Phenotypic and functional characterisation of synovial fluid-derived neutrophils in knee osteoarthritis and knee infection.

OBJECTIVE: An increase in the number of neutrophils (NEUs) has long been associated with infections in the knee joints; however, their impact on knee osteoarthritis (KOA) pathophysiology remains largely unexplored. DESIGN: This study compared the phenotypic and functional characteristics of synovial fluid (SF)-derived NEUs in KOA and knee infection (INF). RESULTS: KOA NEUs were characterised by a lower expression of CD11b, CD54, and CD64 and higher expression of CD62L, TLR2, and TLR4 compared with INF NEUs. Except for CCL2, lower levels of inflammatory mediators and proteases were detected in KOA SF than in INF SF. Functionally, KOA NEUs displayed increased reactive oxygen species production and phagocytic activity compared with INF NEUs. Moreover, KOA and INF NEUs differed in cell sizes, histological characteristics of the surrounding synovial tissues, and their effects on the endothelial cells assessed by human umbilical vein endothelial cells. When KOA patients were subdivided based on the SF NEU abundance, patients with high NEUs (10%-60%) were characterised by i) elevated SF protein levels of TNF-α, IL-1RA, MMP-9, sTREM-1, VILIP-1 and ii) lower CD54, CD64, TLR2 and TLR4 expression compared to patients with low NEUs (<10%). Analysis of paired SF samples suggests that low or high NEU percentages, respectively, persist throughout the course of disease. CONCLUSIONS: Our findings suggest that NEU may play a significant role in KOA pathophysiology. Further studies should explore the mechanisms that contribute to the increased number of NEUs in SF and the clinical consequences of neutrophilic phenotype in KOA.

Odds-ratio network for postoperative factors revealing differences in the 2-year longitudinal pattern of satisfaction between women and men after total knee arthroplasty.

Timely and accurate assessments of the factors influencing satisfaction, a key indicator of success in primary total knee arthroplasty (TKA), may help improve TKA outcomes. Here we performed the longitudinal trend analysis of relation between satisfaction and 12 postoperative factors, which positively or negatively influence the patient satisfaction 2 years after TKA. In a real-world registry cohort (women/men: 1121/650), we showed similarities and differences between women and men in the contribution of postoperative factors to satisfaction 2 years after TKA as assessed by odds-ratio-similarity network. In men, the strongest negative factors were pain and complications, followed by mechanical problems. In women, the strongest negative factors were the pain and knee instability, followed by other mechanical problems, complications and low levels of sports activity. In both sexes, physical activity and the Knee Society Score (general and functional) influenced positively satisfaction; long-distance walking was associated with satisfaction only in women. A trend analysis revealed a reduction in the strength of satisfaction-related factors over 2 years of check-ups, particularly in women. Our study demonstrates that the key check-up for assessing the evolution of satisfaction in the 2 years after TKA was at 3 months in both sexes.

Knee osteoarthritis phenotypes based on synovial fluid immune cells correlate with clinical outcome trajectories.

BACKGROUND: Knee osteoarthritis (KOA) is a highly heterogeneous disease encompassing a wide range of clinical phenotypes. Phenotypes based on immune cells and protein pattern in synovial fluid (SF) and their relationship to clinical trajectories have not been described. OBJECTIVE: To assess phenotypes based on immune cells and protein pattern of SF in KOA. DESIGN: SF-derived immune cells were investigated in 119 patients with KOA using flow cytometry. Immune-phenotypes (iPhen) were determined by multivariate patient similarity network analysis and related to clinical trajectory (3-6 months post-sampling) along with protein pattern and macrophage chemokine receptors. RESULTS: Four iPhen were detected based on the distribution of T-lymphocytes, monocyte-macrophage lineage cells and activated CD8+ T-lymphocytes. The 'activated' phenotype (n = 17) had high T-lymphocytes but low monocyte-macrophage lineage cells and neutrophils, all highly activated, and showed improved symptoms in 70% patients. The 'lymphoid progressive' phenotype (n = 31) had high neutrophils, low lymphocytes and monocyte-macrophage lineage cells, low activation and was associated with lower pain levels. The 'myeloid progressive' phenotype (n = 35) had high NK and monocyte-macrophage lineage cells but low T-lymphocytes and activation. The 'aggressive' phenotype (n = 36) had high lymphocytes, macrophages, NK cells and neutrophils and high activation, and only 39% of patients improved during follow-up. Low CXCR4 and CCR7 expression on macrophages and high CXCL10 in SF were linked to improved clinical trajectory. CONCLUSION: We identified four immune-phenotypes that were associated with different clinical trajectories in KOA patients. How these phenotypes can be targeted therapeutically deserves further investigation.

