Analysis of monoclonal antibodies that recognize γδ T/null cells

Thirty two monoclonal antibodies (mAbs) from the first round of analysis in the Second International Swine CD Workshop were placed together with additional mAb derived from the first workshop in the null cell panel for further evaluation. Preparations of peripheral blood leukocytes, concanavalin A stimulated peripheral blood mononuclear cells, and spleen cells were used in flow cytometric analyses. Nineteen mAbs identified molecules that were not expressed on null cells, not lineage specific, or recognized activation molecules. Sixteen mAbs including control mAbs were identified that were specific for null cells. One of the latter mAbs, 041 (PGBL22A), that recognizes a determinant on a constant region of porcine γδ TcR established the majority of null cells are γδ T cells. Use of this mAb in further comparisons demonstrated the γδ T cell population is comprised of two major subpopulations, one negative and one positive for CD2. Two color analyses demonstrated that 11 of the mAbs formed a broad cluster that included control mAbs 188 (MAC320) that defined the CD2 negative SWC6 cluster in the first workshop and mAb 122 (CC101) that might recognize an orthologue of bovine WC1 and nine mAbs that recognize determinants on one or more molecules with overlapping patterns of expression on subsets of CD2⁻ γδ T cells. Two groups of mAbs formed the previously identified subset clusters SWC4 and SWC5. Two new mAbs formed a third subcluster. Three mAbs did not form clusters. Three mAbs predicted to recognize TcR in the first workshop (020 [PT14A], 021 [PT79A], and 022 [MUC127A]) and mAb PGBL22A were shown to immunoprecipitate a 37, 40 kDa heterodimer.     

Maternal and foetal immune responses of cattle following an experimental challenge with Neospora caninum at day 70 of gestation

The immune responses of pregnant cattle and their foetuses were examined following inoculation on day 70 of gestation either intravenously (iv) (group 1) or subcutaneously (sc) (group 2) with live NC1 strain tachyzoites or with Vero cells (control) (group 3). Peripheral blood mononuclear cell (PBMC) responses to Neospora antigen and foetal viability were assessed throughout the experiment. Two animals from each group were sacrificed at 14, 28, 42 and 56 days post inoculation (pi). At post mortem, maternal lymph nodes, spleen and PBMC and when possible foetal spleen, thymus and PBMC samples were collected for analysis. Inoculation with NC1 (iv and sc) lead to foetal deaths in all group 1 dams (6/6) and in 3/6 group 2 dams from day 28pi; statistically significant (p ≤ 0.05) increases in cell-mediated immune (CMI) responses including antigen-specific cell proliferation and IFN-γ production as well as increased levels of IL-4, IL-10 and IL-12 were observed in challenged dams compared to the group 3 animals. Lymph node samples from the group 2 animals carrying live foetuses showed greater levels of cellular proliferation as well as significantly (p ≤ 0.05) higher levels of IFN-γ compared to the dams in group 2 carrying dead foetuses. Foetal spleen, thymus and PBMC samples demonstrated cellular proliferation as well as IFN-γ, IL-4, IL-10 and IL-12 production following mitogenic stimulation with Con A from day 14pi (day 84 gestation) onwards. This study shows that the generation of robust peripheral and local maternal CMI responses (lymphoproliferation, IFN-γ) may inhibit the vertical transmission of the parasite.     

Identification of Ehrlichia ruminantium (Gardel strain) IFN-gamma inducing proteins after vaccination with a killed vaccine

IFN-γ is considered as a key factor in protection against heartwater of ruminants, caused by the obligate intracellular bacterium Ehrlichia ruminantium. In this study, a better definition of the molecular masses of IFN-γ inducing proteins of the Gardel strain of E. ruminantium was obtained by the use of continuous flow electrophoresis (CFE) and sensitized polyclonal lymphocytes. Out of 15 E. ruminantium CFE fractions tested within the 14–39 kDa region, eight were commonly reacted to by all goats. Interestingly, half of these fractions fall within the 23–29 kDa region, shown previously to contain polymorphic B-cell epitopes. Thus, the results suggest that this region also contains T-cell epitopes potentially involved in protection. Also, several proteins were found to be more immunogenic than the serologically immunodominant MAP1 protein. Finally, high activity within the 15–19 kDa region was observed, which confirms previous work done with CD4+ T-cell lines obtained from cattle immunized with a South African strain of E. ruminantium. The proteins falling within the molecular weight ranges defined in this study may have potential as vaccine antigens.     

