bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal Antibodies to a VWF-A2 Decapeptide with the C-Terminal Residue Tyr1605, Generated by ADAMTS13 Cleavage, Develop a Highly Sensitive ELISA for Its Activity and Characterize Upshaw-Schulman Syndrome.
Authors: Kato Seiji , Matsumoto Masanori , Matsuyama Tomomi , Hiura Hisahide , Fujimura Yoshihiro
Published: October 2019

We prepared murine monoclonal antibodies (mAbs) against a decapeptide of the VWF-A2 domain that has a C-terminal Tyr1605, generated by ADAMTS13 cleavage. These mAbs reacted neither with purified VWF nor the pentadecapeptide 1596-DREQAPNLVYMVTGN-1610, unless the Y1605-M1606 bond was cleaved. Using one of these mAbs, we developed a novel and highly sensitive ELISA for ADAMTS13 activity. In this assay, a recombinant VWF polypeptide (D1596–R1668) tagged with GST and His at the ends (GST-VWF73-His), is used as a substrate; upon cleavage by ADAMTS13, two fragments, GST-VWF10 and VWF63-His, are generated. Thus, the amount of GST-VWF10 can be directly measured using our mAb labeled with peroxidase. The specificity of this assay was confirmed by inhibition with EDTA, IgGs purified from thrombotic thrombocytopenic purpura (TTP) patients, and an anti-ADAMTS13 mAb. The detection limit of this assay was 0.5% of the normal.

Of 20 patients with congenital TTP (Upshaw-Schulman syndrome, USS) whose ADAMTS13 gene mutations were identified, 14 had a severe deficiency (<0.5%), and 6 had a moderate deficiency (0.55~1.3%) of ADAMTS13 activity. No correlation was found between these two groups in terms of the onset of clinical manifestations. Further, a unique asymptomatic carrier of USS with two mutations (R268P/P475S) in the gene showed 4.2 % of normal plasma ADAMTS13 activity in this novel ELISA, indicating that plasma levels of ADAMTS13 activity between 1.3 and 4.2 % represent a gray zone which may lead to clinical manifestations of TTP. Table 1 shows a clinical and laboratory data of USS patients included in this study.

Table 1. Clinical and laboratory data of 20 USS patients
Plasma Fibronectin Enhances Adhesion and Aggregation of Platelets by Both Its Incorporation into Adherent Platelets and Its Crosslinking to Fibrin Networks.
Authors: Cho Jaehyung , Mosher Deane F.
Published: October 2019

Plasma fibronectin has recently been demonstrated to stabilize growth of platelet-rich thrombi in injured mesenteric arterioles in vivo. We have determined the impact of two forms of insolubilized plasma fibronectin on adhesion and aggregation of platelets - fibronectin-crosslinked fibrin by activated factor XIII (fibronectin-fibrin) and fibronectin assembled by adherent platelets. In static adhesion assays, 2-fold more platelets adhered to fibronectin-fibrin than to fibrin. Antibody blocking studies demonstrated that a different mix of αIIbβ3, αvβ3, and α5β1 integrins mediate platelet adhesion to both substrates. At a shear rate of 300 or 1,250s-1 in a parallel plate flow chamber, more platelets adhered and aggregated on surfaces coated with fibronectin-fibrin than surfaces coated with fibrin. The number of adherent platelets and the volume of platelet thrombi increased by 3- to 4-fold when soluble fibronectin was co-perfused with platelets, which resulted in assembly of fibronectin by adherent platelets. Increased adhesion and aggregation of platelets were dependent on the amounts of fibronectin crosslinked to fibrin prior to perfusion or included in the perfusate. The effects of perfused fibronectin were blocked by two inhibitors of fibronectin assembly, the N-terminal 70-kD fragment of fibronectin or the functional upstream domain of protein F1 of Streptococcus pyogenes, indicating that assembly of soluble fibronectin by adherent platelets is mediated by the binding of the N-terminal region of fibronectin to the platelet surface. Thus, plasma fibronectin has the potential to enhance adhesion and aggregation of platelets in vivo as a result of being crosslinked to fibrin networks and its subsequent assembly by adherent platelets.
Interaction of High Molecular Weight Kininogen with Endothelial Cell Receptors suPAR, gC1qR and Cytokeratin 1 by Surface Plasmon Resonance (BiaCore).
Authors: Espinola Ricardo G. , Pixley Robin A. , Ghebrehiwet Berhane , Joseph Kusumam , Kuo Alice , Cines Douglas B. , Colman Robert W.
Published: October 2019

The physiologic activation of the plasma kallikrein kinin system (KKS) requires the assembly of these proteins on the cell membrane. High molecular weight kininogen (HK) binds to endothelial cells through an interaction with a multiprotein receptor complex that consist of: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR) and cytokeratin 1 (CK1). The affinity of HK and cleaved HK (Hka) for endothelial cells is KD=7–52 nM but the affinity for each of the three binding proteins is unknown. We first examined the direct binding of HK and Hka to the soluble receptor form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance (BiaCore). We linked suPAR, gC1qR and CK1 (800–1100 pg/mm2) by amine coupling to a CM-5 chip and perfused HK and Hka at a concentration ranging from 50 to 400 nM in the presence or absence of 10 μM ZnCl2. A Langmuir binding model with local fit (stoichiometry of 1:1) was used to analyse kon (association rate constant), koff (dissociation rate constant) and KD (equilibrium dissociation constant) for HK and Hka.

