Proper histological evaluation of lung tissue and accurate antigen detection by immunodetection techniques require histological tissue processing to preserve tissue reactivity and open alveolar spaces. In this study, we focused on testing and comparing different procedures of tissue cryopreservation. Sucrose or Tissue Tek were used with several methods of freezing samples by supercooled liquids and liquid nitrogen. Changes in tissue caused during the freezing of samples and the effect of cryoprotectants on the tissue were recorded. Rat and porcine pulmonary tissues were used in this experiment. This study aimed to optimize the process of lung cryopreservation with emphasis on enabling proper anatomical evaluation and preserving a high tissue immunoreactivity. The best results were obtained by inflating pulmonary parenchyma with a 1 : 1 mixture of O.C.T. with phosphate buffered saline (PBS) frozen in supercooled n-heptane placed on dry ice. Pulmonary tissue prepared in this way enables to perform proper histological evaluation and to detect target molecules by immunohistochemical analysis.

Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, Actinobacillus pleuropneumoniae (APP) caused infections and still worries many pig breeders around the world. To better understand the influence of pathogenic effect of APP on a respiratory system-lungs and tracheobronchial lymph nodes (TBLN), we aimed to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI). In this study, six pigs were intranasally infected by APP and two were used as non-infected control, and 48 cryosections have been obtained. MALDI-TOF MSI and immunohistochemistry (IHC) were used to study spatial distribution of infectious markers, especially interleukins, in cryosections of porcine tissues of lungs (necrotic area, marginal zone) and tracheobronchial lymph nodes (TBLN) from pigs infected by APP. CD163, interleukin 1β (IL‑1β) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL‑1β were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL‑1β and protegrin‑4 precursor were all significantly (p < 0.001) more expressed in necrotic areas of lungs infected by APP than in marginal zone, TBLN and in control lungs.

• MeSH
• Actinobacillus pleuropneumoniae patogenita   MeSH
• antigeny diferenciační myelomonocytární metabolismus   MeSH
• biologické markery metabolismus   MeSH
• bronchy metabolismus   MeSH
• CD antigeny metabolismus   MeSH
• infekce bakteriemi rodu Actinobacillus metabolismus   mikrobiologie   MeSH
• infekce dýchací soustavy metabolismus   mikrobiologie   MeSH
• interleukin-1beta metabolismus   MeSH
• kationické antimikrobiální peptidy metabolismus   MeSH
• lymfatické uzliny metabolismus   MeSH
• plíce metabolismus   MeSH
• prasata MeSH
• receptory buněčného povrchu metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Dietary supplementation with polyunsaturated fatty acids (PUFA) n-3 can affect cutaneous wound healing; however, recent findings demonstrate the variable extent of their influence on the quality of healing. Here, we compare the effect of several dietary oils, containing different levels of PUFA n-3 and PUFA n-6, on wound healing in the rat model. Rats were fed the feed mixture with 8% palm oil (P), safflower oil (S), fish oil (F) or Schizochytrium microalga extract (Sch) and compared to the animals fed by control feed mixture (C). Dorsal full-thickness cutaneous excisions were performed after 52 days of feeding and skin was left to heal for an additional 12 days. Histopathological analysis of skin wounds was performed, including immune cells immunolabeling and the determination of hydroxyproline amount as well as gene expression analyses of molecules contributing to different steps of the healing. Matrix-assisted-laser-desorption-ionization mass-spectrometry-imaging (MALDI-MSI) was used to determine the amount of collagen α-1(III) chain fragment in healing samples. Treatment by Schizochytrium extract resulted in decrease in the total wound area, in contrast to the safflower oil group where the size of the wound was larger when comparing to control animals. Diet with Schizochytrium extract and safflower oils displayed a tendency to increase the number of new vessels. The number of MPO-positive cells was diminished following any of oil treatment in comparison to the control, but their highest amount was found in animals with a fish oil diet. On the other hand, the number of CD68-positive macrophages was increased, with the most significant enhancement in the fish oil and safflower oil group. Hydroxyproline concentration was the highest in the safflower oil group but it was also enhanced in all other analyzed treatments in comparison to the control. MALDI-MSI signal intensity of a collagen III fragment decreased in the sequence C > S > Sch > P > F treatment. In conclusion, we observed differences in tissue response during healing between dietary oils, with the activation of inflammation observed following the treatment with oil containing high eicosapentaenoic acid (EPA) level (fish oil) and enhanced healing features were induced by the diet with high content of docosahexaenoic acid (DHA, Schizochytrium extract).

