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International guidelines recommend involving various professions and disciplines at an early stage in the event of chronic back pain. In connection with this, terms such as multiprofessional or interprofessional interventions are often mentioned without a uniform idea of what they mean. This article is intended to provide an overview of multiprofessional interventions for patients with chronic back pain and the integration into a meaningful interdisciplinary and interprofessional multimodal treatment concept. This is illustrated in a biopsychosocial pillar model, which should be pursued for each patient individually.Internationale Leitlinien empfehlen bei anhaltenden Rückenschmerzen bereits frühzeitig die Einbindung verschiedener Professionen und Disziplinen. Damit in Verbindung werden häufig Begriffe wie multiprofessionelle oder interprofessionelle Therapieansätze genannt ohne eine einheitliche Vorstellung, was darunter verstanden wird. Der vorliegende Beitrag soll Orientierung geben, welche multiprofessionellen Therapieansätze es bei chronischen Rückenschmerzen gibt und wie diese in ein interdisziplinäres und interprofessionelles multimodales Therapiekonzept integriert werden können. Dies stellen wir in einem biopsychosozialen Säulenmodell dar, das für jeden Patienten individuell erstellt werden sollte.

CME-Rheuma 21: Precision Medicine - Synovial Biopsy in Rheumatology Abstract. Synovial biopsy is increasingly performed in the medicine of the musculoskeletal system. On the one hand it allows the in-depth diagnosis of unclear arthritides. On the other hand, there is an increasing body of publications showing that histology, immunohistochemistry and RNA analysis of synovial tissue may lead to subclassifications within rheumatoid arthritis. This in turn may have predictive value for the treatment response. We herein give a short overview of the joint biopsy technique, the basic evaluation of biopsy samples and the prospects of synovial biopsy.

Objective Primary Sjögren syndrome (pSS) is characterized by T and B cell infiltration of exocrine glands. The cysteine protease cathepsin S (CatS) is crucially involved in MHCII processing and T cell stimulation, and elevated levels have been found in patients with RA, psoriasis and pSS. However, little is known about the functional characteristics and mechanisms of SS-A- and SS-B-specific T cells in pSS patients. We herein investigated the inhibition of CatS activity in different biocompartments of pSS patients including antigen-specific T cell responses. Methods Ex vivo CatS activity was assessed in tears, plasma and saliva of 15 pSS patients and 13 healthy controls (HC) and in the presence or absence of the specific CatS inhibitor RO5459072. In addition, antigen (SS-A (60kD), SS-B, influenza H3N2, tetanus toxoid and SEB)-specific T cell responses were examined using ex vivo IFN-γ/IL-17 Dual ELISPOT and Bromdesoxyuridin (BrdU) proliferation assays in the presence or absence of RO5459072. Supernatants were analysed for IL-1β, IL-6, IL-10, TNF-α, IL-21, IL-22 and IL-23, using conventional ELISA. Results CatS activity was significantly elevated in tear fluid, but not other biocompartments, was inversely associated with exocrinic function in pSS patients and could significantly be suppressed by RO5459072. Moreover, CatS inhibition by RO5459072 led to strong and dose-dependent suppression of SS-A/SS-B-specific T cell effector functions and cytokine secretion by CD14+ monocytes. However, RO5459072 was incapable of suppressing SS-A/SS-B-induced secretion of cytokines in CD14+ monocytes when T cells were absent, confirming a CatS/MHCII-mediated mechanism of suppression. Conclusion CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. Conversely, CatS inhibition diminishes T cell and associated monokine responses towards relevant autoantigens in pSS. Thus, CatS inhibition may represent a promising novel treatment strategy in pSS. Electronic supplementary material The online version of this article (10.1186/s13075-019-1955-2) contains supplementary material, which is available to authorized users.

Vaccines prevent infectious diseases, but vaccination is not without risk and adverse events are reported although they are more commonly reported for biologicals than for vaccines. Vaccines and biologicals must undergo vigorous assessment before and after licensure to minimise safety concerns. Potential safety concerns should be identified as early as possible during the development for vaccines and biologicals to minimize investment risk. State-of-the art tools and methods to identify safety concerns and biomarkers that are predictive of clinical outcomes are indispensable. For vaccines and adjuvant formulations, systems biology approaches, supported by single-cell microfluidics applied to translational studies between preclinical and clinical studies, could improve reactogenicity and safety predictions. Next-generation animal models for clinical assessment of injection-site reactions with greater relevance for target human population and criteria to define the level of acceptability of local reactogenicity at vaccine injection sites in pre-clinical animal species should be assessed. Advanced in silico machine-learning-based analytics, species-specific cell or tissue expression, receptor occupancy and kinetics and cell-based assays for functional activity are needed to improve pre-clinical safety assessment of biologicals. The in vitro MIMIC® system could be used to compliment preclinical and clinical studies for assessing immune-toxicity, immunogenicity, immuno-inflammatory and mode of action of biologicals and vaccines. Sanofi Pasteur brought together leading experts in this field to review the state-of-the-art at a unique 'Safety Biomarkers Symposium' on 28-29 November 2017. Here we summarise the proceedings of this symposium. This unique scientific meeting confirmed the importance for institutions and industrial organizations to collaborate to develop tools and methods needed for predicting reactogenicity and immune-inflammatory reactions to vaccines and biologicals, and to develop more accuracy, reliability safety biomarkers, to inform decisions on the attrition or advancement of vaccines and biologicals.

