Background GLPG1205 is a selective functional antagonist of G-protein-coupled receptor 84, which plays an important role in fibrotic processes. This study assessed the efficacy, safety and tolerability of GLPG1205 for treatment of idiopathic pulmonary fibrosis (IPF). Methods PINTA (ClinicalTrials.gov: NCT03725852) was a phase 2, randomised, double-blind, placebo-controlled, proof-of-concept trial. Patients with IPF were randomised 2:1 to once-daily oral GLPG1205 100 mg or placebo for 26 weeks and stratified to receive GLPG1205 alone or with local standard of care (nintedanib or pirfenidone). The primary end-point was change from baseline in forced vital capacity (FVC); other end-points were safety and tolerability, and lung volumes measured by imaging (high-resolution computed tomography). The study was not powered for statistical significance. Results In total, 68 patients received study medication. Least squares mean change from baseline in FVC at week 26 was −33.68 (95% CI −112.0–44.68) mL with GLPG1205 and −76.00 (95% CI −170.7–18.71) mL with placebo (least squares mean difference 42.33 (95% CI −81.84–166.5) mL; p=0.50). Lung volumes by imaging declined −58.30 versus −262.72 mL (whole lung) and −33.68 versus −135.48 mL (lower lobes) with GLPG1205 versus placebo, respectively. Treatment with GLPG1205 versus placebo resulted in higher proportions of serious and severe treatment-emergent adverse events and treatment-emergent discontinuations, most apparent with nintedanib. Conclusions Treatment with GLPG1205 did not result in a significant difference in FVC decline versus placebo. GLPG1205 demonstrated a poorer safety and tolerability profile than placebo. The PINTA trial (NCT03725852) did not find a significant difference between GLPG1205 and placebo on change in FVC in patients with idiopathic pulmonary fibrosis. GLPG1205 demonstrated a poorer safety and tolerability profile versus placebo. https://bit.ly/3EQGst7

Introduction: G-protein-coupled receptor 84 (GPR84), which is activated by a range of medium chain fatty acids, is involved in fibrotic processes. GLPG1205, a selective GPR84 antagonist, inhibits monocyte and neutrophil migration and activation among other actions. Aim: To assess the efficacy and safety of GLPG1205 vs placebo (PBO) in idiopathic pulmonary fibrosis (IPF). Methods: PINTA (NCT03725852) was a phase 2, randomised, double-blind, PBO-controlled, proof-of-concept trial in Europe and Oman. IPF patients received oral GLPG1205 100mg or PBO once daily, on top of standard of care (nintedanib/pirfenidone/neither) for 26 weeks. Primary endpoint: change from baseline (CFB) in FVC; other endpoints: safety, tolerability and functional respiratory imaging (FRI). PINTA was not powered for statistical significance. Results: 68 patients were dosed. Least squares (LS) mean (SE) CFB in FVC at Week 26 was −34mL (39) with GLPG1205 vs −76mL (47) with PBO (LS mean difference=42mL [95% CI −82, 166; p=0.495]). Total lung volume by FRI declined less with GLPG1205 compared with PBO. The most frequent adverse events (AEs) for GLPG1205 alone were GI disorders, particularly nausea (6/17 patients vs 0/8 on PBO). No relevant safety signals were seen for GLPG1205 alone or with pirfenidone. For GLPG1205 with nintedanib, higher rates of early discontinuations, high-grade treatment-emergent AEs, serious AEs and GI AEs were observed and there was 1 death (IPF exacerbation, unrelated to study drug). Conclusion: GLPG1205 resulted in smaller declines in FVC and total lung volume at Week 26 vs PBO, consistent with a therapeutic effect. This along with the observed safety profile for GLPG1205 alone supports further development. Funding: Galapagos

Regulatory T cells (Tregs) play a crucial role in controlling autoimmune and inflammatory responses. Recent studies have demonstrated that dendritic cells (DCs) contribute to the homeostasis of peripheral Tregs. Autophagy, a critical pathway for cellular homeostasis, is active in DCs and is upregulated in different inflammatory conditions. We have shown that Tregs are expanded and have phenotypic alterations and impaired suppressive functions in mice with autophagy-deficient DCs. RNA profiling of Tregs revealed that autophagy in DCs is required to stabilize Treg expression signatures. This phenotype is linked to the downregulation of ICOS-Ligand expression in autophagy-deficient DCs, a consequence of the accumulation of ADAM10, the metalloproteinase responsible for its cleavage. Upon inflammation, in antigen-induced arthritis, mice with autophagy-deficient DCs exhibit increased synovial inflammation and cartilage and bone erosion correlating with Treg-to-Th17 conversion. Our data reveal a mechanism that couples autophagy deficiency in DCs to the function, homeostasis, and stability of Tregs.

