GLPG1205 for idiopathic pulmonary fibrosis: a phase 2 randomised placebo-controlled trial.

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.

Deficiency in IL-1 Receptor Type 2 Aggravates K/BxN Serum Transfer-Induced Arthritis in Mice but Has No Impact on Systemic Inflammatory Responses.

The biological activity of IL-1 is tightly regulated by the specific receptor antagonist (IL-1Ra) and the decoy receptor IL-1 receptor type 2 (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. To define the functional role of endogenous IL-1R2 in the K/BxN serum transfer arthritis model and in IL-1ß- or LPS-induced systemic inflammation in vivo, IL-1R2-/- mice were created and compared with wild type mice. IL-1R2-/- mice bred habitually and exhibited a normal phenotype. IL-1R2 deficiency aggravated arthritis severity and increased mRNA levels for key cytokines and chemokines such as IL-6, IL-1ß, Cxcl-1, and Cxcl-2 significantly in ankles. There was no effect of IL-1R2 deficiency on the cell-autonomous cytokine response to IL-1ß in the tested cell types, i.e., neutrophils, macrophages, and fibroblasts, but IL-1R2 deficiency on neutrophils increased the IL-1-induced response of fibroblasts in trans. Furthermore, IL-1ß induced shedding of IL-1R2 in vivo. Inflammatory responses to IL-1ß and LPS-induced mortality were not different in IL-1R2-/- compared with wild type mice. Our data demonstrate that the decoy receptor IL-1R2 plays an important inhibitory role in local IL-1- and neutrophil-dependent tissue inflammation as shown in the K/BxN serum transfer arthritis model. In contrast to IL-1Ra, IL-1R2 appears to be less crucial for systemic responses to acute administration of IL-1 or LPS.

Nanocrystal-Polymer Particles: Extended Delivery Carriers for Osteoarthritis Treatment.

An efficient treatment for osteoarthritis (OA) can benefit from the local release of a high therapeutic dose over an extended period of time. Such a treatment will minimize systemic side effects and avoid the inconvenience of frequent injections. To this aim, nanocrystal-polymer particles (NPPs) are developed by combining the advantages of nanotechnology and microparticles. Nanocrystals are produced by wet milling kartogenin (KGN), which is known to promote chondrogenesis and to foster chondroprotection. A fluorescent biodegradable polymer is synthesized for intravital particle tracking. Polymer microparticles with 320 nm embedded KGN nanocrystals (KGN-NPPs) show a high drug loading of 31.5% (w/w) and an extended drug release of 62% over 3 months. In vitro, these particles do not alter mitochondrial activity in cultured human OA synoviocytes. In vivo, KGN-NPPs demonstrate higher bioactivity than a KGN solution in a murine mechanistic OA model based on histological assessment (Osteoarthritis Research Society International score), epiphyseal thickness (microcomputed tomography), OA biomarkers (e.g., vascular endothelial growth factor, Adamts5), and prolonged intra-articular persistence (fluorescence analysis). This work provides proof-of-concept of a novel and innovative extended drug delivery system with the potential to treat human OA.

Modeling the Effect of the Selective S1P1 Receptor Modulator Ponesimod on Subsets of Blood Lymphocytes.

PURPOSE: This analysis aimed at describing the effect of the selective sphingosine-1-phosphate receptor 1 modulator ponesimod on lymphocyte subsets in peripheral blood. As the involvement of different lymphocyte subsets varies among different autoimmune diseases, characterizing the effect of ponesimod on these may be beneficial in better understanding treatment effects. METHODS: Three phase 1 clinical studies in healthy human subjects were pooled. Non-linear mixed-effects modeling techniques were used to study the effect of ponesimod on lymphocyte subsets such as B cells, T helper cells, T cytotoxic cells, and natural killer cells in a qualitative and quantitative manner. RESULTS: Indirect-response Imax models including circadian variation best described the effect of ponesimod on lymphocyte subsets. B cells and T helper cells were shown to be more affected compared to T cytotoxic cells with respect to the maximum possible reduction (100% for B and T helper cells, 95% for T cytotoxic cells) and the concentration required to reach half the maximum effect. Inter-individual variability was found to be larger for T cytotoxic compared to T helper, and B cells. CONCLUSION: These first models for ponesimod on the level of lymphocyte subsets offer a valuable tool for the analysis and interpretation of results from ponesimod trials in autoimmune diseases.

