bluuebunny/crossref_metadata_2025 huggingface dataset:
Thalidomide in Combination with Rituximab Is Active in Waldenström’s Macroglobulinemia and May Overcome Poor Response Determinants Associated with Rituximab Monotherapy.
Authors: Treon Steven P. , Hunter Zachary , Patterson Christopher J. , Branagan Andrew R.
Published: October 2019

INTRODUCTION: Rituximab is active in Waldenstrom’s macroglobulinemia (WM), producing major (CR + PR) response rates of 40–50% using an extended dose schedule. Lower response rates though are observed among patients with the FcgRIIIA-158 F/F polymorphism (JCO 23:474) and higher serum IgM levels (>6,000 mg/dL) (Ann Oncol 16:132). Thalidomide enhances rituximab mediated ADCC killing of lymphoplasmacytic cells and produces response rates of 25% in WM. As such, we conducted this phase II study using combination thalidomide and rituximab in patients naïve to either agent.

METHODS: Intended therapy was as follows: Weeks 1–52 Thalidomide (200 mg po qHS for 2 weeks, then 400 mg po qHS);Weeks 2–5, 13–16 Rituximab (375 mg/m2/week). Twenty-five patients were enrolled, 20 of who were previously untreated, with a baseline median age of 62 (range 44–86 yrs), BM involvement of 40 (range 5–90%), serum IgM of 3670 (range 924–8610 mg/dL), B2M of 2.6 (range 1.4–7.8 mg/L), Hct of 33.0 (range 24.5–37.7%).

RESULTS: Grade 3/4 toxicities to thalidomide included paresthesia (n=10); somnolence (n=8); rash (n=7); neuropathic pain (n=6); confusion (n=4); tremors (n=2); and bradycardia (n=2). Dose reduction occurred in all patients and led to premature discontinuation in 11 patients. All evaluable patients received the intended rituximab therapy. Paradoxical IgM spikes following rituximab occurred during both courses of therapy and were observed in 12 patients. Two patients succumbed, unrelated to therapy, and were unevaluable. Responses were as follows: CR (n=1); PR (n=15); MR (n=2); SD (n=1) for an overall and a major response rate of 78% and 70%, respectively, among evaluable patients. Four patients had no response to therapy. With a median follow-up of 19 months, 6 patients have progressed. The median duration of response was 19.6+ months (range 7 to 26+ months) Overall response was associated with median cumulative dosed thalidomide: CR/PR/MR (29,275 mg) vs. SD/NR (7,400 mg); p=0.004. Importantly, overall responses were unaffected by FcgRIIIA-158 polymorphism status (75% vs. 71% for VV/FV vs. FF); serum IgM (83% vs. 80% for <6,000 vs. >6,000 mg/dL); and B2M levels (86% vs. 78% for <3 vs. ≥ 3 g/dL); p=NS. Lastly, following completion of therapy, 15/18 responding patients reported improvements in quality of life.

CONCLUSION: These studies demonstrate that thalidomide in combination with rituximab is highly active in WM, though additional studies are warranted to determine the ideal dose and schedule for thalidomide in view of toxicities. Moreover, thalidomide in combination with rituximab may overcome unfavorable prognostic determinants previously reported with rituximab monotherapy in WM.
Novel Fusion of the MALT1/MLT and MAP4 Genes Detected in a Diffuse Large B-Cell Lymphoma.
Authors: Murga Penas Eva M. , Hinz Kristina , Behrmann Petra , Hansmann Martin L. , Becher Claudia , Siebert Reiner , Dierlamm Judith ,   on Behalf of the Network Project “Molecular Mechanisms of Malignant Lymphomas”
Published: October 2019

The MALT1/MLT gene codes for a human paracaspase and plays a crucial role in NF-κB activation in response to TCR induction. Rearrangements of MALT1/MLT by the translocations t(11;18)(q21;q21) and t(14;18)(q32;q21) act by activating the NF-κB pathway and are the most frequent structural chromosomal abnormalities in extranodal marginal zone B-cell lymphomas of the MALT-type. They occur in 20 to 50% of low grade MALT lymphomas, but with only rare exception are absent in diffuse large B-cell lymphomas (DLBCL).

By screening 118 diffuse large B-cell lymphomas and 28 Burkitt’s lymphomas by interphase FISH with probes flanking MALT1/MLT (Abbott Vysis), we found two cases with a break within MALT1/MLT. Further experiments with plasmid subclones from the BAMH1 fragments of PAC 152M5, which spans the entire coding region of MALT1/MLT, confirmed the break within MALT1/MLT in one case. This patient presented with a relapse of a DLBCL, centroblastic subtype, without any concomitant low-grade component or lymphoepithelial lesions. During relapse only nodal manifestations were seen (stage IIIA), however, at the time of primary diagnosis a lymphoma infiltration of one adrenal gland was present. FISH experiments with MALT1/MLT, API2 and IGH specific probes did not identify a t(11;18) or a t(14;18). 3′ RACE-PCR revealed a novel in frame fusion of exon 9 of the MALT1/MLT gene and exon 9 of the microtubule-associated protein 4 (MAP4) gene. RT-PCR with specific primers for MALT1/MLT and MAP4 confirmed the presence of the fusion transcript MALT1/MLT-MAP4. In addition, interphase FISH showed that the translocation was accompanied by a deletion of MALT1/MLT sequences distal to the breakpoint including the caspase-like domain, which is essential for activation of the NF-κB pathway in MALT lymphomas. Corresponding to this result, 5′ RACE-PCR with specific primers for MALT1/MLT and RT-PCR with nested primers for MAP4 and MALT1/MLT did not detect the reciprocal 5′MAP4-3′MALT1/MLT transcript. The MAP4 gene on chromosome 3p21 is the major microtubule-associated protein in non-neuronal tissues and promotes microtubule polymerization. Disruption of the microtubule-dynamics is induced by microtubule-interfering drugs, such as taxanes and Vinca alkaloids.

