RESUMEN
Objective: This study aims to characterize differential expressed pathways (DEP) in subjects with preclinical systemic sclerosis (PreSSc) characterized uniquely by Raynaud phenomenon, specific autoantibodies, and/or capillaroscopy positive for scleroderma pattern. Methods: Whole-blood samples from 33 PreSSc with clinical prospective data (baseline and after 4 years of follow-up) and 16 matched healthy controls (HC) were analyzed for global gene expression transcriptome analysis via RNA sequencing. Functional Analysis of Individual Microarray Expression method annotated Reactome individualized pathways. ANOVA analysis identified DEP whose predictive capability were tested in logistic regression models after extensive internal validation. Results: At 4 years, 42.4% subjects progressed (evolving PreSSc), while the others kept stable PreSSc clinical features (stable PreSSc). At baseline, out of 831 pathways, 541 DEP were significant at a false discovery rate <0.05, differentiating PreSSc versus HC with an AUROC = 0.792 ± 0.242 in regression models. Four clinical groups were identified via unsupervised clustering (HC, HC and PreSSc with HC-like features, PreSSc and HC with PreSSc-like features, and PreSSc). Biological signatures changed with disease progression while remaining unchanged in stable subjects. The magnitude of change was related to the baseline cluster, yet no DEP at baseline was predictive of progression. Disease progression was mostly related to changes in signal transduction pathways especially linked to calcium-related events and inositol 1,4,5-triphosphate metabolism. Conclusion: PreSSc had distinguished Reactome pathway signatures compared to HC. Progression to definite SSc was characterized by a shift in biological fingertips. Calcium-related events promoting endothelial damage and vasculopathy may be relevant to disease progression.
Asunto(s)
Esclerodermia Sistémica , Transcriptoma , Humanos , Estudios Prospectivos , Calcio , Progresión de la EnfermedadRESUMEN
OBJECTIVE: Systemic sclerosis (SSc) is an autoimmune disease with health-related quality of life (HRQoL) high impairment. Pain is of paramount importance to be targeted by therapeutical approaches. Our study aim was to perform an add-on device-based non-invasive neuromodulatory treatment through transcutaneous auricular vagal nerve stimulation (tVNS) in patients with SSc, assessing its effects on pain as primary endpoint and on inflammation, cardiovascular autonomic control and HRQoL. METHODS: Thirty-two patients with SSc were enrolled based on reported pain assessed through Numeric Rating Scale (NRS). Twenty-one (90% with limited cutaneous SSc) completed a randomised, cross-over, patient-blind trial, in which interventional and active control were used in random order for 4 weeks, interspersed with 4 weeks washout. NRS, Patient-Reported Outcomes Measurement Information System-29 (PROMIS-29) Item4 for pain interference, heart rate variability (HRV), serum cytokines and HRQoL questionnaires (Health Assessment Questionnaire, Patient Health Questionnaire-9, University of California, Los Angeles Gastrointestinal Tract, Pittsburgh Sleep Quality Index) were assessed at baseline, at T1 (after 1 month of tVNS or active control), at T2 (after washout) and at T3 (after 1 month of active control or tVNS). T-test for paired data and Wilcoxon signed-rank test for non-normally distributed parameters were performed to compare the effect of tVNS and active control. RESULTS: NRS pain was significantly reduced by tVNS and not by active control (Mean±SD: -27.7%±21.3% vs -7.7%±26.3%, p=0.002). Interleukin-6 was downregulated in tVNS versus active control (p=0.029). No significant differences were observed in tVNS versus active control for PROMIS-29 Item4, QoL scales and HRV with both spectral and symbolic analyses. CONCLUSION: tVNS demonstrated to be a safe and non-invasive add-on tool to reduce pain in SSc.
Asunto(s)
Esclerodermia Sistémica , Estimulación Eléctrica Transcutánea del Nervio , Estimulación del Nervio Vago , Humanos , Manejo del Dolor , Calidad de Vida , Dolor/etiología , Esclerodermia Sistémica/complicaciones , Esclerodermia Sistémica/terapiaRESUMEN
Calcification starts with hydroxyapatite (HA) crystallization on cell membranous components, as with aortic valve interstitial cells (AVICs), wherein a cell-membrane-derived substance containing acidic phospholipids (PPM/PPLs) acts as major crystal nucleator. Since nucleic acid removal is recommended to prevent calcification in valve biosubstitutes derived from decellularized valve scaffolds, the involvement of ribosomal RNA (rRNA) and nuclear chromatin (NC) was here explored in three distinct contexts: (i) bovine AVIC pro-calcific cultures; (ii) porcine aortic valve leaflets that had undergone accelerated calcification after xenogeneic subdermal implantation; and (iii) human aortic valve leaflets affected by calcific stenosis. Ultrastructurally, shared AVIC degenerative patterns included (i) the melting of ribosomes with PPM/PPLs, and the same for apparently well-featured NC; (ii) selective precipitation of silver particles on all three components after adapted von Kossa reactions; and (iii) labelling by anti-rRNA immunogold particles. Shared features were also provided by parallel light microscopy. In conclusion, the present results indicate that rRNA and NC contribute to AVIC mineralization in vitro and in vivo, with their anionic charges enhancing the HA nucleation capacity exerted by PPM/PPL substrates, supporting the concept that nucleic acid removal is needed for valve pre-implantation treatments, besides better elucidating the modality of pro-calcific cell death.
