Katja Lakota

Levels of adiponectin, a marker for PPAR-gamma activity, correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker?

IntroductionProgressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.MethodsAdiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.ResultsPatients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.ConclusionsLevels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.

Antiphospholipid antibodies as non-traditional risk factors in atherosclerosis based cardiovascular diseases without overt autoimmunity. A critical updated review

The role of antiphospholipid antibodies (aPL) associated with cardiovascular diseases has been extensively studied in autoimmune patients, however it was largely unknown whether and how aPL associate with coronary artery disease (CAD), ishemic stroke (IS) and peripheral artery disease (PAD) in non-autoimmune patients. The current review attempts to prioritize for the first time clinical studies based on cause-outcome and strengths relationships in reference to aPL and CAD/PAD, in addition to supplementing Breys comprehensive review on IS with other, additional studies. Our overview indicates that all case-control and cross-sectional studies found an aPL association with CAD, PAD and IS, while cohort and nested case-control studies reported a prevailing negative risk association between aPL and IS (confirming Brey), with an unclear/unresolved risk association between aPL and CAD. The only cohort, follow-up study found in PAD reported on positive risk association between aPL and disease. The most frequently associated aPL in all studies reported, irrespective of disease, was aCL, with a less frequent association reported for LA, aβ2GPI and other aPL.

Tenascin-C drives persistence of organ fibrosis

The factors responsible for maintaining persistent organ fibrosis in systemic sclerosis (SSc) are not known but emerging evidence implicates toll-like receptors (TLRs) in the pathogenesis of SSc. Here we show the expression, mechanism of action and pathogenic role of endogenous TLR activators in skin from patients with SSc, skin fibroblasts, and in mouse models of organ fibrosis. Levels of tenascin-C are elevated in SSc skin biopsy samples, and serum and SSc fibroblasts, and in fibrotic skin tissues from mice. Exogenous tenascin-C stimulates collagen gene expression and myofibroblast transformation via TLR4 signalling. Mice lacking tenascin-C show attenuation of skin and lung fibrosis, and accelerated fibrosis resolution. These results identify tenascin-C as an endogenous danger signal that is upregulated in SSc and drives TLR4-dependent fibroblast activation, and by its persistence impedes fibrosis resolution. Disrupting this fibrosis amplification loop might be a viable strategy for the treatment of SSc.

Serum amyloid A activation of human coronary artery endothelial cells exhibits a neutrophil promoting molecular profile.

BACKGROUND Serum amyloid A (SAA) has been shown to be an active participant in atherosclerosis and cardiovascular diseases. SAA-stimulated human coronary artery endothelial cells (HCAEC) were reported to release pro-inflammatory cytokines, chemokines and adhesion molecules; however it remains unclear which putative SAA receptors are present in these cells and how they act. We investigated the effects of inflammatory stimuli on the expression of SAA receptors, signaling pathways and molecular profiles in HCAEC. METHODOLOGY/PRINCIPLE FINDINGS HCAEC were cultured in vitro and stimulated with SAA (1000nM) or IL-1β (1000pg/ml). Expression of mRNA was determined by qPCR, and expression and quantification of proteins were assessed by dot array blots and ELISA, respectively. Protein phosphorylation was determined by dot blot arrays and Western blots. We report that all potential SAA receptors tested (FPR2/ALX, RAGE, TANIS, TLR2, TLR4 and CLA-1/hSR-B1) are expressed in HCAEC. Importantly, IL-1β or SAA significantly increased solely the expression of the innate immune receptor TLR2. SAA upregulated the phosphorylation of ERK1/2, NF-κB (p65, p105) and JNK, as well as expression/release of IL-6, IL-8, G-CSF, GM-CSF, ICAM-1 and VCAM-1, all potent molecules involved in neutrophil-related activities. A TLR2-dependent positive feedback mechanism of SAA expression was found. CONCLUSION/SIGNIFICANCE SAA stimulated responses in HCAEC target neutrophil rather than monocyte/macrophage activation.

Serum amyloid A is a marker for pulmonary involvement in systemic sclerosis.

