FROM DISEASE TO THE GENE - Identification of arthritis-regulating loci in rats

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation of the peripheral joints that eventually leads to cartilage destruction and bone erosion. The causes of RA remain largely unknown, but considerable evidence suggests a multifactorial aetiology involving both environmental and genetic factors. Large efforts have been directed towards the understanding of the molecular mechanisms underlying RA. Because of the complexity of the disease in humans, animal models for RA have become attractive tools for gene-identification. Use of such models not only overcomes genetic complications, but it also permits studies under stable environmental conditions. However, so far genetic studies using animals have had only limited success. In fact, researchers have encountered significant difficulties in the analysis of complex traits. The first part of this thesis is summarizing two major problems we have faced in the past years. In the first study we investigated the genetic setup and the response towards various arthritis models of two DA rat substrains. We detected several genetic and phenotypic differences, suggesting that one of the substrains had been genetically contaminated from another rat strain. The second study is based on the observation that a spontaneous mutation in our DA rat colony results in decreased arthritis susceptibility in the DA rats. We subsequently isolated the mutation in a new substrain of DA rats, called DACP, and using genetic linkage analysis we located the mutation and identified a new quantitative trait locus (QTL) for pristaneinduced arthritis (PIA) at chromosome 9, Pia27. In the second part of this thesis, we were utilizing the traditional congenic rat strain strategy in the identification and characterization of arthritis regulating loci. The third paper investigated the influence of different genetic backgrounds on the detection of previously reported loci for PIA. We found that the arthritis-regulating gene Ncf1 as well as the major histocompatibility complex (MHC) are silent in certain genetic backgrounds, while they can be detected in other genetic setups. The fourth study describes the positional cloning of the immunoglobulin lambda light chain (Igl) locus as one locus controlling rheumatoid factor (RF) production in rats. In addition, evidence suggests that this genetic region may be associated with Ovalbumin-induced airway inflammation, an animal model for allergic bronchitis or asthma. Identification of genes involved in complex disorders such as RA will be extremely valuable in understanding disease regulating mechanisms as well as improve diagnosis and identification of specific targets for therapeutic drugs. However, the findings in this thesis demonstrate that mapping those genes is a complex and challenging process and involving various problems, such as genetic variability and complex genetic interactions

Phrenologyand the Insanity Defence: Medical Jurisprudence in the McNaughtan Trial

This thesis argues that phrenology shaped the defence argument in the McNaughtan trial. The role of this now-discredited science exemplifies the negotiation of scientific, legal and lay knowledge in the early nineteenth century, at a time when science was challenging the primacy of lay understandings of insanity. Phrenological ideas allowed the defence to privilege medical opinion over lay opinion, and propose a model of the mind that could account for McNaughtan’s insanity. This was possible because the medical and professional communities accepted some elements of the science. They applied these principles when explaining and verifying insanity in a courtroom setting

histoneHMM:Differential analysis of histone modifications with broad genomic footprints

BACKGROUND: ChIP-seq has become a routine method for interrogating the genome-wide distribution of various histone modifications. An important experimental goal is to compare the ChIP-seq profiles between an experimental sample and a reference sample, and to identify regions that show differential enrichment. However, comparative analysis of samples remains challenging for histone modifications with broad domains, such as heterochromatin-associated H3K27me3, as most ChIP-seq algorithms are designed to detect well defined peak-like features. RESULTS: To address this limitation we introduce histoneHMM, a powerful bivariate Hidden Markov Model for the differential analysis of histone modifications with broad genomic footprints. histoneHMM aggregates short-reads over larger regions and takes the resulting bivariate read counts as inputs for an unsupervised classification procedure, requiring no further tuning parameters. histoneHMM outputs probabilistic classifications of genomic regions as being either modified in both samples, unmodified in both samples or differentially modified between samples. We extensively tested histoneHMM in the context of two broad repressive marks, H3K27me3 and H3K9me3, and evaluated region calls with follow up qPCR as well as RNA-seq data. Our results show that histoneHMM outperforms competing methods in detecting functionally relevant differentially modified regions. CONCLUSION: histoneHMM is a fast algorithm written in C++ and compiled as an R package. It runs in the popular R computing environment and thus seamlessly integrates with the extensive bioinformatic tool sets available through Bioconductor. This makeshistoneHMM an attractive choice for the differential analysis of ChIP-seq data. Software is available from http://histonehmm.molgen.mpg.de

