Expression of 11ß-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptors in reproductive tissue of male horses at different stages of sexual maturity.

Glucocorticoids (GCs) as mediators of the stress response may affect Leydig cell function by inhibiting either luteinizing hormone receptor expression or testosterone biosynthesis. The isozymes 11ß-hydroxysteroid dehydrogenase (11ßHSD) 1 and 11ßHSD2 control the intracellular cortisol levels. Little is known about the effects of stress on fertility in the equine. The objective of the present study was to determine the presence and cellular localization of glucocorticoid receptors (GCR) and glucocorticoid-metabolizing enzymes (11ßHSD1 and 11ßHSD2) in equine epididymal and testicular tissue with special regard to sexual maturation. Testicular and epididymal tissue was collected from 21 healthy stallions, and four age groups were designed: pre-pubertal, young, mature and older horses. Immunohistochemistry (IHC) analysis and quantitative real-time PCR (qRT-PCR) were used. Pre-pubertal horses showed higher testicular gene expression of 11ßHSD1, 11ßHSD2 and GCR than horses of all other groups (p < 0.05). A positive intranuclear immunoreaction for GCR was seen in epithelial cells of caput, corpus and cauda epididymidis and in Leydig cells. Significant differences (p < 0.05) between age groups occurred. The number of Leydig cells staining positive for GCR was highest in immature stallions (p < 0.05). The enzyme 11ßHSD1 was localized in epithelial cells of the caput and corpus epididymidis and in Leydig cells. As determined by enzyme assay, nicotinamide adenine dinucleotide (NAD)-dependant dehydrogenase (oxidation) activity was not detected in testicular tissue from immature stallions but in all other age groups (n = 3 per group). Results of this study suggest a contribution of GCs to maturation of male reproductive tissue in horses. In mature stallions, expression of 11ßHSD enzymes and the oxidative 11ßHSD activity in Leydig cells and epididymal basal and principal cells suggest a protective role on these tissues contributing to physiological intracellular glucocorticoid concentrations.

Sex-dependent insulin like growth factor-1 expression in preattachment equine embryos.

An adjustment of sex ratio of offspring to the conditions present at conception is seen in many mammals including horses. This depends on preferential survival of male embryos under conditions of high energy intake. In several species, growth factors including insulin like growth factor (IGF)-1 have been shown to promote embryonic development by decreasing apoptosis and increasing cell proliferation. We hypothesized that sex-related differences in IGF-1 expression in equine embryos during the phase of maternal recognition of pregnancy might exist and thus contribute to preferential survival of embryos from either of both sexes under specific environmental conditions. Insulin like growth factor-1 mRNA expression of in vivo-produced equine embryos on different days of pregnancy (Day 8, N = 6; Day 10, N = 8; Day 12, N = 14) was analyzed. Insulin like growth factor-1 mRNA expression was evaluated by reverse transcription quantitative polymerase chain reaction. The sex of the embryo was determined by detection of X-inactivation specific transcript (Xist) RNA and equine sex determining region of the Y chromosome DNA. Embryos positive for Xist expression were classified as female, and Xist negative and equine sex determining region of the Y chromosome positive embryos were classified as male. From 28 embryos tested, 15 (54%) showed positive Xist expression and were thus classified as female. Insulin like growth factor-1 mRNA expression was influenced by sex (P = 0.01) but not by day of pregnancy (relative expression of IGF-1 in relation to ß-actin, Day 8: male 5.1 ± 2.1, female 11.4; Day 10: male 5.2 ± 1.6, female 17.4 ± 6.7; Day 12: male 2.6 ± 0.3, female 11.6 ± 2.4). Results demonstrate an increased expression of IGF-1 in female equine embryos. Sex-related influences on expression of the IGF system are probably related to a gradual X chromosome inactivation.

Progestin treatment does not affect expression of cytokines, steroid receptors, oxytocin receptor, and cyclooxygenase 2 in fetal membranes and endometrium from pony mares at parturition.

