Prion diseases show remarkable clinical and neuropathological heterogeneity. All reported cases with definite variant Creutzfeldt-Jakob disease (vCJD) were homozygous at PRNP codon 129. Heterozygosity at codon 219 has been shown to be protective against sporadic CJD (sCJD). Copy number variants (CNVs) are a novel source of genetic variability associated with susceptibility to neuropsychiatric disorders. Aims: Hypotheses tested: · The clinico-pathological phenotype of prion disease is modified by investigation findings, co-deposition of amyloid beta, tau proteins and/or candidate genetic variation. · The MRC Scale can be used for analysis of disease progression in CJD · Copy number variation alters the risk of prion disease in the UK and Papua New Guinea (PNG) Methods: Case reports illustrated genetic susceptibility and phenotypic heterogeneity. The MRC Scale was used to assess disease progression and study power in sCJD. Real-time PCR and gene sequencing were used to assess the role of candidate genes in clinico-pathological heterogeneity. GWAS were used to assess the role of CNVs as susceptibility loci to prion diseases. Results: Two patients with vCJD were heterozygous at codon 219. The MRC scale could be administered daily requiring only 90 patients to provide sufficient study power. Amyloid-β deposition was significantly influenced by APOE e4 haplotype in definite sCJD. Prion protein and hyperphosphorylated tau deposition were influenced by MAPTH1c haplotype. CNV duplications at chromosome 10 and 14 were significantly enriched in cases when compared to controls. The finding was confirmed using real-time PCR but was not replicated in the German cohort. Analysis using Penn CNV revealed a nominally significant association of CNV deletion at PARK2 gene. Conclusion: Heterozygosity at codon 219 is protective against sCJD but may confer susceptibility to vCJD. Patient stratification and assessments using MRC Scale allowed adequate study power to justify future therapeutic trials. MAPTH1c haplotype played a role in both prion and tau protein deposition. Chromosome 10 and 14 duplications and deletion at PARK2 gene may play a role in prion disease susceptibility.

Leukotrienes (LTs) play major roles in lung immune responses, and LTD4 is the most potent agonist for cysteinyl LT1, leading to bronchoconstriction and tissue remodeling. Here, we studied LT crosstalk between myeloid cells and pulmonary epithelial cells. Monocytic cells (Mono Mac 6 cell line, primary dendritic cells) and eosinophils produced primarily LTC4. In coincubations of these myeloid cells and epithelial cells, LTD4 became a prominent product. LTC4 released from the myeloid cells was further transformed by the epithelial cells in a transcellular manner. Formation of LTD4 was rapid when catalyzed by γ-glutamyl transpeptidase (GGT)1 in the A549 epithelial lung cancer cell line, but considerably slower when catalyzed by GGT5 in primary bronchial epithelial cells. When A549 cells were cultured in the presence of IL-1β, GGT1 expression increased about 2-fold. Also exosomes from A549 cells contained GGT1 and augmented LTD4 formation. Serine-borate complex (SBC), an inhibitor of GGT, inhibited conversion of LTC4 to LTD4. Unexpectedly, SBC also upregulated translocation of 5-lipoxygenase (LO) to the nucleus in Mono Mac 6 cells, and 5-LO activity. Our results demonstrate an active role for epithelial cells in biosynthesis of LTD4, which may be of particular relevance in the lung.

IMPORTANCE A major challenge for drug development in neurodegenerative diseases is that adequately powered efficacy studies with meaningful end points typically require several hundred participants and long durations. Prion diseases represent the archetype of brain diseases caused by protein misfolding, the most common subtype being sporadic Creutzfeldt-Jakob disease (sCJD), a rapidly progressive dementia. There is no well-established trial method in prion disease. OBJECTIVE To establish a more powerful and meaningful clinical trial method in sCJD. DESIGN, SETTING, AND PARTICIPANTS A stratified medicine and simulation approach based on a prospective interval-cohort study conducted from October 2008 to June 2014. This study involved 598 participants with probable or definite sCJD followed up over 470 patient-years at a specialist national referral service in the United Kingdom with domiciliary, care home, and hospital patient visits. We fitted linear mixed models to the outcome measurements, and simulated clinical trials involving 10 to 120 patients (no dropouts) with early to moderately advanced prion disease using model parameters to compare the power of various designs. MAIN OUTCOMES AND MEASURES A total of 2681 assessments were done using a functionally orientated composite end point (Medical Research Council Scale) and associated with clinical investigations (brain magnetic resonance imaging, electroencephalography, and cerebrospinal fluid analysis) and molecular data (prion protein [PrP] gene sequencing, PrPSc type). RESULTS Of the 598 participants, 273 were men. The PrP gene sequence was significantly associated with decline relative to any other demographic or investigation factors. Patients with sCJD and polymorphic codon 129 genotypes MM, VV, and MV lost 10% of their function in 5.3 (95% CI, 4.2-6.9), 13.2 (95% CI, 10.9-16.6), and 27.8 (95% CI, 21.9-37.8) days, respectively (P < .001). Simulations indicate that an adequately powered (80%; 2-sided α = .05) open-label randomized trial using 50% reduction in Medical Research Council Scale decline as the primary outcome could be conducted with only 120 participants assessed every 10 days and only 90 participants assessed daily, providing considerably more power than using survival as the primary outcome. Restricting to VV or MV codon 129 genotypes increased power even further. Alternatively, single-arm intervention studies (half the total sample size) could provide similar power in comparison to the natural history cohort. CONCLUSIONS AND RELEVANCE Functional end points in neurodegeneration need not require long and very large clinical studies to be adequately powered for efficacy. Patients with sCJD may be an efficient and cost-effective group for testing disease-modifying therapeutics. Stratified medicine and natural history cohort approaches may transform the feasibility of clinical trials in orphan diseases.

