MRI quantitative muscle characterization in children with X-linked hypophosphatemia.

INTRODUCTION: Children with X Linked Hypophosphatemia (XLH) suffer from carential ricket, bone deformities and lameness. No previous study demonstrated a morphological distinction in muscles in these patients. The aim of this prospective study was to characterize, using Magnetic Resonance Imaging (MRI), the muscle morphology of pelvis, thigh and leg in children with XLH and to compare it with typically developed (TD) children. HYPOTHESIS: We hypothesized that lower limbs muscles in children with XLH are different from TD children and could explain limp walking. MATERIAL AND METHODS: Three-dimensional reconstructions of the muscles were performed in 11 patients with XLH and 15 TD children. Muscle lengths, sections and volumes were calculated and normalized with height and weight. Mean age was 10. RESULTS: Lengths were all smaller in children with XLH except for the Medius/minimus gluteus muscles (p=0.64). The difference seemed higher in muscles with a long tendinous part as semitendinosus (0.139 vs 0,164; p<0.01). All volumes were significantly inferior in children with XLH. This preliminary study showed significant differences in muscle structures between patients with XLH and TD children. DISCUSSION: Medius/minimus gluteus seemed to be particularly developed in children with XLH. Nevertheless it is not possible to conclude if it is related to XLH or a consequence of bone deformities. LEVEL OF PROOF: IV.

Combined gait analysis and radiologic examination in children with X-linked hypophosphatemia.

BACKGROUND: X-linked hypophosphataemia causes bone deformities and gait abnormalities that tend to worsen with age in the absence of appropriate treatment. However, doctors do not currently use quantitative tools to characterize these symptoms and their possible interactions. METHODS: Radiographs and 3D gait data from 43 non-surgical growing children with X-linked hypophosphataemia were acquired prospectively. Data from age-matched typically developing children were used to form the reference group. Subgroups based on radiological parameters were compared with each other and with the reference population. Linear correlations between radiographic parameters and gait variables were examined. FINDING: X-linked hypophosphatemic patients differed from the control group in pelvic tilt, ankle plantarflexion, knee flexion moment and power. High correlations with tibiofemoral angle were found for trunk lean, knee and hip adduction, and knee abduction moment. The Gait Deviation Index was below 80 for 88% of the patients with a high tibiofemoral angle (varus). Compared to other subgroups, varus patients had augmented trunk lean (+3°) and knee adduction (+10°) and decreased hip adduction (-5°) and ankle plantarflexion (-6°). Femoral torsion was associated with alterations in rotation at the knee, and hip. INTERPRETATION: Gait abnormalities induced in X-linked hypophosphataemia have been described in a large cohort of children. Links between gait alterations and lower limb deformities were found, with varus deformities standing out. Since bony deformities appear when X-linked hypophosphatemic children start walking and have been found to alter gait patterns, we suggest that combining radiology with gait analysis may improve the clinical management of X-linked hypophosphataemia.

Quantitative analysis of lower limb and pelvic deformities in children with X-linked hypophosphatemic rickets.

INTRODUCTION: X-linked hypophosphatemia (XLH) rickets mainly causes leg deformities in children that can worsen as they grow. We hypothesized that quantifying the bone parameters will help to document and monitor these deformities in children with XLH. METHODS: Thirty-five growing children affected by XLH were included in this cross-sectional study. Biplanar radiographs were taken with an EOS system allowing three-dimensional (3D) reconstructions of the pelvis and legs. Sixteen geometric parameters were calculated for the legs and pelvis. A control group of 40 age-matched patients was used to define the reference values for these geometric parameters. RESULTS: For the legs, significant differences (p<0.05) appeared between the XLH patients and the control group in the neck-shaft angle, femur/tibia length ratio and HKS. Among the 70 legs in the XLH group, 23 were in genu varum, 25 were in genu valgum and 22 were straight. There were significant differences between the genu varum and genu valgum subgroups in the femoral mechanical angle and the HKS. A strong correlation was found between the femoral mechanical angle and femorotibial angle (r2=0.73) and between the femoral mechanical angle and HKS (r2=0.69) The sacral slope and acetabular anteversion were significant different from the reference values. DISCUSSION: Quantitative radiological parameters derived from 3D reconstructions show that the deformities in XLH patients are (1) mainly in - but not limited to - the femoral shaft; (2) highly variable from one person to another. Some of these radiological parameters may be useful for the diagnosis and monitoring of XLH patients. LEVEL OF EVIDENCE: III; case control study.

