Welcome to the Autoimmunity 2021 Congress Calendar

Background and Aims

Optineurin (OPTN) is an autophagy receptor with multiple properties and has been reported to act as a negative regulator of osteoclastogenesis in osteoclast precursors. Since RANKL expressed on synovial fibroblasts (SFs) plays a major role in osteoclastogenesis in rheumatoid arthritis (RA) joint, we aimed to explore the role of OPTN in SFs form RA patients (RASFs).

Methods

The expression of OPTN was analyzed in RASFs using RT-qPCR and western blot. Cell surface expression of RANKL was analyzed by flow cytometry. OPTN was downregulated in RASFs using siRNA. Monocytes were cocultured with OPTN-reduced RASFs and stained with Tartrate-Resistant Acid Phosphatase (TRAP) to evaluate osteoclast differentiation. Protein levels of IκBα and nuclear NF-κB1 were measured to evaluate NF-κB signaling.

Results

OPTN was upregulated by TNF-α or IFN-γ in RASFs at both mRNA and protein levels. Cell surface RANKL was significantly increased following treatment with TNF-α or IFN-γ and the effect was further pronounced in OPTN-reduced RASFs compared to control RASFs, while osteoprotegerin levels remained unchanged after OPTN knockdown. Monocytes cocultured with OPTN-reduced RASFs differentiated more into TRAP+ multinucleated cells compared to those cocultured with control RASFs. MMP3 and IL-6 were upregulated while GATA-3, CHST15 and HAS1 were downregulated in OPTN-reduced RASFs. IκBα degradation and nuclear NF-κB1 expression following TNF-α treatment was prolonged in OPTN-reduced RASFs.

Conclusions

OPTN plays a protective role in RA with its upregulation when immersing with pro-inflammatory cytokines. Absence of OPTN might worsen RA by generating joint destructive state including increased RANKL expression on RASFs and subsequent osteoclast differentiation.

Background and Aims

The quality of antigen is believed to be important to an in vitro diagnostic test. Protein purification from human/animal tissue and recombinant expression technology are the two commonly used techniques to acquire antigens. Purification of certain antigens does not completely eliminate the contamination with other cell components which might cause falsely elevated signals. Here, we aim to compare the way how antigens are purified and how this affects the results of routine samples.

Methods

First, we compared two commercially available tests for the measurement of anti-human thyroid peroxidase (TPO) antibodies. While one test applies TPO purified from human thyroid gland, the other applies recombinant human TPO. Various samples containing anti-TPO and/or anti-thyroglobulin (TG) antibodies were measured with both tests.

Results

When testing serum samples solely containing anti-TG antibodies, a strong signal was observed with the test applying purified TPO but not with the test that applies recombinant TPO. This indicates a potential interference of anti-TG antibodies on an anti-TPO diagnostic test that is dependent on the applied TPO antigen. To determine if this signal was indeed caused by anti-TG antibodies, these antibodies were depleted from samples containing anti-TG and/or anti-TPO antibodies by incubating the with soluble TG. Anti-TG-antibody-deplete samples, independent of anti-TPO antibody concentrations, showed a clear decrease in signal in the test using purified TPO, whereas the concentration remained unchanged when TPO was recombinant.

Conclusions

These results highlight that the antigen preparation significantly affects an antibody test’s performance. Test applying purified TPO could give false positive results when samples contain anti-TG antibodies.

Background and Aims

In Australia, lupus nephritis (LN) affects a culturally diverse population of patients. We established the Lupus Nephritis Australian Registry (LUNAR) to better understand the patient population receiving Myfortic and other immunosuppressants, and to analyse the safety and efficacy of the treatments they received.

Methods

A non-interventional, multicentre, registry of patients with biopsy-proven ISN/RPS class III, IV or V LN, treated with Myfortic or other immunosuppressants, was established. We collected baseline demographics and 6-monthly follow-up clinical and safety data over a 5-year period (2013-2018).