Severe neutrophil-dominated inflammation and enhanced myelopoiesis in IL-33-overexpressing CMV/IL33 mice.

IL-33 is a cytokine of the IL-1 family, which signals through the ST2 receptor. Previous studies emphasized a role for IL-33 in shaping innate and adaptive immune responses. IL-33 was also reported to modulate myelopoiesis and myeloid cell activity. In this article, we describe IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, which display an inflammatory phenotype associated with growth retardation and paw swelling. The phenotype of CMV/IL33 mice is dependent on activation of the ST2 receptor and is characterized by extensive neutrophil infiltration into different organs, including the paws. Local or systemic levels of proinflammatory mediators such as IL-1ß, Cxcl-1, G-CSF, and IL-6 are increased. CMV/IL-33 mice also suffer from anemia, thrombocytosis, and a marked dysregulation of myelopoiesis, leading to an important increase in myeloid cell production or accumulation in bone marrow (BM), spleen, and peripheral blood. Consistently, recombinant IL-33 induced proliferation of myeloid lineage cells in BM-derived granulocyte cultures, whereas IL-33 knockout mice exhibited minor deficiencies in spleen and BM myeloid cell populations. Our observations reveal a neutrophil-dominated inflammatory phenotype in IL-33-overexpressing CMV/IL33 and LysM/IL33 mice, and highlight important regulatory effects of IL-33 on myelopoiesis in vitro and in vivo, where excessive IL-33 signaling can translate into the occurrence of a myeloproliferative disorder.

Immune-mediated experimental arthritis in IL-33 deficient mice.

Previous work suggested implication of the interleukin (IL)-1 family cytokine IL-33, signaling through its receptor ST2, in the pathogenesis of human and mouse arthritis. In this study, we directly investigated the role of endogenous IL-33 in antigen-induced arthritis (AIA) and collagen-induced arthritis (CIA) using IL-33 KO mice. AIA was induced by injection of methylated bovine serum albumin (mBSA) into knee joints of previously immunized mice. CIA was induced by immunization with bovine type II collagen. Disease severity was evaluated by clinical and histological scoring and cellular immune responses were assessed in cultured draining lymph node cells. Our results indicate that the development of AIA or CIA, as assessed by clinical or histological evaluation, is not impaired in IL-33 deficient mice. We did not observe any consistent modifications in humoral or cellular immune responses in IL-33 KO mice, although IL-33 deficiency enhanced antigen-specific IFN-γ production, proliferation or IgG2a titers in some experiments, suggesting that endogenous IL-33 may contribute to shaping the adaptive immune response. In conclusion, our data suggest that IL-33 plays a modifying rather than a pivotal role in disease development in two models of immune-mediated arthritis.

Mice deficient in hepatocyte-specific IL-1Ra show delayed resolution of concanavalin A-induced hepatitis.

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-1Ra(ΔH)), we demonstrate that hepatocytes represent the major cellular source of local IL-1Ra. Most interestingly, hepatic necrosis and inflammation were increased in IL-1Ra(ΔH) as compared with wild-type mice during the late phase of the disease, leading to a delayed resolution of hepatitis in IL-1Ra(ΔH) mice. In conclusion, our results show that the local production of IL-1Ra by hepatocytes contributes to the resolution of hepatitis.

Articular inflammation is controlled by myeloid cell-derived interleukin 1 receptor antagonist during the acute phase of arthritis in mice.

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-1Ra(ΔM)) or in both hepatocytes and myeloid cells (IL-1Ra(ΔH+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-1Ra(ΔM) and IL-1Ra(ΔH+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.

Autoimmunity and inflammation are independent of class II transactivator type PIV-dependent class II major histocompatibility complex expression in peripheral tissues during collagen-induced arthritis.

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.

Protein level expression of Toll-like receptors 2, 4 and 9 in renal disease.

