A protocol to guide development of a sensitive ELISA for canine erythropoietin

Background: The determination of canine erythropoietin (EPO) concentration is crucial for monitoring the effect of human recombinant (hr) EPO therapy in dogs with chronic renal failure. Current assays are not specific for canine EPO and not sensitive enough to detect physiologic EPO levels in dogs.
Objective: The objective of this study was to develop a simple and sensitive ELISA for canine EPO that could serve as a starting point for developing a commercially available assay.
Methods: The ELISA was based on a mouse monoclonal antibody (mAb) and a rabbit polyclonal antibody (pAb) using 2 different immunization techniques: gene electrotransfer (GET) to generate the pAb and multiple antigen peptides (MAPs) to generate the mAb. The ELISA was performed using both EPO obtained from HeLa cells transfected with an expression plasmid encoding canine EPO and canine plasma with known concentrations of EPO.
Results: The ELISA standard curve was linear for canine EPO concentrations of 7–66 mU/ml. Coefficients of variation were about 10%. No cross-reactivity between canine EPO and hrEPO was detected.
Conclusions: Using novel GET and MAP technology, we developed a sensitive and specific ELISA for canine EPO that can be used to guide future clinical applications for EPO detection and monitoring in dogs.     

Erythropoietin-producing cells in the liver of ICR-derived glomerulonephritis (ICGN) mice

The ICR-derived glomerulonephritis (ICGN) mouse is an appropriate model for anemia associated with chronic renal disorder (CRD). Insufficient renal production of erythropoietin (EPO) induces the anemia associated with CRD. EPO mRNA is expressed in both kidneys and liver of progressing-stage ICGN mice. Hypoxic stimulation induced the EPO mRNA expression in the liver as well as in the kidneys of ICGN mice. The localization of EPO-producing cells in the liver remains controversial. Present study using an amplified in situ hybridization technique identified that nonparenchymal cells were the source of hepatic EPO production in ICGN mice under both normoxia and hypoxia.     

Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN) mice

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.     

Ontogeny of erythropoietin receptor mRNA expression in various tissues of the foetal and the neonatal pig

Erythropoietin receptor (EPOR) mRNA expression in liver, spleen, bone marrow and testes of foetal and neonatal pigs was analysed using a real-time RT-PCR assay. The results showed that early in the foetal life, EPOR expression is greatest in the liver. Later in foetal life, the spleen has the greatest expression of EPOR, whereas at 2 weeks after birth, the main expression of EPOR is found in the bone marrow. These findings contradict our earlier hypothesis that erythropoietin (EPO) acting in a paracrine fashion can account for an extensive erythropoiesis at birth, a point of time when plasma EPO concentrations are low. Results presented in the present paper suggest that the spleen or, alternatively, the bone marrow is able to respond to very low concentrations of circulating EPO around the time of birth. The testes were found to express significant amounts of EPOR. Since EPO mRNA has previously been found in the testes, a paracrine function of EPO may exist in this organ.     

藏猪促红细胞生成素的表达与低氧适应分析

为了解藏猪肾脏中促红细胞生成素（erythropoietin,EPO）的表达与其低氧适应之间的关系,采用Western blotting技术检测西藏林芝地区（海拔约3000 m）的高海拔藏猪（ZZ）和高海拔大约克夏猪（YZ）、北京中顺景盛养殖场（海拔约100 m）的低海拔藏猪（ZB）和安徽合肥市（海拔约为36 m）的低海拔大约克夏猪（YH）肾脏组织EPO蛋白的表达量。结果发现,ZZ、ZB、YZ、YH的EPO表达量,在A组试验中,高海拔藏猪与高海拔大约克夏猪差异显著（P<0.05）,高海拔大约克夏猪与低海拔藏猪、低海拔大约克夏猪差异极显著（P<0.01）;B组试验中,高海拔藏猪与低海拔藏猪差异不显著（P>0.05）,高海拔藏猪与高、低海拔大约克夏猪差异显著（P<0.05）;C组试验中,高、低海拔藏猪间差异显著（P<0.05）,低海拔藏猪与高、低海拔大约克夏猪差异极显著（P<0.01）。当大约克夏猪处在高海拔应急状态下时,EPO的表达量高于藏猪。而长期生活在高原的大约克夏猪EPO的表达量较藏猪低,但高于低海拔大约克夏猪。EPO的稳定表达是藏猪适应高原低氧环境的一个重要因素,而高原大约克夏猪肾脏组织内EPO的高表达,则是大约克夏猪适应高原低氧环境的生理调节过程。     

