Regulatory interactions between IRG resistance GTPases in the cellular response to Toxoplasma gondii

Members of the immunity-related GTPase (IRG) family are interferon-inducible resistance factors against a broad spectrum of intracellular pathogens including Toxoplasma gondii. The molecular mechanisms governing the function and regulation of the IRG resistance system are largely unknown. We find that IRG proteins function in a system of direct, nucleotide-dependent regulatory interactions between family members. After interferon induction but before infection, the three members of the GMS subfamily of IRG proteins, Irgm1, Irgm2 and Irgm3, which possess an atypical nucleotide-binding site, regulate the intracellular positioning of the conventional GKS subfamily members, Irga6 and Irgb6. Following infection, the normal accumulation of Irga6 protein at the parasitophorous vacuole membrane (PVM) is nucleotide dependent and also depends on the presence of all three GMS proteins. We present evidence that an essential role of the GMS proteins in this response is control of the nucleotide-bound state of the GKS proteins, preventing their GTP-dependent activation before infection. Accumulation of IRG proteins at the PVM has previously been shown to be associated with a block in pathogen replication: our results relate for the first time the enzymatic properties of IRG proteins to their role in pathogen resistance

Importance of the costimulatory molecule CD2 in experimental colitis

Titel und Inhalt, Zusammenfassung I Abkürzungen IV 1. Einleitung 1 2. Fragestellung und Zielsetzung 14 3. Material und Methoden 15 4. Ergebnisse 43 5. Diskussion 87 6. Zusammenfassung 102 7. Anhang 103Chronisch-entzündliche Darmerkrankungen (CED) gehören zu den am häufigsten auftretenden chronisch rezidivierenden Entzündungen. Obwohl in den letzten Jahren viele neue Erkenntnisse zu ihrer Pathogenese gesammelt wurden, sind bislang noch immer viele Aspekte der Entstehung ungeklärt. Daher gibt es bislang keine Therapie, die an den Ursachen ansetzt. Diese Arbeit liefert einen möglichen neuen Ansatzpunkt für die Behandlung chronisch-entzündlicher Darmerkrankungen, da im Verlauf dieser Arbeit zum ersten Mal gezeigt werden konnte, dass durch die Modulation von CD2 der Verlauf experimenteller Colitis positiv beeinflusst werden kann. Durch die Behandlung von Mäusen mit dem anti-CD2 mAk 12-15 konnte eine Verbesserung (Transfer ConA-aktivierter T-Zellblasten) bzw. Verzögerung (Transfer von CD45RBhigh T-Zellen) einer, durch adoptiven Transfer CD4 positiver T-Zellen induzierten Colitis erreicht werden. Bei etablierter Transfercolitis führte die Therapie mit diesem anti-CD2 mAk zu einer Abschwächung der Entzündung. Untersuchungen des Zytokinprofils und der Proliferation von Lymphozyten dieser anti-CD2 mAk behandelten Transfercolitis- Mäuse zeigten sowohl eine signifikant geringere IL-2 Produktion als auch eine gehemmte T-Zellproliferation. Die am Beispiel einer Infektion mit Toxoplasma gondii untersuchte Infektabwehr wurde durch die anti-CD2 mAk Behandlung nicht beeinflusst. CD2 defiziente Mäuse wiesen in diesem Modell sogar signifikant weniger T. gondii Vakuolen im Darm auf als ebenfalls infizierte Kontrollmäuse. Auf Grund ihrer Immunpathologie wird die Toxoplasmen-Infektion auch als Crohn- Modell genutzt. Bei Untersuchung dieses Th1-vermittelten Aspekts einer T. gondii-Infektion zeigte sich ein nicht signifikanter Trend für ein verlängertes Überleben nach anti-CD2 mAk Behandlung. CD2 defiziente Mäuse allerdings überlebten signifikant länger, und wiesen einen niedrigeren histologischen Score sowie eine geringere Produktion von IL-6 und IFN-g auf. Schließlich konnten durch in vitro-Untersuchungen an verschiedenen Populationen humaner Lymphozyten sowie human CD2 transgener Splenozyten 5 humane anti-CD2 mAk identifiziert werden, die sowohl die Proliferation als auch die IFN-g Synthese stimulierter Lymphozyten inhibieren. Damit könnten diese anti-CD2 mAk potentielle Kandidaten für eine CD2 gerichtete Colitis- Immuntherapie beim Menschen sein.Inflammatory bowel disease (IBD) affects millions of people worldwide and is characterized by uncontrolled chronic inflammation of the intestinal mucosa. Although in the last years many new insights into the pathogenesis were gained, the etiology remains to be unknown. Therefore, today exists no therapy, which treats the trigger of this disease. The presented work demonstrates a possible new strategy for the treatment of chronic inflammatory bowel disease, as it could be shown for the first time that the process of experimental colitis can be positively affected by modulation of the costimulatory adhesion molecule CD2. Colitis induced by adoptive transfer of CD4+ T cells, could be improved (transfer of ConA activated T cell blasts) and/or delayed (transfer of CD45RBhigh T cells) by treating mice with the non-depleting anti-CD2 monoclonal antibody (mAb) 12-15. In established transfer colitis treatment with the anti-CD2 mAb attenuated intestinal inflammation. As confirmed by the cytokine profile and proliferation of stimulated lymphocytes. Anti-CD2 mAb treated mice with established transfer colitis showed both a significantly smaller IL-2 production and an inhibited T cell proliferation. Infection of mice with Toxoplasma gondii (T. gondii) leads to a Th1 mediated immuno-pathology of the small intestine. Immune defence was not affected by treatment with anti-CD2 mAb 12-15. CD2 deficient mice exhibited even significantly less pseudocysts of T. gondii in the intestine than infected control mice. Due to their immunopathology T. gondii infections of mice are also used as a model for Crohn´s Disease. With regard to the Th1-mediated aspect of T. gondii infection anti-CD2 mAb treatment effectuated a (not significant) trend for prolonged survival of the mice. Infected CD2 deficient mice however survived significantly longer and exhibited less signs of histological inflammation as well as smaller production of IL-6 and IFN-g. Finally, investigations of different populations of stimulated human lymphocytes and human CD2 transgenic splenocytes were used to identify 5 human anti-CD2 mAk, which in-vitro inhibited proliferation and IFN-g synthesis of stimulated lymphocytes. These anti-CD2 antibodies could be potential candidates for a CD2 directed immunotherapy of colitis in humans

