bluuebunny/crossref_metadata_2025 huggingface dataset:
Deficiency in GATA-4 or GATA-6 Diminishes Definitive Hematopoiesis in Murine Embryonic Stem Cell Derived Embryoid Bodies.
Authors: Pierre Monique S. , Yoder Mervin
Published: April 2020

Formation of mesoderm derived blood islands in the mouse embryonic yolk sac requires the presence of visceral endoderm (VE) and VE derived factors. Murine embryonic stem (ES) cells can be differentiated into embryoid bodies (EBs) which serve as an in vitro model recapitulating many embryonic developmental processes, including formation of early hematopoietic cells. Previous investigators have reported that differentiation of ES cells deficient in either GATA-4 or GATA-6 results in EBs with disrupted differentiation of visceral endoderm and defective blood island formation. In the current study, we have compared GATA-4 and GATA-6 null ES cell derived EBs to wild-type EBs in their ability to commit to early hematopoietic lineages using hematopoietic progenitor colony assays, and used RT-PCR to assess the expression of endoderm genes. As expected, we observed differences in expression of endoderm genes in wild-type and GATA-4 or GATA-6 null EBs. Blast colony forming cell assays and primitive erythroid progenitor assays revealed no difference in the ability of wild-type and GATA-4 or GATA-6 null EBs to form hemangioblast or primitive erythroid progenitor colonies. In contrast, comparisons of definitive hematopoietic progenitor colonies from day 8, 9 and 10 GATA-4 and GATA-6 null EBs revealed a significant reduction in colony numbers at day 8 (p-values < 0.05) compared to wild-type. Strikingly, definitive progenitor colony numbers are rescued nearly to wild-type levels after the addition of the visceral endoderm derived factor vascular endothelial growth factor (VEGF) during EB differentiation. Furthermore, this rescue response can be blocked by the addition of soluble Flk-1 (VEGF receptor) to EB cultures. These results suggest that GATA-4 and GATA-6 transcription factors and/or visceral endoderm are not necessary for hemangioblast, primitive erythroid, or definitive progenitor emergence from EBs but play a role in definitive progenitor expansion in EBs.
Pulmonary Function Tests and Their Correlation with Tricuspid Regurgitant Jet Velocity in Pediatric Sickle Cell Disease Patients.
Authors: Arteta Manuel , Campbell Andrew D. , Minniti Caterina , Ensing Greg , Sable Craig , Rana Sohail , Darbari D. , Onyinye Onyekwere , Kato Greg J. , Gladwin Mark T. , Castro Oswaldo L. , Gordeuk Victor R.
Published: April 2020

Background: Adults and children with sickle cell disease (SCD) often have a restrictive pattern of pulmonary function and decreased DLCO. Echocardiography-determined tricuspid regurgitation jet velocity (TRV) ≥2.5 m/s occurs in about 30% of adults with SCD and is associated with increased mortality. Its prevalence and significance in children has not been established. We prospectively evaluated pulmonary function tests and their relationship to TRV in a multicenter study of children with SCD.

Methods: Patients with SCD (Hb SS, SC, Sb thalassemia or other major sickling phenotypes) aged 7 to 20 years were evaluated clinically and with spirometry, plethysmography, diffusing capacity, and echocardiography under basal circumstances. Controls were matched by ethnicity, sex and age to every sixth patient with SCD studied.

Results: Pulmonary function testing was performed in 115 of 267 children enrolled in the study as of August 2007 (93 had SCD and 22 were controls). TRV was measurable in 71 patients and 18 controls who had pulmonary function tests performed. FEV1 and FVC were significantly lower in children with SCD compared to controls and TRV was significantly higher, but FV1/FVC and TLC were not different. Among children with SCD, a higher TRV was associated with lower values for FEV1, FVC FEV1/FVC and TLC but not DLCO.

Conclusions: Similar to adults, pediatric patients with SCD had lower FEV1 and FVC than controls but almost identical FEV1/FVC suggesting restrictive lung dysfunction. In contrast to adults, DLCO was not lower among pediatric SCD patients than controls. Lower FEV1/FVC, a marker of obstructive lung dysfunction, correlated with higher TRV as did lower FVC and TLC, markers of restrictive lung dysfunction.

Table I. Summary of pulmonary function tests. Results in mean (SD) unless otherwise indicated SCD (n = 71) Control (n = 18) P *N = 52 **N = 9 ***n = 59 ****n = 13 Age (years) 13 (4) 12 (4) 0.2 Females in no. and % 30 (42%) 13 (72%) 0.023 FEV1 (% predicted) 89 (16) 99 (16) 0.025 FVC (% predicted) 93 (16) 104 (17) 0.018 FEV1/FVC (%) 84 (7) 84 (6) 0.9 TLC (% predicted) 89 (15)* 98 (18)** 0.2 Residual volume (% predicted) 105 (39)* 117 (46)** 0.5 DLCO adjusted for hemoglobin (% predicted) 99 (20)*** 88 (12)**** 0.019 TR velocity (m/sec) 2.3 (0.3) 2.1 (0.3) 0.021 Table II. Spearman Correlation of Pulmonary Function Tests with TRV in SCD Patients N R P value FEV1 (% predicted) 71 −0.299 0.011 FVC (% predicted) 71 −0.257 0.030 FEV1/FVC (%) 71 −0.257 0.030 TLC (% predicted) 52 −0.263 0.060 DLCO adj. for hemoglobin (% predicted) 59 −0.098 0.2
Epstein-Barr Virus: Main Pathogenic Agent in Hodgkin Lymphoma Associated Hemophagocytic Syndrome?.
Authors: Menard Fanny , Besson Caroline , Rince Patricia , Lambotte Olivier , Lazure Thierry , Canioni Danielle , Hermine Olivier , Brousset Pierre , Legendre Claire , Martin Antoine , Gaulard Philippe , Raphael Martine , Larroche Claire
Published: April 2020