Erythropoietin receptor expression in canine mammary tumor: an immunohistochemical study

Erythropoietin (EPO) is a cytokine primarily involved in the regulation of the erythropoiesis. Recently, it has been demonstrated that EPO and its receptor (EPOR) are expressed in several neoplastic cell lines and solid tumors. Furthermore, in vitro and in vivo studies have shown that EPO could promote human breast carcinoma growth by means of the binding with its receptor, although a clear function for EPO in this setting has not been yet established. While the human medical literature has been accumulating strong evidence on EPO's role in oncogenesis, to date, there are no veterinary reports focusing on such an issue. The aim of the present study was to investigate the immunohistochemical expression of EPOR in canine mammary gland dysplastic and neoplastic lesions. Our results show a weak to moderate EPOR expression in dysplastic glands, being immunoreactivity enhanced as the lesion shows an increasing malignant pattern. On the basis of these findings, this study describes, for the first time, the evidence for EPOR expression in canine mammary gland tumor and suggests a feasible EPO's role for canine mammary tumor progression.     

Mast cells in canine mammary gland tumour: number, distribution and EPOR positivity

Erythropoietin (EPO)-mediated mitogenic and anti-apoptotic effects involve all the cells expressing functional receptors for EPO (EPOR), as demonstrated by in vitro and in vivo studies. EPO shows pleiotropic effects and acts as an endogenous mediator of adaptive tissue response to metabolic stress protecting tissues from different injuries. Recently, the EPO/EPOR complex has been identified in several neoplastic cell lines and solid tumours. In this study, the authors investigated the mast cells (MCs) number, distribution and their immunoreactivity for EPOR in normal, dysplastic and neoplastic canine mammary gland. The results showed that MCs were more numerous in displastic glands compared with normal and neoplastic glands. As far as the EPOR immunoreactivity is concerned, we did not observe MCs reaction on cancer, in contrast with previously published data where epithelium of neoplastic gland showed an increase in EPOR expression along with the neoplastic progression. Overall, our results might be suggestive for MCs role in oncogenesis and offer new insight regarding to the expression of EPOR in mammary gland cancer in dog.     

Resolution of renal adenocarcinoma-induced secondary inappropriate polycythaemia after nephrectomy in two cats

Two cases of secondary, inappropriate polycythaemia caused by renal adenocarcinoma in domestic shorthair cats, are described. The cats were 9 and 12 years old and both were presented because of generalised seizures presumably due to hyperviscosity. Both cats had a markedly increased haematocrit (0.770 and 0.632 l/l) and thrombocytosis (744 x 10(9)/l and 926 x 10(9)/l). An abdominal ultrasound revealed a mass in the cranial pole of one kidney in both cats. Serum erythropoietin (EPO) concentration was within the reference interval (RI) in both cats but was inappropriately high considering the markedly increased haematocrit. The cats were initially stabilised and managed by multiple phlebotomies and intravenous fluid therapy and underwent nephrectomy of the affected kidney later on. Both the polycythaemia and thrombocytosis resolved following surgery. Postoperative serum EPO concentration, measured in one cat, decreased markedly. Histopathology of the affected kidneys confirmed a diagnosis of renal adenocarcinoma. Both cats were stable for an 8-month follow-up period; however, one cat had developed a stable chronic kidney disease (CKD), while the other was represented 8 months postoperatively due to dyspnoea, and had radiographic evidence of lung metastasis, presumably because of the spread of the original renal tumour and was euthanased. Initial stabilisation of polycythaemic cats should include multiple phlebotomies. Nephrectomy should be considered in cats with secondary, inappropriate, renal adenocarcinoma-related polycythaemia when only one kidney is affected by the tumour, and provided that the other kidney's function is satisfactory. Nephrectomy should be expected to resolve the polycythaemia and lead to normalisation of serum EPO concentration.     

