bluuebunny/crossref_metadata_2025 huggingface dataset:
Activated Protein C-Resistance Induced by a Low Molecular Weight Inhibitor.
Authors: Kalafatis Michael , Chiott Rihard E. , Branda Richard F. , Mann Kenneth G.
Published: October 2019

Activated protein C (APC) inactivates factor Va (fVa) following three cleavages in the heavy chain at R506, R306 and R679. Cleavage at R506 precedes cleavage at R306. Cleavage at Arg306 is strictly lipid-dependent and results in total inactivation of the factor Va molecule with dissociation of fragments from the A2 domain from the rest of the molecule. Factor VLeiden is associated with an R→Q substitution at position 506 and is present at approximately 8% of the Caucasian population. The heterozygous presentation of factor VLeiden results in delayed inactivation of factor Va and “APC-resistance” with attendant increased risk of venous thrombosis. However, not all cases of “APC-resistance” are explained by factor VLeiden. We observed “APC-resistance” in a patient displaying heterozygous factor VLeiden, Waldenstrom’s macroglobulinenemia, systemic lupus erythrematosus (anticoagulant) and a history of coronary artery disease. The patient’s plasma resistance to APC inactivation was not repaired by immunodepletion of his factor VLeiden and replacement by normal plasma factor V. Conversely when the patient’s fVa was returned to factor V immunodepleted normal plasma it did not display APC-resistance. Cleavage of the patient’s plasma fVa at R306 was not detected following prolonged incubation of his clotted plasma at 37°C even when 2 nM APC was added following clotting. These data suggested that the APC-resistance observed in the patient was not due to the presence of factor VLeiden, but due to some property which inhibited the lipid dependent cleavage at Arg306. The patient’s plasma was depleted of IgG/IgM and the purified immunoglobulin fraction assessed for inhibition of APC cleavage and inactivation of fVa in a system using purified reagents. The data were compared with the inhibition of fVa inactivation by APC by an IgG/IgM fraction obtained from normal plasma under similar experimental conditions. No inhibition of APC cleavage and inactivation of fVa by the IgM/IgG fraction obtained from either plasma were observed. In addition, the fVa molecule contained in the IgM/IgG-depleted patient plasma was still resistant to cleavage and inactivation by APC. Following dialysis the patient’s plasma lost its ability to inhibit fVa cleavage and inactivation by APC. Overall these studies indicate that inhibition of fVa cleavage and inactivation by APC in the patient’s plasma is caused by a hitherto undescribed metabolite of low molecular weight. The mechanism of action of this metabolite is not yet known, but the evidence suggests that the metabolite interferes with the lipid-dependent cleavage and inactivation of fVa by APC at R306. These data demonstrate the existence of an as yet unknown APC inhibitor of low molecular weight in the plasma of a patient with lupus anticoagulant and severe thrombotic symptoms.
Compound Heterozygosity in the Novel Mutations W157R and T591K in the γ-Glutamyl Carboxylase Gene Causes Hereditary Combined Vitamin K-Dependent Coagulation Factor Deficiency in a Tunisian Family.
Authors: Darghouth Dhouha , Hallgren Kevin W. , Hain Rebecca L. , Mrad Amel , Gharbi Youssef , Maherzi Ahmed , LeRicousse Sophie , Kastally Radhia , Berkner Kathleen L. , Rosa Jean-Philippe
Published: October 2019

Combined deficiency in vitamin K-dependent (VKD) coagulation factors is an autosomal recessive bleeding disorder associated with defects in either the VKD carboxylase which converts Glus to Glas in VKD proteins to render them active or the vitamin K epoxide reductase (VKORC1) which supplies the reduced vitamin K cofactor required for carboxylation. Such defects are rare, and we now report the fourth case of deficiency caused by mutations in the carboxylase gene. The mutations were identified in a two year old Tunisian girl who exhibited impaired function in several VKD procoagulant and anticoagulant factors that was not restored by vitamin K administration. Sequence analysis of the propositus did not identify any mutations in the VKORC1 gene but, remarkably, revealed three heterozygous mutations in the carboxylase gene, D31N, W157R and T591K within exons 2, 4 and 13, respectively. None of these mutations have previously been reported. Family analysis showed that D31N and T591K were coallelic and transmitted by the mother while W157R was transmitted by the father. The mutations were not found in the genomes of 200 normal subjects, ruling out frequent polymorphisms. Mutational analysis indicated wild type activity for the D31N carboxylase. In contrast, the W157R and T591K enzymes had activities that were, respectively, 7% and 0% that of wild type carboxylase, and their compound heterozygosity can therefore account for defective carboxylation. Residues 157 and 591 are both highly evolutionarily conserved, and residue 157 lies within a region previously suggested to be important for carboxylase binding to VKD Glus or propeptide. However, the hydrophobic nature of this region and inability of vitamin K administration to restore VKD protein function alternatively suggests that residue 157 may be important for vitamin K binding.
Response to Treatment of Helicobacter Pylori in Patients with Immune Thromboctypenic Purpura: A Systematic Review and Metaanalysis.
Authors: Bussel James B. , Ostrow Allison , Segal Jodi B.
Published: October 2019

Background: Patients with immune thrombocytopenic purpura (ITP) infected with Helicobacter pylori (H. pylori) are often treated with antibiotics to eradicate infection and raise platelet counts. We aimed to systematically review the evidence regarding this practice to better quantify response rate.

Study Design and Methods: We searched MEDLINE through April 2005, without language restriction; reviewed reference lists and queried experts for articles describing treatment of ITP with antibiotics targeting H. pylori. We included studies of any design involving more than 5 patients. Response rates were tabulated using the authors’ definitions, and pooled using a random effects model. Techniques of meta-regression were used to assess whether patient or study characteristics influenced response rates.