[Strong Antibacterial Efficacy of Titanium Surfaces Modified by Nanotubes and Silver Nanoparticles]. / Silné antibakteriální vlastnosti kovového povrchu modifikovaného nanotrubicemi titanu a nanostríbrem.

PURPOSE OF THE STUDY Nano-structuring and nano-silver have been extensively studied for improving the antibacterial ability of implants due to their powerful antibacterial activity; however, there is no clinical application as yet. The aim of the study was to determine the antibacterial, antiadhesive and cytotoxic features of Ti6Al4V modified with nano-texturing and silver nano-particles. MATERIAL AND METHODS The nanoparticles were applied on polished and nano-textured Ti6Al4V using sonoreduction. The surface topography, roughness, friction coefficients, hardness and elastic modulus values for prepared top layers were established. The materials were tested for antibacterial and antiadhesion activity using reference bacterial strains (Staphylococcus epidermidis CCM 7221, Staphylococcus aureus MRSA 4591, Enterococcus faecalis CCM 4224, Escherichia coli CCM 3954) and their cytocompatibility. RESULTS A strong antibacterial activity of samples treated with nano-texture and/or silver nanoparticles compared to all the tested bacterial strains at 24 hours was proven. This antibacterial activity was diminishing in relation to Staphylococcus aureusand Enterococcus faecalisat 48 and 72 hours but remained very effective against Staphylococcus epidermidisand Escherichia coli. We also demonstrated antibiofilm activity for samples treated with silver nanoparticles and nano-tubes in experiments lasting 24 and 72 hours. DISCUSSION Our main findings are in agreement with those reported in recent literature. The implant surfaces treated with nano-texture in combination with silver nanoparticles exhibit strong antibacterial and antibiofilm characteristics. Despite there is conclusive evidence of strong antibacterial functioning, why these implant modifications have not been widely applied in clinical practice remains a question. While many obstacles including legislative procedures required for clinical implementation are more or less known, it should be clearly demonstrated that this surface modification does neither harm the patient nor interfere with the long-term survivorship of the implants before their wide-range clinical application. CONCLUSIONS Surface modification of Ti6Al4V with nano-texturing and silver nanoparticles resulted in strong antibacterial and modest antibiofilm effects. Thus, our results confirmed the technological potential of nano-texturing and silver nanoparticles for the improvement of antibacterial properties of implants. Key words:prosthetic joint infection, anti-infective biomaterials, titanium alloy, silver nanoparticles, nanotubes, prevention of infection.

Gender-related differences observed among immune cells in synovial fluid in knee osteoarthritis.