In most mammalian species, progestins have a major function in maintaining pregnancy. In humans, the physiologic initiation of parturition bears similarities with inflammatory processes and anti-inflammatory effects of progestins have been suggested to postpone birth until term. To examine if comparable effects exist in the horse, mares were treated with the synthetic progestin altrenogest from day 280 of gestation until parturition (N = 5) or were left untreated as controls (N = 7). Tissue from the amnion (AMN), allantochorion (AC), and endometrium (EM) was collected at foaling and mRNA expression of interleukin (IL)-6 and -8, cyclooxygenase 2 (COX2), estrogen receptor (ER) α, progesterone receptor, and oxytocin receptor (OTR) was analyzed. Leukocytes, steroid receptors, COX2, and OTR were also investigated by histology and immunohistochemistry. Expression of mRNA for IL-6 was higher in AMN and EM versus AC (P < 0.01). Expression of IL-8 was higher in AMN than AC and EM (P < 0.001). Steroid receptors and OTR were highly expressed in EM but not in AMN and AC (P < 0.001). Expression of COX2 was most pronounced in AC whereas IL expression was not upregulated in AC. No differences in mRNA expression existed between altrenogest-treated and control animals. Endometrial polymorphonuclear leukocytes were increased in altrenogest-treated mares. Epithelial cells of all tissues, except AC chorionic villi stained progesterone receptor-positive. Staining for ER was more pronounced in the amnion facing epithelium of the AC in altrenogest-treated versus control animals (P < 0.01). In conclusion, COX2 is highly expressed in the AC. The fetal membranes thus might play a role in the onset of labor in the horse. Altrenogest did not affect gene expression in the AMN, AC, and EM but had localized effects on inflammatory cells and ER expression. No anti-inflammatory effects of altrenogest in healthy, late pregnant pony mares could be detected.

The morphological basis of proestrus endometrial bleeding in canines.

Endometrial bleeding during proestrus is a well-known phenomenon in the bitch. However, the exact events on the cellular level have not been studied. In the present investigation, immunohistochemical methods and transmission electron microscopy were employed to obtain more information about this cyclic event in canines. Long, stretched blood vessels were seen in H&E stained sections during proestrus. These vessels showed mitotic activity, as evidenced by Ki67 immunostaining. Although the endothelial lining and basement membrane of endometrial blood vessels seemed continuous, as indicated by immunohistochemical staining for laminin and Von Willebrand factor, transmission electron microscopy showed an extreme thinning and even interruption of the vascular wall in endometrial venules. Platelets were frequently seen in those areas, and also detected by immunohistochemistry. Interestingly, all endometrial capillaries examined by electron microscopy had an intact wall. We therefore postulate the endometrial venules to be the blood vessels that are mainly responsible for proestrus endometrial bleeding, rather than subepithelial capillaries.

Mammary gland secretory concretions contain non-collagenous bone matrix proteins.

Secretory concretions in mammary gland alveoli are commonly of microscopical size. However, some concretions reach clinically palpable dimensions and may occlude teat canals and obstruct milk flow. We studied secretory concretions in sheep, goat and cow mammary glands, using routine histological staining methods, conventional histochemistry and electron microscopy. As concretions frequently mineralize, immunostaining for keratan sulphate and calcium-binding non-collagenous bone matrix proteins (bone sialoprotein, osteocalcin, osteonectin and osteopontin) was performed. Concretions consisted of organic matrix (condensed secretions) with calcium precipitates. Mineralized deposits mostly show concentric organization, bound haematoxylin, and were readily identified in H&E-stained sections. Mineral components of concretions reacted for calcium carbonate and phosphate, organic matrix was found to contain sialoglycan material. Immunohistochemistry revealed bone sialoprotein, osteonectin and keratan sulphate in cow and goat concretions. Osteocalcin was detected in sheep, cow and goat concretions, whilst osteopontin was not identified in any of the specimens studied. Our results indicate the presence of non-collagenous bone matrix proteins (except osteopontin) in mammary gland concretions. These glycoproteins are commonly thought to govern mineralization of organic matrix and are assumed also to promote mineral deposition in mammary gland secretory concretions. Besides caseins, these particular glycoproteins have to be considered as calcium-binding milk proteins.

Localization of matrix metalloproteinases, (MMPs) their tissue inhibitors, and vascular endothelial growth factor (VEGF) in growth plates of children and adolescents indicates a role for MMPs in human postnatal growth and skeletal maturation.

