suPAR and WT1 modify the adhesion of podocytes and are related to proteinuria in class IV lupus nephritis.

Introduction: Lupus nephritis (LN) affects up to 60 % of the patients with Systemic Lupus Erythematosus (SLE) and renal damage progression is associated with proteinuria, caused in part by the integrity of the glomerular basement membrane (GBM) and by podocyte injury. The soluble urokinase plasminogen activator receptor (suPAR) and Wilms Tumor 1 (WT1) have been related to podocyte effacement and consequently with proteinuria which raises questions about its pathogenic role in LN. Objective: Define whether suPAR levels and WT1 expression influence in podocyte anchorage destabilization in LN class IV. Materials and methods: This is a cross-sectional study of cases and controls. We studied patients with SLE without renal involvement (n = 12), SLE and LN class IV with proteinuria ≤0.5 g/24 h (n = 12), LN class IV with proteinuria ≥0.5 g/24 h (n = 12) and compared them with renal tissue control (CR) (n = 12) and control sera (CS) (n = 12). The CR was integrated by cadaveric samples without SLE or renal involvement and the CS was integrated by healthy participants. The expression and cellular localization of WT1, urokinase-type plasminogen activator receptor (uPAR), ac-α-tubulin, vimentin, and ß3-integrin was assessed by immunohistochemistry (IHC). The concentration of suPAR in serum was analyzed by enzyme-linked immunosorbent assay (ELISA). Results: In patients with LN, the activation of anchoring proteins was increased, such as podocyte ß3-integrin, as well as the acetylation of alpha-acetyl-tubulin and uPAR, in contrast to the decrease in vimentin; interestingly, the cellular localization of WT1 was cytoplasmic and the number of podocytes per glomerulus decreased. The concentrations of suPAR was increased in patients with LN. Conclusion: The destabilization of podocyte anchorage modulated by ß3-integrin activation, and tubulin acetylation, associated with decreased WT1 cytoplasmic expression, and increased suPAR levels could be involved in kidney damage in patients with LN class IV.

Epidermolysis Bullosa Acquisita: A Rare Autoimmune Blistering Disease in Children.

A 7-year-old girl presented with a 2-year history of recurrent blisters on the skin and oral mucosa. The patient was otherwise healthy, and her family history was unremarkable for any dermatologic or other medical disease. Examination revealed multiple tense vesicles, milia, and atrophic scars present over the extensor surface of the extremities and erosions on the oral mucosa (Figure 1). A skin biopsy established a pauci-inflammatory subepidermal blister (Figure 2a). Direct immunofluorescence (DIF) evidenced the linear deposition of immunoglobulin G (IgG), immunoglobulin M (IgM), and κ and λ chains at the dermal-epithelial junction (DEJ). Indirect immunofluorescence (IIF), using the salt-split technique, established anti-epithelial antibodies on the dermal side (Figure 2b). An enzyme-linked immunosorbent assay (ELISA) was positive for Collagen Type VII (COL7) antibodies. A diagnosis of epidermolysis bullosa acquisita (EBA) was made, and treatment with azathioprine and deflazacort was administered for 8 months with progressive lessening of her symptomatology and complete clinical response at 2-year follow-up. (SKINmed. 2022;20:460-462).

Bronchiolitis Obliterans With Anti-Epiplakin Antibodies in a Boy With Paraneoplastic Pemphigus.

Paraneoplastic pemphigus is a rare and severe autoimmune blistering disease characterized by a recalcitrant and severe mucositis, and polymorphic cutaneous lesions, associated with benign and malignant neoplasms. Paraneoplastic pemphigus is caused by production of autoantibodies against various epidermal proteins involved in cell adhesion. Bronchiolitis obliterans (BO) is one of the leading causes of mortality in these patients. Recent advances have associated the presence of anti-epiplakin antibodies with the development of BO in adult patients. Here we describe the first pediatric patient in whom the association of anti-epiplakin antibodies and BO have been reported so far.

Multiple autoimmunity and epitope spreading in monozygotic twins.

