Institute of Preclinical Sciences

Primerjava mezenhimskih matičnih celic iz maščobnega tkiva psov in mačk

lukam — Mon, 25 Jan 2021 09:51:53 +0000

Comparison of Canine and Feline Adipose-Derived Mesenchymal Stem Cells/Medicinal Signaling Cells lukam Mon, 01/25/2021 - 10:55 Institute of Preclinical Sciences mesenchymal stem cells dog cat comparison proliferation differentiation cell surface marker viability Research

Comparison of Canine and Feline Adipose-Derived Mesenchymal Stem Cells/Medicinal Signaling Cells With Regard to Cell Surface Marker Expression, Viability, Proliferation, and Differentiation Potential.

Remarkable immunomodulatory abilities of mesenchymal stem cells, also called multipotent mesenchymal stromal cells or medicinal signaling cells (MSCs), have entailed significant advances in veterinary regenerative medicine in recent years. Despite positive outcomes from MSC therapies in various diseases in dogs and cats, differences in MSC characteristics between small animal veterinary patients are not well-known. We performed a comparative study of cells' surface marker expression, viability, proliferation, and differentiation capacity of adipose-derived MSCs (ADMSCs) from dogs and domestic cats. The same growth media and methods were used to isolate, characterize, and culture canine and feline ADMSCs. Adipose tissue was collected from 11 dogs and 8 cats of both sexes. The expression of surface markers CD44, CD90, and CD34 was detected by flow cytometry. Viability at passage 3 was measured with the hemocytometer and compared to the viability measured by flow cytometry after 1 day of handling. The proliferation potential of MSCs was measured by calculating cell doubling and cell doubling time from second to eighth passage. Differentiation potential was determined at early and late passages by inducing cells toward adipogenic, osteogenic, and chondrogenic differentiation using commercial media. Our study shows that the percentage of CD44+CD90+ and CD34−/− cells is higher in cells from dogs than in cells from cats. The viability of cells measured by two different methods at passage 3 differed between the species, and finally, canine ADMSCs possess greater proliferation and differentiation potential in comparison to the feline ADMSCs. The results show that in the development of new cell therapies it is necessary to take into account the animal species and the results of stem cell research cannot be easily transferred from one species to another.

Authors: Metka Voga, Valerija Kovač, Gregor Majdič

The full article is published in Frontiers in Veterinary Science

Nitrozativni stres v možganih psov s pasjo kognitivno motnjo

lukam — Mon, 18 Jan 2021 14:28:53 +0000

Nitrosative stress in brains from dogs with canine cognitive dysfunction lukam Mon, 01/18/2021 - 15:47 Institute of Preclinical Sciences Kognitivna motnja pes nitrozativni stres nNOS eNOS iNOS 3-nitrotirozin Research

Nitrosative stress in the frontal cortex from dogs with canine cognitive dysfunction.

Canine cognitive dysfunction (CCD) is an age-related disorder similar to human Alzheimer’s disease (AD) that occurs in elderly dogs. Nitrosative stress has been implicated as one of the causes leading to neurodegenerative diseases, particularly AD. Its involvement in the development of CCD has not been studied so far. In the present study, immunohistochemical staining detected all three isoforms of nitric oxide synthases (nNOS, eNOS, and iNOS) and 3-nitrotyrosine (3-NT) in brains from CCD-affected dogs and non-demented control dogs in all layers of the canine frontal cortex. In CCD-affected and non-demented brains, nNOS was highly expressed in pyramidal-like neurons in the upper cortical layers. nNOS has also been observed in astrocytes in the CCD frontal cortex. The nNOS immunohistochemical staining was statistically significantly elevated in dogs with CCD in comparison to non-demented dogs. Blood vessel wall cells were positive for eNOS, which was also expressed in astrocytes and neurons. Intense 3-NT immunoreactivity was observed in the upper cortical layers, where amyloid-beta deposits spread in the last stage of CCD. Brain cells in the same area were highly
immunoreactive for iNOS. This infers that neuroinflammation and nitrosative stress might exacerbate the neurodegenerative process in CCD-affected brains, ultimately leading to cognitive impairment.

Slika 1. nNOS protein v pasjih možganih. Slika prikazuje značilno obarvane živčne celice (rjavo) v čelnem režnju 16 letnega shih-tzuja s pasjo kognitivno motnjo. Merili sta dolgi 50 µm.

Slika 2. 3-NT v pasjih možganih. 3-NT (zeleno obarvanje) je prisoten v celicah, ki obkrožajo amiloide (Aβ) plake (rdeče obarvanje) pa tudi izven celic v območju plaka. Celična jedra so obarvana modro. Prikazan je čelni reženj 16 letnega jazbečarja s kognitivno motnjo. Merilo je dolgo 10 µm.

