scholarly journals Comparison of Gold Standard Genescan with NGS-Based TCR-Beta Clonality Analysis Using Oncomine TCR Beta-Short Read Assay

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4664-4664
Author(s):  
Cora Husemann ◽  
Karsten Kleo ◽  
Timothy Looney ◽  
Chris Allen ◽  
Bernhard Josef Woermann ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Celiac Disease ◽  
T Cell ◽  
Multiplex Pcr ◽  
Gold Standard ◽  
Dna Assay ◽  
Thermo Fisher Scientific ◽  
Pcr Products ◽  
Primer Sets ◽  
Fisher Scientific

Introduction All mature T-cell-based lymphoid malignancies harbor identical (clonal) rearrangements of their T cell receptor (TCR) genes (van Dongen et al. Clinica Chimica Acta. 1991, 198, 1-92). The clonality assessment of the rearranged TCR gene is particularly important for the identification, characterization and monitoring of T-cell neoplasms since histology and immunophenotyping alone is not enough to make conclusive diagnosis in all cases. Diagnostic clonality testing is currently based on the parallel analysis of the rearranged TCR-gamma and TCR-beta chain genes. This is done using a multiplex PCR developed within the European BIOMED-2/EuroClonality consortium (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis of the resultant PCR products. Although, this method is considered the "gold standard", which is established worldwide in many molecular diagnostic laboratories, the resolution of this approach is limited, especially in cases with low percentage of clonally rearranged TCRs and a high mixture of non-clonally rearranged T-cells. Next generation sequencing (NGS) is a powerful tool to provide resolution at single gene level. This also holds true for NGS assays, which are able to detect all unique TCR rearrangements in a given sample with very high resolution and sensitivity. This approach can be employed for precise assessment of the immune repertoire, minimal residue disease and T-cell clonality. Detailed insights into the clonotypes provides a great potential for early diagnosis of T-cell neoplasms, and the identification of individual clones is essential for monitoring of the disease. Methods We investigated 19 formalin-fixed paraffin-embedded (FFPE) tissue samples from celiac disease patients (n = 14) and routine diagnostic cases suspicious of lymphoma (n = 5). After DNA extraction, we performed multiplex PCRs using the BIOMED-2 TCR-beta primer sets (van Dongen et al. Leukemia. 2003, 17, 2257-2317) followed by capillary electrophoresis. For TCR-beta NGS, the Oncomine™ TCR Beta-SR DNA Assay (Thermo Fisher Scientific) was used according to the manufacturer's instructions and sequencing was performed on the IonTorrent S5. Identification of individual clonotypes and bioinformatics analysis of the data was done with the help of the IonReporter software (Thermofisher Scientific). It is worth noting that the 14 celiac disease samples were previously analysed by a different TCR-beta NGS approach (Ritter et al. Gut. 2018, 67, 644-653) and utilized for the comparison of the NGS-data. Results 17 out of 19 cases analysed by TCR-beta multiplex PCR (BioMed-2) followed by capillary electrophoresis and by the Oncomine™ TCR Beta-SR assay displayed a very similar length distribution of the PCR products. This holds true for samples with a clonal appearance and for samples with an oligo-/polyclonal pattern. In two discrepant cases, the Primerset B of the BioMed-2 approach showed a dominant amplification product, which was not as clear by TCR-beta NGS in which both cases displayed an oligoclonal TCR-beta gene rearrangement pattern with a few dominant T-cell populations. Both samples were from celiac disease patients, which mainly present a pronounced oligoclonal TCR-beta gene rearrangement pattern. Strikingly, the NGS data from the Oncomine™ TCR Beta-SR DNA Assay delivered highly comparable results when compared to the sequencing data of Ritter et al. 2018 despite having completely different primer sets and a different NGS platform. Conclusion Our comparison of the conventional multiplex PCR (BioMed-2) and TCR-beta NGS (Oncomine™ TCR Beta-SR DNA Assay) demonstrated a very high concordance (17/19 cases) of the molecular data. The two discordant cases can be explained by an over-interpretation of dominant species BioMed-2 Primerset B amplifications, which often show up in cases with low T-cell content or oligoclonal T-cell counts. These T-cell clonotypes are detectable by TCR-beta NGS only at a low percentage because of combination of all type of rearrangements in one assay. The robustness and reliability of NGS-based TCR-beta clonality testing was demonstrated by comparison of two completely different assays, leading to very similar results for all celiac disease patients. We are thus very confident that NGS-based clonality testing will be the "gold standard" of the future. Disclosures Looney: Thermo Fisher Scientific: Employment. Allen:Thermo Fisher Scientific: Employment.

