Search

Background Primary Sjogren’s syndrome (pSS) is a systemic and progressive autoimmune disease characterised by lymphoid infiltration and progressive alteration of exocrine glands secretory function. Ectopic germinal center-like structures harbour plasma cells that generate autoantibodies leading to immune complex formation. Leniolisib (CDZ173) is an oral low molecular weight compound that selectively inhibits the lipid kinase PI3Kδ. In animals, leniolisib blocks PI3Kδ-dependent B cell functions, disrupts germinal centre formation and immune cell trafficking, supporting the rationale for a PI3Kδ–targeted therapy in pSS Objectives To evaluate the safety, tolerability, pharmacokinetics (PK) and preliminary clinical efficacy of multiple oral doses of leniolisib, after 12 weeks of treatment, in pSS patients Methods Double-blind, randomised, placebo-controlled, parallel-design study recruited 30 seropositive pSS patients with moderate to severe disease activity as determined by EULAR Sjogren’s Syndrome Disease Activity Index (ESSDAI)≥6, EULAR Sjogren‘s Syndrome Patient Reported Index (ESSPRI)≥5 and stimulated whole salivary flow rate >0 mL/min. Patients were randomised in a 2:1 ratio to receive leniolisib (70 mg b.i.d) or placebo. The primary outcome was change in ESSPRI at week 12. Secondary outcomes included PK, changes in ESSDAI, the Short Form-36 (SF-36), Multidimensional Fatigue Inventory (MFI) and global Visual Analogue Scales (VAS) completed by patients and physicians. Additional assessments included lacrimal gland function and biomarkers relevant to pathway and disease Results Overall safety and tolerability profile of leniolisib was acceptable, but appeared less favourable than placebo. In particular, rash occurred more frequently in the leniolisib group (11/20 patients) compared to placebo (1/10 patients). There was a slight improvement (not statistically significant) in ESSPRI scores (dryness, pain and fatigue) favouring leniolisib. Similar trends were observed in secondary endpoints (SF-36/mental and physical, MFI, VAS completed by patients and physicians). After 12 weeks of treatment, there was a slight improvement (not statistically significant) in the lacrimal gland function in the leniolisib group compared to placebo. The observed PK profile was as expected based on healthy volunteer data. Biomarker results suggest a strong and sustained target and pathway engagement, as evidenced by inhibition of phosphorylated Akt in ex-vivo stimulated B cells, significant decrease in serum CXCL13 and reduced frequency of circulating Follicular T helper-like cells. There was a trend of decreasing autoantibody levels in leniolisib-treated patients Conclusions Leniolisib had an acceptable safety and tolerability profile, but caused rashes a known class effect of PI3K inhibitors. Target and pathway engagement were confirmed, however no clear efficacy signal for leniolisib was seen based on ESSPRI and ESSDAI in this Proof-of-Concept study at the studied dose. Disclosure of Interest T. Dorner: None declared, M. Zeher: None declared, U. Laessing: None declared, F. Chaperon: None declared, S. De Buck: None declared, A. Hasselberg: None declared, M.-A. Valentin Shareholder of: Novartis stock option, Employee of: Novartis, S. Ma: None declared, M. Cabanski: None declared, C. Kalis: None declared, C. Burkhart: None declared, P. Gergely: None declared

Pathogenic gain-of-function variants in the genes encoding phosphoinositide 3-kinase δ (PI3Kδ) lead to accumulation of transitional B cells and senescent T cells, lymphadenopathy, and immune deficiency (activated PI3Kδ syndrome [APDS]). Knowing the genetic etiology of APDS afforded us the opportunity to explore PI3Kδ inhibition as a precision-medicine therapy. Here, we report in vitro and in vivo effects of inhibiting PI3Kδ in APDS. Treatment with leniolisib (CDZ173), a selective PI3Kδ inhibitor, caused dose-dependent suppression of PI3Kδ pathway hyperactivation (measured as phosphorylation of AKT/S6) in cell lines ectopically expressing APDS-causative p110δ variants and in T-cell blasts derived from patients. A clinical trial with 6 APDS patients was conducted as a 12-week, open-label, multisite, within-subject, dose-escalation study of oral leniolisib to assess safety, pharmacokinetics, and effects on lymphoproliferation and immune dysregulation. Oral leniolisib led to a dose-dependent reduction in PI3K/AKT pathway activity assessed ex vivo and improved immune dysregulation. We observed normalization of circulating transitional and naive B cells, reduction in PD-1+CD4+ and senescent CD57+CD4− T cells, and decreases in elevated serum immunoglobulin M and inflammatory markers including interferon γ, tumor necrosis factor, CXCL13, and CXCL10 with leniolisib therapy. After 12 weeks of treatment, all patients showed amelioration of lymphoproliferation with lymph node sizes and spleen volumes reduced by 39% (mean; range, 26%-57%) and 40% (mean; range, 13%-65%), respectively. Thus, leniolisib was well tolerated and improved laboratory and clinical parameters in APDS, supporting the specific inhibition of PI3Kδ as a promising new targeted therapy in APDS and other diseases characterized by overactivation of the PI3Kδ pathway. This trial was registered at www.clinicaltrials.gov as #NCT02435173.