We conclude that the 5′MALT1/MLT-3′MAP4 fusion is the pathogenetically relevant transcript in this non-reciprocal der(18)t(3;18)(p21;q21) translocation. The absence of the caspase like domain distinguishes this novel gene fusion, MALT1/MLT-MAP4, from the API2-MALT1/MLT in the t(11;18) and the IGH-MALT1/MLT in the t(14;18), in which the caspase like domain is invariably present and points to MAP4 as a new target gene with possible therapeutic implications in DLBCL.
RNAi Silencing of TEL-AML1 Reveals a Heat Shock Protein Dependent Mechanism in the Development of Leukemia.
Authors: Diakos Christofer , Gerner Christopher , Inthal Andrea , Gadner Helmut , Panzer-Grümayer Renate
Published: October 2019

The t(12;21) chromosomal translocation generating the TEL-AML1 fusion product is the most frequent genetic aberration of childhood acute lymphoblastic leukemia. This translocation has been shown to occur in utero and thus represents an early or even initiating event in leukemia development. However, additional molecular changes are necessary for the clinical manifestation of the disease. Despite increasing knowledge on TEL and AML1, the role of the TEL-AML1 fusion gene and its contribution to the malignant transformation is largely unknown. In particular, the mechanisms by which TEL-AML1 influences the biology of the affected cell, and thus survival and expansion, are yet to be defined. The aim of this study was to investigate the functional contribution of TEL-AML1 to the leukemic cell phenotype. We particularly sought to search for TEL-AML1 dependent genes as well as for pathways that function in a fusion gene dependent manner. The t(12;21) positive B cell precursor leukemia cell line REH was used as model system and RNA interference technology, a new and highly specific approach, was employed to block the expression of the TEL-AML1 fusion gene. The successful silencing of TEL-AML1 was demonstrated by Western blot analysis. In order to gain a broad overview of the impact of TEL-AML1 silencing upon the leukemic cell, RNA was isolated and subjected to Affymetrix gene chip analysis. In addition, we studied the proteome by high-resolution 2D electrophoresis. Expression profiling resulted in a specific pattern of differentially regulated genes after TEL-AML1 suppression with a particular emphasis on genes involved in the control of apoptosis. Genes with anti-apoptotic properties were down regulated after TEL-AML1 silencing while those that contribute to apoptosis were largely unaffected. Among the affected anti-apoptotic genes were the heat shock proteins, the most prominent representatives of which were HSP90 and HSP70. Their changes were observed at the mRNA but also at the protein level. This is of special interest, since both heat shock proteins interfere with the intrinsic as well as the extrinsic pathway of apoptosis and hence provide a powerful anti-apoptotic signal. These first data support a model in which TEL-AML1, as an early event, is functionally linked to an anti-apoptotic network and especially to the members of the heat shock protein family. Engagement of these mechanisms might rescue a cell from apoptosis and in consequence give rise to the development of a pre-leukemic clone, a reservoir for the acquisition of further mutations that might eventually give rise to leukemia. Thus, decreasing the apoptosis defence by silencing TEL-AML1 or by inhibiting the heat shock proteins might sensitize TEL-AML1 positive cells to apoptosis. This would not only be an option for future therapy of leukemias but also for the eradication of the pre-leukemic clone as a proposed source of relapse.
Immune-Mediated Protection Against BCR/ABL-Induced Leukemia: A Common Pathway Shared between IRF8/ICSBP and IFN alpha and beta.
Authors: Nardi Valentina , Mohammed Azam , Daley George Q.
Published: October 2019

Most Chronic Myeloid Leukemia (CML) patients are treated with the targeted agent imatinib (Gleevec), but cytogenetic remission is rarely achieved and the only cure remains bone marrow transplantation. The mainstay of therapy prior to imatinib was interferon (IFN) alpha, which induces cytogenic remission in a minority of patients. Because clinical response to IFN alpha correlates with immune system reactivity against leukemic clones, immunotherapy may be an important adjunct to imatinib. ICSBP (Interferon Consensus Sequence Binding Protein) is a tumor suppressor of CML. Ectopic expression of ICSBP in Bcr-Abl transformed cells prevents the formation of a lethal leukemia, and we have shown that this anti-leukemic effect is mediated by a long-lasting and potent CD8+ response against unknown epitopes on the leukemic cells. We have begun to characterize the immune response against CML induced by ICSBP and to understand its relationship to the anti-leukemic effects of IFN alpha. IFN alpha and IFN beta can substitute for ICSBP expression in inducing potent anti-leukemic immunity against BCR-ABL transformed cells. We could find little evidence for the direct regulation of the IFN genes by ICSBP; thus we have favored the alternative hypothesis that ICSBP and IFNs alpha and beta activate a common pathway that ultimately triggers an immune-mediated anti-leukemic response. To test this hypothesis, we compared the transcriptional profiles of BaF3 cells to those overexpressing Bcr-Abl, ICSBP, or both (unpublished data). Our data confirm the reported effects of ICSBP on genes that regulate proliferation and apoptosis in cells overexpressing Bcr-Abl (e.g. cyclin D2). In addition, our analysis revealed an interesting new mechanism through which ICSBP may stimulate an anti-leukemic immune response and Bcr-Abl expressing cells may escape immune detection. IFN alpha and beta appear to share the mechanism induced by ICSBP. Our data confirm the differential expression of several known (e.g. Proteinase 3) as well as new potential protein epitopes of the anti-leukemic immune response induced by ICSBP and the IFNs. Our in vivo experiments should shed further light on the role of the immune regulatory genes we have identified in the immune response to CML.
c-MYC Is a Major Downstream Target of NOTCH in T-Cell Acute Lymphoblastic Leukemia.
Authors: Lau Arthur , Aster Jon C. , Weng Andrew P.
Published: October 2019