OBJECTIVE: There is no existing comprehensive report on the cellular composition of synovial fluids (SFs) from knee osteoarthritis (OA). We therefore aimed to characterise the immune cell composition in SFs from knee OA (KOA) and in subgroups according to gender. DESIGN: The immunophenotyping of monocyte/macrophage lineage cells, T and B cells, NK cells, neutrophils, dendritic and mast cells (MC) present in SFs from 53 patients (24 males/29 females) with KOA was performed using 6-colour flow cytometry. RESULTS: SFs from patients with OA contained 90% hematopoietic cells. Lymphocytes were the predominant cell population (44.8%) in the SFs of OA patients, with CD4+ T lymphocytes being more prevalent than CD8+ T cells (CD4+/CD8+ ratio = 1.3). Within the monocyte/macrophage lineage gating, monocytes accounted for 33.9%, macrophages 14.8%, myeloid dendritic cells 16.4%. The rest of the hematopoietic cells were comprised of neutrophils (8%), NK cells (3.8%), T regulatory cells (1.2%), plasmacytoid dendritic cells (1.1%), mast cells (0.3%). In OA females, a higher percentage of CD4+ T cells (P = 0.023), macrophages (P = 0.012), and a lower percentage of monocytes (P = 0.008) and CD8+ T cells (P = 0.002) were detected in comparison to OA males. CONCLUSIONS: Based on the immune cell composition of SFs, data mining analysis revealed distinct phenotypes (monocyte- and lymphocyte-predominant) within each gender group. This first study on the cellular complexity of SFs in KOA showed marked differences between male and female patients. The findings give a rational starting point for patient stratification according to their phenotypes, as is required for phenotype-specific treatment strategies.

Sleep apnoea in patients with nocturnal hypertension - a multicenter study in the Czech Republic.

Sleep apnoea (SA) is common in patients with hypertension. Nowadays, limited data on the prevalence of SA in nocturnal hypertension (NH) exist. Therefore, we studied the occurrence of SA in Czech patients and its association with 24-h ambulatory blood pressure monitoring (ABPM), breathing disturbances in sleep, anthropometric data, Mallampati score and Epworth sleepiness scale (ESS) using the Apnea Link device. Undiagnosed SA was found in 72.9 % patients (29.3 % mild, 26.6 % moderate, 17.0 % severe) of 188 patients with NH measured by ABPM. The median of the apnoea-hypopnoea index (AHI) was 12.0 (25th-75th percentile 5.0-23.8). Moderate/severe SA (AHI>/=15) was associated with BMI, waist circumference, mean night saturation (SpO(2)), t90, oxygen desaturation index (ODI), ESS (daytime BP only) (p0.09). A likelihood of moderate/severe SA was enhanced by ODI>14.5 events/h (odds ratio=57.49, 95 % CI=22.79-145.01), t90>6.5 % (8.07, 4.09-15.92), mean night SpO(2)<93.5 % (3.55, 1.92-6.59), BMI>29.05 kg/m(2) (6.22, 3.10-12.49), circum waist>105.5 cm (3.73, 1.57-8.83), but not by any ABPM parameter. In conclusion, a high incidence of SA (72.9 %) was observed in Czech patients with NH. SA severity was associated with body characteristics and oxygenation parameters, but not with ABMP parameters and Mallampati score.

[Smoking, Preoperative Activity, and Waiting Time for the Surgery Could Predict the Risk of Early Reoperation in Total Knee Arthroplastyy]. / Kourení, predoperacní aktivita a cekání na operaci by mohlo predikovat cetnost casných reoperací u pacientu s TEP kolena.