• MeSH
• antigeny CD8 metabolismus   MeSH
• dietní tuky nenasycené aplikace a dávkování   farmakologie   MeSH
• hojení ran účinky léků   MeSH
• indoly chemie   MeSH
• kolagen typ III metabolismus   MeSH
• krysa rodu rattus MeSH
• kůže účinky léků   zranění   metabolismus   MeSH
• kyseliny mastné omega-3 analýza   MeSH
• kyseliny mastné omega-6 analýza   MeSH
• makrofágy imunologie   MeSH
• modely nemocí na zvířatech MeSH
• palmový olej aplikace a dávkování   chemie   farmakologie   MeSH
• rybí oleje aplikace a dávkování   chemie   farmakologie   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• světlicový olej aplikace a dávkování   chemie   farmakologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

The objective of the present study was to compare the effects of dietary oils containing polyunsaturated fatty acids (PUFA) n-3 and n-6, respectively, on cutaneous wound healing in rats, and to demonstrate the usefulness of the matrix-assisted-laser-desorption-ionization mass spectroscopy-imaging (MALDI MSI) method in this type of experiment. Superiority of PUFA n-3 in this context was the tested hypothesis. Four groups of male Wistar rats by twelve animals each were fed a diet with added 5% of palm oil (P; control), fish oil (F), Schizochytrium microalga oil (Sch) and safflower oil (S), respectively, for eight weeks. Consequently, dorsal full-thickness cutaneous excisions were performed, and selected markers of wound healing were evaluated 18 days post excision. The median of signal intensity corresponding to an amount of collagen α-1 (III) fragment, quantified using MALDI MSI, decreased in a sequence P > F > Sch > S (P < 0.001). Using haematoxylin-eosin staining of the histological preparations, semi-quantitatively assessed epithelium height tended to decrease in the order of P > S > Sch > F; the wound extent in the sequence of P > Sch > S > F; and the sequence of the progress of neo-angiogenesis was assessed as S > P > F ≈ Sch. It was concluded that the tested hypothesis was confirmed only partially: PUFA n-3 showed better results regarding the wound extent, but were inferior in terms of epithelium height and progress of neo-angiogenesis. This was the first time MALDI MSI was successfully employed for evaluating skin wound healing in a rat model.

• MeSH
• dietní tuky nenasycené * terapeutické užití   MeSH
• dietní tuky terapeutické užití   MeSH
• hojení ran * MeSH
• kolagen typu I, řetězec alfa 1 analýza   MeSH
• nenasycené mastné kyseliny terapeutické užití   MeSH
• potkani Wistar MeSH
• rány a poranění dietoterapie   veterinární   MeSH
• reepitalizace MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice statistika a číselné údaje   veterinární   MeSH
• výsledek terapie MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• klinická studie MeSH

• MeSH
• buněčná diferenciace účinky léků   MeSH
• buněčné linie MeSH
• cytokiny genetika   metabolismus   MeSH
• interleukin-10 genetika   metabolismus   MeSH
• interleukin-6 genetika   metabolismus   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé MeSH
• lipopolysacharidy farmakologie   MeSH
• makrofágy metabolismus   účinky léků   MeSH
• nádory prostaty * MeSH
• TNF-alfa genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Lactobacillus, Bifidobacterium and Enterococcus cultures are increasingly used as probiotics for humans and pigs. The aim of this study was to investigate if co-cultivation of porcine and human neutrophils with probiotics can lead to increased apoptosis in vitro. Ten adult Large white pigs and 10 healthy human donors were used in this study. Neutrophils were isolated by dextran sedimentation and cultivated with and without the lactic acid bacteria Bifidobacterium bifidum, Lactobacillus rhamnosus, and Enterococcus faecium for 2, 4, 24 and 48 h. Early and late apoptosis was measured using flow cytometry, and cell lysis was detected based on lactate dehydrogenase activity (LDH). A significant (P < 0.05; P < 0.01) increase in apoptotic neutrophils and LDH was observed at 24 h and 48 h in vitro. All probiotics exerted their greatest effects on the early apoptosis of porcine neutrophils, while the effects of L. rhamnosus were most pronounced on late apoptosis and those of B. bifidumon LDH release of human neutrophils. The increased neutrophil apoptosis caused by probiotic bacteria can be beneficial for more efficient efferocytosis and faster resolution of inflammation and tissue regeneration. In conclusion, we have demonstrated that the interaction of B. bifidum, L. rhamnosus,and E. faecium with human and porcine neutrophils leads to their apoptosis.