HK Hka KD kon koff KD kon koff Immobilized Protein nM M-1s-1 x104 s-1 x10-4 nM M-1s-1 x104 s-1 x10-4 gC1qR 0.8±0.7 12.3±5.0 0.8±0.5 0.7±0.5 24.4±10.9 1.4±0.5 CK1 15.1±1.0 3.6±0.3 5.4±0.7 6.3±1.9 5.8±0.3 3.6±0.9 suPAR 2313.5±1465.3 1.0±0.7 164.8±22.2 43.6±16.6 21.3±11.9 78.5±10.6

The binding of HK and Hka to the three receptors was zinc dependent. The affinity for HK and Hka was gC1qR>CK1>suPAR. The high affinity for gC1qR and CK1 was similar for HK and Hka and was due to a very slow koff. The affinity for Hka compared to HK was 2.5-fold tighter for CK1 but 50-fold tighter for suPAR. A reversed immobiization was then performed. We immobilized HK, Hka and LK (800 pg/mm2) and suPAR, gC1qR and CK1 were flowed over the chip at a concentration ranging from 50 to 700 nM. Only gC1qR bound to immobilized HK and Hka, with a KD of 6.31± 2.8 pM for HK and 2.86±1.5 pM for Hka while the other 2 receptors did not bind. Receptor integrity was shown by preincubating HK or Hka with the pure receptors and observing competition with the immobilized receptors. Among the HK/Hka receptors, complex formation was only observed between gC1qR and immobiized CK1 with or without Hka indicating that a CK1-gC1qR complex can form independently of kininogen. This study indicates that although suPAR has the weakest affinity of the three receptors it is the only one that distinguishes between HK and Hka. Only one binary complex was revealed by this technique between CK1 and gC1qR. These results lay the foundation for anlayzing the affinity of these receptors in a cellular environment.
Mouse Model of MYC-Deregulating Chromosomal Translocations Associated with Human Burkitt Lymphoma and Multiple Myeloma.
Authors: Janz Siegfried , John Shaughnessy D.
Published: October 2019

Purpose: Burkitt lymphoma (BL) is tightly associated with chromosomal t(8;14)(q24;q32) translocations that result in the deregulated expression of the MYC (c-myc) oncogene due to its juxtaposition to enhancers of the immunoglobulin-heavy chain locus IGH. The t(8;14)(q24;q32) MYC-IGH translocation is widely believed to be the initiating event in the pathogenesis of BL. Multiple myeloma (MM) is a malignant neoplasm of plasma cells that is often associated with complex cytogenetic alterations that juxtapose MYC, analogous to the t(8;14)(q24;q32) translocation, to IGH enhancers. However, MYC-IGH rearrangements of this sort occur during the progression of MM, not as tumor-initiating events as in BL. Accurate mouse models of MYC deregulation associated with human BL and MM are needed to study the events involved in tumor initiation and progression and design new approaches for treatment and prevention. To that end, we have generated a new mouse model of human t(8;14)(q24;q32) translocation.

Experimental procedure: We inserted a His6-tagged mouse Myc cDNA, MycHis, into two different locations of the mouse immunoglobulin heavy-chain locus, Igh, just 5′ of Eμ or Cμ. In the latter case, Eμ was deleted during gene targeting. The newly developed gene-insertion strains were designated iMycEμ and iMycCμ. The iMycEμ mice provide a specific model of the t(8;14)(q24;q32) observed in endemic BL (eBL). The iMycCμ mice mimic the t(8;14)(q24;q32) found in sporadic BL (sBL) and immunodeficiency-associated BL (iBL) (Fig. 1).

Figure 1 Figure 1.

Result: Both “iMyc” gene-insertion strains are prone to the spontaneous development of mature B cell and plasma cell tumors. However, in the Eμ-containing iMycEμ mice, tumors arose earlier and tumor incidence was higher compared to the Eμ-deleted iMycCμ (Fig. 2 left).

Figure 2 Figure 2.

This correlated well with the control of MycHis expression by Eμ, as MycHis mRNA levels were higher in B cells from iMycEμ than iMycCμ mice. Interestingly, the iMycCμ transgene was as effective in inducing inflammation-dependent peritoneal plasmacytomas as the iMycCμ transgene (Fig. 2 right). This indicated that host factors, such as chronic inflammation, can greatly enhance the transformation-inducing potency of a deregulated Myc gene that is only weakly oncogenic in spontaneous tumor development.

Conclusion: The “iMyc” mice provide a model of Myc deregulation that is conducive to plasma cell tumors in mice. The mice may thus afford a good opportunity to identify the genes that collaborate with Myc in neoplastic plasma cell development. Combined with the transgenic expression of MM genes, such as DKK1, the “iMyc” mice may also be useful as a platform for modelling human plasma cell neoplasms in mice.
Pharmacological Blockade of NF-kB with AS602868, a Potent Small Molecule Inhibitor of IKK2 Leads to Apoptosis of Sensitive and Resistant Chronic Myeloid Leukemia Cells.
Authors: Imbert Veronique , Gonthier Nadege , Frelin Catherine , Sirvent Nicolas , Hummelsberger Michael , von Bueltzingsloewen Anne Sirvent , Cassuto Jill-Patrice , Mahon Francois-Xavier , Rousselot Philippe , Dreano Michel , Peyron Jean-Francois
Published: October 2019