Changes in adipose bone marrow and bone morphology in X-linked hypophosphatemic rickets.

INTRODUCTION: X-linked hypophosphatemic (XLH) rickets causes significant bone deformities in the lower limbs resulting from a bone mineralization defect. According to Frost's Mechanostat theory, compensatory modeling of the bones takes place during increased mechanical loads. In addition, mechanical stimuli modulate the differentiation of mesenchymal stem cells; common precursors to bone marrow adipocytes and osteoblasts. HYPOTHESIS: Bone deformities of the lower limbs lead to increased femoral bone mass and decreased fatty infiltration of the bone marrow (FIBM) in children with XLH rickets compared to a control group. PATIENTS AND METHODS: Eleven children (10.3years [6-17]) with XLH rickets and 22 healthy children (10.2years [5-15.5]) underwent lower limb Magnetic Resonance Imaging. A calculation of FIBM was performed at the mid-femur, as well as a calculation of the total bone cross-sectional area (CSA), the cortical CSA, the anteroposterior and mediolateral axes of the femur, bone marrow and the thickness of the femoral cortices. RESULTS: Total bone CSA, total cortical CSA and bone marrow CSA were higher in the XLH group than in the control group (p<0.05). The mid-lateral diameters of the femur and bone marrow were more elongated than those of the control group (p<0.001). Only the anterior cortex was thinned in the XLH group (p=0.001), while there was no difference with the control group for the posterior, medial and lateral cortices. The total percentage of FIBM was 72.81% [±3.95] and 77.4% [±5.52] for the XLH and control groups respectively (p<0.001). DISCUSSION: The increase in bone mass in the XLH population reflects an adaptation of bone tissue to the bone deformities present in this pathology. The decrease in FIBM indicates a lower risk of osteoporosis in the XLH population and may constitute a new monitoring parameter in this pathology. LEVEL OF STUDY: III; Case-control study.

Prospective Analysis of Muscle Adiposity in Children With X-linked Hypophosphatemic Rickets vs Control Children.

Context: Children with X-linked hypophosphatemic (XLH) rickets have muscle weakness that severely impairs their function. Intermuscular and intramuscular adipose tissue (IMAT and intraMAT, respectively) may contribute to this muscle weakness. Objective: This work aimed to compare IMAT and intraMAT in XLH children vs typically developing (TD) children. Methods: A prospective, monocentric cohort study was conducted of XLH (n = 11; aged 10.3 years [6-17]) and TD children (n = 22; aged 10.2 years [5-15.5]). All children underwent magnetic resonance imaging of the lower limbs; IMAT and intraMAT percentages were calculated after manual contouring of each muscle of the thigh and the deep fascia at mid-thigh level. Results: XLH children were comparable in age but shorter and heavier than TD children (P = .001 and P = .03, respectively). They had smaller muscle length and volume than TD children (P < .001) but there was no statistically significant difference in muscle cross-sectional area between the groups (P = .833). The total percentage of IMAT was higher in XLH children (8.66% vs 3.60% in TD children; P < .0001). In addition, though the total percentage of intraMAT did not differ significantly (12.58% and 10.85% in XLH and TD children, respectively; P = .143) intraMAT was statistically significantly higher in XLH children than TD children in 4 of the 13 muscles studied. Conclusion: Our results show that IMAT is higher in young children with XLH, independently of obesity and overweight. Further, these results will facilitate both the early prevention of functional and metabolic consequences of the increase in adipose tissue in XLH children.

Fast quasi-automated 3D reconstruction of lower limbs from low dose biplanar radiographs using statistical shape models and contour matching.

Three-dimensional bone reconstructions from medical imaging are essential for biomechanical modelling and are growing tools in clinics. Several methods of lower limbs reconstruction from biplanar radiographs have been proposed in the literature but with significant operator dependence. A novel reconstruction method based on reduced manual annotation, statistical shape models and fully automatic adjustments was proposed in this study. While significantly reducing operator intervention, the proposed method demonstrated similar or better precision than previous approaches on clinical parameters. Meanwhile, shape accuracy was improved to around 1mm. By quasi-automating the 3D reconstruction without loss of accuracy and precision, the proposed approach is a considerable step towards extensive use of 3D personalized models in clinical routine and large cohort biomechanical studies.