Inflammation in systemic sclerosis (SSc) is a prominent, but incompletely characterized feature in early stages of the disease. The goal of these studies was to determine the circulating levels, clinical correlates and biological effects of the acute phase protein serum amyloid A (SAA), a marker of inflammation, in patients with SSc. Circulating levels of SAA were determined by multiplex assays in serum from 129 SSc patients and 98 healthy controls. Correlations between SAA levels and clinical and laboratory features of disease were analyzed. The effects of SAA on human pulmonary fibroblasts were studied ex vivo. Elevated levels of SAA were found in 25% of SSc patients, with the highest levels in those with early-stage disease and diffuse cutaneous involvement. Significant negative correlations of SAA were found with forced vital capacity and diffusion capacity for carbon monoxide. Patients with elevated SAA had greater dyspnea and more frequent interstitial lung disease, and had worse scores on patient-reported outcome measures. Incubation with recombinant SAA induced dose-dependent stimulation of IL-6 and IL-8 in normal lung fibroblasts in culture. Serum levels of the inflammatory marker SAA are elevated in patients with early diffuse cutaneous SSc, and correlate with pulmonary involvement. In lung fibroblasts, SAA acts as a direct stimulus for increased cytokine production. These findings suggest that systemic inflammation in SSc may be linked to lung involvement and SAA could serve as a potential biomarker for this complication.

Antibodies against acute phase proteins and their functions in the pathogenesis of disease: a collective profile of 25 different antibodies.

The acute phase response is a defense system in which the innate immune response is activated following injury or infection. Positive and negative acute phase proteins (APPs) are crucial for protecting the host organism, as well as returning it to homeostatic levels, the first with elevated concentrations and the latter with decreased concentrations during the acute phase. Reports about the presence of antibodies against APPs are known, however their individual, as well as potentially collective, pathological or physiological roles are still emerging. Some of these autoantibodies are specifically connected with diseases (such as pancreatic secretory trypsin inhibitor and C3, C4 nephritic factors), while others have been reported as natural antibodies. The persistent presence (even if only minor) of autoantibodies in healthy blood donors indicates an overlapping category of autoantibodies, which could become pathogenic, depending on the autoantibody characteristics such as avidity, epitope specificity, changes in the microenvironment leading to different oxidative status and others. This review uses the novel approach of studying the overall autoantibody population against APPs, their functions and connections to diseases. The primary function of autoantibodies against APPs (anti-APPs) is thought to promote their clearance, however autoantibodies against negative APPs have also been found and applying the same role to those is doubtful. There is also the theory of consumption in the stage of inflammation, which could be relevant to anti-APPs. Reports about protective roles of autoantibodies are also emerging, showing lowered levels of antibodies in diseases, which could be interesting for therapeutic intervention.

Uropathogenic Escherichia coli induces serum amyloid a in mice following urinary tract and systemic inoculation.

Serum amyloid A (SAA) is an acute phase protein involved in the homeostasis of inflammatory responses and appears to be a vital host defense component with protective anti-infective properties. SAA expression remains poorly defined in many tissues, including the urinary tract which often faces bacterial challenge. Urinary tract infections (UTIs) are usually caused by strains of uropathogenic Escherichia coli (UPEC) and frequently occur among otherwise healthy individuals, many of whom experience bouts of recurrent and relapsing infections despite the use of antibiotics. To date, whether SAA is present in the infected urothelium and whether or not the induction of SAA can protect the host against UPEC is unclear. Here we show, using mouse models coupled with immunofluorescence microscopy and quantitative RT-PCR, that delivery of UPEC either directly into the urinary tract via catheterization or systemically via intraperitoneal injection triggers the expression of SAA. As measured by ELISA, serum levels of SAA1/2 were also transiently elevated in response to UTI, but circulating SAA3 levels were only up-regulated substantially following intraperitoneal inoculation of UPEC. In in vitro assays, physiological relevant levels of SAA1/2 did not affect the growth or viability of UPEC, but were able to block biofilm formation by the uropathogens. We suggest that SAA functions as a critical host defense against UTIs, preventing the formation of biofilms both upon and within the urothelium and possibly providing clinicians with a sensitive serological marker for UTI.