Results: We retrieved 52 potentially eligible studies. Of these, 16 studies met our inclusion criteria. From 10 studies, we had individual level patient data. None of the studies was a controlled trial. The pooled response rate (both partial and complete responses) was 45% [95% confidence interval (C.I.) 32% to 58%]. The number of previous treatments (0–1, 2, 3 or more) influenced the response rate with an 18% (p=0.001) decline in response rate with each higher category; duration of disease did not independently predict response. In analyses of individual patient data, quartile of platelet count upon entry also influenced response rate; 58% of patients in the highest quartile responded compared to 34% in the lowest quartile (p=0.018).

Conclusions: No randomized controlled trial has tested H. pylori eradication with antibiotics as a treatment for ITP. The sixteen observational studies we reviewed were fairly consistent with approximately half of treated patients having a platelet response to therapy. Without control groups, these studies do not provide strong evidence to support practice recommendations. However, given the low toxicity of antibiotic therapy, this treatment may be appropriate for patients who have not demonstrated refractoriness to multiple therapies.

Response Rates by Number of Previous Treatments Response Rates by Number of Previous Treatments
The Effect of Polygonum Multiflorum Extracts on Hematopoiesis and Platelet Production.
Authors: Yang Mo , Huang Wei Zhe , Pong Nga Hin , Liu Ai Guo Liu , Li Chi Kong , Ng Pak Cheung , Fok Tai Fai , Li Karen Kwai Har
Published: October 2019

To date, there is no ideal treatment for thrombocytopenia. We have proposed a possible mechanism of serotonin (5-HT) on megakaryocyte (MK) differentiation and platelet formation (Yang et al, Blood, 2003 suppl). The root of polygonum multiflorum thunb (Heshouwu) is an important ingredient of many commonly used prescriptions in Chinese medicine for promoting blood production. Polygonum multiflorum extracts (PME) also inhibit monoamine oxidase (MAO) and increase the level of 5-HT. Therefore, we hypothesize that polygonum multiflorum may have a promoting effect on thrombopoiesis via inhibition of monoamine oxidase (MAO) to increase 5-HT levels. The objective of this study was to investigate the hematopoietic role of PME in irradiated mice. PME (125 mg/kg/day) and TPO (12.5 ug/kg/day) were given by intra-peritoneal injection daily for 21 days starting from the day after irradiation (4 Gy). Peripheral blood platelets, white blood cells (WBC), and red blood cells (RBC) were analyzed from PME, TPO, and vehicle control groups on day 0, 7, 14 and 21. On day 21, the mice were sacrificed and bone marrow cells were harvested for CFU-MK, CFU-GM, BFU-E, CFU-GEMM and CFU-F (fibroblastoid) assays (n=8). We also investigated the in vitro effect of PME on CFU-F formation. Our results showed that PME enhanced the recovery of platelets, WBC, and RBC counts. Moreover, PME also promoted CFU-MK (30 ± 8 vs 15 ± 3 colonies/2 x 105 cells, p<0.01), CFU-GM (38 ± 7 vs 28 ± 7 colonies/2 x 105 cells, p<0.05), BFU-E (19 ± 3 vs 12 ± 4 colonies/2 x 105 cells, p<0.05), and CFU-F formation (36 ± 11 vs 23 ± 7 colonies/2 x 106 cells, p<0.01). Similar results were obtained in TPO-treated group. In in-vitro study, we further analyzed the effect of PME (0–500 ug/ml) on mouse CFU-F formation. The results showed that PME at 100–500 ug/ml significantly enhanced CFU-F formation (p<0.05, n=6). Our studies showed that PME enhances thrombopoiesis in vivo and the growth of bone marrow stromal cells in vitro. Therefore, we speculate that the thrombopoietic activity of PME may be mediated via promoting the bone marrow stromal cells. Although TPO has been effective as an agent for the recovery of platelet production after the onset of thrombocytopenia, long-term clinical usage of TPO may induce potential side effects such as thrombosis. Here we reported that the effect of PME is comparable with that of TPO on hematopoiesis and the production of platelets.
Monoclonal IgG Specific for the CD44 Antigen Mimics the Action of IVIg in the Amelioration of ITP.
Authors: Song Seng , Crow Andrew R. , Siragam Vinayakumar , Freedman John , Lazarus Alan
Published: October 2019

Previous work in our laboratory has shown that both IVIg and “anti-D like” erythrocyte-reactive antibodies ameliorate immune thrombocytopenia (ITP) in a murine model. However, they appear to function through different mechanisms: IVIg is dependent upon the inhibitory Fc receptor, FcγRIIB in the amelioration of ITP, but the anti-erythrocyte antibodies function independently of FcγRIIB expression. We have also demonstrated that anti-erythrocyte antibodies down-modulate the expression of the activating Fc receptor, FcγRIIIA in splenic macrophages during the amelioration of ITP. A monoclonal antibody to CD44, cell surface protein involved in cell homing, cancer progression and inflammation, can also ameliorate murine ITP. Elucidation of the mechanisms of action of anti-CD44 antibodies in the amelioration of ITP would be critical to the successful application of this potential therapy. In this report, two monoclonal antibodies against CD44, KM114 and IM7 were studied in C57BL/6 and FcγRIIB−/− mice to assess their therapeutic activity in treating ITP. Both KM114 and IM7 bound well to splenic cells from all mice tested. Neither KM114 nor IM7 demonstrated any in vitro anti-idiotypic activity which could neutralize the binding of the thrombocytopenia-inducing anti-platelet antibody. At the dose that protected against thrombocytopenia, KM114 did not mediate RES blockade. In contrast, IM7, which did not at all protect against thrombocytopenia, blocked RES. Since FcγRIIB has been documented to play a key role in the function of IVIg in the amelioration of ITP, we questioned the significance of it in the function of KM114. Surprisingly, KM114 was not able to ameliorate ITP in mice genetically deficient in FcγRIIB. These results suggest that, like IVIg, monoclonal antibody against CD44 mediates amelioration of murine thrombocytopenia in a manner dependent upon the inhibitory FcγRIIB rather than via RES blockade.
Inhibition of IL-1β and TNFα Gene Expression by IVIg or Anti-Red Blood Cell Antibodies in a Mouse Model of ITP.
Authors: Aubin Eric , Bazin Renée , Lemieux Réal
Published: October 2019

