Tattoo Pigment Identification in Inks and Skin Biopsies of Adverse Reactions by Complementary Elemental and Molecular Bioimaging with Mass Spectral Library Matching.

Tattooing has become increasingly popular throughout society. Despite the recognized issue of adverse reactions in tattoos, regulations remain challenging with limited data available and a missing positive list. The diverse chemical properties of mostly insoluble inorganic and organic pigments pose an outstanding analytical challenge, which typically requires extensive sample preparation. Here, we present a multimodal bioimaging approach combining micro X-ray fluorescence (µXRF) and laser desorption ionization-mass spectrometry (LDI-MS) to detect the elemental and molecular composition in the same sample. The pigment structures directly absorb the laser energy, eliminating the need for matrix application. A computational data processing workflow clusters spatially resolved LDI-MS scans to merge redundant information into consensus spectra, which are then matched against new open mass spectral libraries of tattoo pigments. When applied to 13 tattoo inks and 68 skin samples from skin biopsies in adverse tattoo reactions, characteristic signal patterns of isotopes, ion adducts, and in-source fragments in LDI-MS1 scans yielded confident compound annotations across various pigment classes. Combined with µXRF, pigment annotations were achieved for all skin samples with 14 unique structures and 2 inorganic pigments, emphasizing the applicability to larger studies. The tattoo-specific spectral libraries and further information are available on the tattoo-analysis.github.io website.

Effects of B-cell directed therapy on the preclinical stage of rheumatoid arthritis: the PRAIRI study.

OBJECTIVES: We explored the effects of B-cell directed therapy in subjects at risk of developing autoantibodypositive rheumatoid arthritis (RA), who never experienced inflammatory arthritis before, and explored biomarkers predictive of arthritis development. METHODS: Individuals positive for both anti-citrullinated peptide antibodies and rheumatoid factor but without arthritis were included in a randomised, double-blind, placebo-controlled study to receive a single infusion of 1000 mg rituximab or placebo. RESULTS: Eighty-one individuals received treatment and were followed up for a mean of 29.0 (0-54) months, during which 30/81 (37%) individuals developed arthritis. The observed risk of developing arthritis in the placebo-treated group was 40%, which was decreased by 55% (HR 0.45, 95% CI 0.154 to 1.322) in the rituximab-treated group at 12 months. Rituximab treatment caused a delay in arthritis development of 12 months compared with placebo treatment at the point when 25% of the subjects had developed arthritis (p<0.0001). Erythrocyte sedimentation rate and the presence of anti-citrullinated α-enolase peptide 1 at baseline were significant predictors of arthritis development. CONCLUSIONS: A single infusion of 1000 mg rituximab significantly delays the development of arthritis in subjects at risk of developing RA, providing evidence for the pathogenetic role of B cells in the earliest, prearthritis stage of autoantibody positive RA.

Lymph node biopsy analysis reveals an altered immunoregulatory balance already during the at-risk phase of autoantibody positive rheumatoid arthritis.

The balance between proinflammatory and regulatory CD4+ T cells is tightly controlled in lymphoid organs. In autoimmune diseases this balance is altered in the periphery and target tissue of patients. However, not much is known about the balance initiated in lymphoid organs during the development of disease. Since systemic autoimmunity is present years before the clinical manifestations of rheumatoid arthritis (RA), it is possible to study the immunoregulatory balance during the earliest (preclinical) phases of disease. Here, we report for the first time the frequency and phenotype of proinflammatory and regulatory CD4+ T cells in lymph node biopsies obtained from autoantibody positive individuals at risk for developing RA, patients with established disease and healthy controls. The frequency of proinflammatory LN Th1 cells was increased in RA patients compared with HCs, while the frequency of regulatory T cells was lower in LN biopsies of RA-risk individuals. Upon in vitro stimulation LN CD4+ T cells produced lower levels of proinflammatory cytokines, IFN-γ and IL-17A, in both RA-risk individuals and early RA patients. This study shows that already during the earliest phases of systemic autoimmunity the immunoregulatory balance between proinflammatory and regulatory CD4+ T cells is altered in LN tissue.

