Age related еx vivo characteristics of mesenchymal stem cells isolated from canine stifle joint synovial fluid

Cilj ovog rada je bio da se utvrdi: da li ćelije izolovane iz sinovijalne tečnosti (ST) sadrže ćelije koje imaju fenotipske i funkcionalne karakteristike mezenhimalnih matičnih ćelija (MMĆ), kao i da se in vivo ispita njihov hondrogeni potencijal, uporede karakteristike izolovanih matičnih ćelija (MĆ) tokom starenja i okarakterišu zglobne strukture kolenog zgloba pasa kako bi se definisale promene vezane za starenje. Uzorci ST, sinovijalne membrane (SM) i hrskavice sa subhondralnom kosti uzeti su iz kolenog zgloba 28 pasa rase nemački ovčar, muškog pola, koji su prema uzrastu i nivou fizičke aktivnosti svrstani u 4 grupe: mladi radni policijski psi (n = 4) i mladi neradni vlasnički psi (n = 5) starosti 3-5 godina; stari radni policijski psi (n = 13) starosti 10-12 godina i stari neradni vlasnički psi (n = 6) starosti 10-13 godina. Kultura adherentnih ćelija je uspostavljena iz ST 5 mladih i 5 starih radnih pasa. Adherentne ćelije su uzgajane tokom 3 nedelje pre određivanja CD90 i CD34 molekula metodom protočne citometrije. Istovremeno, ispitano je njihovo vreme udvajanja u kulturi (PTD), broj CFU-F i aktivnost markera starenja β-galaktozidaze. Adipogeni, osteogeni i hondrogeni potencijal su ispitani nizom imunocitohemijskih, biohemijskih, imunohistohemijskih i molekularnih metoda. Hondrogeni potencijal reprezentativnog uzorka ćelija je ispitan i metodom ksenotransplantacije umnoženih ćelija imunodeficijentnim miševima. Da bi se stekao sveobuhvatni uvid u promene koštano-zglobnog sistema tokom procesa starenja i da bi se ustanovio mogući značaj fizičke aktivnosti za nastanak ovih promena, pored kliničkog pregleda, izvršeno je radiološko ispitivanje oba kolena zgloba, zgloba kuka i lumbosakralnog dela kičme, makroskopski pregled svih zglobnih struktura kao i histološka i imunohistohemijska (IHH) analiza hrskavice i SM. Sinovijalna tečnost je ispitana sa citološkog i biohemijskog aspekta kako bi se isključilo prisustvo inflamacije. Ukupna debljina hrskavice, broj hondrocita na vertikalnom preseku, njihova gustina i površina određeni su histomorfometrijski. Analiziran je i izgled površine hrskavice, oblik hondrocita, morfologija izogenih grupa, zona koja deli kalcifikovanu od nekalcifikovane hrskavice, subhondralna kost i zastupljenost proteoglikana i glikozamioglikana (PG/GAG). Na SM, procenjivan je broj slojeva ćelija, prisustvo prstolikih nabora i ćelijske infiltracije. Imunohistohemijski je ispitivano prisustvo i distribucija kolagena tipa I, II i X u hrskavici i CD44 molekula u sinovijalnoj membrani...The aim of this study was to determine: whether synovial fluid (SF) of dogs contains cells that have phenotypic and functional characteristics of mesenchymal stem cells (MSCs), to investigate there in vivo chondrogenic potential, to compare MSCs characteristics during aging and to define the changes associated with aging in stifle joints structures. Samples of SF, synovial membrane (SM) and cartilage with subchondral bone were taken from the 28 German Shepherd dogs, all males, who were assigned to four groups according to their age and physical activity: young working military dogs (n = 4) from 3 to 5 years old (3.8 ± 0.9); young non-working client-owned dogs (n = 5) from 3 to 5 years old (3.8 ± 0.8); aged working military dogs (n = 13) from 10 to 12 years old (10.5 ± 0.7) and aged non-working, client-owned dogs (n = 6), from 10 to 13 years old (11 ± 1.5). Canine SF adherent cells (cSFSc) from 5 young and 5 aged dogs were expanded (37ºC, 5% CO2, humidified atmosphere) for three weeks before their phenotypes were characterized by flowcytometry for the presence of CD90 and CD34. At the same time, population doubling time (PDT), number of CFU-F and SA-β-galactosidase activity (marker of cell ageing) have been determined. Adipogenic, osteogenic and chondrogenic potentials have been tested using imunocytochemical, biochemical, immunohistochemical (IHC) and molecular methods. Chondrogenic potential of representative cell samples was examined using xenotransplantation method in immunodeficient mice. In order to gain a comprehensive insight into the age related changes of musculoskeletal system and to determine the possible role of physical activity for the occurrence of these changes, in addition to clinical examination, radiological examination of both stifle and hip joints and lumbosacral part of the spine, macroscopic examination of all stifle structure as well as histological and IHC analysis of cartilage and SM was performed. SF cytological and biochemical analysis were done in order to exclude the presence of joint inflammation. The whole cartilage thickness, total chondrocytes number per vertical section, chondrocyte density and chondrocyte surface area in each cartilage zone were determined by histomorphometry. It was also analyzed: the appearance of the cartilage surface, chondrocyte shape and isogenous group morphology, tidemark morphology, subchondral bone structure and PG/GAG content. On SM, it was evaluated the number of cell layers, the presence of short and finger-like villi and the presents of cell infiltration..

