F Bozić

University of Zagreb, Zagreb, Grad Zagreb, Croatia

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Publications (9)14.9 Total impact

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    ABSTRACT: Enterotoxigenic Escherichia coli (ETEC) infection is the most common type of porcine postweaning colibacillosis (PWC). Among fimbriae of porcine ETEC strains the best studied family of fimbriae are the members of F4 adhesins, existing in at least three variants: ab, ac, ad. Active immunization against porcine PWC is difficult due to: i) ETEC strains are only one of the essential predisposing factors, ii) the success of vaccinal antigen uptake depends on the presence of enterocyte receptors for F4 adhesins, iii) the intestinal immune system may react with tolerance or hypersensitivity to the same antigens depending on the dose and form of the vaccinal immunogen, and iv) kinetics of the specific immune responses may be different in the case of F4 (earlier) and the other ETEC adhesins, particularly F18 (later). The aim of this study was to test the effectiveness of a live attenuated F4ac+ non-ETEC vaccine against porcine PWC by analyzing quantitative differences in the small intestinal lymphoid and myeloid cell subsets of immunized (with or without levamisole given as an adjuvant) vs control non-immunized pigs. Four week-old pigs were intragastrically immunized with a vaccine candidate F4ac+ non-ETEC strain 2407 at day 0, challenged 7 days later with a virulent F4ac+ strain ETEC 11-800/1/94, euthanatized at day 13 and sampled for immunohistology. Non-immunized pigs received saline at day 0 and were processed as the principals. Immuno-phenotypes of lymphoid and myeloid cell subsets were demonstrated within jejunal and ileal mucosa by immunohistochemical avidin-biotin complex method and corresponding morphometric data were analyzed using software program Lucia G for digital image analyses. Monoclonal antibodies reactive with surface molecules on porcine immune cells such as CD3, CD45RA, CD45RC, CD21 and SWC3 enabled clear insight into distribution patterns and amount of these cells within the gut-associated lymphoid tissues (GALT) examined. The numbers of jejunal and ileal cell subsets tested were significantly increased (at P<0.5 or lower) in both principal groups (vaccinated or levamisole primed-vaccinated) of pigs, compared to those recorded in the control non-vaccinated pigs. Based on the histomorphometric quantification of porcine intestinal immune cells from the GALT compartments tested, it is possible to differentiate the responses of pigs immunized by an experimental mucosal vaccine from those of non-immunized pigs.
    European journal of histochemistry: EJH 01/2010; 54(1):e4. · 2.41 Impact Factor
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    ABSTRACT: Levamisole (2, 3, 5, 6-tetrahydro-6-phenylimidazole 2,1-b thiazole) is a well-known nonspecific stimulator of host defence mechanisms. In previous investigations, we have found that levamisole acts on cell-mediated immunity in challenge-induced porcine postweaning colibacillosis (PWC). We assume that levamisole could also act synergistically on humoural immune response when applied as an adjuvant with vaccine candidate strains for oral immunization of weaned pigs against PWC. The influence of levamisole in combination with experimental F4ac(+) nonenterotoxigenic Escherichia coli (non-ETEC) vacinal strain on proliferation of IgA(+) cells was examined in 4-week-old weaned pigs experimentally infected with ETEC. We have performed identification and morphometric quantification of the plasma cell phenotype within jejunal/ileal mucosa. Plasma cells were identified by immunohistochemistry with monoclonal anti-IgA antibodies and quantifying by use of digital image analysis. Quantification of IgA(+) cells from levamisole-primed vaccinated and challenge-infected weaned pigs showed significantly increased number (P < 0.05 for both jejunum and ileum) compared with those observed in unprimed vaccinated/challenge-infected controls. It is suggested from these results that levamisole may contribute in initiation of local humoural immune response to enteric pathogens, such as enterotoxigenic E. coli.
