Immunohistochemical localization of chromogranin A in normal tissues from laboratory animals

To analyze the distribution of Chromogranin A in endocrine cells of various species of laboratory animals (dog, gerbil, guinea pig, hamster, monkey, mouse, and fetal, neonatal, and adult rats), normal tissues were stained immunohistochemically with polyclonal anti-bovine Chromogranin A antiserum (SP-1). Selected tissues (pituitary, adrenal, thyroid, parathyroid, pancreas, brain, peripheral nerve, stomach, small and large intestine, bone marrow, spleen, thymus, lymph node, and liver) from these species and from the rabbit were stained with two monoclonal anti-human Chromogranin A antibodies (LK2H10 and PHE5) to compare the immunoreactivities of the monoclonal antibodies and polyclonal antiserum. Staining with the polyclonal antiserum (SP-1) resulted in a broader spectrum of immunoreactivity but had more nonspecific background staining than either monoclonal antibody. Immunoreactivity and staining intensity with SP-1 varied between species, but most endocrine tissues (pituitary cells in the anterior and intermediate lobes, thyroid "C" cells, adrenal medulla, parathyroid, pancreatic islets, and enterochromaffin cells) from most species stained positively. In some species, pancreatic alpha cells stained more intensely, and two populations of adrenal medullary cells with different staining intensities were observed. Sciatic nerve (axonal area) was immunoreactive with monoclonal antibodies and/or the polyclonal antiserum in several species. The spectrum of immunoreactive tissues from fetal and neonatal rats increased with age. There was good cross-reactivity between species with SP-1, but not with either LK2H10 or PHE5. These results indicate that many endocrine cells with secretory granules in laboratory animals express Chromogranin A and that a polyclonal antiserum, such as SP-1, is more sensitive in detecting this protein in various species than monoclonal antibodies such as LK2H10 or PHE5.     

Molecular cloning of equine chromogranin A and its expression in endocrine and exocrine tissues

Chromogranin A (CGA) is a member of a family of highly acidic proteins co-stored and co-released with catecholamines in the adrenal medullary cells as well as in other neurons and paraneurons. The nucleotide sequence encoding equine CGA was determined using RT-PCR and rapid amplification of complementary DNA (cDNA) ends (RACE) techniques. A total 1,828 bp of the nucleotide sequence reveals that equine CGA is a 448-residue protein preceded by an 18-residue signal peptide. Comparison of the amino acid sequence of equine CGA with those of human, porcine, bovine, mouse, rat and frog CGA showed high conservation at the NH2-terminal 1-77 amino acids regions (94.8%, 93.5%, 92.2%, 81.8%, 83.1% and 66.2%, respectively) and COOH-terminal 314-430 amino acids regions (90.6%, 81.4%, 90.6%, 80.5%, 83.3% and 39.0%, respectively), as well as a potential dibasic cleavage site, whereas the middle portion showed marked sequence variation (52.5%, 49.1%, 38.9%, 26.6%, 27.9% and 6.2%, respectively). Northern blot analysis and RT-PCR elucidated the tissue distribution of equine CGA mRNA. Its expression was confirmed not only in the adrenal medullary cells but also in other organs (cerebrum, cerebellum, pituitary gland, spinal cord, liver, thyroid gland, striated muscle, lung, spleen, kidney, parotid gland and sublingual gland). Further, in adrenal chromaffin cells and pituitary cells of the anterior-intermediate lobe, the expression was confirmed by in situ hybridization with anti-sense CGA cRNA probe.     

Enzyme-linked immunosorbent assay for detection of canine chromogranin A by use of immunological cross-reactivity of rabbit anti-bovine chromogranin A antibody

Bovine and canine chromogranin A were extracted and purified from each specie's adrenal glands. Isolated bovine 70 kDa protein showed 100% identity to bovine CgA reported previously, whereas isolated canine 68 kDa protein showed 83.3% identity to bovine CgA by the NH(2)-terminal amino acid sequence analysis. Rabbit antibody to purified bovine protein (CgA) was found to immunologically cross-reacted with purified canine protein (CgA). In sandwich ELISA with anti-bovine CgA, concentration-dependent curves were obtained ranging from 0.3 to 20 mug/ml for canine CgA. From these findings, sandwich ELISA with anti-bovine CgA is found to be useful to determine the concentration of canine CgA.     