Results

149 patients were enrolled across 8 sites (83.7% female; mean age 38.8yrs). Most were Caucasian (45.2%) - patients of Asian ethnicity (29.6%) and Aboriginal/Torres Strait Islander or Maori/Pacific Islander descent (10.4%) were over-represented compared to the general population. The mean duration of SLE and LN was 8.6yrs and 5.4yrs, respectively. Most patients had class IV LN on initial kidney biopsy (59.3%), with 21.5% having class III and 11.1% having class V LN. Immunosuppressants used prior to screening included corticosteroids (68.9%), mycophenolate mofetil ((MMF) 61.5%), Myfortic (41.5%) and cyclophosphamide (32.6%). During the study, most patients were treated with Myfortic (76.3%), MMF (65.2%) and/or hydroxychloroquine (67.4%). Rituximab and azathioprine were each started in 8.9% of patients during the study. Kidney function was preserved for most patients during the study (eGFR decline tended to be associated with older patients and patients with a longer duration of LN) and mycophenolate-based therapy was well-tolerated.

Conclusions

LUNAR is the first study outlining the demographics, outcomes and practice patterns in the management of Australian LN patients.

Background and Aims

Vitiligo is a disorder of the skin affecting 1% of world population. Even if the role of immune system seems to be well-established, the pathogenesis of the disease remains unclear. We Investigated the role of MIA (melanoma inhibitory protein) in a new mouse model of development of vitiligo-like patchtes after having previously observed that MIA is present in the skin of patients and interacts with proteins which keep melanocytes firmly attached to the basal membrane.

Methods

C57/Bl6 mice (n=10 per conditions) were treated in the tail every 15 days for 3 months with saline (control) or MIA 1% solution to induce pigment loss. Depigmentation spots were scored by blinded observators. Histological examination was performed to evaluate the differences between normal and pigmented skin, melanocytes and inflammatory markers.

Results

The control group did not show any sign of depigmentation. The treated group showed, instead, clear zones of complete depigmentation with the appearance of white patches with whitening of the hair and a clear-cut edge. Histology showed the absence of melanocytes, without presence of inflammatory cells. According to our data, melanocytes are not only directly destroyed by the immune system but detach from the basal membrane toward the stratum corneum and exfoliate together with the surrounding keratinocytes, creating the depigmented macules.

Conclusions

This study demonstrates that MIA protein is able itself to create a vitiligo-like patch by detaching the melanocytes from the basal membrane providing experimental evidence that not only an autoimmune pathway seems to be related to the vitiligo-like depigmentation.

Background and Aims

Automated systems for anti-nuclear antibody (ANA) detection by indirect immunofluorescence analysis (IFA) provide an estimate of the fluorescence intensity (FI). The aim was to evaluate whether variability in FI estimations could be reduced by using a standard preparation.

Methods

Two standards [IUIS/ASC ANA#4 (large speckled; 1/8 dilution) and DFS Megapool] and three serum samples were distributed to 34 laboratories using automated IFA (ZenitPro;n=2, Zenit G-sight;n=5, EUROPattern;n=10, NOVAView;n=14, Image Navigator;n=3). Samples were analysed in duplicate in 5 runs. Sample1 was also distributed by the Belgian External Quality Assessment scheme (EQA). Instrument-specific FI and/or estimated titers were documented. Besides, relative fluorescence or light units (RLU) and titer ratios were calculated based on the two standards.

Results

EQA results revealed an inter-laboratory FI variability that tended to be lower for automated compared to manual IFA analysis.

With almost every automated IFA system, outlier results were observed.

Imprecision results revealed pattern-dependent coefficient of variations (CV%), with lowest CV% for DFS Megapool for all substrates.

The use of the DFS Megapool as a reference material to calculate RLUs and titer ratios reduced inter-laboratory variation (e.g. sample1: CV% without standard: 12.1-16.9% versus CV% with standard: 1.4-8.9%;p<0.05) and aligned results between instruments from the same manufacturer and between instruments from different manufacturers. Such effect was not observed when the IUIS/ASC ANA#4 was used as reference material.

Conclusions

Automated IFA analysis allows to align FI measurements, if a common protocol is applied, a total process iQC program is implemented and a suitable standard is available. The DFS Megapool presented the best reproducibility.

Background and Aims

During the past few years, the safety of silicone breast implants (SBIs) has stirred an intense debate, concerning their potential for induction of autoimmunity. Recently we found that women with SBIs had an increased risk of having an autoimmune disease. The serologic presence of autoantibodies against muscarinic/adrenergic receptors, has been described previously in autoimmune diseases. In the current study, we aimed to evaluate circulating autoantibodies level against muscarinic (M1-M5) and adrenergic (α1, α2, β1, β2) receptors in women with SBIs, and to explore any correlation between autoantibodies level and the severity of clinical symptoms reported by the patients.

