Placental Transfer of a Fully Human IgG2 Monoclonal Antibody in the Cynomolgus Monkey,Rat, and Rabbit: A Comparative Assessment from during Organogenesis to Late Gestation

Understanding species differences in the placental transfer of monoclonal antibodies is important to inform species selection for nonclinical safety assessment, interpret embryo‐fetal changes observed in these studies, and extrapolate their human relevance. Data presented here for a fully human immunoglobulin G2 monoclonal antibody (IgG2X) revealed that, during organogenesis, in both the cynomolgus monkey (gestation day 35 [gd35]) and the rat (gd10) the extent of IgG2X placental transfer (approximately 0.5% maternal plasma concentration, MPC) was similar to the limited published human data for endogenous IgG. At this early gestational stage, IgG2X placental transfer was approximately 6‐fold higher in the rabbit (gd10). By the end of organogenesis, rat embryonic plasma concentrations (gd16) exceeded those in the cynomolgus monkey (gd50) by approximately 3‐fold. These data suggest that relative to the cynomolgus monkey, the rabbit (and to a lesser extent the rat) may overestimate potential harmful effects to the human embryo during this critical period of development. Beyond organogenesis, fetal IgG2X plasma concentrations increased approximately 10‐fold early in the second trimester (gd50–70) in the cynomolgus monkey and remained relatively unchanged thereafter (at approximately 5% MPC). Late gestational assessment was precluded in rabbits due to immunogenicity, but in rats, fetal IgG2X plasma concentrations increased more than 6‐fold from gd16 to gd21 (reaching approximately 15% MPC). In rats, maternal exposure consistent with that achieved by ICH S6(R1) high‐dose selection criteria resulted in embryonic plasma concentrations, reaching pharmacologically relevant levels during organogenesis. Furthermore, dose proportional exposure in both mothers and embryos indicated that this was unlikely to occur at the lower therapeutic dose levels used in humans     

Cardiac diastolic dysfunction in high-fat diet fed mice is associated with lipotoxicity without impairment of cardiac energetics in vivo

Obesity is often associated with abnormalities in cardiac morphology and function. This study tested the hypothesis that obesity-related cardiomyopathy is caused by impaired cardiac energetics. In a mouse model of high-fat diet (HFD)-induced obesity, we applied in vivo cardiac ³¹P magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI) to investigate cardiac energy status and function, respectively. The measurements were complemented by ex vivo determination of oxygen consumption in isolated cardiac mitochondria, the expression of proteins involved in energy metabolism, and markers of oxidative stress and calcium homeostasis. We also assessed whether HFD induced myocardial lipid accumulation using in vivo ¹H MRS, and if this was associated with apoptosis and fibrosis. Twenty weeks of HFD feeding resulted in early stage cardiomyopathy, as indicated by diastolic dysfunction and increased left ventricular mass, without any effects on systolic function. In vivo cardiac phosphocreatine-to-ATP ratio and ex vivo oxygen consumption in isolated cardiac mitochondria were not reduced after HFD feeding, suggesting that the diastolic dysfunction was not caused by impaired cardiac energetics. HFD feeding promoted mitochondrial adaptations for increased utilization of fatty acids, which was however not sufficient to prevent the accumulation of myocardial lipids and lipid intermediates. Myocardial lipid accumulation was associated with oxidative stress and fibrosis, but not apoptosis. Furthermore, HFD feeding strongly reduced the phosphorylation of phospholamban, a prominent regulator of cardiac calcium homeostasis and contractility. In conclusion, HFD-induced early stage cardiomyopathy in mice is associated with lipotoxicity-associated oxidative stress, fibrosis, and disturbed calcium homeostasis, rather than impaired cardiac energetics.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

The Nucleocapsid Domain Is Responsible for the Ability of Spleen Necrosis Virus (SNV) Gag Polyprotein To Package both SNV and Murine Leukemia Virus RNA

