Dog leukocyte antigen genotyping across class I and class II genes in beagle dogs as laboratory animals.

Dog leukocyte antigen (DLA) polymorphisms have been found to be associated with inter-individual variations in the risk, susceptibility, and severity of immune-related phenomena. While DLA class II genes have been extensively studied, less research has been performed on the polymorphisms of DLA class I genes, especially in beagle dogs commonly used as laboratory animals for safety evaluations in drug development. We genotyped four DLA class I genes and four DLA class II genes by locus-specific Sanger sequencing using 93 laboratory beagle dogs derived from two different strains: TOYO and Marshall. The results showed that, for DLA class I genes, 11, 4, 1, and 2 alleles, including a novel allele, were detected in DLA-88, DLA-12/88L, DLA-64, and DLA-79, while, for DLA class II genes, 1, 10, 6, and 7 alleles were detected in DLA-DRA, DLA-DRB1, DLA-DQA1, and DLA-DQB1, respectively. It was estimated that there were 14 DLA haplotypes, six of which had a frequency of ≥ 5%. Furthermore, when comparing the DLA diversity between TOYO and Marshall strains, the most common alleles and haplotypes differed between them. This is the first study to genotype all DLA loci and determine DLA haplotypes including all DLA class I and class II genes in dogs. Integrating information on the DLA diversity of laboratory beagle dogs should reinforce their benefit as an animal model for understanding various diseases associated with a specific DLA type.

Subgenomic T cell receptor alpha and delta (TRA/TRD) loci in common carp.

In jawed vertebrates, the T cell receptor alpha (TRA) and delta (TRD) genes, which encode the TRα and TRδ chains, respectively, are located as a nested structure on a single chromosome. To date, no animal has been reported to harbor multiple TRA/TRD loci on different chromosomes. Therefore, herein, we describe the first full annotation of the TRA/TRD genomic regions of common carp, an allo-tetraploid fish species that experiences cyprinid-specific whole-genome duplication (WGD) in evolution. Fine genomic maps of TRA/TRD genomic regions 1 and 2, on LG30 and LG22, respectively, were constructed using the annotations of complete sets of TRA and TRD genes, including TRA/TRD variable (V), TRA junction (J), and constant (C), TRD diversity (D), and the J and C genes. The structure and synteny of the TRA/TRD genomic regions were highly conserved in zebrafish, indicating that these regions are on individual chromosomes. Furthermore, analysis of the variable regions of the TRA and TRD genes in a monoclonal T cell line revealed that both subgenomic regions 1 and 2 were indeed rearranged. Although carp TRAV and TRDV genes were phylogenetically divided into different lineages, they were mixed and organized into the TRA/TRD V gene clusters on the genome, similar to that in other vertebrates. Notably, 285 potential TRA/TRD V genes were detected in the TRA/TRD genomic regions, which is the most abundant number of genes in vertebrates and approximately two-fold that in zebrafish. The recombination signal sequences (RSSs) at the end of each V gene differed between TRAV and TRDV, suggesting that RSS variations might separate each V gene into a TRα or TRδ chain. This study is the first to describe subgenomic TRA/TRD loci in animals. Our findings provide fundamental insights to elucidate the impact of WGD on the evolution of immune repertoire.

Role of DLA-DRB1 amino acids outside the shared epitope in dachshund susceptibility to immune-mediated polyarthritis.

Canine immune-mediated polyarthritis (IMPA) is an idiopathic disorder encompassing both erosive and non-erosive forms of rheumatoid arthritis (RA), with a clinical picture similar to human RA. Resemblance in major histocompatibility complex (MHC)-associated risk between the two was first noted within the specific amino acid motif known as the shared epitope (SE) on human leukocyte antigen DRB1. Following further identification of amino acids conferring risk for human RA outside the SE, this study was designed to examine amino acids both within and outside the classic SE in dachshunds, a breed with reported susceptibility to IMPA in Japan. Genome-wide association studies have linked positions 11, 13 and 71 with strong risk for human RA and important roles in antigen presentation to T cells. Sequence based genotyping of 16 case and 64 control dachshunds revealed strong associations comparable to human RA between IMPA risk and valine at position 11 (Val-11), phenylalanine at 13 (Phe-13), and arginine at 71 (Arg-71) on the dog leukocyte antigen (DLA)-DRB1 molecule (OR 2.89, 95%CI 1.3-6.4, p = 0.009), while association with the classic SE was significant only regarding homozygote frequency of the QRRAA haplotype-also carrying Val 11 and Phe 13 outside the SE (p = 0.04). Moreover, limited range in possible combinations of amino acids at positions 11, 13 and 71 starting with Val-11 among all DLA-DRB1 alleles registered with the GenBank and IPD-MHC canine databases, suggested potential of further single-breed analyses in dachshunds to clarify the disorder in terms of diagnosis, treatment, and epigenetic control, while clinical and immunopathogenetic similarities between human and dachshund RA also suggested the possibility of gaining insight into RA per se through study of canine IMPA as a spontaneous model of human RA.

