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Background Biopharmaceutical products (BPs) are widely used to treat autoimmune diseases, but immunogenicity limits their efficacy for an important proportion of patients. Our knowledge of patient-related factors influencing the occurrence of antidrug antibodies (ADAs) is still limited. Methods and findings The European consortium ABIRISK (Anti-Biopharmaceutical Immunization: prediction and analysis of clinical relevance to minimize the RISK) conducted a clinical and genomic multicohort prospective study of 560 patients with multiple sclerosis (MS, n = 147), rheumatoid arthritis (RA, n = 229), Crohn's disease (n = 148), or ulcerative colitis (n = 36) treated with 8 different biopharmaceuticals (etanercept, n = 84; infliximab, n = 101; adalimumab, n = 153; interferon [IFN]-beta-1a intramuscularly [IM], n = 38; IFN-beta-1a subcutaneously [SC], n = 68; IFN-beta-1b SC, n = 41; rituximab, n = 31; tocilizumab, n = 44) and followed during the first 12 months of therapy for time to ADA development. From the bioclinical data collected, we explored the relationships between patient-related factors and the occurrence of ADAs. Both baseline and time-dependent factors such as concomitant medications were analyzed using Cox proportional hazard regression models. Mean age and disease duration were 35.1 and 0.85 years, respectively, for MS; 54.2 and 3.17 years for RA; and 36.9 and 3.69 years for inflammatory bowel diseases (IBDs). In a multivariate Cox regression model including each of the clinical and genetic factors mentioned hereafter, among the clinical factors, immunosuppressants (adjusted hazard ratio [aHR] = 0.408 [95% confidence interval (CI) 0.253-0.657], p < 0.001) and antibiotics (aHR = 0.121 [0.0437-0.333], p < 0.0001) were independently negatively associated with time to ADA development, whereas infections during the study (aHR = 2.757 [1.616-4.704], p < 0.001) and tobacco smoking (aHR = 2.150 [1.319-3.503], p < 0.01) were positively associated. 351,824 Single-Nucleotide Polymorphisms (SNPs) and 38 imputed Human Leukocyte Antigen (HLA) alleles were analyzed through a genome-wide association study. We found that the HLA-DQA1*05 allele significantly increased the rate of immunogenicity (aHR = 3.9 [1.923-5.976], p < 0.0001 for the homozygotes). Among the 6 genetic variants selected at a 20% false discovery rate (FDR) threshold, the minor allele of rs10508884, which is situated in an intron of the CXCL12 gene, increased the rate of immunogenicity (aHR = 3.804 [2.139-6.764], p < 1 x 10.sup.-5 for patients homozygous for the minor allele) and was chosen for validation through a CXCL12 protein enzyme-linked immunosorbent assay (ELISA) on patient serum at baseline before therapy start. CXCL12 protein levels were higher for patients homozygous for the minor allele carrying higher ADA risk (mean: 2,693 pg/ml) than for the other genotypes (mean: 2,317 pg/ml; p = 0.014), and patients with CXCL12 levels above the median in serum were more prone to develop ADAs (aHR = 2.329 [1.106-4.90], p = 0.026). A limitation of the study is the lack of replication; therefore, other studies are required to confirm our findings. Conclusion In our study, we found that immunosuppressants and antibiotics were associated with decreased risk of ADA development, whereas tobacco smoking and infections during the study were associated with increased risk. We found that the HLA-DQA1*05 allele was associated with an increased rate of immunogenicity. Moreover, our results suggest a relationship between CXCL12 production and ADA development independent of the disease, which is consistent with its known function in affinity maturation of antibodies and plasma cell survival. Our findings may help physicians in the management of patients receiving biotherapies., Author(s): Signe Hässler 1,2,3,*, Delphine Bachelet 1,4, Julianne Duhaze 1,5, Natacha Szely 6, Aude Gleizes 6,7, Salima Hacein-Bey Abina 7,8, Orhan Aktas 9, Michael Auer 10, Jerôme Avouac 11,12, Mary [...]

