Biomedical subjects
Publications and source records attributed to I Trebichavský.
Probiotics modulate production of both cytokine and antimicrobial peptides. This effect can be regarded as a part of complex interplay between them and the host.
The pig amnion was in vivo intraamniotically infected with E. coli for 10 h at 80-85 d of gestation either with the nonpathogenic O86 strain or enteropathogenic O55 strain. TNF-alpha, IL-10, IL-1beta and IFN-gamma were determined in amniotic fluids by ELISA, the expression of cytokines and some other inflammatory markers was determined by immunohistochemistry. Intraamniotic infection induced high levels of TNF-alpha in amniotic fluids which correlated with bacterial virulence whereas IL-10 was induced only by O86. The IL-1beta level did not increase significantly and was expressed in all infected membranes. IFN-gamma was negligible or absent. TNF-alpha, IL-12p40, calprotectin, HSP65 and gp91phox were found by immunohistochemistry only in amnion membranes infected with the enteropathogenic strain 055.
Calprotectin (MRP8/14, S100A8/S100A9, 27E10 antigen) is a heterodimer of two calcium-binding proteins present in the cytoplasm of neutrophils and expressed on the membrane of monocytes. Upon neutrophil activation or endothelial adhesion of monocytes, calprotectin is released and may be detected in serum or body fluids as potentially useful clinical inflammatory marker. The soluble form of calprotectin provides both bacteriostatic and cytokine-like effects in the local environment. When calprotectin metabolism is affected on a systemic level, the zinc-binding properties of protein may induce severe dysregulation of zinc homeostasis with severe clinical symptoms. The distribution of membrane form of calprotectin is restricted to monocytes and immature macrophages and the presence of calprotectin-positive infiltrating cells reflects the influx of mononuclear phagocytes to the site of inflammation. Calprotectin expression and release seems to be of particular importance in immune and immunopathological reactions.
The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus creating a strong need to develop new antimicrobial agents. These new antibiotics should possess novel mechanisms of action and different cellular targets compared with existing antimicrobials. Recent discoveries and isolations of so-called animal antibiotics, mostly small cationic peptides, which represent a potent branch of natural immunity, offered the possibility to acquire new and effective antibiotics of this provenance. To this date, more than 500 antibiotic peptides have been distinguished and defined. Their antimicrobial properties present new opportunities for their use as antibiotics or for construction of their more effective derivatives, but much research is still required to pave the way to their practical use. This is a survey of substances forming an armamentarium of natural immunity of mammals.
One-week-old breast-fed miniature piglets were orally infected either with virulent LT2 strain or with a non-virulent SF1591 rough mutant of Salmonella Typhimurium for 1 d. Both microorganisms were cultivated from mesenteric lymph nodes but not from the blood of infected piglets. Interleukins (IL) 1 beta, 8, 18, tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were quantified by ELISA in plasma and washes of a terminal part of the small bowel. In plasma, cytokines were mostly missing in non-infected piglets and either missing or low in infected piglets. In the gut of non-infected piglets, IL-1 beta, IL-8 and IL-18 were detected whereas TNF-alpha and IFN-gamma were mostly missing. IFN-gamma levels highly increased (p < 0.05) after infection with nonvirulent salmonellae. The variability of cytokine levels in the gut of suckling piglets is discussed.
With the exception of cyclostomes, all vertebrates share the common immune strategy of adaptive, highly specific immunity, based on the products of recombination-activating genes and recombined noninherited receptors for antigens. In addition, they have retained ancient vectors of innate immunity, such as antimicrobial peptides, which are widespread in all eukaryotic organisms and show a high degree of structural homology across most animal taxa. Recently, these substances have become the objects of intensive study for their outstanding bioactive properties with the aim to be applied as very efficient antibiotics, antimicrobials, and even cancerostatics in clinical practice.
Escherichia coli (2 x 10(4) bacteria) of the non-pathogenic O86 strain or enteropathogenic O55 strain were administered into the pig amniotic cavity at 79 to 86 days of gestation for six or ten hours. Translocation of bacteria into fetal lungs was confirmed by cultivation as well as by light and electron microscopy. Infection caused an influx of macrophages that were immunostained in cryostat sections by monoclonal antibody recognizing calprotectin.
Intraamniotic infections caused by viruses, bacteria or mycoplasmas are frequently followed by damage of fetus or increased perinatal morbidity and mortality. Cytokines are key substances regulating a number of biological processes including reproductive and inflammatory processes. An association between intraamniotic infections, rising concentrations of inflammatory cytokines in amniotic fluid and preterm labor is suggested. A great effort is made to find reliable markers typical for intraamniotic infections with high predictive value that make possible prompt identification of patients with intraamniotic infection. This review concerns inflammatory mediators, especially IL-1, IL-6, IL-8, TNF-alpha, and other important biologically active substances as prostaglandins and NO metabolites and their roles in intraamniotic infections. Finally, we discuss their relevance for diagnosis of intraamniotic infections.
Reactive NO metabolites play a distinct role in the control of Salmonella enterica serovar Typhimurium (ST; a facultative intracellular pathogen) in susceptible host. A significant increase of nitrite and/or nitrate plasma levels, 3-nitro-tyrosine expression and pathological changes in mesenteric lymph nodes have been observed in gnotobiotic piglets orally infected for 1 d with a virulent strain of ST but not in piglets infected with a rough mutant of ST.