We recently reported that activating mutations in the NOTCH1 receptor occur in a high percentage of primary human T-cell acute lymphoblastic leukemias (T-ALL). Withdrawal of NOTCH signals by treatment with γ-secretase inhibitors (GSI) or by transduction with a dominant-negative Mastermind-like-1 polypeptide (a specific NOTCH pathway inhibitor) induces growth arrest of many T-ALL cell lines, suggesting that NOTCH supplies signals that are needed for maintenance of growth of T-ALL cells. In order to identify downstream targets of NOTCH that mediate these effects, we performed gene expression profiling on NOTCH signaling-dependent T-ALL cell lines before and after NOTCH inhibition. Among a number of identified candidate genes was c-MYC, which was of particular interest given its importance in promoting cellular growth and its known dysregulation in a number of hematolymphoid neoplasms. c-MYC was down-regulated following NOTCH inhibition, and rapidly up-regulated following release of NOTCH inhibition, even in the presence of protein synthesis inhibitors, suggesting that it is a direct NOTCH transcriptional target. Further, a subset of murine and human T-ALL cell lines were rescued from GSI-mediated growth arrest by c-MYC-expressing retroviruses. The failure of c-MYC to rescue some NOTCH-dependent cell lines likely stems from differences in cellular context, such as collaborating oncogenic lesions and/or the stage of T cell development the cell lines recapitulate. Nevertheless, these data implicate c-MYC as a major downstream target of NOTCH in T-ALL.
Adenovirus Capsid Hexon Is the Main Target Protein of Adenovirus-Specific CD4+ T-Cells: Fundamentals for Targeting Adenovirus by Adoptive Immunotherapy.
Authors: Arbach Olga , Frentsch Marco , Voigt Sebastian , Chmielewicz Barbara , Kaiser Marco , Ellerbrok Heins , Lauster Roland , Radbruch Andreas , Scheffold Alexander , Ebell Wolfram , Thiel Andreas
Published: October 2019

Introduction: Increasing evidence has emerged that adenoviruses (AdV) can cause severe complications in immunocompromised hosts especially following hematopoietic stem cell transplantation (HSCT). So far protective immune responses in adenovirus infection have not been assessed in detail, but control of infection seems to be dependent on CD4+ T-cells. However, the main antigenic adenovirus targets for specific CD4+ T-cells are unknown.

Methods: We here use a novel technique to directly assess the entire repertoire of CD4+ T-cells specific for a defined antigen according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. Additionally, we evaluated the cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells.

Results: AdV-lysate-specific CD4+ T-cells reacted efficiently with AdV-hexon and penton proteins. Furthermore, CD4+ T-cells specific for hexon protein derived from AdV-serogroup B crossreact with recombinant hexon protein derived from various other AdV-serotypes and are characterized by a Th1-like cytokine profile.

Conclusion: Our results imply that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive proinflammatory CD4+ T-cells for adoptive T-cell therapies.
Study on Methylation of p15INK4B Gene and Its Mechanisms in Patients with Myelodysplastic Syndrome and Leukemia.
Authors: Tong Hongyan , Ling Maofang , Jin Jie
Published: October 2019

The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in the mononuclear cells (MNCs) from bone marrow of 54 cases with hematopoietic malignances were detected by using RT-PCR, Western blot, and methylation-specific PCR. Of the 54 patients, 10 cases were low-risk MDS, 10 cases were high-risk MDS, 10 cases were acute myeloid leukemia (AML), 10 cases were acute lymphocytic leukemia (ALL), 10 cases were chronic myeloid leukemia in chronic phase (CML-CP), and 4 cases were CML in blast phase (CML-BP). 10 normal persons were studied as nective controls. The results showed that the incidence of p15INK4B methylation in cells of high-risk MDS was higher than that in low-risk MDS (6/10 VS 1/10, P=0.003), and the p15INK4B methylation was found to be associated with the down-regulation of the expressions of p15INK4B gene on both mRNA (r=−0.734, p<0.001) and protein (r=−0.664, p=0.001)levels, which indicated that the silencing of p15INK4B gene was in conjunction with hypermethylation in MDS. The expressions of p15INK4B on mRNA level and protein levels were almost detected in the MNCs from bone marrow of normal persons without the p15INK4B methylation. We also found the expression of DNMT3A and DNMT3B in high-risk MDS (densitometry readings respectively: 0.624±0.146, 0.577±0.344) were higher than in low-risk MDS (densitometry readings respectively: 0.487±0.300, 0.338±0.290) (P<0.05). The expression of DNMT1 was higher in the groups of low-risk MDS, high-risk MDS, AL and CML-CP( densitometry readings respectively: 0.487±0.218, 0.697±0.243, 0.706±0.463 and 0.867±0.375) than in normal control (densitometry reading: 0.181±0.312)(P<0.05, figure listed bellow), which indicated that up-regulated DNMTS might contribute to the hypermethylation of p15INK4B, and the higher expressions of de novo methyltransferases DNMT3A and DNMT3B may be related to the disease progression of MDS. The methylation of p15INK4B was also detected in 9/20 of AL cases accompanied by over-expressions of DNMT1, DNMT3A, and DNMT3B (densitometry readings respectively: 0.706±0.463, 1.066±0.547, and 0.530±0.428). The methylation of p15INK4B was detected in 1 of 10 cases of CML-CP patients, but all be detected in 4 case of CML-BP patients. These results indicated that the hypermethylation of p15INK4B gene may be one of the most common genetic event in pathogenesis of high-risk MDS, acute leukemia, and blast phase of CML. Furthermore, DNMT3A and DNMT3B were substantially over-expressed in the bone marrow cells of these patients. which might play an important role in the transformation from MDS to acute leukemia.