The Bcr-Abl inhibitor imatinib mesylate is now the first choice treatment for all newly diagnosed CML patients. Nevertheless not all patients reach a complete cytogenetic response (CCR) and many develop resistance for the drug upon disease progression. Additional cellular targets should be thus identified to develop alternative therapeutic strategies. The transcription factor NF-kB appears as a promising target for the treatment of cancer since it is a pro-survival factor, found abnormaly active in numerous hematologic malignancies and solid tumors. NF-kB activation relies on ubiquitin-mediated degradation of its inhibitor IkB after phosphorylation by 2 specific kinases, IKK1 and IKK2. We disrupted NF-kB signaling pathway by the use of AS602868, a small molecule inhibitor of IKK2 on two CML cell lines, LAMA84 and BaF3/Bcr-Abl and on primary CML cells. First we showed that NF-kB is constitutively activated in both CML cell lines and that this constitutive activation is under the control of the Bcr-Abl kinase. We next demonstrated that IKK2 targeting by AS602868 led to apoptosis of CML cells. AS602868 induced a dose dependent growth inhibition (IC50 3–4μM) of CML cell lines. This inhibition was associated with activation of cellular caspases 3, 8 and 9 and induction of DNA fragmentation. Interestingly AS602868 could induce death of imatinib resistant cellular clones derived from LAMA84 and BaF3-Bcr-Abl cells (IC50 1–2μM). Finally AS602868 selectively inhibited the proliferation and the hematopoietic colony formation of primary CML cells derived from imatinib sensitive or resistant patients. Although the exact mechanism of NF-kB activation by Bcr-Abl remains to be elucidated, our data strongly suggest that targeting NF-kB with the IKK2 inhibitor AS602868 may represent a new promising therapeutic tool for the treatment of CML.
ZAP-70 Expression Is More Predictive Than VH Gene Mutational Status of BCR-Mediated Tyrosine Phosphorylation, NF-κB Activation and CLL Cell Survival.
Authors: Pepper Chris , Alghazal Suhair , Brennan Paul , Rowntree Clare , Pratt Guy , Fegan Chris
Published: October 2019

We analysed tyrosine phosphorylation, NF-κB activation and survival in a cohort of 64 chronic lymphocytic leukemia patients. By graphing VH gene mutational status, CD38 expression and ZAP-70 expression as continuous variables, we investigated correlations between these prognostic markers and cell signaling. There was a strong correlation between CD38 expression and basal levels of tyrosine phosphorylation (r2 = 0.68). VH gene mutational status and ZAP-70 expression correlated with the induction of tyrosine phosphorylation by anti-IgM (r2 = 0.31 and 0.46 respectively). These findings suggest that tyrosine phosphorylation integrates these three prognostic phenotypes. In the context of NF-κB signaling, the percentage change in tyrosine phosphorylation (post anti-IgM) and ZAP-70 expression correlated with NF-κB activation (r2 = 0.2 and 0.19 respectively) and the ability to activate NF-κB was strongly correlated with cell survival (r2 = 0.67). The therapeutic rationale for the inhibition of NF-κB was strengthened by our observations that the NF-κB inhibitor, BAY 11–7082, caused a concentration-dependent increase in apoptosis in CLL samples. Taken together, our data suggest that CD38 is a surrogate marker of basal cellular activation in CLL that is independent of the B-cell receptor. Furthermore, ZAP-70 expression is the most important predictor of tyrosine phosphorylation, downstream NF-κB activation and CLL cell survival following B-cell receptor cross-linking. These findings provide a mechanism-based rationale for the poor prognosis of CD38+ and ZAP-70+ CLL patients.
How Frequently Is Venous Thromboembolism in Heparin-Treated Patients Associated with Heparin-Induced Thrombocytopenia?.
Authors: Hursting Marcie J. , McCollum David , Levine Robert L.
Published: October 2019

Unfractionated heparin (UFH) and low-molecular-weight heparin (LMWH) are routinely used for prophylaxis or treatment of venous thromboembolism (VTE) yet paradoxically can promote VTE in a patient with heparin-induced thrombocytopenia (HIT), an immune-mediated prothrombotic reaction to heparins. The purpose of this meta-analysis was to estimate how frequently new or recurrent VTE in heparin-treated patients is HIT-associated. Studies using UFH or LMWH for prophylaxis or treatment of thrombosis in which an outcome variable was new or recurrent VTE and in which information was available for the determination of HIT were abstracted to determine the proportion of patients with VTE that was associated with HIT. From a comprehensive literature search, we identified 10 studies (6 randomized trials, 3 prospective cohort studies, 1 retrospective cohort study) of intravenous (iv) or subcutaneous (sc) UFH or LMWH therapy that reported objectively confirmed VTE (at least 1 event per study) both overall and in patients with comorbid HIT, which was prospectively defined in each study (typically a platelet count <100–150 x109/L or a 30–50% decrease in count, with serologic confirmation). In each study, which may have included 2 treatment groups, medical or surgical patients received UFH iv (5 studies), UFH sc (3 studies), or LMWH sc (5 studies) therapy typically for 7–10 days and were followed in hospital or for 10 to 90 days for new or recurrent VTE. Across the studies in a total of 6219 heparin-treated patients, 386 patients had new or recurrent VTE, and 34 had HIT-associated VTE. The frequency of HIT-associated VTE among heparin-treated patients with VTE was not different between iv and sc UFH therapy (13.2% versus 12.4%, P>0.99) but was significantly different between UFH and LMWH therapy (12.8% versus 0.7%, P<0.001) (Table 1). These data indicate that new or recurrent VTE is HIT-associated infrequently (<1%) in LMWH-treated patients yet often (approximately 1 in 8 cases) in UFH-treated patients. Because continued or repeated exposure to heparins can be catastrophic in HIT, physicians should have a high degree of suspicion for HIT in patients developing VTE during or soon after UFH therapy and should use alternative anticoagulation until HIT has been excluded.