Hemophagocytic syndrome (HPS) is characterized by an uncontrolled proliferation of macrophages displaying extensive phagocytosis of hematopoietic cells that can be associated with non Hodgkin lymphomas. However, Hodgkin lymphoma associated with hemophagocytic syndrome (HL-HPS) has rarely been published. We report here a large series of 34 patients presenting HL-HPS. Histological analysis and Epstein-Barr Virus (EBV) studies were performed. The 34 patients were enrolled from 1992 to 2006. HPS diagnosis was based on usual clinico-biological criteria. Bone marrow aspiration or biopsy were performed in all cases and confirmed HPS, showing hemophagocytic features in scattered macrophages. HL was documented on histological material in all 34 patients. The biopsies were performed on different localizations: lymph nodes (n=18), bone marrow (n=27), liver (n=2) and tonsil (n=1). All histological slides were reviewed and classified according to the World Health Organization (WHO) Lymphoma classification. The presence of EBV in the tumour cells was detected using EBER RNA in situ hybridization and the expression of viral proteins LMP-1, EBNA-2 by immunoperoxidase. Patients were 26 men and 8 women (sex ratio M/F = 3.3). The median age was 45 years (range 19–84). HIV status was negative in 26 patients and positive in 8 patients. All patients were in clinical stage IVB. HL subtypes (18 lymph nodes) were Mixed Cellularity (n=12, 67%), Nodular Sclerosis (n=2, 11%) and Lymphocyte Depleted (n=4, 22%). Extra nodal tissues were highly infiltrated by tumour cells in all cases. The presence of EBV in tumour cells was detected in 32 out of the 34 patients (94%). In all EBV positive cases, high levels of LMP-1 without EBNA2 expression were detected, defining a latency II. This study reports the largest series to date of HL-HPS. The features of HL-HPS are particular by the high proportion of mixed cellularity subtypes (67%) in contrast to the frequency of nodular sclerosis subtypes observed in the general population of non overt immunosuppressed patients, and by the striking high proportion of EBV associated HL (32 out of the 34 patients, 94%). Our findings suggest not only a pathogenic role of EBV, but also a defective immune system in the control of EBV infection in HL-HPS patients.The high predominance of male in our population of HPS-HL (sex ratio M/F = 4.3 after exclusion of HIV positive patients) and the strong association with EBV could suggest X-linked immune defect in these patients.
Oxygen Levels and HIV-1 Transcription.
Authors: Nekhai Sergei , Charles Sharroya , Ammosova Tatyana , Cardenas Jessica , Jerebtsova Marina , Ayodeji Abisola A. , Niu Xiaomei , Ray Patricio E. , Kashanchi Fatah , Gordeuk Victor R.
Published: April 2020

HIV-1 progression may be faster and survival shorter in HIV-positive patients with higher iron stores. Some studies indicate that iron stimulates HIV-1 viral replication and iron chelation decreases viral replication in cultured cells. Iron depletion may remove iron from prolyl hydroxylases and increases HIF-1a protein level thus mimicking hypoxia. Recent studies indicate that moderate hypoxia (3–6% O2) may be the physiological O2 concentration that macrophages and T cells encounter in certain organs in vivo. Cellular metabolism of macrophages and T cells cultured at atmospheric 21% O2 is significantly different from cells cultured at 3–6% O2 [1] . Interestingly, HIV-1 progression might be slower in sickle cell disease patients who have anemia-related hypoxia [2]. HIV-1 replicates efficiently in activated T cells or macrophages, in part due to the increased levels of NF-kB that enhance HIV-1 transcription in cooperation with HIV-1 Tat. HIV-1 Tat induces transcription of HIV-1 genes by recruiting host cell Cdk9/cyclin T1 to TAR RNA. Our recent studies point to a host cell kinase, CDK2, that phosphorylates HIV-1 Tat and induces HIV-1 transcription [3]. We also showed that iron chelators reduce cellular activity of CDK2 and inhibit HIV-1 transcription [4]. Here we analyzed the effect of 3% versus 21% O2 concentration on HIV-1 transcription and replication and cellular activities of CDK2 and CDK9. Using reporter assays, we found that 3% O2 inhibited HIV-1 Tat-induced transcription and viral replication. CDK9 activity but not CDK2 activity was inhibited in the cells cultured under 3% O2. Thus inhibition of CDK2 by iron chelators that we previously observed might not be directly related to upregulation of HIF1a. Rather, our results indicate that HIV-1 transcription is suppressed at physiological oxygen levels likely due to deregulation of CDK9 thus providing a new insight into the HIV-1 latency. These findings suggest that consideration should be given to studying the effect of small molecular inhibitors of HIF-1 on HIV-1 transcription and replication.
Inhibition of HIV-1 Tat-Induced Interleukin-10 Expression by Mononuclear Cells at 3% O2.
Authors: Edossa Amanuel , Gordeuk Victor R. , Nekhai Sergei
Published: April 2020

The production of IL-10, which counteracts the effect of pro-inflammatory cytokines such as TNF-alpha, is induced by HIV-1 Tat in monocytes/macrophages by a process regulated by CREB-1 transcription factors [1]. IL-10 has been reported to suppress HIV-1 replication in macrophages by inhibiting HIV-1 transcription [2] at a, but it has also been reported to contribute to cell-mediated immunity in the setting of HIV infection [3]. We compared Tat-induced expression of IL-10 by THP-1 monocytes at 3% O2 (20 mmHg), the tension that monocytes see in certain tissues, and atmospheric 21% O2 (150 mmHg), the conventional in vitro culture condition. THP-1 cells were treated with recombinant HIV-Tat protein in combination with or without lipopolysaccharide (LPS) and IL-10 expression was analyzed by RT-PCR and ELISA. Treatment of THP-1 cells with HIV-Tat and LPS resulted in a dose- and time-dependent increase in IL-10 expression in the cells cultured at 21% O2. In contrast under 3% O2 the expression of IL-10 was reduced by 3-fold. Treatment with tautomycin, a protein phosphatase-1 (PP1) inhibitor, prevented IL-10 expression in LPS-stimulated THP-1 cells cultured at 21% O2. Thus, PP1 is likely to participate in Tat-mediated IL-10 production. PP1 has shown to affect CREB activity [4]; therefore we propose that PP1 might be involved in the CREB-mediated IL-10 gene expression. The inability of Tat to induce IL-10 production under 3% O2 might reflect the inadequate response of HIV-1 infected macrophages in vivo that might permit viral replication in infected macrophages. Further investigation of the role of PP1 in IL-10 expression could lead to new therapeutic opportunities for HIV-1 infection.
A Rho-GTPase Effector, Rhotekin-2 (RTKN2) Is Associated with BMP8b and IL-16 Cytokine Expression and Increased Sensitivity to Apoptosis in Lymphocytes.
Authors: Collier Fiona M. , Baker Adele J. , Walder Ken , Stupka Nicole , Martin Sheree D. , Kirkland Mark A.
Published: April 2020

