High-temperature deep-level transient spectroscopy system for defect studies in wide-bandgap semiconductors.

Full investigation of deep defect states and impurities in wide-bandgap materials by employing commercial transient capacitance spectroscopy is a challenge, demanding very high temperatures. Therefore, a high-temperature deep-level transient spectroscopy (HT-DLTS) system was developed for measurements up to 1100 K. The upper limit of the temperature range allows for the study of deep defects and trap centers in the bandgap, deeper than previously reported by DLTS characterization in any material. Performance of the system was tested by carrying out measurements on the well-known intrinsic defects in n-type 4H-SiC in the temperature range 300-950 K. Experimental observations performed on 4H-SiC Schottky diodes were in good agreement with the literature. However, the DLTS measurements were restricted by the operation and quality of the electrodes.

Rare and Common Variants Conferring Risk of Tooth Agenesis.

We present association results from a large genome-wide association study of tooth agenesis (TA) as well as selective TA, including 1,944 subjects with congenitally missing teeth, excluding third molars, and 338,554 controls, all of European ancestry. We also tested the association of previously identified risk variants, for timing of tooth eruption and orofacial clefts, with TA. We report associations between TA and 9 novel risk variants. Five of these variants associate with selective TA, including a variant conferring risk of orofacial clefts. These results contribute to a deeper understanding of the genetic architecture of tooth development and disease. The few variants previously associated with TA were uncovered through candidate gene studies guided by mouse knockouts. Knowing the etiology and clinical features of TA is important for planning oral rehabilitation that often involves an interdisciplinary approach.

Phosphorylation of intracellular signalling molecules in peripheral blood cells from patients with psoriasis on originator or biosimilar infliximab.

BACKGROUND: Psoriasis vulgaris is a chronic, inflammatory skin disease characterized by a dysregulated immune response and it is associated with substantial systemic comorbidities. Biological drugs such as tumour necrosis factor (TNF)-α inhibitors can ameliorate the disease but are expensive. Biosimilar drugs have the same amino-acid sequence as the originator, but differences in manufacturing can affect biological activity, efficacy and tolerability. OBJECTIVES: To explore potential differences in intracellular phosphorylation of signalling molecules in peripheral blood cells from patients with psoriasis treated with the TNF-α inhibitor infliximab compared with healthy controls, and to investigate if the phosphorylation pattern was influenced by switching from the originator infliximab to the biosimilar CT-P13. METHODS: By flow cytometry, we measured phosphorylation of nuclear factor kappa B, extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and signal transducer and activator of transcription 3, before and after TNF-α stimulation in monocytes and T, B, natural killer and CD3+  CD56+ cells from 25 patients with psoriasis treated with infliximab and 19 healthy controls. RESULTS: At inclusion, phosphorylation levels of peripheral blood mononuclear cells (PBMCs) were increased in patients with psoriasis compared with healthy controls, even though clinical remission had already been achieved. Phosphorylation levels declined in patients on both originator infliximab and biosimilar during continued treatment. No significant differences were detected between the two medications after 12 months. CONCLUSIONS: Patients with psoriasis on infliximab have higher activation levels of PBMCs than do healthy controls, possibly reflecting systemic inflammation. Switching from the originator infliximab to biosimilar CT-P13 did not affect phosphorylation levels or clinical parameters, suggesting that CT-P13 is a noninferior treatment alternative to the originator infliximab.

Patients with Primary Sjögren's Syndrome Have Alterations in Absolute Quantities of Specific Peripheral Leucocyte Populations.

An accurate dissection of peripheral blood enumeration is lacking in primary Sjögren's syndrome (pSS). The purpose of this study was to quantify different leucocyte populations in peripheral blood of patients with pSS. Numbers of specific leucocyte subsets were determined in 86 pSS patients and 74 healthy donors quantifying 21 distinct subtypes by flow cytometry. Subgroups of pSS patients were stratified based on presence of extraglandular manifestations (EGMs) and SSA/SSB autoantibodies. Overall, pSS patients manifested decreased lymphocyte subpopulations compared to healthy donors. Such decreases were more pronounced in SSA/SSB positive patients and patients with EGM. Granulocyte and monocyte subpopulations were increased in pSS patients compared to healthy donors, with the greatest increases in SSA/SSB positive patients. Unsupervised hierarchal clustering based on cell quantities was used to further subgroup the pSS patients into four clusters. One of the clusters characterized by higher concentrations of NKT cells, CD56hi NK cells, CD20+ CD38- B cells and CD8+ CD38- T cells was associated with weaker clinical symptoms than the other clusters, possibly marking a milder disease phenotype. In conclusion, our analyses indicate significant alterations in the cellular profiles of peripheral blood leucocytes in patients with pSS and may help to stratify the patients according to disease severity.

