Cellulose, mostly in the form of toilet paper, forms a major component of the particulates in raw municipal wastewater, which could lead to significant consequences due to the potential accumulation of cellulosic fibers and slow biodegradability. Despite the sparse reports on cellulose content and degradation in wastewater and sludge, an accurate and validated method for its quantification in such matrices does not exist. In this paper, four different methods were compared including dilute acid hydrolysis, concentrated acid hydrolysis, enzymatic hydrolysis, and the Schweitzer reagent method. The Schweitzer reagent method, applied to municipal wastewater and sludge, was found to be a very robust and reliable quantification method in light of its reproducibility, accuracy, and ideal (100%) recovery. The determination of cellulose content is critical to understand its fate in wastewater treatment plants as well as improve sludge management and enhance resource recovery.

Background Overexpression of B cell activating factor (BAFF) contributes to the pathogenesis of primary Sjögren9s syndrome (pSS) [1]. Treatment of pSS patients with VAY736, an anti-human BAFF receptor mAb, appears promising and was associated with a positive therapeutic effect [2]. Given the complexity and heterogeneity of pSS, there is a need to further identify molecular mechanisms involved in pSS and in response to new therapeutics. Objectives To address this question, we assessed a panel of biomarkers in 27 patients from a clinical trial and tested their associations with pSS activity and clinical response to VAY736. Methods This study comprised 27 pSS patients treated with a single intravenous dose of VAY736 at 10 mg/kg (n=12), 3 mg/kg (n=6), or placebo (n=9). The disease activity scores included EULAR Sjogren9s Syndrome Disease Activity Index (ESSDAI) and Patient Reported Index (ESSPRI), patient9s and physician9s reported visual analog scales (VAS), Short-Form 36 and Multidimensional Fatigue Inventory (MFI). BAFF and a panel of chemokines in serum and saliva were assessed using immunoassays. Circulating B cells and B cell subsets were measured by flow cytometry. High frequency ultrasound (US) of the parotid and sub-mandibular glands measured gland thickness and quality using a 4 point scoring (de Vita) [3]. Shear wave elastography of the parotid glands was also measured. All biomarkers were measured at baseline (BL) and post-treatment w6, w12, w24. The effect of VAY736 on biomarker levels was assessed by descriptive statistics. Correlations between biomarkers and disease activity scores were calculated at BL and w6, w12, and w24 using levels and relative changes from BL. Results In addition to B cell depletion, serum BAFF increase, and improvements in US and elastography measures [2], a subset of serum chemokine tended to be reduced nine weeks after VAY736 treatment. Pooling all 27 patients, salivary BAFF levels correlated with parotid De Vita scores at BL (left: r=0.75, right: r=0.72, p<10-4 for both) and w6 (left: r=0.72,; right: r=0.78, p<10-4 for both) but not at later time points. Pooling the 18 VAY736 treated patients, increase in salivary BAFF correlated with decrease in MFI at w6 (r=-0.83, p=3×10-4) and high levels of one of the serum chemokines at BL correlated with decrease in ESSPRI at w24 (r=-0.76, p=3×10-4). In the same patients, the B cell count at BL correlated with changes in several clinical outcomes at w12: ESSPRI (r=-0.65, p=0.01), Physician9s VAS (r=-0.6, p=0.01), shear wave (r=-0.63, p=0.02), and parotid thickness (r=-0.6, p=0.03). Conclusions We identified a set of markers correlated with clinical outcomes in pSS after treatment with VAY736, which have the potential to provide additional insight in pSS and treatment-modifying effects. Further large-scale studies are necessary to confirm the value of these markers. References Varin MM et al. Autoimmun Rev 2010;9(9):604–8. Dörner T et al. Arthritis Rheum 2016; 68(suppl S10):4051. De Vita S, et al. Clin Exp Rheumatol 1992;10:351–6. Disclosure of Interest J. Doucet Employee of: Novartis AG, R. Kazma Employee of: Novartis AG, M. Cabanski Employee of: Novartis AG, E. Kamphausen Employee of: Novartis AG, P. Maguire Employee of: Novartis AG, A. Avrameas Employee of: Novartis AG, M.-A. Valentin: None declared, Y. Li Employee of: Novartis AG, A. Auger-Sarrazin Employee of: Novartis AG, S. Kaiser Employee of: Novartis AG, P. Follet Employee of: Novartis AG, S. Oliver Employee of: Novartis AG, A. Vitaliti Employee of: Novartis AG

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.

