Quantification of mRNA encoding cytokines and chemokines in nasal biopsies from dogs with sino-nasal aspergillosis

Canine sino-nasal aspergillosis is usually caused by Aspergillus fumigatus and is similar to human chronic erosive non-invasive fungal sinusitis. The pathogenesis of the disease is poorly understood. We investigated the nature of the local immune response mounted in canine sino-nasal aspergillosis. Quantitative RT-PCR was carried out on RNA isolated from nasal biopsies from diseased and control dogs, using specific assays designed to amplify mRNA encoding a panel of cytokines and chemokines. Canine sino-nasal aspergillosis was associated with significantly increased expression of mRNA encoding MCP-1, -2, -3 and -4, IL-8, IL-10, IL-18 and TNF-alpha relative to controls (P<0.01) but there was no difference between groups with respect to IL-4, IL-5, IL-6, IL-12, TGF-beta, and eotaxin-2 and -3. The up-regulation of proinflammatory cytokines and chemokines related to the influx of phagocytic cells might account for the localisation of this infection to the upper respiratory tract. The up-regulation of the expression of the immunomodulatory cytokine IL-10 in nasal tissue from affected dogs might be important in limiting the extent of local tissue destruction, but might also account for the fact that infected dogs are generally unable to clear this infection spontaneously.     

Quantification of mRNA encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (CRCoV)

One of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type I interferons (IFNs). However some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. Canine respiratory coronavirus (CRCoV) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (CIRD). This study aimed to quantify pro-inflammatory cytokine mRNAs following infection of canine air-interface tracheal cultures with CRCoV. Within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for CRCoV, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. An assay of ciliary function was used to assess potential effects of CRCoV on the mucociliary system. CRCoV was shown to reduce the mRNA levels of the pro-inflammatory cytokines TNF-alpha and IL-6 and the chemokine IL-8 during the 72 h post-inoculation. The mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease.     

Glycohistochemical characterization of histologically normal nasal mucosa and enzootic nasal tumor of sheep

Objective-To determine glycohistochemical characteristics of enzootic nasal tumors (ENTs) of sheep, compare results for ENT with those of histologically normal nasal mucosa of sheep, and identify the histologic origin of ENT. Sample-ENT and nasal mucosa samples obtained from cadavers of 5 adult Lacaune sheep with ENT and 5 Lacaune sheep unaffected by ENT, respectively. Procedures-Samples of ENT and nasal mucosa were collected from cadavers of sheep and sectioned. Conventional and lectin histochemical analyses were used to identify glycoconjugates in tissue sections on the basis of their principal chemical groups and principal terminal or internal oligosaccharidic glucidic residues, respectively. Results-ENTs had papillary and tubular portions. Cells in the papillary portion of ENTs had secretion and surface glycoconjugates, which included sulfated glycosaminoglycans and neutral and sialilated glycoproteins. Cells in the tubular portion of ENTs had surface glycoconjugates, which included neutral and sialilated glycoproteins. Both portions of ENTs had C(4)-acetylated sialoderivatives that were not detected in sections of histologically normal nasal mucosa. Conclusions and Clinical Relevance-The papillary portion of ENTs in sheep may originate from respiratory glands and goblet cells. The tubular portion of ENTs in sheep may originate from olfactory glands. Presence of C(4)-acetylated sialoderivatives in cells of ENTs could confer resistance against pathogens to those cells.     

Real-time RT-PCR quantification of pregnancy-associated plasma protein-A mRNA abundance in bovine granulosa and theca cells: effects of hormones in vitro

Ovarian follicular growth and dominance are controlled by a series of hormonal and intraovarian events including a decrease in intrafollicular IGF-binding proteins −2, −4 and −5 levels. Proteolytic enzymes such as pregnancy-associated plasma protein-A (PAPP-A) degrade IGFBPs and increase bioavailability of IGF-I and -II during follicular development. The objective of this study was to determine the effect of IGF-I, IGF-II, insulin (INS), LH, FSH, estradiol (E2), leptin or cortisol on ovarian PAPP-A mRNA levels. Granulosa (GC) from small (SM) (1–5 mm) and large (LG) (8–22 mm) follicles as well as theca cells (TC) from LG follicles were collected from bovine ovaries and cultured for 48 h in medium containing 10% FCS and then treated with various hormones in serum-free medium for an additional 24 h. Cells were treated with various concentrations (3–500 ng/ml) and combinations of IGF-I, IGF-II, FSH, LH, E2, INS, leptin and (or) cortisol for 24 h (Experiments 1–10). PAPP-A mRNA levels were measured using quantitative real-time RT-PCR. In SM-GC and LG-GC, none of the treatments significantly affected (P > 0.10) PAPP-A mRNA abundance. In LG-TC, IGF-I, LH or cortisol did not affect (P > 0.10) PAPP-A mRNA levels, whereas INS with or without LH decreased (P < 0.05) PAPP-A mRNA. E2 alone decreased PAPP-A mRNA levels in LG-TC, and E2 amplified the insulin-induced inhibition of PAPP-A mRNA abundance in LG-TC. We conclude that control of PAPP-A mRNA abundance in granulosa and theca cells differs, and that E2 may be part of an intraovarian negative feedback system which may reduce the bioavailable IGFs in the theca layer during growth and selection of follicles.     