Tocilizumab (TCZ) has been approved for giant cell arteritis (GCA). Interleukin-6 (IL-6) receptor blockade suppresses clinical disease and is steroid sparing.1 2 Since IL-6 induces the acute-phase response, the clinically used inflammation markers (C reactive protein (CRP), erythrocyte sedimentation rate (ESR)) are suppressed during TCZ treatment. Whether serum IL-6 is useful in monitoring disease activity and detecting infections in TCZ-treated GCA is unknown. We longitudinally measured IL-6 in 23 patients with intravenous TCZ–treated GCA, two patients with polymyalgia rheumatica and one patient with Takayasu arteritis of our GCA cohort (EKBB-239/09), and in 13/26 patients additionally before TCZ treatment. Patient characteristics are shown in table 1. At each visit, clinical and laboratory parameters (white blood cell (WBC), CRP, ESR) were assessed. Relapse was defined as the need for treatment intensification following new or increasing symptoms, or rising CRP/ESR not otherwise explained.2 View this table: Table 1 Patient characteristics of ‘relapser’ and ‘non-relapser’ During TCZ therapy, CRP, ESR and WBC rapidly declined and were sustainably suppressed. IL-6 levels rose and remained elevated throughout TCZ treatment (figure 1). Daily prednisone doses were successfully tapered after TCZ was initiated (figure 1C). During 23.8 patient-years of TCZ therapy, eight infections were documented with available IL-6 levels (online supplementary table S1). IL-6, but not CRP, ESR …

Objective: To characterize patients with both monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in their synovial fluid (SF).Method: Forty-nine gout patients with acute arthritis were included. Those patients with MSU crystals only in their SF were compared to those patients with both MSU and CPP crystals in their SF.Results: A total of 36 out of 49 patients (73.5%) had only MSU crystals, whereas 13 out of 49 (26.5%) had both MSU and CPP crystals in their SF. Co-deposition of CPP crystals was associated with long-standing gout disease (p = 0.022), kidney dysfunction (p = 0.024), and erosive arthritis (p = 0.049), but not with age.Conclusion: Long-standing gout may be a risk factor for CPP deposition disease, and the frequency of CPP co-deposition may be higher than expected.

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the ‘Fc-load’ on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.

Zusammenfassung. Die Gicht ist die häufigste Arthritis weltweit mit zunehmender Prävalenz in den entwickelten Ländern und mit enormen sozioökonomischen Konsequenzen. Das Wissen über die Pathomechanismen, welche zur Arthritis führen und diese einschränken, hat in den letzten Jahren stark zugenommen. Entsprechend zeichnen sich auch neue Therapieansätze und Therapeutika ab. In dieser Übersichtsarbeit werden neben einigen Aspekten der Klinik, Diagnose und Therapie der Gicht, detailliert die intrazellulären Mechanismen, welche zur NLRP3 Inflammasom Aktivierung und IL-1beta Sekretion führen, behandelt. Sie gibt auch einen Ausblick auf ausgewählte, neue therapeutische Ansätze.

Background Primary Sjogren syndrome (pSS) is an autoimmune disease characterised by an infiltration of T and B cells into exocrine gland tissue and its subsequent destruction 1 . Antigen presenting cells, including B cells, foster T cell activation and anti-SS-A/SS-B producing plasma cells, eventually leading to disease progression and systemic complications 2 . Objectives Cathepsin S (CatS) is crucially involved in MHCII processing in pSS mouse models 3 and patients 4 . In this translational study we investigated the ex vivo effects of the CatS inhibitor RO5459072 in different bio-compartments, including specific T cells, of pSS patients and healthy controls. Methods Ex vivo CatS activity was assessed in different bio-compartments of 15 pSS patients and 13 healthy controls and in presence or absence of RO5459072 using commercial activity and quantification assays. In addition, antigen (5 µg/mL SS-A, 5 µg/mL SS-B, 5 µg/mL Influenza H 3 N 2 ; 2 µg/mL Tetanus Toxoid and 100 ng/mL SEB) specific T cell responses were examined using 2 × 10 5 PBMC/well IFN-g/IL-17 Dual ELISPOT (48 hour incubation) and 5 × 10 4 PBMC/well BrdU proliferation assays after (72 hour incubation) in presence or absence of RO5459072. Results pSS patients showed significantly higher CatS activity in tear fluid than healthy controls (two-tailed t-test p ex vivo derived pSS patient cells in Elispot and BrdU assays (table 1). Table 1 Suppression of antigen specific T cell responses (Elispot) and suppression of cell proliferation (BrdU) in the absence or presence of RO5450972 (0–100 µM). One-tailed t-test: *p Conclusions CatS activity in tear fluid seems to be a relevant biomarker for pSS disease activity. RO5459072 is a potent inhibitor of CatS and the pSS associated relevant antigen specific T cell responses. References [1] Voulgarelis M, Tzioufas AG. Pathogenetic mechanisms in the initiation and perpetuation of Sjogren’s syndrome. Nat. Rev. 2010;6:529–537. [2] Brito-Zeron P, Baldini C, Bootsma H, et al. Sjogren syndrome. Nat Rev Dis Primers 2016;2:16047. [3] Saegusa K, Ishimaru N, Yanagi K, et al. Cathepsin S inhibitor prevents autoantigen presentation and autoimmunity. J Clin Invest2002;110(3):361–9. [4] Hamm-Alvarez SF, Janga SR, Edman MC, et al. Tear cathepsin S as a candidate biomarker for Sjogren’s syndrome. Arthritis Rheumatol 2014;66(7):1872–81. Acknowledgements We would like to thank Ms. Evelyn Fischer and Dr. Bettina Bannert for their valuable contribution of patient samples, technical assistance and clinical information. Disclosure of Interest P. Hargreaves Grant/research support from: Roche, M. Theron Employee of: Roche, F. Kolb Employee of: Roche, M. Manchester Employee of: Roche, B. Reis Employee of: Roche, A. Tiaden: None declared, D. Kyburz Grant/research support from: Roche, T. Manigold Grant/research support from: Roche

