Recent compelling evidence has lead to renewed interest in the role of antibodies and immune complexes in the pathogenesis of several autoimmune disorders, such as rheumatoid arthritis. These immune complexes, consisting of autoantibodies to self-antigens, can mediate inflammatory responses largely through binding and activating the immunoglobulin Fc receptors (FcRs). Using cell-based structure activity relationships with cultured human mast cells, we have identified the small molecule R406 [N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine] as a potent inhibitor of immunoglobulin E (IgE)- and IgG-mediated activation of Fc receptor signaling (EC50 for degranulation = 56–64 nM). Here we show that the primary target for R406 is the spleen tyrosine kinase (Syk), which plays a key role in the signaling of activating Fc receptors and the B-cell receptor (BCR). R406 inhibited phosphorylation of Syk substrate linker for activation of T cells in mast cells and B-cell linker protein/SLP65 in B cells. R406 bound to the ATP binding pocket of Syk and inhibited its kinase activity as an ATP-competitive inhibitor (Ki = 30 nM). Furthermore, R406 blocked Syk-dependent FcR-mediated activation of monocytes/macrophages and neutrophils and BCR-mediated activation of B lymphocytes. R406 was selective as assessed using a large panel of Syk-independent cell-based assays representing both specific and general signaling pathways. Consistent with Syk inhibition, oral administration of R406 to mice reduced immune complex-mediated inflammation in a reverse-passive Arthus reaction and two antibody-induced arthritis models. Finally, we report a first-inhuman study showing that R406 is orally bioavailable, achieving exposures capable of inhibiting Syk-dependent IgE-mediated basophil activation. Collectively, the results show R406 potential for modulating Syk activity in human disease.
The CD52 antigen is a lymphocyte glycoprotein with an extremely short polypeptide backbone and a single N-linked glycan, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Treatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of cases, in the appearance and persistence of CD52-negative T cells. Similarly, CD52-negative B cells emerged following in vitro treatment of a CD52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-negative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe defect in the synthesis of a mature GPI precursor in both the B and T cell lines. Therefore the phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-biosynthetic pathway, i.e. synthesis of GlcNAc-phosphatidylinositol, is blocked. In all cases studied to date, this defect maps to a mutation of the phosphatidylinositolglycan class A (PIG-A) structural gene. We therefore amplified the PIG-A gene from both the GPI-negative B and T cells by PCR and determined the nucleotide sequence. No differences from the wild-type sequence were detected; therefore a classical PNH mutation cannot be responsible for the GPI-biosynthesis defect in these cell lines. Significantly, the GPI-negative phenotype of the B cells was reversible upon separation of the positive and negative cells, resulting in a redistribution to a mixed population with either CD52-positive or -negative cells, whereas populations of 100% CD52-negative T cells were stably maintained during culture. Therefore, whereas the GPI-biosynthesis deficiency in the T cell lines may be due to a mutation in another gene required by the GPI-biosynthetic pathway, the reversible nature of this block in the B cell lines suggests a less direct cause, possibly an alteration in a regulatory factor. Overall, these data demonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism for the development of GPI deficiency.
Background: Interleukin-1 beta (IL-1b) is a key mediator of the inflammatory response and is known to exacerbate damage during chronic disease and acute tissue injury. Through association with the adaptor protein Myd88, interleukin receptor associated kinases (IRAK)1 and 4 initiate signaling downstream of IL-1Rs resulting in the activation of the NFkB and MAPK pathways and the production of proinflammatory cytokines (1). IL-1Rs are broadly expressed across cell types and little is known about differences in signaling between cell types and the role of IRAK1 and IRAK4 kinase activity. Objectives: We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recent phase 1 study. The aim of this study was to evaluate the effect of R835 on IL-1R signaling in primary human fibroblasts and endothelial cells. Methods: Human dermal fibroblasts, lung fibroblasts or endothelial cells were stimulated with IL-1b and the effect of R835 on the signaling pathway was evaluated by western blotting. Human dermal fibroblasts were stimulated with different amounts of IL-1b to evaluate both the signaling pathways activated and the cytokines produced. The ability of R835 to inhibit cytokine production induced by high or low amounts of IL-1b in dermal fibroblasts was assessed. Results: In human endothelial cells, inhibition of IRAK1/4 kinases with R835 resulted in a block of IL-1b-induced IRAK4 phosphorylation, IRAK1 degradation and downstream NFkB, p38 and JNK activation. In contrast, in both human primary dermal and lung fibroblasts stimulated with IL-1b, we observed potent inhibition of IRAK4 phosphorylation, IRAK1 degradation, and downstream JNK phosphorylation, but no inhibition of NFkB pathway proteins and only weak inhibition of p38. Upon titration of IL-1b we observed that dermal fibroblasts produced IL-8 and GRO in response to low levels of IL-1b (20pg/ml), and produced additional cytokines including G-CSF and GM-CSF with higher levels of IL-1b (400pg/ml). In the presence of low levels of IL-1b (20pg/ml), we observed a weak activation of NFkB pathway proteins and p38, compared to a very robust NFkB, p38 and additional JNK activation in the presence of higher levels of IL-1b (400pg/ml). Consistent with these results, in dermal fibroblasts, R835 showed little to no inhibition of IL-8 and GRO induced by low levels of IL-1b, but potently inhibited G-CSF and GM-CSF induced by high levels of IL-1b where JNK was activated. Conclusion: This study has elucidated signaling differences between cell types downstream of the IL-1R. In endothelial cells, as in myeloid cells, the kinase activity of IRAK1 and IRAK4 is required for the activation of all downstream signaling. Unexpectedly, in human fibroblasts, IRAK1/4 kinase activity appears to primarily regulate the JNK pathway, and