The aim of the present study was to investigate the expression pattern of claudin-1, -2, -3, -4, -5, -7, -8, -10 and -18 in the intact fundic and pyloric gastric mucosa of dogs. Intense, linear, membranous claudin-18 positivity was detected in the surface gastric cells and in the epithelial cells of the gastric glands both in the fundic and pyloric stomach regions. The mucous neck cells in the apical part of the glands, furthermore the parietal cells and chief cells of the basal part of the gland were all positive for claudin-18, in the same way as the enteroendocrine cells. Cells of the basal part of the pyloric glands showed intense, linear, membranous claudin-2 positivity, but cells of the superficial portion of these glands and the surface gastric cells in this region were claudin-2 negative. Fibroblasts, endothelial cells, lymphocytes of the propria layer, smooth muscle cells and vegetative neurons were all negative for claudin-2. All gastric epithelial cells were negative for claudin-1, -3, -4, -5, -6, -7, -8 and -10. The endothelial cells of the propria layer had intense claudin-5 positivity. We assume that claudin-18 forms a paracellular barrier against gastric acid in the healthy canine stomach, in the same way as in mice.
The recently identified claudins are dominant components of tight junctions, responsible for cell adhesion, polarity and paracellular permeability. Certain claudins have been shown to have relevance in tumour development. The aim of the present study was to analyse the expression of claudin-1,-2,-3,-4,-5,-7 and-10 in normal canine mammary glands. Samples from the inguinal mammary regions of 20 non-castrated, 1–13 years old female dogs were studied. Immunohistochemical analysis was performed on conventional specimens and tissue microarrays. The results of the immunohistochemical reactions detecting claudins in tissue sections were photodocumented. The immunoreactivity of claudins was quantitatively analysed on digital images using Leica QWin morphometry software. Intense membranous immunolabelling was found for claudin-1,-3 and-7, intense membranous with non-granular cytoplasmic immunolabelling for claudin-2, moderate membranous immunolabelling for claudin-4 and-5, and weak membranous immunolabelling for claudin-10. The occurrence of tight junctions was confirmed by ultrathin section electron microscopy. The available data suggested that claudins might be proteins preserved throughout the evolution of mammals. The results of our study support the concept that they are indeed preserved, since the same type of claudins, in identical distribution, could be detected in our canine mammary tissue samples as could be found in human mammary tissue.
Claudins are integral membrane proteins of the tight junction structures expressed by epithelial and endothelial cells. The present study has evaluated the expression of claudin-4 in 10 normal canine hepatoid glands and in 67 hepatoid glands with hyperplastic and neoplastic lesions. The lesions studied included normal hepatoid glands (n = 10), nodular hyperplasias (n = 10), adenomas (n = 12), epitheliomas (n = 15), differentiated carcinomas (n = 15) and anaplastic carcinomas (n = 15). There was an intensive expression of claudin-4 in normal canine hepatoid glands as well as in hyperplasias and adenomas. Claudin-4 was detected as a well-localised linear circumferential membranous staining pattern of epithelial cells (mature hepatoid cells) in normal hepatoid glands, perianal gland hyperplasias and adenomas. In nodular hyperplasia and adenoma, the reserve cells showed membrane positivity for the claudin-4 molecule. There was a weaker expression in hepatoid gland epitheliomas. In the epitheliomas, the basaloid reserve cells never expressed the claudin-4 molecule. The multiple small parts of epitheliomas in which the cells exhibited typical hepatoid features showed a well-localised linear circumferential membranous staining pattern for claudin-4. The numerical score for cellular expression of claudin-4 was higher in differentiated carcinomas than in epitheliomas, but moderately lower than in adenomas. The anaplastic, poorly differentiated hepatoid gland carcinomas showed an overexpression of claudin-4. These results suggest that low claudin-4 expression in epitheliomas is a molecular characteristic indicative of increasing cellular disorientation, detachment motility and invasion by tumour cells, and claudin-4 seems to be helpful in distinguishing undifferentiated carcinomas from differentiated carcinomas and epitheliomas of the hepatoid gland. In addition, claudin-4 can help distinguish epithelioma from differentiated carcinoma of the canine hepatoid gland.