The effect of rituximab on patient reported outcomes in the preclinical phase of rheumatoid arthritis: 2 year data from the PRAIRI study.

OBJECTIVES: Early treatment of individuals at risk of developing rheumatoid arthritis (RA-risk) in the preclinical phase has the potential to positively impact both patients and society by preventing disease onset and improving patients' quality of life. The PRAIRI study was a randomised, double-blind, placebo-controlled trial with the B-cell depleting agent rituximab (RTX), which resulted in a significant delay of arthritis development of up to 12 months in seropositive RA-risk individuals. Here, we report our findings on patient-reported outcomes (PROs) in this study population. METHODS: Seventy-eight RA-risk individuals were treated with one single dose of either placebo (PBO) or 1000 mg RTX plus 100 mg methylprednisolone (MP) and anti-histamines, regardless of treatment allocation, as co-medication. Data on quality of life were collected at baseline and 1, 4, 6, 12 and 24 months using established PRO questionnaires (visual analogue scale (VAS) pain, health assessment questionnaire disability index (HAQ-DI) score, EuroQol five dimension (EQ-5D) and both physical and mental component score of the 36-item short-form heath survey (SF-36)). RESULTS: No significant changes in quality of life over a 2 year follow-up were observed in at-risk individuals treated with RTX compared to PBO given the PRO scores at 24 months (mean difference±SEM: HAQ score=0.07±0.16; EQ-5D=-0.02±0.05; VAS pain=11.11±7.40). Furthermore, no significant effect of treatment on perceived arthritis severity at the time of clinically manifest disease (arthritis) was found. CONCLUSION: One single dose of RTX plus MP administered to RA-risk individuals does not have a meaningful and measurable positive effect on PROs after 2 years of follow-up and/or perceived disease severity at the time of arthritis development. TRIAL REGISTRATION NUMBER: Trial registered at EU Clinical Trial Register, EudraCT Number: 2009-010955-29 (https://www.clinicaltrialsregister.eu/ctr-search/search?query=Prevention+of+RA+by+B+cell+directed+therapy).

Cutaneous non-allergic complications in tattoos: An overview of the literature.

Tattooing is creating a permanent design by placing exogenous pigment particles and additives into the dermis. An adverse reaction may occur due to the act of tattooing and subsequent application of aftercare products. Numerous articles report the wide spectrum of adverse reactions related to tattooing, ranging from superficial infections and vasculitis to Koebner triggered autoimmune diseases. These reactions have different time of onset of symptoms, appearing immediately after placement of the tattoo until several years later. In this article we will give an overview of cutaneous non-allergic adverse reactions of tattoos.

Impaired lymph node stromal cell function during the earliest phases of rheumatoid arthritis.

BACKGROUND: Systemic autoimmunity can be present years before clinical onset of rheumatoid arthritis (RA). Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) regulate immune responses through their intimate connection with leucocytes. We postulate that malfunctioning of LNSCs creates a microenvironment in which normal immune responses are not properly controlled, possibly leading to autoimmune disease. In this study we established an experimental model for studying the functional capacities of human LNSCs during RA development. METHODS: Twenty-four patients with RA, 23 individuals positive for autoantibodies but without clinical disease (RA risk group) and 14 seronegative healthy control subjects underwent ultrasound-guided inguinal lymph node (LN) biopsy. Human LNSCs were isolated and expanded in vitro for functional analyses. In analogous co-cultures consisting of LNSCs and peripheral blood mononuclear cells, αCD3/αCD28-induced T-cell proliferation was measured using carboxyfluorescein diacetate succinimidyl ester dilution. RESULTS: Fibroblast-like cells expanded from the LN biopsy comprised of fibroblastic reticular cells (gp38+CD31-) and double-negative (gp38-CD31-) cells. Cultured LNSCs stably expressed characteristic adhesion molecules and cytokines. Basal expression of C-X-C motif chemokine ligand 12 (CXCL12) was lower in LNSCs from RA risk individuals than in those from healthy control subjects. Key LN chemokines C-C motif chemokine ligand (CCL19), CCL21 and CXCL13 were induced in LNSCs upon stimulation with tumour necrosis factor-α and lymphotoxin α1ß2, but to a lesser extent in LNSCs from patients with RA. The effect of human LNSCs on T-cell proliferation was ratio-dependent and altered in RA LNSCs. CONCLUSIONS: Overall, we developed an experimental model to facilitate research on the role of LNSCs during the earliest phases of RA. Using this innovative model, we show, for the first time to our knowledge, that the LN stromal environment is changed during the earliest phases of RA, probably contributing to deregulated immune responses early in disease pathogenesis.