Inherited gain-of-function mutations in PIK3CD encoding PI3Kδ (phosphoinositide 3-kinase delta) lead to accumulation of senescent T cells, lymphadenopathy, splenomegaly and immune-deficiency (Activated PI3Kδ Syndrome, APDS). Clinical manifestations include multilineage cytopenias and susceptibility to B cell Non-Hodgkins Lymphoma. We recently reported use of leniolisib (CDZ173), a novel, potent and selective oral PI3Kd inhibitor in six APDS patients in a 12-week, open-label, multi-center, within-subject dose-escalation study (Rao et al, Blood 23 November 2017). Leniolisib was safe and well tolerated and led to a dose-dependent reduction in PI3K/AKT pathway activity and improved the immune dysregulation with normalization of circulating transitional and naïve B cells and reduction in PD-1+CD4+ and senescent CD57+CD8+ T cells. Elevated serum IgM and other biomarkers including IFNg, TNF, CXCL13 and CXCL10 normalized or decreased with leniolisib. Cytopenias and lymphoproliferation improved in all patients with index lymph node sizes and spleen volumes reduced by 39% and 40%, respectively. Here we report the long term follow up safety and efficacy data in all six patients (Table 1) who have continued treatment with leniolisib in an extension study at 70 mg b.i.d (ClinicalTrials.gov number NCT02859727). These patients have been treated for up to 949 days (as of July 20th 2018). Three of them were lymphoma survivors. Three patients were treated with rapamycin prior to starting leniolisib. No patient in the 12-week clinical trial or in the long term extension study has experienced any significant adverse events. Notably, side effects prevalent with mTOR or other PI3K inhibitors such as diarrhea/colitis, skin rashes, susceptibility to infections or liver enzyme elevation were absent. Three patients have stopped immunoglobulin supplementation as a reflection of the normalization of their B cell function. Cytopenias, including anemia, thrombocyopenia, neutropenia and lymphopenia have continued to improve. (Figure 1: Showing representative improvement of laboratory data including normalization of IgM for patient 002, following 18 months of treatment with leniolisib; Figure2: Showing improvement of lymphoproliferation in CT scans from 2016 and 2018 for Patient 003. This patient stopped leniolisib for 15 months from October 2016 until January 2018 leading to worsening lymphoproliferation that improved promptly on resumption of therapy). Available data on longterm safety, effects on lymphoproliferation and immune dysregulation in the ongoing leniolisib extension study will be shared during the meeting. The results support safe and efficacious long term inhibition of PI3Kδ protein as a new targeted therapeutic approach in APDS and other disorders of nonmalignant lymphopoliferation associated with hyperactive PI3Kinase pathway. Disclosures Rao: novartis: Research Funding. Dalm:Novartis: Research Funding. Sediva:Novartis: Research Funding. van Hagen:Novartis: Research Funding. Cabanski:Novartis: Employment. Valentin:Novartis: Employment. de Buck:Novartis: Employment. Kalis:Novartis: Employment. Hasselberg:Novartis: Employment. Burkhart:Novartis: Employment. Kucher:Novartis: Employment. Sloth:Novartis: Employment. Uzel:Novartis: Research Funding.

Allergies to nickel (Ni(2+)) are the most frequent cause of contact hypersensitivity (CHS) in industrialized countries. The efficient development of CHS requires both a T lymphocyte-specific signal and a proinflammatory signal. Here we show that Ni(2+) triggered an inflammatory response by directly activating human Toll-like receptor 4 (TLR4). Ni(2+)-induced TLR4 activation was species-specific, as mouse TLR4 could not generate this response. Studies with mutant TLR4 proteins revealed that the non-conserved histidines 456 and 458 of human TLR4 are required for activation by Ni(2+) but not by the natural ligand lipopolysaccharide. Accordingly, transgenic expression of human TLR4 in TLR4-deficient mice allowed efficient sensitization to Ni(2+) and elicitation of CHS. Our data implicate site-specific human TLR4 inhibition as a potential strategy for therapeutic intervention in CHS that would not affect vital immune responses.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam 3 CSK 4 and MALP-2, on Ag (DNP–HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam 3 CSK 4 , but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam 3 CSK 4 , most probably by electrostatic forces, reduced the level of active DNP–HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE + Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam 3 CSK 4 indicate that one must be cautious when interpretating results obtained with “model” substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcɛR1) and TLR2 are examined.

