Expression of genes related to corticotropin production and glucocorticoid feedback in corticotroph adenomas of dogs with Cushing's disease

Cushing's disease caused by pituitary corticotroph adenoma is a common endocrine disease in dogs. A characteristic biochemical feature of corticotroph adenomas is their relative resistance to negative feedback by glucocorticoids. In this study, we examined gene expression related to adrenocorticotropic hormone (ACTH) production and secretion, and the negative feedback by glucocorticoids in canine corticotroph adenoma. We used resected corticotroph adenomas from 10 dogs with Cushing's disease. In order to investigate the alteration of gene expression between corticotroph adenoma and normal corticotrophic cells, ACTH-positive cells in the anterior lobe were microdissected using a laser-capture microdissection system, and mRNA levels of proopiomelanocortin (POMC), corticotropin releasing hormone receptor 1 (CRHR1), glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and 11 beta hydroxysteroid dehydrogenase (11HSD) type 1 and type 2 were determined using real-time RT-PCR. POMC, CRHR1, and 11HSD2 mRNA levels in corticotroph adenoma were greater than those in normal corticotrophic cells (POMC, 5.5-fold; CRHR1, 4.9-fold; 11HSD2, 4.2-fold, P<0.01, respectively). MR and 11HSD1 mRNA levels in corticotroph adenoma were lower than those in normal corticotrophic cells (MR, 2.2-fold; 11HSD1, 2.9-fold, P<0.01, respectively). GR mRNA levels did not differ between corticotroph adenoma and normal corticotrophic cells. Our results may help to understand the increased ACTH production and the resistance to negative feedback suppression by glucocorticoids in canine corticotroph adenomas. These changes in gene expression may have a role in the growth of canine corticotroph adenoma, and help elucidate the pathophysiology of dogs with Cushing's disease.     

Identification and characterisation of side population cells in the canine pituitary gland

To date, stem/progenitor cells have not been identified in the canine pituitary gland. Cells that efficiently exclude the vital dye Hoechst 33342 can be visualised and identified using fluorescence activated cell sorting (FACS) as a 'side population' (SP), distinct from the main population (MP). Such SPs have been identified in several tissues and display stem/progenitor cell characteristics. In this study, a small SP (1.3%, n=6) was detected in the anterior pituitary glands of healthy dogs. Quantitative PCR indicated significantly higher expression of CD34 and Thy1 in this SP, but no differences in the expression of CD133, Bmi-1, Axin2 or Shh. Pro-opiomelanocortin (POMC) and Lhx3 expression were significantly higher in the MP than in the SP, but no differences in the expression of Tpit, GH or PRL were found. The study demonstrated the existence of an SP of cells in the normal canine pituitary gland, encompassing cells with stem cell characteristics and without POMC expression.     

Characterization of canine caspase-3

The canine caspase-3 gene was cloned and sequenced. The canine caspase-3 cDNA clone was 1473 base pairs in length and encoded 277 amino acids. The predicted amino acid sequence showed 88.4%, 88.0%, 85.9%, 65.9% and 60.6% homology with that of human, pig, mouse, chicken and zebra fish caspase-3, respectively. The caspase-3 mRNA was confirmed to express in skin, lymph node, bone marrow, small intestine and lung from a healthy dog by RT-PCR analysis.     

Plasma pro-opiomelanocortin, pro-adrenocorticotropin hormone, and pituitary adenoma size in dogs with Cushing's disease

It is difficult to predict the size of pituitary corticotroph tumors in dogs with Cushing's disease (pituitary-dependent hyperadrenocorticism [PDH]) without pituitary imaging techniques. The purpose of this study was to examine the relationship between plasma adrenocorticotropin hormone (ACTH) precursor concentration and pituitary size in dogs with Cushing's disease. Plasma concentrations of ACTH precursors (pro-opiomelanocortin [POMC]/pro-ACTH) and pituitary tumor height/brain area were measured in 36 dogs with pituitary corticotroph adenomas of various sizes. There was a correlation between tumor size (measured as the pituitary tumor height/brain area ratio [P/B]) and POMC/pro-ACTH concentration (r = .70; P < .0001). Dogs with P/B > or = 0.40 x 10(-2) mm(-1) had higher concentrations of ACTH precursors than dogs with P/B < 0.40 x 10(-2) mm(-1) (median concentration 85 pmol/L, range 15-1,350 pmol/L, n = 14 versus 15 pmol/L, range 15-108 pmol/L, n = 22; P < .0001). With a threshold of 35 pmol/L of POMC/pro-ACTH concentration, the estimated sensitivity and specificity of the kit were 93% (95% confidence interval [CI], 79-100%) and 86% (95% CI, 73-100%), respectively. We interpret these data as indicating that measurement of POMC and pro-ACTH might be of value in the characterization of tumor size in dogs with Cushing's disease. Low POMC/pro-ACTH concentrations make it unlikely that a large pituitary tumor exists in dogs with PDH.     

