BACKGROUND: In view of the ever-increasing representation of Staphylococcus spp. strains resistant to various antibiotics, the development of in vivo models for evaluation of novel antimicrobials is of utmost importance. METHODS: In this article, we describe the development of a fully immunocompetent porcine model of extensive skin and soft tissue damage suitable for testing topical antimicrobial agents that matches the real clinical situation. The model was developed in three consecutive stages with protocols for each stage amended based on the results of the previous one. RESULTS: In the final model, 10 excisions of the skin and underlying soft tissue were created in each pig under general anesthesia, with additional incisions to the fascia performed at the base of the defects and immediately inoculated with Staphylococcus aureus suspension. One pig was not inoculated and used as the negative control. Subsequently, the bandages were changed on Days 4, 8, 11, and 15. At these time points, a filter paper imprint technique (FPIT) was made from each wound for semi-quantitative microbiological evaluation. Tissue samples from the base of the wound together with the adjacent intact tissue of three randomly selected defects of each pig were taken for microbiological, histopathological, and molecular-biological examination. The infection with the inoculated S. aureus strains was sufficient during the whole experiment as confirmed by both FPIT and from tissue samples. The dynamics of the inflammatory markers and clinical signs of infection are also described. CONCLUSIONS: A successfully developed porcine model is suitable for in vivo testing of novel short-acting topical antimicrobial agents.

• Klíčová slova
• Staphylococcus aureus, animal model, antimicrobial agents, porcine model, skin and soft‐tissue infection (SSTI), wound infection,
• MeSH
• antibakteriální látky * aplikace a dávkování   terapeutické užití   farmakologie   MeSH
• aplikace lokální MeSH
• infekce měkkých tkání * farmakoterapie   mikrobiologie   MeSH
• kůže mikrobiologie   patologie   MeSH
• methicilin rezistentní Staphylococcus aureus * účinky léků   MeSH
• modely nemocí na zvířatech * MeSH
• prasata MeSH
• stafylokokové infekce kůže * farmakoterapie   mikrobiologie   MeSH
• stafylokokové infekce * farmakoterapie   mikrobiologie   MeSH
• Staphylococcus aureus * účinky léků   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antibakteriální látky * MeSH

The aim of this study was to establish a cell culture system for the generation of porcine monocyte-derived macrophages (MDMs) under reduced-serum conditions. Cultures based on either the Nu-Serum™ Growth Medium Supplement (NUS) or a conventional fetal bovine serum (FBS) were compared, which included the assessment of FBS from two different providers (FBS1 and FBS2). The data obtained confirmed the significant impact of culture conditions on in vitro-generated MDMs. The MDMs cultured under reduced-serum conditions showed increased levels of IL-1β and CD86 mRNA and a proinflammatory cytokine profile, characterized by the increased mRNA expression of IL-23p19, CXCL10, and CCL5. Phagocytic and respiratory burst activities were not adversely affected. Surprisingly, the difference between the two FBSs was much more pronounced than the effect of the reduced-serum supplement. The FBS1 culture conditions gave rise to macrophages with higher surface levels of CD14, CD16, and CD163, a lower CD80 mRNA expression, and an increased induction of IL-10 gene expression. In contrast, none of these trends were observed in macrophage cultures supplemented with FBS2. Instead, the FBS2 culture showed increased levels of IL-1b and CD86 mRNA. In conclusion, reduced-serum culture is a useful tool for in vitro porcine MDM generation, in line with the current research trend of reducing FBS use in biological research.

