The Caspase Pathway as a Possible Therapeutic Target in Experimental Pemphigus

Apoptosis plays a role in pemphigus IgG-dependent acantholysis; theoretically, the blockade of the caspase pathway could prevent the blistering that is caused by pemphigus autoantibodies. Using this strategy, we attempted to block the pathogenic effect of pemphigus IgG in Balb/c mice by using the caspase inhibitor Ac-DEVD-CMK. This inhibitor was administrated before the injection of pemphigus IgG into neonatal mice. The main results of the present investigation are as follows: (1) pemphigus IgG induces intraepidermal blisters in Balb/c neonatal mice; (2) keratinocytes around the blister and acantholytic cells undergo apoptosis; (3) the caspases inhibitor Ac-DEVD-CMK prevents apoptosis; (4) the inhibition of the caspase pathway prevents blister formation. In conclusion, inhibition of the caspase pathway may be a promising therapeutic tool that can help in the treatment of pemphigus flare ups

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

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

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

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

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

Posttranslational Protein Modification in the Salivary Glands of Sjögren’s Syndrome Patients

The present study investigated posttranslational reactions in the salivary glands of patients with Sjögren’s syndrome. We analysed the biopsies of primary Sjögren’s patients using immunohistochemistry and a tag-purified anticyclic citrullinated protein (CCP) antibody to detect citrullinated peptides, and the presence of peptidylarginine deiminase 2 (PAD2) was assessed simultaneously. The present work demonstrated the weak presence of the PAD2 enzyme in some normal salivary glands, although PAD2 expression was increased considerably in Sjögren’s patients. The presence of citrullinated proteins was also detected in the salivary tissues of Sjögren’s patients, which strongly supports the in situ posttranslational modification of proteins in this setting. Furthermore, the mutual expression of CCP and PAD2 suggests that this posttranslational modification is enzyme dependent. In conclusion, patients with Sjögren’s syndrome expressed the catalytic machinery to produce posttranslational reactions that may result in autoantigen triggering