Different tau fibril types reduce prion level in chronically and de novo infected cells.

Neurodegenerative diseases are often characterized by the codeposition of different amyloidogenic proteins, normally defining distinct proteinopathies. An example is represented by prion diseases, where the classical deposition of the aberrant conformational isoform of the prion protein (PrPSc) can be associated with tau insoluble species, which are usually involved in another class of diseases called tauopathies. How this copresence of amyloidogenic proteins can influence the progression of prion diseases is still a matter of debate. Recently, the cellular form of the prion protein, PrPC, has been investigated as a possible receptor of amyloidogenic proteins, since its binding activity with Aß, tau, and α-synuclein has been reported, and it has been linked to several neurotoxic behaviors exerted by these proteins. We have previously shown that the treatment of chronically prion-infected cells with tau K18 fibrils reduced PrPSc levels. In this work, we further explored this mechanism by using another tau construct that includes the sequence that forms the core of Alzheimer's disease tau filaments in vivo to obtain a distinct fibril type. Despite a difference of six amino acids, these two constructs form fibrils characterized by distinct biochemical and biological features. However, their effects on PrPSc reduction were comparable and probably based on the binding to PrPC at the plasma membrane, inhibiting the pathological conversion event. Our results suggest PrPC as receptor for different types of tau fibrils and point out a role of tau amyloid fibrils in preventing the pathological PrPC to PrPSc conformational change.

Transmission of Norwegian reindeer CWD to sheep by intracerebral inoculation results in an unusual phenotype and prion distribution.

Chronic wasting disease (CWD), a prion disease affecting cervids, has been known in North America (NA) since the 1960s and emerged in Norway in 2016. Surveillance and studies have revealed that there are different forms of CWD in Fennoscandia: contagious CWD in Norwegian reindeer and sporadic CWD in moose and red deer. Experimental studies have demonstrated that NA CWD prions can infect various species, but thus far, there have been no reports of natural transmission to non-cervid species. In vitro and laboratory animal studies of the Norwegian CWD strains suggest that these strains are different from the NA strains. In this work, we describe the intracerebral transmission of reindeer CWD to six scrapie-susceptible sheep. Detection methods included immunohistochemistry (IHC), western blot (WB), enzyme-linked immunosorbent assay (ELISA), real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). In the brain, grey matter vacuolation was limited, while all sheep exhibited vacuolation of the white matter. IHC and WB conventional detection techniques failed to detect prions; however, positive seeding activity with the RT-QuIC and PMCA amplification techniques was observed in the central nervous system of all but one sheep. Prions were robustly amplified in the lymph nodes of all animals, mainly by RT-QuIC. Additionally, two lymph nodes were positive by WB, and one was positive by ELISA. These findings suggest that sheep can propagate reindeer CWD prions after intracerebral inoculation, resulting in an unusual disease phenotype and prion distribution with a low amount of detectable prions.

Unfolding Mechanism and Fibril Formation Propensity of Human Prion Protein in the Presence of Molecular Crowding Agents.

The pathological process of prion diseases implicates that the normal physiological cellular prion protein (PrPC) converts into misfolded abnormal scrapie prion (PrPSc) through post-translational modifications that increase ß-sheet conformation. We recently demonstrated that HuPrP(90-231) thermal unfolding is partially irreversible and characterized by an intermediate state (ß-PrPI), which has been revealed to be involved in the initial stages of PrPC fibrillation, with a seeding activity comparable to that of human infectious prions. In this study, we report the thermal unfolding characterization, in cell-mimicking conditions, of the truncated (HuPrP(90-231)) and full-length (HuPrP(23-231)) human prion protein by means of CD and NMR spectroscopy, revealing that HuPrP(90-231) thermal unfolding is characterized by two successive transitions, as in buffer solution. The amyloidogenic propensity of HuPrP(90-231) under crowded conditions has also been investigated. Our findings show that although the prion intermediate, structurally very similar to ß-PrPI, forms at a lower temperature compared to when it is dissolved in buffer solution, in cell-mimicking conditions, the formation of prion fibrils requires a longer incubation time, outlining how molecular crowding influences both the equilibrium states of PrP and its kinetic pathways of folding and aggregation.