Background Toll-like receptors (TLRs) and macrophages play an important role in rheumatoid arthritis (RA). Currently, it is not clear whether inflammatory M1 or anti-inflammatory M2 predominate among the resident macrophages in the synovium. In the present study, we set out to investigate the impact of TLR stimulation on monocyte-derived M1 and M2 macrophage function and phenotype by mimicking the exposure to abundant TLR agonists as occurs in the context of RA. The response of macrophage subsets to TLR2 and TLR4 activation was evaluated on cluster of differentiation (CD) marker profile; cytokine secretion; gene expression; and NF-κB, interferon regulatory factors 3 and 7 (IRF3/7), and mitogen-activated protein kinase (MAPK) activation. Methods Human monocytes were isolated from peripheral blood of healthy individuals and patients with RA and differentiated into M1-like and M2-like macrophages by granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF), respectively. Cells were either (1) stimulated with TLR ligands Pam3 or lipopolysaccharide (LPS) or (2) classically activated via interferon (IFN)-γ/LPS. Cytokine production was measured by enzyme-linked immunosorbent assay, and gene expression was measured by qPCR. Cells were stained for CD markers and analyzed by fluorescence-activated cell sorting. NF-κB, IRF3/7, and MAPKs were detected by Western blotting. Results Monocyte-derived macrophages of healthy donors (HD) or patients with RA displayed comparable subset-specific phenotypes upon exposure to TLR agonists. CD14 and CD163 marker expression on M2 macrophages did not change upon TLR2 and TLR4 engagement. By contrast, M2 gene markers HMOX1, FOLR2, and SLC40A1 were decreased. Importantly, M2 macrophages derived from HD or patients with RA showed both a decreased ratio of interleukin (IL)-10/IL-6 and IL-10/IL-8 upon stimulation with TLR2 ligand Pam3 compared with TLR4 ligand LPS. Gene expression of TLR2 was increased, whereas TLR4 expression was decreased, by TLR ligand stimulation. MAPKs p38, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase were activated more strongly in M2 than in M1 macrophages by Pam3 or LPS. Conclusions We show that the anti-inflammatory activity of M2 macrophages is reduced in the presence of abundant TLR2 ligands without significant changes in cell surface markers. Thus, the classical M1/M2 paradigm based on cellular markers does not apply to macrophage functions in inflammatory conditions such as RA. Electronic supplementary material The online version of this article (doi:10.1186/s13075-017-1447-1) contains supplementary material, which is available to authorized users.

Die Therapie immunvermittelter und maligner Erkrankungen befindet sich im Umbruch. Zunehmend kommen gezielt mit molekularen Zellstrukturen interagierende therapeutische Proteine, sogenannte Biologika oder Biopharmaka, zum Einsatz. Biologika sind in der Regel Antikorper oder Fusionsproteine und werden gentechnologisch nach dem Vorbild korpereigener Strukturen in zellularen Expressionssystemen, wie zum Beispiel Hamster-Ovarzellen, hergestellt und in hochreiner Form verabreicht. Biologika haben mit den klassischen kleinmolekularen Medikamenten, den sogenannten Synthetika, sowohl in der Wirkungsweise wie auch hinsichtlich der Nebenwirkungen nur noch wenig gemein. Der zunehmende Einsatz von Biologika im klinischen Alltag stellt eine Herausforderung fur die anwendenden Arzte dar, die nebst den neuen Wirkmechanismen auch den Umgang mit komplexen Nebenwirkungsmustern von direkt mit dem Immunsystem interagierenden Substanzen kennen und behandeln lernen mussen. Akute Infusionreaktionen (infusion-related reaction, IRR) stellen mit einer Inzidenz von 0,1 – 3% die haufigste Nebenwirkung dar. Interessanterweise handelt es sich in den meisten Fallen nicht um IgE-vermittelte Reaktionen und lassen sogar eine Reexposition zu. Wahrend regulatorische Behorden und die Pharmaindustrie sich vor allem auf anti-drug-antibodies (ADA)-vermittelte Nebenwirkungen konzentrieren, wollen wir hier einen Uberblick uber das Nebenwirkungsspektrum und mogliche Abklarungsalgorithmen geben.