Myeloid differentiation factor 88 is required for resistance to Neospora caninum infection

Neospora caninum is an intracellular parasite that causes major economic impact on cattle raising farms, and infects a wide range of warm-blooded hosts worldwide. Innate immune mechanisms that lead to protection against this parasite are still unknown. In order to investigate whether myeloid differentiation factor 88 (MyD88) is required for resistance against N. caninum, genetically deficient mice (MyD88^−/−) and wild type littermates were infected with live tachyzoites and the resistance to infection was evaluated. We found that sub-lethal tachyzoite doses induced acute mortality of MyD88^−/− mice, which succumbed to infection due to uncontrolled parasite replication. Higher parasitism in MyD88^−/− mice was associated with the lack of IL-12 production by dendritic cells, delayed IFN-γ responses by NKT, CD4⁺ and CD8⁺ T lymphocytes, and production of high levels of IL-10. MyD88^−/− mice replenished with IL-12 and IFN-γ abolished susceptibility as the animals survived throughout the experimental period. We conclude that protective IFN-γ-mediated immunity to N. caninum is dependent on initial MyD88 signaling, in a mechanism triggered by production of IL-12 by dendritic cells. Further knowledge on Toll-like receptor recognition of N. caninum antigens is encouraged, since it could generate new prophylactic and therapeutic tools to control parasite burden.     

Activation of bovine peripheral blood gammadelta T cells for cell division and IFN-gamma production

Bovine peripheral blood gammadelta T cells have been evaluated for effector function (IFN-gamma production) and clonal expansion in a variety of systems including following activation by mitogens, IL-12, and stimulation, through the T cell receptor (TCR) with anti-CD3 monoclonal antibody (mAb), a cell-bound molecule and a soluble antigenic extract. To evaluate cell division, carboxyfluorescein succinimidyl ester (CFSE) loading of cells and flow cytometric analysis were used, while IFN-gamma production was evaluated by intracytoplasmic staining. It was found that bovine gammadelta T cells produced IFN-gamma and clonally expanded when stimulated through the TCR/CD3 complex by a cell-associated autologous molecule on monocyte, by bacterial components following in vivo sensitization of gammadelta T cells with a leptospira vaccine or by anti-CD3 mAb. In addition, gammadelta T cells were activated efficiently for effector function but not clonal expansion by culturing with IL-12. In contrast, stimulation by Con A or PMA/ionomycin induced efficient replication but only low level IFN-gamma production which was not enhanced by the presence of IL-12. In several systems the amount of IFN-gamma produced per cell by gammadelta T cells was less than that produced by CD4 T cells in the same cultures.     

Analysis of T lymphocyte subsets proliferating in response to infective and UV-inactivated African swine fever viruses.

The proliferative response to infective and UV-inactivated African swine fever virus was analyzed in cells from pigs surviving an experimental infection with attenuated virus. All the pigs showed strong dose-dependent proliferative responses to both infective and UV-inactivated virus. This response was also observed when nitrocellulose-bound solubilized virus proteins were used in the assay. Heterologous isolates also induced proliferation, however it was significantly lower than that induced by the isolate used to infect the animals. The response to infective virus was blocked equally by anti-CD4 and anti-CD8 monoclonal antibodies (mAb); the response to UV-inactivated virus was almost abolished by anti-CD4 and 60% inhibited by anti-CD8 mAb. FACS analysis of 28-day T cell lines derived from peripheral blood mononuclear cells demonstrated the progressive increase of the CD8+ subset when the cells were stimulated with infective virus, whereas the stimulation with UV-inactivated virus induced the increase of both CD4+ and CD8+ subsets. In this case, the sum of CD4+ and CD8+ percentages was higher than the total percentage of T cells, suggesting the presence of cells positive for both CD4+ and CD8+.     

