Biomarkers In Prediction of Acute Mesenteric Ischaemia: a prospective multicentre study (BIPAMI study): a study protocol.

BACKGROUND: Acute mesenteric ischaemia (AMI) is a life-threatening disease where early diagnosis is critical to avoid morbidity and mortality from extensive irreversible bowel necrosis. Appropriate prediction of presence of bowel necrosis is currently not available but would help to choose the optimal method of treatment. The study aims to identify combinations of biomarkers that can reliably identify AMI and distinguish between potentially reversible and irreversible bowel ischaemia. METHODS: This is a prospective multicentre study. Adult patients with clinical suspicion of AMI (n = 250) will be included. Blood will be sampled on admission, at and after interventions, or during the first 48 h of suspicion of AMI if no intervention undertaken. Samples will be collected and the following serum or plasma biomarkers measured at Tartu University Hospital laboratory: intestinal fatty acid-binding protein (I-FABP), alpha-glutathione S-transferase (Alpha- GST), interleukin 6 (IL-6), procalcitonin (PCT), ischaemia-modified albumin (IMA), D-lactate, D-dimer, signal peptide-CUB-EGF domain-containing protein 1 (SCUBE-1) and lipopolysaccharide-binding protein (LBP). Additionally, more common laboratory markers will be measured in routine clinical practice at study sites. Diagnosis of AMI will be confirmed by computed tomography angiography, surgery, endoscopy or autopsy. Student's t or Wilcoxon rank tests will be used for comparisons between transmural vs. suspected (but not confirmed) AMI (comparison A), confirmed AMI of any stage vs suspected AMI (comparison B) and non-transmural AMI vs transmural AMI (comparison C). Optimal cut-off values for each comparison will be identified based on the AUROC analysis and likelihood ratios calculated. Positive likelihood ratio > 10 (> 5) and negative likelihood ratio < 0.1 (< 0.2) indicate high (moderate) diagnostic accuracy, respectively. All biomarkers with at least moderate accuracy will be entered as binary covariates (using the best cutoffs) into the multivariable stepwise regression analysis to identify the best combination of biomarkers for all comparisons separately. The best models for each comparison will be used to construct a practical score to distinguish between no AMI, non-transmural AMI and transmural AMI. DISCUSSION: As a result of this study, we aim to propose a score including set of biomarkers that can be used for diagnosis and decision-making in patients with suspected AMI. TRIAL REGISTRATION: NCT06212921 (Registration Date 19-01-2024).

Expression of CILP-2 and DDR2 and ultrastructural changes in the articular cartilage of patients with knee osteoarthritis undergoing total knee arthroplasty: a pilot morphological study.

The aim of the study was to correlate the immunohistochemical expression of cartilage intermediate layer protein 2 (CILP-2) and discoidin domain receptor 2 (DDR2), and the ultrastructural changes in the cartilage with the degree of articular cartilage damage in osteoarthritis (OA) patients. Cartilage samples were obtained from twenty patients aged from 46 to 68 years undergoing total knee arthroplasty. In each patient, medial and lateral tibial plateau samples were analysed applying OARSI histopathology grading. Positive correlation was noted between the extent of CILP-2 staining intensity and OARSI grades. Abundant staining for CILP-2 was found in the superficial and middle layers and in the pericellular matrix (PCM) of the deep zone. Transmission electron microscopy studies demonstrated strong damage of chondrocytes, the organelles were often diminished or focally aggregated. As a characteristic finding, PCM was frequently expanded, which may reflect a pathogenic step in OA progression. In conclusion, CILP-2 may potentially be a relevant marker of OA progression as its expression correlated better with cartilage damage than the known marker of articular cartilage damage, DDR2.

GADA and anti-ZnT8 complicate the outcome of phenotypic type 2 diabetes of adults.