    Journal of Veterinary Pharmacology and Therapeutics 08/2008; 31(4):328-33. · 1.35 Impact Factor
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    ABSTRACT: Recent findings demonstrate that priming by levamisole of weaned pigs experimentally vaccinated against postweaning colibacillosis (PWC) contributes to immune protection from challenge-induced clinical disease through stimulation of the mesenteric lymph node cells that participate in cell-mediated immunity. With the objective of better understanding the mechanisms by which levamisole induces protective mucosal cell-mediated immune response to vaccination against PWC, it was tested whether the drug synergizes experimental F4ac+ Escherichia coli oral vaccine in stimulating T cells also in the jejunal lamina propria (JLP) and ileal Peyer's patch (IPP) upon virulent challenge. Commercial crossbred pigs weaned at 4 weeks were allocated into two equal groups. The experimental group was i.m. primed with levamisole at an immunostimulatory dose of 2.5 mg/kg once daily, for 3 consecutive days, and controls received saline. Both groups were vaccinated orally with the vaccinal E. coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. The results determined by immunophenotyping of isolated cells indicate that priming by levamisole of the vaccinated weaned pigs selectively recruited and activated T cells in the IPP, a lymphoid organ-generating B lymphocytes. The pig IPP is normally populated with up to 5% of CD3+ T cells and CD6 is an activation antigen expressed exclusively by T cells in swine. Therefore, a significantly higher number of CD3+ (P < 0.01) and CD6+ (P < 0.001) cells observed within the IPP follicles of the primed-vaccinated vs. unprimed-vaccinated challenge-infected pigs suggest enhanced T cell-mediated immunity in this B-cell compartment induced by the potentiating action of the drug and vaccine. The ability of levamisole to influence interaction between activated T cells and B cells in the IPP of primed-vaccinated weaned pigs, and the possibility that this interaction plays a role in regulating B-cell maturation within the IPP follicles, are discussed.
    Journal of Veterinary Pharmacology and Therapeutics 06/2006; 29(3):199-204. · 1.35 Impact Factor
  • F Bozić, V Bilić, I Valpotić
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    ABSTRACT: The present study tested the hypothesis that levamisole exerts its immunopotentiating activity in weaned pigs vaccinated against colibacillosis by priming the lymphocytes and macrophages in the mesenteric lymph node (MLN). Ten weaned piglets were used and allocated into two equal groups. The experimental group was intramuscularly primed with levamisole at an immunostimulatory dose of 2.5 mg/kg given daily, in three consecutive days, and controls received saline according to the same schedule. Both groups were orally vaccinated with the vaccinal Escherichia coli strain on day 0 and challenged with the virulent E. coli strain 7 days later. All pigs were killed on postchallenge day 6. Upon virulent challenge the health status of the two groups was evaluated by clinical observations, and expression of CD25, SWC7 and SWC9 activation antigens by MLN and spleen T and B cells and macrophages, respectively, was tested using flow cytometry. Priming by levamisole significantly contributed to the effectiveness of a live attenuated oral vaccine against porcine postweaning colibacillosis, as evidenced by a good health status of primed vaccinated vs. un-primed vaccinated pigs. The CD3+, CD25+ and SWC9+ MLN but not spleen T cells and macrophages increased in experimental vs. control pigs, implying that levamisole exerts its potentiating activity in the MLN by augmenting both recruitment and activation of cells that participate in cell-mediated immunity.
    Journal of Veterinary Pharmacology and Therapeutics 07/2003; 26(3):225-31. · 1.35 Impact Factor
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    ABSTRACT: To assess the influence of a live attenuated oral vaccine against porcine post-weaning colibacillosis (PWC) induced by enterotoxigenic Escherichia coli (ETEC) on mucosal lymphoid cell CD45 isoforms expression, experimental group of weaned pigs (n=6) was immunized orally with F4ac+ non-ETEC strain (day 0) and challenged with F4ac+ ETEC strain 7 days latter. Non-immunized ETEC-infected pigs (n=6) served as control. All pigs were killed on post-challenge day 7. The small intestine was excised for isolation of jejunal lamina propria (JLP) and ileal Peyer's patch (IPP) lymphocytes and immunohistochemical studies. The results obtained by immunophenotyping of isolated cells show that the proportion of CD45RA+ and CD45RC+ JLP, but not IPP, cells were higher in the non-ETEC-immunized ETEC-infected pigs versus non-immunized infected. Additionally, while CD45RA+ JLP cells increased only slightly, the expression of CD45RC isoform on the JLP cells was significantly higher (P< or =0.01) in the experimental than in the control group. The results of the quantitative phenotypic analysis of isolated lymphocytes were not confirmed by immunohistochemical in situ staining. The majority of intestinal immune cells was found to express CD45RA antigen in situ, but no differences were observed between the two groups of weaned pigs neither in CD45RA+ nor in CD45RC+ cells. Our overall evidence indicates that the increased expression of CD45RC isoform was in fact induced in a limited number of JLP T cells in the vaccinated pigs. This was accompanied with the impaired protection of the vaccinated pigs from challenge-induced PWC.
    Veterinary Immunology and Immunopathology 08/2002; 86(3-4):137-46. · 1.88 Impact Factor
  • F Bozić, V Bilić, I Valpotić
    Journal of Veterinary Pharmacology and Therapeutics 03/2002; 25(1):69-72. · 1.35 Impact Factor
  • F Bozić, E Srebocan, Z Kozarić
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    ABSTRACT: In order to assess the effects of starvation on intestinal intraepithelial lymphocytes (i-IEL) count and histological changes of the carp gut mucosa, one group of fish (n = 10) were fed commercially prepared standard diets and another group of fish (n = 10) were starved for 4 weeks. Carp starved for 4 weeks developed enteropathy, comprising folds atrophy, stratum compactum hyperplasia, significant periodic-acid-Schiff (PAS)-positive (P < 0.00001), but not Alcian blue (ALB)-positive, goblet cell (GC) hyperplasia and a significant decrease (P < 0.00001) in i-IEL numbers. These changes were associated with a dense cellular infiltrate into the lamina propria. Taken together, these data suggest that the pathobiology of starvation-induced i-IELs decrease, matching PAS-positive goblet cell proliferation and inflammatory cells homing to the gut, could be classified as a non-infectious enteropathy induced by starvation.