Assessment of a novel deep learning-based marker-less motion capture system for gait study.

BACKGROUND: Marker-less systems based on digital video cameras and deep learning for gait analysis could have a deep impact in clinical routine. A recently developed system has shown promising results in terms of joint center position but has not been yet evaluated in terms of gait outcomes. RESEARCH QUESTION: How does this novel marker-less system compare to a marker-based reference system in terms of clinically relevant gait parameters? METHODS: The deep learning method behind the developed marker-less system was trained on a dedicated dataset consisting of forty-one asymptomatic and pathological subjects each performing ten walking trials. The system could estimate the three-dimensional position of seventeen joint centers or keypoints (e.g., neck, shoulders, hip, knee, and ankles). We evaluated the marker-less system against a marker-based system in terms of differences in joint position (Euclidean distance), detection of gait events (e.g., heel strike and toe-off), spatiotemporal parameters (e.g., step length, time), kinematic parameters (e.g., hip and knee extension-flexion), and inter-trial reliability for kinematic parameters. RESULTS: The marker-less system was able to estimate the three-dimensional position of joint centers with a mean difference of 13.1 mm (SD = 10.2 mm). 99% of the estimated gait events were estimated within 10 ms of the corresponding reference values. Estimated spatiotemporal parameters showed zero bias. The mean and standard deviation of the differences of the estimated kinematic parameters varied by parameter (for example, the mean and standard deviation for knee extension flexion angle were -3.0° and 2.7°). Inter-trial reliability of the measured parameters was similar to that of the marker-based references. SIGNIFICANCE: The developed marker-less system can measure the spatiotemporal parameters within the range of the minimum detectable changes obtained using the marker-based reference system. Moreover, except for hip extension flexion, the system showed promising results in terms of several kinematic parameters.

Systemic Immune Bias Delineates Malignant Astrocytoma Survival Cohorts.

Patients with grade III anaplastic astrocytomas (AA) separate into survival cohorts based on the presence or absence of mutations in isocitrate dehydrogenase (IDH). Progression to glioblastoma (GBM), morphologically distinguishable by elevated microvascular proliferation, necrosis, and cell division in tumor tissues, is considerably more rapid in IDH wild-type tumors such that their diagnosis as AA is relatively rare. More often initially presenting as GBM, these contain higher numbers of tumor-associated macrophages (TAMs) than most AA, and GBM patients also have higher levels of circulating M2 monocytes. TAM and M2 monocytes share functional properties inhibitory for antitumor immunity. Yet, although there is a wealth of data implicating TAM in tumor-immune evasion, there has been limited analysis of the impact of the circulating M2 monocytes. In the current study, immune parameters in sera, circulating cells, and tumor tissues from patients with primary gliomas morphologically diagnosed as AA were assessed. Profound differences in serum cytokines, glioma extracellular vesicle cross-reactive Abs, and gene expression by circulating cells identified two distinct patient cohorts. Evidence of type 2-immune bias was most often seen in patients with IDH wild-type AA, whereas a type 1 bias was common in patients with tumors expressing the IDH1R132H mutation. Nevertheless, a patient's immune profile was better correlated with the extent of tumor vascular enhancement on magnetic resonance imaging than IDH mutational status. Regardless of IDH genotype, AA progression appears to be associated with a switch in systemic immune bias from type 1 to type 2 and the loss of tumor vasculature integrity.

A novel dataset and deep learning-based approach for marker-less motion capture during gait.

