Immunohistochemical localization of Clostridium perfringens beta2-toxin in the gastrointestinal tract of horses

Clostridia-associated intestinal disease in horses was generally reported to be due to infection with Clostridium perfringens type A, which harbors the cpa-encoded alpha-toxin. A recent study demonstrated a high incidence of beta2-toxigenic C. perfringens in horses suffering or dying from typhlocolitis, suggesting that this novel type of C. perfringens might play an important role in typhlocolitis and possibly other equine intestinal diseases. A retrospective study was conducted to assess the presence of the beta2-toxin in tissues of the equine gastrointestinal tract. Monospecific polyclonal antibodies against recombinant beta2-toxin were produced in rabbits and used to demonstrate the beta2-toxin in sections of the gastrointestinal tract by immunohistochemical methods. Sections from 69 horses were stained and beta2-toxin was observed immunohistochemically in 40 animals. Sections from the stomach, small intestine, and large intestine were positive. Immunopositivity for beta2-toxin was significantly associated with presence of beta2-toxigenic bacteria. This investigation demonstrates local production of beta2-toxin and suggests that immunohistochemistry using antitoxin antibodies represents a useful diagnostic method in those cases where isolation of bacteria and polymerase chain reaction typing is not feasible. Although the association between the presence of beta2-toxin and development of gastrointestinal disease in horses remains uncertain, the findings of this study indicate that the potential causal relationship warrants further investigation.     

Immunohistochemical localization of transforming growth factor alpha in regenerating rat liver

Immunohistochemical localization of TGF-alpha and cell proliferation kinetics during liver regeneration after two-thirds partial hepatectomy (PH) were investigated. Twenty-four to 72 hr after PH, appreciable increase in the number of TGF-alpha-positive hepatocytes was observed in zones 1 and 2. At the peak at 36 hr, almost all positive cells were stained in their nuclei. Considerable increase in the BrdU labeling index was observed 24-36 hr after PH with a peak at 24 hr in zones 1 and 2. These results indicated an association between TGF-alpha expression and hepatocyte regeneration. It is suggested that immunohistochemical localization of TGF-alpha may be a useful marker of cell proliferation activity in rat liver.     

Affinity of the alpha4-beta1 integrin-targeting peptide LLP2A to canine lymphoma

Lymphoma is an important disease in dogs and people, with similar biological characteristics. We tested the binding affinity of a peptidomimetic LLP2A, previously shown to bind the alpha4-beta1 integrin on human lymphoma cell lines, to lymphocytes of dogs with spontaneously occurring lymphoma. Fine needle aspirates of lymph nodes from 32 dogs with B-cell lymphoma and 7 dogs with T-cell lymphoma were evaluated using flow cytometry. For B cells, the lowest MFI levels were in unlabeled, non-neoplastic lymphocytes. The highest median fluorescent intensity (MFI) levels occurred in LLP2A-labeled lymphoma cells from dogs that had not received chemotherapy followed by labeled lymphoma cells from dogs that had received chemotherapy. The fluorescence profile of the T-cell samples was similar although many of the differences were not statistically significant, likely due to low sample number. Specifically, LLP2A-labeled T-cell lymphoma cells had a significantly higher MFI compared to unlabeled non-neoplastic lymphocytes. LLP2A affinity was not significantly different in unlabeled and labeled T-cell lymphoma cells, and labeled non-neoplastic lymphocytes. For both B and T cells, labeling with LLP2A tended to increase MFI in both normal and lymphoma cells. Lymphoma cells had higher mean MFI levels than non-neoplastic lymphocytes, and chemotherapy acted to decrease MFI. In summary, these data demonstrate that LLP2A has affinity to canine lymphoma cells and indicates expression of the alpha4-beta1 integrin on these cells. In fact, LLP2A preferentially binds neoplastic B-cells, suggesting that this small molecule may be of use in cross-species clinical trials of targeted therapeutics.     

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.     

Immunohistochemical localization of IgG1 and IgG2 in prepartum and lactating bovine mammary tissue

The differential distributions of IgG1 and IgG2 were determined in prepartum and lactating bovine mammary tissue by indirect immunofluorescence. IgG1 was found predominately within the alveolar epithelial cells and lumens of prepartum tissue whereas IgG2 was largely confined to the stromal area surrounding the alveoli. Both IgG subclasses were confined predominately to the stroma in lactating tissue. Few IgG containing stromal cells were readily distinguished in any of the mammary tissue used in this study.     