Expression analysis of CCL27 and CCL28 mRNA in lesional and non-lesional skin of dogs with atopic dermatitis

Chemokines are important regulators of the selective recruitment of inflammatory cells into sites of allergic inflammation. Since canine atopic dermatitis (AD) shares many clinical features of human AD, patterns of chemokine production in dogs may also be similar with those in humans. The aim of this study was to examine mRNA expression of CCL27 and CCL28 in lesional skin of dogs with AD to demonstrate similarity of chemokine production with human counterparts. RNA was extracted from skin biopsy specimens of 12 dogs with AD. The mRNA expression of CC chemokines (CCL4, CCL19, CCL20, CCL21, CCL24, CCL27 and CCL28) was analyzed by quantitative real-time PCR and was compared between lesional and non-lesional skin. Seven types of chemokines examined were constitutively expressed in both lesional and non-lesional skin. It was found that mRNA expression levels of CCL27 and CCL28 among the chemokines were significantly different between lesional and non-lesional skin (P<0.05). Expression level of CCL27 mRNA in lesional skin was significantly lower than that in non-lesional skin. On the other hand, CCL28 mRNA expression in lesional skin was found to be higher than that in non-lesional skin. These results suggest that CCL28 but not CCL27 may play important roles in immunopathogenesis of canine AD, indicating that experimental canine study may provide additional information that can be extrapolated to human AD.     

Expression of CC chemokine receptor 4 (CCR4) mRNA in canine atopic skin lesion

CC chemokine receptor 4 (CCR4) is a G protein-coupled seven transmembrane receptor that is selectively expressed on Th2 cells and plays an important role in the trafficking of Th2 cells into inflammatory sites. In this study, a full-length canine CCR4 cDNA was cloned and characterized in order to examine the potential role of CCR4 in allergic responses that produce skin lesions in canine atopic dermatitis (AD). The canine CCR4 cDNA reported in this study contained an open reading frame of 1083 nucleotides encoding 360 amino acids. The predicted amino acid sequence of canine CCR4 showed 91.9, 85.3 and 84.5% similarity with those of the human, mouse and guinea pig counterparts, respectively. Expression of CCR4 mRNA was detected in various tissues including thymus, spleen, heart, small intestine and lymph node. Furthermore, it was found that CCR4 mRNA was preferentially expressed in lesional skin of dogs with AD, together with the mRNA of thymus and activation-regulated chemokine (TARC), which is a ligand for CCR4. The present study demonstrates that CCR4 contributes strongly to the immunopathogenesis of canine AD.     

Gene expressions of canine angiopoietin-1 and -2 in normal tissues and spontaneous tumours

The angiopoietin (Ang) family of proteins are central to the regulation of angiogenesis. The purposes of this study were to determine cDNA sequences of canine Ang-1 and Ang-2 and investigate their expressions in normal tissues and spontaneous tumours. The cDNA sequences of canine Ang-1 and Ang-2 were 1,494 and 1,488 bp, and the deduced amino acid sequences were 497 and 495 residues, respectively. The cDNA sequences of canine Ang-1 and Ang-2 showed high homology with those of the other mammalian species. Canine Ang-1 and Ang-2 mRNA were detectable in all 22 normal tissues and spontaneous tumours. Higher mRNA expression level of canine Ang-2 was demonstrated in mammary simple carcinomas, haemangiosarcoma and hepatocellular carcinoma in comparison with normal tissues.     

Canine Bcl-xL gene and its expression in tumor cell lines

The canine Bcl-xL gene was cloned and sequenced. Canine Bcl-xL cDNA clone was 1252 bp in length, and encoded 233 deduced amino acides. The predicted canine Bcl-xL amino acid sequence shared 99.6%, 97.0%, 97.9%, 98.7% and 98.3% homology with that of human, mouse, rat, sheep and pig Bcl-xL, respectively. RT-PCR analysis revealed that canine Bcl-xL mRNA was constitutively expressed in CL-1 (canine lymphoma) and GL-1 (canine B cell leukemia) cell lines.     

Reference genes for canine skin when using quantitative real-time PCR

Quantitative real-time PCR (qPCR) facilitates the quantification of mRNA expression. Accurate qPCR analysis of gene expression requires the normalisation of data using a reference or housekeeping gene which is expressed at a similar level in all tissues tested. GAPDH is the most well known and most widely used reference gene but many papers have demonstrated that it is not stably expressed in different tissues. The aim of this study was to measure reference gene stability in canine skin using real-time qPCR. Skin samples from healthy control dogs (n=7) and dogs with atopic dermatitis (lesional skin n=7 and non-lesional skin n=7) were used to quantify seven reference genes (IMP, CG14980, S7, HIRA, GAPDH, RPL13A and SDHA) in canine whole skin. Three different statistical programs (Bestkeeper, GeNorm and Normfinder) were used to assess the stability of the reference genes. The results confirmed that GAPDH is not a stably expressed reference gene in canine skin; this finding may influence interpretation of previous qPCR studies on canine skin using this as a reference gene. RPL13A and CG14980 were found to be the most stably expressed genes in canine whole skin and would be more suitable as reference genes in future studies.     