Secondary Protein Aggregates in Neurodegenerative Diseases: Almost the Rule Rather than the Exception.

The presence of protein aggregates is a hallmark of many neurodegenerative diseases, including Parkinson's disease (PD), Alzheimer's disease (AD), and frontotemporal lobar degeneration (FTLD). Traditionally, each disease has been associated with the aggregation of specific proteins, which serve as disease-specific biomarkers. For example, aggregates of α-synuclein (α-syn) are found in α-synucleinopathies such as PD, dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Similarly, AD is characterized by aggregates of amyloid-beta (Aß) and tau proteins. However, it has been observed that these protein aggregates can also occur in other neurodegenerative diseases, contributing to disease progression. For instance, α-syn aggregates have been detected in AD, Down syndrome, Huntington's disease, prion diseases, and various forms of FTLD. Similarly, Aß aggregates have been found in conditions like DLB and PD. Tau aggregates, in addition to being present in primary tauopathies, have been identified in prion diseases, α-synucleinopathies, and cognitively healthy aged subjects. Finally, aggregates of TDP-43, typically associated with FTLD and amyotrophic lateral sclerosis (ALS), have been observed in AD, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), MSA, DLB, and other neurodegenerative diseases. These findings highlight the complexity of protein aggregation in neurodegeneration and suggest potential interactions and common mechanisms underlying different diseases. A deeper understating of this complex scenario may eventually lead to the identification of a better elucidation of the pathogenetic mechanisms of these devastating conditions and hopefully new therapeutic stragegies.

Impact of seed amplification assay and surface-enhanced Raman spectroscopy combined approach on the clinical diagnosis of Alzheimer's disease.

BACKGROUND: The current diagnosis of Alzheimer's disease (AD) is based on a series of analyses which involve clinical, instrumental and laboratory findings. However, signs, symptoms and biomarker alterations observed in AD might overlap with other dementias, resulting in misdiagnosis. METHODS: Here we describe a new diagnostic approach for AD which takes advantage of the boosted sensitivity in biomolecular detection, as allowed by seed amplification assay (SAA), combined with the unique specificity in biomolecular recognition, as provided by surface-enhanced Raman spectroscopy (SERS). RESULTS: The SAA-SERS approach supported by machine learning data analysis allowed efficient identification of pathological Aß oligomers in the cerebrospinal fluid of patients with a clinical diagnosis of AD or mild cognitive impairment due to AD. CONCLUSIONS: Such analytical approach can be used to recognize disease features, thus allowing early stratification and selection of patients, which is fundamental in clinical treatments and pharmacological trials.

Corrigendum: PMCA-based detection of prions in the olfactory mucosa of patients with sporadic Creutzfeldt-Jakob disease.

[This corrects the article DOI: 10.3389/fnagi.2022.848991.].

Ambroxol as a disease-modifying treatment to reduce the risk of cognitive impairment in GBA-associated Parkinson's disease: a multicentre, randomised, double-blind, placebo-controlled, phase II trial. The AMBITIOUS study protocol.