Immunohistochemical localization of CB1 receptor in canine salivary glands

CB1 is a member of the G-protein-linked receptor superfamily that is present in the central nervous system as well as in certain peripheral neuronal and non-neuronal tissues. Recently, the presence of CB1 was found in the ductal system of the major salivary glands of laboratory animals, but no data are available for domestic mammals. Thus, in the present study, we examined the presence and distribution of CB1 in the major salivary glands of dogs using immunohistochemical techniques. CB1 was found in the parotid and mandibular glands of adult dogs; positive immunoreaction was localized to the cells of the striated ducts, with a peculiar localization on or near the apical membrane. This particular localization may be explained by the characteristics of this receptor as membrane-associated. The acinar structures were completely negative for CB1. We conclude that CB1 is involved in the control of dog salivary secretion via endogenous substances, likely endocannabinoids. The localization of CB1 highlights that endocannabinoids promote qualitative and/or quantitative changes of the primary saliva in the ductal system.     

Immunohistochemical localization of oestrogen receptor alpha in the various cell categories of chick embryo ovary

The immunohistochemical (IHC) localization of oestrogen receptor alpha (ERα) was studied in the developing left ovary of 14.5-day-old chick embryos. The study was focused in particular on distinguishing in cortex and medulla the different cell categories that proved positive to the reaction, in order to gain further understanding of gonadal cell interactions during ovarian development. Immunostained cells were observed in both the cortex and medulla, but the reactivity for ERα was discontinuous, probably due to variable cell requirements. In the cortex, positivity was observed in cells of the ovarian surface epithelium, in germ cells and in prefollicular cells. In the medulla, positivity was found in the following cell categories: interstitial cells, poorly differentiated somatic cord cells, including those delimiting lacunae, germ cells and their accompanying cells of epithelial origin. Furthermore, the IHC results showed that the intracellular localization of the antigen was cytoplasmic, nuclear, or both. The significance of ERα presence and intracellular localization was discussed in relation and as supplementary to previous research by various Authors. In particular, as regards the unusual cytoplasmic immunoreactivity, a gradual shift of ERα localization from cytoplasmic to nuclear during the embryonic period is suggested.     

Immunohistochemical localization of neuropeptides in bovine pancreas

The occurrence and density of distribution of nerves and endocrine cells that are immunoreactive for neuropeptides in the bovine pancreas were studied by immunohistochemistry. The six neuropeptides localized were galanin (GAL), substance P (SP), methionine-enkephalin (MENK), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP). The exocrine pancreas was shown to have an appreciable number of GAL- and SP-immunoreactive nerve fibres but few fibres showing immunoreactivity for VIP and CGRP. Numerous MENK-, GAL-, SP-, and NPY-immunoreactive nerve fibres were seen in the endocrine portion of the pancreas. Nerve cell bodies in the intrapancreatic ganglia showed immunoreactivity for all of the neuropeptides except CGRP. Endocrine cells showing immunoreactivity for GAL and SP were observed in the large islets and islets of Langerhans, respectively. The present results indicate a characteristic distribution of neuropeptides in the bovine pancreas, which may regulate both exocrine and endocrine secretions of pancreas.     

Immunohistochemical localization of haptoglobin in porcine lungs

The extravasation of erythrocytes into the lower respiratory tract occurs in numerous lung injuries and may lead to oxidative damages in lung tissues. Haptoglobin (Hp), the major haemoglobin-binding protein, is known to reduce lung injury associated with exposure to blood in mice. In pigs, Hp is a major acute phase protein and its serum concentrations are elevated in various infections of the respiratory tract. However, information on the porcine Hp response towards inflammatory stimuli is restricted to blood. We herein investigated the presence of Hp in lung tissues from pigs with acute and chronic bronchopneumonia via immunohistochemistry. Hp was localized in airway epithelial cells and immigrated leucocytes whereas in alveolar epithelial cells there was no distinct signal. Unaltered lungs showed less Hp-positive cells compared with lungs from pigs with acute or chronic bronchopneumonia.     