Prion diseases are dementing illnesses with poorly defined neuropsychological features. This is probably because the most common form, sporadic Creutzfeldt‐Jakob disease, is often rapidly progressive with pervasive cognitive decline making detailed neuropsychological investigation difficult. This study, which includes patients with inherited, acquired (iatrogenic and variant) and sporadic forms of the disease, is the only large‐scale neuropsychological investigation of this patient group ever undertaken and aimed to define a neuropsychological profile of human prion diseases.

Significance 5-Lipoxygenase (5LO) is a key enzyme in biosynthesis of leukotrienes (LTs), lipid mediators of inflammation. To study the roles of the 5LO accessory proteins coactosin-like protein (CLP) and 5LO-activating protein (FLAP), we knocked down these proteins in human monocytic cells. Our results show that expression of CLP was required for full cellular 5LO activity when cells were activated with Ca2+ ionophore, as well as with a physiological stimulus (lipopolysaccharide followed by N-formylmethionyl-leucyl-phenylalanine). During LT biosynthesis in stimulated cells, 5LO typically translocates to the nuclear membrane. This redistribution, from cytosolic to perinuclear, was clearly compromised in both CLP- and FLAP-deficient cells. Our results suggest that the CLP–5LO interaction may be a target for reduced LT production. 5-Lipoxygenase (5LO) is a key enzyme in leukotriene (LT) biosynthesis. Two accessory proteins, coactosin-like protein (CLP) and 5-lipoxygenase–activating protein (FLAP), can support 5LO activity. To study the roles of CLP and FLAP, we knocked down these proteins in the human monocytic cell line Mono Mac 6 (MM6). Expression of CLP increased MM6 cellular 5LO activity for all stimuli tested. CLP is not absolutely crucial, however; some 5LO activity remained in all incubations of CLP knockdown cells. FLAP knockdown had minor effects in the presence of exogenous arachidonic acid, but led to prominent reductions in 5LO product formation from endogenous substrate. Similar effects were observed after CLP and FLAP knockdown in human primary macrophages as well. In addition, FLAP knockdown reduced conversion of leukotriene A4 to leukotriene C4 (LTC4), suggesting a role for the activity of LTC4 synthase. After stimulation of MM6 cells by phorbol myristate acetate and ionophore A23187, a perinuclear ring pattern was observed for 5LO. This redistribution from cytosolic to perinuclear was clearly compromised in both CLP- and FLAP-deficient cells. In addition, association of CLP with the nucleus was almost absent after 5LO knockdown, and was clearly reduced in FLAP knockdown cells. Coimmunoprecipitation experiments indicated that 5LO-CLP complex formation in MM6 cells was increased by stimulation with ionophore, and that this complex was formed to the same extent in FLAP knockdown cells. A possible interpretation of our findings is that on cell stimulation, formation of the 5LO-CLP complex augments the translocation from cytosol to nucleus, whereas FLAP stabilizes association of this complex with the perinuclear membrane.

The prion diseases are rare neurodegenerative conditions that cause complex and highly variable neuropsychiatric syndromes, often with remarkably rapid progression. Prominent behavioral and psychiatric symptoms have been recognized since these diseases were first described. While research on such symptoms in common dementias has led to major changes in the way these symptoms are managed, evidence to guide the care of patients with prion disease is scarce. The authors review the published research and draw on more than 10 years' experience at the U.K. National Prion Clinic, including two large prospective clinical research studies in which more than 300 patients with prion disease have been followed up from diagnosis to death, with detailed observational data gathered on symptomatology and symptomatic treatments. The authors group behavioral and psychiatric symptoms into psychotic features, agitated features, and mood disorder and describe their natural history, showing that they spontaneously improve or resolve in many patients and are short-lived in many others because of rapid progression of global neurological disability. Diagnostic category, disease severity, age, gender, and genetic variation are or may be predictive factors. The authors review the observational data on pharmacological treatment of these symptoms in the U.K. clinical studies and make cautious recommendations for clinical practice. While nonpharmacological measures should be the first-line interventions for these symptoms, the authors conclude that there is a role for judicious use of pharmacological agents in some patients: antipsychotics for severe psychosis or agitation; benzodiazepines, particularly in the late stages of disease; and antidepressants for mood disorder.

