Prion Strain Characterization of a Novel Subtype of Creutzfeldt-Jakob Disease.

In 2007, we reported a patient with an atypical form of Creutzfeldt-Jakob disease (CJD) heterozygous for methionine-valine (MV) at codon 129 who showed a novel pathological prion protein (PrPTSE) conformation with an atypical glycoform (AG) profile and intraneuronal PrP deposition. In the present study, we further characterize the conformational properties of this pathological prion protein (PrPTSE MVAG), showing that PrPTSE MVAG is composed of multiple conformers with biochemical properties distinct from those of PrPTSE type 1 and type 2 of MV sporadic CJD (sCJD). Experimental transmission of CJD-MVAG to bank voles and gene-targeted transgenic mice carrying the human prion protein gene (TgHu mice) showed unique transmission rates, survival times, neuropathological changes, PrPTSE deposition patterns, and PrPTSE glycotypes that are distinct from those of sCJD-MV1 and sCJD-MV2. These biochemical and experimental data suggest the presence of a novel prion strain in CJD-MVAGIMPORTANCE Sporadic Creutzfeldt-Jakob disease is caused by the misfolding of the cellular prion protein, which assumes two different major conformations (type 1 and type 2) and, together with the methionine/valine polymorphic codon 129 of the prion protein gene, contribute to the occurrence of distinct clinical-pathological phenotypes. Inoculation in laboratory rodents of brain tissues from the six possible combinations of pathological prion protein types with codon 129 genotypes results in the identification of 3 or 4 strains of prions. We report on the identification of a novel strain of Creutzfeldt-Jakob disease isolated from a patient who carried an abnormally glycosylated pathological prion protein. This novel strain has unique biochemical characteristics, does not transmit to humanized transgenic mice, and shows exclusive transmission properties in bank voles. The identification of a novel human prion strain improves our understanding of the pathogenesis of the disease and of possible mechanisms of prion transmission.

Chronic wasting disease and atypical forms of bovine spongiform encephalopathy and scrapie are not transmissible to mice expressing wild-type levels of human prion protein.

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle transmissible spongiform encephalopathies (TSEs) can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several novel TSEs in sheep, cattle and deer have been described and the risk posed to humans by these agents is currently unknown. In this study, we inoculated two forms of atypical BSE (BASE and H-type BSE), a chronic wasting disease (CWD) isolate and seven isolates of atypical scrapie into gene-targeted transgenic (Tg) mice expressing the human prion protein (PrP). Upon challenge with these ruminant TSEs, gene-targeted Tg mice expressing human PrP did not show any signs of disease pathology. These data strongly suggest the presence of a substantial transmission barrier between these recently identified ruminant TSEs and humans.

Mycobacterium smegmatis expressing a chimeric protein MPT64-proteolipid protein (PLP) 139-151 reorganizes the PLP-specific T cell repertoire favoring a CD8-mediated response and induces a relapsing experimental autoimmune encephalomyelitis.

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139-151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMS(p139)). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMS(p139) was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMS(p139) modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4(+) T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMS(p139) because lymph node APCs infected with rMS(p139) selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMS(p139) expanded p139-specific CD8(+) cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross-reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.

Neuroinvasion of the 263K scrapie strain after intranasal administration occurs through olfactory-unrelated pathways.

The olfactory system has been implicated in the pathogenesis of transmissible spongiform encephalopathies (TSEs). To examine this issue and identify the pattern of TSE agent spread after intranasal administration, we inoculated a high-infectious dose of neurotropic scrapie strain 263K into the nasal cavity of Syrian hamsters. All animals allowed to survive became symptomatic with a mean incubation period of 162.4 days. Analysis at different time points revealed deposition of the pathological prion protein (PrP(TSE)) in nasal-associated lymphoid tissues in the absence of brain involvement from 80 days post-infection (50% of the incubation period). Olfactory-related structures and brainstem nuclei were involved from 100 days post-inoculation (62% of the incubation period) when animals were still asymptomatic. Intriguingly, vagal or trigeminal nuclei were identified as early sites of PrP(TSE) deposition in some pre-symptomatic animals. These findings indicate that the 263K scrapie agent is unable to effectively spread from the olfactory neuroepithelium to the olfactory-related structures and that, after intranasal inoculation, neuroinvasion occurs through olfactory-unrelated pathways.