Methods

A test panel of autoantibodies against muscarinic (M1-M5) and adrenergic (α1, α2, β1, β2) receptors was determined in the sera of 25 females with SBIs, using 'Auto-Antibodies against G protein-coupled receptors ELISA' kit.

Results

Patients reported shared clinical symptoms such as myalgia, arthralgia, sleep disturbance, chronic fatigue, memory loss and dry mouth. ELISA examination of circulating autoantibodies revealed 100% of patients with positive levels of anti-muscarinic cholinergic receptor type 3 (anti-M3R) antibodies, 80% of patients with positive levels of anti-α1 adrenergic receptor (anti-α1AR) antibody and 54% of patients shows positive levels of both anti-M2R and anti-α2AR antibodies. More importantly, we found direct correlation between certain anti-muscarinic or adrenergic receptor autoantibody level and the severity of specific reported symptoms which relates to the receptor’s physiological function in the body.

Conclusions

Our results highlight the importance of circulating anti-muscarinic and adrenergic autoantibodies for the diagnosis, pathogenesis and potential therapy for women with SBIs-induced clinical syndrome.

Background and Aims

Anti-DFS70/LEDGFp75 auto-antibodies (Anti-DFS 70 Ab) usually target the dense fine speckled protein of 70kDa (DFS70), commonly known as lens epithelium-derived growth factor p75 (LEDGFp75).

This auto-antigen is thought to be a stress response protein that is ubiquitously expressed in mammalian cells and tissues, with overexpression in cancer cells and tumors such as breast cancer

The aim of our work was to study the frequency of Anti-DFS70 Ab and if they could be a new biomarker in breast cancer.

Methods

Is a prospective descriptive study including 32 patients with a breast cancer in Military Hospital of Tunis

The blood samples and clinical data were collected from all patients. The Serological analyses were performed using Dot Technic EUROIMMUN ^© in the laboratory of immunology. Written informed consent was obtained from all participants

Results

The middle age of patients was 47 years

We found a frequency of 15,63 % of Anti-DFS70 Ab in our breast cancer patients

There was a significant correlation between Anti DFS 70 Ab and the tumor size (p=0.038 <p=0.5).

We have noted also that all patients with lymph node involvement have positive Anti- DFS70 Ab.

Conclusions

Our results suggests that Anti-DFS Ab could be a new biomarker in breast cancer.

A more large study is needed to confirm our results.

Background and Aims

Regulatory features of B-cells have gained increased attention in patients with systemic vasculitis, including granulomatosis with polyangiitis (GPA). The aim with this study was to investigate the frequencies and phenotypes of two putative regulatory B (Breg) cell populations and assess the capacity of B-cells from GPA patients to regulate T-cell activation.

Methods

37 GPA patients (22 remission and 15 active) and 31 healthy controls (HC) were included for measurement of CD19⁺CD24^highCD27⁺ (memory) and CD19⁺CD24^highCD38^high (transitional) Breg cells, and 11 GPA patients in remission and 12 HC for the functional studies of B-cells. HC were matched for age and gender.

Results

Memory Breg cells were reduced in GPA both during active disease and remission compared with HC, whereas transitional Breg cells did not differ between the groups. Memory Breg cells consisted of less CD25⁺ cells during both active disease and remission but expressed more PD-L1 and CD86 during remission compared with active disease and HC. B-cells from GPA patients regulated T-cell proliferation and IL-17a production but failed to regulate IFN-γ production (Fig 1). The levels of IFN-γ and the IFN-γ regulated cytokine CXCL10 were elevated in patient plasma.

Conclusions

Despite reduced levels and altered phenotypes of memory Breg cells, the capacity of GPA patient B-cells to regulate T-cell proliferation and IL-17a production was unaffected. However, we found a profound inability of patient B-cells to regulate T-cell IFN-γ production. This alteration could be important for the persistent inflammation that continues during remission and results in the chronic relapsing nature of the disease.

Background and Aims

Epidemiological data on primary Sjögren syndrome (pSS) are variable and depend on the diagnostic criteria. We determined the incidence rate of pSS in Slovenia using the 2002 American European (AE) and the new ACR/EULAR 2016 classification criteria.

Methods

We included 399 consecutive subjects (365 females, mean age 57.8 +/- 13.8, range: 21.4-88.5) referred for evaluation of pSS at our department, which provides rheumatology services for 704,000 adult residents. Subject assessment included questionnaire about ocular and oral involvement, Schirmer-I test, unstimulated salivary flow (USF) test, Rose Bengal scoring, immunoserological tests, and minor salivary gland biopsy. Gender- and age-specific pSS incidence rates were calculated based on the ACR/EULAR and the AE criteria.