Sweat deposited via latent fingerprints (LFPs) was previously used to detect cocaine, opioids, cannabis and amphetamine via a point-of-care test (POCT). This screening method combined non-invasive sampling with a rapid result turnaround to produce a qualitative result outside of the laboratory. We report the novel application of a LFP drug screening test in a social care setting. Clients were tested on either an ad hoc or a routine basis using the POCT DOA114 (Intelligent Fingerprinting Ltd) drug screening cartridge. Screening cutoff values were 45, 35 and 95 pg/fingerprint for benzoylecgonine (BZE), morphine and amphetamine analytes, respectively. Confirmation LFP samples (DOA150, Intelligent Fingerprinting Ltd) and oral fluid (OF) were analyzed using ultra-performance liquid chromatography with tandem mass spectrometry. Thirty-six clients aged 36 ± 11 years participated (53% females). Individuals self-reported alcohol consumption (39%) and smoking (60%). Of 131 screening tests collected over 8 weeks, 14% tested positive for cocaine, 2% tested positive for opioids and 1% tested positive for amphetamine. Polydrug use was indicated in 10% of tests. Of 32 LFP confirmation tests, 63% were positive for cocaine and BZE. Opioids were also detected (31%), with the metabolite 6-monoacetylmorphine (6-MAM) being the most common (16%). In OF, cocaine was the dominant analyte (9%) followed by 6-MAM (5%). On comparing positive LFP screening tests with positive OF samples, we found that 39% and 38% were cocaine and opiate positive, respectively. Of the drugs screened for via the LFP POCT, cocaine was the most prevalent analyte in LFP and OF confirmation samples. The study is a step change in the routine drug screening procedures in a social care setting, especially useful for on-site cocaine detection in clients whose drug use was being monitored. Additionally, testing was easily accepted by clients and social care workers. [ABSTRACT FROM AUTHOR]

Here, we describe the use of a fluorescence based lateral flow competition assay for the screening of four classes of drugs, viz, Δ9-tetrahydrocannabinol (THC), cocaine (through the detection of benzoylecgonine, BZE), opiates (through the detection of morphine, MOR) and amphetamine (AMP) present in the sweat of a fingerprint. The Drug Screening Cartridge was specifically developed for fingerprint sample collection and analysis. For this study, the cut-offs were set at: 190, 90, 68 and 80 pg/fingerprint for THC, BZE, MOR and AMP, respectively. Working with three UK coroners, the Drug Screening Cartridge, together with its fluorescence reader, was applied to the detection of drugs in the sweat of a fingerprint from deceased individuals. The study shows that there was sufficient sweat present on the fingertips to enable analysis and that the Drug Screening Cartridge could detect the presence, or absence, of each drug. The presence of the drugs was confirmed using LC–MS-MS analysis of a second fingerprint sample collected simultaneously. Excellent correlation was achieved between the results obtained from the Drug Screening Cartridge and the LC–MS-MS analysis of the fingerprint samples obtained from 75 individuals. The accuracy of the results was: 99% for THC; 95% for BZE; 96% for MOR and 93% for AMP. The results obtained using the Drug Screening Cartridge were also compared to toxicological analysis of blood and urine samples with good correlation. The accuracy of the results between the Drug Screening Cartridge and blood was: 96%, 92%, 88% and 97% for THC, BZE, MOR and AMP, respectively. The comparison with urine showed an accuracy ranging between 86% and 92%. This fingerprint sample method has a collection time of just 5 s and a total analysis time of <10 mins. These results show that the lateral flow Drug Screening Cartridge is an excellent screening test to provide information on drug use from the sweat in a single fingerprint sample. [ABSTRACT FROM AUTHOR]