The full article is published in Frontiers in Veterinary Science

Authors: Sonja Prpar Mihevc, Maja Zakošek Pipan, Malan Štrbenc, Boris Rogelj, Gregor Majdič

Poliklorirani bifenili (PCB): prepovedani več kot 40 let, a zelo obstojni in še aktualni

lukam — Tue, 22 Dec 2020 08:55:52 +0000

Polychlorinated biphenyls (PCB): highly persistent chemicals banned for more than 40 years lukam Tue, 12/22/2020 - 10:06 Institute of Preclinical Sciences poliklorirani bifenili PCB trabekularna kostnina epifizni rastni hrustanec histomorfometrija podgana Research

Effects of PBC-169 and PCB-155 on microscopical structure of femur bone tissue of rat offspring.

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent, organic pollutants also considered endocrine-disrupting chemicals. Our study examined the effects of lactational exposure to nondioxin-like PCB-155 and/or dioxin-like PCB-169 on longitudinal femur growth at the distal epiphyseal growth plate (EGP) in young rats at three different ages [postnatal days (PNDs) 9, 22, and 42]. After delivery, lactating rats were divided into four groups (PCB-169, PCB-155, PCB-155 + 169, and control) and administered PCBs intraperitoneally.

The femurs of offspring were used to estimate growth rate (µm/day), and histomorphometric analysis on the distal femur included the thickness of the EGP and zones of proliferation and hypertrophy with calcification. Stereometry was used to determine trabecular bone volume density. In the PCB-169 and PCB-155 + 169 groups, PCB-169 affected longitudinal bone growth in the early postnatal period by interfering with chondrocytes in the EGP zone of proliferation and, to a lesser extent, the zone of hypertrophy. Morphometric alterations in EGP structure diminished until puberty. A slow growth rate persisted in the PCB-169 group until PND 42, while in the PCB-155 group, a fast growth rate between PNDs 9 to 22 was significantly reduced between PNDs 22 to 42. Sterometric assessment showed decreased trabecular bone volume in the PCB-155 + 169 group compared with that in the control on PND 9 and increased in the PCB-169 group compared with that in the PCB-155 group on PND 42. To summarize, studied PCB congeners exerted congener- and age-dependent effects on femur growth rate and its histomorphometric characteristics.

The full article is published in Scientific Reports (Nature).

Authors: Jana Brankovič, Gregor Fazarinc, Janja Jan in Milka Vrecl

Kakšne poti endocitoze uporabljajo herpesvirusni, s proteinom G sklopljeni receptorji in kako jih ugotavljamo?

lukam — Mon, 14 Dec 2020 14:13:44 +0000

Which endocytotic pathways do herpesvirus encoded G-protein coupled receptors use and how do we study them? lukam Mon, 12/14/2020 - 15:23 Institute of Preclinical Sciences endocytosis G-protein coupled receptors herpesvirus Research

Methods for Studying Endocytotic Pathways of Herpesvirus Encoded G Protein-Coupled Receptors.

In mammalian cell, different physiological and pathological cellular processes are regulated by G protein-coupled receptors (GPCRs), a family of receptors expressed at the cell surface. Intriguingly, during evolution, several herpesviruses adopted genes for such receptors (viral GPCRs) from their hosts to benefit their own survival during infection. Viruses use these receptors to evade host immune system, to enter the cell and to transform infected cells and thereby establish widespread and lifelong infection; processes where receptor signaling and/or endocytosis seem central. Endocytosis is a fundamental process involved in trafficking of various extracellular and transmembrane molecules from the cell surface to its interior. This enables cells to communicate and respond to external environments, maintaining cellular homeostasis, and transduce signals. vGPCRs are rising as potential drug targets as exemplified by the cytomegalovirus-encoded receptor US28, where its constitutive internalization has been used for selective drug delivery in virus infected cells. This review provides an overview of the current knowledge of endocytic and cell localization properties of vGPCRs and methodological approaches used for studying receptor internalization. Using novel approaches, we show constitutive internalization of the BILF1 receptor from human and porcine γ-1 herpesviruses and we present motifs from the eukaryotic linear motif (ELM) resources with importance for vGPCR endocytosis. The fact that vGPCRs are only expressed at the surface of infected cells makes them attractive targets for antiviral therapy. With increasing knowledge on their trafficking and signaling properties, we are making step forward in discovering their druggable potential.

Authors: Maša Mavri, Katja Spiess, Mette Marie Rosenkilde, Catrin Sian Rutland, Milka Vrecl, Valentina Kubale

The full article is published in MDPI Molecules

Kdo sodeluje z dopaminskim receptorjem tipa 2 in kaj to povzroči? Kako nanj vplivajo nekatera njegova lastna zaporedja?

lukam — Wed, 07 Oct 2020 12:14:28 +0000

Who cooperates with the type 2 dopamine receptor and what are the consequences? How is D2-R affected by some of its own sequences? lukam Wed, 10/07/2020 - 15:33 Institute of Preclinical Sciences D2 dopamine receptor intracellular trafficking endoplasmic reticulum retention motifs intracellular loop 3 interacting partners Research

How do the preserved motifs in the type 2 dopamine receptor (D2-R) and interacting proteins affect localization and trafficking in the cell of both receptor isofoms?