Stromal cell markers are differentially expressed in the synovial tissue of patients with early arthritis.

INTRODUCTION: Previous studies have shown increased expression of stromal markers in synovial tissue (ST) of patients with established rheumatoid arthritis (RA). Here, ST expression of stromal markers in early arthritis in relationship to diagnosis and prognostic outcome was studied. METHODS: ST from 56 patients included in two different early arthritis cohorts and 7 non-inflammatory controls was analysed using immunofluorescence to detect stromal markers CD55, CD248, fibroblast activation protein (FAP) and podoplanin. Diagnostic classification (gout, psoriatic arthritis, unclassified arthritis (UA), parvovirus associated arthritis, reactive arthritis and RA), disease outcome (resolving vs persistent) and clinical variables were determined at baseline and after follow-up, and related to the expression of stromal markers. RESULTS: We observed expression of all stromal markers in ST of early arthritis patients, independent of diagnosis or prognostic outcome. Synovial expression of FAP was significantly higher in patients developing early RA compared to other diagnostic groups and non-inflammatory controls. In RA FAP protein was expressed in both lining and sublining layers. Podoplanin expression was higher in all early inflammatory arthritis patients than controls, but did not differentiate diagnostic outcomes. Stromal marker expression was not associated with prognostic outcomes of disease persistence or resolution. There was no association with clinical or sonographic variables. CONCLUSIONS: Stromal cell markers CD55, CD248, FAP and podoplanin are expressed in ST in the earliest stage of arthritis. Baseline expression of FAP is higher in early synovitis patients who fulfil classification criteria for RA over time. These results suggest that significant fibroblast activation occurs in RA in the early window of disease.

Neo-Epitopes--Fragments of Cartilage and Connective Tissue Degradation in Early Rheumatoid Arthritis and Unclassified Arthritis.

OBJECTIVE: Tissue destruction in rheumatoid arthritis (RA) is predominantly mediated by matrix metalloproteinases (MMPs), thereby generating protein fragments. Previous studies have revealed that these fragments include MMP-mediated collagen type I, II, and III degradation, citrullinated and MMP-degraded vimentin and MMP degraded C-reactive protein. We evaluated if biomarkers measuring serum levels of specific sequences of the mentioned fragments would provide further information of diagnostic and/or prognostic processes in early arthritis. METHODS: Ninety-two early arthritis patients (arthritis duration<1 year, DMARD naïve) were enrolled. Patients either fulfilled the ACR/EULAR2010 criteria for RA (n = 60) or had unclassified arthritis (UA) (n = 32). Patients fulfilling the RA criteria after 2 years follow-up were classified into non-erosive (n = 25), or erosive disease (n = 13). Concentrations of the biomarkers: C1M, C2M, C3M, VICM and CRPM were measured in baseline serum. RESULTS: C1M, C3M and CRPM were able to discriminate between the UA and RA baseline diagnosis in 92 patients with an AUROC of 0.64 (95%CI 0.517 to 0.762), 0.73 (95%CI 0.622 to 0.838) and 0.68 (95%CI 0.570 to 0.795). C2M showed a potential for discrimination between non-erosive and erosive disease in 38 patients with an AUROC of 0.75 (95%CI 0.597 to 0.910). All of the applied biomarkers correlated with one or more of the disease activity parameters: DAS28, ESR, CRP, SJC66, TJC68 and/or HAQ. CONCLUSION: This is the first study evaluating the applied biomarkers at this early stage of arthritis. C1M, C3M, CRPM might be the best diagnostic marker, whereas high levels of C2M indicated progression of disease at follow-up in early RA patients.