• MeSH
• apoptóza MeSH
• Bifidobacterium bifidum fyziologie   imunologie   MeSH
• Enterococcus faecium fyziologie   imunologie   MeSH
• Lacticaseibacillus rhamnosus fyziologie   imunologie   MeSH
• lidé MeSH
• neutrofily MeSH
• probiotika * aplikace a dávkování   farmakologie   metabolismus   MeSH
• průtoková cytometrie metody   MeSH
• Sus scrofa MeSH
• techniky in vitro MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH

Identification of specific cell death is of a great value for many scientists. Predominant types of cell death can be detected by flow-cytometry (FCM). Nevertheless, the absence of cellular morphology analysis leads to the misclassification of cell death type due to underestimated oncosis. However, the definition of the oncosis is important because of its potential reversibility. Therefore, FCM analysis of cell death using annexin V/propidium iodide assay was compared with holographic microscopy coupled with fluorescence detection - "Multimodal holographic microscopy (MHM)". The aim was to highlight FCM limitations and to point out MHM advantages. It was shown that the annexin V+/PI- phenotype is not specific of early apoptotic cells, as previously believed, and that morphological criteria have to be necessarily combined with annexin V/PI for the cell death type to be ascertained precisely. MHM makes it possible to distinguish oncosis clearly from apoptosis and to stratify the progression of oncosis.

• MeSH
• apoptóza * MeSH
• časové faktory MeSH
• fenotyp MeSH
• fluorescenční mikroskopie metody   MeSH
• holografie metody   MeSH
• lidé MeSH
• multimodální zobrazování metody   MeSH
• nádorové buněčné linie MeSH
• nekróza MeSH
• viabilita buněk MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This paper investigates the association between expression of CD14 and occurrence of apoptosis in blood, resident ((RES)PMN) and inflammatory ((INF)PMN) polymorphonuclear leukocytes (PMN) from heifer mammary glands. The fresh population of (RES)PMN contained a statistically significant higher proportion of CD14+, apoptotic and necrotic cells than did populations of (INF)PMN and blood PMN. In vitro cultivation of (RES)PMN, (INF)PMN and blood PMN led to concurrent increase of apoptotic, necrotic and CD14+ cells. A positive correlation was found between the proportions of both apoptotic and necrotic PMN and CD14+ PMN as determined by three-color flow cytometry analysis. Our study confirmed that expression of CD14 in blood PMN, (RES)PMN and (INF)PMN from heifer mammary glands was accompanied by apoptosis and necrosis.

• MeSH
• antigeny CD14 genetika   metabolismus   MeSH
• buněčná smrt fyziologie   MeSH
• kultivované buňky MeSH
• mléčné žlázy zvířat fyziologie   MeSH
• neutrofily cytologie   metabolismus   MeSH
• regulace genové exprese fyziologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

BACKGROUND: Macrophages may play a prominent role in defense of the bovine mammary gland, and their functionality is necessary for successful eradication of bacterial pathogens. In contrast to necrosis, however, apoptosis has not yet been studied in macrophages from bovine mammary glands. Therefore, the aim of this study was to confirm the occurrence of apoptosis in macrophages from resting heifer mammary glands and during the inflammatory response. METHODS: Inflammatory response was induced by phosphate buffered saline (PBS) and by lipopolysaccharide (LPS). Resident macrophages (RESMAC) were obtained before and inflammatory macrophages (INFMAC) 24, 48, 72 and 168 hours after inducing inflammatory response in mammary glands of unbred heifers. Cell samples were analyzed for differential counts, apoptosis and necrosis using flow cytometry. RESULTS: Populations of RESMAC and INFMAC contained monocyte-like cells and vacuolized cells. Apoptosis was detected differentially in both morphologically different types of RESMAC and INFMAC and also during initiation and resolution of the inflammatory response. In the RESMAC population, approximately one-tenth of monocyte-like cells and one-third of vacuolized cells were apoptotic. In the INFMAC population obtained 24 h after PBS treatment, approximately one-tenth of monocyte-like cells and almost one-quarter of vacuolized cells were apoptotic. At the same time following LPS, however, we observed a significantly lower percentage of apoptotic cells in the population of monocyte-like INFMAC and vacuolized INFMAC. Moreover, a higher percentage of apoptotic cells in INFMAC was detected during all time points after PBS in contrast to LPS. Comparing RESMAC and INFMAC, we observed that vacuolized cells from populations of RESMAC and INFMAC underwent apoptosis more intensively than did monocyte-like cells. CONCLUSIONS: We conclude that apoptosis of virgin mammary gland macrophages is involved in regulating their lifespan, and it is involved in the resolution process of the inflammatory response.

• MeSH
• antigeny CD14 metabolismus   MeSH
• apoptóza MeSH
• kultivované buňky MeSH
• lipopolysacharidy farmakologie   MeSH
• makrofágy cytologie   metabolismus   MeSH
• mléčné žlázy zvířat cytologie   účinky léků   patologie   MeSH
• regulace genové exprese MeSH
• skot MeSH
• viabilita buněk fyziologie   MeSH
• zánět chemicky indukované   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• Publikační typ
• abstrakty MeSH