BACKGROUND: The deep learning-based human pose estimation methods, which can estimate joint centers position, have achieved promising results on the publicly available human pose datasets (e.g., Human3.6 M). However, these datasets may be less efficient for gait study, particularly for clinical applications, because of the limited number of subjects, their homogeneity (all asymptomatic adults), and the errors introduced by marker placement on subjects' regular clothing. RESEARCH QUESTION: How a new human pose dataset, adapted for gait study, could contribute to the advancement and evaluation of marker-less motion capture systems? METHODS: A marker-less system, based on deep learning-based pose estimation methods, was proposed. A new dataset (ENSAM dataset) was collected. Twenty-two asymptomatic adults, one adult with scoliosis, one adult with spondylolisthesis, and seven children with bone disease performed ten walking trials, while being recorded both by the proposed marker-less system and a reference system - combining a marker-based motion capture system and a medical imaging system (EOS). The dataset was split into training and test sets. The pose estimation method, already trained on the Human3.6 M dataset, was evaluated on the ENSAM test set, then reevaluated after further training on the ENSAM training set. The joints coordinates were evaluated, using Bland-Altman bias and 95 % confidence interval, and joint position error (the Euclidean distance between the estimated joint centers and the corresponding reference values). RESULTS: The Bland-Altman 95 % confidence intervals were substantially improved after finetuning the pose estimation method on the ENSAM training set (e.g., from 106.9 mm to 17.4 mm for the hip joint). With the new dataset and approach, the mean joint position error varied from 6.2 mm for ankles to 21.1 mm for shoulders. SIGNIFICANCE: The proposed marker-less system achieved promising results in terms of joint position errors. Future studies are necessary to assess the system in terms of gait parameters.

Brain tissue-resident immune memory cells are required for long-term protection against CNS infection with rabies virus.

Immune memory cells residing in previously infected, nonlymphoid tissues play a role in immune surveillance. In the event that circulating antibodies fail to prevent virus spread to the tissues in a secondary infection, these memory cells provide an essential defense against tissue reinfection. CNS tissues are isolated from circulating immune cells and antibodies by the blood-brain barrier, making the presence of tissue-resident immune memory cells particularly needed to combat recurrent infection by neurotropic viruses. Wild-type and laboratory-engineered rabies viruses are neurotropic, differ in pathogenicity, and have varying effects on BBB functions. These viruses have proven invaluable tools in demonstrating the importance of tissue-resident immune memory cells in the reinfection of CNS tissues. Only Type 1 immune memory is effective at therapeutically clearing a secondary infection with wild-type rabies viruses from the CNS and does so despite the maintenance of blood-brain barrier integrity.

Combined 3D analysis of lower-limb morphology and function in children with idiopathic equinovarus clubfoot: A preliminary study.

INTRODUCTION: In children treated for idiopathic equinovarus clubfoot (EVCF), the relation between morphologic defects on clinical examination and standard X-ray on the one hand and functional abnormalities on the other is difficult to objectify. The aim of the present study was to demonstrate the feasibility of combined 3D analysis of the foot and lower limb based on biplanar EOS radiographs and gait analysis. The study hypothesis was that this provides better understanding of abnormalities in form and function. METHODS: Ten children with unilateral EVCF and "very good" clinical results were included. They underwent gait analysis on the Rizzoli Institute multisegment foot model. Kinematic data were collected for the hip, knee, ankle and foot (hindfoot/midfoot, midfoot/forefoot and hindfoot/forefoot). Biplanar EOS radiographs were taken to determine anatomic landmarks and radiological parameters. RESULTS: Complete acquisition time was around 2hours per patient. No significant differences were found between EVCF and healthy feet except for calcaneal incidence, tibiocalcaneal angle and hindfoot/midfoot and hindfoot/forefoot inversion. DISCUSSION: The feasibility of the combined analysis was confirmed. There were no differences in range of motion, moment or power between EVCF and healthy feet in this series of patients with very good results. The functional results are related to radiological results within the normal range. The protocol provided anatomic and kinematic reference data. A larger-scale study could more objectively assess the contribution of EOS radiography using optoelectronic markers. LEVEL OF EVIDENCE: II, low-power prospective study.

Sphenopalatine ganglion stimulation is a reversible and frequency-dependent modulator of the blood-brain barrier.