Table 1. VTE and HIT-associated VTE in heparin-treated patients Heparin type (#studies) Heparin-treated patients Patients with VTE, n (%) Patients with HIT-associated VTE, n (%) HIT-associated VTE/any VTE, % (95% CI) *P>0.99 vs sc UFH; **P<0.001 vs LMWH UFH, iv (5 studies) 2687 129 (4.8) 17 (0.6) 13.2 (8.3 - 20.3)* UFH, sc (3 studies) 1105 113 (10.2) 14 (1.3) 12.4 (6.9 - 19.9) UFH, iv or sc 3792 242 (6.4) 31 (0.8) 12.8 (8.9 - 17.7)** LMWH, sc (5 studies) 2427 144 (5.9) 1 (0.04) 0.7 (0.02 - 3.8)
DAP10 Costimulates Chimeric Receptor-Mediated T Cell Responses to Tumor Antigen.
Authors: Altvater Bianca , Pscherer Sibylle , Juergens Heribert , Rossig Claudia
Published: October 2019

Chimeric receptors (chRecs) combining extracellular recognition domains with the T cell receptor ζ an redirect the cellular immune response of primary T-cells to tumor cells. T cell activation by chRec induces efficient cytokine release and cytotoxicity, however, it fails to mediate proliferative responses, limiting the usefulness of chRec-gene-modified T cells for adoptive immunotherapy of cancer. Inclusion of a CD28 costimulatory signaling component in the chRec endodomain enhances antigen-specific proliferation. Whereas the signal mediated by ligation of CD28 is of crucial importance for the activation of resting CD4+ T cells, further molecules with costimulatory functions have contributory roles. NKG2D is a stimulatory receptor that was first identified in NK cells, but is also expressed in cytotoxic T cells and positively modulates CD8+ T cell immune responses. We hypothesized that inclusion of the NKG2D-associated signaling domain DAP10 would enhance the capacity of chRecs to induce tumor-specific activation and proliferation of in vitro expanded effector T cells. Based on a GD2-specific scFv, we generated chRecs containing either the DAP10 signaling chain alone (14.G2a-DAP10) or combined with TCRζ 14.G2a-DAP10ζ), and expressed them in nonspecifically activated human peripheral blood T cells of three individual donors by retroviral gene transfer. As controls, T cells were transduced with 14.G2a-ζ and -CD28ζ chRec. High chRec surface expression was obtained with all four constructs (55±11%, ζ; 85±3, CD28ζ; 68±5%, DAP10; 78±1%; DAP10ζ). Immunophenotypes were dominated by a CD3+CD8+ population in all cell cultures. Whereas DAP10 alone failed to mediate specific tumor cell lysis, 51Cr release assays revealed efficient and comparable lysis of GD2+ tumor targets by T cells transduced with all ζ-containing constructs, with 49±8% (ζ), 52±7% (CD28ζ), and 52±18% (DAP10ζ) cytolysis at an effector-to-target ratio of 40:1. Intracellular cytokine secretion by chRec+ T cells was induced in response to tumor targets by 14.G2a-ζ (up to 37% IFN-γ secreting cells), CD28ζ, and DAPζ (both up to 22%), but not by DAP10 alone (0,2%). Weekly stimulation with tumor cells for 6 weeks induced only limited expansion of T cells transduced with 14.G2a-ζ (7–45fold) or with 14.G2a-DAP10 (14–26-fold). Adding CD28 or DAP10 domains significantly enhanced expansion by a comparable degree (270–483-fold and 126–436-fold, respectively). Thus, while neither CD28 nor DAP10 enhances antigen-specific cytokine secretion and cytolysis, DAP10 signaling can completely replace CD28 signaling in costimulating antigen-specific proliferation of peripheral blood T cells. DAP10-containing chRec may be a powerful new tool for adoptive immunotherapy of cancer.
Induction of CML28-Specific Cytotoxic T Cells by Dendritic Cells Cotransfected with CML28 Nucleic Acid Vaccination and SOCS1-Specific siRNA Expression Vector In Vitro.
Authors: Zhang Donghua , Zhou Hongsheng , Zhang Lu , Wang Yaya , Dai Min , Zhang Li
Published: October 2019

CML28 is a new tumor-associated antigen overexpressed in tumor cells, which is an attractive target for antigen-specific immunotherapy. In this study, we evaluated the possibility to induce CML28-specific cytotoxic T cells by cotransfection of CML28 nucleic acid vaccination and SOCS1-specific siRNA expression vector with dendritic cells in vitro. The full length CML28 cDNA was amplified from K562 by RT-PCR and cloned into a bicistronic vector pIRES2-EGFP to construct the CML28 nucleic acid vaccination. Dendritic cells (DCs) were generated from peripheral blood mononuclear cells (PBMNCs) of HLA-A2+ healthy donor. Construct the recombinant plasmid psiRNA-hH1neo-SOCS1 encoding hairpin small interfering RNA (siRNA) targeting to suppressors of cytokine signaling 1 (SOCS1) sequences using a vector-base RNA interference technology. Cotransfect CML28 nucleic acid vaccination and recombinant siRNA vector psiRNA-hH1neo-SOCS1 into DCs by electroporation. Real-time RT-PCR was performed to validate the SCOS1 gene silencing efficacy. The expression products of CML28 nucleic acid vaccination, His-CML28 fusion protein and GFP protein, were measured by Western Blotting and fluorescence microscope respectively. Labeling autologous nonadherent fraction of PBMNCs as responder cells by 5, 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) firstly, and then coculture with cotransfected DCs for mixed lymphocyte reaction (MLR). Autogous T cell proliferative response of MLR was measured by FACS by detecting CFSE. Cotransfected DCs and the PBMNCs of a HLA-A2+ primary leukemia patient were used as target cells, untransfected DCs, K562 and HL60 were used as control. The standard 51Cr-release assay was performed to measure cytotoxicity of stimulated lyphocytes. CML28 nucleic acid vaccination could encode His-CML28 and GFP protein in DCs successfully. SOCS1-specific siRNA expression vector psiRNA-hH1neo-SOCS1 significantly suppress the expression of SOCS1 in DCs. Down regulation of SOCS1 resulted in higher expression level of CD80, CD86 and CD83 in cotranfected DCs and more rounds of cell division of responder cells in CFSE-MLR, which indicates SOCS1 gene silencing greatly contribute to maturation of DCs and enhance the the proliferative response of responder cells. CTLs induced by cotransfected DCs exhibit stronger CML28-specific cytotoxicity against HLA- matched target cells comparing to CTLs induced by CML28 nucleic acid vaccination transfected DCs only. SOCS1 gene silencing in DCs could greatly enhance the anti-tumor efficacy of CML28 nucleic acid vaccination. DCs cotransfected with CML28 nucleic acid vaccination and SOCS1-specific siRNA expression vector could effectively induce autologous CML28-specific cytotoxic T cells that lyse CML28 positive tumor cells in an antigen-specific and major histocompatibility complex (MHC) class I-restricted manner.
p21-Activated Kinase 1 Regulates Hyperproliferation of Nf1 Haploinsufficient Mast Cells In Vitro and In Vivo Via Activation of the MAPK Pathway.
Authors: McDaniel Andrew S.
Published: October 2019