PURPOSE OF THE STUDY Information that would help physicians make decisions and improve the likelihood of achieving the desired results of medical interventions is sought as part of the concept of the individualized approach to patients. The primary purpose of our study was to identify which features determine the higher/lower likelihood of the need for early reoperation after a TKA (total knee arthroplasty). The successful preoperative identification of high risk patients could lead to the adjustment of the surgical procedure and thus lower the percentage of revision surgeries. MATERIAL AND METHODS In total, 826 patients (296 men and 530 women) were included in our prospective study; these patients underwent TKA implantation at our department between September 2010 and March 2015. The average age of the patients at the time of primary TKA implantation was 68.9 years. Over 60 preoperative and perioperative parameters were tracked and continuously recorded in our arthroplasty register. First, conventional analysis of individual parameters was carried out and odd ratios for their relationship with revision surgeries were set. Subsequently, the data were transformed into a graph and methods of complex network analysis were applied to identify such combinations of features (parameters) that would significantly separate the operated patients into homogeneous subgroups. The observed patient subgroups were then reanalyzed for parameters related to reoperations. RESULTS Thirty-three patients (4% of those studied) required early TKA revision (within 3 years of primary implantation). The most frequent reason for revision surgery was an early postoperative infection. The analysis of observed characteristics proved that the likelihood of revision surgery was by 80% lower in women in comparison with men. Other parameters associated with a higher frequency of reoperations were the level of preoperative activity, smoking and the waiting time for the first operation. Patients waiting for primary TKA implantation for more than 3 months showed a 2.7 times greater likelihood of revision surgery when compared to those who were operated within 3 months after the indication to surgery. Patients declaring medium or high activity levels (assessed by means of the UCLA scale) had a 2.1 times higher likelihood of revision surgery in comparison to patients with low physical activity levels. Smoking meant up to 3.2 times greater likelihood of revision in comparison with nonsmokers. Conversely, no correlation between a greater risk of reoperation and age, BMI (body mass index) or the level of comorbidities evaluated by means of the Charlson scale was confirmed. No correlation between the risk of revision and primary diagnosis was found either. DISCUSSION The frequency of early TKA revision surgeries (within 3 years after the primary surgery) in the evaluated sample is relatively high (4%). On the contrary, the reasons for early revisions correspond with recent publications. The risks of TKA infection overlap with the predictors of wound healing disorders to a great extent. Smoking, obesity and comorbidities decreasing the efficiency of the immune system are mentioned most frequently. Patients waiting for TKA implantation longer were more inclined to require early revision surgery too. Awareness of this fact is reflected in the tendency to shorten the waiting time for TKA surgery. A number of studies have pointed out the negative influence of longer waiting times on postoperative results. In our study, it was men who required revision most frequently, specifically the group of those having smoking and higher physical activity in their case histories. The influence of smoking on early postoperative morbidity is also well known. A significant finding is that stopping smoking can decrease the probability of early reoperation. However, we failed to explain the influence of higher physical activity. The influence of patients' age, BMI, level of comorbidities or primary diagnosis on the frequency of revision surgeries were not demonstrated. CONCLUSIONS We proved that women definitely show a lower risk of early TKA revision surgeries in comparison with men. A higher frequency of reoperations was related to modifiable factors such as smoking, longer waiting times for the primary operation, and a higher preoperative level of physical activity. A significant finding is that stopping smoking could decrease the probability of early TKA revision. Nonetheless, we do not recommend decreasing preoperative physical activity at this point; it will require further studies and verification of this finding. Also, the potential mechanism of the influence of greater preoperative load on the particular reason for revision is yet to be explained. Key words: total knee arthroplasty, complication, early reoperation, risk factors, multivariate analysis, smoking, preoperative activity, waiting times for surgery.

[Low expression of Tlr-1, -2, -4 a il-2, -2r, -10, -10r and TgFb1 in pseudosynovial tissue collected at revision total knee arthroplasty for aseptic loosening]. / Nízká exprese Tlr-1, -2, -4 a il-2, -2r, -10, -10r, TgFb1 v pseudosynoviální tkáni odebrané pri reoperaci asepticky uvolnené totální endoprotézy kycle a kolena.

