CD163+ macrophage density in perimysial connective tissue associated with prognosis in IMNM.

OBJECTIVE: The pathological features of immune-mediated necrotizing myopathy (IMNM) are dominated by the infiltration of macrophages. We aimed to perform a histopathologic semiquantitative analysis to investigate the relationship between macrophage markers and prognosis. METHODS: Semiquantitative analysis of histologic features was performed in 62 samples of IMNM. Independent risk factors were identified through univariate and multivariate regression analysis. Cluster analysis was performed using the partitioning around the medoids (PAM) method. Decision tree modeling was utilized to efficiently determine cluster labels for IMNM patients. The validity of the developmental cohort was assessed by accuracy in comparison with the validation cohort. RESULTS: The most enriched groups in patients with IMNM were macrophages expressing CD206 and CD163. In the multivariate logistic regression model, the high density of CD163+ macrophages in perimysial connective tissue increased the risk of unfavorable prognosis (p = 0.025, OR = 1.463, 95% CI: 1.049-2.041). In cluster analysis, patients in Cluster 1, with lower CD163+ macrophage density and inflammatory burden, had a more favorable prognosis. Conversely, patients in Cluster 3, which were enriched for CD163+ macrophages in the perimysial connective tissue, had the most severe clinical features and the worst prognosis. Correlations were found between the density of CD163+ macrophages in connective tissue and symptom duration (R2 = 0.166, p < 0.001), dysphagia (p = 0.004), cardiac involvement (p = 0.021), CK (R2 = 0.067, p = 0.042), CRP (R2 = 0.117, p < 0.001), and ESR (R2 = 0.171, p < 0.001). CONCLUSION: The density of CD163+ macrophages in perimysial connective tissue may serve as a potential marker for the prediction of IMNM prognosis.

Differential expression and alternative splicing of transcripts in orbital adipose/connective tissue of thyroid-associated ophthalmopathy.

Thyroid-associated ophthalmopathy is a typical autoimmune disease of orbital tissues. Alternative splicing significantly influences many diseases progression, including cancer, age-related macular degeneration, and multiple sclerosis, by modulating the expression of transcripts. However, its role in thyroid-associated ophthalmopathy is still unclear. In this study, differential expression transcripts and differential alternative splicing genes in orbital adipose/connective tissues of thyroid-associated ophthalmopathy patients were detected using RNA sequencing, Cuffdiff, and replicate multivariate analysis of transcript splicing. Three thousand ninety six differential expression transcripts and 2355 differential alternative splicing genes were screened out, while functional enrichment analysis indicated that differential expression transcript and differential alternative splicing genes were associated with immune modulation, extracellular matrix remodeling, and adipogenesis. The expression of the SORBS1, SEPT2, COL12A1, and VCAN gene transcripts was verified by qRT-PCR. In conclusion, prevalent alternative splicing is involved in the disease development in thyroid-associated ophthalmopathy. More attention should be paid to the mechanism of alternative splicing to explore more potential therapeutic targets in thyroid-associated ophthalmopathy.

SNAP23 is essential for platelet and mast cell development and required in connective tissue mast cells for anaphylaxis.

Degranulation, a fundamental effector response from mast cells (MCs) and platelets, is an example of regulated exocytosis. This process is mediated by SNARE proteins and their regulators. We have previously shown that several of these proteins are essential for exocytosis in MCs and platelets. Here, we assessed the role of the SNARE protein SNAP23 using conditional knockout mice, in which SNAP23 was selectively deleted from either the megakaryocyte/platelet or connective tissue MC lineages. We found that removal of SNAP23 in platelets results in severe defects in degranulation of all three platelet secretory granule types, i.e., alpha, dense, and lysosomal granules. The mutation also induces thrombocytopenia, abnormal platelet morphology and activation, and reduction in the number of alpha granules. Therefore, the degranulation defect might not be secondary to an intrinsic failure of the machinery mediating regulated exocytosis in platelets. When we removed SNAP23 expression in MCs, there was a complete developmental failure in vitro and in vivo. The developmental defects in platelets and MCs and the abnormal translocation of membrane proteins to the surface of platelets indicate that SNAP23 is also involved in constitutive exocytosis in these cells. The MC conditional deletant animals lacked connective tissue MCs, but their mucosal MCs were normal and expanded in response to an antigenic stimulus. We used this mouse to show that connective tissue MCs are required and mucosal MCs are not sufficient for an anaphylactic response.