Expression of Toll-Like Receptors in Peripheral Blood Mononuclear Cells of Patients with Primary Sjögren's Syndrome.

Toll-like receptors (TLRs) are pattern recognition receptors important for the detection of pathogen-associated molecular patterns. They are localized on cellular membranes, on either the cell surface or the endosomes. Primary Sjögren's syndrome (pSS) is a systemic rheumatic autoimmune disease characterized by lymphocytic infiltrations in exocrine glands resulting in dryness in eyes and mouth. In a majority of patients, autoantibodies against Ro/SSA and/or La/SSB are present. Here we analysed mRNA levels of TLR1-10 and protein expression levels of most of them in human peripheral blood mononuclear cells from 20 patients with pSS and 20 healthy controls. Patients with pSS showed significantly higher mRNA levels of TLR8 than controls, while transcript levels of TLR9 were significantly lower. At the protein level, patients with pSS expressed significantly less TLR5 and significantly more TLR7 compared with healthy controls. TLR7 and 8 are encoded by genes localized on the X chromosome, which is especially interesting regarding the gender imbalance of pSS. The differential expression of various TLR in PBMC of patients with pSS might contribute to an altered recognition of nucleic acids, eventually resulting in the development of autoimmune disease.

Calprotectin (S100A8/A9) and S100A12 are associated with measures of disease activity in a longitudinal study of patients with rheumatoid arthritis treated with infliximab.

OBJECTIVES: The pro-inflammatory proteins calprotectin (a heterocomplex of S100A8/A9) and S100A12 have been associated with disease activity in rheumatoid arthritis (RA). The aim of this study was to compare their potential as biomarkers in a prospective study of RA patients starting with infliximab as their first biological disease-modifying anti-rheumatic drug (DMARD). METHOD: Thirty-nine RA patients were examined and serum samples collected when starting with infliximab and after 3, 6, and 12 months. Calprotectin and S100A12 were analysed by enzyme-linked immunosorbent assays (ELISAs) and, together with C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), measured at all time points. A disease activity score of 28 joints (DAS28) was calculated. Radiographs of the hands, wrists, and feet were taken at baseline and after 3 years, and assessed according to the modified Sharp/van der Heijde (SvH) score. Responsiveness was evaluated according to the European League of Associations for Rheumatology (EULAR) response criteria based on 28 joints. RESULTS: Both S100 proteins were significantly higher in seropositive than in seronegative patients (p = 0.01). Calprotectin correlated significantly with CRP (ρ = 0.51-0.75), ESR (ρ = 0.32-0.52), and DAS28 (ρ = 0.32-0.62). S100A12 correlated with calprotectin (ρ = 0.62-0.77) and CRP (ρ = 0.32-0.63). The S100 proteins, and especially calprotectin (ρ = 0.23-0.39), showed weak associations with radiographic progression, unlike CRP/ESR. None of the S100 proteins could predict responsiveness. CONCLUSIONS: Calprotectin showed the strongest correlation with measures of disease activity and may be better than S100A12 when evaluating disease activity in RA patients. More extensive studies are needed to further compare the predictive value of the S100 proteins relative to radiographic progression.

Low Protein A20 in Minor Salivary Glands is Associated with Lymphoma in Primary Sjögren's Syndrome.