Pulmonary thromboembolism

Objective – To review the pathophysiology, clinical signs, diagnosis, and treatment of pulmonary thromboembolism (PTE) in small animals. Data Sources – Human and veterinary clinical studies, reviews, texts, and recent research in canine and feline PTE diagnosis and thromboembolic therapeutics. Human Data Synthesis – In humans, clinical probability assessment and point‐of‐care D‐dimer‐based algorithms are widely used. Computed tomography pulmonary angiography is the gold standard for PTE diagnosis in humans. Echocardiography is increasingly used for bedside assessment of affected patients. In low‐risk human patients anticoagulants alone are recommended while patients with cardiogenic shock are treated with thrombolytics followed by anticoagulation. Veterinary Data Synthesis – PTE is associated with numerous predisposing conditions causing hypercoagulability, blood flow stasis, or endothelial injury. Identifying at‐risk patients is key to diagnosis in small animals. Thromboelastography provides a method for identifying hypercoagulable patients. Computed tomography pulmonary angiography may replace selective pulmonary angiography as the imaging technique of choice for PTE diagnosis. PTE therapy consists of supportive treatment combined with appropriate, individualized thromboembolic pharmacotherapy for acute treatment and chronic management. Thrombolytic therapy for PTE remains controversial but may be indicated in hemodynamically unstable acute PTE. Thromboprophylaxis in specific conditions is rational although evidence of efficacy is limited. Prognosis depends upon degree of cardiopulmonary compromise and patient response to therapy. Mortality rates in small animals are unknown. Conclusions – New diagnostic techniques and advances in therapy offer significant potential for improvements in the identification and treatment of PTE in small animals. Further study must be directed to validating new diagnostic modalities and evaluating therapeutic regimes.     

Transvenous cardiac pacing

Transvenous pacing therapy is a life-saving technique for patients with clinically significant bradyarrhythmias. For most symptomatic bradyarrhythmias in small animals, there is no effective substitute for cardiac pacing. The methods employed for pacemaker placement, although potentially time-consuming, are not technically difficult. This article discusses the indications, techniques, clinical decision-making, and potential complications associated with temporary and permanent transvenous cardiac pacing.     

Improvement of anemia associated with chronic renal failure by recombinant human erythropoietin treatment in ICR-derived glomerulonephritis (ICGN) mice

The ICR-derived glomerulonephritis (ICGN) mouse, a novel inbred mouse strain with a hereditary nephrotic syndrome, develops severe anemia associated with chronic renal failure. To reveal the pathogenic mechanism of anemia in ICGN mice, we subcutaneously administered recombinant human erythropoietin (rhEPO; 5 IU/mouse/day) or saline for 5 days to ICGN mice. In terminal-stage ICGN mice with severe anemia, rhEPO significantly increased hematocrit (Ht), red blood cells (RBC) and hemoglobin levels. Endogenous EPO levels in peripheral blood were reduced by rhEPO injection. No histopathological changes in bone marrow and kidneys were induced by rhEPO injection. Insufficiency of EPO may cause anemia in ICGN mice.     

Evaluation of weight loss in canine cancer bearing patients undergoing radiation therapy

Critical weight loss, as defined by ≥5% decrease in body weight, has been associated with increased morbidity and mortality in human patients with cancers of the head and neck. Weight loss has anecdotally been reported to occur frequently in veterinary patients undergoing radiation therapy and is hypothesized to be more severe in patients with cancers of the head and neck, along with those hospitalized during radiation therapy. The primary objective of this retrospective study was to evaluate the occurrence of critical weight loss in canine cancer bearing patients undergoing either definitive or palliative radiation protocols and to determine if weight changes were associated with radiation toxicity, tumour location or patient hospitalization status. Data from 47 dogs who underwent definitive and 43 dogs who underwent palliative radiation protocols at the University of Tennessee were included for analysis. Dogs were categorized based on tumour location (head/neck or other), hospitalization status (boarded or non‐boarded) and radiation toxicity score. Weight recorded at the start of treatment, midway through treatment and at the final treatment was used for analysis. No significant differences were found in regard to weight change over time, location or hospitalization status when evaluated for both protocols. Overall, 5/90 dogs (5.5%) lost 5% or more of their body weight during therapy, and 7/90 dogs (7.7%) gained 5% or more of their body weight. The results of the current study suggest that critical weight loss occurs in a small percentage of canine patients undergoing radiation therapy, contrary to what is often anecdotally reported.     

