bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia Increases IL-8 Secretion of Mesenchymal Stroma Cells Affecting Migratory Capacity in An Autocrine Manner
Authors: Wobus Manja , Mueller Katrin , Ehninger Gerhard , Bornhaeuser Martin
Published: April 2020

Hypoxia increases IL-8 secretion of mesenchymal stroma cells affecting migratory capacity in an autocrine manner Manja Wobus, Katrin Müller, Gerhard Ehninger, Martin Bornhäuser Department of Hematology/Oncology, University Hospital Dresden, Fetscherstr. 74, 01307 Dresden, Germany Adult bone marrow-derived stem cells represent an important source of cells for regeneration and repair of a number of damaged tissues. Mesenchymal stromal cells (MSCs) give rise to the cellular components of the bone marrow microenvironment and support expansion and differentiation of hematopoeitic stem cells in the respective niche. Low oxygen tension is thought to be an integral component of the endosteal niche microenvironment. When used for therapeutic purposes, MSCs cultured in standard conditions must adapt from 21% oxygen to less than 1% oxygen in the ischemic tissue. Understanding the mechanisms by which the production of cytokines and growth factors by MSCs is regulated may represent an important way to optimize their beneficial paracrine and autocrine effects. Human primary MSCs were incubated under normoxic (20% O2) and hypoxic (0.5% O2) conditions over five days and the pattern of released cytokines in the cell culture supernatants was compared using a human cytokine array (R & D Systems). Amongst others, we found upregulated IL-8 levels under hypoxic conditions leading us to further investigation of IL-8 expression in MSCs and its role for in-vitro migration. As expected, IL-8 mRNA levels were significantly higher in hypoxic MSCs. The result of the cytokine array was confirmed by examination of secreted IL-8 in the cell culture supernatant by ELISA (PeproTech). The migration capacity was investigated in a 24-well transwell chamber assay with 8 μm pore size. Using recombinant IL-8 as a chemoattractant in the lower chamber, we detected an almost twofold enhanced MSC migration rate after 24 hours under hypoxic conditions. As a different approach to investigate the migratory capacity, we used an in-vitro scratch assay. A wound was applied to a MSC monolayer in absence or presence of IL-8 which clearly enhanced the migration of MSCs into the wound area after 24 hours. In summary, IL-8 secretion by human primary MSCs is clearly increased under hypoxic conditions. IL-8 in turn seems to be a chemotactic factor for MSCs and enhances their migratory capacity in an autocrine manner.
A Novel Mechanism Underlying the Expression Regulation of An IFN-Inducible Gene RIG-G
Authors: Lou Ye Jiang , Pan Xiao Rong , Jia Pei Min , Li Dong , Xiao Shu , Chen Sai-Juan , Chen Zhu , Tong Jian Hua
Published: April 2020

The RIG-G gene, originally identified due to its markedly up-regulated transcriptional expression in acute promyelocytic leukemia cell line NB4 after treament with all-trans retinoic acid (ATRA) for 72 hours, is actually a member of Interferon (IFN)-inducible genes family. It acts as an important molecular mediator of the antiproliferative activity of IFN-related pathways through upregulation of cell cycle inhibitors p21 and p27. The fact of a synergistic induction of RIG-G mRNA in NB4 cells by the treatment with ATRA and IFNs implies a possible role of RIG-G in cross-talk between these two signaling pathways. However, the mechanism underlying the expression regulation of RIG-G gene remains unknown. In this work, we show that IRF-9 and STAT2 are two basic components required for RIG-G expression, although the tyrosine phosphorylation of STAT2 could further enhance the transactivation activity of the complex of IRF-9 and STAT2 by facilitating their interaction. In addition, we demonstrate the ability of ATRA to induce IFNa synthesis and secretion along with subsequent phosphorylation of STAT2 in NB4 cells, which may not only account for the dramatic expression of RIG-G after ATRA treatment but also provide a new view of signaling cross-talk between IFNa and ATRA. Moreover, we also observe that IRF-1, a primary response gene of both IFNa and ATRA, can modulate the transcription of RIG-G in both STAT2/IRF-9 dependent and independent ways. Taken together, we report for the first time that the complex of IRF-9 and STAT2 is sufficient for RIG-G expression even without the phosphorylation, and suggest a novel mechanism, different from the classical IFN-induced JAK-STAT pathways, through which IFN modulates its target genes. Our studies regarding the transcription regulation of RIG-G gene enrich our knowledge on IFN signaling pathways and will be beneficial to further better understanding of the complexity of biological responses to IFNs.
Molecular Mechanisms Underlying the Regulation of Alternate Macrophage and Neutrophil Gene Programs by the Egr Transcription Factors
Authors: Cheng Jason X , Laslo Peter , Spooner Chauncey , Bertolino Eric , Singh Harinder
Published: April 2020

Cell fate determination in the hematopoietic system involves the onset and resolution of mixed lineage patterns of gene expression. Molecular mechanisms underlying the concerted activation and repression of alternate lineage genes remain to be elucidated. We have proposed a gene regulatory network for macrophage development in which the transcription factors PU.1 and the Egr’s function in a feed forward loop to activate macrophage genes. In the network, the Egr’s counteract the neutrophil regulator Gfi-1 and also repress alternate lineage genes. Using mutant alleles of Egr-1 and Egr-2 we validate the model and demonstrate that the Egr’s are required for initiating but not maintaining the repression of neutrophil genes during macrophage differentiation. Employing a PU.1−/− progenitor cell line we show that PU.1 transiently binds to and activates both macrophage and neutrophil gene promoters thereby leading to the onset of mixed lineage gene activity. While inducing macrophage cell fate determination, PU.1 remains persistently bound to macrophage gene promoters aided by the Egr’s. Surprisingly, the Egr’s displace PU.1 from neutrophil gene promoters by interacting with a co-repressor Nab-2 thereby repressing the alternate lineage program. We propose that the Egr’s have evolved to molecularly discriminate distinct sets of target genes and delineate divergent patterns of gene activity.
A Novel B220+ NK Cell Progenitor Found in the Murine Lung with Potent in Vitro NK Potential Gives Rise to Mature NK Cells with Distinct NK Cell-Surface Receptor Expression
Authors: Halim Timotheus You Fu , Takei Fumio
Published: April 2020

