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Background The phospholipase domain-containing 3 gene (PNPLA3)-148M variant is associated with liver steatosis but its influence on the metabolism of triglyceride-rich lipoproteins remains unclear. Here, we investigated the kinetics of large, triglyceride-rich very-low-density lipoprotein (VLDL), (VLDL1), and smaller VLDL2 in homozygotes for the PNPLA3-148M variant. Methods and results The kinetics of apolipoprotein (apo) B100 (apoB100) and triglyceride in VLDL subfractions were analysed in nine subjects homozygous for PNPLA3-148M and nine subjects homozygous for PNPLA3-148I (controls). Liver fat was >3-fold higher in the 148M subjects. Production rates for apoB100 and triglyceride in VLDL1 did not differ significantly between the two groups. Likewise, production rates for VLDL2-apoB100 and -triglyceride, and fractional clearance rates for both apoB100 and triglyceride in VLDL1 and VLDL2, were not significantly different. Conclusions Despite the higher liver fat content in PNPLA3 148M homozygotes, there was no increase in VLDL production. Equally, VLDL production was maintained at normal levels despite the putative impairment in cytosolic lipid hydrolysis in these subjects.

Publisher Copyright: © 2021 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd. Copyright: Copyright 2021 Elsevier B.V., All rights reserved. Aim: To elucidate the impact of liraglutide on the kinetics of apolipoprotein (apo)B48- and apoB100-containing triglyceride-rich lipoproteins in subjects with type 2 diabetes (T2D) after a single fat-rich meal. Materials and Methods: Subjects with T2D were included in a study to investigate postprandial apoB48 and apoB100 metabolism before and after 16 weeks on l.8 mg/day liraglutide (n = 14) or placebo (n = 4). Stable isotope tracer and compartmental modelling techniques were used to determine the impact of liraglutide on chylomicron and very low-density lipoprotein (VLDL) production and clearance after a single fat-rich meal. Results: Liraglutide reduced apoB48 synthesis in chylomicrons by 60% (p

Objective: Increased risk of atherosclerotic cardiovascular disease in subjects with type 2 diabetes is linked to elevated levels of triglyceride-rich lipoproteins and their remnants. The metabolic effects of PCSK9 (proprotein convertase subtilisin/kexin 9) inhibitors on this dyslipidemia were investigated using stable-isotope-labeled tracers. Approach and Results: Triglyceride transport and the metabolism of apos (apolipoproteins) B48, B100, C-III, and E after a fat-rich meal were investigated before and on evolocumab treatment in 13 subjects with type 2 diabetes. Kinetic parameters were determined for the following: apoB48 in chylomicrons; triglyceride in VLDL 1 (very low-density lipoprotein) and VLDL 2 ; and apoB100 in VLDL 1 , VLDL 2 , IDL (intermediate-density lipoprotein), and LDL (low-density lipoprotein). Evolocumab did not alter the kinetics of apoB48 in chylomicrons or apoB100 or triglyceride in VLDL 1 . In contrast, the fractional catabolic rates of VLDL 2 -apoB100 and VLDL 2 -triglyceride were both increased by about 45%, which led to a 28% fall in the VLDL 2 plasma level. LDL-apoB100 was markedly reduced by evolocumab, which was linked to metabolic heterogeneity in this fraction. Evolocumab increased clearance of the more rapidly metabolized LDL by 61% and decreased production of the more slowly cleared LDL by 75%. ApoC-III kinetics were not altered by evolocumab, but the apoE fractional catabolic rates increased by 45% and the apoE plasma level fell by 33%. The apoE fractional catabolic rates was associated with the decrease in VLDL 2 - and IDL-apoB100 concentrations. Conclusions: Evolocumab had only minor effects on lipoproteins that are involved in triglyceride transport (chylomicrons and VLDL 1 ) but, in contrast, had a profound impact on lipoproteins that carry cholesterol (VLDL 2 , IDL, LDL). Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02948777.

Background Certain periodontopathogenic bacteria have been linked to cancers. Treponema denticola (Td) is associated with severe periodontitis. Chymotrypsin-like proteinase (CTLP), a major virulence factor of Td, can degrade various host proteins and peptides, and modulate inflammatory responses. However, the role of Td in the tongue carcinogenesis remains unknown. This study aimed to investigate the presence of Td-CTLP in early-stage mobile tongue squamous cell carcinoma (MTSCC) and its relation to clinical and pathological characteristics. Methods The immunopositivity of Td-CTLP was assessed in samples obtained from 60 patients with MTSCC and associated with their clinicopathological data. Additionally, Td-CTLP expression was compared with immunoexpression of matrix metalloproteinases (MMP-8 and MMP-9), toll-like receptors (TLR-2, TLR-4, TLR-7 and TLR-9), c-Myc, Ki-67, Bmi-1 and Snail. Results Treponema denticola-chymotrypsin-like proteinase was present in 95% of MTSCC tumours of which many (40.4%) showed high immunopositivity. Td-CTLP positivity was significantly associated with invasion depth, tumour diameter and the expression of TLR-7, TLR-9 and c-Myc. High Td-CTLP immunopositivity in younger patients (≤ 60 years old) predicted early relapse. Conclusion Our data indicate that Td and its CTLP are present in early-stage MTSCC carcinoma and may contribute to carcinogenesis, and therefore provide novel perspectives into intervention and therapeutic measures of MTSCC.

Background: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. Methods: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. Results: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and alpha-1-antichymotrypsin, as well as complement C1q. Conclusions: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.

Copyright © 2017 Inês P. Perpétuo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited., Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16- monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6., This work was supported by Fundação para a Ciência e Tecnologia (SFRH/BD/70533/2010 to Inês P. Perpétuo) and by a research grant from Investigator-Initiated Studies Program of Merck Sharp & Dohme Corp. (Merck_P08574 to João E. Fonseca).

Aseptic loosening and osteolysis of joint replacements are driven by macrophage-mediated inflammatory reactions to implant-derived wear debris, but many aspects of these events remain poorly characterized. To better understand the relationships among inflammatory and chemotactic mediators, macrophage phenotype and polarizing cytokines, osteoclast activity, and Toll-like receptors (TLRs) in the pathogenesis of aseptic loosening, we determined how the relative expressions of these factors in the peri-implant tissues correlate to each other and to the life span of the implants using Pearson correlation. The expression of pro-inflammatory mediators and chemokines showed positive correlations among themselves, and with TLR4. Furthermore, M1-polarizing IFN-γ showed positive correlations with a number of pro-inflammatory and chemotactic mediators, whereas M2-polarizing IL-4 showed no such association. IL-8 expression significantly correlated with early time to revision. Similar trends were observed for TNF-α, IFN-γ and CCL3, while the opposite was detected for IL-4. However, none of the inflammatory mediators correlated with the markers of osteoclast activity or the RANKL/OPG ratio. The results highlight the importance of the inflammatory mediators, IFN-γ and TLR4 in the pathogenesis of aseptic loosening; increased pro-inflammatory status was associated with early time to revision, whereas IL-4 correlated with longer implant survival. This article is protected by copyright. All rights reserved.

Sjögren's syndrome (SS) is an autoimmune disease characterized by lymphoplasmacytoid focal adenitis leading to mucosal dryness, with 9:1 female dominance and peak incidence at menopause. Due to autoimmune adenitis it can be speculated that the normal epithelial cell renewal has failed, possibly as a result of local intracrine failure to process dehydroepiandrosterone (DHEA) to dihydrotestosterone (DHT). Local intracrine/-cellular DHT deficiency seems to predispose to SS if estrogens are low, in menopausal women and in men. This intracrine failure could be the initial noxious stimulus, factor X, initiating the development of SS. Abnormal release and presentation of exocrine gland-derived antigens (Ag-epitopes), in a complex with major histocompatibility complex class II (MHC II), by migratory dendritic cells (DC) activates T-cells in the regional lymph nodes. B-cells with the same specificity capture and present self-Ag to Th-cells which provide T-cell help. B-cells transform to plasma cells and start to produce autoantibodies (Ab) against these T-cell-dependent Ag. Ab against SS-A/Ro and SS-B/La ribonucleoproteins occur only in HLA-DQw2.1/DQw6 heterozygous individuals, but hY-RNA and RNA polymerase III transcripts in these Ag may in all SS patients stimulate toll-like receptors (TLR) 7 and 9 of the plasmacytoid DCs, because IFN-α and IFN-signature are produced. CD8+αEβ7+cytotoxic T-cells activated via cross-presentation recirculate to attack intracrine-deficient, apoptotic epithelial cells expressing self-Ag on their surface. Exocrine glands fall into the sphere of mucosal/gut-associated lymphatic tissue. This together with immune complexes spreads the immunological memory/aggression to extra-glandular sites explaining the systemic nature of the syndrome. Secondary SS could be explained by disturbed lymphocyte recirculation. There is no conclusive evidence that SS in those few men affected is more severe than in women, suggesting that sex steroid endo-/intracrinology has its major impact on the target tissue, not on immune modulation. This article is part of a Special Issue entitled 'Essential role of DHEA'.

The expression of clock genes 'ARNTL2', 'NPAS2' and 'DEC2' are disturbed in rheumatoid arthritis, an autoimmune disease with circadian variation of symptoms. We have shown that TNF is a potent inducer of these genes. We investigated the regulation of 'ARNTL2' and 'NPAS2' by TNF and elucidated their effect on other clock gene expressions. Additionally, we studied the effect of 'DEC1' and 'DEC2' on 'ARNTL', 'ARNTL2' and 'NPAS2'. Cultured primary human fibroblasts were stimulated with TNF and the effects on ARNTL2 and NPAS2 were studied with RT-qPCR and immunofluorescence staining. The role of NF-κB was analyzed using IKK-2 inhibitor IMD-0354. TNF promoted ARNTL2 localization into the nuclei. Similar to 'DEC2', the effects of TNF on 'ARNTL2' and 'NPAS2' expressions were mediated via NF-κB. Cloned 'ARNTL', 'ARNTL2', 'NPAS2', 'DEC1' and 'DEC2' were transfected into HEK293. The ARNTL2/NPAS2 dimer was a weaker inducer of 'PER3' and 'DBP' than ARNTL/NPAS2. ARNTL2 and NPAS2 are regulated by TNF via the same mechanism as DEC2. Compared to their paralogs they have unique effects on other circadian components. Our data suggest that these genes are responsible, at least in fibroblasts, for the accurate adaptation of circadian timekeeping in individual cells during inflammation.

Copyright: © 2017 Perpétuo, Caetano-Lopes, Vieira-Sousa, Campanilho-Marques, Ponte, Khmelinskii, Canhão, Ainola and Fonseca. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms., Introduction: Ankylosing spondylitis (AS) is typically characterized by focal bone overgrowth and also by systemic bone loss. We hypothesize that the increased osteoproliferation found in AS might be partially due to reduced ability of osteoclast precursors (OCPs) to differentiate into osteoclasts (OCs). Therefore, our aim was to characterize bone remodeling and pro-osteoclastogenesis inflammatory environment, monocytes’ phenotype, and in vitro osteoclast differentiation in AS patients. Methods: Patients with active AS without any ongoing therapy and age- and gender-matched healthy donors were recruited. Receptor activator of nuclear factor-κβ (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations were assessed. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and quantitative reverse transcription real-time PCR for OC-specific genes were performed. Results: Pro- and anti-inflammatory cytokine serum levels were higher in AS patients than in controls. RANKL neutrophil expression was higher in AS patients when compared to healthy donors, but CD51/CD61 expression was lower in the classical monocyte subpopulation. Concerning osteoclastogenesis, we found no differences in the in vitro osteoclast differentiating potential of these cells when compared to healthy donors. However, we observed low expression of CSF1R, RANK, and NFATc1 in AS OCPs. Conclusion: Despite the high levels of pro-inflammatory cytokines present in AS patients, no differences in the number of OC or resorbed area were found between AS patients and healthy donors. Moreover, we observed that OCPs have low OC-specific gene expression. These findings support our hypothesis of an impaired response of OCPs to pro-osteoclastogenic stimuli in vivo in AS patients.

Objective Evidence of increased apoptosis is observed in periodontitis, and may be associated with destruction of the periodontal tissue caused by the increased cell death, with the release of danger signals and subsequent stimulation of the proinflammatory processes. However, the exact mechanisms associated with these processes remain unclear. This study aimed to investigate the presence of the periodontal pathogen Treponema denticola, apoptosis, High Mobility Group Box1 as a damage-associated molecular pattern and several inflammatory markers in periodontitis and gingivitis subjects. Materials and methods Soft tissue specimens from gingival tissues of periodontitis and gingivitis patients were used for immunohistochemical and immunofluorescence staining of Treponema denticola chymotrypsin-like proteinase (CTLP), apoptosis markers, High Mobility Group Box1, Toll-like receptor 4, inflammatory cell markers and proinflammatory cytokines. Results Treponema denticola was detected in all periodontitis-affected tissues. This was associated with a significant increase in the number of apoptotic cells, including macrophages, alterations in the expression of High Mobility Group Box1 and its receptor, and increased levels of proinflammatory cytokines compared with gingivitis. Conclusions In summary, the presence of Treponema denticola (especially its CTLP), apoptosis, High Mobility Group Box1, and inflammatory markers suggest their potential involvement in the pathogenesis of periodontitis. This article is protected by copyright. All rights reserved.

Objective. T helper 17 (Th17) and mast cells produce IL-17A in RA and critically contribute to the pathogenesis of RA. However, the complete IL-17 cytokine profile in RA is unknown. The aim of the study was to systematically study the expression of IL-17 family cytokines in RA. Methods. The expression of all IL-17 cytokines in RA synovium and pannus as well as in the synovium of OA was determined using quantitative RT-PCR (qRT-PCR). IL-17A and IL-17B were immunostained. Peripheral blood neutrophils were analysed for IL-17B. The effect of IL-17B alone or in combination with TNF-a was tested in vitro on fibroblasts and endothelial cells. Results. In all tissues IL-17B was the most expressed IL-17 family cytokine, found in lining but most strongly expressed in human neutrophil elastase containing polymorphonuclear cells. This pattern was distinct from that of IL-17A, which was found in mast cell tryptase immunoreactive cells. Circulating neutrophils contained IL-17B, verifying the in vivo results. Fibroblasts up-regulated the expression of IL17RB, a putative receptor of IL-17B, after TNF-a stimulation. IL-17B significantly enhanced TNF-a-induced production of G-CSF and IL-6 in fibroblasts. Conclusion. IL-17B, which is present in synovium, may contribute to the pathogenesis of RA. IL-17B can enhance the effects of TNF-a on the production of cytokines and chemokines that control immune cell trafficking and neutrophil homeostasis in the inflamed tissues.

Regenerative medicine is a multidisciplinary field utilizing the potential of stem cells and the regenerative capability of the body to restore, maintain, or enhance tissue and organ functions. Stem cells are unspecialized cells that can self-renew but also differentiate into several somatic cells when subjected the appropriate environmental cues. The ability to reliably direct stem cell fate would provide tremendous potential for basic research and clinical therapies. Proper tissue function and regeneration rely on the spatial and temporal control of biophysical and biochemical cues, including soluble molecules, cell-cell contacts, cell-extracellular matrix contacts, and physical forces. The mechanisms involved remain poorly understood. This review focuses on the stem cell-extracellular matrix interactions by summarizing the observations of the effects of material variables (such as overall architecture, surface topography, charge, ζ-potential, surface energy, and elastic modulus) on the stem cell fate. It also deals with the mechanisms underlying the effects of these extrinsic, material variables. Insight in the environmental interactions of the stem cells is crucial for the development of new material-based approaches for cell culture experiments and future experimental and clinical regenerative medicine applications.

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.

Objectives It was hypothesized that beta 2 defensin (BD-2) is increased in RAU lesions compared with healthy controls to promote anti-microbial host defence. Methods RAU and control mucosa samples were subjected to quantitative real-time PCR and immunostained for BD-2, CD68, mast cell tryptase and 4-hydroxynonenal (4HNE). The effect of tumour necrosis factor-α (TNF-α) ± interleukin-17C (IL-17C), without and with vitamin K3, was studied on BD-2 expression in epithelial SCC-25 cells. Results Although BD-2 mRNA did not differ between healthy and RAU mucosa, BD-2 stained strongly in acute-phase RAU epithelium (P = 0.001). In controls, subepithelial BD-2+ cells were mast cells and macrophages, whereas in RAU, most infiltrating leucocytes were BD-2+ (P = 0.004). In cell culture, BD-2 was increased 124-fold by TNF-α (P

Objectives Oral lichen planus (OLP) is an autoimmune disease characterized by a band-like T-cell infiltrate below the apoptotic epithelial cells and degenerated basement membrane. We tested the hypothesis that the high-affinity histamine H4 receptors (H4Rs) are downregulated in OLP by high histamine concentrations and proinflammatory T-cell cytokines. Materials and Methods Immunohistochemistry and immunofluorescence staining, image analysis and quantitative real-time polymerase chain reaction of tissue samples and cytokine-stimulated cultured SCC-25 and primary human oral keratinocytes. Results H4R immunoreactivity was weak in OLP and characterized by mast cell (MC) hyperplasia and degranulation. In contrast to controls, H4R immunostaining and MC counts were negatively correlated in OLP (P = 0.003). H4R agonist at nanomolar levels led to a rapid internalization of H4Rs, whereas high histamine concentration and interferon-γ decreased HRH4-gene transcripts. Conclusion Healthy oral epithelial cells are equipped with H4R, which displays a uniform staining pattern in a MC-independent fashion. In contrast, in OLP, increased numbers of activated MCs associate with increasing loss of epithelial H4R. Cell culture experiments suggest a rapid H4R stimulation-dependent receptor internalization and a slow cytokine-driven decrease in H4R synthesis. H4R may be involved in the maintenance of healthy oral mucosa. In OLP, this maintenance might be impaired by MC degranulation and inflammatory cytokines.

Aseptic loosening of prosthetic components, the most common long-term complication after total hip replacement (THR), is characterized by the formation of a synovial membrane-like interface tissue (SMLIT). It was hypothesized that the hyaluronan synthase (HAS)/hyaluronan (HA)/HA receptor CD44 signalling system is responsible for the synovial-like differentiation of the interface membrane. SMLIT was therefore compared with osteoarthritis (OA) synovial membrane by using reverse transcriptase polymerase chain reaction (RT-PCR) of HAS 1, 2 and 3, histochemical HA assay, and immunohistochemistry of CD44 and its non-HA ligands. All three isoforms of HAS were found in these samples. HA and CD44 were most abundant in the lining, but the signal was actually stronger in aseptic loosening than in OA (p

Background Recurrent aphthous ulcer (RAU) is an ulcerative disease of non-keratinized oral mucosa. Colon and bronchial epithelial cells produce interleukin-17C (IL-17C) upon stimulation of Toll-like receptor 2 (TLR2), TLR3 and TLR5, which are highly expressed in epithelial cells in RAU lesions. We therefore investigated the eventual presence and function of IL-17C in cultured human oral keratinocytes (HOK) and control biopsies compared to RAU lesions. Methods Expression of IL-17A, IL-17C, IL-17RA and IL-17RE was analysed in cultured HOK cells using quantitative real-time polymerase chain reaction (qRT-PCR). HOK cells were stimulated with IL-17C and analysed for IL-8 and tumour necrosis factor-α (TNF-α) using qRT-PCR. Control mucosa (n = 5) was immunostained for IL-17A, IL-17C, IL-8, TNF-α and mast cell tryptase and compared with RAU lesions (n = 5) using the mean grey scale value. Results IL-17C, but no IL-17A, mRNA was found in cultured HOK cells. Components of the heterodimeric IL-17RA/IL-17RE receptor for IL-17C were also highly expressed. Stimulation of HOK with IL-17C increased TNF-α mRNA (P = 0.03; IL-8 increase was not statistically significant). HOK in RAU lesions stained intensively for IL-17C compared to controls (P = 0.006). This was associated with increased epithelial immunostaining of TNF-α (P = 0.04) and IL-8 (P = 0.02). Most of the inflammatory cells which stained for IL-17A in control mucosa and RAU lesions were also mast cell tryptase positive. Conclusion IL-17C is highly expressed in epithelial cells in RAU lesions, where it seems to stimulate oral keratinocytes via IL-17RA/IL-17RE to produce pro-inflammatory cytokines. Human oral epithelial cells are probably important inflammatory cells in RAU.

Five different laminin (LM) alpha, four LM-beta, and three LM-gamma chains form the 15-16 currently known approximately 400-900 kDa heterodimeric LM-monomers, which self-assemble in the lamina lucida of the basement membrane (BM) to a network, connected with nidogens and perlecans with the underlying type IV collagen network. In labial salivary glands (LSG), the structurally organizing/polarizing BM separates the tubuloacinar epithelium from the connective tissue stroma but plays regulatory roles as well. Tissue distribution of LM-alpha, -beta, and -gamma chains is described, and application of the known combinatorial rules allows some conclusions also on the corresponding distribution of the LM-trimers. Currently, known integrin (Int) and non integrin (e.g., dystroglycans and Lutheran blood group antigens) LM-receptors are described. LMs are regulated at transcriptional, translational, and posttranslational levels, together with the regulation of alternative splicing, binding partners (assembly), secretion, and degradation. In LSGs, LM-alpha1, -alpha2, and -alpha4 are only found in the acinar (not ductal) BM, LM-alpha4 also in the periductal/ interstitial stroma. Pattern recognition disclosed irregular expression in the acinar BM, suggesting some dynamic and/or regulatory role. It seems that in a female-dominant autoimmune exocrinopathy, Sjogren's syndrome (SS), LM-alpha1 and -alpha2 are decreased, together with their Int alpha1beta1 and alpha2beta1 receptors. Because LM-111/211-to-Int-alpha1beta1/alpha2beta1 interactions play a crucial role in the transdifferentiation of the intercalated duct progenitors to secretory acinar cells, acinar remodeling is impaired in SS. Disturbed hemidesmosomal Int alpha6beta4/LM-332 interactions in SS may lead to acinar cell anoikis. Interestingly, dehydroepiandrosterone (DHEA) prohormone and its intracrine androgenic dihydrotestosterone (DHT) end product upregulate at least Int alpha1beta1/alpha2beta1, whereas LM-alpha1 is upregulated by outside-in LM-111/211-to-Int-alpha1beta1/alpha2beta1 signaling. It seems that LM alterations precede the lymphocyte infiltration, suggesting that acinar BM-Int pathology, perhaps related to endo- and intracrine sex steroid metabolism, represents an early pathogenic phases in SS.

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease of the axial skeleton, characterised by systemic osteoporosis along with new local bone formation. Previous studies have shown that serum levels of TNF, IL-6 and IL-17 are increased in AS patients and may be implicated in the development of secondary osteoporosis, since these cytokines are able to induce osteoclast (OC) differentiation and, therefore, bone resorption. In this work we aimed to assess the effects of TNF-blocking therapy in the systemic inflammatory environment of AS patients with active disease as well as in OC differentiation and activity. Patients with AS starting TNF-blocking therapy were recruited for this study and blood was collected at baseline and 6 months after the first treatment administration. We performed ELISAs in the serum of these patients to assess cytokine levels and bone turnover markers. We also characterised RANKL surface expression in circulating neutrophils, B and T lymphocytes and monocyte subpopulation frequency and phenotype by flow cytometry. We cultured circulating monocytes from AS patients, before and after therapy, and from healthy controls under osteoclastogenic conditions. We performed two functional assays (TRAP staining and resorption pit assay) and analysed the expression of osteoclast specific genes. We found no differences (before and after treatment) in any of the circulating monocytes’ subpopulations regarding frequency or cell death assessed by annexin staining. Phenotype analysis only revealed differences in the classical monocyte subpopulation where there was a significant decrease in HLA-DR surface expression after treatment. The frequency of RANKL positive B lymphocytes was reduced after treatment. No changes were observed on RANKL expression in neutrophils or T lymphocytes after treatment. Before TNF-blocking treatment AS patients have increased levels of pro-inflammatory cytokines when compared with healthy subjects. After TNF-blocking therapy IL-17, TGF-β and osteoprotegerin were significantly decreased. Interestingly, we observed that after TNF-blocking therapy, gene expression was favouring osteoclastogenesis and that differentiated OCs have increased resorption activity. Our results suggest that in AS patients there might be a paradoxical effect of TNF blocking therapy that induces OC activity.