Introduction The biological activity of interleukin (IL)-1 is tightly regulated by a specific receptor antagonist (IL-1Ra) and the decoy receptor IL-1R2. The role of IL-1Ra has been well demonstrated in IL-1Ra deficient mice. In contrast, the role of endogenous IL-1R2 remains widely unknown. Methods We generated IL-1R2 deficient mice in a C57BL/6 background and investigated the role of IL-1R2 in both lipopolysaccharide (LPS)-induced lethality by injecting 10 mg/kg E. coli LPS intraperitoneally (i.p.), and in systemic inflammatory responses induced by i.p. injections of 10 µg/kg IL-1β. Lethality was monitored daily and serum cytokine levels were measured at different time points by ELISA, respectively. Arthritis was induced by injecting i.p. 1.5 mg purified arthritogenic IgGs purified from K/BxN serum. Arthritis severity was assessed by clinical and histological scoring. Tissue mRNA levels of cytokines were measured by real-time PCR. Results IL-1R2 deficient mice bred normally and exhibited a grossly normal phenotype. IL-1R2 was selectively expressed by wild-type (WT), but not IL-1R2 deficient, neutrophils. The number of immune cells in bone marrow, spleen and peripheral blood was similar in IL-1R2 deficient and WT mice. The phagocytic function and oxidative burst of IL-1R2 deficient neutrophils were normal. The rate of LPS-induced mortality and the level of inflammatory responses to IL-1b were similar in IL-1R2 deficient mice and their WT littermates. In contrast, IL-1R2 deficiency was associated with aggravated arthritis severity. Levels of IL-6, IL-1β, CXCL-1 and CXCL-2 mRNA were significantly increased in ankles of IL-1R2 deficient mice. Immunohistochemical analyses indicated that IL-1R2 is mainly expressed by infiltrating neutrophils. Conclusions These data show that the decoy receptor IL-1R2 plays an important role during K/BxN serum transfer-induced inflammatory arthritis and suggest that neutrophils exert anti-inflammatory activities in arthritis by expressing IL-1R2. However, in contrast to IL-1Ra, IL-1R2 is not essential for normal homeostasis and systemic responses to LPS and acute IL-1 administration.

INTRODUCTION:Interleukin (IL)-36 refers to three related IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, that bind to the IL-36 receptor (IL-36R). IL-36 exerts proinflammatory effects in skin and lung and stimulates T cell responses. In the present study, we examined the expression and function of IL-36R and its ligands in experimental arthritis.METHODS:Collagen-induced arthritis (CIA), antigen-induced arthritis (AIA), and K/BxN serum transfer-induced arthritis were induced according to standard protocols. Messenger RNA levels for IL-36R and its ligands in the joints of mice with CIA were determined by RT-qPCR. Mice with CIA were injected with a blocking monoclonal anti-IL-36R, a blocking anti-IL-1RI, or their isotype-matched control antibodies at the time of arthritis onset. Anti-IL-36R or control antibodies were also injected at the time of AIA induction. Finally, IL-36R-deficient mice were examined in AIA and serum transfer-induced arthritis. The development and severity of arthritis were assessed by clinical and histological scoring.RESULTS:IL-36R, IL-36Ra and IL-36γ mRNA were detected in the joints of mice with CIA, but their levels did not correlate with arthritis severity. As opposed to anti-IL-1RI antibody treatment, the injection of an anti-IL-36R antibody was devoid of effect on the development and severity of CIA. The severity of joint inflammation and structural damage in AIA was also unaltered by anti-IL-36R antibody treatment. Finally, the severity of AIA and K/BxN serum transfer-induced arthritis was similar in IL-36R-deficient and wild-type mice.CONCLUSIONS:The development and severity of experimental arthritis are independent of IL-36R signaling.

