Study on the Fatigue Crack Initiation and Growth Behavior in Bismuth- and Lead-Based Free-Cutting Brasses.

Several studies have been conducted on the fatigue behavior of copper and 7-3, and 6-4 brasses. However, there have been fewer studies on the fatigue behavior and fatigue crack growth (FCG) properties of free-cutting brass, primarily because emphasis has been placed on the development of lead-free free-cutting brass. In this study, fatigue experiments were performed in the atmosphere at room temperature using three types of free-cutting, two types of bismuth (Bi)-based (with different grain sizes), and lead (Pb)-based brasses. It was found that lead-free Bi-based free-cutting brass had approximately the same fatigue performance as that of Pb-based free-cutting brass. It was also clarified that the addition of Bi or Pb initiated fatigue cracks, and that the crack growth period occupied most of the fatigue life. Differences in the FCG behavior of the three free-cutting brasses were observed in the low ΔK range. The modified linear fracture mechanics parameter M was used to quantitatively analyze the fatigue life and FCG behavior (short surface cracks). A comparison between the calculated and experimental results showed that M was useful.

Elasto-Plastic Fatigue Crack Growth Behavior of Extruded Mg Alloy with Deformation Anisotropy Due to Stress Ratio Fluctuation.

Fatigue crack growth (FCG) experiments were performed using a low-temperature extruded magnesium alloy AZ31 with texture. Under a constant maximum stress intensity factor (Kmax), the stress ratio R was changed from 0.1 to -1 during the fatigue crack growth process, and the FCG behavior before and after the R change was investigated. As a result, tensile twins were generated owing to the fatigue load on the compression side of R = -1, and the FCG velocity was accelerated. In addition, when the maximum compressive stress at R = -1 (|(σmin)R = -1|) exceeded the compressive yield strength of the material (σcy), the FCG velocity after R fluctuation greatly accelerated. On the other hand, under the condition |(σmin)R = -1| < σcy, the degree of acceleration of the FCG velocity due to R fluctuation was small. In either case, the degree of acceleration in the FCG increased as the Kmax value increased. The above FCG acceleration mechanism due to the R fluctuation was considered based on the observation of the deformation and twinning states of the fatigue crack tip, the fatigue crack closure behavior, and the cyclic stress-strain curve of the fatigue process. The FCG acceleration mechanism was as follows: First, the driving force of the FCG increased owing to the increase in crack opening displacement due to the generation of tensile twins. Second, the coalescence of the main crack and a plurality of microcracks were generated at the twin interface. The elasto-plastic FCG behavior after the stress ratio fluctuations is defined by the effective J-integral range ΔJeff.

Effect of Rod-like Structure on Fatigue Life, Short Surface Crack Initiation and Growth Characteristics of Extruded Aluminum Alloy A2024 (Analysis via Modified Linear Elastic Fracture Mechanics).

In the Al alloy A2024-T3 extruded material, a rod-like structure is generated parallel to the extrusion direction. In this study, the effects of rod-like structures on fatigue crack initiation and growth behavior were comprehensively investigated. Two types of specimens were used in a fatigue experiment, in which the direction of the load stress amplitude was parallel (specimen P) and perpendicular (specimen V) to the rod-like structure. Based on the experimental and analytical results, the following findings were obtained regarding the fatigue life, location of crack initiation, and fatigue crack growth behavior. Because the fatigue life of specimen P was longer than that of specimen V, it is inferred that the rod-like structure significantly affects the fatigue life. In specimen P, fatigue cracks were generated from the grain boundaries of the Al matrix. By contrast, in specimen V, cracks were generated from the Cu-Mg-based intermetallic compound in the Al matrix. In specimen P, fatigue cracks were more likely to propagate across the rod-like structure, which decreased the fatigue crack growth rate. In specimen V, fatigue cracks did not propagate across the rod-like structure; instead, they propagated through the Al matrix. Therefore, the fatigue crack growth resistance of specimen V was lower than that of specimen P. The relationship between the fatigue crack growth rate and the modified linear elastic fracture mechanics parameter could be used to predict the S-N curve (stress amplitude vs. fatigue life) and fatigue crack growth behavior. The predicted results agreed well with the experimental results.

Development and characterization of a unique anti-IgE mouse monoclonal antibody cross-reactive between human and canine IgE.