PURPOSE OF THE STUDY: Aseptic loosening (AL) and periprosthetic osteolysis (PPOL) in total hip (THA) and knee (TKA) arthroplasty are linked to an inflammatory process initiated by wear debris released from artificial joints. There is still limited information about the contribution of Toll-like receptors (TLRs) and distinct regulatory cytokines to AL/PPOL in both joints. METHODS: In this study, we investigated mRNA expression of TLR-1,-2,-4 and cytokines/receptors (IL-2,-2R,-10,-10R, TGFb1) in pseudosynovial tissue obtained from 55 patients with aseptically failed THAs/TKAs and 37 control patients with hip/knee primary osteoarthritis (OA) using quantitative RT-PCR. Immunohistochemical staining was used to detect the corresponding proteins. Non-parametric Kruskal-Wallis and Mann-Whitney tests were used to determine differences between the patient groups. RESULTS: When comparing expression profiles between patients with aseptically failed THA and TKA, higher amounts of TLR-1,- 2,-4 and IL-2R mRNA transcripts were detected in THA patients. The mRNA expression of studied molecules (TLR-1,-2,-4, IL-2, IL-10, IL-2R, IL-10R, TGFb1) did not differ between THA and OA hip tissues. Lower mRNA expression of TLR-1,-2,- 4, IL-10, and IL-10R was detected in TKA when compared to control knee OA. Similar mRNA profiles of IL-2, IL-2R, and TGFb1 were observed in TKA and knee OA. Using immunohistochemistry, we detected low expression of TLR-1 protein in failed THA/TKA, whereas TLR-2 protein levels were higher in TKA/THA patients than in OA controls. High individual variability in TLR-4 protein levels was detected among patients with aseptically loosened THA and TKA. IL-10 protein levels were similar in THA and TKA patient subgroups and control subjects, whereas IL-10R protein level was higher in failed TKAs and OA controls than in THAs. No difference in IL-2 protein levels was detected between patients with THA/TKA and those with OA. DISCUSSION: Our data indicate close similarity between the expression patterns in aseptically failed THA and TKA. However, certain differences were observed which also suggest unique pathways associated with the end-stage of aseptic loosening in THA and TKA. For instance, differences in the size, shape and load of polyethylene particles between THA and TKA could play some role. The composition of THA and TKA and differences in terms of mechanical forces might also be involved. CONCLUSIONS: This is the fist study comparing the gene expression profile of a particular set of innate immunity regulatory molecules between tissues from aseptically failed THA and TKA. Low expression of TLR-1,-2,-4 and cytokines/receptors (IL-2, IL-2R, IL-10, IL-10R, and TGFb1) was observed in pseudosynovial tissues obtained from aseptically failed THAs and TKAs. Higher amount of TLR transcripts was detected in THA as compared to TKA. These findings indicate certain differences in the mechanism of aseptic loosening occurring at the site of THA and TKA. Further research is warranted.

Comparison of periprosthetic tissues in knee and hip joints: differential expression of CCL3 and DC-STAMP in total knee and hip arthroplasty and similar cytokine profiles in primary knee and hip osteoarthritis.

OBJECTIVE: To identify expression profiles (EP) associated with aseptic loosening of total knee arthroplasty (TKA) and to compare them with EP observed in total hip arthroplasty (THA), and primary knee and hip osteoarthritis (OA). DESIGN: Gene EP of TNF, IL-6, IL-8, CHIT1, BMP4, CCL3, CCL18, MMP9, RANKL, OPG, DC-STAMP and SOCS3 were assessed using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) on tissues retrieved from patients with aseptically failed TKA (n = 21), THA (n = 41) and primary knee (n = 20) and hip (n = 17) OA. Immunohistochemistry was applied to localize the proteins. RESULTS: When compared to knee OA, the pseudosynovial tissue in TKA exhibit (1) elevation of alternative macrophage activation marker (CHIT1), chemokine (IL-8), and a proteolytic enzyme (MMP9); (2) downregulation of pro-inflammatory cytokine (TNF), osteoclastic regulator (OPG) and a stimulator of bone formation (BMP4); (3) no difference in IL-6, CCL3, CCL18, RANKL, DC-STAMP and SOCS3. The EP in TKA differed from EP in aseptically failed THA by lower CCL3 and DC-STAMP mRNA and protein expression. EP of all studied inflammatory and osteoclastogenic molecules were similar in knee and hip OA. CONCLUSIONS: Comparing to OA, aseptic loosening of TKA is associated with upregulated expression of CHIT1, IL-8 and MMP9, dysregulated RANKL:OPG ratio and low levels of inflammatory cytokines. Similar cytokine profiles were associated with primary knee and hip OA. Further research is required to explain the differences in CCL3 and DC-STAMP expression between failed TKA and THA.

