Bilateral Leydig cell tumor in a six-year-old intersex goat affected by Polled Intersex Syndrome.

A 6-year-old, sterile, Blanca Celtibérica breed adult doe was referred to our faculty. The doe had external female genitalia, a short anogenital distance, and normally shaped udders. Masculinization signs in the head shape and male behavior were also noted at the time of referral. Genetic analysis demonstrated normal 2n = 60 XX karyotype and an absence of the sex-determining region Y (SRY). The animal was homozygous for a DNA deletion responsible for the Polled Intersex Syndrome (PIS). A uterus and 2 uterine horns were present at the postmortem examination. Gartner's ducts and degenerated Wolffian derivatives persisted. There were 2 intra-abdominal testicle-like structures, one of which consisted of epididymal and deferent ducts. An advanced Leydig cell tumor, resulting in almost total destruction of the intratesticular structures, was also observed. Leydig cell tumors usually produce testosterone. Thus, these histologic findings are compatible with the evident virilization.

Identification of avian dendritic cells in the spleen using a monoclonal antibody specific for chicken follicular dendritic cells.

BACKGROUND: In the chicken, circulating antigens enter the splenic white pulp via the Schweigger-Seidel sheaths (ellipsoids), where they are bound by cells, the ellipsoid associated cells (EAC), which are located on the periphery of the ellipsoid. There is an increasing body of evidence that these antigen-binding cells move through the PALS, to be finally located within the germinal centers, where these antigen-transporting EAC function as follicular dendritic cells (FDC). The aim of the current study was to further study the relationship between the EAC, the FDC, and the antigen-bearing EAC which migrate through the splenic white pulp. METHODS: In order to identify the splenic FDC and their presumed migrating EAC precursors in the chicken, we used a monoclonal antibody produced against chicken FDC and an antiserum anti-S-100 protein which identifies chicken dendritic cells in lymphoid organs. RESULTS: Cells reacting with the 74.3 monoclonal antibody, which identifies FDC, were found within the germinal center, around the penicilliform capillary, in the periellipsoidal white pulp, and in the periarteriolar lymphatic sheaths (PALS). S-100+ cells were found in these same locations. CONCLUSIONS: A comparison between the staining patterns obtained with both antibodies strongly suggested that the intrasplenic distribution of 74.3+ cells was identical with that of FDC, EAC, and antigen-binding EAC migrating in the PALS. Therefore, the 74.3 monoclonal antibody identified not only FDC but also the splenic precursor cells of FDC, in accordance with the hypothesis of the migration of the EAC through the white pulp. S-100+ cells were more numerous than 74.3+ cells, which is in accordance with the fact that S-100 protein antibody stains both FDC and interdigitating dendritic cells (ID). This has allowed us to suggest that 74.3- EAC may represent precursors of ID. The current findings reinforce previous investigations, which provided evidence supporting the migration of EAC through the PALS and further supported the hypothesis which considers EAC precursors of FDC.

Distribution of bursal secretory dendritic cells in the chicken.

BACKGROUND: The bursa of Fabricius provided the microenvironment for B-cell differentiation. Continuous contact between lymphoid cells and antigen in the bursa further suggested that antigenic material has an important influence on the maintenance and development of B cells in the bursa. In addition, a dendritic cell, the bursal secretory dendritic cell (BSDC), has been identified in the medulla. The hypothesis that, in the bursal follicles, the contact between the lymphoid cells and the antigen may be mediated by dendritic cells prompted us to identify a bursal dendritic cell that becomes activated after contact with the antigen. METHODS: A polyclonal antiserum to S-100 protein was used to identify bursal dendritic cells because S-100 protein, a calcium-binding protein, has been shown to be a marker for the identification of chicken dendritic cells following recent contact with antigen. RESULTS: At every age investigated, S-100-positive cells showed a location and shape identical to those described for BSDCs. Positive cells were found within and under the follicle-associated epithelial cells (FAE), indicating that these cells were strategically placed where they would encounter the antigen. In addition, positive cells were found arranged along the corticomedullary junction, which is a regenerative zone for the BSDC. After 10 weeks of age, the number of positive cells dramatically decreased, suggesting that the endocytic activity of the FAE may become impaired as the bursa regresses. CONCLUSIONS: The polyclonal antiserum to S-100 protein identified the BSDCs in the bursal follicles. Positive cells may be BSDCs that have undergone a functional activation after contact with the antigen. These cells may have a role as antigen-presenting cells in the bursal follicles. Hence, these cells may be involved in the events that lead to B-cell differentiation.

Ultrastructural identification of the splenic follicular dendritic cells in the chicken.