Asunto(s)
Estenosis de la Válvula Aórtica , Válvula Aórtica , Humanos , Animales , Bovinos , Porcinos , Válvula Aórtica/metabolismo , Durapatita/metabolismo , ARN Ribosómico/metabolismo , Estenosis de la Válvula Aórtica/metabolismo , Modelos Animales , Cromatina/metabolismo , Células CultivadasRESUMEN
BACKGROUND: The study of molecular mechanisms characterizing disease progression may be relevant to get insights into systemic sclerosis (SSc) pathogenesis and to intercept patients at very early stage. We aimed at investigating the proteomic profile of preclinical systemic sclerosis (PreSSc) via a discovery/validation two-step approach. METHODS: SOMAcan aptamer-based analysis was performed on a serum sample of 13 PreSSc (discovery cohort) according to 2001 LeRoy and Medsger criteria (characterized solely by Raynaud phenomenon plus a positive nailfold capillaroscopy and SSc-specific antibodies without any other sign of definite disease) and 8 healthy controls (HCs) age, gender, and ethnicity matched. Prospective data were available up to 4±0.6 years to determine the progression to definite SSc according to the EULAR/ACR 2013 classification criteria. In proteins with relative fluorescence units (RFU) > |1.5|-fold vs HCs values, univariate analysis was conducted via bootstrap aggregating models to determine the predicting accuracy (progression vs non-progression) of categorized baseline protein values. Gene Ontologies (GO terms) and Reactome terms of significant proteins at the adjusted 0.05 threshold were explored. Significant proteins from the discovery cohort were finally validated via ELISAs in an independent validation cohort of 50 PreSSc with clinical prospective data up to 5 years. Time-to-event analysis for interval-censored data was used to evaluate disease progression. RESULTS: In the discovery cohort, 286 out of 1306 proteins analyzed via SomaScan, were differentially expressed versus HCs. Ten proteins were significantly associated with disease progression; analysis through GO and Reactome showed differentially enriched pathways involving angiogenesis, endothelial cell chemotaxis, and endothelial cell chemotaxis to fibroblast growth factor (FGF). In the validation cohort, endostatin (HR=10.23, CI95=2.2-47.59, p=0.003) was strongly associated with disease progression, as well as bFGF (HR=0.84, CI95=0.709-0.996, p=0.045) and PAF-AHß (HR=0.372, CI95=0.171-0.809, p=0.013) CONCLUSIONS: A distinct protein profile characterized PreSSc from HCs and proteins associated with hypoxia, vasculopathy, and fibrosis regulation are linked with the progression from preclinical to definite SSc. These proteins, in particular endostatin, can be regarded both as markers of severity and molecules with pathogenetic significance as well as therapeutic targets.
Asunto(s)
Proteómica , Esclerodermia Sistémica , Humanos , Biomarcadores , Progresión de la Enfermedad , Endostatinas , Angioscopía Microscópica , Estudios Prospectivos , Esclerodermia Sistémica/sangre , Esclerodermia Sistémica/genética , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patologíaRESUMEN
OBJECTIVE: To investigate peripheral blood cell (PBCs) global gene expression profile of SSc at its preclinical stage (PreSSc) and to characterize the molecular changes associated with progression to a definite disease over time. MATERIAL AND METHODS: Clinical data and PBCs of 33 participants with PreSSc and 16 healthy controls (HCs) were collected at baseline and follow-up (mean 4.2 years). Global gene expression profiling was conducted by RNA sequencing and a modular analysis was performed. RESULTS: Comparison of baseline PreSSc to HCs revealed 2889 differentially expressed genes. Interferon signalling was the only activated pathway among top over-represented pathways. Moreover, 10 modules were significantly decreased in PreSSc samples (related to lymphoid lineage, cytotoxic/NK cell, and erythropoiesis) in comparison to HCs. At follow-up, 14 subjects (42.4%) presented signs of progression (evolving PreSSc) and 19 remained in stable preclinical stage (stable PreSSc). Progression was not associated with baseline clinical features or baseline PBC transcript modules. At follow-up stable PreSSc normalized their down-regulated cytotoxic/NK cell and protein synthesis modules while evolving PreSSc kept a down-regulation of cytotoxic/NK cell and protein synthesis modules. Transcript level changes of follow-up vs baseline in stable PreSSc vs evolving PreSSc showed 549 differentially expressed transcripts (336 up and 213 down) with upregulation of the EIF2 Signalling pathway. CONCLUSIONS: Participants with PreSSc had a distinct gene expression profile indicating that molecular differences at a transcriptomic level are already present in the preclinical stages of SSc. Furthermore, a reduced NK signature in PBCs was related to SSc progression over time.
