Peripheral blood mononuclear cell responses to Dermatophagoides farinae in canine atopic dermatitis

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans that is characterised by the presence of allergen-specific IgE. Data from skin tests and serological analysis suggest that the house dust mite Dermatophagoides farinae is the most important allergen in dogs with atopic dermatitis. The aim of this study was to determine if D. farinae specific peripheral blood mononuclear cell (PBMC) responses could be detected in dogs with atopic dermatitis. PBMCs were isolated by the density centrifugation from dogs with atopic dermatitis that were skin test positive for D. farinae, dogs with atopic dermatitis that were skin test negative for D. farinae, and healthy dogs. Cells were cultured with increasing concentrations of the D. farinae extract, no antigen, vaccine antigens or concanavalin A (ConA). There was significantly greater responsiveness of PBMCs from the D. farinae positive dogs than from either the D. farinae negative or healthy dogs (ANOVA, P<0.05). In contrast, no significant differences were observed in the control responses between the three groups. This is the first study to demonstrate that D. farinae specific circulating memory cells are involved in the pathogenesis of canine house dust mite hypersensitivity.     

Peripheral blood mononuclear cell responses to major and minor Dermatophagoides allergens in canine atopic dermatitis.

Atopic dermatitis is a chronic inflammatory and pruritic skin disease commonly seen in dogs and humans. Most cases involve hypersensitivity to the house dust mites (HDM) Dermatophagoides farinae and Dermatophagoides pteronyssinus. Human atopic dermatitis is associated with the HDM derived allergens Der f 1 and 2, and Der p 1 and 2. Serological data, however, suggest that a 98/104kD protein is the most important allergen in dogs with atopic dermatitis. The aim of this study was to characterise the specificity of circulating T-cells in canine atopic dermatitis for HDM derived allergens. Peripheral blood mononuclear cells (PBMCs) from dogs with atopic dermatitis that were skin test positive for D. farinae and D. pteronyssinus were cultured with crude extracts of D. farinae, D. pteronyssinus and D. microceras, a 98/104kD allergen purified from D. farinae, Der f 1 and Der f 2. There was significantly greater responsiveness of PBMCs to the D. farinae and D. pteronyssinus extracts compared to the D. microceras extract, and similarly to the purified 98/104kD allergen compared to Der f 1 and Der f 2. The close association between serological findings and PBMC proliferation implies that the 98/104kD HDM protein is a major target of immune recognition and that T-cells also participate in the pathogenesis of canine atopic dermatitis by supporting IgE production.     

In vitro production of antibodies to bovine virus diarrhoea virus by peripheral blood mononuclear cells from cattle immunized by infection

A method for in vitro production of antibodies to bovine virus diarrhoea virus (BVDV) by peripheral blood mononuclear cells (PBMC) was developed. The PBMC were cultured in microtitre plates coated with detergent-solubilized BVDV and the supernatants were tested in an enzyme-linked immunosorbent assay which detects IgG antibodies to BVDV. Following incubation of PBMC with an optimal concentration of pokeweed mitogen for 5 days, antibodies to BVDV were detected in culture supernatants of PBMC from immune cattle, but not in supernatants of PBMC from seronegative cattle, from persistently BVDV-infected cattle or from a 5-day-old calf that received BVDV antibodies via colostrum. This antibody-production assay may therefore be used as a tool to discriminate between passively acquired antibodies and those actively induced.     

Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.     

Bovine virus diarrhoea virus induces in vitro a proliferative response of peripheral blood mononuclear cells from cattle immunized by infection.

The ability of bovine peripheral blood mononuclear cells (PBMC) to mount a proliferative response to bovine virus diarrhoea virus (BVDV) in vitro was examined. After culturing PBMC in the presence of a non-cytopathic strain of BVDV for 6 days, the magnitude of PBMC proliferation was measured as incorporation of radiolabelled thymidine, present during the last 18 h. Live, but not heat-inactivated, BVDV evoked a proliferative response of PBMC obtained from cattle seropositive to the virus. However, PBMC from seronegative or persistently BVDV-infected animals were not stimulated by BVDV. The presence of live BVDV did not alter the proliferative response of PBMC to stimulation with concanavalin A.     

Recombinant canine IL-13 receptor alpha2-Fc fusion protein inhibits canine allergen-specific-IgE production in vitro by peripheral blood mononuclear cells from allergic dogs

Human IL-13, like IL-4, is involved in the regulation of B-cell development, IgE synthesis and allergic responses. However, because IL-13 does not affect either murine Ig class switching or IgE production in vitro, the use of murine models to study the role of IL-13 in IgE-mediated diseases has been limited. In this communication, we report that recombinant protein of canine IL-13 (rcaIL-13) stimulates production of allergen-specific-IgE in vitro by peripheral blood mononuclear cells (PBMC) from flea allergen-sensitized dogs, and that this stimulation activity is specifically inhibited by recombinant protein of canine IL-13Ralpha2 and Fc fragment of canine IgG heavy chain (rcaIL-13Ralpha2-Fc). The data suggest that the regulatory effects of IL-13 on IgE production in canine PBMC are similar to those reported in humans. Thus, canine IL-13 may be a central mediator of allergic diseases in dogs, and allergic dogs may be excellent models for research on IgE-mediated diseases in humans.     

