BACKGROUND: As a step towards clinical use of AAV-mediated gene therapy, brains of large animals are used to settle delivery parameters as most brain connections, and relative sizes in large animals and primates, are reasonably common. Prior to application in the clinic, approaches that have shown to be successful in rodent models are tested in larger animal species, such as dogs, non-human primates, and in this case, minipigs. NEW METHOD: We evaluated alternate delivery routes to target the basal ganglia by injections into the more superficial corona radiata, and, deeper into the brain, the thalamus. Anatomically known connections can be used to predict the expression of the transgene following infusion of AAV5. For optimal control over delivery of the vector with regards to anatomical location in the brain and spread in the tissue, we have used magnetic resonance image-guided convection-enhanced diffusion delivery. RESULTS: While the transduction of the cortex was observed, only partial transduction of the basal ganglia was achieved via the corona radiata. Thalamic administration, on the other hand, resulted in widespread transduction from the midbrain to the frontal cortex COMPARISON WITH EXISTING METHODS: Compared to other methods, such as delivery directly to the striatum, thalamic injection may provide an alternative when for instance, injection into the basal ganglia directly is not feasible. CONCLUSIONS: The study results suggest that thalamic administration of AAV5 has significant potential for indications where the transduction of specific areas of the brain is required.
- MeSH
- Dependovirus genetika MeSH
- genetická terapie metody MeSH
- genetické vektory MeSH
- konvekce * MeSH
- magnetická rezonanční tomografie MeSH
- miniaturní prasata genetika MeSH
- prasata MeSH
- psi MeSH
- thalamus * diagnostické zobrazování MeSH
- zvířata MeSH
- Check Tag
- psi MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Major histocompatibility complex (MHC) is an important complex that presents antigen to T cells. The second exon of swine MHC (SLA) class II genes has antigen binding sites that bind with extracellular antigen. Populations with high MHC gene diversity result in low gut microbiota, and individuals with MHC gene heterozygote have lower gut microbiota diversity than that of homozygote. The pig is an animal with organs physiologically and anatomically similar to humans than any other mammal, and the pig is also suitably developed as a laboratory animal to establish the animal models of human disease. However, the relationship between SLA genetic diversity and the gut microbes of the pig is ambiguous. We studied the characterization of SLA class II genes and calculated the genetic diversity, and then we selected experimental animal groups divided by different SLA genotypes to investigate the gut microbiota composition by sequencing V3 to V4 hypervariable regions of bacterial 16 s rRNA from fecal samples. Our results showed that Guizhou minipigs had a low SLA genetic diversity, which may be due to the small founder population. The Guizhou minipig population deviated from neutral selection and balancing selection, which shows that Guizhou minipigs experience a strong artificial selection in recent years. We observed that the sex differences influenced gut microbiota much more deeply than that of genetics. Our results also showed that the individual with heterozygote of genes at the SLA class II region had much higher abundant gut microbiota than that of the homozygote.
National cancer databases document that melanoma is the most aggressive and deadly cutaneous malignancy with worldwide increasing incidence in the Caucasian population. Around 10% of melanomas occur in families. Several germline mutations were identified that might help to indicate individuals at risk for preventive interventions and early disease detection. More than 50% of sporadic melanomas carry mutations in Ras/Raf/mitogen-activated protein kinase (MAPK/MEK) pathway, which may represent aims of novel targeted therapies. Despite advances in targeted therapies and immunotherapies, the outcomes in metastatic tumor are still unsatisfactory. Here, we review animal models that help our understanding of melanoma development and treatment, including non-vertebrate, mouse, swine, and other mammal models, with an emphasis on those with spontaneously developing melanoma. Special attention is paid to the melanoma-bearing Libechov minipig (MeLiM). This original swine model of hereditary metastatic melanoma enables studying biological processes underlying melanoma progression, as well as spontaneous regression. Current histological, immunohistochemical, biochemical, genetic, hematological, immunological, and skin microbiome findings in the MeLiM model are summarized, together with development of new therapeutic approaches based on tumor devitalization. The ongoing study of molecular and immunological base of spontaneous regression in MeLiM model has potential to bring new knowledge of clinical importance.
- MeSH
- melanom genetika MeSH
- miniaturní prasata genetika MeSH
- modely nemocí na zvířatech MeSH
- nádory kůže genetika MeSH
- prasata genetika MeSH
- progrese nemoci MeSH
- sekundární malignity genetika MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
BACKGROUND: While several novel therapeutic approaches for HD are in development, resources to conduct clinical trials are limited. Large animal models have been proposed to improve assessment of safety, tolerability and especially to increase translational reliability of efficacy signals obtained in preclinical studies. They may thus help to select candidates for translation to human studies. We here introduce a battery of novel tests designed to assess the motor, cognitive and behavioral phenotype of a transgenic (tg) HD minipig model. NEW METHODS: A group of tgHD and wildtype (wt) Libechov minipigs (n=36) was available for assessment with (1) a gait test using the GAITRite(®) automated acquisition system, (2) a hurdle-test, (3) a tongue coordination test, (4) a color discrimination test, (5) a startbox back and forth test and (6) a dominance test. Performance of all tests and definition of measures obtained is presented. RESULTS: Minipigs were able to learn performance of all tests. All tests were safe, well tolerated and feasible. Exploratory between group comparisons showed no differences between groups of tgHD and wt minipigs assessed, but low variability within and between groups. COMPARISON WITH EXISTING METHOD(S): So far there are no established or validated assessments to test minipigs in the domains described. CONCLUSIONS: The data shows that the tests presented are safe, well tolerated and all measures defined can be assessed. Prospective longitudinal application of these tests is warranted to determine their test-retest reliability, sensitivity and validity in assessing motor, cognitive and behavioral features of tg and wt minipigs.
