shRNA targeting caspase-3 inhibits apoptosis and cell detachment induced by Pemphigus Vulgaris autoantibodies

Pemphigus is an organ-specific autoimmune disease that affects the skin and mucous membranes. It is induced by the deposition of pemphigus IgG autoantibodies, which mainly target Dsg1 and 3 and cause a loss of cell adhesion in a phenomenon known as acantholysis, and clinically is reflected as intraepidermal blistering. The present work assessed the effect of pemphigus vulgaris IgG (PV-IgG) on cell adhesion and caspase 3- dependent apoptosis in HaCaT cells. The expression of caspase-3 induced by PV-IgG was silenced in cells pre-treated with caspase 3-shRNA. PV-IgG induced cell detachment and apoptotic changes as demonstrated by the annexin-FITC assays. Treatment of cell cultures with normal IgG (control; N-IgG) did not have relevant effects on the aforementioned parameters. Then, the effect of PV-IgG on cells previously treated with shRNA was tested. The results demonstrated that shRNA reduced apoptotic features and the relative expression of caspase-3 measured by qRT-PCR, which showed a decrease of 96%. In conclusion shRNA prevented cell detachment and apoptosis of HaCaT cells induced by PV-IgG. The presented results further our understanding of the molecular pathophysiologic mechanisms involved in pemphigus diseases.Pemphigus is an organ-specific autoimmune disease that affects the skin and mucous membranes. It is induced by the deposition of pemphigus IgG autoantibodies, which mainly target Dsg1 and 3 and cause a loss of cell adhesion in a phenomenon known as acantholysis, and clinically is reflected as intraepidermal blistering. The present work assessed the effect of pemphigus vulgaris IgG (PV-IgG) on cell adhesion and caspase 3- dependent apoptosis in HaCaT cells. The expression of caspase-3 induced by PV-IgG was silenced in cells pre-treated with caspase 3-shRNA. PV-IgG induced cell detachment and apoptotic changes as demonstrated by the annexin-FITC assays. Treatment of cell cultures with normal IgG (control; N-IgG) did not have relevant effects on the aforementioned parameters. Then, the effect of PV-IgG on cells previously treated with shRNA was tested. The results demonstrated that shRNA reduced apoptotic features and the relative expression of caspase-3 measured by qRT-PCR, which showed a decrease of 96%. In conclusion shRNA prevented cell detachment and apoptosis of HaCaT cells induced by PV-IgG. The presented results further our understanding of the molecular pathophysiologic mechanisms involved in pemphigus diseases

An Activin Receptor IA/Activin-Like Kinase-2 (R206H) Mutation in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is an exceptionally rare genetic disease that is characterised by congenital malformations of the great toes and progressive heterotopic ossification (HO) in specific anatomical areas.This disease is caused by a mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2). A Mexican family with one member affected by FOP was studied. The patient is a 19-year-old female who first presented with symptoms of FOP at 8 years old; she developed spontaneous and painful swelling of the right scapular area accompanied by functional limitation of movement. Mutation analysis was performed in which genomic DNA as PCR amplified using primers flanking exons 4 and 6, and PCR products were digested with Cac8I and HphI restriction enzymes.The most informative results were obtained with the exon 4 flanking primers and the Cac8I restriction enzyme, which generated a 253 bp product that carries the ACVR1 617G>A mutation, which causes an amino acid substitution of histidine for arginine at position 206 of the glycine-serine (GS) domain, and its mutation results in the dysregulation of bone morphogenetic protein (BMP) signalling that causes FO

Autoimmune vitiligo in rheumatic disease in the mestizo Mexican population

Vitiligo is a chronic disease characterized by the dysfunction or destruction of melanocytes with secondary depigmentation. The aim of the present study was to determine the prevalence of vitiligo associated with autoimmune rheumatic diseases. The clinical records from a 10-year database of patients with rheumatic diseases and associated vitiligo was analysed, with one group of patients having autoimmune rheumatic disease and another non-autoimmune rheumatic disease. Available serum samples were used to assess the anti-melanocyte antibodies. A total of 5,251 individual clinical files were archived in the last 10 years, and these patients underwent multiple rheumatology consultations, with 0.3% of the group presenting with vitiligo. The prevalence of vitiligo in the autoimmune rheumatic disease group was 0.672%, which was mainly associated with lupus and arthritis. However, patients with more than one autoimmune disease had an increased relative risk to develop vitiligo, and anti-melanocyte antibodies were positive in 92% of these patients. By contrast, the prevalence was 0.082% in the group that lacked autoimmune rheumatic disease and had negative autoantibodies. In conclusion, the association between vitiligo and autoimmune rheumatic diseases was relatively low. However, the relative risk increased when there were other autoimmune comorbidities, such as thyroiditis or celiac disease. Therefore, the presence of multiple autoimmune syndromes should be suspected