Background: Anti-drug antibodies (ADA) against natalizumab develop early during treatment. ADA persistency is defined by two consecutive positive results as performed by the current qualitative ELISA assay (positive/negative). Very little is known about the magnitude of the natalizumab ADA response and persistency. Design/methods: We developed a highly sensitive natalizumab ADA titration assay on the Meso Scale Discovery (MSD) platform and a pharmacokinetic (PK) assay. We included 43 patients with a positive ELISA-ADA result within 6 months of treatment initiation (baseline) of whom a follow-up serum sample was available 12–30 months after treatment start. MSD-ADA titres and drug levels were measured. Results: Median MSD-ADA titre at baseline was 4881 and 303 at follow-up. A titre of >400 at baseline had a 94% sensitivity and 89% specificity to predict ADA persistency. Reversion to ADA negativity occurred in 10 patients with mean drug levels of 10.8 μg/mL. The median trough drug level in ADA-positive samples was 0 µg/mL. PK levels and ADA titres correlated strongly negatively (r = −0.67). Conclusion: High baseline natalizumab ADA titres accurately predict persistency. Despite continuous treatment, the majority of patients with persistent ADA had no detectable drug levels indicating loss of efficacy in line with phase 3 study results. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a family of noncoding RNAs that play critical roles in the posttranscriptional regulation of gene expression. Accumulating evidence supports their involvement in the pathogenesis of multiple sclerosis (MS). Here, we compare miR-17 expressions in CD4+ T cells from relapsing-remitting (RR) MS patients treated with natalizumab versus untreated patients. miR-17 was downregulated under natalizumab treatment and upregulated during relapse, therefore supporting a possible role of miR-17 in MS immunopathogenesis. Downregulation of miR-17 was associated with upregulation of PTEN, BIM, E2F1, and p21 target genes. In vitro miR-17 inhibition was associated with upregulation of the same targets and resulted in impaired CD4+ T cell activation and proliferation. We further describe deregulated TGFBR2 expression in untreated patients versus healthy volunteers (HVs) and confirm in vitro the link between miR-17 and TGFBR2 expressions. These findings support an effect of natalizumab on expression of specific miRNA and subsequent expression of genes involved in proliferation and control of the cell cycle. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are a family of noncoding RNAs that play critical roles in the posttranscriptional regulation of gene expression. Accumulating evidence supports their involvement in the pathogenesis of multiple sclerosis (MS). Here, we compare miR-17 expressions in CD4+ T cells from relapsing-remitting (RR) MS patients treated with natalizumab versus untreated patients. miR-17 was downregulated under natalizumab treatment and upregulated during relapse, therefore supporting a possible role of miR-17 in MS immunopathogenesis. Downregulation of miR-17 was associated with upregulation of PTEN, BIM, E2F1, and p21 target genes. In vitro miR-17 inhibition was associated with upregulation of the same targets and resulted in impaired CD4+ T cell activation and proliferation. We further describe deregulated TGFBR2 expression in untreated patients versus healthy volunteers (HVs) and confirm in vitro the link between miR-17 and TGFBR2 expressions. These findings support an effect of natalizumab on expression of specific miRNA and subsequent expression of genes involved in proliferation and control of the cell cycle. [ABSTRACT FROM AUTHOR]

A correction to the article "A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference" published in a 2007 issue of "Nature Genetics" periodical.

Background: Anti-drug antibodies (ADA) against natalizumab develop early during treatment. ADA persistency is defined by two consecutive positive results as performed by the current qualitative ELISA assay (positive/negative). Very little is known about the magnitude of the natalizumab ADA response and persistency., Design/methods: We developed a highly sensitive natalizumab ADA titration assay on the Meso Scale Discovery (MSD) platform and a pharmacokinetic (PK) assay. We included 43 patients with a positive ELISA-ADA result within 6 months of treatment initiation (baseline) of whom a follow-up serum sample was available 12-30 months after treatment start. MSD-ADA titres and drug levels were measured., Results: Median MSD-ADA titre at baseline was 4881 and 303 at follow-up. A titre of >400 at baseline had a 94% sensitivity and 89% specificity to predict ADA persistency. Reversion to ADA negativity occurred in 10 patients with mean drug levels of 10.8 μg/mL. The median trough drug level in ADA-positive samples was 0 µg/mL. PK levels and ADA titres correlated strongly negatively ( r = -0.67)., Conclusion: High baseline natalizumab ADA titres accurately predict persistency. Despite continuous treatment, the majority of patients with persistent ADA had no detectable drug levels indicating loss of efficacy in line with phase 3 study results.