p21-activated kinases (Paks) are downstream mediators of Rho GTPase proteins and have been implicated in yeast and immortalized cells as positive regulators of MAPK pathway members in modulating cell growth and cytoskeletal functions. However, their role in primary mammalian cells has not been described.

NF1 encodes neurofibromin, which negatively regulates p21Ras activity by stimulating its intrinsic GTPase activity, and accelerating hydrolysis of Ras from the GTP to the GDP confirmation. Disruption of the NF1 locus results in neurofibromatosis type I (NF1), an inherited disorder characterized by the development of neurofibromas that contain large numbers of degranulating mast cells that have been implicated in tumor progression. Utilizing a genetic intercross of Pak 1−/− mice with mice haploinsufficient at the Nf1 locus, we studied the role of Pak1 in the context of normal and hyperactivated Ras-MAPK signaling in primary inflammatory mast cells. Pak1 was found to directly contribute to Ras-dependent signaling by modulating both Raf-1, Mek-1 and ERK1/2 activation. Loss of Pak1 fully corrects the hyperphosphorylation of ERK1/2 found in Nf1+/− mast cells to that of wild type controls. Deletion of Pak1 in Nf1+/− mast cells is associated with a correction of Kit ligand mediated proliferation to wild type levels in vitro. Further, after subcutaneous administration of Kit ligand via micro osmotic pumps, which is an established model that stimulates local proliferation of mast cells in vivo (Ingram, JEM 2001), we confirmed that genetic disruption of Pak1 corrects the proliferation of Nf1+/− mast cells in vivo to that of wild type controls. These data provide direct genetic evidence that Pak1 modulates the Ras-Raf-Mek-Erk pathway and identifies a specific molecular target within the inflammatory tumor microenvironment for the treatment or prevention of neurofibromas.
Mutant CXCR4 Identified in Neutropenic Patients with Myelokathexis Impairs Survival of Human Myeloid Cells.
Authors: Makaryan Vahagn , Dale David C. , Aprikyan Andrew A.
Published: October 2019

Myelokathexis (WHIM syndrome) is a very rare hematopoietic congenital disorder that is characterized by extremely low level of circulating neutrophils in peripheral blood. It is inherited as an autosomal dominant disease and is diagnosed in early childhood. These patients may have hypogammaglobulinemia and suffer from recurrent infections associated with warts. The hallmark of myelokathexis is a hyperplastic bone marrow and hypersegmented neutrophils with nuclear lobs connected with thin filaments. Myelokathexis is due to a characteristic retention of mature neutrophils in bone marrow, which are not being released to peripheral circulation. We and others reported abnormal cell survival characteristics and impaired bcl-x expression in bone marrow myeloid cells of myelokathexis patients that was partially restored by G-CSF treatment. Recently, it has also been reported that heterozygous truncation mutations in the carboxyterminal domain of the CXCR4 gene, a sole receptor for SDF-1 chemokine, were observed in most, but not all of the families with WHIM syndrome. Subsequently, an impaired receptor internalization and increased chemotaxis towards SDF-1 have been observed in cells expressing truncated CXCR4. Nevertheless, the mechanism of mutant CXCR4 induced myelokathexis remains largely unknown.

We performed mutational analysis of the CXCR4 gene in 3 unrelated families with myelokathexis and identified a previously reported R334ter truncation mutation in exon 2 in two of the families. In addition, two silent polymorphisms have been identified in exon 2 of the CXCR4 gene in one of these patients. The third family with afflicted mother and son had a new mutation in the CXCR4 carboxyterminal domain, which resulted in deletion of the last 16 amino acids and subsequent frame shift. None of these mutations were observed in healthy volunteers examined. Since the morphological examination by electron microscopy and flow cytometry analysis of bone marrow cells from some of these patients revealed characteristic apoptotic features, we examined the effect of mutant CXCR4 gene expression on survival of human promyelocytic HL-60 cells. Preliminary data demonstrated that human promyelocytic cells transfected with truncated CXCR4 exhibited impaired cell survival characteristics compared with control HL-60 cells transfected with intact CXCR4. The truncated, but not wild type CXCR4 also increased apoptosis in HL-60 cells induced to differentiate along the granulocytic pathway as determined by flow cytometry of annexin V labeled cells. Thus, these data link together the abnormal survival of proliferating and differentiating myeloid cells in WHIM syndrome with mutant CXCR4 expression. Current studies are focused on elucidation of specific signaling pathways mediating mutant CXCR4-triggered myelokathexis.
In Vitro and In Vivo Immunomodulatory Effects of Mesenchymal Stem Cells from Adipose Tissue.
Authors: Yañez Rosa M. , Lamana Maria L. , Garcia-Castro Javier , Ramirez Manuel , Bueren Juan A.
Published: October 2019