scholarly journals Simplified Fluorescent Multiplex PCR Method for Evaluation of the T-Cell Receptor Vβ-Chain Repertoire

2005 ◽  
Vol 12 (4) ◽  
pp. 477-483 ◽  
Author(s):  
Sanjit Fernandes ◽  
Surendra Chavan ◽  
Vivek Chitnis ◽  
Nina Kohn ◽  
Savita Pahwa
Keyword(s):  
T Cell ◽  
Multiplex Pcr ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
T Cell Subsets ◽  
Clinical Samples ◽  
Cdr3 Length ◽  
Pcr Products ◽  
Pcr Method ◽  
T Cell Receptor Vβ

ABSTRACTRationale: evaluation of the T-cell receptor (TCR) Vβ-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR Vβ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream Vβ primers instead of the conventionally labeled downstream Cβ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a Cβ primer and an Omniscript RT kit. The 24 Vβ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 Vβ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled Cβ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: Vβ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled Vβ primers. The resolution was superior to that obtained with the labeled Cβ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the Cβ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor Vβ-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR Vβ repertoire analyses in clinical trials.

203 SINGLE-STEP GENE EDITING OF 3 XENOANTIGENS IN PORCINE FIBROBLASTS USING PROGRAMMABLE NUCLEASES

2017 ◽  
Vol 29 (1) ◽  
pp. 210 ◽  
Author(s):  
A. Perota ◽  
I. Lagutina ◽  
C. Quadalti ◽  
R. Duchi ◽  
P. Turini ◽  
...  
Keyword(s):  
Gene Editing ◽  
Magnetic Beads ◽  
Single Step ◽  
High Rate ◽  
Pcr Analysis ◽  
Thermo Fisher Scientific ◽  
Protective Genes ◽  
Pcr Products ◽  
Programmable Nucleases ◽  
Fisher Scientific

Programmable nucleases (ZFN, Tal Effector Nucleases, and CRISPR) opened a new era for mammal genome editing, in particular for the pigs used for xenotransplantation. Multiple gene editing events are required both for knockout (KO) of xenoantigens and for targeted integration of human protective genes (Perota et al. 2016 J. Genet. Genomics 43, 233–23). The objective of the present work was to edit selected pig lines to KO the enzymes coding for the most relevant xenoantigens (i.e. GGTA1, CMAH, and B4GalNT2), combining Talens and CRISPR/Cas9 technologies to magnetic beads selection (Li et al. 2013 Xenotransplantation 22, 20–31). Primary porcine adult fibroblasts were transfected using Nucleofector (V-024 program). In a single reaction 2 × 106 fibroblasts were co-transfected using 2 different sets of TALENS (4 μg/set) specific for CMAH (Conchon et al., 2013) and GGTA1 (Perota et al., 2015) genes together with B4GalNT2-specific CRISPR/Cas9 expression vector (2 μg; pX330-B4GalNT2; Estrada et al., 2015). Eight days post-transfection (DPT), Gal–/– cells were selected initially using biotin-conjugated IB4 lectin (Sigma, St. Louis, MO, USA) and magnetic beads (Dynabeads M-280, Thermo Fisher Scientific, Waltham, MA, USA). The selected cells were then plated on 150-mm Petri dishes (200 cells/dish) and cultured for 10 days. Selected colonies were expanded for PCR analysis and cryopreserved for somatic cell nuclear transfer (SCNT). All colonies were analysed by PCR for CMAH gene and their resulting products were digested with HindIII (HindIII-RFLP). Colonies that lost wild-type HindIII as a consequence of Talens effected deletion were PCR characterised for GGTA1, selecting those that had detectable Indels after gel electrophoresis and finally analysed by PCR for B4GalNT2. All PCR products were validated by sequencing for all the 3 genes of interest (TopoTA, Thermo Fisher Scientific). Selected colonies were used as nuclear donors for SCNT (Lagutina et al., 2006). Eight DPT we obtained 3.45 ×106 cells. About 6.0 × 103 Gal-negative cells (0.17%) were collected from the supernatant after magnetic beads separation. Eighteen DPT, 120 colonies were picked up and their HindIII-RFLP analyses on CMAH gene revealed that 22 colonies (18.3%) were KO for both CMAH alleles. Of these 22 colonies following electrophoretic analyses of GGTA1-PCR products, 13 colonies had detectable Indels. These 13 colonies were finally PCR analysed and sequenced for B4GalNT2 and sequenced. Final sequencing results confirmed that 2 colonies (1.6%) resulted in KO for the 3 genes. Three different zona-free SCNT experiments were done and 579 reconstructed embryos were obtained. On Day 7, 322 morulae or blastocysts (56%) were transferred in 3 synchronised sows and 2 (66%) became pregnant. In conclusion, after gene editing with programmable nucleases, combining beads-mediated selection with well-designed molecular analyses, we developed a multistep assay that can be used efficiently to detect desired gene edited events in cell colonies suitable for the SCNT. Embryos generated after SCNT were able to establish pregnancies at a high rate. This work is supported by European FP7 grants Translink (n° 603049) and Xenoislet (n° 601827).