Human Mast Cell Proteome Reveals Unique Lineage, Putative Functions, and Structural Basis for Cell Ablation.

Mast cells are rare tissue-resident cells of importance to human allergies. To understand the structural basis of principle mast cell functions, we analyzed the proteome of primary human and mouse mast cells by quantitative mass spectrometry. We identified a mast-cell-specific proteome signature, indicative of a unique lineage, only distantly related to other immune cell types, including innate immune cells. Proteome comparison between human and mouse suggested evolutionary conservation of core mast cell functions. In addition to specific proteases and proteins associated with degranulation and proteoglycan biosynthesis, mast cells expressed proteins potentially involved in interactions with neurons and neurotransmitter metabolism, including cell adhesion molecules, ion channels, and G protein coupled receptors. Toward targeted cell ablation in severe allergic diseases, we used MRGPRX2 for mast cell depletion in human skin biopsies. These proteome analyses suggest a unique role of mast cells in the immune system, probably intertwined with the nervous system.

Anti-centrosome antibodies: Prevalence and disease association in Chinese population.

Anti-centrosome antibodies are rare findings with undefined clinical significance in clinical research. We aimed at investigating the prevalence and clinical significance of anti-centrosome antibodies in Chinese population. Testing results of total of 281,230 ANA-positive sera were retrospectively obtained from West China Hospital Sichuan University in China between 2008 and 2017. We retrospectively collected and analysed the clinical and laboratory data of the patients with positive anti-centrosome antibody. Of the 356 453 patients tested, 281 230 patients had positive antinuclear antibodies (ANAs, 78.9%), but only 78 patients with positive anti-centrosome antibodies (0.022%), of which 74.4% are females. Diagnoses were established in 69 of 78 patients: 37 cases were autoimmune diseases, mainly including undifferentiated connective tissue diseases (UCTD, 9/37), rheumatoid arthritis (RA, 6/37), Sjögren's syndrome (SS, 5/37) and primary biliary cirrhosis (PBC, 5/37), and the remaining were other autoimmune conditions. The most frequent clinical symptoms of the anti-centrosome-positive patients were arthralgia and eyes and mouth drying. Additionally, 86.7% of anti-centrosome antibodies were not associated with other ANA profiles; however, when associated, the most frequent ANA was anti-U1RNP. Anti-centrosome antibodies are featured by a low prevalence and female gender predominance. They are correlated with some specific diseases, both autoimmune diseases, especially UCTD, RA, SS and PBC, and non-autoimmune diseases, such as infection and cancer, which suggests that they might be potential supporting serological markers of these diseases.

A Connective Tissue Mast-Cell-Specific Receptor Detects Bacterial Quorum-Sensing Molecules and Mediates Antibacterial Immunity.

Quorum-sensing molecules (QSMs) are secreted by bacteria to signal population density. Upon reaching a critical concentration, QSMs induce transcriptional alterations in bacteria, which enable virulence factor expression and biofilm formation. It is unclear whether mammalian hosts can recognize QSMs to trigger responsive antibacterial immunity. We report that mouse mast-cell-specific G-protein-coupled receptor Mrgprb2 and its human homolog MRGPRX2 are receptors for Gram-positive QSMs, including competence-stimulating peptide (CSP)-1. CSP-1 activates Mrgprb2 and MRGPRX2, triggering mast cell degranulation, which inhibits bacterial growth and prevents biofilm formation. Such antibacterial functions are reduced in Mrgprb2-deficient mast cells, while wild-type mast cells fail to inhibit the growth of bacterial strains lacking CSP-1. Mrgprb2-knockout mice exhibit reduced bacterial clearance, while pharmacologically activating Mrgprb2 in vivo eliminates bacteria and improves disease score. These findings identify a host defense mechanism that uses QSMs as an "Achilles heel" and suggest MRGPRX2 as a potential therapeutic target for controlling bacterial infections.

Automatic classification of IgA endomysial antibody test for celiac disease: a new method deploying machine learning.