    Berliner und Münchener tierärztliche Wochenschrift 01/2001; 114(3-4):134-8. · 0.89 Impact Factor
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    ABSTRACT: The potential role of intestinal intraepithelial lymphocytes (i-IELs) in the generation of host protective immunity after helminth infection was investigated using the Trichinella spiralis (Owen, 1835)/mouse model. In this study we found a significant rise of TCRgamma(delta)+ i-IELs (P < 0.001) concurrent with the jejunal goblet cells (GC) hyperplasia in T. spiralis-infected C57BL mice on day 4 p.i. However, no direct relationship between the kinetics of the increase in TCRgamma(delta)+ i-IELs and T. spiralis expulsion was observed in infected mice. Taken together, these results implicate that gamma(delta) i-IELs probably perform a unique functions related to the regulation of the GC proliferation accompanying T. spiralis gut infection. As is known, these TCRgamma(delta)+ i-IELs may release mediators or growth factors that in turn influence GC differentiation. With the use of dexamethason (DEX), a potent anti-inflammatory agent which also induces apoptotic cell death in i-IELs, we have confirmed that the expulsion of T. spiralis from the mouse gut is accompanied by an inflammatory response. Indeed, the GC are clearly involved in these phenomena, apparently under the regulation by TCRgamma(delta)+ i-IEL-mediated responses, since DEX abrogated GC proliferation in T. spiralis-infected C57BL mice and subsequently augmented adult worm burden. Our data also show that the rejection of adult worms starts concurrently with a significant increase in TCRalpha(beta)+ and CD8+ i-IELs (P < 0.05 and P < or = 0.01, respectively), namely by day 7 p.i. At the same time, CD4+ cells significantly decreased (P < 0.05) in the intestinal epithelium of T. spiralis-infected, vs uninfected mice. These results may indicate that the TCRalpha(beta)+ and CD8+ i-IELs act as effectors of anti-T spiralis defence reactions. The implications of these findings for the potential role of intestinal intraepithelial CD8+ and TCRalpha(beta)+ cells in the pathogenesis of the intestinal lesions during T. spiralis gut infection are discussed.
    Folia parasitologica 02/2000; 47(1):55-9. · 2.52 Impact Factor
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    ABSTRACT: To assess the gamma delta TCR T cells in the control of the timing of the mucosal response to enteric parasitic infections, we used C57BL mice, orally infected with 200 viable T. spiralis larvae. The small intestine, spleens and Peyer's patches (PP) were excised on 1, 4, 7, 14, 21 and 29 postinfection days (p.i.) for immunophenotyping and histological studies. Uninfected mice served as control. Characterization of isolated lymphocytes of C57BL control mice, confirmed that T cell immunophenotype differs in spleen, PP and i-IEL. Practically all i-IEL were CD3+ cells (83%). In addition, most of the i-IEL expressed Ly-2 (65%). Among the i-IEL, the level of gamma delta TCR+ cells was significantly higher (29%) than that found in spleen (3%) and PP (3%). The expression was high on CD3+ and Ly-2+ (26 and 21%, respectively) and low on L3T4+ i-IEL (< 1%). During T. spiralis infection alpha beta TCR+ CD3+, gamma delta TCR+ CD3+ and gamma delta TCR+ Ly-2+ i-IEL increased on day 4 and 7. However, infected mice displayed a reduction in i-IEL number from 14 to 29 p.i. day. At the same time the proportion of gamma delta TCR on spleen Ly-2+ and on PP CD3+ and Ly-2+ cells increased on 14 and 21 p.i. day. Adult worms were expelled from the gut by day 14. Thus, the kinetics of gamma delta TCR+ i-IEL, but not spleen and PP gamma delta TCR, corresponded to the kinetics of worm expulsion in C57BL mice. Most murine i-IEL of the gamma delta T cell lineage tend to be cytolytic when activated. We speculated that gamma delta T cells of i-IEL during the early stages of infection recognize and eliminate damaged epithelial cells generated by parasite antigens, simultaneously accelerating the worm expulsion.
    Comparative Immunology Microbiology and Infectious Diseases 07/1998; 21(3):201-14. · 1.81 Impact Factor