Outbreak of imported diphtheria with Corynebacterium diphtheriae among migrants arriving in Germany, 2022.

From July 2022, cases of imported diphtheria with toxigenic Corynebacterium diphtheriae remarkably increased among migrants arriving in Germany. Up to 30 September 2022, 44 cases have been reported to the national public health institute, all laboratory-confirmed, male, and mainly coming from Syria (n = 21) and Afghanistan (n = 17). Phylogeny and available journey information indicate that most cases (n = 19) were infected along the Balkan route. Active case finding, increased laboratory preparedness and epicentre localisation in countries along this route are important.

SARS-CoV-2 Omicron variants BA.1 and BA.2 both show similarly reduced disease severity of COVID-19 compared to Delta, Germany, 2021 to 2022.

German national surveillance data analysis shows that hospitalisation odds associated with Omicron lineage BA.1 or BA.2 infections are up to 80% lower than with Delta infection, primarily in ≥ 35-year-olds. Hospitalised vaccinated Omicron cases' proportions (2.3% for both lineages) seemed lower than those of the unvaccinated (4.4% for both lineages). Independent of vaccination status, the hospitalisation frequency among cases with Delta seemed nearly threefold higher (8.3%) than with Omicron (3.0% for both lineages), suggesting that Omicron inherently causes less severe disease.

Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients.

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients.

Evaluation of Latent Fingerprints for Drug Screening in a Social Care Setting.

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.

Clinicogenomic factors of biotherapy immunogenicity in autoimmune disease: A prospective multicohort study of the ABIRISK consortium.

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 × 10-5 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.

Establishment of a Highly Sensitive Assay for Detection of Hepatitis E Virus-Specific Immunoglobulins.

Hepatitis E, a liver disease caused by infection with the hepatitis E virus (HEV), is a worldwide emerging disease. The diagnosis is based on the detection of viral RNA and of HEV-specific immunoglobulins (Ig). For the latter, various assays are commercially available but still lack harmonization. In this study, a Luminex-based multiplex serological assay was established that measures the presence of total IgG, IgA, and IgM antibodies, targeting a short peptide derived from the viral E2 protein. For the validation, 160 serum samples with a known HEV serostatus were used to determine the assay cutoff and accuracy. Thereby, HEV IgG- and RNA-positive sera were identified with a sensitivity of 100% and a specificity of 98% (95% confidence interval [CI], 94% to 100%). Application of the assay by retesting 514 serum samples previously characterized with different HEV-IgG or total antibody tests revealed a high level of agreement between the assays (Cohen's kappa, 0.58 to 0.99). The established method is highly sensitive and specific and can be easily implemented in a multiplex format to facilitate rapid differential diagnostics with a few microliters of sample input.

Serological Analysis of Herpes B Virus at Individual Epitope Resolution: From Two-Dimensional Peptide Arrays to Multiplex Bead Flow Assays.

Macacine herpesvirus or B Virus (BV) is a zoonotic agent that leads to high mortality rates in humans if transmitted and untreated. Here, BV is used as a test case to establish a two-step procedure for developing high throughput serological assays based on synthetic peptides. In step 1, peptide microarray analysis of 42 monkey sera (30 of them tested BV positive by ELISA) revealed 1148 responses against 369 different peptides. The latter could be grouped into 142 different antibody target regions (ATRs) in six different glycoproteins (gB, gC, gD, gG, gH, and gL) of BV. The high number of newly detected ATRs was made possible inter alia by a new preanalytical protocol that reduced unspecific binding of serum components to the cellulose-based matrix of the microarray. In step 2, soluble peptides corresponding to eight ATRs of particularly high antigenicity were synthesized and coupled to fluorescently labeled beads, which were subsequently employed in immunochemical bead flow assays. Their outcome mirrored the ELISA results used as reference. Hence, convenient, fast, and economical screening of arbitrarily large macaque colonies for BV infection is now possible. The study demonstrates that a technology platform switch from two-dimensional high-resolution peptide arrays used for epitope discovery to a readily available bead array platform for serology applications is feasible.

Herpes zoster incidence in Germany - an indirect validation study for self-reported disease data from pretest studies of the population-based German National Cohort.

