Hematology of equine fetuses with comparisons to their dams

The Hematologic values of 19 equine fetuses between 202 and 238 days gestation were compared with those of their dams. The red blood cell (RBC) count, hemoglobin concentration, hematocrit, and mean corpuscular hemoglobin concentration were significantly lower in fetal blood, while the mean corpuscular volume, mean corpuscular hemoglobin, and red cell distribution width were significantly higher. Mares had a significantly higher nucleated blood cell count than fetuses, and all nucleated cells were leukocytes (WBC). Most WBC in mare blood were segmented neutrophils and lymphocytes. In contrast, over one-half of the nucleated cells in fetal blood were nucleated RBC, and the majority of WBC in fetal blood were lymphocytes. Mares also had significantly higher plasma protein and fibrinogen concentrations than their fetuses. Mild macrocytosis and mild polychromasia were observed in most fetal blood samples, but not in blood samples from mares. All fetal blood contained reticulocytes, and most samples contained Heinz bodies and Howell-Jolly bodies. The results of this study will contribute to the development of hematologic reference values that may be useful in equine fetal research and, possibly, in the diagnosis of equine fetal disease.     

Cloning and pharmacological characterization of the equine adenosine A3 receptor

The aim of this study was to establish a heterologous expression system for the equine adenosine A(3) receptor (eA(3)-R) in an effort to ascertain its pharmacologic profile. Initially, radioligand binding assays identified clones expressing the eA(3)-R in human embryonic kidney cells (HEK) based on the specific binding of [(125)I]AB-MECA. Subsequently, adenylate cyclase assays were utilized to demonstrate functional coupling of the eA(3)-R to the G-protein/adenylate cyclase system. Equilibrium competition binding assays were then performed using selective and non-selective A(3) agonists and antagonists. Results from these experiments revealed a rank order of agonist potency to be IB-MECA > NECA > CGS21680, and an antagonist potency of MRS1220 > ZM241385 > 8-p-sulphophenyltheophylline; these rank orders were in agreement with that of other mammalian A(3)-R's. Lastly, NF-kappaB reporter gene assays revealed an IB-MECA concentration-dependent inhibition of TNFalpha-stimulated NF-kappaB activity. These results indicate that the heterologously expressed eA(3)-R is functional, has a pharmacological profile similar to that of other mammalian A(3) receptors, and its activation has an inhibitory effect on a key regulatory pathway in the inflammatory response. Thus, the eA(3)-R may serve as a pharmacological target in the treatment of equine inflammatory disease.     

Cloning and expression of equine insulin-like growth factor binding proteins in normal equine tendon

OBJECTIVES: To define a portion of the nucleotide sequences of each of the 6 insulin-like growth factor (IGF) binding proteins (IGFBPs) in horses and describe patterns of messenger RNA (mRNA) and protein expression for IGFBPs in normal equine tendons. ANIMALS: 7 horses. PROCEDURE: Total RNA was extracted from the tensile region of normal superficial digital flexor tendons and reverse transcribed into complimentary DNA (cDNA). The cDNA was amplified via PCR, and products representing portions of each IGFBP were cloned and sequenced. Nucleotide sequences were used to deduce the amino acid sequences, and both nucleotide and predicted amino acid sequences were compared with those published for bovine, human, mouse, and ovine IGFBPs. Gene expression was quantitated by real-time PCR assay, and protein expression was evaluated by western ligand blot (WLB). RESULTS: Clones ranged in size from 262 to 522 bp and had high degrees of sequence homology with other mammalian species. Sequence homology was highest between bovine and equine IGFBPs (86% to 95%) and amongst the IGFBP-5 sequences from the various species (92% to 95%). Message for IGFBP-2 to -6, but not IGFBP-1, was expressed in normal tendon. Protein expression for IGFBP-2, -3, and -4 was detected byWLB in normal tendon and markedly increased in damaged tendons. CONCLUSIONS AND CLINICAL RELEVANCE: Results provide basic information and tools needed for further characterization of the role of the IGF system in tendon healing and may lead to the ability to potentiate the response of healing tendon to exogenous IGF-I via concurrent manipulation of IGFBPs.     