Patients with primary Sjögren's syndrome (pSS) have an increased risk of developing lymphomas, particularly the subtype mucosa-associated lymphoid tissue (MALT) lymphoma. Chronic antigen stimulation and increased activation of nuclear factor-κB (NF-κB) are important factors for the pathogenesis of MALT lymphomas. Protein A20 is an inhibitor of NF-κB. A recent study of pSS-associated MALT lymphomas identified potential functional abnormalities in the TNFAIP3 gene, which encodes protein A20. The present study aimed to assess protein A20 by immunohistochemistry (IHC) in minor salivary glands (MSGs) and lymphoma tissue sections of patients with pSS and investigate a potential association with lymphoma development. Protein A20 staining in lymphocytes was scored in four categories (0 = negative, 1 = weak, 2 = moderate and 3 = strong). For statistical purposes, these scores were simplified into negative (scores 0-1) and positive (scores 2-3). We investigated associations between protein A20-staining, focus scores, germinal centre (GC)-like structures and monoclonal B-cell infiltration in MSGs. MSG protein A20 staining was weaker in pSS patients with lymphomas than in those without lymphomas (P = 0.01). Weak protein A20 staining was also highly associated with a lack of GC formation (P < 0.01). Finally, weaker A20 staining was observed in the majority of pSS-associated MALT lymphoma tissues. In conclusion, we found absent or weak protein A20 immunoreactivity in MSGs of patients with pSS with lymphomas. This finding indicates that protein A20 downregulation in lymphocytes might be a mechanism underlying lymphoma genesis in patients with pSS.

TLR-7 and -9 Stimulation of Peripheral Blood B Cells Indicate Altered TLR Signalling in Primary Sjögren's Syndrome Patients by Increased Secretion of Cytokines.

Primary Sjögren's syndrome (pSS) is a chronic, inflammatory autoimmune disease characterised by lymphocytic infiltrations in the exocrine glands, resulting in destruction of salivary and lacrimal glands. B cells have an important role in the disease, as detection of autoantibodies against SSA/Ro or SSB/La is one of the diagnostic criteria, being found in a majority of the patients. Toll-like receptors (TLR) are pattern recognition receptors. TLR-7 and -9 are found in endosomes and bind microbial nucleic acids. We have previously shown that pSS patients and healthy controls have similar expression pattern of TLR-7 and -9 in various B-cell populations. In this study we further analysed the responsiveness of B cells upon TLR stimulation. B cells isolated from peripheral blood of 21 pSS patients and 18 healthy controls were stimulated with TLR-7 and -9 ligands for 24 h before being analysed for the expression of certain surface markers and intracellular cytokine levels by flow cytometry. Secreted cytokines were measured by a multiplex cytokine assay. Patients with pSS had more naïve and less preswitched memory B cells compared to controls in unstimulated as well as via TLR-7 stimulated cells. Unstimulated and via TLR-7 stimulated B cells from pSS patients also had fewer IL-10(+) preswitched memory B cells. Moreover, TLR-7 and -9 stimulated B cells of pSS patients secreted increased amounts of several cytokines. B cells of pSS patients show a different responsiveness upon stimulation of TLR-7 and -9 compared to controls.

Peritumoral dermis of squamous cell carcinomas in renal transplant recipients contains less CD11c+ myeloid dendritic cells and FoxP3+ T cells compared to immunocompetent controls.

BACKGROUND: Renal transplant recipients (RTR) have an increased risk of developing cutaneous squamous cell carcinomas (SCC). These SCC are often more aggressive than SCC in immunocompetent individuals. OBJECTIVES: In this comparative study, we analysed the cell composition in the tissue immediately surrounding invasive SCC in immunosuppressed RTR and immunocompetent controls in an effort to further elucidate the role of the local immune system. METHODS: Morphology and quantity of various dendritic cell (DC) subsets, macrophages and FoxP3+ T cells were analysed by immunohistochemical staining. RESULTS: The number of CD11c+ myeloid DC and FoxP3+ T cells was significantly reduced in RTR, whereas the number of plasmacytoid DC, Langerhans cells and macrophages was similar in RTR and controls. CONCLUSIONS: A reduction in CD11c+ mDC in peritumoral dermis in RTR might contribute to impaired immunosurveillance thus giving rise to an increased risk to develop aggressive SCC in these patients.

Passive transfer of antibodies to the linear epitope 60 kD Ro 273-289 induces features of Sjögren's syndrome in naive mice.