IL-33 is the most recently described member of the IL-1 family of cytokines. Expression studies and data obtained with ST2 KO mice, ST2 blockade and recombinant IL-33 have suggested implication of the IL-33/ST2 axis in the pathogenesis of human and mouse arthritis. In the present study we investigated the role of endogenous IL-33 in K/BxN serum transfer-induced arthritis by using IL-33 KO mice and compared the results to those obtained using ST2 KO mice. Arthritis was induced in IL-33 KO and ST2 KO mice, backcrossed to the C57BL/6 background, or in WT C57BL/6 control mice, by injection of arthritogenic K/BxN serum or purified total IgG. The development of arthritis was followed by clinical scoring of the paws and confirmed by histological evaluation. Cytokine mRNA expression was assessed by RT-qPCR. Cytokine protein levels were monitored by ELISA or Milliplex. IL-33 mRNA and protein expression in synovial tissues, as well as circulating IL-33 levels, were similar in arthritic WT and ST2 KO mice, and absent in IL-33 KO mice. The incidence and severity of K/BxN serum transfer-induced arthritis was identical in IL-33 KO and WT mice, while disease development was significantly reduced in ST2 KO mice. Similar data were obtained when injecting purified arthritogenic total IgG instead of complete K/BxN serum, excluding a potential contribution of IL-33 contained in the serum of donor mice to explain this difference. IL-33 is expressed locally in the inflamed synovium and is detected in the circulation during serum transfer-induced arthritis. However IL-33 KO mice displayed identical arthritis development and severity as compared to WT controls. In contrast, disease severity was reduced in ST2 KO mice. The reasons for the difference between IL-33 KO and ST2 KO mice, which might suggest IL-33 independent effects of ST2, or reveal the existence of confounding variables in these KO strains, are currently under investigation.

Objectives To define the cell type (myeloid vs other cells) specific effect of interleukin 1 (IL-1) receptor antagonist (IL-1Ra) deficiency on the acute inflammatory phase of arthritis. Methods Arthritis was induced by K/BxN serum transfer in wild-type (WT), IL-1Ra-deficient (IL-1Ra−/−) and conditional knockout mice. In the latter, IL-1Ra production was specifically targeted in myeloid cells (IL-1RaΔM) or in both hepatocytes and myeloid cells (IL-1RaΔH+M). Arthritis severity was clinically evaluated and ankle sections were scored for synovial inflammation and cartilage erosion. Quantitative RT-PCR, western blot and immunohistochemical analyses measured expression, localisation and cellular sources of the different IL-1Ra isoforms in arthritic joints. Results Total and myeloid cell-specific IL-1Ra deficiency was associated with increased arthritis severity, although disease incidence was similar to that of WT mice. Increased clinical scores were associated with exacerbated synovial inflammation. All IL-1Ra isoforms, except for intracellular (ic)IL-1Ra2, were expressed in arthritic joints of WT mice. In contrast, production of secreted (s)IL-1Ra and icIL-1Ra3 isoforms was markedly decreased in arthritic joints of both IL-1RaΔM and IL-1RaΔH+M mice. Immunohistochemical and western blot analyses suggested that the icIL-1Ra1 isoform is produced primarily by synovial fibroblasts. Conclusion Myeloid cell-derived IL-1Ra, including both sIL-1Ra and icIL-1Ra3 isoforms, controls articular inflammation during the acute phase of K/BxN serum transfer-induced arthritis.

Interleukin‐1 receptor antagonist (IL‐1Ra) is a specific IL‐1 inhibitor that possesses anti‐inflammatory activities. Several studies in human and mouse suggested a protective role for IL‐1Ra in liver inflammation, and we previously demonstrated that hepatocytes produce high levels of IL‐1Ra in response to inflammatory challenge in vitro and in vivo. In the present study, we investigated the production and the biological function of hepatocyte‐derived IL‐1Ra in concanavalin A (ConA)‐induced hepatitis in mice. We show that the injured liver produces large amounts of IL‐1Ra and that secreted and intracellular IL‐1Ra isoforms are produced with different kinetics during the course of hepatitis. By using hepatocyte‐specific IL‐1Ra‐deficient mice (IL‐1RaΔH), we demonstrate that hepatocytes represent the major cellular source of local IL‐1Ra. Most interestingly, hepatic necrosis and inflammation were increased in IL‐1RaΔH as compared with wild‐type mice during the late phase of the disease, leading to a delayed resolution of hepatitis in IL‐1RaΔH mice. In conclusion, our results show that the local production of IL‐1Ra by hepatocytes contributes to the resolution of hepatitis.