BACKGROUND: Until now, herpes zoster (HZ)-related disease burden in Germany has been estimated based on health insurance data and clinical findings. However, the validity of self-reported HZ is unclear. This study investigated the validity of self-reported herpes zoster (HZ) and its complication postherpetic neuralgia (PHN) using data from the pretest studies of the German National Cohort (GNC) in comparison with estimates based on health insurance data. METHODS: Data of 4751 participants aged between 20 and 69 years from two pretest studies of the GNC carried out in 2011 and 2012 were used. Based on self-reports of physician-diagnosed HZ and PHN, age- and sex-specific HZ incidence rates and PHN proportions were estimated. For comparison, estimates based on statutory health insurance data from the German population were considered. RESULTS: Eleven percent (95%-CI, 10.4 to 12.3, n = 539) of the participants reported at least one HZ episode in their lifetime. Our estimated age-specific HZ incidence rates were lower than previous estimates based on statutory health insurance data. The PHN proportion in participants older than 50 years was 5.9% (1.9 to 13.9%), which was in line with estimates based on health insurance data. CONCLUSION: As age- and sex-specific patterns were comparable with that in health insurance data, self-reported diagnosis of HZ seems to be a valid instrument for overall disease trends. Possible reasons for observed differences in incidence rates are recall bias in self-reported data or overestimation in health insurance data.

PARP-1 deregulation in multiple sclerosis.

BACKGROUND: Poly (ADP-ribose) polymerase 1 (PARP-1) plays pivotal roles in immune and inflammatory responses. Accumulating evidence suggests PARP-1 as a promising target for immunomodulation in multiple sclerosis and natalizumab-associated progressive multifocal leukoencephalopathy. OBJECTIVE: This study explores expression of PARP-1 and downstream effectors in multiple sclerosis and during natalizumab treatment. METHODS: Transcriptional expressions were studied by real-time reverse transcriptase polymerase chain reaction on CD4+T/CD8+T/CD14+/B cells and peripheral blood mononuclear cells from healthy volunteers, untreated and natalizumab-treated non-progressive multifocal leukoencephalopathy and progressive multifocal leukoencephalopathy multiple sclerosis patients. RESULTS: PARP-1 expression was higher in CD4+T, CD8+T and B cells from untreated patients compared to healthy volunteers. Natalizumab treatment restored deregulated PARP-1 expression in T cells but not in B cells. Sustained upregulation of PARP-1 was associated with decreased expression of downstream PARP-1 factors such as TGFBR1/TGFBR2/BCL6 in B cells. Notably, a higher expression of PARP-1 was detected in progressive multifocal leukoencephalopathy patients. CONCLUSIONS: Given the importance of PARP-1 in inflammatory processes, its upregulation in multiple sclerosis lymphocyte populations suggests a potential role in the immune pathogenesis of multiple sclerosis. Strikingly higher PARP-1 expression in progressive multifocal leukoencephalopathy cases suggests its involvement in progressive multifocal leukoencephalopathy disease pathomechanisms. These results further support the value of PARP-1 inhibitors as a potential novel therapeutic strategy for multiple sclerosis and natalizumab-associated progressive multifocal leukoencephalopathy.

Prediction of natalizumab anti-drug antibodies persistency.

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.

Drug screening using the sweat of a fingerprint: lateral flow detection of Δ9-tetrahydrocannabinol, cocaine, opiates and amphetamine.

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.

Monocyte NOTCH2 expression predicts IFN-ß immunogenicity in multiple sclerosis patients.

Multiple sclerosis (MS) is an autoimmune disease characterized by CNS inflammation leading to demyelination and axonal damage. IFN-ß is an established treatment for MS; however, up to 30% of IFN-ß-treated MS patients develop neutralizing antidrug antibodies (nADA), leading to reduced drug bioactivity and efficacy. Mechanisms driving antidrug immunogenicity remain uncertain, and reliable biomarkers to predict immunogenicity development are lacking. Using high-throughput flow cytometry, NOTCH2 expression on CD14+ monocytes and increased frequency of proinflammatory monocyte subsets were identified as baseline predictors of nADA development in MS patients treated with IFN-ß. The association of this monocyte profile with nADA development was validated in 2 independent cross-sectional MS patient cohorts and a prospective cohort followed before and after IFN-ß administration. Reduced monocyte NOTCH2 expression in nADA+ MS patients was associated with NOTCH2 activation measured by increased expression of Notch-responsive genes, polarization of monocytes toward a nonclassical phenotype, and increased proinflammatory IL-6 production. NOTCH2 activation was T cell dependent and was only triggered in the presence of serum from nADA+ patients. Thus, nADA development was driven by a proinflammatory environment that triggered activation of the NOTCH2 signaling pathway prior to first IFN-ß administration.