BACKGROUND: The sphenopalatine ganglion (SPG) is a vasoactive mediator of the anterior intracranial circulation in mammals. SPG stimulation has been demonstrated to alter blood-brain barrier (BBB) permeability, although this phenomenon is not well characterized. OBJECTIVE: To determine the effect of SPG stimulation on the BBB using rat models. METHODS: Extravasation of fluorescent tracer 70 kDa FITC-dextran into rat brain specimens was measured across a range of stimulation parameters to assess BBB permeability. Tight junction (TJ) morphology was compared by assessing differences in the staining of proteins occludin and ZO-1 and analyzing ultrastructural changes on transmission electron microscopy (TEM) between stimulated and unstimulated specimens. RESULTS: SPG stimulation at 10 Hz maximally increased BBB permeability, exhibiting a 6-fold increase in fluorescent traceruptake (1.66% vs 0.28%, p < 0.0001). This effect was reversed 4-hours after stimulation (0.36% uptake, p = 0.99). High-frequency stimulation at 20 Hz and 200 Hz did not increase tracer extravasation, (0.26% and 0.28% uptake, p = >0.999 and p = 0.998, respectively). Stimulation was associated a significant decrease in the colocalization of occludin and ZO-1 with endothelial markers in stimulated brains compared to control (74.6% vs. 39.7% and 67.2% vs. 60.4% colocalization, respectively, p < 0.0001), and ultrastructural changes in TJ morphology associated with increased BBB permeability were observed on TEM. CONCLUSION: This study is the first to show a reversible, frequency-dependent increase in BBB permeability with SPG stimulation and introduces a putative mechanism of action through TJ disruption. Bypassing the BBB with SPG stimulation could enable new paradigms in delivering therapeutics to the CNS. Further study of this technology is needed.

Clearance of attenuated rabies virus from brain tissues is required for long-term protection against CNS challenge with a pathogenic variant.

Rabies virus is a neurotropic lyssavirus which is 100% fatal in its pathogenic form when reaching unprotected CNS tissues. Death can be prevented by mechanisms delivering appropriate immune effectors across the blood-brain barrier which normally remains intact during pathogenic rabies virus infection. One therapeutic approach is to superinfect CNS tissues with attenuated rabies virus which induces blood-brain barrier permeability and immune cell entry. Current thinking is that peripheral rabies immunization is sufficient to protect against a challenge with pathogenic rabies virus. While this is undoubtedly the case if the virus is confined to the periphery, what happens if the virus reaches the CNS is less well-understood. In the current study, we find that peripheral immunization does not fully protect mice long-term against an intranasal challenge with pathogenic rabies virus. Protection is significantly better in mice that have cleared attenuated virus from the CNS and is associated with a more robust CNS recall response evidently due to the presence in CNS tissues of elevated numbers of lymphocytes phenotypically resembling long-term resident immune cells. Adoptive transfer of cells from rabies-immune mice fails to protect against CNS challenge with pathogenic rabies virus further supporting the concept that long-term resident immune cell populations must be established in brain tissues to protect against a subsequent CNS challenge with pathogenic rabies virus.

Protection Against CNS-Targeted Rabies Virus Infection is Dependent upon Type-1 Immune Mechanisms Induced by Live-Attenuated Rabies Vaccines.

Rabies remains a major public health issue worldwide, especially in developing countries where access to medical care can represent a real challenge. While there is still no cure for rabies, it is a vaccine-preventable disease with pre- and post-exposure prophylaxis regimens approved by the World Health Organization (WHO). However, many rabies-exposed individuals have limited access to vaccines and virus-neutralizing antibodies approved for post-exposure prophylaxis. Unfortunately, any delay in the administration of these reagents can have lethal consequences. This highlights the need to develop cost-effective immunological reagents with a greater window of efficacy. Live-attenuated vaccine strains of rabies virus presents a potential treatment in filling this gap. We show here that immunization with live-attenuated vaccines provide long-lasting rabies immunity, superior to the protection induced by inactivated vaccines. In the absence of an immunostimulatory adjuvant, vaccination with multiple doses of inactivated rabies virus induces a type-2 immune response. This type of immunity is highly effective at inducing neutralizing antibody but has limited efficacy in clearing the virus from central nervous system (CNS) tissues. In contrast, a single infection with live-attenuated rabies vaccine safely drives a type-1 immune response, associated with both the production of a neutralizing antibody and the clearance of wild-type rabies virus from CNS tissues. These results indicate that live-attenuated rabies strains have the potential to be more effective in post-exposure prophylaxis than conventional inactivated vaccines.

Blockade of Glutamine Synthetase Enhances Inflammatory Response in Microglial Cells.

