Clinical disease upregulates expression of CD40 and CD40 ligand on peripheral blood mononuclear cells from cattle naturally infected with Mycobacterium avium subsp. paratuberculosis.

CD40 and CD40 ligand (CD40L) have costimulatory effects as part of a complex series of events in host immunity. In this study, the expression of CD40 and CD40L on peripheral blood mononuclear cells (PBMCs) isolated from cattle with Johne's disease were measured on freshly isolated PBMCs and on cells cultured for 8, 24, and 72 h in the presence or absence of live Mycobacterium avium subsp. paratuberculosis and exogenous gamma interferon, interleukin 10, and transforming growth factor ß. Results demonstrated greater CD40 and CD40L expression on fresh PBMCs obtained from animals in the clinical stage of disease (symptomatic) than those from healthy control animals or cows in the subclinical stage of disease (asymptomatic). A similar expression profile with greater magnitude was noted for cultured PBMCs, with increased CD40 expression after 8 and 24 h of culture and increased CD40L expression between 24 and 72 h on PBMCs obtained from clinically infected animals. The addition of live M. avium subsp. paratuberculosis to cell cultures resulted in downregulation of CD40L expression in naturally infected cows, regardless of the disease stage. In contrast, the addition of live M. avium subsp. paratuberculosis to cultures resulted in upregulation of CD40 expression on cells obtained from clinically infected animals, while a decrease in expression was noted for healthy and subclinically infected cows. No effects of exogenous cytokines on CD40 or CD40L expression were observed. These results clearly point for the first time to a disparity in the expression of these costimulatory molecules on immune cells from cattle in different stages of Johne's disease and suggest further investigation into their roles in paratuberculosis pathogenesis.

Effect of supplementation of probiotics and phytosterols alone or in combination on serum and hepatic lipid profiles and thyroid hormones of hypercholesterolemic rats.

Probiotic bacteria and phytosterols are natural hypocholesterolemic agents with potential cardiovascular benefits. Accordingly, the present study was conducted to evaluate the effect of supplementation of probiotics and phytosterols alone or in combination on serum and hepatic lipid profiles and thyroid hormones of hypercholesterolemic rats. Mixed probiotics treatment consisted of 8 probiotic strains: 2 strains of each of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, and Lactobacillus reuteri. The rats were fed for 8 wk with the given treatments in addition to a high-fat-high-cholesterol basal diet to induce hypercholesterolemia. Results showed that supplementation significantly reduced serum total cholesterol, low-density-lipoprotein cholesterol (LDL-C), high-density-lipoprotein cholesterol, and triglycerides compared with the controls. The symbiotic treatment was more effective in lowering LDL-C, whereas mixed probiotics treatment more effectively lowered serum total cholesterol and LDL-C than the phytosterol-containing treatment. The phytosterol-containing treatments induced the increased activity of thyroid glands, as evident by elevated levels of serum total thyroxine, total triiodothyronine, and free triiodothyronine. In conclusion, the lipid profile can effectively be reduced to lower the incidence of cardiovascular disease using combinations of Lactobacillus-based probiotics and phytosterols in functional foods.

Effects of topically applied agents on intra-oral wound healing in a rat model: a clinical and histomorphometric study.

OBJECTIVES: Topically applied chlorhexidine and hyaluronan have many studies supporting their use to enhance oral wound healing. Allantoin is widely used topically to promote epithelial proliferation and wound healing, with very little scientific evidence to support such uses. This study investigated and compared the influence of these agents on the healing of intra-oral excisional wounds with large epithelial and connective tissue defects. METHODS: Excisional wounds, 3 mm in diameter, were made at the centre of the palate of 125 Wistar male albino rats. Five animals constituted the baseline group at time 0. The remaining animals were divided into four experimental and one control groups, in which chlorhexidine digluconate gel 0.2% (Perio.Kin®), hyaluronan gel (Gengigel®), allantoin 0.5% in vehicle gel, vehicle gel alone and nothing were applied daily to the wounds. The wound areas were measured photographically and the epithelialization rates were determined histologically at 0, 3, 7, 14 and 21 days post-surgery. RESULTS: The mean wound area and mean distance between the epithelial margins decreased significantly with time in all experimental and control groups (P < 0.05). A significant rate of wound area reduction was observed following the use of Perio.Kin® and Gengigel® at 7 and 14 days. Perio.Kin® showed a significant rate of wound epithelialization at 7 days. Allantoin did not positively or negatively affect wound healing. CONCLUSIONS: None of the tested agents had a negative effect on the rate of wound healing when applied on an excisional wound with epithelial and connective tissue defect. Positive results were achieved with Perio.Kin® and Gengigel®.

Investigation of the role of tumour necrosis factor-{alpha}, interleukin-1{beta}, interleukin-10, nitric oxide and rheumatoid factor-immunoglobulin M in a rat model of arthritis.