Natural Killer (NK) cells are important effector cells in innate immunity, and play a vital role in antiviral defense, tumor surveillance and modulation of the adaptive immune response. NK cells were originally believed to arise from a lin−NK1.1−CD122+ bone marrow (BM) progenitor. However, recent findings have identified NK progenitors (NKP) and distinct NK differentiation pathways in the thymus, lymph node, spleen and liver. The physiological role of these extra-BM developmental pathways remains to be determined. We hypothesized that these alternative pathways on NK development would have an impact on the generation of the phenotypic heterogeneity observed in cell-surface receptor expression and functional NK cell subsets. Here, we demonstrate the identification of a small population of cells in the lung of C57Bl/6 mice (0.02% of lung leukocytes) that have a lin−NK1.1−CD122+B220+ cell surface phenotype. These cells also show potent in vitro NK cell activity when cultured on OP-9 stromal cells with IL-7, mSCF, Flt3L and IL-15, as well as in vivo NK cell potential upon adoptive transplant into RAG-2−/− IL2Rγ−/− and NOD/SCID IL2Rγ−/− hosts. Mature NK cells (CD3−NK1.1+) derived in vitro from conventional BM NKP and lung B220+ NKP were characterized for cell-surface receptor expression after 16–18 days (figure 1, mean with SEM). Clear differences in activating and inhibitory NK cell-surface marker expression were observed between NK cells derived in vitro from conventional BM NKP and lung B220+ NKP (Ly49D p<0.05, Ly49G2 p<0.05, NKG2A/C/E p<0.05). These findings suggest that B220+ NKP may generate phenotypically and functionally distinct NK cell types.

Figure 1: Cell-surface receptor expression on in vitro derived NK cells Figure 1:. Cell-surface receptor expression on in vitro derived NK cells
Up Regulation of transendothelial migration on Hematopoietic Progenitor Cells (HPC) and neutrophils by Leukotreine B4 (LTB4) via elevated Endothelial Permeability
Authors: Mun Yeung-Chul , Oh Soo A. , Choi Moon Young , Ahn Jee-Young , Lee Kyoung-Eun , Yoo Eun-Sun , Seong Chu-Myong
Published: April 2020

Backgrounds : The mechanisms on HPC mobilization by Hematopoietic growth factors seem to be multifactorial processes. Though the roles of protease (ie. NE, MMPs) and cell adhesion molecules (CAMs) seem to be important, the precise mechanisms on HPC mobilization are still unknown. LTB4 is lipid mediator derived from membrane phospholipids during the process of inflammation, and have many roles (ie; inducer of chemotaxis, the production of nitric oxide, transepithelial migration of neutrophil). The major activities of LTB4 include the recruitment and activation of leukocytes, suggesting that it has considerable functional overlap with the chemokine family of chemoattractant peptides, which also direct the recruitment of leukocytes. We previously had reported that LTB4 was able to mobilize HPC of 8.5-fold baseline within only 4 hours without significant side effects in the murine models (Blood 2005, 106;11(2):401b-402b) and LTB4 receptor and reactive oxygen species (ROS) were involved in the pathway of mobilization induced by LTB4 or G-CSF using C57BL/6 mice (Blood 2006, 108;11(2):395b, Blood 2007, 110;11(2):303B). In this study, we investigated the cellular mechanisms of HPC mobilization which was induced by LTB4.

Methods : We studied the expression of LTB4 receptors (BLT1 and BLT2) on the surface of murine HPC using RT-PCR. To elucidate the effects of LTB4 on the cells which compose the bone marrow environment for the HPC mobilization, we studied the influence of LTB4 on proliferation, transmigration, permeability, and expression of CAMs on HPC and neutrophils from C57BL/6 mice and MS5 cells, murine stromal cell line cells, and bEnd3 cells as well.

Results : Proliferations of b.End3 cell and MS-5 cell were not affected by treatment of LTB4. But, permeability of b.End3 cell monolayer were increased 1.3-fold by LTB4 treatment (100 nM) compared with control. After LTB4 treatment, transendothelial migrations of total nucleated cells (TNC) (22.2% vs 9.4%), HPC (2.7% vs 0%) and neutrophils (75.6% vs 13.6%) from the C57BL/6 mice bone marrow were increased compared with control group respectively. These effects were reversed by LTB4 receptor antagonists (ie; LTB4APA and U75302). Transendothelial migration of neutrophil was increased quickly compared with those of HPCs and total nucleated cells from the bone marrow. However, LTB4 didn’t affect on the expression of ICAM-1 and VCAM-1 of b.End3 cell, murine microvascular endothelial cell.