BACKGROUND: A proportion of phenotypic type 2 diabetes (T2D) patients produce pancreatic autoantibodies and a majority of T2D patients develop serious life-disabling complications over time despite the implementation of adequate clinical interventions. This study determined whether the presence of pancreatic autoantibodies (GADA, IA-2A, anti-ZnT8, or ICA) was associated with serious complications or concomitant diseases of adult patients diagnosed with T2D (N = 305). MAIN RESULTS: In the study population, 22.3% (N = 68) of subjects were positive for at least 1 of the 4 of the markers associated with autoimmune diabetes (presence of pancreatic autoantibody - pAb), followed by GADA (14.1%, N = 43), ICA (8.9%, N = 27), anti-ZnT8 (5.6%, N = 17) and IA-2A (2.0%, N = 6). Logistic regression analysis adjusted for patient's age, gender and duration of T2D revealed that (i) pAb was associated with higher prevalence of adiposity (odds ratio of adjusted regression model (adOR) 2.51, P = 0.032); (ii) pAb, GADA and anti-ZnT8 were associated with autoimmune thyroid disease (adORs 3.07, P = 0.012; 6.29, P < 0.001 and 3.52, P = 0.052, respectively); (iii) pAb and GADA, in particular, were risk factors for neurological complications (adORs 2.10, P = 0.036; 2.76, P = 0.009, respectively) and polyneuropathy in particular (adORs 2.60, P = 0.012; 3.10, P = 0.007, respectively); and (iv) anti-ZnT8 was a risk factor for developing nephropathy (adOR 4.61, P = 0.022). In addition, adiposity was associated with 5.3-year earlier onset of disease (adjusted linear regression model, P = 0.006). CONCLUSIONS: These results suggest that GADA and anti-ZnT8 are associated with progression of serious T2D complications, including polyneuropathy and nephropathy. In addition, adiposity represents a significant risk for autoimmunity development in T2D patients.

Cartilage Collagen Neoepitope C2C Expression in the Articular Cartilage and Its Relation to Joint Tissue Damage in Patients with Knee Osteoarthritis.

Pathological cleavage of type II collagen (Col2) and generation of Col2 neoepitopes can serve as useful molecular markers of the progression of osteoarthritis (OA). One of such potential biomarkers is type II collagen neoepitope C2C. The aim of this study was to correlate the degree of articular cartilage damage in OA patients with C2C expression in histological samples of tissues removed during total knee replacement. Cartilage samples were obtained from 27 patients ranging in age from 55 to 66 years. In each patient, medial and lateral tibia plateau samples were analyzed according to the OARSI histopathology grading system. The C2C expression was evaluated on histological slides by semi-quantitative analysis using ImageJ Fiji 2.14.0 software. Spearman's rank correlation analysis revealed a positive weak correlation (rho = 0.289, p = 0.0356) between the histological grade of tissue damage and the percentage of C2C staining. In addition, a highly significant positive correlation (rho = 0.388, p = 0.0041) was discovered between the osteoarthritis score (combining the histological grade of damage with the OA macroscopic stage) and the percentage of C2C staining in the samples. The C2C expression was detected in all the regions of the articular cartilage (i.e., the superficial zone, mid zone, deep zone and tidemark area, and the zone of calcified cartilage). Our findings imply that local expression of C2C correlates with the articular cartilage damage in OA-affected knees. This confirms that C2C can be used as a prospective marker for assessing pathological changes in the OA course and OA clinical trials.

Comparison of Antigen Retrieval Methods for Immunohistochemical Analysis of Cartilage Matrix Glycoproteins Using Cartilage Intermediate Layer Protein 2 (CILP-2) as an Example.