Numerous studies have focused on the expression, regulation, and biological significance of matrix metalloproteinases (MMPs) in the growth plate. Findings in mouse knockout models and in vitro data from various species indicate that MMPs not only degrade extracellular matrix components but may regulate the activity of local growth factors. In this study we investigated the presence, distribution, and activity of various MMPs and inhibitors, tissue transglutaminase (tTG or TG2) and vascular endothelial growth factor (VEGF) in the human child and adolescent growth plates by means of immunohistochemistry and gelatin zymography. Tissue was derived during orthopedic surgery (epiphysiodesis) in two prepubertal and four pubertal patients.MMP-2 and MMP-14 were present in reserve cell chondrocytes. MMP-14 was the most prominent MMP within all zones of the growth plate including proliferating chondrocytes. MMP-1 and MMP-13 (collagenases 1 and 3), MMP-9 (gelatinases B), MMP-10, and MMP-11 (stromelysins) and VEGF were positive in hypertrophic chondrocytes and osteoblasts. MMP-2 showed the same expression pattern but was negative in osteoblasts. Osteoclasts stained positive for MMP-9, MMP-2, and TG2. Tissue inhibitor of MMP (TIMP)-1 was present in all zones of the growth plate, osteoblasts, and osteoclasts; TIMP-2 was found in hypertrophic chondrocytes and osteoblasts. In summary, the presence of MMPs, TIMPs, TG2, and VEGF in our study indicated that the MMPs are relevant in growth plate physiology during the postnatal period in humans. The specific location of MMP expression within the growth plate may be the basis for further studies on the role of MMPs in the local regulation of chondrocyte differentiation, proliferation, and ossification at the chondroosseus junction.

Digital cushions in horses comprise coarse connective tissue, myxoid tissue, and cartilage but only little unilocular fat tissue.

Digital cushions were studied in horses with particular reference to vascularization, tissue constituents and matrix components. The cushions mainly resembled a network of coarse collagen bundles. The areas inbetween the bundles were replenished with loosely woven interstitial connective tissue, myxoid tissue, and fibrocartilage. Expected masses of fat lobules were missing: only solitary adipocytes or small groups of adipocytes were seen. Vascular supply to the cushions was remarkably poor. The mucinous myxoid matrix largely consisted of hyaluronan with little sulphated glycosaminoglycans. Myxoid cells were stellate or ramified in shape and showed a tendency to store glycogen and lipid droplets. Myxoid cells reacted for vimentin and stained for S-100 protein. Moreover, myxoid cells often reacted for neuron specific enolase and glial fibrillary acidic protein. Myxoid tissue continuously transformed into loosely organized interstitial connective tissue with fibroblasts, which remained unreactive when tested for neuroectodermal markers. Myxoid tissue also was not clearly demarcated against irregularly interspersed islets of fibrocartilage or hyaline cartilage. Chondrocytes did not stain for neuron specific enolase but reactivity for S-100 protein and glial fibrillary acidic protein was noted in peripheral regions of fibrocartilage. Single or grouped unilocular fat cells were rarely placed into myxoid areas. Unilocular fat cells stained for vimentin, S-100 protein, and occasionally for glial fibrillary acidic protein but not for neuron specific enolase. Continuous transformation of myxoid tissue into cartilage together with corresponding reactivity for neuroectodermal marker proteins of myxoid cells and peripherally located chondrocytes suggest close relationship between myxoid cells and chondrocytes. The same criteria indicate relationship between myxoid cells and adipocytes. Coarse connective tissue, myxoid tissue, fibrous cartilage, and fat cells are functionally combined to absorb mechanical shock in the horse digital cushions.

Mineralised deposits in the uterine glands of mares with chronic endometrial degeneration.

Chronic degenerative disease of the mare's endometrium is characterised by changes in the uterine glands, including cystic dilation, hyperplasia and periglandular fibrosis. Endometrial biopsies were taken from 23 mares with different grades of endometrial degeneration. Solid structures were identified within the lumina of the uterine glands and shown to be calcified by histochemical staining. Most of them were not homogenous but composed of a mixture of mineral and organic substances. Further examinations of these mineralised structures by immunohistochemical methods revealed the presence of the non-collagenous matrix proteins osteopontin, osteonectin and bone sialoprotein, which are known to be involved in calcification processes such as urolithiosis. Osteopontin and bone sialoprotein were identified within the calculi, frequently arranged in concentric layers. Osteonectin was the only matrix protein that was also present in the glandular epithelium. Osteocalcin was not found in either the calculi or the glandular epithelial cells.

Estrogen receptor-alpha and estrogen receptor-beta are present in the human growth plate in childhood and adolescence, in identical distribution.