BACKGROUND: The efficacy assessment of human anti-IgE monoclonal antibodies (mAbs) in animal models before clinical trials is hampered due to the lack of cross-reactivity of anti-IgE mAbs between species. OBJECTIVE: We developed CRE-DR (an anti-dog IgE monoclonal antibody), an anti-IgE mouse mAb that recognizes canine and human IgE, and then examined its IgE specificity and cross-reactivity between three animal and human species. METHODS: After mouse immunization with a synthetic peptide derived from canine IgE (282 NTNDWIEGETYYC294 ), we generated a hybridoma producing CRE-DR. The CRE-DR purified from the ascites of hybridoma-inoculated mice was used for ELISA and Western blot analysis to examine reactivity to dog, human, and rodent IgEs as well as recombinant bovine serum albumin (BSA)-conjugated to canine, human, and rodent IgE amino acid peptides corresponding to the immunizing sequence. We then performed enzyme-linked immunosorbent assays (ELISAs) for dog IgE using sera from dogs with atopic dermatitis (AD) after inhibition with canine IgE and IgG. The amino acid sequence recognized by CRE-DR was identified by ELISA using synthetic peptides. RESULTS: CRE-DR is a monoclonal mouse IgG1κ specific for dog IgE, and the ELISA values in atopic dog sera were inhibited by dog IgE, but not dog IgG. The binding of CRE-DR to human IgE was relatively maintained, but not to rodent IgEs, which results were confirmed with the BSA-conjugated IgE peptides of the various species. The CRE-DR reactivity was supported by the comparison of amino acid sequence of CRE-DR epitope, DWIEGETYYC, in dog IgE; one, two, and three amino acids were substituted in the human, rat, and mouse IgE epitopes, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: CRE-DR is a mAb cross-reactive to dog and human IgEs, which can allow the use of a dog model of allergy to test the efficacy of a CRE-DR-derived anti-IgE therapeutic mAb before human clinical trials.

Effect of Specimen Thickness and Stress Intensity Factor Range on Plasticity-Induced Fatigue Crack Closure in A7075-T6 Alloy.

Fatigue crack growth experiments are performed using A7075-T6 compact tension (CT) specimens with various thicknesses t (1-21 mm). The stress intensity factor at the crack opening level Kop is measured, and the effects of t and the stress intensity factor range ΔK on Kop are investigated. In addition, the change in Kop value due to specimen surface removal is investigated. Furthermore, we clarify that the radius of curvature of the leading edge of the fatigue crack decreases as t becomes thinner. Using the three-dimensional elastoplastic finite element method, the amount of plastic lateral contraction (depression depth d) at the crack tip after fatigue loading is calculated quantitatively. The following main experimental results are obtained: In the region where ΔK is 5 MPam1/2 or higher, the rate of fatigue crack growth da/dN at a constant ΔK value increases as t increases from 1 to 11 mm. The da/dN between t = 11 and 21 mm is the same. Meanwhile, in the region where ΔK is less than 5 MPam1/2, the effect of t on da/dN is not observed. The effects of t and ΔK on the da/dN-ΔK relationship are considered physically and quantitatively based on d.

Hydrolyzed diets may stimulate food-reactive lymphocytes in dogs.

Hydrolyzed proteins are often prescribed for dogs with food hypersensitivity in food elimination programs. However, the potential of these diets to stimulate lymphocyte-mediated hypersensitivity is currently unknown. In this study, two commercially available hydrolyzed diets for dogs, D-1 (Aminopeptide Formula Dry, Royal Canin Japon, Tokyo, Japan), and D-2 (Canine z/d Ultra Dry, Hill's-Colgate (Japan) Ltd., Tokyo, Japan), were analyzed to identify residual proteins or peptides, as well as activated helper T-lymphocyte reactions in dogs with suspected food hypersensitivity. Proteins and peptides with molecular weights >1 kDa (majority 1.5-3.5 kDa) were detected in both diet extracts with sodium dodecyl sulfate polyacrylamide gel electrophoresis, and size exclusion chromatography. When peripheral blood mononuclear cells (PBMC's) from 316 dogs with suspected food allergies were cultured with hydrolyzed diet extracts, flow cytometry analysis revealed detectable levels of CD25low helper T-lymphocytes stimulated by D-1 and D-2 in 91 of 316, (28.8%), and 75 of 316 (23.7%) samples, respectively. These data indicated that the extracts contained proteins or peptides large enough to activate the lymphocytes. The percentages of CD25low helper T-lymphocytes stimulated by D-1 and D-2 extracts increased to 38.7% and 29.6%, respectively, in 186 of the original 316 samples (186/316, 58.9%), also reactive to poultry-related antigens. Thus, both poultry-related antigens, and D-1 and D-2 diet extracts may activate helper T-lymphocytes. These results demonstrate that hydrolyzed diets may contain proteins that stimulate helper T-lymphocytes, and may not be effective for treating all dogs with food hypersensitivity.

Lower trunk muscle activity-induced alignment and cop position during single-leg standing.