Activation of Peptidylarginine Deiminase in the Salivary Glands of Balb/c Mice Drives the Citrullination of Ro and La Ribonucleoproteins

The goal of the present study was to determine whether peptidylarginine deiminase PAD2 and PAD4 enzymes are present in Balb/c mouse salivary glands and whether they are able to citrullinate Ro and La ribonucleoproteins. Salivary glands from Balb/c mice were cultured in DMEM and supplemented with one of the following stimulants: ATP, LPS, TNF, IFNγ, or IL-6. A control group without stimulant was also evaluated. PAD2, PAD4, citrullinated peptides, Ro60, and La were detected by immunohistochemistry and double immunofluorescence. PAD2 and PAD4 mRNAs and protein expression were detected by qPCR and Western blot analysis. PAD activity was assessed using an antigen capture enzyme-linked immunosorbent assay. LPS, ATP, and TNF triggered PAD2 and PAD4 expression; in contrast, no expression was detected in the control group (p < 0 001). PAD transcription slightly increased in response to stimulation. Additionally, PAD2/4 activity modified the arginine residues of a reporter protein (fibrinogen) in vitro. PADs citrullinated Ro60 and La ribonucleoproteins in vivo. Molecular stimulants induced apoptosis in ductal cells and the externalization of Ro60 and La ribonucleoproteins onto apoptotic membranes. PAD enzymes citrullinate Ro and La ribonucleoproteins, and this experimental approach may facilitate our understanding of the role of posttranslational modifications in the pathophysiology of Sjögren’s syndrome

Apoptosis and cell proliferation: the paradox of salivary glands in sjögren’s disease

Este estudo avalia a apoptose e a proliferação nas glândulas salivares dos doentes com Síndroma de Sjögren primária. Métodos: A apoptose foi estudada por imunohistoquímica utilizando anticorpos monoclonais anti- -Fas, FasL e Caspase 3 e as características apoptóticas por TUNEL. Os estudos foram executados em vinte e quatro glândulas salivares minor de doentes com Síndroma de Sjögren primária e num igual número de controlos. A proliferação foi avaliada com anticorpos monoclonais anti-PCNA e anti-Ki67. Resultados:Todas as glândulas salivares dos doentes com Sjögren apresentavam moléculas apoptoticas no epitélio dos ductos salivares, e menos no tecido acinar, consequentemente a presença do caspase 3, Fas/FasL eram concordantes com a expressão da apoptose por TUNEL. Os marcadores de proliferação foram encontrados nas células inflamatórias presentes, mas não no epitélio ductal nem nos acinos. A expressão de marcadores de apoptose ou de proliferação nos tecidos das biopsias dos controlos foi escassa. Conclusão: Os dados actuais sugerem que as células do epitélio ductal e dos acinos das glândulas salivares dos doentes com doença de Sjögren têm aumento da apoptose. A proliferação foi observada principalmente no infiltrado celular linfóide. Em conjunto, estes eventos constituem um paradoxo biológico relacionado com o processo inflamatório das glândulas salivares na Síndrome de Sjögren.Aim: To assess apoptosis and proliferation in salivary glands of patients with primary Sjögren’s syndrome. Methods: Studies were performed in twenty four minor salivary glands from patients with primary Sjögren’s syndrome and an equal number of controls. Apoptosis was studied by immunohistochemistry using monoclonal antibodies anti-Fas, FasL and Caspase 3 and apoptotic features by TUNEL. Proliferation was assessed with monoclonal anti-PCNA and anti-Ki67 antibodies. Results: All salivary glands from Sjögren’s display apoptotic molecules along the epithelia of salivary ducts, and in a smaller amount in acinar tissue. The presence of Caspase 3, Fas/FasL was concordant with the expression of apoptosis by TUNEL. Proliferation markers were encountered in inflammatory emigrant cells, but not in ductal epithelia nor in acini. Control biopsies poorly expressed apoptotic or proliferation markers. Conclusion: Present data suggests that the ductal epithelial and acinar cells of salivary glands from Sjögren’s disease patients exhibit increased apoptosis. Proliferation was mainly observed in infiltrating lymphoid cells. Both events constitute a biological paradox related to the inflammatory process of salivary glands in Sjögren’s disease

Apoptosis and redistribution of the Ro autoantigen in Balb/c mouse like in subacute cutaneous lupus erythematosus