• Klíčová slova
• in vitro, monocyte-derived macrophages, pig, porcine, serum reduction,
• Publikační typ
• časopisecké články MeSH

In Glässer's disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

• Klíčová slova
• Glaesserela parasuis, Haemophilus parasuis, antibodies, antioxidants, capsule, catalase, porcine alveolar macrophages, reactive oxygen species,
• MeSH
• alveolární makrofágy imunologie   metabolismus   mikrobiologie   MeSH
• antigeny bakteriální imunologie   MeSH
• bakteriální pouzdra imunologie   MeSH
• fagocytóza MeSH
• Haemophilus parasuis imunologie   patogenita   MeSH
• hemofilové infekce imunologie   metabolismus   mikrobiologie   MeSH
• kinetika MeSH
• kultivované buňky MeSH
• neutralizující protilátky imunologie   metabolismus   MeSH
• protilátky bakteriální imunologie   metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• séroskupina MeSH
• specificita protilátek MeSH
• Sus scrofa MeSH
• virulence MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny bakteriální MeSH
• neutralizující protilátky MeSH
• protilátky bakteriální MeSH
• reaktivní formy kyslíku MeSH

Deoxynivalenol (DON) is a mycotoxin frequently found in cereals, and pigs are one of the most sensitive farm species to DON. The aim of this study was to determine the effects of DON in very low doses on peripheral blood mononuclear cells (PBMC) and on particular lymphocyte subpopulations. The cells were exposed to 1, 10 and 100 ng/mL of DON and lymphocyte viability, proliferation, and cytokine (Interleukin (IL)-1β, IL-2, IL-8, IL-17, Interferon (IFN) γ and tumor necrosis factor (TNF) α production were studied. Cells exposed to DON for 5 days in concentrations of 1 and 10 ng/mL showed higher viability compared to control cells. After 18 h of DON (100 ng/mL) exposure, a significantly lower proliferation after mitogen stimulation was observed. In contrast, an increase of spontaneous proliferation induced by DON (100 ng/mL) was detected. After DON exposure, the expression of cytokine genes decreased, with the exception of IL-1β and IL-8, which increased after 18 h exposure to 100 ng/mL of DON. Among lymphocyte subpopulations, helper T-cells and γδ T-cells exhibiting lower production of IL-17, IFNγ and TNFα were most affected by DON exposure (10 ng/mL). These findings show that subclinical doses of DON lead to changes in immune response.

• Klíčová slova
• PBMC, animal health, cytokines, deoxynivalenol, immunotoxicity, lymphocytes, pig, subclinical dose,
• MeSH
• cytokiny biosyntéza   genetika   MeSH
• exprese genu účinky léků   MeSH
• kultivované buňky MeSH
• leukocyty mononukleární účinky léků   imunologie   MeSH
• podskupiny lymfocytů účinky léků   imunologie   MeSH
• prasata MeSH
• proliferace buněk účinky léků   MeSH
• trichotheceny toxicita   MeSH
• viabilita buněk účinky léků   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokiny MeSH
• deoxynivalenol MeSH Prohlížeč
• trichotheceny MeSH

BACKGROUND: Lactoferrin (LF) is an 80 kDa glycoprotein which is known for its effects against bacteria, viruses and other pathogens. It also has a high potential in nutrition therapy and welfare of people and a variety of animals, including piglets. The ability to bind lipopolysaccharide (LPS) is one of the described anti-inflammatory mechanisms of LF. Previous studies suggested that cells can be stimulated even by LPS-free LF. Therefore, the aim of our study was to bring additional information about this possibility. Porcine monocyte derived macrophages (MDMF) and human embryonic kidney (HEK) cells were stimulated with unpurified LF in complex with LPS and with purified LF without bound LPS. RESULTS: Both cell types were stimulated with unpurified as well as purified LF. On the other hand, neither HEK0 cells not expressing any TLR nor HEK4a cells transfected with TLR4 produced any pro-inflammatory cytokine transcripts after stimulation with purified LF. This suggests that purified LF without LPS stimulates cells via another receptor than TLR4. An alternative, TLR4-independent, pathway was further confirmed by analyses of the NF-kappa-B-inducing kinase (NIK) activation. Western blot analyses showed NIK which activates different NFκB subunits compared to LF-LPS signaling via TLR4. Though, this confirmed an alternative pathway which is used by the purified LF free of LPS. This stimulation of MDMF led to low, but significant amounts of pro-inflammatory cytokines, which can be considered as a positive stimulation of the immune system. CONCLUSION: Our results suggest that LF's ability is not only to bind LPS, but LF itself may be a stimulant of pro-inflammatory pathways.