UNLABELLED: Collagen-induced arthritis (CIA) in rats is a widely used preclinical animal model of rheumatoid arthritis (RA). However, CIA development in Sprague-Dawley (SD) rats is less severe in terms of inflammatory response compared with other strains. Therefore, a modified CIA model called MCIA, using N-acetylmuramyl dipeptide (MDP), has been developed in the less sensitive SD rat strains. This work was conducted to better understand the immunopathological role and contributions of the pro-inflammatory T-helper type 1 (Th-1) cytokines and inflammatory mediators, interleukin-1 (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO); the anti-inflammatory T-helper type 2 (Th-2) cytokine, IL-10 and autoantibodies such as rheumatoid factor (RF)-immunoglobulin M (IgM) in this newly developed RA model. TNF-alpha, NO and RF-IgM levels were significantly increased, while IL-1beta levels were not affected in this MCIA rat model. The levels of IL-10 were lower than the baseline when compared with controls. IN CONCLUSION: (1) the immunological features represented in the MCIA rat model favour the Th-1 cytokine profile over Th-2 and (2) RF-IgM can be used as a diagnostic test in preclinical RA models.

Assessment of humoral and cellular-mediated immune response in chickens treated with tilmicosin, florfenicol, or enrofloxacin at the time of Newcastle disease vaccination.

The effect of tilmicosin, florfenicol, or enrofloxacin on humoral and cell-mediated immune response induced by Newcastle disease (ND) vaccination was evaluated in 20-wk-old specific-pathogen-free layer chickens. Humoral immunity was measured by detection of ND virus (NDV) antibody titer and anti-NDV IgG response using the hemagglutination inhibition (HI) test and ELISA, respectively, whereas cell-mediated immunity was evaluated by measurement of chicken interferon gamma (ChIFN-gamma) produced in splenocytes cell culture stimulated with concanavalin A, inactivated NDV antigen, or live attenuated La Sota strain using ELISA. Florfenicol hampered the ND antibody production measured by both HI and ELISA. Tilmicosin and enrofloxacin reduced the production of ND antibody in the first 3 wk after the last ND vaccination measured by HI test, which suggests that these antibiotics exert their effect mainly on the IgM isotype. The ND-vaccinated, but not treated group, showed an increase in ChIFN-gamma production after NDV antigen-specific stimulation above the nonstimulated cell culture, whereas this effect was masked in all the antibiotic-treated groups due to the stronger ChIFN-gamma production background value reported in the nonstimulated cell culture. In conclusion, our results showed, for the first time, that tilmicosin, florfenicol, or enrofloxacin reduced the humoral immune response and had beneficial effects on the cell-mediated immune response. In addition, we demonstrated that the combination of both inactivated and attenuated ND vaccine gave a strong immune response at both the humoral and cellular level.

Role of nitric oxide production in dairy cows naturally infected with Mycobacterium avium subsp. paratuberculosis.

Nitric oxide (NO) is a crucial mediator in host defense and is one of the major killing mechanisms within macrophages. Its induction is highly affected by the types of cytokines and the infectious agents present. In the current study, NO production was evaluated after in vitro infection of unfractionated peripheral blood mononuclear cells (PBMCs) with Mycobacterium avium subsp. paratuberculosis (MAP) after 8h, 3 and 6 days of culture for cows in different stages of disease. In addition, the effects of in vitro exposure to inhibitory cytokines such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta) as well as the pro-inflammatory cytokine IFN-gamma were correlated with the level of NO production. Nitric oxide production was consistently higher in cell cultures from subclinically infected animals at all time points. An upregulation of NO production was demonstrated in unfractionated cell cultures from healthy control cows after exposure to MAP infection as compared to noninfected cell cultures. A similar increase in NO due to the addition of MAP to cell cultures was also noted for clinically infected cows. NO level among subclinically infected cattle was greater at all time points tested and was further boosted with the combination of both in vitro MAP infection and IFN-gamma stimulation. Alternatively, nonspecific stimulation with LPS from Escherichia coli O111:B4-W resulted in an upregulation of NO production in all infected groups at 3 and 6 days after in vitro infection. Finally, the in vitro exposure to inhibitory cytokines such as IL-10 and TGF-beta prior to MAP infection or LPS stimulation resulted in the downregulation of this inflammatory mediator (NO) in all experimental groups at all time points. In summary, a higher level of NO production was associated with cows in the subclinical stage of MAP infection. As well, the results demonstrated an increase in NO production upon infection with MAP and in the presence of exogenous IFN-gamma. Finally, the results suggest an important role of IL-10 and TGF-beta on the profile of NO production which may explain the low NO production in MAP clinically infected cows.

Differential expression of CD5 on B lymphocytes in cattle infected with Mycobacterium avium subsp. paratuberculosis.

