Clinical characterisation of polydactyly in Maine Coon cats.

Objectives Polydactyly has been reported in a number of vertebrate species, including the domestic cat. It is a common characteristic in some breeding lines of the Maine Coon. The aim of this study was to assess the limb phenotype of polydactyl cats using physical and radiographic examinations. Methods We used physical examination and radiography to characterise the polydactyly phenotype in a cohort of 70 Maine Coon cats, including 48 polydactyl cats from four different breeding lines from Europe, Canada and the USA. Results The phenotypic expression of polydactyly showed great variability, not only in digit number and conformation, but also in the structure of the carpus and tarsus. Comparison of the size of the radius in polydactyl and non-polydactyl 3-month-old kittens and adult females did not reveal any difference between polydactyl and non-polydactyl cats. Conclusions and relevance We conclude that polydactyly in Maine Coon cats is characterised by broad phenotypic diversity. Polydactyly not only affects digit number and conformation, but also carpus and tarsus conformation, with no apparent deleterious consequence on feline welfare.

Novel association between vasoactive intestinal peptide and CRTH2 receptor in recruiting eosinophils: a possible biochemical mechanism for allergic eosinophilic inflammation of the airways.

We explored the relation between vasoactive intestinal peptide (VIP), CRTH2, and eosinophil recruitment. It is shown that CRTH2 expression by eosinophils from allergic rhinitis (AR) patients and eosinophil cell line (Eol-1 cells) was up-regulated by VIP treatment. This was functional and resulted in exaggerated migratory response of cells against PGD2. Nasal challenge of AR patients resulted in a significant increase of VIP contents in nasal secretion (ELISA), and the immunohistochemical studies of allergic nasal tissues showed significant expression of VIP in association with intense eosinophil recruitment. Biochemical assays showed that VIP-induced eosinophil chemotaxis from AR patients and Eol-1 cells was mediated through the CRTH2 receptor. Cell migration against VIP was sensitive to protein kinase C (PKC) and protein kinase A (PKA) inhibition but not to tyrosine kinase or p38 MAPK inhibition or calcium chelation. Western blot demonstrated a novel CRTH2-mediated cytosol-to-membrane translocation of PKC-ε, PKC-δ, and PKA-α, -γ, and -IIαreg in Eol-1 cells upon stimulation with VIP. Confocal images and FACS demonstrated a strong association and co-localization between VIP peptide and CRTH2 molecules. Further, VIP induced PGD2 secretion from eosinophils. Our results demonstrate the first evidence of association between VIP and CRTH2 in recruiting eosinophils.

The human epidermal growth factor receptor (EGFR) gene in European patients with advanced colorectal cancer harbors infrequent mutations in its tyrosine kinase domain.

BACKGROUND: The epidermal growth factor receptor (EGFR), a member of the ErbB family of receptors, is a transmembrane tyrosine kinase (TK) activated by the binding of extracellular ligands of the EGF-family and involved in triggering the MAPK signaling pathway, which leads to cell proliferation. Mutations in the EGFR tyrosine kinase domain are frequent in non-small-cell lung cancer (NSCLC). However, to date, only very few, mainly non-European, studies have reported rare EGFR mutations in colorectal cancer (CRC). METHODS: We screened 236 clinical tumor samples from European patients with advanced CRC by direct DNA sequencing to detect potential, as yet unknown mutations, in the EGFR gene exons 18 to 21, mainly covering the EGFR TK catalytic domain. RESULTS: EGFR sequences showed somatic missense mutations in exons 18 and 20 at a frequency of 2.1% and 0.4% respectively. Somatic SNPs were also found in exons 20 and 21 at a frequency of about 3.1% and 0.4% respectively. Of these mutations, four have not yet been described elsewhere. CONCLUSIONS: These mutation frequencies are higher than in a similarly sized population characterized by Barber and colleagues, but still too low to account for a major role played by the EGFR gene in CRC.

One stage functional end-to-end stapled intestinal anastomosis and resection performed by nonexpert surgeons for the treatment of small intestinal obstruction in 30 dogs.

