Identification of bovine doppel protein in testis, ovary and ejaculated spermatozoa

Doppel (Dpl) protein is a recently identified prion-like protein. Although Dpl might be expressed in the brain after prion gene deletion, in both human and mice Dpl is normally expressed only in testis and spermatozoa, where it appears to be involved in male fertility. Little information is available so far about the expression pattern of Dpl in bovines, thus, hampering possible research on the role of this protein in bovine infertility. We have thus, designed, produced and validated through Western blotting a polyclonal antibody against bovine Dpl. With this antibody we then screened bovine tissues for Dpl expression by immunohistochemistry. Ejaculated spermatozoa were screened by flow cytometry and immunocytochemistry. Bovine Dpl was expressed in all the developing stages of germinal cells, from spermatogones to ejaculated spermatozoa, in Sertoli cells and in ovarian follicles (granulosa cells and follicular fluid). Dpl immunoreactivity was also found on other tissues, where endothelial cells, peripheral nerves and scattered lymphocytes stained positive. This distribution pattern suggests that Dpl might be involved in sperm maturation/capacitation in bovines, like it might be in mice. This hypothesis needs to be verified by widespread application of the flow cytometric protocol established in this paper on spermatozoa from animals with reduced fertility

Probable role of Brain Natriuretic Peptide (BNP) in lung hypertension secondary to scleroderma

BACKGROUND Scleroderma, when complicated with pulmonary hypertension (PHT), presents a worse prognosis; recently treatment with new drugs seems to offer good perspectives, especially in early diagnosis and treatment. The standard approach for diagnosing PHT consists in measurement of the pulmonary artery pressure (PAP) by means of echodoppler. AIM OF INVESTIGATION Aim of this work is evaluating the significance of the NT-proBNP parameter, matched to echodoppler, in diagnosing scleroderma PHT. MATERIALS AND METHODS Sixty (60) patients, who came to observation for progressive systemic sclerosis underwent echodoppler in order to measure the PAP (normal values up to 30 mmHg). NT-proBNP was determined on serum sample using ECLIA method by Modular E170 (Roche Diagnostics); manufacturer reference values for age and gender were used. Forty-three (43) patients underwent a further NT-proBNP sampling 5 days later in order to assess parameter stability. RESULTS PHT and non- PHT patients showed statistically different (p < 0,001) medians (126 vs 69 pg/ml). No pathologic values of NT-proBNP were measured in the group with PAP < 30 mmHg, while 27% of cases who had PAP between 30 and 40 showed pathologic concentrations. The positivity ratio increases to 57% in patients showing PAP > 40 mmHg. No relevant correlation (r = 0,2) was found between PAP and NT-proBNP. Mean average between the two sampling groups was 31%. CONCLUSIONS In scleroderma patients, combination of NT-proBNP and PAP seems to improve the diagnosis of pulmonary hypertension, especially in presence of borderline pulmonary pressure values. We therefore propose the biochemical observation of NT-proBNP when PAP is > 30 mmHg and in monitoring the evolution of the pathology

Production and characterization of a polyclonal antibody against the bovine prion-like doppel protein

PRODUCTION AND CHARACTERIZATION OF A POLYCLONAL ANTIBODY AGAINST THE BOVINE PRION-LIKE DOPPEL PROTEIN Marco Rondena, Fabrizio Ceciliani, Chiara Bazzocchi, Saverio Paltrinieri Dipartimento di Patologia Animale, Igiene e Sanit\ue0 Pubblica Veterinaria \u2013 Milano AIM OF THE STUDY: Doppel (Dpl) is a prion-like protein recently identified in mammalian. The sequence is notably homologous to that of PrP. Dpl is associated to neurodegeneration when ectopically expressed in transgenic mouse. The aim of the study is to raise and characterize a polyclonal antibody (pAb) against bovine Dpl to study the tissue distribution of this protein. MATERIAL AND METHODS. A synthetic peptide spanning from residues 67 to 81 of bovine Dpl sequence was synthetized. Using this peptide, a pAb (Dpl 67-81), was raised in rabbit and purified by means of Protein A affinity chromatography. In order to test the specificity of Dpl 67-81, bovine Dpl was cloned and overexpressed in E.coli in the expression vector PQE30 and purified following methal chelate affinity and bovine PrP was purified by means of cation-exchange chromatography and cobalt-chelate affinity chromatography. Homogenate of tissues were tested for the presence of Dpl after SDS-PAGE and Western Blotting. Blots were visualized using ECL. Immunohistochemistry on cryostatic sections using the avidin-biotin complex methods was performed. RESULTS: Dpl 67-81 strongly react against rDpl (titre =1:20000) and not against bovine PrP. Dpl was detected in testis, spleen, limphnodes by both immunoblotting and immunohistochemistry. In particular endothelial and stromal cells showed a granular cytoplasmic positivity. CONCLUSIONS: Dpl 67-81 can detect the expression of Dpl in tissues using both Western Blotting and immunohistochemistry. Moreover, it does not crossreact with PrP, and therefore it could be also used in experiments of functional blocking of Dpl in order to study its function and its relationship with PrP