Germ-free piglets were orally infected with either enteropathogenic E. coli 055 or a virulent strain of Salmonella typhimurium. Orally applied antiserum against E. coli protected infected animals in spite of the fact that the bacteria were consistently found in mesenteric lymph nodes and other organs. By contrast, the application of an antiserum against S. typhimurium was without any effect on the outcome of infection. The treatment with anti-bacterial antiserum prevented inflammation of ileal mucosa (TNF-alpha and heat shock protein 65 expression) only in piglets infected with E. coli. A decrease in the frequency of ileal MAC320+ cells was observed in all infected piglets treated with antiserum.
Before the onset of specific immune response, the host defence against Salmonella infection is regulated by cytokines characteristic for immunity to intracellular bacteria. Cytokine response to non-typhoidal Salmonellae is described.
The germ-free pig model is shown to be useful for studying salmonellosis. The immune status of germ-free and infected gnotobiotic piglets is described. The regulatory role of cytokine is discussed and compared with our experimental findings.
The effect of controlled antigenic stimulation in immunologically virgin organisms, i.e. pig fetuses treated with NDCM (Nocardia delipidated cell mitogen) and germ-free (GF) piglets associated with a non-pathogenic E. coli 086, on peripheral blood lymphocyte subsets defined by the expression of CD5 and CD8 was studied by double color flow cytometry. Stimulation of both fetuses and GF piglets increased the frequency of CD8low+ lymphocytes. A prominent subset of CD5-CD8low+NK cells was present in GF and E. coli associated piglets and their frequency was slightly higher in E. coli associated animals. The most pronounced difference between stimulated and non-stimulated animals was in a relative proportion of an ill-defined lymphocyte subset with an unusual CD5low+CD8low+ expression. Both NDCM injection into fetal blood circulation and association of GF piglets with E. coli resulted in a marked increase of frequency of CD5low+CD8low+ lymphocytes in peripheral blood.
We have recently reported that thymic B lymphocytes (TBL) are the first B-cell subpopulation undergoing isotype switching to IgG and IgA during embryonic life. The aim of this study is to analyze the influence of antigenic stimulation on TBL location and activity using a germ-free (GF) newborn pig model, in which maternal antibodies and antigens do not affect B-cell development. Immunohistological analysis showed that TBL were disseminated mainly in the thymic medulla. There were no differences in the distribution of TBL, both in GF newborn piglets before and after colonization with Escherichia coli and in older conventionally reared (CONV) piglets. The number of immunoglobulin (Ig)-secreting cells measured by the ELISPOT method was not influenced by microflora and food antigens. IgM-positive cells secreting IgM and CD45RC-positive cells spontaneously producing IgM, IgG, and IgA were detected in newborn thymus. Our findings suggest that TBL differentiation and Ig switching to IgG and IgA-secreting cells is not influenced by external antigens and that the thymic microenvironment plays an important role in this process.
Prenatal development of cord blood monocytes and tissue macrophages was studied in pig foetuses by immunophenotyping and functional assays. The function of peripheral blood monocytes was compared in germ-free and conventional piglets. First macrophages were identified by electron microscopy in foetal liver on the 25th day of gestation. Monoclonal antibodies against porcine CD45 and SWC3 antigens were used for flow cytometric identification of myelomonocytic cells in cell suspensions prepared from the yolk sac, foetal liver, spleen and cord blood. Leukocytes expressing the common myelomonocytic antigen SWC3 were found in all organs studied since the earliest stages of development. Opsonized zymosan ingestion assay was used to determine the phagocytic capacity of foetal mononuclear phagocytes isolated from cord blood, liver and spleen. In the foetal liver, avid phagocytosis of apoptic cells had been found to occur before cells were able to ingest zymosan in vitro. The first cells capable of ingesting zymosan particles were found on the 40th day of gestation in umbilical blood and 17 days later in foetal spleen and liver. Their relative proportion increased with age. Cord blood monocytes and peripheral blood monocytes in germ-free piglets had low oxidatory burst activity as shown by iodonitrophenyl tetrazolium reduction assay. A remarkable increase of oxidatory burst activity was observed in conventional piglets, probably due to activation of immune mechanisms by the microflora colonizing gastrointestinal tract.
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3-day-old miniature piglets were stimulated in vivo with Bacillus firmus by the intraperitoneal or intragastric route for 1 d. Cells containing IgA and IgG2 were detected in the ileum in all stimulated but not in control animals. The frequency of blood CD3+ cells increased after intraperitoneal administration of B. firmus, the ratio of polymorphonuclears to lymphocytes increased in all stimulated piglets. B. firmus induced antitumor immunity in rats with transplanted Yoshida sarcoma cells. Granular lymphocytes and dead tumor cells were found in peritoneal exudate of stimulated animals. B. firmus induced IFN-gamma synthesis in human blood lymphocytes stimulated in vitro for 1 d. The amount of TNF-alpha produced by these stimulated human peripheral blood mononuclears (PBMC) was lower than that of PBMC stimulated with some other bacterial immunomodulators. Cells containing TGF-beta or IL-8 were not found in human PBMC stimulated with B. firmus.
Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFN alpha) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFN alpha secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFN alpha-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFN alpha was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFN alpha in utero inducibility by mutant virus as compared with wild virus. Flow cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFN alpha secretion. The present data provided in vivo evidence of IFN alpha secretion at the cell level in foetal lymphohaematopoietic organs. Such IFN alpha-secreting cells in lymphohaematopoietic tissues may be the source of IFN alpha detected during foetal infections.