Pathogenic gain-of-function variants in the genes encoding PI3Kδ (phosphoinositide 3-kinase delta) 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 a dose-dependent suppression of PI3Kδ pathway hyperactivation (measured as phosphorylation of AKT/S6) in cell lines ectopically expressing APDScausative p110δ variants and in T cell blasts derived from patients. A clinical trial with six APDS patients was conducted as a 12-week, open-label, multi-site, within-subject dose-escalation study of oral leniolisib to assess safety, pharmacokinetics and effects on lymphoproliferation and immune dysregulation. Oral leniolisib led to a dosedependent reduction in PI3K/AKT pathway activity assessed ex vivo and improved immune dysregulation. We observed normalization of circulating transitional and naïve B cells, reduction in PD-1+CD4+ and senescent CD57+CD4T cells, and decreases in elevated serum IgM and inflammatory markers including IFNγ, TNF, 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. The study is registered at ClinicalTrials.gov, number NCT02435173.

Background Overexpression of B cell activating factor (BAFF) in salivary glands contributes to the pathogenesis of primary Sjögren9s syndrome (pSS) by promoting autoantibody (AAB) production. Treatment of pSS patients with VAY736, an anti-BAFF receptor mAb, appears promising and was associated with a depletion of circulating B cells and a positive therapeutic effect [1]. In addition to the classical anti-SS-A/Ro and anti-SS-B/La, a broader set of AABs may reflect B cell disturbances in pSS and could serve as markers during clinical development of novel pSS therapeutics. Objectives In this study, we explored novel AABs in pSS patients and healthy controls (HCs) and we tested their associations with the disease, disease activity, and clinical response to VAY736. Methods Reactivity of AABs to 1,596 antigens was measured in serum samples from 27 pSS patients from a placebo-controlled trial at baseline and post-treatment week 12 and from 50 age and gender-matched HCs. Patients were treated at baseline with a single dose of VAY736 at 10 mg/kg (n=12), 3 mg/kg (n=6), or placebo (n=9). First, to identify AABs associated with pSS, 3 different methods compared AAB levels at baseline between pSS patients and HCs: Wilcoxon rank sum test, significance analysis of microarrays, and comparison of the 90th quantiles between groups. Second, to identify AABs associated with pSS activity, Pearson correlation of AABs with EULAR Sjögren9s Syndrome Disease Activity Index, secondary outcomes, and salivary and serum BAFF were tested, using baseline and week 12 levels as well as relative changes. Third, VAY736 treatment-specific changes in AAB levels were investigated using linear mixed-effects models adjusting for dosage, age, and gender effects. Results Of 1,596 antigens, 36 were statistically different between pSS patients and HCs for at least one of the 3 tests, including the known SS-A/Ro and SS-B/La (significant for all 3 tests) as well as novel antigens. SS-A/Ro and SS-B/La AABs were not associated with disease activity or response to treatment. However, 48 AABs were significantly correlated with pSS activity combining all treatment arms, and 12 AABs had baseline values that correlated with change in pSS activity upon VAY736 treatment (unadjusted p<0.05). Interestingly, 51 serum AABs correlated with BAFF saliva levels (|r|>0.55), but not with BAFF serum levels. The genes encoding novel antigens are involved in apoptotic, anti-viral, metabolic, inflammatory, blood coagulation and B-cell processes, suggesting a possible link to the disease pathology. Finally, there was no reduction in AABs levels in response to VAY736, possibly because the 12 weeks post-treatment analysis was too short to identify large effects. Conclusions In conclusion, we identified new AABs in pSS patients that have the potential to serve as markers of diagnosis, pSS activity, or as predictors of clinical outcome measures. Further large-scale studies are needed to confirm the value of these markers. References Dörner T et al. Arthritis Rheum 2016; 68(suppl S10):4051. Disclosure of Interest P. Budde Employee of: Protagen AG, J. Doucet Employee of: Novartis AG, H.-D. Zucht Employee of: Protagen AG, R. Kazma Employee of: Novartis AG, P. Maguire Employee of: Novartis AG, A. Avrameas Employee of: Novartis AG, M.-A. Valentin Employee of: Novartis AG, S. Oliver Employee of: Novartis AG, P. Schulz-Knappe Employee of: Protagen AG, A. Vitaliti Employee of: Novartis AG