Here, we describe the use of a fluorescence based lateral flow competition assay for the screening of four classes of drugs, viz, Δ9-tetrahydrocannabinol (THC), cocaine (through the detection of benzoylecgonine, BZE), opiates (through the detection of morphine, MOR) and amphetamine (AMP) present in the sweat of a fingerprint. The Drug Screening Cartridge was specifically developed for fingerprint sample collection and analysis. For this study, the cut-offs were set at: 190, 90, 68 and 80 pg/fingerprint for THC, BZE, MOR and AMP, respectively. Working with three UK coroners, the Drug Screening Cartridge, together with its fluorescence reader, was applied to the detection of drugs in the sweat of a fingerprint from deceased individuals. The study shows that there was sufficient sweat present on the fingertips to enable analysis and that the Drug Screening Cartridge could detect the presence, or absence, of each drug. The presence of the drugs was confirmed using LC-MS-MS analysis of a second fingerprint sample collected simultaneously. Excellent correlation was achieved between the results obtained from the Drug Screening Cartridge and the LC-MS-MS analysis of the fingerprint samples obtained from 75 individuals. The accuracy of the results was: 99% for THC; 95% for BZE; 96% for MOR and 93% for AMP. The results obtained using the Drug Screening Cartridge were also compared to toxicological analysis of blood and urine samples with good correlation. The accuracy of the results between the Drug Screening Cartridge and blood was: 96%, 92%, 88% and 97% for THC, BZE, MOR and AMP, respectively. The comparison with urine showed an accuracy ranging between 86% and 92%. This fingerprint sample method has a collection time of just 5 s and a total analysis time of <10 mins. These results show that the lateral flow Drug Screening Cartridge is an excellent screening test to provide information on drug use from the sweat in a single fingerprint sample., (© The Author(s) 2018. Published by Oxford University Press.)

Antibodies against biopharmaceuticals (anti-drug antibodies, ADA) have been a well-integrated part of the clinical care of multiple sclerosis (MS) in several European countries. ADA data generated in Europe during the more than 10 years of ADA monitoring in MS patients treated with interferon beta (IFNβ) and natalizumab have been pooled and characterized through collaboration within a European consortium. The aim of this study was to report on the clinical practice of ADA testing in Europe, considering the number of ADA tests performed and type of ADA assays used, and to determine the frequency of ADA testing against the different drug preparations in different countries. A common database platform (tranSMART) for querying, analyzing and storing retrospective data of MS cohorts was set up to harmonize the data and compare results of ADA tests between different countries. Retrospective data from six countries (Sweden, Austria, Spain, Switzerland, Germany and Denmark) on 20,695 patients and on 42,555 samples were loaded into tranSMART including data points of age, gender, treatment, samples, and ADA results. The previously observed immunogenic difference among the four IFNβ preparations was confirmed in this large dataset. Decreased usage of the more immunogenic preparations IFNβ-1a subcutaneous (s.c.) and IFNβ-1b s.c. in favor of the least immunogenic preparation IFNβ-1a intramuscular (i.m.) was observed. The median time from treatment start to first ADA test correlated with time to first positive test. Shorter times were observed for IFNβ-1b-Extavia s.c. (0.99 and 0.94 years) and natalizumab (0.25 and 0.23 years), which were introduced on the market when ADA testing was already available, as compared to IFNβ-1a i.m. (1.41 and 2.27 years), IFNβ-1b-Betaferon s.c. (2.51 and 1.96 years) and IFNβ-1a s.c. (2.11 and 2.09 years) which were available years before routine testing began. A higher rate of anti-IFNβ ADA was observed in test samples taken from older patients. Testing for ADA varies between different European countries and is highly dependent on the policy within each country. For drugs where routine monitoring of ADA is not in place, there is a risk that some patients remain on treatment for several years despite ADA positivity. For drugs where a strategy of ADA testing is introduced with the release of the drug, there is a reduced risk of having ADA positive patients and thus of less efficient treatment. This indicates that potential savings in health cost might be achieved by routine analysis of ADA., Competing Interests: J. Link, M. Auer, R. Ramanujam, S. Haässler, D. Bachelet, C. Mbogning, P.E. Hyldgaard