Gata3 hypermethylation and Foxp3 hypomethylation are associated with sustained protection and bystander effect following epicutaneous immunotherapy in peanut-sensitized mice.

BACKGROUND: Epicutaneous immunotherapy (EPIT) is a promising method for treating food allergies. In animal models, EPIT induces sustained unresponsiveness and prevents further sensitization mediated by Tregs. Here, we elucidate the mechanisms underlying the therapeutic effect of EPIT, by characterizing the kinetics of DNA methylation changes in sorted cells from spleen and blood and by evaluating its persistence and bystander effect compared to oral immunotherapy (OIT). METHODS: BALB/c mice orally sensitized to peanut proteins (PPE) were treated by EPIT using a PPE-patch or by PPE-OIT. Another set of peanut-sensitized mice treated by EPIT or OIT were sacrificed following a protocol of sensitization to OVA. DNA methylation was analyzed during immunotherapy and 8 weeks after the end of treatment in sorted cells from spleen and blood by pyrosequencing. Humoral and cellular responses were measured during and after immunotherapy. RESULTS: Analyses showed a significant hypermethylation of the Gata3 promoter detectable only in Th2 cells for EPIT from the 4th week and a significant hypomethylation of the Foxp3 promoter in CD62L+ Tregs, which was sustained only for EPIT. In addition, mice treated with EPIT were protected from subsequent sensitization and maintained the epigenetic signature characteristic for EPIT. CONCLUSIONS: Our study demonstrates that EPIT leads to a unique and stable epigenetic signature in specific T-cell compartments with downregulation of Th2 key regulators and upregulation of Treg transcription factors, likely explaining the sustainability of protection and the observed bystander effect.

Glenoid Bone Reaction to All-Soft Suture Anchors Used for Shoulder Labral Repairs.

BACKGROUND: All-soft suture anchors (ASSAs) are commonly used for shoulder labral repair and capsulorrhaphy in patients with shoulder instability. While these anchors may have some specific advantages over other types of suture anchors, little is known about the prevalence and time-dependence of bone cyst formation and tunnel expansion after implantation of ASSAs. The aim of this study was to quantify the proportions of cyst formation and tunnel expansion around ASSAs and to characterize and test for differences in abnormalities observed at different postoperative time points. METHODS: Thirty patients who were treated with arthroscopic shoulder stabilization surgery with ASSAs (1.4 mm; JuggerKnot, Biomet) underwent a computed tomography (CT) scan of the operatively treated shoulder at 1 month (10 patients), 6 months (10 patients), or 12 months (10 patients) postoperatively. Demographic and operative data were collected, and CT scans were evaluated for cyst formation, tunnel expansion, and tunnel volume measured in cubic millimeters. Statistical analyses were performed to detect differences in these outcomes among the follow-up groups. All shoulders were stable at all time points of the study, and there were no incidents of recurrent instability during the study period. RESULTS: Ninety-one suture anchors were evaluated in 30 patients. Tunnel expansion was identified in the large majority of patients in the 6-month and 12-month follow-up groups, with a significant increase in these proportions compared with the 1-month follow-up group (p = 0.002). Mean tunnel volumes also significantly increased over the study period (p < 0.001). The presence of cyst formation was negligible in all 3 follow-up cohorts. CONCLUSIONS: This study demonstrated low rates of cyst formation but a significantly increased tunnel volume 6 and 12 months after shoulder labral surgery with ASSAs. There was no association with the initial tunnel location. Additional well-controlled studies with longer follow-up are needed to identify potential associations among tunnel expansion, intraoperative technique, and clinical outcomes. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Branched-chain fatty acids, increased in tears of blepharitis patients, are not toxic for conjunctival cells.

AIM: The composition of the meibum of blepharitis patients is characterised by increased levels of branched-chain fatty acids (BCFAs) that return to normal values in patients treated with cyclins and lid hygiene. The aim of this study was to determine if BCFAs had toxic effects on conjunctival cells related to the disease. METHODS: Chang and IOBA-NHC conjunctival human cells were treated with BCFAs (isoC16 and isoC20) or palmitic acid as a control for 4 h or 24 h at 50 microM or 100 microM. Morphological and functional changes were investigated by measuring mitochondrial dehydrogenase activity, cell permeability, mitochondrial depolarisation, chromatin condensation, IL-1beta and reactive oxygen species production. RESULTS: None of the fatty acids modified the parameters of cytotoxicity in conjunctival cells in Chang or IOBA-NHC cell lines. Only the mitochondrial dehydrogenase activity was significantly decreased in relation to the isoC20 concentration increase. CONCLUSIONS: The increase in BCFAs in the tears of blepharitis patients does not consistently participate in the conjunctival cell changes throughout the course of the disease. Instead, it is likely an adaptive response of the ocular surface to the lack of tears, possibly increasing meibum fluidity, thus enhancing lacrimal film stability.