• Klíčová slova
• Inflammatory cytokines, LPS, NFκB, NIK, TLR4,
• MeSH
• cytokiny genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• laktoferrin izolace a purifikace   farmakologie   MeSH
• lidé MeSH
• lipopolysacharidy farmakologie   MeSH
• makrofágy účinky léků   MeSH
• prasata MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• regulace genové exprese účinky léků   MeSH
• signální transdukce účinky léků   MeSH
• toll-like receptor 4 genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zánět MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny MeSH
• intracelulární signální peptidy a proteiny MeSH
• laktoferrin MeSH
• lipopolysacharidy MeSH
• Nik related kinase MeSH Prohlížeč
• protein-serin-threoninkinasy MeSH
• toll-like receptor 4 MeSH

Poultry is the most frequent reservoir of non-typhoid Salmonella enterica for humans. Understanding the interactions between chickens and S. enterica is therefore important for vaccine design and subsequent decrease in the incidence of human salmonellosis. In this study we therefore characterized the interactions between chickens and phoP, aroA, SPI1 and SPI2 mutants of S. Enteritidis. First we tested the response of HD11 chicken macrophage-like cell line to S. Enteritidis infection monitoring the transcription of 36 genes related to immune response. All the mutants and the wild type strain induced inflammatory signaling in the HD11 cell line though the response to SPI1 mutant infection was different from the rest of the mutants. When newly hatched chickens were inoculated, the phoP as well as the SPI1 mutant did not induce an expression of any of the tested genes in the cecum. Despite this, such chickens were protected against challenge with wild-type S. Enteritidis. On the other hand, inoculation of chickens with the aroA or SPI2 mutant induced expression of 27 and 18 genes, respectively, including genes encoding immunoglobulins. Challenge of chickens inoculated with these two mutants resulted in repeated induction of 11 and 13 tested genes, respectively, including the genes encoding immunoglobulins. In conclusion, SPI1 and phoP mutants induced protective immunity without inducing an inflammatory response and antibody production. Inoculation of chickens with the SPI2 and aroA mutants also led to protective immunity but was associated with inflammation and antibody production. The differences in interaction between the mutants and chicken host can be used for a more detailed understanding of the chicken immune system.

• MeSH
• atenuované vakcíny imunologie   MeSH
• buněčné linie MeSH
• cékum imunologie   mikrobiologie   MeSH
• kur domácí MeSH
• makrofágy imunologie   MeSH
• mutace MeSH
• nemoci drůbeže imunologie   mikrobiologie   MeSH
• Salmonella enteritidis * genetika   imunologie   MeSH
• salmonelová infekce u zvířat imunologie   mikrobiologie   MeSH
• salmonelové vakcíny imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• atenuované vakcíny MeSH
• salmonelové vakcíny MeSH

Chickens can be infected with Salmonella enterica at any time during their life. However, infections within the first hours and days of their life are epidemiologically the most important, as newly hatched chickens are highly sensitive to Salmonella infection. Salmonella is initially recognized in the chicken caecum by TLR receptors and this recognition is followed by induction of chemokines, cytokines and many effector genes. This results in infiltration of heterophils, macrophages, B- and T-lymphocytes and changes in total gene expression in the caecal lamina propria. The highest induction in expression is observed for matrix metalloproteinase 7 (MMP7). Expression of this gene is increased in the chicken caecum over 4000 fold during the first 10 days after the infection of newly hatched chickens. Additional highly inducible genes in the caecum following S. Enteritidis infection include immune responsive gene 1 (IRG1), serum amyloid A (SAA), extracellular fatty acid binding protein (ExFABP), serine protease inhibitor (SERPINB10), trappin 6-like (TRAP6), calprotectin (MRP126), mitochondrial ES1 protein homolog (ES1), interferon-induced protein with tetratricopeptide repeats 5 (IFIT5), avidin (AVD) and transglutaminase 4 (TGM4). The induction of expression of these proteins exceeds a factor of 50. Similar induction rates are also observed for chemokines and cytokines such as IL1β, IL6, IL8, IL17, IL18, IL22, IFNγ, AH221 or iNOS. Once the infection is under control, which happens approx. 2 weeks after infection, expression of IgY and IgA increases to facilitate Salmonella elimination from the gut lumen. This review outlines the function of individual proteins expressed in chickens after infection with non-typhoid Salmonella serovars.