Background: Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme ß-glucocerebrosidase (GCase), are the most frequent genetic risk factor for Parkinson's disease (PD). GBA-related PD (GBA-PD) patients have higher risk of dementia and reduced survival than non-carriers. Preclinical studies and one open-label trial in humans demonstrated that the chaperone ambroxol (ABX) increases GCase levels and modulates α-synuclein levels in the blood and cerebrospinal fluid (CSF). Methods and analysis: In this multicentre, double-blind, placebo-controlled, phase II clinical trial, we randomise patients with GBA-PD in a 1:1 ratio to either oral ABX 1.2 g/day or placebo. The duration of treatment is 52 weeks. Each participant is assessed at baseline and weeks 12, 26, 38, 52 and 78. Changes in the Montreal Cognitive Assessment score and the frequency of mild cognitive impairment and dementia between baseline and weeks 52 are the primary outcome measures. Secondary outcome measures include changes in validated scales/questionnaires assessing motor and non-motor symptoms. Neuroimaging features and CSF neurodegeneration markers are used as surrogate markers of disease progression. GCase activity, ABX and α-synuclein levels are also analysed in blood and CSF. A repeated-measures analysis of variance will be used for elaborating results. The primary analysis will be by intention to treat. Ethics and dissemination: The study and protocols have been approved by the ethics committee of centres. The study is conducted according to good clinical practice and the Declaration of Helsinki. The trial findings will be published in peer-reviewed journals and presented at conferences. Trial registration numbers: NCT05287503, EudraCT 2021-004565-13.

Approaching the Gut and Nasal Microbiota in Parkinson's Disease in the Era of the Seed Amplification Assays.

Parkinson's disease (PD) is a neurodegenerative disorder often associated with pre-motor symptoms involving both gastrointestinal and olfactory tissues. PD patients frequently suffer from hyposmia, hyposalivation, dysphagia and gastrointestinal dysfunctions. During the last few years it has been speculated that microbial agents could play a crucial role in PD. In particular, alterations of the microbiota composition (dysbiosis) might contribute to the formation of misfolded α-synuclein, which is believed to be the leading cause of PD. However, while several findings confirmed that there might be an important link between intestinal microbiota alterations and PD onset, little is known about the potential contribution of the nasal microbiota. Here, we describe the latest findings on this topic by considering that more than 80% of patients with PD develop remarkable olfactory deficits in their prodromal disease stage. Therefore, the nasal microbiota might contribute to PD, eventually boosting the gut microbiota in promoting disease onset. Finally, we present the applications of the seed amplification assays to the study of the gut and olfactory mucosa of PD patients, and how they could be exploited to investigate whether pathogenic bacteria present in the gut and the nose might promote α-synuclein misfolding and aggregation.

PMCA-Based Detection of Prions in the Olfactory Mucosa of Patients With Sporadic Creutzfeldt-Jakob Disease.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare neurodegenerative disorder caused by the conformational conversion of the prion protein (PrPC) into an abnormally folded form, named prion (or PrPSc). The combination of the polymorphism at codon 129 of the PrP gene (coding either methionine or valine) with the biochemical feature of the proteinase-K resistant PrP (generating either PrPSc type 1 or 2) gives rise to different PrPSc strains, which cause variable phenotypes of sCJD. The definitive diagnosis of sCJD and its classification can be achieved only post-mortem after PrPSc identification and characterization in the brain. By exploiting the Real-Time Quaking-Induced Conversion (RT-QuIC) assay, traces of PrPSc were found in the olfactory mucosa (OM) of sCJD patients, thus demonstrating that PrPSc is not confined to the brain. Here, we have optimized another technique, named protein misfolding cyclic amplification (PMCA) for detecting PrPSc in OM samples of sCJD patients. OM samples were collected from 27 sCJD and 2 genetic CJD patients (E200K). Samples from 34 patients with other neurodegenerative disorders were included as controls. Brains were collected from 26 sCJD patients and 16 of them underwent OM collection. Brain and OM samples were subjected to PMCA using the brains of transgenic mice expressing human PrPC with methionine at codon 129 as reaction substrates. The amplified products were analyzed by Western blot after proteinase K digestion. Quantitative PMCA was performed to estimate PrPSc concentration in OM. PMCA enabled the detection of prions in OM samples with 79.3% sensitivity and 100% specificity. Except for a few cases, a predominant type 1 PrPSc was generated, regardless of the tissues analyzed. Notably, all amplified PrPSc were less resistant to PK compared to the original strain. In conclusion, although the optimized PMCA did not consent to recognize sCJD subtypes from the analysis of OM collected from living patients, it enabled us to estimate for the first time the amount of prions accumulating in this biological tissue. Further assay optimizations are needed to faithfully amplify peripheral prions whose recognition could lead to a better diagnosis and selection of patients for future clinical trials.