Conclusions : Through our data, it is suggested that transendothelial migration of HPCs and neutrophils were upregulated by LTB4 via elevated endothelial permeability. Further studies are necessary to find the molecular mechanisms on the mobilization by LTB4. Prompt mobilization by LTB4, which takes only 4 hours may be very useful in the clinic if safty profiles are completed in human.
Hematopoietic Differentiation of Human Embryonic Stem Cells in Vitro : Comparison of Three Cell Lines
Authors: Brenot Marion , Bennaceur-Griscelli Annelise , Peschanski Marc , Mitjavila-Garcia Maria Teresa
Published: April 2020

Human embryonic stem cells (hES) isolated from the inner cell mass of a blastocyst have the ability to self renew indefinitely while maintaining their pluripotency to differentiate into multiple cell lineages. Therefore, hES represent an important source of cells for perspective cell therapies and serve as an essential tool for fundamental research, specifically for understanding pathophysiological mechanisms of human diseases for the development of novel pharmacological drugs. The generation of hematopoietic stem cells from hES may serve as an alternative source of cells for hematopoietic reconstitution following bone marrow transplantation and an interesting approach to understand early stages of hematopoietic development which are difficult to study in human embryos. Using two different methods, we have differentiated three hES cell lines (SA01, H1 and H9) into hematopoietic cells by generating embryoid bodies and co-culturing on the murine Op9 cell line. In both experimental approaches, we obtain cells expressing CD34 and when cultured in hematopoietic conditions, SA01 and H1 cell lines differentiate into various hematopoietic lineages as demonstrated by BFU-E, CFU-GM and CFU-GEMM colony formation, whereas H9 have almost exclusively granulo-macrophage differentiation. Cells composing these hematopoietic colonies express CD45, CD11b, CD31, CD41 and CD235 and staining with May Grundwald-Giemsa demonstrate neutrophil and erythrocyte morphology. These results demonstrate the capacity of hES to differentiate into mature hematopoietic cells in vitro. Nevertheless, there exist some quantitative and qualitative differences about hematopoietic differentiation between the hES cell lines used. However, we still have to evaluate their capacity to reconstitute hematopoiesis in vivo in an immune deficient mouse model. We will also be interested in developing in vitro methods to expand these hematopoietic precursor cells derived from hES which may be used as a viable source for future cell therapy.
Plasma Adrenomedullin Levels Are Elevated in Pediatric Patients with Severe Sickle Cell Anemia
Authors: Piccin Andrea , Murphy Ciaran , Eakins Elva , Murphy William , Kunde Jan , McMahon Corrina , Smith Owen
Published: April 2020

Nitric Oxide (NO) and endothelin-1 (ET-1) are key modulators of endothelial vessels and actively participate in the pathophysiology of vaso-occlusive events. Sickle cell anemia (SCA) is an important model for the study of blood cells/endothelium interactions. In SCA vaso-occlusive events may be severe and cumulative. Adrenomedullin (ADM) is a strong hypotension-inducer and mediates diuresis, natriuresis and vasodilation. Adrenomedullin levels are raised in chronic cardiovascular conditions. The aim of this study was to investigate the role of adrenomedullin in SCA and its relationship to endothelin-1 and nitric oxide. A cohort of 111 children with HbSS SCA was studied with 18 age-matched controls of sub-Sahara African ethnicity. The patients were in four clinical groups:

steady state (n=55, mean age 5.3 years), patients undergoing chest crisis (n=9, mean age 5.3 years), on hydroxyurea (n=30, mean age 7.2 years), on transfusion therapy (n=17, mean age 6.5 years).

A third cohort of patients with HbSC genotype (n=13, mean age 6 years) all in steady state was also included. Pro-adrenomedullin and c-terminal endothelin-1 precursor (CT-proET-1) were measured by coated tube assays according to the method by Morgenthaler et al (Clin Chem.2005;51:1823–29) and Papassotiriou et al (Clin Chem.2006;52(6):1144–51), nitric oxide was quantified according to the Griess reaction. Results (median and interquartile range) are shown in table 1. Overall pro-adrenomedullin levels were raised in chest crisis: 0.38 nmol/l (0.31–0.49) vs steady state: 0.27 nmol/l (0.25–0.32; p<0.01) and control: 0.28 nmol/l (0.27–0.31; p <0.01). CT-proET-1 levels were reduced in patients on hydroxyurea therapy: 43.6 pmol/l (12.6–49.6) vs control: 55.1 pmol/l (45.2–63.9; p<0.05). Nitric oxide levels were significantly lower in chest crisis (19.3 mmol/l plasma; 10.7–19.9) compared to hydroxyurea (22.2 mmol/l plasma; 18.3–28.4; p<0.05), and HbSC (30.6 mmol/l plasma; 20.8–39.5; p<0.05) and approach significance when compared to steady state (22.5mmol/l plasma; 16.9–28.2; p=0.07). This study indicates that during chest crisis plasma adrenomedullin levels are elevated compared to normal controls and SCA patients in steady state (see figure 1). Plasma nitric oxide levels are diminished. These observations indicate that markers of ongoing vascular damage are evident in children with SCA with chest crises, and suggest that treatments that prevent onset or progression of pulmonary hypertension, such as bosentan, may also have a role in the prevention of pulmonary hypertension in children with SCA.

Table 1. ADM, ET-1, NO and hematological/biochemical values in SCA patients and controls. IR= interquartile range.

Figure 1. ADM and NO levels in sickle cell patients and controls. Bars indicate median and interquartile range. Figure 1. ADM and NO levels in sickle cell patients and controls. Bars indicate median and interquartile range.
Increased Plasma Levels of NGAL and IL-18 Correlate with Reduced GFR Providing Two Novel Biomarkers for Kidney Injury Assessment: Evidence of Early Tubuloglomerular Defect in Patients with HbS/b-Thalassemia
Authors: Papassotiriou Ioannis , Margeli Alexandra , Hantzi Eugenia , Terpos Evangelos , Voskaridou Ersi
Published: April 2020