OBJECTIVES: To describe stapled 1-stage functional end-to-end intestinal anastomosis for treatment of small intestinal obstruction in dogs and evaluate outcome when the technique is performed by nonexpert surgeons after limited training in the technique. STUDY DESIGN: Case series. ANIMALS: Dogs (n=30) with intestinal lesions requiring an enterectomy. METHODS: Stapled 1-stage functional end-to-end anastomosis and resection using a GIA-60 and a TA-55 stapling devices were performed under supervision of senior residents and faculty surgeons by junior surgeons previously trained in the technique on pigs. Procedure duration and technical problems were recorded. Short-term results were collected during hospitalization and at suture removal. Long-term outcome was established by clinical and ultrasonographic examinations at least 2 months after surgery and from written questionnaires, completed by owners. RESULTS: Mean±SD procedure duration was 15±12 minutes. Postoperative recovery was uneventful in 25 dogs. One dog had anastomotic leakage, 1 had a localized abscess at the transverse staple line, and 3 dogs developed an incisional abdominal wall abscess. No long-term complications occurred (follow-up, 2-32 months). CONCLUSION: Stapled 1-stage functional end-to-end anastomosis and resection is a fast and safe procedure in the hand of nonexpert but trained surgeons.

Anatomic study of cranial nerve emergence and associated skull foramina in cats using CT and MRI.

Magnetic resonance (MR) images of the brain of four normal cats were reviewed retrospectively to assess the emergence and course of the cranial nerves (CNs). Two-millimeter-thick images were obtained in transverse, sagittal, and dorsal planes using a 1.5 T unit. CN skull foramina, as anatomic landmarks for MR imaging, were identified by computed tomography performed on an isolated cat skull using thin wire within each skull foramen. Thin slice (1 mm slice thickness) images were obtained with a high-resolution bone filter scan protocol. The origins of CNs II, V, VII, and VIII and the group of IX, X, XI, and XII could be identified. The pathway and proximal divisions of CNs V were described. CNs III, IV, and VI were not distinguished from each other but could be seen together in the orbital fissure. CN V was characterized by slight contrast enhancement.

Silencing of E7 oncogene restores functional E-cadherin expression in human papillomavirus 16-transformed keratinocytes.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with cancer of the uterine cervix. The persistence or progression of cervical lesions suggests that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most squamous intra-epithelial lesions show quantitative and functional alterations of Langerhans cells (LCs). Moreover, E-cadherin-dependent adhesion of LC to keratinocytes (KCs) is defective in cervical HPV16-associated (pre)neoplastic lesions. The possible role of viral oncoprotein E7 in the reduced levels of cell surface E-cadherin was investigated by silencing HPV16 E7 by RNA interference (siRNA). This treatment induced an increased cell surface E-cadherin expression in HPV16-positive KC and a significant adhesion of LC to these squamous cells. The E-cadherin re-expression following HPV16 E7 silencing was associated with increased detection levels of retinoblastoma protein and the activating protein (AP)-2alpha transcription factor. These data suggest that HPV16 E7-induced alterations of LC/KC adhesion may play a role in the defective immune response during cervical carcinogenesis.

The combined immunodetection of AP-2alpha and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors.

INTRODUCTION: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line. METHODS: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting. RESULTS: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene. CONCLUSION: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels.

Unusual case of osteopenia associated with nutritional calcium and vitamin D deficiency in an adult dog.

A 6-year-old, spayed female rottweiler was presented for facial enlargement from swelling of the maxilla and mandible. The dog was fed a homemade diet deficient in calcium and vitamin D, suggesting that rubber jaw syndrome was a secondary nutritional disorder. Radiographic and tomodensitometric examinations revealed diffuse bone resorption in the skull. The plasma parathormone concentration was high, and serum 25-hydroxycholecalciferol concentration was low. Based on these findings, nutritional calcium and vitamin D deficiency associated with secondary hyperparathyroidism was diagnosed. Dietary correction resulted in clinical and biological improvement, with an increase in skull mineralization.

Anatomical study of cranial nerve emergence and skull foramina in the dog using magnetic resonance imaging and computed tomography.

Twenty-two magnetic resonance imaging (MRI) brain studies of different breeds of dogs were reviewed to assess the anatomy of cranial nerve (CN) origins and associated skull foramina. These included five anatomic studies of normal brains using 2-mm-thick slices and 17 studies using conventional clinical protocols with 3- or 4-mm slices on both normal and abnormal brains. Images were obtained in transverse, sagittal, and dorsal planes to allow a thorough comparison between studies. CNs II, III, V (and its divisions), and VIII were observed consistently on conventional studies. On the thin-slice studies, the origins and proximal portions of CNN IV, VII, and the group of IX, X, and XI could be seen. The origins of CNN VI and XII were not observed with certainty. In parallel, a computed tomography study of an isolated skull was performed with a thin copper wire within each of the skull foramina to determine precisely each CN exit and to facilitate recognition of the course of CNs when exiting the skull on MRI images.