Background In rheumatoid arthritis (RA), non-professional antigen presenting cells (APCs) such as fibroblast-like synoviocytes (FLS) can express MHC class II (MHCII) molecules and function as non-professional APCs in vitro. Objective To examine the regulation of MHCII expression in FLS and to investigate the role of FLS as non-professional APCs in collagen-induced arthritis (CIA). Methods Expression of MHCII, CIITA and Ciita isoforms pI, pIII and pIV was examined by RT-qPCR, immunohistochemistry and flow cytometry in human synovial tissues, arthritic mouse joints and human as well as mouse FLS. CIA was induced in mice knockout for the isoform IV of Ciita (pIV-/-), in pIV-/- mice transgenic for CIITA in the thymus (pIV-/- K14 CIITA) and in control littermates in the DBA/1 background by immunising with bovine collagen type II (CII) in complete Freund9s adjuvant. Results HLA-DRA, total CIITA and CIITA pIII mRNA levels were significantly increased in the synovial tissues from RA compared to osteoarthritis patients. Human FLS expressed surface MHCII via CIITA pIII and pIV, while MHCII expression in murine FLS was entirely mediated by pIV. pIV-/- mice lacked both inducible MHCII expression on non-professional APCs including FLS, and in the thymic cortex. The thymic defect in pIV-/- mice impaired CD4+ positive selection, thus protecting pIV-/- mice from CIA by preventing CD4+ T cells immune responses against CII and blocking the release of IFN-γ and IL-17 in ex vivo stimulated lymph node cells. The production of T dependent, arthritogenic anti-CII antibodies was also impaired in pIV-/- mice. A normal thymic expression of MHCII and CD4+ T cell repertoire was obtained in pIV-/- K14 CIITA Tg mice. Immune responses against CII were restored in pIV-/- K14 CIITA Tg mice, as well as the arthritis incidence and clinical severity despite the lack of MHCII expression by mouse FLS. At histology, inflammation and neutrophils infiltration scores were not reduced in pIV-/- K14 CIITA Tg mice, while the bone erosion score was significantly lower than in controls. Conclusion Over expression of MHCII is tightly correlated with CIITA pIII in the arthritic human synovium. MHCII is induced via CIITA pIII and pIV in human FLS. In the mouse, MHCII expression in the thymic cortex and in FLS is strictly dependent upon Ciita pIV. The lack of Ciita pIV in the periphery of pIV-/- K14 CIITA Tg mice lowered the bone erosion score but did not significantly protect from inflammation and autoimmune responses in CIA. Funding Supported by grants from the Albert Böni foundation (to JMW), Carlos et Elise de Reuters foundation (to JMW), and the Swiss National Science Foundation (grant No 310030_122477 to JMW; No 320000-119728 to CG; No 320000-120319 (to GP).

OBJECTIVE To determine the regulation of class II major histocompatibility complex (MHC) expression in fibroblast-like synoviocytes (FLS) in order to investigate their role as nonprofessional antigen-presenting cells in collagen-induced arthritis (CIA). METHODS Expression of class II MHC, class II MHC transactivator (CIITA), and Ciita isoforms PI, PIII, and PIV was examined by real-time quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry in human synovial tissues, arthritic mouse joints, and human and murine FLS. CIA was induced in mice in which isoform PIV of Ciita was knocked out (PIV(-/-) ), in PIV(-/-) mice transgenic for CIITA in the thymus (K14 CIITA), and in their control littermates. RESULTS HLA-DRA, total CIITA, and CIITA PIII messenger RNA levels were significantly increased in synovial tissue samples from patients with rheumatoid arthritis compared with the levels in tissue from patients with osteoarthritis. Human FLS expressed surface class II MHC via CIITA PIII and PIV, while class II MHC expression in murine FLS was entirely mediated by PIV. Mice with a targeted deletion of CIITA PIV lack CD4+ T cells and were protected against CIA. The expression of CIITA was restored in the thymus of PIV(-/-) K14 CIITA-transgenic mice, which had a normal CD4+ T cell repertoire and normal surface levels of class II MHC on professional antigen-presenting cells, but did not induce class II MHC on FLS. Synovial inflammation and immune responses against type II collagen were similar in PIV(-/-) K14 CIITA-transgenic mice and control mice with CIA, but bone erosion was significantly reduced in the absence of PIV. CONCLUSION Overexpression of class II MHC is tightly correlated with CIITA expression in arthritic synovium and in FLS. Selective targeting of Ciita PIV in peripheral tissues abrogates class II MHC expression by murine FLS but does not protect against inflammation and autoimmune responses in CIA.