BACKGROUND: Toll-like receptors (TLR) recognize a variety of ligands, including pathogen-associated molecular patterns and link innate and adaptive immunity. Individual receptors can be up-regulated during infection and inflammation. We examined the expression of selected TLRs at the protein level in various types of renal disease. METHODS: Frozen sections of renal biopsies were stained with monoclonal antibodies to TLR-2, -4 and -9. RESULTS: Up-regulation of the three TLRs studied was seen, although the extent was modest. TLR-2- and -4-positive cells belonged to the population of infiltrating inflammatory cells; only in the case of TLR-9 were intrinsic glomerular cells positive in polyoma virus infection and haemolytic uraemic syndrome (HUS). CONCLUSIONS: Evidence for the involvement of the three TLRs tested in a variety of human renal diseases was found. These findings add to our understanding of the role of the innate immune system in kidney disease.

Adeno-associated virus type 5-mediated intraarticular administration of tumor necrosis factor small interfering RNA improves collagen-induced arthritis.

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.

Macroautophagy in Dendritic Cells Controls the Homeostasis and Stability of Regulatory T Cells.

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.

BK virus large T and VP-1 expression in infected human renal allografts.

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.

[Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma.

PURPOSE: Although metabolic changes make diagnosis of insulinoma relatively easy, surgical removal is hampered by difficulties in locating it, and there is no efficient treatment for malignant insulinoma. We have previously shown that the high density of glucagon-like peptide-1 receptors (GLP-1R) in human insulinoma cells provides an attractive target for molecular imaging and internal radiotherapy. In this study, we investigated the therapeutic potential of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4, an (111)In-labeled agonist of GLP-1, in a transgenic mouse model of human insulinoma. EXPERIMENTAL DESIGN: [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 was assessed in the Rip1Tag2 mouse model of pancreatic beta-cell carcinogenesis, which exhibits a GLP-1R expression comparable with human insulinoma. Mice were injected with 1.1, 5.6, or 28 MBq of the radiopeptide and sacrificed 7 days after injection. Tumor uptake and response, the mechanism of action of the radiopeptide, and therapy toxicity were investigated. RESULTS: Tumor uptake was >200% injected activity per gram, with a dose deposition of 3 Gy/MBq at 40 pmol [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4. Other GLP-1R-positive organs showed > or =30 times lower dose deposition. A single injection of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 resulted in a reduction of the tumor volume by up to 94% in a dose-dependent manner without significant acute organ toxicity. The therapeutic effect was due to increased tumor cell apoptosis and necrosis and decreased proliferation. CONCLUSIONS: The results suggest that [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 is a promising radiopeptide capable of selectively targeting insulinoma. Furthermore, Auger-emitting radiopharmaceuticals such as (111)In are able to produce a marked therapeutic effect if a high tumor uptake is achieved.

Nanocrystals of a potent p38 MAPK inhibitor embedded in microparticles: Therapeutic effects in inflammatory and mechanistic murine models of osteoarthritis.

This study aimed to formulate nanocrystal-polymer particles (NPPs) containing the potent p38α/ß MAPK inhibitor PH-797804 (PH-NPPs) and to test their extended-release properties over months in comparison to those of conventional PH microparticles for the intra-articular treatment of inflammatory and mechanistic murine models mirroring aspects of human osteoarthritis (OA). The steps of the study were (i) to formulate PH nanocrystals (wet milling), (ii) to encapsulate a high payload of PH nanocrystals in fluorescent particles (spray drying), (iii) to assess in vitro drug release, (iv) to evaluate PH-NPP toxicity to human OA synoviocytes (MTT test), (v) to investigate the in vivo bioactivity of the particles in mice in an inflammatory antigen-induced arthritis (AIA) model (using histology and RT-qPCR) and (vi) to investigate the in vivo bioactivity of the particles in the OA model obtained by mechanistic surgical destabilization of the medial meniscus (DMM) (using histology, micro-CT, and multiplex ELISA). The PH nanocrystals stabilized with vitamin E TPGS had a monomodal size distribution. The PH-NPPs had a mean diameter of 14.2 µm and drug loading of ~31.5% (w/w), and ~20% of the PH was released over 3 months. The NPPs did not exhibit toxicity to cultured human OA synoviocytes at 100 × IC50. Finally, in vivo studies showed good retention of PH-NPPs in the joint and adjacent tissues for up to 2 months, and the PH-NPPs exhibited good functional relevance by significantly reducing inflammation and joint destruction and by inhibiting several biomarkers (e.g., IL-1ß). In conclusion, local treatment with PH-NPPs, used as an extended-release drug delivery system, improved inflammation and joint degradation in two distinct mouse models, indicating treatment potential for human OA.

