Establishment of a quantitative ELISA for the measurement of allergen-specific IgE in dogs using anti-IgE antibody cross-reactive to mouse and dog IgE.

As IgE plays a pivotal role in type I hypersensitivity-mediated allergic diseases, it is valuable to measure absolute quantity of serum antigen-specific IgE for clinical and research purposes. Here we describe a novel ELISA system that enables quantification of antigen-specific IgE in ng/ml in dogs. A newly developed monoclonal antibody (CRE-DM) was shown to recognize canine and mouse IgE equally in a dose dependent manner, but it did not recognize canine IgG. The reactivity of CRE-DM to canine IgE was also confirmed by an inhibition ELISA using canine IgE as an inhibitor and the maximum inhibition rate was 91.3%. In order to know whether canine IgE specific to an allergen could be quantitatively measured with an ELISA using CRE-DM, we established a quantitative ELISA that could measure canine IgE recognizing Cry j 1, one of the major allergens of Japanese cedar pollen. In this ELISA, a standard curve was created by using concentration-predetermined Cry j 1-specific monoclonal mouse IgE. According to the standard curve, the concentration of Cry j 1-specific IgE in dogs that were experimentally sensitized to Japanese cedar pollen could be calculated and determined in ng/ml. The specificity of the Cry j 1-specific IgE ELISA using CRE-DM was also confirmed by inhibition ELISA using canine IgE as an inhibitor and the inhibition rate was 97.0%. Reproducibility of the ELISA in three independent assays was determined using groups of pooled canine sera whose Cry j 1-IgE titers ranged from 155.9 to 888.2 ng/ml. Intra- and inter-assay reproducibility was determined with coefficient of variation ranging between 3.1-5.2% and 2.2-8.0%, respectively. These results demonstrated that the ELISA utilizing CRE-DM was a specific, reliable and robust new laboratory test that could quantify absolute amount of antigen-specific IgE in canine serum. The ELISA will serve as a useful tool in the clinics to evaluate the change of serum IgE titers during anti-allergic treatments as well as during seasonal fluctuation of allergen exposure.

Clinical efficacy of a novel elimination diet composed of a mixture of amino acids and potatoes in dogs with non-seasonal pruritic dermatitis.

There has been a need for improvement of the elimination diet used for diagnosis of adverse food reaction (AFR) in dogs. Recently, a novel elimination diet composed of a mixture of amino acids and potatoes was developed. We evaluated the efficacy of the elimination diet for diagnosis of AFR in dogs. Twenty dogs that were suspected to have allergic dermatitis were enrolled in a 2-month food elimination trial using the diet. Before and after the trial, the clinical symptoms were evaluated based on the change in canine atopic dermatitis extent and severity index (CADESI), pruritus score and medication score. Of the 20 dogs, 15 completed the food elimination trial. The remaining 5 dogs were removed from the trial because of diet unpalatability, skin disease progression or diarrhea. On the basis of evaluation of the clinical scores, we observed that the clinical symptoms improved in 11 of the 15 dogs that completed the food elimination trial. Provocative challenge was performed in 10 of the 11 dogs that showed improvement in their clinical symptoms. Of the 10 dogs, 7 were diagnosed as having AFR against food ingredients such as pork, beef, chicken and wheat because their skin symptoms reappeared after intake of these ingredients. The results of the food elimination trial and the provocative challenge indicated the usefulness of the novel elimination diet for diagnosis of AFR.

Cultivation and characterization of canine skin-derived mast cells.

It is essential to develop a technique to culture purified skin-derived mast cells (SMCs) to facilitate immunological research on allergic diseases in dogs. This study was performed to develop an efficient culture system for canine SMCs and to characterize the cells in comparison to canine bone marrow-derived mast cells (BMMCs). Enzymatically digested skin biopsy samples were cultivated in serum-free AIM-V medium supplemented with recombinant canine stem cell factor. Three to five weeks after the initiation of culture, mast cells were collected by a magnetic activated cell separation system using anti-c-Kit antibody. The collected cells were composed of a uniform population showing morphological characteristics of mast cells with a round or oval nucleus and abundant toluidine blue-positive metachromatic granules in the cytoplasm. The results of flow cytometric analysis for the presence of cell membrane c-Kit and Fc epsilon receptor I (FcepsilonRI) indicated that approximately 90% of the cells were mast cells. The cytoplasmic granules were positive for both tryptase and chymase. Apparent dose-dependent degranulation was induced by antibody-mediated cross-linking of immunoglobulin E (IgE) bound to the cells. These cytological and immunological characteristics observed in SMCs were mostly similar to those observed in BMMCs; however, IgE-mediated degranulation was significantly lower in SMCs than BMMCs. The culture system for canine SMCs developed in this study would be useful in understanding the pathophysiology and developing anti-allergic therapeutics in canine allergic dermatitis.

Identification of canine natural CD3-positive T cells expressing an invariant T-cell receptor alpha chain.

