Antiviral Protection via RdRP-Mediated Stable Activation of Innate Immunity

For many emerging and re-emerging infectious diseases, definitive solutions via sterilizing adaptive immunity may require years or decades to develop, if they are even possible. The innate immune system offers alternative mechanisms that do not require antigen-specific recognition or a priori knowledge of the causative agent. However, it is unclear whether effective stable innate immune system activation can be achieved without triggering harmful autoimmunity or other chronic inflammatory sequelae. Here, we show that transgenic expression of a picornavirus RNA-dependent RNA polymerase (RdRP), in the absence of other viral proteins, can profoundly reconfigure mammalian innate antiviral immunity by exposing the normally membrane-sequestered RdRP activity to sustained innate immune detection. RdRP-transgenic mice have life-long, quantitatively dramatic upregulation of 80 interferon-stimulated genes (ISGs) and show profound resistance to normally lethal viral challenge. Multiple crosses with defined knockout mice (Rag1, Mda5, Mavs, Ifnar1, Ifngr1, and Tlr3) established that the mechanism operates via MDA5 and MAVS and is fully independent of the adaptive immune system. Human cell models recapitulated the key features with striking fidelity, with the RdRP inducing an analogous ISG network and a strict block to HIV-1 infection. This RdRP-mediated antiviral mechanism does not depend on secondary structure within the RdRP mRNA but operates at the protein level and requires RdRP catalysis. Importantly, despite lifelong massive ISG elevations, RdRP mice are entirely healthy, with normal longevity. Our data reveal that a powerfully augmented MDA5-mediated activation state can be a well-tolerated mammalian innate immune system configuration. These results provide a foundation for augmenting innate immunity to achieve broad-spectrum antiviral protection.     

Localization of EB1, IFT polypeptides, and kinesin-2 in Chlamydomonas flagellar axonemes via immunogold scanning electron microscopy

Intraflagellar transport (IFT) refers to the bi-directional movement of particles and associated cargo along the axonemes of eukaryotic flagella and cilia. To provide a new perspective on the morphology of IFT particles, their association with the axoneme, and their composition, we have used immunogold localization coupled to detection via scanning electron microscopy. Here we co-localize in the Chlamydomonas flagellar axoneme polypeptides labeled with specific antibodies. Chlamydomonas EB1 localizes to the distal tip of the axoneme, as expected from previous immunofluorescent data (Pedersen et al. Curr Biol2003;13(22):1969-1974), thus demonstrating the utility of this approach. Using antibodies to IFT-related polypeptides, particles can be identified associated with the axoneme that fall into one of two classes: The first class is composed of IFT particles labeled with polyclonal antibodies to kinesin-2 and monoclonal antibodies to either IFT139 (an IFT complex A polypeptide) or IFT172 (a complex B polypeptide). The second class is comprised of particles that label with antibodies to IFT139 alone; thus, discrete particles are present associated with the axoneme that are composed only of complex A polypeptides. When IFT particles were purified by sucrose gradient ultracentrifugation, they appeared as more or less spherical aggregates of varying dimensions labeled with antibodies to IFT139 and to the motor protein kinesin-2. By contrast, isolated IFT particles that were labeled with IFT172 antibodies were not labeled with kinesin-2 antibodies. The data are discussed in terms of the total polypeptide composition of an IFT particle and the interaction of the particles with the motors that power IFT.     

Ultrastructure of spore development in <Emphasis Type="Italic">Scutellospora heterogama</Emphasis>

The ultrastructural detail of spore development in Scutellospora heterogama is described. Although the main ontogenetic events are similar to those described from light microscopy, the complexity of wall layering is greater when examined at an ultrastructural level. The basic concept of a rigid spore wall enclosing two inner, flexible walls still holds true, but there are additional zones within these three walls distinguishable using electron microscopy, including an inner layer that is involved in the formation of the germination shield. The spore wall has three layers rather than the two reported previously. An outer, thin ornamented layer and an inner, thicker layer are both derived from the hyphal wall and present at all stages of development. These layers differentiate into the outer spore layer visible at the light microscope level. A third inner layer unique to the spore develops during spore swelling and rapidly expands before contracting back to form the second wall layer visible by light microscopy. The two inner flexible walls also are more complex than light microscopy suggests. The close association with the inner flexible walls with germination shield formation consolidates the preferred use of the term ‘germinal walls’ for these structures. A thin electron-dense layer separates the two germinal walls and is the region in which the germination shield forms. The inner germinal wall develops at least two sub-layers, one of which has an appearance similar to that of the expanding layer of the outer spore wall. An electron-dense layer is formed on the inner surface of the inner germinal wall as the germination shield develops, and this forms the wall surrounding the germination shield as well as the germination tube. At maturity, the outer germinal wall develops a thin, striate layer within its substructure.     

