Einfluss einer Anthracyclin- und Taxanbehandlung auf die immunologische Tumorabwehr unter Antikörpertherapie

Brustkrebs ist noch immer die häufigste Krebserkrankung bei Frauen. Da bei etwa einem Viertel der Patient*innen, die an Brustkrebs leiden, eine Überexpression von Her2/neu besteht, die mit einem schlechteren klinischen Outcome korreliert, rückte Her2/neu sehr schnell als Target zielgerichteter Therapien in den Fokus. Der erste dieser Anti-Her2-Antikörper, der in die Klinik Einzug hielt, war Trastuzumab. Durch die Her2-Bindung vermittelt Trastuzumab dabei neben direkten, auch indirekte immunvermittelte Effekte, die u. a. von NK-Zellen getragen werden. Denn sie können die Her2+ Zielzellen antikörperabhängig attackieren. Diese antikörperabhängige zelluläre Zytotoxizität (ADCC) wird als wesentlicher Wirkmechanismus der Trastuzumab-Therapie diskutiert. Leitliniengerecht erfolgt die Anti-Her2-Antikörpertherapie bei Patient*innen mit Her2+ Brustkrebs aber i. d. R. in Kombination mit einer (neo-)adjuvanten Chemotherapie. Um deren immunsuppressiven Effekte wissend, wäre auch eine Kompromittierung der NK-Zellen und der NK-Zell-vermittelten ADCC denkbar. Da einige konventionelle Chemotherapeutika aber entgegen der allgemeinen Annahme auch immunogen agieren, stellt sich die Frage, ob und inwieweit die kombinierten Therapien – v. a. im Hinblick auf ADCC – tatsächlich interferieren. Um dem nachzugehen, ahmten wir die (neo-)adjuvante Therapie in einem in-vitro-Kokulturmodell nach, indem wir den Anti-Her2-Antikörper (Trastuzumab) entweder mit einem Anthracyclin (Epirubicin) oder einem Taxan (Paclitaxel) kombinierten. Vorrangig untersucht wurde das in einem Anti-Her2-Antikörper-sensiblen Her2+ Tumormodell (BT-474) mit NK-Zellen der Linie NK3.3, aber auch mit cord-blood derived MNC, die polyklonale NK-Zellen enthalten. In beiden Modellen zeichnete sich – bemessen an der Induktion von Zelltod (Annexin V/FITC-Assay) – ein (mindestens tendenziell) positiver Effekt der additiven Anthracyclin (Epirubicin)- und geringer auch der Taxan (Paclitaxel)-Therapie auf die direkte und indirekte antikörperabhängige zelluläre Zytotoxizität (ADCC) der NK-Zellen ab. Der beobachtete Benefit warf die Folgefrage auf, wie v. a. Epirubicin die antikörperab- und -unabhängige NK-Zell-Zytotoxizität akzentuieren könnten: indirekt, indem sie die Her2+ Tumorzellen „sensibilisieren“ (bereits beschrieben), und/oder direkt, indem sie die NK-Zellen „konditionieren“. Nach Änderungen auf NK-Zell-Ebene fahndend, analysierten wir deren Zytokin- und Proteinprofil in Abhängigkeit der Behandlungen (intra- und extrazelluläre FACS-Analysen, Antibody-Array). Das Protein-Profiling (scioCD antibody array, Sciomics) förderte dabei die differentielle Expression einiger Proteine zu Tage, denen aber – anders als erwartet – eher eine immunregulierende (und weniger eine zytotoxische) Bedeutung zukommt. Über diese könnte die NK-Zelle mit anderen Immunzellen interagieren und nicht nur als „Killer“ agieren – eine Hypothese, die jedoch in dieser in-vitro-Studie nicht weiter verfolgt werden konnte

Phonological awareness in German-speaking preschool children with cochlear implants – 3 case examples

