A homozygous mutation in the tight-junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts

The tight junction, or zonula occludens, is a specialized cell-cell junction that regulates epithelial and endothelial permeability, and it is an essential component of the blood-brain barrier in the cerebrovascular endothelium. In addition to functioning as a diffusion barrier, tight junctions are also involved in signal transduction. In this study, we identified a homozygous mutation in the tight-junction protein gene JAM3 in a large consanguineous family from the United Arab Emirates. Some members of this family had a rare autosomal-recessive syndrome characterized by severe hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts. Their clinical presentation overlaps with some reported cases of pseudo-TORCH syndrome as well as with cases involving mutations in occludin, another component of the tight-junction complex. However, massive intracranial hemorrhage distinguishes these patients from others. Homozygosity mapping identified the disease locus in this family on chromosome 11q25 with a maximum multipoint LOD score of 6.15. Sequence analysis of genes in the candidate interval uncovered a mutation in the canonical splice-donor site of intron 5 of JAM3. RT-PCR analysis of a patient lymphoblast cell line confirmed abnormal splicing, leading to a frameshift mutation with early termination. JAM3 is known to be present in vascular endothelium, although its roles in cerebral vasculature have not been implicated. Our results suggest that JAM3 is essential for maintaining the integrity of the cerebrovascular endothelium as well as for normal lens development in humans.     

Circulating mediators of bone remodeling in psoriatic arthritis: implications for disordered osteoclastogenesis and bone erosion

Introduction

Rheumatoid arthritis (RA) is a systemic disease manifested by chronic inflammation in multiple articular joints, including the knees and small joints of the hands and feet. We have developed a unique modification to a clinically accepted method for delivering therapies directly to the synovium. Our therapy is based on our previous discovery of an analog peptide (A9) with amino acid substitutions made at positions 260 (I to A), 261 (A to B), and 263 (F to N) that could profoundly suppress immunity to type II collagen (CII) and arthritis in the collagen-induced arthritis model (CIA).

Methods

We engineered an adenoviral vector to contain the CB11 portion of recombinant type II collagen and used PCR to introduce point mutations at three sites within (CII_{124-402, 260A, 261B, 263D}), (rCB11-A9) so that the resulting molecule contained the A9 sequence at the exact site of the wild-type sequence.

Results

We used this construct to target intra-articular tissues of mice and utilized the collagen-induced arthritis model to show that this treatment strategy provided a sustained, local therapy for individual arthritic joints, effective whether given to prevent arthritis or as a treatment. We also developed a novel system for in vivo bioimaging, using the firefly luciferase reporter gene to allow serial bioluminescence imaging to show that luciferase can be detected as late as 18 days post injection into the joint.

Conclusions

Our therapy is unique in that we target synovial cells to ultimately shut down T cell-mediated inflammation. Its effectiveness is based on its ability to transform potential inflammatory T cells and/or bystander T cells into therapeutic (regulatory-like) T cells which secrete interleukin (IL)-4. We believe this approach has potential to effectively suppress RA with minimal side effects.     

Reproduction of in vivo motion using a parallel robot

Although alterations in knee joint loading resulting from injury have been shown to influence the development of osteoarthritis, actual in vivo loading conditions of the joint remain unknown. A method for determining in vivo ligament loads by reproducing joint specific in vivo kinematics using a robotic testing apparatus is described. The in vivo kinematics of the ovine stifle joint during walking were measured with 3D optical motion analysis using markers rigidly affixed to the tibia and femur. An additional independent single degree of freedom measuring device was also used to record a measure of motion. Following sacrifice, the joint was mounted in a robotic/universal force sensor test apparatus and referenced using a coordinate measuring machine. A parallel robot configuration was chosen over the conventional serial manipulator because of its greater accuracy and stiffness. Median normal gait kinematics were applied to the joint and the resulting accuracy compared. The mean error in reproduction as determined by the motion analysis system varied between 0.06 mm and 0.67 mm and 0.07 deg and 0.74 deg for the two individual tests. The mean error measured by the independent device was found to be 0.07 mm and 0.83 mm for the two experiments, respectively. This study demonstrates the ability of this system to reproduce in vivo kinematics of the ovine stifle joint in vitro. The importance of system stiffness is discussed to ensure accurate reproduction of joint motion.     

Epitope cross-reactivity frequently differs between central and effector memory HIV-specific CD8+ T cells

HIV diversity may limit the breadth of vaccine coverage due to epitope sequence differences between strains. Although amino acid substitutions within CD8(+) T cell HIV epitopes can result in complete or partial abrogation of responses, this has primarily been demonstrated in effector CD8(+) T cells. In an HIV-infected Kenyan cohort, we demonstrate that the cross-reactivity of HIV epitope variants differs dramatically between overnight IFN-gamma and longer-term proliferation assays. For most epitopes, particular variants (not the index peptide) were preferred in proliferation in the absence of corresponding overnight IFN-gamma responses and in the absence of the variant in the HIV quasispecies. Most proliferating CD8(+) T cells were polyfunctional via cytokine analyses. A trend to positive correlation was observed between proliferation (but not IFN-gamma) and CD4 counts. We present findings relevant to the assessment of HIV vaccine candidates and toward a better understanding of how viral diversity is tolerated by central and effector memory CD8(+) T cells.     

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G-protein-coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G-protein-independent manner. Here, we show that at low concentrations of an agonist, beta(2)-adrenergic receptors (beta(2)-ARs) signal through Galpha(s) to activate the mitogen-activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through beta(2)-ARs via an additional pathway that is G-protein-independent but tyrosine kinase Src-dependent. This new dosage-dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.     

