Improved monovalent TNF receptor 1-selective inhibitor with novel heterodimerizing Fc

The development of alternative therapeutic strategies to tumor necrosis factor (TNF)-blocking antibodies for the treatment of inflammatory diseases has generated increasing interest. In particular, selective inhibition of TNF receptor 1 (TNFR1) promises a more precise intervention, tackling only the pro-inflammatory responses mediated by TNF while leaving regenerative and pro-survival signals transduced by TNFR2 untouched. We recently generated a monovalent anti-TNFR1 antibody fragment (Fab 13.7) as an efficient inhibitor of TNFR1. To improve the pharmacokinetic properties of Fab 13.7, the variable domains of the heavy and light chains were fused to the N-termini of newly generated heterodimerizing Fc chains. This novel Fc heterodimerization technology, designated “Fc-one/kappa” (Fc1κ) is based on interspersed constant Ig domains substituting the CH3 domains of a γ1 Fc. The interspersed immunoglobulin (Ig) domains originate from the per se heterodimerizing constant CH1 and CLκ domains and contain sequence stretches of an IgG1 CH3 domain, destined to enable interaction with the neonatal Fc receptor, and thus promote extended serum half-life. The resulting monovalent Fv-Fc1κ fusion protein (Atrosimab) retained strong binding to TNFR1 as determined by enzyme-linked immunosorbent assay and quartz crystal microbalance, and potently inhibited TNF-induced activation of TNFR1. Atrosimab lacks agonistic activity for TNFR1 on its own and in the presence of anti-human IgG antibodies and displays clearly improved pharmacokinetic properties.     

Rescue of cytochrome P450 oxidoreductase (Por) mouse mutants reveals functions in vasculogenesis, brain and limb patterning linked to retinoic acid homeostasis

Cytochrome P450 oxidoreductase (POR) acts as an electron donor for all cytochrome P450 enzymes. Knockout mouse Por(-/-) mutants, which are early embryonic (E9.5) lethal, have been found to have overall elevated retinoic acid (RA) levels, leading to the idea that POR early developmental function is mainly linked to the activity of the CYP26 RA-metabolizing enzymes (Otto et al., Mol. Cell. Biol. 23, 6103-6116). By crossing Por mutants with a RA-reporter lacZ transgene, we show that Por(-/-) embryos exhibit both elevated and ectopic RA signaling activity e.g. in cephalic and caudal tissues. Two strategies were used to functionally demonstrate that decreasing retinoid levels can reverse Por(-/-) phenotypic defects, (i) by culturing Por(-/-) embryos in defined serum-free medium, and (ii) by generating compound mutants defective in RA synthesis due to haploinsufficiency of the retinaldehyde dehydrogenase 2 (Raldh2) gene. Both approaches clearly improved the Por(-/-) early phenotype, the latter allowing mutants to be recovered up until E13.5. Abnormal brain patterning, with posteriorization of hindbrain cell fates and defective mid- and forebrain development and vascular defects were rescued in E9.5 Por(-/-) embryos. E13.5 Por(-/-); Raldh2(+/-) embryos exhibited abdominal/caudal and limb defects that strikingly phenocopy those of Cyp26a1(-/-) and Cyp26b1(-/-) mutants, respectively. Por(-/-); Raldh2(+/-) limb buds were truncated and proximalized and the anterior-posterior patterning system was not established. Thus, POR function is indispensable for the proper regulation of RA levels and tissue distribution not only during early embryonic development but also in later morphogenesis and molecular patterning of the brain, abdominal/caudal region and limbs.     

High-fat diets: modeling the metabolic disorders of human obesity in rodents

Research Methods and Procedures: High‐fat (HF) diet feeding can induce obesity and metabolic disorders in rodents that resemble the human metabolic syndrome. However, this dietary intervention is not standardized, and the HF‐induced phenotype varies distinctly among different studies. The question which HF diet type is best to model the metabolic deterioration seen in human obesity remains unclear. Therefore, in this review, metabolic data obtained with different HF diet approaches are compiled. Both whole‐body and organ‐specific diet effects are analyzed. Results: On the basis of these results, we conclude that animal fats and ω‐6/ω‐9‐containing plant oils can be used to generate an obese and insulin‐resistant phenotype in rodents, whereas fish oil‐fed animals do not develop these disorders. Discussion: Looking at the present data, it does not seem possible to define an ideal HF diet, and an exact definition of diet composition and a thorough metabolic characterization of the HF diet effects in a researcher's specific laboratory setting remains essential for metabolic studies with this model.     