Gout is the most frequent arthritis worldwide with increasing prevalence in industrialized countries and massive socioeconomic consequences. The knowledge regarding the pathomechanisms which lead to arthritis has substantially increased during the last decade. Consistently, new therapeutic approaches and substances appear at the horizon. This review covers aspects of clinical presentation, diagnosis and current treatment. The pathomechanisms leading to NLRP3 inflammasome activation and IL-1beta secretion are reviewed in detail. Finally, selected new therapeutic targets and substances are discussed.

Peripheral nerve biopsy remains the gold standard for diagnosing vasculitic neuropathy (VN).1 This procedure is invasive, of limited sensitivity and not always feasible.2 A biomarker indicating acute axonal loss in patients with systemic vasculitis would allow rapid screening and adjustments of diagnostic or treatment strategies, potentially improving clinical outcome.3 Neurofilaments are structural proteins specific to neurons that are released into blood and cerebrospinal fluid following neuronal damage. They have been suggested as a biomarker in various neurological diseases, mainly affecting the central nervous system.4–6 In patients with multiple sclerosis, serum neurofilament light chain (sNfL) levels correlated with MRI and clinical disease activity/severity, were lower in patients under treatment and showed potential to predict future disease activity and disability.5 In this retrospective study we aimed to test whether sNfL could serve as a marker of vasculitic damage of peripheral nerves and/or disease activity in patients with VN. Patients were recruited from our prospective local ethical board-approved vasculitis …

In the past few years, we have witnessed extraordinary advances in the understanding of the functions of regulatory T (Treg) cells in immunity against pathogens. However, controversy exists over the part that these cells play in determining the outcome of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections, the two main causes of chronic liver inflammation worldwide. Treg-cell responses may be either beneficial or detrimental to those infected with HBV and HCV, by either limiting liver immunopathology or suppressing protective T-cell responses. We review the latest research on CD4 Treg cells, dissect much of the Treg-related HBV and HCV literature, and discuss how new insights in Treg immunobiology apply to human and primate models of HBV and HCV infections. Moreover, we discuss the limitations of the conclusions drawn from current studies on Treg cells, and suggest experimental approaches that can resolve current conflicts and improve our understanding of the roles of Treg-cell subsets in HBV and HCV infections.

Hepatitis C virus (HCV) poses a global health problem because it readily establishes persistent infection and a vaccine is not available. CD4(+)CD25(+) T cells have been implicated in HCV persistence because their frequency is increased in the blood of HCV-infected patients and their in vitro depletion results in increased IFN-gamma production by HCV-specific T cells. Studying a well-characterized cohort of 16 chimpanzees, the sole animal model for HCV infection, we here demonstrate that the frequency of Foxp3(+)CD4(+)CD25(+) regulatory T cells (T(Regs)) and the extent of suppression was as high in spontaneously HCV-recovered chimpanzees as in persistently HCV-infected chimpanzees. Foxp3(+)CD4(+)CD25(+) T(Regs) suppressed IFN-gamma production, expansion, and activation-induced cell death of HCV-specific T cells after recovery from HCV infection and in persistent HCV infection. Thus, T(Reg) cells control HCV-specific T cells not only in persistent infection but also after recovery, where they may regulate memory T-cell responses by controlling their activation and preventing apoptosis. However, Foxp3(+)CD4(+)CD25(+) T(Reg) cells of both HCV-recovered and HCV-infected chimpanzees differed from Foxp3(+)CD4(+)CD25(+)T(Reg) cells of HCV-naive chimpanzees in increased IL-2 responsiveness and lower T-cell receptor excision circle content, implying a history of in vivo proliferation. This result suggests that HCV infection alters the population of Foxp3(+)CD4(+)CD25(+) T(Reg) cells.

Clearance of hepatitis B virus infection (HBV) infection implies a polyclonal vigorous T-helper 1 (Th1) and cytotoxic T-lymphocyte (CTL) response. Interleukin-18 (IL-18), a monokine that shares functional abilities with IL-12, is a potent inductor of interferon-gamma (IFN-gamma) by Th1 and natural killer (NK) cells. However, the role and regulation in HBV infection of IFN-gamma have not been defined. This study therefore sought to determine hepatitis B core antigen (HBcAg)-mediated regulation of IL-18 production in peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) and patients with chronic hepatitis B (CHB) or acute hepatitis B (AHB); 31 HC, 27 patients with CHB and 8 patients with AHB infection were included in the study. HBcAg-mediated induction of IL-18 was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and specific enzyme-linked immunosorbent assay (ELISA). HBcAg induced IL-18 gene transcription and dose-dependent secretion of mature IL-18 protein in HC, CHB, and AHB. HBcAg-dependent IL-18 levels were abrogated by inhibition of Caspase-1, but not by blockade of CD40-CD154 interaction. Serum levels of IFN-gamma correlated inversely with viremia in patients with CHB (rho = - 0.54, P < 0.05), but not with serum levels of IL-12 or IL-18. Interestingly, in PBMCs of HBeAg-negative patients, HBcAg induced significantly higher amounts of IL-18 than in those of HBeAg-positive patients. A variant, lacking the histone-like arginine-rich domain, did not induce IL-18 in either HC or CHB in vitro. Taken together, these results indicate that HBcAg induces IL-18 secretion by induction of Caspase-1. Differential regulation in HBeAg-negative and positive patients suggests an important role of IL-18 in CHB infection.