Essential fatty acids and skin disease

Essential fatty acids (EFAs) are important in the maintenance of epidermal barrier function, as components of cell membranes and as precursors of prostaglandins, leukotrienes, thromboxanes and other substances involved in mediating inflammation. There is increasing evidence that dietary supplementation with certain EFAs, notably gamma-linolenic acid (GLA) and eicosapentanoic acid (EPA), can have anti-inflammatory actions. In man, it has been shown that supplementation with evening primrose oil (EPO), a source of GLA, can ameliorate atopy. Recent studies in the dog indicate that both EPO and cold water marine fish oil (containing EPA) can ameliorate allergic skin disease and improve skin and coat condition. EFA supplementation also promises to be useful in the treatment of seborrhoea and other conditions in the cat.     

管花肉苁蓉醇提物对大鼠血液生化指标及心血管功能的影响

旨在探讨管花肉苁蓉醇提物对大鼠血液生化指标及心血管功能的影响,为管花肉苁蓉对心血管功能不全的辅助治疗作用提供现代医学研究佐证。将24只大鼠随机分为低、中、高剂量组和对照组,分别灌胃0.1、0.2、0.3 g/（100 g·BW）的管花肉苁蓉乙醇提取物和同体积的生理盐水,连续给药30 d。试验结束时,测定并比较各给药组与对照组的主要血液生化指标、心血管功能相关酶和激素水平、脏器系数以及颈动脉平滑肌的运动指标。结果表明,低剂量管花肉苁蓉醇提物显著提高大鼠血清ALT含量（P〈0.05）,但低、中、高剂量管花肉苁蓉醇提物对其他血液生化指标影响均不显著（P〉0.05）;对大鼠3种心肌酶活力的影响不显著（P〉0.05）;低、中剂量组的Ang-Ⅱ水平显著低于对照组（P〈0.05）,但EPO活力、ALD水平的变化不显著（P〉0.05）;对大鼠的增重率、脏器系数影响不显著（P〉0.05）,对颈动脉平滑肌运动指标有一定的降低趋势（P〉0.05）。综上提示,总体来看,管花肉苁蓉醇提物对大鼠的血液生化指标及心血管功能有一定的影响。     

Double-blinded Study Examining the Effects of Evening Primrose Oil on Feline Pruritic Dermatitis

Abstract— The object of this study was to evaluate the effectiveness of Evening Primrose Oil (EPO) liquid (Efamol Vet; Guildford, U.K.) to decrease the signs of feline pruritic skin disease. Cats were accepted into the study if their dermatologic signs were related to probable flea allergy and/or atopy, their signs did not abate with increased flea control alone and they were not on any concurrent anti-inflammatory drugs or supplements. They were initially placed on 4 weeks of intense flea control then randomly in a double-blinded manner placed on either 8 weeks of EPO liquid or olive oil (control). Pruritus, erythema, self-trauma, alopecia and overall dermatologic condition were monitored and scored at 4-week intervals for the entire 12 weeks of study. The mean of each parameter for each group (EPO or olive oil) was compared to itself over time and to the other group using an analysis of variance with repeated measures. No significant difference ( P ≪ 0.05) was noted over time or between groups for any of the five parameters.     

Anemia with chronic renal disorder and disrupted metabolism of erythropoietin in ICR-derived glomerulonephritis (ICGN) mice

The ICR-derived glomerulonephritis (ICGN) mouse, a new inbred mouse strain with a hereditary nephrotic syndrome, is considered to be a good model of human idiopathic nephrotic syndrome and notably exhibits proteinuria and hypoproteinemia from the neonatal stage. In chronic renal disorder (CRD), anemia is a major subsequent symptom (renal anemia). The precise cause of renal anemia remains unclear, primarily owing to the lack of appropriate spontaneous animal models for CRD. To establish adequate animal models for anemia with CRD, we examined the hematological-biochemical properties and histopathological characteristics. With the deterioration of renal function, ICGN mice developed a normochromic and normocytic anemia, and exhibited normochromic and microcytic at the terminal stage. The expression of erythropoietin (EPO) mRNA both in the kidneys and liver and the EPO leak into the urine were observed in ICGN mice, indicating a disrupted metabolism of EPO in ICGN mice. In addition, a lack of iron induced by the hemolysis in the spleen and the leak of transferrin into urine as proteinuria aggravated the anemic condition. In conclusion, the ICGN mouse is a good model for anemia with CRD.     

