scholarly journals 55: Alloanatigenic reactions after hematopoietic cell transplantation (HCT) induce genomic alterations in epithelial cells as shown in human studies and in an in vitro model

2007 ◽  
Vol 13 (2) ◽  
pp. 23
Author(s):  
A. Spyridonidis ◽  
P. Faber ◽  
L. Petrikkos ◽  
H. Bertz ◽  
J. Finke
Keyword(s):  
Epithelial Cells ◽  
Cell Transplantation ◽  
In Vitro Model ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Human Studies ◽  
Genomic Alterations ◽  
Vitro Model

Alloanatigenic Reactions after Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelial Cells as Shown in Human Studies and in an In Vitro Model.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3213-3213
Author(s):  
Alexandros Spyridonidis ◽  
Philipp Faber ◽  
Loizos Petrikkos ◽  
Jurgen Finke
Keyword(s):  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Protein Translation ◽  
Hematopoietic Cell ◽  
Hygromycin B ◽  
Hygromycin Resistance ◽  
Hacat Cells ◽  
Genomic Alterations ◽  
Allogeneic Hct

Abstract Previous studies from our group demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of patients who underwent allogeneic hematopoietic cell transplantation (HCT) (Blood2006;107:3389–3396). These genomic alterations were found only after allogeneic but not after autologous HCT, and therefore we hypothesized that an “allogeneic” effect is substantially involved in the mutation process. We extended our previous analyses by examining 210 bucall swabs obtained from 70 patients between day (d+) 26 and d+3514 after allogeneic HCT for the presence of MSI. MSI analysis was performed by PCR and denaturing capillary electrophoresis at three tetranucleotide (THO-1, SEE33, D14S120) and three mononucleotide microsatellite (ZP3, BAT26, SRY) loci. MSI was found in the buccal smears of 38% allografted patients (median time of occurence 322 days). In a prospective trial, in which patients were followed from time before transplantation until d+365, 5 out of 14 (35%) patients exhibited MSI after transplantation although all of them showed stable microsatellites before transplantation. We are currently pefroming statistical analyses in order to identify which clinical factors influence the presence of MSI and we will present the data in the meeting. To test the hypothesis that an “alloantigenic” effect is responsible for th induction of MSI, we developed a model system in which keratinocyte (HaCaT) cells were transfected with a palsmid vector which carries a G418 (neo) selectable marker and a microsatellite repeat (CA) that places the sequence for Hygromycin Resistance (HygR) out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the HygR reading frame and become detectable by hygromycin treatment as hygromycin resistant (HygR+) colonies. Pools of stably transfected (neo+) HaCaT cells were treated with supernatant (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLC) and assayed for HygR+ colonies 48h later. Cells transfected with a control, in-frame hygromycin B gene construct (p12) were used as positive controls. Using this system, we found that HaCaT cells aquire hygromycin resistance after treatment with supernatatant from MLC. Treatment of cells with hydrogen hyperoxid which has been shown in a E. Coli system to induce MSI (PNAS1998, 95:12468–12473) generated HygR+ colonies at a >80% lower frequency than the SN-MLC treatment. Control p12 transfected cells were grown with high efficiency in the presence of hygromycin B. In summary, our in vivo data confirm our previous results and provide evidence of genomic alterations after allogeneic HCT and our in vitro data are compatible with the hypothesis that an “alloantigenic” factor is the driving force in producing detectable MSI in the allografted patients. Elucidating the ultimate mechanisms underlying the genomic instability following allogeneic HCT may prove to be of major therapeutic value.

Alloantigeneic Reactions after Human Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelium: A Confirmation in Vivo and in Vitro Study and Implications to Secondary Neoplasia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 452-452
Author(s):  
Maria Themeli ◽  
Loizos Petrikkos ◽  
Miguel Waterhouse ◽  
Hartmut Bertz ◽  
Eleni Lagadinou ◽  
...  
Keyword(s):  
Cell Transplantation ◽  
Hematopoietic Cell Transplantation ◽  
Hematopoietic Cell ◽  
Secondary Malignancy ◽  
Hacat Cells ◽  
Genomic Alterations ◽  
Allogeneic Hct ◽  
Dna Strand