Pathogenic gain-of-function variants in the genes encoding phosphoinositide 3-kinase delta (PI3K delta) lead to accumulation of transitional B cells and senescent T cells, lymphadenopathy, and immune deficiency (activated PI3K delta syndrome [APDS]). Knowing the genetic etiology of APDS afforded us the opportunity to explore PI3K delta inhibition as a precision-medicine therapy. Here, we report in vitro and in vivo effects of inhibiting PI3K delta in APDS. Treatment with leniolisib (CDZ173), a selective PI3K delta inhibitor, caused dosedependent suppression of PI3Kd pathwayhy peractivation (measured as phosphorylation of AKT/S6) in cell lines ectopically expressing APDS-causative p110 delta 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 naiveBcells, reduction in PD-11 CD41 and senescent CD57(+) CD4(-) T cells, and decreases in elevated serum immunoglobulin M and inflammatory markers including interferon gamma, 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 PI3Kd as a promising new targeted therapy in APDS and other diseases characterized by overactivation of the PI3Kd pathway.

Background VAY736 is a novel, defucosylated, human IgG1 mAb targeting the receptor for B cell activating Factor of the TNF family (BAFF-R), providing both enhanced antibody-dependent cellular cytotoxicity (ADCC)-mediated depletion of B cells and blockade of BAFF:BAFF-R signaling that drives B cell differentiation, proliferation and survival. Objectives To clinically evaluate the clinical efficacy of the dual mechanisms of action of VAY736 in patients with pSS, a highly BAFF-driven, systemic autoimmune disease involving lymphocytic infiltration and progressive dysfunction of exocrine glands along with various extra-glandular manifestations. Methods A single center, randomized, parallel group, double-blind, placebo-controlled trial recruited 27 seropositive pSS patients with EULAR Sjögren9s Syndrome Disease Activity Index (ESSDAI) ≥6 and residual salivary flow over a 10-month period and randomized for treatment with intravenous VAY736 at either a single high dose, (n=12), a single lower dose (n=6), or with placebo (n=9). Outcomes were measured at baseline and at weeks 6, 12 and 24. The primary outcome was change in ESSDAI at week 12. Secondary outcomes included the EULAR Sjögren9s Syndrome Patient Reported Index (ESSPRI), Short Form-36 (SF-36), Multidimensional Fatigue Inventory (MFI) and global VAS assessments. Additional outcomes included salivary flow rates, Ocular Staining Score (OSS), high resolution salivary gland ultrasound (US) de Vita scores, serum markers of B cell hyperactivity and flow cytometry-determined lymphocyte subsets. Results Data analyses included all patients. VAY736 was safe and well-tolerated with no drug-related SAE, drop outs or discontinuations. Mean age was 50.5 years with 4 males, 2 in placebo and 1 in each treated arm. Baseline mean ESSDAI scores (range) were 11.5 (6–18), 14.5 (6–31) and 11.1 (6–19) in the high dose, lower dose and placebo arms, respectively. Rapid, profound depletion of circulating B cells was observed in all VAY736-treated patients. The primary endpoint of ESSDAI was reduced within 12 weeks but did not reach clinical or statistical significance. Improvements in VAY736-treated subjects were seen across the clinical secondary outcome measures, particularly for scores of patient and physician global assessments and SF-36 physical. Of note, the higher dose group had more sustained effects on clinical outcomes (e.g., ESSPRI, MFI) at weeks 6 and 12, while maximal effects in the lower dose group were more evident at the earlier week 6 time point, suggesting reduced VAY736 tissue exposure in some patients by week 12. PD measurements (serum BAFF levels, circulating B cells) confirmed target engagement. A trend in improvement of US scores occurred in the high dose group. There were no consistent changes in salivary flow rate or OSS. Analyses of additional biomarkers are pending. Conclusions Despite a limited, single infusion, VAY736 achieved in this early phase trial trends in the primary outcome and across all secondary outcomes. Thus, this treatment was safe and suggests a positive therapeutic effect for this dual mechanisms of action in pSS that warrant further evaluation. Disclosure of Interest T. Dörner: None declared, M. Posch: None declared, F. Wagner: None declared, A. Hüser: None declared, T. Fischer: None declared, L. Mooney Employee of: Novartis Pharma AG, O. Petricoul Employee of: Novartis Pharma AG, P. Maguire Employee of: Novartis Pharma AG, P. Pal Employee of: Novartis Pharma AG, J. Doucet Employee of: Novartis Pharma AG, M. Cabanski Employee of: Novartis Pharma AG, E. Kamphausen Employee of: Novartis Pharma AG, S. Oliver Employee of: Novartis Pharma AG

