Promene u limfatičnim organima komercijalnih pilića nakon vakcinacije protiv Marekove bolesti - histološka i stereološka analiza

The aim of this study was to investigate histomorphometrical characteristics of the thymus, bursa of Fabricius and spleen in the chickens vaccinated with a vaccine against Marek’s disease. For this purpose, we used newly hatched chickens of the light hybrid line, obtained from a local hatchery. The chickens were vaccinated on the 5th day after hatching with a bivalent cell-associated Marek's disease vaccine (PFU-2000 per dose). On day 13 both vaccinated chickens and unvaccinated controls were sacrificed, and thymus, bursa and spleen were removed and processed for light microscopy. The serial tissue sections, hematoxylin-eosine stained, were used for histomorphometric analysis. Vaccination against Marek’s disease decreased the relative mass of the lymphoid organs, and caused significant damage of the thymus and spleen in experimental chicken. In addition, vaccination, similar to Marek's disease virus, induced morphometric changes in the lymphoid organs. Namely, it significantly decreased the diameter and volume of lymphoid follicles, volume of follicular medulla and number of cells in the follicular cortex in the bursa of Fabricius. In the thymus, vaccination reduced the thymus volume and the absolute number of thymocytes. However, vaccination against Marek’s disease caused an increase in the diameter, number and volume of lymphoid follicles in the spleen. The present data suggest that vaccination against Marek’s disease was able to induce the immune response in processed organs, although it reduced the mass and number of lymphocytes in the major lymphoid organs.Cilj ovih ispitivanja su bile histomorfometrijske karakteristike timusa, burze Fabricii i slezine pilića vakcinisanih protiv Marekove bolesti. Za istraživanja su korišćeni jednodnevni pilići lake hibridne linije, gajeni u standardnim uslovima. Pilići su vakcinisani 5. dana od izleganja, bivalentnom vakcinom protiv Marekove bolesti (PFU 2000 po dozi). Trinaestog dana od izleganja kontrolni i vakcinisani pilići su žrtvovani, uzeti su im timus, burza Fabricii i slezina koji su pripremljeni za svetlosno mikroskopsku analizu. Za analizu su korišćeni serijski preseci limfatič nih organa, bojeni metodom hematoksilin-eozin. Dobijeni rezultati su ukazali da vakcinacija protiv Marekove bolesti dovodi do smanjenja relativne mase timusa, burze i slezine, kao i povećanja indeksa oštećenja timusa i slezine. Takođe je ustanovjleno da vakcinacija dovodi do promena morfometrijskih parametara u limfatičnim organima. U burzi Fabricii je uočeno značajno smanjenje dijametra i zapremine limfocitnih folikula, zapremine medule folikula i broja ćelija u korteksu folikula. U timusu vakcinacija dovodi do redukcije zapremine timusa i apsolutnog broja timocita. Međutim, vakcinacija protiv Marekove bolesti u slezini dovodi do povećanja dijametra limfocitnih folikula, kao i do povećanja njihovog broja i zapremine. Rezultati ove studije su ukazali da vakcinacija pilića protiv Marekove bolesti, iako redukuje broj T i B limfocita, indukuje razvoj imunskog odgovora