OBJECTIVE: To localize estrogen receptor-alpha (ER-alpha) and estrogen receptor-beta (ER-beta) within the growth plate and adjacent bony tissue of children in the prepubertal and pubertal age period. METHODS: Tissue was taken during orthopedic surgery (epiphysiodesis) for correction of congenital or traumatic leg length difference in 2 prepubertal females and 2 adolescent males. Immunohistochemistry was performed on paraffin-embedded or cryostat sections by using commercially available rabbit polyclonal antibodies for ER-alpha and ER-beta. RESULTS: Both ER-alpha and ER-beta were detected within the growth plate in all sections investigated. Immunostaining was restricted to hypertrophic chondrocytes. In the bony tissue adjacent to the growth plate, osteoblasts stained positive for both ER-alpha and ER-beta, whereas osteocytes and osteoclasts were negative. Staining with ER-alpha was mainly nuclear but some cells also showed cytoplasmic signals, while ER-beta staining was predominantly cytoplasmic, only few nuclei stained positive. There was no difference in the local distribution of both ERs between tissue from prepubertal and pubertal patients. CONCLUSION: Our findings indicate that the hypertrophic chondrocyte is the main target cell for estrogen action within the growth plate. The presence of ER in prepubertal children suggests that estrogens play a role in skeletal maturation under physiological conditions also in this age-group.

Integrins and extracellular matrix proteins in the human childhood and adolescent growth plate.

Interaction of chondrocytes with the surrounding matrix significantly influences differentiation and growth. These processes involve cell surface proteins, particularly integrins. The aim of this study was to compare the expression of integrins (alpha1, alpha2, alpha3, alpha5, alpha6, alphav, beta1, beta3, and beta5 subunits) together with matching binding proteins in human childhood and adolescent growth plate cartilage using immunohistochemistry. Integrin beta1 was detected in all chondrocytes of the growth plate cartilage, beta3 only in osteoclasts of the opening zone, and beta5 in hypertrophic chondrocytes and osteoblasts. Integrin alpha1, alpha2, and alpha5 subunits were expressed by chondrocytes in the proliferative and hypertrophic zone as well as in osteoblasts and osteoclasts. Integrin av and alpha6 subunits were present in chondrocytes of all zones, alpha3 only in osteoclasts. Collagen type II and fibronectin were seen throughout the growth plate, collagen type X in the hypertrophic zone, collagen type I in the ossifying trabecules. Laminin was expressed by chondrocytes in the resting zone and more weakly in the proliferative zone, collagen VI was present in the pericellular and interterritorial matrix in all zones of the growth plate. These results differ from previous reports on the distribution of integrins in the fetal growth plate. However, there was no difference in integrin expression in children before and during puberty. Our results indicate that integrin expression is not influenced by endocrine factors during sexual maturation and suggest that the process of skeletal maturation is not regulated via altered integrin expression.

Evidence for distinct mechanisms in the shaping of the CD4 T cell repertoire in histologically distinct myasthenia gravis-associated thymomas.

The major histocompatibility complex (MHC) class II is involved both in thymocyte maturation and peptide presentation and might thus play a key role in the pathogenesis of paraneoplastic myasthenia gravis (MG) in thymomas. To further investigate this issue, we analyzed and scored the expression of epithelial class II expression in 35 thymomas (medullary, MDT; mixed, MXT; cortical and well differentiated thymic carcinoma, CT/WDTC) and correlated it with the histological tumor subtype, prevalence of MG and thymocyte maturation, which was analyzed by flow cytometry and RT-PCR. Our results show that both MHC class II expression and thymocyte maturation are highly dependent on the histological tumor subtype. CT/WDTC retain features of the normal outer thymic cortex, namely substantial MHC class II expression together with normal early thymocyte maturation until late phases of positive selection, but disturbed terminal thymopoiesis. By contrast, MDT and MXT retain features of the normal inner cortex and the medulla with low to absent class II expression and highly abnormal early thymocyte maturation including impaired positive selection, while terminal T cell maturation in MXT appeared undisturbed. There was no correlation between MHC class II expression and MG status for a given tumor subtype. In conclusion, our results provide evidence for a different histogenesis of cortical thymomas and well differentiated carcinomas on the one hand and mixed and medullary thymomas on the other. Decreased expression levels of MHC class II, although of crucial importance for abnormal intratumorous maturation, are not sufficient to explain the emergence of paraneoplastic MG.