Plasma-derived human IgG (IVIg) have been used for more than 2 decades for the treatment of several autoimmune diseases such as ITP, even if their mechanism of action is still not fully understood. Recently, several studies with IVIg-treated patients have shown that IVIg can modulate the serum level of both pro-inflammatory and anti-inflammatory cytokines. In ITP, it is known that the destruction of autoantibody-coated platelets occurs mainly in the spleen. We thus used a mouse model of ITP to evaluate the modulatory effects of IVIg on the expression of several cytokine genes in the spleen of the animals by quantitative RT-PCR. The results showed that the induction of platelet destruction following injection of monoclonal anti-platelet antibodies (MWReg30) in BALB/c mice did not change the expression level of IL-1ra and IL-10 (anti-inflammatory cytokines) and IL-6, IFNγ and MCP-1 (pro-inflammatory cytokines). In contrast, MWReg30-treated mice showed an increase in IL-1β and TNFα mRNA expression by up to 4.2 and 2.1 fold respectively compared to control mice. Treatment of thrombocytopenic mice with IVIg (2g/Kg) prevented the increase of IL-1β mRNA but not TNFα mRNA. In parallel, the effect of anti-red blood cell (anti-RBC) antibodies, used to mimic anti-D treatment of ITP patients, was analysed. The results were similar to those observed with IVIg for IL-1β mRNA, but in contrast to IVIg, anti-RBC treatment also prevented the increase in TNFα gene expression, suggesting that the mechanisms of action of IVIg and anti-D in ITP treatment are different, in agreement with previous studies. In summary, we showed that IVIg and anti-RBC treatments attenuate the expression of important pro-inflammatory cytokines. IL-1β and TNFα are known to increase the phagocytic activity of macrophages and also to enhance the production of antibodies by B cells. Therefore, IVIg and anti-D could contribute to the prevention of platelet destruction in ITP by limiting the activation of phagocytes and the production of autoantibodies. This study was assisted by a grant from the Bayer/CBS/HQ partnership Fund to RB and RL.
BLIMP-1 Is a Target of Cellular Stress and Downstream of the Unfolded Protein Response.
Authors: Doody Gina , Stephenson Sophie , Tooze Reuben
Published: October 2019

Human B lymphocyte-induced maturation protein-1 (BLIMP-1) was originally described as a repressor of the interferon-beta response to viral infection. Subsequently, the murine orthologue was identified as a regulator of plasma cell differentiation. The involvement of BLIMP-1 in hemopoietic differentiation is not restricted to the B-cell lineage as BLIMP-1 is induced during differentiation of myeloid progenitors. During in vitro macrophage and plasma cell differentiation the expression of BLIMP-1 is cytokine driven. However, the BLIMP-1 response to virus infection can be reproduced by transfection with double-stranded RNA (dsRNA), indicating that BLIMP-1 is a target of dsRNA responsive signaling pathways. A central regulator of the intracellular response to viral infection is the interferon-inducible double-stranded RNA activated kinase, PKR. PKR belongs to a family of kinases that phosphorylate the eukaryotic translation initiation factor 2-alpha (eIF2α) and activate common downstream signaling pathways. PERK, the endoplasmic reticulum (ER) PKR-homologue is activated during the unfolded protein response (UPR), a stress response involved in both macrophage activation and terminal B-cell differentiation. This suggested the hypothesis that BLIMP-1 may represent a shared target of signaling pathways in the response to cellular stresses such as virus infection and the UPR. In this study we demonstrate that BLIMP-1 is rapidly upregulated during the UPR in human myeloid and B-cell lines. This response is conserved in primary murine macrophages, in which mimics of physiological stress and classical activation stimuli also induce Blimp-1. During the UPR, BLIMP-1 mRNA is induced at the level of transcription, with enhanced recruitment of RNA polymerase II to the BLIMP-1 promoter. Furthermore the stress response is limited to induction of BLIMP-1α mRNA and does not affect levels of an alternate transcript encoding a truncated protein, BLIMP-1β. The common induction of BLIMP-1 mRNA by stimuli which trigger the UPR supports the hypothesis that BLIMP-1 is a target of the eIF2α kinase family. To test this hypothesis directly, we employed a dominant negative mutant PERK. Our data demonstrate that the BLIMP-1 response to UPR stress is dependent on an intact PERK signaling pathway. Collectively our results provide evidence for a novel link between cellular stress, the eIF2α kinase family and a regulator of differentiation in macrophages and B-cells.
Reciprocal Interaction of Monocytes and NK Cells by Activation-Induced Expression of Ligands for the Activating Immunoreceptor NKG2D.
Authors: Krusch Matthias , Kloss Mercedes , Peterfi Andrea , Kumbier Ingrid , Kanz Lothar , Kiener Peter A. , Salih Helmut R.
Published: October 2019