The aim of this study was to compare different antigen retrieval methods to improve the outcome of immunohistochemistry (IHC) performed on osteoarthritic (OA) cartilage obtained from total knee replacement operation. A voluminous and dense extracellular matrix of articular cartilage inhibits antibody penetration, and therefore, proteins present at low concentrations and masked during fixation may need antigen retrieval to enhance an IHC outcome. We focused on the IHC detection of a minor but diagnostically promising cartilage glycoprotein, CILP-2 (cartilage intermediate layer protein 2), to demonstrate the effect of four different protocols: (1) heat-induced epitope retrieval (HIER), (2) proteolytic-induced epitope retrieval applying proteinase K and hyaluronidase (PIER), (3) HIER combined with PIER, and (4) no antigen retrieval (control). A semi-quantitative staining assessment based on the CILP-2 staining extent was applied. Out of the tested antigen retrieval protocols, the best CILP-2 IHC staining results were achieved by PIER. Combining PIER with HIER did not improve CILP-2 staining in the given experimental setting. Rather the opposite, the application of heat reduced the positive effect of PIER on CILP-2 staining and resulted in the frequent detachment of sections from the slides. Our findings emphasize the need for proper adaptation of antigen retrieval protocols for IHC to maximize the quantitative evaluation of minor matrix proteins in OA articular cartilage samples.

Autoantibodies Neutralizing Type III Interferons Are Uncommon in Patients with Severe Coronavirus Disease 2019 Pneumonia.

Autoantibodies (AABs) neutralizing type I interferons (IFN) underlie about 15% of cases of critical coronavirus disease 2019 (COVID-19) pneumonia. The impact of autoimmunity toward type III IFNs remains unexplored. We included samples from 1,002 patients with COVID-19 (50% with severe disease) and 1,489 SARS-CoV-2-naive individuals. We studied the prevalence and neutralizing capacity of AABs toward IFNλ and IFNα. Luciferase-based immunoprecipitation method was applied using pooled IFNα (subtypes 1, 2, 8, and 21) or pooled IFNλ1-IFNλ3 as antigens, followed by reporter cell-based neutralization assay. In the SARS-CoV-2-naive cohort, IFNλ AABs were more common (8.5%) than those targeting IFNα2 (2.9%) and were related with older age. In the COVID-19 cohort the presence of autoreactivity to IFNλ did not associate with severe disease [odds ratio (OR) 0.84; 95% confidence interval (CI) 0.40-1.73], unlike to IFNα (OR 4.88; 95% CI 2.40-11.06; P < 0.001). Most IFNλ AAB-positive COVID-19 samples (67%) did not neutralize any of the 3 IFNλ subtypes. Pan-IFNλ neutralization occurred in 5 patients (0.50%), who all suffered from severe COVID-19 pneumonia, and 4 of them neutralized IFNα2 in addition to IFNλ. Overall, AABs to type III IFNs are rarely neutralizing, and do not seem to predispose to severe COVID-19 pneumonia on their own.

Novel Early Pregnancy Multimarker Screening Test for Preeclampsia Risk Prediction.

Preeclampsia (PE) is a common pregnancy-linked disease, causing preterm births, complicated deliveries, and health consequences for mothers and offspring. We have previously developed 6PLEX, a multiplex assay that measures PE-related maternal serum biomarkers ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 in a single test tube. This study investigated the potential of 6PLEX to develop novel PE prediction models for early pregnancy. We analyzed 132 serum samples drawn at 70-275 gestational days (g days) from 53 pregnant women (PE, n = 22; controls, n = 31). PE prediction models were developed using a machine learning strategy based on the stepwise selection of the most significant models and incorporating parameters with optimal resampling. Alternative models included also placental FLT1 rs4769613 T/C genotypes, a high-confidence risk factor for PE. The best performing PE prediction model using samples collected at 70-98 g days comprised of PTX3, sFlt-1, and ADAM12, the subject's parity and gestational age at sampling (AUC 0.94 [95%CI 0.84-0.99]). All cases, that developed PE several months later (onset 257.4 ± 15.2 g days), were correctly identified. The model's specificity was 80% [95%CI 65-100] and the overall accuracy was 88% [95%CI 73-95]. Incorporating additionally the placental FLT1 rs4769613 T/C genotype data increased the prediction accuracy to 93.5% [AUC = 0.97 (95%CI 0.89-1.00)]. However, 6PLEX measurements of samples collected at 100-182 g days were insufficiently informative to develop reliable PE prediction models for mid-pregnancy (accuracy <75%). In summary, the developed model opens new horizons for first-trimester PE screening, combining the easily standardizable 6PLEX assay with routinely collected antenatal care data and resulting in high sensitivity and specificity.