This article examines the role of different types of the evolutionarily conserved retention motifs and dopamine receptor interacting proteins (DRIPs) on the type 2 dopamine receptor D₂ (D₂-R) and its routing regulation. D₂-R is a member of protein-coupled receptors (GPCRs), the largest family of membrane-bound receptors. Two isoforms, short (D_2S-R) and long (D_2L-R) and differ in their intracellular localization and trafficking functionality as D_2L-R is retained inside cell, mostly in the endoplasmic reticulum (ER) and therefore not accessible to drugs as desirable drug target. These motifs might be involved in D₂-R exit from the ER and have an impact on cell-surface and intracellular localization and, therefore, also play a role in the function of dopamine receptor signaling, ligand binding and possible dimerization.

Our recent bioinformatic data on potential new interaction partners for the ICL3 of D2-Rs are also presented. Described motifs and DRIPs are highly relevant, and have clinical impacts on the pathophysiology of several diseases such as Parkinson’s disease, schizophrenia, Tourette’s syndrome, Huntington’s disease, manic depression, and others, as they are connected to a variety of essential motifs and differences in communication with interaction partners.

Full article is available at the following link.

Authors: Kaja Blagotinšek Cokan, Maša Mavri, Catrin Sian Rutland, Sanja Glišić, Milan Senćanski, Milka Vrecl, Valentina Kubale

Potencialni vzroki za spolne razlike v obolevnosti in umrljivosti bolnikov s Covid-19

lukam — Wed, 10 Jun 2020 09:01:06 +0000

Potential causes for sex differences in morbiditiy and mortality in Covid-19 patients lukam Wed, 06/10/2020 - 11:08 Institute of Preclinical Sciences lung ACE2 COVID 19 sex difference androgens Research

Could sex/gender differences in the ACE2 expression in the lungs contribute to the large gender disparity in morbidity and mortality of patients infected with Sars-CoV-2 virus?

Image captured at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID

Could sex/gender differences in the ACE2 expression in the lungs contribute to the large gender disparity in morbidity and mortality of patients infected with Sars-CoV-2 virus?

The severity and especially mortality of the new coronavirus disease Covid-19 shows significant gender differences, as men with this disease often become more seriously ill and die more often than women. The cause of the sex differences is not yet known, but in our article we presented the hypothesis that the cause is a sex difference in the expression of the ACE2 gene in the lungs.

The ACE2 gene is the entry point for the Sars-Cov-2 virus, and although no data are yet available for the lungs, this gene is differently expressed between the sexes under the influence of sex hormones in some other tissues such as the kidneys and fat tissue. If men have a higher expression of ACE2 in their lungs, this could mean that more virus can enter their body, which could lead to a more serious course of the disease and higher mortality. If such differences are modulated by sex hormones, they may also be able to affect ACE2 expression in patients with Covid-19 disease and could be thus used to influence the disease progress in human patients.

The full article is available at the following link.

Author: prof. dr. Gregor Majdič

Genetsko preverjanje rodovnikov psov

lukam — Fri, 04 Jan 2019 10:11:09 +0000

Gene Verification of Pedigrees of Dogs lukam Sun, 04/05/2020 - 08:39 Institute of Preclinical Sciences Diagnostics

For several years the Faculty of Veterinary Medicine in Ljubljana has routinely performed genetic checks on the pedigrees of dogs. The method is well-established and integrated into ISAG inter-laboratory controls and can therefore ensure correct and verifiable results. In 2018 the institute signed a contract with the Kennel Club of Slovenia for the production of DNA profiles in dogs. Thus, the institute is certified by the Kennel Club as an official pedigree checker and can offer you and your clients this service which is mandatory for the owners/breeders of dogs.

The institute is certified by the Kennel Club as an official pedigree checker and can offer you and your clients this service which is mandatory for the owners/breeders of dogs.

Instructions for preparation of samples

The test requires DNA that is isolated from the blood, or it may be taken from a swab of the oral mucosa if blood cannot be drawn.

Blood

The blood sample is taken into an EDTA test tube (at least 1ml). The name of the dog, owner and date of blood collection is written on the test tube.

Swab

Swabs are taken by rubbing a cotton bud on the mucous membrane, i.e. the gums or the inside of the cheeks. They are then air dried.

The dried samples are placed in a clean envelope (it must not be polyvinyl and must be air-tight). When sending samples from different dogs, the cotton buds should be stored in different envelopes. It is not recommended to take a swab from puppies under eight weeks old due to the possibility of contamination from the mother.

On the envelope we write down the name of the dog, the owner and the date that the swab was taken are written on the envelope.

Form

When submitting a sample please ensure you also send a completed DNA profile form with information about the dog, owner, payer and veterinarian who took the sample. The form and the sample should be sent by post or courier to the Faculty of Veterinary Medicine, Gerbičeva 60, Ljubljana, and marked ‘for the genetic testing of dogs’.

Gene Testing Form (456.2 KB)

Results are normally available within 10 working days of receipt of the sample.

The cost of producing a DNA profile for an individual dog is:

28,00 € excl. DDV
34,16 € incl. DDV

We can also send you a test tube with EDTA or a kit for taking a sample.

For more information call: 01 4779 120 (Dr. Marko Cotman).

Institute of Preclinical Sciences

Department of Biochemistry, Molecular Biology and Genetics