Murine leukemia virus (MLV)-based vector RNA can be packaged and propagated by the proteins of spleen necrosis virus (SNV). We recently demonstrated that MLV proteins cannot support the replication of an SNV-based vector; RNA analysis revealed that MLV proteins cannot efficiently package SNV-based vector RNA. The domain in Gag responsible for the specificity of RNA packaging was identified using chimeric gag-pol expression constructs. A competitive packaging system was established by generating a cell line that expresses one viral vector RNA containing the MLV packaging signal (Psi) and another viral vector RNA containing the SNV packaging signal (E). The chimeric gag-pol expression constructs were introduced into the cells, and vector titers as well as the efficiency of RNA packaging were examined. Our data confirm that Gag is solely responsible for the selection of viral RNAs. Furthermore, the nucleocapsid (NC) domain in the SNV Gag is responsible for its ability to interact with both SNV E and MLV Psi. Replacement of the SNV NC with the MLV NC generated a chimeric Gag that could not package SNV RNA but retained its ability to package MLV RNA. A construct expressing SNV gag-MLV pol supported the replication of both MLV and SNV vectors, indicating that the gag and pol gene products from two different viruses can functionally cooperate to perform one cycle of retroviral replication. Viral titer data indicated that SNV cis-acting elements are not ideal substrates for MLV pol gene products since infectious viruses were generated at a lower efficiency. These results indicate that the nonreciprocal recognition between SNV and MLV extends beyond the Gag-RNA interaction and also includes interactions between Pol and other cis-acting elements.     

Modes of Action of Five Different Endopectate Lyases from Erwinia chrysanthemi 3937

Five endopectate lyases from the phytopathogenic bacterium Erwinia chrysanthemi, PelA, PelB, PelD, PelI, and PelL, were analyzed with respect to their modes of action on polymeric and oligomeric substrates (degree of polymerization, 2 to 8). On polygalacturonate, PelB showed higher reaction rates than PelD, PelI, and PelA, whereas the reaction rates for PelL were extremely low. The product progression during polygalacturonate cleavage showed a typical depolymerization profile for each enzyme and demonstrated their endolytic character. PelA, PelI, and PelL released oligogalacturonates of different sizes, whereas PelD and PelB released mostly unsaturated dimer and unsaturated trimer, respectively. Upon prolonged incubation, all enzymes degraded the primary products further, to unsaturated dimer and trimer, except for PelL, which degraded the primary products to unsaturated tetramer and pentamer in addition to unsaturated dimer and trimer. The bond cleavage frequencies on oligogalacturonates revealed differences in the modes of action of these enzymes that were commensurate with the product progression profiles. The preferential products formed from the oligogalacturonates were unsaturated dimer for PelD, unsaturated trimer for PelB, and unsaturated tetramer for PelI and PelL. For PelA, preferential products were dependent on the sizes of the oligogalacturonates. Whereas PelB and PelD displayed their highest activities on hexagalacturonate and tetragalacturonate, respectively, PelA, PelI, and PelL were most active on the octamer, the largest substrate used. The bond cleavage frequencies and reaction rates were used to estimate the number of subsites of each enzyme.     

Nucleotide Binding by Lhs1p Is Essential for Its Nucleotide Exchange Activity and for Function in Vivo

Protein translocation and folding in the endoplasmic reticulum of Saccharomyces cerevisiae involves two distinct Hsp70 chaperones, Lhs1p and Kar2p. Both proteins have the characteristic domain structure of the Hsp70 family consisting of a conserved N-terminal nucleotide binding domain and a C-terminal substrate binding domain. Kar2p is a canonical Hsp70 whose substrate binding activity is regulated by cochaperones that promote either ATP hydrolysis or nucleotide exchange. Lhs1p is a member of the Grp170/Lhs1p subfamily of Hsp70s and was previously shown to function as a nucleotide exchange factor (NEF) for Kar2p. Here we show that in addition to this NEF activity, Lhs1p can function as a holdase that prevents protein aggregation in vitro. Analysis of the nucleotide requirement of these functions demonstrates that nucleotide binding to Lhs1p stimulates the interaction with Kar2p and is essential for NEF activity. In contrast, Lhs1p holdase activity is nucleotide-independent and unaffected by mutations that interfere with ATP binding and NEF activity. In vivo, these mutants show severe protein translocation defects and are unable to support growth despite the presence of a second Kar2p-specific NEF, Sil1p. Thus, Lhs1p-dependent nucleotide exchange activity is vital for ER protein biogenesis in vivo.     

