Stability of αB-crystallin gene expression in canine mammary gland neoplasms. Should it be considered as circulating tumor cell genetic marker?

αB-crystallin is a member of a small family of thermal shock proteins that protects cells from stress. Because of lack of its expression in peripheral blood leukocytes, it was proposed as a molecular marker of circulating tumor cells in canine mammary gland tumors. The aim of the present study was to determine if αB-crystallin shows stability of expression, what is the requirement for this type of marker. It was also assessed whether there is co-expression of αB-crystallin with the basal marker, cytokeratin 17. For this purpose, samples of various types of canine mammary gland tumors of epithelial origin, were selected. Using RT-qPCR, we have found αB-crystallin and cytokeratin 17 co-expression in benign and malignant canine mammary gland tumors. It has been demonstrated that the expression of αB-crystallin in tested neoplastic samples is not stable in comparison to the control group. Furthermore αB-crystallin overor down- expression was associated witch the same cytokeratin 17 pattern. αB-crystallin can be a marker of circulating tumor cells in the bloodstream, but for cancers in which basal marker expression occurs and thus not universal for all cancers originating from the mammary gland tissue.

Expression of VPAC1 receptor at the level of mRNA and protein in the porcine female reproductive system.

The presence and distribution of vasoactive intestinal polypeptide (VIP) receptor VPAC1 was studied in the ovary, oviduct and uterus (uterine horn and cervix) of the domestic pig using methods of molecular biology (RT-PCR and immunoblot) and immunohistochemistry. The expression of VPAC1 receptor at mRNA level was confirmed with RT-PCR in all the studied parts of the porcine female reproductive system by the presence of 525 bp PCR product and at the level of proteins by the detection of 46 kDa protein band in immunoblot. Immunohistochemical stainings revealed the cellular distribution of VPAC1 receptor protein. In the ovary it was present in the wall of arterial blood vessels, as well as in the ovarian follicles of different stages. In the tubular organs the VPAC1 receptor immunohistochemical stainings were observed in the wall of the arterial blood vessels, in the muscular membrane, as well as in the mucosal epithelium. The study confirmed the presence of VPAC1 receptor in the tissues of the porcine female reproductive tract what clearly shows the possibility of influence of VIP on the porcine ovary, oviduct and uterus.

The application of circulating tumor cells detecting methods in veterinary oncology.

Cancers are one of the most common diseases affecting dogs. Many of them develop spontaneously and their biology and histopathology shows many similarities to human cancers. What more, it is proved that there are much more analogies in molecular mechanisms of cancer development between these two species. Human oncology is seeking more and more efficient methods for an early disease detection which results directly in the extended life expectancy of patients affected. One of the most modern trends in the diagnosis of cancer is to detect circulating tumor cells (CTC) in the blood of patients. It is known that these cells are responsible for the formation of metastases in distant organs what results in the patient death. Moreover, it's confirmed that CTC are already present in patients' bloodstream in the early stages of tumor development. There is no doubt that mechanism of metastasis development in dogs is identical and thus the CTC are also present in their bloodstream. Despite the intense researches there is still no optimal method of isolating cancer cells from the blood where they occur extremely rarely. The purpose of this study is to analyze the implications of the detection methods of tumor cells in the blood in veterinary oncology.

Nitric oxide in the bovine oviduct: influence on contractile activity and nitric oxide synthase isoforms localization.