Claudin-5 is an endothelium-specific tight junction protein. The aim of the present study was to detect the expression pattern of this molecule in intact pancreatic tissues and in well-differentiated and poorly differentiated pancreatic acinar cell carcinomas from dogs by the use of cross-reactive humanised anticlaudin-5 antibody. The necropsy samples taken from dogs included 10 nonneoplastic pancreatic tissues, 10 well-differentiated pancreatic acinar cell carcinomas, 10 poorly differentiated pancreatic acinar cell carcinomas, 5 intrahepatic metastases of well-differentiated and 5 intrahepatic metastases of poorly differentiated acinar cell carcinomas. A strong lateral membrane claudin-5 positivity was detected in exocrine cells in all intact pancreas samples. The endocrine cells of the islets of Langerhans and the epithelial cells of the ducts were negative for claudin-5. The endothelial cells of vessels and lymphatic channels in the stroma of the intact pancreas showed strong membrane positivity for this claudin. All well-differentiated exocrine pancreas carcinomas and all poorly-differentiated pancreatic acinar cell carcinoma samples showed a diffuse loss of claudin-5 expression. The claudin-5-positive peritumoural vessels and lymphatic channels facilitated the detection of vascular invasion of the claudin-5-negative cancer cells. In liver metastasis samples, the pancreatic carcinomas were negative for claudin-5. It seems that the loss of expression of claudin-5 may lead to carcinogenesis in canine exocrine pancreatic cells.
Claudins are key tight junctional proteins between adjacent epithelial, mesothelial or endothelial cells, which are responsible for the permeability of the paracellular space. This paper describes that the endothelial cells of normal hepatic arterioles, portal venules and portal lymphatics as well as the endothelium of sinusoids from dogs show strong membranous claudin-5 cross-reactivity. In 25 liver biopsy samples taken from dogs with portal vein hypoperfusion, an increased number of arterioles was detected in the portal areas (PAs) by the use of humanised anti-claudin-5 antibody. The increased number of hyperplastic hepatic arterioles per PA was 5–6, 8–12 and 15–20 in the case of small, medium-sized and large PAs, respectively. It is suggested that the claudin-5 marker can improve the detection of hepatic arteriolar proliferation in the PAs of liver samples.
A 5-year-old female degu (
) showed the clinical sign of metrorrhagia. During ovariohysterectomy a circumscribed tumoural lesion was found in the right uterine horn. The histopathological diagnosis of this soft tissue mass was primary benign cavernous angioleiomyoma of the uterus. During immunohistochemical analysis the neoplastic endothelial cells of this mixed mesenchymal tumour showed strong membrane positivity for the endothelial marker claudin-5 but were negative for CD31 (another endothelial marker). The endothelial cells of the internal positive control tissues such as intact peritumoural vessels were positive for claudin-5 but negative for the CD31 endothelial marker. As it has been described also in other species, it seems that claudin-5 is a better endothelial marker than CD31 for the detection of normal and neoplastic endothelial cells in different tissues of degus.
In this study, synchronous spontaneous, independent liver and gallbladder tumours were detected in a Bearded dragon (Pogona vitticeps). The multiple tumours consisted of intrahepatic cholangiocarcinoma as well as in situ adenocarcinoma and two adenomas of the gallbladder. The biliary epithelial cells and the cholangiocarcinoma showed membranous cross-immunoreactivity for claudin-7. The gallbladder epithelial cells, its adenoma and adenocarcinoma showed basolateral cross-reactivity for claudin-7. We think that the humanised anti-claudin-7 antibody is a good marker for the detection of different primary cholangiocellular and gallbladder tumours in Bearded dragons. The cholangiocytes, the cholangiocarcinoma, the endothelial cells of the liver and the epithelial cells and gallbladder tumours all showed claudin-5 cross-reactivity. The humanised anti-cytokeratin AE1–AE3 antibody showed cross-reactivity in the biliary epithelial cells, cholangiocarcinoma cells, epithelial cells and tumour cells of the gallbladder. It seems that this humanised antibody is a useful epithelial marker for the different neoplastic lesions of epithelial cells in reptiles. The humanised anti-α-smooth muscle actin (α-SMA) antibody showed intense cross-reactivity in the smooth muscle cells of the hepatic vessels and in the muscle layer of the gallbladder. The portal myofibroblasts, the endothelial cells of the sinusoids and the stromal cells of the cholangiocarcinoma and gallbladder tumours were positive for α-SMA. The antibovine anti-vimentin and humanised anti-Ki-67 antibodies did not show crossreactivity in the different samples from the Bearded dragon.
In our recent investigation, angiogenesis was evaluated and quantified by immunohistochemical evaluation of microvessel density (MVD) using claudin-5 (CLDN-5) as a marker for vascular endothelium in 67 canine mammary gland tumours. Computer image analysis was used to measure the intratumoural MVD. Higher intratumoural MVD was detected in malignant simple neoplasms compared with benign tumours. Furthermore, the results of MVD were correlated with histological grade, higher grades being accompanied by higher MVD. In simple adenomas and grade I tubular-tubulopapillary simple carcinomas the intratumoural microvessels were wide and regular in shape with evident erythrocytes in their lumen. In grade III solid carcinomas the microvessels were smaller, less regular and had irregular shape, often without a distinct lumen, and isolated endothelial cells were frequently present. In the complex carcinomas MVD was low and the intratumoural microvessels were mostly irregular in shape without a distinct lumen. The evaluation of MVD by CLDN-5 immunohistochemistry may give useful additional information on the angiogenic potential of breast cancers in dogs.