Promising novel biomarkers for the assessment of the acute kidney injury (AKI) or chronic kidney injury (CKI) include plasma levels of cystatin C, Neutrophil Gelatinase-associated Lipocalin (NGAL) and Interleukin-18 (IL-18) and urinary levels of NGAL, IL-18 and Kidney Injury Molecule-1 (KIM-1). These biochemical indices have been reported to be useful for evaluating the duration and severity of kidney disease and predicting progression and adverse clinical outcomes. Furthermore, these markers have given promising results in differentiating the various causes of AKI or CKI. Progressive renal failure is one of the main complications in HbS/β-thalassemia (HbS/β-thal). Early identification of patients at high risk of developing renal failure is of great importance as it may allow specific measures to delay the progression of renal damage and thus to reduce the incidence of end-stage renal failure and mortality. Continuing our previous studies (Voskaridou et al, Kidney Int2006;69:2037–42), we explored the activation of tubuloglomerular feedback in 58 adult patients (age 42±17years, males/females 20/58) with HbS/b-thal by measuring the above mentioned parameters, along with the more classical ones such us b2-microglobulin and N-acetyl-b-D-glucosaminidase (NAG) in blood and urine using standard methodology. GFR values were calculated according to the recently proposed cystatin C–based prediction equation using only each concentration in mg/L (nephelometric assay, Dade Behring Siemens Healthcare Diagnostics): GFR [mL/min/1.73m2] = 76.7 x Cystatin C−1.18. The main results of the study were:

impairment of GFR in 21/58 patients (36%); increased plasma concentrations of NGAL and IL-18 in 34/58 (58%) and 58/58 (100%) patients, respectively; increased and/or detectable urine concentrations of NGAL and IL-18 in 54/58 (93%) and 46/58 (79%) patients, respectively; significant negative correlations between GFR and plasma NGAL and IL-18 (r=−0.735, p<0.0001 and r=−0.350, p<0.007, respectively) and significant positive correlations between cystatin C and urine NGAL and IL-18 (binomial, p<0.005 and r=0.412, p<0.03, respectively).

These findings suggest that tubuloglomerular feedback is activated in almost all studied patients with HbS/b-thalassemia. Measurements of plasma and urine NGAL and IL-18 contribute significantly to the early detection and risk stratification of renal disease in these patients. However, prior to their clinical use, these biomarkers must undergo through rigorous validation in multiple cohorts.
Hydroxyurea Therapy Increases Expression of BCAM/LU and Adhesion to Laminin in Children with Sickle Cell Disease
Authors: Johnson Clarissa E , Telen Marilyn J.
Published: April 2020

Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease. The tendency of red blood cells (RBCs) to adhere to extracellular matrix molecules and the vascular endothelium is believed to be a significant contributor to the vaso-occlusive process. Some published studies have shown that hydroxyurea decreases sickle (SS) RBC adhesion to some ligands, although the mechanism by which this occurs is not completely understood. SS RBCs demonstrate increased expression of several adhesion molecules, especially BCAM/LU, and also conserve functional signaling pathways that are associated with upregulation of adhesion. BCAM/LU mediates adhesion to the extracellular matrix protein laminin. We hypothesized that patients responsive to hydroxyurea (HU) therapy would exhibit reduced adhesion to laminin as well as a decrease in adhesion molecule expression. Our subjects included patients with Hb SS between the ages of 5 to 18. They were divided into three groups:

children not receiving HU therapy (n = 3); children receiving HU therapy for over 6 months (n = 5), and children initially not receiving HU but who were initiating therapy at the time of study enrollment (n = 5).

Adhesion to laminin was examined using a graduated height flow chamber to quantitate the adhesion of SS RBCs. Expression of adhesion molecules was analyzed by western blot and densitometry, using monoclonal antibody to BCAM/LU. We found that HU therapy was associated with significantly increased expression of BCAM/LU (HU: 145.8 ± 14.0 SEM; no HU: 60.8 ± 11.0 SEM densitometry units, p = .0014). This somewhat unexpected finding confirms results published earlier this year by Odievre et al. (2008). Adhesion to laminin was also increased for patients on HU (HU: 9.3 ± 5.9; no HU: .3 ± .3, p=.2), although this increase was not significant, given the variability in adhesion seen among patients and the small number of subjects. Nevertheless, the increase in adhesion corresponded to the increase in BCAM/LU expression. In contrast, adhesion to endothelial cells was decreased, although not significantly, in patients on HU (HU: 38.1 ± 38; no HU: 127.2 ± 122.5, p=.6). Our findings thus confirm earlier published data showing that HU increases the expression of BCAM/LU measured by flow cytometry and further shows that this increased expression is associated with increased adhesion to laminin but not to endothelial cells. Potential mechanisms by which HU affects adhesion molecule expression and activity merit further investigation, as does the physiologic role of these alterations. Comparison of results from patient-matched pre-treatment and post-treatment samples should also help define the effects of HU.

Figure Figure
Dermatofibrosarcoma Protuberans in 3 Patients with ADA-SCID
Authors: Kesserwan Chimene , Sokolic Robert A. , Heselmeyer Kerstin , Ortiz-Melendez Clarymar , Cohen Jeffrey , Jagadeesh Jaya , Garabedian Elizabeth , Baird Kristin , Wayne Alan S. , Cowen Edward W. , Pittaluga Stefania , Lee Chyi-Chia Richard , Sherry Richard , Bridge Julia , Finlayson Laura , Walsh Noreen , Issekutz Andrew , Candotti Fabio
Published: April 2020