Dynamic Contrast-Enhanced Magnetic Resonance Imaging Using Pharmacokinetic Modeling: Initial Experience in Patients With Early Arthritis.

OBJECTIVE: Analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using pharmacokinetic modeling (PKM) provides quantitative measures that mirror microvessel integrity and can be used as an objective marker of the level of synovial inflammation. The aim of this study was to investigate the PKM parameters K(trans) , kep , and ve in a prospective cohort of disease-modifying antirheumatic drug (DMARD)-naive patients with early arthritis, and to validate the results by assessing their correlation with the number of synovial endothelial cells (ECs). METHODS: Forty-seven patients with early arthritis (arthritis duration <1 year, DMARD naive; comprising 14 patients with rheumatoid arthritis, 22 with unclassified arthritis, 6 with spondyloarthritis [SpA], and 5 with other arthritides) were included. At baseline, DCE-MRI was performed on an inflamed knee joint of each patient. These images were used to calculate the K(trans) (volume transfer constant between the plasma and extracellular extravascular space [EES]), the kep (transfer constant between the EES and plasma), and the ve (fractional volume of the EES). Second, markers of disease activity were collected. Finally, vascularity was evaluated by immunohistochemical analysis of synovial tissue samples obtained from the inflamed knee joints, using antibodies to detect von Willebrand factor (vWF), a marker of ECs. RESULTS: The 3 PKM parameters differed significantly between diagnostic groups at baseline, with the highest K(trans) value being observed in patients with SpA (median 0.050/minute, interquartile range [IQR] 0.041- 0.069). Furthermore, the K(trans) , kep , and ve values correlated significantly with markers of disease activity. Finally, the PKM parameters K(trans) and kep , but not ve , correlated significantly with synovial expression of vWF (r = 0.647, P = 0.004 for K(trans) ; r = 0.614, P = 0.007 for kep ; r = 0.398, P = 0.102 for ve ). CONCLUSION: These results suggest that the K(trans) , kep , and ve can be used to detect synovial inflammation in patients with early arthritis, and these PKM parameters may be helpful in differential diagnosis. This approach may also be useful in translational research analyzing tissue microcirculation and angiogenesis.

Nuclear Factor-κB-inducing Kinase Is Expressed in Synovial Endothelial Cells in Patients with Early Arthritis and Correlates with Markers of Inflammation: A Prospective Cohort Study.

OBJECTIVE: The nuclear factor-κB (NF-κB) family of transcription factors is strongly involved in synovial inflammation. We have previously shown that NF-κB-inducing kinase (NIK) is a key regulator of inflammation-induced angiogenesis in rheumatoid arthritis (RA) synovial tissue (ST). Here, we investigated synovial NIK expression in patients with early arthritis and in autoantibody-positive individuals at risk of developing RA. METHODS: ST biopsies were obtained by arthroscopy from 154 patients with early arthritis (duration < 1 yr) with various diagnoses and 54 IgM rheumatoid factor-positive and/or anticitrullinated protein antibodies-positive individuals without evidence of arthritis. ST was stained for NIK and endothelial cell (EC) markers. Additionally, measures of disease activity were collected and contrast-enhanced magnetic resonance imaging (MRI) was performed in a subset of these patients. RESULTS: In patients with early arthritis, NIK was predominantly expressed in EC of small blood vessels. Further, NIK expression correlated with erythrocyte sedimentation rate (r 0.184, p = 0.024), C-reactive protein (r 0.194, p = 0.017), joint swelling (r 0.297, p < 0.001), synovial immune cell markers (lining r 0.585, p < 0.001; sublining macrophages r 0.728, p < 0.001; T cells r 0.733, p < 0.001; and B cells r 0.264, p = 0.040), MRI effusion (r 0.665, p < 0.001), MRI synovitis (r 0.632, p < 0.001), and MRI total score (r 0.569, p < 0.001). In 18.5% of autoantibody-positive individuals, ST NIK(+)EC were present, but this was not predictive of the development of arthritis. CONCLUSION: NIK(+)EC are present in the earliest phase of synovial inflammation and may be indicative of high angiogenic activity in the inflamed ST. Therefore, NIK(+)EC may play an important role in the persistence of synovitis. Collectively, our data underscore the importance of angiogenesis in synovial inflammation and identify NIK as a potential therapeutic target in arthritis.