• MeSH
• cékum metabolismus   MeSH
• exprese genu * MeSH
• kur domácí * MeSH
• nemoci drůbeže genetika   mikrobiologie   MeSH
• ptačí proteiny genetika   metabolismus   MeSH
• Salmonella fyziologie   MeSH
• salmonelová infekce u zvířat genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ptačí proteiny MeSH

BACKGROUND: Following infection and initial multiplication in the gut lumen, Salmonella Typhimurium crosses the intestinal epithelial barrier and comes into contact with cells of the host immune system. Mononuclear phagocytes which comprise macrophages and dendritic cells (DC) are of key importance for the outcome of Salmonella infection. Although macrophages and DC may differentiate from a common precursor, their capacities to process and present antigen differ significantly. In this study, we therefore compared the response of porcine macrophages and DC differentiated from peripheral blood monocytes to S. Typhimurium and one of the most potent bacterial pathogen associated molecular patterns, bacterial lipopolysaccharide. To avoid any bias, the expression was determined by protein LC-MS/MS and verified at the level of transcription by quantitative RT-PCR. RESULTS: Within 4 days of culture, peripheral blood monocytes differentiated into two populations with distinct morphology and expression of MHC II. Mass spectrometry identified 446 proteins in macrophages and 672 in DC. Out of these, 433 proteins were inducible in macrophages either after infection with S. Typhimurium or LPS exposure and 144 proteins were inducible in DC. The expression of the 46 most inducible proteins was verified at the level of transcription and the differential expression was confirmed in 22 of them. Out of these, 16 genes were induced in both cell types, 3 genes (VCAM1, HMOX1 and Serglycin) were significantly induced in macrophages only and OLDLR1 and CDC42 were induced exclusively in DC. Thirteen out of 22 up-regulated genes contained the NF-kappaB binding site in their promoters and could be considered as either part of the NF-kappaB feedback loop (IkappaBalpha and ISG15) or as NF-kappaB targets (IL1beta, IL1alpha, AMCF2, IL8, SOD2, CD14, CD48, OPN, OLDLR1, HMOX1 and VCAM1). CONCLUSIONS: The difference in the response of monocyte derived macrophages and DC was quantitative rather than qualitative. Despite the similarity of the responses, compared to DC, the macrophages responded in a more pro-inflammatory fashion.

• MeSH
• buněčná diferenciace MeSH
• dendritické buňky účinky léků   imunologie   mikrobiologie   MeSH
• kultivované buňky MeSH
• lipopolysacharidy farmakologie   MeSH
• makrofágy cytologie   účinky léků   imunologie   mikrobiologie   MeSH
• prasata * MeSH
• regulace genové exprese účinky léků   imunologie   MeSH
• Salmonella typhimurium fyziologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• lipopolysacharidy MeSH