Dermatofibrosarcoma protuberans (DFSP) is a rare malignant skin tumor with characteristic cytogenetic findings (t(17;22)(q22;q13)) resulting in a fusion COL1A-PDGFB gene. Here we report an association between DFSP and Adenosine Deaminase Deficient Severe Combined Immunodeficiency (ADA-SCID), a rare congenital immunodeficiency syndrome. We observed DFSP in 3 ADA-SCID patients. The first patient is a 3.6-year-old boy who developed a 1.5 cm soft tissue mass on the right medial thigh. The second patient is a 2.4-year-old girl with 12 plaques (0.4– 0.8 cm) distributed on the trunk, thigh and lower extremities. The third patient is a 6-year-old boy with 5 depressed plaques ranging from 1–2 cm. The diagnosis in the first two patients was confirmed by immunohistochemistry (CD34 positive/Factor XIIIa negative) and demonstration of the COL1A1-PDGFB fusion by Fluorescent In Situ Hybridization (FISH). In the third patient, lesional tissue was positive for CD34 and negative for Factor XIIIa. FISH was inconclusive and further molecular analysis (RT-PCR) is ongoing. We came across two additional ADA-SCID patients in the literature who developed DFSP and we conclude therefore that this unique and unreported association between two rare disease entities merits further study. Importantly, our observations describe the first 2 cases presenting with multiple multicentric DFSPs. DFSP may be overlooked particularly in children who may present with an indolent plaque-like lesion mistaken as birthmark or benign vascular proliferation. DFSP in ADA-SCID may be caused by an unknown virus in the setting of defective immune surveillance. The search for a viral etiology is ongoing. Alternatively, the increased DNA breakage characteristic of ADA-SCID may play a role in the onset of this malignancy.
Analysis of Chromosomal Aberrations and DNA Mutations in Bone Marrow Mesenchymal Stroma Cells from Patients with Myelodysplastic Syndrome Amd Acute Myeloid Leukemia
Authors: Blau Olga , Hofmann Wolf-Karsten , Blau Igor W. , Baldus Claudia D , Nolte Florian , Thiel Gundula , Schuemann Elke , Benlasfer Ouidad , Thiel Eckhard
Published: April 2020

Introduction: The biology of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) is not well understood. In MDS, ineffective hematopoiesis may result from disturbed interactions between hematopoietic cells (HC) and the hematopoietic microenvironment. Bone marrow mesenchymal stroma cells (BMSC) are key components of the hematopoietic microenvironment. BMSC from patients with hematological disorders display functional and quantitative alterations. However, the question whether BMSC in MDS/AML have cytogenetic abnormalities is discussed controversially.

Methods: In the present study, we have collected BMSC from 51 MDS and 42 AML patients at the time of initial diagnosis. Chromosome preparation was performed after cell culture for 30 days and analyzed by conventional cytogenetic (G-banding) and by different types of FISH-techniques. Furthermore, FLT3 and NPM1 mutation analysis was performed in HC and BMSC. As a control we have studied BMSC from 25 healthy individuals.

Results: Cytogenetic analysis of BMSC was successfully performed in 90 of the 93 cases. Clonal structural chromosomal aberrations, including t(1;7), t(1;10), t(1;2), t(7;9), i(1q), inv(X), del(7q), del(13q), del(17p), and others, were detectable in BMSC of 15% of patients. All cytogenetic markers were confirmed by FISH with specific probes and M-FISH. Interestingly, cytogenetic markers in BMSC differed from the aberrations in HC from the same individual. We have found cytogenetic abnormalities in BMSC from patients presenting with cytogenetic alterations in their HC as well as from those with normal karyotype. In BMSC we could not detect specific mutations of NPM1 and FLT3 (ITD and TKD), independent from the mutation status of HC. For control analysis, BMSC cultures from 25 healthy donors were prepared under the same conditions (time of culture, number and frequency of passages). BMSC from healthy donors did show normal diploid karyotypes and absence of DNA-mutations of NPM1 and FLT3.

Conclusions: Our data indicate that BMSC from MDS and AML patients are characterized by genetic instability. Lack of aberrations as detected in HC and appearance of novel clonal rearrangements in BMSC may suggest enhanced genetic susceptibility and potential involvement of BMSC in the pathogenesis of MDS and AML.
Prognostic Value of Minimal Residual Disease (MRD) Detection in Adult Acute Lymphoblastic Leukemia (ALL) – HYPER-CVAD Protocol
Authors: Tsagarakis Nikolaos , Kentrou Nektaria , Pergaminou Mirsini , Marinakis Theodore , Papadhimitriou Stefanos I , Gortzolidis Georgios , Galanopoulos Athanasios , Michali Evridiki , Papadimitriou Konstantinos , Skoumi Dimitra , Anagnostopoulos Nikolaos , Paterakis Georgios
Published: April 2020

Objective: HYPER-CVAD is an intensive treatment protocol of short duration, for ALL and other lymphoid neoplasms. It is consisted of 8 alternating cycles (parts A and B): in part A (cycles 1, 3, 5, 7) fractionated doses of cyclophosphamide, vincristine, doxorubicin and dexamethazone are administrated, while in part B (cycles 2, 4, 6, 8) high doses of methotrexate and aracytin. The two first cycles compose the induction therapy, while the next 6 cycles constitute the consolidation therapy, followed by two years of maintenance therapy. Protection of CNS is achieved with intradorsal injections, whereas in Ph+ ALL patients, imatinib is also administered. The aim of this study was the clinical evaluation of MRD detection in adult patients with ALL, during chemotherapy with HYPER-CVAD.

Patients/Methods: During the period 1999–2008, 30 patients were hospitalized in our hospital for ALL and were treated with HYPER-CVAD therapeutic protocol. Among them, 14/30 (46,7%) were males and 16/30 (53,3%) females (median age 43,5 years, range 16–70). Median follow-up time was 12,8 months (range 0,5–100). ALL of T-origin had 8/30 patients and of B-origin, 22/30 (1 B1-EGIL/pro-B, 17 B2-EGIL/B-common, 4 B3-EGIL/pre-B). Caryotypic analysis and FISH was done in all patients (7/30 bcr/abl+). According to classical prognostic markers of ALL: 21/30 were classified as high, 4/30 as medium and 5/30 low risk, respectively. MRD presence was detected in bone marrow samples, with flow cytometric panels, at three particular treatment time-points: completion of induction therapy (T1), completion of consolidation therapy (T2) and at the end of maintenance therapy (T3). Overall survival (OS) and disease free survival (DFS) were investigated, especially in relation to the influence of MRD presence in (OS) and (DFS), respectively. For statistical analysis, Kaplan-Meier was used.