The oviducts of 64 Holstein cows in luteal (early I, early II and late) and follicular phases were evaluated to determine the protein expression and mRNA transcription of different nitric oxide synthase isoforms (eNOS, iNOS, nNOS) as well as the effect of nitric oxide (NO) on spontaneous contractility in vitro. The expression patterns of nitric oxide synthase (NOS) isoforms in isthmus and ampulla (n = 6 for each phase) were determined by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. In the contractility studies, longitudinal and circular isolated strips of isthmus and ampulla (n = 10 for each phase) of oviducts located ipsilateral to the luteal structure or preovulatory follicle were treated as follows: a) L-arginine, an endogenous NO donor (10(-8) to 10(-3)m), b) N(ω)-nitro-L-arginine methyl ester (L-NAME), a NOS inhibitor (10(-5)m) and L-arginine (10(-3)m), c) methylene blue (MB), an inhibitor of soluble guanylate (10(-5)m) and L-arginine (10(-3)m) and d) sodium nitroprusside (SNP), an exogenous NO donor (10(-8) to 10(-4)m). Immunohistochemical evaluation revealed that endothelial NOS (eNOS) expression detected in epithelial layer of isthmus and ampulla was strong in early I luteal phase, moderate in follicular phase and weak in other phases. Neuronal NOS (nNOS) immunoreactivity was strong in isthmus and moderate in ampulla, and staining of nerve fibers was observed mostly in early I luteal and follicular phases. All eNOS, nNOS and inducible NOS (iNOS) isoforms were detected by RT-PCR. eNOS and iNOS proteins were evident, whereas nNOS was undetectable by Western blot analysis in the tissue examined. L-arginine applied alone or after L-NAME did not alter or increase the contractile tension of the strips in most tissues examined. However, L-arginine applied after MB increased contractile tension in the strips of ampulla and longitudinal isthmus from early I luteal phase and circular isthmus from follicular phase but decreased it in isthmus from early II luteal phase. SNP differentially modulated oviductal contraction depending on the type of muscular strips and period examined. These results showed the estrous phase-dependent changes related to endogenous NO system which might be of physiological importance to the oviduct for secretory and ciliary functions involved in gametes and embryo(s) transportation.

Analysis of the chemical coding of neurons in the intermediate thoracic ganglion of the pig.

The pig has been widely used as a model in cardiovascular research. A unique feature of the porcine extrinsic sympathetic cardiac nerves is that they arise from intermediate ganglia in the thoracic cavity. The localization and pattern of distribution of nerve cell bodies and fibers containing tyrosine hydroxylase (TH), dopamine B-hydroxylase (DBH), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), methionine-enkephalin (MET) as well as calcitonin gene-related peptide (CGRP), substance P (SP) and pituitary adenylate cyclase-activating peptide (PACAP) was studied with immunohistochemistry. Almost all the neurons showed immunoreactivity to TH. Immunoreactivity to NPY, VIP, SOM, GAL, MET and PACAP was displayed by nerve cell bodies while nerve fibers exhibited immunoreactivity to all the neuropeptides studied. Therefore, it seems that the chemical coding of neurons and especially nerve fibers in the porcine intermediate ganglion share general similarities (with certain neurochemical variability), with porcine prevertebral ganglia (e.g., celiacomesenteric and caudal mesenteric ganglia).

Partial sequence analysis of the L1 gene of bovine papillomavirus type 1 detected by PCR with MY09/MY11 primers in equine sarcoids in Poland.

BPV-1 is now recognized as a main etiological agent of equine sarcoids. The etiopathogenesis of the equine sarcoids is equivocal and is not yet fully understood. The aim of the present study was to analyse a partial sequence of the L1 gene of BPV associated with equine sarcoids in Polish horses. After clinical diagnosis, 40 skin lesions obtained from 29 horses were collected. The amplicons of a fragment of BPV L1 DNA were detected using PCR with MY09/MY11 primers in 31 specimens. All of them were recognized as BPV-1. Phylogenetic analysis has allowed the amplicons of partial L1 gene to be divided into 3 phylogenetic groups (A, B, C) and one separate isolate (20c). Sequence variants from phylogenetic groups B, C and isolate 20c represented new genetic variants of BPV-1 L1. Sequence variants from groups B and C were submitted to GenBank NCBI.

Distribution and chemical coding of intramural neurons in the porcine ileum during proliferative enteropathy.

Enteric neurons are highly adaptive in their response to various pathological processes including inflammation, so the aim of this study was to describe the chemical coding of neurons in the ileal intramural ganglia in porcine proliferative enteropathy (PPE). Accordingly, juvenile Large White Polish pigs with clinically diagnosed Lawsonia intracellularis infection (PPE; n=3) and a group of uninfected controls (C; n=3) were studied. Ileal tissue from each animal was processed for dual-labelling immunofluorescence using antiserum specific for protein gene product 9.5 (PGP 9.5) in combination with antiserum to one of: vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), somatostatin (SOM), neuropeptide Y (NPY) or galanin (GAL). In infected pigs, enteric neurons were found in ganglia located within three intramural plexuses: inner submucosal (ISP), outer submucosal (OSP) and myenteric (MP). Immunofluorescence labelling revealed increases in the number of neurons containing GAL, SOM, VIP and CGRP in pigs with PPE. Neuropeptides may therefore have an important role in the function of porcine enteric local nerve circuits under pathological conditions, when the nervous system is stressed, challenged or afflicted by disease such as PPE. However, further studies are required to determine the exact physiological relevance of the observed adaptive changes.

