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.     

Establishment and characterization of eight feline mammary adenocarcinoma cell lines

Eight new feline mammary adenocarcinoma cell lines derived from either primary or metastatic lesions were established. The morphology of all the cell lines was epithelioid and round to spindle in shape, with cell growth occurring in a monolayer fashion. On immunohistochemistry, these cells reacted with anti-keratin and anti-vimentin antisera. The doubling time of these cells was between 19 and 54 hr. Tumor masses were developed in nude mice by subcutaneous inoculation of the cells that were histologically identical to their original mammary tumor lesions. Telomerase activities measured using the telomeric repeat amplification protocol assay revealed high telemetric activity in all of the cells.     

Expression of RANTES mRNA in skin lesions of feline eosinophilic plaque

Abstract One of the mechanisms of eosinophil infiltration is its induction by chemoattractants such as regulated upon activation, normal T-expressed and secreted (RANTES) which is a cysteine–cysteine chemokine that mediates chemotaxis and activation of eosinophils in humans and mice. Skin lesions of feline eosinophilic plaque are characterized by a predominant infiltration of eosinophils. The mechanism(s) of eosinophilic infiltration in the skin and/or mucosa of cats is unknown. It is possible that RANTES is involved. To investigate the presence of RANTES in the skin of cats with eosinophilic plaques and nonaffected skin, we cloned and sequenced the full-length feline RANTES cDNA gene, in order to determine whether it is present in the skin of cats with eosinophilic plaques and/or if it is present in normal adjacent skin. We were able to document the the expression of RANTES mRNAs in skin with feline eosinophilic plaque as well as in normal cat skin. The full-length cDNA sequence of the RANTES gene (742 bp) contained a single open reading frame of 276 bp encoding a protein of 92 amino acids. The amino acid sequence of feline RANTES shared 67 and 74% sequence identity with that of bovine and mouse RANTES genes, respectively. RT–PCR analysis on RANTES mRNA in the skin of cats with eosinophilic plaque revealed that its expression was higher in the eosinophilic plaque skin lesions than in the normal skin. The result suggested that RANTES might play a role to induce eosinophil infiltration in feline eosinophilic plaque lesions.     

Expression of E-cadherin in normal, hyperplastic and neoplastic feline mammary tissue

This paper describes an immunohistochemical study of the expression of E-cadherin in four samples of normal, eight samples of hyperplastic and 19 samples of neoplastic feline mammary tissue. In the normal tissues, the luminal epithelial cells showed a strong pattern of staining for E-cadherin at the cell-cell boundaries, whereas the myoepithelium showed no immunoreactivity. In the hyperplastic tissues and the five benign neoplasms, there were disturbances in the expression of E-cadherin in the luminal epithelium, in the form of the coexistence of membranous and cytoplasmic staining, together with immunoreactivity in a small percentage of myoepithelial cells. In 11 of 14 carcinomas, there was a reduction or absence of E-cadherin expression and abnormalities in the pattern of immunostaining; these changes were more pronounced in cribriform and solid carcinomas.     

Expression of cysteine sulfinate decarboxylase mRNA in rat mammary gland

Our previous report demonstrated that high concentration of taurine is present in rat milk for the first few days of lactation and plays an important role in the body growth of rat pups. In the present study, gene expression of rate-limiting enzyme for taurine biosynthesis, cysteine sulfinate decarboxylase (CSD) were examined in rat mammary gland. By RT-PCR, CSD mRNA was found to be expressed in rat mammary gland like that in the liver. The expression level of CSD mRNA in the mammary gland was higher in the earlier lactational stage (days 1 and 6 of lactation) than that in the later lactational stage (day 14). CSD mRNA expression in the mammary gland of non-pregnant rats was only a trace level. By in situ hybridization analysis, CSD mRNA was demonstrated in the epithelial cells of the mammary gland. These results suggest that high concentrations of taurine in the milk are at least partially resulted from de novo synthesis of taurine in mammary gland epithelial cells and that the expression pattern of CSD mRNA may be responsible for the changes in taurine levels in the milk during a lactational period.     

Ultrastructure of feline mammary hypertrophy

The ultrastructure of feline mammary hypertrophy was studied in a five-month-old female which had aborted recently, a ten-year-old female which was one month postestrus, and a four-year-old progestin-treated neutered male. Morphologic comparisons were made to normal mammary tissue from a one-year-old female cat. Hypertrophied mammary tissue had the same cell types and spatial relationships as did the normal gland. Major differences included a more highly developed duct system composed of metabolically active cells which often were arranged in multiple cell layers, and periductular stroma with increased fibroblasts and vascularization. Hypertrophied epithelial cells were characterized generally by smooth-contoured nuclear membranes, more evenly dispersed heterochromatin, prominent nucleoli, increased polyribosomes, and elongated mitochondria. Secretory activity was developed significantly only in the cat that had aborted recently. Modifications in myoepithelial cells included: more evenly dispersed nuclear heterochromatin, thicker bundles of cytoplasmic filaments, more straight plasma membranes along the basal lamina, and elongated hemidesmosomes. Multilayering of the basal lamina was accentuated. Stromal fibroblasts had nuclear heterochromatin distributed similarly to that of epithelial and myoepithelial cells, and increased rough endoplasmic reticulum. Myoepithelial cells did not contribute to the increased stromal cellularity. No significant ultrastructural differences were noted between mammary hypertrophy in young, old, and progestin-treated cats.     