Phenotypic and molecular characterization of novel pulmonary adenocarcinoma cell lines established from a dog.

Canine pulmonary adenocarcinoma (PAC) resembles human lung tumors in never-smokers, but it is rarer than human pulmonary adenocarcinoma. Therefore, research on canine PAC is challenging. In the present study, we successfully established various novel canine PAC cell lines from a single lesion in a dog, including two parent cell lines and fourteen cloned cell lines, and characterized their cellular properties in vitro. Several of these cell lines showed epithelial-mesenchymal transition (EMT)-like and/or cancer stem cell (CSCs)-like phenotypes. We additionally assessed the sensitivity of the cells to vinorelbine in vitro. Three clonal lines, two of which showed EMT- and CSC-like phenotypes, were resistant to vinorelbine. Furthermore, we evaluated the expression and activation status of EGFR, HER2, and Ras signaling factors. The findings indicated that the cell lines we established preserved the expression and activation of these factors to varying extents. These novel canine PAC cell lines can be utilized in future research for understanding the pathogenesis and development of treatments for canine PAC.

Treatment with Ligilactobacillus murinus lowers blood pressure and intestinal permeability in spontaneously hypertensive rats.

One feature of hypertension is a microbial imbalance with increased intestinal permeability. In this study, we examined whether an alteration in the microbiota affects blood pressure and intestinal permeability in spontaneously hypertensive rats (SHRs). We performed a 16S metagenome analysis of feces from 10- to 15-week-old SHRs using a synthetic long-read sequencing approach, and found a candidate for the microbiome treatment, Ligilactobacillus murinus (L. murinus), that was robustly decreased. Oral administration of L. murinus to SHRs for 2 weeks significantly inhibited blood pressure elevation and improved endothelium-dependent vasodilation but did not attenuate enhanced vascular contraction in SHR mesenteric arteries. The proximal colon of SHRs exhibited increased intestinal permeability with decreased levels of the tight junction protein claudin 4, morphological changes such as decreased intestinal crypts and elevated TNF-α levels, which was reversed by treatment with L. murinus. Consistent with these intestinal phenotypes, plasma lipopolysaccharides levels were elevated in SHR but decreased following L. murinus administration. We concluded that oral administration of L. murinus to SHRs exerts protective effects on intestinal permeability via restoration of claudin 4 expression and reversal of morphologic disorder, which may improve low-grade endotoxemia and thus reduce development of hypertension via recovery of endothelial vasodilating functions.

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.

Expression profile of immunoregulatory factors in canine tumors.

Cancers utilize a variety of molecules to escape host immune responses. Better understanding the immune environment surrounding cancer may facilitate application of innovative cancer immunotherapies, such as immune checkpoint inhibitors, to dogs as well as humans. In this study, we screened the expression of 20 immune regulatory molecules in diverse canine tumors (n = 59). Quantitative RT-PCR (qPCR) analysis revealed that some immune regulatory molecules, such as LGALS9 (coding Galectin-9) and CD48, were expressed in most canine tumors, but other molecules, such as CD274 (coding PD-L1), IL4I1, PVR, TNFSF18, ICOSLG, and TNFSF4, were rarely expressed. NECTIN2 was highly expressed in epithelial tumors but was low in non-epithelial tumors. In contrast, VSIR and CD200 expressions were low in epithelial tumors but high in non-epithelial tumors. Interestingly, several tumors expressed distinctive immunoregulatory factors. Hepatocellular carcinomas expressed FGL1, mast cell tumors expressed PDCD1LG2 (coding PD-L2), transitional cell carcinomas expressed VTCN1 (coding B7x), and lymphomas and squamous cell carcinomas expressed CD70. Consistent with qPCR results, immunofluorescence staining confirmed that hepatocellular carcinomas expressed FGL-1 protein. Thus, this study reveals the expression profile of immunoregulatory molecules in canine tumors and opens the door to better understanding the relationship between canine tumors and host immunity.