Efficient transmission and characterization of Creutzfeldt-Jakob disease strains in bank voles.

Transmission of prions between species is limited by the "species barrier," which hampers a full characterization of human prion strains in the mouse model. We report that the efficiency of primary transmission of prions from Creutzfeldt-Jakob disease patients to a wild rodent species, the bank vole (Clethrionomys glareolus), is comparable to that reported in transgenic mice carrying human prion protein, in spite of a low prion protein-sequence homology between man and vole. Voles infected with sporadic and genetic Creutzfeldt-Jakob disease isolates show strain-specific patterns of spongiform degeneration and pathological prion protein-deposition, and accumulate protease-resistant prion protein with biochemical properties similar to the human counterpart. Adaptation of genetic Creutzfeldt-Jakob disease isolates to voles shows little or no evidence of a transmission barrier, in contrast to the striking barriers observed during transmission of mouse, hamster, and sheep prions to voles. Our results imply that in voles there is no clear relationship between the degree of homology of the prion protein of the donor and recipient species and susceptibility, consistent with the view that the prion strain gives a major contribution to the species barrier. The vole is therefore a valuable model to study human prion diversity and, being susceptible to a range of animal prions, represents a unique tool for comparing isolates from different species.

Ralstonia mannitolilytica infections in an oncologic day ward: description of a cluster among high-risk patients.

BACKGROUND: Ralstonia spp, an environmental microorganism, has been occasionally associated with healthcare infections. The aim of this study was to investigate an outbreak caused by Ralstonia mannitolilytica in oncology patients. METHODS: Case definition: Oncology outpatients attending a day ward, with positive blood and/or central venous catheter (CVC) culture for Ralstonia spp from September 2013 - June 2014. We analysed medical records, procedures and environmental samples. R. mannitolilytica was identified by 16S rRNA sequencing, and typed by Pulsed Field Gel Electrophoresis (PFGE); resistance to carbapenemes was investigated by phenotypic and molecular methods. RESULTS: The patients (N = 22) had different malignancies and received different therapy; all had a CVC and 16 patients presented chills and/or fever. R. mannitolilytica was isolated from both blood and CVC (n = 12) or only blood (n = 6) or CVC tips (n = 4). The isolates had indistinguishable PFGE profile, and showed resistance to carbapenems. All the isolates were negative for carbapenemase genes while phenotypic tests suggests the presence of an AmpC ß-lactamase activity,responsible for carbapenem resistance. All patients had had CVC flushed with saline to keep the venous access pervious or before receiving chemotherapy at various times before the onset of symptoms. After the first four cases occurred, the multi-dose saline bottles used for CVC flushing were replaced with single-dose vials; environmental samples were negative for R. mannitolilytica. CONCLUSIONS: Although the source of R. mannitolilytica remains unidentified, CVC flushing with contaminated saline solution seems to be the most likely origin of R. mannitolilytica CVC colonization and subsequent infections. In order to prevent similar outbreaks we recommend removal of any CVC that is no longer necessary and the use of single-dose solutions for any parenteral treatment of oncology patients.

Scrapie infectivity is quickly cleared in tissues of orally-infected farmed fish.

BACKGROUND: Scrapie and bovine spongiform encephalopathy (BSE) belongs to the group of animal transmissible spongiform encephalopathy (TSE). BSE epidemic in the UK and elsewhere in Europe has been linked to the use of bovine meat and bone meals (MBM) in the feeding of cattle. There is concern that pigs, poultry and fish bred for human consumption and fed with infected MBM would eventually develop BSE or carry residual infectivity without disease. Although there has been no evidence of infection in these species, experimental data on the susceptibility to the BSE agent of farm animals other than sheep and cow are limited only to pigs and domestic chicken. In the framework of a EU-granted project we have challenged two species of fish largely used in human food consumption, rainbow trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus), with a mouse-adapted TSE strain (scrapie 139A), to assess the risk related to oral consumption of TSE contaminated food. In trout, we also checked the "in vitro" ability of the pathological isoform of the mouse prion protein (PrPSc) to cross the intestinal epithelium when added to the mucosal side of everted intestine. RESULTS: Fish challenged with a large amount of scrapie mouse brain homogenate by either oral or parenteral routes, showed the ability to clear the majority of infectivity load. None of the fish tissues taken at different time points after oral or parenteral inoculation was able to provoke scrapie disease after intracerebral inoculation in recipient mice. However, a few recipient mice were positive for PrPSc and spongiform lesions in the brain. We also showed a specific binding of PrPSc to the mucosal side of fish intestine in the absence of an active uptake of the prion protein through the intestinal wall. CONCLUSION: These results indicate that scrapie 139A, and possibly BSE, is quickly removed from fish tissues despite evidence of a prion like protein in fish and of a specific binding of PrPSc to the mucosal side of fish intestine.