Expression of IL-18 in intestinal epithelial cells (IEC) has been implicated in Th1 cell-mediated chronic intestinal inflammation and anti-tumor immunity. However, physiological regulatory factors have not been identified. Besides their effects on proliferation and restitution, immunomodulatory functions have been attributed to short chain fatty acids (SCFA). We investigated the effect of SCFA (butyrate, propionate, acetate) on expression of IL-18 in IEC in vitro and in vivo. Expression of IL-18 mRNA and protein in human carcinoma-derived HT-29 and Caco-2 cells was analyzed by reverse transcription-PCR and Western blot. Transcriptional regulation of IL-18 gene expression was determined by transient transfection of wild-type and mutated IL-18 promoter. Further, in vivo expression of IL-18 in the intestine from butyrate-treated and untreated mice was assessed by immunohistochemistry. IL-18 mRNA and the IL-18 protein were expressed in IEC, while IL-18 secretion was not observed. Butyrate and acetate increased intracellular IL-18 content in a time- and dose-dependent fashion. In contrast to proinflammatory stimuli butyrate potently activated the IL-18 promoter, indicating that IL-18 is regulated at the transcriptional level by SCFA. Furthermore, a 108-bp sequence in the proximal region was identified to be essential for IL-18 promoter activation by butyrate. As proof of principle butyrate effects were confirmed in vivo by demonstration of increased IL-18 protein expression in IEC from butyrate-treated mice. In conclusion, SCFA up-regulate IL-18 protein expression in IEC, suggesting a potential regulatory contribution of these luminal constituents to T cell mediated inflammatory and neoplastic intestinal conditions.

In humans, hantaviruses can cause haemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS). Currently it is estimated that 150,000 to 200,000 cases of hantavirus disease occur each year, the majority being reported in Asia. However, human hantavirus infections are increasingly reported in the Americas and Europe. Although many of the underlying pathogenic mechanisms still remain unclear, recent evidence rather argues against a purely immune-mediated pathophysiology of human disease. Despite the high morbidity and case-fatality rates of HFRS and HCPS, respectively, no vaccine or drug is currently proven to be preventive or therapeutic. This review summarises clinical features and current epidemiological findings, as well as concepts regarding the immunology, pathogenesis and intervention strategies of human hantaviral diseases.

Vascular leakage is a serious side effect of therapies based on monoclonal antibodies or cytokines which may lead to life-threatening situations. With the steady increase of new drug development programs for large molecules, there is an urgent need for reliable tools to assess this potential liability of new medicines in a rapid and cost-effective manner. Using human umbilical vein endothelial cells (HUVECs) as a model for endothelium, we established an impedance-based assay measuring the integrity of the endothelial cell monolayer in real time. We could demonstrate that the HUVEC monolayer in our system was a relevant model as cells expressed major junctional proteins known to be responsible for maintaining tightness as well as receptors targeted by molecules known to induce vascular leakage in vivo. We assessed the time-dependent loss of barrier function using impedance and confirmed that signals obtained corresponded well to those from standard transwell assays. We assayed a series of reference molecules which led to the expected change of barrier integrity. A nonspecific cytotoxic effect could be excluded by using human fibroblasts as a nonresponder cell line. Finally, we could show reversibility of vascular permeability induced by histamine, IL-1β, or TNF-α by coincubation with established antagonists, further demonstrating relevance of this new model. Taken together, our results suggest that impedance in combination with HUVECs as a specific model can be applied to assess clinically relevant vascular leakage on an in vitro level.

Introduction In 2006 the anti-CD28 superagonistic IgG4 TGN1412, having passed pre-clinical safety screens, caused a severe ‘cytokine storm’ in 6 healthy volunteers. Others have shown that for TGN1412 to induce an inflammatory signal in human peripheral blood mononuclear cells (PBMCs) or in human diluted blood, endothelial cells or bound monoclonal antibody (mAb) is required as part of a bioassay complex. These types of protocols rely on different donor cells and therefore have limitations as bioassays for pre-clinical testing. Methods We performed studies using human PBMC/endothelial cell co-cultures, whole blood/endothelial cell co-cultures and human whole blood alone. We bracketed responses of a CD28 superagonist antibody with mAbs against CD52 (alemtuzumab, MabCampath-1H) or epidermal growth factor receptor (cetuximab, Erbitux) and with the immunostimulant lipopolysaccharide. We detected cytokine responses at the level of protein release (using ELISAs and Luminex assays) and gene induction (using real-time PCR arrays). Results Here we confirm that IL-8 release was induced in a mixed endothelial cell-PBMC system by the anti-CD28 mAb. We go on to show that an alemtuzumab and an anti-CD28 mAb, but not cetuximab induced the release of a range of cytokines including IL-8, IL-6, IFNγ, IL-2 and IL10 after 24 h and induced cytokine gene induction after 1 h. Co-cultures of whole blood and HUVECS showed larger variability but no superiority over whole blood alone at a range of time points (0.5–48 h). Discussion We suggest that, whilst limitations exist, human blood-based in vitro assays may prove useful in assessing the potential of mAbs and other biotherapeutics to cause release of cytokines in humans.

