Kai 1 and Kai 2: Characterization of these dog erythrocyte antigens by monoclonal antibodies

<div><p><i>Dog Erythrocyte Antigens (DEA)</i> have thus far been found by sensitizing dogs with canine allogeneic blood and are clinically important regarding blood transfusion incompatibilities, but remain poorly defined. The goals of this study were to discover and characterize two <i>DEAs</i>, named as <i>Kai 1</i> and <i>Kai 2</i>. The monoclonal antibodies were produced by mouse hybridoma techniques and examined by ELISA isotyping, immunoblotting, and affinity chromatography. Canine blood samples were typed and the development of alloantibodies was examined in transfused dogs. The monoclonal <i>Kai 1</i> and <i>Kai 2</i> antibodies were isotyped as IgM kappa and IgG3 lamda, respectively, and identified two different erythrocyte membrane proteins of 200 kDa and 80 kDa in molecular weights, respectively. Either <i>Kai 1</i> or <i>Kai 2</i> can be expressed but not both in an individual dog. There were no naturally occurring anti-<i>Kai 1</i> or <i>Kai 2</i> alloantibodies. In addition, <i>Kai 1-</i> and/or <i>Kai 2-</i> dogs developed <i>Kai 1</i> and <i>Kai 2</i> alloantibodies, respectively, when transfused with mismatched blood. This is the first discovery of canine blood types by screening monoclonal antibodies. <i>Kai 1</i> and <i>Kai 2</i> are novel blood types which can induce anti-<i>Kai 1</i> or anti-<i>Kai 2</i> alloantibodies when <i>Kai 1-</i> and/or <i>Kai 2-</i> dogs are transfused with <i>Kai 1+</i> or <i>Kai 2+</i> blood. These canine blood types may explain some of the blood incompatibilities and transfusion reactions observed in dogs in clinical practice.</p></div

Comparison of <i>Kai</i> and <i>DEA 1</i> in 50 dogs.

<p>Comparison of <i>Kai</i> and <i>DEA 1</i> in 50 dogs.</p

SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) of purified (A) <i>anti-Kai 1</i> and (B) <i>anti-Kai 2</i> monoclonal antibodies.

<p>M: Marker (kDa); Lane 1: Normal mouse IgG, Lane 2 and 3: Purified mAb.</p

Affinity purification and chromatography of <i>Kai 1</i> and <i>Kai 2</i> antigens from erythrocyte membrane extracts.

<p>(A) Schematic diagram of affinity purification procedure. (B) Membrane proteins from <i>Kai 1+</i> RBCs bound with <i>anti-Kai 1</i> mAbs and IgM heavy & light chains of <i>anti-Kai 1</i> antibody. M: marker (kDa), Lane 1: purified membrane protein (10 μg) from <i>Kai 1+</i> RBCs. (C) Membrane proteins <i>Kai 2+</i> from RBCs bound with <i>anti-Kai 2</i> antibody and IgG heavy & light chains of <i>Kai 2</i> antibody. M: marker, Lane 1: bovine serum albumin (2 μg), Lane 2: purified membrane protein (10 μg) from <i>Kai 2+</i> blood. Molecular weights are indicated on the left side in kDa.</p

Isotyping of (A) <i>anti-Kai 1</i> and (B) <i>anti-Kai 2</i> antibodies by ELISA.

<p>Results were expressed as optical density (O.D.) values in ELISA, and values >0.2 (horizontal dashed line) were considered positive.</p

Effects of Exposure to Ozone on the Ocular Surface in an Experimental Model of Allergic Conjunctivitis

<div><p>Based on previous findings that ozone can induce an inflammatory response in the ocular surface of an animal model and in cultured human conjunctival epithelial cells, we investigated whether exposure to ozone exacerbates symptoms of allergic conjunctivitis. We evaluated the effects of exposure to ozone on conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, production of inflammatory cytokines in tears, and aqueous tear production in a mouse model of allergic conjunctivitis. To validate our <i>in vivo</i> results, we used interleukin (IL)-1α-pretreated conjunctival epithelial cells as an <i>in vitro</i> substitute for the mouse model. We evaluated whether exposure to ozone increased the inflammatory response and altered oxidative status and mitochondrial function in IL-1α-pretreated conjunctival epithelial cells. In the <i>in vivo</i> study, ozone induced increases in conjunctival chemosis, conjunctival injection, corneal and conjunctival fluorescein staining scores, and production of inflammatory cytokines, accompanied by a decrease in tear volume. In the <i>in vitro</i> study, exposure to ozone led to additional increases in IL-6 and tumor necrosis factor-α mRNA levels, which were already induced by treatment with IL-1α. Ozone did not induce any changes in cell viability. Pretreatment with IL-1α increased the expression of manganese superoxide dismutase, and exposure to ozone led to additional increments in the expression of this antioxidant enzyme. Ozone did not induce any changes in mitochondrial activity or expression of mitochondrial enzymes and proteins related to mitochondrial function, with the exception of phosphor-mammalian target of rapamycin. Treatment with butylated hydroxyanisole, a free radical scavenger, attenuated the ozone-induced increases in IL-6 expression in IL-1α-pretreated conjunctival epithelial cells. Therefore, we conclude that exposure to ozone exacerbates the detrimental effects on the integrity of the ocular surface caused by conjunctival allergic reactions, and further increases the inflammatory response in IL-1α-pretreated conjunctival epithelial cells.</p></div

Major cross-match results in the transfused dogs related to <i>DEA 1</i> and <i>Kai</i>.

<p>Major cross-match results in the transfused dogs related to <i>DEA 1</i> and <i>Kai</i>.</p

Major crossmatch test incompatibility in two dogs 21 days after receiving <i>Kai</i> mismatched transfusions.

<p>(A) Microscopic agglutination in case of a <i>Kai 2-/DEA 1+</i> dog receiving <i>Kai 2+/DEA 1+</i> blood. (B) Microscopic agglutination in case of a <i>Kai 1-/DEA 1+</i> dog receiving <i>Kai 1+/DEA 1+</i> blood. Bar = 200 μm.</p

Changes in corneal and conjunctival fluorescein staining scores after exposure to ozone in a mouse model of experimental allergic conjunctivitis.

<p>(A) Corneal fluorescein staining scores. (B) Conjunctival fluorescein staining scores. N/C, negative control; 1 week, after 1 week of exposure; 2 weeks, after 2 weeks of exposure. Error bars represent the standard error of the mean (**<i>p</i> < 0.01, ***<i>p</i> < 0.001).</p

Changes in aqueous tear production after exposure to ozone in a mouse model of experimental allergic conjunctivitis.

<p>N/C, negative control. Error bars represent the standard error of the mean (**<i>p</i> < 0.01, ***<i>p</i> < 0.001).</p