Distal ERBB2 promoter fragment displays specific transcriptional and nuclear binding activities in ERBB2 overexpressing breast cancer cells.

Overexpression of the ERBB2 gene occurs in 30% of human breast cancers and is correlated with poor prognosis. The deregulation is the consequence of an increased transcription level and gene amplification. Several laboratories, including our own, have identified, in the proximal promoter, enhancers implicated in the gene overexpression. However, our previous studies of a 6-kb ERBB2 promoter fragment revealed the presence of repressing fragments, which were able to overcome the effect of the proximal enhancers. These repressing elements were functional in all cell lines, regardless of their endogenous ERBB2 expression level. Here, we show that a distal ERBB2 promoter region restores high transcription rates specifically in ERBB2 overexpressing breast cancer cells. This distal promoter region thus contains enhancers essential for the overexpression of the gene. By EMSA, performed with nuclear extract of cells overexpressing (BT-474) or not (MDA-MB-231) the ERBB2 gene, we show that at least two sequences of the distal promoter region are bound exclusively by BT-474 extract. Further experiments reveal that AP-2 transcription factors contribute to this differential binding activity, by binding recognition sequences located 4500 bp and 4000 bp upstream of the transcription start site. These sites are occupied by AP2 in vivo, as demonstrated by ChIP assay. Inactivation of AP-2 proteins in ERBB2 overexpressing cells reduces the distal promoter activity up to 70%, indicating the AP-2 factors are implicated in the strong distal enhancing effect. Moreover, we identified a 54-bp fragment that is bound specifically by BT-474 nuclear extract. Further experiments did not lead to the identification of the protein responsible for this binding. Our results thus highlight the importance of ERBB2 distal promoter region and further implicate AP-2 in ERBB2 overexpression in breast cancer cells.

Yin Yang 1 cooperates with activator protein 2 to stimulate ERBB2 gene expression in mammary cancer cells.

Overexpression of the ERBB2 oncogene is observed in about 30% of breast cancers and is generally correlated with a poor prognosis. Previous results from our and other laboratories indicated that elevated transcriptional activity contributes significantly to the overexpression of ERBB2 mRNA in mammary adenocarcinoma cell lines. Activator protein 2 (AP-2) transcription factors account for this overexpression through two recognition sequences located 215 and 500 bp upstream from the transcription start site. Furthermore, AP-2 transcription factors are highly expressed in cancer cell lines overexpressing ERBB2. In this report, we examined the cooperative effect of Yin Yang 1 (YY1) on AP-2-induced activation of ERBB2 promoter activity. We detected high levels of YY1 transcription factor in mammary cancer cell lines. Notably, we showed that YY1 enhances AP-2alpha transcriptional activation of the ERBB2 promoter through an AP-2 site both in HepG2 and in HCT116 cells, whereas a carboxyl-terminal-truncated form of YY1 cannot. Moreover, we demonstrated the interaction between endogenous AP-2 and YY1 factors in the BT-474 mammary adenocarcinoma cell line. In addition, inhibition of endogenous YY1 protein by an antisense decreased the transcription of an AP-2-responsive ERBB2 reporter plasmid in BT-474 breast cancer cells. Finally, we detected in vivo AP-2 and YY1 occupancy of the ERBB2 proximal promoter in chromatin immunoprecipitation assays. Our data thus provide evidence that YY1 cooperates with AP-2 to stimulate ERBB2 promoter activity through the AP-2 binding sites.

Significance of a common extended mucosal interface observed in transverse small intestine sonograms.