Results

We identified 109 incipient cases of pSS (104 females) according to the ACR/EULAR criteria. Annual incidence rate was 5.2/100,000 adults (95% CI 4.3-6.2), 9.6/100,000 females (95% CI 7.9-11.6) and 0.5/100,000 males 0.5 (95% CI 0.2-1.1). Only 90/109 subjects also fulfilled the AE criteria. The discrepancy in the criteria fulfillment was mainly due to lack of objective sicca symptoms. The 109 subjects with pSS had anti-Ro antibodies in 72%, histologically positive focus score in 83%, while 57 % were positive for both.

Conclusions

The pSS incidence is higher than previously reported in Slovenia, possibly due to the higher sensitivity of the ACR/EULAR 2016 classification criteria in our cohort.

Background and Aims

Membranous glomerulonephritis (MG) causes about 25% of nephrotic syndromes in the adult. Etiology is autoimmune and it is due to the deposit of anti-phospholipase A2 receptor (Anti-PLA2R) immune complexes in the glomerular basement membrane. Anti-PLA2R is highly specific for primary membranous glomerulonephritis (PMG).

The aim was to implement the Anti-PLA2R (ELISA) in our laboratory and study its correlation with PMG, being a request from the department of Nephrology, as our hospital hosts a reference center for transplants.

Methods

Values of Anti-PLA2R were determined in 40 patients by ELISA, using a commercialized kit (Anti-PLA2R ELISA (IgG), by EUROIMMUN, Germany). Patients’ age ranged from 18 to 80. 22 individuals with Systemic Lupus Erythematosus (SLE), Diabetes Mellitus or other autoimmune diseases, alongside 2 patients with known Anti-PLA2R positive PMG, formed the problem group. 20 of them had nephropathy or proteinuria. The other 16 were healthy controls or had no autoimmune disease.

Results

39 presented negative Anti-PLA2R levels, except for one with PMG, with similar levels to the previous ones (53.58 RU/ml) from an external laboratory. The second PMG patient had 8.84 RU/ml, lower than previous results, which could indicate a good control of the disease.

3 patients with MG and SLE had levels of <0.1 RU/ml, implying a secondary cause of the MG.

Conclusions

Anti-PLA2R detection by ELISA allows quantification, which is necessary for the long-term follow-up of PMG, as it correlates with clinical manifestations and biochemical data of the associated renal failure. It is also useful for the evaluation of transplanted patients with PMG.

Background and Aims

The pathophysiology of inflammatory bowel diseases (IBD) reflects a balance between mucosal injury and reparative mechanisms. Some regenerating gene (Reg) family members (REG Iα, REG Iβ, and REG IV) are expressed in Crohn’s disease (CD) and ulcerative colitis (UC) and involved as proliferative mucosal factors in IBD. We revealed that REG Iαand REG Iβ were induced by IL-6/IL-22 via MZF1, RTEF1/TEAD4, and STAT3 in REG Iα and HLTF/FOXN2F in REG Iβ, respectively. Although REG IV was up-regulated in IBD biopsy samples, the up-regulation of REG IV was not at all observed in cell culture system by autoimmune-related cytokines such as IL-6, IL-8, IL-17A, IL-22, TNFα, HGF, bFGF, and EGF.

Methods

Here, we analyzed REG IV expression in LS-174T and HT-29 human colonic epithelial cells by real-time RT-PCR and ELISA.

Results

REG IV expression was induced by lipopolysaccharide (LPS) in both cells. However, LPS did not activate REG IVpromoter (-1,053~+22) activity. As the LPS-induced up-regulation of REG IV was considered to be regulated post-transcriptionally, we searched targeted microRNA (miR) using the MicroRNA.org program (http://www.microrna.org/microrna/home.do), which revealed that REG IV mRNA have a potential target sequence for miR-24. We measured the miR-24 level of LPS-treated cells by RT-PCR and found that the level was significantly lower (0.0278±0.00665 fold in LS-174T cells [P=0.0368]; 0.1400±0.0618 fold in HT-29 cells [P=0.0490]).

Conclusions

The LPS-induced increase of REG IV mRNA was abolished by the introduction of miR-24 mimic but not by non-specific control RNA in both LS-174T and HT-29 cells.