Colchicine modulates expression of pro-inflammatory genes in neutrophils from patients with familial Mediterranean fever and healthy subjects.

Colchicine (Col) is a microtubule depolymerizing drug, widely used for treatment of familial Mediterranean fever (FMF). Mechanisms by which Col exerts its beneficial effects are not yet completely understood, especially with respect to gene expression in polymorphonuclear neutrophils (PMNs), the main effector cells in acute inflammatory attacks of FMF. This study was, therefore, designed to elucidate possible modulatory effect of Col on expression of inflammation-related genes in circulating PMNs from 16 FMF patients in the remission period and 11 healthy subjects. In vitro effect of Col exposure (1 microg/ml) on expression of 8 selected genes was examined using quantitative real-time RT-PCR. Col up-regulated expression of IL-8 and IL-1beta genes in FMF (13-fold and 2.7-fold, p less than 0.05, respectively) and healthy (3-fold and 6.5-fold, p less than 0.05, respectively) PMNs, and down-regulated caspase-1 in FMF neutrophils (3-fold, p less than 0.05). In FMF PMNs treated with Col mRNAs of IL-8 (51-fold, p less than 0.01) and c-FOS (7-fold, p less than 0.05) transcripts were elevated compared to those from healthy subjects. By contrast, caspase-1 mRNA was decreased in FMF neutrophils compared to healthy cells (1.6-fold, p less than 0.05). Hereby, we provide evidence that, at least in vitro, Col displays pro-inflammatory potential in respect to IL-1beta and IL-8 genes. At the same time, our findings implicate suppression of caspase-1 expression by Col as a potential mechanism for its effects in FMF treatment.

MicroRNAs: Emerging Regulators of Immune-Mediated Diseases.

MicroRNAs (miRNAs) represent the most abundant class of regulators of gene expression in humans: they regulate one-third of human protein-coding genes. These small noncoding ∼22-nucleotides (nt)-long RNAs originate by multistep process from miRNA genes localized in the genomic DNA. To date, more than 1420 miRNAs have been identified in humans (miRBase v17). The main mechanism of miRNA action is the posttranscriptional regulation via RNA interference with their target mRNAs. The majority of target mRNAs (more than 80%) undergo degradation after recognition by complementary miRNA; the translational inhibition with little or no influence on mRNA levels has been also reported. Each miRNA may suppress multiple mRNA targets (average ∼200), and at the same time, one mRNA can be targeted by many miRNAs enabling to control a spectrum wide range of cellular processes. Recently, the role of miRNAs in the development of immune cells and the maintenance of immune system homeostasis gained attention, and the involvement of miRNAs in the pathogenesis of several immune system diseases has emerged. This review focuses on the role of miRNAs in autoimmune disorders (systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease and psoriasis), inflammatory pathologies of distinct organ (atherosclerosis, osteoarthritis and atopic eczema) and/or systemic locations such as allergy. The role of miRNAs, their predicted and known mRNA targets and description of their actions in physiological immune reactions and in the pathological processes ongoing in immune-mediated human disorders will be discussed. Finally, miRNA-based diagnostics and therapeutic potentials will be highlighted.

T-helper cell type-1 transcription factor T-bet is upregulated in pulmonary sarcoidosis.