Abstract We previously demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of pts who underwent allogeneic Hematopoietic Cell Transplantation (HCT) but not after autologous transplantation (Blood2006;107:3389–3396). Confirmation in a larger and independent patient cohort and demonstration in an in vitro system was needed. In total 176 buccal samples obtained from 71 unselected patients who underwent allogeneic HCT were analyzed for MSI with standard PCR for microsatellite markers (THO-1, SEE33, D14S120 and D1S80) and analysis by capillary electrophoresis. MSI was observed in 37 (52%) allo-transplanted pts but never in the 31 controls (16 healthy and 15 pts after auto-HCT, 47 samples). MSI+ pts were significantly older than MSI-pts (median age 60y vs 48y, p<0.05). Other clinical features were not significantly different between MSI+ and MSI− pts: gender, myeloid vs lymphoid disease, sibling vs unrelated HCT, myeloablative vs reduced intensity conditioning, sex mismatched transplantation, bone marrow vs mobilized peripheral blood graft, T-cell depletion of the graft, use of methotrexate for GvHD prophylaxis, number of CD3+ cells/kg b.w. infused, occurrence and grade of mucositis during neutropenia, CMV reactivation, median ferritin levels post-transplant and occurrence of GvHD vs no GvHD. When GVHD was analyzed according to its severity we found that there was a trend for increasing risk of MSI for pts who experienced severe GvHD (RR=1.8) as compared to pts with no GvHD (RR=1). Secondary malignancy (5 skin-, 1 adeno-Ca) was diagnosed in 5 (14%) of the MSI+ pts and only in 1 (3%) MSI− pts. In an in vitro model of mutation analysis we tested the hypothesis that an alloantigenic stimulus is substantially involved in the mutation process. Briefly, keratinocyte DNA-repair proficient HaCaT cells were transfected with a reporter plasmid and genomic instability (GI) was detected and measured as hygromycin resistant clones. Treatment of HaCaT cells with H2O2 resulted in a time and dose dependent GI induction (1.9–5.7 fold). Cytokines abundant in alloreactions such as IFN-γ, TNF-α, TGF-β didn’t result in any GI in various concentrations and incubation times. Also, treatment with supernatants from different MHC non-matched Mixed Lymphocyte Cultures (MLC-SN) or with MLC separated from HaCaT cells with a semipermeable membrane didn’t induce any significant GI. In contrast, exposure of HaCaT cells to the whole MLC which includes activated cellular components (measured as BrdU+) resulted in significant induction of GI in 6 out of 8 different cases (mean 2.4-fold). GI was also evaluated in terms of DNA strand break formation by using the neutral comet assay. Treatment of HaCaT cells with whole MLC resulted in significant increase of DNA strand breaks as compared to HaCaT cells exposed to MLC-SN (p=0.039) or untreated controls (p=0.048). Western blot analysis revealed no significant alterations in the levels of MMR proteins upon treatment with MLC or MLC-SN. Exposure of HaCaT cells to MLC or MLC-SN resulted in increase of intracellular ROS levels as compared to untreated cells. The MLC treatment caused significantly higher increase of ROS levels than MLC-SN. The results of the present study confirm that GI of epithelial cells is a frequent phenomenon in patients after allogeneic HCT and give in vivo and in vitro support that alloantigeneic processes can be the cause of such a phenomenon. Progress in understanding DNA damage and repair after allogeneic HCT can potentially provide molecular biomarkers and therapeutic targets.

Abstract 135: Endothelial Dysfunction in Acute Graft-versus-host Disease After Allogeneic Hematopoietic Cell Transplantation. In vitro Evidence of the Protective Effect of Defibrotide

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Enrique Mir ◽  
Marta Palomo ◽  
Enric Carreras ◽  
Maribel Diaz-Ricart ◽  
Montse Rovira ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Protective Effect ◽  
Cell Transplantation ◽  
Graft Versus Host Disease ◽  
Hematopoietic Cell Transplantation ◽  
Endothelial Damage ◽  
Hematopoietic Cell ◽  
Graft Versus Host ◽  
Acute Graft

Acute Graft-Versus-Host Disease (aGVHD) is the most common early complication after allogeneic Hematopoietic Cell Transplantation (allo-HCT). We demonstrated endothelial dysfunction (ED) in association with allo-HCT. According to this data, aGVHD has been linked to an inflammatory process that may affect the endothelium. To investigate the differential degree of endothelial damage in patients developing or not aGVHD, to identify potential biomarkers, and to explore the protective effect of defibrotide (DF) in this scenario. DF has orphan designation for GVHD prevention. Patients blood samples were collected before allo-HCT, at day 0, and every week till day 28 after HCT. Plasma proteins (sTNFR1, sVCAM-1, VWF and ADAMTS-13) were measured as biomarkers of ED in individual samples from patients developing (GVHD, n=24), or not (NoGVHD, n=13), aGVHD. In in vitro assays, endothelial cells (EC) in culture were exposed to media containing pooled sera from patients to evaluate changes in the: a) expression of VCAM-1 and ICAM-1 on cell surfaces; b) presence of VWF on the extracellular matrix (ECM) and c) reactivity of the ECM towards platelets, under flow. The effect of DF was explored in the in vitro experiments by previous exposure of the EC (for 24h) followed by continuous incubation (100 μg/ml, added every 24h). Levels of sTNFRI, sVCAM-1 and VWF in samples from group GVHD were significantly higher than in NoGVHD (increases of 100, 37 and 150% respectively, at diagnose, p<0.01). ADAMTS-13 activity and VWF levels were inversely related. In in vitro studies, cell surface expression of VCAM-1 and ICAM-1, presence of VWF and platelet adhesion on the ECM in response to GVHD samples were always superior (increases vs NoGVHD of 80, 40, 100 and 21%, respectively, at diagnose). In vitro exposure of EC to DF attenuated signs of endothelial injury reducing significantly (p<0.05) the expression of VCAM-1, ICAM-1 and VWF (reductions of 22, 30 and 30%, respectively) in the GVHD condition. Our results demonstrate endothelial damage in association with aGVHD, as evidenced by elevated plasma levels of several biomarkers. The in vitro approach showed a marked proinflammatory and prothrombotic phenotype in association with aGVHD, which could be significantly prevented by defibrotide.

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