Henning Locht

Soluble urokinase plasminogen activator receptor in plasma of patients with inflammatory rheumatic disorders: increased concentrations in rheumatoid arthritis

OBJECTIVE Urokinase type plasminogen activator (uPA) catalyses the formation of the proteolytic enzyme plasmin, which is involved in matrix degradation in the processes of tissue remodelling. Because of a surface bound uPA receptor (uPAR), expressed by some cell types (for example, macrophages, malignant cells and inflammatory activated synoviocytes), the action of uPA can be localised and intensified. uPAR seems to have a role in the mechanisms leading to invasive growth of malignant tissue and the rheumatoid pannus. uPAR may become cleaved at its cell surface anchor, thus forming a free soluble receptor (suPAR). suPAR is detectable in low but constant values in plasma of healthy people, while increased concentrations are found in patients with disseminated malignant disease, so that suPAR may be an indicator of invasive growth and tissue remodelling. suPAR concentrations in plasma have not previously been measured in rheumatic patients. A controlled cross sectional measurement was performed of suPAR in plasma of patients with various inflammatory rheumatic disorders with special reference to rheumatoid arthritis (RA). METHODS suPAR in plasma was measured by ELISA technique in patients with RA (n=51), reactive arthritis (ReA) (n=23), primary Sjögren’s syndrome (PSS) (n=42) and sex and age matched healthy controls (n=53). RESULTS In the control group suPAR (median) was 0.91 (range 0.56–1.94) μg/l. Median suPAR value in RA was 1.47 (range 0.65–6.62) μg/l; in ReA 0.68 μg/l (range 0.52–1.48) and in PSS 1.12 μg/l (range 0.76–1.92); p versus controls <0.001 in all patient groups. suPAR values in RA were also significantly increased compared with ReA (p<0.001) and PSS (p=0.004) groups. suPAR in RA was positively correlated to C reactive protein (CRP) (p<0.01) and erythrocyte sedimentation rate (p<0.05) and number of swollen joints (p<0.05). The ReA group had the highest CRP values of all groups, but at the same time the lowest suPAR concentrations in plasma. CONCLUSIONS Increased suPAR concentrations were found in plasma in RA, and to a smaller extent also in PSS, but not in ReA. In RA suPAR is related to disease activity. suPAR seems though not merely to be an acute phase reactant like CRP. Increased suPAR values might reflect erosive activity in RA.

International multicenter evaluation of autoantibodies to ribosomal P proteins.

ABSTRACT Autoantibodies to the ribosomal phosphoproteins (Rib-P) are a serological feature of patients with systemic lupus erythematosus (SLE). The reported prevalence of anti-Rib-P antibodies in SLE ranges from 10 to 40%, being higher in Asian patients. The variation in the observed frequency may be related to a number of factors but is dependent in large part on the test system used to detect the autoantibodies. An association of anti-Rib-P with central nervous system involvement and neuropsychiatric manifestations of SLE has been controversial. In the present international multicenter study, we evaluated the clinical accuracy of a new sensitive Rib-P-specific enzyme-linked immunosorbent assay based on recombinant Rib-P polypeptides. The results showed that 21.3% of 947 SLE patients, but only 0.7% of 1,113 control patients, had a positive test result (P < 0.0001). The sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency were determined to be 21.3%, 99.3%, 95.6%, 62.2%, and 65.3%, respectively. When evaluated in the context of participating centers, the prevalence of anti-Rib-P antibodies was found in descending frequency, as follows: China (35%) > Poland (34%) > Japan (28%) > United States (26%) > Germany (Freiburg; 23.3%) > Denmark (20.5%) > Germany (Berlin; 19%) > Mexico (15.7%) > Israel (11.7%) > Brazil (10%) > Canada (8%). The substantial data from this study indicate that the prevalence of anti-Rib-P antibodies may not be restricted to the genetic background of the patients or to the detection system but may depend on regional practice differences and patient selection. We confirm previously reported associations of antiribosomal antibodies with clinical symptoms and serological findings. Remarkably, we found a lower occurrence of serositis in Rib-P-positive lupus patients.