Self-assembled thermoresponsive nanostructures of hyaluronic acid conjugates for osteoarthritis therapy.

Under pathological conditions, joints and skin are often affected by an imbalance in the breakdown and production of hyaluronic acid (HA). The unique biochemical and biomechanical properties provided by HA must be restored for the long-term lubrication and cushioning effects. To overcome the inconvenience of repeated injections and the rapid degradation of exogenous HA treatments, HA is conjugated to a thermosensitive polymer, enabling the spontaneous formation of nanoparticles (HA Nano) at body temperature. Three HA Nano preparations are tested for their injectability, sensitivity to enzymatic degradation and cytocompatibility. One of them is delivered via subcutaneous and intra-articular injections to healthy mice and tested in a murine osteoarthritis (OA) model. It is found to be biocompatible, to offer a prolonged residence time at the injection site, have the ability to protect cartilage, to reduce pro-inflammatory cytokines and to preserve epiphysis thickness. In this study, HA Nano spontaneously forms nanoparticles at body temperature in vivo and is a promising candidate for the next generation of the sustainable/long-lasting treatment of OA and potentially also dermatological conditions.

Low prevalence of SV40 in Swiss mesothelioma patients after elimination of false-positive PCR results.

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.

In situ hybridization of synovial tissue.

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.

Effect of particle size on the biodistribution of nano- and microparticles following intra-articular injection in mice.

Intra-articular (IA) injection of extended drug release forms based on biodegradable microparticles holds promise for the treatment of joint diseases. However, the fate of microparticles following intra-articular injection is controversial and has not been thoroughly investigated. The aim of this work was therefore to evaluate the biodistribution of fluorescent poly(lactic acid) particles of different sizes after IA injection in arthritic or healthy mice. Regardless of the inflammatory status of the joint, 300 nm-nanoparticles leaked from the joint. Due to inflammation and related increase of vascular permeability, 3 µm-microparticles that were retained in the non-inflamed synovial membrane leaked from the inflamed joint. Complete retention of 10 µm-microparticles was observed independently of the joint inflammatory status. Embedding particles in a hyaluronic acid gel prolonged the retention of the formulations only in inflamed joints. Depending on particle's size, formulations were preferentially eliminated by blood vessels or lymphatic pathways. Poly(lactic acid) particles of 3 µm were biocompatible and retained in knee joints at least for 6 weeks. This work highlights the need to deliver hyaluronic acid-embedded particles of at least 3 µm to guarantee their retention in inflamed joints. These results will contribute to the rational design of long-lasting formulations to treat acute and chronic joint diseases.

Intra-articular bioactivity of a p38 MAPK inhibitor and development of an extended-release system.

In the treatment of arthritic diseases, oral or systemic administration of anti-inflammatory substances, such as p38 MAPK inhibitors, is hampered by numerous side effects. To overcome them, formulations of rapid and extended drug delivery systems were studied in intra-articular administration. For the first time, VX-745, a highly selective p38 MAPK inhibitor, demonstrated in vivo bioactivity, similar to dexamethasone activity, following intra-articular administration in an antigen-induced arthritic (AIA) mouse model. The in vitro bioactivity of VX-745 was also shown on synoviocytes, reducing the IL-6 concentration. Process and formulation parameters (i.e., polymer concentration, aqueous/organic phase ratio, emulsification speed and process, and evaporation pressure) and particle characterisation (i.e., drug loading, size of particle, and surface aspect) were extensively examined to produce optimised formulations. Indeed, a burst release provides a rapid saturation of intracellular p38 MAPK to relieve patients from pain and inflammation. Then, drug diffusion would be sufficient to maintain an effective dose over 2-3 months. This study confirms the effectiveness of encapsulated p38 MAPK inhibitors in extended drug delivery systems and seems to be a promising strategy for intra-articular treatment.