AIMS: Microglial cells are brain-resident macrophages engaged in surveillance and maintained in a constant state of relative inactivity. However, their involvement in autoimmune diseases indicates that in pathological conditions microglia gain an inflammatory phenotype. The mechanisms underlying this change in the microglial phenotype are still unclear. Since metabolism is an important modulator of immune cell function, we focused our attention on glutamine synthetase (GS), a modulator of the response to lipopolysaccharide (LPS) activation in other cell types, which is expressed by microglia. RESULTS: GS inhibition enhances release of inflammatory mediators of LPS-activated microglia in vitro, leading to perturbation of the redox balance and decreased viability of cocultured neurons. GS inhibition also decreases insulin-mediated glucose uptake in microglia. In vivo, microglia-specific GS ablation enhances expression of inflammatory markers upon LPS treatment. In the spinal cords from experimental autoimmune encephalomyelitis (EAE), GS expression levels and glutamine/glutamate ratios are reduced. INNOVATION: Recently, metabolism has been highlighted as mediator of immune cell function through the discovery of mechanisms that (behind these metabolic changes) modulate the inflammatory response. The present study shows for the first time a metabolic mechanism mediating microglial response to a proinflammatory stimulus, pointing to GS activity as a master modulator of immune cell function and thus unraveling a potential therapeutic target. CONCLUSIONS: Our study highlights a new role of GS in modulating immune response in microglia, providing insights into the pathogenic mechanisms associated with inflammation and new strategies of therapeutic intervention. Antioxid. Redox Signal. 26, 351-363.

T-bet Is Required for the Rapid Clearance of Attenuated Rabies Virus from Central Nervous System Tissue.

Much of our understanding of CNS immunity has been gained from models involving pathological inflammation. Attenuated rabies viruses (RABV) are unique tools to study CNS immunity in the absence of conventional inflammatory mechanisms, as they spread from the site of inoculation to the CNS transaxonally, thereby bypassing the blood-brain barrier (BBB), and are cleared without neutrophil or monocyte infiltration. To better understand the role of CD4 T cell subsets in the clearance of the virus from CNS tissues, we examined the development of antiviral immunity in wild-type (WT) and T-bet knockout mice (T-bet(-/-)), which lack Th1 cells. Early control of RABV replication in the CNS tissues of WT mice is associated with the production of IFN-γ, with antiviral effects likely mediated through the enhanced expression of type I IFNs. Of interest, IFN-α and -γ are overexpressed in the infected T-bet(-/-) by comparison with WT CNS tissues, and the initial control of RABV infection is similar. Ultimately, attenuated RABV are cleared from the CNS tissues of WT mice by Ab locally produced by the activities of infiltrating T and B cells. Although T and B cell infiltration into the CNS of infected T-bet(-/-) mice is comparable, their activities are not, the consequence being delayed, low-level Ab production and prolonged RABV replication. More importantly, neither T-bet(-/-) mice immunized with an attenuated virus, nor WT mice with Th2 RABV-specific immunity induced by immunization with inactivated virus, are protected in the long term against challenge with a pathogenic RABV.

Expression of interferon gamma by a recombinant rabies virus strongly attenuates the pathogenicity of the virus via induction of type I interferon.

UNLABELLED: Previous animal model experiments have shown a correlation between interferon gamma (IFN-γ) expression and both survival from infection with attenuated rabies virus (RABV) and reduction of neurological sequelae. Therefore, we hypothesized that rapid production of murine IFN-γ by the rabies virus itself would induce a more robust antiviral response than would occur naturally in mice. To test this hypothesis, we used reverse engineering to clone the mouse IFN-γ gene into a pathogenic rabies virus backbone, SPBN, to produce the recombinant rabies virus designated SPBNγ. Morbidity and mortality were monitored in mice infected intranasally with SPBNγ or SPBN(-) control virus to determine the degree of attenuation caused by the expression of IFN-γ. Incorporation of IFN-γ into the rabies virus genome highly attenuated the virus. SPBNγ has a 50% lethal dose (LD50) more than 100-fold greater than SPBN(-). In vitro and in vivo mouse experiments show that SPBNγ infection enhances the production of type I interferons. Furthermore, knockout mice lacking the ability to signal through the type I interferon receptor (IFNAR(-/-)) cannot control the SPBNγ infection and rapidly die. These data suggest that IFN-γ production has antiviral effects in rabies, largely due to the induction of type I interferons. IMPORTANCE: Survival from rabies is dependent upon the early control of virus replication and spread. Once the virus reaches the central nervous system (CNS), this becomes highly problematic. Studies of CNS immunity to RABV have shown that control of replication begins at the onset of T cell entry and IFN-γ production in the CNS prior to the appearance of virus-neutralizing antibodies. Moreover, antibody-deficient mice are able to control but not clear attenuated RABV from the CNS. We find here that IFN-γ triggers the early production of type I interferons with the expected antiviral effects. We also show that engineering a lethal rabies virus to express IFN-γ directly in the infected tissue reduces rabies virus replication and spread, limiting its pathogenicity in normal and immunocompromised mice. Therefore, vector delivery of IFN-γ to the brain may have the potential to treat individuals who would otherwise succumb to infection with rabies virus.

