Large-Scale Polymorphism Analysis of Dog Leukocyte Antigen Class I and Class II Genes (DLA-88, DLA-12/88L and DLA-DRB1) and Comparison of the Haplotype Diversity between Breeds in Japan.

Polymorphisms of canine leukocyte antigen (DLA) class I (DLA-88 and DLA-12/88L) and class II (DLA-DRB1) genes are important for disease susceptibility studies, but information on the genetic diversity among dog breeds is still lacking. To better elucidate the polymorphism and genetic diversity between breeds, we genotyped DLA-88, DLA-12/88L, and DLA-DRB1 loci using 829 dogs of 59 breeds in Japan. Genotyping by Sanger sequencing identified 89, 43, and 61 alleles in DLA-88, DLA-12/88L, and DLA-DRB1 loci, respectively, and a total of 131 DLA-88-DLA-12/88L-DLA-DRB1 haplotypes (88-12/88L-DRB1) were detected more than once. Of the 829 dogs, 198 were homozygotes for one of the 52 different 88-12/88L-DRB1 haplotypes (homozygosity rate: 23.8%). Statistical modeling suggests that 90% of the DLA homozygotes or heterozygotes with one or other of the 52 different 88-12/88L-DRB1 haplotypes within somatic stem cell lines would benefit graft outcome after 88-12/88L-DRB1-matched transplantation. As previously reported for DLA class II haplotypes, the diversity of 88-12/88L-DRB1 haplotypes varied remarkably between breeds but was relatively conserved within most breeds. Therefore, the genetic characteristics of high DLA homozygosity rate and poor DLA diversity within a breed are useful for transplantation therapy, but they may affect biological fitness as homozygosity progresses.

Dog leukocyte antigen class II alleles and haplotypes associated with meningoencephalomyelitis of unknown origin in Chihuahuas.

Idiopathic non-infectious meningoencephalomyelitis (NIME), which is thought to be an immune-mediated disease, is a common inflammatory disease in dogs. Meningoencephalomyelitis of unknown origin (MUO), a subgroup of NIME, consists of necrotizing meningoencephalitis (NME), necrotizing leukoencephalitis, and granulomatous meningoencephalomyelitis. Recent studies have shown associations between disease development and dog leukocyte antigen (DLA) class II genes in NME in Pugs and in NIME in Greyhounds. This study focused on Chihuahuas, which have a high incidence of MUO and are one of the most common dog breeds in Japan. Because the development of MUO seems to be associated with DLA class II genes, we aimed to evaluate the association between DLA class II genes and MUO development in Chihuahuas. Blood samples were obtained from 22 Chihuahuas with MUO (MUO group) and 46 without neurological diseases (control). The allele sequences of three DLA class II loci were determined, and haplotypes were estimated from these data. In total, 23 haplotypes were detected. The frequency of one haplotype (DLA-DRB1*015:01--DQA1*006:01--DQB1*023:01) was significantly higher in the MUO group than in the control group (odds ratio, 7.11; 95% confidence interval, 1.37-36.81; P=0.0141). The results suggest that the development of MUO in Chihuahuas may be associated with DLA class II genes. Because the identified risk haplotypes differed from those of other breeds, the pathogenesis of NIME-related diseases may differ among dog breeds.

A fish cytokine related to human IL-3, IL-5, and GM-CSF, induces development of eosinophil/basophil/mast-cell type (EBM) granulocytes.

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are related cytokines that signal through receptors possessing the ß common (ßc) chain. As a family, these cytokines combine rather non-specific hematopoietic growth factor properties with a special importance for eosinophils, basophils, and mast cells. In fish the cytokines of this family are called IL-5fam, and the present study, using carp, constitutes their first functional analysis. Carp il-5fam expression was enhanced by stimulation with phytohemagglutinin and killed bacteria. Reminiscent of mammalian IL-3/IL-5/GM-CSF family members, recombinant carp IL-5fam (rcIL-5fam) induced activation of transcription factor STAT5 and efficiently promoted proliferation and colony-formation of eosinophil/basophil/mast-cell type (EBM) granulocytes. Upon addition of recombinant carp ßc the growth effect of rcIL-5fam was reduced, suggesting ßc participation in the signaling route. In summary, despite differences in individual cytokines and cell populations, fish and mammalian IL-3/IL-5/GM-CSF family members share growth factor functions for non-neutrophil granulocytes.

Evaluation of alloreactive T cells based on the degree of MHC incompatibility using flow cytometric mixed lymphocyte reaction assay in dogs.

