In-depth analysis of data from the RAS-ALS study reveals new insights in rasagiline treatment for amyotrophic lateral sclerosis.

BACKGROUND AND PURPOSE: In 2016, we concluded a randomized controlled trial testing 1 mg rasagiline per day add-on to standard therapy in 252 amyotrophic lateral sclerosis (ALS) patients. This article aims at better characterizing ALS patients who could possibly benefit from rasagiline by reporting new subgroup analysis and genetic data. METHODS: We performed further exploratory in-depth analyses of the study population and investigated the relevance of single nucleotide polymorphisms (SNPs) related to the dopaminergic system. RESULTS: Placebo-treated patients with very slow disease progression (loss of Amyotrophic Lateral Sclerosis Functional Rating Scale-Revised [ALSFRS-R] per month before randomization of ≤0.328 points) showed a per se survival probability after 24 months of 0.85 (95% confidence interval = 0.65-0.94). The large group of intermediate to fast progressing ALS patients showed a prolonged survival in the rasagiline group compared to placebo after 6 and 12 months (p = 0.02, p = 0.04), and a reduced decline of ALSFRS-R after 18 months (p = 0.049). SNP genotypes in the MAOB gene and DRD2 gene did not show clear associations with rasagiline treatment effects. CONCLUSIONS: These results underline the need to consider individual disease progression at baseline in future ALS studies. Very slow disease progressors compromise the statistical power of studies with treatment durations of 12-18 months using clinical endpoints. Analysis of MAOB and DRD2 SNPs revealed no clear relationship to any outcome parameter. More insights are expected from future studies elucidating whether patients with DRD2CC genotype (Rs2283265) show a pronounced benefit from treatment with rasagiline, pointing to the opportunities precision medicine could open up for ALS patients in the future.

Effect of High-Caloric Nutrition on Survival in Amyotrophic Lateral Sclerosis.

OBJECTIVE: Weight loss has been identified as a negative prognostic factor in amyotrophic lateral sclerosis, but there is no evidence regarding whether a high-caloric diet increases survival. Therefore, we sought to evaluate the efficacy of a high-caloric fatty diet (HCFD) for increasing survival. METHODS: A 1:1 randomized, placebo-controlled, parallel-group, double-blinded trial (LIPCAL-ALS study) was conducted between February 2015 and September 2018. Patients were followed up at 3, 6, 9, 12, 15, and 18 months after randomization. The study was performed at 12 sites of the clinical and scientific network of German motor neuron disease centers (ALS/MND-NET). Eligible patients were randomly assigned (1:1) to receive either HCFD (405kcal/day, 100% fat) or placebo in addition to riluzole (100mg/day). The primary endpoint was survival time, defined as time to death or time to study cutoff date. RESULTS: Two hundred one patients (80 female, 121 male, age = 62.4 ± 10.8 years) were included. The confirmatory analysis of the primary outcome survival showed a survival probability of 0.39 (95% confidence interval [CI] = 0.27-0.51) in the placebo group and 0.37 (95% CI = 0.25-0.49) in the HCFD group, both after 28 months (point in time of the last event). The hazard ratio was 0.97, 1-sided 97.5% CI = -∞ to 1.44, p = 0.44. INTERPRETATION: The results provide no evidence for a life-prolonging effect of HCFD for the whole amyotrophic lateral sclerosis population. However, post hoc analysis revealed a significant survival benefit for the subgroup of fast-progressing patients. ANN NEUROL 2020;87:206-216.

Associations between histogram analysis parameters derived from morphological sequences and histopathological tissue alterations in myositis and other myopathies: a preliminary study.