Biohemijske promene u krvnom serumu pasa tretiranih fenobarbitonom

Despite being described as the safest antiepileptic drug of first choice the presented literature data are much varied as far as dog blood serum biochemical parameters are considered. The aim of this study was to investigate the effect of phenobarbitone at different per os doses on the values of selected blood serum biochemical parameters in dogs during both short and long term application. The study was conducted on 30 dogs of different races, both sexes, ranging from 2 to 8 years of age. A total of 15 healthy and 15 dogs suffering from idiophatic epilepsy were observed. During the short term per os application of phenobarbitone (given at 3 week intervals) to the healthy population in varied doses a statistically significant increase in ALT and AP was recorded. Application of 16 mg/kg/day of phenobarbitone to the healthy population during 14 days resulted in a significant increase of ALT and AP. This increase was dependant on the duration of the treatment. During chronic application of phenobarbitone to dogs suffering from idiopathic epilepsy a significant increase in values of AP and ALT depending on the given dose was recorded. In two of the studied epileptic dogs treated with high therapeutic doses of phenobarbitone clinical signs of hepatotoxicity were recorded. Hepatotoxicity resulted in decreased albumin and total protein concentrations, as well as increased blood serum total bilirubin, AST, ALP and AP. The obtained results indicate that a long term application of phenobarbitone at high therapeutic doses can cause hepatotoxicity. However, there was no relationship between phenobarbitone dosage and duration of therapy and blood glucose, urea, creatinine, total proteins, albumins, total bilirubin, triglycerides and cholesterol.Iako označen kao antiepileptik prvog izbora i veoma bezbedan lek, u literaturi su prezentovani različiti podaci o uticaju fenobarbitona na vrednosti biohemijskih parametara krvnog seruma kod pasa. S obzirom na to, cilj ovog istraživanja bio je da se ispita uticaj fenobarbitona pri različitim per os dozama na vrednosti biohemijskih parametara krvnog seruma kod pasa, tokom kratkotrajne i dugotrajne aplikacije. Istraživanje je sprovedeno na 30 pasa različitih rasa, oba pola, starosne kategorije od dve do osam godina, od kojih su 15 bili zdravi psi a 15 psi oboleli od idiopatske epilepsije. Tokom kratkotrajne per os aplikacije fenobarbitona (intervali od po tri nedelje) zdravoj populaciji pasa pri različitim per os dozama, u krvnom serumu je registrovano značajno povećanje aktivnosti ALT i vrlo značajno povećanje aktivnosti AP u zavisnosti od doze leka. Aplikacija fenobarbitona u dozi od 16 mg/kg/dnevno tokom 14 nedelja zdravoj populaciji pasa ukazala je, na vrlo značajno povećanje aktivnosti ALT i AP u zavisnosti od dužine trajanja aplikacije. Tokom hronične aplikacije fenobarbitona psima obolelim od idiopatske epilepsije, ustanovljeno je statistički vrlo značajno povećanje aktivnosti AP i ALT u krvnom serumu u zavisnosti od doze leka. Kod dve jedinke u bolesnoj grupi pasa pri visokim terapeutskim dozama fenobarbitona registrovana je hepatotoksičnost, što je dovelo do statistički značajnog smanjenja koncentracije albumina i ukupnih proteina, statistički značajnog povećanja koncentracije ukupnog bilirubina, statistički značajnog povećanja aktivnosti AST i statistički vrlo značajnog povećanja aktivnosti ALT i AP u krvnom serumu Dobijeni rezultati ukazuju da dugotrajna aplikacija fenobarbitona, pri visokim terapeutskim dozama može izazvati hepatotoksičnost. Nije ustanovljeno postojanje veze između koncentracije glukoze, uree, kreatinina, ukupnih proteina, albumina, ukupnog bilirubina, triglicerida i holesterola u krvnom serumu pasa sa per os dozom fenobarbitona, niti sa dužinom trajanja terapije

Importance of N-terminal proline for the promiscuous activity of 4-oxalocrotonate tautomerase (4-OT)