Progesterone receptors in feline mammary adenocarcinomas

Estradiol and progesterone receptors were measured in tumor cytosols from 3 intact and 4 neutered female cats with spontaneously occurring mammary adenocarcinomas. Serum from 2 of the intact cats which had been in estrus 4 and 4 to 6 weeks before tumor excision contained progesterone concentrations of 16.2 and 2.2 ng/ml, respectively; serum progesterone in the other cats was less than 2 ng/ml. Estradiol receptors were not detected in any cytosols. Progesterone receptors were detected in all of the cytosols, in concentrations ranging from 4.0 to 11.7 (mean = 7.2) fmol/mg of protein. Scatchard plot analysis of tumor cytosol from an 8th cat with mammary adenocarcinoma revealed presence of high affinity progesterone binding with a dissociation constant (Kd) of 3.47 nM. Tumor receptor content could not be correlated with stage of the estrous cycle nor with whether the cat was intact or neutered.     

Activation of AKT in feline mammary carcinoma: a new prognostic factor for feline mammary tumours

The PI3K/AKT/PTEN pathway is involved in the pathogenesis of several human cancers. This study investigated the biological and prognostic value of PI3K/AKT/PTEN pathway dysregulation in feline mammary tumours. Expression of p-AKT, HER2, PTEN and steroid receptors was assessed by immunohistochemistry (IHC) in 27 malignant and 12 benign mammary tumours from 39 female cats followed up over a 24-month period. Feline mammary carcinoma (FMC) cell lines were analyzed by Western blot and the feline AKT gene sequence was characterized. p-AKT expression statistically correlated with tumour malignancy, histological dedifferentiation and clinical recurrence. The animals with tumours expressing p-AKT had a shorter disease-free period than those with p-AKT-negative tumours. AKT activation was associated with HER2 expression and PTEN down-regulation, as occurs in human breast cancer, and feline AKT sequencing showed high homology with the human AKT gene. No AKT activation was observed in relation to either oestrogen receptor α (ERα) or progesterone receptor expression. Taken together, these data offer an explanation for AKT signalling and its role in FMC pathogenesis and prognosis, shedding new light on similarities between feline mammary tumours and hormone-independent breast cancer.     

Expression of NOS and VEGF in feline mammary tumours and their correlation with angiogenesis

In order to define the role of nitric oxide (NO) in feline mammary tumours, the expression of endothelial or inducible nitric oxide synthase (e/iNOS) and vascular endothelial growth factor (VEGF), and their relationship with angiogenesis, was investigated in 23 feline mammary tumours (two hyperplastic, 19 adenocarcinoma, one osteosarcoma and one squamous cell carcinoma) by immunohistochemistry. Tumour angiogenesis was assessed by CD31 immunostaining and was expressed as microvessel density (MVD). In general, iNOS immunoreactivity was localised in tumour cells and occasionally in stromal myofibroblasts, whereas eNOS and VEGF were localised in the cytoplasm of tumour epithelial cells and endothelium. In malignancy, expression of iNOS increased from well- to less-differentiated phenotypes (Grades 1-3) and was significantly higher in G3 and G2 when compared with G1 cases. However, increasing eNOS expression was limited only in hyperplastic lesions and showed no significant changes among the grades. In addition, expression of iNOS was positively correlated with VEGF and MVD in feline mammary tumours and both measures were significantly greater in less differentiated phenotypes (P<0.05). In conclusion, the expression of NOS isoforms in feline mammary tumours depended on tumour grade, and the positive correlations between iNOS and angiogenic markers suggests that iNOS synthesised by tumour cells promotes tumour growth. Thus, iNOS can be used as an important immunohistochemical marker to determine the degree of malignancy and prognosis of feline mammary carcinoma.     

Retrospective evaluation of adjunctive doxorubicin for the treatment of feline mammary gland adenocarcinoma: 67 cases

Medical records for 67 cats with histologically confirmed mammary gland adenocarcinomas treated with adjunctive doxorubicin from June 1994 through December 2002 were reviewed. Data were examined to evaluate factors influencing disease-free interval (DFI) and survival time. The Kaplan-Meier median survival time of cats that received surgery and doxorubicin was 448 days. The Kaplan-Meier median DFI was 255 days. Significant univariate prognostic factors for DFI included histological subtype, completion of initial chemotherapy, development of metastatic disease, and location of metastatic disease. Significant univariate prognostic factors for survival included tumor volume, the development of metastatic disease, and location of metastatic disease.     

