Niels Kruse

A polymorphism in the human cytotoxic T-lymphocyte antigen 4 ( CTLA4) gene (exon 1 +49) alters T-cell activation.

Abstract. The cytotoxic T-lymphocyte antigen 4 (CTLA4) is an important modifier of T-cell activation with down-regulatory properties upon B7 engagement. An allelic polymorphism in exon 1 of the CTLA4 gene coding for the peptide leader sequence of CTLA4 was recently described. This polymorphism was detected in association with several autoimmune diseases. In this study, we investigated the functional impact of the CTLA4 exon 1 +49 A/G dimorphism on T-cell activation and cellular localization. We examined the T-cell response from healthy donors either homozygous for A or G at position +49 of the exon 1. Under suboptimal stimulation conditions we found a greater proliferative response of cells from donors homozygous for G at position +49. FACS analysis of CTLA4 expression revealed a reduced up-regulation of CTLA4 from G/G donors upon T-cell activation, if compared with wild-type cells. Intracellular CTLA4 distribution demonstrated qualitatively different staining patterns between the two genotypes as determined using confocal fluorescence microscopy. Our results suggest that the G allele at position +49 of exon 1 affects the CTLA4-driven down-regulation of T-cell activation and may be an important factor in the pathogenesis of autoimmune diseases.

Characterizing mild cognitive impairment in incident Parkinson disease: The ICICLE-PD Study

Objective: To describe the frequency of mild cognitive impairment (MCI) in Parkinson disease (PD) in a cohort of newly diagnosed incident PD cases and the associations with a panel of biomarkers. Methods: Between June 2009 and December 2011, 219 subjects with PD and 99 age-matched controls participated in clinical and neuropsychological assessments as part of a longitudinal observational study. Consenting individuals underwent structural MRI, lumbar puncture, and genotyping for common variants of COMT, MAPT, SNCA, BuChE, EGF, and APOE. PD-MCI was defined with reference to the new Movement Disorder Society criteria. Results: The frequency of PD-MCI was 42.5% using level 2 criteria at 1.5 SDs below normative values. Memory impairment was the most common domain affected, with 15.1% impaired at 1.5 SDs. Depression scores were significantly higher in those with PD-MCI than the cognitively normal PD group. A significant correlation was found between visual Pattern Recognition Memory and cerebrospinal β-amyloid 1–42 levels (β standardized coefficient = 0.350; p = 0.008) after controlling for age and education in a linear regression model, with lower β-amyloid 1–42 and 1–40 levels observed in those with PD-MCI. Voxel-based morphometry did not reveal any areas of significant gray matter loss in participants with PD-MCI compared with controls, and no specific genotype was associated with PD-MCI at the 1.5-SD threshold. Conclusions: In a large cohort of newly diagnosed PD participants, PD-MCI is common and significantly correlates with lower cerebrospinal β-amyloid 1–42 and 1–40 levels. Future longitudinal studies should enable us to determine those measures predictive of cognitive decline.

Recommendations to standardize preanalytical confounding factors in Alzheimer's and Parkinson's disease cerebrospinal fluid biomarkers: an update

Early diagnosis of neurodegenerative disorders such as Alzheimers (AD) or Parkinsons disease (PD) is needed to slow down or halt the disease at the earliest stage. Cerebrospinal fluid (CSF) biomarkers can be a good tool for early diagnosis. However, their use in clinical practice is challenging due to the high variability found between centers in the concentrations of both AD CSF biomarkers (Aβ42, total tau and phosphorylated tau) and PD CSF biomarker (α-synuclein). Such a variability has been partially attributed to different preanalytical procedures between laboratories, thus highlighting the need to establish standardized operating procedures. Here, we merge two previous consensus guidelines for preanalytical confounding factors in order to achieve one exhaustive guideline updated with new evidence for Aβ42, total tau and phosphorylated tau, and α-synuclein. The proposed standardized operating procedures are applicable not only to novel CSF biomarkers in AD and PD, but also to biomarkers for other neurodegenerative disorders.

Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis

Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects. We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease. In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed. In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection. Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.

A Practical Guide to Immunoassay Method Validation

Biochemical markers have a central position in the diagnosis and management of patients in clinical medicine, and also in clinical research and drug development, also for brain disorders, such as Alzheimer’s disease. The enzyme-linked immunosorbent assay (ELISA) is frequently used for measurement of low-abundance biomarkers. However, the quality of ELISA methods varies, which may introduce both systematic and random errors. This urges the need for more rigorous control of assay performance, regardless of its use in a research setting, in clinical routine, or drug development. The aim of a method validation is to present objective evidence that a method fulfills the requirements for its intended use. Although much has been published on which parameters to investigate in a method validation, less is available on a detailed level on how to perform the corresponding experiments. To remedy this, standard operating procedures (SOPs) with step-by-step instructions for a number of different validation parameters is included in the present work together with a validation report template, which allow for a well-ordered presentation of the results. Even though the SOPs were developed with the intended use for immunochemical methods and to be used for multicenter evaluations, most of them are generic and can be used for other technologies as well.