[Purpose] The purpose of this study was to clarify fundamental changes induced by lower trunk muscle contraction during single-leg standing. [Subjects and Methods] Ten healthy normal males participated in this study. All subjects could accurately perform lower trunk muscle contraction-type Abdominal Expansion (AE), Abdominal Bracing (AB), and Abdominal Cave-in (AC). The alignment and position of the center of foot pressure (COP) during single-leg standing with SLR and step position after rotating the body from single-leg standing with maximum SLR were measured in each lower trunk muscle contraction type. [Results] When AC was performed during single-leg standing with SLR, the SLR angle increased, COP shifted backward, and the posterior tilt angle of the trunk and cross step distance decreased. [Conclusion] It was assumed that AC during wind-up increases the angle of lower limb elevation and decreases the posterior tilt angle of the trunk and cross step distance.

A photoresponsive soft interface reversibly controls wettability and cell adhesion by conformational changes in a spiropyran-conjugated amphiphilic block copolymer.

The functionalities of soft interfaces including cell adhesion can be enhanced by dynamic conversion of polymer properties and movement via external stimuli. Light is a superior stimulus, and various surfaces modified with photoreactive molecules have been prepared. However, in most of these studies, the surface properties are irreversibly changed due to photo-degradation, and reversible adhesion and collection of cells is not feasible. In this study, we developed a photoresponsive polymer soft interface that was able to spatiotemporally control wettability, cell adhesion, and detachment in a reversible manner. Spiropyran molecules were introduced into the hydrophobic block of an amphiphilic diblock copolymer consisting of poly(methyl methacrylate) and polyethylene glycol, and the monomer unit numbers of these components were optimized. The copolymer was immobilized on a glass substrate as a nanofilm. With alternating irradiation using UV and visible light, the surface exhibited reversible changes in hydrophobicity and hydrophilicity, and the direction of change was opposite to the polarity change in photo-isomerization of spiropyran. We also achieved photo-control of effective cell adhesion and detachment with sequential irradiation with UV and visible light. These remarkable functions could be ascribed to conformational changes triggered by photo-isomerization of spiropyran. This photoresponsive polymer soft interface may have applications as a powerful tool in biological studies by facilitating sequential changes in wettability and bioaffinity. STATEMENT OF SIGNIFICANCE: We developed a photoresponsive polymer soft interface, which was able to spatiotemporally control wettability and cell adhesion/detachment in a reversible manner, by introducing spiropyran into the hydrophobic block of an amphiphilic diblock copolymer. With alternating irradiation using UV and visible light, the surface exhibited unique reversible wettability changes; the direction of hydrophobicity and hydrophilicity change was opposite to the polarity change in spiropyran photo-isomerization. Light-dependent reversible control of spatiotemporal cell adhesion and detachment was also achieved with sequential UV (adhesion) and visible light irradiation (detachment). Cell detachment using noncytotoxic visible light was realized for the first time. Cell-patterning capability stably lasted for 25days. This photoresponsive surface could be applied to fabrication of engineered tissues comprised of several cellular species.

IgE reactivity to hen egg white allergens in dogs with cutaneous adverse food reactions.

Dogs with cutaneous adverse food reactions (CAFR) often have specific IgE to food allergens. Egg white, which is majorly composed of ovomucoid, ovalbumin, ovotransferrin, and lysozyme, is a food allergen in dogs. Information of the IgE reactivity to purified egg white allergens supports accurate diagnosis and efficiency treatment in humans. However, to the best of our knowledge, there have been no studies on the IgE reactivity to purified egg white allergens in dogs. Here, we investigated the IgE reactivity to crude and purified allergens of hen egg white in dogs with CAFR. First, when we examined serum samples from 82 dogs with CAFR for specific IgE to crude egg white by ELISA, 9.8% (8/82) of the dogs with CAFR showed the IgE reactivity to crude egg white. We then used sera from the eight dogs with positive IgE reactivity to crude egg white to examine the IgE reactivity to four purified allergens, ovomucoid, ovalbumin, ovotransferrin, and lysozyme, by ELISA. We found that 75% (6/8) of the dogs showed IgE reactivity to both ovomucoid and ovalbumin, and that 37.5% (3/8) of the dogs showed IgE reactivity to ovotransferrin. None (0/8) showed IgE reactivity to lysozyme. Moreover, validating these results, the immunoblot analyses were performed using the sera of the three dogs showing the highest IgE reactivity to crude egg white. Both anti-ovomucoid and anti-ovalbumin IgE were detected in the sera of these dogs, while anti-ovotransferrin IgE was not detected. Considering these, ovomucoid and ovalbumin appears to be the major egg white allergens in dogs with CAFR.

Food allergens inducing a lymphocyte-mediated immunological reaction in canine atopic-like dermatitis.