Asunto(s)
Esclerodermia Sistémica , Transcriptoma , Humanos , Perfilación de la Expresión Génica , Esclerodermia Sistémica/complicaciones , Regulación hacia Arriba , Progresión de la EnfermedadRESUMEN
Calcium-dependent cytosolic phospholipase A2α (cPLA2α) had been previously found to be overexpressed by aortic valve interstitial cells (AVICs) subjected to in vitro calcific induction. Here, cPLA2α expression was immunohistochemically assayed in porcine aortic valve leaflets (iAVLs) that had undergone accelerated calcification subsequent to 2- to 28-day-long implantation in rat subcutis. A time-dependent increase in cPLA2α-positive AVICs paralleled mineralization progression depending on dramatic cell membrane degeneration with the release of hydroxyapatite-nucleating acidic lipid material, as revealed by immunogold particles decorating organelle membranes in 2d-iAVLs, as well as membrane-derived lipid byproducts in 7d- to 28d-iAVLs. Additional positivity was detected for (i) pro-inflammatory IL-6, mostly exhibited by rat peri-implant cells surrounding 14d- and 28d-iAVLs; (ii) calcium-binding osteopontin, with time-dependent increase and no ossification occurrence; (iii) anti-calcific fetuin-A, mostly restricted to blood plasma within vessels irrorating the connective envelopes of 28d-iAVLs; (iv) early apoptosis marker annexin-V, limited to sporadic AVICs in all iAVLs. No positivity was found for either apoptosis executioner cleaved caspase-3 or autophagy marker MAP1. In conclusion, cPLA2α appears to be a factor characterizing AVL calcification concurrently with a distinct still uncoded cell death form also in an animal model, as well as a putative target for the prevention and treatment of calcific valve diseases.
Asunto(s)
Estenosis de la Válvula Aórtica/metabolismo , Válvula Aórtica/metabolismo , Calcio/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Osteogénesis/fisiología , Animales , Apoptosis/fisiología , Autofagia/fisiología , Calcinosis/metabolismo , Células Cultivadas , Modelos Animales de Enfermedad , Células Intersticiales de Cajal/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
The involvement of calcium-dependent cytosolic phospholipase A2α (cPLA2α) in aortic valve calcification is not exhaustively elucidated. Here, cPLA2α expression in aortic valve interstitial cell (AVIC) pro-calcific cultures simulating either metastatic or dystrophic calcification was estimated by qPCR, Western blotting, and counting of cPLA2α-immunoreactive cells, with parallel ultrastructural examination of AVIC calcific degeneration. These evaluations also involved pro-calcific AVIC cultures treated with cPLA2α inhibitor dexamethasone. cPLA2α over-expression resulted for both types of pro-calcific AVIC cultures. Compared to controls, enzyme content was found to increase by up to 300% and 186% in metastatic and dystrophic calcification-like cultures, respectively. Increases in mRNA amounts were also observed, although they were not as striking as those in enzyme content. Moreover, cPLA2α increases were time-dependent and strictly associated with mineralization progression. Conversely, drastically lower levels of enzyme content resulted for the pro-calcific AVIC cultures supplemented with dexamethasone. In particular, cPLA2α amounts were found to decrease by almost 88% and 48% in metastatic and dystrophic calcification-like cultures, respectively, with mRNA amounts showing a similar trend. Interestingly, these drastic decreases in cPLA2α amounts were paralleled by drastic decreases in mineralization degrees, as revealed ultrastructurally. In conclusion, cPLA2α may be regarded as a crucial co-factor contributing to AVIC mineralization in vitro, thus being an attractive potential target for designing novel therapeutic strategies aimed to counteract onset or progression of calcific aortic valve diseases.