Immunohistochemical study on the secretory host defense system of bactericidal peptides in rat digestive organs

To clarify the fundamental regulation mechanism against indigenous bacterial proliferation in the alimentary tract, we immunohistochemically examined the localization of 4 bactericidal peptides (BP) in the rat digestive exocrine glands. In the upper alimentary tract, lysozyme was detected in the gustatory, extraorbital lacrimal and parotid glands. Secretory phospholipase A2 (sPLA2) was detected in the extraorbital lacrimal glands. β-defensin1 was detected in the gustatory and extraorbital lacrimal glands. β-defensin2 was detected in the Harderian glands. In the stomach, β-defensins were detected in the gastric superficial epithelial cells. In the small and large intestines, only lysozyme and sPLA2 were detected in the Paneth cells. In the cecum, all 4 BP were detected in the middle to apical portions of the crypts, and only sPLA2 was detected in the basal portion. No BP were localized in other exocrine glands associated with the alimentary tract. In addition, all 4 BP were also detected in the columnar epithelial cells of the apical portions of intestinal villi. In the intestinal superficial epithelial cells, lysozyme and β-defensins were detected in the ascending colon, whereas only β-defensin1 was detected in the descending colon and rectum. These results suggest that BP are mainly secreted from exocrine tissues in the initial portion of the digestive tract and play a role in host defense against indigenous bacteria throughout the digestive tract. Part of the BP in the chyme might be absorbed by the epithelium at the most inner sites of mucosae in the small and large intestines.     

Immunohistochemical localisation of alpha 2-macroglobulin in the horse

A peroxidase antiperoxidase technique was used to identify alpha 2-macroglobulin in formalin-fixed sections of normal equine lung and liver and in tissue sections and bronchoalveolar lavage fluid from horses with various lung diseases. Equine peripheral blood leucocytes and bronchoalveolar lavage samples from clinically normal horses were negative for alpha 2-macroglobulin. It was concluded that liver and pulmonary macrophages may be potential sources of alpha 2-macroglobulin. Although alpha 2-macroglobulin may play a role in various chronic bronchointerstitial pneumonias of the horse, it is doubtful that it is of importance in equine chronic small airway disease.     

A review of diazepam and its use in the horse

The application of diazepam in clinical equine practice has come into favor in recent years. This review covers the relevant studies concerning the pharmacological properties, pharmacodynamics and pharmacokinetics of diazepam and its clinical use in the horse. As a benzodiazepinic drug diazepam potentates GABA-mediated inhibition in the CNS. It exerts its principal action in the brain stem reticular formation and produces hypnotic, sedative, anxiolytic, anticonvulsant and skeletal muscle-relaxant effects. However, Diazepam is largely used in equine practice in combination with ketamine and xylazine hydrochloride for induction of anesthesia and alone as a sedative/ataractic/neuroleptic. We aimed to present a general discussion of the relevant data from pharmacokinetic, pharmacodynamic and clinical studies and subsequently to evaluate their results. The discussion is based on our results, reported partially in previous publications including an evaluation of the literature. Available knowledge concerning the pharmacology of diazepam and its application in the horse is presented. The relative merits and demerits of a variety of agents intended for use in horses are discussed, with the aim of providing practitioners and clinicians with a wider selection of safe drugs for equine sedation and premedication.     

Immunohistochemical demonstration of keratin in canine neuroepithelioma

Morphological features and immunoreactivity for cytokeratin (CK), glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE) of three canine neuroepitheliomas and three canine ependymomas were investigated. Neuroepitheliomas were in three German shepherds as intradural-extramedullary solitary masses, with spinal cord displacement between T10 and L2. Histologically, they contained tubules and acini, lined by epithelial cells with focal squamous metaplasia, rosette-like structures, and polygonal to spindle-shaped cells between tubules. Acini were empty or filled with a homogeneous, eosinophilic periodic acid-Schiff (PAS)-positive material. Mitotic indices varied from low to moderate. Ependymomas occurred in the third (two cases) and fourth ventricle in adult boxers. Histologically, they were composed of cells with an ill-defined, scant amphophilic cytoplasm, with a central round euchromatic nucleus; cells formed pseudorosettes, with a central fibro-vascular stroma. Neuroepitheliomas stained for CK, but ependymomas did not. Both failed to stain for GFAP, NSE, or phosphotungstic acid hematoxylin (PTAH). Thus, antibodies to cytokeratin are useful to distinguish neuroepitheliomas from ependymomas.     

Foetal development of endocrine organs in dog

The canine adenohypophysis starts to be identifiable from 25 day of pregnancy. ACTH-immunoreactive cells migrate until day 38 after which the number of ACTH-producing cells increases but their distribution does not change. The STH- and LH-producing cells first appear on day 38 of pregnancy. The primordium of the adrenal glands appears as a slender structure on day 27 and forms the definitive cortical structure on day 35. The histological pattern of the foetal adrenal cortex differs from the post-natal structure in so far as the three cortical zones (definitive zone, transitional zone and foetal zone) extend from the outside towards the inside of gland. The mass of foetal and neonatal adrenals is more than 10 times larger than the adult adrenals relative to body weight. The cortisol concentration in the amnion is slightly lower than in the allantois but the foetal serum cortisol concentration is significantly higher than in the maternal and foetal fluid compartments. The thyroxine concentrations in the allantois and amnion fluids exceed the foetal serum concentrations except in the ninth week of pregnancy, but thyroxine levels in foetal fluids and serum are below the physiological levels of adult animals. The exocrine and endocrine functions of the pancreas develop and act in parallel. Pancreatic cells are first detected at 30 days when the branched structure is clearly detectable immunohistochemically, and at that time, insulin-positive β-cells and α-cells are visible as well. The foetal serum glucose concentration exceeds the healthy adult range, but the glucose concentration in the allantois and amnion fluid remains below the physiological blood glucose concentration of mature dogs. The insulin concentration in the allantois fluid greatly exceeds the foetal serum and amnion insulin concentrations.     