Dimethyl fumarate influences innate and adaptive immunity in multiple sclerosis.

INTRODUCTION: The mode of action of dimethyl fumarate (DMF), an immunomodulatory treatment for relapsing-remitting multiple sclerosis (RRMS), has not yet been fully elucidated. While in-vitro experiments and animal studies suggest effects on immune cell survival, proliferation, migration and oxidative stress response, corresponding observations from human studies are lacking. This study aims to characterize ex-vivo and in-vivo effects in a cohort of DMF treated RRMS patients. METHODS: Blood samples were collected from twenty well-characterized RRMS patients at baseline and after 3, 6 and 12 months of DMF treatment and an age- and gender-matched cohort of 20 healthy individuals at 0 and 3 months. Leukocyte subpopulations, immunoglobulin levels and cytokine secretion were measured. T cells were assessed for their levels of reactive oxygen species (ROS), metabolic status and their proliferative capacity. Levels of antioxidants were determined in serum by mass spectrometry. Responses of monocyte activation markers as well as NFkB and MAPK pathways to DMF were analysed. RESULTS: Upon DMF treatment, all lymphocyte subpopulations dropped significantly over the course of 12 months with cytotoxic and effector T cells being affected most significantly. DMF induced cell death and inhibited proliferation of T cells in-vitro. Interestingly, this anti-proliferative effect decreased under treatment. In-vivo DMF treatment led to decreased T cell glycolysis and higher turn-over of antioxidants. In line with these results a significant increase of cytosolic ROS levels after 3 months treatment was detected in T cells. In-vitro DMF treatment reduced NFkB (p65) translocation to the nucleus and MAPK (p38) levels decreased upon stimulation with monomethyl fumarate (MMF) in-vitro and ex-vivo. Consequently, the expression of co-stimulatory molecules like CD40 and CD150 was decreased in antigen presenting cells both in-vitro and ex-vivo. CONCLUSION: This study translates knowledge from in-vitro and animal studies on DMF into the clinical setting. Our data suggest that DMF not only alters lymphocyte composition, but also has profound effects on proliferation and induces oxidative stress in T cells. It also acts on innate immunity by reducing the activation status of antigen presenting cells (APCs) via NFkB and MAPK inactivation.

Validation of HAV biomarker 2A for differential diagnostic of hepatitis A infected and vaccinated individuals using multiplex serology.

BACKGROUND: Worldwide about 1.5 million clinical cases of hepatitis A virus (HAV) infections occur every year and increasingly countries are introducing HAV vaccination into the childhood immunization schedule with a single dose instead of the originally licenced two dose regimen. Diagnosis of acute HAV infection is determined serologically by anti-HAV-IgM detection using ELISA. Additionally anti-HAV-IgG can become positive during the early phase of symptoms, but remains detectable after infection and also after vaccination against HAV. Currently no serological marker allows the differentiation of HAV vaccinated individuals and those with a past infection with HAV. Such differentiation would greatly improve evaluation of vaccination campaigns and risk assessment of HAV outbreaks. Here we tested the HAV non-structural protein 2A, important for the capsid assembly, as a biomarker for the differentiation of the immune status in previously infected and vaccinated individuals. METHODS: HAV antigens were recombinantly expressed as glutathione-S-transferase (GST) fusion proteins. Using glutathione tagged, magnetic fluorescent beads (Luminex®), the proteins were affinity purified and used in a multiplex serological assay. The multiplex HAV assay was validated using 381 reference sera in which the immune status HAV negative, vaccinated or infected was established using the Abbott ARCHITECT® HAVAb-IgM or IgG, the commercial HAV ELISA from Abnova and documentation in vaccination cards. RESULTS: HAV multiplex serology showed a sensitivity of 99% and specificity of 95% to detect anti-HAV IgG/IgM positive individuals. HAV biomarker 2A allowed the differentiation between previously infected and vaccinated individuals. HAV vaccinated individuals and previously infected individuals could be identified with 92% accuracy. CONCLUSION: HAV biomarker 2A can be used to differentiate between previously HAV-vaccinated and naturally infected individuals. Within a multiplex serological approach this assay can provide valuable novel information in the context of outbreak investigations, longitudinal population based studies and evaluations of immunization campaigns.