Asunto(s)
Estenosis de la Válvula Aórtica/patología , Válvula Aórtica/patología , Calcinosis/patología , Calcio/metabolismo , Fosfolipasas A2 Grupo IV/metabolismo , Células Intersticiales de Cajal/patología , Animales , Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/metabolismo , Calcinosis/metabolismo , Bovinos , Células Cultivadas , Fosfolipasas A2 Grupo IV/genética , Células Intersticiales de Cajal/metabolismoRESUMEN
OBJECTIVES: TLR3 mediates skin solar injury by binding nuclear material released from apoptotic keratinocytes, resulting in the production of pro-inflammatory cytokines. Because the TLR3 gene is located in 4q35, a known systemic lupus erythematosus (SLE) susceptibility locus, we wondered whether TLR3 single nucleotide polymorphisms (SNPs) were associated with inflammatory mechanisms relevant to the development of SLE, and disease susceptibility. METHODS: Functional assays were carried out in TLR3-transfected HEK293 cells and in monocyte-derived dendritic cells (moDCs). TLR3 and IFNß immunofluorescence studies were performed in skin samples from 7 SLE patients and 3 controls. We performed a SNP association study in a discovery cohort of 153 patients and 105 controls, followed by a confirmation study in an independent cohort of 1,380 patients and 2,104 controls. RESULTS: TLR3 and IFNß are overexpressed in SLE skin lesions. TLR3 overexpression in HEK293 cells amplifies their sensitivity to a pro-apoptotic stimulus. Taking advantage of a naturally occurring polymorphic TLR3 variant (rs3775291) that weakly versus strongly responds to poly I:C stimulation, we found that TLR3 is associated with amplified apoptotic responses, production of the Ro/SSA autoantigen and increased maturation of myeloid-derived dendritic cells (moDC) after exposure to UV irradiation. However, TLR3 SNPs are not associated with susceptibility to SLE in a large population of patients and controls. CONCLUSIONS: TLR3 is overexpressed in SLE skin lesions and amplifies apoptotic and inflammatory responses to UV-irradiation in antigen-presenting cells in vitro. However, TLR3 SNPs do not impact susceptibility to the development of the disease.
Asunto(s)
Lupus Eritematoso Sistémico , Receptor Toll-Like 3 , Células Presentadoras de Antígenos , Apoptosis , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/genética , Polimorfismo de Nucleótido Simple , Receptor Toll-Like 3/genéticaRESUMEN
Ectopic calcification of native and bioprosthetic heart valves represents a major public health problem causing severe morbidity and mortality worldwide. Valve procalcific degeneration is known to be caused mainly by calcium salt precipitation onto membranes of suffering non-scavenged cells and dead-cell-derived products acting as major hydroxyapatite nucleators. Although etiopathogenesis of calcification in native valves is still far from being exhaustively elucidated, it is well known that bioprosthesis mineralization may be primed by glutaraldehyde-mediated toxicity for xenografts, cryopreservation-related damage for allografts and graft immune rejection for both. Instead, mechanical valves, which are free from calcification, are extremely thrombogenic, requiring chronic anticoagulation therapies for transplanted patients. Since surgical substitution of failed valves is still the leading therapeutic option, progressive improvements in tissue engineering techniques are crucial to attain readily available valve implants with good biocompatibility, proper functionality and long-term durability in order to meet the considerable clinical demand for valve substitutes. Bioengineered valves obtained from acellular non-valvular scaffolds or decellularized native valves are proving to be a compelling alternative to mechanical and bioprosthetic valve implants, as they appear to permit repopulation by the host's own cells with associated tissue remodelling, growth and repair, besides showing less propensity to calcification and adequate hemodynamic performances. In this review, insights into valve calcification onset as revealed by in vivo and in vitro procalcific models are updated as well as advances in the field of valve bioengineering.
RESUMEN
Vaginal mucosa represents a rare site for primary melanoma. These neoplasms more commonly occur in the postmenopausal period, usually presenting with vaginal discharge, bleeding, or palpable mass. We report a case of an 89-year-old woman presenting with vaginal bleeding and a non-pigmented lesion in the lower third of the vagina at the gynaecological examination. A PAP smear and tissue incisional biopsy were concurrently performed. The cytological sample showed a subpopulation of non-cohesive, atypical epithelioid cells suggestive for malignancy. The histological examination showed the same morphological characteristics in the neoplastic population widely underlying the vaginal epithelium, with scattered intraepithelial nests and single elements. In both samples, there was no evidence of melanin pigment within the malignant cells. Immunohistochemical analysis performed on the tissue biopsy demonstrated a strong and diffuse positivity for melanocytic markers (HMB-45, S-100, Melan-A), confirming the diagnosis of primary amelanotic vaginal melanoma.