犬瘟热病毒荧光定量RT-PCR检测方法的建立及初步应用

根据犬瘟热病毒的核衣壳蛋白(N)基因保守序列,设计特异性引物,建立犬瘟热病毒荧光定量RT-PCR检测方法。试验结果表明,该方法敏感性比普通RT-PCR高出100倍,重复性良好,变异系数在1.01%以下,对犬的常见病原核酸提取物检测均为阴性,证明特异性良好。对32份临床样本进行检测,27份为阳性,高于普通RT-PCR方法。试验结果表明该方法可用于犬瘟热感染情况的检测。     

Relative quantification of canine CD56 mRNA expression by real-time polymerase chain reaction method in normal tissues and activated lymphocytes

Real-time PCR was optimized for the quantification of canine CD56 mRNA expression. This study was conducted to easily quantify canine CD56 expression and to identify its expression in normal tissues, peripheral blood mononuclear cells and activated lymphocytes in dogs. This assay revealed the highest level of CD56 mRNA expression in the normal canine brain, followed by the lung, kidney and liver. CD56 mRNA expression level in peripheral blood mononuclear cells was considerably lower; among activated lymphocytes in vitro, CD56 mRNA expression was increased.     

Immunohistochemical expression of calretinin in canine testicular tumours and normal canine testicular tissue

Calretinin is a calcium-binding protein expressed abundantly in the central and peripheral neural tissues. It has been demonstrated to be a valuable marker in human testicular neoplasia. The immunohistochemical expression of calretinin has been studied in 102 samples of normal (n=25) and three different neoplastic canine testicular tumours (n=77). In normal canine testis, calretinin expression was restricted to Leydig and Sertoli cells of the testis. In tumour tissues, calretinin expression was detected in all tumours investigated (interstitial cell tumours, seminoma, and Sertoli cell tumours), with a cytoplasmic and nuclear pattern of cellular distribution. The present work reports, for the first time, calretinin immunohistochemical expression in normal and neoplastic canine testis.     

Cloning of cDNA encoding canine endothelin receptors and their expressions in normal tissues

The receptors for endothelin (ET) family, ETA and ETB, were molecularly cloned and the expression of ETA and ETB as well as preproendothelin-1 (PPET-1, precursor of ET-1) was examined in normal canine tissues by RT-PCR. The entire open reading frames of the canine ETA and ETB were shown to encode 427 and 442 amino acid residues, respectively, showing from 87.4 to 97.3% sequence similarity to human, mouse, and rat counterparts. ETA and ETB mRNAs were ubiquitously expressed in a variety of canine tissues in this study and PPET-1 mRNA was detected in the tissues except for heart and liver. It was speculated that ET could play an important role in physiological events in most of the organs.     

水貂IFN-α、IFN-β及IFN-γmRNA荧光定量RT-PCR检测方法的建立及应用

为采用SYBR GreenⅠ染料建立检测水貂IFN-α、IFN-β和IFN-γmRNA荧光定量RT-PCR检测方法,根据GenBank中登录的MiIFN-α和MiIFN-β、MiIFN-γ和3-磷酸甘油脱氢酶(GAPDH)基因序列,分别设计合成特异性引物,通过RT-PCR扩增MiIFN-α、MiIFN-β、MiIFN-γ和MiGAPDH的基因片段,构建到pMD18-T载体中制备质粒标准品,建立了相应基因mRNA的荧光定量RT-PCR检测方法并建立标准曲线。结果表明,MiGAPDH、MiIFN-α和MiIFN-β和MiIFN-γ基因的Ct值与标准品稀释度在10拷贝质粒/μL~107拷贝质粒/μL内均呈良好的线性关系,相关系数(R2)均为1.00,熔解曲线均呈单一熔解峰,检测下限为10拷贝质粒/μL,重复性试验表明组内、组间变异系数均小于4.5%。临床样品检测结果表明水貂感染肠炎病毒后,MiIFN-α、MiIFN-β和MiIFNγ相对表达水平均在6d达到最高峰值,MiIFN-α相对表达量要比MiIFN-β和MiIFN-γ相对表达量高两个数量级,并且在感染期(4d~6d)MiIFN-α相对表达量明显提高。本研究为水貂IFN mRNA的定量分析提供了有效工具。     

猪脂肪组织中UCP-2 mRNA表达产物的半定量分析

解偶联蛋白基因是新近发现的能够增加能量消耗，与脂肪代谢和能量调控密切相关的一组基因。本实验室建立了以18s rRNA为内标的RT—PCR法，半定量分析了猪脂肪组织中UCP-2 mRNA的表达水平。提取猪脂肪组织总RNA，经反转录和PCR扩增，PCR产物的电泳条带于VDS摄像系统下扫描灰度找出PCR线性扩增范围，确定RT—PCR的最佳循环数和Mg^2＋浓度。通过UCP-2 mRNA与18s rRNA PCR产物的灰度之比，即可计算出UCP-2 mRNA的相对含量。     