Figure Figure
Creation of a Ribosomal Protein Sa/LAMR1 Heterozygous Mouse.
Authors: Ellis Steven R. , Logsdon Paula M. , Han Junying
Published: October 2019

Diamond Blackfan anemia (DBA) is a severe hypoplastic anemia that generally presents early in infancy. Approximately 25% of DBA cases have been linked to mutations in the gene encoding ribosomal protein S19. The remaining cases are of unknown etiology. Our studies in yeast have identified a specific role for Rps19 in the maturation of 40S ribosomal subunits. While only one other small subunit ribosomal protein, Rps18, has a function virtually identical to Rps19 in subunit maturation, several others have functions closely related to Rps19. If the involvement of Rps19 in DBA is linked to its role in the synthesis of 40S ribosomal subunits, we would expect that one or more of these other ribosomal proteins may be responsible for DBA in patients with normal RPS19. To address a potential role for ribosomal proteins other than Rps19 in DBA we have created a transgenic mouse heterozygous at the LAMR1 locus. LAMR1 encodes ribosomal protein Sa, the mammalian homolog of the yeast ribosomal protein S0. Rps0, like Rps19, is required for the maturation of the 3′ end of 18S rRNA. We are currently in the fourth generation of out-crossing the original 129SvEv/C57BL6J chimeras to a C57BL6 background. The frequency of heterozygous pups is about that expected by Mendelian segregation suggesting that haploinsufficiency for ribosomal protein Sa does not lead to significant embryonic lethality. The heterozygous mice do, however, exhibit a higher frequency of craniofacial abnormalities and premature deaths relative to their wild-type littermates. The overall fitness of the heterozygous mice appears to be decreasing with each generation of outcrossing to the C57/BL6 background. Efforts are underway to understand the nature of the premature deaths and to obtain detailed hematological profiles on the LAMR1 heterozygous mice.
The Molecular Signature of Iron Metabolism in Polycythaemia Mice.
Authors: Schumacher Armin , Mok Henry , Mlodnicka Agnieszka E. , Hentze Matthias W. , Muckenthaler Martina
Published: October 2019

Recent positional cloning of the radiation-induced polycythaemia (Pcm) mutation revealed a 58-bp microdeletion in the promoter region of ferroportin 1 (Fpn1), the sole cellular iron exporter identified to date. The microdeletion causes aberrant transcription initiation and results in the absence of the iron-responsive element in the 5′ untranslated region of the vast majority of Fpn1 transcripts, thereby disrupting translational regulation of Fpn1 expression. Pcm mutant mice exhibit the gamut of iron balance disorders, ranging from iron deficiency at birth to tissue iron overload by young adulthood. Consistent with the perinatal iron deficiency, Pcm pups display a microcytic, hypochromic anemia. Strikingly, the majority of young adult Pcm heterozygous animals display a transient erythropoietin (Epo)-dependent polycythemia with peak hematocrits of up to 80%, eponymous of the mutant strain. Here we report a molecular definition of the regulatory mechanisms governing the dynamic changes in iron balance in Pcm heterozygous mice between 3 and 12 weeks of age. Therein, hepatic and/or duodenal response patterns of iron transporters, such as Trfr, cybrd1 and Slc11a2, defined the transition from early postnatal iron deficiency to iron overload by 12 weeks of age. A significant delay in developmental upregulation of hepcidin (Hamp), the pivotal hormonal regulator of iron homeostasis, correlated with high levels of Fpn1 expression in hepatic Kupffer cells during postnatal development. Conversely, upon upregulation of Hamp expression at 12 weeks of age, Fpn1 expression decreased, indicative of a Hamp-mediated homeostatic loop. Aged cohorts of Pcm mice exhibited low levels of Fpn1 expression in the context of an iron-deficiency erythropoiesis and profound iron sequestration in reticuloendothelial macrophages, duodenum and other tissues. Similar to the anemia of chronic disease, these findings are consistent with decreased iron bioavailability due to sustained downregulation of Fpn1 levels by Hamp. Therefore, iron-deficiency erythropoiesis marks both the beginning and the endpoint of the hematopoietic defects in Pcm mice. However, whereas the embryonic/perinatal anemia results from primary organismal iron deficiency, adult Pcm mice develop anemia due to decreased iron bioavailability despite organismal iron overload. The polycythemia develops at the transition phase between the two disease states, governed by unimpeded Epo signaling. We conclude that regulatory alleles, such as Pcm, with highly dynamic changes in iron balance are ideally suited to interrogate the genetic circuitry regulating iron metabolism.
Cooperative Silencing of Chicken ρ- and βH- Globin Gene Expression by Chicken β-Globin Regulatory Elements: Involvement of RNAi Mediated Repression.
Authors: Epner Elliot M. , Wang Jin , Huang Jing
Published: October 2019