High salt reduces the activation of IL-4- and IL-13-stimulated macrophages

A high intake of dietary salt (NaCl) has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. We have recently shown that salt has a proinflammatory effect and boosts the activation of Th17 cells and the activation of classical, LPS-induced macrophages (M1). Here, we examined how the activation of alternative (M2) macrophages is affected by salt. In stark contrast to Th17 cells and M1 macrophages, high salt blunted the alternative activation of BM-derived mouse macrophages stimulated with IL-4 and IL-13, M(IL-4+IL-13) macrophages. Salt-induced reduction of M(IL-4+IL-13) activation was not associated with increased polarization toward a proinflammatory M1 phenotype. In vitro, high salt decreased the ability of M(IL-4+IL-13) macrophages to suppress effector T cell proliferation. Moreover, mice fed a high salt diet exhibited reduced M2 activation following chitin injection and delayed wound healing compared with control animals. We further identified a high salt-induced reduction in glycolysis and mitochondrial metabolic output, coupled with blunted AKT and mTOR signaling, which indicates a mechanism by which NaCl inhibits full M2 macrophage activation. Collectively, this study provides evidence that high salt reduces noninflammatory innate immune cell activation and may thus lead to an overall imbalance in immune homeostasis

Natural genetic variation of the cardiac transcriptome in non-diseased donors and patients with dilated cardiomyopathy

Background: Genetic variation is an important determinant of RNA transcription and splicing, which in turn contributes to variation in human traits, including cardiovascular diseases. Results: Here we report the first in-depth survey of heart transcriptome variation using RNA-sequencing in 97 patients with dilated cardiomyopathy and 108 non-diseased controls. We reveal extensive differences of gene expression and splicing between dilated cardiomyopathy patients and controls, affecting known as well as novel dilated cardiomyopathy genes. Moreover, we show a widespread effect of genetic variation on the regulation of transcription, isoform usage, and allele-specific expression. Systematic annotation of genome-wide association SNPs identifies 60 functional candidate genes for heart phenotypes, representing 20% of all published heart genome-wide association loci. Focusing on the dilated cardiomyopathy phenotype we found that eQTL variants are also enriched for dilated cardiomyopathy genome-wide association signals in two independent cohorts. Conclusions: RNA transcription, splicing, and allele-specific expression are each important determinants of the dilated cardiomyopathy phenotype and are controlled by genetic factors. Our results represent a powerful resource for the field of cardiovascular genetics

A trans-acting locus regulates an anti-viral expression network and type 1 diabetes risk

Combined analyses of gene networks and DNA sequence variation can provide new insights into the aetiology of common diseases that may not be apparent from genome-wide association studies alone. Recent advances in rat genomics are facilitating systems-genetics approaches. Here we report the use of integrated genome-wide approaches across seven rat tissues to identify gene networks and the loci underlying their regulation. We defined an interferon regulatory factor 7 (IRF7)-driven inflammatory network (IDIN) enriched for viral response genes, which represents a molecular biomarker for macrophages and which was regulated in multiple tissues by a locus on rat chromosome 15q25. We show that Epstein-Barr virus induced gene 2 (Ebi2, also known as Gpr183), which lies at this locus and controls B lymphocyte migration, is expressed in macrophages and regulates the IDIN. The human orthologous locus on chromosome 13q32 controlled the human equivalent of the IDIN, which was conserved in monocytes. IDIN genes were more likely to associate with susceptibility to type 1 diabetes (T1D)-a macrophage-associated autoimmune disease-than randomly selected immune response genes (P = 8.85 x 10(-6)). The human locus controlling the IDIN was associated with the risk of T1D at single nucleotide polymorphism rs9585056 (P = 7.0 x 10(-10); odds ratio, 1.15), which was one of five single nucleotide polymorphisms in this region associated with EBI2 (GPR183) expression. These data implicate IRF7 network genes and their regulatory locus in the pathogenesis of T1D

Monitoring of analgesia in pigs with general anaesthesia induced by ketamine- azaperone with special regard to the nociceptive flexor reflex (resp. RIII- Reflex)