Type 1 diabetes (T1D) is an autoimmune disease in which an inappropriate self-directed immune response affects and destroys insulin-producing β-cells in pancreatic islets leading to dysregulated blood glucose levels. T1D may affect people of any age, its clinical presentation is highly variable, and its incidence is increasing worldwide (1). The initial triggering events of T1D are unknown and their elucidation is of pivotal importance. Several factors might lead to the breakdown of β-cell–specific T-cell tolerance, including genetics, exogenous infectious pathogen, noninfectious environment agents, endogenous superantigens, or physiological stress events (2). The hallmark of autoimmune diabetes is insulitis, which progresses to a destruction of β-cells that results in clinical T1D. An altered balance between proinflammatory T-helper type 1 (Th1)/Th17 cells (γ-interferon [IFN-γ], interleukin [IL]-17) and Th2 immune response (IL-4, IL-10) leads to T1D (3,4). Evidence also suggests that both genetic and environmental factors may induce local inflammatory response, where activated intraislet dendritic cells (DCs) prevent peripheral T-cell tolerance (5). Moreover, it has been recently demonstrated that the development of destructive insulitis is partly due to impaired islet-resident Foxp3+ regulatory T cells (6,7). Several animal experimental models have been used for the investigation of the pathogenesis of T1D and it appears that a NOD mouse is the model of choice. NOD mice spontaneously develop early peri-insulitis and later intraislet insulitis caused by autoreactive T cells, …

Progress in therapeutics for rare disorders like prion disease is impeded by the lack of validated outcome measures and a paucity of natural history data derived from prospective observational studies. The first analysis of the U.K. National Prion Monitoring Cohort involved 1337 scheduled clinical assessments and 479 telephone assessments in 437 participants over 373 patient-years of follow-up. Scale development has included semi-quantitative and qualitative carer interviews, item response modelling (Rasch analysis), inter-rater reliability testing, construct analysis and correlation with several existing scales. The proposed 20-point Medical Research Council prion disease rating scale assesses domains of cognitive function, speech, mobility, personal care/feeding and continence, according to their relative importance documented by carer interviews. It is quick and simple to administer, and has been validated for use by doctors and nurses and for use over the telephone, allowing for frequent assessments that capture the rapid change typical of these diseases. The Medical Research Council Scale correlates highly with widely used cognitive and single item scales, but has substantial advantages over these including minimal floor effects. Three clear patterns of decline were observed using the scale: fast linear decline, slow linear decline (usually inherited prion disease) and in some patients, decline followed by a prolonged preterminal plateau at very low functional levels. Rates of decline and progress through milestones measured using the scale vary between sporadic, acquired and inherited prion diseases following clinical expectations. We have developed and validated a new functionally-oriented outcome measure and propose that future clinical trials in prion disease should collect data compatible with this scale, to allow for combined and comparative analyses. Such approaches may be advantageous in orphan conditions, where single studies of feasible duration will often struggle to achieve statistical power.

The need to find a prion disease neuroimaging biomarker is important with the development of therapeutic agents. Diffusion tensor imaging (DTI) is an MRI sequence that can visualise white matter changes in the brain. Voxel-based analysis of DTI and voxel based morphometry (VBM) was performed on 17 asymptomatic prion protein gene mutation carriers, 14 symptomatic inherited prion disease patients, seven sporadic CJD patients (sCJD) and 24 healthy controls. There were significant differences found in grey matter voxels between the symptomatic and the control patients in the cortex bilaterally. In addition there was reduced fractional anisotropy in the corpus callosum, frontal white matter, internal capsule, optic radiation and cerebellum; these regions did not overlap with areas of brain atrophy. In the asymptomatic patients there were directional changes seen in keeping with the symptomatic patients, but due to the small patient number no statistically significant differences were found with VBM and voxel based analysis of DTI. In sCJD grey matter changes were found in the thalamus on VBM but voxel based analysis of DTI demonstrated change in the corpus callosum, thalamus and cerebellar white matter. These data show that voxel based analysis of DTI can detect significant microstructural white matter changes in the absence of its loss. DTI may prove to be a useful biomarker in prion disease.