Large Granular Lymphocyte Leukemia: Clonality Reconsidered.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3102-3102
Author(s):  
Wesley Witteles ◽  
Bing Zhang ◽  
Iris Schrijver ◽  
Daniel Arber ◽  
Jason Gotlib ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
T Cell ◽  
High Resolution ◽  
Clinical Characteristics ◽  
Cell Receptor ◽  
Concordance Rate ◽  
Large Granular Lymphocyte ◽  
Third Generation ◽  
Primer Sets ◽  
Lgl Leukemia

Abstract Background: T-cell large granular lymphocyte (LGL) leukemia is widely considered to represent a monoclonal proliferation of lymphocytes. Clonality assessment methods have evolved from Southern blots (first-generation) to polymerase chain reaction with heteroduplex electrophoresis (second-generation) to high-resolution capillary electrophoresis (third-generation) testing. Aims: To determine if third-generation T-cell clonality assays result in a higher frequency of oligoclonal results, to compare the concordance for testing at the T-cell receptor (TCR) gamma (TCRG) and TCR beta (TCRB) loci, and to compare the clinical characteristics of patients with monoclonal vs. oligoclonal TCRs. Methods: The study population consisted of patients from August 1999-April 2007 with elevated circulating LGLs and cytopenia(s). TCRG locus clonality was determined by both the heteroduplex method and capillary electrophoresis in 35 patients. 89 samples were tested for TCRG and TCRB clonality using the Biomed II PCR primer sets and capillary electrophoresis on an ABI 3100 automated DNA sequencer. Determinations of clonality were made independently by three pathologists blinded to the clinical characteristics of the patients. Results: A total of 93 patients (median age 50 years, 53% female) were evaluated. Median absolute neutrophil count was 1.56 × 109/L (range 0.2–7.8 × 109/L), median lymphocyte count was 1.81 × 109/L (range 0.6–13 × 109/L), and median hemoglobin was 13 g/dL (range 6.3–17.4 g/dL). The concordance rate for TCRG clonality testing by the heteroduplex and capillary electrophoresis methods was only 40%. The primary difference was a striking increase in the frequency of oligoclonal results by the capillary electrophoresis method (p= 0.00007). All of these samples appeared monoclonal by the lower resolution heteroduplex assay (Table 1). Concordance for clonality for TCRG vs. TCRB was 54% (Table 2). All samples had monoclonality or oligoclonality demonstrated at TCRG or TCRB, but only 26% were monoclonal at both loci. The clinical characteristics for the 23 patients with monoclonal TCRG and TCRB appeared similar to the 23 patients with oligoclonal TCRG and TCRB. The median age in both groups was 53 years, with 61% of patients in each group requiring treatment after a median of 36.8 and 38.6 months of follow-up, respectively. Discussion: The high resolution of capillary electrophoresis appears to result in a much greater proportion of oligoclonal TCRG results, which by the older heteroduplex method would have been considered monoclonal. Furthermore, the concordance rate at TCRG and TCRB appears to be remarkably low. Though oligoclonal T-cell populations are generally believed to be transient and reactive processes, the clinical characteristics of our oligoclonal and monoclonal cohorts did not differ significantly. Conclusion: Capillary electrophoresis frequently identifies patients with oligoclonal TCR whose clinical features are indistinguishable from those of patients with classic monoclonal LGL leukemia. Heteroduplex Monoclonal Negative Oligoclonal Total Monoclonal 12 1 1 14 Capillary Electrophoresis Negative 4 2 0 6 Oligoclonal 15 0 0 15 Total 31 3 1 35 TCRG Monoclonal Negative Oligoclonal Total Monoclonal 23 3 12 38 TCRB Negative 7 0 1 8 Oligoclonal 15 3 25 43 Total 45 6 38 89