Effect of total or partial uterus extirpation on sympathetic uterus-projecting neurons in porcine inferior mesenteric ganglion. B. Changes in expression of neuropeptide Y, galanin, vasoactive intestinal polypeptide, pituitary adenylate-cyclase activating peptide, somatostatin and substance P.

The expression of neuropeptide Y (NPY), galanin (GAL), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), somatostatin (SOM) and substance P (SP) was studied in the neurons of the inferior mesenteric ganglion (IMG) projecting to the uterine horn and uterine cervix after uterus extirpation-induced axotomy in sexually immature gilts. The expression was studied with immunohistochemistry, in situ hybridization and RT-PCR. Uterus-projecting neurons were identified by retrograde tracing with Fast Blue (FB). Immunohistochemistry revealed that FB-positive (FB+) uterus-projecting neurons in control animals contained only immunoreactivities to NPY (ca. 50%) and GAL (single neurons). Uterus extirpation increased the occurrence of NPY and GAL in FB+ neurons. No other studied neuropeptides were found in axotomized uterus-projecting neurons. Hybridization in situ revealed the reduction of NPY expression and induction of GAL expression in FB+ neurons. RT-PCR detected induction of GAL expression in the IMG after uterus extirpation. The expression level of NPY and SOM was significant and was not affected by axotomy. The expression level of PACAP was very low and did not differ between IMG of control, partially and totally hysterectomized animals. No VIP and SP expression was detected in all ganglia. The presented data show clear axotomy-related changes in the expression of GAL and NPY in the uterus-projecting neurons of the porcine IMG.

Effect of total or partial uterus extirpation on uterus-projecting neurons in porcine inferior mesenteric ganglion. C. Changes in expression of apoptosis-associated (Bcl-2 and Bax) and regeneration-associated (GAP-43) proteins.

The expression of Bcl-2 and Bax proteins was studied with immunohistochemistry, immuoblotting and RT-PCR in the uterine horn- and uterine cervix-projecting neurons of the inferior mesenteric ganglion (IMG) of the sexually immature gilts after partial or total hysterectomy. Additionally, the expression of regeneration-associated protein GAP-43 was studied in these neurons with immunohistochemistry. The uterus-projecting neurons were identified with retrograde fluorescent tracer Fast Blue (FB). The weak immunoreactivity to Bcl-2 and GAP-43 and moderately intense immunoreactivity to Bax was revealed in all FB+ (FB+) neurons of control and hysterectomized pigs. No difference in the intensity of immunostaining for Bcl-2, Bax and GAP-43 was found between control and hysterectomized gilts. Immunoblotting revealed the presence of Bcl-2 and Bax proteins in IMGs of control and hysterectomized animals and no difference in the band intensities between control and experimental groups was detected. RT-PCR detected weak induction of bcl-2 and bax only in the ganglia of animals which had undergone total hysterectomy. It was found that the axotomy of the uterus-projecting neurons located in the porcine IMG did not change the expression of the studied substances (Bcl-2, Bax and GAP-43) at protein level and only the induction of bcl-2 and bax at the level of RNA was visible.

Uterus-innervating neurones in porcine inferior mesenteric ganglion: an immunohistochemical characteristic.

The presence of tyrosine hydroxylase, neuropeptide Y, vasoactive intestinal polypeptide, galanin, Met-enkephalin-Arg-Gly-Leu, substance P and calcitonin gene-related peptide was studied with immunohistochemistry in uterus-innervating neurones found in the inferior mesenteric ganglia after fluorescent tracer (Fast Blue) injection into different regions of the porcine uterus (uterine cervix, paracervical, middle and paraoviductal part of the uterine horn). Virtually all Fast Blue-positive neurones found in the inferior mesenteric ganglia after tracer injection into all studied parts of the uterus contained tyrosine hydroxylase and ca. 45% of them contained neuropeptide Y. Single galanin-positive/Fast Blue-positive cells were found in the ganglia only after tracer injections into uterine cervix. No other studied substances were found in the Fast-Blue positive neurones of the inferior mesenteric ganglia.

Immunohistochemical study of the otic ganglion in the pig.

