Myofiber stress-response in myositis: parallel investigations on patients and experimental animal models of muscle regeneration and systemic inflammation

Introduction: The endoplasmic reticulum (ER) stress-response, evoked in mice by the overexpression of class I major histocompatibility complex antigen (MHC-I), was proposed as a major mechanism responsible for skeletal muscle damage and dysfunction in autoimmune myositis. The present study was undertaken to characterize in more detail the ER stress-response occurring in myofibers of patients with inflammatory myopathies, focusing on the expression and distribution of Grp94, calreticulin and Grp75, three ER chaperones involved in immunomodulation. Methods: Muscle biopsies were obtained from seven healthy subjects and 29 myositis patients, who were subdivided into groups based on the morphological evidence of inflammation and/or sarcolemmal immunoreactivity for MHC-I. Biopsies were analyzed by means of immunohistochemistry and western blot using anti-Grp94, anti-calreticulin and anti-Grp75 specific antibodies. Parallel analyses on these ER chaperones were conducted in rabbit and/or murine skeletal muscle after experimental induction of regeneration or systemic inflammation. Results: Upregulation of Grp94 characterized regenerating myofibers of myositis patients (P = 0.03, compared with values detected in biopsies without signs of muscle regeneration) and developing and regenerating myofibers of mouse muscles. Conversely, levels of calreticulin and Grp75 increased about fourfold and twofold, respectively, in patient biopsies positive for sarcolemmal MHC-I immunoreactivity, compared with healthy subjects and patients negative for both inflammation and MHC-I labeling (P < 0.005). Differently from calreticulin, the Grp75 level increased significantly also in patient biopsies that displayed occasional sarcolemmal MHC-I immunoreactivity (P = 0.002), suggesting the interference of other mechanisms. Experimental systemic inflammation achieved in mice and rabbits by a single injection of bacterial lipopolysaccharide significantly increased Grp75 and calreticulin but not MHC-I expression in muscles. Conclusions: These results indicate that, in myositis patients, muscle regeneration and inflammation, in addition to MHC-I upregulation, do evoke an ER stress-response characterized by the increased expression of Grp94 and Grp75, respectively. The increase in the muscle Grp75 level in patients showing occasional immunoreactivity for sarcolemmal MHC-I might be considered further as a broader indicator of idiopathic inflammatory myopathy

The role of autophagy in vernal kerato-conjunctivitis

Autophagy is involved in many biological aspects, including cell survival and death, innate and adaptive immunity and cancer. An involvement of autophagy is also reported in some inflammatory diseases such as asthma. In the present study we explored the role of autophagy in vernal keratoconjunctivitis (VKC), a severe inflammatory disease mainly found in children and adolescents. Autophagy and apoptosis markers (LC3A, LC3B, Beclin-1, cathepsin B, BCL-2, BAX, caspase 3) expression in conjunctival biopsies from 9 active VKC patients and 9 healthy age matched normal subjects were analyzed using immunohistochemistry and qPCR techniques. Conjunctival cells cultures were treated with inflammatory stimuli (IL-1b, histamine, IL-4, TNFa) and analysed by western blotting for autophagy markers expression. LC3B, Catepsin D and B and Beclin-1expression strongly increased in the stroma of VKC whereas the epithelium was consistently negative for all of the molecules studied but positive for Beclin-1 in VKC. qPCR analysis demonstrated a similar mRNAs expression in VKC and normal subjects. In “in vitro” experiments autophagy induction revealed that only LC3B expression was changed in conjunctival fibroblasts by inflammatory stimuli. In particular, both LC3BI, the LC3B free form, and LC3BII, the phosphatidyl-ethanolamine-conjugated form, involved in the autophagosome formation, were decreased in fibroblast cultures at 24h after TNFα stimulation. However, since LC3B-II is normally degraded by lysosomes and the total amount of LC3B-II depends on the balance between its formation and degradation, we analyzed the expression of LC3B-II in the presence and absence of chloroquine, an inhibitor of lysosomal degradation. We found a significant increased amount of LC3BII compared to the control, indicating an over-expression of this protein in stimulated fibroblasts that is quickly damped by its degradation. Since one of the key steps in autophagy is the conversion of LC3B from LC3B-I to LC3B-II, our results suggest that autophagy may be involved in the pathogenesis of VKC

Electronic structure and vertical transport in random dimer GaAs-Al_xGa_(1-x)As superlattices

We report a systematic study of several GaAs-AlxGa1-xAs semiconductor superlattices grown by molecular-beam epitaxy specifically designed to explore the existence of extended states in random dimer superlattices. We have confirmed our previous results [V. Bellani et al., Phys. Rev. Lett. 82, 2159 (1999)] with much additional evidence that allows us to lay claim to a clear-cut experimental verification of the presence of extended states in random dimer superlattices due to the short-range correlations (dimers) that inhibit the localization effects of the disorder