Role of CXCR4 and SDF-1 in mammary tumor metastasis in the cat

It has recently been suggested that the chemokine receptor CXCR4 and its ligand SDF-1 (CXCL12) promote metastasis of various cancers in humans. Since feline mammary tumors also metastasize to distant organs frequently, we used real-time quantitative PCR to examine the expression of feline CXCR4 (fCXCR4) in ten feline mammary tumor cell lines and seven feline mammary tumor tissues, and also the expression of feline SDF-1 (fSDF-1) in various organs. Cell lines derived from metastatic regions expressed more fCXCR4 than those derived from primary tumors. Mammary tumor tissues overexpressed more fCXCR4 than normal mammary tissues. Organs with high levels of fSDF-1 expression represent common sites of metastasis. Migration assays using the feline mammary tumor cell line NAC were also performed to test the activity of TN14003 and TC14012, antagonists of human CXCR4, to antagonize fCXCR4 expressed on NAC cells. TN14003 and TC14012 inhibited migration of NAC cells. We conclude that fCXCR4 may be a therapeutic target for feline mammary tumors.     

Expression patterns of the slit subfamily mRNA in canine malignant mammary tumors

Slit, a secreted protein, functions as a chemorepellent factor in axon guidance and neuronal migration and as an inhibitor in leukocyte chemotaxis. In humans, slit2 protein attracts endothelial cells and promotes tube formation in the tumor angiogenic mechanism. In this study, we cloned a part of the canine slit subfamily and examined the expression of slit subfamily mRNAs in 3 normal canine mammary glands and 11 mammary tumor samples by RT-PCR. The cloned part of the slit gene sequences showed high similarity to those of the human, mouse, and rat. The mRNAs were expressed at low levels in the normal mammary gland. The expression levels of slit1 mRNA were low in both the normal and tumor tissues. In contrast, the expression of slit2 mRNA increased in most of the malignant mammary tumors, and an increase in slit3 mRNA expression was observed in 2 of the malignant mixed tumors. These results suggest that the expression of slit2 plays an important role in tumor angiogenesis in canine mammary gland tumors and that slit2 can be a putative marker for malignancy diagnosis of these tumors.     

胰岛素受体mRNA在新生犊牛组织中的表达

应用半定量RT-PCR方法检测了新生犊牛中枢神经系统和外周组织中胰岛素受体（insulin receptor，InsR）基因的表达。结果表明，InsR基因在肝、皮下脂肪、半腱肌、胰、肾皮质、脾、心、肺、下丘脑、肠系膜淋巴结、主动脉、十二指肠、结肠、垂体、大脑皮质、小脑皮质中都有表达。其中，肝、半腱肌、下丘脑、胰、主动脉、垂体中InsR基因的表达量显著多于其他组织（P〈0．05）。InsR基因在各组织中的广泛分布表明胰岛素在体内具有广泛的生理功能。     

Expression of estrogen receptor 1 and progesterone receptor in primary goat mammary epithelial cells

The exact role and sensitivity of cells to estrogen and progesterone mediated through the steroid receptors during lactation is not known. Expression of estrogen receptor 1 (ESR1) and progesterone receptor (PGR) was quantified in mammary tissue‐derived primary goat mammary epithelial cells (pgMECs) to determine the influence of donor tissue physiology (lactating and juvenile) and cell culture growth conditions (basal and lactogenic) on ESR1 and PGR expression in the derived cells. Relative messenger RNA (mRNA) levels for both receptors were the highest in cell lines derived from mammary tissue of juvenile goats. Maintaining pgMECs in lactogenic conditions resulted in up‐regulation of ESR1 (1.36‐ to 12.35‐fold) and in down‐regulation of PGR (‐2.53‐ to ‐3.62‐fold), compared to basal conditions. Based on Western blotting analysis we suggest that the differences in mRNA expression are translated to the protein level. We suggest that differential expression in lactating conditions is correlated with terminal differentiation of the pgMECs. Double immunostainings showed that estrogen receptor alpha (ER‐α) positive cells do not exclusively belong to the luminal lineage and that ER‐α and PGR can be expressed individually or co‐expressed in the pgMECs. The derived primary cultures/lines in early passages are hormone‐responsive and represent a useful surrogate for mammary tissue in research experiments.     

Expression and functional analysis of chemokine receptor 7 in canine lymphoma cell lines

C-C chemokine receptor 7 (CCR7) contributes to cell homing to lymph nodes (LNs). Recent studies reported that CCR7 is also expressed in tumor cells, which correlates with LN metastasis in various cancers. However, the expression of CCR7 in tumor cells is unknown in dogs due to the lack of appropriate antibodies. In the present study, a fusion protein of C-C chemokine ligand 19 (CCL19) was employed as an alternative method to CCR7 antibodies. The fusion CCL19 protein specifically detected CCR7 expressed in canine lymphoma cell lines, which showed active chemotaxis to both canine and mouse ligands. The present study will help further research on the involvement of canine CCR7 in LN metastasis.