BACKGROUND: There is a need to identify the follicular dendritic cells (FDC) of the chicken spleen at the ultrastructural level during a secondary immune response. METHODS: The cells were identified after intravenous priming BSA and boosting with biotinylated BSA conjugated to colloidal gold particles. Monoclonal antibodies raised specifically either to chicken IgG or IgM were used to characterize these immune complex-trapping cells. RESULTS: The FDC had an irregular morphology which varied through time, supporting the existence of two types of FDC in the chicken spleen, one showing filiform cell processes, the other provided with beaded dendrites. When the filiform dendrites were observed, the FDC bound the antigen on their surfaces. These dendrites showed an intrincate convoluted configuration, forming tightly wrapped networks near the cell body. The networks had the same features as those described in mammals as antigen retaining reticulum (ARR). In chickens, the ARR, which represents sites of antigen localization on FDC, reached maximum development on day 5 after the second injection of BSA and had disappeared by day 8. At this time FDC had beaded dendrites. CONCLUSIONS: Antigen is retained on FDC in the chicken spleen for long periods of time.

Antigen-binding cells in the cecal tonsil and Peyer's patches of the chicken after bovine serum albumin administration.

It has been previously reported in the chicken that the ellipsoid-associated cells (EAC), which are considered to be a type of splenic dendritic cell, migrate from the spleen into the blood after binding antigen on their surface. In the current study we traced the localization of these cells within two peripheral lymphoid organs, the cecal tonsil (CT) and the Peyer's patches (PP). The migration of the cells was followed by light microscopy using bovine serum albumin bound to biotin and conjugated to gold particles as a histochemically identifiable antigen detected as peroxidase reaction. The observations showed that the EAC after entering the circulating blood migrated into the lymphoid tissue of the CT and the PP. As a consequence, the antigen-binding cells were found in the diffuse lymphoid tissue and the germinal centers in both lymphoid organs. In the former location they were seen 24 h after the second antigen administration and in the germinal centers on Day 3. In addition, antigen-binding cells started to be observed in the lymphoid tissue at the same time as T and B cells were found to proliferate by using 5-bromo-2'-deoxyuridine. Based upon these findings, we suggested that the EAC have a role as antigen-transporting cells from the spleen to the CT and the PP via the blood stream. Furthermore, our results provided evidence that after the antigen-transporting EAC entered the above-mentioned organs, these cells behaved as antigen-presenting cells in both the T- and B-dependent areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of splenic cells with antigen-transporting capability in the chicken.

BACKGROUND: The objective of the present study is to investigate the migration pattern of the splenic dendritic cell of the chicken named the ellipsoid-associated cell (EAC) from the site of initial location at the periphery of the ellipsoid to the splenic T- and B-dependent areas. METHODS: Bovine serum albumin bound to biotin and conjugated to gold particles was used as a histochemically identifiable antigen detected as a peroxidase reaction. The antigen was intravenously injected, and subsequently its pattern of distribution in a time sequence and within the tissue was examined at the light and electron microscopy levels. In addition, an hour prior to sacrifice, the chickens received a single injection of the thymidine analogue 5-bromo-2'-deoxyuridine, in order to quantify the number of DNA synthesizing cells and to establish a relationship between the migrating EAC and the rate of mitosis in the white pulp. RESULTS: The observations showed that between 12 hours and 3 days after the second antigen administration the labeled EAC, which was first located around the ellipsoid, progressively reached further areas with time towards the periarteriolar lymphoid sheaths, where newly formed germinal centers appeared. Furthermore, the rate of cell proliferation within the white pulp was associated with the arrival of the antigen-transporting EAC. CONCLUSIONS: The results suggest that migrating EAC have a role as both antigen-transporting cell and antigen-presenting cell in the T- and B-dependent areas, as a result of which migrating EAC is transiently found in periellipsoidal white pulp, then periarteriolar lymphoid sheaths, and finally germinal centers, where it may function as an interdigitating cell or as a follicular dendritic cell, depending on its location. Thus, we conclude that the EACs are precursors of both interdigitating and follicular dendritic cells.

Immunocytochemical and immunoelectron microscopy study of dendritic cells in the caecal tonsils of chickens infected with Eimeria tenella.

In the present study S-100 protein containing cells in the caecal tonsil were investigated, both at light microscopic and at electron microscopic levels, after oral coccidia inoculation (Eimeria tenella). The birds were infected with a single (Day 0) or two (Days 0 and 21) infective doses of 500 oocysts. Immunoelectron reactivity for S-100 protein was demonstrated in infected chickens, but not in controls. It was found in follicular dendritic cells and interdigitating dendritic cells which were present in the germinal center and diffuse lymphoid tissue respectively, both of them being located in the deep lymphoid tissue near the muscular layer and around the deep glands. Outside the lymphoid tissue, immunoreactivity for S-100 protein was found in cells lying between the epithelial cells of the deep crypt epithelium. Positivity for S-100 protein was observed at 3, 12, 18, 24 and 48 h after the first inoculation as well as on Days 3, 5, 6, 7, 8, 25 and 30 of the experiment. Positive reaction for S-100 protein was detected both while schizont (the more immunogenic stage) development occurs and the number of sporozoites in the caeca lumen was higher, as well as when the production of oocysts reached a maximum. Complementary studies demonstrated that S-100 positive dendritic cells gave a negative reaction for esterase activity, whereas a subset of S-100 negative intraepithelial lymphocytes located between epithelial cells lining the deep glands exhibited esterase activity. These esterase positive cells are hypothesized to be involved in the regulation of local defences.