In man, infection with South American Andes virus (ANDV) causes hantavirus cardiopulmonary syndrome (HCPS). HCPS due to ANDV is endemic in Southern Chile and much of Argentina and increasing numbers of cases are reported all over South America. A case-fatality rate of about 36% together with the absence of successful antiviral therapies urge the development of a vaccine. Although T-cell responses were shown to be critically involved in immunity to hantaviruses in mouse models, no data are available on the magnitude, specificity and longevity of ANDV-specific memory T-cell responses in patients. Using sets of overlapping peptides in IFN-γ ELISPOT assays, we herein show in 78 Chilean convalescent patients that Gn-derived epitopes were immunodominant as compared to those from the N- and Gc-proteins. Furthermore, while the relative contribution of the N-specific response significantly declined over time, Gn-specific responses remained readily detectable ex vivo up to 13 years after the acute infection. Tetramer analysis further showed that up to 16.8% of all circulating CD3+CD8+ T cells were specific for the single HLA-B*3501-restricted epitope Gn465–473 years after the acute infection. Remarkably, Gn465–473–specific cells readily secreted IFN-γ, granzyme B and TNF-α but not IL-2 upon stimulation and showed a ‘revertant’ CD45RA+CD27−CD28−CCR7−CD127− effector memory phenotype, thereby resembling a phenotype seen in other latent virus infections. Most intriguingly, titers of neutralizing antibodies increased over time in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest intrinsic, latent antigenic stimulation of Gn-specific T-cells. However, it remains a major task for future studies to proof this hypothesis by determination of viral antigen in convalescent patients. Furthermore, it remains to be seen whether Gn-specific T cells are critical for viral control and protective immunity. If so, Gn-derived immunodominant epitopes could be of high value for future ANDV vaccines., Author Summary In man, hantavirus cardiopulmonary syndrome (HCPS) caused by Andes Virus (ANDV) is endemic in the Southern cone of Chile and Argentina but cases of HCPS are being increasingly reported all over South America since 1995. HCPS is characterized by fulminant pulmonary edema which progresses to shock and death in about 36% of patients with HCPS. Nevertheless, to date, neither antiviral treatments nor vaccines inducing neutralizing antibodies (NAb) have proven effective against HCPS-causing hantaviruses. We set out for the first study on human cellular immunity towards ANDV in 78 convalescent survivors of ANDV infection. We found that Gn-specific responses were predominant as compared to N- and Gc-specific responses, even up to 13 years after the infection. Surprisingly, most of the Gn-specific responses were restricted to two neighboring epitopes within the Gn carboxyterminus. Interestingly, among HLA-B*3501+ patients, Gn465−473-specific CD8+ T-cells showed highly differentiated but resting phenotype and functions. It remains to be seen in future studies whether the immunodominace of Gn-specific T-cells is crucial for protective immunity. Most intriguingly, titers of neutralizing antibodies increased in 10/17 individuals months to years after the acute infection and independently of whether they were residents of endemic areas or not. Thus, our data suggest viral persistence or latency in part of ANDV-convalescent patients. However, it remains a major task for future studies to proof the concept of latent/persistent human ANDV infection by the determination of viral antigen in convalescent patients.

Hantavirus Cardiopulmonary Syndrome (HCPS) due to Andes virus (ANDV) is endemic in Chile and Argentina and currently demonstrates a case-fatality rate of 37% in humans. By contrast to the chronically infected rodents, it is believed that ANDV in humans is cleared during the acute phase. Moreover, to date, both magnitude and quality of human T-cell responses during ANDV infection and clearance are unknown. Using IFN-γ and granzyme B ELISPOT assays as well as flow cytometry, we prospectively studied the ANDV-specific T-cell responses in a 56-year-old convalescing survivor of severe HCPS, whose blood cells remained PCR-positive for ANDV-RNA until day 53 after hospital admission, that is, 67 days after infection and 42 days after discharge. PCR-negativity was closely related to the increase and function of (Gn46–60)-specific IFN-γ+ granzyme B+ CD8+ T-cells, but not to neutralizing antibody titers. Concurrently, the phenotype of CD45RA+CCR7− Gn46–60-specific T-cells shifted from a CD28−CD27+ “intermediate” to a CD28− CD27− “late” effector memory beyond day 53 after hospital admission. This is the first report that shows that ANDV can persist in the human hosts for more than 2 months. Moreover, the kinetics of T-cell responses during ANDV clearance may indicate a major role of T-cells for clearance of ANDV and human immunity to this pathogen. J. Med. Virol. 80:1947–1951, 2008. © 2008 Wiley-Liss, Inc.