Antibody levels remain high to one-year's follow-up after moderate and severe COVID-19, but not after mild cases.

BACKGROUND: Understanding the longevity of antibodies against SARS-CoV-2 infection is of utmost importance in predicting the further course of the pandemic and to plan vaccination strategies. Here we report a cohort of COVID-19 patients with different disease severities whose antibody dynamics we evaluated during one-year of follow-up. METHODS: We conducted a longitudinal study of 123 COVID-19 patients and 45 SARS CoV-2 negative outpatients with upper respiratory tract infection. We analyzed the demographic and clinical features of the patients with COVID-19 in relation to different disease severities according to the WHO classification. The antibody response was evaluated by a Luciferase Immunoprecipitation System (LIPS) assay at 3, 6, and 12 months after the acute infection. RESULTS: Amongst the enrolled COVID-19 patients, 15 (12%) had mild, 42 (34%) had moderate, 39 (32%) had severe and 27 (22%) had critical disease courses; 79% of the patients were hospitalized. During follow-up, all patients had anti-SARS RBD-IgG levels above the cut-off value on all visits, but the antibody levels varied significantly between the different disease severity groups. Between the six- and 12-month follow-up visits, 41% of patients were vaccinated, which enhanced their antibody levels significantly. CONCLUSION: Our data demonstrate sustained antibody levels at one-year after moderate and severe COVID-19 infection. Vaccination of patients with the mild disease is important to raise the antibody levels to a protective level.

Meta-analysis of genome-wide association studies confirms a susceptibility locus for knee osteoarthritis on chromosome 7q22.

OBJECTIVES: Osteoarthritis (OA) is the most prevalent form of arthritis and accounts for substantial morbidity and disability, particularly in older people. It is characterised by changes in joint structure, including degeneration of the articular cartilage, and its aetiology is multifactorial with a strong postulated genetic component. METHODS: A meta-analysis was performed of four genome-wide association (GWA) studies of 2371 cases of knee OA and 35 909 controls in Caucasian populations. Replication of the top hits was attempted with data from 10 additional replication datasets. RESULTS: With a cumulative sample size of 6709 cases and 44 439 controls, one genome-wide significant locus was identified on chromosome 7q22 for knee OA (rs4730250, p=9.2 × 10⁻9), thereby confirming its role as a susceptibility locus for OA. CONCLUSION: The associated signal is located within a large (500 kb) linkage disequilibrium block that contains six genes: PRKAR2B (protein kinase, cAMP-dependent, regulatory, type II, ß), HPB1 (HMG-box transcription factor 1), COG5 (component of oligomeric golgi complex 5), GPR22 (G protein-coupled receptor 22), DUS4L (dihydrouridine synthase 4-like) and BCAP29 (B cell receptor-associated protein 29). Gene expression analyses of the (six) genes in primary cells derived from different joint tissues confirmed expression of all the genes in the joint environment.

Impact of cryopreservation on serum concentration of matrix metalloproteinases (MMP)-7, TIMP-1, vascular growth factors (VEGF) and VEGF-R2 in Biobank samples.