The innate immune system is essential for host defense; it senses the presence of potentially pathogenic-invading microorganisms, and the contribution of Toll-like receptors (TLRs) to this response is increasingly recognized. In the present study, we investigated the contribution of TLR4 to the course of cutaneous leishmaniasis in vivo. We used C57BL/10ScNCr (TLR40/0) and C57BL/10ScCr [TLR4/interleukin-12 (IL-12)Rβ20/0] mice and compared the course of Leishmania major infection, parasite load, cell recruitment, and cytokine profile with those of wild-type C57BL/10ScSn mice. Our results confirm the importance of IL-12 receptor-mediated signaling in resistance to L. major infections. Importantly, we show that the lack of TLR4 results in an increased permissiveness for parasite growth during the innate and adaptive phase of the immune response and in delayed healing of the cutaneous lesions. The use of the tlr4 transgenic mouse strain TCr5 demonstrated unequivocally that TLR4 contributes to the efficient control of Leishmania growth in vivo.

The innate immune response to Gram-negative bacteria depends mainly on the ability of the host to respond to the LPS component. Consequently, the state of LPS sensitivity at the time of infection and the numbers of invading bacteria (i.e. the amounts of LPS) are primary factors determining the innate responses provoked by Gram-negative pathogens. LPS sensitivity increases following treatment of mice with live or killed micro-organisms. Two types of sensitization have been recognized, strong, IFN-gamma-dependent and moderate IFN-gamma-independent. IL-12 and IL-18 are intimately involved in the induction of IFN-gamma by bacteria. We showed that Gram-negative bacteria induce IFN-gamma in mice also by an IFN-beta-dependent pathway that requires IL-18 and is independent of IL-12 signaling. This pathway is STAT4 dependent, the activation of which is directly linked to IFN-beta. Further, IFN-beta can be replaced by IFN-alpha. While different components of Gram-negative bacteria induce IL-12 and IL-18, LPS seems to be the only component in these bacteria capable of inducing IFN-beta. Therefore, the IFN-beta pathway of IFN-gamma induction, unlike the IL-12 pathway, proceeds only in LPS responder mice. The IFN-alpha/beta-dependent pathway is expected to play a role whenever IFN-alpha or IFN-beta, and IL-18 are produced concomitantly during infection.

The contribution of murine Toll-like receptors 2 and 4 (TLR2 and -4, respectively) to cytokine induction by heat-killed bacteria was analyzed in vitro and in vivo. Gram-negative bacteria induced cytokines primarily via TLR4; the contribution of TLR2 was only minor. Neither TLR4 nor, surprisingly, TLR2 was required in the MyD88-dependent response toStaphylococcus aureus.

C57BL/10ScCr (Cr) mice carry a deletion of the Toll-like receptor 4 (tlr4) gene (i.e. they are tlr4(0/0)) and are thus refractory to LPS effects. Insertion of wild-type tlr4 transgene into the tlr4(0/0) Cr germ line endowed LPS susceptibility in the two transgenic lines created, indicating that TLR4 is the only limiting factor for LPS responsiveness in Cr mice. The absolute levels of tlr4 mRNA expressed by the heterozygous transgenic (tlr4(Tr/0)), wild-type C57BL/10ScSn (Sn) (tlr4(+/+)) and heterozygous F1 (Sn x Cr) (tlr4(+/0)) mice varied markedly. However, the pattern of distribution of expression in the different organs was the same in all strains. In different biological assays (B cell mitogenicity, cytokine induction and lethal toxicity) the degree of LPS response obtained in the different strains of mice correlated with the levels of tlr4 mRNA expression. In macrophages, investigation of the LPS-induced cytokine (IL-6) response revealed a linear relationship between the response and the logarithm of TLR4-MD-2 levels.

