Immunome database for marsupials and monotremes

Background: To understand the evolutionary origins of our own immune system, we need to characterise the immune system of our distant relatives, the marsupials and monotremes. The recent sequencing of the genomes of two marsupials (opossum and tammar wallaby) and a monotreme (platypus) provides an opportunity to characterise the immune gene repertoires of these model organisms. This was required as many genes involved in immunity evolve rapidly and fail to be detected by automated gene annotation pipelines.Description: We have developed a database of immune genes from the tammar wallaby, red-necked wallaby, northern brown bandicoot, brush-tail possum, opossum, echidna and platypus. The resource contains 2,235 newly identified sequences and 3,197 sequences which had been described previously. This comprehensive dataset was built from a variety of sources, including EST projects and expert-curated gene predictions generated through a variety of methods including chained-BLAST and sensitive HMMER searches. To facilitate systems-based research we have grouped sequences based on broad Gene Ontology categories as well as by specific functional immune groups. Sequences can be extracted by keyword, gene name, protein domain and organism name. Users can also search the database using BLAST.Conclusion: The Immunome Database for Marsupials and Monotremes (IDMM) is a comprehensive database of all known marsupial and monotreme immune genes. It provides a single point of reference for genomic and transcriptomic datasets. Data from other marsupial and monotreme species will be added to the database as it become available. This resource will be utilized by marsupial and monotreme immunologists as well as researchers interested in the evolution of mammalian immunity

In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals

BACKGROUND: Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals. RESULTS: The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations. CONCLUSION: The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals

Identification and analysis of divergent immune gene families within the Tasmanian devil genome

Background: The Tasmanian devil (Sarcophilus harrisii) is being threatened with extinction in the wild by a disease known as devil facial tumour disease (DFTD). In order to prevent the spread of this disease a thorough understanding of the Tasmanian devil immune system and its response to the disease is required. In 2011 and 2012 two genome sequencing projects of the Tasmania devil were released. This has provided us with the raw data required to begin to investigate the Tasmanian devil immunome in depth. In this study we characterise immune gene families of the Tasmanian devil. We focus on immunoglobulins, T cell receptors and cytokine families. Results: We identify and describe 119 cytokines including 40 interleukins, 39 chemokines, 8 interferons, 18 tumour necrosis family cytokines and 14 additional cytokines. Constant regions for immunoglobulins and T cell receptors were also identified. The repertoire of genes in these families was similar to the opossum, however devil specific duplications were seen and orthologs to eutherian genes not previously identified in any marsupial were also identified. Conclusions: By using multiple data sources as well as targeted search methods, highly divergent genes across the Tasmanian devil immune system were identified and characterised. This understanding will allow for the development of devil specific assays and reagents and allow for future studies into the immune response of the Tasmanian devil immune system to DFTD

Analysis of the platypus genome suggests a transposon origin for mammalian imprinting

Comparisons between the platypus and eutherian mammalian genomes provides new insights into how epigenetic imprinting may have evolved in mammalian genomes

Telomere dynamics and homeostasis in a transmissible cancer

Devil Facial Tumour Disease (DFTD) is a unique clonal cancer that threatens the world\u27s largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii) with extinction. This transmissible cancer is passed between individual devils by cell implantation during social interactions. The tumour arose in a Schwann cell of a single devil over 15 years ago and since then has expanded clonally, without showing signs of replicative senescence; in stark contrast to a somatic cell that displays a finite capacity for replication, known as the “Hayflick limit”

Devil Facial Tumor Disease, A Potential Model of the Cancer Stem-Cell Process?

Tasmanian devil facial tumor disease (DFTD) is a naturally occurring contagious cancer which is transmitted as a clonal cell line between devils. The malignant cell line evolved from a Schwann cell or precursor prior to 1996 and since then has undergone continuous division without exhausting its replicative potential, suggesting a profound capacity for self renewal. It is therefore important to elucidate whether DFTD may have a stem cell origin. Deciphering the pathways regulating DFT cell proliferation and survival could lead to increased understanding of this transimissible cancer and to the development of successful therapies to halt the disease. We investigated whether DFT cells have originated from transformed stem cells by measuring the expression levels of thirteen genes characteristic to embryonic stem and/or pluripotent germ cells. No differences in gene expression were observed between DFT cells and peripheral nerve controls, and therefore our results provide additional support for Schwann cell or peripheral nerve origin of DFTD. Although our dataset is preliminary, it does not suggest that DFTs have cancer stem cells (CSCs) origin. We provide details of further experiments needed to ultimately confirm the role of cancer stem cells in DFTD progression