An electronic patient-reported outcome mobile app for data collection in type a hemophilia:Design and usability study

BACKGROUND: There is currently limited evidence on the level and intensity of physical activity in individuals with hemophilia A. Mobile technologies can offer a rigorous and reliable alternative to support data collection processes but they are often associated with poor user retention. The lack of longitudinal continuity in their use can be partly attributed to the insufficient consideration of stakeholder inputs in the development process of mobile apps. Several user-centered models have been proposed to guarantee that a thorough knowledge of the end user needs is considered in the development process of mobile apps. OBJECTIVE: The aim of this study is to design and validate an electronic patient-reported outcome mobile app that requires sustained active input by individuals during POWER, an observational study that aims at evaluating the relationship between physical activity levels and bleeding in patients with hemophilia A. METHODS: We adopted a user-centered design and engaged several stakeholders in the development and usability testing of this mobile app. During the concept generation and ideation phase, we organized a need-assessment focus group (FG) with patient representatives to elicit specific design requirements for the end users. We then conducted 2 exploratory FGs to seek additional inputs for the app’s improvement and 2 confirmatory FGs to validate the app and test its usability in the field through the mobile health app usability questionnaire. RESULTS: The findings from the thematic analysis of the need-assessment FG revealed that there was a demand for sense making, for simplification of app functionalities, for maximizing integration, and for minimizing the feeling of external control. Participants involved in the later stages of the design refinement contributed to improving the design further by upgrading the app’s layout and making the experience with the app more efficient through functions such as chatbots and visual feedback on the number of hours a wearable device had been worn, to ensure that the observed data were actually registered. The end users rated the app highly during the quantitative assessment, with an average mobile health app usability questionnaire score of 5.32 (SD 0.66; range 4.44-6.23) and 6.20 (SD 0.43; range 5.72-6.88) out of 7 in the 2 iterative usability testing cycles. CONCLUSIONS: The results of the usability test indicated a high, growing satisfaction with the electronic patient-reported outcome app. The adoption of a thorough user-centered design process using several types of FGs helped maximize the likelihood of sustained retention of the app’s users and made it fit for data collection of relevant outcomes in the observational POWER study. The continuous use of the app and the actual level of engagement will be evaluated during the ongoing trial. TRIAL REGISTRATION: ClinicalTrials.gov NCT04165135; https://clinicaltrials.gov/ct2/show/NCT0416513

Possible Autophagy induction in Vernal Keratoconjunctivitis via Tumor Necrosis Factor Alpha Stimulation

Tumor necrosis factor alpha (TNFα) is one of the main mediators of inflammatory response in many pathological diseases, involved in a widespread biological functions, including autophagy. Previous data obtained in our laboratory demonstrated that TNFα and some autophagy markers (which markers please indicate) are overexpressed in a severe inflammatory disease such as vernal keratoconjunctivitis (VKC). In the present study we explored the role of TNFα in the induction of autophagy in VKC, using an in vitro model. Primary conjunctival cell cultures were treated with TNFa and analysed by qPCR and western blotting for expression of some autophagy and lysosomial markers at 4, 10 and 24 hours after exposure. qPCR results demonstrated that LC3B, Beclin-1, LAMP1 and p62 strongly increased from 4 to 24 hours, whereas the expression of Catepsin D, a protein implicated in lysosomial apototic pathway, was comparable to that of untreated control. Western blotting analysis revealed lipidation of LC3B quantified as an increased LC3BII/LC3BI ratio. Moreover, double immunofluorescence for Cathepsin D and LAMP1 showed that Cathepsin D was localized within the lysosomes at 4, 10, 24 hours after cell exposure to inflammatory stimuli. In conclusion, our data demonstrated that TNFα significantly induce in VKC LC3B lipidation, LC3BII/LC3BI ratio and p62 (qPCR) in the cells exposed to inflammatory stimuli which shows possible activation of autophagy pathway

The role of heat shock proteins in the inflammatory state of vernal keratoconjunctivitis