Cloning and expression of the extra-cellular part of the alpha chain of the equine high-affinity IgE receptor and its use in the detection of IgE

The high-affinity receptor for IgE (FcepsilonRI) plays a central role in IgE-mediated allergic reactions. Cross-linking of FcepsilonRI by IgE-antigen complexes results in the activation of mast cells and basophils and is thought to contribute to the immunopathology of Heaves, a chronic obstructive pulmonary disease of horses. Recombinant protein corresponding to the extra-cellular portion of the FcepsilonRI alpha subunit, cloned and sequenced previously, was expressed using both mammalian cells and insect cells. The yield of expressed protein was considerably greater using insect cells and the baculovirus expression system. The recombinant proteins differed in size between the two systems, presumably due to differences in the extent of glycosylation. However, recombinant protein from both cell systems bound equine IgE present in bronchoalveolar lavage fluid from horses with Heaves. These results suggest that the recombinant extra-cellular part of FcepsilonRI should be a useful tool with which to study equine IgE responses.     

Evaluation of the erythroid regenerative response in two different models of experimentally induced iron deficiency anemia

Anemia was induced in weanling Sprague Dawley rats either by feeding an iron-deficient diet or by chronic phlebotomy. The erythroid regenerative response was then evaluated before and after a hemolytic event, and results were compared with those of a third group of control nonphlebotomized rats fed an iron-replete diet. Diet and phlebotomy groups developed a similar degree of anemia (mean hemoglobin concentration 7.9 g/dL and 7.8 g/dL, respectively; controls, 13.9 g/dL) and hypoferremia (mean serum iron concentration 25.4 microgram/dL and 34.9 microgram/dL, respectively; controls, 222.0 microgram/dL). However, the anemia in diet rats was nonregenerative (reticulocyte count, 83.1 X 10(3) cells/microliter) and associated with bone marrow erythroid hypoplasia; whereas the anemia in phlebotomy rats was regenerative (reticulocyte count, 169.6 X 10(3) cells/microliter) and associated with bone marrow erythroid hyperplasia. Thrombocytosis was seen in diet rats (1,580 X 10(3) cells/microliter) but not phlebotomy rats (901 X 10(3) cells/microliter) when compared with controls (809 X 10(3) cells/microliter). To further evaluate the regenerative capability, phenylhydrazine (PHZ) was administered to induce hemolysis. Erythrocyte mass declined approximately 25% in all groups, including controls. The reticulocytosis (265.3 X 10(3) cells/microliter) seen in phlebotomy rats was earlier and significantly greater than that seen in either diet or control rats. Hemoglobin concentration returned to pre-PHZ concentrations (7.9 g/dL) in phlebotomy rats within 4 days posthemolysis. In diet rats, the maximal regenerative response (176.3 X 10(3) cells/microliter) was not seen until 8 days posthemolysis, and hemoglobin (7.5 g/dL) did not return to pre-PHZ concentrations during the 8-day study. In many aspects, the anemia seen following diet- or phlebotomy-induced iron deficiency was similar. However, the erythroid regenerative capability varied depending on the mechanism by which anemia was induced and furthermore altered the efficiency of hemoglobin production following a hemolytic event. These results suggest that the availability of iron in the diet may modulate the pathogenesis of iron deficiency anemia.     

Cloning and pharmacological characterization of the equine adenosine A2A receptor: a potential therapeutic target for the treatment of equine endotoxemia

The aim of the current study was to clone the equine adenosine A(2A) receptor gene and to establish a heterologous expression system to ascertain its pharmacologic profile via radioligand binding and functional assays. An eA(2A)-R expression construct was generated by ligation of the eA(2A) cDNA into the pcDNA3.1 expression vector, and stably transfected into human embryonic kidney cells (HEK). Binding assays identified those clones expressing the eA(2A)-R, and equilibrium saturation isotherm experiments were utilized to determine dissociation constants (K(D)), and receptor densities (B(max)) of selected clones. Equilibrium competition binding revealed a rank order of agonist potency of ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of antagonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenyltheophylline > caffeine. Furthermore, adenylate cyclase assays using selective A(2A)-R agonists revealed that the eA(2A)-R functionally coupled to Galpha(s) as indicated by an increase in intracellular [(3)H]cAMP upon receptor activation. Finally, NF-kappaB reporter gene assays revealed a CGS21680 concentration-dependent inhibition of NF-kappaB activity. These results indicate that the heterologously expressed eA(2A)-R has a pharmacological profile similar to that of other mammalian A(2A) receptors and thus can be utilized for further characterization of the eA(2A)-R to ascertain whether it can serve as a suitable pharmacological target for equine inflammatory disease.     