BACKGROUND: Blood biomarkers are subject to pre-analytical variability. In many cases, the stability of important new tissue biomarkers during freeze cycles and storage has not been studied sufficiently. METHODS: To test the stability of matrix metalloproteinases-7 (MMP-7) and their tissue inhibitors (TIMP-1), vascular growth factors (VEGF) and VEGF-receptor, serum samples were frozen and then thawed up to six times. The impact of storage temperature was investigated using an accelerated stability testing protocol. Stability at -20°C and -75°C was calculated using the Arrhenius equation. RESULTS: The average concentration of TIMP-1 was stable, even after six freeze/thaw cycles. One thawing did not change the concentration of MMP-7 and VEGF-receptor. However, repeated freeze/thaw cycles increased the measured values significantly. Decreases in VEGF concentrations were dramatic, even after the first freeze/thaw cycle. According to the Arrhenius calculation, MMP-7 showed excellent stability, at least 5 years at -20°C and several 100 years at -75°C. The VEGF-receptor maintains 90% of its initial concentration at -20°C over 3 months, and decades at -75°C. TIMP-1 and VEGF showed poor stability with cryopreservation, even at -75°C. CONCLUSIONS: The stability of MMP-7, TIMP-1, VEGF or VEGF-receptor in biobanking is highly variable, and this should be taken into account in the interpretation of results. A temperature -20°C is unsuitable for prolonged storage of the biomarkers investigated, and repeated thawing of sera is not recommended. VEGF is especially unstable and should be quantitated using serum that has never been frozen.

Genetic variation in the SMAD3 gene is associated with hip and knee osteoarthritis.

OBJECTIVE: Smad3 (or, MADH3) is a key intracellular messenger in the transforming growth factor beta signaling pathway. In mice, Smad3 deficiency accelerates growth plate chondrocyte maturation and leads to an osteoarthritis (OA)-like disease. We undertook this study to investigate the role of genetic variation in SMAD3 in the risk of large-joint OA in humans. METHODS: Ten tag single-nucleotide polymorphisms (SNPs) in the SMAD3 gene region were tested in a discovery set: 313 patients who had undergone total knee replacement, 214 patients who had undergone total hip replacement, and 520 controls from the UK. The SNP associated with both hip and knee OA was subsequently genotyped in 1,221 controls and 1,074 cases from 2 cohorts of patients with hip OA and 2,537 controls and 1,575 cases from 4 cohorts of patients with knee OA. RESULTS: A SNP (rs12901499) mapping to intron 1 of SMAD3 was associated with both knee and hip OA (P < 0.0022 and P < 0.021, respectively) in the discovery set. In all study cohorts, the major allele (G) was increased among OA patients relative to controls. A meta-analysis for knee OA yielded an odds ratio (OR) of 1.22 (95% confidence interval [95% CI] 1.12-1.34), P < 7.5 x 10(-6). For hip OA, the OR was 1.22 (95% CI 1.09-1.36), P < 4.0 x 10(-4). No evidence for heterogeneity was found (I(2) = 0%). CONCLUSION: Our data indicate that genetic variation in the SMAD3 gene is involved in the risk of both hip OA and knee OA in European populations, confirming the results from animal models on the potential importance of this molecule in the pathogenesis of OA.

Risk Assessment of the Progression of Early Knee Osteoarthritis by Collagen Neoepitope C2C: A Longitudinal Study of an Estonian Middle-Aged Cohort.

One of the unmet needs to be addressed is prognostic biomarkers for early knee osteoarthritis (kOA). We aimed to study the association of urinary collagen type-II C-terminal cleavage neoepitope (uC2C) with the emergence and progression of kOA. The longitudinal data of 330 subjects (aged 32-60 years) from an Estonian population-based cohort were used. The radiographic progression was evaluated by the grading system of Nagaosa et al. of knee compartments at baseline and three years later. The emerging kOA consisted of subjects with developing osteophytes or joint space narrowing, whereas kOA progressors showed aggravation of radiographic grade. Baseline uC2C levels were measured by the IBEX-uC2C assay. At baseline, the subjects were middle-aged (mean age, 47.6 years) and overweight (mean BMI, 28.0 kg/m2), and the majority of them (51.2%) had a diagnosis of kOA grade 1. Multiple logistic regression models adjusted for sex, age, and BMI were used for risk calculations. We demonstrate that increased uC2C accurately predicted the risk of emerging of kOA (OR = 5.87 (1.71-20.22); AUC = 0.79) compared with controls without radiographic kOA over 12 years. However, the most accurate prediction of progression by the biomarker was found in women (OR = 23.0 (2.2-245), AUC = 0.91). In conclusion, uC2C may be a promising candidate as a prognostic biomarker for kOA progression, particularly of emerging kOA in women.

