Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Recently , several studies demonstrated the feasibility of a real-time quantitative PCR (qPCR) approach for chimerism monitoring. qPCR offers a fast , sensitive, and elegant quantification of genotypes. However, before it becomes an established method for routine chimerism monitoring , a qPCR marker set for every transplant pair should be available. This requirement poses a major challenge since the genetic markers for qPCR-short insertions/deletions (Indels) and single nucleotide polymorphisms (SNPs)-published to-date do not guarantee applicability for every transplant pair. The aim of our study was to design and validate a new SNP allele-specific system to supplement an already existing Indel primer panel and improve applicability of the qPCR approach for chimerism status monitoring. Here , we present an approach for an economical in-house design of SNP allele-specific qPCR primers/probe sets with a locus-individualized reference system that allows for the accurate quantification of the respective informative locus using a simple ⌬⌬Ct method. We designed primers/probe sets specific for seven biallelic SNP loci and validated them in a population of 30 transplant pairs. Repeatability varied depending on the amount of quantifiable genotype. The combination of our SNPqPCR system and Indel primers increased recipient genotype identification from 86.6% to 96.6% when tested in a population of our transplant pairs. These results demonstrate the feasibility of our SNP-based qPCR approach to improve the applicability of a qPCR for chimerism monitoring.
Bacteriophage T4 encodes three ADP-ribosyltransferases, Alt, ModA, and ModB. These enzymes participate in the regulation of the T4 replication cycle by ADP-ribosylating a defined set of host proteins. In order to obtain a better understanding of the phage-host interactions and their consequences for regulating the T4 replication cycle, we studied cloning, overexpression, and characterization of purified ModA and ModB enzymes. Site-directed mutagenesis confirmed that amino acids, as deduced from secondary structure alignments, are indeed decisive for the activity of the enzymes, implying that the transfer reaction follows the Sn1-type reaction scheme proposed for this class of enzymes. In vitro transcription assays performed with Altand ModA-modified RNA polymerases demonstrated that the Alt-ribosylated polymerase enhances transcription from T4 early promoters on a T4 DNA template, whereas the transcriptional activity of ModA-modified polymerase, without the participation of T4-encoded auxiliary proteins for middle mode or late transcription, is reduced. The results presented here support the conclusion that ADP-ribosylation of RNA polymerase and of other host proteins allows initial phage-directed mRNA synthesis reactions to escape from host control. In contrast, subsequent modification of the other cellular target proteins limits transcription from phage early genes and participates in redirecting transcription to phage middle and late genes.Posttranslational ADP-ribosylation of proteins is catalyzed by ADP-ribosyltransferases (ADP-RTs), which have been identified in viral, bacterial, and eukaryotic systems. Transfer of the ADP-ribose moiety from the substrate NAD ϩ to a specific amino acid residue, frequently histidine or arginine within a target protein, modulates the activity of the acceptor. ADP-ribosylation changes the electrostatic potential of a target protein by introducing two phosphate groups and may affect protein-DNA as well as protein-protein interactions. ADP-RTs were initially discovered as the exotoxins of pathogenic bacteria. Therefore, most of our knowledge concerning these proteins has been gained by biochemical, genetic, and structural studies performed on bacterial toxins, such as those produced by Corynebacterium diphtheriae (15, 30), Bordetella pertussis (34), Vibrio cholerae (46), Pseudomonas aeruginosa (33), and Escherichia coli (45). New putative bacterial toxins were found to be encoded in the genomes of Streptococcus pyogenes and Salmonella enterica serovar Typhi (50).To date, the family of ADP-RTs comprises more than 40 enzymes, including bacterial exotoxins as well as a variety of other enzymes, such as the eukaryotic mono-and poly-ADPRTs, which include T-cell differentiation alloantigens like RT6 (9), poly-ADP-RTs (56), the dinitrogenase reductase regulation factor DraT (51,77), and enzymes encoded by T-even bacteriophages. The bacteriophage T4 gene products Alt (76 kDa), ModA (23 kDa), and ModB (24 kDa) appear to actively regulate gene expression during the transition from host t...
This study evaluated the efficacy and safety of dose-dense high-dose methylprednisolone (HDMP) plus rituximab (Rtx) in patients with high-risk CLL. Twenty-nine patients with relapsed or progressive CLL with adverse cytogenetics (17p deletion, TP53 mutation, 11q deletion, and/or trisomy 12) and/or progression within 12 months of fludarabine treatment were included. HDMP (1 g/m(2)) was administered daily for 5 days of each treatment course. Rtx was administered on days 1 (375 mg/m(2)) and 5 (500 mg/m(2)) of the first treatment course, on days 1 (500 mg/m(2)) and 5 (500 mg/m(2)) of the second course, and on day 1 (500 mg/m(2)) of courses 3-6. The cycles were repeated every 21 days. The overall response rate (ORR) was 62%, and 28% of patients had stable disease. In 13 patients with 17p deletion/TP53 mutation, ORR was 69%. After 22 months, the median progression-free and overall survivals were 12 and 31 months, respectively. The most frequent toxicity was hyperglycemia, and three deaths occurred in the study. Dose-dense treatment with HDMP and Rtx is an effective therapy with a favorable safety profile in patients with high-risk CLL, including those with 17p deletion/TP53 mutation.
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