Exercise-induced alterations in pro-inflammatory cytokines and prostaglandin F2alpha in horses

Using an established standardized exercise test on a high-speed treadmill, thirteen Thoroughbred racehorses were exercised to fatigue (failure); blood samples were obtained before exercise, at failure, and at 2, 6, 24, 48, and 72 h after exercise. The exercise test induced a systemic inflammatory response characterized by a mild transient endotoxemia, leukocytosis, increased leukocyte expression of mRNA for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, and IL-6, and increased circulating concentrations of TNF-alpha and prostaglandin F2 alpha (PGF 2 alpha), with the most pronounced changes being evident at failure and 2h after exercise. Expression of mRNA for IL-6, TNF-alpha, and IL-1 beta was increased by 120-fold, three-fold, and four-fold, respectively, when compared to pre-exercise values. Plasma concentrations of 6-keto-PGF1alpha and PGE2 did not change in response to the exercise test. Collectively, these findings indicate that brief, strenuous exercise induces endotoxemia and a systemic pro-inflammatory response in horses that persists for at least 2h.     

Bronchoconstrictive properties of inhaled 8-epi-PGF2alpha in healthy and heaves-susceptible horses

The 8-epi-PGF2alpha is a marker of oxidative stress which is increased in lungs of asthmatic humans and heaves-susceptible horses. 8-Epi-PGF2alpha has also been demonstrated to be an in vitro and in vivo bronchoconstrictor in humans and rodents. We hypothesised that inhaled 8-epi-PGF2alpha was a bronchoconstrictor in healthy and heaves-susceptible horses in clinical remission. The effect on ventilatory mechanics of nebulised 8-epi-PGF2alpha was compared to that of PGF2alpha and U46619, a thromboxane A2 agonist. Pulmonary resistance (R(L)) and dynamic compliance (Cdyn) were assessed in six healthy horses and in six heaves-susceptible horses in clinical remission before (baseline) and immediately after a single inhalation challenge of 1 mg 8-epi-PGF2alpha PGF2alpha, or U46619 and placebo. R(L) and Cdyn were unchanged after inhalation of 8-epi-PGF2alpha in healthy horses. In heaves-susceptible horses, 8-epi-PGF2alpha induced a significant increase of R(L) and a significant decrease of Cdyn when compared to baseline values. Differences between R(L) and Cdyn values after 8-epi-PGF2alpha inhalation and those of placebo inhalation were not significant. Differences with healthy horses were not significant. PGF2alpha and U46619 induced a significant bronchoconstriction in healthy (R(L) and Cdyn versus baseline) and heaves-susceptible horses (R(L) and Cdyn, versus baseline and placebo), the R(L) increase in heaves-susceptible horses after PGF2alpha inhalation was significantly higher than that in healthy horses. Our results suggest that 8-epi-PGF2alpha is not a bronchoconstrictor in healthy horses, and a bronchoconstrictor far less efficient than PGF2alpha and U46619 at the same dose in heaves-susceptible horses.     

Effects of human alpha interferon on experimentally induced equine herpesvirus-1 infection in horses

The immunotherapeutic effect of low-dose human alpha interferon on viral shedding and clinical disease was evaluated in horses inoculated with equine herpesvirus-1 (EHV-1). Eighteen clinically healthy weanling horses, 5 to 7 months old, were allotted to 3 equal groups. Two groups were treated orally with human alpha-2a interferon (0.22 or 2.2 U/kg of body weight), on days 2 and 1 before inoculation with EHV-1, the day of inoculation, and again on postinoculation day 1. The horses of the remaining group were given a placebo orally on the same days. The horses were monitored daily for changes in body temperature and for clinical signs of respiratory tract disease. Blood and nasal swab specimens were collected daily for virus isolation. Blood was also collected at intervals throughout the monitoring period for evaluation of CBC, serum IgG and IgM concentrations, and antibody titers to EHV-1. Febrile responses, nasal discharge, viral shedding, changes in CBC, and an increase in antibody titers to EHV-1 were noticed in all horses after inoculation. There was no significant difference (P greater than 0.05) in mean values of the factors measured between treatment and control groups.     