IgG leakage may contribute to neuronal dysfunction in drug-refractory epilepsies with blood-brain barrier disruption.

Focal epilepsies are often associated with blood-brain barrier disruption. In 4 entorhinal cortex tissue samples and 13 hippocampal samples from patients with pharmacoresistent temporal lobe epilepsy, we observed immunoglobulin G (IgG) leakage in the parenchyma and IgG-positive neurons that had evidence of neurodegeneration, such as shrinkage and eosinophilia. These findings were not present in samples from 12 nonepileptic control subjects. To complement these findings, we used a rat in vivo model that mimics the development of limbic epilepsy with blood-brain barrier disruption. During epileptogenesis, IgG leakage and neuronal IgG uptake increased concomitantly with the occurrence of seizures. Immunoglobulin G accumulation in neurons was selective, particularly for interneurons and pyramidal neurons. Immunohistochemistry and electron microscopy showed that IgG uptake in the rat neurons was associated with eosinophilia, shrinkage, and ultrastructural degenerative changes. Moreover, IgG-positive neurons lost their NeuN immunohistochemical staining. Together, these observations suggest that IgG leakage is related to neuronal impairment and may be a pathogenic mechanism in epileptogenesis and chronic epilepsy.

Sodium-23 solid-state nuclear magnetic resonance of commercial sodium naproxen and its solvates.

We report on the investigation of sodium coordination environments with solid-state ²³Na nuclear magnetic resonance (NMR) spectroscopy of various hydrates and solvates of sodium naproxen (SN), a commercially available anti-inflammatory drug sold over the counter as Aleve®, among other names. The ²³Na quadrupolar coupling constant is found to change significantly depending on the hydration state, and subtle changes in oxygen coordination environment about the sodium cations were apparent in the NMR spectra. High-resolution double-rotation NMR experiments are also performed on powdered samples to obtain solution-like ²³Na NMR spectra. Our attempts at crystallizing various solvates of SN have led to the characterization of the first crystal structure for the heminonahydrated form. The composition of commercial SN is also investigated and it is shown that Aleve® is composed of approximately 80% monohydrate solvate. Density-functional theory calculations, using the gauge-including projector-augmented-wave formalism, allow for the assignment of ²³Na NMR peaks to specific sodium sites in the reported X-ray crystal structure.

Epileptiform activity induces vascular remodeling and zonula occludens 1 downregulation in organotypic hippocampal cultures: role of VEGF signaling pathways.

Recent studies suggest that blood-brain barrier (BBB) permeability contributes to epileptogenesis in symptomatic epilepsies. We have previously described angiogenesis, aberrant vascularization, and BBB alteration in drug-refractory temporal lobe epilepsy. Here, we investigated the role of vascular endothelial growth factor (VEGF) in an in vitro integrative model of vascular remodeling induced by epileptiform activity in rat organotypic hippocampal cultures. After kainate-induced seizure-like events (SLEs), we observed an overexpression of VEGF and VEGF receptor-2 (VEGFR-2) as well as receptor activation. Vascular density and branching were significantly increased, whereas zonula occludens 1 (ZO-1), a key protein of tight junctions (TJs), was downregulated. These effects were fully prevented by VEGF neutralization. Using selective inhibitors of VEGFR-2 signaling pathways, we found that phosphatidylinositol 3-kinase is involved in cell survival, protein kinase C (PKC) in vascularization, and Src in ZO-1 regulation. Recombinant VEGF reproduced the kainate-induced vascular changes. As in the kainate model, VEGFR-2 and Src were involved in ZO-1 downregulation. These results showed that VEGF/VEGFR-2 initiates the vascular remodeling induced by SLEs and pointed out the roles of PKC in vascularization and Src in TJ dysfunction, respectively. This suggests that Src pathway could be a therapeutic target for BBB protection in epilepsies.