An echogenic stripe can be seen through the mucosal layer on either side of the ultrasonographic image of a bowel loop in cross-section. The mucosa is a continuous layer surrounding the intestinal lumen; thus it should appear as a continuous hypoechoic band with no bright stripe in it. The purpose of this study was to investigate the origin of this stripe and to determine whether it is an artifact. Intestinal cross-sectional images were obtained using different transducer types and frequencies. In vivo and in vitro, different angles of insonification were used and, in vitro, bowel loops were also fluid filled during an ultrasonographic examination, to assess the potential influence of intestinal shape and position on this stripe. Some of these loops were evaluated histologically to determine if the echogenic stripe had a histologic basis. The echogenic stripe was present only when the loop was flattened. It remained on each side of the maximal cross-sectional width and disappeared when this width was parallel to the ultrasound beam, or when the bowel loop had a completely round shape and was dilated. There was no influence of transducer type, shape, or frequency on the appearance of the line. Histologically, uneven and larger distance between the mucosal villi could be seen on either side of the bowel loop corresponding to the location of the echogenic stripe seen on the ultrasonographic images. In conclusion, the occasional echogenic stripe represents an interface within the mucosa due to altered position of villi on either side of the maximal intestinal cross-sectional width in collapsed bowel segment.

Informative evaluation of the teaching skills of the faculty at Alfort veterinary school.

A quantitative method for the informative evaluation of teaching activities was devised using a survey from 33 experts. It is based on an evaluation, by students and two peers, of the four steps of the instructional methodology: analysis of training needs, statement of learning objectives, delivery of teaching session, and testing of student performance. Faculty evaluation is optional, and results are strictly confidential. Results of a three-year trial of this protocol at the Alfort veterinary school are presented and discussed. Each year the method has been assessed and some changes have been implemented. It is now felt that evaluation questionnaires for lectures and tutorial sessions have been validated, while those for laboratories and clinical teaching have to be tested through a larger number of settings. A the same time, a change from informative to summative evaluation is under consideration.

Spinal subarachnoid cysts in 13 dogs.

Thirteen dogs, including 6 Rottweiler dogs, exhibiting clinical signs of spinal cord dysfunction and myelographically confirmed subarachnoid space enlargement were investigated. To characterize the lesions and to get a better understanding of their pathogenesis, different imaging techniques were used in association with explorative surgical procedures (12 dogs) and histopathologic techniques (5 dogs). All subjects underwent preoperative myelography, five of which were examined by computed tomography (CT) scanning and one by magnetic resonance imaging (MRI) as well as cerebrospinal fluid (CSF) flow measurement (velocimetry). Most animals were <12 months old (7/13 dogs) and Rottweilers were over-represented (6/13 dogs). The lesions were mainly located dorsally with respect to the spinal cord (10/13 dogs) and in the cranial cervical area (8/13 dogs). MRI suggested spinal cord deviation with signs of ventral leptomeningeal adhesion opposite the enlarged space. In one dog, velocimetry confirmed that the "cyst" was freely communicating with the surrounding CSF space. Surgical investigation confirmed leptomeninges-induced ventral adhesion in 4/5 dogs. Follow-up studies, carried out from 6 months to 2.5 years postoperatively, showed there was full recovery in 8/13 dogs. This study suggests that the compression of the spinal cord is possibly not caused by a cyst. Adhesion resulting from a combination of microtrauma and chronic inflammatory processes induces a secondary enlargement of the subarachnoid space and may be a significant causative factor in spinal cord compression and dysfunction. The over-representation of Rottweilers and the young age of the animals in the study suggest a possible genetic predisposition and an inherited etiology.

Identification of HTF (HER2 transcription factor) as an AP-2 (activator protein-2) transcription factor and contribution of the HTF binding site to ERBB2 gene overexpression.

The ERBB2 gene is overexpressed in 30% of human breast cancers and this is correlated with poor prognosis. Overexpression of the ERBB2 gene is due to increased transcription and gene amplification. Our previous studies have identified a new cis element in the ERBB2 promoter which is involved in the gene's overexpression. This cis element, located 501 bp upstream from the main ERBB2 transcription initiation site, binds a transcription factor called HTF (HER2 transcription factor). We report here the identification of HTF as an AP-2 (activator protein-2) transcription factor. The new cis element is bound by AP-2 with high affinity, compared with a previously described AP-2 binding site located 284 bp downstream. Co-transfection of an AP-2alpha expression vector with a reporter vector containing the newly identified AP-2 binding site in front of a minimal ERBB2 promoter induced a dose-dependent increase in transcriptional activity. We examined the contribution of the new AP-2 binding site to ERBB2 overexpression. For this purpose we abolished the new and/or the previously described AP-2 binding sequence by site-directed mutagenesis. The results show that the two functional AP-2 sites in the first 700 bp of the ERBB2 promoter co-operate to achieve maximal transcriptional activity.