We report clinical, serologic, and immunogenetic studies of a set of monozygotic male twin patients who develop autoimmune thyroiditis and vitiligo associated with the HLA-DRB1*04-DQB1*03:02 and HLA-DRB1*03-DQB1*0201 haplotypes. The patients had detectable anti-thyroid and anti-melanocyte autoantibodies. A critical review is presented regarding the role of MHC II molecules linked to clinical manifestations of various autoimmune diseases displayed in a single patient, as is the case in the twin patients reported here.

Cytoplasmic Localization of WT1 and Decrease of miRNA-16-1 in Nephrotic Syndrome.

Nephrotic syndrome (NS) is a glomerular disease that is defined by the leakage of protein into the urine and is associated with hypoalbuminemia, hyperlipidemia, and edema. Steroid-resistant NS (SRNS) patients do not respond to treatment with corticosteroids and show decreased Wilms tumor 1 (WT1) expression in podocytes. Downregulation of WT1 has been shown to be affected by certain microRNAs (miRNAs). Twenty-one patients with idiopathic NS (68.75% were SSNS and 31.25% SRNS) and 10 healthy controls were enrolled in the study. Podocyte number and WT1 location were determined by immunofluorescence, and the serum levels of miR-15a, miR-16-1, and miR-193a were quantified by RT-qPCR. Low expression and delocalization of WT1 protein from the nucleus to the cytoplasm were found in kidney biopsies of patients with SRNS and both nuclear and cytoplasmic localization were found in steroid-sensitive NS (SSNS) patients. In sera from NS patients, low expression levels of miR-15a and miR-16-1 were found compared with healthy controls, but only the miR-16-1 expression levels showed statistically significant decrease (p = 0.019). The miR-193a expression levels only slightly increased in NS patients. We concluded that low expression and delocalization from the WT1 protein in NS patients contribute to loss of podocytes while modulation from WT1 protein is not associated with the miRNAs analyzed in sera from the patients.

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.

Potential protein targets of the peptidylarginine deiminase 2 and peptidylarginine deiminase 4 enzymes in rheumatoid synovial tissue and its possible meaning.

OBJECTIVE: The molecular mechanism of citrullination involves the calcium-dependent peptidylarginine deiminase (PAD) family of enzymes. These enzymes induce a stereochemical modification of normal proteins and transform them into autoantigens, which in rheumatoid arthritis trigger a complex cascade of joint inflammatory events followed by chronic synovitis, pannus formation, and finally, cartilage destruction. By hypothesizing that PAD2 and PAD4 enzymes produce autoantigens, we investigated five possible synovial protein targets of PAD enzymes. MATERIAL AND METHODS: We measured PAD2, PAD4, and citrullinated proteins in 10 rheumatoid and 10 osteoarthritis synovial biopsies and then assessed the post-translational modifications of fibrinogen, cytokeratin, tubulin, IgG, and vimentin proteins using a double-fluorescence assay with specific antibodies and an affinity-purified anti-citrullinated peptide (CCP) antibody. The degree of co-localization was analyzed, and statistical significance was determined by ANOVA, Fisher's exact test, and regression analysis. RESULTS: The principal results of this study demonstrated that citrullinated proteins, such as fibrinogen, IgG, and other probed proteins, were targets of PAD2 and PAD4 activity in rheumatoid synovial biopsies, whereas osteoarthritis biopsies were negative for this enzyme (p<0.0001). An analysis of citrullination sites using the UniProtKB/Swiss-Prot data bank predicts that the secondary structure of the analyzed proteins displays most of the sites for citrullination; a discussion regarding its possible meaning in terms of pathogenesis is made. CONCLUSION: Our results support the conclusion that the synovial citrullination of proteins is PAD2 and PAD4 dependent. Furthermore, there is a collection of candidate proteins that can be citrullinated.

Pemphigus foliaceus with circinated plaques and neutrophil pustules.

Pemphigus is a group of autoimmune intraepidermal bullous diseases; being pemphigus foliaceus (PF) and pemphigus vulgaris (PV) the most common subtypes. Pustular variants are scarcely reported for both PV and PF. The purpose of this manuscript was to describe the clinical, microscopic and immunologic findings of an atypical case of PF presenting with pustules, including a review of the literature. PF is described as blisters and because this entity is rare, it is not known for the general medical community that they are other clinical features that can be seen as this one we present here with pustules.