Rho GTPases are molecular switches controlling a broad range of cellular processes in lymphocytes including activation, immune response and gene transcription. They rapidly cycle between a guanosine diphosphate (GDP)-bound and guanosine triphosphate (GTP)-bound state, and the GTP-bound state is the active conformation that initiates downstream signaling pathways via effector proteins. We identified a novel Rho effector, RTKN2, that is highly expressed in lymphocytes, particulary freshly isolated CD4 T-cells; and is switched off in activated T-cells. When the protein was over-expressed in an unrelated cell line, it conferred resistance to apoptosis, and this resistance was maintained using the media alone from the over-expressing cells. In the current study we investigated genes relating to high or low expression of RTKN2 in lymphocytes. In human samples, RTKN2 expression was down regulated in PHA activated T-cells and it correlated with the expression of Zap-70, a Rho-guanine nucleotide exchange factor (GEF). We then utilized a real time cytokine array (Superarray Bioscience Corporation) to compare high versus low RTKN2 expression levels in the same population of lymphocytes. The real time array linked high levels of RTKN2 with cytokines BMP8b, (a TGF-beta family protein) and IL-16 (a modulator of T cell activation, and an inhibitor of HIV replication relationship), and this observation was confirmed in a series of T-cell samples. Further, we studied the effect of down regulation of RTKN2 in primary lymphocytes, using short hairpin (sh)RNA plasmid that also expressed GFP. Transfected GFP-positive cells had lower survival than cells transfected with scrambled (sh)RNA, and also demonstrated increased sensitivity to the the induction of apoptosis by oxysterols. These findings indicate that RTKN2 plays a role in survival of T-lymphocytes and this may be mediated through the secretion of cytokines.
Regulation of TLR Signaling by USP7.
Authors: Tan Yaohong , Rosenblatt Joseph D. , Federoff Howard J. , Bowers William J. , Tolba Khaled A.
Published: April 2020

USP7 is a 1102 animo acid de-ubiquitinating enzyme (DUB) that belongs to the ubiquitin-specific protease (USP) family. By virtue of its interaction with the tumor suppressor p53 and its negative regulator MDM2, USP7 plays a key role in regulation of cell survival and stress response. USP7 also interacts with two viral proteins, namely HSV-1 ICP0 and EBV EBNA-1, however the precise significance of this has not been fully understood. We have previously shown that HSV-1 ICP0 specifically inhibits TLR-mediated innate response to viral infection. In this work, we further investigated this observation, showing that ICP0 recruits USP7, a nuclear protein to the peri-nuclear zone where the latter interacts with TRAF6 and IKKγ. USP7 interaction with TRAF6 and IKKγ were mediated by its amino terminal TRAF domain and resulted in de-ubiquitination of both TRAF6 and IKKγ. Over-expression of USP7 but not the catalytically-inactive mutant C223S (where the active site cysteine was mutated to serine) inhibited TLR response by de-ubiquitinating both TRAF6 and IKKγ, and this effect was augmented by co-expression of ICP0. On the other hand, RNAi-mediated knock-down of endogenous USP7 augmented TLR response, comparable to that seen with A20 and Cyld knockdown and suggesting a significant degree of redundancy at the level of DUB-mediated regulation of TLR signaling. Confocal microscopy showed that association between ICP0 and USP7 alters the sub-cellular localization of USP7 from nuclear to cytoplasmic where it predominantly accumulates in the peri-nuclear region. We hypothesized that translocation of USP7 from nuclear to cytosolic would bring it into close proximity of its two substrates, TRAF6 and IKKγ. In support of this, over-expression of a modified USP7 encoding a nuclear-export signal (NES) and thus expressed in both cytosol and nucleus was associated with enhanced ability to suppress TLR response and de-ubiquitinate IKKγ. In summary, our results identify a novel function for the de-ubiquitinating enzyme USP7 as well as reveal an additional mechanism by which HSV evades host innate immunity.
The Axl/Gas6 Pathway Is Required for Human Natural Killer Cell Development.
Authors: Park Il-Kyoo , Giovenzana Chiara , Hughes Tiffany L. , Yu Jianhua , Trotta Rossana , Caligiuri Michael A.
Published: April 2020

Natural killer (NK) cells play an important role in host defense against microbial infection and tumors. Previous studies showed that IL-15 is essential for NK cell differentiation in vitro and in vivo. However the molecular mechanisms by which IL-15 is able to drive NK differentiation remain poorly understood. Here we show that blocking interaction between the receptor tyrosine kinase Axl and its ligands (Gas6 and protein S) by either soluble Axl/immunoglobulin Fc fusion protein (Axl-Fc) or warfarin, a vitamin K inhibitor, diminished the number and percentage of CD3−CD56+ NK cells differentiated from CD34+ human hematopoietic progenitors (HPCs) in blood and secondary lymphoid tissues in the presence of IL-15. Axl-Fc or warfarin increased HPC apoptosis, reduced the frequency of NK precursors, and resulted in impaired IFN-γ production. Mechanistically, in human CD34+ HPCs, Axl-Fc significantly inhibited IL-15-induced signaling events, such as phosphorylation of STAT5 and ERK. Taken together, our results suggest that the Axl/Gas6 pathway is important for IL-15-induced differentiation of NK cells from human CD34+ HPCs.
MicroRNA Expression Signatures Accurately Discriminate Acute Lymphoblastic Leukemia from Acute Myeloid Leukemia.
Authors: Mi Shuangli , Lu Jun , Sun Miao , Li Zejuan , Zhang Hao , Neilly Mary Beth , Wang Yungui , Qian Zhijian , Jin Jie , Zhang Yanming , Bohlander Stefan K. , Le Michelle M. , Larson Richard , Golub Todd R. , Rowley Janet D. , Chen Jianjun
Published: April 2020