Jensen, C. Sievers, K. Ingenhoven, N. Fissolo, V. Grummel, M. Pallardy, P. Broeët: have nothing to disclose. M. Ryner: has received research support from Biogen Idec and Sanofi-Aventis, and received speaker honoraria from Biogen Idec. C. Warnke: has received honoraria for consulting from Novartis, Biogen, Bayer and TEVA. D. Buck: has received compensation for activities with Bayer HealthCare, Biogen Idec, MerckSerono, and Novartis and she is supported by the ABIRISK Consortium. R. Lindberg: has received research support from the Swiss MS Society, Swiss National Science Foundation, European FP6 and IMI JU programs, Roche Postdoc Fellowship Program (RPF-program), unrestricted research grants from Novartis and Biogen. M Comabella: has received compensation for consulting services and speaking honoraria from Bayer Schering Pharma, Merk Serono, Biogen-Idec, Teva Pharmaceuticals, Sanofi-Aventis, Genzyme, and Novartis. X. Montalban: has received speaking honoraria and travel expenses for scientific meetings, has been a steering committee member of clinical trials or participated in advisory boards of clinical trials in the past years with Bayer Schering Pharma, Biogen Idec, EMD Merck Serono, Genentech, Genzyme, Novartis, Sanofi-Aventis, Teva Pharmaceuticals, Almirall and Roche. B. Kieseier has received honoraria for lecturing, travel expenses for attending meetings, and financial support for research from Bayer Health Care, Biogen Idec, Genzyme/Sanofi Aventis, Grifols, Merck Serono, Mitsubishi Europe, Novartis, Roche, Talecris, and TEVA. P. S. Sørensen: has served on scientific advisory boards for Biogen Idec, Merck Serono, Novartis, Genzyme, Teva Pharmaceutical Industries Ltd., GlaxoSmithKline, medDay Pharmaceuticals and Forward Pharma; has been on steering committees or independent data monitoring boards in clinical trials sponsored by Merck Serono, Teva Pharmaceutical Industries Ltd., and GlaxoSmithKline; and has received speaker honoraria from Biogen Idec, Merck Serono, Teva Pharmaceutical Industries Ltd., Genzyme, and Novartis. His department has received research support from Biogen Idec, Bayer Schering, Merck Serono, TEVA, Baxter, Sanofi-Aventis, BioMS, Novartis, Bayer, RoFAR, Roche, Genzyme, from the Danish Multiple Sclerosis Society, the Danish Medical Research Council, and the European Union Sixth Framework Programme: Life sciences, Genomics and Biotechnology for health. H-P. Hartung: has received honoraris for consulting, serving on steering committees and speaking from Biogen, GeNeuro, Genzyme, Merck, Novartis, Opexa, Receptos, Roche, Sanofi, Teva with approval by the president of Heinrich-Heine University. T. Derfuss: serves on scientific advisory boards for Novartis Pharmaceuticals, Merck Serono, Biogen Idec, Genzyme, GeNeuro, Mitsubishi Pharma, Teva Pharmaceuticals and Bayer Schering Pharma; has received funding for travel and/or speaker honoraria from Biogen Idec, Genzyme, Novartis, Merck Serono and Bayer Schering Pharma; and receives research support from Biogen Idec, Novartis Pharma, the European Union, the Swiss National Foundation and the Swiss MS Society. B. Hemmer: has served on scientific advisory boards for Roche, Novartis, Bayer Schering, Merck Serono, Biogen Idec, GSK, Chugai Pharmaceuticals, Micromet, Genentech and Genzyme Corporation; serves on the international advisory board of Archives of Neurology and Experimental Neurology; has received speaker honoraria from Bayer Schering, Novartis, Biogen Idec, Merck Serono, Roche, and Teva Pharmaceutical Industries Ltd.; and has received research support from Biogen Idec, Bayer Schering, Merck Serono, Five prime, Metanomics, Chugai Pharmaceuticals and Novartis. He has filed a patent for the detection of antibodies and T cells against KIR4.1 in a subpopulation of MS patients and genetic determinants of neutralizing antibodies to interferon-beta. F. Deisenhammer: participated in meetings sponsored by or received honoraria for acting as an advisor/speaker for Bayer Healthcare, Biogen Idec, Genzyme-Sanofi, Merck, Novartis Pharma, and Roche. A. Fogdell-Hahn: has received funding and speaking honoraria from Biogen Idec and Pfizer. A. Lawton is employed by GlaxoSmithKline. At the time of writing D. Sikkema and J. Davidson were employed by GlaxoSmithKline, in which J. Davidson also held stocks/shares. N. Donnellan is employed by IPSEN. P. Doönnes is an employee of SciCross AB and has received support from the Innovative Medicines Initiative Joint Undertaking under grant agreement no. 115303, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007–2013) and EFPIA companies' in kind contribution. All the company-employed authors declare that this does not alter their adherence to PLOS ONE policies on sharing data and materials.