Pathologic and immunohistochemical findings in a feline aortic body tumor

The presence of a heart-base tumor was diagnosed by ultrasound imaging in a 10-year-old, female, domestic shorthaired cat presenting with dyspnea and pleural effusion because of the presence of a modified transudate. Hematology and clinical chemistry were unremarkable. The owner elected euthanasia. At necropsy, a locally extensive, firm, multilobulated nodule surrounded the pulmonary vein. The tumor was composed of lobules of large polygonal cells separated by a fine fibrovascular stroma. Tumor cells infiltrated the myocardium, and neoplastic emboli were present, but no metastases were macroscopically detectable. Tumor cells were immunohistochemically positive for chromogranin A, for synaptophysin and, faintly, for neuron-specific enolase and negative for vimentin, cytokeratin, alpha smooth muscle actin, glial fibrillary acidic protein, thyreoglobulin, and calcitonin. Based on histologic and immunohistochemical findings, the diagnosis of chemodectoma was made

Immunohistochemical study on the relationship between doppel protein and bovine leukocytes.

Doppel (Dpl) is a prion-like protein whose sequence is notably similar to that of prion protein (PrP). Dpl is mainly expressed on testis and ovary and it is associated to neurodegeneration when ectopically expressed in transgenic mouse. PrP expression has been identified on tissue and blood leukocytes. Also Dpl has been detected on leukocytes located in germinal centers of lymphoid organs. Aim of this work is to immunohistochemically verify if also Dpl is expressed on leukocytes. This would help us to understand the biology and the mechanism of invasion of prion related protein. Samples of spleen and lymph nodes were collected from clinically healthy bovine at slaugthering. Tissues were frozen in liquid nitrogen. Cryostatic sections were stained by immunohistochemstry using the ABC technique: bovine Dpl was identified using a polyclonal antibody; leukocyte subsets were identified with monoclonal antibodies against the following antigens: CD1 (dendritic cells); CD4 (T4 cells); CD8 (T8 cells); CD11b (monocytes and granulocytes); CD11c (myeloid cells); CD14 (macrophages) CD21 (B cells); TCR \uf067/\uf064 (T cell subpopulations); MHC II (activated leukocytes). Bovine Dpl was occasionally identified on spleen and lymph nodes. The distribution of the different leukocyte subsets in these organs, as defined by using the specific monoclonal antibodies, was normal: the Dpl-expressing leukocytes were morphologically identifiable as lyymphocytes and were localised at the periphery of germinal centers, where most of the lymphocytes were B-positive. The large part of the cells detectable on lymphoid organs, however, was Dpl negative. Based on their morphology, localisation and immunophenotype, the Dpl positive cells in spleen and lymph nodes might be interpreted as B lymphocytes. Nevertheless, most of the cells from lymphoid organs did not express, Dpl. This suggests that the positive cells might be derived from blood. The flow cytometric identification of Dpl on blood leukocytes is required to confirm this hypothesis