Three new species of deep-sea Gromia (Protista, Rhizaria) from the bathyal and abyssal Weddell Sea, Antarctica

We describe three new species of the genus Gromia from bathyal and abyssal depths in the Weddell Sea. The new species are characterized by a combination of morphological and molecular criteria. All three species possess a distinct oral capsule and a layer of `honeycomb membranes', which form the inner part of the organic test wall. Both these features are typical of gromiids. Their identification as gromiids is confirmed by analyses of partial small subunit ribosomal DNA (SSU rDNA) gene sequences. Gromia marmorea sp. nov. is a rounded species with a prominent oral capsule and a characteristically mottled appearance. In Gromia melinus sp. nov., the test surface exhibits a polygonal pattern of ridges, with a layer of clay particles coating the surface between the ridges. Gromia winnetoui sp. nov. represents an elongate morphotype in which the organic test is enclosed within an agglutinated case, a feature previously unknown in gromiids. Phylogenetic analysis using the maximum-likelihood method revealed that all three species form distinct clades, reflecting the morphological differences among Weddell Sea species, as well as between deep-water Southern Ocean Gromia and previously described gromiids

Laboratory-Generated DNA Can Cause Anomalous Pathogen Diagnostic Test Results

The coronavirus disease 2019 (COVID-19) pandemic has brought about the unprecedented expansion of highly sensitive molecular diagnostics as a primary infection control strategy. At the same time, many laboratories have shifted focus to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research and diagnostic development, leading to large-scale production of SARS-CoV-2 nucleic acids that can interfere with these tests. We have identified multiple instances, in independent laboratories, in which nucleic acids generated in research settings are suspected to have caused researchers to test positive for SARS-CoV-2 in surveillance testing. In some cases, the affected individuals did not work directly with these nucleic acids but were exposed via a contaminated surface or object. Though researchers have long been vigilant of DNA contaminants, the transfer of these contaminants to SARS-CoV-2 testing samples can result in anomalous test results. The impact of these incidents stretches into the public sphere, placing additional burdens on public health resources, placing affected researchers and their contacts in isolation and quarantine, removing them from the testing pool for 3 months, and carrying the potential to trigger shutdowns of classrooms and workplaces. We report our observations as a call for increased stewardship over nucleic acids with the potential to impact both the use and development of diagnostics

Is the Scotia Sea a centre of Antarctic marine diversification? Some evidence of cryptic speciation in the circum-Antarctic bivalve Lissarca notorcadensis (Arcoidea: Philobryidae)

The bivalve Lissarca notorcadensis is one of the most abundant species in Antarctic waters and has colonised the entire Antarctic shelf and Scotia Sea Islands. Its brooding reproduction, low dispersal capabilities and epizoic lifestyle predict limited gene flow between geographically isolated populations. Relationships between specimens from seven regions in the Southern Ocean and outgroups were assessed with nuclear 28S rDNA and mitochondrial cytochrome oxidase subunit I (COI) genes. The 28S dataset indicate that while Lissarca appears to be a monophyletic genus, there is polyphyly between the Limopsidae and Philobryidae. Thirteen CO1 haplotypes were found, mostly unique to the sample regions, and two distinct lineages were distinguished. Specimens from the Weddell and Ross Sea form one lineage while individuals from the banks and islands of the Scotia Sea form the other. Within each lineage, further vicariance was observed forming six regionally isolated groups. Our results provide initial evidence for reproductively isolated populations of L. notorcadensis. The islands of the Scotia Sea appear to act as centres of speciation in the Southern Ocean