The chicken β-globin locus represents a well characterized, model system where the relationship between chromatin structure, transcription and DNA replication can be studied. The locus contains several regulatory elements including an intergenic enhancer as well as upstream regulatory elements that may function either alone or in combination with the intergenic enhancer as an LCR. The availability of the recombination proficient chicken B cell line DT40 has allowed the introduction of mutations into the endogenous chicken β-globin locus and phenotypic analysis after microcell mediated chromosome transfer into human erythroleukemia (K562) cells. Using this system, we have introduced deletions in the chicken β-globin intergenic enhancer as well as 5′ HS 1,2, and 3. Expression of the embryonic ρ and fetal βH chicken globin genes were repressed by the intergenic enhancer, 5′ HS1, or 5′HS2. No ρ or βH globin gene expression was detected in K562 cells containing control chicken chromosomes, while ρ and βH mRNA were activated when the intergenic enhancer, 5′ HS1, or 5′HS2 were deleted. Chromatin immunoprecipitation (ChIP) experiments that assayed RNA polmerase II (pol II), GATA-1 and NF-E2 p45/ p18 binding at regulatory elements and gene promoters in targeted cell lines supported this hypothesis and suggested a potential role for 5′HS3 in gene activation. However, targeted deletion of 5′ HS3, unlike the other chicken β-globin regulatory elements, showed no transcriptional phenotype. Our results demonstrate the intergenic enhancer, 5′HS1, and 5′ HS2 function through a common silencing mechanism involving pol II, GATA-1, and NF-E2/P18. The recent demonstration of the involvement of Pol II in the synthesis of miRNA’s prompted us to investigate the role of miRNA’s in gene silencing in this system. A small miRNA was identified at the intergenic enhancer region. ChIP assays showed the binding of two components of the RISC (Dicer and Ago2) at the chicken globin regulatory elements. These results are consistent with the involvement of RISC and miRNA’s in gene silencing in this system.
Over-Expression of Prolylcarboxypeptidase Enhances Plasma Prekallikrein Activation on Chinese Hamster Ovary Cell Membranes.
Authors: Shariat-Madar Zia , Rahimy Ehsan , Mahdi Fakhri , Schmaier Alvin H.
Published: October 2019

Recent investigations indicate that there is a physiologic plasma prekallikrein (PK) activator on the membrane of endothelial cells (HUVEC). PK complexes with high molecular weight kininogen (HK) and HK is PK’s receptor on HUVEC. When assembled on HUVEC, PK is activated to plasma kallikrein independent of FXIIa by the serine protease prolylcarboxypeptidase (PRCP) (Km=9 nM). PRCP has been shown to be a PK activator when isolated from HUVEC (JBC277:17962, 2002) and produced as a recombinant protein (Blood103:4554, 2004). To additionally confirm that human PRCP is a physiologic PK activator, PRCP was over-expressed in Chinese hamster ovary cells (CHO). CHO were transfected with full-length PRCP under the control of a CMV promoter and CHO rPRCP was expressed as a fusion protein with C-terminal enhanced green fluorescent protein (EGFP). The presence of rPRCP in transfected CHO was detected by real time RT-PCR, immunoblot and immunoprecipitation. In CHO cells, PRCP mRNA and PK activation are 2–3-fold higher than controls. The increase in PRCP-induced PK activation on the transfected CHO cells parallels the increase in PRCP antigen expression on the transfected cells, as determined by anti-PRCP and anti-GFP antibodies. PK activation of the transfected cells is blocked by siRNA to the translation initiation site of PRCP, anti-PRCP antibody, and Z-Pro-Pro-aldehyde dimethyl acetate, with IC50 of the last 2 inhibitors of 0.01 and 7.0 mM respectively. Cellular localization of PRCP in intact cells using confocal microscopy and flow cytometry indicate that its over-expression results in its placement on the CHO cell plasma membrane. These investigations independently confirm that prolylcarboxypeptidase is expressed on cell membranes and its expression increases PK activation. The endothelial cell membrane expression of PRCP resulting in PK activation contributes to the constitutive level of bradykinin in the intravascular compartment to regulate blood pressure homeostasis.
Low Levels of Zinc and Anti-Oxidant Vitamines (A, C & E) in Patients with Sickle Cell Anemia.
Authors: Hasanato Rana M.
Published: October 2019

Background : Patients with sickle cell anemia have a higher potential for oxidative damage due to chronic redox imbalance in their red blood cells that often leads to hemolysis, endothelial injury and recurrent vaso-occlusive episodes. In this study, we evaluated plasma levels of the anti-oxidant vitamins A, C,& E and serum level of Zinc along with serum level of Copper (pro-oxidant ).

Patients and methods: 25 adult patients with documented severe sickle cell anemia and frequent painful episodes (12 males & 13 females, aged 29.27 +/− 12.94 years) and 25 matched normal controls were studied. Plasma levels of Vitamins A, C, & E were measured by HPLC technique and serum levels of zinc & copper were measured by atomic absorption spectrometry.

Results: (Table ): Conclusion These results indicate that levels of Vitamines A, C, and E and Zinc are significantly low in patients with sickle cell anemia in comparison with matched normal controls. Besides, Copper level, which is a well known pro-oxidant, is significantly higher in sickle cell anemia patients. Therefore, supplementing sickle cell anemia patients with anti-oxidant vitamins and trace elements may contribute to the amilioration of symptoms and severity of sickle cell anemia

Levels of Vitamine A, C, & E , Zinc & Copper in patients with Sickle cell anemia and normal controls Tested Items SCS patients Control group P-value Results are shown as mean +/− standard deviation., SCA = Sickle cell anemia Vitamin A level mcg/l 0.13 +/− 0.03 0.33 +/−0.05 <0.0001 Vitamin C level mg/l 9.00 +/− 3.43 20.32 +/−3.74 <0.0001 Vitamin E level, mcg/l 1.95 +/− .0.69 4.30 +/−0.80 <0.0001 Zinc level mmol/l 9.00 +/− 1.89 11.81 +/−1.93 <0.0001 Copper level mmol/l 24.58 +/− 4.87 18.42 +/−3.06 <0.0001
Expression of Transcription Factors NFAT1, c-Jun, c-Fos, and Bach2 in Umbilical Cord Blood and Adult Blood T-Cells.
Authors: Huang Ying , Leahy Patrick , Kaminski Beth A. , Daum-Woods Kathy , Veigl Martina , Laughlin Mary J.
Published: October 2019