Results: At treatment time-point (T1), MRD was detected in 10/28 (35,7%) patients, at time-point (T2) in 7/23 (30,4%) and at treatment-point (T3) in 9/17 (52,94%). The influence of MRD detection in (OS) was statistically significant (p<0,05) at (T2) and (T3), while in (DFS) the detection of MRD presence in any of the standardized treatment time-points resulted in decreased DFS (T1/p<0,05, T2 and T3/p<0,001).

Conclusions: Our results suggest that MRD detection during therapy of adult ALL is a negative prognostic indicator for (OS) and (DFS), the independency of which has to be confirmed after prolonged follow-up time and increased number of cases.
Automated Classification of Normal and Leukemic B Cells on the ImageStream® Imaging Flow Cytometer Using Immunocytomorphological Image Characteristics
Authors: Venkatachalam Vidya , Martin Catherine Claude , Jayat-Vignoles Chantal , Faucher Jean-Luc , Feuillard Jean , Morrissey Phil , George Thaddeus
Published: April 2020

Leukemic B cells can be distinguished from normal B cells based on their surface staining patterns and their morphology, but the differences can be subtle and the abnormal events rare. In order to reliably differentiate normal from leukemic B cells, objective quantitation of large data sets is required. This is accomplished with the ImageStream cytometer, which automatically acquires multispectral images of thousands of individual cells in flow at very high speeds. The associated IDEAS data analysis software measures hundreds of photometric and morphometric features per cell, thereby enabling simultaneous immunophenotyping and morphology-based measurements. The features quantify individual image characteristics such as shape, size, signal strength and texture. We have leveraged these features by implementing an automated classification routine that identifies the feature combination that best separates different cell types of interest. Our automated classification routine utilizes a modification of Fisher’s Linear Discriminant criterion to measure discrimination power between two or more populations of cells. The classifier generates a composite feature consisting of a weighted linear combination of basic image features that provides maximum discrimination. Thus, our method transforms a multi-parameter classification task that can be difficult to conceptualize into a classification based on a single composite feature that can be readily conceptualized and visualized using a simple histogram output. Since the composite feature is generated from, and tested and optimized on, large numbers of cells, we obtain results that are highly objective, repeatable, statistically significant and scalable with data size. We used this classification approach to study normal and tumor cells of the hematopoietic lineage with the goal of improving diagnosis of hematological disorders. Here we show the results of using automated classification to distinguish the subtle differences between B cells of CLL patients and those of normal patients. We also used the classifier to distinguish B-cell ALL from B-cell CLL. In both cases, we used a combination of shape, size, texture and signal strength features from the SSC, brightfield and nuclear imagery generated by the ImageStream system. The data presented demonstrate the application of a novel automated cell classification method to the discrimination of normal and tumor cells within large and heterogeneous cell samples.
Genomic Modifications Occur during Maturation in Myeloid Cells
Authors: Shirasaki Ryosuke , Tashiro Haruko , Akiyama Nobu , Kawasugi Kazuo , Shirafuji Naoki
Published: April 2020

Objective: A chromosomal translocation plays an important role on the leukemia-generation from the normal hematopoietic cells. Various kinds of translocation-patterns and -partners have been reported; however, a typical translocation has been reported like AML-1 and MTG8 in acute myelogenous leukemia (AML) (M2), and PML and RARα in AML (M3). This implies that some common mechanism exists on the chromosomal double strand breaks and the generation of translocations. We make a hypothesis that, when normal immature cells come into a maturation process, a specific event may occur on DNA level to induce irreversible changes toward cell-death. And a target region for the irreversible change may be the near site to chromosomal translocations. We compared the digested fragment-size for restriction-endonucleases using southern blotting among hematopoietic cell lines, normal bone marrow immature progenitor cells and normal blood mature neutrophils.

Materials and Methods: Bone marrows and bloods were obtained from informed healthy persons, and mononuclear cells were prepared after Ficoll-Paque sedimentation method, and neutrophil-rich fractions were prepared with density gravity centrifugation and hypotonic RBC lyses method. Various kinds of human cell lines, cultured in DMEM with 10% FCS, were also prepared. High molecular weight DNA was extracted from these cells, and they were digested with several kinds of restriction endonucleases. After electrophoresis, the digested DNA was transferred to a nitrocellulose membrane, and southern blot-hybridization was examined. The probe used was AML-1, MTG-8, PML, and RARα, in which the 5′ and 3′ breakpoint regions observed in chromosomal translocations were selected, respectively. Results and Discussion: No changes on the blotted fragment-size were observed among in the cultured cell lines, bone marrow immature mononuclear cells, and blood mononuclear cells; however, when 5′-PML probe was hybridized after DNA was digested with HindIII, the observed blot size was changed between normal immature myeloid progenitor cells and normal blood mature neutrophilic granulocytes. This change was not due to the personal polymorphism. These observations indicate that when myeloid cells become mature to neutrophilic granulocytes, some change occurs at DNA level. PML works on the maintenance of nucleobody for cell division, and it is very important to determine whether PML gene is maintained in neutrophilic granulocytes. We now prepare a genomic library from blood neutrophils to isolate the target clone, and determine DNA sequence near at the HindIII site to identify the modification of DNA during maturation of myeloid cells.
A Case of Acute Myeloid Leukemia with Maturation (AML-M2) with C-MYC Amplification in Homogenous Staining Region (hsr) and Double Minutes (dmin)
Authors: Lee Seungok , Kim Myungshin , Lim Jihyang , Kim Yonggoo , Han Kyungja , Kim Heeje , Min Woo-Sung
Published: April 2020