Ziel dieser klinischen Studie war es, erstmalig ein quantitatives Analgesiemonitoring mittels elektromyografischer Aufzeichnung (EMG) des Nozizeptiven Flexorreflexes (NFR) und der elektroenzephalografischen Aufzeichnung (EEG) des Bispektralindex (BIS) unter der praxisüblichen Ketamin- Azaperon-Allgemeinanästhesie zu etablieren und mittels des traditionellen schmerzspezifischen Abwehrverhaltens zu überprüfen. Bei Eignung des NFR sollte insbesondere der in der Praxis zur Feststellung der chirurgischen Toleranz verwendete Zwischenklauenreflex (ZKR) als Analgesieindikator kontrolliert werden. Darüber hinaus sollte auch erstmalig die Möglichkeit der Bewusstseinskontrolle beim Schwein mithilfe des BIS erfasst werden. Mit dem NFR und den Wirkstoffspiegeln von Ketamin, Norketamin und Azaperon im Blut sollte auch geprüft werden, ob Kortisol unter der Ketamin-Azaperon- Allgemeinanästhesie als Stress- bzw. Schmerzparameter geeignet ist. Begleitend sollten mögliche unerwünschte Nebenwirkungen dieser Allgemeinanästhesie auf die Vitalparameter von Herz, Kreislauf und Atmung, insbesondere aber auch das Auftreten eines mit Ketamin assoziierten Katalepsierisikos untersucht werden. Als Modell zur Kontrolle der somatischen und viszeralen Analgesie diente die Kastration 30 klinisch gesunder, männlicher Schweine zwischen 42 und 60 kg Körpergewicht. Als somatische und viszerale Schmerzstimuli dienten die Inzsion in die Skrotalhaut, die Tunica vaginalis und die Hoden, der Zug am Samenstrang und das Quetschen und Abtrennen desselben mit einem Emaskulator sowie die Abschlussdesinfektion der Wunde. Die Allgemeinanästhesie wurde mit einer Ausgangsdosis von 20 mg/kg Ketamin (Ursotamin®) und 2 mg/kg Azaperon (Stresnil®) intramuskulär eingeleitet. Sofort nach dem Niederlegen der Tiere wurde mit der elektromyografischen Aufzeichnung des NFR über dem Musculus deltoideus im minütlichen Abstand und exakt zum Zeitpunkt einer Manipulation bzw. einem chirurgischen Schmerzstimulus begonnen. Zur Stimulation eines NFR wurde der Nervus ulnaris distal des Karpalgelenkes elektrisch gereizt. Ein Reiz bestand aus 5 Zügen, im Abstand von je 0,5 s mit je 5 Rechteckimpulsen von je 1 ms Dauer und einem Abstand von je 4 ms. Die Stimulationsstärke war supramaximal auf 45 mA begrenzt. Als Grenzwert für die Schmerzwahrnehmung wurden 40 µV festgelegt. Dieser wurde aus dem Mittelwert des Nulllinienrauschens (15 µV) plus der sechsfachen Standardabweichung berechnet. Zur Bewusstseinskontrolle und zusätzlich zum Analgesiemonitoring wurde auch der BIS mittels EEG bestimmt, das von der Stirn abgeleitet wurde. Die Kortisol-, Ketamin-, Norketamin- und Azaperonkonzentrationen wurden im Serum von 13 venenkatheterisierten Schweinen vor, während und bis 4 Stunden nach der Operation mit der Flüssigchromatografie mit Massenspektrometrie-Kopplung (LC- MS-Methode) bestimmt. Das schmerzspezifische Abwehrverhalten wurde mittels eines standardisierten semiquantitativen Scorings auf der Basis von Abwehrbewegungen und Lautäußerungen beurteilt, wobei das Fehlen beider als Score 0 (chirurgische Toleranz) definiert ist. Der Zwischenklauenreflex wurde manuell, wie unter Praxisbedingungen, überprüft. Die Sauerstoffsättigung, die endtidale Kohlendioxidkonzentration, die Herzfrequenz, der mittlere arterielle Blutdruck und die Atemfrequenz wurden zur Überprüfung möglicher anästhesiebedingter Nebenwirkungen mit dem Standardüberwachungsequipment in der Humananästhesieologie laufend aufgezeichnet. Zur Katalepsiekontrolle wurde das EMG ebenfalls laufend auf einem weiteren Kanal aufgezeichnet. Die Ergebnisse des NFR-Monitorings zeigten, dass die Ausgangsdosierung von Ketamin und Azaperon zur Unterbrechung der Schmerzweiterleitung zum Effektor (Muskel) sowohl nach somatischen als auch viszeralen Stimuli führt. Bezogen auf die chirurgische Toleranz (Score 0) lag die Testsensitivität und -spezifität des NFR bei 93% bzw. 98%. Auch postoperativ, bis mindestens zwei Stunden nach Anästhesieeinleitung (max. Aufzeichnungszeit) konnte noch eine deutliche Schmerzreduktion anhand des NFR (74 µV) festgestellt werden, die in etwa dem Score 1 entsprach. Die konstant hohen BIS-Werte im perioperativen Zeitraum zeigten, dass sich dieser nicht zur Beurteilung des Bewusstseinszustandes und der Analgesie bei Schweinen eignet. Als Analgesieindikatoren ungeeignet erwiesen sich auch die Vitalparameter und das Kortisol. Die Sensitivität und Spezifität des ZKR unter Berücksichtigung des NFR-Grenzwertes (40 µV) betrugen 92% bzw. 89%. Das EMG zur Katalepsiekontrolle lag konstant auf dem Niveau des Nulllinienrauschens. Alle Tiere zeigten in Übereinstimmung damit auch klinisch einen schlaffen Muskeltonus. Selbst bei wiederholter Nachdosierung mit Ketamin (bis 60 mg/kg insgesamt) konnten anhand der Vitalparameter keine