Serum Vaspin Levels Are Associated with the Development of Clinically Manifest Arthritis in Autoantibody-Positive Individuals.

OBJECTIVES: We have previously shown that overweight may increase the risk of developing rheumatoid arthritis (RA) in autoantibody positive individuals. Adipose tissue could contribute to the development of RA by production of various bioactive peptides. Therefore, we examined levels of adipokines in serum and synovial tissue of subjects at risk of RA. METHODS: Fifty-one individuals positive for immunoglobulin M rheumatoid factor (IgM-RF) and/or anti-citrullinated protein antibodies (ACPA), without arthritis, were included in this prospective study. Levels of adiponectin, vaspin, resistin, leptin, chemerin and omentin were determined in baseline fasting serum samples (n = 27). Synovial tissue was obtained by arthroscopy at baseline and we examined the expression of adiponectin, resistin and visfatin by immunohistochemistry. RESULTS: The development of clinically manifest arthritis after follow-up was associated with baseline serum vaspin levels (HR1.5 (95% CI 1.1 to 2.2); p = 0.020), also after adjustment for overweight (HR1.7 (95% CI 1.1 to 2.5); p = 0.016). This association was not seen for other adipokines. Various serum adipokine levels correlated with BMI (adiponectin r = -0.538, leptin r = 0.664; chemerin r = 0.529) and systemic markers of inflammation such as CRP levels at baseline (adiponectin r = -0.449, omentin r = -0.557, leptin r = 0.635, chemerin r = 0.619, resistin r = 0.520) and ESR (leptin r = 0.512, chemerin r = 0.708), p-value<0.05. Synovial expression of adiponectin, resistin and visfatin was not associated with development of clinically manifest arthritis. CONCLUSIONS: In this exploratory study, serum adipokines were associated with an increased inflammatory state in autoantibody-positive individuals at risk of developing RA. Furthermore, serum vaspin levels may assist in predicting the development of arthritis in these individuals.

Expression of Prostaglandin E2 Enzymes in the Synovium of Arthralgia Patients at Risk of Developing Rheumatoid Arthritis and in Early Arthritis Patients.

OBJECTIVE: Arthralgia may precede the development of synovial inflammation in autoantibody-positive individuals at risk of developing rheumatoid arthritis (RA). A major pathway involved in pain is the prostaglandin (PG) E2 pathway. We investigated this pathway in the synovium of individuals with RA-specific autoantibodies and in early arthritis patients. METHODS: Nineteen autoantibody-positive individuals (IgM-rheumatoid factor and/or anti-cyclic citrullinated peptide antibodies) with arthralgia (n=15) and/or a positive family history of RA (n=8), who had been prospectively followed for at least 2 years, were included. In addition, we included early arthritis patients (disease-modifying antirheumatic drug naïve) who after 2 years follow up fulfilled classification criteria for RA (n=63), spondyloarthritis (SpA; n=14), or had unclassified arthritis (UA; n=27). In all subjects we assessed pain and performed synovial biopsy sampling by mini-arthroscopy at baseline. Tissue sections were examined by immunohistochemistry to detect and quantify PGE2 pathway enzymes expression levels (mPGES-1; COX-1 and -2; 15-PGDH). RESULTS: In both study groups synovial expression of PGE2 enzymes was not clearly related to pain sensation. Expression levels at baseline were not associated with the development of arthritis after follow up (6 out of 19 autoantibody-positive individuals). However, in early SpA patients the expression levels of mPGES-1 and COX-1 were significantly increased compared to RA and UA patients. CONCLUSION: Pain in autoantibody-positive individuals without synovial inflammation who are at risk of developing RA and in early arthritis patients may be regulated by pathways other than the PGE2 pathway or originate at sites other than the synovium. In contrast, in SpA, the PGE2 pathway may be inherently linked to the pathophysiology/etiology of the disease.