Animal Models of Autosomal Recessive Parkinsonism.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder. The neuropathological hallmark of the disease is the loss of dopamine neurons of the substantia nigra pars compacta. The clinical manifestations of PD are bradykinesia, rigidity, resting tremors and postural instability. PD patients often display non-motor symptoms such as depression, anxiety, weakness, sleep disturbances and cognitive disorders. Although, in 90% of cases, PD has a sporadic onset of unknown etiology, highly penetrant rare genetic mutations in many genes have been linked with typical familial PD. Understanding the mechanisms behind the DA neuron death in these Mendelian forms may help to illuminate the pathogenesis of DA neuron degeneration in the more common forms of PD. A key step in the identification of the molecular pathways underlying DA neuron death, and in the development of therapeutic strategies, is the creation and characterization of animal models that faithfully recapitulate the human disease. In this review, we outline the current status of PD modeling using mouse, rat and non-mammalian models, focusing on animal models for autosomal recessive PD.

Sporadic Creutzfeldt-Jakob disease: Real-Time Quaking Induced Conversion (RT-QuIC) assay represents a major diagnostic advance.

Sporadic Creutzfeldt-Jakob disease (sCJD) is a rare and fatal neurodegenerative disorder with an incidence of 1.5 to 2 cases per million population/year. The disease is caused by a proteinaceous infectious agent, named prion (or PrPSc), which arises from the conformational conversion of the cellular prion protein (PrPC). Once formed, PrPSc interacts with the normally folded PrPC coercing it to undergo similar structural rearrangement. The disease is highly heterogeneous from a clinical and neuropathological point of view. The origin of this variability lies in the aberrant structures acquired by PrPSc. At least six different sCJD phenotypes have been described and each of them is thought to be caused by a peculiar PrPSc strain. Definitive sCJD diagnosis requires brain analysis with the aim of identifying intracerebral accumulation of PrPSc which currently represents the only reliable biomarker of the disease. Clinical diagnosis of sCJD is very challenging and is based on the combination of several clinical, instrumental and laboratory tests representing surrogate disease biomarkers. Thanks to the advent of the ultrasensitive Real-Time Quaking-Induced Conversion (RT-QuIC) assay, PrPSc was found in several peripheral tissues of sCJD patients, sometimes even before the clinical onset of the disease. This discovery represents an important step forward for the clinical diagnosis of sCJD. In this manuscript, we present an overview of the current applications and future perspectives of RT-QuIC in the field of sCJD diagnosis.

PMCA-generated prions from the olfactory mucosa of patients with Fatal Familial Insomnia cause prion disease in mice.

Background: Fatal Familial Insomnia (FFI) is a genetic prion disease caused by the D178N mutation in the prion protein gene (PRNP) in coupling phase with methionine at PRNP 129. In 2017, we have shown that the olfactory mucosa (OM) collected from FFI patients contained traces of PrPSc detectable by Protein Misfolding Cyclic Amplification (PMCA). Methods: In this work, we have challenged PMCA-generated products obtained from OM and brain homogenate of FFI patients in BvPrP-Tg407 transgenic mice expressing the bank vole prion protein to test their ability to induce prion pathology. Results: All inoculated mice developed mild spongiform changes, astroglial activation, and PrPSc deposition mainly affecting the thalamus. However, their neuropathological alterations were different from those found in the brain of BvPrP-Tg407 mice injected with raw FFI brain homogenate. Conclusions: Although with some experimental constraints, we show that PrPSc present in OM of FFI patients is potentially infectious. Funding: This work was supported in part by the Italian Ministry of Health (GR-2013-02355724 and Ricerca Corrente), MJFF, ALZ, Alzheimer's Research UK and the Weston Brain Institute (BAND2015), and Euronanomed III (SPEEDY) to FM; by the Spanish Ministerio de Economía y Competitividad (grant AGL2016-78054-R [AEI/FEDER, UE]) to JMT and JCE; AM-M was supported by a fellowship from the INIA (FPI-SGIT-2015-02).