Follow-Up of Exocrine Pancreatic Function in Type-1 Diabetes mellitus

In a previous study, mild to moderate exocrine pancreatic insufficiency, as measured by the secretin-pancreozymin test, was found in 23 (43%) of 53 patients with type-1 diabetes mellitus. Of these 53 patients, 20 (7 of whom initially had an abnormal secretin-pancreozymin test) were available for a follow-up examination 11 years later. Of the 7 patients with abnormal exocrine pancreatic function at the first test, 5 remained abnormal and 2 became normal, whereas of the 13 patients with initially normal pancreatic function the test result remained normal in 11 patients and became abnormal in 2. In these 2 groups the test result did not differ significantly between both tests. However, exocrine pancreatic function had returned to normal or had become abnormal in 2 patients, respectively, at the second test. In the 3 patients with exocrine pancreatic insufficiency at the first and second tests, the lipase level had not fallen below 10% or less than the normal level at which steatorrhea occurs and therapy is required. There was no significant correlation between the duration of the diabetes and the test results for both time points of investigation. The data suggest that mild to moderate exocrine pancreatic insufficiency found in type-1 diabetes is due to an early event in the course of the diabetes and does not progress. Therefore, this finding is of minor clinical importance and expensive pancreatic enzyme substitution will not be required.

NOD2/CARD15 Gene Polymorphisms in Crohn’s Disease: A Genotype-Phenotype Analysis in Danish and Portuguese Patients and Controls

Background: A North-South gradient in Crohn’s disease (CD) implying a higher incidence in northern Europe compared to southern Europe has been established. Aims: To investigate whether there is a difference between Denmark and Portugal in the frequency of CARD15 mutations in CD patients compared to a healthy background population and to compare genotype-phenotype relations in the two countries. Methods: 58 Danish patients and 29 Portuguese patients with CD were matched for age, sex and disease behaviour at time of diagnosis and compared with 200 healthy Danish and Portuguese controls. Phenotypes were recorded at year of diagnosis, 3 years after diagnosis and at end of follow-up. Patients were genotyped for Arg702Trp, Gly908Arg and Leu1007InsC. Results: 22% of the Danish patients vs. 9% of Danish controls compared to 21% of the Portuguese patients vs. 16% had at least one mutation. Mutation rates in Danish patients were significantly different (p = 0.02) compared with Danish controls, no difference (p = 0.51) was found between Portuguese patients and controls. However, a possible relationship between CD and presence of genetic mutations was found when comparing the two countries (p = 0.03) using the Mantel-Haenszel test. No difference in evolution of phenotypes and the CARD15 status in CD was found during follow-up between the two matched populations. Ileal disease correlated to high occurrence of CARD15. Conclusion: No North-South gradient regarding occurrence of CARD15 was revealed. Although a trend towards more mutations in the Portuguese controls was seen, a relationship between CD and CARD15 mutations was observed in both countries.

Characterisation of autoantibodies to neutrophil granule constituents among patients with reactive arthritis, rheumatoid arthritis, and ulcerative colitis

