Guanine-Modified Inhibitory Oligonucleotides Efficiently Impair TLR7- and TLR9-Mediated Immune Responses of Human Immune Cells

Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX_3–5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.     

Pre-Use Susceptibility to Ceftaroline in Clinical Staphylococcus aureus Isolates from Germany: Is There a Non-Susceptible Pool to be Selected?

Ceftaroline is a new cephalosporin active against Methicillin-resistant Staphylococcus aureus (MRSA). Based on a representative collection of clinical S. aureus isolates from Germany, supplemented with isolates of clonal lineages ST228 and ST239, we demonstrate the in-vitro susceptibility towards ceftaroline prior to its introduction into clinical use for a total of 219 isolates. Susceptibility testing was performed by broth microdilution, disc diffusion and Etest, respectively. Results were interpreted according to EUCAST guidelines and showed considerable variance in dependence on clonal affiliation of the isolates tested. Among isolates of widespread hospital-associated lineages we found a high proportion of clinical isolates with MICs close to the EUCAST breakpoint (MIC_50/90 1.0/1.5 mg/L); currently, interpretation of these “borderline” MICs is complicated by a lack of concordant susceptibility testing methods and reasonable breakpoint determination. Isolates of clonal lineages ST228 and ST239 demonstrated increased MIC_50/90 values of 2.5/3.33 mg/L. Sequencing of mecA revealed no association of resistance to a specific mecA polymorphism, but rather reveals two regions in the non-penicillin-binding domain of PbP2a which displayed different combinations of mutations putatively involved in resistance development. This study provides national baseline data to (i) adjust susceptibility testing methods and current breakpoints to clinical and epidemiological requirements, (ii) evaluate current breakpoints with respect to therapeutic outcome and (iii) monitor further resistance evolution.     

Genotypic Tannin Levels in Populus tremula Impact the Way Nitrogen Enrichment Affects Growth and Allocation Responses for Some Traits and Not for Others

Plant intraspecific variability has been proposed as a key mechanism by which plants adapt to environmental change. In boreal forests where nitrogen availability is strongly limited, nitrogen addition happens indirectly through atmospheric N deposition and directly through industrial forest fertilization. These anthropogenic inputs of N have numerous environmental consequences, including shifts in plant species composition and reductions in plant species diversity. However, we know less about how genetic differences within plant populations determine how species respond to eutrophication in boreal forests. According to plant defense theories, nitrogen addition will cause plants to shift carbon allocation more towards growth and less to chemical defense, potentially enhancing vulnerability to antagonists. Aspens are keystone species in boreal forests that produce condensed tannins to serve as chemical defense. We conducted an experiment using ten Populus tremula genotypes from the Swedish Aspen Collection that express extreme levels of baseline investment into foliar condensed tannins. We investigated whether investment into growth and phenolic defense compounds in young plants varied in response to two nitrogen addition levels, corresponding to atmospheric N deposition and industrial forest fertilization. Nitrogen addition generally caused growth to increase, and tannin levels to decrease; however, individualistic responses among genotypes were found for height growth, biomass of specific tissues, root:shoot ratios, and tissue lignin and N concentrations. A genotype’s baseline ability to produce and store condensed tannins also influenced plant responses to N, although this effect was relatively minor. High-tannin genotypes tended to grow less biomass under low nitrogen levels and more at the highest fertilization level. Thus, the ability in aspen to produce foliar tannins is likely associated with a steeper reaction norm of growth responses, which suggests a higher plasticity to nitrogen addition, and potentially an advantage when adapting to higher concentrations of soil nitrogen.     

U.S. National Institutes of Health Core Consolidation–Investing in Greater Efficiency

The U.S. National Institutes of Health (NIH) invests substantial resources in core research facilities (cores) that support research by providing advanced technologies and scientific and technical expertise as a shared resource. In 2010, the NIH issued an initiative to consolidate multiple core facilities into a single, more efficient core. Twenty-six institutions were awarded supplements to consolidate a number of similar core facilities. Although this approach may not work for all core settings, this effort resulted in consolidated cores that were more efficient and of greater benefit to investigators. The improvements in core operations resulted in both increased services and more core users through installation of advanced instrumentation, access to higher levels of management expertise; integration of information management and data systems; and consolidation of billing; purchasing, scheduling, and tracking services. Cost recovery to support core operations also benefitted from the consolidation effort, in some cases severalfold. In conclusion, this program of core consolidation resulted in improvements in the effective operation of core facilities, benefiting both investigators and their supporting institutions.     

Biochemical Characterization of Human Anti-Hepatitis B Monoclonal Antibody Produced in the Microalgae Phaeodactylum tricornutum

Monoclonal antibodies (mAbs) represent actually the major class of biopharmaceuticals. They are produced recombinantly using living cells as biofactories. Among the different expression systems currently available, microalgae represent an emerging alternative which displays several biotechnological advantages. Indeed, microalgae are classified as generally recognized as safe organisms and can be grown easily in bioreactors with high growth rates similarly to CHO cells. Moreover, microalgae exhibit a phototrophic lifestyle involving low production costs as protein expression is fueled by photosynthesis. However, questions remain to be solved before any industrial production of algae-made biopharmaceuticals. Among them, protein heterogeneity as well as protein post-translational modifications need to be evaluated. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals including mAbs are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. In this paper, we assess the quality of the first recombinant algae-made mAbs produced in the diatom, Phaeodactylum tricornutum. We are focusing on the characterization of their C- and N-terminal extremities, their signal peptide cleavage and their post-translational modifications including N-glycosylation macro- and microheterogeneity. This study brings understanding on diatom cellular biology, especially secretion and intracellular trafficking of proteins. Overall, it reinforces the positioning of P. tricornutum as an emerging host for the production of biopharmaceuticals and prove that P. tricornutum is suitable for producing recombinant proteins bearing high mannose-type N-glycans.     