Haplotype structures and polymorphisms of dog leukocyte antigen (DLA) class I loci shaped by intralocus and interlocus recombination events.

The dog leukocyte antigen (DLA) class I genomic region is located on chromosome 12, and the class I genomic region is composed of at least two distinct haplotypic gene structures, DLA-88-DLA-12 and DLA-88-DLA-88L. However, detailed information of the genomic differences among DLA-88, DLA-12, and DLA-88L are still lacking at the full-length gene level, and therefore, DLA allelic sequences classified for each of these loci are limited in number so far. In this study, we determined the DNA sequence of a 95-kb DLA class I genomic region including DLA-88, DLA-12/88L, and DLA-64 with three DLA homozygous dogs and of 37 full-length allelic gene sequences for DLA-88 and DLA-12/88L loci in 26 DLA class I homozygous dogs. Nucleotide diversity profiles of the 95-kb regions and sequence identity scores of the allelic sequences suggested that DLA-88L is a hybrid gene generated by interlocus and/or intralocus gene conversion between DLA-88 and DLA-12. The putative minimum conversion tract was estimated to be at least an 850-bp segment in length located from the 5´flanking untranslated region to the end of intron 2. In addition, at least one DLA-12 allele (DLA-12*004:01) was newly generated by interlocus gene conversion. In conclusion, the analysis for the occurrence of gene conversion within the dog DLA class I region revealed intralocus gene conversion tracts in 17 of 27 DLA-88 alleles and two of 10 DLA-12 alleles, suggesting that intralocus gene conversion has played an important role in expanding DLA allelic variations.

Dog leukocyte antigen (DLA) class II genotypes associated with chronic enteropathy in French bulldogs and miniature dachshunds.

Canine chronic enteropathy (CE) is a group of immunogenetic disorders of unclear etiology characterized by chronic or recurrent gastrointestinal signs and inflammation. Diagnosis of CE subtypes by treatment response is a lengthy and challenging process, particularly in refractory cases of the disease. Given known association of dog leukocyte antigen (DLA) class II genotype and various immunogenetic disorders between and across breeds, this study was designed to examine the potential of determining susceptibility to refractory CE through identification of risk and protective genotypes in French bulldogs and miniature dachshunds-two popular dog breeds in Japan. Sequence-based genotyping of three DLA class II genes in 29 French bulldogs and 30 miniature dachshunds with refractory CE revealed a protective haplotype DLA-DRB1*002:01-DQA1*009:01-DQB1*001:01 against CE in French bulldogs (OR 0.09, 95 % CI 0.01-0.71, p = 0.0084). No statistical difference was noted between miniature dachshund cases and controls. These findings, largely disparate from a previous study on German shepherd dogs in the UK, were taken as possible indication of etiological differences in the refractory CE noted between and within breeds, and by extension, the potential of identifying such disease heterogeneity by DLA typing. The DLA-DQA1/DQB1 haplotype, protective against CE in our French bulldogs, has been reported as protective in various immune-mediated disorders such as Doberman hepatitis (Dyggve et al., 2011). Likewise, the DLA-DRB1*006:01 risk allele for Doberman hepatitis was noted in more French bulldogs with CE compared to controls, in line with reports on genotypes associated with both risk and protection being shared across various autoimmune diseases and breeds. These findings support an immunogenetic basis to the French bulldog-CE in our analysis, calling for further DLA studies working with larger samples and different breeds towards phenotypic clarification that may aid in early diagnosis, treatment, and prophylaxis through epigenetic approaches and breeding.

Identification of Novel Alleles and Structural Haplotypes of Major Histocompatibility Complex Class I and DRB Genes in Domestic Cat (Felis catus) by a Newly Developed NGS-Based Genotyping Method.