- MeSH
- chování zvířat * MeSH
- chůze (způsob) fyziologie MeSH
- design vybavení MeSH
- diskriminace (psychologie) fyziologie MeSH
- fenotyp MeSH
- geneticky modifikovaná zvířata * MeSH
- Huntingtonova nemoc genetika MeSH
- jazyk patofyziologie MeSH
- lidé MeSH
- miniaturní prasata genetika fyziologie psychologie MeSH
- modely nemocí na zvířatech * MeSH
- neuropsychologické testy MeSH
- pohybová aktivita fyziologie MeSH
- prasata MeSH
- protein huntingtin genetika metabolismus MeSH
- studie proveditelnosti MeSH
- učení fyziologie MeSH
- vidění barevné fyziologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Maligní melanom je jedním z nejzávažnějších kožních nádorů u lidí i zvířat. Je vysoce odolný vůči konvenčním terapiím a i přes velké úsilí při vývoji nových imunoterapií došlo jen k jejich drobným vylepšením. Proto se jako ideální směr dalšího výzkumu jeví možnost vývoje imunoterapií, které by byly schopné navodit kompletní regresi nádoru, což je ideální výsledek při léčbě jakéhokoliv nádor. Bohužel podmínky potřebné k dosažení úplné regrese nejsou dosud dobře známé. V laboratoři biologie nádorů v Ústavu živočišné fyziologie a genetiky AV ČR je k dispozici model melanomu u miniaturních prasat, u kterého dochází k spontánní regresi nádorů po období progrese spojené s metastázami především do sleziny, plic a lymfatických uzlin, kdy dojde k vyléčení většiny zvířat a pouze asi 5 % umírá na progresi nádoru nebo přidružené komplikace. Další výzkum MeLiM modelu umožní získat nové poznatky o spontánní regresi melanomu a nabídne nové možnosti pro tvorbu účinnějších imunoterapií.
Malignant melanoma is one of the most serious skin cancer diseases in humans and animals. It is highly resistant to conventional therapies and despite major efforts in development of novel immunotherapies there have been only minor improvements. Therefore, an ideal further research should be led towards developing immunotherapies that would be capable of inducing a complete tumor regression. This is the ideal result of the treatment of all tumor. Unfortunately, the conditions required to achieve complete regressions are not well known yet. In the laboratory of tumor biology at the Institute of Animal Physiology and Genetics AS CR an animal model of melanoma is being researched, ie melanoma bearing Libechov minipigs, in which spontaneous tumor regression occurs after a period of progression associated with organ metastases - mainly in the spleen, lungs and lymph nodes. Most animals are completelycured, but around 10 % die of tumor progression or associated complications. Further research of MeLiM model will provide new knowledge about the spontaneous regression of melanoma and offer new possibilities for creating more effective immunotherapy.
- MeSH
- Cyprinodontiformes MeSH
- lidé MeSH
- melanom * MeSH
- miniaturní prasata * genetika MeSH
- modely nemocí na zvířatech MeSH
- myši MeSH
- spontánní regrese nádoru MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5' long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5'LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.
- MeSH
- DNA virů genetika MeSH
- endogenní retroviry genetika MeSH
- epigeneze genetická * MeSH
- koncové repetice genetika MeSH
- kultivované buňky MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- ledviny metabolismus virologie MeSH
- lidé MeSH
- messenger RNA genetika MeSH
- metylace DNA * MeSH
- miniaturní prasata genetika virologie MeSH
- nemoci prasat genetika přenos virologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- prasata MeSH
- proviry genetika MeSH
- replikace viru * MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
S tím, jak se zvyšuje průměrný věk obyvatelstva, roste i počet lidí trpících neurodegenerativními onemocněními. Bohužel, dodnes nebyl nalezen žádný způsob terapie těchto chorob. Připraveno je mnoho produktů, které by mohly být použité k jejich léčení, avšak pro jejich verifikaci v pre-klinických testech je potřebný vhodný zvířecí model. Ačkoli malé zvířecí modely poskytují mnoho informací, čast o dostatečně nesimulují průběh nemoci u pacientů. Proto jsme vytvořili transgenní miniaturní prase jako model pro studium Huntingtonovy choroby, která je způsobena mutací v genu zvaném huntingtin. Naše snaha je zaměřena na charakterizaci její pre-klinického i klinického stádia. Dlouhodobé sledování fenotypu umožní získat potřebná data o průběhu choroby na molekulárně genetické, metabolické i behaviorální úrovni a poskytne informace o možnosti použití tohoto modelu pro testování potenciálních terapií neurodegenerativních onemocnění.
As aged population grows, the number of people with age-related neurodegenerative diseases is rising fast. Unfortunately, no known therapy is available to treat these diseases. The list of compounds could be used in treatment of neurodegenerative disorders but suitable animal model for pre-clinical tests is necessary to be generated. Although, small animal models of human diseases are highly informative they often do not faithfully mimic the human conditions. Miniature pig seems to be suitable large animal model for simulating of neurodegenerative diseases. We have generated transgenic miniature pig as a model of Huntington’s Disease which is caused by the mutation in huntingtin gene. Our current effort is focused on characterization of both pre-manifest and manifest stages of HD. Phenotyping will allow the collection of longitudinal molecular data, to complement metabolic, behavioral and MRI assessments that will yield detailed information of the disease process over a decade or more and will provide information about possibilities of translational research.