A representative T-cell subset exclusively using an invariant TCRalpha chain (iTCRalpha) is natural killer T (NKT) cells which are becoming an emerging topic for cancer and immune disorder in humans and mice. However, NKT cells in dogs have not yet been identified. In this study, CD3(+) T-lymphocyte population reactive to alpha-galactosylceramide-loaded mouse CD1d (alpha-GalCer/CD1d) were identified with flow cytometric analysis in mononuclear cells from spleen, liver, and peripheral blood of a dog with percentages of 0.028%, 0.045%, and 0.004%, respectively. Using cDNA library synthesized from mRNAs of the alpha-GalCer/CD1d reactive CD3(+) lymphocytes in the spleen cells, molecular analysis of canine iTCRalpha was carried out. Consequently, Variable (Valpha) and Joining (Jalpha) regions of iTCRalpha cDNA were found to be homogeneous to both mouse Valpha14-Jalpha281 and human Valpha24-JalphaQ. Characteristic features of iTCRalpha of NKT cells, such as the amino acid sequence of complementarity-determining region (CDR) 3 and extra cysteine residue, were well conserved among dogs, mice, and humans. In quantitative real-time PCR analysis, relative expression of the canine iTCRalpha mRNA in alpha-GalCer/CD1d reactive CD3(+) lymphocytes was 271-fold higher than that in CD3(+) lymphocytes unbound to alpha-GalCer/CD1d, indicating that the iTCRalpha mRNA was preferentially expressed in alpha-GalCer/CD1d-reactive CD3(+) lymphocytes in the dog. Together, the results strongly suggested that alpha-GalCer/CD1d-binding CD3(+) T lymphocytes identified in this study could be considered to be canine NKT cells.

The antibody against human CD25, ACT-1, recognizes canine T-lymphocytes in the G2/M and G0/G1 phases of the cell cycle during proliferation.

Because of the lack of an appropriate antibody against the canine CD25 molecule, we investigated whether anti-human CD25 antibody, ACT-1, could be useful in detecting canine T-lymphocyte proliferation. Peripheral mononuclear cells from a dog were cultured for 4 days with or without concanavalin A stimulation. In the last 24 hr, bromodeoxyuridine (BrdU) and human recombinant IL-2 were added. While the cell cycle was detected using anti-BrdU antibody and 7-amino-actinomycine (7-AAD), the cultured cells were stained with anti-canine CD4 antibody and ACT-1. The results showed that T-lymphocytes reactive to ACT-1 were present in the G2/M and G0/G1 phases in 94.4% and 70.0% of CD4-positive T-lymphocytes, respectively, suggesting that flow cytometory with ACT-1 might be useful in detecting canine T-lymphocytes during and after activation.

CC chemokine receptor 4-positive CD4(+) lymphocytes in peripheral blood increases during maturation in healthy beagles.

In this study, percentages of CCR4(+) cells in peripheral CD4(+) T-lymphocytes were examined with flow cytometry in 46 healthy beagles between 3 months and 7 years of age. The percentage of CCR4(+) cells varied from 9.9% to 33.5%. The mean percentage was significantly lower in the group with ages of less than 1 year than those with ages equal to or more than 1 year (p<0.05), suggesting that maturation might increase the CCR4(+) T-lymphocyte subset. No influence of aging on the percentages was detected among the groups with ages equal to or more than 1 year. The findings are useful for establishing a reference value for the percentage of peripheral CCR4(+)CD4(+) lymphocytes in dogs.

Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells.

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.

Identification of bovine serum albumin as an IgE-reactive beef component in a dog with food hypersensitivity against beef.

IgE-reactive beef components were examined by an immunoblot analysis using a serum from a dog with food hypersensitivity against beef. The immunoblot analysis revealed a distinct band at approximately 66 kDa and a faint band at approximately 50 kDa. The immunoblot analysis for serum IgE reactivity to bovine serum albumin (BSA) also revealed a positive band at 66 kDa. Serum IgE reactivity to the 66-kDa protein of beef was diminished by pre-incubating the serum sample with BSA. Furthermore, a positive reaction to BSA was detected in intradermal testing in the dog. These results clearly indicated that BSA was an IgE-reactive beef component in the dog with food hypersensitivity against beef.

Regulation of B1 cell migration by signals through Toll-like receptors.

Peritoneal B1 cells are known to generate large amounts of antibodies outside their residential site. These antibodies play an important role in the early defense against bacteria and viruses, before the establishment of adaptive immune responses. Although many stimuli, including antigen, lipopolysaccharide, or cytokines, have been shown to activate B1 cells and induce their differentiation into plasma cells, the molecular signals required for their egress from the peritoneal cavity are not understood. We demonstrate here that direct signals through Toll-like receptors (TLRs) induce specific, rapid, and transient down-regulation of integrins and CD9 on B1 cells, which is required for detachment from local matrix and a high velocity movement of cells in response to chemokines. Thus, we revealed an unexpected role for TLRs in governing the interplay between integrins, tetraspanins, and chemokine receptors required for B1 cell egress and, as such, in facilitating appropriate transition from innate to adaptive immune responses.

Cloning of cDNA encoding canine endothelin receptors and their expressions in normal tissues.

The receptors for endothelin (ET) family, ETA and ETB, were molecularly cloned and the expression of ETA and ETB as well as preproendothelin-1 (PPET-1, precursor of ET-1) was examined in normal canine tissues by RT-PCR. The entire open reading frames of the canine ETA and ETB were shown to encode 427 and 442 amino acid residues, respectively, showing from 87.4 to 97.3% sequence similarity to human, mouse, and rat counterparts. ETA and ETB mRNAs were ubiquitously expressed in a variety of canine tissues in this study and PPET-1 mRNA was detected in the tissues except for heart and liver. It was speculated that ET could play an important role in physiological events in most of the organs.