OBJECTIVES: Magnetic resonance imaging (MRI) is a cornerstone in diagnosis of myopathies. Recently, imaging techniques, such as histogram analysis are used to obtain novel imaging biomarkers. The present study sought to elucidate possible associations between histopathology derived from muscle biopsies and histogram parameters derived from clinical MRI in myositis and other myopathies. METHODS: 20 patients with myopathies were included in this retrospective study. MRI was performed using a 1.5T MRI scanner including T2- and T1- weighted images. The histogram parameters of the MRI sequences were obtained of the biopsied muscle. The histopathology analysis included the scoring systems proposed by Tateyama et al., Fanin et al., Allenbach et al. and immunohistochemical stainings for MHC-I, CD68, CD8 and CD4. RESULTS: Entropy derived from T2-weighted images showed strong positive associations with the inflammation scores (r=0.71, p=0.0005 with Allenbach et al score and r=0.68, p=0.001 with Tateyama score). Furthermore, there were strong associations between entropy derived from T2-weighted images with MHC-I staining (r=0.67, p=0.022), with the amount of CD20 cells (r=0.70, p=0.022), with the amount of CD4 positive cells (r=0.78, p=0.0075) and with the amount of CD8 positive cells (r=0.79, p=0.004). Other parameters showed no associations with the investigated histopathology features. CONCLUSIONS: Entropy derived from T2-weighted images showed strong associaitions with inflammation scores and with the sole amount of immune cells in myopathies. These results need to be confirmed by clinical studies, whether it is also related to clinical performance or can predict treatment response.

Jitter patterns in conventional concentric needle electromyography recordings of regenerating motor units.

BACKGROUND: The time interval between two potential components of the same motor unit potential (MUP) is measured for jitter analysis. Enhanced jitter is generally thought to result from impaired neuromuscular transmission as occurs in myasthenia gravis or during reinnervation. METHODS: Within a database of conventional video-electromyography (EMG) recordings 4 MUP with peculiar jitter patterns were identified. In 1 spontaneously discharging MUP, massive and chaotic jitter was seen with a mean consecutive difference (MCD) of 9.3 ms. In 2 spontaneously discharging MUP a certain potential subgroup jittered relative to the other part(s) of the MUP (MCD 2.0 and 3.3 ms). A jittering satellite was detected in a fourth voluntarily recruited MUP (MCD 0.6 ms). RESULTS: These different jitter patterns recorded with conventional EMG technique may mainly result from dysmyelination. CONCLUSIONS: A new look at the contribution of dysmyelination to abnormal jitter is also warranted in single fiber EMG recordings.

Spontaneous continuous motor unit single discharges.

INTRODUCTION: Motor unit hyperexcitability (MUH) may become manifest in needle electromyography (EMG) recordings as fasciculation potentials, myokymic discharges, or neuromyotonic discharges. Here, we describe a further MUH phenomenon. METHODS: Needle EMG recordings of the Neurology Hospital of Halle (Saale) stored in a video mode as .wav data between 2000 and 2015 were screened for spontaneous continuous motor unit single discharges (SCMUSD). RESULTS: We identified 23 video needle EMG waveforms from 14 patients with SCMUSD. The corresponding motor units discharged at a rate of about 6 H Z (6.3 ± 4.0; range, 1.3-18.1). The coefficient of variation of the discharge rate was 3.5% ± 1.7%. Neurogenic disorders were diagnosed in 12 patients, limb girdle muscle dystrophy was diagnosed in one patient, and stiff-limb syndrome was diagnosed in one patient. DISCUSSION: Spontaneous continuous motor unit single discharge, as described here, widens the spectrum of MUH phenomena.

Associations between magnetic resonance imaging and EMG findings in myopathies.

OBJECTIVES: Magnetic resonance imaging (MRI) is a cornerstone in diagnosis of myopathies. The present study sought to elucidate possible associations between electromyography (EMG) findings and histogram parameters derived from clinical MRI in myositis and other myopathies. MATERIALS AND METHODS: Twenty six patients with myopathies were included in this retrospective study. Clinical MRI was performed with a 1.5T MRI scanner including T2- and T1-weighted images. EMG analysis was performed during clinical diagnostic workup. The histogram parameters of the MRI sequences were obtained of the same muscle, which was investigated with EMG. RESULTS: Several correlations were identified between mean duration of the motor unit potentials (MUP) and histogram parameters derived from T1- and T2-weighted images. The highest for T1-weighted images was mode (r = -.73, P < .0001) and for T2-weighted images was p25 (r = -.57, P = .022). There were significant differences for several histogram parameters between muscles with pathological spontaneous activity and without. So, for T1-weighted images, the best discrimination was achieved with mean (P = .096), and for T2-weighted images for p10 (P = .05). Mean SI values derived from T1-weighted images achieved an AUC of 0.84 with a sensitivity of 0.81 and a specificity of 0.86 to discriminate patients with and without pathological spontaneous activity (PSA). CONCLUSIONS: The present study identified strong associations between histogram analysis derived from morphological MRI sequences and the duration of the MUP derived from EMG in myopathies strengthening the fact that both diagnostic modalities can reflect disease state in a similar fashion. Histogram parameters can predict muscles with PSA.