Fundamentals of host immune response against Brucella abortus: what the mouse model has revealed about control of infection

The studies reviewed here evaluated the role cellular immune system components play in control of brucellosis by conducting comparative studies with brucella-resistant C57BL/10 or C57BL/6 mice and susceptible BALB/c mice. We have shown by both in vitro and in vivo studies that activation of macrophages with interferon-gamma (IFN-γ) is an important factor for control of infection with B. abortus in the mouse model and that the mechanism of anti-brucella activity largely involved reactive oxygen intermediates. Differences in control of the organism by resistant and susceptible mice was not related to inherent differences in the ability of their macrophages to control infection either with or without IFN-γ activation nor was it attributable to NK cells since we found no role for them in control of brucellosis in either mouse strain. However, relative resistance to brucellosis did correlate with increased production of IFN-γ by CD4 T cells during the first weeks after infection while IL-10 contributed to susceptibility in BALB/c mice. Moreover, by 3 weeks post-infection splenocytes from the susceptible BALB/c mice failed to produce IFN-γ and relied on TNF- as well as CD8 T cells to control infection until the end of the plateau phase around 6 weeks post-infection when IFN-γ production resumed and clearance began. In contrast, IFN-γ was crucial for control throughout the infection in the more resistant C57BL/6 mice and the mice died in its absence by 6 weeks post-infection compared to 12 weeks for the more susceptible mice that relied on additional mechanisms of control. In contrast to the IFN-γ knock-out mice, both β2 microglobulin knock-out C57BL/6 mice, which do not express conventional MHC class I molecules and thus cannot present antigen to CD8 T cells, or perforin knock-out C57BL/6 mice, which have no T cell cytotoxic activity, controlled and cleared the infection as well as normal C57BL/6 mice. The hiatus of IFN-γ production in BALB/c mice correlated with very high levels of total IL-12 and it was postulated that the lack of IFN-γ was a consequence of p40 homodimer blocking activity. However, reduction of p40 IL-12 in vivo through administration of indomethacin reduced the infection without a concomitant measurable increase in IFN-γ. Current studies are aimed at elucidating the mechanism of the IFN-γ hiatus.     

CD4+CD25+ T cells expressing FoxP3 in Icelandic horses affected with insect bite hypersensitivity

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis caused by bites of midges from the genus Culicoides. We have shown previously that peripheral blood mononuclear cells (PBMC) from IBH-affected horses produce higher levels of IL-4 and lower levels of IL-10 and TGF-β1 than those from healthy horses, suggesting that IBH is associated with a reduced regulatory immune response. FoxP3 is a crucial marker of regulatory T cells (Tregs). Here we have determined the proportion of CD4(+)CD25(+)FoxP3(+) T cells by flow cytometry in PBMC directly after isolation or after stimulation with Culicoides extract or a control antigen (Tetanus Toxoid). There were no differences between healthy and IBH horses either in the proportion of FoxP3(+)CD4(+)CD25(+) cells in freshly isolated PBMC or in the following stimulation with Tetanus Toxoid. However, upon stimulation of PBMC with the allergen, expression of FoxP3 by CD4(+)CD25(+high) and CD4(+)CD25(+dim) cells was significantly higher in healthy than in IBH horses. Addition of recombinant IL-4 to PBMC from healthy horses stimulated with the allergen significantly decreased the proportion of FoxP3 expressing cells within CD4(+)CD25(+high). These results suggest that IBH is associated with a decreased number of allergen-induced Tregs. This could be a consequence of the increased IL-4 production by PBMC of IBH-affected horses.     