T cell-mediated protection against HCV depends on constantly activated effector CD8+T cells that control emergence, spread and expansion of the virus. Why these cells fail to contain HCV replication in 70–80% of the individuals who develop persistent viremia is not clear. Although many reviews have focused on HCV's ability to interfere with the process of antigen presentation by dendritic cells (DC), only few have discussed the mechanisms whereby HCV-derived antigens become available for presentation to naive CD8+T cells. The importance of these mechanisms has been recently brought to light by new insight into DC biology, antigen processing, HCV replication and the immune system's functional anatomy. This review explores the different immunological scenarios in which CD8+T cell responses against HCV may be initiated. It describes the critical factors limiting antigen sensing and capture by APC and antigen recognition by T cells, and discusses how these factors may favor chronicity of HCV infection. Despite the lack of critical detail and hard experimental proof, this review proposes a model whereby liver seclusion, unproductive infection of professional antigen presenting cells and lack of direct tissue damage hamper the launch of a virus-specific CD8+T cell response. The implications for vaccine development are also discussed.

Clearance of hepatitis B virus infection (HBV) infection implies a polyclonal vigorous T-helper 1 (Th1) and cytotoxic T-lymphocyte (CTL) response. Interleukin-18 (IL-18), a monokine that shares functional abilities with IL-12, is a potent inductor of interferon-gamma (IFN-gamma) by Th1 and natural killer (NK) cells. However, the role and regulation in HBV infection of IFN-gamma have not been defined. This study therefore sought to determine hepatitis B core antigen (HBcAg)-mediated regulation of IL-18 production in peripheral blood mononuclear cells (PBMCs) from healthy controls (HC) and patients with chronic hepatitis B (CHB) or acute hepatitis B (AHB); 31 HC, 27 patients with CHB and 8 patients with AHB infection were included in the study. HBcAg-mediated induction of IL-18 was determined by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and specific enzyme-linked immunosorbent assay (ELISA). HBcAg induced IL-18 gene transcription and dose-dependent secretion of mature IL-18 protein in HC, CHB, and AHB. HBcAg-dependent IL-18 levels were abrogated by inhibition of Caspase-1, but not by blockade of CD40-CD154 interaction. Serum levels of IFN-gamma correlated inversely with viremia in patients with CHB (rho = - 0.54, P0.05), but not with serum levels of IL-12 or IL-18. Interestingly, in PBMCs of HBeAg-negative patients, HBcAg induced significantly higher amounts of IL-18 than in those of HBeAg-positive patients. A variant, lacking the histone-like arginine-rich domain, did not induce IL-18 in either HC or CHB in vitro. Taken together, these results indicate that HBcAg induces IL-18 secretion by induction of Caspase-1. Differential regulation in HBeAg-negative and positive patients suggests an important role of IL-18 in CHB infection.

OBJECTIVE Toll-like receptors (TLR) 2 and 4 were shown recently to mediate lipopolysaccharide (LPS)/endotoxin effects in vivo. Absence of clinical features, such as fever and leucocytosis, frequent infections, and up-regulation of anti-inflammatory cytokines suggest systemic differential regulation of LPS effects in patients with chronic endotoxinaemia due to liver cirrhosis. DESIGN Regulation of TLR2 and TLR4 represents a possible pathway to control LPS-induced immune responses in liver cirrhosis. METHODS We compared the expression of TLR2 and TLR4 in peripheral blood mononuclear cells (PBMC) (n = 28) and in liver biopsies (n = 20) of controls and of patients with liver cirrhosis by applying the reverse transcriptase polymerase chain reaction technique. The data were correlated to serum levels of LPS and CD14. RESULTS Expression of TLR2 was up-regulated (P < 0.01 to P < 0.05) in the PBMC of patients with high serum endotoxin levels, while TLR4 expression in patients at Child-Pugh stage A was down-regulated, irrespective of the origin (alcoholic or viral) of cirrhosis. A strong and significant correlation between expression of TLR2 and serum LPS (r = 0.638, P < 0.01) and soluble CD14 (r = 0.550, P < 0.05) was observed. Intrahepatic expression of TLR2/4 was not altered significantly in patients with liver cirrhosis. CONCLUSION Our data indicate LPS-driven regulation of TLR2/4 in patients with liver cirrhosis, suggesting involvement in mechanisms of systemic LPS hyporesponsiveness.

Background and aims. Luminal bacteria have been implicated in the pathogenesis of inflammatory bowel diseases. Exposure of intestinal epithelial cells (IEC) to bacterial components potentially initiates intestinal inflammation by release of chemokines and recruitment of inflammatory cells. We analyzed receptor expression and signaling pathways involved in activation of human primary IEC and carcinoma-derived cell lines by lipopolysaccharide (LPS). Materials and methods. HT-29/p, HT-29/MTX, and Caco-2 cells were stimulated by LPS. IL-8 content in supernatants was analyzed by ELISA, and expression of CD14, Toll-like receptor (TLR) 2 and TLR 4 was determined by RT-PCR. Presence of TLR 4 protein was assessed by western blot analysis. LPS response was modulated by sCD14, LPS-binding protein, neutralization of CD14, and inhibitors of early signal activation. Results. LPS dose-dependently induced secretion of IL-8 in undifferentiated HT-29/p cells while Caco-2 and permanently differentiated HT-29/MTX cells were unresponsive. Differently to HT-29/MTX, both HT-29/p and Caco-2 cells constitutively expressed transcripts for CD14. However, CD14 was not required for LPS-mediated induction of IL-8 in HT-29/p cells since neutralizing anti-CD14 antibodies left IL-8 levels unchanged. Unresponsiveness of Caco-2 and HT-29/MTX cells to LPS persisted in the presence of sCD14 and/or LPS-binding protein. Neither cell line expressed TLR 2 transcripts while only responsive HT-29/p cells expressed TLR 4 mRNA and TLR 4 protein. Butyrate down-regulated TLR 4 expression and significantly diminished LPS-dependent IL-8 secretion. Inhibition of G protein dependent kinase activation reduced IL-8 levels to 50%; the phosphatidyl-inositol-3′-kinase inhibitor LY294002 abrogated the response. Conclusion. Responsiveness of IEC lines to LPS is positively correlated with TLR 4 expression. Strategies targeting TLR 4 expression or TLR 4 mediated signaling may antagonize IEC activation by LPS.