Paraneoplastic autoimmunity in thymus tumors.

Autoimmune phenomena are more frequent in thymic epithelial tumors (TET) than in any other human tumor. Mysthenia gravis (MG) is by far the most common autoimmune disease in thymoma patients. MG is characterized by muscle weakness due to autoantibodies against the acetylcholine receptor (AChR), and CD4+ AChR-specific T cells play a pivotal role for the production of these autoantibodies. About 10% of MG patients have a thymoma and, interestingly, only such thymomas exhibit an MG association that maintains thymuslike morphological and functional features with respect to the homing and differentiation of immature T cells. Since AChR protein is not expressed in thymomas, the specificity of the autoimmunity in thymoma-associated MG is thought to be determined by nonreceptor proteins with AChR epitopes. Such proteins are overexpressed in cortical-type MG-associated thymomas, and medullary thymomas express these proteins at barely detectable levels. Aside from this quantitative difference, the pathogenesis of anti-AChR autoimmunity might be qualitatively different in these thymoma subtypes. Our findings suggest that an antigen-specific abnormal T-cell selection by cortical-type TET may contribute to the pathogenesis of paraneoplastic MG. In contrast, an abnormal (intratumorous) activation of autoreactive T cells may be operative in medullary thymomas.

Abnormal thymocyte development and generation of autoreactive T cells in mixed and cortical thymomas.

To gain insight into the pathogenesis of thymoma-associated myasthenia gravis, thymocyte maturation was investigated in mixed and cortical thymomas by three-color flow cytometry. Although we detected cells at all recognizable stages, we noted an unusual increased percentage of early CD4+/CD3- thymocytes--especially in mixed thymoma--and a pronounced decreased percentage of mature CD4+/CD3+ cells in cortical thymomas as well. The percentage of CD3+/CD69+ cells that arose after positive selection was reduced in both thymoma subtypes compared with control thymuses, which suggests differences in the rate or efficiency of positive selection particularly in mixed thymomas. Mature T cells in 10 of 11 thymomas were not activated in situ as shown by the absence of CD25 expression. After stimulation with recombinant human acetylcholine receptor alpha-subunit fragments, thymocytes from 8 of 11 thymomas of both subtypes proliferated more strongly than those from controls, regardless of whether the donors were myasthenic. Responses of residual thymus cells to tetanus toxoid correlated well with those of autologous blood T cells, whereas those from the thymomas clearly did not--implying minimal colonization of thymomas by mature recirculating T cells. In conclusion, our results show that cortical and mixed thymomas exhibited differences in thymocyte maturation. Nevertheless, both thymoma subtypes seem to contribute to the pathogenesis of paraneoplastic myasthenia gravis by generating naive but potentially autoaggressive T cells; in some thymomas, these cells may then be actively immunized inside the tumor.

Experiencia con tratamiento OMS en enfermedad de Hansen en pacientes multibacilares

El presente trabajo tiene como finalidad la evoluación del tratamiento OMS en pacientes multibacilares a través de dos grupos. Un grupo conformado por 18 pacientes tratados con el plan OMS, y un grupo de 7 pacientes tratados con el plan OMS modificado, siendo este último una variante del plan OMS en el cual la rifampicina es utilizada con una dosis de 600 mg. por día durante un lapso de dos meses y posteriormente continuada con una dosis de 600 mg. por mes. Asimismo, se evalúan en el presente trabajo las baciloscopías con muestras de mucus nasal, lóbulo de orega, musclo y de la impronta de piel, en relación al índice morfológico y al número de bacilos encontrados; las histopatologías, teniendo en cuenta los cambios histológicos y el número y morfología bacilar;los episodios reaccionales; las reacciones reversales y los colaterales de la medicación empleada.

Espiroadenoma ecrino: a propósito de dos casos / Eccrine spiradenoma: apropos of 2 cases

Se comunican dos casos de espiroadenoma ecrino en dos mujeres de 52 años de edad. El primero se localiza en la región axilar y es un tumor hipodérmico, cubierto de piel normal, con dolor punzante a la palpación. El segundo se ubica en la región lumbar, de 5 mm de diámetro, cubierto por piel normal algo pigmentada y doloroso espontáneamente. Se aprovecha la oportunidad para realizar una revisión bibliográfica de las características clínicas, histológicas, patogenia, diagnósticos diferenciales y tratamiento del espiroadenoma ecrino