Whole blood and tissue fungal DNA quantification in the diagnosis of canine sino-nasal aspergillosis

Various combinations of tests are used to confirm the diagnosis of canine sino-nasal aspergillosis (SNA) because false-positive and false-negative results can occur with each test. Therefore, the aim of this study was to evaluate whether detection of fungal DNA in blood and nasal tissue samples was of value in the clinical diagnosis of this disease. Four groups were included in the study (dogs with SNA, lymphoplasmacytic rhinitis or nasal neoplasia, and control animals). Real-time PCR assays detecting DNA from all Penicillium and Aspergillus species (PenAsp assay) or species-specific DNA from A. fumigatus, A. terreus, A. flavus and A. niger were applied to whole blood and nasal tissue samples. Results obtained by PCR were compared between the groups. Sensitivity, specificity, positive and negative predictive values (PPV and NPV) for fungal DNA detection were compared with those for alternative diagnostic procedures including histopathology, serology and fungal culture. Significantly more fungal DNA was detected by the PenAsp assay in tissue biopsies from dogs with SNA than in the three other groups. Sensitivity, specificity, PPV and NPV for this method were 1.00, 0.06, 0.32 and 1.00. A. fumigatus DNA was detected in seven tissue biopsies from dogs with SNA and in one biopsy from a dog with a nasal tumour. Sensitivity, specificity, PPV and NPV for this diagnostic test were 0.50, 0.97, 0.87 and 0.82. No significant difference was found between the groups with respect to the amount of DNA detected in blood by the PenAsp assay. Sensitivity, specificity, PPV and NPV for this method were 0.71, 0.24, 0.31 and 0.64. A. fumigatus DNA was detected in the blood of three dogs with SNA and sixteen dogs without SNA. Sensitivity, specificity, PPV and NPV for this diagnostic tool were 0.21, 0.45, 0.15 and 0.54. Detection of A. fumigatus DNA in nasal tissue had the highest specificity, PPV and NPV but sensitivity of this method was low. Detection of fungal DNA in whole blood was of no value in the diagnosis of SNA.     

荧光定量RT-PCR检测鸡CD4、CD8基因表达水平

白细胞分化抗原CD4和CD8在机体免疫应答及其信号传递过程中发挥着重要作用。本试验建立了定量检测鸡CD4和CD8 mRNA表达水平的SYBR Green I实时荧光定量RT-PCR（RRT-PCR）方法,并采用该方法对26-50日龄商品鸡外周血淋巴细胞（PBL）中CD4和CD8 mRNA表达水平进行了检测。结果显示：所建立的RRT-PCR对CD4和CD8 mRNA的扩增效率分别为93%和91%;线性范围分别在10^-4-10^-9和10^-3-10^-9;相关系数分别为0.998 0和0.999 9;最低分别能检测105和120拷贝;熔解曲线分别在78.2和86.7℃附近出现1个单特异峰;组内变异系数分别在1.13%-2.15%和1.17%-3.68%,组间变异系数分别在1.16%-3.25%和1.66%-2.86%。26-50日龄鸡PBL CD4和CD8的mRNA表达水平有小幅波动,与采用流式细胞术检测结果的报道一致。本试验建立的RRT-PCR方法敏感性高、稳定性和再现性好,为检测鸡CD4、CD8基因表达水平提供了精确定量的新方法。     

Protein expression profiling of normal and neoplastic canine prostate and bladder tissue

Prostatic carcinoma is an important cancer in both men and dogs. Dogs have been a valuable animal model for investigating prostate cancer, but their relevance is unclear as the origin of canine prostatic carcinomas is unknown. We hypothesized that a proteomic approach for diagnosis of these neoplasms might provide quantitative data useful for more complete characterization of their origin. Protein expression profiles were prepared from normal canine prostate glands and bladders. The normal protein profiles were then compared with protein expression profiles of three canine prostatic carcinomas. Two‐dimensional differential in‐gel electrophoresis (2‐D DIGE) was used to analyse an average of approximately 1000 proteins per carcinoma. When compared with normal prostate tissue, the carcinomas exhibited greater than 2.5‐fold difference in expression for an average of 230 proteins. Similar proteomic comparisons between the carcinomas and the normal bladder revealed a greater than 2.5‐fold difference in expression for an average of 208 proteins. Mass spectrometry and protein database homology were used to identify nine proteins (α‐enolase, vimentin, GRP78, endoplasmin (GRP94), albumin, keratins 7 and 8, haptoglobin, and transferrin) overexpressed by the carcinomas. Statistical testing demonstrated that keratin 7, GRP78, and endoplasmin were significantly overexpressed in the carcinomas compared with normal prostate or bladder. Principal components analysis revealed that the carcinomas formed a unique cluster distinct from either the normal prostate or normal bladder. In conclusion, proteomic analysis revealed that whereas the majority of proteins expressed by canine prostatic carcinomas are also expressed by normal and neoplastic bladder and prostate tissue, the carcinomas contained unique protein components that allowed their segregation as a distinct group separate from normal canine prostate and bladder. Additionally, several proteins uniquely expressed by canine prostatic carcinomas were also identified.