Human acute leukemias include acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML). It is estimated that 5,200 and 13,410 cases will be diagnosed with and 1,420 and 8,990 will die of ALL and AML, respectively, in the United States in 2007. Although remarkable progress has been made in the past decades in the treatment and in the understanding of the biology of acute leukemias, the 5-year overall survival rate of patients with AML is only approximately 22%, which is much lower than that of ALL patients (65%; http://seer.cancer.gov). One of the most exciting recent findings is the discovery of an abundant class of small (∼22 nt), non-(protein-)coding RNAs, called microRNAs (miRNAs, miRs), which can function as oncogenes and tumor suppressors, whose deregulation is clearly associated with the development of cancer. To understand the distinct mechanisms in leukemogenesis between ALL and AML and to identify novel markers for diagnosis and treatment of acute leukemia, we have performed a large-scale miRNA expression profiling assay with a bead-based flow cytometric method and identified 27 differentially expressed miRNAs. Among them, miR-128a and b are significantly overexpressed while let-7b and miR-223 are significantly down-regulated in ALL compared to AML. They are the most discriminatory miRNAs between ALL and AML. Using the expression signatures of any two of the four most significantly discriminatory miRNAs in diagnosis of ALL and AML resulted in an accuracy rate of 97–100%. The differential expression patterns of these four miRNAs were validated further through quantitative real-time PCR on 98 acute leukemia samples covering most of the common cytogenetic subtypes of AML and B- and T-cell ALL, along with 10 normal controls. Furthermore, we found that overexpression of miR-128a and b in ALL was at least partly associated with hypomethylation, rather than amplification of DNA locus copy. Moreover, several important target genes of these four miRNAs have also been validated. We are currently exploring the role of these four miRNAs and their critical target genes in leukemogenesis and in the determination of lineage fate during leukemia development using in vitro and in vivo models. This work will enhance our understanding of the biological role of these miRNAs and their targets in leukemogenesis, and in determining the lineage fate of acute leukemia.
Mechanism by Which Exogenous Administration of Flt3 Ligand (FL) Expands Natural Killer Cells In Vivo.
Authors: Guimond Martin , Freud Aharon G. , Mao Hsiaoyin C. , Blaser Bradley W. , Lagemann Gerritt Gerritt , Vandeusen Jeffrey B. , Dorrance Adrienne , Ferketich Amy K. , Cavalcanti Marta , Mackall Crystal L. , Caligiuri Michael A.
Published: April 2020

The mechanism underlying the robust expansion of natural killer (NK) cells during exogenous administration of FL is unknown. Endogenous IL-15 had no impact on the in vivo expansion of NK cell precursors during FL administration but was required for the FL-mediated expansion of mature NK cells in the spleen and blood. Studies performed using in vivo BM chimeras showed that cells derived from hematopoietic precursors (HPC), not stromal cells, provided the endogenous IL-15 required for mature NK cell expansion by FL administration. Exogenous administration of FL significantly increased both CD11b(+)CD11c(-) and CD11b(+)CD11c(+) populations but not their relatively abundant expression of IL-15 or IL-15 receptor alpha on a per cell basis. This increase preceded and correlated with NK cell expansion, the latter of which largely resulted from enhanced survival and proliferation of an existing pool of mature NK cells rather than increased de novo production of NK cells from bone marrow precursors. Finally, in vivo elimination of CD11c+ cells during the course of FL treatment significantly decreased NK cell expansion. In summary, FL administration increases NK cells in vivo by expanding antigen presenting cells which in turn provide the requisite IL-15 to enhance survival and proliferation of mature NK cells.
FDG-PET/CT Is Superior to Anatomic Imaging for the Staging and Follow Up of Bony Involvement in Patients with Lymphoma.
Authors: Elstrom Rebecca L. , Brown Richard K.J.
Published: April 2020

Background and Objectives: Positron tomission tomography using 18fluoro-2-deoxyglucose in combination with low dose non-enhanced computed tomography (FDG-PET/CT) is increasingly utilized in the management of patients with lymphoma. Numerous studies have demonstrated improved accuracy for both staging and restaging as compared to standard diagnostic CT. However, there is a paucity of data on the significance of bone uptake in patients with lymphoma. This is one area in which FDG-PET has the potential to dramatically influence care of lymphoma patients. However, false positive FDG-PET has been shown in patients with traumatic or benign bone lesions. The aim of this study was to evaluate the utility and accuracy of FDG-PET/CT in comparison with standard anatomic imaging with CT and MRI in the staging and follow up of patients with Hodgkin lymphoma (HL) or diffuse large B cell lymphoma (DLBCL).

Design and Methods: We reviewed a database of 75 lymphoma patients who underwent concurrent FDG-PET/CT and standard diagnostic CT scans or MRI, and identified those with bone involvement by lymphoma. Involvement of bone was demonstrated by either biopsy of a bone lesion or radiologic appearance and clinical follow up highly suggestive of bone involvement. Follow up studies were evaluated for resolution of FDG avid lesions on PET, and anatomic lesions on CT or MRI.