Diversity,structure and dynamics of a gallery forest in central Brazil

The structure and dynamics of approximately 64 ha of undisturbed gallery forest were studied over six years. Trees from 31 cm gbh (c. 10 cm dbh) were measured every three years from 1985. They were in 151 (10×20 m) permanent plots in the Gama forest in the Federal District of Brazil. Natural regeneration (individuals under 31 cm gbh) was measured in subplots (of 2×2 m, 5×5 m and 10×10 m) within the 200 m² plots. The total tree flora (gbh31 cm) consisted of 93 species, 81 genera and 44 families in 1985. The Leguminosae, Myrtaceae and Rubiaceae were the families richest in number of species. Most individuals and species were under 45 cm diameter and 20 m high while the maximum diameter per species ranged from 30 to 95 cm. The density structure of trees and natural regeneration was similar, in which the densities of c. 80% of the species represented less than 1% of the total density. The periodic mean annual diameter increment for trees from 10 cm dbh, was c. 0.25 cm/year. Variability was high with coefficients of variation c. 100% or more. The Gama community may maintain tree diversity and structure in undisturbed conditions. Regeneration of c. 80% of the species was found in the establishing phase (poles); the diameter structure was typical of native forests with the number of individuals decreasing with increasing size classes and showing little change over the six years; recruitment compensated for the mortality of most of the abundant species. The soils in Gama gallery forest were dystrophic with high aluminium content. Multivariate analysis suggested the stream, natural gaps and edges as the main causes of floristic differentiation at the community level.     

GENETIC DIVERGENCE CORRELATES WITH MORPHOLOGICAL AND ECOLOGICAL SUBDIVISION IN THE DEEP-WATER ELK KELP, PELAGOPHYCUS PORRA (PHAEOPHYCEAE)

Pelagophycus porra (Leman) Setchell has a narrow distribution confined to deep water from the Channel Islands off the southern California coast to central Baja California, Mexico. Distinct morphotypes are consistently correlated with distinctive habitats, that is, windward exposures characterized by strong water motion and rocky substrates, and sheltered areas with soft substrates found on the lee sides of the islands. We tested the hypothesis that morphologically and ecologically distinct forms reflect genetically distinct stands. Individuals representing populations from three islands and the mainland were compared using RFLP analyses of the nuclear rDNA internal transcribed spacers (ITS1 and ITS2), chloroplast trn L (UAA) intron sequences, and random amplified polymorphic DNA (RAPDs). No variation was found in a survey of 20 restriction sites of ITS1 (ca. 320 base pair [bp]) and ITS2 (ca. 360 bp) among individuals from six populations. Likewise, comparisons of trn L intron (241 bp) sequences among nine individuals from seven populations were identical with the exception of a CATAGT insert in two adjacent stands. A RAPD analysis of 24 individuals from nine populations (4 windward and 5 leeward) using 16 primers generated 166 bands. Thirty-eight percent of the bands did not vary, 16% were unique to a given individual, and 46% were variable. Neighbor joining analysis produced a well-resolved tree with moderately high bootstrap support in which windward and leeward populations were easily distinguished. The lack of divergence in both the fast evolving nuclear rDNA-ITS and the chloroplast trn L intron does not support the morphotypes as different species. However, the compartmentalized differentiation shown in the RAPD data clearly points to isolation. This, and previous ecological studies that demonstrate habitat specificity suggest that leeward stands probably comprise a species in statu nascendi.