Immunoexpression of aromatase in immature and adult males of the European bison (Bison bonasus, Linnaeus 1758).

Based on recent literature dealing with the role of oestrogens in the male gonad, attempts were undertaken to reveal the site of aromatization within the testis of the European bison (Bison bonasus). Testes were collected from culled animals living in free-ranging populations in Bialowieza Forest, Poland (nine males aged 8 months to 10 years). Moreover, to check for any alterations in the expression of testicular aromatase between American bison (Bison bison) and European bison, testes from one adult 10-year-old individual were also chosen for this study. For immunohistochemistry, 4% formaldehyde fixative was used. Both qualitative and quantitative evaluations of immunohistochemical staining were performed. Leydig cells, Sertoli cells and germ cells exhibited a positive immunoreaction for aromatase in testes of immature and sexually mature bison. A marked increase in aromatase expression was observed in three adult European individuals with impaired spermatogenesis. Consistent with recent data and those of our own, it might be suggested that the strong expression of aromatase negatively affects spermatogenic function in bison testes and may serve as a possible explanation of specific sperm defects observed in European bison bulls. On the contrary, one cannot exclude that differences in the aromatase immunoexpression levels are attributed to the homozygosity, the cause of frequent disease in European bison.

Morphology and function of human Leydig cells in vitro. Immunocytochemical and radioimmunological analyses.

The aim of our study was to show whether the cells isolated from testes of patients underwent bilateral orchiectomy for prostatic cancer are able to grown in vitro, and if so, are functionally active. Immuncytochemistry was performed to show the functional status of human cultured cells. In detail, immunolocalization of luteinizing hormone receptors (LHR), mitochondria, and cytoskeletal elements was demonstrated. Moreover, radioimmunological assay was used to measure testosterone secretion by cultured Leydig cells. Using Nomarski interference contrast and fine immunofluorescence analysis the positive immunostaining for LHR was observed in almost all Leydig cells, however it was of various intensity in individual cells. Testosterone measurement revealed significant difference between testosterone secretion by hCG-stimulated and unstimulated Leydig cells (p<0.05). Moreover, testosterone levels were significantly higher in 24- and 48- hour-cultures than in those of 72 hrs (p<0.05). Morphological analysis of Leydig cells in culture revealed the presence of mononuclear and multinucleate cells. The latter cells occurred in both hCG-stimulated and unstimulated cultures. In Leydig cells labeled with a molecular marker MitoTtracker, an abundance of mitochondria and typical distribution of microtubules and microfilaments were observed irrespective of the number of nuclei within the cell, suggesting no functional differences between mono- and multinucleate human Leydig cells in vitro. Since the percentage of multinucleate cells was similar in both hCG-stimulated and unstimulated cultures (23.70% and 22.80%), respectively, the appearance of these cell population seems to be independent of hormonal stimulation.

Losartan metabolite EXP3174 reduces the weight of formed thrombus in 2K1C hypertensive rats.

The aim of the study was to evaluate the effect of an active metabolite of losartan - EXP3174 - on a performed venous thrombus in hypertensive rat. The contribution of coagulation and fibrinolytic systems as well as platelets in the EXP3174 action was also determined. Male Wistar rats with renovascular hypertension (2K1C) were used in the study. Stasis-induced venous thrombus was allowed to age for 7 hours before intravenous injection of EXP3174 (10 mg/kg and 30 mg/kg). Thrombus reduction was then evaluated as the difference in thrombus weight 1 hour after drug or its vehiculum administration (8 h-aged thrombus). Euglobulin clot lysis time (ECLT), overall hemostasis potential (OHP), overall coagulation potential (OCP), the time to fibrin generation (TFG), and thrombin amidolytic activity were measured in blood. Collagen-induced platelet aggregation was measured in whole blood. Bleeding time (BT) and systolic blood pressure (SBP) were measured 1 hour after drug administration. EXP3174 in hypotensive doses caused a marked, dose-dependent decrease in venous thrombus weight. Shortened ECLT and decreased OCP, OHP and thrombin amidolytic activity were also observed. Platelet aggregation was significantly decreased after a higher dose of EXP3174, while no changes in BT were observed. Our study provides evidence for reduction of the weight of a formed venous thrombus by losartan metabolite - EXP3174 - in the mechanism involving enhancement of plasma fibrinolytic activity as well as reduced platelet reactivity and coagulation inhibition in 2K1C hypertensive rats.

Connexin 43 expression in human and mouse testes with impaired spermatogenesis.