OBJECTIVE To study the frequency and distribution of antineutrophil cytoplasmic autoantibodies (ANCA) among patients with reactive arthritis (ReA), rheumatoid arthritis (RA), and ulcerative colitis (UC) using different immunological methods. METHODS Fifty serum samples from patients with reactive arthritis (26 with acute disease and 24 with chronic disease—that is disease of more than one year) were analysed for ANCA with indirect immunofluorescence, enzyme linked immunosorbent assay (ELISA) with six different neutrophil granule proteins as antigens, and immunoblotting on whole neutrophil extract and extracts of azurophil and specific granules. Thirty serum samples from patients with RA and UC served as controls in ELISA and indirect immunofluorescence. RESULTS Sixteen per cent of patients with ReA were positive in immunofluorescence compared with 30% of patients with RA, and 70% of patients with UC. Thirty two per cent of patients with ReA were positive in ELISA. Antibodies directed against lactoferrin occurred in 20%, antibodies against bactericidal permeability increasing protein (BPI), elastase, cathepsin G, myeloperoxidase, and proteinase 3 were found in 8%, 2%, 2%, 8%, and 6%, respectively. Overall, 50% of RA sera and 53% of UC sera were positive in one or more ELISA assays, the corresponding figures for antibodies against individual antigens were for RA 7%, 3%, 0%, 13%, 47%, 17% and for UC 13%, 20%, 0%, 23%, 10%, and 17%. In immunoblotting, bands corresponding to lactoferrin and BPI were recognised in 44% and 22% of ReA sera. CONCLUSION Antibodies against neutrophil granule antigens are often found in patients with ReA, primarily among those with chronic disease. The different methods detect various subsets of antibodies, with immunoblotting being the most and immunofluorescence the least sensitive.

Anti-lactoferrin antibodies and other types of anti-neutrophil cytoplasmic antibodies (ANCA) in reactive arthritis and ankylosing spondylitis.

Fifty‐five serum samples from patients with reactive arthritis (ReA), 40 from patients with ankylosing spondylitis (AS) and three from patients with chronic sacroiliac joint arthritis were analysed for the presence of ANCA of IgG class by means of enzyme immunosorbent assay using lactoferrin (Lf), myeloperoxidase (MPO) and antigen extracted from azurophil granules (‘α‐antigen’) containing proteinase 3 (PR3) as substrate. IgG‐ANCA were found in 31 (56%) patients with ReA. Twenty‐three (42%) had anti‐Lf antibodies, nine (16%) had anti‐MPO and eight (15%) had anti‐α‐antigen antibodies, none of which reacted with PR3. Only six (14%) AS or sacroiliac joint arthritis patients had ANCA (P < 0.001). Three (7%) had anti‐Lf, two (5%) anti‐MPO and two (5%) anti‐α‐antigen antibodies. Yersinia and Salmonella bacteria were separated by SDS–PAGE and blots were incubated with serum from rabbits immunized with human Lf. The hyperimmune serum recognized a band of 78 kD from both bacteria which was not seen when preimmune serum was used. The reaction to the 78‐kD antigen could be completely inhibited when anti‐Lf antibodies were absorbed on Lf coupled to cyanogen bromide‐activated Sepharose, possibly indicating cross‐reacting epitopes in Lf and enterobacterial antigen.

Genetic and environmental factors as predictors of disease severity and extent at time of diagnosis in an inception cohort of inflammatory bowel disease, Copenhagen County and City 2003-2005.

BACKGROUND AND AIMS The etiology of the inflammatory bowel diseases (IBD), Crohns disease (CD) and ulcerative colitis (UC) remains unknown. We aimed to investigate the influence of genetic, serological, and environmental factors on phenotypic presentation of IBD at diagnosis in a population-based Danish inception cohort from 2003-2005. METHODS Three-hundred-forty-seven (62%) of 562 cohort patients were genotyped. ASCA and p/c-ANCA were determined and patients answered a questionnaire concerning environmental factors with possible influence on IBD. RESULTS Fourteen percent of CD patients vs. 11% of controls were positive for common CARD15 mutation (ns), whereas more CD patients than healthy controls were homozygous for the OCTN-TC haplotype (p=0.03). ASCA was more common in CD (22%) than UC (14%) (p=0.045) and was related to age and localization of CD. p-ANCA was more frequent in UC (p=0.00001) but was related to pure colonic CD (p=0.0001). Sugar consumption was significantly higher in CD patients than in UC patients (p=0.0001) and more CD patients than UC patients had undergone appendectomy prior to IBD diagnosis (p=0.03). A possible relation between tonsillectomy and disease severity in CD, and a relation between use of oral contraception and disease localization of UC to rectum/left-sided colon were found. CONCLUSIONS In this cohort of unselected IBD patients we found a very low frequency of mutations in IBD susceptibility genes and observed a greater impact of ASCA and ANCA than of genetic factors on disease phenotypes. In addition, several environmental factors seemed to influence disease occurrence and disease presentation in both UC and especially CD.

