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Prion Diseases

#B-1440


Surveillance of Transmissible Spongiform Encephalopathies in the Republic of Belarus, Study of Astrocyte Role in the Disease Pathogenesis, and Development of Proteome- and Nanotechnology-Based Method for Pre-Clinical PrPd Detection

Tech Area / Field

  • AGR-DIS/Disease Surveillance/Agriculture
  • AGR-DIG/Diagnostics/Agriculture
  • MED-DID/Diagnostics & Devices/Medicine

Status
3 Approved without Funding

Registration date
03.08.2006

Leading Institute
Belarussian Research Institute for Epidemiology and Microbiology, Belarus, Minsk

Collaborators

  • Canadian Food Inspection Agency / National Centre for Foreign Animal Disease / National BSE Reference Laboratory, Canada, MB, Winnipeg\nAmerican Red Cross, USA, MD, Rockville

Project summary

Project goal: to implement epidemiological surveillance of transmissible spongiform encephalopathies (TSE)/prion diseases in Belarus, to study a role of astrocytes in TSE pathogenesis, and to develop new diagnostic approach for early detection of animal and human TSEs using the proteome- and nanotechnology-based methods.

Formulation of task: Epidemiological situation on occurrence of TSEs in Belarus is unknown. Presently, the laboratory diagnosis of Creutzfeldt-Jakob disease (CJD) and other TSEs is based on post-mortem detection of abnormal disease-associated prion protein (PrPd) in the brain of afflicted inpiduals. There is no evidence that BSE or vCJD have occurred in Belarus but a theoretical possibility of their presence exists because of trading practices and tourism. However, deficiencies in continuous and extensive surveillance of these diseases due to the lack of appropriate effective methods for TSE detection did not permit to claim with certainty their absolute absence. The situation in the world and CIS states calls for development of reliable, sensitive and fast methods for detection of TSEs in humans and animals.

The role of astroglial cells, a CNS structural component, in pathogenesis of TSEs has not been fully investigated, determining the need for their study. Primary astrocyte cell cultures may serve as a sensitive model for studying the disease pathogenesis and demonstration of TSE infectivity in various samples of human and animal origin, thus replacing the more expensive animal models.

Influence of proposed project on progress in the field of development:

  1. Epidemiological situation on prion infections in the Republic of Belarus will be studied;
  2. Knowledge concerning the pathogenesis of TSEs will be expanded. The mechanisms of the astrocyte reaction to the TSE agents will be elucidated in vitro. Biological and biochemical properties of new and archived TSE isolates will be described;
  3. Method, based on application of proteomic and nanotechnology for detection of PrPd will be developed and samples of different tissues, including blood, CSF, brain, and lymphoreticular tissues from humans and animals will be tested.

Project participants: Project will be carried out at the State Institution "Research Institute for Epidemiology and Microbiology" (RIEM) of the Ministry of Public Health of the Republic of Belarus, which will serve as a Reference Center for the epidemiological monitoring and studies of TSEs in Belarus. The majority of specialists, which will carry out the project, have a long-standing experience in the area of "weapons" knowledge (code 3.4). The laboratories involved with the project have well established structures of highly intellectual, professional and skilled experts in the fields of virological, microbiological and immunological research, and adequate space capacities and the significant portion of capital equipment necessary for the project.

Expected results: Research includes 3 categories of development of technologies: basic researches, applied researches and development. We expect to achieve the following results by carrying out research in the each of three basic tasks:

First task:

  1. epidemiological surveillance of TSE in Belarus will be implemented;
  2. compliance with WHO recommendations on detection, registration, and diagnosis of CJD will be assured on the territory of Belarus;
  3. training on TSE diseases for different specialists including epidemiologists, neurologists, neuropathologists, psychiatrists, pediatricians, virologists, biochemists and laboratory technicians will be organized and provided;

Second Task:
  1. cell models for studies of the TSE isolates and their characterization will be established;
  2. the ability of prions (2-3 strains) to reproduce in primary and continuous astrocyte cultures will be studied;
  3. the kinetics of prion propagation and accumulation within long-term passages (5-7 passages, up to 270 days) will be studied;
  4. cell and sub-cell PrPd localization in astrocytes at different passage levels will be investigated;
  5. prion-induced changes in cell morphology and function, including metabolic changes and mechanisms of apoptosis, will be determined;
  6. samples of blood, cerebrospinal fluid (CSF) and brain tissues (if possible) from possible and probable CJD cases will be collected and a final diagnosis will be histopathologically confirmed (when possible);
  7. collaboration with foreign laboratories-partners for exchange of materials and information will be established.

Third task:
  1. proteome-based approaches for pre-clinical diagnosis of human TSEs, using scanning microscopy and laser scanning nanotechnology will be developed;
  2. methods of prion detection in humans and animals will be improved with use of modern nanotechnology-based approach and high specific monoclonal antibodies to PrPd will be developed and tested;
  3. tests for human TSEs detection will be standardized in different geographical regions of Belarus;
  4. experiments on detection of PrPd in blood lymphocytes and plasma from TSE patients will be undertaken for evaluation of transmission risk associated with blood transfusion and use of plasma-derived products.

Project results application: Epidemiological surveillance for human and animal TSE will elucidate the occurrence of human disease and distribution of the animal diseases within the borders of Belarus. Specimens of new human and animal TSE isolates will be collected, archived and characterized by biochemical, histochemical, immunological methods and by inoculation into appropriate cell cultures and rodent models.