Objective The aim was to explore PA skills German-speaking preschool children with cochlea implants (CIs) and how these skills may be related to their speech and language skills. Methods Three monolingual German-speaking pre-school children aged 5;04–6;01 with bilateral CIs were tested. Their cognitive, speech and language skills were assessed. Six subtests of a standardized PA test battery were administered (i.e. rhyme identification, rhyme production; phoneme identification- input and -output; phoneme blending-input and -output). Results All three children showed distinctive PA profiles. One boy, who had no spoken language deficits, struggled to complete the rhyme tasks but performed well on three phoneme tasks. However, he showed a discrepancy between expressive and receptive phoneme blending skills, scoring poorly on the expressive subtest. The second boy, who displayed grammar comprehension and expressive vocabulary difficulties, showed a mixed profile, with a below average performance on rhyme production. The girl who had significant speech and language deficits scored below average on all six PA subtests. Conclusions PA profiles in children with CI vary considerably and PA testing should include a range of different PA tasks. The assumed link between spoken language deficits and PA difficulties shown in children with normal hearing could be confirmed

Functional DNA methylation signatures for autism spectrum disorder genomic risk loci: 16p11.2 deletions and CHD8 variants

Background: Autism spectrum disorder (ASD) is a common and etiologically heterogeneous neurodevelopmental disorder. Although many genetic causes have been identified (\u3e 200 ASD-risk genes), no single gene variant accounts for \u3e 1% of all ASD cases. A role for epigenetic mechanisms in ASD etiology is supported by the fact that many ASD-risk genes function as epigenetic regulators and evidence that epigenetic dysregulation can interrupt normal brain development. Gene-specific DNAm profiles have been shown to assist in the interpretation of variants of unknown significance. Therefore, we investigated the epigenome in patients with ASD or two of the most common genomic variants conferring increased risk for ASD. Genome-wide DNA methylation (DNAm) was assessed using the Illumina Infinium HumanMethylation450 and MethylationEPIC arrays in blood from individuals with ASD of heterogeneous, undefined etiology (n = 52), and individuals with 16p11.2 deletions (16p11.2del, n = 9) or pathogenic variants in the chromatin modifier CHD8 (CHD8 +/-, n = 7). Results: DNAm patterns did not clearly distinguish heterogeneous ASD cases from controls. However, the homogeneous genetically-defined 16p11.2del and CHD8 +/- subgroups each exhibited unique DNAm signatures that distinguished 16p11.2del or CHD8 +/- individuals from each other and from heterogeneous ASD and control groups with high sensitivity and specificity. These signatures also classified additional 16p11.2del (n = 9) and CHD8 (n = 13) variants as pathogenic or benign. Our findings that DNAm alterations in each signature target unique genes in relevant biological pathways including neural development support their functional relevance. Furthermore, genes identified in our CHD8 +/- DNAm signature in blood overlapped differentially expressed genes in CHD8 +/- human-induced pluripotent cell-derived neurons and cerebral organoids from independent studies. Conclusions: DNAm signatures can provide clinical utility complementary to next-generation sequencing in the interpretation of variants of unknown significance. Our study constitutes a novel approach for ASD risk-associated molecular classification that elucidates the vital cross-talk between genetics and epigenetics in the etiology of ASD

Intercellular network structure and regulatory motifs in the human hematopoietic system.

The hematopoietic system is a distributed tissue that consists of functionally distinct cell types continuously produced through hematopoietic stem cell (HSC) differentiation. Combining genomic and phenotypic data with high-content experiments, we have built a directional cell-cell communication network between 12 cell types isolated from human umbilical cord blood. Network structure analysis revealed that ligand production is cell type dependent, whereas ligand binding is promiscuous. Consequently, additional control strategies such as cell frequency modulation and compartmentalization were needed to achieve specificity in HSC fate regulation. Incorporating the in vitro effects (quiescence, self-renewal, proliferation, or differentiation) of 27 HSC binding ligands into the topology of the cell-cell communication network allowed coding of cell type-dependent feedback regulation of HSC fate. Pathway enrichment analysis identified intracellular regulatory motifs enriched in these cell type- and ligand-coupled responses. This study uncovers cellular mechanisms of hematopoietic cell feedback in HSC fate regulation, provides insight into the design principles of the human hematopoietic system, and serves as a foundation for the analysis of intercellular regulation in multicellular systems

Prevalence and Clinical Features of Inflammatory Bowel Diseases Associated With Monogenic Variants, Identified by Whole-Exome Sequencing in 1000 Children at a Single Center