Immunohistochemical localisation of alpha-1-protease inhibitor in the horse

Using a peroxidase anti-peroxidase technique alpha-1-protease inhibitor (alpha-1-PI) was identified in normal equine hepatocytes in formalin-fixed liver sections, and in airway secretions and macrophages in formalin-fixed lung sections of horses with chronic small airway disease and chronic bronchointerstitial pneumonia. In addition, it was identified occasionally in macrophages in bronchoalveolar lavage samples from clinically healthy horses and from horses with chronic small airway disease. Equine peripheral blood leucocytes and formalin-fixed lung sections with normal histology were negative for alpha-1-PI.     

Immunohistochemical demonstration of lumbar intervertebral disc innervation in the dog

Low back pain is a common ailment in dogs, particularly in specific breeds such as the German shepherd dog. A number of structures such as facet joint capsules, ligaments, dorsal root ganglia, periosteum, vertebral endplates and meninges have been associated with this condition. Yet, in spite of all diagnostic efforts, the origin of pain remains obscure in a substantial proportion of all cases. A further structure often being involved in vertebral column disorders is the intervertebral disc. The presence of nerves, however, is a precondition for pain sensation and, consequently, structures lacking innervation can be left out of consideration as a cause for low back pain. Nerve fibres have been demonstrated at the periphery of the intervertebral disc in man, rabbit and rat. With regard to the dog, however, the extent of intervertebral disc innervation is still being disputed. The goal of the present study, therefore, was to substantiate and expand current knowledge of intervertebral disc innervation. Protein gene product (PGP) 9.5 was used for immunohistochemical examination of serial transversal and sagittal paraffin sections of lumbar discs from adult dogs. This general marker revealed nerve fibres to be confined to the periphery of the intervertebral discs. These results indicate that even limited pathological processes affecting the outer layers of the intervertebral disc are prone to cause low back pain.     

Immunohistochemical identification and localization of orexin A and orexin type 2 receptor in the horse gastrointestinal tract

The aim of the present study was to investigate the presence and the distribution of cells containing orexin A and orexin type 2 receptor in the horse stomach and gut, by means of immunohistochemical techniques.Orexin A was identified in the stomach fundic and pyloric regions and in the duodenum. In the same stomach regions, a large subset of orexin A-positive cells also showed orexin type 2 receptor-like immunoreactivity. Moreover, in the duodenum, many of them, seemed to store serotonin.Characteristically, enteric neurons or ganglia also displayed orexin A and, sometimes, orexin type 2 receptor immunoreaction.Orexin A and orexin type 2 receptor immunoreactivity was also found in the nerve fibers in the enteric submucosal layer.Our results, together with data present in the literature, could contribute to the understanding of complex mechanisms regulating the horse gut functionality that are depending very likely on the consequence of the co-operation of both a central and a peripheral control.     

IgE and IgG antibodies in skin allergy of the horse

In horses, allergies have been characterized by clinical signs and/or intradermal (i.d.) allergen testing. Our aim was to find the first direct evidence that immunoglobulin E (IgE) mediates equine allergy. In addition, we tested the hypothesis that immediate skin reactions in horses can also be mediated by IgG. Anti-IgE affinity columns were used to purify IgE from serum of one healthy horse and three horses affected with summer eczema, an allergic dermatitis which is believed to be induced by Culicoides midges. A modified Prausnitz-Küstner experiment was performed in four clinical healthy horses by i.d. injection of the purified serum IgE antibodies. The following day, Culicoides allergen was injected at the same sites. Skin reactions were not observed in response to allergen alone, and in two horses after stimulation at any previous IgE injection site. However, the other two horses showed an immediate skin reaction at the previous injection sites of IgE obtained from allergic horses. In addition, purified monoclonal antibodies to various equine immunoglobulin isotypes were injected i.d. into six healthy horses. Immediate skin reactions were observed in response to anti-IgE (6/6 horses) and anti-IgG(T) injections (5/6 horses). The specificities of both antibodies for IgE and IgG(T), respectively, were confirmed by enzyme linked immunosorbent assays. The results provide the first direct evidence that IgE mediates classical Type-I allergy in horses and plays a major role in the pathogenesis of summer eczema. The data also suggest that IgG(T) can bind to skin mast cells and might contribute to clinical allergy.