Old World arenavirus infection interferes with the expression of functional alpha-dystroglycan in the host cell

alpha-Dystroglycan (alpha-DG) is an important cellular receptor for extracellular matrix (ECM) proteins as well as the Old World arenaviruses lymphocytic choriomeningitis virus (LCMV) and the human pathogenic Lassa fever virus (LFV). Specific O-glycosylation of alpha-DG is critical for its function as receptor for ECM proteins and arenaviruses. Here, we investigated the impact of arenavirus infection on alpha-DG expression. Infection with an immunosuppressive LCMV isolate caused a marked reduction in expression of functional alpha-DG without affecting biosynthesis of DG core protein or global cell surface glycoprotein expression. The effect was caused by the viral glycoprotein (GP), and it critically depended on alpha-DG binding affinity and GP maturation. An equivalent effect was observed with LFVGP. Viral GP was found to associate with a complex between DG and the glycosyltransferase LARGE in the Golgi. Overexpression of LARGE restored functional alpha-DG expression in infected cells. We provide evidence that virus-induced down-modulation of functional alpha-DG perturbs DG-mediated assembly of laminin at the cell surface, affecting normal cell-matrix interactions.     

Hepatocyte growth factor enhances IL-1β stimulated IL-8 secretion by Caco-2 epithelial cells

Hepatocyte growth factor (HGF) can induce proliferation and migration of intestinal epithelial cells and has also been shown to be important in wound healing of inflamed mucosal tissues. HGF is known to be expressed along with interleukin-1 (IL-1) by inflamed mucosal tissues, yet the effect of HGF on IL-1-induced proinflammatory cytokine responses by colonic epithelial cells is unknown. In this report, we have examined the effect of HGF on IL-1-induced secretion of IL-8 by the Caco-2 colonic epithelial cell line. HGF stimulation alone had no effect on the secretion of IL-8 by the Caco-2 cells. However, culture of the cells with HGF and suboptimal levels of IL-1 resulted in a significant enhancement of IL-8 secretion compared to cells cultured with IL-1 alone. A similar effect was seen with HGF and IL-1 simulation of monocyte chemoattractant protein-1 secretion by the rat IEC-6 intestinal epithelial cell line. The enhancing effect of HGF was seen regardless of whether the culture medium contained serum or not. Simultaneous stimulation with HGF and IL-1 was required for the enhancing effect as cells pretreated with HGF for 24 h and then stimulated with IL-1 alone secreted IL-8 levels similar to that of cells stimulated with IL-1 alone. These results suggest that in addition to wound healing, HGF may play a role in the IL-1-induced chemokine response of epithelial cells in inflamed mucosal tissues.     

Crystal structure of the pregnane X receptor-estradiol complex provides insights into endobiotic recognition

The human nuclear pregnane X receptor (PXR) responds to a wide variety of xenobiotic and endobiotic compounds, including pregnanes, progesterones, corticosterones, lithocholic acids, and 17beta-estradiol. In response to these ligands, the receptor controls the expression of genes central to the metabolism and excretion of potentially harmful chemicals from both exogenous and endogenous sources. Although the structural basis of PXR's interaction with small and large xenobiotics has been examined, the detailed nature of its binding to endobiotics, including steroid-like ligands, remains unclear. We report the crystal structure of the human PXR ligand-binding domain (LBD) in complex with 17beta-estradiol, a representative steroid ligand, at 2.65 A resolution. Estradiol is found to occupy only one region of PXR's expansive ligand-binding pocket, leaving a notable 1000 A3 of space unoccupied, and to bridge between the key polar residues Ser-247 and Arg-410 in the PXR LBD. Positioning the steroid scaffold in this way allows it to make several direct contacts to alphaAF of the receptor's AF-2 region. The PXR-estradiol complex was compared with that of other nuclear receptors, including the estrogen receptor, in complexes with analogous ligands. It was found that PXR's placement of the steroid is remarkably distinct relative to other members of the nuclear receptor superfamily. Using the PXR-estradiol complex as a guide, the binding of other steroid- and cholesterol-like molecules was then considered. The results provide detailed insights into the manner in which human PXR responds to a wide range of endobiotic compounds.     

Colour and Escape Behaviour in Polymorphic Populations of an Aposematic Poison Frog

The phenomenon of aposematism, or the pairing of antipredator defence with conspicuous or distinctive signals, serves as an excellent example of how traits act in concert to shape fitness. Not only does this complex phenotype require the integration of multiple traits, it alters the fitness pay‐offs of yet others. The protection offered by aposematism may, for example, reduce the costs associated with foraging or sexual display. Thus, well‐protected aposematic lineages should be bolder, more active and less likely to respond to perceived threats of predation than more cryptic lineages. Comparisons of differently coloured morphs of the polytypic strawberry poison frog (Oophaga pumilio) have supported the predicted behavioural correlates of aposematism, with the exception of those regarding responsiveness to simulated predators. We tested the key prediction that aposematic coloration will be associated with reduced sensitivity to predators in two polymorphic O. pumilio populations. The novel approach of studying polymorphic populations allowed us to assess the effect of colour in the absence of potentially confounding habitat differences. We found that colour was associated with the probability that a frog would attempt escape and the distance at which it fled, but only in one population, and not in the predicted direction. An overall comparison of the two populations superficially supported our predictions, but this pattern actually arose because frogs occupying higher perches were less likely to respond, a pattern that may reflect the value of high perches and the costs associated with returning to them after attempted escape. These results highlight the complexity of the relationship between predators and prey, the challenges associated with understanding how and why traits are correlated, and the intimate ties between behaviour and morphological evolution.