重组截短型ELR1蛋白在昆虫表达系统中的分泌表达与结晶

为获得马传染性贫血病毒单一受体(ELR1)的结晶体,本研究通过PCR方法获得ELR1胞外区基因片段,利用基因重组技术构建包含ELR1胞外区基因的重组杆状病毒表达质粒(Bacmid-ELR1).将其转染至昆虫细胞sf9中进行蛋白表达,western blot检测结果显示ELR1胞外区截短型重组蛋白在昆虫表达系统中获得表达.目的蛋白经亲和层析和凝胶过滤方法纯后化,通过多组结晶条件筛选获得了ELR1胞外区重组蛋白结晶体.本研究为ELR1立体结构和功能的解析提供了重要基础.     

Cloning and expression of recombinant equine interleukin-3 and its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes

Interleukin-3 is a growth and differentiation factor for various hematopoietic cells. IL-3 also enhances stimulus-dependent release of mediators and cytokine production by mature basophils. Function of IL-3 has not been studied in horses because of lack of horse-specific reagents. Our aim was to produce recombinant equine IL-3 and test its effect on sulfidoleukotriene and cytokine production by equine peripheral blood leukocytes (PBL).Equine IL-3 was cloned, expressed in E. coli and purified. PBL of 19 healthy and 20 insect bite hypersensitivity (IBH)-affected horses were stimulated with Culicoides nubeculosus extract with or without IL-3. Sulfidoleukotriene (sLT) production was measured in supernatants by ELISA and mRNA expression of IL-4, IL-13 and thymic stromal lymphopoietin (TSLP) assessed in cell lysate by quantitative real-time PCR.Recombinant equine IL-3 (req-IL-3) had a dose dependent effect on sLT production by stimulated equine PBL and significantly increased IL-4, IL-13 and TSLP expression compared to non-primed cells.IL-3 priming significantly increased Culicoides-induced sLT production in IBH-affected but not in non-affected horses and was particularly effective in young IBH-affected horses (≤3 years).A functionally active recombinant equine IL-3 has been produced which will be useful for future immunological studies in horses. It will also allow improving the sensitivity of cellular in vitro tests for allergy diagnosis in horses.     

Cloning,expression and biological activity of equine interleukin (IL)-5

The cytokine, interleukin (IL)-5 stimulates eosinophil differentiation, activation and survival and can prime these cells, increasing the response to other mediators. In view of its many effects on eosinophils, IL-5 has been implicated in the pathogenesis of allergic disease in man. Here we report the cloning of equine IL-5 and expression of the recombinant protein by transfection of Chinese hamster ovary (CHO) cells. The cloned cDNA sequence consisted of 405 nucleotides and encoded a protein of 135 amino acids. There is >85% identity with feline, bovine, ovine, canine, and human IL-5 sequences at the nucleotide and protein level. Supernatants containing equine IL-5 were also examined for biological activity. CHO supernatant containing equine recombinant (eqr) IL-5, like the human ortholog (hrIL-5), induced concentration dependent equine eosinophil adherence to autologous serum-coated plastic (9.7+/-1.5% with a 1:100 dilution of eqrIL-5 and 9.1+/-1.6% adherence with 1 nM hrIL-5; n = 4). The eqr protein also caused concentration dependent superoxide production (11.9+/-2.4 nmol (reduced cytochrome (cyt) C)/10(6) cells at a 1:50 dilution, n = 4). In contrast, hrIL-5 only caused significant superoxide production when diluted in conditioned CHO medium, an effect that was inhibited by the anti-human mAb, TRFK5 (4.4+/-0.3 versus 0.3+/-0.4 nmol/10(6) cells for 0.5 nM hrIL-5 in the presence of the isotype matched IgG1 control (10 microM) and TRFK5 (10 microM), respectively). TRFK5 also significantly inhibited hrIL-5 induced adherence at concentrations of 0.3 microg/ml and above but had no significant inhibitory effect on either superoxide or adherence caused by eqrIL-5. These results demonstrate that equine IL-5 expressed by CHO cells stimulates equine eosinophils, suggesting that this cytokine could play a role in eosinophil recruitment and activation in equine allergic disease. The anti-human and murine moAb TRFK5 does not appear to recognise the equine protein.     