Connexin 43 (Cx43) belongs to a family of proteins that form gap junction channels. The aim of this study was to examine the expression of Cx43 in the testis of a patient with Klinefelter's syndrome and of mice with the mosaic mutation and a partial deletion in the long arm of the Y chromosome. These genetic disorders are characterized by the presence of numerous degenerated seminiferous tubules and impaired spermatogenesis. In mouse testes, the expression and presence of Cx43 were detected by means of immunohistochemistry and Western blot analysis, respectively. In testes of Klinefelter's patient only immunoexpression of Cx43 was detected. Regardless of the species Cx43 protein was ubiquitously distributed in testes of reproductively normal males, whereas in those with testicular disorders either a weak intensity of staining or no staining within the seminiferous tubules was observed. Moderate to strong or very strong staining was confined to the interstitial tissue. In an immunoblot analysis of testicular homogenates Cx43 appeared as one major band of approximately 43 kDa. Our study adds three more examples of pathological gonads in which the absence or apparent decrease of Cx43 expression within the seminiferous tubules was found. A positive correlation between severe spermatogenic impairment and loss of Cx43 immunoreactivity observed in this study supports previous data that gap junctions play a crucial role in spermatogenesis. Strong Cx43 expression detected mostly in the interstitial tissue of the Klinefelter's patient may presumably be of importance in sustaining Leydig cell metabolic activity. However, the role of gap junction communication in the control of Leydig cell function seems to be more complex than originally thought.

Expression of connexin 43 protein in testes, epididymides and prostates of stallions.

REASONS FOR PERFORMING STUDY: Connexin 43 (Cx43) is a ubiquitously distributed gap junction protein in testes and other reproductive tissues. Adjacent cells share ions and small metabolites through intercellular channels, which are present in gap junctions. Previously, Cx43 has not been reported in testes, epididymides and prostates either in healthy stallions or cryptorchid horses. OBJECTIVES: To demonstrate the expression pattern of Cx43 in the reproductive tissues of stallions and examine whether naturally occurring bilateral cryptorchidism has any influence on distribution and expression of Cx43. METHODS: The expression and the presence of Cx43 protein were detected by means of immunohistochemistry and Western blot analysis using a polyclonal rabbit anti-Cx43 antibody. RESULTS: In stallions, gap junctions appeared as structures localised to cell-cell contacts between adjacent cells. In testes, Cx43 expression was detected in the interstitial tissue and seminiferous tubules, between Leydig and Sertoli, as well as Sertoli and germ cells. In epididymides, Cx43 was localised between epithelial cells, whereas in prostates, between secretory cells of the glandular epithelium. In the cryptorchid, a clear reduction of Cx43 signal was observed in all reproductive tissues. CONCLUSIONS: Coupling of Leydig cells via gap junctions may suggest that steroidogenic function of the testis is under the influence of these intercellular channels. Within seminiferous tubules, the expression was found to be stage-specific, pointing to its role in coordinating spermatogenesis. Differential distribution of Cx43 protein in the reproductive tract of normal and cryptorchid stallions indicates that expression is clearly dependent on the physiological status of the horse. POTENTIAL RELEVANCE: Detection of Cx43 expression in equine testicular, epididymal, and prostatic cells is important for a better understanding of the role of intercellular membrane channels in direct cell communication within the reproductive tract of stallions.

Immunoexpression of androgen receptors in the reproductive tract of the stallion.

The objective of this study was to visualize androgen receptors (ARs) in the testis, epididymis, and prostate of the stallion by means of immunohistochemistry. Nuclear immunostaining was found in all somatic cells in the testis--Leydig, Sertoli, and peritubular myoid cells; in both types of epithelial cells of the epididymis; and in the secretory cells of the prostate. These results indicate that ARs are distributed throughout the reproductive tract cells of the stallion.

The expression of aromatase, estrogen receptor alpha and estrogen receptor beta in mouse Leydig cells in vitro that derived from cryptorchid males.

A broad expression of aromatase and estrogen receptors (ERs) in the testis suggests an important role for estrogens in regulating testicular cell function and reproductive events. The aim of the present study was to show whether Leydig cells in vitro isolated from cryptorchid testes of two inbred strains of mice, KE and CBA, are a site of estrogen synthesis. Using immunocytochemistry, aromatase, estrogen receptor alpha(ERalpha), and estrogen receptor beta(ERbeta) were localized in cultured Leydig cells. Immunoreactive aromatase was found in the cytoplasm of control Leydig cells and those isolated from cryptorchid males, however the intensity of immunostaining was different, being stronger in Leydig cells deriving from cryptorchid mice. The strongest aromatase immunostaining was found in cryptorchid-KE Leydig cells. Strong immunoexpression of ERalpha was detected in the nuclei of both KE-and CBA-Leydig cells. The intensity of ERalpha immunostaining was stronger in cultured cells deriving from cryptorchid testes. ERbeta immunoexpression was detected predominantly in KE-Leydig cells. Control CBA-Leydig cells were negative for ERbeta or the result was inconclusive, whereas in cryptorchid CBA-Leydig cells a weak immunostaining was present in their nuclei. Western blot analysis confirmed the results obtained by immunocytochemistry. In KE- and CBA-Leydig cells aromatase as a band of 55 kDa protein was present, whereas ERalpha molecular weight was 67 kDa on Western blots. No band was detected for ERbeta. Radioimmunological analysis revealed that androgen and estrogen levels secreted by Leydig cells in vitro were strain-dependent. Additionally, in KE-Leydig cells that derived from cryptorchid mice estrogen level was distinctly higher in comparison with that of the respective control.

Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.

A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.