Allelic variants in the PHTF1-PTPN22, C12orf30 and CD226 regions as candidate susceptibility factors for the type 1 diabetes in the Estonian population.

BACKGROUND: Type 1 diabetes is a multifactorial disease with a strong genetic component. The aim of the study was to assess the impact of single nucleotide polymorphisms (SNPs) in several genes as susceptible markers in the risk of type 1 diabetes in the Estonian population. METHODS: The rs6679677 (1p13), rs17696736 (12q24) and rs763361 (18q22) were genotyped in a total of 230 controls and 154 type 1 diabetes patients of Estonian origin. RESULTS: The rs6679677 A (OR = 2.13, 95%CI = 1.48-3.08, p = 0.00001), rs17696736 G (OR = 1.53, 95%CI = 1.14-2.04, p = 0.0046) and rs763361 T (OR = 1.48, 95%CI = 1.11-1.98, p = 0.0084) alleles were associated with risk of type 1 diabetes. CONCLUSIONS: The current study supports the rs6679677 (PHTF1-PTPN22), rs17696736 (C12orf30) and rs763361 (CD226) SNPs as susceptibility factors for type 1 diabetes outside the major histocompatibility region (MHC) region. The full study had 80% or above to detect an odds ratio of 1.8 under the assumption of an additive model at type 1 error rate, alpha = 0.05.

Cartilage collagen neoepitope C2C in urine as an integrative diagnostic marker for early knee osteoarthritis.

Objective: To investigate the suitability of urinary collagen type-II C-terminal cleavage neoepitope (uC2C) as a marker for early knee osteoarthritis (kOA). Design: We examined 302 Estonian subjects (mean age, 49 years): 186 subjects with and 20 control subjects without knee symptoms, and 96 patients treated by arthroscopy. For the latter, cartilage lesions were characterized using Société Francaise d'Arthroscopie (SFA) scores. Standardized radiographs of bilateral tibiofemoral (TF) and patellofemoral (PF) joints were assessed for osteoarthritis (OA) features. Osteophytes (Ophs) and joint space narrowing (JSN) were graded separately. uC2C was measured by the uC2C-HUSA assay. Logistic and linear regression models were used for data analysis. Results: Of the kOA cases, 50% were isolated (TF or PF) grade 1; 10% were grade 2. JSN with Ophs was more frequent in females than in males (52% vs. 34%, p = 0.01). Increased uC2C level was associated with gradual increase in the risk of kOA grade of severity (odds ratio = 2.14-3.7) including grade 1 vs. 0. TF-OA and PF-OA equally predicted uC2C concentration (R 2 = 0.33-0.35). uC2C prediction was better for females than for males (R 2 = 0.42 vs. 0.22 by TF-OA). The best predictive model for uC2C level (R 2 = 0.75) included three OA features: macroscopic cartilage lesions, TF Ophs, and PF-JSN. Conclusions: uC2C as an integrative marker of kOA is associated with cartilage degradation and Oph formation in the PF- and TF-joints. Increased uC2C concentration could be used as an early diagnostic marker for kOA in clinical studies.

Single-Tube Multimarker Assay for Estimating the Risk to Develop Preeclampsia.