Vaccination of calves with Mycobacteria bovis Bacilli Calmete Guerin (BCG) induced rapid increase in the proportion of peripheral blood γδ T cells

Changes in the proportion of peripheral blood T cell subsets after subcutaneous inoculation of cattle with Mycobacterium bovis Bacille Calmette-Guerin (BCG) were studied. Calves were injected with approximately 8 × 10⁶ BCG bacillus and blood samples collected at weekly intervals for flow-cytometric analyses to determine the proportion of CD4⁺, CD8⁺ and γδ T cells. In addition, whole blood samples were stimulated in vitro with M. bovis purified protein derivative (PPD) and the secreted IFN-γ quantified by ELISA. Results showed cellular and cytokine changes which could be categorized into three phases. The first phase occurred within the first 2 weeks after vaccination involving an increase in proportion of WC1⁺ γδ T cells and a concomitant increase in the secretion of IFN-γ. These two responses peaked at 2 weeks and waned thereafter. The second phase involved an increase in the CD4/CD8 ratio as a result of an increase in the proportion of CD4⁺ T cells between 4 and 6 weeks. The third phase involved a decrease in the CD4/CD8 ratio due to an increase in the proportion of CD8⁺ T cells between 8 and 10 weeks. Surprisingly, the IFN-γ response was associated with changes in the γδ rather than the CD4⁺ or CD8⁺ T cells, suggesting that this cytokine was secreted by γδ-T cells. These results are consistent with the reported ability of γδ T cells to act rapidly and bridging the innate and classically adaptive immune responses.     

Differentiation of porcine myeloid bone marrow haematopoietic cell populations.

The myeloid panel of monoclonal antibodies (mAbs) submitted to the Third Swine CD Workshop were analysed for reactivity with bone marrow haematopoietic cells (BMHC). Using single and triple immunofluorescence labelling by flow cytometry (FCM), the mAbs were grouped according to their capacity to recognise myeloid cell populations and/or maturation stages. Group 1 consisted of mAbs labelling the majority of myeloid BMHC, including neutrophilic, eosinophilic and monocytic cells. The ligands for SWC3 and CD11b-like mAbs of group 1 showed a maturation-dependent intensity of expression. The other antibodies of group 1 reacted with BMHC to give a sharp, single peak. Group 2 mAbs reacted only with monocytic cells. The anti-human CD49e mAb Sam-1 was the only mAb detecting the majority of monocytic cells, but not other BMHC. The mAbs in group 3 recognised antigens expressed on granulocytes, but not monocytes. The previously identified SWC8 in this group proved to be useful in differentiating major population of BMHC when cells were double labelled with the pan-myeloid SWC3. Other mAbs within group 3, such as MIL4 and TMG6-5 (an anti-human CD11b), only recognised subsets of neutrophils and eosinophils. Group 4 mAbs reacted with the more mature subpopulations of neutrophils and monocytes. Some of these antibodies might prove useful for assessment of cell maturity, such as anti-CD14 and the anti-human CD50 mAb HP2/19.     

Bovine CD4+ T-cells lines reactive with soluble and membrane antigens of Cowdria ruminantium.

Cowdria-specific CD4⁺ T-cell lines generated from immunised cattle respond to both soluble and membrane proteins of the agent. Furthermore, the lines produced the Cowdria-inhibitory cytokine IFN-γ in response to soluble antigens fractionated by gel filtration and FPLC. Activity eluted as a single peak around fraction 15 for all T-cell lines tested. This fraction induced the highest production of IFN-γ by the lines and was shown by SDS-polyacrylamide gel electrophoresis and silver staining analysis to contain less than 10 different bands ranging from 22 to 32 kDa. Given their high sensitivity and specificity, these short-term CD4⁺ T-cell lines will be valuable tools for the identification of Cowdria antigens for incorporation in a subunit vaccine.     