Results: Fourteen patients with either HL or DLBCL who underwent both FDG-PET/CT and diagnostic CT were identified to have bone involvement by lymphoma. FDG-PET identified bone involvement in all 14 patients, whereas CT imaging identified bone involvement in seven. One patient in whom CT did not detect bone involvement had evidence of bone lymphoma by MRI. Eight patients had confirmation of bone lymphoma by biopsy, while 6 were confirmed by clinical criteria (radiologic appearance and clinical follow up). Thirteen of the patients had follow up FDG-PET/CT scans, and 12 had follow up CT and/or MRI. All follow up FDG-PET scans showed resolution of FDG avid bone lesions after anti-lymphoma therapy. In contrast, all CT and MRI scans which originally showed evidence of bone involvement had persistent abnormality on follow up, with only 2 showing improvement. At a median follow up of 9 months (range 0–20 months), 11 patients remain in remission, while 2 patients subsequently showed progression by FDG-PET, CT and biopsy in soft tissue sites, but not bone. One patient remains on therapy. No patient in our series was found to have a benign etiology of a lesion initially thought due to lymphoma.

Conclusion: FDG-PET/CT is useful in the staging and follow up of patients with lymphoma with bone involvement. The lack of sensitivity of CT combined with the delayed resolution of anatomic abnormalities limit the utility of standard anatomic imaging, making FDG-PET/CT the imaging modality of choice for patients with bone lymphoma.
Front-Line Treatment of Low-Grade Non-Follicular Non-Hodgkin Lymphoma (Final Report of Gisl LL02 Trial).
Authors: Goldaniga Maria , Merli Francesco , Stelitano Caterina , Callea Vincenzo , Ilariucci Fiorella , Russo Mario , Mazza Patrizio , Luminari Stefano , Rossi Francesca , Brugiatelli Maura , Luisi Dimitri , Olivero Barbara , Deliliers Giorgio Lambertenghi , Federico Massimo , Baldini Luca
Published: April 2020

Indolent Non-follicular non-Hodgkin Lymphoma (NFo-NHL) is a group of relatively frequent lymphoproliferative diseases, nevertheless extended clinical and prognostic studies are still lacking. In 2002 the Gruppo Italiano Studio Linfomi (GISL) initiated a LL02 prospective multicenter phase II trial, with the aim to evaluate the efficacy and safety of FC combination in the first-line therapy of NFo-NHL patients younger than 70 years. Between July 2002 and September 2006, 58 adult patients (35 males and 23 females, median age 64 yrs, range 40–75) affected by NFo-NHL in active disease phase, were consecutively enrolled in 12 GISL Hematological Centres. Patients were treated with a dose of 25 mg/mq Fludarabine plus 250 mg/mq Cyclophosphamide administred intravenously daily for 3 days; each cycle was repeated every 28 days for 6 courses. During the treatment patients received oral thrimethoprim-sulphametoxazole prophylaxis. After the intermediate evaluation, 48/58 patients (82.8%) had an objective response (ORR) with a 20.7% of complete remission (CR) plus 62.1% of partial remission (PR); at the final evaluation the ORR percentage was 84.5% with a 41.4% of CR (24 pts) and 43.1% of PR (25 pts); three patients were in progressive disease (5.2%) and one in stable disease (1.7%). The median overall survival (OS) was not reached with an 88% and 84% at 12 and 24 months; the progression free survival (PFS) was 89% and 77% and the event free survival (EFS) was 81% and 66% at 12 and 24 months respectively.About the toxicity profile, the major toxicity was hematological with a 18% cases of WHO grade III or IV anemia, 40% leucopenia, 33% neutropenia and 10% piastrinopenia. The 12% of patients had an infective episode wich a 7.7% of WHO grade III–IV.In conclusion the FC chemotherapy is a useful chance for advanced untreated non follicular low-grade NHL, with an optimal ORR, CR and PFS. The crucial point of FC remains OS, that not seems to be significantly improved in comparison with fludarabine alone or with standard therapy, even though the better quality of responses; Rituximab plus FC association is growing in literature as the probably key to find a real improvement also in this aspect.
Chemotherapy-Refractory Diffuse Large B-Cell Lymphomas (DLBCL) Are Effectively Killed by Ofatumumab-Induced Complement-Mediated Cytoxicity.
Authors: Cillessen Saskia A.G.M. , Mackus Wendy J.M. , Castricum Kitty C.M. , Vos Wim , Kortman Pim C. , van de Winkel Jan G.J. , Parren Paul W.H.I. , Meijer Chris J.L.M. , Oudejans Joost J. ,   (Intr. by Ole Baadsgaard)
Published: April 2020

The combination of chimeric CD20 monoclonal antibody (mAb) rituximab (R) with chemotherapy (CHOP) has significantly improved clinical outcome in patients with diffuse large B-cell lymphoma (DLBCL). However, the efficacy of R-CHOP is variable and DLBCL remains fatal in 30–40% of the patients. We recently developed the novel fully human CD20 mAb, ofatumumab (2F2) that is currently in clinical development for CLL and follicular lymphoma (phase III) and rheumatoid arthritis (phase II). In this study, we compared ofatumumab with rituximab for their ability to induce complement dependent cytotoxicity (CDC) of isolated lymphoma cells of chemotherapy-refractory DLBCL patients. Human type II CD20 mAb 11B8 does not induce CDC and was used as a control. Ofatumumab and rituximab induced CDC of all DLBCL samples tested, including the DLBCL cell lines SUDHL4, SUDHL5 and HT and lymphoma cells derived from ten chemotherapy-refractory DLBCL patients. Ofatumumab was significantly more effective in inducing CDC in nine out of ten DLBCL tumor samples when compared to rituximab (p=0.001, Two-way-Anova). The lethal doses (LD50) for ofatumumab (0.1 ± 2.8 μg/ml) was significantly lower when compared to the LD50 for rituximab (6.4 ± 4.9 μg/ml, p=0.04, Mann-Whitney test). The control CD20 mAb 11B8 showed no CDC induction of DLBCL cells. Sensitivity of DLBCL patient cells to ofatumumab- and rituximab-induced CDC negatively correlated with expression of complement defense molecule CD59, but not with expression of CD46, CD55, as determined by immunohistochemistry or apoptosis inhibitors Bcl-2 and XIAP. Functional inhibition of CD55 and CD59 using blocking mAb demonstrated that ofatumumab-induced CDC of DLBCL tumor cells was less sensitive to expression of these complement defense molecules than rituximab-induced CDC. We conclude that chemotherapy-refractory DLBCL cases are sensitive to CD20 mAb induced CDC with ofatumumab being the most effective mAb, especially in patients expressing high levels of CD59. Based on our results ofatumumab should be considered as a valuable therapy option for chemotherapy-refractory DLBCL patients.
Tumour Burden Influence on Individual Rituximab Exposure and Response: Pharmacokinetic - Pharmacodynamic Approach by Using a Syngenic Bioluminescent Murine Model.
Authors: Daydé David , Ternant David , Lerondel Stéphanie , Watier Hervé , Le Pape Alain , Bardos Pierre , Paintaud Gilles , Cartron Guillaume ,   (Intr. by Jean-Francois Rossi)
Published: April 2020