The Alpha-Synuclein RT-QuIC Products Generated by the Olfactory Mucosa of Patients with Parkinson's Disease and Multiple System Atrophy Induce Inflammatory Responses in SH-SY5Y Cells.

Parkinson's disease (PD) and multiple system atrophy (MSA) are caused by two distinct strains of disease-associated α-synuclein (αSynD). Recently, we have shown that olfactory mucosa (OM) samples of patients with PD and MSA can seed the aggregation of recombinant α-synuclein by means of Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC). Remarkably, the biochemical and morphological properties of the final α-synuclein aggregates significantly differed between PD and MSA seeded samples. Here, these aggregates were given to neuron-like differentiated SH-SY5Y cells and distinct inflammatory responses were observed. To deepen whether the morphological features of α-synuclein aggregates were responsible for this variable SH-SY5Y inflammatory response, we generated three biochemically and morphologically distinct α-synuclein aggregates starting from recombinant α-synuclein that were used to seed αSyn_RT-QuIC reaction; the final reaction products were used to stimulate SH-SY5Y cells. Our study showed that, in contrast to OM samples of PD and MSA patients, the artificial aggregates did not transfer their distinctive features to the αSyn_RT-QuIC products and the latter induced analogous inflammatory responses in cells. Thus, the natural composition of the αSynD strains but also other specific factors in OM tissue can substantially modulate the biochemical, morphological and inflammatory features of the αSyn_RT-QuIC products.

Discrimination of MSA-P and MSA-C by RT-QuIC analysis of olfactory mucosa: the first assessment of assay reproducibility between two specialized laboratories.

BACKGROUND: Detection of the pathological and disease-associated alpha-synuclein (αSynD) in the brain is required to formulate the definitive diagnosis of multiple system atrophy (MSA) and Parkinson's disease (PD). We recently showed that αSynD can be detected in the olfactory mucosa (OM) of MSA and PD patients. For this reason, we have performed the first interlaboratory study based on α-synuclein Real-Time Quaking-Induced Conversion (αSyn_RT-QuIC) analysis of OM samples collected from PD and MSA patients with the parkinsonian (MSA-P) and cerebellar (MSA-C) phenotypes. METHODS: OM samples were prospectively collected from patients with a probable diagnosis of MSA-P (n = 20, mean disease duration 4.4 years), MSA-C (n = 10, mean disease duration 4 years), PD (n = 13, mean disease duration 8 years), and healthy control subjects (HS) (n = 11). Each sample was analyzed by αSyn_RT-QuIC in two independent specialized laboratories, one located in Italy (ITA-lab) and one located in the USA (USA-lab). Both laboratories have developed and used harmonized αSyn_RT-QuIC analytical procedures. Results were correlated with demographic and clinical data. RESULTS: The αSyn_RT-QuIC analysis reached a 96% interrater agreement of results (IAR) between laboratories (Kappa = 0.93, 95% CI 0.83-1.00). In particular, αSyn_RT-QuIC seeding activity was found in the OM of 9/13 patients with PD (sensitivity 69%, IAR 100%) and 18/20 patients with MSA-P (sensitivity 90%, IAR 100%). Interestingly, samples collected from patients with MSA-C did not induce αSyn_RT-QuIC seeding activity, except for one subject in USA-lab. Therefore, we found that MSA-P and MSA-C induced opposite effects. Regardless of disease diagnosis, the αSyn_RT-QuIC seeding activity correlated with some clinical parameters, including the rigidity and postural instability. CONCLUSIONS: Our study provides evidence that OM-αSynD may serve as a novel biomarker for accurate clinical diagnoses of PD, MSA-P, and MSA-C. Moreover, αSyn_RT-QuIC represents a reliable assay that can distinguish patients with MSA-P from those with MSA-C, and may lead to significant advancements in patients stratification and selection for emerging pharmacological treatments and clinical trials.