Diferentovanje timocita u organ kulturi timusa odraslih pacova

To investigate the differences between thymocytes development in vivo and in vitro, thymus lobe fragments from 12-weeks old male Albino Oxford rats were cultivated over a 7-days period. In the controls and cultivated thymic lobes fragments were evaluated and the viability, apoptosis and cell cycle of thymocytes, as well as the histological characteristics of thymic tissue. Additionally, we analyzed the expression of CD4, CD8 and TCRαβ on thymocytes by flow cytometry. The obtained results showed that thymus cellularity decreased during cultivated time due to expanded apoptosis, decreased proliferation and the absence of progenitors reseeding thymus. The relative proportion of thymocyte subsets in the first 24 hours of culture remained similar as in the control. However, cultivation for 3 and 7 days modulated the relative proportions between thymoctye subsets. The percentage of DP TCRαβlow increased, DP TCRαβhi subset remained unchanged, both SP TCRαβhi subsets decreased while the same mature SP phenotype dominated in culture media. These results demonstrate that cultivated thymic fragments retain the capacity for T cell development, although cultivation modulates this process.Sa namerom da ispitamo razlike između in vivo i in vitro sazrevanja timocita gajili smo fragmente lobusa timusa poreklom od mužjaka Albino Oksford pacova, starih dvanaest nedelja, u vremenskom periodu od sedam dana. Nakon kultivacije određivani su vijabilnost, apoptoza i ćelijski ciklus timocita, kao i histološke osobine timusnog tkiva. Takođe je analizirano ispoljavanje markera diferentovanja CD4, CD8 and TCRαβ na površini timocita metodom tečne citofluorometrije. Dobijeni rezultati su ukazali da sedmodnevna kultivacija dovodi do smanjenja broja ćelija u timusu usled povećane apoptoze, smanjene proliferacije i odsustva ulaska progenitora timocita. Tokom prvih 24 sata kultivacije ne dolazi do promena u odnosima timocitnih populacija. Međutim, duže vreme kultivacije - 3 i 7 dana moduliše relativne odnose između timocitnih subpopulacija - povećava se procenat DP TCRαβlow, procenat DP TCRαβhi timocita ostaje nepromenjen, dok su procenti ćelija oba subseta SP TCRαβhi smanjeni, mada je prisustvo pomenutih SP subsetova dominantno u medijumu za kultivaciju. Navedeni rezultati pokazuju da kultivisani fragmenti timusnog tkiva zadržavaju sposobnost da podrže sazrevanje timocita u jednostruko pozitivne T ćelije, mada je diferentovanje timocita donekle modulisano kultivacijom

Network connectivity is shown to change in C57BL/6 mice during a continuing immune response subsequent to tetanus toxoid hyperimmunization

We have already demonstrated (Stojanovic et al., 2009) a connection between tetanus toxoid (TTd) hyperimmunization and the induction of anti-phospholipid syndrome (APS) in BALB/c mice. Here we show that C57BL/6 mice subjected to an identical procedure do not exhibit any like pathology attributable to anti-phospholipid antibodies; we explain that this absence results from idiotypic connectivity. Six groups of C57BL/6 mice were hyperimmunized with TTd in aluminum hydroxide or glycerol, with or without pretreatments. Pretreated mice had been injected with polyclonal or nonspecific immune stimulators, such as complete Freund's adjuvant (CFA) or glycerol. The epitope specificity of induced antibodies was tested by indirect ELISA using a tetanus toxoid immunogen and these autoantigens: phospholipids, gangliosides, laminin. Idiotypic connectivity was tested by competitive ELISA and gauged from the degree to which the interaction of idiotypic/anti-idiotypic complementary antibodies was inhibited in the presence of immunized sera antibodies. Higher idiotypic connectivity was noted amongst pretreated mice. There was a positive correlation between higher connectivity and autoantibody levels that acted to favor the participation of natural autoantibodies in the inhibitory process. We conclude that idiotypic connectivity plays a protective role in immunization-induced autoimmunity

Optimizacija metode za izolaciju epitelnih stanica iz nežljezdanog dijela želuca štakora za protočnu citometriju

Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results.Tradicionalne metode u ispitivanju stanične proliferacije temelje se na imunohistokemijskom otkrivanju proliferacijskih stanica u ciljanom tkivu. Kako su dugotrajne, optimizacija novih i učinkovitijih metoda važna je za velika istraživanja o proliferaciji. U ovom smo radu željeli optimizirati izolaciju epitelnih stanica prednjeg želuca štakora za protočnu citometriju. Kao marker stanične proliferacije koristili smo Ki-67 protutijelo za otkrivanje ovoga nuklearnog proteina izraženog u proliferacijskim stanicama. Također smo učinili imunohistokemijsku detekciju Ki- 67 pozitivnih stanica i bojenje propidij-jodidom kako bismo potvrdili rezultate. Butil-hidroksianizol korišten je kao pozitivna kontrola da se osigura stanična proliferacija. Rezultati su pokazali da izolacija epitelnih stanica s kolagenazom, tripsinom i staničnim cjedilom osigurava veliku vijabilnost stanica (> 95 %) i čistoću uzoraka. Protočna citometrija i Ki-67 bojenje pokazali su da tretman butil-hidroksianizolom dovodi do znakovitog porasta proliferacije. Primijećena je znakovita pozitivna korelacija između rezultata dobivenih imunohistokemijom i protočnom citometrijom, dok su protočni citometrijski podaci imali manju pogrešku mjerenja, što upućuje na jednaku osjetljivost i veću točnost ove metode. Bojenje propidij-jodidom pokazalo je da postotak stanica u G2+S fazi staničnog ciklusa pozitivno korelira s postotkom Ki-67 pozitivnih stanica procijenjenih protočnom citometrijom, što upućuje na to da Ki-67 oslikava stanice u aktivnoj diobi. Zaključujemo da je opisana izolacija epitelnih stanica prednjeg želuca štakora jednostavna i pouzdana metoda za dobivanje održivih stanica za upotrebu u protočnoj citometriji. U usporedbi s imunohistokemijom, protočna citometrijska detekcija antigena Ki-67 jednako je osjetljiva, ali mnogo brža i daje točnije rezultate

Ghrelin endocrine cells in the human stomach during prenatal and early postnatal development

The aim of this study was to investigate the appearance, localization and density of ghrelin cells in the human stomach during prenatal development. For this purpose the antrum and corpus of embryos, fetuses and infants are stained immunohistochemically by the streptavidin-biotin technique. The presence of P/D1 cells at 11 weeks of fetal development, their highest density during the first detection and higher density in the corpus than in antrum, and their localization in the glandular base of stomach gland, all suggest that ghrelin plays a major role in the early stages of the developing stomach

BRCA1 and TOP2A gene amplification and protein expression in four molecular subtypes of breast cancer

We studied TOP2A amplification (using FISH methods), and TOP2A and BRCA1 protein overexpression (immunohistochemistry) in four molecular subtypes of breast cancer. Of 53 patients, 32 showed TOP2A and 38 showed BRCA1 overexpression. The highest percentage of TOP2A amplification (47.8%) and deletion (13%) was detected in Luminal B subtypes. Of 11 Luminal B tumors with TOP2A amplification, 9 (81.8%) overexpressed TOP2A. BRCA1 protein overexpression showed significant positive correlation with TOP2A protein expression. BRCA1 and TOP2A proteins exhibited similar patterns of expression in Luminal B and triple-negative breast cancer, suggesting the same prognosis in those patients

Somatostatin-14 izaziva različite promene u timusima prepubertalnih i mladih odraslih pacova