Background: Nuclear factor of activated T cells 1 (NFAT1 or NFATc2) is proposed to plays a role in Graft versus host disease (GVHD) because it regulates the transcription of many cytokines and surface regulatory proteins including: cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), IL-13, IFN-γ, CD40L, granulocyte-macrophage colony-stimulating factor (GM-CSF), and TNF-α. NFAT1 regulates the expression of these genes through direct interaction with the leucine zipper region of AP-1 (Fos/Jun) and through cooperative binding to a 15-bp DNA sequence that contains both NFAT1 and AP-1 sites. The highly conserved basic regions of AP-1 (Fos/Jun heterodimer) bind to the TGTTTCA consensus sequence in DNA. Human Bach2 is also a basic-leucine zipper (bZip) protein. The amino acid residues in contact with the DNA in the basic regions of c-Jun and c-Fos are also identical in Bach2 (human and mouse). The high conservation of DNA-contacting amino acid residues between Bach2 and AP-1 strongly suggests that Bach2 (homodimers) and AP-1 (Fos/Jun heterodimers) can bind to the same DNA site, which may block the formation of NFAT1/AP-1/DNA complex and the subsequent expression of cytokine genes. Clinical studies have determined that umbilical cord blood (UCB) elicits reduced incidence and severity of GVHD, compared to Bone Marrow (BM), due in part to reduced donor T-cell cytokine production.

Methods: We compared the RNA and protein levels of NFAT1, c-Jun, c-Fos, and Bach2 in human UCB CD4+ T-cells with Adult Blood (AB) CD4+ T-cells by Quantitative RT-PCR analyses and Western blots. This comparison was done in both stimulated and non-stimulated conditions. Our ongoing interest is to test whether Bach2 binds the same DNA site as AP-1, and to test the potential of Bach2 and AP-1 competing for the same DNA site using Electrophoretic mobility shift assay (EMSA) with purified Protein of Bach2, c-Jun, and c-Fos from E. coli.

Results: Our data showed a high expression of Bach2 RNA in both non-stimulated and stimulated UCB CD4+T-cells. In both the non-stimulated comparison and stimulated comparison samples, we detected no significant changes in RNA levels of NFAT1, c-Jun, or c-Fos.

Conclusions: Our data support the hypothesis that Bach2 acts as a transcriptional repressor of cytokine genes due to the fact that highly expressed Bach2 in UCB competes with AP-1 for same DNA binding site.
Clinical Application Study of Plasma Thrombmodulin.
Authors: Lu Xingguo , Tong Jiefeng , Zhang Qiang , Zhang Mao , Zhao Xiaoying , Fu Gousheng , Xu Genbo
Published: October 2019

Objective: To investigate the clinical value of plasma thrombmoclulin (PTM).

Methold: PTM was determined in 979 patients and 60 healthy controls with ELISA method.

Result: The PTM of the control group was 20.40±7.72 μg/L, there was no difference in genders and ages. The PTM of the patients with microangiopathy were higher than that of the patients with macroangiopathy (P <0.01). We defined PTM level higher than its normal upper limit (35.54μg/L) as PTM positive, in microangiopathy diseases, the sensitivity of PTM was 77.7%, the specificity was 71.2% and the positive predictive value was 75.6%. In chronic primary glomerular disease, the PTM of chronic renal failure (CRF) group was higher than that of non-CRF group (P <0.01), the sensitivity was 85.7%, the specificity was 82.4%, the positive predictive value was 77.8%. The PTM level in septemia group was higher than that of non-septemia group(P <0.01),the sensitivity of PTM was 86.6%, the specificity was 89.5% and the positive predictive value was 76.5%. In multiple trauma, the PTM of multiple organ failare (MOF) group was higher than that of non-MOF group (P <0.01), the sensitivity of PTM was 77.8%, the specificity was 77.3% and the positive predictive value was 73.7%. In systemic lupus erythematosus (SLE), the PTM of the patients with albuminuria was higher than that of the patients without albuminuria(P <0.01), the sensitivity was 77.8%, the specificity 92.3% and the positive predictive value was 93.3%. In diabetes, the PTM of complication group was higher than that of no complication group, the sensitivity was 53.4%, the specificity was 97.1% and the positive predictive value was 98.6%, the PTM of microangiopathy group was higher than that of macroangiopathy group (P <0.01), the sensitivity was 71.2%, the specificity was 97.1%, and the positive predictive value was 97.9%. Compared the acute stage with restoration stage in stroke, pre-chemotherapeutics with post-operation in AL and MM, and pre-operation with post-operation in cancer, the PTM was connected with clinical development.

Conclusion: PTM was a good criterion in evaluating the microangiopathy in clinical application. PTM was also a valuable index in prediction or assessment of the severity of diseases or in the observation of therapeutic effectiveness.
Thoracal Carcinoma Surgery in FVII Deficient Patient Managed with rFVIIa.
Authors: Erdlenbruch Kerstin , Stephan Bernhard , Bakchoul Tamam , Schenk Joachim , Pindur Gerhard
Published: October 2019

Introduction: In patients with congenital FVII deficiency and positive bleeding history surgical procedures should be covered by an sufficient substitution therapy of FVII concentrate. For the first three postoperative days trough level of at least >0,2U are recommended. We report haemostasis management of a complex thoracic surgery procedure in a 14 year old boy with FVII deficiency and diagnosis of a mucoepidermoid bronchogenic carcinoma of the left upper lobe. Extended thoracic surgery without extra corporal circulation was performed as a lobectomy of the left upper lobe including extended preparation of lymphnodes.

Preoperative Lab:

Platelets 285000/μl, INR 1,6, aPTT 28s, fibrinogen 239 mg/dl, FVII 0.33IU

Because of positve bleeding anamnesis, the extended surgical procedure and possibility of further extension of the planned procedure we decide to cover surgery by factor treatment to be on the safe side though FVII activity was 0.33IU. Because of virus safety and safety profile we decide to give rFVIIa (NovoSevenR) as bolus injections. Frequency of treatment was chosen according to regular lab controls and clinical course.