scholarly journals Fully Automated Workflows Quantify and Report Key T-Cell and B-Cell Receptor Biomarkers Relevant to Immuno-Oncology and Heme-Oncology Research

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 4002-4002
Author(s):  
Shrutii Sarda ◽  
Geoffrey Lowman ◽  
Michelle Toro ◽  
Loni Pickle ◽  
Timothy Looney ◽  
...  
Keyword(s):  
T Cell ◽  
B Cell ◽  
Complex Disease ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Oncology Research ◽  
Gene Usage ◽  
Thermo Fisher Scientific ◽  
V Gene ◽  
Fisher Scientific

Abstract Background T-cell and B-cell repertoire analysis is used in oncology research, to understand the etiology of complex disease phenotypes, for the identification of biomarkers predictive of disease burden, outcome, and response to treatment, and for research in diagnosis and recurrence monitoring. Key predictors include secondary and tertiary repertoire features not reported by existing sequencing software solutions. For example, due to ongoing somatic hypermutation in mature B-cell receptors, the underlying sequence of a given clone can accumulate base differences and appear as several distinct clones with smaller frequencies, thereby hampering the ability of analysis software to detect its presence as a single dominant clone with the highest frequency. This has particularly detrimental implications for research in disorders such as follicular lymphoma and may require clonal lineage analysis for proper mitigation. Therefore, to aid the downstream analytics of biomarker identification and the study of complex disease, we developed fully automated analysis solutions that directly compute and report several key features (clonal lineage, amongst several others described below) pertinent to this area of research. Results We developed the Oncomine™ TCR Beta-SR, TCR Gamma-SR, BCR IGH-SR and BCR IGKL-SR workflows on Ion Reporter™ to characterize T-cell (β, γ chains) and B-cell (heavy and light (κ, δ) chains) repertoires. These workflows generate output tables and visualizations for primary repertoire features such as detected clones (viz., unique rearrangements in the receptor DNA sequence), their frequencies, as well as their somatic hypermutation levels in the case of B-cells (Figure 1a & 1b) for clonality assessment and rare clone detection. The software also quantifies and reports several secondary and tertiary repertoire features in a sample, such as clonal diversity, evenness of the clonal population, and B-cell lineage groupings useful in identifying related sub-clones. It includes spectratyping format plots to simultaneously assess the above features as a function of v-gene usage and CDR3 length combinations (Figure 1c & 1d), thereby providing users a complete snapshot of the repertoire, and also the capability to quickly determine CDR3 lengths and V-gene usage of highly expanded or mutated clones. A separate CDR3 lengths histogram is included, as well as a heatmap that depicts the distributions/intensity of Variable-Joining gene combinations (Figure 1e & 1f). Furthermore, the TCR workflows also report (i) convergence frequencies (fraction of clones with different nucleotide sequences, but identical amino acid sequences), and (ii) haplotype grouping for an analyzed sample, based on V-gene allele genotyping and clustering (Figure 1g). In addition, the long read Oncomine™ BCR IGH-LR workflow uniquely reports the isotype class for every detected clone, and includes a visualization of total reads, clones and lineages in the sample represented by isotype (Figure 1h). Conclusion The Oncomine™ immune repertoire workflows for T-cell and B-cell receptor sequencing were designed to be of high utility in distinct areas of malignancy research, and we expect them to greatly simplify complex downstream analyses. The unique capabilities of the workflows to automatically report secondary and tertiary repertoire features such as (i) clonal lineages for improved dominant clone detection in blood cancers, (ii) TCR clone convergence for prediction of response to immune checkpoint inhibitors [1,2], (iii) TCR haplotype grouping for evaluation of risk factors for autoimmunity and immune-related adverse events [3], and (iv) isotype classification in BCRs for studying pan-cancer immune evasion mechanisms, demonstrate the clear advantages of using these automated workflows over other existing solutions. For research use only. References 1) Looney TJ et al. (2020) TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer. Front. Immunol. 10:2985. 2) Naidus et al. (2021) Early changes in the circulating T cells are associated with clinical outcomes after PD-L1 blockade by durvalumab in advanced NSCLC patients. Cancer Immunology, Immunotherapy 70:2095-2102 3) Looney TJ et al. (2019) Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing. Journal of Immunotherapy and Precision Oncology. 2 (4): 137-143. Figure 1 Figure 1. Disclosures Sarda: Thermo Fisher Scientific: Current Employment. Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Looney: Thermo Fisher Scientific: Ended employment in the past 24 months; Singular Genomics: Current Employment. Hyland: Thermo Fisher Scientific: Current Employment.