Variations in cytokine mRNA expression during normal human pregnancy

Epidemiological data provide evidence that disease activity of T cell‐mediated, organ‐specific autoimmune diseases is reduced during pregnancy. Although there are several experimental animal studies on the effect of pregnancy on the immune system, the situation in humans is less clear. We therefore performed a prospective analysis of cytokine mRNA expression in whole blood by a new on‐line reverse transcriptase‐polymerase chain reaction technique and of serum hormone levels during pregnancy in healthy women. The control group included age‐matched non‐pregnant healthy women. Quantitativecytokine mRNA expression revealed significantly reduced IL‐18, interferon‐gamma (IFN‐γ), and IL‐2 mRNA levels in the first and second trimester in pregnancy compared with non‐pregnant women. No difference between groups was detected for tumour necrosis factor‐alpha (TNF‐α) mRNA. IL‐4 and IL‐10 mRNA were detected at low levels in only 20% of pregnant women and were reduced to a statistically significant extent in the second and third trimester compared with the control group. Changes in IL‐18 mRNA expression correlated inversely with serum values for human choriogonadotropin (HCG) and IL‐10 serum levels correlated with increases in serum 17β‐oestradiol levels. These data indicate immunomodulatory effects of pregnancy at the cytokine level which may be related to the variations in the clinical course of organ‐specific, T cell‐mediated autoimmune diseases during pregnancy.

Induction of α-synuclein aggregate formation by CSF exosomes from patients with Parkinson’s disease and dementia with Lewy bodies

Stuendl et al. show that CSF exosomes of patients with Parkinson’s disease or dementia with Lewy bodies contain α-synuclein and induce α-synuclein aggregation in a reporter cell line. Thus, exosomes may support inter-neuronal transmission of α-synuclein pathology. CSF exosomal α-synuclein may serve as a biomarker in α-synuclein-related neurodegeneration.

ABC-transporter gene-polymorphisms are potential pharmacogenetic markers for mitoxantrone response in multiple sclerosis

Escalation therapy with mitoxantrone (MX) in highly active multiple sclerosis is limited by partially dose-dependent side-effects. Predictors of therapeutic response may result in individualized risk stratification and MX dosing. ATP-binding cassette-transporters ABCB1 and ABCG2 represent multi-drug resistance mechanisms involved in active cellular MX efflux. Here, we investigated the role of ABC-gene single nucleotide polymorphisms (SNPs) for clinical MX response, corroborated by experimental in vitro and in vivo data. Frequencies of ABCB1 2677G>T, 3435C>T and five ABCG2-SNPs were analysed in 832 multiple sclerosis patients (Germany, Spain) and 264 healthy donors. Using a flow-cytometry-based in vitro assay, MX efflux in leukocytes from individuals with variant alleles in both ABC-genes (designated genotype ABCB1/ABCG2-L(ow), 22.2% of patients) was 37.7% lower than from individuals homozygous for common alleles (ABCB1/ABCG2-H(igh), P < 0.05, 14.8% of patients), resulting in genotype-dependent MX accumulation and cell death. Addition of glucocorticosteroids (GCs) inhibited MX efflux in vitro. ABC-transporters were highly expressed in leukocyte subsets, glial and neuronal cells as well as myocardium, i.e. cells/tissues potentially affected by MX therapy. In vivo significance was further corroborated in experimental autoimmune encephalomyelitis in Abcg2(-/-) animals. Using a MX dose titrated to be ineffective in wild-type animals, disease course and histopathology in Abcg2(-/-) mice were strongly ameliorated. Retrospective clinical analysis in MX monotherapy patients (n = 155) used expanded disability status scale, relapse rate and multiple sclerosis functional composite as major outcome parameters. The clinical response rate [overall 121 of 155 patients (78.1%)] increased significantly with genotypes associated with decreasing ABCB1/ABCG2-function [ABCB1/ABCG2-H 15/24 (62.5%) responders, ABCB1/ABCG2-I(ntermediate) 78/98 (79.6%), ABCB1/ABCG2-L 28/33 (84.8%), exact Cochran-Armitage test P = 0.039]. The odds ratio for response was 1.9 (95% CI 1.0-3.5) with each increase in ABCB1/ABCG2 score (from ABCB1/ABCG2-H to -I-, and -I to -L). In 36 patients with severe cardiac or haematological side effects no statistically relevant difference in genotype frequency was observed. However, one patient with biopsy proven cardiomyopathy only after 24 mg/m2 MX exhibited a rare genotype with variant, partly homozygous alleles in 3 ABC-transporter genes. In conclusion, SNPs in ABC-transporter genes may serve as pharmacogenetic markers associated with clinical response to MX therapy in multiple sclerosis. Combined MX/GC-treatment warrants further investigation.

Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

CTLA4 exon 1 dimorphism is associated with primary progressive multiple sclerosis

The cytotoxic T-lymphocyte antigen 4 (CTLA4) is an important modifier of T-cell activation with down-regulatory properties upon B7 engagement. We investigated the association of the CTLA4 A/G dimorphism in exon 1 (+49) with disease susceptibility, disease course and severity. No differences in the allelic distribution of the G(49) allele between multiple sclerosis (MS) patients and the control group was found. However, the G(49) allele occurred in a significant higher percentage of patients with primary progressive MS compared to patients with bout onset of disease. The results suggest that dysregulation of CTLA4-driven down-regulation of T-cell function due a genetic dimorphism in exon 1 may be involved in the pathogenesis of different MS disease subtypes.