Haemostasis management during surgery:

Preoperative rFVIIa dosage: 30 μg/kg BW, given immediately before skin cut as a bolus. Intraoperativly was no significant bleeding and no transfusion requirements though it was a complex and extended procedure.

Haemostasis management postoperatively:

Regular control of FVII activity and Hb was done every 6 hours to decide if substitution therapy is required. In the early phase after surgery FVII activity remained >1.0 IU, so additional factor treatment was not necessary. Hb remained stable in normal range. 24h after surgery FVII activity fell to 0.16IU. Substitution was done with dosage of 15μg/kgBW rFVIIa as a bolus (NovoSevenR). Frequency: once daily, duration: 2 days Clinical course: no bleeding complications. For remove of drainges a last bolus of 30μg/kg was given. Residual FVII activity: >0,2IU in all regular controls during the first three days.

Figure 1: thromboplastin time, F VII activity and tFVIIa substitution in the course of time Figure 1:. thromboplastin time, F VII activity and tFVIIa substitution in the course of time

Results:

Bleeding/Transfusion requirements: No bleeding complications or wound healing complications occurred, no blood products has been transfused.

Adverse events: no adverse events, no thrombembolic events and no inhibitor development.

Conclusions: Regular control of FVII activity and clinical bleeding signs led to an efficient use of factor concentrates. rFVIIa given as bolus in FVII deficiency seem to be a save and efficient therapy for haemostasis management in complex thoracic cancer surgery.
Self-Managed Long-Term Low-Molecular-Weight Heparin Therapy Compared with Usual-Care Anticoagulation for the Treatment of Patients with Proximal Venous Thrombosis: Harm Associated with Thrombocytopenia.
Authors: Hull Russell D. , Pineo Graham F. , Brant Rollin F. ,   for the LITE Trial Investigators
Published: October 2019

Context: A substantial clinical need exists for an alternate to vitamin-K-antagonists for treating deep-vein thrombosis in many patients. Long-term low-molecular-weight heparin, body-weight adjusted, avoids anticoagulant monitoring and may be associated with less harm due to thrombocytopenia.

Objective: Considerable evidence suggests that reporting of harms-related data* from randomized clinical trials needs improvement. In accordance with CONSORT* we present a harm analysis regarding thrombocytopenia.

Design: Randomized clinical trial using objective outcome measures.

Setting: 30 centres across Canada.

Participants: Acute symptomatic proximal-vein thrombosis patients.

Intervention: Therapeutic tinzaparin subcutaneously once-daily, or intravenous unfractionated heparin/oral vitamin-K-antagonist therapy for 3 months.

Main Outcome Measures: Benefit was assessed by assessing the frequency of symptomatic objectively documented recurrent venous thromboembolism and harm by objectively documented hemorrhagic complications and the outcome of thrombocytopenia.

Results: Benefit and bleeding harm are reported elsewhere. Long-term low-molecular-weight heparin treatment was at least as effective but associated with less harm due to bleeding than usual care. Thrombocytopenia occurred in 21 of 369 (5.7%) patients receiving tinzaparin versus 9 of 368 (2.4%) patients receiving usual care (absolute difference 3.3). Six of 9 patients with thrombocytopenia receiving usual care died (66.7 percent, 95 percent confidence interval 29.9 to 92.5) versus 4 of 21 with thrombocytopenia receiving low-molecular-weight heparin (19 percent, 95 percent confidence interval 5.4 to 41.9 percent) (absolute difference 47.6 percent, 95 percent confidence interval 82.7 to 12.5 percent, p=0.03). Recurrent venous thromboembolism occurred infrequently in patients with thrombocytopenia.

Conclusion: Our findings suggest that harm associated with thrombocytopenia in patients receiving intravenous unfractionated heparin versus low-molecular-weight heparin favors low-molecular-weight heparin use. Further evaluation in rigorous trials is required.
TGF-β1 Affects the Proliferation of Normal Human Bone Marrow Fibroblasts and Its Expresssion of Smad3 and Smad7 mRNA.
Authors: Huang Jinwen , Jin Xing
Published: October 2019

Current drug therapy in myelofibrosis with myeloid metaplasia (MMM) still can not get great progress, the development of the myelofibrosis eventually causes bone marrow failure. The histopathology of MMM patients showed that the bone marrow tissue were full of marrow fibroblasts and an extensive deposition of collagens. Further researches suggested that the proliferation of the fibroblasts was controlled by many factors. One of the most important factor was TGF-β1, and its effects largely depended on Smad pathway. In order to understand the effects of TGF-β1 on the proliferation of marrow fibroblasts, we stimulated the normal human bone marrow fibroblasts with different concentrations of TGF-β1 in the current study. The proliferation test with direct cell counting and MTT assay revealed that different concentrations of TGF-β1 had various effects and it could get the maximal proliferation effect at 0.1ng/ml. Then we detected the Smad3 mRNA and Smad7 mRNA in one normal human bone marrow fibroblasts with RT-PCT methods at above concentration (0.1ng/ml). We found that the expression level of Smad3 mRNA decreased with the time, and that the expression level of Smad7 mRNA reached the highest at 30 min after stimulation by TGF-β1, then it began to reduce. These results suggested that TGF-β1 activate the Smads pathway in normal human marrow fibroblasts, and its proliferation effect require the activation of this pathway. We also noticed that the changes of the smad3 mRNA and smad7 mRNA were similar in skin fibroblasts stimulated by TGF-β1. These changes were considered as a feedback modulation mechanism. Whether the over-proliferation of marrow fibroblasts in MMM patients is correlated with the imbalance of this feedback warrants further research. It may provide a more specific and targeted therapy for blocking myelofibrosis.