Immunotherapy with the humanised monoclonal anti-CD20 antibody rituximab represents an interesting therapeutic option for patients with B-cell Non-Hodgkin’s Lymphoma. Early clinical trials have shown that rituximab exposure was variable in patients receiving similar doses and that clinical response was related to rituximab concentrations. The aim of our study was to evaluate the influence of tumour burden on rituximab exposure and response to treatment. 8.103 cells of murine T-lymphoma cells (EL4) transduced with human CD20 cDNA (EL4-hCD20) and stably transfected with luciferase plasmid (EL4-hCD20-luc) were injected to C57Bl6J mice by i.v. route. Tumour detection, disease dissemination and progression were evaluated by in vivo bioluminescence imaging. Quantitative imaging analysis was performed after background noises and cosmic rays elimination. Rituximab concentrations were carried out by ELISA method. One dose of rituximab was injected thirteen day after lymphoma cell infusion, a time need for the development of disseminated disease. In the control group without rituximab, all mice developed disseminated lymphoma and died within 30 days whereas we found a significant dose/response relationship with increased doses of rituximab (from 150 μg to 1000 μg). At 500μg of rituximab, we found an important variability in response to treatment with about 23% of mice cured, 59% in partial response and 18% with progressive diseases. For all of these mice, tumour burden, determined before rituximab injection were calculated taking into account their size and bioluminescent activities corrected by the absorption coefficient of light according to the site localisation. Thus, we have demonstrated a significant correlation between tumour burden and response to rituximab and survival. Mice with the lower tumour burden having increased remission rates and survival. Using a pharmacokinetic-pharmacodynamic (PK-PD) model we also demonstrated that tumour burden influence rituximab exposure and response. There is a connection between tumour burden and rituximab efficiency (r2 = 0.938). Such results should have consequences for the rituximab use in human and in pre-clinical assessment of new anti-CD20 antibodies. D.D. is supported by a grant from Région Centre. This study has been supported by Association pour la Recherche contre le Cancer (Grant number: 3229), by the Institut National du Cancer and Cancéropôle Grand Ouest (MAb IMPACT - IMProving ACTivation of FcγRIIIa-expressing effector cells, pharmacogenetic-based optimisation of monoclonal antibody therapy for cancer-federative project) and by Roche France.
Rituximab Sensitizes TRAIL-Resistant B-NHL Lines to Apoptosis by Both TRAIL and Fully Humanized Antibodies Targeting TRAIL-R1 (Mapatumumab) and TRAIL-R2 (Lexatumumab).
Authors: Vega Mario I. , Huerta-Yepez Sara , Baritaki Stavroula , Martinez-Paniagua Melisa A. , Gonzalez-Bonilla Cesar R. , Bonavida Benjamin
Published: April 2020

Clinical trials are currently in progress to evaluate the therapeutic efficacy of recombinant human TRAIL and fully humanized monoclonal antibodies that target TRAIL-R1 (DR4, mapatumumab) and TRAIL-R2 (DR5, lexatumumab). Although these proteins are cytotoxic to sensitive tumor cells, many tumors are resistant and require a sensitization stimulus. We have reported that rituximab inhibits anti-apoptotic survival pathways such as NF-κB activity and sensitizes tumor cells to both chemotherapeutic drugs and Fas-L- induced apoptosis (Vega et al., J. Immunol175(4):2174–83, 2005). Sensitization to TRAIL is also regulated by NF-κB. Thus, we hypothesize that rituximab-induced inhibition of NF-κB also sensitizes B-NHL cells to TRAIL apoptosis. Treatment of Ramos, Daudi, 2F7 and Raji with rituximab sensitized the cells to TRAIL apoptosis in a concentration dependant feature. Sensitization to TRAIL by Rituximab involved the type II mitochondrial apoptosis pathway. Examination of the mechanism of rituximab sensitization to TRAIL revealed that inhibition of NF-κB was associated with inhibition of the DR5 transcription repressor Yin Yang 1 (YY1) and up regulation of DR5 (Vega et al., Blood 108(11):2387, ASH Annual Meeting Abstract 2006). There was no up regulation of DR4 by rituximab. The direct role of YY1 in the rituximab induced sensitization to TRAIL was corroborated by YY1siRNA. These findings suggested that one mechanism of Rituximab sensitization to TRAIL involved up regulation of DR5 expression. However, since TRAIL binds to both DR4 and DR5, the role of DR4 in rituximab induced sensitization to TRAIL needed further clarification. This was addressed by the use of the specific fully humanized monoclonal antibodies directed against DR4 (TRAIL-R1) (HGS-ETR1, mapatumumab) and DR5 (TRAIL-R2) (HGS-ETR2, lexatumumab) provided by Human Genome Sciences. Treatment of Ramos with rituximab sensitized the cells to both HGS-ETR1 and HGS-ETR2 induced apoptosis in a concentration dependant fashion. The sensitization by these antibodies was comparable to that achieved with TRAIL. These findings demonstrate that rituximab sensitizes B-NHL cells to both TRAIL and DR4/DR5 agonist-antibodies. The studies also suggest that, while the up regulation of DR5 expression by rituximab via inhibition of YY1 was critical for TRAIL apoptosis, up regulation of DR4 by rituximab was not required for sensitization with HGS-ETR1 monoclonal antibody. Further, these results suggest that rituximab may affect cell signaling induced by both DR4 and DR5. The molecular mechanism by which rituximab sensitizes B-NHL cells to TRAIL and both HGS-ETR1 and HGS-ETR2 will be presented.
Activated T Cell-Mediated Immunotherapy with a Chimeric Receptor Against CD38 in B-Cell Malignancies.
Authors: Mihara Keichiro , Yanagihara Kazuyoshi , Takigahira Misato , Ochiya Takahiro , Imai Chihaya , Kitanaka Akira , Takihara Yoshihiro , Kimura Akiro
Published: April 2020