Dissecting Long-Term Glucose Metabolism Identifies New Susceptibility Period for Metabolic Dysfunction in Aged Mice

Metabolic disorders, like diabetes and obesity, are pathogenic outcomes of imbalance in glucose metabolism. Nutrient excess and mitochondrial imbalance are implicated in dysfunctional glucose metabolism with age. We used conplastic mouse strains with defined mitochondrial DNA (mtDNA) mutations on a common nuclear genomic background, and administered a high-fat diet up to 18 months of age. The conplastic mouse strain B6-mt^FVB, with a mutation in the mt-Atp8 gene, conferred β-cell dysfunction and impaired glucose tolerance after high-fat diet. To our surprise, despite of this functional deficit, blood glucose levels adapted to perturbations with age. Blood glucose levels were particularly sensitive to perturbations at the early age of 3 to 6 months. Overall the dynamics consisted of a peak between 3–6 months followed by adaptation by 12 months of age. With the help of mathematical modeling we delineate how body weight, insulin and leptin regulate this non-linear blood glucose dynamics. The model predicted a second rise in glucose between 15 and 21 months, which could be experimentally confirmed as a secondary peak. We therefore hypothesize that these two peaks correspond to two sensitive periods of life, where perturbations to the basal metabolism can mark the system for vulnerability to pathologies at later age. Further mathematical modeling may perspectively allow the design of targeted periods for therapeutic interventions and could predict effects on weight loss and insulin levels under conditions of pre-diabetic obesity.     

A Small Motor Cortex Lesion Abolished Ocular Dominance Plasticity in the Adult Mouse Primary Visual Cortex and Impaired Experience-Dependent Visual Improvements

It was previously shown that a small lesion in the primary somatosensory cortex (S1) prevented both cortical plasticity and sensory learning in the adult mouse visual system: While 3-month-old control mice continued to show ocular dominance (OD) plasticity in their primary visual cortex (V1) after monocular deprivation (MD), age-matched mice with a small photothrombotically induced (PT) stroke lesion in S1, positioned at least 1 mm anterior to the anterior border of V1, no longer expressed OD-plasticity. In addition, in the S1-lesioned mice, neither the experience-dependent increase of the spatial frequency threshold (“visual acuity”) nor of the contrast threshold (“contrast sensitivity”) of the optomotor reflex through the open eye was present. To assess whether these plasticity impairments can also occur if a lesion is placed more distant from V1, we tested the effect of a PT-lesion in the secondary motor cortex (M2). We observed that mice with a small M2-lesion restricted to the superficial cortical layers no longer expressed an OD-shift towards the open eye after 7 days of MD in V1 of the lesioned hemisphere. Consistent with previous findings about the consequences of an S1-lesion, OD-plasticity in V1 of the nonlesioned hemisphere of the M2-lesioned mice was still present. In addition, the experience-dependent improvements of both visual acuity and contrast sensitivity of the open eye were severely reduced. In contrast, sham-lesioned mice displayed both an OD-shift and improvements of visual capabilities of their open eye. To summarize, our data indicate that even a very small lesion restricted to the superficial cortical layers and more than 3mm anterior to the anterior border of V1 compromised V1-plasticity and impaired learning-induced visual improvements in adult mice. Thus both plasticity phenomena cannot only depend on modality-specific and local nerve cell networks but are clearly influenced by long-range interactions even from distant brain regions.     

Dose-Dependent Mutation Rates Determine Optimum Erlotinib Dosing Strategies for EGFR Mutant Non-Small Cell Lung Cancer Patients

Background

The advent of targeted therapy for cancer treatment has brought about a paradigm shift in the clinical management of human malignancies. Agents such as erlotinib used for EGFR-mutant non-small cell lung cancer or imatinib for chronic myeloid leukemia, for instance, lead to rapid tumor responses. Unfortunately, however, resistance often emerges and renders these agents ineffective after a variable amount of time. The FDA-approved dosing schedules for these drugs were not designed to optimally prevent the emergence of resistance. To this end, we have previously utilized evolutionary mathematical modeling of treatment responses to elucidate the dosing schedules best able to prevent or delay the onset of resistance. Here we expand on our approaches by taking into account dose-dependent mutation rates at which resistant cells emerge. The relationship between the serum drug concentration and the rate at which resistance mutations arise can lead to non-intuitive results about the best dose administration strategies to prevent or delay the emergence of resistance.

Methods

We used mathematical modeling, available clinical trial data, and different considerations of the relationship between mutation rate and drug concentration to predict the effectiveness of different dosing strategies.

Results

We designed several distinct measures to interrogate the effects of different treatment dosing strategies and found that a low-dose continuous strategy coupled with high-dose pulses leads to the maximal delay until clinically observable resistance. Furthermore, the response to treatment is robust against different assumptions of the mutation rate as a function of drug concentration.

Conclusions

For new and existing targeted drugs, our methodology can be employed to compare the effectiveness of different dose administration schedules and investigate the influence of changing mutation rates on outcomes.     

Modelling parasite aggregation: disentangling statistical and ecological approaches

The overdispersion in macroparasite infection intensity among host populations is commonly simulated using a constant negative binomial aggregation parameter. We describe an alternative to utilising the negative binomial approach and demonstrate important disparities in intervention efficacy projections that can come about from opting for pattern-fitting models that are not process-explicit. We present model output in the context of the epidemiology and control of soil-transmitted helminths due to the significant public health burden imposed by these parasites, but our methods are applicable to other infections with demonstrable aggregation in parasite numbers among hosts.