Rapid classification of partial waxy wheats using PCR-based markers

Genome ◽  
2002 ◽  
Vol 45 (6) ◽  
pp. 1150-1156 ◽  
Author(s):  
T Nakamura ◽  
P Vrinten ◽  
M Saito ◽  
M Konda
Keyword(s):  
Capillary Electrophoresis ◽  
Multiplex Pcr ◽  
Wheat Line ◽  
Wild Type ◽  
Waxy Wheat ◽  
Sds Page ◽  
Primer Sets ◽  
Pcr Conditions ◽  
Single Set

Mutations in the three homeologous waxy loci Wx-A1, Wx-B1, and Wx-D1 of a waxy wheat line have previously been characterized at the molecular level. Using combinations of these mutations, six types of partial waxy wheat plus wild type and waxy wheat (types 1–8) can be produced. Here, we describe primer sets for all three loci that can be used under a single set of conditions, allowing 32 lines to be characterized as types 1–8 in a single PCR run using a 96-well plate. Using multiplex PCR, mutations at the Wx-B1 and Wx-D1 loci can be identified in a single PCR, reducing the number of reactions necessary to identify and select the desired partial waxy wheat line. A single multiplex PCR can be used to detect all three mutations when products are analyzed using capillary electrophoresis on a microchip device. The PCR conditions and primers are effective with a number of cultivars from other countries, indicating that the mutations found at the Wx-A1 and Wx-B1 loci of these cultivars likely have the same origins as the mutations in the corresponding loci of the waxy wheat line used in this study. The PCR selection method described here is an easy and effective alternative to the commonly used SDS–PAGE methods for identification of null alleles.Key words: partial waxy wheat, amylose, PCR, capillary electrophoresis, marker-assisted selection.

scholarly journals miR106a-363 Cluster Has Oncogenic Potential in Childhood T-Cell Acute Lymphoblastic Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5142-5142
Author(s):  
Monika Drobna ◽  
Bronislawa Szarzynska-Zawadzka ◽  
Maria Kosmalska ◽  
Roman Jaksik ◽  
Tomasz Szczepanski ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Lines ◽  
Target Genes ◽  
Lymphoblastic Leukemia ◽  
Luciferase Reporter ◽  
Pathway Enrichment Analysis ◽  
Thermo Fisher Scientific ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Fisher Scientific