CD38 is expected to be one of the most useful molecular targets on the surface of malignant B-cells. T cell-mediated immunotherapy with a chimeric receptor could provide a powerful tool for treating cancer. However, since CD38 is also expressed on effector cells such as activated T cells, NK cells, and monocytes, these cells with the chimeric receptor could be eliminated by autologous cytotoxicity through the interaction with the antigen. In this study, we developed a novel methodology for enhancing the survival and clonal expansion of T lymphocytes expressing an anti-CD38 chimeric receptor. Hut78 T cells, which express very little CD38, retrovirally transduced with the anti-CD38 chimeric receptor showed powerful cytotoxic activity against B cell lines expressing CD38, such as HT (lymphoma), RPMI8226 (myeloma), 380 (ALL-Ph1−) and OP-1 (ALL-Ph1+) cells (mean specific cytotoxicity was 97.94% ± 0.31% after four days of culture in vitro). However, in activated human T cells and Jurkat cells constitutively expressing CD38, the recovery rate of cells transduced with the chimeric receptor was extremely low, because the cells eradicated each other and/or themselves by inducing apoptosis. To block the interaction of the anti-CD38 chimeric receptor with CD38 antigen, we incubated activated T cells and Jurkat cells in medium supplemented with an anti-CD38 antibody before the transduction. The number of viable cells harvested after the transduction was dramatically increased by the antibody in a dose-dependent manner. Using this method, we prepared human peripheral T cells bearing the chimeric receptor and injected them into NOD/SCID mice, which were transplanted with HT cells labeled with luciferase. Lucuferase activity was not detectable in 13 days in five of six mice with T cells transduced with the chimeric receptor. In contrast, the activity had a rapid and steady increase in all of the mice injected with vector-transduced T cells. These results clearly showed that even though human peripheral T cells express any molecule on their surface, an antibody could protect T cells transduced with a chimeric receptor-containing vector from cytolysis, and apoptosis. These findings may provide us with a powerful tool for improving T cell-mediated targeting therapy.
Pharmacokinetics of Anti-CD22 Antibody Conjugates with Uncleavable and Cleavable Linkers in Rats.
Authors: Xie Dong , Deng Rong , Baudys Jakub , Chan Pam , Dere Randy , Ebens Allen , Fielder Paul , Fuji Reina , Gray Alane , Hung Catherine , Iyer Suhas , McKeever Kathleen , Kenrick Margaret , Leach Will , Polson Andrew , Saad Ola , Stephan Jean-Philippe , Shen Ben Quan , Tibbitts Jay , Yu Shang-Fan , Prabhu Saileta
Published: April 2020

CD22 is a B cell specific antigen and is expressed in Non-Hodgkin lymphoma (NHL) with limited expression on normal tissues. Currently, we are evaluating anti-CD22 antibody drug conjugates (anti-CD22 ADC) consisting of a humanized monoclonal IgG antibody conjugated to mitotic inhibitors such as DM1 (a maytansinoid), Monomethylauristatin F (MMAF), or Monomethylauristatin E (MMAE). The antibody binds to human and cynomolgus monkey CD22, but does not bind to rodent CD22. The cytotoxic drugs were conjugated to the antibody via uncleavable linkers (MCC-DM1, MC-MMAF) or cleavable linkers (SPP-DM1, vc-MMAE). These conjugates have demonstrated anti-tumor activity in mouse xenograft models of human B-cell tumors. This study was to assess and to compare the antigen-independent pharmacokinetics of anti-CD22 ADCs with uncleavable linkers (anti-CD22-MCC-DM1 and anti-CD22-MC-MMAF) and cleavable linkers (anti-CD22-SPP-DM1 and anti-CD22-vc-MMAE) in rats, and to support a pilot rat safety study. Following a single IV dose, the anti-CD22 ADCs exhibited a biphasic disposition profile. The conjugates with cleavable linkers had faster clearance compared to those with uncleavable linkers (59.4 mL/day/kg for anti-CD22-SPP-DM1 vs. 18.0 mL/day/kg for anti-CD22-MCC-DM1). The faster clearance for anti-CD22 ADCs with cleavable linker has also been observed with other ADCs (different target). In addition, it was found that free DM1 in plasma that was deconjugated from anti-CD22-SPP-DM1 had ∼70 fold higher exposure compared with that from anti-CD22-MCC-DM1 (area under curve (AUC) for free DM1 in plasma: 562 day*ng/mL for anti-CD22-SPP-DM1 vs. 8.18 day*ng/mL for anti-CD22-MCC-DM1). Furthermore, anti-CD22-SPP-DM1 had a different DM1 release profile compared with anti-CD22-MCC-DM1 when incubated with human plasma in vitro. Maximum concentrations of free DM1 were reached at ∼6 hr and ∼24 hr for anti-CD22-SPP-DM1 and anti-CD22-MCC-DM1, respectively. These observations were consistent with the safety findings in rats: anti-CD22-SPP-DM1 had greater toxicity (body weight loss, renal toxicity) compared with anti-CD22-MCC-DM1. The data described here indicate that the anti-CD22 ADCs with uncleavable linkers are cleared slower than, and have a better safety profile than the conjugates with cleavable linkers.
De Novo Expression of ATP Binding Cassette C1 (ABCC1) on T-ALL Cells Confers Resistance to Vincristine Independent of an MDR1 Phenotype.
Authors: Winter Stuart S. , Khawaja Hadya M. , Lovato Debbie M. , Larson Richard S.
Published: April 2020