Homogeneous staining region (hsr) and double minutes (dmin), cytogenetic indicators of gene amplification, are rare findings in hematologic malignancies, and most frequently associated with acute myeloid leukemias (AML). Herein, we report a 74-year-old female presenting AML with maturation (AML-M2) with c-MYC amplification in hsr and dmin. Conventional cytogenetic techniques showed 46, XX, hsr(4)(q25)[15]/46, XX,~20dmin[5]. The c-MYC amplification in hsr was suspected by multicolor fluorescence in situ hybridization (mFISH) using 24XCyte Human mFISH Painting Kit. Amplification of c-MYC in hsr and dmin were confirmed by fluorescence in situ hybridization (FISH) using LSI MYC(8q24) Dual color, Break Apart Rearrangement Probe. The presentation of both dmin and hsr was rare in AML cases. Majority of AML cases with c-MYC or MLL amplification in dmin or hsr were women older than 50 years, had de novo AML with complex karyotypes and had a short survival. But, this case had no chromosomal aberration besides c-MYC amplification and showed a good response to chemotherapy with a complete remission for 21 months.
The Frequency and Clonality of γδ+T Cells, distribution of TRGV and TRDV Repertoire in Cord Blood T Cells
Authors: Li Yang Qiu , Chen Shaohua , Yang Lijian , Li Bo , Cai Dongqing
Published: April 2020

Our previous study has showed the feature of distribution and clonality of T-cell receptor alpha variable region (TRAV) and TRBV subfamily T cells. Recently, data indicated the γδ+T cells may play an important role in mediated graft versus leukemia effect after stem cells transplantation and anti-cancer response. In order to farther characterize the repertoire of CB T-cells, the frequency of αβ+ and γδ+T cells were detected in cord blood (CB) by FACS, the CDR3 size of 4 TRGV and 8 TRDV subfamily genes were analyzed in mononuclear cells (MCs) from 16 cord blood samples, using RT-PCR and genescan technique. Ten healthy individuals served as control. To determine the expression level of TRGV subfamily genes, quantitative analysis of TRGV I–III subfamilies was performed by real-time PCR. Low percentage of CD3+TCRγδ+ cells (1.33±0.58%) was observed in CB. The frequency expression was found in TRGVI (93.75%) and TRGVII (81.25%) in CBMCs, whereas the TRGVIII was detected in 9 out of 16 samples (56.25%). In TRDV subfamilies, the mean value of the number of expressed subfamilies in CBMCs is higher than that from adult peripheral blood (PB) group. The most frequently expressed in CB were TRDV1 (100%), TRDV2(93.75), TRDV8 (93.75%) and TRDV3 (81.25%). The frequency of TRDV5 and TRDV8 in CBMCs were significant higher that that from PBMC. The most PCR products of TRGV and TRDV subfamilies from 10 cord blood displayed polyclonal rearrangement pattern, whereas one or two PCR products from 6 CB samples displayed oligoclonality or biclonality. In contrast, PCR products from 9 of 10 adult healthy controls contained at least an oligoclonal peak in different TRGV or TRDV subfamilies respectively. In the present study, all PCR products form TRGVIV were showed monoclonality, the direct sequencing results showed that the V region of TRGVIV joint direct to C region, indicating that there is no rearrangement in TRGVIV and it is a pseudogene. The sequence data have been submitted to genebank and the Accession Number is EU395807. The pattern of TRGV subfamily expression level in CBMCs was TRGVI>TRGVIII>TRGVII, in contrast, TRGVII>TRGVI>TRGVII was found in PBMCs. In conclusion, our results indicate polyclonal and more diverse TRDV segment usage in CB γδ+ T-cells. The pattern of TRGV expression levels was different between T cells from PB and CB.
Characterization of MHC Class-I Restricted TCRαβ+CD4−CD8− Double-Negative T Cells Recognizing the gp100 Antigen from a Melanoma Patient after gp100 Vaccination
Authors: Voelkl Simon , Moore Tamson , Rehli Michael , Nishimura Michael , Fischer Karin , Mackensen Andreas
Published: April 2020

The immune attack against malignant tumors requires the concerted action of CD8+ cytotoxic T lymphocytes (CTL) as well as CD4+ T helper cells. The contribution of T cell receptor (TCR)αβ+ CD4− CD8− double-negative (DN) T cells to anti-tumor immune responses is widely unknown. In previous studies, we have demonstrated that DN T cells with a broad TCR repertoire are present in humans in the peripheral blood and the lymph nodes of healthy individuals. Here we characterize a human DN T cell clone (T4H2) recognizing an HLA-A2-restricted melanoma-associated antigenic gp100-peptide isolated from the peripheral blood of a melanoma patient. Antigen recognition by the T4H2 DN clone resulted in specific secretion of IFN-γ and TNF. Although lacking the CD8 molecule the gp100-specifc DN T cell clone was able to confer antigen-specific cytotoxicity against gp100-loaded target cells as well as HLA-A2+ gp100 expressing melanoma cells. The cytotoxic capacity was found to be perforin/granzymeB-dependent. Together, these data indicate that functionally active antigen-specific DN T cells recognizing MHC class I-restricted tumor-associated antigen (TAA) may contribute to anti-tumor immunity in vivo.
Allergen-Specific B Cell Quantity Is Similar in Allergic and Nonallergic Individuals, Whereas Allergen-Specific T Cells May Be Increased in Allergic Individuals
Authors: Khan Faisal , Ueno-Yamanouchi Aito , Serushago Bazir , Bowen Tom , Lu Cathy , Luider Joanne , Storek Jan
Published: April 2020