Michael addition of aldehydes to nitro-olefins is an effective method to obtain useful chiral gamma-nitroaldehydes. gamma-Nitroaldehydes are precursors for chiral gamma-aminobutyric acid analogues, which have numerous pharmacological activities and are used for the treatment of neurological disorders. A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) was developed and shown to be an effective biocatalyst for the Michael addition of branched aldehydes to beta-nitrostyrenes. The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine, both containing a reactive epsilon-amino group, on the Michael addition catalyzed by 4-OT. First, the effects of these mutations were examined by in silico analysis, followed by the generation of three terminal lysine mutants. The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for their ability to utilise beta-nitrostyrene (1), (E)-1-nitro-2-(2-thienyl)ethene (2) and trans-p-chloro-beta-nitrostyrene (3) as Michael acceptors with isobutanal (2-methylpropanal) as the donor. For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields. However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for 4-OT-catalysed Michael-additions, but it is not essential

Retrospective analysis of clinical pathological findings in obstruction of lower urinary pathways in cats

Diseases of the lower urinary pathways with urethral obstruction are frequent urological conditions demanding urgent treatment that lead to the occurrence of post renal azotemia due to difficult or disabled urine elimination. The aim of these investigations was to establish the most frequent causes for the occurrence of obstructions of the lower urinary pathways in cats, the most frequent clinical signs, as well as the changes in the hematological and biochemical blood and urine parameters. The obtained results indicate that the most frequent cause of urethral obstruction in cats is the presence of urethra stones, sand, and urethra plugs caused by the aggregation of struvite crystals on organic matrix. The laboratory findings showed that all animals were dehydrated, azotemia was established in half the animals, and signs of uremia were evident in 10% cats. Over 90% cats had disrupted tubular function, and only 50% had clear laboratory signs of inflammation. It can be concluded from the presented results that all cats with urethra obstruction exhibit a certain degree of disrupted kidney function, which is, in most cases, a reversible pathological change when treated with timely and adequate therapy. [Projekat Ministarstva nauke Republike Srbije, br. 175061

Canine babesiosis in different seasons - a fertile ground for multiple questions

Background: Canine babesiosis is a disease with a seasonal character. We have previously observed that in Belgrade, the capital of the Republic of Serbia, 60% of cases with the acute-phase response (APR) give a notable maximum between mid- February and May, the so-called “high season”. The other two peak seasons with less pronounced maxima are in winter, from January to mid-February, and in autumn, from October to December. Objectives: Based on clinical observations, we postulate that dogs infected with B. canis in the “high season” develop a more intense APR than dogs in the other two seasons. Material and Methods: To test this hypothesis, we compare the relative parasite load and the level of acute-phase proteins and reactants, their indexes (calculated as a combination of concentrations of fast and slow positive, and fast and slow negative acute phase reactants), as well as the concentration of coagulation factors, complete blood count, and biochemistry among dogs that developed the disease in the autumn, winter, and “high season”. Results: The relative parasite load is the lowest, and APR Indexes are the highest in dogs infected in “high season”. These findings are accompanied by relatively low creatinine and urea, implying that dogs in “high season” had hemodilution, thus further suggesting short incubation. Unlike other acute-phase proteins, fibrinogen showed the lowest concentration in “high season”. Conclusion: Future research will help unveil the connection between more intense APR and relatively low parasite load and fibrinogen concentration

Histološke karakteristike mekih tkiva posle implantacije biokeramičkih materijala i procena biokompatibilnosti

Histological evaluations of soft tissue reactions in implanted biomaterials is a long established method of investigating their biocompatibility. It is a very important procedure convenient for various biomaterials, as appropriate for soft as for hard tissue, but at the same time this method is not sufficient for a prediction of their clinic performance. The wide spread practice of this method is in the simplicity of its use in subcutaneous or intramuscular implantations on the animal model, and consequently exploring principal and surrounding tissue is very simple with screening tests for esimating the irritaton index and interaction between tissue and biomaterials. The screening tests (evaluation tests) involve implantation of biomaterials in various locations in the body, different tissue treatment as immunohistochemical tissue analysis, transmissional, scanning and confocal microscopy. Interactions among soft tissue with bioceramics and the evaluation of their cellular and tissue performance in inflammation and immunological response are the first step in the estimation of biocompatibility.Histološko ispitivanje reakcije mekih tkiva u odnosu na biomaterijal je davno ustanovljena metoda procenjivanja biokompatibilnosti. To je istovremeno i veoma važna metoda koja je primenjiva na sve oblike biomaterijala, bilo da su namenjeni za kontakt sa mekim tkivom ili ne, ali i metoda koja ima ograničeni značaj za predviđanje kliničkih performansi. Rasprostranjenost korišćenja ove metode proizilazi iz činjenice da je potkožna ili intramuskularna implantacija potencijalnog biomaterijala na životinje jednostavna, a dalja ispitivanja reakcije osnovnog i susednih tkiva su u principu jednostavan skrining test za određivanje iritacije tkiva i interakcije tkiva i biomaterijala. Skrining tehnike (testovi procenjivanja) uključuju implantaciju materijala na različitim lokalizacijama, kao i različite načine obrade tkiva, primenu imunohistohemijskih metoda bojenja, transmisione, skening i konfokalne mikropskopije i primenu drugih sofisticiranih metoda u obradi i interpretaciji podataka. Interakcija mekog tkiva sa biokeramikom i procena tkivnog i ćelijskog prikaza u procesu inflamacije i imunološkom odgovoru prvi je korak u određivanju njihove biokompatibilnosti