Background Alfa-interferons (IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b) are effective treatments for chronic hepatitis C infection. However, their usage has been associated with a variety of adverse events, including interstitial pneumonitis and pulmonary arterial hypertension. Although rare, these adverse events can be severe and potentially life-threatening, emphasizing the need for simple biomarkers of IFN-induced lung toxicity. Methods Human lung microvascular endothelial cells (HLMVEC), human pulmonary artery smooth muscle (HPASM) cells and A549 cells were grown under standard conditions and plated into 96- or 6-well plates. Cells were stimulated with various concentrations of different IFNs in hydrocortisone-free medium. After 24 and 48 hours, IP10 and ET-1 were measured by ELISA in conditioned medium. In a second set of experiments, cells were pre-treated with tumour necrosis factor-α (TNF-α) (10 ng/mL). Results IFNα2a, IFNα2b, 40KDa-PEGIFNα2a and 12KDa-PEGIFNα2b, but not IFNλ, induced IP10 (CXCL10) release and increased IP10 gene induction in HLMVEC. In addition, all four IFNα preparations induced IP10 release from HPASM cells and A549 cells pre-treated with TNFα. In each of these cell types, 40KDa-PEGIFNα2a was significantly less active than the native forms of IFNα2a, IFNα2b or 12KDa-PEGIFNα2b. Similarly, IFNα2a, IFNα2b and 12KDa-PEGIFNα2b, but not 40KDa-PEGIFNα2a, induced endothelin (ET)-1 release from HPASM cells. Conclusions Consistent with other interstitial pulmonary diseases, both IP10 and ET1 may serve as markers to monitor IFN-induced lung toxicity in patients. In addition, both markers may also serve to help characterize the risk associated with IFNα preparations to induce lung toxicity.

MCSP/CSPG4 is a large transmembrane proteoglycan identified in melanomas as HMW-MAA. In the mouse it is known as neurite growth factor 2 (NG2), a marker of pericyte recruitment. MCSP has been used as a target for clinical imaging of (uveal) melanomas by immunoscintigraphy. MCSP shows uniform and abundant expression in ca. 60-80% of melanoma, and was described in lobular breast carcinoma, glioblastoma, osteo- & chondrosarcoma, and basal cell carcinoma. It is present at high levels on pericytes of tumor neovasculature, but down-regulated as vessels mature. Normal tissue expression is low and it is not detected on PBMCs. We have generated human/Cynomolgus cross-reactive antibodies against a membrane-proximal MCSP epitope by mouse immunization with a linear peptide derived from the membrane proximal D3 domain followed by boosting with melanoma cells. The mouse antibody LC007 was selected for humanization due to its potent induction of ADCC as a chimeric antibody, compared to antibodies to membrane distal epitopes of MCSP. LC007 as chimeric IgG1 and its humanized IgG1 derivative M4-3-ML2 are characterized by the following properties: i) Specific binding to the native epitope on MCSP+ melanoma cells, but no induction of internalization; ii) Specific IHC staining of MCSP+ cells in FFPET samples; iii) ca 10 nM monovalent affinity for hMCSP D3 domain. Moreover, glycoengineering of LC007 and M4-3-ML2 antibodies using GlycoMab technology resulted in increased binding affinity for hFcgRIIIa and enhanced ADCC potency and absolute killing of melanoma cell lines. As expected, neither up to 10 ug/mL wildtype, nor glycoengineered M4-3-ML2 induced relevant cytokine (IL-6, TNF-α, IFN-γ) release in human whole blood supporting that MCSP is not expressed there. Subsequently, we studied anti-tumoral efficacy of the chimeric antibody LC007 and the humanized antibody M4-3-ML2 in disseminated models of MV3 and MDA-MB435 melanoma after i.v. injection of tumor cells in hCD16 transgenic Scid mice, which express the functional human high affinity FcgRIIIa receptor on NK cells. Both, glycoengineered LC007 and M4-3-ML2 mediated efficacy in terms of enhanced median and overall survival in both disseminated xenograft models, and were superior to the respective non-glycoengineered antibodies. Taken together, our studies support MCSP/CSPG4 as an attractive target for antibody-based cancer immunotherapy. Further studies investigating the anti-angiogenic effect of MCSP antibodies via their action on pericytes/vascular smooth muscle cells are ongoing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-236. doi:1538-7445.AM2012-LB-236