Widespread use of endomysial autoantibody (EmA) test in diagnostics of celiac disease is limited due to its subjectivity and its requirement of an expert evaluator. The study aimed to determine whether machine learning can be applied to create a new observer-independent method of automatic assessment and classification of the EmA test for celiac disease. The study material comprised of 2597 high-quality IgA-class EmA images collected in 2017-2018. According to standard procedure, highly-experienced professional classified samples into the following four classes: I - positive, II - negative, III - IgA deficient, and IV - equivocal. Machine learning was deployed to create a classification model. The sensitivity and specificity of the model were 82.84% and 99.40%, respectively. The accuracy was 96.80%. The classification error was 3.20%. The area under the curve was 99.67%, 99.61%, 100%, and 99.89%, for I, II, III, and IV class, respectively. The mean assessment time per image was 16.11 seconds. This is the first study deploying machine learning for the automatic classification of IgA-class EmA test for celiac disease. The results indicate that using machine learning enables quick and precise EmA test analysis that can be further developed to simplify EmA analysis.

Adult Connective Tissue-Resident Mast Cells Originate from Late Erythro-Myeloid Progenitors.

Tissue-resident mast cells are associated with many inflammatory and physiological processes. Although mast cells arise from the yolk sac, the exact ontogeny of adult mast cells remains unclear. Here we have investigated the hematopoietic origin of mast cells using fate-mapping systems. We have shown that early erythro-myeloid progenitors (EMPs), late EMPs, and definitive hematopoietic stem cells (HSCs) each gave rise to mast cells in succession via an intermediate integrin ß7+ progenitor. From late embryogenesis to adult, early EMP-derived mast cells were largely replaced by late EMP-derived cells in most connective tissues except adipose and pleural cavity. Thus, mast cells with distinct origin displayed tissue-location preferences: early EMP-derived cells were limited to adipose and pleural cavity and late EMP-derived cells dominated most connective tissues, while HSC-derived cells were a main group in mucosa. Therefore, embryonic origin shapes the heterogeneity of adult mast cells, with diverse functions in immunity and development.

Changes in the Structure and Cell Composition of Human Carinal Lymph Nodes during Aging.

Changes in the structure and cell composition of carinal lymph nodes were studied in humans during aging. Replacement of node parenchyma with fibrous connective tissue progressing with age was demonstrated. The medullary matter significantly prevailed over the cortical substance. The lymph nodes in the cortical substance were small and had no light centers; the concentration of mature CD20+ B cells was high; the paracortical area was fragmented and thinned and contained no CD4+ T helpers. Ki-67+ cells were absent in all structural components of the lymph nodes reflecting exhaustion of lymphopoietic function, which was determined by the replacement of the reticular tissue of the microenvironment with the connective tissue and by the absence of CD4+ T cells regulating cellular and humoral immunity. The disintegration of the reticular stroma in the sinus system that acts as a biological filter impairs the function of lymph purification.

Mast Cell Activation Protects Cornea by Promoting Neutrophil Infiltration via Stimulating ICAM-1 and Vascular Dilation in Fungal Keratitis.

The role of mast cells (MCs) in fungal infection is largely unknown. This study was to explore a protective role and mechanism of MCs in fungal keratitis. Experimental fungal keratitis (FK) mouse model was developed. Mice untreated (UT) or receiving corneal wound without fungal infection (Mock) were used as controls. Large number of connective tissue MCs was found in normal mice. MC activation with degranulation was largely observed, and the percentage of degranulated/total cells was high in FK. Dilated limbal vasculature with increased permeability, as well as largely infiltrated neutrophils with stimulated ICAM-1 protein levels were observed in corneas of FK mice, when compared with Mock and UT mice. Interestingly, pretreatment with cromolyn sodium (Block) significantly blocked MC degranulation, dramatically suppressed vascular dilation and permeability, and markedly reduced neutrophil infiltration with lower ICAM-1 levels in FK mice at 6-24 hours. Furthermore, the Block mice manifested prolonged disease course, increased pathological damage, and vigorous fungus growth, with much higher corneal perforation rate than FK mice at 72 h. These findings reveal a novel phenomenon that MCs play a vital role in protecting cornea against fungal infection through degranulation that promotes neutrophil infiltration via stimulating ICAM-1 production and limbal vascular dilation and permeability.

Rutin and meloxicam attenuate paw inflammation in mice: Affecting sorbitol dehydrogenase activity.