Clinical practice of analysis of anti-drug antibodies against interferon beta and natalizumab in multiple sclerosis patients in Europe: A descriptive study of test results.

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 (IFNß) 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 IFNß preparations was confirmed in this large dataset. Decreased usage of the more immunogenic preparations IFNß-1a subcutaneous (s.c.) and IFNß-1b s.c. in favor of the least immunogenic preparation IFNß-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 IFNß-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 IFNß-1a i.m. (1.41 and 2.27 years), IFNß-1b-Betaferon s.c. (2.51 and 1.96 years) and IFNß-1a s.c. (2.11 and 2.09 years) which were available years before routine testing began. A higher rate of anti-IFNß 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.

Natalizumab-induced POU2AF1/Spi-B upregulation: A possible route for PML development.

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.

Evaluation of a questionnaire to assess selected infectious diseases and their risk factors : findings of a multicenter study.

BACKGROUND/OBJECTIVES: The risk to die from an infectious disease in Germany has been continuously decreasing over the last century. Since infections are, however, not only causes of death but risk factors for diseases like cardiovascular diseases, it is essential to monitor and analyze their prevalence and frequency, especially in consideration of the increased life expectancy. To gain more knowledge about infectious diseases as risk factors and their implications on the condition and change of the immune status, the German National Cohort (GNC), a population-based prospective cohort study, will recruit 200,000 subjects between 2014 and 2017. In Pretest 1, a feasibility study for the GNC, we evaluated a self-administered and self-report questionnaire on infectious diseases and on the use of health care facilities (hereinafter called "ID Screen") for feasibility and validity. METHODS: From August-November 2011, 435 participants between the ages of 20-69 completed the ID Screen. All subjects had been recruited via a random sample from the local residents' registration offices by 4 of the 18 participating study centers. The questionnaire encompasses 77 variables in six sections assessing items such as 12-month prevalence of infections, cumulative prevalence of infectious diseases, visit of health care facilities and vaccination. The feasibility was amongst others evaluated by assessing the completeness and comprehensiveness of the questionnaire. To assess the questionnaires ability to measure "immune status" and "susceptibility to infections", multivariate analysis was used. RESULTS: The overall practicability was good and most items were well understood, demonstrated by < 2/33 missing questions per questionnaire and only three variables: vaccination for influenza and pneumococci and infection with chickenpox had a frequency > 5 % of missing values. However, direct comparison of the items 12-month prevalence and lifetime prevalence of nephritis/pyelitis showed poor agreement and thereby poor understanding by 80 % of the participants, illustrating the necessity for a clear, lay person appropriate description of rare diseases to increase comprehensibility. The questionnaire will be used to support the assessment of immune dysfunction and frequency of infection. An analysis of these constructs in an exploratory factor analysis revealed limited applicability due to low interitem correlation (Cronbach's α < 0.5). This is corroborated by the extraction of more than one factor with a Kaiser-Meyer-Olkin measure of 0.6 instead of a unidimensional latent construct for "immune status". CONCLUSION: All in all, the ID Screen is a good and reliable tool to measure infectious diseases as risk factors and outcome in general, but requires a better translation of infection specific terms into lay person terms. For the assessment of the overall immune status, the tool has strong limitations. Vaccinations status should also rather be assessed based on vaccination certificates than on participants' recall.

Unraveling natalizumab effects on deregulated miR-17 expression in CD4+ T cells of patients with relapsing-remitting multiple sclerosis.

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.

MiR-126: a novel route for natalizumab action?

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.