Reciprocal Interactions of NK cells with Dendritic Cells (DC) can induce activation of NK cells, maturation or lysis of DC and influence subsequent adaptive immune responses. However, little is known about the interaction of peripheral blood monocytes with NK cells, especially regarding involved immunoregulatory surface molecules. Here we report that monocytes express ligands for the activating immunoreceptor NKG2D expressed on NK cells upon treatment with various stimuli. Incubation of monocytes with TNF, GM-CSF, IFN-g and various TLR ligands (LPS, Pam3Cys, R848, PolyI:C) induced surface expression of the NKG2D ligands (NKG2DL) MICA and to a lesser extent MICB, but no relevant changes of ULBP molecules, as determined by FACS analysis. Expression was confirmed by quantitative PCR analysis of NKG2DL mRNA induction. To elucidate the functional consequences of NKG2DL expression on monocytes for NK cell functions we performed coculture assays of monocytes and autologous NK cells. NKG2DL expression on stimulated monocytes lead to a significant induction of IFN-g release into the culture supernatant by NK cells as determined by ELISA. This IFN-g release was blocked by addition of a NKG2D-Ig fusionprotein, but not by isotype control demonstrating that the induction of NK cell IFN-g production was in fact specifically due to NKG2DL expression on monocytes. Since both monocytes and NK cells rapidly migrate to sites of inflammation, and monocytes display a high plasticity regarding their function and maturation which is influenced by IFN-g, our data indicate that NKG2DL expression on monocytes might not only mediate reciprocal activation of NK cells and monocytes but also might influence other components of the innate and adaptive immune system and thereby determine the course of subsequent immune reactions.
Platelet Binding to Monocytes Induces Changes in Integrin Functionality Promoting Monocyte Adhesion and Transendothelial Migration. Do Platelets Migrate Along?.
Authors: Martins Paula da Costa , van Gils Janine M. , Mol Anita , Hordijk Peter L. , Zwaginga Jaap-Jan
Published: October 2019

Monocyte adhesion to and transmigration across the endothelium are essential steps in atherogenesis. We have shown that the adhesive interactions between monocytes and the activated endothelium are increased when platelets bind to the monocyte surface and form platelet-monocyte complexes (PMC). PMC formation is dependent on interactions between platelet-displayed P-selectin and PSGL-1 on the monocyte surface. To better understand the effect of platelet binding on the capacity of monocytes to adhere to activated endothelium the P-selectin-PSGL-1 interaction - induced changes in integrin functionality were studied. The binding of platelets to monocytes via P-selectin-PSGL-1 interactions was shown to increase expression and activity of α4 beta;1 - and αM β2 - integrins which, resulted in increased monocyte adhesion to ICAM-1, VCAM-1, fibronectin and subsequently to stimulated endothelial cells. Platelet binding also induced monocyte migration (up to a 3-fold increase), compared to monocytes without platelets on their surface. To investigate the role of platelets in this process we determined the fate of platelets (within the PMC) during monocyte transendothelial migration. After forming PMC by mixing freshly isolated and fluorescently labeled platelets and monocytes, PMC were seeded on endothelial cells cultured on top of a fibrin gel. The cells were allowed to migrate across the endothelial layer into the gel where afterwards the position of platelets and monocytes was analyzed by confocal laser scanning microscopy. We found that the platelets were retained at the endothelium, suggesting that they detach from the PMC and are left behind on the endothelial layer upon monocyte transendothelial migration. In line with this we observed that the monocytes that were in the fibrin gel, underneath the endothelial layer, did not carry any platelets. This was confirmed by PMC migration over endothelial layered transwell filters since almost only monocytes were found in the lower compartment after migration. After testing different migration barriers, platelets seem to be shed from the monocyte surface upon monocyte migration by mechanical stress rather than only endothelial interaction. Our data suggest that monocytes bound to platelets are in a higher state of activation and have an increased atherogenic capacity. Furthermore, platelets seem to mainly play a role in the monocyte recruitment to the endothelium because once the monocytes cross the endothelial layer, platelets detach from their surface. Altogether, our findings, by showing that PMC have a strong atherogenic capacity, might be helpful in finding new therapeutic ways to prevent atherosclerosis and inflammation.
Mutation of Mouse MAYP/PSTPIP2 Causes a Macrophage Autoinflammatory Disease.
Authors: Chitu Violeta , Grosse Johannes , Marquardt Andreas , Hanke Petra , Schmittwolf Carolin , Zeitlmann Lutz , Schropp Patricia , Barth Bettina , Yu Philipp , Paffenholz Rainer , Stumm Gabriele , Nehls Michael , Stanley E. Richard
Published: October 2019

Macrophage actin-associated tyrosine phosphorylated protein (MAYP), a Pombe Cdc15 Homology (PCH) family protein also known as proline serine threonine phosphatase-interacting protein 2 (PSTPIP2), is involved in the regulation of macrophage motility (Chitu et al., 2005). Mutations in a closely related gene, PSTPIP1/CD2 binding protein 1 (CD2BP1) cause a dominantly inherited autoinflammatory disorder known as PAPA syndrome. A mutant mouse obtained by chemical mutagenesis exhibited an autoinflammatory disorder characterized by macrophage infiltration and inflammation leading to osteolysis and necrosis in paws and necrosis of ears. Positional cloning of this recessive mutation, termed Lupo, identified a T to A nucleotide exchange leading to an amino acid substitution (I282N) in the sequence of MAYP. MaypLp/Lp disease was transferable by bone marrow transplantation and developed in the absence of lymphocytes. Consistent with the involvement of macrophages, lesion development could be prevented by the administration of chlodronate liposomes. MAYP is expressed in monocytes/macrophages and a Mac1+ sub-fraction of granulocytes. LPS stimulation increases its expression in macrophages. Due to instability of the mutant protein, MAYP expression is reduced 3-fold in MaypLp/Lp macrophages and upon LPS stimulation does not rise above the level of unstimulated wt cells. MaypLp/Lp mice expressed elevated circulating levels of IL-4, Rantes, TGF-beta and MCP-1 and in vitro their macrophages exhibit altered cytokine production. These studies suggest that MAYP plays an anti-inflammatory role in macrophages.
Down-Regulation of Cellular Vascular Endothelial Growth Factor (VEGF) Levels Induces Differentiation of Leukemic Cells into Leukemic-Dendritic Cells in Patients with Acute Myeloid Leukemia.
Authors: Kang Hyun-Kyu , Park Jung-Sun , Choi Bo-Hwa , Nguyen Thanh-Nhan , Zhu Xiao-Wei , Cho Duck , Nam Jong-Hee , Kim Young-Jin , Kim Hyeoung Joon , Lee Je-Jung
Published: October 2019