Rutin, naturally occurring flavonoid, has reported to cover interesting multiple pharmacological properties. This study evaluated rutin or/and meloxicam effects in paw inflammation induced by formalin in mice. Mice were divided into four groups: I-Formalin group, II-Rutin 60 mg/kg (p.o.), III-Meloxicam 10 mg/kg (p.o.), plus IV-Combined rutin and meloxicam. Therapies were administered once a day for 7 days. The curative effects were assessed on inflammatory, oxidative stress, and apoptosis. Both rutin and/or meloxicam induced marked improvement in paw licking time on the 1st day and by combined treatment only on the 3rd day as well reduction in paw edema% on the 3rd day. Moreover, noticeable progress in liver malondialdehyde content, superoxide dismutase, and sorbitol dehydrogenase activities as well decline in paw interleukin-1ß level and extent of apoptosis. The results spot light on the good influence of combined rutin and meloxicam in formalin-induced mice paw inflammation to a better extent than either rutin or meloxicam lonely.

Celiac antibodies in children with type 1 diabetes - A diagnostic validation study.

INTRODUCTION: Autoimmune diseases, such as celiac disease (CD) and diabetes mellitus type 1, tend to co-occur within the same patient. The prevalence of CD in diabetic children is higher than in the general population, and is estimated to be 0.6-16.4%. The diagnosis of CD is based on histopathological examination and serological testing, however, these methods are still imperfect and new diagnostic algorithms should be considered. AIM: The aim of the study was to assess the diagnostic value of serological tests detecting antibodies against deamidated gliadin peptide, endomysium, tissue transglutaminase, neo-epitope tissue transglutaminase and to identify HLA-related genetic predisposition to CD in patients with type 1 diabetes mellitus (DM1). METHODS: Autoantibodies were measured in the sera of 392 children suffering from DM1 aged 1-19 years old (mean 11.76 ± 4.14 years old). Additionally, PCR-based assessment of HLA DQ2/DQ8 genotyping was performed. RESULTS: A positive result of at least one serological test was obtained from 81 children (20.66%). The sensitivity and specificity were 76.47% and 91.67% for anti-DGP IgA, 70.59% and 58.33% for IgG anti-DGP, respectively. A positive predictive value was 100% for the anti-TG IgA at cutoff levels of 5 and 10 times higher than upper limit of reference values. HLA DQ2 and/or DQ8 were found in 97.6% of examined children. CONCLUSIONS: Tests based on anti-TG IgA are more accurate for detecting CD in children with type 1 diabetes than anti-DGP IgA. A high percentage of diabetic children carry HLA alleles predisposing to CD, which indicates that genetic screening in this group of patients is not obligated.

A Caucasian American patient with celiac disease diagnosed in Japan and successfully treated with a gluten-free diet.

We report the case of a 33-year-old Caucasian American man diagnosed with celiac disease in Japan. He presented to a community hospital because of chronic watery diarrhea and weight loss for 6 months. The laboratory data showed low serum albumin and serum cholesterol. A colonoscopy was normal. He was referred to our hospital for further work-up. Serum tissue transglutaminase immunoglobulin A (IgA) and endomysial antibody were positive. The HLA type was DQ2. Esophagogastroduodenoscopy (EGD) revealed nodular and mosaic-patterned mucosa from the bulb to the second part of the duodenum. The histopathological findings were consistent with Marsh type 3c of the modified Marsh classification for celiac disease. The patient was instructed to follow a gluten-free diet (GFD). Six months after the initiation of the GFD, his symptom and the levels of serum albumin and cholesterol were improved, and the serum tissue transglutaminase IgA and endomysial antibody became negative. However, EGD showed little improvement. Capsule endoscopy also revealed mosaic-patterned mucosa, nodular mucosa, and scalloping of the folds of the duodenum and proximal small intestine. There was no definite improvement in histopathological findings. Collectively, the GFD was effective in this patient with celiac disease, but it should be maintained to achieve endoscopic and histopathologic healing.

Epigenetics: The Future Direction in Systemic Sclerosis.

Systemic sclerosis (SSc) is an immune-mediated connective tissue disease of which the aetiology is still unclear. Previous genetic studies including candidate-gene studies and genomewide association studies have identified a number of genetic variations that confer risk to SSc. However, these variants, such as single nucleotide polymorphisms, cannot completely explain the SSc susceptibility and the diversity in the clinical symptoms of SSc patients. The contribution of epigenetic mechanisms as a link between genetics and environmental triggers represents promising field in understanding the pathogenesis of SSc. The aim of this review was to present the current knowledge on epigenetic mechanisms and highlight novel directions in diagnostic and therapeutical approaches.