Bearing in mind the role of somatostatin in thymus functions, and changes of somatostatin level and expression of its receptors during postnatal life, the aim of this study was to investigate whether centrally applied SRIH-14 induces different changes in the thymic compartments and thymocyte profile in peripubertal and young adult rats. To this end, 4- and 10-week-old male AO rats were cannulated and treated intracerebroventriculary with three doses of SRIH-14, applied every other day. In peripubertal rats, SRIH-14 decreases thymic relative weight and volume, as well as the volume of thymic compartments, especially of deep cortex, as a result of thymocytes loss by apoptosis. Also, SRIH-14 increases the percentage of immature thymocytes preceding the DPTCRαβlow cells (DNTCRαβ-/low, DPTCRαβ-, SPCD8TCRαβ-/low and SPCD4TCRαβ-/low), decreases the percentages of DPTCRαβlow and DPTCRαβhi cells, while the relative proportion of CD4+/CD8+TCRαβhi cells remained unaltered. In young adult rats, SRIH-14 does not lead to changes in relative thymus weight, although decreases the thymic cortex cellularity and volume. In addition, decreases the percentage of DPTCRαβ-/hi cells and increases the percentages of cells within DNTCRαβhi and both SP subpopulations, but much more of the CD8+TCRαβhi subset. These results suggest that the effects of SRIH-14 on the thymus and thymocytes subpopulations are age-dependent.Sa obzirom da literaturni podaci ukazuju da somatostatin ima uticaja na funkciju timusa, kao i da se tokom postnatalnog života menja nivo somatostatina i ekspresija njegovih receptora u timusu, cilj ovog rada je bio da se ispita da li SRIH- 14 davan intracerebroventrikularno u prepubertalnih i mladih odraslih pacova, dovodi do različitih promena u zonama timusa i profilu timocitnih subsetova. Mužjacima pacova AO soja, starim 4 i 10 nedelja, ugrađene su kanile u treću moždanu komoru i oni su tretirani somatostatinom, ukupno tri doze, ubrizgane svakog drugog dana. U prepubertalnih pacova tretman somatostatinom dovodi do smanjenja relativne mase i zapremine timusa, kao i zapremine timusnih zona, naročito dubokog korteksa, što je bila posledica gubitka timocita apoptozom. Takođe, SRIH-14 je doveo do povećanja procenta timocita nezrelog fenotipa (DNTCRαβ-/low, DPTCRαβ-, SPCD8TCRαβ-/low i SPCD4TCRαβ-/low) prekusora DPTCRαβlow subpopulacije, smanjenja procenta DPTCRαβlow i DPTCRαβhi subsetova, dok je relativni odnos najzrelijih CD4TCRαβhi i CD8TCRαβhi timocita ostao neizmenjen. U mladih adulta primena somatostatina, iako dovodi do smanjena zapremine i celularosti korteksa ne dovodi do promena u relativnoj masi timusa. Procenat DPTCRαβ-/hi timocita je bio smanjen, dok je procentualno učešće DNTCRαβhi timocita i obe jednostruko pozitivne subpopulacije (CD4TCRαβhi i CD8TCRαβhi, više CD8TCRαβhi) u ukupnom broju timocita bilo povećano. Navedeni rezultati ukazuju da SRIH-14 različito utiče na morfologiju timusa i na subpopulacije timocita pacova u zavisnosti od uzrasta tretiranih životinja

Factors effecting the induction of rat forestomach hyperplasia induced by Swedish oral smokeless tobacco (snus)

Long term exposure to oral smokeless tobacco may induce lesions in the oral cavity characterized by a hyperplastic epithelium. The possible role of nicotine and the physical properties of oral tobacco for developing these lesions, as well as of dysplasia and neoplasia is unclear. Low nitrosamine Swedish snus as well as non-genotoxic butylated hydroxyanisole induces increased cellular proliferation in the rat forestomach epithelia. Using this model, we report here on the effects of nicotine, pH, and particle size. Snus with different properties had no impact on oxidative stress as determined by 8-oxo-7,8-dihydro-2′-deoxyguanosine, or on interleukin IL-1b. Whereas BHA boosted IL-6, probably due to the presence of nicotine. there was no significant enhancement of cell divisions with increasing particle size, although in individual samples the variations in proliferation rates increased greatly with increasing particle size. Conforming to human experience, the enhanced cell proliferation caused by snus was found to be completely reversible. A cacao bean extract had a protective action similar to that previously found for blueberries. The main cause of the observed tobacco induced cell proliferation could be mechanical irritation, possibly in combination with nicotine, whereas within the studied range, pH did not affect the rate of cell division. © 201

Lokalizacija monoaminskih neurotransmitera u jajnicima i jajovodima pacova tokom polnog sazrevanja