It has become anticipated that regenerative medicine will extend into the field of veterinary medicine as new treatments for various disorders. Although the use of allogeneic stem cells for tissue regeneration is more attractive than that of autologous cells in emergencies, the therapeutic potential of allogeneic transplantation is often limited by allo-immune responses inducing graft rejection. Therefore, a methodology for quantifying and monitoring alloreactive T cells is necessary for evaluating allo-immune responses. The mixed lymphocyte reaction (MLR) is widely used to evaluate T cell alloreactivity. In human, flow cytometric MLR with carboxyfluorescein diacetate succinimidyl ester has been established and used as a more useful assay than conventional MLR with radioisotope labeling. However, the available information about alloreactivity based on the differences of dog major histocompatibility complex (MHC) (dog leukocyte antigen, DLA) is quite limited in dog. In this paper, we describe our established flow cytometric MLR method that can quantify the T cell alloreactivity while distinguishing cell phenotypes in dog, and T cell alloreactivity among DLA-type matched pairs was significantly lower than DLA-mismatched pairs, suggesting that our developed flow cytometric MLR method is useful for quantifying T cell alloreactivity. In addition, we demonstrated the advantage of DLA homozygous cells as a donor (stimulator) for allogeneic transplantation. We also elucidated that the frequency of alloreactive T cell precursors was almost the same as that of mouse and human (1-10%). To our knowledge, this is the first report to focus on the degree of allo-immune responses in dog based on the differences of DLA polymorphisms.

Paralogs of Common Carp Granulocyte Colony-Stimulating Factor (G-CSF) Have Different Functions Regarding Development, Trafficking and Activation of Neutrophils.

Mammalian granulocyte colony-stimulating factor (G-CSF; CSF3) is a primary cytokine that promotes the development, mobilization, and activation of neutrophils and their precursors. Teleosts have been reported to possess two paralogs as a likely result of the teleost-wide whole genome duplication (WGD) event, but functional divergence of G-CSF paralogs remains poorly understood. Common carp are an allotetraploid species owing to an additional WGD event in the carp lineage and here, we report on genomic synteny, sequence similarity, and phylogeny of four common carp G-CSF paralogs (g-csfa1 and g-csfa2; g-csfb1 and g-csfb2). G-csfa1 and g-csfa2 show differential and relatively high gene expression levels, while g-csfb1 and g-csfb2 show low basal gene expression levels in most tissues. All paralogs are expressed higher in macrophages than in other leukocyte sub-types and are highly up-regulated by treatment of macrophages with mitogens. Recombinant G-CSFa1 and G-CSFb1 both promoted the proliferation of kidney hematopoietic cells, while only G-CSFb1 induced the differentiation of kidney cells along the neutrophil-lineage. Colony-forming unit assays revealed that G-CSFb1 alone stimulates the formation of CFU-G colonies from head- and trunk-kidney whereas the combination of G-CSFa1 and G-CSFb1 stimulates the formation of both CFU-G and CFU-GM colonies. Recombinant G-CSFa1 and G-CSFb1 also exhibit chemotactic activity against kidney neutrophils and up-regulation of cxcr1 mRNA expression was highest in neutrophils after G-CSFb1 stimulation. Furthermore, G-CSFb1 more than G-CSFa1 induced priming of kidney neutrophils through up-regulation of a NADPH-oxidase component p47 phox . In vivo administration of G-CSF paralogs increased the number of circulating blood neutrophils of carp. Our findings demonstrate that gene duplications in teleosts can lead to functional divergence between paralogs and shed light on the sub-functionalization of G-CSF paralogs in cyprinid fish.

Thrombopoietin (TPO) induces thrombocytic colony formation of kidney cells synergistically with kit ligand A and a non-secretory TPO variant exists in common carp.

The development of mammalian megakaryocytes and platelets is regulated by numerous cytokine signals, primarily through the thrombopoietin (TPO)/c-MPL axis. Although non-mammalian vertebrates are known to possess nucleated thrombocytes functionally equivalent to mammalian platelets, the dynamics of the thrombocyte development remains unclear. Here we identified TPO and a splice variant (TPO-v) caused by the intron retention in common carp (Cyprinus carpio). Both the tpo and its variant transcripts were highly expressed in heart and liver. Recombinant carp TPO (rcTPO) was produced and purified in HEK293T cells stably expressing tpo, but TPO-v was shown not to be secreted from the transfectants. rcTPO induced the formation of colony-forming unit-thrombocyte (CFU-T) colonies which were recognized by a monoclonal antibody against carp thrombocytes expressing c-mpl and cd41, in a dose-dependent manner. The combination of rcTPO and recombinant carp Kit ligand A (rcKITLA) exerted a significant synergistic effect on three types of colony formation: thrombocytic colonies, thrombocytic burst colonies and thrombocytic/erythroid colonies. Utilizing this colony assay to examine the distribution of thrombocytic progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in thrombopoiesis, while the spleen does not. Our results indicate that carp possess mechanisms of TPO- and KITLA-dependent thrombopoiesis similar to those in other vertebrates and the sites of thrombopoiesis are restricted to the kidney, the primary hematopoietic organ in the teleost fish.