OBJECTIVE:RNA interference (RNAi) is a powerful tool for sequence-specific gene silencing, and interest in its application in human diseases is growing. Given the success of recent strategies for administering gene therapy in rheumatoid arthritis using recombinant vectors such as adeno-associated virus type 5 (rAAV5) for optimized intraarticular gene transfer, we undertook the present study to determine the feasibility of using rAAV5-mediated RNAi-based therapy in arthritis.METHODS:We developed rAAV5 vectors expressing short hairpin small interfering RNA (shRNA) against tumor necrosis factor alpha (TNFalpha) under H1 promoter, and carrying the enhanced green fluorescent protein (eGFP) reporter gene under cytomegalovirus promoter (rAAV5-shTNF). TNFalpha gene silencing was validated in vitro with mouse macrophages. Mice with collagen-induced arthritis were injected in the ankle and knee joints, at disease onset, with either rAAV5-shTNF or control rAAV5-eGFP vectors (5 x 10(9) particles). Arthritis severity was assessed clinically and histologically, and immunologic response was examined. Local and systemic transgene expression was monitored using quantitative reverse transcriptase-polymerase chain reaction, immunohistochemical analysis, and enzyme-linked immunosorbent assay.RESULTS:After a single injection of rAAV5-shTNF into inflamed joints, local TNFalpha gene silencing provided rapid and long-term suppression of arthritis progression and reduced joint damage compared with that observed in control groups. Treatment with rAAV5-shTNF was associated with decreased proliferation and interferon-gamma production by antigen-stimulated T cells from draining lymph nodes, and the potency of this treatment was similar to that observed with other treatment strategies targeting TNFalpha at the protein level, either locally or systemically.CONCLUSION:Our data present the first proof-of-concept for the application of rAAV5-mediated RNAi-based gene therapy for local blockade of inflammation in experimental arthritis.

Objective. The balance between interleukin-1 (IL-1) and its specific inhibitor, the IL-1 receptor antagonist (IL-1Ra), plays a major role in the development of arthritis. The purpose of this study was to investigate the role of IL-1Ra produced specifically by myeloid cells in the control of collagen-induced arthritis (CIA) by using myeloid cell-specific IL-1Ra-deficient mice (IL-1Ra(Delta M)).Methods. IL-1Ra(Delta M) mice were generated by using the loxP/Cre recombinase system. CIA was induced in IL-1Ra(Delta M) mice and littermate control mice by a single immunization with bovine type II collagen (CII) in Freund's complete adjuvant. Arthritis severity was assessed by clinical and histologic scoring. Draining lymph node (DLN) cell responses were examined ex vivo, and ankle extracts were used in the quantification of cytokines and chemokines.Results. Clinical and histopathologic evaluations revealed an early disease onset and a severe form of CIA in IL-1Ra(Delta M) mice. This was characterized by increased production of interferon-gamma (IFN gamma) and IL-17 by CII-stimulated DLN cells. We also observed that the CII-specific CD4+ T cell response shifted in vivo, from a dominant Th1 response early in the course of the arthritis to the presence of both Th1 and Th17 cytokines later in the disease course. Interestingly, IL-1Ra levels were higher in the arthritic joints of IL-1Ra(Delta M) mice as compared with the controls, indicating that nonmyeloid cells strongly contribute to the local production of IL-1Ra. However, this enhanced IL-1Ra production was not sufficient to limit joint inflammation and tissue damage.Conclusion. Our results suggest that myeloid cell-derived IL-1Ra plays a critical role in the control of the development and the severity of CIA by modulating Th1 and Th17 responses in lymphoid organs.