The innate host response in caries and periodontitis.

INTRODUCTION: Innate immunity rapidly defends the host against infectious insults. These reactions are of limited specificity and exhaust without providing long-term protection. Functional fluids and effector molecules contribute to the defence against infectious agents, drive the immune response, and direct the cellular players. AIM: To review the literature and present a summary of current knowledge about the function of tissues, cellular players and soluble mediators of innate immunity relevant to caries and periodontitis. METHODS: Historical and recent literature was critically reviewed based on publications in peer-reviewed scientific journals. RESULTS: The innate immune response is vital to resistance against caries and periodontitis and rapidly attempts to protect against infectious agents in the dental hard and soft tissues. Soluble mediators include specialized proteins and lipids. They function to signal to immune and inflammatory cells, provide antimicrobial resistance, and also induce mechanisms for potential repair of damaged tissues. CONCLUSIONS: Far less investigated than adaptive immunity, innate immune responses are an emerging scientific and therapeutic frontier. Soluble mediators of the innate response provide a network of signals to organize the near immediate molecular and cellular response to infection, including direct and immediate antimicrobial activity. Further studies in human disease and animal models are generally needed.

Assessment of immunologic, proangiogenic and neurogenic properties of human peripheral nerve epineurium for potential clinical application.

The epineural sheath is a promising naturally occurring material for enhancement of peripheral nerve regeneration. Based on a literature search there is a limited number of reports on the biological and immunological properties of human epineurium. The goal of this study was to assess, using immunocytochemical methods, the immunological (HLA class I and II antigens, T lymphocytes, macrophages), proangiogenic (VEGF, CD31), and neurogenic (GFAP, S-100) properties of human epineurium isolated from ilioinguinal nerves (n=19) taken from deceased donors, and from sciatic nerves (n=12) taken from limbs amputated due to critical ischemia. Our studies confirmed reduced expression of HLA class II antigens on the infiltrating cells, a reduced number of T lymphocytes, and greater vessel density in the epineurium obtained from deceased organ donors. Macrophages were more abundant in the epineurium isolated from the amputated limbs. We found that the epineurium harvested from peripheral nerves of the deceased donors showed negligible immunogenic and increased proangiogenic properties compared to the epineurium of nerves taken from amputated limbs. These findings support the rationale to use human epineurium obtained from deceased donors as a new biological material for enhancement of peripheral nerve repair for potential clinical application in regenerative medicine.

Diagnosis of celiac disease and applicability of ESPGHAN guidelines in Mediterranean countries: a real life prospective study.

BACKGROUND: We assessed how the diagnosis of Celiac Disease (CD) is made and how the new ESPGHAN guidelines can be applied in children from countries with different resources. METHODS: A real life prospective study was performed in 14 centres of 13 different Mediterranean countries. Participants were asked to apply the usual diagnostic work-up for CD according to their diagnostic facilities. RESULTS: There were 1974 patients enrolled in the study, mean age 4 years, 10 months; 865 male, 1109 female. CD was confirmed in 511 (25.9%) and was unconfirmed in 1391 (70.5%) patients; 14 patients were diagnosed as having CD according to the new ESPGHAN guidelines, 43 patients were classified as having potential CD. In all participating countries the diagnosis of CD relied on histology of duodenal biopsy; in 5 countries, HLA, and in one country endomysial antibodies (EMA) were not available. Symptoms did not add a significant increase to the pre-test probability of serological tests. The positive predictive value of tissue transglutaminase type 2 (tTG) antibodies performed with different kits but all corresponding to those recommended by ESPGHAN was 96.1% (95% CI 94-97.9%) in presence of tTG > 10xULN. In 135 patients with tTG >10xULN, HLA genotyping was performed and in all it was compatible with CD. CONCLUSIONS: The results of our study show that CD diagnosis still relies on intestinal biopsy in the Mediterranean area. New ESPGHAN criteria are not applicable in 5 countries due to lack of resources needed to perform HLA genotyping and, in one country, EMA assay. Further simplification of the new ESPGHAN guidelines might be made according to what preliminarily the present results suggest if confirmed by new prospective studies.