Acquired drug resistance eventually leads to treatment failure in T-cell acute lymphoblastic leukemia (T-ALL). To define mechanisms of vincristine (VCR)-resistance, and to address whether inducible resistance in T-ALL might be mechanistically-dependent on ABC drug transporter expression, we developed VCR-resistant Sup T1 and Jurkat by incrementally growing cells in increasing drug concentrations. Jurkat (CD1a−, sCD3+) and Sup T1 (CD1a+, sCD3−) are arrested at “mature” and “cortical” stages of T-cell development and, respectively, showed 1190-fold (0.0006 μM to 0.714 μM) and 790-fold (0.0006 μM to 0.474 μM) increased resistance to VCR. Microarray analysis showed that expression of the ATP Binding Cassette C1 (ABCC1; Multidrug Resistance Protein, MDR2) was increased more than 29-fold in VCR-resistant cells as compared to the parental controls; fold increases in mRNA of ABCC1 were confirmed by RT-PCR, and cell surface expression by flow cytometric analysis. We then employed a flow cytometry-based assay that measures the ability of ABC pumps to extrude fluorescent dyes (Calcein AM). We confirmed that VCR-resistant Jurkat and Sup T1 actively extrude Calcein AM; drug extrusion was also effectively blocked by verapamil and other known ABCB1 antagonists. In contrast to our previous observations for ABCB1-mediated multidrug resistance (Estes, et al., British Journal of Haematology, in press), ABCC1-mediated drug resistance conferred a more narrow spectrum of multidrug resistance for the chemotherapeutic agents commonly used in T-ALL induction. To investigate whether ABC expression might impact patient outcome, we next examined ABCC1 and ABCB1 expression (>1.5-fold) in 80 T-ALL patients treated on COG studies 8704 and 9404. Different from our earlier findings that ABCB1 expression in diagnostic marrows conferred a 100% risk for on-therapy relapse, we found that ABCC1 expression only predicted treatment failure in approximately 25% of affected patients. These results suggest that ABC transporters are differentially regulated in Sup T1 and Jurkat, and that substrate-specific ABC protein regulation results in unique multidrug resistance phenotypes in T-ALL. This study contributes to the repertoire of substrate-specific, drug resistant T-ALL cells lines that are available for modeling treatment failure, provides mechanistically-based insights into relapse, and establishes an experimental means for investigating compounds that reverse ABCC1 mediated drug resistance.
Multi-Drug Resistant Leukemic Cells Highly Express HLA Class I Molecules and Single-Chain Fv Diabody Specific to HLA-A Overcomes Drug Resistance in These Cells.
Authors: Jalili Ali , Ozaki Shuji , Kimura Naoki , Abe Masahiro , Matsumoto Toshio
Published: April 2020

Many tumor cells become resistant to commonly used cytotoxic drugs due to the overexpression of ATP-binding cassette (ABC) transporters. Specifically, p-glycoprotein (MDR-1) is frequently up-regulated in chemotherapy-resistant tumor cells, which is associated with poor prognosis. On the other hand, human leukocyte antigen (HLA) class I molecules are known to be significantly down-regulated in advanced tumor cells to escape from immune surveillance. However, the relationship between MDR-1 expression and HLA expression is not fully understood. Recently, we have developed a recombinant single-chain Fv diabody specific to HLA-A and demonstrated that this agent mediates cell death in HLA-overexpressing lymphoid tumor cells but not in normal cells (Cancer Res2007; 67:1184). Here, we investigated the expression levels of HLA class I in chemo-resistant leukemic cells and evaluated the therapeutic potential of single-chain Fv diabody specific to HLA-A, C3B3-DB (Chugai Pharmaceutical Co. Ltd., Tokyo, Japan). Chemotherapy-resistant cells were established by subculturing of myeloid leukemia cell line HL60 and Burkitt’s lymphoma cell line BL-TH in increasing doses of vincristine (VCR), and named HL60/VCR and BL-TH/VCR, respectively. MDR-1 is strongly expressed in HL60/VCR and BL-TH/VCR cells both at the mRNA and protein levels, but not in the parental cells. Interestingly, expression levels of HLA class I molecules are 8 times higher in HL60/VCR and BL-TH/VCR cells than in the parental cells, suggesting that MDR-1 modulates cell surface expression of HLA by its transporter function. Next, we examined the cytotoxic activity of C3B3-DB on these chemo-resistant cell lines. C3B3-DB induced apoptosis in HL60/VCR and BL-TH/VCR cells and these chemo-resistant cell lines were more sensitive to C3B3-DB than the parent cells. Combination of C3B3-DB with chemotherapeutic agents such as VCR and daunorubicin (DNR) resulted in enhanced cytotoxicity against HL60/VCR and BL-TH/VCR cells. Importantly, pretreatment of these chemo-resistant cell lines with C3B3-DB reduced expression levels of MDR-1 and increased drug retention in these cells as detected by flow cytometry and confocal microscopy. Furthermore, combination of C3B3-DB with VCR significantly blocked the cell cycle at the G2 phase compared with VCR alone. Similar results were obtained with primary acute myeloid leukemia cells from 2 patients, resulting in up-regulation of both HLA class I and MDR-1 molecules at relapse phase compared at diagnosis. These results suggest that C3B3-DB enhances cytotoxicity of chemotherapeutic agents and provides a novel approach for overcoming drug resistance in hematological malignancies.