Babesia gibsoni infection in a dog from indiana

Abstract: A 10-year-old spayed female mixed-breed dog was presented to the Purdue University Veterinary Teaching Hospital (PUVTH) with complaints of persistent anemia with occasional exacerbations, anorexia, and lethargy. The dog had been presented to the referring veterinarian 2 months prior with multiple bite wounds received during a fight with 3 Pit Bull Terriers. The dog was discharged after the wounds were cleaned and surgically closed. Upon admission to the PUVTH, blood was collected for a complete blood count and biochemical analysis. Microscopic examination of peripheral blood smears revealed intraerythrocytic protozoal parasites consistent with Babesia gibsoni . Molecular analysis confirmed that the organism was B gibsoni and that its 18S ribosomal RNA sequence was identical to that of other B gibsoni isolates from Oklahoma, North Carolina, and Okinawa, Japan. Hematologic changes included moderately severe, regenerative, macrocytic, normochromic anemia, with poikilocytosis, polychromasia, anisocytosis, and a marked increase in nucleated RBCs. Biochemical changes included increased serum alanine aminotransferase, alkaline phosphatase, and gamma glutamyl-transferase activities. The dog was treated with imidocarb, but despite initial clinical improvement, the dog died 2 weeks after the first dose. A necropsy was not performed. The infection in this dog is the first reported case of B gibsoni infection in Indiana. Because of the widespread geographical distribution of the organism, veterinarians and veterinary clinical pathologists throughout the United States should carefully examine Romanowsky-stained blood smears from patients with acute hemolytic anemia for small intraerythrocytic babesial parasites.     

猪转化生长因子β受体Ⅱ基因克隆与组织表达特征分析

为了研究猪转化生长因子β受体Ⅱ(transforming growth factor beta receptorⅡ,TGFBR2)基因序列特征和组织表达特征,利用克隆测序技术获得二花脸猪TGFBR2基因编码区序列,采用RT-PCR技术分析二花脸猪TGFBR2基因的组织表达特征。结果显示:二花脸猪TGFBR2基因编码区序列长度为1 461 bp,编码蛋白含486个氨基酸残基,含有ec TbetaR2结构域、蛋白激酶结构域等典型的功能域;RT-PCR发现TGFBR2基因在二花脸猪下丘脑、子宫、卵巢和肌肉等12种组织中广泛表达,特别是在卵巢组织中高表达。结果表明:猪TGFBR2基因为进化上相对保守且在卵巢组织中高表达的基因。     

Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period

To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0 = day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was found in the extracts of day 25 gravid endometrium and was immunochemically localized to the glandular epithelium/luminal cavity of the pregnant uterus. High concentrations of estradiol-17β (E₂) were detected in day 25 conceptuses. Concentrations of E₂ were higher in the gravid endometrial portion than in other endometrial regions. On the other hand, progesterone concentrations did not differ among endometrial samples analyzed. Furthermore, the expression of IL-1RN mRNA was up-regulated in endometrium culture samples treated with 10 ng/mL E₂ and 10 ng/mL progesterone. In the analysis of related gene expression, increased amounts of IL-1α and IL-6 mRNA were also found in the day 25 gravid endometrium; however, these expressions in endometrial culture samples were not up-regulated by the steroid treatment. These results indicate that expression of IL-1RN in the endometrium is likely regulated by E₂ and progesterone and suggest that IL-1RN regulates the degree of IL-1 signal transduction and thereby plays an important role in the establishment of equine pregnancy.     

Cloning,expression, and tissue distribution of bovine interleukin-21

Bovine interleukin-21 (IL-21) cDNA was cloned and sequenced from bovine peripheral blood lymphocytes (PBLs) stimulated with 10 microg/ml concanavalin A (ConA), 10 microg/ml phytohemagglutinin (PHA), and 50 ng/ml phorbol 12-myristate 13-acetate (PMA) for 48 h. The open reading frame of the bovine IL-21 cDNA is 459 bp in length and encodes 152 amino acids. The predicted amino acid sequence is 78.2 and 58.5% homologous to the human and murine IL-21 amino acid sequences, respectively. Recombinant bovine IL-21 was expressed by a baculovirus expression system. The bovine IL-21 was processed to the mature form in insect cells and secreted to the supernatant confirmed by N-terminal amino acid sequencing. The recombinant bovine mature IL-21 induced the proliferation of human IL-2-dependent cells, ILT-MAT. The mRNA expression for bovine IL-21 was observed in the spleen, but not in the brain, heart, lung, liver, and kidney. The bovine IL-21 identified in this study may provide new methods for the enhancement of innate immunity in cows.     