Fibronectin synthesis in the harderian gland of the chicken.

Immunoelectron microscopic staining with a monoclonal antibody against fibronectin demonstrated the presence of this extracellular matrix glycoprotein in the avian Harderian gland. Fibronectin was detected as a component of the electron-dense material, which has been observed both between the epithelial cells lining the ducts of the gland and between the lymphoid cells within the subepithelial lymphoid tissue. Additionally, intracellular fibronectin was detected in the rough endoplasmic reticulum, Golgi complex, and secretory vacuoles in the cytoplasm of a cell, showing the ultrastructural features of a myofibroblast. These findings indicate that the Harderian gland myofibroblasts secrete and release fibronectin into the extracellular matrix.

Follicular dendritic cell activation in the harderian gland of the chicken.

The avian follicular dendritic cell changes that occur in the germinal center of the Harderian gland during the course of the immune response were studied by electron microscopy and the immunoperoxidase method was employed for the detection of S-100 protein. The chickens were injected twice with Salmonella O Antigen into the nictitating membrane at 9-day intervals. The follicular dendritic cells exhibited filiform processes at between 24 and 96 h after the second antigen administration. Filiform dendrites tended to convolute near the cell body. Therefore, it can be assumed that these processes make it more difficult for macrophages and B cells to make contact with the immune complexes retained by the follicular dendritic cells and, as a consequence, the period of antigen handling by these cells increases. Evidence is provided that the dendritic processes are closely associated with both lymphoblasts and lymphocytes. Furthermore, S-100 protein was found in the abovementioned cells exclusively and only in those cells where filiform dendrites were observed. These findings suggest that, during a secondary immune response, the follicular dendritic cell undergoes a functional activation which involves morphological changes and the phenotypic expression of the S-100 protein. This activation is hypothesized to be similar to that described for follicular dendritic cells in mammals after fixing immune complex.

Quantitative assessment of ATPase-positive epidermal dendritic (Langerhans) cells in piglets and adult swine.

To assess the distribution pattern of Langerhans cells (LC) in normal porcine skin, epidermal sheets from six anatomical sites from three age-matched groups of male and female pigs were stained for ATPase activity. This histoenzymological technique is considered specific for Langerhans cells in normal epidermis. No statistically significant differences were observed between mean Langerhans cell density per mm2 of epidermis from male and female pigs, nor between different anatomical sites in the same age group. Statistically significant differences (P less than 0.0005) were observed when comparing group A one- to two-week-old piglets (463 to 518 LC mm-2) with group B six month olds (641 to 804 LC mm-2) and group C two years and over sows (741 to 830 LC mm-2). Morphological variations in the skin of young piglets, much thinner and with rudimentary or no epidermal rete pegs, could account for this significant variation.

Early in-vitro histological chondral differentiation.

In vitro chondrogenesis is possible in the chick embryo from stage 4 of Hamburger and Hamilton (1951), only 18-19 hours of incubation, before somite formation. In stage 4 of Hamburger and Hamilton (1951) the chondroblasts are placed laterally to the primitive streak and notochord cells are not necessary for cartilage differentiation.

Ultrastructural localization of a soluble antigen in the chicken Harderian gland.

The relationship between plasma cells, macrophages, B and T cells, dendritic cells, and epithelium in the chicken Harderian gland have been studied by means of ultrastructural localization of the horseradish peroxidase following local immunization. After 5 d, peroxidase activity was found in vesicles located in macrophages and immature plasma cells. On day 7, peroxidase-antiperoxidase complexes were found in vesicles of the epithelial cells lining the secondary ducts and the acini, in the lumina of the ducts, and on the surface of lymphocytes located among these epithelial cells. Dendritic cells showing peroxidase activity on their surface were seen in the subepithelial lymphoid tissue and in the lymphoid follicles. On day 9, peroxidase activity was found as iccosomes on the surface of dendritic cells and lymphoblasts. These results indicate that immature plasma cells in the Harderian gland can take up antigen and may have a role in presenting it to T cells. Further, our results suggest that intraepithelial lymphocytes might be involved in antigen transportation from the epithelium to the subepithelial lymphoid tissue.