Canine atopic-like dermatitis (ALD) is suspected to be associated with food allergies, particularly those mediated by lymphocytes. In this study, 54 cases were included as ALD dogs, based on the negative IgE test results. In the dogs, the percentage of activated cells in helper-T lymphocytes was measured by flow cytometry using cultured peripheral lymphocytes under food allergen stimulation. We observed that 49 of the 54 ALD dogs (90.7%) had positive lymphocyte reactions against one or more food allergens. The most common food allergen was soybean, showing positive results in 21 dogs (42.9%), while the allergen to cause the lowest number of reactions was catfish (only 5 dogs, 10.2%). These results may be useful in considering elimination diets for ALD dogs.

Vaccination with antigen-transfected, NKT cell ligand-loaded, human cells elicits robust in situ immune responses by dendritic cells.

Both innate and adaptive immunity are crucial for cancer immunosurveillance, but precise therapeutic equations to restore immunosurveillance in patients with cancer patients have yet to be developed. In murine models, α-galactosylceramide (α-GalCer)-loaded, tumor antigen-expressing syngeneic or allogeneic cells can act as cellular adjuvants, linking the innate and adaptive immune systems. In the current study, we established human artificial adjuvant vector cells (aAVC) consisting of human HEK293 embryonic kidney cells stably transfected with the natural killer T (NKT) immune cell receptor CD1d, loaded with the CD1d ligand α-GalCer and then transfected with antigen-encoding mRNA. When administered to mice or dogs, these aAVC-activated invariant NKT (iNKT) cells elicited antigen-specific T-cell responses with no adverse events. In parallel experiments, using NOD/SCID/IL-2rγc(null)-immunodeficient (hDC-NOG) mouse model, we also showed that the human melanoma antigen, MART-1, expressed by mRNA transfected aAVCs can be cross-presented to antigen-specific T cells by human dendritic cells. Antigen-specific T-cell responses elicited and expanded by aAVCs were verified as functional in tumor immunity. Our results support the clinical development of aAVCs to harness innate and adaptive immunity for effective cancer immunotherapy.

Lymphocyte blastogenic responses to food antigens in cats showing clinical symptoms of food hypersensitivity.

Three cats were diagnosed as having food hypersensitivity by food elimination and oral food provocation tests. Twelve allergenic food ingredients were identified by oral food provocation test in the 3 cats. Of the 12 food ingredients, 9 offending food antigens were shown to be positive in a lymphocyte stimulation test; however, none of them were positive in antigen-specific IgE testing, and only four food antigens were positive in intradermal testing. The stimulation indices in the lymphocyte stimulation tests for the 9 food ingredients were found to be decreased after the cats were fed elimination diets. The present study demonstrates that the lymphocyte stimulation test reflects an immunologic reaction involved in food hypersensitivity and can help identify allergenic food ingredients in feline food hypersensitivity.

Measurement for canine IgE using canine recombinant high affinity IgE receptor α chain (FcεRIα).

To detect allergen-specific IgE in dogs with allergic diseases, we developed a recombinant canine high affinity IgE receptor α chain (FcεRIα)-based IgE detection system. Using the recombinant protein of canine FcεRIα expressed by an Escherichia coli expression system, we could detect house dust mite (Dermatophagoides farinae) allergen-specific IgE in sera from dogs naturally and experimentally sensitized to this allergen with ELISA and western blotting. The IgE binding activity of recombinant canine FcεRIα on ELISA was impaired by heat treatment of these sera. The specificity of this recombinant canine FcεRIα-based IgE detection system was confirmed by inhibition assays with canine IgE. The recombinant canine FcεRIα-based IgE detection system established in this study offers an alternative tool to measure allergen-specific IgE in dogs.

Flow cytometric analysis of lymphocyte proliferative responses to food allergens in dogs with food allergy.

Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5- to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.

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.

[A case of squamous carcinoma in the buccal region treated with Mohs chemosurgery in a patient with senile dementia of Alzheimer type].

An 89-year-old woman was given a diagnosis of senile dementia of Alzheimer type around 2003. In 2006, the patient was examined at a dermatology clinic complaining of tumor in the right buccal region. At the time, possible squamous carcinoma was pointed, but nothing was done for it. In March 2008, home care became difficult due to progression of dementia, and the patient was admitted to our hospital. On admission, a protruding skin tumor (20 x 20 x 2 mm, cauliflower-like surface) was observed in the right buccal region. Surgical ablation was recommended, however, the family strongly requested to let it follow its in natural course. In December 2008, the tumor increased rapidly to 40 x 55 x 25 mm with bleeding and a bad smell, which decreased her QOL markedly. After obtaining consent from the patient and her family, Mohs chemosurgery was performed. As the result, the tumor resolved and the QOL improved. Mohs chemosurgery is considered as a very effective treatments for rapidly advancing progressive skin cancer in patients with severe cognitive impairment.

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.