Asunto(s)
Melanoma Amelanótico/patología , Neoplasias Vaginales/patología , Anciano de 80 o más Años , Femenino , HumanosRESUMEN
Systemic sclerosis (SSc), an autoimmune disorder, is characterized by vasculopathy, inflammation, progressive perivascular and interstitial fibrosis. Its pathogenesis is largely unknown, however strong evidences suggest that genetic predisposition may contribute to SSc development. Several gene polymorphisms involved in regulatory T cell function have been identified in many autoimmune diseases, including SSc. Moreover, dysregulation of co-stimulatory and/or co-inhibitory signals, including ICOS signalling, can lead to autoimmunity. The aim of the present study was to investigate the association of the FOXP3 rs2294020, ICOS rs6726035 and ICOSL rs378299 SNPs with both the susceptibility and the progression to SSc in an Italian case-series of patients. SNP genotyping results were successfully obtained from a total of 350 subjects including 166 individuals with SSc and 184 healthy controls. Although analysis tests did not show any significant associations between the SNPs under study and susceptibility to SSc, the occurrence of FOXP3 rs2294020 in female patients was associated with decreased time to progression from early to definite SSc (allelic model: HR=1.43; CI=1.03-1.99; p=0.03; dominant model: HR=1.54; CI=1.04-2.28; p=0.03). The inclusion of presence of ACA autoantibodies in the model did not significantly change the estimates. No conclusions can be drawn for the susceptibility to the disease or the time to progression in men due to the low statistical power. This study provides evidence of the association of rs2294020 with SSc evolution in female patients, modulating the time of progression from the diagnosis of early SSc to the diagnosis of definite SSc, while no effect on SSc susceptibility per se was found. rs2294020 may be considered a disease-modifying gene-variant rather than a disease-susceptibility SNP in SSc.
Asunto(s)
Factores de Transcripción Forkhead/genética , Genotipo , Esclerodermia Sistémica/genética , Estudios de Casos y Controles , Progresión de la Enfermedad , Femenino , Frecuencia de los Genes , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Humanos , Ligando Coestimulador de Linfocitos T Inducibles/genética , Proteína Coestimuladora de Linfocitos T Inducibles/genética , Italia , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
Several animal models are currently used for the surgical implantation of either biologic or biopolymeric scaffolds in order to provide in vivo assessment of tissue-engineered heart valves. The Vietnamese pig (VP) is herein proposed as a suitable recipient to test the function of novel bioengineered valve substitutes, in the reconstruction of the right ventricular outflow tract (RVOT). This review aims to provide a complete and exhaustive panel of physiological parameters and methodological information for preclinical studies of tissue-engineered heart valves in the VP animal model.
Asunto(s)
Prótesis Valvulares Cardíacas , Ingeniería de Tejidos , Aloinjertos , Animales , Válvula Aórtica/cirugía , Ecocardiografía , Regeneración Tisular Dirigida , Modelos Animales , Diseño de Prótesis , PorcinosRESUMEN
Valve dystrophic calcification is a common disorder affecting normophosphatemic subjects. Here, cultured aortic valve interstitial cells (AVICs) were treated 3 to 28 days with phosphate (Pi) concentrations spanning the normal range in humans (0.8, 1.3, and 2.0 mM) alone or supplemented with proinflammatory stimuli to assess possible priming of dystrophic-like calcification. Compared with controls, spectrophotometric analyses revealed marked increases in calcium amounts and alkaline phosphatase activity for 2.0-mM-Pi-containing cultures, with enhancing by proinflammatory mediators. Ultrastructurally, AVICs treated with low/middle Pi concentrations showed an enormous endoplasmic reticulum (ER) enclosing organelle debris, so apparently executing a survival-related atypical macroautophagocytosis, consistently with ultracytochemical demonstration of ER-associated acid phosphatase activity and decreases in autophagosomes and immunodetectable MAP1LC3. In contrast, AVICs cultured at 2.0-mM Pi underwent mineralization due to intracellular release and peripheral layering of phospholipid-rich material acting as hydroxyapatite nucleator, as revealed by Cuprolinic Blue and von Kossa ultracytochemical reactions. Lack of immunoblotted caspase-3 cleaved form indicated apoptosis absence for all cultures. In conclusion, fates of cultured AVICs were crucially driven by Pi concentration, suggesting that serum Pi levels just below the upper limit of normophosphatemia in humans may represent a critical watershed between macroautophagy-associated cell restoring and procalcific cell death.
Asunto(s)
Válvula Aórtica/citología , Válvula Aórtica/patología , Calcinosis/patología , Fosfatos/metabolismo , Fosfatasa Alcalina/metabolismo , Animales , Válvula Aórtica/metabolismo , Válvula Aórtica/ultraestructura , Autofagia , Calcinosis/metabolismo , Calcio/metabolismo , Bovinos , Supervivencia Celular , Células Cultivadas , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructuraRESUMEN
Autoimmune diseases often share common susceptibility genes. Most genetic variants associated with susceptibility to systemic lupus erythematosus are also associated with other autoimmune diseases. The X-linked variant rs2294020 is positioned in exon 7 of the CCDC22 gene. The encoded protein functions in the regulation of NF-κB, a master regulator in immune response. The aim of this study is to investigate whether the rs2294020 polymorphism may be a general susceptibility factor for autoimmunity. We evaluated case-control association between the occurrence of rs2294020 and different autoimmune diseases, including new data for systemic lupus erythematosus and previous genome-wide association studies (GWAS) (though most did not analyse the X chromosome) of psoriasis, celiac disease, Crohn's disease, ulcerative colitis, multiple sclerosis, vitiligo, type-1 diabetes, rheumatoid arthritis, and ankylosing spondylitis. Cases from patients affected by amyotrophic lateral sclerosis and type-2 diabetes were also included in the study. We detected nominal significant associations of rs2294020 with systemic lupus erythematosus (additive model test: p=0.01), vitiligo (p=0.016), psoriasis (p=0.038), and in only one of two studies of multiple sclerosis (p=0.03). Our results suggest that rs2294020 is associated with the risk of several autoimmune diseases in European populations, specifically with diseases that present themselves, among else, in the skin.