Previous studies have shown the immunomodulatory properties of bone marrow mesenchymal stem cells (BM-MSCs), opening the possibility of using these cells for the treatment of graft-versus-host disease (GVHD) in patients transplanted with allogeneic hematopoietic grafts. Additionally, Phase I studies in patients with Crohn’s disease suggested the efficacy of adipose tissue-derived mesenchymal stem cells (Ad-MSCs) for the healing of Crohn’s fistulas. In the present study we have investigated in vitro and in vivo, the immunomodulatory effects of Ad-MSCs, compared to BM-MSCs. We observed that both BM-MSCs and Ad-MSCs were negative for CD34, CD45, CD14, CD31 and MHC class I expression, while positive for CD29, CD44, CD90 and CD105. When studying the immunomodulatory effects of these cells in vitro, we found that - as happened with BM-MSCs - Ad-MSCs did not induce proliferation of allogeneic lymphocytes and were not lysed by cytotoxic T cells or alloreactive natural killer cells, indicating that Ad-MSCs are non-immunogenic. Additionally, the presence of Ad-MSCs inhibited in a dose-dependent fashion, both the mixed lymphocyte reaction (MLR) and the T cell proliferation induced by mitogens. To determine whether cell-to-cell contact between Ad-MSCs and PBMNCs was required for immunosuppression, transwell experiments were conducted. Phytohaemagglutinin (PHA)-stimulated lymphocytes were cultured in the upper chamber of a transwell, while irradiated Ad-MSCs remained in the lower chamber. As observed with BM-MSCs, Ad-MSCs were also capable of suppressing the lymphocytes proliferation in this transwell assay. When conditioned medium from Ad-MSCs was added to the MLR, the immuno-suppressive effect persisted, although at a lower level than that observed in a cell-to-cell contact system. Next we studied whether our in vitro findings were of significance in an in vivo mouse model of haploidentical transplantation. In these experiments irradiated F1(C57Bl/Balbc) recipient mice received 1x107 bone marrow cells from C57Bl mice, together with 2x107 splenocytes from the donor, to induce GVHD. One cohort of recipient mice received additional i.v. infusions of 5x105 mouse Ad-MSCs, administered at periodic intervals for up to 28 days post-transplant. When compared to the control group, the severity of the GVHD was significantly reduced in mice receiving Ad-MSCs. Our results suggest that Ad-MSCs obtained from adipose tissue may constitute a new and readily available source of immunomodulatory cells for the prophylaxis and/or treatment of GVHD in patients transplanted with allogeneic grafts.
Resting B Cells Suppress CD8+ T Cell Function and Prevent the Induction of Graft Versus Host Disease.
Authors: Ritchie David S. , Watt Victoria
Published: October 2019

B cells have been variously shown to induce direct tolerance of antigen specific CD8+ T cells, induce T cell anergy via TGF-b production, down regulate IL-12 production by dendritic cells (DC) and influence Th1/Th2 differentiation via the production of regulatory cytokines. Through these mechanisms, B cells can exert a regulatory function in in vivo models of T cell immunity including, experimental autoimmune encephalitis (EAE) and rheumatoid arthritis (RA). Recently, B cells have been shown to be essential in the prevention of effector T cell differentiation in a model of autoimmunity.

We have previously shown that resting B cells inhibited tumor protection induced by dendritic cells vaccination. Inhibition of DC immunity by B cells was independent of presentation of major histocompatibility molecule (MHC) class-I bound tumor antigen but dependent on the expression of class-II MHC. Furthermore the inhibitory effect of B cells was lost if the B cells were activated by CD40L or if CD4+/CD25+ regulatory T cells (Treg) were depleted. These studies have been further extended to examine the role of resting B cells on the induction and severity of graft versus host disease (GVHD) induced in a major MHC mismatch model. We have found that mice transplanted with B cell depleted marrow revealed more rapid CD8+ T cell engraftment, higher IL-2 and IFN-γ production, more severe GVHD and shorter survival. Conversely, those who received additional resting B cells at the time of marrow infusion were substantially protected from GVHD. These findings indicate that resting B cells may regulate T cell activation, in part via the suppressive effects of Treg, but also through their important role in T cell homeostasis. Resting B cells may therefore limit the efficacy of DC based immunotherapy or alternatively be used therapeutically to limit CD8+ T cell autoimmunity including GVHD.
Delayed Versus Initial Risk-Categorization of Pediatric Cancer Patients with Prolonged Febrile Neutropenia. A Randomized, Prospective Clinical Trial.
Authors: Ahmed Nabil M. , Mahallawy Hadir El , El Haddad Alaa M.
Published: October 2019

Purpose:

Hospitalization with empiric broad spectrum intravenous antibiotics has been the standard of care for treatment of high-risk febrile neutropenia. In an attempt to improved hospital utilization we attempt to identify a group of patients at a lower risk of sepsis that would be eligible for outpatient therapy. We hypothesized that patients initially considered at high risk for sepsis can be candidates for outpatient therapy if at 72 hours satisfied certain selection criteria.

Patients and methods:

A prospective, randomized clinical trial was conducted in 119 febrile neutropenic pediatric cancer patients. Patients were randomized into two groups depending on the time of risk-categorization: Initial (control) vs. delayed (study) groups. Patients in the study group were risk-assessed at 72 hours and accordingly considered for outpatient therapy if afebrile, had an absolute neutrophil count (ANC) > 100/uL, negative blood cultures, and no signs of infection among other carefully defined “Early Discharge Criteria”. Those randomized to the control group were treated in the hospital. We analyzed the clinical features, microbiological data, hospital course, duration of hospitalization and treatment cost. Intention-to-treat analysis was used. Results: We found a statistically significant difference between the duration of hospitalization in the group risk-assessed at 72 hours and control group (p<0.01). “Delayed Risk-categorization” correlated better with the duration of neutropenia, need for antibiotic change and episode outcome. Duration of hospitalization and per-episode median treatment cost were significantly less in the study group vs. control (p< 0.001). There was a significant tendency for blood cultures to cluster around the first 48 hours. Comparing the blood culture yield between patients treated in the hospital to those treated as outpatients after risk-assessment at 72 hours there was no statistically significant difference demonstrated [p=0.35].