The major histocompatibility complex (MHC) is a highly polymorphic and duplicated genomic region that encodes transplantation and immune regulatory molecules. Although it is well-known that particular MHC allelic polymorphisms and haplotypes are genetically relate to immune-mediated diseases detailed information of the cat MHC (Feline Leukocyte Antigen; FLA) genetic and haplotypic structure and diversity is limited in comparison to humans and many other species. In this study, to better understand the degree and types of allele and allelic haplotype diversity of FLA-class I (FLA-I) and FLA-DRB loci in domestic cats, we identified six expressible FLA-I loci in peripheral white blood cells by in silico estimation of the coding exons and NGS-based amplicon sequencing using five unrelated cats. We then used a newly developed NGS-based genotyping method to genotype and annotate 32 FLA-I and 16 FLA-DRB sequences in two families of 20 domestic cats. A total of 14 FLA-I and seven FLA-DRB were identified as novel polymorphic sequences. Phylogenetic analyses grouped the sequences into six FLA-I (FLA-E/H/K, FLA-A, FLA-J, FLA-L, FLA-O and a tentatively named FLA-E/H/K_Rec) and four FLA-DRB (FLA-DRB1, FLA-DRB3, FLA-DRB4, and FLA-DRB5) lineages. Pedigree analysis of two cat families revealed eight distinct FLA structural haplotypes (Class I - DRB) with five to eight FLA-I and two to three FLA-DRB transcribed loci per haplotype. It is evident that the eight FLA haplotypes were generated by gene duplications and deletions, and rearrangements by genetic recombination with the accumulation and/or inheritance of novel polymorphisms. These findings are useful for further genetic diversity analysis and disease association studies among cat breeds and in veterinary medicine.

The utility of DLA typing for transplantation medicine in canine models.

Transplantation medicine is used for the treatment of severe canine diseases, and the dog leukocyte antigen (DLA) is considered to be important in graft rejection. However, the utility of direct sequencing of both DLA classes I and II has not been assessed thoroughly. Eight healthy beagles with identified DLA genes were divided into two sets of four dogs, each including one donor and three recipients for skin transplantation. The following recipients were selected: one dog with a complete match, one with a haploidentical match, and one with a complete mismatch of the DLA gene with the donor. Full-thickness skin segments were obtained from each donor and transplanted to the recipients. A mixed lymphocyte reaction (MLR) assay was performed and analyzed by flow cytometry. Skin grafts of DLA haploidentical and mismatched pairs were grossly rejected within 14 days, whereas in fully matched DLA pairs, survival was as long as 21 days. Histopathological evaluation also showed moderate to severe lymphocytic infiltration and necrosis in DLA mismatched pairs. As seen in the MLR assay, the stimulation index of DLA mismatched pairs was significantly higher than that of fully matched DLA pairs in both sets (P<0.001). The allogeneic transplantation results suggested that it is possible to prolong transplant engraftment by completely matching the DLA genotype between the donor and recipient. Additionally, the MLR assay may be used as a simplified in vitro method to select donors.

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.

Identification of novel polymorphisms and two distinct haplotype structures in dog leukocyte antigen class I genes: DLA-88, DLA-12 and DLA-64.

The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88-DLA-12 and DLA-88-DLA-88L, were identified, and the novel haplotype DLA-88-DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

Exploring erythropoiesis of common carp (Cyprinus carpio) using an in vitro colony assay in the presence of recombinant carp kit ligand A and erythropoietin.

The use of in vitro colony assays in mammals has contributed to identification of erythroid progenitor cells such as burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E) progenitors, and serves to examine functions of erythropoietic growth factors like Erythropoietin (Epo) and Kit ligand. Here, we established an in vitro colony-forming assay capable of investigating erythropoiesis in carp (Cyprinus carpio), cloned and functionally characterized recombinant homologous molecules Epo and Kit ligand A (Kitla), and identified three distinct erythroid progenitor cells in carp. Recombinant carp Epo induced the formation of CFU-E-like and BFU-E-like erythroid colonies, expressing erythroid marker genes, ß-globin, epor and gata1. Recombinant carp Kitla alone induced limited colony formation, whereas a combination of Kitla and Epo dramatically enhanced erythroid colony formation and colony cell growth, as well as stimulated the formation of thrombocytic/erythroid colonies expressing not only erythroid markers but also thrombocytic markers, cd41 and c-mpl. Utilizing this colony assay to examine the distribution of distinct erythroid progenitor cells in carp, we demonstrated that carp head and trunk kidney play a primary role in erythropoiesis, while the spleen plays a secondary. Furthermore, we showed that presumably bi-potent thrombocytic/erythroid progenitor cells localize principally in the trunk kidney. Our results indicate that teleost fish possess mechanisms of Epo- and Kitla-dependent erythropoiesis similar to those in other vertebrates, and also help to demonstrate the diversity of erythropoietic sites among vertebrates.