Search

ObjectiveRefractory coeliac disease (RCD) is a potentially hazardous complication of coeliac disease (CD). In contrast to RCD type I, RCD type II is a precursor entity of enteropathy-associated T-cell lymphoma (EATL), which is associated with clonally expanding T-cells that are also found in the sequentially developing EATL. Using high-throughput sequencing (HTS), we aimed to establish the small-intestinal T-cell repertoire (TCR) in CD and RCD to unravel the role of distinct T-cell clonotypes in RCD pathogenesis.DesignDNA extracted from duodenal mucosa specimens of controls (n=9), active coeliacs (n=10), coeliacs on a gluten-free diet (n=9), RCD type I (n=8), RCD type II (n=8) and unclassified Marsh I cases (n=3) collected from 2002 to 2013 was examined by TCRβ-complementarity-determining regions 3 (CDR3) multiplex PCR followed by HTS of the amplicons.ResultsOn average, 106sequence reads per sample were generated consisting of up to 900 individual TCRβ rearrangements. In RCD type II, the most frequent clonotypes (ie, sequence reads with identical CDR3) represent in average 42.6% of all TCRβ rearrangements, which was significantly higher than in controls (6.8%; p<0.01) or RCD type I (6.7%; p<0.01). Repeat endoscopies in individual patients revealed stability of clonotypes for up to several years without clinical symptoms of EATL. Dominant clonotypes identified in individual patients with RCD type II were unique and not related between patients. CD-associated, gliadin-dependent CDR3 motifs were only detectable at low frequencies.ConclusionsTCRβ-HTS analysis unravels the TCR in CD and allows detailed analysis of individual TCRβ rearrangements. Dominant TCRβ sequences identified in patients with RCD type II are unique and not homologous to known gliadin-specific TCR sequences, supporting the assumption that these clonal T-cells expand independent of gluten stimulation.

Trotz der bekannten Vorteile der Raman-Spektroskopie, wie bspw. eine höhere chemische Selektivität gegenüber Messmethoden im nahen Infrarot (NIR) oder die im Vergleich zum mittleren Infrarotbereich (MIR) niedrigen Matrixeinflüsse des Wassermoleküls, ist diese optische Messtechnik in der Online-Prozessanalysentechnik nicht weit verbreitet. Ein wesentliches Problem besteht in einem oftmals kostenintensiven Nachrüsten einer Messstelle durch den Einbau sogenannter Immersionssonden in eine produktführende Rohrleitung oder einen Behälter. Eine praktikable Alternative stellt das hier entwickelte neuartige Sondensystem dar, welches eine Strahlführung über Linsen mit relativ großen Durchmessern beinhaltet, da dieses an vorhandene Schauglasarmaturen angekoppelt werden kann. Mit diesem robusten Sondenaufbau sind Brennweiten weit über 25 mm möglich, welche Echtzeit-Messungen von außerhalb der produktführenden Leitungen durch optische Schaugläser gestatten. Die dadurch entstehenden Messoptionen werden exemplarisch am Nachweis von Ethanol durch Schaugläser unterschiedlicher Dicken sowie bei einer quantitativen Echtzeit-Verfolgung eines Propylencarbonat-Wasser-Gemisches durch eine Schauglasarmatur (Nenndruck PN 16, Nennweite DN 50) im Technikumsmaßstab untersucht. Die vorgestellte Raman-Sonde hat durch einfache Adaption an bereits vorhandene Armaturen industrieller Anlagen das Potential einer preiswerten und kontaktlosen Inline-Messlösung mit hoher Standzeit in der Prozessanalysentechnik (PAT).

Im mittleren infraroten Spektralbereich (Wellenlänge von 3–12 Mikrometer) zeigen Flüssigkeiten spektrale Signaturen. Oft sind diese Absorptionsbanden jedoch breitbandig ausgeprägt und sehr stark im Vergleich zu etwa Strukturen in gasförmigen Species. Daher verwendet man bei klassischen Transmissionsmessverfahren mit üblichen FTIR-Spektrometern nur sehr kurze Absorptionswege (optische Pfadlänge OPL von einigen zehn Mikrometern), damit vom Detektor noch ein vom Rauschen differenzierbares Signal detektiert werden kann. Je nach Signatur lässt sich gegebenenfalls durch eine Reduktion auf eine Signal- und Referenzbande ein photometrischer Nachweis führen.