Small mammal responses to biosolids on grazed rangelands in British Columbia

Grasslands are globally declining due to habitat conversion, overgrazing, climate change, and changes in fire and drought regimes. Degraded grasslands are less able to support wildlife species, which contributes to the imperilment of many species. Biosolids are used by some ranchers as an organic amendment to support more plant growth and in turn more livestock. We worked on a large cattle ranch in central British Columbia, Canada, to determine how biosolids amendment affected small mammal populations, as mice and voles are major prey for many predators and contribute to seed dispersal and underground dynamics. We found that biosolids-amended pastures had more grass cover and supported fewer deermice (Peromyscus maniculatus) than did unamended pastures. Voles were scarce, likely due to a cyclic low. Although we sampled sites that had had biosolids applied 1 or 3 years prior to our work started, deermouse populations were similarly low across these sites. The reduction in deermice on sites with biosolids relative to unamended sites could be a signal of habitat restoration, because deermice prefer disturbed habitats rather than ones with high plant cover. Grass cover was more than twice as high on sites amended with biosolids, making these sites less suitable for deermice.     

The Role of Fibroblasts in Pancreatic Cancer: Extracellular Matrix Versus Paracrine Factors

BACKGROUND AND AIM: Desmoplasia is a characteristic feature and a suspected mechanism of tumor progression in pancreatic ductal adenocarcinoma (PDAC). Main constituents of the stroma involve cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM). The aim of this study was to dissect the interaction of CAFs, ECM, and PDAC cells in both an in vitro setting and a large-scale clinical cohort study. METHODS AND MATERIAL: Patients operated for PDAC were identified from our prospectively maintained clinical database. A standard pathology protocol was applied for pancreatoduodenectomy specimens also assessing CAF activation as either CAF grade 0 or CAF grade +. Interaction between a spectrum of pancreatic cancer cell lines (PCCs) and mouse embryonic fibroblasts (NIH 3T3) was assessed in a conditioned medium experimental setup. RESULTS: One hundred eleven patients operated for PDAC from 2001 to 2011 were identified. Univariate analysis disclosed CAF grade + (P = .030), positive M status (P < .001), and lymph node ratio (LNR) > 0.1 (P = .045) to impair overall survival. Independent prognostic factors were CAF grade (P = .050) and positive M status (P = .002). CAF grade correlated with N status (CC = 0.206, P = .030), LNR (CC = 0.187, P = .049), tumor size (CC = ?0.275, P = .003), and M status (CC = 0.190, P = .045). In the in vitro setting, paracrine effects of pancreatic cancer cell resulted in morphological activation of fibroblasts and tumor cell differentiation–dependent increase of fibroblast growth. Paracrine effects of poorly differentiated PCCs led to an upregulation of Vimentin in NIH 3T3 fibroblasts. Paracrine effects of fibroblasts on their part promoted cancer cell motility in all PCCs. As the second stromal component, fibroblast-derived ECM resulted in significantly decreased proliferation depending on density and led to upregulation of ZEB1 in poorly differentiated PCCs. CONCLUSION: In PDAC patients, positive CAF grading was identified as a negative prognostic parameter correlating with positive N status, high LNR, positive M status, and smaller tumor size. Whereas bilateral interaction of PCCs and CAFs promotes tumor progression, ECM poses PCC growth restrictions. In summary, our study discloses differential effects of stromal components and may help to interpret heterogeneous results of former studies.     

Risk Adjustment for Smoking Identified through Tobacco Use Diagnoses in Hospital Data: A Validation Study

Adjustment for the differing risk profiles of patients is essential to the use of administrative hospital data for epidemiological research. Smoking is an important factor to include in such adjustments, but the accuracy of the diagnostic codes denoting smoking in hospital records is unknown. The aims of this study were to measure the validity of current smoking and ever smoked status identified from diagnoses in hospital records using a range of algorithms, relative to self-reported smoking status; and to examine whether the misclassification of smoking identified through hospital data is differential or non-differential with respect to common exposures and outcomes. Data from the baseline questionnaire of the 45 and Up Study, completed by 267,153 residents of New South Wales (NSW), Australia, aged 45 years and older, were linked to the NSW Admitted Patient Data Collection. Patients who had been admitted to hospital for an overnight stay between 1 July 2005 and the date of completion of the questionnaire (1 January 2006 to 2 March 2009) were included. Smokers were identified by applying a range of algorithms to hospital admission histories, and compared against self-reported smoking in the questionnaire (‘gold standard’). Sensitivities for current smoking ranged from 59% to 84%, while specificities were 94% to 98%. Sensitivities for ever smoked ranged from 45% to 74% and specificities were 93% to 97%. For the majority of algorithms, sensitivities and/or specificities differed significantly according to principal diagnosis, number of comorbidities, socioeconomic status, residential remoteness, Indigenous status, 28 day readmission and 365 day mortality. The identification of smoking through diagnoses in hospital data results in differential misclassification. Risk adjustment based on smoking identified from these data will yield potentially misleading results. Systematic capture of information about smoking in hospital records using a mandatory item would increase the utility of administrative data for epidemiological research.