Trilostane-induced inhibition of cortisol secretion results in reduced negative feedback at the hypothalamic–pituitary axis

Cushing's disease caused by pituitary corticotroph adenoma in dogs is usually treated by medical treatment, and the efficacy of this treatment has been reported. However, controversy remains as to whether reduced negative feedback through the inhibition of cortisol secretion, similar to Nelson's syndrome, may appear as an adverse effect. The purpose of this study was to investigate the effect of reduced negative feedback through the inhibition of cortisol secretion by daily trilostane administration on the pituitary–adrenal axis in clinically normal dogs. Dogs were administered 5 mg/kg trilostane twice a day every day for 8 weeks (n = 8) or 16 weeks (n = 3). After the initiation of trilostane administration, plasma adrenocorticotropic hormone (ACTH) concentrations were increased remarkably. As assessed by magnetic resonance imaging (MRI) during administration, the pituitary became enlarged. After trilostane administration, the cytoplasmic areas of the pituitary corticotrophs were increased and the ratio of pituitary corticotrophs to all cells in the anterior lobe was greater in the trilostane-treated dogs than that in untreated animals. In addition, histological examinations revealed bilateral adrenal cortical hyperplasia. Using real-time PCR quantification, the expression of proopiomelanocortin (POMC) mRNA in the pituitary and ACTH receptor (ACTH-R) mRNA in the adrenal gland was greater in the dogs treated with trilostane than in untreated dogs. These results indicate that reduced negative feedback induced hyperfunction of the pituitary corticotrophs and pituitary enlargement in healthy dogs. These changes suggest that the inhibition of cortisol secretion by trilostane may increase the risk for accelerating the growth of corticotroph adenomas in dogs with Cushing's disease.     

猪垂体特异性转录因子1基因cDNA的克隆及序列分析

本试验根据公开发表的猪POU1F1基因序列设计1对引物,提取猪垂体总RNA,通过RT-PCR方法扩增出POU1F1基因cDNA全序列,扩增产物用琼脂糖凝胶检测为预期的876 bp特异性条带。将扩增产物克隆入PTZ57R/T载体进行序列测定。经DNAStar软件分析序列与已发表序列同源性为99.7%,克隆获得的序列为进一步对猪POU1F1基因不同拼接形式功能性研究奠定了基础。     

Identifying innate immune pathways of the chicken may lead to new antiviral therapies

Fractalkine, also known as CX(3)CL1, is a unique chemokine that mediates inflammatory responses and is involved in the pathogenesis of several inflammatory disorders, including inflammatory bowel disease (IBD) in humans. In this study, we isolated cDNAs encoding canine fractalkine and its receptor CX(3)CR1, and assessed the biological activity of these molecules. The deduced amino acid sequence of the canine fractalkine cDNA showed 66% and 57% identity to human and mouse homologs, respectively. The N-terminal chemokine domain of the canine fractalkine showed 68% and 65% identity to human and mouse counterparts, respectively. The canine CX(3)CR1 amino acid sequence showed close homology to its human (83% identity) and mouse (81% identity) counterparts. Fractalkine and CX(3)CR1 mRNA were detected in all tissues in this study. Relatively higher expression levels of fractalkine mRNA were observed in the brain, medulla spinalis, small intestine, and mesenteric lymph nodes (MLNs), whereas higher expression levels of CX(3)CR1 mRNA were observed in the medulla spinalis, brain, liver, small intestine, and MLNs. The cross-reactivities of anti-human fractalkine antibody and anti-rat CX(3)CR1 antibody to canine proteins were confirmed using recombinant canine fractalkine and a cell line overexpressing canine CX(3)CR1, respectively. A transwell chemotaxis assay showed that the recombinant canine fractalkine induced migration in canine lymphoid cells expressing CX(3)CR1. The present study will be useful in understanding the canine immune system and the immunopathogenesis of canine inflammatory diseases.     

Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell–cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus‐secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.     

Molecular cloning and expression of the canine metallothionein-III gene.

We have isolated and determined the complete nucleotide sequence of canine metallothionein-III (MT-III) cDNA. The predicted amino acid sequence of the canine MT-III showed a high homology (93%, 87% identity) to that of human and mouse MT-III. The canine MT-III had 2 insertions relative to known mammalian MT-I and MT-II: a threonine after the 4th amino acid and a block of 6 amino acids near the carboxyl terminus. Expression of the canine MT-III mRNA was found exclusively in the central nervous system, where neurons in the olfactory bulb, hippocampus and cerebral cortex showed predominant signals.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.