ZusammenfassungTypische Spektrometer im mittleren Infrarot verwenden aufgrund der starken Absorption in flüssigen Medien Transmissionsküvetten mit kleinsten Schichtdicken. Durch die damit einhergehende Verstopfungsproblematik werden diese im Vergleich zu Messzellen oder Sonden in anderen Spektralbereichen wartungsintensiver. Der Einsatz der ATR (abgeschwächte Totalreflexion) Technik erlaubt dahingegen eine Messung mit effektiv kleinen Absorptionslängen in Reflexionsgeometrie. Zusätzlich wird hier am Beispiel der Isocyanate eine Anwendung vorgestellt, die es ermöglicht, die breitbandige Spektroskopie auf die bekannte Photometrie zu reduzieren, um eine gleichzeitig sensitive und robuste Messapplikation zu erhalten. Dieser vorgestellte Ansatz der ATR Photometrie hat das Potential einer preiswerten inline Messlösung für verschiedenste Anwendungen in der Prozessanalysentechnik (PAT).

We describe the use of oligonucleotide fingerprinting for the generation of a normalized cDNA library from unfertilized sea urchin eggs and report the preliminary analysis of this library, which resulted in the establishment of a partial gene catalogue of the sea urchin egg. In an analysis of 21,925 cDNA clones by hybridization with 217 oligonucleotide probes, we were able to identify 6291 clusters corresponding to different transcripts, ranging in size from 1 to 265 clones. This corresponds to an average 3.5-fold normalization of the starting library. The normalized library represents about one-third of all genes expressed in the sea urchin egg. To generate sequence information for the transcripts represented by the clusters, representative clones selected from 711 clusters were sequenced. The construction and preliminary analysis of the normalized library are the first steps in the assembly of an increasingly complete collection of maternal genes expressed in the sea urchin egg, which will provide a number of insights into the early development of this well-characterized model organism.

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith–Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith–Magenis syndrome.

A prerequisite to understanding the evolution of the human X chromosome is the analysis of synteny of X-linked genes in different species. We have focused on the spermine synthase gene in human Xp22.1. We show that whereas the human gene spans a genomic region of 54 kb, theFugu rubripesgene is encompassed in a 4.7-kb region. However, we could not find conserved synteny between this region of human Xp22 and the equivalentF. rubripesregion. A cosmid clone containing theF. rubripesgene does not contain other X-linked genes. Instead we identified homologs of human genes that are autosomally localized: the ryanodine receptor type I (RYRI), which is implicated in malignant hyperthermia and central core disease, and the HE6 gene. Comparison of theF. rubripes, Tetraodon fluviatilis,mouse, human, andDanio rerio5′UTRs of spermine synthase highlights conserved sequences potentially involved in regulation. Interestingly, pseudogenes of this gene that are present in the human and mouse genomes seem to be absent in the compactF. rubripesgenome. Analysis of aD. rerioPAC clone containing spermine synthase shows an intermediate genomic size in this fish. Sequence analysis of this PAC clone did not reveal other known genes: neither the RYRI gene, nor the HE6 gene, nor other human Xp22 genes were identified.

Hennig: HS Diagnomics GmbH: Employment, Equity Ownership.

Background: Reactivation of CMV and EBV negatively impacts on outcome after allogeneic stem cell transplantation (aSCT). Specific antiviral therapy is only available for CMV. With the exception of ganciclovir all drugs are being used off-label. 40-50% of patients reactivate CMV following aSCT. For the 20-30% of patients reactivating EBV, only rituximab is available to control EBV. Rituximab leads to long term B-cell depletion requiring frequent administration of immunoglobulins. To cover the unmet medical need of CMV and EBV control after aSCT, we investigate a somatic cell therapy approach by means of CMV- and EBV-specific peptide-stimulated T cells. We have set up a prospective randomized controlled phase I/IIa multi-center clinical trial to evaluate the preventive and preemptive adoptive transfer of this ATMP in patients after aSCT (EudraCT number 2012-004240-30). The multi-center trial is currently recruiting.