IgG and IgM isotypes of anti-cardiolipin and anti-β2-glycoprotein i antibodies reflect different forms of recent thrombo-embolic events

We correlated the distribution and levels of serum anti-cardiolipin (aCL) and anti-β2-glycoprotein-1 antibodies (anti-β2-GPI) of the IgG and IgM isotypes to the clinical spectrum of recent (<6 months) thromboembolic events in a cohort of 162 patients. Clinical information was obtained by questionnaires from the referring physicians. Cerebro-vascular infarction (CVI) had taken place in 82 patients, deep venous thrombosis (DVT) in 34, pulmonary embolism (PE) in 14, myocardial infarction (MI) in four, and other thromboses in 28 patients. SLE was the most commonly associated rheumatic disease and accounted for 20 (12%) patients. In 124 (77%) patients no underlying rheumatic disease was identified. Isolated IgG aCL was found in 31 of 48 patients with DVT/PE (65%), but in only 21 of 82 patients with CVI (26%); p < 0.0001. IgG anti-β2-GPI were detected in 23 (48%) DVT/PE patients, but in only 13 (16%) CVI patients; p < 0.001. The IgG class anti-β2-GPI positive patients had significantly higher levels of IgG aCL (mean 65 units) compared to IgG anti-β2-GPI negative patients (mean 29 units); p < 0.0001. In contrast, isolated IgM aCL was found in nine (19%) patients with DVT/PE, but in 46 (56%) CVI patients; p < 0.0001. Only ten patients had IgM anti-β2-GPI. The present study shows that the IgG and IgM aCL isotypes seem to define different clinical subsets of patients with thrombo-embolic events with IgG aCL being most prevalent in the group having DVT/PE, IgM aCL being found primarily among CVI patients.

The role of Campylobacter jejuni cytolethal distending toxin in gastroenteritis: toxin detection, antibody production, and clinical outcome

Mortensen NP, Schiellerup P, Boisen N, Klein BM, Locht H, AbuOun M, Newell D, Krogfelt KA. The role of Campylobacter jejuni cytolethal distending toxin in gastroenteritis: toxin detection, antibody production and clinical outcome. APMIS 2011.

Genetic and Environmental Factors in Monozygotic Twins with Crohn’s Disease and Their First-Degree Relatives: A Case Report

Background/Aims: Familial Crohn’s disease has shown concordance concerning location and clinical type of the disease especially among monozygotic twins. Susceptibility to Crohn’s disease is both based on genetic and environmental factors. We investigated polymorphisms of CARD15, TLR4, and OCTN, and environmental factors in a monozygotic twin pair with Crohn’s disease and their first-degree relatives. Methods: 22-year-old monozygotic female twins with ileocolonic Crohn’s disease and their healthy brother and parents were examined. DNA samples from patients and relatives were genotyped for CARD15, TLR4,and OCTN polymorphisms. ASCA and p-ANCA analyses were performed. Additionally, patients and relatives filled out a questionnaire concerning multiple environmental factors. Results: Both twins presented in the same year with identical Vienna Classification phenotypes: stenotic behavior (B2) and localization in terminal ileum and colon (L3). Both carried a CARD15 R702W variant, but had normal alleles in TLR4 and OCTN. They were smokers since the age of 15, used oral contraceptives and had undergone appendectomy. The healthy father and brother were CARD15 R702W positive, were non-smokers and had not undergone appendectomy. Conclusion: This case report is the first to describe complete concordance in CARD15 status, phenotypic appearance, and smoking, appendectomy and oral contraceptive use in a pair of monozygotic twins with CD.