Readmission rate after early discharge was 3.3%.

Conclusion: We conclude that pediatric febrile-neutropenic patients initially at considered at “high risk for sepsis” can be reevaluated at 72 hours for outpatient therapy. The practice of “Delayed Risk-Categoriztion” could be particularly useful to reduce the overall treatment costs, and duration of hospitalization. In addition, this practice could potenitally offer a superior quality of life.
Response to Immunizations Following Allogeneic HCT with Adjuvant Rituximab Therapy.
Authors: Pao Mary , Papadopoulos Esperanza B. , Boulad Farid , Castro-Malaspina Hugo , Jakubowski Ann , Kernan Nancy A. , Perales Miguel , Prockop Susan , Ford Ruth , Young James W. , O’Reilly Richard J. , Small Trudy N.
Published: October 2019

Use of anti-CD20 monoclonal antibody after allogeneic HCT for the treatment EBV lymphoma or viremia, autoimmune cytopenias, and the prevention or treatment of recurrent NHL has become more frequent. There is limited data on the effect of this therapy on specific antibody production following allogeneic HCT. We therefore examined the response to standard childhood immunizations of 30 patients, including 20 adults (>18 years of age) who received rituximab following a T cell depleted (n=26) or unmodified (n=4) HCT at our institution. T cell function was assessed in vitro by proliferative response to PHA and B cell reconstitution was confirmed by recovery of CD19+ and CD20+ circulating lymphocytes. Patients received a median (range) of 4 (1–12) doses of rituximab, with 15 patients receiving 4–6 doses. The median age of the patient population was 28.5 years with a range of 0.8–63.0 years. Children (median age: 8.5 yrs) received a transplant from an unrelated (n=7), HLA mismatched related (n=2), or HLA matched related (n=1) donor and adults (median age: 42.5 yrs), received a transplant from an unrelated (n=7), HLA mismatched related (n=2), or HLA-matched related (n=11) donor. Patients were vaccinated when their PHA response was at least 75% of the lower limit of normal and CD20+ cells were >100/ul. Vaccination was initiated at a median of 270 and 525 days following the last dose of rituximab in children and adults, respectively. A two to 3-fold rise in titers following a series of 3 tetanus and IPV vaccinations occurred in 19 of 21 and 19 of 19 evaluable patients, respectively. Although 8 of 9 children responded to the H flu conjugate vaccine, only 5 of 13 adults mounted an adequate response. Eighteen patients received the recombinant Hepatitis B vaccine. Nine responded following the initial series (5/7 children, 4/11 adults). Two of three patients who failed initial Hepatitis B vaccination, responded to a second series (1 child, 1 adult). Response to pneumococcal vaccination was the least consistent. None of 11 adults who received the unconjugated 23-valent pneumococcal vaccine developed an adequate response. In view of this observation, children given rituximab were immunized with the conjugated pneumococcal vaccine. Four of eight children responded to the initial series, and two additional children responded following a second series. These preliminary data suggest that both adult and pediatric patients given rituximab following an allogeneic HCT can mount successful antibody responses to standard T-helper cell dependent vaccines. However, B cell responses to T cell independent vaccines may be particularly impaired. Larger prospective trials, stratified by age, comparing vaccine responses in patients following allogeneic transplant in the presence or absence of post transplant rituximab therapy are warranted.
Assessment of Minimal Residual Disease (MRD) by Qualitative RT-PCR (Q-PCR) vs Real-Time Quantitative RT-PCR (RQ-PCR) in a Phase II Study of Acute Promyelocytic Leukemia (APL).
Authors: Gallagher Robert E. , Schachter-Tokarz Esther L. , Jones Dan A. , Estey Elihu H.
Published: October 2019

Assessment of MRD in APL by Q-PCR analysis for the presence or absence of PML-RARα mRNA after the completion of consolidation therapy in treatment regimens employing all-trans retinoic acid (ATRA) and chemotherapy (CT) has provided a reasonably accurate but not infallible prognostic test for disease recurrence (DR). This assessment has been made at detection sensitivity levels up to 1 in 104, which is considered to have optimal predictive value, since non-quantified lower levels of PML-RARα mRNA may not be relevant to clinical outcome. In the current study, we compared the results of Q-PCR (touchdown PCR for 40 cycles followed by post-PCR blot hybridization with a RARα chemiluminescent probe) to results from RQ-PCR in 15 patients on a Phase II clinical trial designed to eliminate CT (low-risk/LR patients, WBC <10K) or minimize CT (high-risk/HR patients, WBC >10K), using combined ATRA and arsenic trioxide therapy. All patients achieved complete remission (CR) and have been followed for a median of 1.2 years. DR occurred in 0/8 LR, 0/1 unclassified and 2/6 HR patients. At CR, all Q-PCR and RQ-PCR assays were positive. For RQ-PCR, the normalized quotient (NQ) value of PML-RARα, i.e., the PML-RARα copy number divided by the housekeeping gene glyceraldehyde-3′-phosphate dehydrogenase (GAPDH) copy number, ranged widely from 1.5 x 10−6 to 7.0 x 10−3. Post-CR follow-up data were:

Patient Group Number Q-PCR+ RQ-PCR+ NQ Range LR/Unclassified 9 0/24 16/24 9 x 10−9 – 1 x 10−7 HR/no DR 4 0/23 8/23 2 x 10−9 – 3 x 10−7 HR/DR 2 1/7 5/7 1 x 10−8 – 5 x 10−5

