AML. The incidence of secondary MDS/AML was statistically higher in abnormal cytogenetics group compared with normal cytogenetics group (p<0.001). The incidence of secondary MDS/AML was not influenced by immunosuppressive therapy (IST) (p=0.715). The patients with trisomy 8 responded poorly to immunosuppressive therapy (IST) and showed statistically significant lower response rate compared with the patients with other cytogenetics (p=0.033). However, response rates of IST were not statistically different in the patients with normal cytogenetics group and the patients with abnormal cytogenetics other than trisomy 8 (p=1.000). Four patients with abnormal cytogenetics received allogeneic hematopoietic stem cell transplantations (allo-HSCT) with the same conditioning as the patients with normal cytogenetics. Three of them are still alive with normal peripheral blood counts. One of them died of acute GVHD and infection after successful engraftment. Our analysis suggested that cytogenetic abnormalities at diagnosis of aplastic anemia could be a risk factor for development of secondary MDS/AML and the patients with trisomy 8 at diagnosis of aplastic anemia might hardly respond to IST. Outcomes of allo-HSCT for aplastic anemia with abnormal cytogenetics probably are not different compared with aplastic anemia with normal cytogenetics.
Significance of Copy Number Alterations for Molecular Treatment Response in Childhood Acute Lymphoblastic Leukemia.
Authors: Steinemann Doris , Cario Gunnar , Stanulla Martin , Karawajew Leonid , Tauscher Marcel , Weigmann Anja , Göhring Gudrun , Ludwig Wolf-Dieter , Harbott Jochen , Radlwimmer Bernhard , Bartram Claus-Rainer , Lichter Peter , Schrappe Martin , Schlegelberger Brigitte
Published: April 2020

In vivo response to initial therapy, as assessed by determination of minimal residual disease after five and 12 weeks of treatment, has evolved as one of strong prognostic factors in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. It is currently not known if the individual treatment response might be influenced by copy number alterations (CNA) leading to altered gene expression. We compared leukemic genomic profiles of 25 treatment sensitive (MRD-SR) and 25 resistant (MRD-HR) childhood ALL patients by means of high-resolution array-CGH. CNA were found in 46 patients (92%) of both treatment response groups. Microscopic alterations affecting the whole or nearly whole chromosome arm were frequently found, e.g. gain of 21 in 11/50, loss of 9p in 5/50, loss of 8p in 3/50, loss of 20q in 3/50 and loss of 7p in 2/50 or gain of 1q in 2/50. The most significant difference was a gain of chromosome 1q23-qter due to an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (p<0.002). The most frequent CNA in the MRD-HR group were deletions of genomic regions harboring the immunoglobulin genes (Ig), e.g. 2p11.2 in 15 of 25 cases (60%) compared to 7 of 25 in the MRD-SR group (28%) (p=0.045). Combining all Ig loci, significantly more MRD-HR than MRD-SR patients were affected with deletions (17 versus 8 patients, p=0.02). The frequency of other CNA, like loss of 9p21 or gains of 21q, did not differ strongly between the two patient groups. This is the first study evaluating the clinical significance of CNA as detected by array-CGH in childhood ALL and may lead to improved risk classification.