Despite recent identification of specific pattern recognition receptors (PRR) for distinct microbial structures, data indicating their relevance in human infectious diseases are limited. We determined the expression levels of the Toll-like receptor (TLR)2 and TLR4 by flow cytometry on granulocytes and monocytes of healthy neonates compared with healthy adults. The basal expression of TLR2 was only slightly lower in neonatal phagocytes, whereas no differences could be detected for TLR4. Analyzing neonates with sepsis, we found an impressive up-regulation of TLR2 on blood phagocytes already at initial presentation of symptoms. Comparison with C-reactive protein, IL-8, and IL-6 suggested that TLR2 expression on monocytes is comparably valuable as an early sepsis marker. Neonatal sepsis remains a leading cause of neonatal morbidity and mortality despite recent advances in neonatal intensive care (1,2). Although several deficiencies in the neonatal immune system have been revealed, including qualitative and quantitative deficits in phagocytes and humoral components (3-8), they may only partly explain the overwhelming nature of neonatal sepsis. The key for differences in the course of neonatal and adult sepsis might be molecular mechanisms involved in the initiation process of systemic inflammatory response syndromes, i.e. the activation of innate immune cells through invading microbes. In the innate immune system, many types of receptors participate in microbe detection. An exciting discovery was the finding that host organisms have developed a set of specific receptors (PRR) for the recognition of highly conserved and essential molecular structures of microbes [pathogen-associated molecular patterns, PAMP)] (9). TLR2 was identified as a receptor for Gram-positive peptidoglycan and bacterial lipopeptides (10,11), whereas TLR4 is part of a receptor complex recognizing Gram-negative bacterial LPS and is required for LPS signal transduction (12). Both finally trigger inflammatory responses through an IL-1 receptor-like pathway that uses an adapter protein (MyD88), IL-1 receptor associated kinase (IRAK), and tumor necrosis factor receptor-associated factor (TRAF)-6 signaling to activate nuclear factot (NF)B and mitogen-activated protein kinase dependent signaling pathways (13). They initiate distinct genetic programs, including the induction of a subset of chemokines like macrophage inflammatory protein (MIP)-1␣, MIP-1␤, and RANTES (regulated upon activation, normal t-cell expressed and secreted) mediated by TLR2 and TLR4, whereas IP-10 is preferentially induced by TLR4 and IL-8 preferentially by TLR2 (14). The model of microbial recognition has now evolved into one in which immune cells use multiple PRR to detect several features of a microbe simultaneously. The signal pattern transduced by the combination of different innate immune receptors may be the key to the kind of immune response that is elicited (15).Except for in vitro experiments or murine models, data regarding the expression and in vivo relevance of TLR in ...

Objective. Systemic juvenile idiopathic arthritis (JIA) features characteristics of autoinflammation and autoimmunity, culminating in chronic arthritis. In this study, we hypothesized that aberrant or incomplete polarization of T helper cells contributes to disease pathology.Methods. Cells or serum samples were obtained from healthy controls (n = 72) and systemic JIA patients (n = 171). Isolated naive T helper cells were cultured under Th1, Th17, and T follicular helper (Tfh) or T peripheral helper (Tph)-polarizing conditions and were partly cocultured with allogenic memory B cells. Cell samples were then analyzed for surface marker, transcription factor, and cytokine expression, as well as plasmablast generation. Serum samples were subjected to multiplexed bead and self-antigen arrays and enzyme-linked immunosorbent assays, and all data were compared to retrospective RNA profiling analyses.Results. Differentiation of systemic JIA-naive T helper cells toward Th1 cells resulted in low expression levels of interferon-γ (IFNγ) and eomesodermin, which was associated in part with disease duration. In contrast, developing Th1 cells in patients with systemic JIA were found to produce elevated levels of interleukin-21 (IL-21), which negatively correlated with cellular expression of IFNγ and eomesodermin. In both in vitro and ex vivo analyses, IL-21 together with programmed cell death 1 (PD-1), inducible T cell costimulator (ICOS), and CXCR5 expression induced naive T helper cells from systemic JIA patients to polarize toward a Tfh/Tph cell phenotype. Retrospective analysis of whole-blood RNAsequencing data demonstrated that Bcl-6, a master transcription factor in Tfh/Tph cell differentiation, was overexpressed specifically in patients with systemic JIA. Naive T helper cells from systemic JIA patients which were stimulated in vitro promoted B cellular plasmablast generation, and self-antigen array data indicated that IgG reactivity profiles of patients with systemic JIA differed from those of healthy controls.Conclusion. In the pathogenesis of systemic JIA, skewing of naive T helper cell differentiation toward a Tfh/Tph cell phenotype may represent an echo of autoimmunity, which may indicate the mechanisms driving progression toward chronic destructive arthritis.