The liver plays an important physiological role in lipopolysaccharide (LPS) detoxification and, in particular, hepatocytes are involved in the clearance of endotoxin of intestinal derivation. In experimental shock models, tumor necrosis factor (TNF)-alpha induces hepatocyte apoptosis and lethal effects are due to secreted TNF-alpha and not to cell-associated TNF-alpha. An exaggerated production of TNF-alpha has been reported in murine viral infections, in which mice become sensitized to low amounts of LPS and both interferon (IFN)-gamma and IFN-alpha/beta are involved in the macrophage-induced release of TNF-alpha. The prominent role of LPS and TNF-alpha in liver injury is also supported by studies of ethanol-induced hepatic damage. In humans, evidence of LPS-induced hepatic injury has been reported in cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis and a decreased phagocytic activity of the reticulo-endothelial system has been found in these diseases. The origin of endotoxemia in hepatitis C virus (HCV) infected patients seems to be multifactorial and LPS may be of exogenous or endogenous derivation. In endotoxemic HCV-positive patients responsive to a combined treatment with IFN-alpha/ribavirin (RIB), endotoxemia was no longer detected at the end of the therapeutic regimen. By contrast, 48% of the non-responders to this treatment were still endotoxemic and their monocytes displayed higher intracellular TNF-alpha and interleukin (IL)-1beta levels than responders. Moreover, in responders, an equilibrium between IFN-gamma and IL-10 serum levels was attained. In the non-responders, serum levels of IL-10 did not increase following treatment. This may imply that an imbalance between T helper (Th)1 and Th2 derived cytokines could be envisaged in the non-responders.

Lipopolysaccharide is an important recognition marker by virtue of which the innate immune system senses and reacts against Gram-negative bacteria invading the LPS susceptible host. This review deals with the factors affecting LPS susceptibility and with the role of the latter in the course and outcome of Salmonella typhimurium infection.

Use of a synergistic effect of DMSO together with a chaotropic salt (NaSCN or MgCl 2 ) allowed to drastically reduce matrix interferences in an ELISA for therapeutic monoclonal antibodies. Optimum combinations were found to be 0.4 M NaSCN together with 10.0% DMSO, and 1.0 M MgCl 2 with 15.0% DMSO. At this optimum combination, quality controls spiked with mAb at 50.0 ng/ml in eighteen individual human sera and plasmas were quantified with an overall accuracy of 102.0%. All of these QCs fulfilled the acceptance criteria of 80.0–120.0% accuracy and precision below 20.0%. The assay was also successfully applied to the quantification of two other mAbs in human serum. Furthermore, the use of the assay was extended to pre-clinical species (cynomolgus monkey and rat serum). Here, the performed validation experiments confirmed the utility of the assay and demonstrated that the assay allowed quantification of mAb from 50.0 ng/ml to 100.0 μg/ml in cynomolgus monkey serum. The method has then been applied to a pharmacokinetic study in cynomolgus monkeys. In summary, this work demonstrates the efficacy of the combination of a chaotropic salt with DMSO to minimize matrix interferences in an ELISA. The robustness thus obtained allowed the successful establishment of a cost effective, target-based ELISA format for use in pharmacokinetic studies, that is easily applicable for the quantification of mAbs in various matrices such as human, cynomolgus monkey or rat serum and plasma.

Little is known about the interplay between pathophysiological processes of allergy and infection, particularly with respect to mast cell (MC)-mediated responses. The presence and recognition of pathogen-associated molecular patterns (PAMPs) might have broad impact on the development and severity of diseases. In this study, we assessed the influence of toll-like receptor 2 (TLR 2)-dependent synthetic analogs of bacterial lipopeptides (LPs), Pam(3)CSK(4) and MALP-2, on Ag (DNP-HSA)-triggered responses in bone marrow-derived MCs (BMMCs). Both LPs strongly synergized with sub-optimal amounts of Ag in the stimulation of cytokine release. Intriguingly, Pam(3)CSK(4), but not MALP-2 suppressed Ag-induced degranulation of BMMCs (together with early tyrosine phosphorylation and calcium mobilization) in a TLR2-independent manner. Further analysis revealed that Pam(3)CSK(4), most probably by electrostatic forces, reduced the level of active DNP-HSA and that this, in turn, was responsible for the suppression of Ag-induced degranulation. Thus, our work demonstrates that LPs can synergize with IgE+Ag in stimulating the production of IL-6 by BMMCs. As well, our findings with Pam(3)CSK(4) indicate that one must be cautious when interpretating results obtained with "model" substances and the combination of ligands must be carefully chosen when functional interactions between the high-affinity receptor for IgE (FcepsilonR1) and TLR2 are examined.

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-alpha levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.

The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.

IFN-αβ is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections. In this study, we describe an as yet undefined IFN-αβ-dependent pathway of IFN-γ induction in mice. This pathway is based on a synergism of IFN-αβ and IL-18, and is independent of IL-12 signaling yet dependent on STAT4. In contradiction to current dogma, we show further that IFN-αβ alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains. This pathway participates in the induction of IFN-γ by Gram-negative bacteria and is therefore expected to play a role whenever IFN-α or IFN-β and IL-18 are produced concomitantly during bacterial, viral, or other infections.