Colocalization of Serum Amyloid A with Microtubules in Human Coronary Artery Endothelial Cells

Serum amyloid A (SAA) acts as a major acute phase protein and represents a sensitive and accurate marker of inflammation. Besides its hepatic origin, as the main source of serum SAA, this protein is also produced extrahepatically. The mRNA levels of SAA become significantly elevated following proinflammatory stimuli, as well as, are induced through their own positive feedback in human primary coronary artery endothelial cells. However, the intracellular functions of SAA are so far unknown. Colocalization of SAA with cytoskeletal filaments has previously been proposed, so we analyzed the colocalization of SAA with all three cytoskeletal elements: actin filaments, vimentin filaments, and microtubules. Immunofluorescent double-labeling analyses confirmed by PLA method revealed a strict colocalization of SAA with microtubules and a very infrequent attachment to vimentin while the distribution of actin filaments appeared clearly separated from SAA staining. Also, no significant colocalization was found between SAA and endomembranes labeled with the fluorescent lipid stain DiO6. However, SAA appears to be located also unbound in the cytosol, as well as inside the nucleus and within nanotubes extending from the cells or bridging neighboring cells. These different locations of SAA in endothelial cells strongly indicate multiple potential functions of this protein.

Comparison and evaluation of different methodologies and tests for detection of anti-dsDNA antibodies on 889 Slovenian patients’ and blood donors’ sera

Aim To evaluate four different commercially available assays for anti-double stranded DNA (dsDNA) detection and compare them with the in-house radioimmunoassay according to Farr (FARR-RIA) in order to select the optimal primary method for use in combination with FARR-RIA. Methods Sera from 583 consecutive patients sent to our laboratory for routine diagnosis, 156 selected patients with autoimmune diseases (76 systemic lupus erythematosus [SLE] patients and 80 patients with other autoimmune diseases), and 150 blood donors were tested for anti-dsDNA antibodies with two enzyme-linked immunoassays (ELISA), two Crithidia luciliae immunoflourescence tests (CLIFT), and FARR-RIA. The specificities and sensitivities of the tests were calculated and compared. Results FARR-RIA and CLIFT 2 showed the highest specificity for SLE (100%), with CLIFT 2 showing higher sensitivity (33% vs 47%). Both ELISAs showed higher sensitivities (>53%) than FARR-RIA but lower specificities (<93%), whereas CLIFT 1 showed the lowest overall agreement with FARR-RIA. Conclusion CLIFT 2 was selected as the primary test for use in combination with FARR-RIA. The use of CLIFT 2 reduced the number of sera that needed to be tested by FARR-RIA, the time needed to report the results, and environmental toxicity, cancerogenicity, and radioactivity.

Uteroglobin, a Possible Ligand of the Lipoxin Receptor Inhibits Serum Amyloid A-Driven Inflammation

Serum amyloid A (SAA) production is increased by inflamed arthritic synovial tissue, where it acts as a cytokine/chemoattractant for inflammatory and immune cells and as an inducer of matrix degrading enzymes. SAA has been shown to bind lipoxin A4 receptor, a member of the formyl-peptide related 2 G-protein coupled receptor family (ALX) and elicit proinflammatory activities in human primary fibroblast-like synoviocytes (FLS). We report on the identification of uteroglobin, a small globular protein with potent anti-inflammatory activities, as a possible ligand of ALX. Uteroglobin-specific association with ALX was demonstrated by an enzyme immunoassay experiment employing a cell line engineered to express the human ALX receptor. Uteroglobins interaction with ALX resulted in the inhibition of SAA responses, such as attenuation of phospholipase A2 activation and cellular chemotaxis. In FLS, uteroglobin showed an antagonism against SAA-induced interleukin-8 release and decreased cell migration. These novel roles described for uteroglobin via ALX may help elucidate genetic and clinical observations indicating that a polymorphism in the uteroglobin promoter is linked to disease outcome, specifically prediction of bone erosion in patients with rheumatoid arthritis or severity of IgA glomerulonephritis and sarcoidosis.