Asunto(s)
Alelos , Enfermedades Autoinmunes/genética , Genes Ligados a X , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Lupus Eritematoso Sistémico/genética , Proteínas/genética , Adulto , Enfermedades Autoinmunes/diagnóstico , Estudios de Casos y Controles , Exones , Femenino , Genotipo , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Masculino , Oportunidad Relativa , Fenotipo , Polimorfismo de Nucleótido SimpleRESUMEN
Decellularized porcine aortic valve conduits (AVCs) implanted in a Vietnamese Pig (VP) experimental animal model were matched against decellularized and then cryopreserved AVCs to assess the effect of cryopreservation on graft hemodynamic performance and propensity to in vivo repopulation by host's cells. VPs (n = 12) underwent right ventricular outflow tract substitution using AVC allografts and were studied for 15-month follow-up. VPs were randomized into two groups, receiving AVCs treated with decellularization alone (D; n = 6) or decellularization/cryopreservation (DC; n = 6), respectively. Serial echocardiography was carried out to follow up hemodynamic function. All explanted AVCs were processed for light and electron microscopy. No signs of dilatation, progressive stenosis, regurgitation, and macroscopic calcification were echocardiographically observed in both D and DC groups. Explanted D grafts exhibited near-normal features, whereas the presence of calcification, inflammatory infiltrates, and disarray of elastic lamellae occurred in some DC grafts. In the unaltered regions of AVCs from both groups, almost complete re-endothelialization was observed for both valve cusps and aorta walls. In addition, side-by-side repopulation by recipient's fibroblasts, myofibroblasts, and smooth muscle cells was paralleled by ongoing tissue remodeling, as revealed by the ultrastructural identification of typical canals of collagen fibrillogenesis and elastogenesis-related features. Incipient neo-vascularization and re-innervation of medial and adventitial tunicae of grafted aortic walls were also detected for both D and DC groups. Cryopreservation did not affect post-implantation AVC hemodynamic behavior and was topically propensive to cell repopulation and tissue renewal, although graft deterioration including calcification was present in several areas. Thus, these preliminary data provide essential information on feasibility of decellularization and cryopreservation coupling in the perspective of treatment optimization and subsequent clinical trials using similarly treated human allografts as innovative heart valve substitutes.
Asunto(s)
Aorta/trasplante , Válvula Aórtica/trasplante , Bioprótesis , Implantación de Prótesis Vascular/instrumentación , Prótesis Vascular , Criopreservación , Implantación de Prótesis de Válvulas Cardíacas/instrumentación , Prótesis Valvulares Cardíacas , Aloinjertos , Animales , Aorta/fisiopatología , Aorta/ultraestructura , Válvula Aórtica/fisiopatología , Válvula Aórtica/ultraestructura , Implantación de Prótesis Vascular/efectos adversos , Proliferación Celular , Ecocardiografía , Supervivencia de Injerto , Implantación de Prótesis de Válvulas Cardíacas/efectos adversos , Hemodinámica , Microscopía Electrónica de Transmisión , Modelos Animales , Complicaciones Posoperatorias/patología , Complicaciones Posoperatorias/fisiopatología , Porcinos , Factores de TiempoRESUMEN
OBJECTIVE: To improve knowledge of vasculopathy in SSc through the assessment of serum levels of circulating angiogenetic and endothelial dysfunction markers in patients at different stages of the disease. METHODS: Sera from 224 subjects were obtained and concentrations of angiopoietin-2, chemokine (C-X-C motif) ligand (CXCL)-16 (CXCL16), E-selectin, soluble intercellular adhesion molecule-1, IL-8 (CXCL8), soluble vascular adhesion molecule-1 and VEGF were determined by a Luminex assay. Subjects included 43 healthy controls, 47 early SSc patients according to LeRoy and Medsger without other signs and symptoms of evolutive disease, 48 definitive SSc (defSSc) patients according to the 2013 ACR/EULAR criteria without skin or lung fibrosis, 51 lcSSc subjects and 35 dcSSc subjects. RESULTS: The four groups of patients showed well-distinct clinical and laboratory characteristics, with a linear decreasing trend in forced vital capacity and diffusing capacity for carbon monoxide % predicted values from early SSc to defSSc to lcSSc and to dcSSc, and a linear increasing trend in ESR, and in the prevalence of abnormal CRP, serum gamma globulins and lung fibrosis (all P < 0.0001). Highly significant linear trends pointing to an increase in angiopoietin-2 (P < 0.0001), CXCL16 (P < 0.0001), E-selectin (P = 0.001) and soluble intercellular adhesion molecule-1 (P = 0.002) in relation to the different disease subsets were observed. CONCLUSION: Markers characterizing vascular activation are found to be increased in SSc patients from the earliest stages of disease when clinical and laboratory findings of advanced disease cannot yet be detected. These abnormalities progress with the appraisal of the first sclerodermatous manifestation in defSSc and further increase with the onset of fibrotic manifestations.