Upregulation of genes for interferon (IFN)-γ and CXC chemokine receptor (CXCR)3 expression, two crucial molecules in sarcoid inflammation and granuloma formation, is directly controlled by the T-helper (Th)1 transcription factor T-bet (T-box, expressed in T-cells). However, there is no information on T-bet expression in sarcoidosis or its relationship with "sarcoidosis-associated" genes. Therefore, we investigated expression of T-bet mRNA and, in parallel, a spectrum of genes known to be involved in sarcoidosis pathogenesis. Transcripts were determined in bronchoalveolar lavage (BAL) cells from 62 sarcoidosis patients and 25 controls by quantitative RT-PCR; T-bet protein was localised by immunohistochemistry. Patient's BAL cells expressed higher mRNA T-bet levels than those of controls (mean ± sd fold change 3.64 ± 1.72; p = 0.00006). T-bet mRNA expression did not vary between clinical phenotypes as assessed by chest radiography stage, presence/absence of Löfgren's syndrome, extrapulmonary/pulmonary involvement or progressing/remitting disease (p > 0.05). T-bet mRNA expression correlated with expression of IFN-γ, CC chemokine ligand 5, CXC chemokine ligand (CXC)10, interleukin (IL)-2 receptor/IL-15 receptor ß, CXCR3 and CXCR6 (p < 0.01). T-bet protein was localised to alveolar macrophages and lymphocytes, tissue multinucleated giant cells, macrophages and lymphocytes. In pulmonary sarcoidosis, T-bet upregulation is associated with changes in expression of IFN-γ, CXCR3 and chemokines/receptors involved in the pathogenesis of sarcoidosis, which suggests a role for T-bet in this Th1 disease, including modulation of some sarcoidosis-associated genes.

Functional variant ANXA11 R230C: true marker of protection and candidate disease modifier in sarcoidosis.

In the recent genome-wide association study the polymorphisms of annexin A11 (ANXA11) gene were associated with susceptibility to sarcoidosis. Beside the replication of this finding and analysis of local ANXA11 expression in bronchoalveolar lavage cells, we wondered whether 'leading' ANXA11 rs1049550 (R230C) variant might also be related to the clinical manifestation of sarcoidosis. The study included 245 Czech patients with sarcoidosis and 254 healthy control subjects. The frequency of ANXA11(*)T allele was significantly lower in patients with sarcoidosis (35%) compared with controls (42%, P=0.04, odds ratio=0.77). Furthermore, ANXA11(*)T allele was less frequent in patients with the infiltration of lung parenchyma by comparison with those with isolated hilar lymphadenopathy (P=0.01). In line with the previous observation, ANXA11 mRNA expression was not deregulated in sarcoidosis and was independent from rs1049550 variant. In conclusion, ANXA11 rs1049550 single nucleotide polymorphism is the susceptibility marker in sarcoidosis, at least in Caucasians. Its role as a disease modifier should be independently replicated.

Protein levels of CC chemokine ligand (CCL)15, CCL16 and macrophage stimulating protein in patients with sarcoidosis.

The objective of this study was to assess protein levels for candidate cytokines, chemokines, growth factors, matrix metalloproteinases and their inhibitors in bronchoalveolar lavage fluid (BALF) in patients with polar forms of pulmonary sarcoidosis, i.e. Löfgren's syndrome (LS) and more advanced chest X-ray (CXR) stage III disease. Twenty-four inflammatory molecules were analysed in unconcentrated BALF samples from 10 sarcoidosis patients with CXR stage III and 10 patients with LS by semiquantitative protein array. Four novel molecules [CC chemokine ligand (CCL)15, CCL16, macrophage migration inhibitory factor (MIF) and macrophage stimulating protein (MSP)], detected for the first time in association with sarcoidosis, were then quantified by enzyme-linked immunosorbent assay in a second cohort of 68 sarcoidosis patients and 17 control subjects. The protein levels of CCL15, CCL16, CCL24, CXCL8, CXCL9, CXCL10, interleukin-16, MIF, MSP and matrix metallopeptidase 1 were increased in CXR stage III patients when compared with patients with LS. CCL15 and MSP up-regulation in CXR stage III patients in comparison with LS patients and controls was confirmed by enzyme-linked immunosorbent assay. Moreover, MSP was associated with treatment requirement (P = 0.001) and CCL15 was elevated in patients with disease progression at 2-year follow-up (P = 0.016). CCL16 levels were increased in sarcoidosis versus controls (P < 0.05), but no difference was observed between patient subgroups. MIF up-regulation was not confirmed in a larger patient group. In conclusion, chemokines CCL15, CCL16 and MSP were found elevated for the first time in BALF from sarcoidosis patients; our results showed that CCL15 and MSP may affect disease course.