Sjögren's syndrome (SS) is an autoimmune inflammatory disease that primarily affects the lacrimal and salivary glands causing dry eyes and mouth. Antibodies to Ro60 are observed frequently in patients with SS; however, the role of these antibodies in SS initiation and progression remains unclear. The sequence Ro60 273-289 (Ro274) is a known B cell epitope of Ro60 and antibodies to this epitope have been observed in a subset of SS patients and in animals immunized with Ro60 protein. Animals immunized with Ro274 linear peptide develop a Sjögren's-like illness. We hypothesized that passive transfer of anti-Ro274-specific immunoglobulin (Ig)G would induce a Sjögren's-like phenotype. To evaluate this hypothesis, we adoptively transferred affinity-purified Ro274 antibodies into naive BALB/c animals, then evaluated salivary gland histology, function and IgG localization 4 days post-transfer. At this time-point, there was no demonstrable mononuclear cell infiltration and salivary glands were histologically normal, but we observed a functional deficit in stimulated salivary flow of animals receiving Ro274 antibodies compared to animals receiving control IgG. Cellular fractionation and enzyme-linked immunosorbent assay revealed Ro274-specific antibodies in the nucleus and cytoplasmic fractions of isolated parotid salivary gland cells that was confirmed by immunohistochemistry. These data support the hypothesis that antibodies to Ro274 deposit in salivary glands can enter intact salivary gland cells and are involved in the dysregulation of salivary flow in SS.

Ductal epithelial expression of Ro52 correlates with inflammation in salivary glands of patients with primary Sjögren's syndrome.

Ro52 is an E3 ubiquitin ligase with a prominent regulatory role in inflammation. The protein is a common target of circulating autoantibodies in rheumatic autoimmune diseases, particularly Sjögren's syndrome (SS). In this study we aimed to investigate the expression of the SS target autoantigen Ro52 in salivary glands of patients with primary Sjögren's syndrome (pSS). Ro52 expression was assessed by immunohistochemical staining of paraffin-embedded and frozen salivary gland biopsies from 28 pSS patients and 19 non-pSS controls from Swedish and Norwegian registries, using anti-human Ro52 monoclonal antibodies. The degree and pattern of staining and inflammation was then evaluated. Furthermore, secreted Ro52 protein was measured in saliva and serum samples from the same individuals through a catch-enzyme-linked immunosorbent assay (ELISA). Ro52 was highly expressed in all the focal infiltrates in pSS patients. Interestingly, a significantly higher degree of Ro52 expression in ductal epithelium was observed in the patients compared to the non-pSS controls (P < 0·03). Moreover, the degree of ductal epithelial expression of Ro52 correlated with the level of inflammation (Spearman's r = 0·48, P < 0·0120). However, no secreted Ro52 protein could be detected in serum and saliva samples of these subjects. Ro52 expression in ductal epithelium coincides with degree of inflammation and is up-regulated in pSS patients. High expression of Ro52 might result in the breakage of tolerance and generation of Ro52 autoantibodies in genetically susceptible individuals. We conclude that the up-regulation of Ro52 in ductal epithelium might be a triggering factor for disease progression in SS.

Characterization of monocyte-derived dendritic cells from immunosuppressed renal transplant recipients with and without squamous cell carcinomas.

Renal transplant recipients (RTR) have a high risk of tumour development, especially cutaneous squamous cell carcinomas (SCC), due to long-term immunosuppressive therapy. RTR may develop multiple lesions over short time periods, and these are often more aggressive with a higher risk of local recurrence and metastasis resulting in increased morbidity and mortality in these patients. Therefore, we took the first step towards evaluating the possibility of generating a therapeutic vaccine based on monocyte-derived dendritic cells (moDC) for these patients. We analysed the phenotype and cytokine/chemokine profile of moDC from long-term immunosuppressed RTR with and without previous SCC. The number of peripheral blood mononuclear cells (PBMC) isolated per ml blood as well as the efficiency of generating moDC from peripheral blood mononuclear cells (PBMC) was similar in patients and immunocompetent controls. Phenotype and cytokine/chemokine profile of the moDC from immunosuppressed patients were similar to those from immunocompetent controls, making moDC-based immunotherapy a potential future treatment option for RTR with multiple SCC.