Susceptibility of astrocytes to TSE isolates will be tested in vitro and pathological changes caused by the infection will be registered and correlated with in vivo findings. Role of astrocytes in pathogenesis of TSE diseases will be clarified. The value of primary astrocyte cell cultures, as a model replacing experimental animals, for bioassays will be revealed.

Developed new diagnostic methods for detection of PrPd will be used for epidemiological monitoring of TSEs and interpretation and verification of human (iatrogenic, sporadic, hereditary) and animal cases of the disease at the Reference Laboratories of the Ministry of Public Health, Ministry of the Veterinary Science and Ministry of Agriculture and Food.

Meeting ISTC goals and objectives: Proposed project meets the following ISTC goals and objectivities:

  1. provides “weapons” scientists and engineers in the Belarus with opportunities to redirect their talents to peaceful activities;
  2. promotes integration of scientists of CIS states into international scientific community;
  3. supports basic research for peaceful purposes, especially in the field of public health and veterinary sciences;
  4. contributes to solution of national or international problems for preservation of the population health against infectious diseases.

Outline scope of activities: The duration of the project is planned for 36 months.
  1. Infectious agents to be investigated belong to prions that have been identified as a cause of transmissible spongiform encephalopathies of human and animals. Archived and/or new isolates of TSEs that will be used for the studies include: agent scrapie, the strain 263K (prototype strain); agent CJD, strain Fukuoka-1 (prototype strain); agent CJD, strain CJD-DV (author's strain was deposited under No. GKV 891 at the State Virus Collection of Ivanovsky Institute of Virology, Moscow); homogenates of brain from human with CJD and lab animals with experimental CJD, whole blood, plasma and lymphocytes from CJD patients and laboratory animals with experimental scrapie and CJD; clinical material received from people with various immunodeficiency and neurodegenerative diseases;
  2. Cell culture to be used: cultures of highly differentiated astrocytes obtained from the brains of intact mice and mice with the experimental scrapie and CJD that have been long-term cultivated in subpassages (5-7 passages, up to 270 days); continuous rat glioma cell line, C-6;
  3. Laboratory animals to be used: outbreed mice; transgenic mice carrying human prion protein (PrP) gene (will be obtained through collaboration or purchased from Dr. Jean Manson, UK); Syrian golden hamsters.

Research at the laboratories will be performed according to Standard Operating Procedures (SOP) and protocols that were discussed with consultants and approved by institutional authorities. Ethical criteria will be implemented when handling confidential human subject information and experimental animals. Collected data will be stored as a hard copies in laboratory journals and electronically and will be made available to collaborators and inspectors from ISTC upon request.

Role of foreign collaborators: According to the volume of activity within the framework of suggested project the collaboration with foreign collaborators from the USA
(
Dr. L. Cervenakova, Transmissible Diseases Department, the American National Red Cross) and Canada (Dr. S. Czub, National BSE Reference Laboratory, Pathology National Centre for Foreign Animal Diseases) will be carried out in the following directions:

  1. scientific consultations with information exchange in the course of project realization;
  2. consultation on writing of SOPs and laboratory protocols and training;
  3. review and discussion of technical reports (quarterly, annual, final, etc.) that will be submitted by project leader to the ISTC;
  4. exchange of biological materials according to institutional, regional, national and international rules;
  5. testing and evaluation of technologies, developed in the course of the project;
  6. participation in seminars and workshops;
  7. writing of joint scientific publications.

Technical approach and methodology: Epidemiological surveillance in different geographical regions of Belarus and diagnosis of human TSE will be performed according to international standards. The working group of epidemiologists, neurologists, neuropathologists and researchers (virologists, microbiologists, molecular biologists) will be formed. The objective of the group will be to introduce the TSE diagnostic criteria based on clinical and laboratory data and establish the registry of all TSE cases using medical consultative services and by the analysis of inpidual’s "death certificates" with diagnosis of neurodegenerative disease. The seminars and practical training on TSE diagnosis for physicians of different specialties will be provided. This will allow for continued surveillance of each suspected TSE case. With such integral approach the true morbidity and mortality from TSEs will be reveled and control on emerging new variant of CJD and other transmissible spongiform encephalopathies (genetic and iatrogenic forms) will be established.

To obtain new fundamental knowledge on TSEs etiopathogenesis the agent isolation from each diagnosed TSE cases, with subsequent study of their biological properties on primary culture model of highly differentiated astroglial cells and laboratory animals will be attempted. The accumulation of PrPd in different organs of experimental animals and the ability of isolated agents to propagate in astrocytes and will be investigated, and agent-induced metabolic "shifts" in these cells will be detected. The study of PrPd kinetics of accumulation, its cellular and subcellular localization, and prion-induced changes in metabolism and ultrastructure of astrocytes by long-term cultivation in subpassages (5-10 passages, up to 270 days) will be conducted.

We will be used: light, phase contrast, immunofluorescent, scanning probe, and laser microscopy, electronic and immune electronic microscopy; immunocytochemical methods; polyacrylamide gel electrophoresis and immunoblots; morphologic and densitometry methods; etc. Method of scanning probe microscopy (SPM) and the specifically developed immunosensors (biochips) carrying the antibodies against PrP will be used and tested. The main steps in development of new technologies will include optimization of the method for nano-lithographic immobilization of PrP-specific antibodies on polymeric layers with spatial resolution not less than 1 m; optimization of the processes for immuno-assay of complexes (antibody-PrP + antibody-PrPd + targeted secondary antibodies) on polymeric layers of biochips with following counts of formed nano-marker particles. It is expected that the new type of biosensor systems of PrPd will allow detection of low protein concentrations in blood, CSF, spleen, muscles and other organs and tissues.


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