Vascular endothelial growth factor (VEGF), produced by most tumors, not only plays an important role in tumor angiogenesis but also can inhibit the maturation of dendritic cells (DCs) from hematopoietic progenitors. In this study, we investigated the relationship between the clinical characteristics of patients with acute myeloid leukemia (AML) and the generation of DCs from the leukemic cells, and found that cellular VEGF level in leukemic cells affected the generation of leukemic-DCs in vitro. Leukemic-DCs were successfully generated from 16 of 21 AML patients, and generated leukemic DCs exhibited high level of HLA-DR and CD54 expression, and intermediate level of CD80, CD86, CD83, and CD1a expression. Leukemic-DCs also were able to stimulate allogeneic T cell proliferation and cytotoxic activities against autologous leukemic cells. The failure of generation of leukemic-DCs from leukemic cells in some AML patients was related with its cellular VEGF levels. The leukemic cells that failed to generate leukemic-DCs had significantly higher cellular VEGF levels than that of generated leukimic-DCs (p = 0.005), and neutralizing of VEGF with anti-VEGF Ab inhibited the cellular VEGF level at the transcription level as determined by RT-PCR analysis, and at the protein level determined by West blotting and ELISA. Inhibition of cellular VEGF levels induced the generation of leukemic-DCs determined by increased levels of surface markers, namely, CD40, CD80, CD86, and HLA-DR. Moreover, inhibition of cellular VEGF levels also increased IL-12 production and allosimulatory capacity of leukemic-DCs, suggesting functional differentiation of leukemic-DCs. Interestingly, cellular VEGF levels also are inversely related to the achievement of complete remission for induction therapy (p = 0.02). Taken together, these results suggest that cellular VEGF levels during the differentiation of AML cells to leukemic-DCs are a predictive marker of the positive response of the AML patients to the remission-inducing therapy, and down-regulation of cellular VEGF levels can be one target of cancer immunotherapy by generating of sufficient number of DCs in AML patients.
Proteasome Inhibitors Affect the Function of Human Dendritic Cells and Induce Caspase-Mediated Apoptosis.
Authors: von Schwarzenberg Karin , Nencioni Alessio , Bringmann Anita , Kanz Lothar , Patrone Franco , Ballestrero Alberto , Brossart Peter
Published: October 2019

Proteasome inhibitors (PI) show antitumor activity against a broad spectrum of human malignancies both in vitro and in vivo. Yet, the consequences of exposure to these compounds on the immune system still have to be clearly determined. In the present study, we have investigated the effect of the proteasome inhibitors on human monocyte-derived dendritic cells (DCs). Exposure to PI results in a reduced activation induced DC maturation and cytokine production, inhibition of their migratory capacity and impaired ability to stimulate T-cell responses. These functional and phenotypic alterations were paralleled by a decreased phosphorylation of the MAP kinase member ERK1/2 while not affecting p38. Furthermore, incubation of DC with bortezomib inhibited the expression of nuclear localized transcription factors that were shown to be important for DC differentiation and function like IRF3, Rel-b and c-rel. Addition of PI to culture medium induced apoptosis of differentiated DCs and strongly reduced the yield of viable DCs when given to monocytes before differentiation to DCs was induced. DC apoptosis was accompanied by caspase activation as detected by caspase-3 and PARP cleavage. Cytochrome c cytosolic relocalization was detectable following exposure to bortezomib and was not prevented by caspase inhibition. This points to the mitochondrial damage as to an upstream event in DC apoptosis via proteasome inhibition. While not affecting Bcl-2 levels, bortezomib was found to promote Bax upregulation in DCs, thus providing a possible explanation for mitochondria dysfunction in response to this compound. In conclusion, this study shows that besides the inhibition of Nf-kB bortezomib is affecting several other pivotal signal transduction pathways in human cells and suggests that inhibition of DC function and induction of apoptosis in DCs may represent a mechanism by which bortezomib can affect the immune responses in patients treated with this compound.
Short-Term Effect of Oral Treatment with Miglustat in Type 1 Gaucher Disease.
Authors: Giraldo Pilar , Alonso Dora , Acedo M. , Franco Rafael , Barez Alberto , Corrales Alejandro , Serrano D. , Roldan Vanessa , Latre Paz , Alfonso Pilar , Pocovi Miguel
Published: October 2019

Background and objective Miglustat (Zavesca®), an inhibitor of the enzyme glucosylceramide synthase, is an orally active molecule that has been shown to be a safe and effective therapy for type I GD. Here we report on our experience of miglustat monotherapy for the treatment of type 1 Gaucher disease (GD) patients in Spain.