Objectives: To assess messenger RNA (mRNA) expression of POU2AF1 and Spi-B and their potential regulatory microRNAs (miRNAs) in natalizumab-treated patients with multiple sclerosis and in therapy-associated progressive multifocal leukoencephalopathy (PML)., Methods: Expression of POU2AF1/Spi-B was analyzed by using real-time reverse transcription PCR assays on isolated B/CD8(+) T lymphocytes and peripheral blood mononuclear cells (PBMCs) from cohorts of untreated and natalizumab-treated patients with and without PML. Longitudinal expression analysis was performed on CD4(+), CD8(+) T and B cells from 14 patients who interrupted natalizumab therapy for 8 weeks. The miRNA profiling was conducted in PBMCs from 5 untreated and 5 natalizumab-treated patients using low-density arrays followed by validation with single miRNAs assays in untreated and natalizumab-treated patients., Results: POU2AF1 and Spi-B mRNAs were upregulated in B and CD8(+) T cells from natalizumab-treated patients, which was validated in PBMCs from different cohorts of natalizumab-treated patients with and without PML, with a noteworthy higher expression of Spi-B in patients with PML. In contrast, downregulation of POU2AF1/Spi-B expression was measured in B and CD8(+) T cells after natalizumab discontinuation. Seventeen differentially expressed miRNAs including miR-10b, a regulator of POU2AF1 mRNA, were identified in long-term natalizumab-treated patients compared with untreated ones., Conclusions: Upregulation of POU2AF1 and Spi-B, known transactivators of the JC virus, the causative agent for PML, and its association with occurrence of PML in natalizumab-treated patients, corroborates POU2AF1/Spi-B as potential biomarkers for PML risk, which merits further evaluation.

Background: MicroRNAs (miRNAs) have emerged as a family of post-transcriptional regulators of gene expression that mediate diverse aspects of immunity. MiRNA dysregulation has been found in multiple sclerosis (MS), reflecting the growing need to identify disease-specific miRNA expression signatures. Our previous low-density array studies reveal differential miR-126 expression in the CD4(+)T cells of untreated relapsing-remitting MS (RRMS) patients. Here, we investigated miR-126 expression in natalizumab-treated patients., Methods: We isolated CD4(+) T cells from untreated (n = 12) and natalizumab-treated MS patients (n = 24), and from healthy volunteers (n = 12). We analyzed the expression of miRNAs and potential targets by real time reverse transcription polymerase chain reaction (RT-PCR). We assessed specific inhibition of miR-126, in vitro., Results: MiR-126 was down-regulated in cells of patients under natalizumab treatment and up-regulated during relapse, supporting a regulatory role in MS immunopathogenesis. MiR-126 expression correlated with the expression of POU2AF1, a regulator of Spi-B that binds to the promoter/enhancer sequences of JC virus (JCV), the pathogen of progressive multifocal leukoencephalopathy (PML), a rare complication of natalizumab treatment. The same trend was found for Spi-B. Strong up-regulation of both genes appeared to be treatment duration-dependent. Specific inhibition experiments supported the link between the expression of miR-126 and POU2AF1/Spi-B., Conclusions: Our findings provided deeper insight into the mode of action of natalizumab, with possible implications for understanding both the effects of natalizumab on MS activity and its specific adverse event profile., (© The Author(s) 2014.)