not the NFkB pathway. Concomitant with that, only the cytokines induced by the additional activation of JNK in fibroblasts are regulated by a dual IRAK1/4 inhibitor. Clinically, an IRAK1/4 inhibitor may show select inhibition of IL-1b-induced cytokines depending on the tissue and cell type involved in inflammation. References: [1]Flannery S, Bowie A G. The interleukin-1 receptor-associated kinases: Critical regulators of innate immune signaling. Biochemical Pharmacology, Volume 80, Issue 12, 15 December 2010, Pages 1981-1991. Disclosure of Interests: Sylvia Braselmann Shareholder of: Shareholder of Rigel Pharmaceuticals, Employee of: Employee of Rigel Pharmaceuticals, Ernest Tai Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Roy Frances Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Chi Young Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vadim Markovtsov Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Esteban Masuda Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals
For 10 years, the refugee now known by the pseudonym XFJ attempted to gain accreditation to drive a taxi-cab. After many internal reviews and rejections by the Victorian Taxi Directorate, XFJ appealed to the Victorian Civil and Administrative Tribunal (VCAT). The difficulty for XFJ was that shortly after arriving in Australia, he had killed his estranged wife. The Supreme Court of Victoria subsequently found him not guilty of murder on the grounds of insanity. Since XFJ's mental health has been stable for many years, much of the legal argument at VCAT and the court cases that followed centred around whether he was "suitable in other respects to provide the service" of driving a taxi, as required by s 169(1)(b)(ii) of the Transport Act 1983 (Vic). This article looks at the tension between the expert medical evidence and the concept of "suitable in other respects" which XFJ's opponents claimed included the maintenance of public confidence and the meeting of community expectations.
Background: Interleukin receptor associated kinases (IRAK) 1 and 4 are kinases involved in Toll-Like Receptor (TLR) and Interleukin-1 Receptor (IL-1R) signaling pathways, which regulate innate immunity and inflammation. Dysregulation of IRAK1/4 signaling can lead to a variety of inflammatory conditions including rheumatoid and gouty arthritis. As a result, IRAK1/4 are promising therapeutic targets for rheumatic diseases (1). We have identified a potent and selective IRAK1/4 inhibitor, R835, that substantially suppressed the elevation of LPS (TLR4 agonist)-induced serum cytokines in healthy human volunteers in a recently completed phase 1 study. Objectives: The aim of our study was to investigate the effect of IRAK1/4 selective inhibition as a potential therapeutic approach for rheumatological diseases. We evaluated the inhibition by our clinical candidate, R835, on TLR-, IL-1R- and NLRP3 inflammasome-induced cytokine production, as well as in preclinical models of arthritis. Methods: The effect of R835 on TLR- or IL-1R-induced cytokine production was evaluated in vitro using THP-1, human primary endothelial cells and human primary dendritic cells. The activity of R835 on the NLRP3 inflammasome was also tested in vitro using THP-1 cells. The pharmacokinetic-pharmacodynamic relationship of R835 was evaluated in a mouse model of IL-1b-induced cytokine release. Mice were pre-treated orally with vehicle or R835 prior to challenge; serum cytokine and plasma compound levels were determined. The efficacy of IRAK1/4 inhibition by R835 in rodent models of joint inflammation was evaluated in a mouse model monosodium (MSU)-induced peritonitis, in rat model of MSU-induced gouty arthritis and in a rat model of collagen-induced arthritis (CIA). Results: In human cells, R835 blocked proinflammatory cytokine production in response to TLR, IL-1R and NLRP3 inflammasome activation. In mice, R835 dose-dependently decreased serum cytokines in response to administration of IL-1b. Mice pre-treated with R835 demonstrated dose-dependent reductions in MSU crystal-induced serum and peritoneal cytokine levels, as well as neutrophil influx in the peritoneal cavity. Prophylactic and therapeutic treatment with R835 also resulted in significant inhibition of MSU crystal-induced knee edema and pain in a rat model of human gouty arthritis. In the rat model of CIA, R835 blocked both onset and progression of disease, by reducing inflammation, cartilage degeneration and synovial inflammation. Conclusion: R835 is a promising clinical candidate for the treatment of a range of cytokine-driven rheumatological diseases. R835 has proven to have desirable pharmacokinetic properties, was well tolerated and suppressed LPS-induced serum cytokines in healthy volunteers in a recent phase 1 study. References: [1]Bahia M S, Kaur M, Silakari P, Silakari O. Interleukin-1 receptor associated kinase inhibitors: Potential therapeutic agents for inflammatory- and immune-related disorders. Cellular Signalling 27 (2015) 1039–1055. Disclosure of Interests: Chrystelle Lamagna Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Meagan Chan Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Ernest Tai Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Stacey Siu Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Roy Frances Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Sothy Yi Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Chi Young Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vadim Markovtsov Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Yan Chen Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Lu Chou Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Gary Park Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Esteban Masuda Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals, Vanessa Taylor Shareholder of: Rigel Pharmaceuticals, Employee of: Rigel Pharmaceuticals
The unexpected transmission of donor-derived infection through organ transplantation is a rare event with current donor screening practices. In this case report we describe a probable donor-derived transmission of Herpes Simplex Virus (HSV)-2 via deceased donor kidney transplantation resulting in HSV hepatitis in the recipient. This manifested as acute liver failure which resolved with appropriate anti-viral therapy. Following recovery from the acute liver insult, the patient developed fibrotic liver morphology and portal hypertension, an unusual departure from the typical course.
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