Cholera toxin transiently inhibits porcine T cell proliferation in vitro

Cholera toxin (Ctx) is an important mucosal adjuvant with potential experimental applications in pigs. However, little is known about the direct effects of Ctx on porcine immune cells. Therefore, we analysed the influence of Ctx on mitogen-induced lymphocyte proliferation. Ctx inhibited peripheral blood mononuclear cell (PBMC) proliferation with an IC₅₀ of 34±17 ng/mL. This inhibition was not due to increased cell death. Lymphoblast formation in cultures stimulated with concanavalin A and Ctx was decreased at 24 h, but had reached the levels of control cultures again at 72 and 120 h, indicating that suppression was transient. Analysis of T cell subsets revealed that Ctx treatment specifically reduced the percentage of CD4⁻CD8⁺ and γδ T cells, whereas the proportion of CD4⁺CD8⁻ increased. Furthermore, Ctx caused secretion of IL-10 by PBMC cultures, but depressed TNFα secretion.     

The immune response of sheep infected with larvae of the sheep blowfly Lucilia cuprina monitored via efferent lymph

Changes in lymphocyte traffic in efferent lymph from the prescapular lymph node of sheep were monitored during local primary and secondary infection with blowfly, Lucilia cuprina. During primary infections the response was characterised by an increase in the output of CD4⁺ T cells over CD8⁺ T cells for the first 48 h after wound initiation. By 72 h the output of CD8⁺ T cells exceeded that of CD4⁺ T cells. During secondary infections the increased output of CD8⁺ T cells was more pronounced and occurred earlier at approximately 48 h. The percentage of B lymphocytes as measured by sIg, CD45R and MHC class II expression increased at approximately 96–120 h after both primary and secondary infections, with the secondary response being greater than the primary. This increase in B cells corresponded with peak antibody titres recorded in the efferent lymph to a first instar antigen preparation as measured by ELISA. An increase in IFN-γ and soluble IL-2 receptor was recorded after both primary and secondary infections, with the response after secondary infection being greater than that recorded after primary larval infections.     

A monoclonal antibody to equine interleukin-4

Interleukin-4 (IL-4) is secreted by T helper type 2 cells, mast cells, basophils and eosinophils. Detection of IL-4 can contribute the evaluation of cellular immune responses during infectious diseases, immunological disorders or vaccination. We used recombinant equine IL-4 to generate a monoclonal antibody (mAb) to equine IL-4. The mAb detected recombinant IL-4 in mammalian cells transfected with different plasmids containing IL-4 cDNA. After mitogen stimulation of equine peripheral blood mononuclear cells, an intracellular protein was recognized by the new mAb in 1–2% of lymphocytes using flow cytometric analysis. In the presence of the secretion blocker Brefeldin A, the protein accumulated and was detected in 4–8% of lymphocytes stimulated with phorbol 12-myristate 13-acetate and ionomycin. Double staining with the new mAb and T-cell or B-cell markers identified a subpopulation of CD4⁺ T-cells expressing the protein recognized by the mAb. In addition, the protein was detectable in cell culture supernatants of mitogen stimulated cells by ELISA when using the new mAb for coating of the plates and a polyclonal antiserum to equine IL-4 for detection. In conclusion, the new mAb detects equine IL-4 and can be used for intracellular staining and ELISA to measure this important cytokine.     

Effect of cortisol infusion patterns and castration on metabolic and immunological indices of stress response in cattle