In subacute cutaneous lupus eryhematosus (SCLE) the cutaneous antigens constitute the main source of Ro and La autoantigens. The aim of this investigation was to demonstrate if UV light increases the availability of Ro autoantigen in the skin, also the blocking effect of Ac-DEVD-CMK a caspase inhibitor was assessed. For this purpose newborn Balb/c mice were UVB irradiated (5–30 mJ/cm2) equivalent to a moderate to severe sunburn. Animals were injected with monoclonal anti-Roantibodies from SCLE patients. Apoptosis was also induced by anti-Fas antibody injection. Skin samples were examined by direct immunofluoresence, by TUNEL, and the expression of caspase 3 by RT-PCR. Major findings of present studies were: 1. UVB irradiation and anti-Fas induced apoptosis of keratinocytes. 2. Apoptosis redistribute the Ro antigen on cell surface and is better triggered by Ro antibody. 3. The caspase 3 inhibitor Ac-DEVD-CMK decreases the availability of Ro autoantigen in epidermis and prevents deposition of anti-Ro. In conclusion, the caspase pathway would be blocked to avoid anti-Ro deposition along skin; this finding would be a prospect in the treatment of SCLE patients

Aquacultural Homoeopathy: A Focus on Marine Species

Homoeopathy is an alternative medical system proposed by Samuel Hahnemann in the eighteenth century. It uses highly diluted and agitated substances that derived from plants, minerals or animals, which have shown to be effective in human medicine, agronomy, veterinary, and as a novelty, in marine aquaculture. Aquacultural homoeopathy has developed rapidly in recent years, partially motivated by the misuse of powerful drugs (hormones, antibiotics, disinfectants) that when solving a problem generate undesirable side effects. In the last 10 years, scientific articles have been published on its application in freshwater fish native to Brazil, obtaining beneficial effects on growth, survival, hepatosomatic index, development of muscle fibres and lipid content in muscle. At Centro de Investigaciones Biológicas del Noroeste (CIBNOR, Mexico: www.cibnor.mx), we have studied the effects of homoeopathy to improve the culture of economically important marine species of molluscs, fish and shrimp. In this chapter, we show a selection of different research with preliminary or advanced results, related to the use of homoeopathy and its impact on zootechnic, biochemical, genomic and transcriptomic parameters in marine molluscs, fish and crustaceans. The results obtained suggest that homoeopathy is an eco-friendly alternative applicable in aquaculture industry to improve various productive and health aspects

An Activin Receptor IA/Activin-Like Kinase-2 (R206H) Mutation in Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP) is an exceptionally rare genetic disease that is characterised by congenital malformations of the great toes and progressive heterotopic ossification (HO) in specific anatomical areas. This disease is caused by a mutation in activin receptor IA/activin-like kinase-2 (ACVR1/ALK2). A Mexican family with one member affected by FOP was studied. The patient is a 19-year-old female who first presented with symptoms of FOP at 8 years old; she developed spontaneous and painful swelling of the right scapular area accompanied by functional limitation of movement. Mutation analysis was performed in which genomic DNA as PCR amplified using primers flanking exons 4 and 6, and PCR products were digested with Cac8I and HphI restriction enzymes. The most informative results were obtained with the exon 4 flanking primers and the Cac8I restriction enzyme, which generated a 253 bp product that carries the ACVR1 617G>A mutation, which causes an amino acid substitution of histidine for arginine at position 206 of the glycine-serine (GS) domain, and its mutation results in the dysregulation of bone morphogenetic protein (BMP) signalling that causes FOP

Transcriptome analysis of Catarina scallop (Argopecten ventricosus) juveniles treated with highly-diluted immunomodulatory compounds reveals activation of non-self-recognition system.

Marine bivalve hatchery productivity is continuously challenged by apparition and propagation of new diseases, mainly those related to vibriosis. Disinfectants and antibiotics are frequently overused to prevent pathogen presence, generating a potential negative impact on the environment. Recently, the use of highly diluted compounds with immunostimulant properties in marine organisms has been trailed successfully to activate the self-protection mechanisms of marine bivalves. Despite their potential as immunostimulants, little is known about their way of action. To understand their effect, a comparative transcriptomic analysis was performed with Argopecten ventricosus juveniles. The experimental design consisted of four treatments formulated from pathogenic Vibrio lysates at two dilutions: [(T1) Vibrio parahaemolyticus and Vibrio alginolyticus 1D; (T2) V. parahaemolyticus and V. alginolyticus 7C]; minerals [(T3) PhA+SiT 7C], scorpion venom [(T4) ViT 31C]; and one control (C1) hydro-alcoholic solution (ethanol 1%). The RNA sequencing (RNAseq) analysis showed a higher modulation of differentially expressed genes (DEG) in mantle tissue compared to gill tissue. The scallops that showed a higher number of DEG related to immune response in mantle tissue corresponded to T1 (V. parahaemolyticus and V. alginolyticus lysate) and T3 (Silicea terra® - Phosphoric acid®). The transcriptome analysis allowed understanding some interactions between A. ventricosus juveniles and highly-diluted treatments

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY-NC-ND 4.0 licenseBackground: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit