Thomas Gelsing Carlsen

Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies

Background:The etiology of the inflammatory bowel diseases, including ulcerative colitis (UC), remains incompletely explained. We hypothesized that an analysis of the UC colon proteome could reveal novel insights into the disease etiology. Methods:Mucosal colon biopsies were taken by endoscopy from noninflamed tissue of 10 patients with UC and 10 controls. The biopsies were either snap-frozen for protein analysis or prepared for histology. The protein content of the biopsies was characterized by high-throughput gel-free quantitative proteomics, and biopsy histology was analyzed by light microscopy and confocal microscopy. Results:We identified and quantified 5711 different proteins with proteomics. The abundance of the proteins calprotectin and lactotransferrin in the tissue correlated with the degree of tissue inflammation as determined by histology. However, fecal calprotectin did not correlate. Forty-six proteins were measured with a statistically significant differences in abundances between the UC colon tissue and controls. Eleven of the proteins with increased abundances in the UC biopsies were associated with neutrophils and neutrophil extracellular traps. The findings were validated by microscopy, where an increased abundance of neutrophils and the presence of neutrophil extracellular traps by extracellular DNA present in the UC colon tissue were confirmed. Conclusions:Neutrophils, induced neutrophil extracellular traps, and several proteins that play a part in innate immunity are all increased in abundance in the morphologically normal colon mucosa from patients with UC. The increased abundance of these antimicrobial compounds points to the stimulation of the innate immune system in the etiology of UC.

Increased Levels of IgG Antibodies against Human HSP60 in Patients with Spondyloarthritis

Spondyloarthritis (SpA) comprises a heterogeneous group of inflammatory diseases, with strong association to human leukocyte antigen (HLA)-B27. A triggering bacterial infection has been considered as the cause of SpA, and bacterial heat shock protein (HSP) seems to be a strong T cell antigen. Since bacterial and human HSP60, also named HSPD1, are highly homologous, cross-reactivity has been suggested in disease initiation. In this study, levels of antibodies against bacterial and human HSP60 were analysed in SpA patients and healthy controls, and the association between such antibodies and disease severity in relation to HLA-B27 was evaluated. Serum samples from 82 patients and 50 controls were analysed by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G1, IgG2, IgG3 and IgG4 antibodies against human HSP60 and HSP60 from Chlamydia trachomatis, Salmonella enteritidis and Campylobacter jejuni. Disease severity was assessed by the clinical scorings Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI) and Bath Ankylosing Spondylitis Metrology Index (BASMI). Levels of IgG1 and IgG3 antibodies against human HSP60, but not antibodies against bacterial HSP60, were elevated in the SpA group compared with the control group. Association between IgG3 antibodies against human HSP60 and BASMI was shown in HLA-B27+ patients. Only weak correlation between antibodies against bacterial and human HSP60 was seen, and there was no indication of cross-reaction. These results suggest that antibodies against human HSP60 is associated with SpA, however, the theory that antibodies against human HSP60 is a specific part of the aetiology, through cross-reaction to bacterial HSP60, cannot be supported by results from this study. We suggest that the association between elevated levels of antibodies against human HSP60 and disease may reflect a general activation of the immune system and an increased expression of human HSP60 in the synovium of patients with SpA.

Proteome analysis of rheumatoid arthritis gut mucosa

Rheumatoid arthritis (RA) is an inflammatory joint disease leading to cartilage damage and ultimately impaired joint function. To gain new insight into the systemic immune manifestations of RA, we characterized the colon mucosa proteome from 11 RA-patients and 10 healthy controls. The biopsies were extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage of methotrexate (MTX), the most frequently prescribed immunosuppressive drug for RA. Additionally, our data suggest that treatment with Leflunomide, a common alternative to MTX, increases DHFR. The findings were supported by immunohistochemistry with confocal microscopy, which furthermore demonstrated that DHFR was located in the cytosol of the intestinal epithelial and interstitial cells. Finally, we identified 223 citrullinated peptides from 121 proteins. Three of the peptides were unique to RA. The list of citrullinated proteins was enriched in extracellular and membrane proteins and included known targets of anticitrullinated protein antibodies (ACPAs). Our findings support that the colon mucosa could trigger the production of ACPAs, which could contribute to the onset of RA. The MS data have been deposited to ProteomeXchange with identifiers PXD001608 and PXD003082.

IgG subclass antibodies to human and bacterial HSP60 are not associated with disease activity and progression over time in axial spondyloarthritis.

IntroductionSpondyloarthritis (SpA), an interrelated group of rheumatic diseases, has been suggested to be triggered by bacterial infections prior to the development of an autoimmune response that causes inflammation of the spinal and peripheral joints. Because human heat shock protein 60 (HSP60), recently renamed HSPD1, and bacterial HSP60 are highly homologous, immunological cross-reactivity has been proposed as a mechanism of disease initiation. However, previous investigations of the humoral immune response to HSP60 in SpA patients have lacked determination of immunoglobulin G (IgG) subclasses and patient follow-up. In this study, we have focused on these parameters in a cohort of axial SpA patients with a well-established set of clinical characteristics, including MRI changes and human leukocyte antigen B27.MethodsIgG subclass antibodies (IgG1, IgG2, IgG3 and IgG4) against recombinant HSP60 of three reactive arthritis-related bacteria; human HSP60; and the microorganisms Chlamydia trachomatis and C. pneumoniae were determined by ELISA. Serum samples collected from 2004 to 2006 and in 2010 and 2011 from 39 axial SpA patients were analyzed and compared with samples from 39 healthy controls. The Mann-Whitney U test and Wilcoxon matched pairs test were used to compare the antibody levels in different and paired groups, respectively. P < 0.01 was considered significant. The Spearman nonparametric correlation was used to determine correlation between antibody levels and between antibody levels and the disease parameters.ResultsElevated levels of IgG1 and IgG3 to human HSP60 and IgG1 to HSP60 of Salmonella enterica Enteritidis were observed in SpA patients compared with healthy controls at both time points. The antibody levels were almost constant over time for IgG1, whereas high levels of IgG3 to human HSP60 tended to decrease over time. The antibody response to human HSP60 was predominantly of the IgG3 subclass, and patients with high levels of IgG3 to this antigen had low levels of IgG1, indicating an inverse association. Different IgG subclasses were produced against bacterial and human HSP60 in the same serum sample, IgG1 and IgG3, respectively, indicating that there was no cross-reaction.ConclusionsA significant association was observed between axial SpA and the presence of IgG1/IgG3 antibodies to human HSP60 and of IgG1 to S. enterica Enteritidis and C. trachomatis. Generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60. No association was observed between clinical and MRI changes with antibodies over time. Altogether, such antibodies do not reflect the disease activity in these patients.This study has been approved by the Regional Research Ethics Committee of Central Jutland, Denmark. Trial registration numbers: 20050046 and 20100083

Interleukin-1α activation and localization in lipopolysaccharide-stimulated human monocytes and macrophages

BACKGROUND Interleukin-1α (IL-1α) is a proinflammatory cytokine belonging to the IL-1 family. It is synthesized as a 33kDa precursor peptide that is cleaved by a calpain-like protease to a 16 kDa propiece and a 17 kDa mature IL-1α peptide. In contrast to its close relative, IL-1β, the role of IL-1α in inflammation is only partly understood. RESULTS Human monocyte derived macrophages, stimulated with lipopolysaccharide (LPS) were analysed for production and localization of IL-1α by use of a monoclonal antibody (MAb) generated against recombinant precursor IL-1α. We found that the MAb detected IL-1α within the nuclei of the cells 2h (hours) after LPS stimulation and production continued for up to 20 h. At no time could we demonstrate cleavage of the IL-1α precursor. The MAb was conjugated to fluorescein isothiocyanate (FITC) for use in flow cytometry. Based on the flow cytometric analysis CD68 positive cells were positive for IL-1α in agreement with CD68 being a marker for monocytes. CONCLUSIONS Here, we demonstrate, for the first time, a method to visualize and measure the production of IL-1α in both human monocytes and macrophages.

Proteomics dataset: The colon mucosa from inflammatory bowel disease patients, gastrointestinal asymptomic rheumatoid arthritis patients, and controls

The datasets presented in this article are related to the research articles entitled “Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies” (Bennike et al., 2015 [1]), and “Proteome Analysis of Rheumatoid Arthritis Gut Mucosa” (Bennike et al., 2017 [2]). The colon mucosa represents the main interacting surface of the gut microbiota and the immune system. Studies have found an altered composition of the gut microbiota in rheumatoid arthritis patients (Zhang et al., 2015; Vaahtovuo et al., 2008; Hazenberg et al., 1992) [5], [6], [7] and inflammatory bowel disease patients (Morgan et al., 2012; Abraham and Medzhitov, 2011; Bennike, 2014) [8], [9], [10]. Therefore, we characterized the proteome of colon mucosa biopsies from 10 inflammatory bowel disease ulcerative colitis (UC) patients, 11 gastrointestinal healthy rheumatoid arthritis (RA) patients, and 10 controls. We conducted the sample preparation and liquid chromatography mass spectrometry (LC-MS/MS) analysis of all samples in one batch, enabling label-free comparison between all biopsies. The datasets are made publicly available to enable critical or extended analyses. The proteomics data and search results, have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples.

Can a functional assay on cytokine kinetics be used for the identification of a disease-related role for Single Nucleotide Polymorphisms (SNPs) in Ankolysing spondylitis?