This study tested the hypotheses that: (1) either acute stress induced by Burdizzo castration, or cortisol infusion would modulate plasma glucose, insulin and growth hormone (GH) concentrations; and (2) immune modulation induced by cortisol would be dependent on the pattern, intensity and duration of circulating cortisol concentrations. Fifty 9.2-month-old Holstein×Friesian bulls (232±2.0 kg) were blocked by weight and randomly assigned to one of five treatments (n=10 per treatment): (1) sham handled control; (2) Burdizzo castration; (3) hydrocortisone infusion to mimic the castration-induced secretion pattern of cortisol; (4) hourly pulse infusion of hydrocortisone; and (5) sustained infusion of hydrocortisone for 8 h. Blood samples were collected intensively on day 0, and weekly from days 1 to 35. Castration acutely increased plasma cortisol, GH and haptoglobin concentrations, suppressed lymphocyte in vitro interferon-γ (IFN-γ) production, but had no effect on plasma glucose and insulin concentrations. Cortisol infusion to simulate the castration-induced secretion pattern of cortisol, and pulse infusion of cortisol did not suppress the IFN-γ production. A sustained infusion of cortisol resulted in the transient suppression of IFN-γ production. Moreover, the sustained cortisol infusion resulted in increased plasma glucose, insulin and GH concentrations. The overall 14-day feed intakes and 35-day growth rates were not affected by treatments. In conclusion, cortisol infusion to induce immune suppression in vivo occurred only at pharmacological doses. Within physiological ranges, cortisol was not associated with the suppression of immune function, indicating that during castration cortisol per se is not responsible for the suppression of in vitro IFN-γ production.     

Blockade of bovine PD-1 increases T cell function and inhibits bovine leukemia virus expression in B cells in vitro

Programmed death-1 (PD-1) is a known immunoinhibitory receptor that contributes to immune evasion of various tumor cells and pathogens causing chronic infection, such as bovine leukemia virus (BLV) infection. First, in this study, to establish a method for the expression and functional analysis of bovine PD-1, hybridomas producing monoclonal antibodies (mAb) specific for bovine PD-1 were established. Treatment with these anti-PD-1 mAb enhanced interferon-gamma (IFN-γ) production of bovine peripheral blood mononuclear cells (PBMC). Next, to examine whether PD-1 blockade by anti-PD-1 mAb could upregulate the immune reaction during chronic infection, the expression and functional analysis of PD-1 in PBMC isolated from BLV-infected cattle with or without lymphoma were performed using anti-PD-1 mAb. The frequencies of both PD-1⁺ CD4⁺ T cells in blood and lymph node and PD-1⁺ CD8⁺ T cells in lymph node were higher in BLV-infected cattle with lymphoma than those without lymphoma or control uninfected cattle. PD-1 blockade enhanced IFN-γ production and proliferation and reduced BLV-gp51 expression and B-cell activation in PBMC from BLV-infected cattle in response to BLV-gp51 peptide mixture. These data show that anti-bovine PD-1 mAb could provide a new therapy to control BLV infection via upregulation of immune response.     

Cytokine response in Balb/c mice infected with Francisella tularensis LVS and the Pohang isolate

We investigated the immune response induced by the Francisella (F.) tularensis live vaccine strain (LVS) and the Pohang isolate. After the Balb/c mice were infected intradermally (i.d) with 2 × 10⁴ cfu of F. tularensis LVS and Pohang, respectively, their blood and organs were collected at different times; 0, 3, 6, 24, 72, 96, 120 and 168 h after infection. Using these samples, RT-PCR and ELISA analysis were carried out for the comparative study of the cytokines, including TNF-α, INF-γ, IL-2, IL-4, IL-10 and IL-12. In the Pohang-infected mice at 120 h, the liver showed a 53 times higher level of TNF-α and a 42 times higher level of IFN-γ than the respective levels at the early time points after infection. The levels of TNF-α and IFN-γ induced by LVS were 5 times lower than those induced by the Pohang isolate. Also, the organs from the Pohang-infected mice showed higher levels of TNF-α, IFN-γ, IL-10 and IL-12 than the levels in the LVS-infected mice. The blood from the Pohang-infected mice at 120 h revealed about a 40 times increased level of IFN-γ, and IL-10 was also increased by 4 times at 96 h compared to an early infection time point, while IL-4 was not induced during the whole infection period. These results suggest that F. tularensis may induce a Th1-mediated immune response to in vivo infection and the Pohang isolate has a higher capacity than the LVS to induce an acute immune response in Blab/c mice.     