Cell-free amplification of prions: Where do we stand?

Neurodegenerative diseases (NDs) such as Alzheimer's disease (AD), Parkinson's disease (PD), atypical parkinsonisms, frontotemporal dementia (FTLD) and prion diseases are characterized by the accumulation of misfolded proteins in the central nervous system (CNS). Although the cause for the initiation of protein aggregation is not well understood, these aggregates are disease-specific. For instance, AD is characterized by the intraneuronal accumulation of tau and extracellular deposition of amyloid-ß (Aß), PD is marked by the intraneuronal accumulation of α-synuclein, many FTLD are associated with the accumulation of TDP-43 while prion diseases show aggregates of misfolded prion protein. Hence, misfolded proteins are considered disease-specific biomarkers and their identification and localization in the CNS, collected postmortem, is required for a definitive diagnosis. With the development of two innovative cell-free amplification techniques named Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking-Induced Conversion (RT-QuIC), traces of disease-specific biomarkers were found in CSF and other peripheral tissues (e.g., urine, blood, and olfactory mucosa) of patients with different NDs. These techniques exploit an important feature shared by many misfolded proteins, that is their ability to interact with their normally folded counterparts and force them to undergo similar structural rearrangements. Essentially, RT-QuIC and PMCA mimic in vitro the same pathological processes of protein misfolding which occur in vivo in a very rapid manner. For this reason, they have been employed for studying different aspects of protein misfolding but, overall, they seem to be very promising for the premortem diagnosis of NDs.

Prion Efficiently Replicates in α-Synuclein Knockout Mice.

Prion diseases are a group of neurodegenerative disorders associated with the conformational conversion of the cellular prion protein (PrPC) into an abnormal misfolded form named PrPSc. Other than accumulating in the brain, PrPSc can bind PrPC and force it to change conformation to PrPSc. The exact mechanism which underlies the process of PrPC/PrPSc conversion still needs to be defined and many molecules or cofactors might be involved. Several studies have documented an important role of PrPC to act as receptor for abnormally folded forms of α-synuclein which are responsible of a group of diseases known as synucleinopathies. The presence of PrPC was required to promote efficient internalization and spreading of abnormal α-synuclein between cells. In this work, we have assessed whether α-synuclein exerts any role in PrPSc conversion and propagation either in vitro or in vivo. Indeed, understanding the mechanism of PrPC/PrPSc conversion and the identification of cofactors involved in this process is crucial for developing new therapeutic strategies. Our results showed that PrPSc was able to efficiently propagate in the brain of animals even in the absence of α-synuclein thus suggesting that this protein did not act as key modulator of prion propagation. Thus, α-synuclein might take part in this process but is not specifically required for sustaining prion conversion and propagation.

Reduction of Immune Activation and Partial Recovery of Staphylococcal Enterotoxin B-Induced Cytokine Production After Switching to an Integrase Strand Transfer Inhibitor-Containing Regimen: Results from an Observational Cohort Study.