Objective: Systemic juvenile idiopathic arthritis (sJIA) features characteristics of autoinflammation and autoimmunity, culminating in chronic arthritis. Previous work indicated decreased IFNg-expression by T helper (Th) cells in sJIA. Here, we hypothesized this to result from aberrant or incomplete Th cell polarization. Methods: Cells or sera were obtained from healthy controls (HC, n=26) and sJIA patients (n=61). Isolated naive Th cells were cultured under Th1, Th17, and T follicular or T peripheral helper (Tf/ph) polarizing conditions and were partly co-cultured with allogenic memory B cells. Surface marker, transcription factor, and/or cytokine expression in peripheral or polarized Th cells or sera as well as B cellular plasma blast generation were studied by flow cytometry, multiplexed bead array assays, ELISA and retrospective RNA profiling analyses. Results: SJIA naive Th cell differentiation towards Th1 resulted in low IFNg; and Eomesodermin expression. Instead, developing sJIA Th1 cells revealed elevated IL-21 release that negatively correlated with cellular IFNg; and Eomesodermin expression. Both In vitro and ex vivo, IL-21 together with PD-1, ICOS and CXCR5 expression indicated naive sJIA Th cell differentiation to rather polarize towards a Tf/ph phenotype. Retrospective analysis of whole blood RNA sequencing data demonstrated sJIA-specific overexpression of Bcl-6 as respective master transcription factor. Compared to controls, in vitro generated sJIA Tf/ph cells promoted enhanced B cellular plasma-blast generation. Conclusion: In sJIA pathogenesis skewing of sJIA naive Th cell differentiation towards a Tf/ph phenotype may represent an echo of autoimmunity, which could shed light on the mechanisms driving the progression towards chronic destructive arthritis.

Background Only few biomarkers are useful for the therapy monitoring of patients suffering from inflammatory diseases, like inflammatory bowel disease (IBD) or rheumatoid arthritis (RA). Next to calprotectin C-Reactive Protein (CRP) is the best described and usually measured in the central laboratory. However, most patients are regularly checked at GP’s offices or small ambulatories. These sites need fast results with simple technology to react immediately. Our quantitative CRP rapid test based on lateral flow technology combined with a cost-effective reader fulfils all these criteria. It can be used with serum and whole blood. Here, we report the validation of the Quantum Blue® hsCRP in capillary blood and show first clinical applications of this new test. Methods The sandwich-type lateral flow test employing anti-CRP antibody-coated gold nanoparticles was calibrated with the ERM-DA474 CRP reference material. Fingerprick blood was collected and diluted 1:10 with a dedicated capillary blood preparation set and then applied onto the test cassette. After a 15-min incubation, the readout was performed with the Quantum Blue® Reader resulting in a quantitative measuring range of 1 to 20 mg/l. 122 capillary blood/serum pairs were collected to compare this rapid hsCRP test with the fully automated serum CRPL3 (PETIA) test on Roche cobas® c501. Clinical performance data together with concomitant CRP measurements were collated from 20 IBD and 69 RA patients. The resulting capillary blood CRP values including those from 28 healthy subjects were used to establish preliminary clinical cut-off values. Results 56 capillary blood/serum sample pairs with values within the measuring range of both the hsCRP and the CRPL3 PETIA could be elected for a quantitative comparison. The capillary blood values were corrected against a mean haematocrit of 42%. A strong correlation (R = 0.941) with a minor bias of 6.1% by Bland-Altman plot was observed. Non-parametric analyses of capillary blood CRP levels from 20 IBD and 69 RA patients vs. their disease activity scores (CDAI, Mayo, DAS28, active joints) resulted in an upper reference limit of 5.8 mg/l and a 95%ile reference range from 1.0 to 19.7 mg/l. Patients with moderate (IBD) to severe (IBD, RA) disease activity could be significantly discriminated by capillary blood CRP from those with low activity or remission. Conclusion The BÜHLMANN Quantum Blue® hsCRP rapid test allows fast and easy detection of CRP in human capillary blood within 15 min in ambulatory settings. The assay shows an excellent absolute correlation to a fully automated serum CRP reference method. A preliminary clinical cut-off of 5.8 mg/l was established in two cohorts of IBD and RA patients to discriminate high from low disease activity.