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.

Apoptosis in chronic cutaneous lupus erythematosus, discoid lupus, and lupus profundus.

INTRODUCTION: Lupus erythematosus is a multisystemic disease that is characterized by autoantibody production and immune complex deposition in such tissues as the mucosa, joints, the central nervous system, and skin. Cutaneous lupus erythematosus is categorized as acute, subacute, and chronic. Chronic cutaneous lupus erythematosus comprises discoid lupus erythematosus (DLE) and lupus profundus (LP). AIM: To analyze the expression of proapoptotic molecules in patients with lupus erythematosus discoid and lupus profundus. MATERIAL AND METHODS: Descriptive study, the study groups comprised 10 cases of LP and 10 cases of DLE, and a control. Skin samples of cases and controls were processed for immunohistochemistry and by TUNEL technique. The database and statistical analysis was performed (statistical test X(2)) SPSS (Chicago, IL, USA). RESULTS: Apoptotic features were broadly distributed along the skin biopsies in epidermal keratinocytes as well as at dermis. By immunohistochemistry the expression of Fas receptor and Fas-L was higher in the skin of lupus patients compared with controls. We also noted differences in Fas-L, -Fas, and -Bax proteins expression intensity in discoid lupus erythematosus patients in the epidermis, and hair follicles. CONCLUSIONS: Fas and Fas-L are expressed similarly in LP and DLE.

An Animal Model Using Metallic Ions to Produce Autoimmune Nephritis.

Autoimmune nephritis triggered by metallic ions was assessed in a Long-Evans rat model. The parameters evaluated included antinuclear autoantibody production, kidney damage mediated by immune complexes detected by immunofluorescence, and renal function tested by retention of nitrogen waste products and proteinuria. To accomplish our goal, the animals were treated with the following ionic metals: HgCl2, CuSO4, AgNO3, and Pb(NO3)2. A group without ionic metals was used as the control. The results of the present investigation demonstrated that metallic ions triggered antinuclear antibody production in 60% of animals, some of them with anti-DNA specificity. Furthermore, all animals treated with heavy metals developed toxic glomerulonephritis with immune complex deposition along the mesangium and membranes. These phenomena were accompanied by proteinuria and increased concentrations of urea. Based on these results, we conclude that metallic ions may induce experimental autoimmune nephritis.

Soluble fas and the -670 polymorphism of fas in lupus nephritis.

This study was performed to clarify the role of soluble Fas (sFas) in lupus nephritis (LN) and establish a potential relationship between LN and the -670 polymorphism of Fas in 67 patients with systemic lupus erythematosus (SLE), including a subset of 24 LN patients with proteinuria. Additionally, a group of 54 healthy subjects (HS) was included. The allelic frequency of the -670 polymorphism of Fas was determined using PCR-RFLP analysis, and sFas levels were assessed by ELISA. Additionally, the WT-1 protein level in urine was measured. The Fas receptor was determined in biopsies by immunohistochemistry (IHC) and in situ hybridization (FISH) and apoptotic features by TUNEL. Results. The -670 Fas polymorphism showed that the G allele was associated with increased SLE susceptibility, with an odds ratio (OR) of 1.86. The sFas was significantly higher in LN patients with the G/G genotype, and this subgroup exhibited correlations between the sFas level and proteinuria and increased urinary WT-1 levels. LN group shows increased expression of Fas and apoptotic features. In conclusion, our results indicate that the G allele of the -670 polymorphism of Fas is associated with genetic susceptibility in SLE patients with elevated levels of sFas in LN with proteinuria.

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.

Autoantibodies in senear-usher syndrome: cross-reactivity or multiple autoimmunity?