Association of IL-6 gene polymorphism with the outcome of allogeneic haematopoietic stem cell transplantation in Czech patients.

Interleukin-6 (IL-6) is an important pro-inflammatory mediator implicated in immune-mediated complications of allogeneic haematopoietic stem cell transplantation (aHSCT). In accord with previous reports, this preliminary study on 56 donor-recipient pairs revealed IL-6-174 single nucleotide polymorphisms as a risk factor for the development of acute graft-versus-host disease and decreased survival after aHSCT.

The CR1 C5507G polymorphism is not involved in susceptibility to idiopathic pulmonary fibrosis in two European populations.

Idiopathic pulmonary fibrosis (IPF), a severe lung disease with unknown aetiology, is thought to have an important genetic component. Single nucleotide polymorphism, C5507G, of the complement receptor 1 (CR1) gene, which affects the number of CR1 molecules on erythrocytes, has been associated with susceptibility to IPF in a single European population. To replicate this finding, 53 Czech IPF patients with 203 Czech healthy control subjects and 70 English IPF patients with 149 English controls were investigated. In both populations, there were no significant differences in distribution of CR1 C5507G variants between IPF patients and their appropriate control groups. In conclusion, the association of the CR1 C5507G polymorphism with susceptibility to IPF was not reproducible in Czech and English populations.

Complement receptor 1 single nucleotide polymorphisms in Czech and Dutch patients with sarcoidosis.

A single nucleotide polymorphism (SNP) C5507G of the complement receptor 1 (CR1) gene has been associated with genetic susceptibility to sarcoidosis in an Italian population. In order to provide further data on the possible involvement of CR1 gene polymorphisms in sarcoidosis, CR1 SNPs C5507G and A3650G were investigated in Czech (n = 210) and Dutch (n = 116) patients with sarcoidosis with ethnically matched groups of healthy control subjects (Czech, n = 203; Dutch, n = 112). CR1 C5507G and A3650G SNPs were not associated with susceptibility to sarcoidosis or its clinical course. Further, CR1 messenger RNA expression in bronchoalveolar lavage cells investigated by quantitative reverse transcriptase-polymerase chain reaction did not differ between sarcoidosis patients and control subjects and was not associated with the presence of the CR1 5507*G allele.

Expression of CCX CKR in pulmonary sarcoidosis.

OBJECTIVE: CCX CKR is a decoy chemokine receptor that specifically binds the chemokines CCL19, CCL25 and CCL21. CCL19 was previously found to be upregulated in pulmonary sarcoidosis. We have, therefore, investigated CCX CKR expression in this inflammatory disease. METHODS AND RESULTS: CCX CKR mRNA was semiquantitated using RT-PCR in unseparated bronchoalveolar (BAL) cells from sarcoidosis patients (S, n = 29) and healthy control subjects (C, n = 9). CCX CKR transcripts were upregulated in patients (mean +/- SEM); S, 0.82 +/- 0.10; C, 0.44 +/- 0.04; p = 0.01; this upregulation paralleled the disease course as assessed by the chest radiographic stage (p < 0.02). Immunocytochemistry localised the CCX CKR protein to ciliated bronchial cells. Flow cytometric fluorescent ligand uptake assay showed that these cells are able to internalize its ligand. CONCLUSION: These observations implicate CCX CKR in the modulation of the inflammatory response in sarcoidosis.