The aim of this study was to investigate the localization and distribution of sympathetic positive structures in the female gonads and oviducts of 15 30 and 60-day-oldAO rats. Histofluorescence and immunohistochemistry methods were used to determine their distribution in the ovary and oviduct, as well as for identification of dopamine and serotonin within sympathetic positive structures. Total monoamine positive structures (fibers and cells) were mainly localized under the surface epithelium, between follicles, especially surrounding groups of primary follicles and between secondary interstitial cells. The distribution of dopamine (DA) immunolabelled fibers and cells was in accordance with the distribution of total monoaminergic structures detected using histofluorescence. However, the density of structures labeled with DA antibodies was lower compared to total catecholamine positive structures detected by the fluorescence method. Delicate serotonin (5-HT) positive fibers were present mainly under the surface epithelium and in interstitial tissue between the cortex and medulla. In the hilum and medulla 5-HT positive cells were found perivascularly, while a dense population of 5-HT positive cells was found in the oviduct and mesovarium in 60-day-old animals. No corpus luteum cells were labelled using either of the two methods. Our findings corroborate observations that the monoamine supply of the ovary is not exclusively via extrinsic innervation and that intragonadal sources have an important role in their synthesis. Furthermore, the intraovarian localization of these elements suggests that monoamines exert direct or indirect effects on ovarian function.Cilj našeg rada je bio ispitivanje lokalizacije i distribucije struktura pozitivnih na monoaminske neurotransmitere u jajnicima i jajovodima pacova. Za identifikaciju neurotransmitera u jajnicima i jajovodima 15, 30 i 60 dana starih AO pacova koristili smo histofluorescenciju (ukupni monoamini) i imunocitohemiju (dopamin i serotonin). Pozitivne monoaminergičke strukture prisutne su između folikula, naročito oko primarnih folikula, kao i između sekundarnih intersticijalnih ćelija. U mezoovarijumu i jajovodu 30 i 60 dana starih životinja zapažaju se fluorescentne ćelije i vlakna. Dopaminergične strukture su prisutne na istim mestima, ali njihova gustina je manja ako se uporede sa strukturama pozitivnim na ukupne monoamine. Retka serotonin-pozitivna vlakna zapažaju se uglavnom ispod klicinog epitela i u intersticijumu između kore i srži jajnika. Kod 30 i 60 dana starih ženki u hilusu i srži, pretežno oko krvnih sudova, nalaze se serotonin pozitivne ćelije, dok se u mezoovarijumu i jajovodu nalazi gusta populacija pozitivnih ćelija. Ni jednom od primenjenih metoda nije uočeno prisustvo monoamina u žutom telu. Naši rezultati potvrduju da neurotransmiteri u jajnicima i jajovodima ne potiču samo iz nervnih terminala, već da značajnu ulogu u njihovoj sintezi imaju intragonadalni izvori. Lokalizacija ovih elemenata pokazuje da monoaminski neurotransmiteri mogu ispoljavati direktan i!i indirektan uticaj na funkciju jajnika

Changes in the thymus of peripubertal rats induced by centrally applied somatostatin-28

The aim of this study was to investigate the effects of centrally applied somatostatin- 28 on morphometric characteristics of the thymus, the thymocyte subpopulations, as well as, on apoptosis and phases of cell cycle in thymocytes. For this purpose, peripubertal male rats were cannulated intracerebroventriculary and treated with repeated, nanomolar concentrations of somatostatin- 28 ( experimental group) or saline ( control group). Animals were sacrificed and their thymuses were used for the analysis of thymocyte subpopulations, cell cycle and apoptosis by flow cytometry and for the evaluation of morphometric parameters by stereological analysis. Our results showed that somatostatin- 28 caused decrease of the thymic mass and volume, as well as total thymocytes number. Stereological analysis revealed volume decrease of thymic cortex and medulla accompanied with cellularity decrease. Somatostatin in the deeper cortex decreased the number of thymocytes, per volume unit, while in outer cortex raised their number. A significant increase in the percentage of double-negative and both single-positive thymocyte subpopulations, in parallel with a diminished percentage of double- positive cells was found. The cellularity of double- positive and single-positive thymocyte subpopulations was decreased. Somatostatin-28 treatment augmented the percentage of apoptotic cells, while the percentage of the cells represented in phases of cell cycle was reduced. These results suggest that somatostatin- 28 induce thymus hypotrophy as result of decreasing cortex and medulla volume and cellularity. Changes in the percentage and cellularity of thymocyte subpopulations and numerical density of thymocytes in outer and deeper cortex, indicate that somatostatin- 28 evoked disturbance in transition of double- negative to double- positive thymocytes