Results: 305 Revision were total hip arthroplasty replacement surgeries between the years 2006 2013. Endoscopic visualization and pneumatically-powered ballistic chisels in 45 cases were used. Average age of these patients was 67.4 and most of the femoral bone defects were scored as Paprosky type 1,2a (45%). It was able to use primary stem even for revision purposes in 45% of patients, thanks to minimal bone damage. Transfemoral approach had to be used only in two cases.T experiments were performed after protocol approval by the Ethics Committee of the University of Southern Santa Catarina. The animals (male swissmice) were subjected to an intraplantar injection of Freud’s complete adjuvant (CFA, 20 μl, 70%). For treatment a far-infrared emitting ceramic pad (80% BioCorn PVC 20% ceramic materials BioPowerTM) was placed inside the animals boxes. After 24 h exposure, mechanical and thermal hyperalgesia were assessed (Von Frey test and hot plate test). In addition, edema formation and the temperature of the right hind paws were evaluated with a micrometer and a digital thermometer respectively. Control animals were placed on a Sham Pad consisting of 100% BioCorn PVC (without bioceramics) and underwent the same experimental protocol. The results demonstrate that CFA i.pl. injection induced mechanical hyperalgesia (P<0.001) which was significantly reduced by acute exposure to the ceramic pad. Analgesia lasted for up to 2 hours with peak effect 30 min after treatment (P<0.001 maximum inhibition of 53±11%). Chronic treatment with the ceramic pad reduced mechanical hyperalgesia on all evaluation days and thermal hyperalgesia on days 1 and 3. In addition, the treatment significantly decreased paw temperature on days 1 and 3 day, 8±1% (P<0.001) and 5±1% (P<0.05) respectively, when compared with control group. Treatment with a far-infrared emitting ceramic pad reduced mechanical and thermal hyperalgesia of inflammatory origin as well as the decrease of paw temperature induced by CFA intraplantar injection in mice.R arthritis (RA) is characterized by chronic joint inflammation and bone damage in which IL-17-producing T helper (Th17) and osteoclast (OC) cells play important roles. Although the etiology of RA is not well understood, it has been long observed that the vast majority of RA patients carry HLA-DRB1 alleles that code a five amino acid sequence motif called the ‘shared epitope’ (SE). The SE not only confers a higher risk for RA, but also increases the likelihood of developing a more erosive disease. The underlying mechanisms by which the SE affects susceptibility to and severity of RA are unknown. It has been recently demonstrated that the SE is a signal transduction ligand that binds specifically to the innate immune system receptor calreticulin, and activates a signaling pathway that facilitates differentiation of Th17 cells and OCs. When administered in vivo to mice with collagen-induced arthritis, the SE increased joint swelling, synovialtissue OC abundance and erosive bone damage. Thus, the SE contributes directly to arthritis severity by facilitating Th17 differentiation and activating OC-mediated bone destruction. In this talk, the new pathway will be described and a proposed therapeutic strategy that specifically targets it will be discussed. To illustrate the potential utility of the proposed therapeutic strategy,recent experimentswithsmall chemical compounds that specifically and potently inhibit the SE-activated pathway and ameliorate inflammatory arthritis in mice will be presented.I 1α(IL-1α) is a proinflammatory cytokine that belongs to the IL-1 family. It is produced mainly by macrophages at sites of infection and regarded as an essential regulator of acute inflammation.IL-1α is synthesized as a 33 kDa precursor peptide that is cleaved by a calpain-like protease to a nuclear -associated 16 kDa propiece and a secreted 17 kDa mature IL-1α peptide. However, the full understanding of its dual function is missing. Recently, SNPs in the gene for IL-1α was also associated with the risk of developing ankylosing spondylitis (AS), a subgroup of the spondyloarthropathies. These findings lead us to produce antibodiestowards the Nand C-terminal region of IL-1α to investigate IL-1α kinetics in human macrophages. This would eventually be used to asses any correlation between a defect in the production of the cytokine and a disease related SNP found in the IL-1α gene in patients suffering from AS. In the present study, human macrophages (Mf) from blood monocytes, stimulated the cells with lipopolysaccharide (LPS) and analysed the production and localization of IL-1α by use of monoclonal antibodies (MAbs) was generated against recombinant precursor IL-1α. It was obtained a MAb specific for the N-terminal propiece and for the C-terminal mature form of IL-1α, respectively. Assays, including DNA sequencing, immunofluorescence microscopy, qPCR and FACS are now available for the analysis of IL-1α kinetics in blood samples from AS patients.Results: Ninety-four patients are included in this study. Eighty-one percent are females, with mean age of 52 (± SD 14.12) years old. Majority (73%) have low level of education. Forty-one percent have rheumatoid arthritis, 21% have osteoarthritis and 12% have gout. NRS is preferred and ranked easiest to use by 41.5% of patients. FACES is a close second; preferred by 39.4% and considered easy to use by 36.2% VAS ranks last on over-all preference and ease of use. On subgroup analysis, VDS was preferred by male patients while FACES was preferred by those with low educational status. The pain score obtained using NRS was significantly correlated with VDS, VAS, and FACES (p=<0.005).Fracture neck of femur is a common orthopaedic condition in the elderly populations of developing as well as the developed world. With the world population aging fast this is a huge burden on the health care systems all over the globe. In this study we wanted to find out whether the infiltration of local anaesthetic agents into the operative wound of patients who had their hip fractures surgically operated on will have an effect on the pain score of the post operative period: overall hospital stay and on the ability to mobilize early.

A role for anti-HSP60 antibodies in arthritis: a critical review

Abstract Introduction As a result of the high sequence similarity between 60-kDa heat shock proteins (HSP60), found in both prokaryotic and eukaryotic cells, it has been suggested, but never concluded, that anti-HSP60 antibodies could be of importance in the pathology of arthritis diseases explained by a concept named molecular mimicry. In a number of clinical studies, antibodies to both human and bacterial HSP60 have been detected in serum from patients with different inflammatory diseases, but divergent results have been published. Recent progress, however, has increased the specificity in tests used to determine the humoral response to HSP60. In this review, these new findings are compared with old questioning the durability of molecular mimicry as a hypothesis for arthritis pathogenesis. The current literature was examined through a thorough PubMed search using appropriate keywords in relation to anti-HSP60 antibodies and arthritisrelated disease. Main research articles were selected for review. Discussion Recent reports have added new information to the interpretation of the humoral immune response to HSP60. By introducing determination of IgG A role for anti-HSP60 antibodies in arthritis: a critical review