Combined measure of salivary alpha-synuclein species as diagnostic biomarker for Parkinson's disease

Parkinson's disease (PD) diagnosis is still vulnerable to bias, and a definitive diagnosis often relies on post-mortem neuropathological diagnosis. In this regard, alpha-synuclein (αsyn)-specific in vivo biomarkers remain a critical unmet need, based on its relevance in the neuropathology. Specifically, content changes in αsyn species such as total (tot-αsyn), oligomeric (o-αsyn), and phosphorylated (p-αsyn) within the cerebrospinal fluid (CSF) and peripheral fluids (i.e., blood and saliva) have been proposed as PD biomarkers possibly reflecting the neuropathological outcome. Here, we measured the p-αsyn levels in the saliva from 15 PD patients along with tot-αsyn, o-αsyn and their ratios, and compared the results with those from 23 healthy subjects (HS), matched per age and sex. We also calculated the optimal cutoff values for different αsyn species to provide information about their capability to discriminate PD from HS. We found that p-αsyn was the most abundant alpha-synuclein species in the saliva. While p-αsyn concentration did not differ between PD and HS when adjusted for total salivary proteins, the ratio p-αsyn/tot-αsyn was largely lower in PD patients than in HS. Moreover, the concentration of o-αsyn was increased in the saliva of PD patients, and tot-αsyn did not differ between PD and HS. The ROC curves indicated that no single αsyn form or ratio could provide an accurate diagnosis of PD. On the other hand, the ratio of different items, namely p-αsyn/tot-αsyn and o-αsyn, yielded more satisfactory diagnostic accuracy, suggesting that the combined measure of different species in the saliva may show more promises as a diagnostic means for PD

Occurrence and traceability of Salmonella spp. in five Sardinian fermented sausage facilities

The aims of the present study were to evaluate the presence of Salmonella in five fermented sausage processing plants and their products during the production process, and to trace the possible sources of contamination. A total of 270 samples were collected: mixture of ground pork meat and fat, products at the end of acidification, sausages at the end of ripening and, during production stages, surfaces in contact with meat and surfaces not in contact with meat. For samples of ground meat, product at the end of acidification and sausages at the end of ripening, the pH and water activity (aw), were determined. All the samples were tested for the presence of Salmonella. Thirtytwo Salmonella isolates were obtained, subjected to serotyping and PFGE. The sausages at the end of ripening pH and aw mean values were 5.39±0.24 and 0.91±0.03, respectively. Salmonella was detected in three processing plants with an overall prevalence of 16.7% in food samples and 5.8% in environmental samples. Salmonella prevalence was 24% in ground meat and products at the end of acidification and was also detected in a sample of sausage at the end of ripening (2%). In environmental samples, Salmonella was detected in 6.6% of surfaces in contact with meat and 5% of surfaces not in contact with meat. Five serotypes were identified among 32 isolates: S. Derby (37.5%), S. Typhimurium and S. Rissen (both 25%), S. Give and monophasic S. Typhimurium (both 6.25%). Six different pulsotypes were obtained with PFGE. The serotypes and the PFGE pattern of the strains were specific for each facility with no overlapping between different processing plants. The same observation can be pointed out considering different sampling days for the same processing plants, thus presumably indicating the raw material (ground pork meat and fat) as the source of contamination. The detection of Salmonella in a sample of sausage at the end of ripening highlights the ability of the pathogen to survive during manufacturing process

Evaluation of vacuum packaging for extending the shelf life of Sardinian fermented sausage

Salsiccia sarda or Sardinian fermented sausage is a traditional dry-fermented sausage included in the list of traditional food products of Sardinia (Italy). At the request of some producing plants, the possibility of extending the shelf life of the vacuum-packed product up to 120 days was evaluated. Manufacturing of 90 samples, representing 3 different batches of Sardinian fermented sausage was carried out in two producing plants (A and B). In the packaged product and subsequently every 30 days for four months (T0, T30, T60, T120), the following analyses were conducted on all samples: physicochemical characteristics, total aerobic mesophilic count, Enterobacteriaceae count, detection of Listeria monocytogenes, Salmonella spp., mesophilic lactic acid bacteria, and coagulase-positive Staphylococci. Moreover, surfaces in contact and surfaces not in contact with food were sampled in both producing plants. Sensory profile analysis was also performed for every analysis time. At the end of the extended shelf life, pH values were equal to 5.90±0.11 (producing plant A) and 5.61±0.29 (producing plant B). Water activity mean values at T120 were 0.894±0.02 (producing plant A) and 0.875±0.01 (producing plant B). L. monocytogenes was detected in 73.3% (33/45) of the samples from producing plant A, with mean levels of 1.12±0.76 log10 CFU/g. In producing plant B, L. monocytogenes was never detected. Enterobacteriaceae were detected in 91.1% (41/45) of samples in producing plant A with mean values of 3.15±1.21 log10 CFU/g, and in 35.5% (16/45) samples in producing plant B samples with mean values of 0.72±0.86 log10 CFU/g. Salmonella and Staphylococcus aureus were never detected. Regarding environmental samples, the sites that were most contaminated by L. monocytogenes were the bagging table (contact surface) and processing room floor drains (non-contact surface) with a prevalence of 50% each (8/16 positive samples for both sampling sites). Sensory analysis results showed that at T30 the overall sensory quality was at its highest; moreover, the visual-tactile aspect, the olfactory characteristics, the gustatory aspects, and the texture showed significant differences in samples throughout the shelf life, with a decreased intensity at 120 days of storage. Overall, the quality and sensory acceptance of the vacuum-packed Sardinian fermented sausage were not affected until 120 days of shelf-life. However, the possible contamination by L. monocytogenes calls attention to the hygienic management of the entire technological process. The environmental sampling was confirmed as a useful verification tool during control