BACKGROUND: Preeclampsia (PE) affects 2%-8% of all pregnancies worldwide. The predictive value of the currently used maternal serum fms-like tyrosine kinase-1/ placental growth factor (sFlt-1/PlGF) test is < 40% for PE onset within 4 weeks. We aimed to develop an innovative multiplex assay to improve PE prediction. METHODS: The 6PLEX assay combining the measurements of ADAM12, sENG, leptin, PlGF, sFlt-1, and PTX3 was developed for the Luminex® xMAP platform. Assay performance was evaluated using 61 serum samples drawn from 53 pregnant women between 180 and 275 gestational days: diagnosed PE cases, n = 4; cases with PE onset within 4-62 days after sampling, n = 25; controls, n = 32. The B·R·A·H·M·S Kryptor sFlt-1/PlGF test (Thermo Fisher Scientific, Hennigsdorf, Germany) was applied as an external reference. Alternative PE prediction formulae combining 6PLEX measurements with clinical parameters were developed. RESULTS: There was a high correlation in sFlt-1/PlGF estimated for individual sera between the 6PLEX and B·R·A·H·M·S Kryptor immunoassays (Spearman's r = 0.93, P < 0.0001). The predictive power of the 6PLEX combined with gestational age and maternal weight at sampling reached AUC 0.99 (95% CI 0.97-1.00) with sensitivity 100.0% and specificity 96.9%. In all models, sFlt-1/PlGF derived from the B·R·A·H·M·S immunoassays exhibited the lowest AUC value (<0.87) and sensitivity (<80%) with broad confidence intervals (13%-92%). The estimated prognostic yield of the 6PLEX compared to the B·R·A·H·M·S assay was significantly higher (96.5% vs 73.7%; P = 0.0005). CONCLUSIONS: The developed single-tube multimarker assay for PE risk estimation in combination with clinical symptoms reached high prognostic yield (96.5%) and exhibited superior performance compared to the sFlt-1/PlGF test.

Patients with down syndrome have increased prevalence of rheumatoid factor but not autoantibodies to anti-cyclic citrullinated peptide.

The association between Down syndrome (DS), a genetic disorder resulting from trisomy of the 21st chromosome, and the autoantibodies of rheumatoid arthritis (RA) has been proposed but not unequivocally proven. The aim of this study was to determine whether adult patients with DS present higher levels of anti-cyclic citrullinated peptide (anti-CCP) antibodies and/or rheumatoid factor (RF) than the general population. Our results showed that none of the 68 patients with DS had anti-CCP antibodies, whereas among 204 age- and sex-matched controls these autoantibodies were present in one person. However, DS patients presented a higher number of RF positive cases than controls (11.7% to 3.2% respectively; Fisher's exact test, p = .027). The higher number of RF positive cases in the DS group without increase of anti-CCP antibodies may be indicative of immune disturbances in general rather than RA in these patients. Our study supports the view that RA does not occur with higher frequency in patients with DS than in the general population.

Celiac Disease in Children, Particularly with Accompanying Type 1 Diabetes, Is Characterized by Substantial Changes in the Blood Cytokine Balance, Which May Reflect Inflammatory Processes in the Small Intestinal Mucosa.