The aim of the study was to analyse the role of heat shock proteins (HSPs) in vernal keratoconjunctivitis (VKC), a recurrent allergic ocular inflammatory disease. We evaluated the expression of some HSPs (Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90) in the mucosal biopsies of VKC patients by immunohistochemistry, and in conjunctival cells cultures treated with inflammatory stimuli (IL-1β, histamine, IL-4, TNFβ, UVB irradiation) by western blotting. Immunohistochemical analysis revealed that Hsp10, Hsp27, Hsp40, Hsp70 and Hsp90 expression was significantly increased in VKC whereas the Hsp60 level was unaltered. In vitro induction by inflammatory stimuli in Chang epithelial conjunctival cells revealed that Hsp70 protein expression was significantly increased in epithelial cells line after 4-10 h from histamine and IL-4 stimulation. The same molecule was also overexpressed in conjunctival fibroblast cultures after TNFβ treatment. Hsp90 protein level was increased in the same cell cultures by IL-1β at 4-10-24 h. The Hsp40 protein expression was increased both in epithelial and fibroblast cultures induced by all inflammatory stimuli. Moreover, UVB irradiation significantly increased Hsp90 expression in primary fibroblast culture and Hsp27 in conjunctival epithelial cells after 10 hours. These results indicate that HSPs levels increase in VKC. In particular, Hsp40 expression is up-regulated by all the typical inflammatory stimuli involved in VKC pathogenesis. The specific role of each one of these chaperonins to further induce or counteract inflammation need to be further investigated

Neuroactive Steroids in First-Episode Psychosis: A Role for Progesterone?

Neuroactive steroids may play a role in the pathophysiology of psychotic disorders, but few studies examined this issue. We compared serumlevels of cortisol, testosterone, dehydroepiandrosterone, and progesterone between a representative sample of firstepisode psychosis (FEP) patients and age- and gender-matched healthy subjects. Furthermore, we analyzed the associations between neuroactive steroids levels and the severity of psychotic symptom dimensions.Male patients had lower levels of progesterone than controls

Heat shock proteins levels and expression in chronic obstructive pulmonary disease and vernal keratoconjunctivitis

Inflammatory response in different organs share many similarities, but site-specific signs. Symptoms can be related to mucosal structure changes. The aim of the study was to compare heat shock proteins (HSPs) levels and expression in chronic obstructive pulmonary disease (COPD) to other inflammatory status of mucosa, such as vernal keratoconjunctivitis (VKC), a recurrent ocular inflammatory disease in which autoimune aggression may have a pathogenetic role. We examined bronchial mucosal biopsies from COPD patients (moderate to severe stage) and conjunctival biopsies from VKC patients; age-matched controls were selected for each group. We evaluated levels (by immunohistochemistry) and expression (by RT-PCR) of a panel of HSPs, among which Hsp10, Hsp27, Hsp40, Hsp60, Hsp70, Hsp90, and of the main heat shock transcription factor (both HSF-1 and pHSF-1). Hsp10 levels and expression increased in all pathological conditions, Hsp27 in VKC, Hsp40 in COPD and VKC, Hsp60 in COPD, Hsp70 and Hsp90 in VKC, as compared to their appropriate controls. Transcription factor pHSF-1 positive cells were significantly increased in COPD compared to controls, while was unaltered in VKC. Moreover, all pathological tissues showed increased levels of macrophages (CD68 positive) in lamina propria, COPD showed increased levels of neutrophils (elastase positive) and VKC increased levels of eosinophils (EG2 positive). Finally, Hsp60 colocalize with elastase positive cells in COPD. These results indicate that HSPs levels and expression change during development of different types of inflammation. Further studies will prove their active involvement and functions in triggering and/or maintaining the inflammatory status

First-episode psychosis and migration in Italy (PEP-Ita migration): a study in the Italian mental health services

BACKGROUND: It has been frequently reported a higher incidence of psychotic disorders in immigrants than in native populations. There is, however, a lack of knowledge about risk factors which may explain this phenomenon. A better understanding of the causes of psychosis among first-generation migrants is highly needed, particularly in Italy, a country with a recent massive migration. METHODS/DESIGN: The "Italian study on first-episode psychosis and migration (PEP-Ita)" is a prospective observational study over a two-year period (1 January 2012-31 December 2013) which will be carried out in 11 Italian mental health centres. All participating centres will collect data about all new cases of migrants with first-episode psychosis. The general purpose ("core") of the PEP-Ita study is to explore the socio-demographic and clinical characteristics, and the pathways to care of a population of first-episode psychosis migrants in Italy. Secondary aims of the study will be: 1) to understand risk and protective factors for the development of psychotic disorders in migrants; 2) to evaluate the correlations between psychopathology of psychotic disorders in migrants and socio-demographic characteristics, migration history, life experiences; 3) to evaluate the clinical and social outcomes of first-episode psychoses in migrants. DISCUSSION: The results of the PEP-Ita study will allow a better understanding of risk factors for psychosis in first-generation migrants in Italy. Moreover, our results will contribute to the development of prevention programmes for psychosis and to the improvement of early intervention treatments for the migrant population in Italy