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

Imaging and micro-invasive analyses of black stains on the passepartout of Codex Atlanticus Folio 843 by Leonardo da Vinci

: This paper accounts for the diagnostic campaign aimed at understanding the phenomenon of black stains appeared on the passepartout close to the margins of Folio 843 of Leonardo da Vinci's Codex Atlanticus. Previous studies excluded microbiological deterioration processes. The study is based on a multi-analytical approach, including non-invasive imaging measurements of the folio, micro-imaging and synchrotron spectroscopy investigations of passepartout fragments at different magnifications and spectral ranges. Photoluminescence hyperspectral and lifetime imaging highlighted that black stains are not composed of fluorescent materials. μATR-FTIR imaging of fragments from the passepartout revealed the presence of a mixture of starch and PVAc glues localized only in the stained areas close to the margin of the folio. FE-SEM observations showed that the dark stains are localized inside cavities formed among cellulose fibers, where an accumulation of inorganic roundish particles (∅100-200&nbsp;nm in diameter size), composed of Hg and S, was detected. Finally, by employing synchrotron μXRF, μXANES and HR-XRD analyses it was possible to identify these particles as metacinnabar (β-HgS). Further research is needed to assess the chemical process leading to the metacinnabar formation in the controlled conservation condition of Leonardo's Codex

Accelerating functional gene discovery in osteoarthritis.

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease

Accelerating functional gene discovery in osteoarthritis.

Osteoarthritis causes debilitating pain and disability, resulting in a considerable socioeconomic burden, yet no drugs are available that prevent disease onset or progression. Here, we develop, validate and use rapid-throughput imaging techniques to identify abnormal joint phenotypes in randomly selected mutant mice generated by the International Knockout Mouse Consortium. We identify 14 genes with functional involvement in osteoarthritis pathogenesis, including the homeobox gene Pitx1, and functionally characterize 6 candidate human osteoarthritis genes in mouse models. We demonstrate sensitivity of the methods by identifying age-related degenerative joint damage in wild-type mice. Finally, we phenotype previously generated mutant mice with an osteoarthritis-associated polymorphism in the Dio2 gene by CRISPR/Cas9 genome editing and demonstrate a protective role in disease onset with public health implications. We hope this expanding resource of mutant mice will accelerate functional gene discovery in osteoarthritis and offer drug discovery opportunities for this common, incapacitating chronic disease

Twelve Variants Polygenic Score for Low-Density Lipoprotein Cholesterol Distribution in a Large Cohort of Patients With Clinically Diagnosed Familial Hypercholesterolemia With or Without Causative Mutations

: Background A significant proportion of individuals clinically diagnosed with familial hypercholesterolemia (FH), but without any disease-causing mutation, are likely to have polygenic hypercholesterolemia. We evaluated the distribution of a polygenic risk score, consisting of 12 low-density lipoprotein cholesterol (LDL-C)-raising variants (polygenic LDL-C risk score), in subjects with a clinical diagnosis of FH. Methods and Results Within the Lipid Transport Disorders Italian Genetic Network (LIPIGEN) study, 875 patients who were FH-mutation positive (women, 54.75%; mean age, 42.47±15.00 years) and 644 patients who were FH-mutation negative (women, 54.21%; mean age, 49.73±13.54 years) were evaluated. Patients who were FH-mutation negative had lower mean levels of pretreatment LDL-C than patients who were FH-mutation positive (217.14±55.49 versus 270.52±68.59 mg/dL, P<0.0001). The mean value (±SD) of the polygenic LDL-C risk score was 1.00 (±0.18) in patients who were FH-mutation negative and 0.94 (±0.20) in patients who were FH-mutation positive (P<0.0001). In the receiver operating characteristic analysis, the area under the curve for recognizing subjects characterized by polygenic hypercholesterolemia was 0.59 (95% CI, 0.56-0.62), with sensitivity and specificity being 78% and 36%, respectively, at 0.905 as a cutoff value. Higher mean polygenic LDL-C risk score levels were observed among patients who were FH-mutation negative having pretreatment LDL-C levels in the range of 150 to 350 mg/dL (150-249 mg/dL: 1.01 versus 0.91, P<0.0001; 250-349 mg/dL: 1.02 versus 0.95, P=0.0001). A positive correlation between polygenic LDL-C risk score and pretreatment LDL-C levels was observed among patients with FH independently of the presence of causative mutations. Conclusions This analysis confirms the role of polymorphisms in modulating LDL-C levels, even in patients with genetically confirmed FH. More data are needed to support the use of the polygenic score in routine clinical practice

Osteocyte transcriptome mapping identifies a molecular landscape controlling skeletal homeostasis and susceptibility to skeletal disease.