For the 2 HR/DR patients, the last pre-DR results were, respectively: Q-PCR, positive and negative; RQ-PCR, NQ = 5 x 10−5 and NQ = 2.3 x 10−7. In overall comparisons to RQ-PCR results, all Q-PCR assays were positive for NQ >10−5, 3/4 were positive in the NQ 10−6 to 10−5 range, and all were negative for NQ <10−6. Additionally, dilution-reconstruction experiments confirmed the stochastic nature of PCR assays near the detection limit and indicated that RQ-PCR was about 10-fold more sensitive than Q-PCR. Although larger studies are needed, our results suggest that HR patients who are Q-PCR-negative but RQ-PCR-positive in the >10−7 – <10−5 range at a critical post-CR checkpoint may be at increased risk of DR and, more generally, they suggest that the results of RQ-PCR testing at a critical checkpoint can be used in combination with clinical risk assessment to stratify the intensity of subsequent MRD monitoring in individual patients.
Splenectomy for Splenic Marginal B-Cell Lymphoma.
Authors: Segota Ena , Shaheen Philip , Hsi Eric , Kalaycio Matt
Published: October 2019

Background: Splenic marginal zone lymphoma (SMZL) is an uncommon indolent B-cell malignancy usually involving the spleen, bone marrow and blood. Splenectomy is frequently performed for both diagnostic and therapeutic purposes, but it is unclear if it affects outcome. To further explore this issue, we reviewed clinical and pathological characteristics, and treatment outcomes of SMZL patients (pts) who underwent splenectomy in our institution.

Methods: Medical records of pts who had splenectomy at the Cleveland Clinic and were diagnosed with a B-cell lymphoproliferative disorder between 1994 and 2004 were reviewed. Pathology slides were reviewed to confirm the diagnosis of SMZL by WHO criteria. Data collection included: clinical and biochemical characteristics at diagnosis including symptoms; reason for and date of splenectomy; hematological response following surgery; additional therapies other than surgery including rationale, regimen used and outcome; and follow-up data.

Results: We identified 18 pts with SMZL having splenectomy in our institution. Twelve (67%) were females and 6 (33%) males. The median age was 65 years (range 43–85). At the time of diagnosis, 9 pts had splenic and bone marrow involvement; 8 splenic, bone marrow and nodal; 1 spleen involvement only. Four pts were treated with chemotherapy prior to splenectomy for presumed CLL, hairy-cell leukemia or unspecified B-cell lymphoproliferative disorder, but none achieved CR. None of the pts received radiation. Splenectomy was performed in 9 pts for diagnostic, and in 9 pts for therapeutic purposes. The most common reason for therapeutic splenectomy was cytopenia (n=5) and abdominal discomfort (n=4). Median follow-up from splenectomy to death or last follow-up is 30 months (range 1–99). Of the 9 pts who had splenectomy for diagnostic purposes the median time to subsequent systemic therapy (n=5) was 7 months (range 0–33). Of the 9 pts treated for therapeutic purposes abdominal symptoms resolved in all (n=4) and 4/5 with cytopenias achieved hematologic response. Seven of 9 pts with only spleen and marrow involvement, achieved complete hematologic remission following surgery lasting from 8+ to 46+months. Of 8 pts presenting with spleen and lymph node involvement, 6 had cytopenias that resolved in only 3 pts following splenectomy. Six of these 8 pts received chemotherapy after splenectomy with median time from surgery to systemic treatment of 16 months (range 3–60). One pt continues to have stable lymphadenopathy observed clinically for 33 months, and 1 pt had stable lymphadenopathy for 24 months and was subsequently lost to follow-up.

Conclusion: Splenectomy, whether performed diagnostically or therapeutically, may result in long remissions in the absence of chemotherapy. Splenectomy appears to be a viable treatment alternative for SMZL.
Identification and Hierarchy of Clonogenic Factors for Human Myeloma Cells: IL-6 for CD45+ Versus IL-6 and Growth Factors for CD45-.
Authors: Amiot Martine , Bataille Regis , Collette Madeleine , Descamps Geraldine , Pellat-deceunynck Catherine
Published: October 2019

Multiple Myeloma (MM) is a fatal plasma-cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow. IL-6 and IGF-1 are known to be essential growth and survival factors in this malignancy. Beside these well characterized growth factors, other growth factors such as HGF, HB-EGF and FGF have been involved in this malignancy. Even though all of them seem to be involved in myeloma cell proliferation, the hierarchy of each growth factor remains to be established. In the present study, a serum-free cytokine-free and collagen-based assay which does not allow the generation of endogeneous myeloma colonies was used to identify the clonogenic factors of fourteen myeloma cell lines. We selected seven myeloma cell lines expressing CD45 on 100% of cells and seven other cell lines lacking CD45 expression. IL-6 is the only clonogenic factor able to stimulate both CD45+ and CD45- myeloma cell lines, generating myeloma colonies from 10 of 14 myeloma cell lines. For some cell lines (LP-1, L363 and XG-2) the percentage of clonogenic cells reached 40% to 50% indicating that clonogenic cells are CD138+ and do not represent a particular CD138-subpopulation. In contrast, the other growth factors (IGF-1, FGF, HGF and HB-EGF) stimulate only some CD45-myeloma cell lines and at a less extend than IL-6. Among them, IGF-1 is the most potent, generating myeloma colonies from 5 of 8 CD45- myeloma cell lines. We searched for a correlation between myeloma-colony formation and the signaling pathway induced by IGF-1 and FGF in MM cells. It appears that activation of both the PI3Kinase and ERK pathway by IGF-1 and FGF fully correlates with their capacity to stimulate the clonogenicity of CD45-myeloma cell lines. In conclusion, the CD45 phenotype of myeloma cells discriminates their signaling and proliferative response to IL-6 and growth factors. These results give a strong rational to the stratification of prognostic value discriminating CD45+ from CD45- MM. Furthermore, treatment strategies for CD45- patients should combined the disruption of signaling induced by both IL-6 and IGF-1 (or other growth factors) whereas CD45+ patients could be targeted through IL-6 only.