Immunohistochemical characterization of estrogen and progesterone receptors in lymphoma of horses

Abstract: Immunohistochemical techniques were used to examine 29 cases of equine lymphoma for estrogen receptor (ER) and progesterone receptor (PR) expression. The lymphomas examined included T-cell-rich large B-cell lymphomas, B-cell neoplasms, and T-cell lymphomas. The individual cases were also classified according to the anatomic location of the tumors. One normal equine lymph node was also examined for ER and PR expression. All of the cases of equine lymphoma and the normal lymph node were negative for ER. A total of 16/29 (55%) PR-positive lymphomas were identified. Seven of the 12 (58%) T-cell-rich large B-cell lymphomas were positive, 7/11 (64%) B-cell tumors were positive, and 2/6 (33%) T-cell neoplasms were positive. Anatomically, 6/9 (66%) subcutaneous lymphomas were PR positive, 3/5 (60%) intrathoracic lymphomas were positive, 1/4 (25%) intra-abdominal lymphomas were positive, 2/5 (40%) intra-abdominal/intrathoracic lymphomas were positive, 1/2 (50%) upper airway lymphomas were positive, and 3/3 (100%) splenic lymphomas were positive. One case involving abdominal and thoracic tumors and leukemia was negative for PR expression. The normal lymph node contained a low percentage (1.9%) of PR-positive lymphocytes. The presence of PR in neoplastic equine lymphoid tissue indicates that these tumors may be responsive to serum progesterone. Also, identification of PR-positive cells in the normal lymph node suggests that PR may be constitu-tively expressed in normal equine lymphocytes. Further studies are needed to quantify PR levels in normal and malignant equine lymphoid tissue and to determine the usefulness of either progestin or antiprogestin drugs in the management of equine lymphoma.     

Immunohistochemical and ultrastructural detection of intestinal spirochetes in Thoroughbred horses.

Studies of equine intestinal spirochetes have long focused on intestinal contents alone, but intestinal spirochetosis has been reported recently in a 21-month-old Thoroughbred colt in Japan. To define the clinical and pathological significances of intestinal spirochetosis in several horses, an epizootiologic survey with histologic, immunohistochemical, and ultrastructural methods was conducted for Brachyspira antigen-containing intestinal spirochetes in 12 diseased or injured Thoroughbred horses, aged from 35 days to 17 years. Brachyspira antigen-containing spirochetes were found in 7 of 12 horses (58.3%) and were more frequent in the cecum than in other parts of the bowel. It was not clear whether the infection was clinically related to diarrhea or dysentery, but histopathology revealed a close association between the bacterial infection and epithelial hyperplasia. Crypt epithelium consisted mainly of goblet cells and showed frequent mitosis throughout its length. Inflammatory cells and congestion were also present. There were numerous spirochetes in the crypts, and some invaded the cecal and colonic epithelia and underlying lamina propria. Ultrastructurally, the spirochetes were divided into 4 types. Three types were identified in degenerative epithelial cells or intracellularly. Brachyspira antigen-containing intestinal spirochetes invading the mucosa were capable of causing epithelial hyperplasia in the cecum and colon in the horses. The findings in this study will increase awareness of the importance of intestinal spirochetosis and may also be helpful for diagnosis and treatment of this condition.     

GnRH受体在山羊结状神经节的分布

取山羊结状神经节5对,采用免疫组织化学SP法观察促性腺激素释放激素(Gonadotropin releasing hormon,GnRH)受体在结状神经节的分布特点。结果显示,GnRH受体在结状神经节上广泛分布,神经元、卫星细胞、神经纤维、血管内皮细胞均有不同程度的免疫阳性染色。在神经元胞体中,细胞膜和细胞质有GnRH受体强阳性产物分布,核不着色;卫星细胞、血管内皮细胞也有强阳性产物分布;神经纤维存在弱阳性产物分布。图像分析结果表明,神经元中GnRH受体的相对表达量与其他非神经结构相比差异性显著(P<0.05)。结果表明,山羊结状神经节中的GnRH受体主要存在于神经元中,山羊结状神经节中的内脏感觉传入神经元对GnRH具有反应性,提示GnRH结状神经节内脏感觉传入神经元的GnRH受体可能作为GnRH对内脏器官的内分泌调节和自主神经对内脏器官的神经调节这2种途径协调作用的节点。     