BACKGROUND: This study aims to investigate the anti-inflammatory effect of biologically active phospholipids (BAP) used in preparations for clinical practice in humans. Until date, except anti-neoplastic ability, little is known about anti-inflammatory property of the phospholipids. METHODS: While the course of bacterially induced acute pneumonia and markers of inflammation were studied in in vivo system in pigs orally supplemented with BAP, the pro- and anti-inflammatory response of lipopolysaccharide-stimulated porcine monocyte-derived macrophages to 24 h- and 48 h-treatment by BAP was investigated in in vitro system. In vivo, the animal health status was monitored and pro-inflammatory IL-1β and IL-8 in sera were detected by ELISA during the experiment, while bronchoalveolar lavage fluids (BALF) and the lungs were examined post-mortem. Total and differential counts of white blood cell (WBC) were determined in blood and BALF. In vitro, mRNA expression of pro-inflammatory (TNF-α, IL-1β, CXCL10) and anti-inflammatory (IL-10 and Arg1) cytokines, and level of activated caspase 1 and phosphorylated protein kinase C epsilon (pPKCϵ), were studied using qRT-PCR and Western blot, respectively. For the purposes of both systems, 6 animals were used in each of the BAP-supplemented and the control groups. RESULTS: In vivo, BAP had a positive influence on the course of the disease. The immunomodulatory effects of BAP were confirmed by lower levels of IL-1β, IL-8, and a lower WBC count in the supplemented group in comparison with the control group. A lower percentage of lung parenchyma was affected in the supplemented group comparing to the control group (on average, 4% and 34% of tissue, respectively). In vitro, BAP suppressed mRNA expression of mRNA for IL-10 and all pro-inflammatory cytokines tested. This down-regulation was dose- and time-dependent. Arg1 mRNA expression remained unaffected. Further dose- and time-dependent suppression of the activated caspase 1 and pPKCϵ was detected in macrophages when treated with BAP. CONCLUSIONS: Our results demonstrate that BAP has anti-inflammatory and immunomodulatory properties, thus emphasizing the potential of this compound as a natural healing agent.

• MeSH
• antiflogistika farmakologie   MeSH
• bakteriální pneumonie metabolismus   patologie   MeSH
• bronchoalveolární lavážní tekutina cytologie   MeSH
• cytokiny krev   MeSH
• fosfolipidethery farmakologie   MeSH
• kultivované buňky MeSH
• leukocyty MeSH
• lipopolysacharidy MeSH
• makrofágy účinky léků   MeSH
• plíce účinky léků   patologie   MeSH
• prasata MeSH
• zánět farmakoterapie   metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antiflogistika MeSH
• cytokiny MeSH
• fosfolipidethery MeSH
• lipopolysacharidy MeSH

The prevalence of Salmonella enterica serovar Enteritidis is gradually decreasing in poultry flocks in the EU, which may result in the demand for a vaccine that allows for the differentiation of vaccinated flocks from those infected by wild-type S. Enteritidis. In this study, we therefore constructed a (Salmonella Pathogenicity Island 1) SPI1-lon mutant with or without fliC encoding for S. Enteritidis flagellin. The combination of SPI1-lon mutations resulted in attenuated but immunogenic mutant suitable for oral vaccination of poultry. In addition, the vaccination of chickens with the SPI1-lon-fliC mutant enabled the serological differentiation of vaccinated and infected chickens. The absence of fliC therefore did not affect the immunogenicity of the vaccine strain and allowed for serological differentiation of the vaccinated chickens. The SPI1-lon-fliC mutant is therefore a suitable marker vaccine strain for oral vaccination of poultry.

• MeSH
• bakteriální proteiny genetika   imunologie   MeSH
• flagelin genetika   imunologie   MeSH
• kur domácí MeSH
• mutace * MeSH
• nemoci drůbeže prevence a kontrola   MeSH
• proteasa La genetika   imunologie   MeSH
• protilátky bakteriální imunologie   MeSH
• Salmonella enteritidis genetika   růst a vývoj   imunologie   ultrastruktura   MeSH
• salmonelová infekce u zvířat prevence a kontrola   MeSH
• salmonelové vakcíny aplikace a dávkování   imunologie   MeSH
• vakcinace veterinární   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• flagelin MeSH
• proteasa La MeSH
• protilátky bakteriální MeSH
• salmonelové vakcíny MeSH
• Spi1 protein, Salmonella MeSH Prohlížeč