Common bile duct obstruction due to a duodenal gastrinoma in a dog

in dogs gastrinomas are rare endocrine neoplasms that have always been reported to arise from the pancreas. We report here what we believe to be the first case of a duodenal gastrinoma in a dog. A nine-year-old, male, Pekinese dog was presented with a three-day history of anorexia, vomiting and mucous diarrhoea. Clinical examination and laboratory findings suggested the presence of a severe hepatobiliary disorder. Abdominal ultrasonography showed a diffuse increase in echogenicity of the liver, with severe gallbladder dilation and marked dilation of the cystic duct, common bile duct and extrahepatic bile ducts. Based on these findings, an extrahepatic biliary tract obstruction (EBTO) of unknown cause was suspected. At laparotomy, the gallbladder and the extrahepatic bile ducts appeared severely dilated. The gallbladder was tense and could not be compressed suggesting an outflow obstruction. The duodenum at the level of the common duct orifice appeared slightly thickened and severely hardened for a length of 1 cm. Biopsies from the duodenum and liver were obtained and a cholecystoduodenostomy was performed. The duodenal biopsy revealed severe fibrosis of the submucosa and a infiltrate of small pockets and cords of round to polygonal cells with granular cytoplasm. Based on this appearance the differential diagnoses included neuroendocrine tumours and poorly differentiated carcinoma. Despite surgery and supportive therapy the dog continued to be anorexic and to vomit 3-6 times daily. After euthanasia and necropsy, histopathology showed the presence of a neuroendocrine neoplasia involving the duodenal wall with focal invasion of the adjacent pancreas and small liver metastases. On immunohistochemistry, the cytoplasm of approximately 90% of neoplastic cells intensely expressed neuron specific enolase and gastrin. These findings were consistent with a diagnosis of gastrimoma. (c) 2004 Elsevier Ltd. All rights reserved

Immunohistochemical characterization of the leucocytic infiltrate associated with canine seminomas

Both canine and human seminomas are typically associated with leucocytic infiltration, the role of which remains poorly understood. In this study, leucocytes infiltrating 10 canine seminomas were characterized. Monoclonal antibodies directed against CD18, CD11a, CD11b, CD11c, CD21, CD3, CD4, CD8 and Major Histocompatibility Complex class I and II (MHC I and MHC II) were also employed. Infiltrating leucocytes were located around vessels, adjacent to the thin fibrous septa between neoplastic lobules, and were also scattered singly amongst neoplastic cells. The more profuse infiltrates often had the appearance of follicular aggregates. Immunohistochemically, all the samples showed generally similar results. Most of the infiltrating cells were positive for CD18 and CD11a. Infiltrating cells were mainly T lymphocytes (CD3\ufe), particularly of the CD8\ufe subset. B lymphocytes (CD21\ufe) were detectable in almost all infiltrates; in the follicular aggregates they were centrally located, whereas T lymphocytes (CD3\ufe) lined the periphery. CD11c\ufe cells were always more numerous than CD11b\ufe cells, demonstrating that if macrophages and antigen-presenting cells (APCs) were well represented, monocytes and granulocytes were practically absent. Almost all of the infiltrating cells were positive for both MHC I and MHC II antigens and, in nine samples, a large number of neoplastic cells expressing MHC I were detected. A few neoplastic cells expressing MHC II were observed in seven cases. The presence of CD8\ufe lymphocytes, together with the large number of both infiltrating and neoplastic cells expressing MHC I, suggests that the lymphocytes mediate a cytotoxic reaction against the neoplastic cells. This hypothesis may underlie the favourable prognosis frequently associated with canine seminomas

Bovine Doppel (Dpl) and prion protein (PrP) expression on lymphoid tissue and circulating leukocytes

Doppel (Dpl) protein shares some structural features with prion protein (PrP), whose pathologic isoform (PrPSC) is considered to be the causative agent of transmissible spongiform encephalopathies. Dpl is mainly expressed in testes but, when ectopically expressed in the central nervous system, is neurotoxic. We have examined the expression pattern of Dpl and PrP on bovine lymphoid tissues and circulating leukocytes. A polyclonal anti-Dpl antibody along with a panel of monoclonal antibodies specific for leukocyte membrane antigens or PrP were used to examine frozen sections from spleen, lymph nodes, and bone marrow by immunohistochemistry. Blood was analyzed by flow cytometry. Double staining was used to study the possible coexpression of the two proteins and to characterize cells expressing Dpl and/or PrP. Dpl was expressed in B-cells, in dendritic cells within lymphoid follicles, bone marrow, circulating myeloid cells, and circulating B-cells. The distribution of Dpl was quite similar to that of PrP. The only differences in expression observed concerned the low number of Dpl+ cells in lymph nodes and the strong Dpl positivity of circulating granulocytes. The two proteins were rarely co-expressed, suggesting an independent expression mechanism in resting cells. The role of Dpl+ leukocytes in the pathogenesis of Dpl- or PrP-induced diseases merits further investigation