Formation of HERV-K and HERV-Fc1 Envelope Family Members is Suppressed on Transcriptional and Translational Level.

The human genome comprises 8% sequences of retroviral origin, so-called human endogenous retroviruses (HERVs). Most of these proviral sequences are defective, but some possess open reading frames. They can lead to the formation of viral transcripts, when activated by intrinsic and extrinsic factors. HERVs are thought to play a pathological role in inflammatory diseases and cancer. Since the consequences of activated proviral sequences in the human body are largely unexplored, selected envelope proteins of human endogenous retroviruses associated with inflammatory diseases, namely HERV-K18, HERV-K113, and HERV-Fc1, were investigated in the present study. A formation of glycosylated envelope proteins was demonstrated in different mammalian cell lines. Nevertheless, protein maturation seemed to be incomplete as no transport to the plasma membrane was observed. Instead, the proteins remained in the ER where they induced the expression of genes involved in unfolded protein response, such as HSPA5 and sXBP1. Furthermore, low expression levels of native envelope proteins were increased by codon optimization. Cell-free expression systems showed that both the transcriptional and translational level is affected. By generating different codon-optimized variants of HERV-K113 envelope, the influence of single rare t-RNA pools in certain cell lines was demonstrated. The mRNA secondary structure also appears to play an important role in the translation of the tested viral envelope proteins. In summary, the formation of certain HERV proteins is basically possible. However, their complete maturation and thus full biologic activity seems to depend on additional factors that might be disease-specific and await elucidation in the future.

Myositis in Lewis rats induced by the superantigen Staphylococcal enterotoxin A.

The aetiology of inflammatory myopathies is not clearly known. A predominance of activated Cd8+ T lymphocytes in inflammatory infiltrates has already been detected. Superantigens activate lymphocytes in an oligoclonal manner. In the present investigation, we investigated local effects after injection of the superantigen (Sag) Staphylococcus enterotoxin A (SEA) in the quadriceps femoris muscle of Lewis rats. Histopathology and gene expression profiling was performed after injection of SEA or saline (control group) after one, three and 10 days. Histology revealed focal myositis predominated by Cd8+ T lymphocytes with a perimysial, endomysial and perivascular distribution, peaking 3 days after SEA injection. Using DNA microarray analysis (Affymetrix Rat Genome 230 2.0) genes that were differentially over-expressed at least 15 times at days one, three or ten after SEA injection were further analysed. One day after SEA injection over-expressed genes were related to the immune response (e.g. Fcnb, CD8a) but also to cell proliferation, differentiation and migration (e.g. Mpp2). Three days after SEA injection, differentially overexpressed genes were mainly related to the immune reaction with a clear signature for a Cd8+ T lymphocyte response (e.g. Cd3d, Cd8, Prf1, Gzmb). Ten days after SEA injection, the differentially overexpressed genes were again associated with the immune reaction (e.g. Cd3d, Il2) but also with regenerative processes and wound healing (e.g. Tgfa, Tpm1, Ripply1). The inflammatory response induced by SEA in Lewis rats shares histological and molecular similarities to polymyositis in humans. Therefore, SEA induced myositis can be taken as a new and apt model for polymyositis.

Expression of A New Endogenous Retrovirus-Associated Transcript in Hodgkin Lymphoma Cells.