Evolution and comparative analysis of the MHC Class III inflammatory region

BACKGROUND: The Major Histocompatibility Complex (MHC) is essential for immune function. Historically, it has been subdivided into three regions (Class I, II, and III), but a cluster of functionally related genes within the Class III region has also been referred to as the Class IV region or "inflammatory region". This group of genes is involved in the inflammatory response, and includes members of the tumour necrosis family. Here we report the sequencing, annotation and comparative analysis of a tammar wallaby BAC containing the inflammatory region. We also discuss the extent of sequence conservation across the entire region and identify elements conserved in evolution. RESULTS: Fourteen Class III genes from the tammar wallaby inflammatory region were characterised and compared to their orthologues in other vertebrates. The organisation and sequence of genes in the inflammatory region of both the wallaby and South American opossum are highly conserved compared to known genes from eutherian ("placental") mammals. Some minor differences separate the two marsupial species. Eight genes within the inflammatory region have remained tightly clustered for at least 360 million years, predating the divergence of the amphibian lineage. Analysis of sequence conservation identified 354 elements that are conserved. These range in size from 7 to 431 bases and cover 15.6% of the inflammatory region, representing approximately a 4-fold increase compared to the average for vertebrate genomes. About 5.5% of this conserved sequence is marsupial-specific, including three cases of marsupial-specific repeats. Highly Conserved Elements were also characterised. CONCLUSION: Using comparative analysis, we show that a cluster of MHC genes involved in inflammation, including TNF, LTA (or its putative teleost homolog TNF-N), APOM, and BAT3 have remained together for over 450 million years, predating the divergence of mammals from fish. The observed enrichment in conserved sequences within the inflammatory region suggests conservation at the transcriptional regulatory level, in addition to the functional level

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites

Targeting of the Tumor Suppressor GRHL3 by a miR-21-Dependent Proto-Oncogenic Network Results in PTEN Loss and Tumorigenesis

SummaryDespite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC

Die Ballon-Okklusionsangiographie der Pulmonalarterien bei chronischer pulmonaler Hypertonie