Design and Methods: We designed a prospective, open-label study of 21 patients with mild-to- moderate type 1 GD, to investigate the efficacy and tolerability of miglustat over a 12-month period in therapy-naïve patients and in patients who have previously received enzyme replacement therapy (ERT). Clinical assessments, including haematological parameters and organomegaly, were carried out prior to and after 6 and 12 months of miglustat therapy. In addition, the results were compared with data analysed retrospectively from 40 patients with type 1 GD who had been treated for 6 months with ERT. The data following 6 months of miglustat therapy are presented here.

Results At present, 16 patients have completed 6 months of miglustat treatment. All patients with anaemia had improved haemoglobin concentrations, and in the treatment-naive group a mean increase of 1 g/dl was observed. Platelet counts improved in patients with thrombocytopenia and were maintained in patients with counts within normal limits at baseline. Chitotriosidase activity was maintained in switched patients and decreased in naïve patients. Miglustat was well tolerated, and the efficacy after 6 months therapy was comparable to that observed in the clinical trials and in patients treated with ERT for 6 months. Interpretation and conclusions In our experience, properly selected patients with mild to moderate type I GD had a satisfactory clinical and biochemical response to 6 months of miglustat therapy. The therapy was well tolerated, and clinical benefits were comparable to those obtained with ERT.
Mild Renal Impairment as a Potential Prognostic Factor in Patients with Solid Tumors and Hematological Malignancies.
Authors: Kleber Martina , Bauchmüller Kris , Ihorst Gabriele , Koch Bernd , Engelhardt Monika
Published: October 2019

The outcome of cancer patients (pts) is influenced by a variety of coexisting conditions. Renal impairment has been shown to be one major risk factor in a number of diseases, such as myocardial infarction (MI), and is associated with dismal clinical outcome. However the influence of milder degrees of renal disease is less well defined, especially not yet in pts with solid tumors (ST) or hematological malignancies (HM). Between 8/2004-3/2005, we analyzed 167 ST and HM pts. Of 108 male and 59 female pts, 94 had ST and 73 HM. Their median age was 59 years (range; 18–91). Local vs. advanced (=metastatic) disease was present in 36 vs. 131 pts, respectively. Disease specific parameters, such as tumor diagnoses, sex, age, local vs. advanced disease, performance status (Karnofsky index=KI), number of concurrent diagnoses, cardiovascular risk factors such as body-mass index (BMI), presence of stroke or transient ischemic attack (TIA) and comorbidities (Satariano index=SI) were determined, also serum creatinine (creat), cystatin C (cC) and ProBNP (BNP). The glomerular filtration rate (GFR) was estimated by use of the ’Modification of Diet in Renal Disease’ (MDRD) equation (NEJM2004; 351:1285). The SI included heart diseases, diabetes, cancer, respiratory, gallbladder, and liver deteriorations. Pts were compared within GFR-, cC-, creat- and comorbidity-groups. Despite a median of 3 (range; 0–8) concurrent diagnoses, the KI in all pts was almost normal with a median of 90% (range; 30–100) and the median SI was 1 (range; 0–3). The median MDRD of all pts was 88ml/min/1.73m2 (14–240), and groups with GFR ≥75 (I), 60–74.9 (II), 45–59.9 (III) and <45 (IV) consisted of 105, 26, 23 and 13 pts, respectively. Of note was that pts showing a decreased GFR (<75ml/min/1.73m2) were older, had a significantly higher BMI (p=0.013), increased number of concurrent diagnoses (p=0.0486), and elevated creat- (p<0.0001), cC- (p<0.0001) and BNP-levels (p<0.0001; all Wilcoxon 2-sample tests). GFR - as compared to creat- or cC-levels - demonstrated this correlation best. Renal impairment (determined by elevated creat levels) was associated with increased odds ratios of 1.4 (95%CI 0.5–4.2), 1.5 (95%CI 0.7–3.2), 2.3 (95%CI 0.1–38.3) and 7.2 (95%CI 1.8–28.3) for comorbidities, such as diabetes, hypertension, apoplex and MI, respectively, which for the latter proved to be of statistical significance (p=0.0044). These observations indicate that grouping pts according to their renal function may be one way of determining specific risk groups. Comparing creat-, cC- and GFR-levels, the latter seems most valuable for determining mild or moderate renal impairment. Especially prior MI and elevated BNP levels will be studied further, since both seem closely associated with risks for renal impairment. Ongoing studies will determine, if mild renal disease is also a risk factor for adverse clinical outcome in ST and HM pts.
A Distinct Molecular Signature of Purified Cancer Stem Cells: Evidence for a Hematopoietic Stem Cell Origin of the 5q- Syndrome.
Authors: Nilsson Lars , Edén Patrik , Olsson Eleonor , Månsson Robert , Åstrand-Grundström Ingbritt , Strömbeck Bodil , Tehranchi Ramin , Theilgaard-Mönch Kim , Hast Robert , Hellström-Lindberg Eva , Samuelsson Jan , Bergh Gösta , Johansson Bertil , Sigvardsson Mikael , Borg Åke , Jacobsen Sten-Eirik W.
Published: October 2019