TOLERABILITY AND TUMOR RESPONSE OF A NOVEL LOW‐DOSE PALLIATIVE RADIATION THERAPY PROTOCOL IN DOGS WITH TRANSITIONAL CELL CARCINOMA OF THE BLADDER AND URETHRA

Previously reported radiation protocols for transitional cell carcinoma of the canine lower urinary tract have been ineffective or associated with increased side effects. Objectives of this retrospective, cross‐sectional study were to describe safety of and tumor responses for a novel palliative radiation protocol for transitional cell carcinoma in dogs. Included dogs had cytologically or histologically confirmed transitional cell carcinoma of the bladder or urethra, and were treated with 10 once‐daily fractions (Monday–Friday) of 2.7 Gy. Thirteen dogs were sampled, with six treated using radiation as first‐line (induction) therapy and seven treated using radiation as rescue therapy after failing previous chemotherapy. Within 6 weeks of radiation, 7.6% (1/13) dogs had a complete response, 53.8% (7/13) partial response, 38.5% (5/13) stable disease, and none had progressive disease. Three patients presenting with urethral obstruction had spontaneous micturition restored during the treatment protocol. A single patient with unilateral ureteral obstruction was patent at recheck examination. Median survival time from time of initial diagnosis was 179 days. Median survival time from start of radiation was 150 days. Acute radiation side effects occurred in 31% (4/13) patients and were classified as grade 1 or 2. No significant late side radiation side effects were reported. No variables examined were identified as prognostic factors. Findings indicated that the reported radiation protocol was safe in this sample of dogs with bladder and urethral transitional cell carcinoma. Future prospective studies are needed to determine utility of this treatment as a rescue therapy in patients with complete urinary tract obstruction.     

The porcine erythropoietin gene: cDNA sequence, genomic sequence and expression analyses in piglets

The porcine erythropoietin (EPO) gene and its cDNA have been cloned and characterized. The cDNA encodes a protein of 194 amino acids. The gene structure and sequence show a high degree of homology to the corresponding human and murine gene. Steroid hormone receptor binding sites are present both in the promoter and in the 3' flanking region of the gene, which also contains an oxygen-sensing sequence. The promoter lacks classical promoter elements such as TATA and CAAT boxes. Expression analyses using a competitive RT-PCR assay showed that the kidneys contain about ten times more erythropoietin mRNA than the liver in five-week-old piglets, thus indicating that the shift from mainly hepatic to mainly renal EPO production has taken place at this age. The testes showed a higher ratio of EPO mRNA to total RNA than the liver. Spleen showed very low levels of expression, while no expression of erythropoietin mRNA was detected in brain tissue, bone marrow, lung, lymph nodes, and ovaries.     

Anti‐platelet therapy in small animal medicine

Thromboembolism is a significant complication in many commonly encountered diseases, and can be a devastating sequel to otherwise treatable conditions. Platelets play an essential role in the hemostatic process and, consequently, are associated with thrombus formation. Platelets adhere to denuded vascular subendothelium, recruit additional platelets and cells, aggregate, and provide the catalytic surface for thrombin production and fibrin formation. Therapy to prevent unwanted thrombus formation and thromboembolic crises is essential in the management of hypercoagulable patients. Unfortunately, many of the medications used in veterinary medicine that inhibit or modulate coagulation factors, such as the heparins, are cost prohibitive, only effective when administered by injection or require frequent drug monitoring, and are therefore poor choices for long term at home therapy. While the role of the platelet in pathologic thrombus formation is not fully understood, veterinarians often resort to anti‐platelet therapy in the management of patients at risk for thromboembolic complications, because many anti‐platelet medications are inexpensive, require minimal drug monitoring, and can be given orally. The aim of this review is to discuss the anti‐platelet therapies that are currently being used or being considered for use to inhibit platelet function and reduce thromboembolic complications in hypercoagulable dogs and cats.