During characterization of a cDNA library from the Hodgkin lymphoma (HL) cell line L-1236, we discovered a new transcript derived from chromosome 1 at the long intergenic non-protein coding RNA 1768 (LINC01768)/colony stimulating factor 1 (CSF1) region. The first exon of this transcript from Hodgkin lymphoma cells (THOLE) starts in the predicted exon 4 of LINC01768 and is part of an endogenous retrovirus (ERV) from the HUERS-P1/LTR8 family. High expression of THOLE was only detectable in HL cell line L-1236. The expression of THOLE in L-1236 cell is another example for ERV/LTR-associated gene expression in HL cells. At the genome level, the HUERS-P1/LTR8 region including THOLE is only present in Hominoidea. The influence of ERV/LTRs on gene expression might explain the characteristic phenotype of human HL.

Apparent diffusion coefficient (ADC) does not correlate with different serological parameters in myositis and myopathy.

Background Magnetic resonance imaging (MRI) is widely used in several muscle disorders. Diffusion-weighted imaging (DWI) is an imaging modality, which can reflect microstructural tissue composition. The apparent diffusion coefficient (ADC) is used to quantify the random motion of water molecules in tissue. Purpose To investigate ADC values in patients with myositis and non-inflammatory myopathy and to analyze possible associations between ADC and laboratory parameters in these patients. Material and Methods Overall, 17 patients with several myositis entities, eight patients with non-inflammatory myopathies, and nine patients without muscle disorder as a control group were included in the study (mean age = 55.3 ± 14.3 years). The diagnosis was confirmed by histopathology in every case. DWI was obtained in a 1.5-T scanner using two b-values: 0 and 1000 s/mm2. In all patients, the blood sample was acquired within three days to the MRI. The following serological parameters were estimated: C-reactive protein, lactate dehydrogenase, alanine aminotransferase, aspartate aminotransferase, creatine kinase, and myoglobine. Results The estimated mean ADC value for the myositis group was 1.89 ± 0.37 × 10-3 mm2/s and for the non-inflammatory myopathy group was 1.79 ± 0.33 × 10-3 mm2/s, respectively. The mean ADC values (1.15 ± 0.37 × 10-3 mm2/s) were significantly higher to unaffected muscles (vs. myositis P = 0.0002 and vs. myopathy P = 0.0021). There were no significant correlations between serological parameters and ADC values. Conclusion Affected muscles showed statistically significantly higher ADC values than normal muscles. No linear correlations between ADC and serological parameters were identified.

Genetic Determinants of Antibody Levels in Cerebrospinal Fluid in Multiple Sclerosis: Possible Links to Endogenous Retroviruses.

The pathogenesis of multiple sclerosis (MS) has not been clarified. In addition to environmental factors; genetic determinants have been implicated in the pathogenesis of MS. Furthermore, endogenous retroviruses (ERV) might play a role in MS. The presence of oligoclonal immunoglobulin in cerebrospinal fluid (CSF) is a typical feature of MS. Recently, genetic polymorphisms in loci on human chromosomes 6, 14 and 18 have been identified as major determinants of CSF antibody levels in MS. The functional relevance of these single nucleotide polymorphisms (SNPs) remains unclear and none of them is located in an open reading frame. In previous studies, we identified ERV sequences in the vicinity of MS associated SNPs. Here, we describe the identification of ERV sequences in the neighborhood of SNPs associated with CSF antibody levels. All of the identified SNPs are located in the vicinity of ERV sequences. One of these sequences has very high homology to a sequence derived from the so-called MS-associated retrovirus (MSRV). Another cluster of three ERV sequences from the immunoglobulin heavy chain locus has retained the typical organization of retroviral genomes. These observations might shed new light on a possible association between ERVs and MS pathogenesis.

A study on viral CNS inflammation beyond herpes encephalitis.