Immunocytochemical detection of dendritic cell by S-100 protein in the chicken.

Positivity for S-100 protein in paraffin embedded chicken lymphoid tissue was found by using a polyclonal antibody against whole bovine S-100 protein. The S-100 protein-containing cells were observed in the locations which have been reported to contain avian dendritic cells such as the medulla of the bursal follicles, and the germinal centers and T-dependent areas in the spleen, Peyer's patches, caecal tonsil and Harderian gland. Positive cells were also found in the location where ellipsoid associated cell have been described, and between epithelial cells covering the Peyer's patches and the caecal tonsil, as well as between the cells lining the ducts within the Harderian gland. Macrophages were devoid of immunostaining. Our results confirm the location described elsewhere for chicken dendritic cells and indicate that S-100 protein can be considered as a cell marker for the identification of the chicken dendritic cell. Intraepithelial positive cells may be interdigitating dendritic cells in an unusual location (their function being the transport of the antigen from the epithelium to the diffuse lymphoid tissue), or cytotoxic T-lymphocytes which, in mammals, are immunoreactive for S-100 protein.

Local immune response in the chicken Harderian gland to antigen given by different ocular routes.

The effectiveness of three ocular routes of antigen administration to produce a local immune response in the Harderian gland was studied. The routes were by eyedrop, injection into the ocular conjunctiva and injection into the nictitating membrane. The antigen was observed in the cytoplasm of macrophages located within the lymphoid tissue only after the injection into the nictitating membrane. The numbers of germinal centres and plaque forming cells found in the gland after injection into the nictitating membrane was higher than the numbers observed following the other two ocular applications. These findings indicate that the injection of the antigen into the nictitating membrane is the most effective ocular route for producing a local immune response in the Harderian gland.

Immunoglobulin classes synthesised by the chicken Harderian gland after local immunisation.

The immunoglobulin (Ig) levels in tears and sera were compared after antigen administration (salmonella O antigen) by eyedrop and injection into the nictitating membrane, to determine the Ig classes synthesised by the plasma cells in the chicken Harderian gland. Samples of tears and sera were collected from immunised and control birds between 24 hours and 24 days after the antigen or sterile saline was administered. Samples were assayed for IgA, IgG and IgM concentrations using radial immunodiffusion. It is suggested that most of the IgG found in tears after local immunisation has an extraglandular origin.

Myofibroblasts and myoepithelial cells in the chicken harderian gland.

An electron microscopic study of the myoepithelial cells in the chicken Harderian gland provides evidence that these cells can be transformed into myofibroblasts. After the application of a Brucella ovis suspension in sterile saline onto the eyeball, every 5 minutes for half an hour, myoepithelial cells gradually develop over a 90-minute period the characteristic features of myofibroblasts: bundles of intracytoplasmic microfilament; abundant rough endoplasmic reticulum; prominent Golgi complex; and surface membrane differentiations, that provide attachment to neighbouring epithelial cells. No typical desmosomes are observed. Besides, the intercellular space between epithelial cells and myofibroblasts increases and the basement membrane adjacent to myofibroblasts disappears. Hypoxia is hypothesized to be involved in the transformation of myoepithelial cells into myofibroblasts.

Granulopoiesis in the pineal gland of chickens.

Foci of differentiating heterophilic granulocytes in the pineal gland were studied by light and electron microscopy in chickens, from hatching until 56 weeks of age. Foci of granulopoiesis could be seen in the first 24 hours after hatching. Thereafter, their number and cellular density increased, becoming highest at 2 weeks. From then on, numbers decreased progressively until foci disappeared at 18 weeks. Granulopoietic cells established local associations with fibroblast-like cells. Mature granulocytes reached the bloodstream by the 2 mechanisms described for hematopoietic cells in the bone marrow, either passing between lining cells or through the cytoplasm of lining cells.

Results and histological development of various surgical techniques for correcting eversion of the third eyelid in dogs.

An experimental study was made of 20 dogs in order to compare various surgical techniques used to correct eversion of the third eyelid, namely resection of most of the cartilage, resection of the central portion of the cartilage, and cartilage homotransplantation. An analysis was made of histological results obtained 45 days after operation, the most satisfactory result being recorded for homotransplantation of the third eyelid cartilage.

Histopathological changes in ewe lambs exposed to prolonged diet on lucerne.

Histopathological changes were observed in 10 ewe lambs reared during nine months on lucerne as compared with 10 controls reared on ryegrass. These changes consisted of periglandular oedema and subacute endometritis in the uterus, secretory-cell hyperplasia with prevalence of neutral mucopolysaccharides in the cervix and hyperkeratinisation and colpitis in the vagina. The mammary glands of lambs reared on lucerne were significantly heavier and showed secretory activity.