OBJECTIVE Interleukin-33 (IL-33; or, IL-1F11) was recently identified as the ligand of the IL-1 family receptor T1/ST2. The aim of this study was to examine IL-33 production in human and mouse joints and to investigate the role of IL-33 and T1/ST2 in experimental arthritis. METHODS IL-33 expression was examined in human synovial tissue, rheumatoid arthritis (RA) synovial fibroblasts, and arthritic mouse joints. Mice with collagen-induced arthritis (CIA) were treated with blocking anti-ST2 antibody or control antibody beginning at the onset of disease. Arthritis severity was assessed by clinical and histologic scoring. Draining lymph node (LN) cell responses were examined ex vivo, and joint messenger RNA (mRNA) was used for expression profiling. RESULTS IL-33 was highly expressed in human RA synovium. In cultured synovial fibroblasts, IL-33 expression was strongly induced by IL-1beta and/or tumor necrosis factor alpha. Furthermore, IL-33 mRNA was detected in the joints of mice with CIA and increased during the early phase of the disease. Administration of a blocking anti-ST2 antibody at the onset of disease attenuated the severity of CIA and reduced joint destruction. Anti-ST2 antibody treatment was associated with a marked decrease in interferon-gamma production as well as with a more limited reduction in IL-17 production by ex vivo-stimulated draining LN cells. Finally, RANKL mRNA levels in the joint were reduced by anti-ST2 treatment. CONCLUSION IL-33 is produced locally in inflamed joints, and neutralization of IL-33 signaling has a therapeutic effect on the course of arthritis. These observations suggest that locally produced IL-33 may contribute to the pathogenesis of joint inflammation and destruction.

Superparamagnetic iron oxide nanoparticles (SPIONs) are attractive materials that have been widely used in medicine for diagnostic imaging and therapeutic applications. In our study, SPIONs and the corticosteroid dexamethasone acetate (DXM) are co-encapsulated into PLGA microparticles for the aim of locally treating inflammatory conditions such as arthritis. The magnetic properties conferred by the SPIONs could help to maintain the microparticles in the joint with an external magnet. The aim of this study was to investigate the interaction between magnetic microparticles and human synovial fibroblasts in terms of microparticle uptake (FACS, confocal and optical microscopy), internalization mechanism (Prussian Blue staining, TEM, immunofluorescence), cell toxicity (MTT) and tissue reaction after intra-articular injection (histology). The results show that the microparticles have an excellent biocompatibility with synoviocytes and that they are internalized through a phagocytic process, as demonstrated by fluorescence-activated cell sorting and morphological analyses of cells exposed to microparticles. Histological analysis showed that the prepared microparticles did not induce any inflammatory reaction in the joint. This type of carrier could represent a suitable magnetically retainable intra-articular drug delivery system for treating joint diseases such as arthritis or osteoarthritis.

Conventional corticosteroid suspensions for the intra-articular treatment of arthritis suffer from limitations such as crystal formation or rapid clearance from the joint. The purpose of this study was to investigate an innovative alternative consisting of corticosteroid encapsulation into magnetically retainable microparticles.

The chapter focuses on the detection of specific mRNA by in situ hybridization (ISH) in synovial tissue specimens. This technique is widely applied, reliable, specific, and sensitive, because even small quantities of mRNA can be detected. Presented here contemporary protocols for ISH using a combined nonradioactive immunohistochemical detection system. In overview, the following steps have to be covered to perform ISH. (1) mRNA probes (sense and antisense) are generated by in vitro transcription of cDNA utilizing digoxigenin-labeled UTP nucleotides, (2) fixed tissue sections are digested with trypsin and treated consecutively with prehybridization solutions, (3) hybridization with labeled riboprobes takes place at 50 degrees C overnight in a humid chamber, (4) unbound riboprobe is removed by incubation with RNase A and additional washing with buffers, (5) stringent washing steps are performed with solutions of different sodium dodecyl sulfate, SSC, and formamide concentrations, (6) digoxigenin-labeled probes are detected immunohistochemically using antidigoxigenin antibodies linked with alkaline phosphatase and NBT/BCIP as detection system.

The association of simian virus 40 (SV40) with malignant pleural mesothelioma is currently under debate. In some malignancies of viral aetiology, viral DNA can be detected in the patients' serum or plasma. To characterize the prevalence of SV40 in Swiss mesothelioma patients, we optimized a real-time PCR for quantitative detection of SV40 DNA in plasma, and used a monoclonal antibody for immunohistochemical detection of SV40 in mesothelioma tissue microarrays. Real-time PCR was linear over five orders of magnitude, and sensitive to a single gene copy. Repeat PCR determinations showed excellent reproducibility. However, SV40 status varied for independent DNA isolates of single samples. We noted that SV40 detection rates by PCR were drastically reduced by the implementation of strict room compartmentalization and decontamination procedures. Therefore, we systematically addressed common sources of contamination and found no cross-reactivity with DNA of other polyomaviruses. Contamination during PCR was rare and plasmid contamination was infrequent. SV40 DNA was reproducibly detected in only 4 of 78 (5.1%) plasma samples. SV40 DNA levels were low and not consistently observed in paired plasma and tumour samples from the same patient. Immunohistochemical analysis revealed a weak but reproducible SV40 staining in 16 of 341 (4.7%) mesotheliomas. Our data support the occurrence of non-reproducible SV40 PCR amplifications and underscore the importance of proper sample handling and analysis. SV40 DNA and protein were found at low prevalence (5%) in plasma and tumour tissue, respectively. This suggests that SV40 does not appear to play a major role in the development of mesothelioma.