The presence of choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), substance P (SP) and calcitonin gene-related peptide (CGRP) was studied in neurons and nerve fibers of the porcine otic ganglion. ChAT-positive neurons were very numerous while VAChT-positive nerve cells were moderate in number. The number of neurons containing NPY and VIP was lower and those containing SOM, GAL, SP or CGRP were observed as scarce, or single nerve cells. The above mentioned substances (except SOM) were present in nerve fibers of the ganglion. ChAT- and VAChT-positive nerve fibers were numerous, while the number of nerve terminals containing NPY, VIP and SP was lower. GAL- and CGRP-positive nerve fibers were scarce.

Influence of active immunization against GnRH on VIP- and NPY-positive innervation of the porcine testis.

The influence of an anti-GnRH vaccine on VIP- and NPY-positive innervation of testes was studied in the pig. The immunization prevented the occurrence of changes in the pattern of VIP- and NPY-positive testicular innervation associated with the sexual maturation: it maintained the density of innervation at the high level characteristic for sexually immature animals. The effect was dependent on the method of immunization: the application of two doses of the vaccine was more efficient than application of only one dose, and vaccination with adjuvant was more efficient than vaccination with the plain vaccine. The studies on VIP and NPY concentration in the testicular tissue with radioimmunoassay (RIA) revealed immunization-dependent changes in the peptide concentration, however, some discrepancies between morphological changes and peptide levels were observed.

Distribution of some neuropeptides in the porcine inferior mesenteric ganglion.

The occurrence and distribution of vasoactive intestinal polypeptide, substance P, and bombesin/gastrin-releasing peptide in neuronal cell bodies and nerve fibers of the porcine inferior mesenteric ganglion were studied with the indirect immunohistochemical technique. Of all substances studied only vasoactive intestinal polypeptide was found in principal ganglionic neurons. The presence of nerve fibers immunoreactive to vasoactive intestinal polypeptide, bombesin/gastrin-releasing peptide, and substance P was also found in the ganglion. There were differences in the pattern of distribution and density of the nerve fibers immunoreactive to the particular peptides. Fibers containing vasoactive intestinal polypeptide and bombesin/gastrin-releasing peptide were numerous, while fibers containing substance P were comparatively scarce. The present results revealed both similarities and specific differences in the occurrence and localisation of various neuropeptides in the porcine inferior mesenteric ganglion in comparison with that of other mammalian species.

Distribution of neuropeptides in the porcine stellate ganglion.

The localization and distribution of neuropeptides including neuropeptide Y (NPY), [Met5]enkephalin-Arg6-Gly7-Leu8 (MEAGL), vasoactive intestinal polypeptide (VIP), calcitonin gene-related peptide (CGRP), substance P and somatostatin (SOM) were analyzed in the stellate ganglion of the pig by use of the indirect immunofluorescence technique. NPY, MEAGL, SOM, VIP and CGRP immunoreactivities were found to exist in subpopulations of neuronal cell bodies of the stellate ganglion. A population of the small intensely fluorescent (SIF) cells showed MEAGL immunoreactivity. In addition, the presence of NPY-, MEAGL-, CGRP-, SP-, SOM- and VIP-immunoreactive nerve fibers and axonal varicosities were observed in the stellate ganglion. The localization and pattern of distribution of these peptides in the porcine stellate ganglion were compared with studies carried out on stellate ganglia of other mammalian species.

Neuropeptides in the porcine coeliac-superior mesenteric ganglion.

The occurrence and distribution of tyrosine hydroxylase, neuropeptide Y, somatostatin, Met5-enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, bombesin gastrin releasing peptide and galanin were immunohistochemically studied in the perikarya and nerve fibres of the porcine coeliac-superior mesenteric ganglion of untreated juvenile pigs. Subpopulations of neurons containing immunoreactivities to tyrosine hydroxylase, neuropeptide Y, Met5-enkephalin-Arg6-Gly7-Leu8, somatostatin, vasoactive intestinal polypeptide and galanin were disclosed in the studied ganglion, whereas principal ganglionic cells were non-immunoreactive for other investigated peptides. Double-immunofluorescence and analysis of consecutive sections revealed a partial colocalization of tyrosine hydroxylase and neuropeptide Y, Met5-enkephalin-Arg6-Gly7-Leu8 and somatostatin, whereas immunoreactivity to vasoactive intestinal polypeptide and/or to neuropeptide Y was found in non-noradrenergic neurons in this ganglion. All of neuropeptides studied were found in nerve fibres in this ganglion. The results of this study were compared with those of previous studies performed on other species.