BACKGROUND AND OBJECTIVE: Despite integrase strand transfer inhibitor (INSTI)-containing regimens now being considered a preferred option for both initial therapy and switching strategies in virologically suppressed patients, their effects on lymphocyte phenotypes and functions in the course of effective combination antiretroviral therapy (cART) are still unclear. Thus, we investigated the effect of a 24-week elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate (EVG/c/FTC/TDF) regimen on the T cell compartment and HIV reservoirs in HIV-infected patients switching from a suppressive protease inhibitor-based regimen. METHODS: Thirty HIV-positive patients receiving suppressive tenofovir disoproxil fumarate/emtricitabine (TDF + FTC) (for a median of 5 years) in association with either darunavir/ritonavir (DVR/r) (47%) or atazanavir/ritonavir (ATV/r) (53%) were followed up for 24 weeks after switching to EVG/c/FTC/TDF. At baseline (week 0 [W0]) and after 12 (W12) and 24 (W24) weeks we analyzed HLA-DR (human leukocyte antigen-DR isotype)/CD38/Ki67/CCR7 (C-C chemokine receptor type 7)/CD45RA/CD127/PD-1 (programmed cell death-1) on CD4/CD8, interferon (IFN)-γ/interleukin (IL)-2 after HIV/Staphylococcal enterotoxin B (SEB) exposure (flow cytometry); total, integrated, and unintegrated HIV-DNA; and residual low-level HIV viremia (quantitative polymerase chain reaction [qPCR]). RESULTS: While EVG/c/FTC/TDF introduction resulted in a stable CD4+ and CD8+ count, residual low-level HIV-RNA viremia, and HIV reservoirs, we observed a significant reduction in both activated CD4+ (p = 0.016) and CD8+ (p = 0.048) T cells, coupled with an increase in IL-2 and IFN-γ release by CD4+ and CD8+ effector memory T cells, and a decrease in cytokine production by terminally differentiated CD8+ T cells following SEB exposure. Furthermore, the magnitude of the reduction of activated HLA-DR + CD38 + CD8+ T cells (r = - 0.63, p = 0.014) inversely correlates with the amount of total HIV-DNA at W24. CONCLUSIONS: Our data show a favorable effect of EVG/c/FTC/TDF switch to preserve immune activation-driven damage to T cell homeostasis, restore the multifunctional properties of effector T cells, and possibly contain cell-associated HIV viral burden in already virologically suppressed patients.

Use of different RT-QuIC substrates for detecting CWD prions in the brain of Norwegian cervids.

Chronic wasting disease (CWD) is a highly contagious prion disease affecting captive and free-ranging cervid populations. CWD has been detected in United States, Canada, South Korea and, most recently, in Europe (Norway, Finland and Sweden). Animals with CWD release infectious prions in the environment through saliva, urine and feces sustaining disease spreading between cervids but also potentially to other non-cervids ruminants (e.g. sheep, goats and cattle). In the light of these considerations and due to CWD unknown zoonotic potential, it is of utmost importance to follow specific surveillance programs useful to minimize disease spreading and transmission. The European community has already in place specific surveillance measures, but the traditional diagnostic tests performed on nervous or lymphoid tissues lack sensitivity. We have optimized a Real-Time Quaking-Induced Conversion (RT-QuIC) assay for detecting CWD prions with high sensitivity and specificity to try to overcome this problem. In this work, we show that bank vole prion protein (PrP) is an excellent substrate for RT-QuIC reactions, enabling the detection of trace-amounts of CWD prions, regardless of prion strain and cervid species. Beside supporting the traditional diagnostic tests, this technology could be exploited for detecting prions in peripheral tissues from live animals, possibly even at preclinical stages of the disease.