Molecular diagnostics of transmissible spongiform encephalopathies.

Clinical criteria for the diagnosis of sporadic, iatrogenic and variant Creutzfeldt-Jakob diseases are now available and show an excellent sensitivity and specificity ( approximately 98%). Post-mortem diagnosis, based upon the identification in the brain of the pathological conformer of the prion protein (PrP(Sc)), is also very accurate, and several diagnostic kits are now available that facilitate the immunochemical measurement of PrP(Sc). Several new molecular diagnostic techniques aimed at increasing the sensitivity and specificity of PrP(Sc) detection, and at identifying markers of disease that are other than PrP(Sc), are the subject of ongoing studies. The aim of these studies is to develop preclinical screening tests for the identification of infected, but still healthy, individuals. These tests are also badly needed to check the safety of blood or blood-derived products, and to ensure meat safety in European countries.

A desk review on institutional and non-institutional organizations active in the field of migrant's health in the WHO European Region.

BACKGROUND: Migrants have problematic access to health-care; non-institutional organizations (NGOs), as well as institutional bodies may play a role in facilitating their access to mainstream health care. AIM: Our research reviews actions that address the need of migrants in terms of health care in order to understand how, where, and who participates in this effort. METHOD: Data were from desk or web research, declaration from organisations and their websites, information from WHO Country Offices. RESULTS: 154 NGOs were identified in the WHO European Region. 58% were direct health care providers while the remaining provided either mediation services or were part of a network organization. 173 national institutes (GOVs) were found; less than the 20% were directly or indirectly involved in health care, whereas the majority were involved in research, policy development, international relations and human rights. CONCLUSION AND RECOMMENDATION: Some gaps, a certain fragmentation and lack of coordination were identified. WHO can play an overarching role in the exchange of expertise and harmonisation of the efforts in this field.

Risk factors for tuberculosis in foreign-born people (FBP) in Italy: a systematic review and meta-analysis.

In Italy, TB notifications in foreign-born people (FBP) are steadily increasing. To investigate this issue we did a meta-analysis on risk factors for FBP people. A systematic search was performed in PubMed and EMBASE from Jan-1980 to Jan-2013. We analysed HIV status, previous TB-treatment, intravenous drug use and alcohol abuse, and multidrug resistant TB. Odd ratio was used as a measure of effect. One and two-stages approaches were used. In the main analysis we used a 2-stages approach to include studies with only aggregate estimates. Among 1996 references, 18 fulfilled inclusion criteria. In TB-affected FBP people positive HIV-status was about 3 times higher than among Italians, after 1996 when combined antiretroviral therapy for HIV was introduced (OR: 2.91; 95%CI: 1.37; 6.17). No association was found between FBP and intravenous drug users in adults; after 1-stage meta-analysis foreign born people from highly endemic countries had a 4 times higher risk to be multidrug resistant TB than Italian people. Finally, TB-affected FBP were less likely than Italians to be alcoholics (OR: 0.10 95%CI: 0.01; 0.84) or of having received previous TB-treatment (OR: 0.55; 95%CI: 0.43; 0.71). An association of multidrug resistant TB with immigrant status as well as an association of Tuberculosis with HIV-positive status in foreign-born people are major findings of this analysis. Drugs and alcohol abuse do not appear to be risk factors for TB in FBP, however they cannot be discharged since may depend on cultural traditions and their role may change in the future along with the migratory waves. An effective control of TB risk factors among migrants is crucial to obtain the goal of TB eradication.