The Family of Chloride Channel Regulator, Calcium-activated Proteins in the Feline Respiratory Tract: A Comparative Perspective on Airway Diseases in Man and Animal Models.

Members of the chloride channel regulator, calcium-activated (CLCA) family are considered to be modifiers in inflammatory, mucus-based respiratory conditions such as asthma and cystic fibrosis. Previous work has shown substantial differences between human and murine CLCA orthologues that limit the value of mouse models. As an alternative, the cat is an unfamiliar but powerful model of human asthma. We therefore characterized the expression profiles of CLCA proteins in the feline respiratory tract. Identical to other species, the feline CLCA1 protein was immunohistochemically localized to virtually all goblet cells and found to be secreted into the mucus. However, it was not detected in submucosal glands where it is expressed in other species. In contrast to all other species studied to date, feline CLCA2 was not found in submucosal glands or any other airway cells. Similar to mice, but in contrast to man and pigs, the feline respiratory tract was devoid of CLCA4 expression. In the airways of asthmatic cats, CLCA1 was strongly overexpressed, similar to human patients. Therefore, despite some similarities in CLCA1 protein expression and secretion, substantial differences were identified between several feline CLCA family members and their respective orthologues in man, mice and pigs, which must be considered in comparative medicine.

Soft X-ray microscopy for probing of topical tacrolimus delivery via micelles.

The penetration of topically applied tacrolimus formulated in micelles into murine skin is reported, measured by X-ray microscopy. Tacrolimus and micelles are probed for the first time by this high spatial resolution technique by element-selective excitation in the C 1s- and O 1s-regimes. This method allows selective detection of the distribution and penetration depth of drugs and carrier molecules into biologic tissues. It is observed that small, but distinct quantities of the drug and micelles, acting as a drug carrier, penetrate the stratum corneum. A comparison is made with the paraffin-based commercial tacrolimus ointment Protopic®, where local drug concentrations show to be low. A slight increase in local drug concentration in the stratum corneum is observed, if tacrolimus is formulated in micelles, as compared to Protopic®. This underscores the importance of the drug formulations for effective drug delivery. Time-resolved penetration shows presence of drug in the stratum corneum 100 min after formulation application, with penetration to deeper skin layers at 1000 min. High resolution micrographs give indications for a penetration pathway along the lipid membranes between corneocytes, but also suggest that the compound may penetrate corneocytes.

[A gastrointestinal stromal tumour of the caecum in a pony with colic]. / Gastrointestinaler stromaler Tumor im Zäkum bei einem Pony mit Kolik.

A 25-year-old pony mare was presented to the clinic with preliminarily reported severe acute colic. The pony during the previous week had shown inappetence, apathy and fever of unknown origin. Clinical examination and placement of a gastric tube were indicative of a secondary gastric dilation. Rectal exploration found moderate caecal meteorism with a tensed and painful medial taenia as well as a dilated and fluid-filled small intestine. In addition, a solid, mobile, non-painful structure of approximately 10 cm diameter was palpated ventrally. A hyperechogenic mass close to the caecum was detected using ultrasonography of the abdomen in the right flank. The peritoneal fluid was an exudate with cytological signs of an acute to subacute peritonitis. Blood analysis showed markedly increased plasma lactate concentration and a marginal neutrophilia and lymphopenia, with a total leukocyte count of 6 G/l. Because the owners refused consent for a laparotomy and the pony showed increased signs of severe pain despite conservative medical treatment, it was euthanized. The main findings on necropsy were extensive adherence of the caecal apex to the right and left colon and the ileum as well as a neoplasia in the lumen of the caecal apex. The mass, which was covered with a mucous membrane, had a tough consistency. The cut surface was grey-white to beige-coloured and multilobular with numerous necrotic and acute haemorrhagic areas. According to histopathological and immunohistochemical findings, the mass was characterized as a gastrointestinal stromal tumour (GIST), a rare neoplasia in horses.

Iron overload syndrome in the black rhinoceros (Diceros bicornis): microscopical lesions and comparison with other rhinoceros species.

The African black rhinoceros (Diceros bicornis) has adapted to a low iron diet during evolution and is thus prone to iron overload in captivity, which is associated with a number of serious disorders. A S88T polymorphism in the HFE gene has been suggested as a potential genetic basis of increased iron uptake in the black rhinoceros, while the Indian rhinoceros is thought to be unaffected by iron overload in captivity. In the present study, the histopathology and distribution of iron accumulations in five black rhinoceroses with iron overload syndrome were characterized and compared with three Indian rhinoceroses (Rhinoceros unicornis) and one African white rhinoceros (Ceratotherium simum). At necropsy examination, iron storage in black rhinoceroses was not associated with gross lesions. Microscopically, the most consistent and highest degree of iron load was found in the spleen, liver, small intestine and lung. There was minimal fibrosis and single cell necrosis in the liver. Endocrine organs, lymph nodes, heart and kidney were less often and less markedly affected. Unexpectedly, Indian rhinoceroses also showed iron load in the spleen and smaller amounts in organs similar to the black rhinoceros except for in the heart, while the white rhinoceros had only minor detectable iron storage in intestine, liver and lung. Sequence analysis confirmed the HFE S88T polymorphism in black but not in Indian rhinoceroses. The results indicate that Indian rhinoceroses may also be affected by iron storage in captivity, although in a milder form than the black rhinoceros, and therefore challenge the relevance of the S88T polymorphism in the HFE gene of black rhinoceroses as the underlying cause for iron overload.