Asunto(s)
Inhibidores de la Angiogénesis/sangre , Mediadores de Inflamación/sangre , Esclerodermia Sistémica/sangre , Adulto , Anciano , Amina Oxidasa (conteniendo Cobre)/sangre , Angiopoyetina 2/sangre , Biomarcadores/sangre , Estudios de Casos y Controles , Moléculas de Adhesión Celular/sangre , Quimiocina CXCL16 , Quimiocinas CXC/sangre , Selectina E/sangre , Endotelio Vascular/fisiopatología , Femenino , Humanos , Molécula 1 de Adhesión Intercelular/sangre , Interleucina-8/sangre , Masculino , Persona de Mediana Edad , Receptores Depuradores/sangre , Esclerodermia Sistémica/fisiopatología , Índice de Severidad de la Enfermedad , Factor A de Crecimiento Endotelial Vascular/sangreRESUMEN
OBJECTIVE: HLAs have been extensively associated with SSc susceptibility but their role in the progression of the disease is poorly understood. In 2013 the ACR and European League Against Rheumatism (EULAR) jointly defined criteria for the classification of SSc that allow the early identification of definite SSc patients. In this study we investigated the role of HLA class II antigens in the progression from early to definite SSc. METHODS: One hundred and fifty-eight subjects with early SSc according to LeRoy and Medsger criteria and no other manifestation indicative of definite SSc at referral were considered. All the patients underwent high-resolution HLA class II typing and the appraisal of definite SSc was retrospectively conducted in a prospective manner. Lifetime analysis was conducted to gauge the effect of genetic and clinical characteristics on progression of the disease. RESULTS: The median estimated time to progression was 45 months from referral; the 5 and 10 year estimates of progression were 59.8% and 80%, respectively. ACAs were associated with a reduced risk of progression [median survival 55 vs 23 months for ACA-positive vs ACA-negative patients, P = 0.035; hazard ratio (HR) 0.67 (95% CI 0.458, 0.979)]. HLA alleles within the HLA DQ5-DR1 haplotype [HLA-DRB1*0101-HLA-DQA1*0101(4)-HLA-DQB1*0501] reduced the risk of progression of the disease [median survival 108 vs 44 months for DQ5-DR1 carriers vs DQ5-DR1 non-carriers; HR 0.388 (CI 0.211, 0.712), P = 0.001, corrected P = 0.014]. In multivariate models, the effect of genetics was found to be independent of ACA positivity or other baseline factors; additive risks were observed when the DQ5-DR1 haplotype and ACA were jointly considered. CONCLUSION: HLA class II alleles within the HLA DQ5-DR1 haplotype are associated with lower rates of progression from early to definite SSc.