unerwünschten Nebenwirkungen festgestellt werden. Lediglich die Atemfrequenz lag perioperativ geringfügig über dem Referenzbereich, was auf die zentral stimulierende Wirkung von Ketamin zurückgeführt werden kann.The objective of this clinical study was to establish for the first time a quantitative assessment of analgesia under practice common general anaesthesia of pigs induced by Ketamine-Azaperone using electromyography (EMG) to record the Nociceptive Flexor Reflex (NFR) but also using Elektroencephalography (EEG) to derive the Bispectral Index (BIS), both under surveillance of the previous traditional pain specific defense behaviour. In case of suitability the NFR should be applied also to test the Interdigital Claw Reflex (ZKR) which is in practice commonly used to assertain surgical tolerance before operations. Furthermore, the possibility to control the consciousness in pigs with aid of the BIS should be investigated for the first time, too. With the NFR and the blood levels of Ketamine, Norketamine and Azaperone it should be checked, if Cortisol can function as a reliable parameter for pain or stress during Ketamine-Azaperone-anaesthesia. Possible negative side effects of the anaesthesia on heart, cardiovascular system and respiration, particularly the risk of catalepsis by Ketamine should be controlled in addition. As a model for monitoring somatic and visceral analgesia the castration of 30 clinically healthy male pigs with a body weight between 42 and 60 kg was used. As somatic and visceral pain stimuli served the incision of the scrotal skin, of the tunica vaginalis and of the testis, the draw, the squeezing and the cutting off the spermatic cord with an emasculator, and finally the disinfection of the wound. The general anaesthesia was induced with an initial intramuscular dose of 20 mg/kg Ketamine (Ursotamine®) and 2 mg/kg Azaperone (Stresnil®). Right after laying down the NFR was recorded every minute using the EMG of the deltoid muscle. Additional records were taken exactly at the time of a certain manipulation or a specific pain stimulus.The stimulation of the NFR was performed by electric irritation of the ulnar nerve distal from the carpal joint.The irritation consisted of five terms of 0.5 s distance, each, with five square impulses of 1 ms duration and a distance of 4 ms. The strength of irritation was limited to 45 mA. The threshold for nociception of 40 µV was determined from the arithmetic mean of the baseline-rushing (15 µV) plus it´s sixfold standard deviation. For control of consciousness and/or analgesia the BIS was evaluated from current EEG derivation of the frontal region. The serum concentrations of Cortisol, Ketamine and Azaperone were determined in 13 pigs with ear vein catheters before and during surgery, and up to four hours afterwards with the Liquid Chromatography coupled Mass Spectrometry (LC-MS). The pain-specific defense behaviour was evaluated using a standardized semi- quantitative scoring on the basis of defense-movements and vocalization. The lack of both is defined as surgical tolerance (Score 0). The ZKR was manually tested as common in practice. The vital parameters oxygen saturation, end- tidal carbon dioxide saturation, heart rate, mean arterial pressure and respiratory rate were currently recorded using the usual surveillance equipment in human anaesthesiology. To control catalepsis the EMG was also currently recorded from the deltoid muscle using another record channel. As an important result the NFR monitoring demonstrated that the initial dosage of Ketamine-Azaperone interrupts the conduction of pain signals to the effectors (muscles) following somatic as well as visceral pain stimuli. Related to surgical tolerance (Score 0) the test sensitivity and specifity of the NFR were 93% and 98%. Post operationem, up to two hours after inducing anaesthesia (maximum time of recording), a significant pain relief (74 µV) was also shown by NFR, corresponding with Score 1. The persistent high BIS values over time showed that this monitoring was not suited to estimate the state of analgesia and of consciousness in pigs. The vital parameters and Cortisol were also inappropiate indicators for analgesia. The test sensitivity and specifity of the ZKR with regard to the NFR-threshold (40 µV) were 92% and 89%. The EMG- values for control of catalepsis remained persistently on the low level of the baseline-rushing. All pigs demonstrated in accordance with this clinically an atonic muscle tone. Even with repeated post-dosing of Ketamine (up to 60 mg/kg in total) no negative side effects were observed regarding the vital parameters. Only the respiratory rate during the monitoring was marginally above the reference range due to the central-stimulating effect of Ketamine