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors and is characterized by high genetic, immunophenotypic, and clinical heterogeneity. MicroRNAs (miRNAs) belong to the class of small noncoding RNAs and are implicated in the regulation of hematopoiesis and in the development of leukemia. miRNAs control expression of their target genes at the post-transcriptional level by blocking translation of messenger RNAs (mRNAs) or promoting their degradation. Some miRNAs are encoded within clusters, giving rise to policistronic transcripts. Such miRNAs are co-expressed and may co-regulate the expression of genes involved in certain biological processes and pathways. In our recent study we performed miRNA profiling in pediatric T-ALL using Next-Generation Sequencing (Dawidowska M et al. Blood 2017; 130:1443) and identified miRNAs differentially expressed in T-ALL. The set of overexpressed miRNAs included, among others, miR-20b-5p, miR-363-3p and miR-92a-2-5p, belonging to a cluster of six miRNAs: miR-106a-363 (ChrXq26.2). miR-106a-363 cluster is a paralog of miR-17-92 cluster (Chr13q31.3), a prototypic oncogenic cluster of eminent importance in human hematopoietic cancers, with reported role in T-ALL pathogenesis (Mavrakis KJ et al., Nature Cell Biology 2010, 12:4). Despite the similarity of seed sequences between miRNAs from miR-17-92 and miR-106a-363 clusters, the significance of miR-106a-363 cluster in T-ALL remains to be elucidated. In this study we investigated the expression of the miR-20b-5p, miR-363-3p and miR-92a-2-5p in children with T-ALL, healthy donor thymocytes, normal bone marrow samples and 6 T-ALL cell lines. RT-qPCR analysis (TaqMan Advanced miRNA Assays; Thermo Fisher Scientific) confirmed overexpression of 2 miRNAs from cluster miR-106a-363 (miR-20b-5p and miR-363-3p) in children with T-ALL and in T-ALL cell lines, suggesting their oncogenic function. To predict potential target genes of overexpressed miRNAs belonging to miR106a-363 cluster, we applied 8 target prediction algorithms and pathway enrichment analysis. This revealed the enrichment of miR-20b-5p and miR-363-3p target genes in GO term: positive regulation of apoptosis. We further validated predicted miRNA-mRNA interactions (Dual Luciferase Reporter Assays; Promega) confirming the majority of them (e.g. PTEN, FBXW7, BCL2L11). Finally, we assessed the effect of mimicry/inhibition (miRVana, Thermo Fisher Scientific) of overexpressed miRNAs from miR-106a-363 cluster on proliferation, cell cycle distribution and apoptosis in 3 T-ALL cell lines. Overexpression of miR-20b-5p and miR-363-3p in CCRF-CEM, DND-41 and P12-Ichikawa cells resulted in increased proliferation and inhibited apoptosis. To summarize, in this study we showed that miRNAs belonging to miR-106a-363 cluster directly interact with mRNAs implicated in the regulation of apoptosis and that miR-20b-5p and miR-363-3p have pro-proliferative and anti-apoptotic effects in T-ALL cells in vitro. These results indicate that miR-106a-363 cluster may have an oncogenic role in the pathogenesis of T-ALL via suppression of pro-apoptotic genes. Research funded by National Science Centre, Poland grants: 2014/15/B/NZ2/03394, 2017/25/N/NZ2/01132 and National Centre of Research and Development (NCRD) grant STARTEGMED3/304586/5/NCBR/2017. Disclosures No relevant conflicts of interest to declare.

Serendipity in Refractory Celiac Disease: Full Recovery of Duodenal Villi and Clinical Symptoms after Fecal Microbiota Transfer

2016 ◽  
Vol 25 (3) ◽  
pp. 385-388 ◽  
Author(s):  
Yvette H. Van Beurden ◽  
Tom Van Gils ◽  
Nienke A. Van Gils ◽  
Zain Kassam ◽  
Chris J.J. Mulder ◽  
...  
Keyword(s):  
Celiac Disease ◽  
T Cell ◽  
Clostridium Difficile ◽  
Clostridium Difficile Infection ◽  
Cell Lymphoma ◽  
Fecal Microbiota ◽  
Disease Type ◽  
T Cell Lymphoma ◽  
Type Ii ◽  
Refractory Celiac Disease

Treatment of refractory celiac disease type II (RCD II) and preventing the development of an enteropathy associated T-cell lymphoma in these patients is still difficult. In this case report, we describe a patient with RCD II who received fecal microbiota transfer as treatment for a recurrent Clostridium difficile infection, and remarkably showed a full recovery of duodenal villi and disappearance of celiac symptoms. This case suggests that altering the gut microbiota may hold promise in improving the clinical and histological consequences of celiac disease and/or RCD II. Abbreviations: CDI: Clostridium difficile infection; EATL : enteropathy associated T-cell lymphoma; FMT: fecal microbiota transfer; IEL: intraepithelial lymphocytes; RCD II: refractory celiac disease type II; TPN: total parenteral nutrition.

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