Osteocytes are master regulators of the skeleton. We mapped the transcriptome of osteocytes from different skeletal sites, across age and sexes in mice to reveal genes and molecular programs that control this complex cellular-network. We define an osteocyte transcriptome signature of 1239 genes that distinguishes osteocytes from other cells. 77% have no previously known role in the skeleton and are enriched for genes regulating neuronal network formation, suggesting this programme is important in osteocyte communication. We evaluated 19 skeletal parameters in 733 knockout mouse lines and reveal 26 osteocyte transcriptome signature genes that control bone structure and function. We showed osteocyte transcriptome signature genes are enriched for human orthologs that cause monogenic skeletal disorders (P = 2.4 × 10-22) and are associated with the polygenic diseases osteoporosis (P = 1.8 × 10-13) and osteoarthritis (P = 1.6 × 10-7). Thus, we reveal the molecular landscape that regulates osteocyte network formation and function and establish the importance of osteocytes in human skeletal disease

Spectrum of mutations in Italian patients with familial hypercholesterolemia: New results from the LIPIGEN study

Background Familial hypercholesterolemia (FH) is an autosomal dominant disease characterized by elevated plasma levels of LDL-cholesterol that confers an increased risk of premature atherosclerotic cardiovascular disease. Early identification and treatment of FH patients can improve prognosis and reduce the burden of cardiovascular mortality. Aim of this study was to perform the mutational analysis of FH patients identified through a collaboration of 20 Lipid Clinics in Italy (LIPIGEN Study). Methods We recruited 1592 individuals with a clinical diagnosis of definite or probable FH according to the Dutch Lipid Clinic Network criteria. We performed a parallel sequencing of the major candidate genes for monogenic hypercholesterolemia (LDLR, APOB, PCSK9, APOE, LDLRAP1, STAP1). Results A total of 213 variants were detected in 1076 subjects. About 90% of them had a pathogenic or likely pathogenic variants. More than 94% of patients carried pathogenic variants in LDLR gene, 27 of which were novel. Pathogenic variants in APOB and PCSK9 were exceedingly rare. We found 4 true homozygotes and 5 putative compound heterozygotes for pathogenic variants in LDLR gene, as well as 5 double heterozygotes for LDLR/APOB pathogenic variants. Two patients were homozygous for pathogenic variants in LDLRAP1 gene resulting in autosomal recessive hypercholesterolemia. One patient was found to be heterozygous for the ApoE variant p.(Leu167del), known to confer an FH phenotype. Conclusions This study shows the molecular characteristics of the FH patients identified in Italy over the last two years. Full phenotypic characterization of these patients and cascade screening of family members is now in progress

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease

Influence of Antisynthetase Antibodies Specificities on Antisynthetase Syndrome Clinical Spectrum TimeCourse

Introduction: Increased cardiovascular (CV) morbidity and mortality is observed in inflammatory joint diseases (IJDs) such as rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the management of CV disease in these conditions is far from being well established.Areas covered: This review summarizes the main epidemiologic, pathophysiological, and clinical risk factors of CV disease associated with IJDs. Less common aspects on early diagnosis and risk stratification of the CV disease in these conditions are also discussed. In Europe, the most commonly used risk algorithm in patients with IJDs is the modified SCORE index based on the revised recommendations proposed by the EULAR task force in 2017.Expert opinion: Early identification of IJD patients at high risk of CV disease is essential. It should include the use of complementary noninvasive imaging techniques. A multidisciplinary approach aimed to improve heart-healthy habits, including strict control of classic CV risk factors is crucial. Adequate management of the underlying IJD is also of main importance since the reduction of disease activity decreases the risk of CV events. Non-steroidal anti-inflammatory drugs may have a lesser harmful effect in IJD than in the general population, due to their anti-inflammatory effects along with other potential beneficial effects.This research was partially funded by FOREUM—Foundation for Research in Rheumatolog