Histopathological and immunohistochemical study of type 3 complement receptors (CD11b/CD18) in livers and spleens of asymptomatic and symptomatic dogs naturally infected with Leishmania (Leishmania) chagasi

Leishmania promastigotes interact with macrophages through the association of multiple membrane surface receptors. Macrophage complement receptor CR3 (CD11b/CD18 or Mac-1) has been implicated in the interaction of both human and murine macrophages with serum-opsonized promastigotes. The aim of this study was to determine CR3 expression in the livers and spleens of dogs naturally infected with Leishmania (Leishmania) chagasi. CR3 expression in liver was higher in asymptomatic than in symptomatic animals. Moreover, the hepatic parasitism load determined by immunocytochemical analysis was lower in parallel with higher numbers of granulomas. In contrast, in spleens, CR3 expression was higher in symptomatic animals than in asymptomatic ones. However, the tissue parasite load was greater in spleens of symptomatic dogs. There was a strict correlation between the parasite load and cellular CR3 expression in the spleens of dogs naturally infected with L. chagasi. CR3 macrophage integrins could be essential receptors for Leishmania survival. Considering that the symptomatic animals showed higher parasite loads and higher CD11b/CD18 expression in their spleens, we can conclude that these splenic cells (monocyte-macrophages) might serve to perpetuate intracellular infection.     

Onset and duration of immunity against Babesia canis infection in dogs vaccinated with antigens from culture supernatants

It has previously been shown that dogs can be vaccinated against heterologous Babesia canis infection using a vaccine containing soluble parasite antigens (SPA) from in vitro cultures of B. canis and B. rossi that are adjuvanted with saponin. In the present study the onset and duration of immunity of vaccinated dogs were studied. Results showed that 3-26 weeks after initial vaccination, dogs effectively limit the level of SPA in plasma upon challenge infection, which was reflected in limited duration and extent of clinical manifestations. There was no statistically significant effect of vaccination on the parasite load in the circulation, which was determined from blood smears. It was further shown that the level of immunity of primary vaccinated dogs (priming and booster vaccination with a 6-week interval) and that of repeatedly vaccinated dogs (a single additional vaccination 6 months after primary vaccination) is comparable. From this study it is concluded that vaccination with this preparation induces protective immunity against clinical babesiosis from 3 weeks after booster vaccination onwards, and remains effective for a period of at least another 6 months. A single booster vaccination is sufficient to maintain immunity for at least another 6 months.     

Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.     

In vitro release of interferon-gamma by trichophytin-stimulated whole blood cell cultures from ringworm-vaccinated and control calves experimentally inoculated with Trichophyton verrucosum

Of a group of seven calves, four were vaccinated against bovine ringworm with the vaccine Ringvac bovis LTF-130 (Alpharma, Oslo, Norway), while three calves were left unvaccinated. All calves were inoculated epicutaneously with a virulent strain of Trichophyton verrucosum . Clinical signs were recorded. In response to stimulation with trichophytin, in vitro interferon-γ (IFN-γ) production in whole blood cell cultures was assessed in samples obtained pre-and post-vaccination and pre- and post-inoculation. A commercial enzyme immunoas-say kit was used to measure IFN-γ levels (Bovigam, CSL, Victoria, Australia). Control calves developed typical ringworm lesions, whereas vaccinated calves did not. Following vaccination, release of IFN-γ in whole blood cell cultures indicated the presence of circulating trichophytin-specific lymphocytes. After inoculation with T. verrucosum , IFN-γ production was demonstrated in samples from both vaccinated and control calves. This study showed that vaccination with Ringvac bovis LTF-130 elicits a protective immune response suggesting involve-ment of the cellular branch of the immune system. Experimental infection of naïve nonvaccinated calves with T. verrucosum , also indicated stimulation of a cell-mediated immune response essential for resolution of lesions.     

Comparison of monoclonal and polyclonal antibodies for the detection of canine IgG1 and IgG2, and associations with infection outcome in Leishmania infantum naturally infected dogs

In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P ≤ 0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies.     

BHV-1 infection and inflammatory cytokines amplify the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood mononuclear cells in vitro

Bovine herpesvirus-1 (BHV-1) has been reported to increase the susceptibility of cattle to respiratory disease caused by Mannheimia (Pasteurella) haemolytica A1. The principal virulence factor of M. haemolytica is a leukotoxin (LKT) that can specifically kill ruminant leukocytes following its binding to the beta2-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 (LFA-1)). In this study, we investigated the effects of experimental infection of bovine peripheral blood mononuclear cells (MNCs) with BHV-1 in vitro, on the subsequent interaction of these cells with the M. haemolytica LKT. We found that BHV-1 infection increased LFA-1 expression (as assessed by flow cytometry), and subsequently enhanced LKT binding and cytotoxicity to bovine MNCs. We also found that BHV-1 infection increased CD18, IL-1beta, and IFN-gamma mRNA expression by MNCs. As previously reported for bovine polymorphonuclear neutrophils (PMNs), MNCs increased their expression of LFA-1, and their LKT binding and cytotoxicity, following exposure to IL-1beta, TNF-alpha, and IFN-gamma. These findings suggest that BHV-1 infection, and the resulting release of inflammatory cytokines, can stimulate expression of LFA-1 in bovine MNCs, thus enhancing the binding and biological effects of LKT. If such a mechanism occurs in vivo it might explain, in part, the increased susceptibility of BHV-1 infected cattle to bovine pasteurellosis.     