Objective To investigate the involvement of a disintegrin and the metalloproteinase ADAM9 (meltrin-gamma) in the formation of multinuclear giant cells and osteoclasts in aseptic loosening of hip replacement implants. Methods We used in situ hybridization, immunohistochemical staining and western blotting of interface membrane surrounding loosened hip implants, macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL) costimulation and polymethyl methacrylate (PMMA) particle stimulation of human monocytes followed by immunofluorescence staining and flow cytometric analysis. Results Morphometric analysis revealed that the ADAM9+ area in the revision total hip replacement (THR) interface was larger than in primary THR samples (37.6+/-5.1 vs 5.2+/-0.8%, P=0.002). Double immunofluorescence staining showed that CD68+ interface tissue macrophages and multinuclear giant cells were ADAM9+. ADAM9 mRNA containing mononuclear and multinuclear cells was often seen in a close spatial relationship with other ADAM9+ cells. Western blotting disclosed a 50 kDa ADAM9 band in tissue extracts. Upon M-CSF and RANKL costimulation of human monocytes, the ADAM9 staining pattern changed over time and ADAM9+ cells formed bi- and multinuclear cells. Flow cytometry disclosed that cells of the monocyte/macrophage lineage changed from ADAM9-negative cells into strongly positive cells during a 3-day culture. Conclusion ADAM9 is expressed in interface tissues around aseptically loosened THR implants. ADAM9 may play a role as a fusion molecule in the formation of multinuclear giant cells and osteoclasts from mononuclear precursors in diseases characterized by bone tissue destruction.

Interface tissue between the bone and loosening total hip implant is acidic and highly osteolytic. It is characterized by the formation of cathepsin K positive foreign body giant cells. Similar structures to those found in the normal joint surround the artificial hip joint. Cells in synovial membrane of the artificial hip generate synovial fluid that is called pseudosynovial fluid. Interface tissue fibroblasts are able to produce receptor activator of NF-B ligand (RANKL), which can induce osteoclastogenesis during the loosening process. Western blot analysis indicated that RANKL is present in the pseudosynovial fluid. Pseudo- synovial fluid induced cultured peripheral blood mononuclear cells to form multinuclear TRAP positive giant cells. In the presence of osteoprotegerin, the soluble RANKL decoy receptor, the number of TRAP positive multinuclear cells was reduced to half (p < 0.05). The multinuclear cells induced with pseudosynovial fluid contained active cathepsin K protein and were capable of bone matrix resorption in vitro. The cells were shown to express osteoclast phenotype markers, such as mRNA for cathepsin K, TRAP, and calcitonin receptor. It is therefore apparent that pseudosynovial fluid from patients with aseptic loosening of total hip prosthesis contains a potent osteoclastogenic factor RANKL that further suggests a favorable environment for osteoclast formation in the peri-implant tissues. It is thus concluded that suppression of RANKL activity may be beneficial in terms of increasing the lifetime of total hip prostheses. © 2005 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 74B: 582-588, 2005

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are systemic, immune-mediated diseases. In RA the main target are the peripheral joints while in AS the axial skeleton and enthesis are affected. RA is characterised by bone repair impairment and AS by exaggerated repair, leading to joint ankylosis. The cause for these differences is not yet understood, however we hypothesize that monocytes from AS patients exhibit reduced responses to osteoclastogenic and/or migratory stimulus. The aim of this work was to characterise bone remodelling regulators and the inflammatory signals that promote osteoclastogenesis and the circulating monocyte subpopulations to understand their response to inflammation and osteoclastogenic signals in untreated AS patients and compare them to RA patients and healthy controls. Untreated RA or AS patients with active disease and age and gender-matched healthy donors were recruited for this study. Blood was collected for flow cytometry measurements of RANKL expression and monocyte subpopulation characterisation. Serum was collected for cytokine and bone turnover marker quantification. We found that RANKL lymphocyte expression is higher in AS and RA patients when compared to healthy donors. When analyzing the monocyte sub-populations we found that both in the classical and the intermediate subpopulations, RANK expression was decreased in AS patients as compared to RA patients and healthy controls. In accordance, CTX-I serum level was also lower in AS patients than in healthy controls. In conclusion, and despite comparable osteoclastogenic stimuli in AS and RA patients, RANK expression is reduced in AS circulating monocytes which may contribute to the bone forming phenotype observed in AS patients.