GnRH受体在山羊结状神经节的分布

取山羊结状神经节5对,采用免疫组织化学SP法观察促性腺激素释放激素（Gonadotropin releasing hormon,GnRH）受体在结状神经节的分布特点。结果显示,GnRH受体在结状神经节上广泛分布,神经元、卫星细胞、神经纤维、血管肉皮细胞均有不同程度的免疫阳性染色。在神经元胞体中,细胞膜和细胞质有GnRH受体强阳性产物分布,核不着色;卫星细胞、血管内皮细胞也有强阳性产物分布;神经纤维存在弱阳性产物分布。图像分析结果表明,神经元中GnRH受体的相对表达量与其他非神经结构相比差异性显著（P〈0．05）。结果表明,山羊结状神经节中的GnRH受体主要存在于神经元中,山羊结状神经节中的内脏感觉传入神经元对GnRH具有反应性,提示GnRH结状神经节内脏感觉传入神经元的GnRH受体可能作为GnRH对内脏器官的内分泌调节和自主神经对内脏器官的神经调节这2种途径协调作用的节点。     

Immunohistochemical evaluation of cyclooxygenase expression in corneal squamous cell carcinoma in horses

OBJECTIVE: To evaluate expression of cyclooxygenase (COX)-1 and COX-2 in the cornea, eyelid, and third eyelid of healthy horses and those affected with squamous cell carcinoma (SCC) by use of immunohistochemical techniques. ANIMALS: 15 horses with SCC involving ocular tissues and 5 unaffected control horses. PROCEDURES: SCC-affected tissues were obtained from the cornea (n = 5 horses), eyelid (5), and third eyelid (5). Site-matched control tissues were obtained from 5 horses unaffected with SCC. Tissue sections of affected and control cornea, eyelid, and third eyelid were stained immunohistochemically for COX-1 and COX-2 via standard techniques. Stain uptake was quantified by use of computer-assisted image analysis of digital photomicrographs. RESULTS: Immunoreactivity for both COX-1 and COX-2 was significantly greater in equine corneas with SCC than in control corneas. No significant differences in COX-1 or COX-2 immunoreactivity were detected in eyelid and third-eyelid SCC, compared with site-matched control tissues. CONCLUSIONS AND CLINICAL RELEVANCE: Immunoreactivity for COX-1 and COX-2 is high in equine corneal SCC, possibly indicating that COX plays a role in oncogenesis or progression of this tumor type at this site. Pharmacologic inhibition of COX may represent a useful adjunctive treatment for corneal SCC in horses.     

Isolation and characterization of alpha 1-acid glycoprotein from horses, and its evaluation as an acute-phase reactive protein in horses.

Equine alpha 1-acid glycoprotein (alpha 1AG) was isolated from equine serum by successive ammonium precipitation, anion- and cation-exchange chromatographies, and gel filtration. Purified equine alpha 1AG had a molecular weight of 46,000 +/- 1,000, and contained 31.4% carbohydrate. Gel isoelectric focusing revealed an isoelectric point range of 2.8 to 3.7. With immunoelectrophoresis, it was found that alpha 1AG migrated to the alpha 1-globulin region. Single radial immunodiffusion was used for quantitative measurement of alpha 1AG in equine serum. In clinically normal foals, serum alpha 1AG was undetectable (less than or equal to 20 micrograms/ml) in less than or equal to 7-day-old foals, but was detected by 14 days. The alpha 1AG concentration (mean +/- SD) increased to reach mean adult values of 99.23 +/- 26.90 micrograms/ml by 1 year of age. The alpha 1AG concentration in pregnant mares decreased at 2 to 3 months before parturition, then gradually increased until 1 day after parturition, when a brief decrease was observed. The concentration increased again at 2 weeks after foaling, then a decrease was observed, after which the alpha 1AG concentration increased again by 2 to 4 months after parturition. The concentration of serum alpha 1AG quickly rose to peak values 2 to 3 days after castration and jejunojejunostomy in adult horses, returning to baseline values by 14 to 28 days after surgery. The alpha 1AG was concluded to be an acute-phase reactive protein in horses.