Herein we present the first global gene expression profile of purified cancer stem cells (CSCs). Constituting 0.08% of bone marrow cells, CD34+CD38−Thy-1+ cells represent the CSCs in 5q− myelodysplastic syndromes. The global gene expression pattern of purified 5q− CD34+CD38−Thy-1+ stem cells was overall very similar to the global gene expression of normal hematopoietic stem cells and not progenitor cells, supporting that 5q− stem cells originate in and outcompete normal CD34+CD38−Thy-1+ stem cells. However, a few distinct differences in gene expression distinguish 5q− from normal stem cells and might therefor be of potential pathogenetic importance. BMI1, a critical regulator of self-renewal is up-regulated in most 5q− patients stem cells, which could explain the obvious advantage of the 5q− stem cells over normal stem cells. The myeloid transcription factor CEBPα was clearly down-regulated in 5q− CD34+CD38+Thy-1− progenitors compared to normal progenitors, consistent with the typical deficient myeloid differentiation seen in 5q− syndrome. In contrast, CEBPα was rather up-regulated in 5q− stem cells from several patients. Thus, these studies demonstrate the importance of specifically identifying, characterizing and eventually targeting CSCs.
Mechanism of Altered Nucleo-Cytoplasmic Traffic of Nucleophosmin in Acute Myelogenous Leukemia Carrying Exon-12 NPM Mutations (NPMc+ AML).
Authors: Falini Brunangelo , Bolli Niccolo , Shan Jing , Martelli Maria Paola , Nicoletti Ildo , Liso Arcangelo , Pasqualucci Laura , Rosati Roberto , Roti Giovanni , Diverio Daniela , Mannucci Roberta , Cazzaniga Giovanni , Biondi Andrea , Saglio Giuseppe , Pane Fabrizio , Lo-Coco Francesco , Pelicci Pier-Giuseppe , Martelli Massimo F. , Gu Wei , Mecucci Cristina
Published: October 2019

We recently found that about one-third of adult AML (60% of all AML with normal karyotype) display aberrant cytoplasmic expression of nucleophosmin (NPM) which is due to mutations occurring at the exon-12 of NPM gene (Falini B, et al., Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype..N Engl J Med2005; 352: 254–266). Hereby, we clarify the molecular mechanism underlying cytoplasmic NPM accumulation that yet remained to be elucidated. AML-associated mutated NPM alleles encode abnormal NPM proteins (25 mutants so far identified) which have acquired at the C-terminus a nuclear export signal-(NES) motif and lost at least one of tryptophan residues 288 and 290 which determine nucleolar localization. Both alterations are crucial for mutant NPM export from nucleus to cytoplasm. In fact, the cytoplasmic NPM accumulation is blocked by leptomycin-B and ratjadones which are specific inhibitors of exportin-1/CRM1, and by re-insertion of tryptophan residues 288 and 290, which respectively relocate NPM mutants in the nucleoplasm and nucleoli. Thus, for cytoplasmic accumulation of NPM to occur, the NES motif and tryptophan mutations must act in concert. Possibly, when NPM mutans enter the nucleus by virtue of their nuclear localization signals (NLS), their capability to bind nucleoli must be hindered at least partially to become a CRM-1 target. Specific antibodies anti-NPM mutant proteins showed that the mutants localized exclusively in the cytoplasm and recruited in that site the wild-type NPM protein which is physiologically located in the nucleoli. These findings suggest that the NPM mutants may interfere with the functions of wild-type NPM and possibly contribute to leukemogenesis. Immunostaining of 393 AML cases using anti-NPM monoclonal antibodies predicted the presence of NPM exon-12 mutations in all 191 NPM-cytoplasmic positive cases. This finding is consistent with the fact that, despite genomic heterogeneity, all NPM mutants contain a NES motif which, in the presence of altered tryptophans, promotes their rapid export from the nucleus to the cytoplasm. The immunohistochemical test is diagnostically relevant since it can be used as simple first-step procedure in molecular-genetic characterization of AML and as a surrogate for mutational analysis in selected cases. These findings are also clinically relevant since cytoplasmic NPM/NPM mutations are predictors of good response to induction therapy and favourable prognosis in AML with a normal karyotype.
Array-CGH and Gene Expression Profiling Based Molecular Characterization of Myeloid Leukemia Cell Lines.
Authors: Rucker Frank G. , Sander Sandrine , Dohner Konstanze , Dohner Hartmut , Pollack Jonathan R. , Bullinger Lars
Published: October 2019

Myeloid leukemias are characterized by cytogenetic and molecular-genetic aberrations resulting in altered gene expression. Nevertheless, so far still little is known regarding the underlying mechanisms of leukemogenesis. To model and investigate the different aspects of leukemia pathogenesis a widely accepted approach is to use immortalized leukemia cell lines. While these provide powerful tools for the investigation of gene function, the dissection of signal transduction pathways and the analysis of drug effects, to our knowledge only few studies have addressed the question whether hematopoietic cell lines represent reliable model systems.

In order to improve the molecular characterization of these model systems we therefore analyzed 18 myeloid leukemia cell lines using DNA microarray technology. To determine the secondary aberrations acquired in addition to their characteristic primary cytogenetic aberrations during numerous passages in vitro, we first analyzed all cell lines by array-CGH (comparative genomic hybridization). Using a BAC/PAC platform with an average resolution of ~ 1.5 Mb across the entire genome we identified recurrent losses and gains, as well as high level amplifications like e.g. an amplification in 4q12 (Kasumi1) and in 8q24 (HL60). The parallel analysis of gene expression using a whole genome cDNA microarray platform helped to further delineate potential candidate genes in the affected regions (e.g. overexpression of KIT in the 4q12 and MYC in the 8q24 amplicon). Furthermore, unsupervised hierarchical cluster analysis revealed distinct gene signatures pointing towards dysregulated transcriptional networks. Comparison of our findings with acute myeloid leukemia patient data (Bullinger et al. 2004) showed the signatures underlying characteristic cytogenetic aberrations like e.g. t(15;17) were conserved in cell lines. Interestingly, these signatures were also quite robust as they displayed a highly significant correlation with published cell line data profiled on a different DNA microarray platform in a different laboratory.