Fig A The expression of Smad3 mRNA stimulated by TGF-β 1(0.1ng/ml) at different time. Fig B The expression of Smad7 mRNA stimulated by TGF-β 1(0.1ng/ml) at different time. Fig A The expression of Smad3 mRNA stimulated by TGF-β 1(0.1ng/ml) at different time. . / Fig B The expression of Smad7 mRNA stimulated by TGF-β 1(0.1ng/ml) at different time.
Molecular Responders (<3-log Reduction) among CML Patients in Complete Cytogenetic Remission Show a Lower Number of BCR-ABL+ Hematopoietic Progenitors Compared to Non-Responders.
Authors: Ibatici Adalberto , Fugazza Giusy , Sessarego Nadia , Podesta Marina , Agliano Stefania , Pozzi Sarah , Corselli Mirko , Garuti Anna , Sessarego Mario , Frassoni Francesco
Published: October 2019

Background. BCR-ABL+ leukaemic hematopoiesis in CML patients treated with the tyrosyn kinase inhibitor Imatinib mesylate can remarkably diminish up to undetectable levels by cytogenetic or molecular analysis. This treatment seems to target leukemic progenitors cells while sparing some quiescent BCR-ABL+ leukaemic stem cells. In this study, we investigated the number of residual BCR-ABL+ hematopoietic progenitors in bone marrow (BM) samples from CML patients who reached a complete cytogenetic remission (CCR) with Imatinib.

Materials and Methods. BM samples (15–20ml) were obtained from 18 CML patients in CCR under Imatinib treatment. All of them were in 1st chronic phase when Imatinib was started. A CD34+ cell-enriched population was selected from BM mononuclear cells using a double immunomagnetic column separation. CFCs and LTC-ICs were obtained by seeding the CD34+ selected population using standard culture methods. FISH studies for the BCR-ABL fusion gene (Dual color, Dual Fusion DNA probe form Vysis, INC, Downer Grove, IL) were performed on freshly selected CD34+ cells, pooled CFCs and LTC-ICs. A total of 100–200 nuclei were scored for each sample. The normal cut-off value used for these probes (0–2%) was established by our lab validation studies. MMR were defined as BCR-ABL/ABL ratios less than 0.05% (roughly equivalent to a 3-log reduction). Real-time QRT-PCR was performed using a TAQ-Man system (ABI Prism 7700 Perkin Elmer, CA) for BCR-ABL and ABL genes. Results. The median number and purity of selected CD34+ cells was 0.51 (0.17–2.1) x 106 cells and 95% (72–99). MMR was present in 10/18 (55.5%) CML patients. The median follow-up for patients in MMR or not in MMR was 34 (14–54) and 36 (6–59) months, respectively. None of the MMR-CML showed residual BCR-ABL+ cells detected by FISH on freshly selected CD34+ cells (MMR vs not MMR P=.0039 by Fisher’s test), CFCs and LTC-ICs. Conversely, 6 out of the 8 CMLs not in MMR showed residual BCR-ABL+ CD34+ cells, CFCs and LTC-ICs.

Conclusions. According to previous studies (Blood2003; 101: 4701–7), we found that CML patients in CCR under Imatinib may have residual BCR-ABL+ hematopoietic progenitors. However, the size of leukemic progenitors may differ substantially between MMR and not MMR. CML patients with more than 3-log reduction of disease show undetectable BCR-ABL+ progenitors (CD34, CFC and LTC-IC) by FISH analysis. QRT-PCR on bone marrow samples may provide a reliable estimation of the number of residual leukaemic progenitors and may identify different clusters of CML patients in CCR.
Identification of the Novel Chromosomal Translocation t(17;19)(q23;q13) in a Pediatric Patient with Acute Myeloid Leukemia.
Authors: Shardy Deborah L. , Azim Mohammed F. , Naeem Rizwan C. , Plon Sharon E.
Published: October 2019

Chromosomal rearrangements have been associated with many hematologic malignancies. Identification of the genes involved in several of these rearrangements has provided information about the development of malignancy and has led to therapeutic interventions. Historically, a considerable number of pediatric acute myeloid leukemia (AML) cases have been reported as cytogenetically normal. However, with improved cytogenetic techniques and the use of fluorescent in situ hybridization (FISH), new translocations are now being identified. We present the case of a 10-year-old male with AML (FAB subtype M1) and a subtle chromosomal translocation. G-band karyotype analysis revealed a balanced, reciprocal translocation between chromosomes 17 and 19 involving bands 17q23 and 19q13. This translocation was present in 20 out of 20 bone marrow cells examined. Peripheral blood chromosome analysis ruled out a constitutional chromosomal abnormality. Metaphase FISH with telomere-region specific probes for chromosomes 17 and 19 confirmed the reciprocal translocation between 17q and 19q. This patient was treated according to the SJCRH AML 2002 protocol and was randomized to receive high-dose cytarabine. Because he had minimal residual disease following induction therapy, he also received Gemtuzumab Ozogamicin. The patient was in cytogenetic remission for one year after completion of therapy, and then he relapsed with the original leukemic clone and additional cytogenetic abnormalities. The t(17;19)(q23;q13) has not been reported previously in malignancies or other disorders, and therefore identification of the genes at the chromosomal breakpoints may provide new insights into the pathogenesis of AML. As an initial step to map the breakpoint regions, we performed FISH with a commercially available probe encompassing the CRX, GLTSCR2, and GLTSCR1 loci on 19q13 (Vysis, Downers Grove, IL). This revealed that the 19q breakpoint is centromeric to these loci. We are further mapping the translocation breakpoint region on chromosome 19q using FISH-mapped bacterial artificial chromosomes (BACs).