Senear-Usher syndrome or pemphigus erythematosus is a pathology that overlaps clinically and serologically with pemphigus foliaceus and lupus erythematosus. Skin biopsies of patients with pemphigus erythematosus reveal acantholysis and deposits of immunoglobulins in desmosomes, and they are positive in the lupus band test. In the present paper, we determined whether the autoantibodies associated with pemphigus erythematosus targeted a single antigen or multiple antigens as a result of the stimulation of independent B cell clones. Our present paper demonstrates that patients with pemphigus erythematosus produce both antiepithelial antibodies specific for desmoglein 1 and 3 and antinuclear antibodies specific for Ro, La, Sm, and double-stranded DNA antigens. After eluting specific anti-epithelial or anti-nuclear antibodies, which were recovered and tested using double-fluorescence assays, a lack of cross-reactivity was demonstrated between desmosomes and nuclear and cytoplasmic lupus antigens. This result suggests that autoantibodies in pemphigus erythematosus are directed against different antigens and that these autoantibodies are produced by independent clones. Given these clinical and serological data, we suggest that pemphigus erythematosus behaves as a multiple autoimmune disease.

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.

Tacrolimus therapy for circinate balanitis associated with reactive arthritis.

BACKGROUND: Reactive arthritis is the current name for the clinical triad characterized by arthritis, urethritis, and conjunctivitis, which develops over the course of a month or more. In some patients, this clinical triad is accompanied by circinate balanitis and keratoderma on the palms and soles. Balanitis, in some cases, is refractory to conventional therapy and can be recurrent, becoming a therapeutic challenge. METHODS: A total of 4 clinical cases of male patients, with ages ranging from 21 to 36 years old, presented with arthritis, conjunctivitis, urethritis, and recurrent circinate balanitis, which developed over a period of 1 to 6 months. All patients were treated with corticosteroids and sulfasalazine. In addition, one was treated with ciprofloxacin; however, balanitis was resistant to treatment in 3 patients. RESULTS: As other treatments had failed, topical 0.1% tacrolimus was used, with excellent results, as the balanitis lesions were cleared during the first week of topical therapy. CONCLUSION: We propose the complementary use of topical tacrolimus in cases of reactive arthritis associated with circinate balanitis.

Apoptosis and cell proliferation: the paradox of salivary glands in Sjögren's disease.

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.

Production of anti-epithelial antibodies and acantholysis by vaccination with an anti-idiotypic antibody, mimicking desmoglein 1.

Theoretically, the immunization of experimental animals with an anti-idiotype antibody may elicit antibodies that recognize epitopes like the original idiotype; this is archived via internal images. Using this strategy, we attempted to produce anti-epithelial antibodies in Balb/c mice immunized with a pemphigus anti-idiotypic determinant. First, when an anti-idiotype antibody was produced in rabbits by immunization with pemphigus immunoglobulin G (IgG), the anti-idiotypic activity was tested successfully. The anti-idiotype IgG was digested with pepsin and purified by gel filtration chromatography to obtain F(ab')(2) fragments, which were used to immunize Balb/c mice. A control group was immunized with normal IgG. The experimental animals immunized with anti-idiotype F(ab')(2) fragments developed anti-epithelial antibodies in the following two months. The elicited antibodies had anti-desmoglein 1 specificity. Additionally, the skin biopsies of these animals exhibited antibody deposition along intercellular spaces of epidermis, and 25% of them developed blisters. Sera and skin biopsies of control Balb/c mice group were negative. In conclusion, the immunization with pemphigus anti-idiotype antibody may elicit anti-epithelial antibodies via internal images. This experimental approach can be used to understand the pathogenic mechanisms of pemphigus.

GW bodies: from RNA biology to clinical implications in autoimmunity.

Evaluation of: Lian S, Fritzler M, Katz J et al. Small interfering RNA-mediated silencing induces target-dependent assembly of GW/P bodies. Mol. Biol. Cell 18, 3375-3387 (2007). GW bodies (GWBs) are also known as mammalian processing bodies and are involved in 5 -3 mRNA degradation. Conversely, siRNA is a powerful tool for silencing genes. Recently, components of RNAi have been associated with GWBs, but as more components of this complex pathway become known, such relationships remain to be clarified. This paper evaluates the induction of GWBs by siRNA transfection. The main results of these studies indicate that siRNA increased the GWBs, such an increase is also dependent on the endogenous expression of the target mRNA; siRNA increases require GW182 or Ago-2 proteins, but not rck/p54 or LSm1. Results of the present studies propose a regulatory function of RNAi in GWB assembly; therefore, cell biology implications of GWBs may open a new area in pathogenic mechanisms of autoimmunity.