Revisiting estimates of CTL killing rates in vivo

Recent experimental advances have allowed the estimation of the in vivo rates of killing of infected target cells by cytotoxic T lymphocytes (CTL). We present several refinements to a method applied previously to quantify killing of targets in the spleen using a dynamical model. We reanalyse data previously used to estimate killing rates of CTL specific for two epitopes of lymphocytic choriomeningitis virus (LCMV) in mice and show that, contrary to previous estimates the "killing rate" of effector CTL is approximately twice that of memory CTL. Further, our method allows the fits to be visualized, and reveals one potentially interesting discrepancy between fits and data. We discuss extensions to the basic CTL killing model to explain this discrepancy and propose experimental tests to distinguish between them.     

Epidemiological Panel Studies of Older Adults: New Frontiers in the Research on Human– Animal Interaction

ABSTRACT

This paper discusses epidemiological studies of older adults and human–animal interaction (HAI), and potential relevance to future research on HAI and animal-assisted interventions (AAI) in aging populations. Key issues in epidemiological work are discussed, including target populations, treatment/exposures, follow up, and endpoint measures as they relate to interacting with or owning companion animals. In outlining the limits in our current state of knowledge, we also make recommendations for the design and analysis of epidemiological studies to advance research on HAI and aging.     

Validation-based model selection for 13C metabolic flux analysis with uncertain measurement errors

Accurate measurements of metabolic fluxes in living cells are central to metabolism research and metabolic engineering. The gold standard method is model-based metabolic flux analysis (MFA), where fluxes are estimated indirectly from mass isotopomer data with the use of a mathematical model of the metabolic network. A critical step in MFA is model selection: choosing what compartments, metabolites, and reactions to include in the metabolic network model. Model selection is often done informally during the modelling process, based on the same data that is used for model fitting (estimation data). This can lead to either overly complex models (overfitting) or too simple ones (underfitting), in both cases resulting in poor flux estimates. Here, we propose a method for model selection based on independent validation data. We demonstrate in simulation studies that this method consistently chooses the correct model in a way that is independent on errors in measurement uncertainty. This independence is beneficial, since estimating the true magnitude of these errors can be difficult. In contrast, commonly used model selection methods based on the χ²-test choose different model structures depending on the believed measurement uncertainty; this can lead to errors in flux estimates, especially when the magnitude of the error is substantially off. We present a new approach for quantification of prediction uncertainty of mass isotopomer distributions in other labelling experiments, to check for problems with too much or too little novelty in the validation data. Finally, in an isotope tracing study on human mammary epithelial cells, the validation-based model selection method identified pyruvate carboxylase as a key model component. Our results argue that validation-based model selection should be an integral part of MFA model development.     

Extracellular enzyme activities during humic acid degradation by the white rot fungi Phanerochaete chrysosporium and Trametes versicolor

Abstract The relationship between humic acid biodegradation and extracellular lignin peroxidase and Mn-dependent peroxidase activities of two white rot fungi, Phanerochaete chrysosporium and Tranetes versicolor , reported to be lignin degraders, was examined. In experimental conditions promoting culture aeration, particularly with T. versicolor no extracellular peroxidase activity could be detected unless humic acids were included in the culture medium. In the presence of humic acids, appreciable enzymatic activities were determined in the culture filtrate of the two fungi. However, T. versicolor was a more effective degrader than P. chrysosporium , and mineralization assays on synthetic humic acids with culture filtrates showed the important role played by Mn²⁺. The surfactant properties of humic acids are suggested to be responsible for the increase of enzymatic activities.