The early diagnosis of herpes simplex virus encephalitis (HSVE) enables induction of antiviral therapy in this potentially life-threatening disease. The study aimed to determine clinical findings including cerebrospinal fluid (CSF) data and MRI imaging in HSVE patients and to identify features distinguishing HSVE from encephalitis of other viral etiologies. We retrospectively reviewed consecutive patients who were diagnosed with viral encephalitis between 2000 and 2014 at the University Hospital Halle. Forty-nine patients with viral encephalitis were identified. A viral etiology could be confirmed by PCR or antibody testing in 22/49 (44.9 %) of patients (15 (30.6 %) HSV, 5 (10.2 %) VZV, 2 (4.1 %) EBV). In HSVE, typical findings were focal slowing in electroencephalophy (EEG) (80 %, p = 0.021) and presence of cortical (86.7 %, p = 0.030) lesions in MRI. Restricted diffusion was particularly helpful in detection of early signal abnormalities in HSVE (p = 0.014). In 27/49 (55.1 %) of patients, no causative agent could be elucidated. In these patients, 15/27 (55.6 %) experienced a rather "benign" disease course with no MRI pathology despite initially HSVE mimicking clinical picture. However, CSF was significantly different showing a higher amount of granulocytes and activated lymphocytes. The remaining 12/27 (44.4 %) patients developed MRI changes consistent with encephalitis, in 4 of these patients, disease course was fatal. Beside PCR-based serology as standard procedure, MRI including diffusion-weighted images and EEG represent additional tools in early HSVE diagnosis. CSF cytology might be particularly supportive in differentiating likely benign forms of encephalitis.

A survey of endogenous retrovirus (ERV) sequences in the vicinity of multiple sclerosis (MS)-associated single nucleotide polymorphisms (SNPs).

Although multiple sclerosis (MS) is one of the most common central nervous system diseases in young adults, little is known about its etiology. Several human endogenous retroviruses (ERVs) are considered to play a role in MS. We are interested in which ERVs can be identified in the vicinity of MS associated genetic marker to find potential initiators of MS. We analysed the chromosomal regions surrounding 58 single nucleotide polymorphisms (SNPs) that are associated with MS identified in one of the last major genome wide association studies. We scanned these regions for putative endogenous retrovirus sequences with large open reading frames (ORFs). We observed that more retrovirus-related putative ORFs exist in the relatively close vicinity of SNP marker indices in multiple sclerosis compared to control SNPs. We found very high homologies to HERV-K, HCML-ARV, XMRV, Galidia ERV, HERV-H/env62 and XMRV-like mouse endogenous retrovirus mERV-XL. The associated genes (CYP27B1, CD6, CD58, MPV17L2, IL12RB1, CXCR5, PTGER4, TAGAP, TYK2, ICAM3, CD86, GALC, GPR65 as well as the HLA DRB1*1501) are mainly involved in the immune system, but also in vitamin D regulation. The most frequently detected ERV sequences are related to the multiple sclerosis-associated retrovirus, the human immunodeficiency virus 1, HERV-K, and the Simian foamy virus. Our data shows that there is a relation between MS associated SNPs and the number of retroviral elements compared to control. Our data identifies new ERV sequences that have not been associated with MS, so far.

The retrovirus/superantigen hypothesis of multiple sclerosis.

The pathogenesis of multiple sclerosis (MS) is as yet unknown. Commonly, MS is assumed to be due to an autoimmune inflammation of the central nervous system (CNS). Neurodegeneration is regarded to be a secondary reaction. This concept is increasingly being challenged. Human endogenous retroviruses (HERV) that could be locally activated in the CNS have been proposed as an alternative concept. HERV-encoded envelope proteins (env) can act as strong immune stimulators (superantigens). Thus, slow disease progression following neurodegeneration might be induced by re-activation of HERV expression directly, while relapses in parallel to inflammation might be secondary to the expression of HERV-encoded superantigens. It has been shown previously that T-cell superantigens are capable to induce a cellular inflammatory reaction in the CNS of experimental animals similar to that in MS. Furthermore, B-cell superantigens have been shown to activate blood leucocytes in vitro to produce immunoglobulin in an oligoclonal manner. It remains to be established, whether the outlined hypothesis accords with all known features of MS. Furthermore, anti-HERV agents may be taken into consideration to enrich and improve MS therapy.

Clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations.

An expansion of the GGGGCC hexanucleotide in the non-coding region of C9orf72 represents the most common cause of familial amyotrophic lateral sclerosis. The objective was to describe and analyse the clinical and genetic features of amyotrophic lateral sclerosis patients with C9orf72 mutations in a large population. Between November 2011 and December 2020, clinical and genetic characteristics of n = 248 patients with amyotrophic lateral sclerosis carrying C9orf72 mutations were collected from the clinical and scientific network of German motoneuron disease centres. Clinical parameters included age of onset, diagnostic delay, family history, neuropsychological examination, progression rate, phosphorylated neurofilament heavy chain levels in CSF and survival. The number of repeats was correlated with the clinical phenotype. The clinical phenotype was compared to n = 84 patients with SOD1 mutations and n = 2178 sporadic patients without any known disease-related mutations. Patients with C9orf72 featured an almost balanced sex ratio with 48.4% (n = 120) women and 51.6% (n = 128) men. The rate of 33.9% patients (n = 63) with bulbar onset was significantly higher compared to sporadic (23.4%, P = 0.002) and SOD1 patients (3.1%, P < 0.001). Of note, 56.3% (n = 138) of C9orf72, but only 16.1% of SOD1 patients reported a negative family history (P < 0.001). The GGGGCC hexanucleotide repeat length did not influence the clinical phenotypes. Age of onset (58.0, interquartile range 52.0-63.8) was later compared to SOD1 (50.0, interquartile range 41.0-58.0; P < 0.001), but earlier compared to sporadic patients (61.0, interquartile range 52.0-69.0; P = 0.01). Median survival was shorter (38.0 months) compared to SOD1 (198.0 months, hazard ratio 1.97, 95% confidence interval 1.34-2.88; P < 0.001) and sporadic patients (76.0 months, hazard ratio 2.34, 95% confidence interval 1.64-3.34; P < 0.001). Phosphorylated neurofilament heavy chain levels in CSF (2880, interquartile range 1632-4638 pg/ml) were higher compared to sporadic patients (1382, interquartile range 458-2839 pg/ml; P < 0.001). In neuropsychological screening, C9orf72 patients displayed abnormal results in memory, verbal fluency and executive functions, showing generally worse performances compared to SOD1 and sporadic patients and a higher share with suspected frontotemporal dementia. In summary, clinical features of patients with C9orf72 mutations differ significantly from SOD1 and sporadic patients. Specifically, they feature a more frequent bulbar onset, a higher share of female patients and shorter survival. Interestingly, we found a high proportion of patients with negative family history and no evidence of a relationship between repeat lengths and disease severity.

Spectrum and frequency of genetic variants in sporadic amyotrophic lateral sclerosis.

Therapy of motoneuron diseases entered a new phase with the use of intrathecal antisense oligonucleotide therapies treating patients with specific gene mutations predominantly in the context of familial amyotrophic lateral sclerosis. With the majority of cases being sporadic, we conducted a cohort study to describe the mutational landscape of sporadic amyotrophic lateral sclerosis. We analysed genetic variants in amyotrophic lateral sclerosis-associated genes to assess and potentially increase the number of patients eligible for gene-specific therapies. We screened 2340 sporadic amyotrophic lateral sclerosis patients from the German Network for motor neuron diseases for variants in 36 amyotrophic lateral sclerosis-associated genes using targeted next-generation sequencing and for the C9orf72 hexanucleotide repeat expansion. The genetic analysis could be completed on 2267 patients. Clinical data included age at onset, disease progression rate and survival. In this study, we found 79 likely pathogenic Class 4 variants and 10 pathogenic Class 5 variants (without the C9orf72 hexanucleotide repeat expansion) according to the American College of Medical Genetics and Genomics guidelines, of which 31 variants are novel. Thus, including C9orf72 hexanucleotide repeat expansion, Class 4, and Class 5 variants, 296 patients, corresponding to ∼13% of our cohort, could be genetically resolved. We detected 437 variants of unknown significance of which 103 are novel. Corroborating the theory of oligogenic causation in amyotrophic lateral sclerosis, we found a co-occurrence of pathogenic variants in 10 patients (0.4%) with 7 being C9orf72 hexanucleotide repeat expansion carriers. In a gene-wise survival analysis, we found a higher hazard ratio of 1.47 (95% confidence interval 1.02-2.1) for death from any cause for patients with the C9orf72 hexanucleotide repeat expansion and a lower hazard ratio of 0.33 (95% confidence interval 0.12-0.9) for patients with pathogenic SOD1 variants than for patients without a causal gene mutation. In summary, the high yield of 296 patients (∼13%) harbouring a pathogenic variant and oncoming gene-specific therapies for SOD1/FUS/C9orf72, which would apply to 227 patients (∼10%) in this cohort, corroborates that genetic testing should be made available to all sporadic amyotrophic lateral sclerosis patients after respective counselling.