No association of primary Sjögren's syndrome with Fcγ receptor gene variants.

The genetic background of primary Sjögren's syndrome (pSS) is partly shared with systemic lupus erythematosus (SLE). Immunoglobulin G Fc receptors are important for clearance of immune complexes. Fcγ receptor variants and gene deletion have been found to confer SLE risk. In this study, four Fcγ receptor single-nucleotide polymorphisms (SNPs) and one copy number variation (CNV) were studied. Swedish and Norwegian pSS patients (N=527) and controls (N=528) were genotyped for the Fcγ receptor gene variant FCGR2A H131R (rs1801274) by the Illumina GoldenGate assay. FCGR3A F158V (rs396991) was analysed in 488 patients and 485 controls, FCGR3B rs447536 was analysed in 471 patients and 467 controls, and FCGR3B rs448740 was analysed in 478 cases and 455 controls, using TaqMan SNP genotyping assays. FCGR3B CNV was analysed in 124 patients and 139 controls using a TaqMan copy number assay. None of the SNPs showed any association with pSS. Also, no FCGR3B CNV association was detected. The lack of association of pSS with Fcγ receptor gene variants indicates that defective immune complex clearance may not be as important in pSS pathogenesis as in SLE, and may point to important differences between SLE and pSS.

Type 1 regulatory T cells and regulatory B cells induced by tolerogenic dendritic cells.

Dendritic cells (DC) are professional antigen-presenting cells that are capable of both activating immune responses and inducing tolerance. Several studies have revealed efficiency of therapeutic vaccination with tolerogenic DC (tolDC) in inhibition of experimental autoimmunity. The purpose of this study was to compare four different protocols for generation of tolDC - the antidiabetic drug troglitazone (TGZ DC), NF-κB inhibitor BAY 11-7082 (BAY DC), prostaglandin D2 metabolite 15d-PGJ2 (PGJ DC) and a combination of dexamethasone and 1α,25-dihydroxyvitamin D3 (DexVD3 DC) regarding phenotype, cytokine production and T cell stimulatory capacity. TGZ DC and BAY DC had a phenotype comparable to immature DC, while DexVD3 DC were more macrophage like. Analysis of cytokine production using cell culture supernatants from all DC populations revealed that DexVD3 DC were efficient producers of IL-10 and produced less pro-inflammatory cytokines. T cells primed with DexVD3 DC showed reduced proliferation, and further analyses of these T cells revealed that functionally effective type 1 regulatory T cells (Tr1) but not FoxP3(+) Treg were induced. Furthermore, DexVD3 DC promoted the induction of regulatory B cells (Breg). Together, these results indicate that DexVD3 DC have the best potential to be used in a tolerogenic antigen-presenting cell-based immunotherapy setting.

Phenotypic diversity of peripheral blood plasma cells in primary Sjögren's syndrome.

Production of autoantibodies is one of the main features of primary Sjögren's syndrome (pSS). Long-lived plasma cells (PC) can produce autoantibodies for prolonged period of times without being affected by immunosuppressive therapies. As of today, little is known about the long-lived PC subset and their contribution to autoimmunity. We have characterized the phenotypic and migratory properties of peripheral blood PC isolated from pSS patients (grouped by focus score, FS) and compared them to PC from rheumatoid arthritis (RA) patients and normal non-autoimmune subjects. We observed two populations of PC in all study groups, CD19+ PC and CD19- PC. Interestingly, the CD19- PC subset was most prominent in autoimmune patients (pSS and RA) compared to normal controls. Further investigation of the PC phenotype revealed that a high percentage of both CD19+ and CD19- PC isolated from pSS and RA patients did not express the CD27 marker, which is normally highly expressed on all types of PC. Differences in the expression of markers such as IgM, IgG, CD95 and CXCR3 in the group with high FS compared to FS = 1, underscore the heterogeneity of pSS patient group and demonstrate that phenotypic pattern of circulating PC associates with the severity of inflammation in the salivary glands of these patients. Our migration experiments show that addition of CXCL12 to PC in vitro, do not alter the migration potential of PC in any group tested. However, we observed an overall higher spontaneous migration of PC from pSS compared to both RA and normal controls.