Metastatic endocervical adenocarcinoma in a western lowland gorilla (Gorilla g. gorilla): no evidence of virus-induced carcinogenesis.

BACKGROUND: Cervical Cancer is the second most common cancer among women. Nevertheless, similar tumours have only been rarely described in Great Apes. This report characterizes the pathological and molecular features of a metastatic endocervical adenocarcinoma in a Western lowland gorilla (Gorilla g. gorilla). METHODS: Necropsy and histopathology was performed to identify the cause of the disease in an cachectic 50-year-old western lowland gorilla. Immunohistochemistry for Ki67, oestrogen receptor alpha and ERBB2 was performed to characterize the tumor. In addition, Pan-herpesvirus and Pan-papillomavirus PCR were used to identify a possible viral cause. RESULTS: The endoccervical carcinoma showed a severe metastatic spread to the lung, brain and bone and was herpesvirus and papillomavirus-negative. Most tumor cells were ERBB2-positive, 15% of tumor cells were Ki67-positive and only few tumor cells had oestrogen receptor alpha expression. CONCLUSIONS: Histopathologically and immunohistochemically, the tumour had striking similarities to human endocervicial adenocarcinomas of the common type. However, PCR analysis failed to identify herpes- or papillomaviral DNA in the tumor at the time of necropsy, thus leaving the question for cause of the disease open.

Sequential targeting of CFTR by BAC vectors generates a novel pig model of cystic fibrosis.

Cystic fibrosis (CF) is the most common lethal inherited disease in Caucasians and is caused by mutations in the CFTR gene. The disease is incurable and medical treatment is limited to the amelioration of symptoms or secondary complications. A comprehensive understanding of the disease mechanisms and the development of novel treatment options require appropriate animal models. Existing CF mouse models fail to reflect important aspects of human CF. We thus generated a CF pig model by inactivating the CFTR gene in primary porcine cells by sequential targeting using modified bacterial artificial chromosome vectors. These cells were then used to generate homozygous CFTR mutant piglets by somatic cell nuclear transfer. The homozygous CFTR mutants lack CFTR protein expression and display severe malformations in the intestine, respiratory tract, pancreas, liver, gallbladder, and male reproductive tract. These phenotypic abnormalities closely resemble both the human CF pathology as well as alterations observed in a recently published CF pig model which was generated by a different gene targeting strategy. Our new CF pig model underlines the value of the CFTR-deficient pig for gaining new insight into the disease mechanisms of CF and for the development and evaluation of new therapeutic strategies. This model will furthermore increase the availability of CF pigs to the scientific community.

Exploiting Fluorescence Lifetime Plasticity in FLIM: Target Molecule Localization in Cells and Tissues.

The mechanisms of drug-receptor interactions and the controlled delivery of drugs via biodegradable and biocompatible nanoparticulate carriers are active research fields in nanomedicine. Many clinically used drugs target G-protein coupled receptors (GPCRs) due to the fact that signaling via GPCRs is crucial in physiological and pathological processes and thus central for the function of biological systems. In this letter, a fast and reliable ratiometric fluorescence lifetime imaging microscopy (rmFLIM) approach is described to analyze the distribution of protein-ligand complexes in the cellular context. Binding of the fluorescently labeled antagonist naloxone to the G-protein coupled µ-opioid receptor is used as an example. To show the broad applicability of the rmFLIM method, we extended this approach to investigate the distribution of polymer-based nanocarriers in histological liver sections.

A soluble secreted glycoprotein (eCLCA1) is overexpressed due to goblet cell hyperplasia and metaplasia in horses with recurrent airway obstruction.

The equine putative chloride channel protein eCLCA1 is thought to be critically involved in the pathogenesis of recurrent airway obstruction (RAO) via modulation of the hydration of airway mucins. A recent study revealed a strong increase of eCLCA1 messenger ribonucleic acid (mRNA) in the lungs of horses with RAO. In this study, eCLCA1 protein and mRNA expression were quantified in airway goblet cells of 9 horses affected with RAO and 9 control horses by using immunohistochemistry and laser microdissection followed by real-time quantitative reverse transcription polymerase chain reaction, respectively. Horses affected by RAO had strong goblet cell metaplasia in bronchioles and goblet cell hyperplasia in bronchi and the trachea. Expression of the eCLCA1 protein was tightly linked to all airway goblet cells in both groups. No differences were detected in the ratio of eCLCA1 mRNA copy numbers to the mRNA copy numbers of the housekeeping gene EF-1a per goblet cell between horses affected with RAO and unaffected horses, suggesting that the increase in eCLCA1 expression is because of increased numbers of goblet cells and not transcriptional upregulation of the eCLCA1 gene. In addition, biochemical analyses of the eCLCA1 protein after in vitro translation and heterologous expression in cultured cells revealed that eCLCA1 is a secreted glycoprotein and not an integral membrane protein. Taken together, the results suggest that eCLCA1 mediates its effect as a soluble constituent of airway mucins that is overexpressed in RAO airways because of goblet cell hyperplasia and metaplasia, not transcriptional upregulation.