Hodgkin's lymphoma RNA-transfected dendritic cells induce cancer/testis antigen-specific immune responses.

Cytotoxic T lymphocytes (CTL) can kill Hodgkin's lymphoma (HL) cells, and CTL have been used for the treatment of Epstein-Barr virus (EBV)-positive HL. For patients with EBV-negative HL, this strategy cannot be employed and alternative target structures have to be defined. In order to establish a system for the stimulation of HL-reactive T cells, we used dendritic cells (DC) as antigen-presenting cells for autologous T cells and transfected these DC with RNA from established HL cell lines. After stimulation of peripheral blood mononuclear cells (PBMC) with RNA-transfected DC, we analyzed the reactivity of primed PBMC by interferon gamma enzyme-linked immunospot. Our results suggest the presence of antigens with expression in HL cell lines and recognition of these antigens in combination with DC-derived human leukocyte antigen molecules. By the analysis of Gene Expression Omnibus microarray data sets from HL cell lines and primary HL samples in comparison with testis and other normal tissues, we identified HL-associated cancer testis antigens (CTA) including the preferentially expressed antigen in melanoma (PRAME). After stimulation of PBMC with RNA-transfected DC, we detected PRAME-reactive T cells. PRAME and other HL-associated CTA might be targets for HL-specific immune therapy or for the monitoring of HL-directed immune responses.

GFPT1-Associated Congenital Myasthenic Syndrome Mimicking a Glycogen Storage Disease - Diagnostic Pitfalls in Myopathology Solved by Next-Generation-Sequencing.

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.

Surfactant Protein-G in Wildtype and 3xTg-AD Mice: Localization in the Forebrain, Age-Dependent Hippocampal Dot-like Deposits and Brain Content.

The classic surfactant proteins (SPs) A, B, C, and D were discovered in the lungs, where they contribute to host defense and regulate the alveolar surface tension during breathing. Their additional importance for brain physiology was discovered decades later. SP-G, a novel amphiphilic SP, was then identified in the lungs and is mostly linked to inflammation. In the brain, it is also present and significantly elevated after hemorrhage in premature infants and in distinct conditions affecting the cerebrospinal fluid circulation of adults. However, current knowledge on SP-G-expression is limited to ependymal cells and some neurons in the subventricular and superficial cortex. Therefore, we primarily focused on the distribution of SP-G-immunoreactivity (ir) and its spatial relationships with components of the neurovascular unit in murine forebrains. Triple fluorescence labeling elucidated SP-G-co-expressing neurons in the habenula, infundibulum, and hypothalamus. Exploring whether SP-G might play a role in Alzheimer's disease (AD), 3xTg-AD mice were investigated and displayed age-dependent hippocampal deposits of ß-amyloid and hyperphosphorylated tau separately from clustered, SP-G-containing dots with additional Reelin-ir-which was used as established marker for disease progression in this specific context. Semi-quantification of those dots, together with immunoassay-based quantification of intra- and extracellular SP-G, revealed a significant elevation in old 3xTg mice when compared to age-matched wildtype animals. This suggests a role of SP-G for the pathophysiology of AD, but a confirmation with human samples is required.