Detection of arthritis-susceptibility loci, including Ncf1, and variable effects of the major histocompatibility complex region depending on genetic background in rats.

OBJECTIVE: To characterize the arthritis-modulating effects of 3 non-major histocompatibility complex (MHC) quantitative trait loci (QTLs) in rat experimental arthritis in the disease-resistant E3 strain, and to investigate the disease-modulating effects of the MHC region (RT1) in various genetic backgrounds. METHODS: A congenic fragment containing Ncf1 along with congenic fragments containing the strongest remaining loci, Pia5/Cia3 and Pia7/Cia13 on chromosome 4, were transferred from the arthritis-susceptible DA strain into the background of the completely resistant E3 strain. The arthritis-regulatory potential of the transferred alleles was evaluated by comparing the susceptibility to experimental arthritis in congenic rats with that in E3 rats. The RT1(u) haplotype from the E3 strain was transferred into the susceptible DA strain (RT1(av1)), and various F(1) and F(2) hybrids were generated to assess the effects of RT1 on arthritis susceptibility. RESULTS: The DA allele of Ncf1 did not break the arthritis resistance of the E3 rats, although it led to enhanced autoimmune B cell responses, as indicated by significantly elevated levels of anticollagen antibodies in congenic rats. Introgressing Pia5 and Pia7 loci on chromosome 4 broke the resistance to arthritis, and the MHC locus on chromosome 20 in DA rats enhanced arthritis when RT1 interacted with E3 genes. CONCLUSION: The findings in these congenic lines confirm the existence of 3 major QTLs that regulate the severity of arthritis and are sufficient to induce the transformation of a completely arthritis-resistant rat strain into an arthritis-susceptible strain. This study also reveals a dramatic difference in the arthritis-regulatory potential of the rat MHC depending on genetic background, suggesting that strong epistatic interactions occur between MHC and non-MHC genes

DA rats from two colonies differ genetically and in their arthritis susceptibility.

The arthritis-susceptible DA rat is one of the most commonly used rat strains for genetic linkage analysis and is instrumental for the identification of many genetic loci. Even though DA rats were kept as inbred lines at different institutes and suppliers, it became obvious that the various breeding stocks differed genetically. To be able to compare the results from different linkage studies it is very import to verify the genetic background of the substrains used in those studies. We performed a genetic and phenotypic analysis of two DA substrains, DA/ZtmRhd and DA/OlaHsd, and found several genetic differences. One of the allelic differences between the DA/ZtmRhd and the DA/OlaHsd strain was located at rat chromosome 3, a 17-Mb large fragment, including the peak marker of a previously identified quantitative trait locus (QTL) for collagen-induced arthritis, Cia11. In addition, the substrains exhibited a significant difference in the susceptibility to pristane-induced arthritis (PIA) and disease severity of collagen-induced arthritis and PIA. However, by generating and testing a congenic line, we could demonstrate that phenotypic differences were not due to the contaminating fragment on chromosome 3. Nevertheless, we conclude that DA substrains show distinct genetic differences and caution should be taken when comparing arthritis data from different DA substrains