Cloning and functional characterization of recombinant equine P-selectin

The recent molecular characterization and sequencing of equine P-selectin (ePsel), and its glycoprotein ligand, P-selectin glycoprotein ligand-1 (PSGL-1), have provided the tools for further investigation into their role in leukocyte trafficking. Here, we report the generation of a genetically engineered chimeric protein (ePsel-IgG) in which the equine P-selectin lectin and epithelial growth factor (EGF) domains were covalently linked to the equine IgG1 heavy chain constant region. The soluble ePsel-IgG was observed to bind to equine monocytes by confocal microscopy and flow cytometry. Furthermore, equine monocytes bound to immobilized ePsel-IgG in a time course and dose dependent manner. Not only did ePsel-IgG act as an adhesion molecule, it was also found to activate ERK1/2 kinase and induce IL-8 mRNA expression in equine monocytes. That all of the aforementioned ePsel-IgG-induced cell binding and cell signaling were abolished by the addition of EDTA, suggested that ePsel-IgG chimera mediated events occurred via the P-selectin ligand, PSGL-1. We were able to demonstrate that 78% of equine monocytes cross-reacted with anti-human HECA-452 antibody, which recognizes the sialy-Lewis X (sLex) epitope, a well-known carbohydrate binding site on human PSGL-1. Pre-incubation of equine PBMC with neuraminidase or O-sialoglycoprotein endopeptidase (OSGP) reduced ePsel-IgG monocyte binding to 36% or 60%, respectively. Taken together, these data suggest that there might be two ligand recognition sites on P-selectin, one of which recognizes sLex and another which recognizes P-selectin ligand core protein. The ePsel-IgG chimera can be a useful as a reagent for further studies on the role of equine P-selectin and signal transduction in inflammatory events in horse.     

Hematologic evaluation of normal and anemic lambs with the Technicon H*1 using EDTA or heparin as anticoagulants

Abstract: A study was performed to evaluate blood from young lambs using the Technicon H*1 hematology analyzer, with emphasis on RBC parameters, comparison of tripotassium EDTA and heparin, and the effects of storage on heparinized blood. Blood samples from lambs 2 days to 18 weeks of age were analyzed within 6 hours, revealing a high precision, except for WBC counts in heparinized blood. The HCT values estimated by the H*1 correlated well (r²= .90) with those obtained by the microhematocrit method. Mean hematologic values obtained for heparinized blood differed by up to 4% from values obtained for blood collected into EDTA. WBC counts decreased 8.4% in heparinized blood stored at 4°C for 1 day, but differences observed in RBC counts were ≤ 2%. Problems occurred when analyzing blood from young lambs with low hemoglobin values because the H*1 incorrectly counted microcytes with volumes of < 10 fL as platelets. When the necessary corrections were performed, the H*1 was useful for analyzing RBC parameters in lamb blood collected both into EDTA and into heparin.     

奶山羊CIDEa基因的克隆、序列分析与组织表达

为获得奶山羊CIDEa基因序列并检测其在乳腺组织中的表达,采用RT-PCR技术从奶山羊乳腺组织扩增CIDEa基因序列,利用生物信息学软件对其进行预测分析,并采用荧光定量PCR检测CIDEa在干奶期和不同泌乳时期乳腺组织中的表达。结果表明:通过克隆测序得到奶山羊CIDEa基因CDS区660 bp,编码219个氨基酸,与GenBank公布的绵羊、牛、人和猪的核苷酸序列同源性分别为99%、96%、85%、85%。奶山羊CIDEa蛋白属于碱性、不稳定亲水蛋白,不存在跨膜结构和信号肽;蛋白主要定位在细胞质、线粒体和细胞核。CIDEa蛋白结构主要由α螺旋、β折叠和不规则卷曲组成。奶山羊CIDEa在泌乳前期、盛期和中期乳腺组织中表达量均显著高于干奶期(P<0.05)。说明CIDEa可能参与奶山羊乳腺的泌乳过程,为进一步研究其在奶山羊乳腺组织中的功能及对乳品质的调控作用奠定基础。