Interferon-gamma, interleukin-4 and interleukin-10 production by T helper cells reveals intact Th1 and regulatory TR1 cell activation and a delay of the Th2 cell response in equine neonates and foals

Cytokines produced by T helper (Th) cells are important in orchestrating the immune response during health and disease. Recent reports indicated that cytokine mRNA expression in foals is often quantitatively lower than that of adult horses suggesting that foal T cells are not fully mature. Here, peripheral blood mononuclear cells from foals and adult horses were stimulated with phorbol 12-myristate 13-acetate and analyzed for intracellular interferon-gamma (IFN-γ), interleukin-4 (IL-4) and IL-10 production, representing the Th1, Th2 and regulatory T_R1 cell phenotypes respectively, by flow cytometry. In agreement with previous reports, all three cytokines were quantitatively reduced in foals compared to adults. However, the balance between Th1 and Th2 cytokines (IFN-γ/IL-4 ratio) showed a clear Th1-biased response in foals by 6 and 12 weeks of life, while similar IFN-γ/IL-10 ratios were found in foals and adult horses. By day 5 after birth, intracellular IFN-γ production by foal CD4⁺ and CD8⁺ T cells resembled that in adults. Overall, IL-4 production was low in foals. IL-4⁺ cells peaked at day 5 of age when IL-4 was mainly produced by IgE⁺ cells. Relative percentages of IL-4⁺ Th2 cells were significantly lower in foals at all time points. The data suggested that equine neonates and young foals have an impaired Th2 response, that the immune response of foals is Th1 biased, that IFN-γ production by Th and cytotoxic T cells is qualitatively similar to adult horses, and regulatory IL-10 production by T cells is developmentally mature in foals during the first three months of life.     

Cytokine expression in an ex vivo culture system of duodenal samples from dogs with chronic enteropathies: modulation by probiotic bacteria

There is evidence that probiotics have immune-modulating effects on intestinal inflammation during chronic enteropathies (CE). In an ex vivo culture system we investigated the influence of probiotics on mRNA and protein expression levels of cytokines in intestinal samples from dogs suffering from CE. Duodenal samples of client-owned dogs with CE (group CE; n = 12) were collected during diagnostic endoscopy. Additional duodenal samples of gastrointestinally healthy dogs (group C; n = 4) from an unrelated study were available. Based on histopathological analyses, no pathological changes or only mild to moderate eosinophilic and/or lymphoplasmacytic duodenitis were diagnosed. Tissue samples were cultured: (1) with cell culture medium alone (negative control), (2) with a probiotic cocktail (PC), constituted of three Lactobacilli spp. from healthy canine fecal isolates, (3) with the individual strains of PC, and (4) with a placebo powder. Viability of intestinal tissue and probiotic bacteria before and after culture was evaluated. The mRNA abundance of interleukin (IL)-10, IL-12p40, interferon (IFN)-γ, tumor necrosis factor (TNF)-, and transforming growth factor (TGF)-β1 was analyzed by real-time polymerase chain reaction (RT-PCR). Protein concentrations of IFN-γ and IL-10 were measured in culture supernatant by ELISA. Results of RT-PCR were expressed as 2^{(−2ΔCrossing Point)} × 100 after normalization with β-actin. There was a loss of about 1 log CFU/mL of probiotic bacteria during the incubation period. Viability of tissue was maintained as confirmed by non-significant release of lactate dehydrogenase. In C, addition of PC increased IL-10 mRNA levels (P < 0.1). In CE, PC increased mRNA and protein levels of IL-10 (P < 0.05). On the mRNA level, the ratio of TNF-/IL-10, IFN-γ/IL-10, and IL-12p40/IL-10 decreased after addition of PC (P < 0.05). The results demonstrate favorable effects of PC on regulatory cytokines relative to inflammatory cytokines that might contribute to reduction of intestinal inflammation.