Die chronische pulmonale Hypertonie ist eine seltene Erkrankung, die heute noch als unheilbar gilt. Die therapeutischen Möglichkeiten haben sich in den letzten Jahren rasant weiterentwickelt und orientieren sich an der Pathogenese und an den Befunden aus der bildgebenden Diagnostik. Diese muss in ihren Möglichkeiten der differentialdiagnostischen Eingrenzung zugrundeliegender Ursachen der pulmonalen Hypertonie Schritt halten. Insbesondere die Ergebnisse der intraarteriellen Pulmonalisangiographie als Referenzmethode in der Bildgebung der Lungen-strombahn haben meist unmittelbare therapeutische Konsequenz, z.B. ob ein Patient für eine pulmonale Thrombendarteriektomie gegeignet ist oder nicht. Wir haben überprüft, ob die selektive pulmonale Ballon-Okklusionsangiographie als Erweiterung der standardisierten Übersichtsangiographie die Differentialdiagnose z.B. der embolischen (CTEPH) gegenüber der nichtembolischen (NoCTEPH) Erkrankung verbessert. Zu diesem Zweck wurden Untersuchungen von 50 Patienten bestehend aus jeweils einer konventionellen Übersichtsangiographie und der ergänzenden Ballon-Okklusionsangiographie retrospektiv nach einem standardisierten Studienprotokoll ausgewertet. Zunächst wurde eine digitale Subtraktionsangiographie der Pulmonalarterien angefertigt. Anschließend führten wir einen weichen Latex-Ballonkatheter in Segment- oder Subsegmentarterien ein. Dann entfalteten wir den Ballon, um die sondierte Arterie zu verschließen und injizierten jeweils 10 bis 15 ml Kontrastmittel, um die kleinen peripheren Gefäße sichtbar zu machen. 13 Patienten litten an einer nichtthrombembolischen Form der chronischen pulmonalen Hypertonie. Bei 36 von 37 Patienten mit CTEPH fanden wir organisiertes embolisches Material als irreguläre Stenosen, Verschlüsse oder Strickleitersysteme (Webs und Bands). In der Darstellung dieser pathologischen Befunde war die Ballon-Okklusionsangiographie der Übersichtsangiographie sowohl qualitativ als auch quantitativ überlegen. Nach unseren Daten entdeckt die Ballon-Okklusionsangiographie in etwa bei jedem fünften Patienten mit negativer Übersichtsangiographie wenigstens ein thrombembolisches Residuum, sie steigert somit als Verfeinerung der Methode die Sensitivität der Pulmonalisangiographie. Generell stellte sie 2,7 bis 3,6 Aufteilungsgenerationen der peripheren Gefäße mehr dar als die konventionelle selektive DSA. Außerdem fanden wir bei 17 Patienten Kollateralgefäße zu den peripheren Segmenten von zentral verschlossenen Pulmonal-arterien. Dieses Phänomen war nur in der Ballon-Okklusionsangiographie zu beobachten und erwies sich als spezifisch für Patienten mit thrombembolischer pulmonaler Hypertonie. Der Befund ist insofern erstaunlich, als dass Pulmonalarterien eigentlich als funktionelle Endarterien ohne Anastomosen zu Nachbararterien beschrieben werden. Bei 11 Patienten fanden sich Anastomosen zu subpleuralen Bronchialarterien. Dieses bereits bekannte Phänomen steht nach unseren Daten in keinem Zusammenhang mit einer bestimmten Erkrankung und ist somit als unspezifisches Merkmal der chronischen pulmonalen Hypertonie zu deuten. In drei Fällen konnten histologisch postkapilläre Formen der pulmonalen Hypertonie (zweimal pulmonale veno-okklusive Erkrankung (PVOD), einmal primäre kapilläre Hämangiomatose) gesichert werden. Bei diesen Patienten zeigte die Ballon-Okklusionsangiographie eine Füllung der Lungenvenen ohne angiographisch sichtbare Anfärbung des Kapillarbettes (fehlende Parenchymanfärbung). Die geschilderten Erkenntnisse aus unserer Studie lassen sich für die Praxis wie folgt zusammenfassen: 1. Die Ballon-Okklusionsangiographie verbessert die Visualisierung der peripheren Pulmonalarterien. 2. Sie erleichtert die Detektion und Lokalisation thrombembolischer Residuen 3. Sie hilft bei der Differentialdiagnose zwischen thrombembolischer und nicht-thrombembolischer chronischer pulmonaler Hypertonie. 4. Vorher unsichtbare Anastomosen und Kollateralgefäße werden sichtbar. 5. Venöse Füllung ohne Parenchymanfärbung ist offensichtlich ein Zeichen der Parenchymerkrankung; diese Konstellation ist bei Patienten mit chronischer pulmonaler Hypertonie möglicherweise ein Hinweis auf das Vorliegen der pulmonalen venookklusiven Erkrankung (PVOD) oder der primären kapillären Hämangiomatose (PCH). 6. Die selektive Ballon-Okklusionsangiographie segmentaler Pulmonalarterien verbessert in Zusammenschau mit der Computertomographie die Zuverlässigkeit in der Selektion von Kandidaten für eine pulmonale Thrombendarteriektomie oder eine Prostazyklintherapie.Purpose: Test the ability of selective balloon occlusion angiography of pulmonary segmental arteries in the differential diagnosis of chronic pulmonary hypertension: embolic vs. non-embolic disease, pulmonary capillary hemangiomatosis, and venoocclusive disease. Methods and Materials: In 50 patients with pulmonary hypertension, digital subtraction angiography (DSA) of pulmonary arteries were used to assist in the selection of candidates appropriate for thrombo-endarterectomy. In addition to these standard methods, we introduced a soft latex balloon catheter into segmental arteries, inflated the balloon to occlude the artery, and injected 10 to 15 ml contrast medium to visualize small peripheral vessels as completely as possible. Results: 13 patients suffered from non-embolic pulmonary hypertension. In 36 of 37 patients with embolic pulmonary hypertension organizing embolic material was depicted as irregular narrowing or occlusion of pulmonary arteries, and weblike strictures. In all of these patients occlusion technique revealed more tiny webs or organized micro emboli in small peripheral arteries. According to our data balloon occlusion angiography discovers in every fifth patient showing a negative conventional pulmonary angiography at least one thromboembolic residuum and thus increases as a sophisticated method the sensitivity of the pulmonary angiography. Generally, occlusion technique revealed additional 2,7 to 3,6 ramifications of peripheral vessels in comparison to conventional selective DSA. Unexpectedly, we found in 11 patients anastomoses to bronchial arteries and in 17 patients collateral vessels to the peripheral segments of centrally occluded pulmonary arteries. These findings are astonishing, because pulmonary arteries are believed ramifying dichotomically without anastomoses. Obviously, there are alterations of pulmonary perfusion, which overcome normal anatomy. 3 patients with characteristic CT signs of interstitial disease (poorly defined nodular opacities and septal lines) underwent lung biopsy: 2 cases of venoocclusive disease, 1 case of pulmonary capillary hemangiomatosis. Occlusion angiography in these 3 patients revealed filling of veins without opacification of capillaries (failing parenchymal phase). Conclusion: Balloon occlusion technique improves the visualization of peripheral pulmonary arteries. Differential diagnosis of embolic and non-embolic pulmonary hypertension is facilitated. Previously invisible anastomoses and collateral vessels become visible. Venous filling without capillary opacification is apparently a sign of parenchymal disease; in patients with chronic pulmonary hypertension it might be a hint at venoocclusive disease or pulmonary capillary hemangiomatosi