Importance of N-terminal proline for the promiscuous activity of 4-oxalocrotonate tautomerase (4-OT)

Michael addition of aldehydes to nitro-olefins is an effective method to obtain useful chiral gamma-nitroaldehydes. gamma-Nitroaldehydes are precursors for chiral gamma-aminobutyric acid analogues, which have numerous pharmacological activities and are used for the treatment of neurological disorders. A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) was developed and shown to be an effective biocatalyst for the Michael addition of branched aldehydes to beta-nitrostyrenes. The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine, both containing a reactive epsilon-amino group, on the Michael addition catalyzed by 4-OT. First, the effects of these mutations were examined by in silico analysis, followed by the generation of three terminal lysine mutants. The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for their ability to utilise beta-nitrostyrene (1), (E)-1-nitro-2-(2-thienyl)ethene (2) and trans-p-chloro-beta-nitrostyrene (3) as Michael acceptors with isobutanal (2-methylpropanal) as the donor. For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields. However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for 4-OT-catalysed Michael-additions, but it is not essential

Proliferation and differentiation potential of canine synovial fluid cells

The aim of this study was to determine whether synovial fluid (SF) of dogs contains cells that have characteristics of MSCs and to describe their differentiation potential. SF adherent cells from 5 young German shepherd dogs (average 3.8 +/- 0.9 years) were expanded (37 degrees C, 5% CO2, humidified atmosphere) three weeks before their phenotype was characterized by flow-cytometry for the presence of CD90 and CD34. Population doubling time (PDT), number of CFU-F and adipogenic, osteogenic and chondrogenic potentials have been determined in vitro. In early passages PTD was 31 +/- 10 hours and expansion fold after 3 sub cultivations (9 days) theoretically could be 372 +/- 134. At P1, 0.55 +/- 0.05% of SF cells had the ability to form CFU-F. Sixty-six percent of cells expressed CD90 and none of the cells expressed markers of hematopoietic cells. Oil Red O staining has shown accumulation of fat droplets in cells grown in adipogenic medium, while deposits of calcium in the osteogenic medium were evidenced with Alizarin red staining. SF cultured in hondrogenic and control medium in three-dimensional conditions formed a cartilage-like tissue. Alcian blue staining of pellets' slides have shown a significant amount of glycosaminoglycans (GAGs) and immunohistochemistry analysis documented collagen type II expression. The amount of GAGs in pellets grown in both conditions showed no difference. SF cells in vitro exhibited osteogenic, adipogenic and chondrogenic differentiation potentials, suggesting the presence of different mesenchymal progenitors. These results also demonstrated that SF cells have a spontaneous chondrogenic potential that should be further explored for possible tissue engineering protocols

Selected hematology ratios in cats with non-septic effusions highly suspected of feline infectious peritonitis

In veterinary medicine, knowledge about hematologic ratios (neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and mean platelet volume-to-platelet ratio (MPV/PLT)) is limited, particularly in cats. While the roles of these ratios have been proven in oncology, systemic inflammation with or without systemic inflammatory response syndrome (SIRS), and sepsis, information is lacking about their alterations in non-septic effusions, like feline infectious peritonitis (FIP). This study aimed to describe whether NLR, PLR, and MPV/PLT were changed and whether they correlated with routine hematologic and biochemical parameters in 16 cats with non-septic effusions, highly suspected to be the effusive form of FIP without SIRS, compared to nine clinically healthy cats. The NLR was calculated as the absolute count of neutrophils divided by the absolute count of lymphocytes, PLR by calculating the absolute platelet divided by the absolute lymphocyte count, and MPV/PLT by dividing mean platelet volume by absolute platelet count. The NLR, MPV, and MPV/PLT ratios were higher in cats with non-septic effusions suspected to be FIP, but PLR did not differ, when compared to healthy cats. Correlation analysis did not show any association between the selected ratios and hematological and biochemical parameters. In the absence of leukocytosis, increased NLR could help us to confirm the presence of systemic inflammation in cats with non-septic effusions indicative of FIP. However, a high MPV/PLT ratio should be interpreted with caution, especially in cats