OBJECTIVE:We investigated the expression of early and late phase BK virus (BKV) proteins and their interactions with host cell proteins in renal allografts, with ongoing polyomavirus associated nephropathy (PVAN), and correlated this with the nuclear and cell morphology.METHODS:Frozen sections from three patients with renal allografts (two biopsies, one explant) with PVAN were analysed by indirect immunofluorescence using BKV specific anti-polyoma large T-antigen and anti-VP-1 antibodies, as well as anti-p53, anti-Ki67, anti-caspase-3, anti-bcl2 and anti-cytokeratin 22 antibodies. Nuclear morphology and size were estimated by DNA Hoechst staining.RESULTS:In infected tubular cells the early and late phases of infection could be distinguished according to expression of large T-antigen or VP-1. The early phase revealed almost normal nuclear proportions, whereas in later phases nuclear size increased about 2 to 3 fold. Expression of large T-antigen was strongly associated with accumulation of p53 in the nucleus, accompanied by the activation of the cell cycle associated cell protein Ki67. In contrast, expression of BKV VP1 correlated only weakly with p53. Virus dependent cell lysis was due to necrosis, since neither caspase 3 nor nuclear nor cytoskeleton changes indicated apoptosis.CONCLUSION:In our selected patients with PVAN a clear distinction between early and late phases was possible, according to the protein expression patterns of BKV markers. Striking nuclear enlargement is only present in the late phase of infection. In the inflammatory setting of PVAN, BKV dependent effects appear to be mediated by the inhibition of p53, resulting in the activation of the cell cycle. We assume that in PVAN similar BKV mechanisms are operative as in certain in vitro systems.

The relationship between HER-2 overexpression and gene amplification is well evaluated in breast cancers but remains unclear or controversial in many other tumor entities. Therefore, we tested the HER-2 status in more than 120 different tumor entities. 5751 tumor samples were analyzed on TMAs by immunohistochemistry (Hercept-Test, DAKO) and fluorescence in situ hybridization (PathVysion, Abbott-Vysis) under highly standardized conditions. HER-2 overexpression (score 2/3+) and amplification occurred most often in breast cancers but was also seen in 18 other tumor entities including cancers of the urinary bladder (amplification in 14.3%, overexpression in 6.7%), stomach (8.3/4.9%), endometrium (6.6/6.8%), lung (2.8/3.1%) and ovary (2.3/1.2%). Remarkably, a strong association between overexpression and amplification was seen in all examined cancer entities. Trastuzumab therapy is highly efficient in HER-2 amplified breast cancer both in metastatic disease and as an adjuvant therapy. A variety of other tumor entities including frequent neoplasms and cancers with often limited therapeutic options have similar patterns of HER-2 alterations as observed in breast cancer (ie high overexpression due to high level gene amplification). Such tumor entities should be carefully evaluated for a possible utility of trastuzumab treatment.

AIM:Reactivation of latent BK virus in kidney-transplanted patients results in severe graft dysfunction. The role of retroviruses infecting also latently target cells is not investigated so far in this setting. We determined the presence or induction of retroviruses in sera of immunosuppressed patients with renal allografts at the timepoint of organ rejection or ongoing polyomavirus nephropathy.PATIENTS AND METHODS:Sera of patients with acute kidney rejection or polyomavirus nephropathy (n=25) and controls (n=8) were tested for reverse transcriptase activity by the ultrasensitive product enhanced reverse transcriptase (PERT) assay. In parallel, kidney biopsies were investigated for histological signs of kidney rejection or polyomavirus nephropathy confirmed by either immunofluorescence or immunohistochemistry.RESULTS:None of the investigated sera, specifically those of patients with ongoing BK virus nephropathy, indicated reverse transcriptase activity.CONCLUSION:Our results do not support the idea of the induction of known or unknown retroviruses in patients with kidney transplantation, even under highly immunosuppressive therapies.