A novel sandwich ELISA for the quantitative and sensitive determination of IL-13 in human serum and plasma was established. The assay employs an incubation step at acidic pH, which was shown to decrease nonspecific binding and interference from IL-13 binding proteins. The assay was validated and was shown to be accurate and precise over the entire quantification range (0.59 to 68.4 pg/mL in human EDTA plasma). The validated assay was successfully applied to samples from healthy volunteers and patients with atopic seasonal rhinitis. The assay is suitable for use in clinical trials to monitor efficacy or pharmacodynamic effects of drug candidates.

Over the past decades, the use of therapeutic monoclonal antibodies (mAbs) has become an important strategy in the treatment of various diseases. To enable pharmacokinetic (PK) assessment, specific immunoassays need to be developed to quantify mAbs in blood. In these assays, the presence of bound target protein can lead to severe underestimation of mAb concentration. Here we describe a novel approach for the quantification of total (free plus bound) human mAb concentration, in human and non-human primate serum, in the presence of a high level of target protein. The method is based on sample digestion with pepsin under optimized conditions to fully digest the target while keeping the mAb in the form of immunoreactive fragments. The quantification of mAb is then performed by ELISA without interference from the target. This method allows accurate quantification of as low as 50ng/ml mAb in the presence of up to 100-fold target molar excess. Intra- and inter-run precision is better than 10%, and intra- and inter-run accuracy in the range of 89.3-106.7%. In conclusion, this general and simple approach allows the accurate and sensitive measurement of preclinical and clinical samples avoiding target interference.

Use of a synergistic effect of DMSO together with a chaotropic salt (NaSCN or MgCl2) allowed to drastically reduce matrix interferences in an ELISA for therapeutic monoclonal antibodies. Optimum combinations were found to be 0.4M NaSCN together with 10.0% DMSO, and 1.0M MgCl2 with 15.0% DMSO. At this optimum combination, quality controls spiked with mAb at 50.0ng/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 50.0ng/ml to 100.0μg/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.

Adipocytes are insulin sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type II diabetes, cardiovascular disease, and metabolic syndrome. Obesity and its related disorders results in dysregulation of the mechanisms that control adipocyte gene expression and function. To identify potential novel therapeutic modulators of adipocytes, we screened 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We observed that less than 2% of the extracts had substantial effects on adipocyte differentiation. Two of the positive hit botanicals we screened that inhibited adipogenesis were extracts from St. John's Wort (SJW). Our studies revealed that two different SJW extracts inhibited adipogenesis as judged by lipid staining and marker gene expression. We also examined the effects of these SJW extracts on insulin sensitivity in mature adipocytes. One of the SJW extracts substantially inhibited insulin sensitive glucose uptake in a time and dose dependent manner. SJW is commonly used in the treatment of depression. However, our studies have revealed that SJW may have a negative impact on adipocyte related diseases by limiting adipocyte differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.

Objective: Peroxisome proliferator‐activated receptor γ (PPARγ) is a transcription factor that plays an important role in adipocyte gene expression. Previous studies from our laboratory and others have shown that PPARγ can be ubiquitin modified and targeted to the proteasome for degradation in response to transcriptional activation. In this study, we determined whether other degradation pathways contributed to the tumor necrosis factor α (TNFα)–induced degradation of PPARγ proteins in 3T3‐L1 adipocytes.Methods and Procedures: In these studies, 3T3‐L1 cells were studied by performing subcellular fractionations and western blotting after various TNFα treatments. Whole tissue extracts from rat adipose tissue were also used to examine PPARγ degradation.Results: We observed that TNFα can induce a caspase‐mediated degradation of PPARγ proteins in the presence of cycloheximide. The caspase‐mediated degradation of both PPARγ1 and PPARγ2 resulted in the generation of a specific 44‐kd cleavage product. The specific cleavage product was unaffected by proteasome inhibitors, but was repressed by a general caspase inhibitor. Use of several specific caspase inhibitors revealed that caspase‐1 was activated following treatment with TNFα and cycloheximide (CH), and inhibition of caspase‐1 blocked the cleavage of PPARγ proteins in cultured adipocytes. In addition, a similar PPARγ degradation product was observed in rodent adipose tissue.Discussion: In summary, this is the first study to demonstrate that PPARγ levels can be modulated by caspase activity.