Efficient RT-QuIC seeding activity for α-synuclein in olfactory mucosa samples of patients with Parkinson's disease and multiple system atrophy.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder whose diagnosis is often challenging because symptoms may overlap with neurodegenerative parkinsonisms. PD is characterized by intraneuronal accumulation of abnormal α-synuclein in brainstem while neurodegenerative parkinsonisms might be associated with accumulation of either α-synuclein, as in the case of Multiple System Atrophy (MSA) or tau, as in the case of Corticobasal Degeneration (CBD) and Progressive Supranuclear Palsy (PSP), in other disease-specific brain regions. Definite diagnosis of all these diseases can be formulated only neuropathologically by detection and localization of α-synuclein or tau aggregates in the brain. Compelling evidence suggests that trace-amount of these proteins can appear in peripheral tissues, including receptor neurons of the olfactory mucosa (OM). METHODS: We have set and standardized the experimental conditions to extend the ultrasensitive Real Time Quaking Induced Conversion (RT-QuIC) assay for OM analysis. In particular, by using human recombinant α-synuclein as substrate of reaction, we have assessed the ability of OM collected from patients with clinical diagnoses of PD and MSA to induce α-synuclein aggregation, and compared their seeding ability to that of OM samples collected from patients with clinical diagnoses of CBD and PSP. RESULTS: Our results showed that a significant percentage of MSA and PD samples induced α-synuclein aggregation with high efficiency, but also few samples of patients with the clinical diagnosis of CBD and PSP caused the same effect. Notably, the final RT-QuIC aggregates obtained from MSA and PD samples owned peculiar biochemical and morphological features potentially enabling their discrimination. CONCLUSIONS: Our study provide the proof-of-concept that olfactory mucosa samples collected from patients with PD and MSA possess important seeding activities for α-synuclein. Additional studies are required for (i) estimating sensitivity and specificity of the technique and for (ii) evaluating its application for the diagnosis of PD and neurodegenerative parkinsonisms. RT-QuIC analyses of OM and cerebrospinal fluid (CSF) can be combined with the aim of increasing the overall diagnostic accuracy of these diseases, especially in the early stages.

Stimulation of PBMC and Monocyte-Derived Macrophages via Toll-Like Receptor Activates Innate Immune Pathways in HIV-Infected Patients on Virally Suppressive Combination Antiretroviral Therapy.

In HIV-infected, combination antiretroviral therapy (cART)-treated patients, immune activation and microbial translocation persist and associate with inadequate CD4 recovery and morbidity/mortality. We analyzed whether alterations in the toll-like receptor (TLR) pathway could be responsible for the immune hyperactivation seen in these patients. PBMC/monocyte-derived macrophages (MDMs) of 28 HIV+ untreated and 35 cART-treated patients with HIV-RNA < 40 cp/mL [20 Full Responders (FRs): CD4 ≥ 350; 15 Immunological Non-Responders (INRs): CD4 < 350], as well as of 16 healthy controls were stimulated with a panel of TLR agonists. We measured: CD4/CD8/CD14/CD38/HLA-DR/Ki67/AnnexinV/CD69/TLR4/8 (Flow Cytometry); PBMC expression of 84 TLR pathway genes (qPCR); PBMC/MDM cytokine release (Multiplex); and plasma lipopolysaccharide (LPS)/sCD14 (LAL/ELISA). PBMC/MDM from cART patients responded weakly to LPS stimulation but released high amounts of pro-inflammatory cytokines. MDM from these patients were characterized by a reduced expression of HLA-DR+ MDM and failed to expand activated HLA-DR+ CD38+ T-lymphocytes. PBMC/MDM from cART patients responded more robustly to ssRNA stimulation; this resulted in a significant expansion of activated CD38 + CD8 and the release of amounts of pro-inflammatory cytokines comparable to those seen in untreated viremic patients. Despite greater constitutive TLR pathway gene expression, PBMC from INRs seemed to upregulate only type I IFN genes following TLR stimulation, whereas PBMC from full responders showed a broader response. Systemic exposure to microbial antigens drives immune activation during cART by triggering TLRs. Bacterial stimulation modifies MDM function/pro-inflammatory profile in cART patients without affecting T-lymphocytes; this suggests translocating bacteria as selective stimulus to chronic innate activation during cART. High constitutive TLR activation is seen in patients lacking CD4 recovery, suggesting an exhausted immune milieu, anergic to further antigen encounters.