Allergen-specific IgE production is a hallmark of allergic asthma, rhinitis or eczema. Theoretically this could be due to a high number of allergen-specific B cells, a high number of allergen-specific T cells helping allergen-specific B cells to differentiate into IgE plasma cells, or other mechanisms. In this study, we compared the number of allergenspecific B cells and T helper (Th) cells in 41 patients with allergic asthma/rhinitis/eczema (allergic individuals) and 34 nonallergic individuals. Allergen-specific B and Th cells were enumerated by culturing CFSE-loaded blood mononuclear cells for 7 days with allergen (cat, dog, D.pteronyssimus, Timothy or birch), and determining by flow cytometry the number of B or Th cells that had proliferated (diluted CFSE). The quantities of B cells specific for each of the 5 allergens were similar in individuals allergic to the allergen (per skin prick test result) compared to nonallergic individuals. The quantity of Timothy-specific Th cells was 3-times higher in Timothy-allergic individuals compared to nonallergic individuals (p=0.023). In contrast, the quantity of cat-specific Th cells was similar in cat-allergic and nonallergic individuals. The quantities of dog, D.pteronyssimus and birch-specific Th cells were slightly higher in dog, D.pteronyssiumus and birch-allergic compared to nonallergic individuals. No significant change in the number of allergen specific B and Th cells was found when blood drawn from the same individual at different time points was compared for 3 allergic and 3 non-allergic individuals. We conclude that for some allergens (eg, Timothy), a high number of allergen-specific Th cells, but not B cells, may play a role in the pathogenesis of allergic asthma/rhinitis/eczema. For other allergens (eg, cat), the pathogenesis may be different.
CD16/FcãRIII Antigen Expression Determines Oxidative Sensitivity in Cytotoxic Lymphocyte Subsets
Authors: Aurelius Johan , Askarieh Galia , Romero Ana I. , Thorén Fredrik B. , Brune Mats L , Hellstrand Kristoffer
Published: April 2020

Oxidative stress, referring to cellular toxicity induced by reactive oxygen metabolites (ROM), has been proposed as a mechanism of immunosuppression in hematological malignant diseases. ROM produced by myeloid cells such as normal or malignant phagocytes have been shown to compromise functions of lymphocytes including natural killer (NK) cells and CD4+ or CD8+ T cells. Phagocyte-derived ROM induce downmodulation of the CD3/CD16 zeta; transduction antigen in these lymphocyte subsets with ensuing loss of function and lack of responsiveness to activating cytokines such as IL- 2. In addition, ROM exposure triggers poly(ADP-ribose) polymerase 1- and apoptosisinducing factor-dependent apoptosis in NK and T cells, but the molecular details of the ROM/lymphocyte interaction are not fully understood. The present study sought to determine the role of the CD16/FcγRIII (CD16) antigen, a signal transduction molecule expressed by NK and T cells and a receptor for the Fc portion of IgG, for oxidative stress in lymphocytes. Human NK (CD56+) or T cells (CD4+ or CD8+) were sorted into pure (>99%) CD16+ or CD16− populations, and incubated overnight with ROM-producing autologous mononuclear phagocytes or exogenous hydrogen peroxide. For all subsets of lymphocytes, the expression of CD16 was a strong determinant of the propensity of cells to acquire features of apoptosis after ROM exposure, as reflected by a pronounced difference of ROM sensitivity between CD16+ and CD16− NK or T cell populations. The higher degree of ROM-induced apoptosis in the CD16+ subsets was observed regardless of whether lymphocytes were incubated with autologous mononuclear phagocytes or exposed to exogenous hydrogen peroxide (p=0.04–0.001 for CD4+/16+, CD8+/16+ and CD16+/56+ lymphocytes as compared with corresponding CD16− subsets). Similar experiments using unsorted T and NK cells suggested that the differential sensitivity to oxidative stress in CD16+ and CD16− lymphocyte subsets was not explained by anti- CD16/CD16 interaction. In conclusion, our findings imply that oxidant-induced apoptosis is preferentially induced in lymphocytes expressing the CD16/FcγRIII antigen.
Clonal CD27+ CD19+ B-Cell Expansion through Inhibition of FCgIIR in HCV+ Lymphorpoliferative Disorders
Authors: De Re Valli , Pavan Alessandro , Racanelli Vito , Sansonno Silvia , Toffoli Giuseppe
Published: April 2020

HCV infection may or may not be associated with extra-hepatic manifestations such as type-II mixed cryoglobulinemia (MC), a clonal B cell proliferative disorder. In persistent HCV infection without MC a increase in serum immunoglobulins (Ig) is commonly observed. We found this increase is polyclonal and is determined primarily by increased levels of both HCV-specific and nonspecific IgG. Despite this hypergammaglobulinemia, memory CD27+ do not accumulate, depending on a heightened sensitivity of memory B cells to BCR-independent noncognate T cell help which speeds up their terminal differentiation into antibody secreting cells and make them more prone to activation induced cell death. In persistent HCV infection with MC, elevation of Ig is a general occurrence too. However, it is attributable to IgG and IgM. The latter include antibodies with rheumatoid factor (RF) activity, which are essential for the development of circulating, cryoprecipitable immune complexes. We found Hypergammaglobulinemia is sustained by a peripheral expansion of IgMk+restriction indicating that a limited number of antigens drives their proliferation through BCR interaction. We shown that IgM RF and their counterparts on the surface of bone marrow-resident monoclonal B cells react against a Fc-epitope of IgG and the HCV-NS3 protein. Based on the above findings, we propose a model whereby BCR, by binding the Fc of IgM/IgG/HCVNS3 immune complexes deprives FcgIIR of its natural ligand. This takes the brake off RF+CD27+ B cell proliferation and promotes their selective accumulation which is otherwise prevented by increased apoptosis susceptibility in persistent HCV infection without MC. By confocal microscopy analysis we found that both Fc and NS3 peptides are able to clusterize BCR. Cytofluorimetric analysis found activation of both SYK/ERK phosphorilation pathways. Both these observations are in agreement with the hypothesis of BCR activation in these samples. Data regarding FcR are in course.