Molecular analyses of the LRRK2 gene in European and North African autosomal dominant Parkinson's disease.

BACKGROUND: Mutations in the leucine-rich-repeat kinase 2 (LRRK2) gene have been identified in families with autosomal dominant Parkinson's disease (ADPD), the most common of which is the p.G2019S substitution that has been found at varying frequencies worldwide. Because of the size of the LRRK2 gene, few studies have analysed the entire gene in large series of ADPD families. METHODS: We performed extensive mutation analyses of all 51 coding exons of the LRRK2 gene in index cases from 226 Parkinson's disease families compatible with autosomal dominant inheritance, mostly from France (n = 182) and North Africa (n = 14). RESULTS: We found 79 sequence variants, 29 of which were novel. Eight potentially or proven pathogenic mutations were found in 22 probands (9.7%). There were four novel amino acid substitutions that are potentially pathogenic (p.S52F, p.N363S, p.I810V, p.R1325Q) and two novel variants, p.H1216R and p.T1410M, that are probably not causative. The common p.G2019S mutation was identified in 13 probands (5.8%) including six from North Africa (43%). The known heterozygous p.R1441H and p.I1371V mutations were found in two probands each, and the p.E334K variant was identified in one single patient. Most potentially or proven pathogenic mutations were located in the functional domains of the Lrrk2 protein. CONCLUSION: This study leads us to conclude that LRRK2 mutations are a common cause of autosomal dominant Parkinson's disease in Europe and North Africa.

Incorporation and metabolism of dietary trans isomers of linolenic acid alter the fatty acid profile of rat tissues.

To study the influence on lipid metabolism and platelet aggregation of the fatty acid isomerization that occurs during heat treatment, weanling rats were fed for 8 wk a diet enriched with 5% isomerized (experimental group) or normal (control group) canola oil. Geometrical isomers of alpha-linolenic acid representing 0.2 g/100 g of the experimental diet were incorporated into liver, platelets, aorta and heart, at the expense of their cis homologue and of 18:2(n-6). The major isomer, 9c,12c,15t-18:3, was also metabolized to 5c,8c,11c,14c,17t-20:5 and to an unknown compound, found in liver, platelets and aorta, which has been identified tentatively as 7c, 10c,13c,16c,19t-22:5. The greater 20:4(n-6)/18:2(n-6) ratio in the liver, platelets and heart of the experimental group than the control group indicated an enhancement of desaturation activities. This induced a higher content of long-chain (n-6) fatty acids in the experimental group. Platelet aggregation tended to be slightly higher (P: = 0.065) in the experimental group. We conclude that 0.2 g of trans isomers of alpha-linolenic acid per 100 g of diet was sufficient to be incorporated and metabolized, thus altering the fatty acid profile of rat tissues.

Incorporation and metabolism of trans 20:5 in endothelial cells. Effect on prostacyclin synthesis.

To study the ability of long-chain trans fatty acids (FA) to be incorporated and metabolized into endothelial cells, bovine aortic endothelial cells were incubated with medium enriched eicosapentaenoic acid (EPA) bound to albumin (M2) or one of its geometrical isomers: 20:5 5c,8c,11t,14c,17c (M3), 20:5 5c,8c,11c,14c,17t (M4), or 20:5 5c,8c,11t,14c,17t (M5). After 48 h of incubation, supernatant and cells were harvested and their lipids were analyzed, including prostacyclin synthesis. EPA and 22:5n-3 of endothelial cells incubated with M2 were, respectively, three and two times higher than in control cells (incubated in M1, without any fatty acid added), whereas 22:6n-3 increased only in the supernatant, suggesting its release after biosynthesis. However, 18:2n-6 and 22:4n-6 decreased (about 30%). Trans 20:5 isomers represented 4.7, 3.9, and 5.2% of total phospholipid FA in endothelial cells incubated with M3, M4, and M5, respectively. They were elongated into trans 22:5 and trans 24:5, as revealed by gas chromatography-mass spectrometry and gas chromatography-Fourier transform infrared analysis. In cells incubated with M2, M3, M4, and M5, prostacyclin synthesis was inhibited by 49.0, 62.5, 60.5, and 72.0%, respectively. This effect may be due to less available arachidonic acid in the cells and to a competition between EPA isomers and AA at the level of cyclooxygenase pathway, as it was demonstrated that 20:5 delta17t was metabolized by this enzyme.