Modeling Parkinson's Disease Neuropathology and Symptoms by Intranigral Inoculation of Preformed Human α-Synuclein Oligomers.

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson's disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies

The double-sided of human leukocyte antigen-G molecules in type 1 autoimmune hepatitis

The immunomodulatory effects of HLA-G expression and its role in cancers, human liver infections and liver transplantation are well documented, but so far, there are only a few reports addressing autoimmune liver diseases, particularly autoimmune hepatitis (AIH). Method and materialsWe analyzed the genetic and phenotypic characteristics of HLA-G in 205 type 1 AIH patients (AIH-1) and a population of 210 healthy controls from Sardinia (Italy). ResultsAnalysis of the HLA-G locus showed no substantial differences in allele frequencies between patients and the healthy control population. The HLA-G UTR-1 haplotype was the most prevalent in both AIH-1 patients and controls (40.24% and 34.29%). Strong linkage was found between the HLA-G UTR-1 haplotype and HLA-DRB1*03:01 in AIH-1 patients but not controls (D' = 0.92 vs D' = 0.50 respectively; P = 1.3x10(-8)). Soluble HLA-G (sHLA-G) levels were significantly lower in AIH-1 patients compared to controls [13.9 (11.6 - 17.4) U/mL vs 21.3 (16.5 - 27.8) U/mL; P = 0.011]. Twenty-four patients with mild or moderate inflammatory involvement, as assessed from liver biopsy, showed much higher sHLA-G levels compared to the 28 patients with severe liver inflammation [33.5 (23.6 - 44.8) U/mL vs 8.8 (6.1 - 14.5) U/mL; P = 0.003]. Finally, immunohistochemistry analysis of 52 liver biopsies from AIH-1 patients did not show expression of HLA-G molecules in the liver parenchyma. However, a percentage of 69.2% (36/52) revealed widespread expression of HLA-G both in the cytoplasm and the membrane of plasma cells labeled with anti-HLA-G monoclonal antibodies. ConclusionThis study highlights the positive immunomodulatory effect of HLA-G molecules on the clinical course of AIH-1 and how this improvement closely correlates with plasma levels of sHLA-G. However, our results open the debate on the ambiguous role of HLA-G molecules expressed by plasma cells, which are pathognomonic features of AIH-1

Neuroprotection by the Immunomodulatory Drug Pomalidomide in the Drosophila LRRK2WD40 Genetic Model of Parkinson’s Disease

The search for new disease-modifying drugs for Parkinson’s disease (PD) is a slow and highly expensive process, and the repurposing of drugs already approved for different medical indications is becoming a compelling alternative option for researchers. Genetic variables represent a predisposing factor to the disease and mutations in leucine-rich repeat kinase 2 (LRRK2) locus have been correlated to late-onset autosomal-dominant PD. The common fruit fly Drosophila melanogaster carrying the mutation LRRK2 loss-of-function in the WD40 domain (LRRK2WD40), is a simple in vivo model of PD and is a valid tool to first evaluate novel therapeutic approaches to the disease. Recent studies have suggested a neuroprotective activity of immunomodulatory agents in PD models. Here the immunomodulatory drug Pomalidomide (POM), a Thalidomide derivative, was examined in the Drosophila LRRK2WD40 genetic model of PD. Mutant and wild type flies received increasing POM doses (1, 0.5, 0.25 mM) through their diet from day 1 post eclosion, until postnatal day (PN) 7 or 14, when POM’s actions were evaluated by quantifying changes in climbing behavior as a measure of motor performance, the number of brain dopaminergic neurons and T-bars, mitochondria integrity. LRRK2WD40 flies displayed a spontaneous age-related impairment of climbing activity, and POM significantly and dose-dependently improved climbing performance both at PN 7 and PN 14. LRRK2WD40 fly motor disability was underpinned by a progressive loss of dopaminergic neurons in posterior clusters of the protocerebrum, which are involved in the control of locomotion, by a low number of T-bars density in the presynaptic bouton active zones. POM treatment fully rescued the cell loss in all posterior clusters at PN 7 and PN 14 and significantly increased the T-bars density. Moreover, several damaged mitochondria with dilated cristae were observed in LRRK2WD40 flies treated with vehicle but not following POM. This study demonstrates the neuroprotective activity of the immunomodulatory agent POM in a genetic model of PD. POM is an FDA-approved clinically available and well-tolerated drug used for the treatment of multiple myeloma. If further validated in mammalian models of PD, POM could rapidly be clinically tested in humans