Cytokines play a pivotal role in the maintenance of intestinal homeostasis inducing pro- or anti-inflammatory response and mucosal barrier function in celiac disease (CD) and type 1 diabetes (T1D). We aimed to compare the levels of pro- and anti-inflammatory cytokines in CD patients without and with coexisting T1D, as well as to evaluate its association with the presence of enteroviruses (EV), regulatory T cells (Tregs), and dendritic cells (DCs) in small bowel mucosa. Altogether, 72 patients (median age 10.1 years) who had undergone small bowel biopsy were studied. The study group consisted of 24 patients with CD (median age 6.5 years), 9 patients with CD and concomitant T1D (median age 7.0 years), two patients with T1D (median age 8.5 years), and 37 patients (median age 14.0 years) with functional gastrointestinal disorders (FGD) and a normal small bowel mucosa as controls. The levels of 33 cytokines in serum were measured by multiple analysis using the Milliplex® MAP Magnetic Bead assay. The densities of FOXP3+ Tregs, CD11c+ DC, indoleamine 2,3-dioxygenase+ (IDO+) DC, langerin+ (CD207+) DCs, and EV were evaluated by immunohistochemistry as described in our previous studies. Circulating anti-EV IgA and IgG were evaluated using ELISA. The most important finding of the study is the significant increase of the serum levels of IL-5, IL-8, IL-13, IL-15, IL-17F, IL-22, IL-27, IP-10, MIP-1ß, sIL-2Rα, sTNFRII, and TNFα in CD patients compared to controls and its correlation with the degree of small bowel mucosa damage graded according to the Marsh classification. The leptin level was higher in females in all study groups. The levels of IL-2, IL-6, IL-12 (P70), IL-15, IP-10, and IFNγ correlated significantly with the density of FOXP3+ Tregs in lamina propria of the small bowel mucosa, which supports the evidence about the signaling role of these cytokines in the peripheral maintenance of FOXP3+ Tregs. At the same time, a significant negative correlation occurred between the level of IL-4 and density of FOXP3+ Tregs in controls. Another important finding of our study was the correlation of IL-17F, IP-10, sTNFRII, MCP-1, and GM-CSF with the density of EV-positive cells in the lamina propria of the small bowel mucosa. Correlation of MIP-1 (CCL-4) with CD103+ DC and langerin+ DC densities may point to their significance in the recruitment of immune cells into the lamina propria and in driving the inflammatory response in CD patients. Our results suggest the predominance of Th1 and Th17 immune responses over EV VP1 protein in CD and T1D patients. The significant elevation of Th2 cytokines, like IL-5 and IL-13, but not IL-4, in CD patients and its correlation with the degree of small bowel mucosa damage could reflect the role of these cytokines in gut defense and inflammation.

Propensity to excessive proinflammatory response in chronic Lyme borreliosis.

The clinical course of Lyme borreliosis is extremely variable. However, all the clinical manifestations, acute or chronic, are characterized by strong inflammation. Borrelia burgdorferi can induce the production of several proinflammatory and anti-inflammatory cytokines. The aim of our study was to find out whether the balance between inflammatory and regulatory mechanisms is important in determining the course of Lyme borreliosis. 13 patients with early Lyme borreliosis, 8 patients with chronic Lyme disease with neurological or joint manifestations, and 15 age- and sex-matched healthy controls were studied. Chronic forms of Lyme borreliosis were characterized by stronger TNF-alpha response by monocytes to lipopolysaccharide as well as to borrelia antigen compared to early Lyme borreliosis and the healthy state. The percentage of IL-10-secreting monocytes in response to borrelia lysate was lower in the Lyme borreliosis patients than in healthy controls. The percentage of CD4(+) CTLA-4(+) regulatory T cells showed the highest values in early Lyme borreliosis. We conclude that chronic forms of Lyme borreliosis can evolve due to an aberrant innate proinflammatory response.

Demographic associations for autoantibodies in disease-free individuals of a European population.

The presence of autoantibodies usually precedes autoimmune disease, but is sometimes considered an incidental finding with no clinical relevance. The prevalence of immune-mediated diseases was studied in a group of individuals from the Estonian Genome Project (n = 51,862), and 6 clinically significant autoantibodies were detected in a subgroup of 994 (auto)immune-mediated disease-free individuals. The overall prevalence of individuals with immune-mediated diseases in the primary cohort was 30.1%. Similarly, 23.6% of the participants in the disease-free subgroup were seropositive for at least one autoantibody. Several phenotypic parameters were associated with autoantibodies. The results suggest that (i) immune-mediated diseases are diagnosed in nearly one-third of a random European population, (ii) 6 common autoantibodies are detectable in almost one-third of individuals without diagnosed autoimmune diseases, (iii) tissue non-specific autoantibodies, especially at high levels, may reflect preclinical disease in symptom-free individuals, and (iv) the incidental positivity of anti-TPO in men with positive familial anamnesis of maternal autoimmune disease deserves further medical attention. These results encourage physicians to evaluate autoantibodies in addition to treating a variety of patient health complaints to detect autoimmune-mediated disease early.