Analytical FcRn affinity chromatography for functional characterization of monoclonal antibodies

The neonatal Fc receptor (FcRn) is important for the metabolic fate of IgG antibodies in vivo. Analysis of the interaction between FcRn and IgG in vitro might provide insight into the structural and functional integrity of therapeutic IgG that may affect pharmacokinetics (PK) in vivo. We developed a standardized pH gradient FcRn affinity liquid chromatography method with conditions closely resembling the physiological mechanism of interaction between IgG and FcRn. This method allows the separation of molecular IgG isoforms, degradation products and engineered molecules based on their affinity to FcRn. Human FcRn was immobilized on the column and a linear pH gradient from pH 5.5 to 8.8 was applied. FcRn chromatography was used in comparison to surface plasmon resonance to characterize different monoclonal IgG preparations, e.g., oxidized or aggregated species. Wild-type and engineered IgGs were compared in vitro by FcRn chromatography and in vivo by PK studies in huFcRn transgenic mice. Analytical FcRn chromatography allows differentiation of IgG samples and variants by peak pattern and retention time profile. The method can distinguish: 1) IgGs with different Fabs, 2) oxidized from native IgG, 3) aggregates from monomer and 4) antibodies with mutations in the Fc part from wild-type IgGs. Changes in the FcRn chromatographic behavior of mutant IgGs relative to the wild-type IgG correlate to changes in the PK profile in the FcRn transgenic mice. These results demonstrate that FcRn affinity chromatography is a useful new method for the assessment of IgG integrity.     

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.     

重度妊娠期肝内胆汁淤积症对母婴结局影响的临床分析

目的：分析重度妊娠期肝内胆汁淤积症（intrahepatic cholestasis of pregnancy,ICP）对母婴结局的影响,以提高对ICP的认识与治疗水平。方法：选择ICP产妇58例,根据ICP分度标准分为轻度组（n=26例）和重度组（n=32例）,比较两组产妇的母婴结局。结果：与轻度组相比,重度组产妇剖宫产率、产后出血与平均产后出血量显著升高或增加,阴道分娩率显著降低,组间比较差异均具有统计学意义（P〈0.05）;重度组新生儿早产、胎儿窘迫、羊水污染和新生儿窒息的发生率均显著上升,新生儿平均出生体重显著降低,组间比较差异均具有统计学意义（P〈0.05）。结论：ICP对产妇及新生儿均有不良影响,对ICP严重程度进行划分,有利于临床处理。对于重度ICP产妇,应尽早采取剖宫产分娩的方式终止妊娠。     

pH‐selective mutagenesis of protein–protein interfaces: In silico design of therapeutic antibodies with prolonged half‐life

Understanding the effects of mutation on pH‐dependent protein binding affinity is important in protein design, especially in the area of protein therapeutics. We propose a novel method for fast in silico mutagenesis of protein–protein complexes to calculate the effect of mutation as a function of pH. The free energy differences between the wild type and mutants are evaluated from a molecular mechanics model, combined with calculations of the equilibria of proton binding. The predicted pH‐dependent energy profiles demonstrate excellent agreement with experimentally measured pH‐dependency of the effect of mutations on the dissociation constants for the complex of turkey ovomucoid third domain (OMTKY3) and proteinase B. The virtual scanning mutagenesis identifies all hotspots responsible for pH‐dependent binding of immunoglobulin G (IgG) to neonatal Fc receptor (FcRn) and the results support the current understanding of the salvage mechanism of the antibody by FcRn based on pH‐selective binding. The method can be used to select mutations that change the pH‐dependent binding profiles of proteins and guide the time consuming and expensive protein engineering experiments. As an application of this method, we propose a computational strategy to search for mutations that can alter the pH‐dependent binding behavior of IgG to FcRn with the aim of improving the half‐life of therapeutic antibodies in the target organism. © Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

METAMORPHOSIS OF THE LARVA OF THE POLYCHAETE ARENICOLA CRISTATA. MORPHOLOGY OF THE ‘HOUR-GLASS’ STAGE

The constricted ‘waist’ of the metamorphosing larva of the polychaete Arenicola cristata is described, using light and electron microscopy. The constriction is shown to be the consequence of the discharge and collapse of a post-trochal ring of epithelial cells which remain as functional components of the post-metamorphic juvenile. Morphological differentiation of neuro-effector and interneuronal contacts is initiated at this time. Muscular and neural changes are discussed in terms of their role in effecting metamorphosis.     

A Revision of South American Austroperlidae (Plecoptera)

The South American Austroperlidae are revised. Klapopteryx kuscheli and Penturoperla barbata are redescribed. The male and nymph of K. armillata are described and figured for the first time and the female genitalia redescribed. K. costalis is declared a junior synonym of K. armillata. K. barilochensis is transferred to Andesobius new genus and redescribed. New family and generic diagnoses and keys to genera and species are given.     

The larva of Rhyacophila ferox Graf, 2006 (Trichoptera: Rhyacophilidae) from the Eastern Alps (Carinthia,Austria)

The hitherto unknown larva of Rhyacophila ferox Graf, 2006, is described and discussed in the context of contemporary Rhyacophilidae keys. In addition, zoogeographical and ecological notes are included.