BACKGROUND & AIMS: A proportion of infants and young children with inflammatory bowel diseases (IBDs) have subtypes associated with a single gene variant (monogenic IBD). We aimed to determine the prevalence of monogenic disease in a cohort of pediatric patients with IBD. METHODS: We performed whole-exome sequencing analyses of blood samples from an unselected cohort of 1005 children with IBD, aged 0-18 years (median age at diagnosis, 11.96 years) at a single center in Canada and their family members (2305 samples total). Variants believed to cause IBD were validated using Sanger sequencing. Biopsies from patients were analyzed by immunofluorescence and histochemical analyses. RESULTS: We identified 40 rare variants associated with 21 monogenic genes among 31 of the 1005 children with IBD (including 5 variants in XIAP, 3 in DOCK8, and 2 each in FOXP3, GUCY2C, and LRBA). These variants occurred in 7.8% of children younger than 6 years and 2.3% of children aged 6-18 years. Of the 17 patients with monogenic Crohn\u27s disease, 35% had abdominal pain, 24% had nonbloody loose stool, 18% had vomiting, 18% had weight loss, and 5% had intermittent bloody loose stool. The 14 patients with monogenic ulcerative colitis or IBD-unclassified received their diagnosis at a younger age, and their most predominant feature was bloody loose stool (78%). Features associated with monogenic IBD, compared to cases of IBD not associated with a single variant, were age of onset younger than 2 years (odds ratio [OR], 6.30; P = .020), family history of autoimmune disease (OR, 5.12; P = .002), extra-intestinal manifestations (OR, 15.36; P \u3c .0001), and surgery (OR, 3.42; P = .042). Seventeen patients had variants in genes that could be corrected with allogeneic hematopoietic stem cell transplantation. CONCLUSIONS: In whole-exome sequencing analyses of more than 1000 children with IBD at a single center, we found that 3% had rare variants in genes previously associated with pediatric IBD. These were associated with different IBD phenotypes, and 1% of the patients had variants that could be potentially corrected with allogeneic hematopoietic stem cell transplantation. Monogenic IBD is rare, but should be considered in analysis of all patients with pediatric onset of IBD

Expanding the Landscape of Chromatin Modification (CM)-Related Functional Domains and Genes in Human

Chromatin modification (CM) plays a key role in regulating transcription, DNA replication, repair and recombination. However, our knowledge of these processes in humans remains very limited. Here we use computational approaches to study proteins and functional domains involved in CM in humans. We analyze the abundance and the pair-wise domain-domain co-occurrences of 25 well-documented CM domains in 5 model organisms: yeast, worm, fly, mouse and human. Results show that domains involved in histone methylation, DNA methylation, and histone variants are remarkably expanded in metazoan, reflecting the increased demand for cell type-specific gene regulation. We find that CM domains tend to co-occur with a limited number of partner domains and are hence not promiscuous. This property is exploited to identify 47 potentially novel CM domains, including 24 DNA-binding domains, whose role in CM has received little attention so far. Lastly, we use a consensus Machine Learning approach to predict 379 novel CM genes (coding for 329 proteins) in humans based on domain compositions. Several of these predictions are supported by very recent experimental studies and others are slated for experimental verification. Identification of novel CM genes and domains in humans will aid our understanding of fundamental epigenetic processes that are important for stem cell differentiation and cancer biology. Information on all the candidate CM domains and genes reported here is publicly available

Cyclic AMP metabolism and adenylate cyclase concentration in patients with advanced hepatic cirrhosis

Glucagon was tested for its effect on plasma adenosine 3′,5′-cyclic monophosphate (cyclic AMP), insulin, and glucose in healthy subjects and in patients with advanced cirrhosis of the liver. In the normal subjects, intravenous infusion of glucagon caused a significant increase in plasma cyclic AMP, glucose, and insulin. In advanced cirrhotics, plasma cyclic AMP, glucose, and insulin did not increase. Adenylate cyclase concentration was measured in liver tissue from end stage cirrhotic patients and from brain-dead organ donors whose cardiovascular function was maintained in a stable state. Basal and total adenylate cyclase concentration were not different in the two groups. Adenylate cyclase from the livers of advanced cirrhotics was, however, significantly less responsive to glucagon stimulation than was that from donor livers. Hepatocytes in advanced cirrhosis have abnormal metabolic behavior characterized by abnormal adenylate cyclase-cyclic AMP response to hormonal stimulation. © 1978