In vitro susceptibility to antimonials and amphotericin B of Leishmania infantum strains isolated from dogs in a region lacking drug selection pressure

The aim of this study was to evaluate the susceptibility to anti-leishmanial agents of 24 strains isolated from dogs living in the urban area of Alger lacking drug selection pressure. Two different Leishmania infantum zymodemes, MON-1 and MON-281, were identified in these dogs. The in vitro susceptibility to the main forms of antimonial and amphotericin were assessed on promastigote and amastigote life stages in culture. The results obtained for both parasite life stages were concordant whatever the molecule tested. Moreover, our data showed that isolates belonging to the relatively rare zymodeme of L. infantum, MON-281, were less susceptible to antimony than MON-1, when at the same time there was no significant difference for amphotericin B.     

Assessment of IgE binding to native and hydrolyzed soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity

OBJECTIVE: To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity. ANIMALS: 8 na?ve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs). PROCEDURES: 6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein. RESULTS: In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.     

Suppressive effect of culture supernatant of erythrocytes and serum from dogs infected with Babesia gibsoni on the morphological maturation of canine reticulocytes in vitro

The present study evaluated the effects of infected culture supernatant of erythrocytes, fractionation of culture supernatant and serum from dogs infected with Babesia gibsoni (B. gibsoni) on the maturation of canine reticulocytes in vitro. The SDS-PAGE demonstrated that significantly broader bands were generated by both the infected culture supernatant of erythrocytes and the serum from dogs chronically infected with B. gibsoni. The culture supernatant of erythrocytes infected with B. gibsoni strongly suppressed the maturation of reticulocytes. Prior studies showed that chronically infected serum had inhibitory effects on both the maturation of reticulocytes and the canine pyrimidine 5''-nucleotidase subclass I and purine-specific 5''-nucleotidase activity. In addition, serum free infected culture supernatant of erythrocytes had an inhibitory effect on the morphological maturation of reticulocytes. These results suggest that infected serum and culture supernatant of erythrocytes might accumulate excess proteins and/or metabolites as a result of the inhibited maturation of reticulocytes and decreased activity of erythrocyte 5''-nucleotidase. Furthermore, the fractions observed at >150 kDa- and 150-70 kDa- in the infected culture supernatant and serum retarded the maturation of canine reticulocytes in vitro. The results obtained from the in vitro examinations, in the present study, suggested that B. gibsoni itself and/or its metabolites might release certain proteins in the infected culture supernatant and serum from infected dogs and as a result delay morphological maturation of canine reticulocytes.     

Use of canine red blood cell with high concentrations of potassium, reduced glutathione, and free amino acid as host cells for in vitro cultivation of Babesia gibsoni

OBJECTIVE: To determine the usefulness of canine RBC with high concentrations of potassium, reduced glutathione (GSH), and amino acid(i.e., HK cells) for in vitro cultivation of Babesia gibsoni. ANIMALS: RBC were obtained from 3 dogs that had inherited HK cells and from 3 genetically unaffected dogs that, therefore, had RBC with lower potassium (LK) concentrations (i.e., LK cells). PROCEDURES: First, B. gibsoni were cultivated using HK or LK cells in alpha-modification of Eagle medium, consisting of Earle salts with glutamine and without ribosides, deoxyribosides, and sodium bicarbonate under a humidified atmosphere containing 5% CO2 at 37 C. Second, parasites were cultivated with LK- or HK-cell lysates. Finally, HK cells were separated into 3 fractions (bottom, middle, top layers) by density gradient centrifugation, and B. gibsoni were cultivated with each of the HK-cell fractions. In addition, the concentrations of free amino acids and reduced glutathione (GSH) in each HK-cell fraction were measured. RESULTS: B. gibsoni preferentially multiplied in HK-cell cultures rather than in LK-cell cultures. Furthermore, the addition of HK-cell lysate to the culture medium resulted in enhanced multiplication of the parasites. Higher multiplication of the parasites was observed in HK cells from the top layer, compared with HK cells from the middle and bottom layers. The HK cells from the top layer had higher concentrations of glutamate, aspartate, and GSH, compared with HK cells from the middle and bottom layer. CONCLUSIONS: Canine HK cells are useful host cells for in vitro cultivation of B. gibsoni, and the high concentrations of glutamate, aspartate, and GSH may result in enhancement of multiplication of the parasites in HK cells.