CD5 is a cell surface molecule involved in antigen recognition and is present on all T lymphocytes and a subset of B lymphocytes. The purpose of this study was to examine CD5+ expression on peripheral blood B cells from healthy, noninfected cattle and cattle with subclinical and clinical paratuberculosis. Peripheral blood mononuclear cells (PBMC) were freshly isolated or cultured for 7 days in the presence or absence of live Mycobacterium avium subsp. paratuberculosis (M. avium subsp. paratuberculosis), and then analyzed by flow cytometry for CD5 expression within the B cell subpopulation. Analysis demonstrated a significant increase (P<0.01) in B cells in clinical animals as compared to healthy control cows and subclinically infected cows. In addition, three subpopulations within the CD5+ B cell population were identified: CD5dim, CD5bright, and a minor population that was characterized as CD5extra bright. A decrease in the CD5dim B cell population along with a concomitant increase in CD5bright B cells was observed in infected cows, an effect that was highly significant (P<0.01) for subclinically infected cows in cultured PBMC. In vitro infection with live M. avium subsp. paratuberculosis did not affect CD5+ expression patterns on B cells, regardless of animal infection status. Addition of exogenous IL-10 to PBMC cultures resulted in decreased numbers of CD5(bright) B cells for healthy control cows, whereas, a synergistic effect of IL-10 and infection with live M. avium subsp. paratuberculosis resulted in increased CD5bright B cells for subclinically infected cows. These results suggest that differential expression of CD5bright and CD5dim subpopulations on B cells in animals with paratuberculosis may reflect a shift in host immunity during the disease process.

Upregulation of transforming growth factor-beta and interleukin-10 in cows with clinical Johne's disease.

Johne's disease progresses through distinct stages including a protracted subclinical stage in which the infection appears to be controlled; followed by a more acute stage in which the host animal demonstrates clinical signs such as diarrhea and weight loss. Little is known about the dynamics of the host immune response during these two phases of disease, however, it is possible that immune modulation in the early stages of disease may play an important role in disease progression. We hypothesized that the clinical stage of Johne's disease is mediated by the expression of cytokines such as transforming growth factor-beta (TGF-beta) and interleukin-10 (IL-10) that may be accompanied by the downregulation of IFN-gamma gene expression. In the present study, tissue samples were collected from the ileum, ileocecal junction, ileocecal lymph node, and mesenteric lymph nodes of healthy, subclinically or clinically infected cows. The expression of TGF-beta, IL-10, and IFN-gamma genes in these tissues was determined by quantitative competitive RT-PCR. The results demonstrate that TGF-beta and IL-10 mRNA levels are higher in cows that have progressed to the clinical stage of disease compared to subclinically infected or healthy cows. In contrast, IFN-gamma gene expression was significantly higher in subclinically infected cows. These results suggest that a change in the balance of cytokines at the site of infection may contribute to the ability of the host to control Mycobacterium avium subsp. paratuberculosis infection.

Effects of gamma interferon, interleukin-10, and transforming growth factor beta on the survival of Mycobacterium avium subsp. paratuberculosis in monocyte-derived macrophages from naturally infected cattle.

Gamma interferon (IFN-gamma) plays a significant role in the control of mycobacterial infections, including Mycobacterium avium subsp. paratuberculosis. However, the contribution of other immunoregulatory cytokines, such as interleukin-10 (IL-10) and transforming growth factor beta (TGF-beta), in Johne's disease has not been investigated as yet. In this study, we examined the effects of in vivo and in vitro infection with M. avium subsp. paratuberculosis on the production of IFN-gamma, IL-10, and TGF-beta by peripheral blood mononuclear cells (PBMC). We also examined the effects of exogenous IFN-gamma, IL-10, and TGF-beta on M. avium subsp. paratuberculosis survival in the cell cultures. PBMC obtained from naturally infected cows, regardless of their disease status, specifically upregulated IL-10 and TGF-beta in culture supernatants in response to stimulation with live M. avium subsp. paratuberculosis. Nonstimulated PBMC recovered from subclinically infected animals secreted the lowest levels of TGF-beta, but after stimulation with live M. avium subsp. paratuberculosis, TGF-beta levels in the culture supernatants increased to levels similar to that produced by PBMC from healthy animals. The numbers of viable M. avium subsp. paratuberculosis recovered from cultures from naturally infected animals were higher than those from healthy cows after in vitro infection with M. avium subsp. paratuberculosis. The addition of exogenous IL-10 and TGF-beta to PBMC isolated from healthy cows inhibited the bactericidal activity of these cells as evidenced by the increased number of viable M. avium subsp. paratuberculosis recovered from these cultures compared to cell cultures containing medium alone. These data suggest important immune regulatory roles for IL-10 and TGF-beta during infection with M. avium subsp. paratuberculosis that may be directly related to their effects on macrophage activation and killing of M. avium subsp. paratuberculosis.