Effects of memantine on mania-like phenotypes exhibited by Drosophila Shaker mutants

Introduction: Increased glutamate levels and electrolytic fluctuations have been observed in acutely manic patients. Despite some efficacy of the non-competitive NMDA receptor antagonist memantine (Mem), such as antidepressant-like and mood-stabilizer drugs in clinical studies, its specific mechanisms of action are still uncertain. The present study aims to better characterize the Drosophila melanogaster fly Shaker mutants (SH), as a translational model of manic episodes within bipolar disorder in humans, and to investigate the potential anti-manic properties of Mem. Methods and results: Our findings showed typical behavioral abnormalities in SH, which mirrored with the overexpression of NMDAR-NR1 protein subunit, matched well to glutamate up-regulation. Such molecular features were associated to a significant reduction of SH brain volume in comparison to Wild Type strain flies (WT). Here we report on the ability of Mem treatment to ameliorate behavioral aberrations of SH (similar to that of Lithium), and its ability to reduce NMDAR-NR1 over-expression. Conclusions: Our results show the involvement of the glutamatergic system in the SH, given the interaction between the Shaker channel and the NMDA receptor, suggesting this model as a promising tool for studying the neurobiology of bipolar disorders. Moreover, our results show Mem as a potential disease-modifying therapy, providing insight on new mechanisms of action

Modeling Parkinson’s Disease Neuropathology and Symptoms by Intranigral Inoculation of Preformed Human α-Synuclein Oligomers

The accumulation of aggregated α-synuclein (αSyn) is a hallmark of Parkinson’s disease (PD). Current evidence indicates that small soluble αSyn oligomers (αSynOs) are the most toxic species among the forms of αSyn aggregates, and that size and topological structural properties are crucial factors for αSynOs-mediated toxicity, involving the interaction with either neurons or glial cells. We previously characterized a human αSynO (H-αSynO) with specific structural properties promoting toxicity against neuronal membranes. Here, we tested the neurotoxic potential of these H-αSynOs in vivo, in relation to the neuropathological and symptomatic features of PD. The H-αSynOs were unilaterally infused into the rat substantia nigra pars compacta (SNpc). Phosphorylated αSyn (p129-αSyn), reactive microglia, and cytokine levels were measured at progressive time points. Additionally, a phagocytosis assay in vitro was performed after microglia pre-exposure to αsynOs. Dopaminergic loss, motor, and cognitive performances were assessed. H-αSynOs triggered p129-αSyn deposition in SNpc neurons and microglia and spread to the striatum. Early and persistent neuroinflammatory responses were induced in the SNpc. In vitro, H-αSynOs inhibited the phagocytic function of microglia. H-αsynOs-infused rats displayed early mitochondrial loss and abnormalities in SNpc neurons, followed by a gradual nigrostriatal dopaminergic loss, associated with motor and cognitive impairment. The intracerebral inoculation of structurally characterized H-αSynOs provides a model of progressive PD neuropathology in rats, which will be helpful for testing neuroprotective therapies

Evolution of the microbiological profile of vacuum-packed ricotta salata cheese during shelf-life

Ricotta salata cheese is a salted variety of ricotta traditionally made in Sardinia (Italy) from the whey remaining after the production of Pecorino Romano protected designation of origin or other sheep milk cheeses. Ricotta salata cheese is very critical for the possible growth of pathogenic and spoilage microorganisms. Sporadic cases of listeriosis associated with ricotta salata cheese have been reported over recent years. The objective of the present study was to assess the evolution of spoilage and pathogen microorganism of vacuum-packed ricotta salata cheese during the entire product shelf-life. The durability study was conducted on 18 vacuum-packed ricotta salata cheese samples analysed at the beginning of the shelf-life and after 60 and 90 days of refrigerated storage. Pathogens as Listeria monocytogenes and Bacillus cereus were never detected. During shelf-life total bacterial counts ranged between 7.90±0.64 and 9.19±0.58 CFU g-1 on the rind and between 2.95±0.68 and 4.27±1.10 CFU g-1 in the inner paste, while Enterobacteriaceae ranged between 4.22±0.66 and 5.30±0.73 CFU g-1 on the rind and 3.13±1.80 and 2.80±0.88 CFU g-1 in the inner paste. By considering the technology used, the intrinsic properties and the almost total absence of competing microflora, ricotta salata cheese can support the growth of spoilage and pathogen microorganisms originating from the processing environment. The high level of total bacterial counts and Enterobacteriaceae observed both on the rind and in the inner paste suggests contamination of the product from the processing environment. Therefore, a strict implementation of hygiene during processing is essential in order to reduce the load of environmental contaminants that may grow during refrigerated storage