Thus, our analyses demonstrate that cell lines exhibit conserved signatures correlating with the primary balanced cytogenetic aberrations, and that most cell lines even when grown and analyzed under different conditions provide highly robust signatures. Therefore, our refined molecular characterization of myeloid cell lines supports the utility of cell lines as powerful model systems and provides additional insights into the molecular mechanisms of leukemogenesis.
Risk Factors of Outcomes after Haploidentical Hematopoietic Stem Cell Transplants for Children with High Risk Acute Leukaemia. A Survey on Behalf of the EBMT.
Authors: Cornish Jaqueline , Klingebiel Thomas , Labopin Myriam , Darbyshire Phillip J. , Hough Rachel , Locatelli Franco , Niethammer Dietrich , Dini Giorgio , Rocha Vanderson
Published: October 2019

In the absence of an HLA identical donor, T-cell depleted haploidentical hematopoietic stem cell transplantation (HSCT) is an alternative option to treat children with high risk or relapsed acute leukaemia. However very few data is available in a large series of children. With the aim to study risk factors of outcomes we have analyzed 196 children (<16 years old) with ALL (n=131) or AML (n=65) transplanted with a T-cell depleted bone marrow (n=18) or peripheral blood related haploidentical HSCT from 1995 to 2004 in Europe. The median age was 8 years and median follow-up 22 months. In the AML group, 13 (20%) children were transplanted in CR1, 22 (34%) in CR2 and 30 (46%) in advanced phase and in ALL group, 28 (21%) in CR1, 74 (56%) in CR2 and 81 (62%) in more advanced phase. The majority of the patients did not receive drugs for GVHD prophylaxis and all received myeloablative conditioning (61% of TBI). Cumulative incidence with competing risk and KM estimates were used to calculate outcomes probabilities. The median days of neutrophil recovery was 14 days (4–72) and 85% of patients had signs of engraftment. Acute GVHD II–IV was observed in 17% of the patients (8% had grade III–IV). Two-years overall LFS, relapse incidence and TRM were 27±4%, 43±3%, 30±3%, respectively. Patients transplanted with AML or ALL had similar outcomes. LFS was 28±6%for AML and 27±4% for ALL. Among the risk factors analysed only the disease status at transplantation was associated with LFS and relapse incidence. Outcomes are listed below according to disease status at transplant.

Outcomes at two years CR1 (n=41) CR2 (n=74) Advanced (n=81) p value Transplant related mortality 32+/−8% 26+/−5% 33+/−5% 0.44 Relapse 32+/−8% 40+/−6% 51+/−6% 0.03 Leukaemia free survival 36+/−8% 34+/−6% 16+/−4% <0.0001

In fact, in a multivariate analysis for LFS and relapse only patients transplanted in remission had better LFS and decreased relapse incidence compared with non remission patients (p<0.001 and p=0.006, respectively). No risk factor was found to be associated with TRM. Most frequently, causes of death were relapse (60%) or infections (22%). In conclusion, haploidentical HSCT is an alternative option to treat children with high risk acute leukaemia in the absence of HLA identical donor.
Effects of Gossypol Acetate on Proliferation and Apoptosis in Lymphoblastoid Cell Line and Primary ALL and CLL Cells.
Authors: Zhao Yongqiang , Cheng Wei , Hua Baolai , Wang Shujie , Yang Dajun
Published: October 2019

Gossypol is a polyphenolic compound extracted from the cotton plant of Malvaceae family. It was originally demonstrated as a male antifertility agent. In the 1960’s, its anticancer efficacy was identified. In the last few years, gossypol has been approved to have antiproliferative and apoptosis-inducing effects on some kinds of cancer cell lines in vitro. Very recently, it has been showed that gossypol is a potent small-molecule inhibitor of Bcl-2 and Bcl-XL proteins. It can bind to Bcl-2/Bcl-XL and block the heterodimerization of Bcl-2/Bcl-XL with proapoptotic proteins, such as Bax, Bak and Bad. Such a blocking of antiapoptotic-proapoptotic protein heterodimerization in turn may inhibit the antiapoptotic function of Bcl-2/Bcl-XL, and induce apoptosis of cancer cells. Overexpression of Bcl-2 has been observed in B-cell lymphoma, B-ALL, B-CLL and correlates closely with the occurrence, progress, recurrence and multidrug-resistance of these malignancies. In this study, we found that gossypol acetate was able to inhibit the proliferation and survival of Raji cells in vitro at concentrations of more than 5 μmol/L. The effect was dose and time dependent. IC50 were 11.9±1.2 μmol/L at 24h and 6.8±0.4 μmol/L at 72h, which were 7.1 and 9.1 folds lower than that of normal MNCs, respectively. Gossypol acetate can remarkably increased pro-apoptosis activity of dexamethasone in Raji cells. Cell cycle analysis by flow cytometry (FCM) indicated that gossypol acetate could induce G0/G1 arrest. Apoptosis was demonstrated both morphologically by Wright-Giemsa staining, Hoechst 33342 staining and by DNA ladder formation in agarose-gel electrophoresis. By determination the percentage of subdiploid amount of DNA, apoptosis was quantified. The result showed that gossypol acetate could increase the percentage of cells with fragmented DNA in time and dose dependent manners at the concentrations of more than 1μmol/L. The fraction of annexin V+/PI− cells was also increased with dose. Colorimetric assay showed that an activation of caspase-3 was seen at 12h and peaked at 24h at the concentration of 25μmol/L. Protein expression of Bcl-2 decreased 13.6±3.8% at 12h and 69.5±6.2% at 24h. Gossypol acetate also had apoptosis-inducing effects in primary leukemia cells from patients with ALL (n=7) and CLL (n=3). The concentration inducing apoptosis in CLL cells was higher than that in ALL cells. It was concluded that gossypol acetate could inhibit the proliferation and induce the apoptosis of Raji cells and primary cultured ALL and CLL cells in a dose and time dependent manner. It seems that down-regulation of Bcl-2 protein expression may play a role in the apoptosis.