Asunto(s)
Antígenos HLA-D/genética , Esclerodermia Sistémica/genética , Adulto , Alelos , Progresión de la Enfermedad , Femenino , Antígenos HLA-D/inmunología , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Cadenas alfa de HLA-DQ/genética , Cadenas alfa de HLA-DQ/inmunología , Cadenas beta de HLA-DQ/genética , Cadenas beta de HLA-DQ/inmunología , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , Antígenos de Histocompatibilidad Clase II , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Modelos de Riesgos Proporcionales , Estudios Prospectivos , Esclerodermia Sistémica/inmunologíaRESUMEN
Tissue-engineered heart valves are proposed as novel viable replacements granting longer durability and growth potential. However, they require extensive in vitro cell-conditioning in bioreactor before implantation. Here, the propensity of non-preconditioned decellularized heart valves to spontaneous in body self-regeneration was investigated in a large animal model. Decellularized porcine aortic valves were evaluated for right ventricular outflow tract (RVOT) reconstruction in Vietnamese Pigs (nâ=â11) with 6 (nâ=â5) and 15 (nâ=â6) follow-up months. Repositioned native valves (nâ=â2 for each time) were considered as control. Tissue and cell components from explanted valves were investigated by histology, immunohistochemistry, electron microscopy, and gene expression. Most substitutes constantly demonstrated in vivo adequate hemodynamic performances and ex vivo progressive repopulation during the 15 implantation months without signs of calcifications, fibrosis and/or thrombosis, as revealed by histological, immunohistochemical, ultrastructural, metabolic and transcriptomic profiles. Colonizing cells displayed native-like phenotypes and actively synthesized novel extracellular matrix elements, as collagen and elastin fibers. New mature blood vessels, i.e. capillaries and vasa vasorum, were identified in repopulated valves especially in the medial and adventitial tunicae of regenerated arterial walls. Such findings correlated to the up-regulated vascular gene transcription. Neoinnervation hallmarks were appreciated at histological and ultrastructural levels. Macrophage populations with reparative M2 phenotype were highly represented in repopulated valves. Indeed, no aspects of adverse/immune reaction were revealed in immunohistochemical and transcriptomic patterns. Among differentiated elements, several cells were identified expressing typical stem cell markers of embryonic, hematopoietic, neural and mesenchymal lineages in significantly higher number and specific topographic distribution in respect to control valves. Following the longest follow-up ever realized in preclinical models, non-preconditioned decellularized allogeneic valves offer suitable microenvironment for in vivo cell homing and tissue remodeling. Manufactured with simple, timesaving and cost-effective procedures, these promising valve replacements hold promise to become an effective alternative, especially for pediatric patients.
Asunto(s)
Implantación de Prótesis de Válvulas Cardíacas , Prótesis Valvulares Cardíacas , Regeneración/fisiología , Aloinjertos/ultraestructura , Animales , Forma de la Célula , Supervivencia Celular , Células Cultivadas , Perfilación de la Expresión Génica , Inmunohistoquímica , Inmunofenotipificación , Sus scrofa , Trasplante HomólogoRESUMEN
INTRODUCTION: We aimed to replicate a recent study which showed higher genetic risk load at 15 loci in men than in women with systemic lupus erythematosus (SLE). This difference was very significant, and it was interpreted as indicating that men require more genetic susceptibility than women to develop SLE. METHODS: Nineteen SLE-associated loci (thirteen of which are shared with the previous study) were analyzed in 1,457 SLE patients and 1,728 healthy controls of European ancestry. Genetic risk load was calculated as sex-specific sum genetic risk scores (GRS(s)). RESULTS: Our results did not replicate those of the previous study at either the level of individual loci or the global level of GRS(s). GRS(s) were larger in women than in men (4.20 ± 1.07 in women vs. 3.27 ± 0.98 in men). This very significant difference (P < 10(-16)) was more dependent on the six new loci not included in the previous study (59% of the difference) than on the thirteen loci that are shared (the remaining 41%). However, the 13 shared loci also showed a higher genetic risk load in women than in men in our study (P = 6.6 × 10(-7)), suggesting that heterogeneity of participants, in addition to different loci, contributed to the opposite results. CONCLUSION: Our results show the lack of a clear trend toward higher genetic risk in one of the sexes for the analyzed SLE loci. They also highlight several limitations of assessments of genetic risk load, including the possibility of ascertainment bias with loci discovered in studies that have included mainly women.
Asunto(s)
Carga Genética , Predisposición Genética a la Enfermedad/genética , Lupus Eritematoso Sistémico/genética , Polimorfismo de Nucleótido Simple , Medición de Riesgo/métodos , Alelos , Estudios de Casos y Controles , Europa (Continente) , Femenino , Frecuencia de los Genes , Predisposición Genética a la Enfermedad/etnología , Humanos , Lupus Eritematoso Sistémico/etnología , Masculino , Oportunidad Relativa , Medición de Riesgo/estadística & datos numéricos , Factores de Riesgo , Factores Sexuales , Población Blanca/genéticaRESUMEN
TREX1 (DNase III) is an exonuclease involved in response to oxidative stress and apoptosis. Heterozygous mutations in TREX1 were previously observed in patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS). We performed a mutational analysis of the TREX1 gene on three autoimmune diseases: SLE (210 patients) and SS (58 patients), to confirm a TREX1 involvement in the Italian population, and systemic sclerosis (SSc, 150 patients) because it shares similarities with SLE (presence of antinuclear antibodies and connective tissue damage). We observed 7 variations; two of these are novel nonsynonymous variants (p.Glu198Lys and p.Met232Val). They were detected in one SS and in one SSc patient, respectively, and in none of the 200 healthy controls typed in this study and of the 1712 published controls. In silico analysis predicts a possibly damaging role on protein function for both variants. The other 5 variations are synonymous and only one of them is novel (p.Pro48Pro). This study contributes to the demonstration that TREX1 is involved in autoimmune diseases and proposes that the spectrum of involved autoimmune diseases can be broader and includes SSc. We do not confirm a role of TREX1 variants in SLE.