Interleukin-1 and tumour necrosis factor alpha induced polymorphonuclear leukocyte-endothelial cell adhesion and transendothelial migration in vitro: the effect of apical versus basal monolayer stimulation.

The cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) enhance polymorphonuclear leukocyte (PMNL) adhesion to vascular endothelium by an endothelial cell dependent mechanism in vitro and induce PMNL infiltration in vivo In this study, we employed human umbilical vein endothelium (HUVE) cultured on microporous membrane filters to form a monolayer, a system in which PMNL adherence and PMNL transendothelial migration could be measured using 51Cr-labelled human PMNL. In this system, it was found that PMNL adhesion and migration were dependent on prior treatment of the HUVE monolayer with IL-1 or TNF alpha for at least 2 h and that cytokine could be removed prior to the addition of PMNL without any effect on the response. PMNL adherence to the HUVE was maximal by 30 min and was followed by progressive migration of PMNL across the monolayer and the membrane filter into the lower chamber. The effect of apical surface versus basal surface exposure of the HUVE monolayer to IL-1 alpha and TNF alpha on subsequent PMNL interaction with the HUVE monolayer in the absence of cytokine was examined. Apical or basal stimulation induced comparable PMNL adherence at 30 min following addition of PMNL (35.5% and 43.1%). However, basal (i.e., abluminal) exposure to IL-1 or TNF alpha of the HUVE induced significantly greater PMNL transendothelial migration (e.g., 27.8% vs. 15.4%; P less than 0.01). The expression of endothelial-leukocyte adhesion molecules ELAM-1 and ICAM-1 following apical versus basal stimulation was determined by ELISA on viable cells. These adhesion molecules were upregulated to a similar extent under both conditions. These observations suggest that spacial localization or orientation of adhesion molecules may be influenced by basal versus apical cytokine stimulation or that other mechanisms are responsible for the preferential PMNL migration with basal stimulation. These findings may have implications for the in vivo interactions of PMNL with vascular endothelium, depending on whether the endothelium is exposed to IL-1 of TNF alpha via the blood on the luminal (apical) surface or via the extravascular space on the abluminal (basal) surface.

The effect of testicular macrophages, macrophage-conditioned medium and interleukin-1alpha on the cytoskeleton of bank vole Leydig cells.

Recently, morphological and functional interactions between cytoskeletal elements and their involvement in cell movements, shape changes and/or translocation of organelles have been intensively studied. Thus, the aim of our work was to determine whether testicular macrophages and/or their products have an influence on Leydig cell cytoskeleton. The source of Leydig cells and macrophages were male bank voles from spring and autumn generations, reared in different regime of light for 7-8 weeks. The Leydig cells were growing in monocultures or in co-cultures with testicular macrophages. All cell cultures were divided to controls or human chorionic gonadotropin-stimulated ones. To some of the cultures testicular macrophage-conditioned medium or interleukin-1alpha were added. The cells were analysed immunocytochemically and radioimmunologically. In Leydig cells obtained from animals kept in a long day, grown in co-cultures with macrophages as well as in those stimulated by testicular macrophage-conditioned medium, distinct rearrangements of microtubules and microfilaments were observed. This phenomenon was strengthened in the presence of hCG in culture media. Concomitantly, basal and hCG-stimulated level of testosterone was enhanced, which indicates the possible involvement of the cytoskeleton in the process of androgen biosynthesis. The influence of IL-1alpha on reorganization of cytoskeletal structures was not observed, suggesting that in the modulation of steroidogenesis by this cytokine cytoskeletal elements do not play an important role.

Lysosomal acid phosphatase activity and progesterone secretion by porcine corpora lutea at various periods of the luteal phase.

Four types of porcine corpora lutea were excised at four different stages of the luteal phase. One group of corpora lutea was homogenized and the concentration of progesterone was assayed. The second part was homogenized for measurement of acid phosphatase (ACPase) activity. The third part was sectioned at -20 degrees C in a cryostat and stained histochemically for acid phosphatase activity. The product of acid phosphatase reaction indicated fewer lysosomes in the luteal cells of early developing corpora lutea, increasing as luteal phase progressed. As the level of progesterone declined in the terminal phase of the cycle, there was a maximal increase in the number of lysosomes and ACPase activity. These events resulted in final regression of corpora lutea. The decrease of the lysosomal reaction in the late regressing corpora lutea is probably due to the extreme fragility of the lysosomes at that stage.

Immunofluorescent localization of the StAR protein in mitochondria of mouse Leydig cells in vitro.

The Steroidogenic Acute Regulatory (StAR) protein is assumed to enhance the rate-limiting step of the steroid biosynthesis. Now, it is the most likely candidate, responsible for acutely regulating transfer of cholesterol from the outer to the inner mitochondrial membrane. In this study, the immunoreactive StAR protein was observed in the mitochondria of mouse cultured Leydig cells stimulated by hCG andtesticular macrophage-conditioned medium. Immunocytochemistry was performed using a polyclonal rabbit antibody against the StAR protein. For selective staining of mitochondria in Leydig cells, the Mito Tracker dye was used. Computerized, superimposed images from double-fluorescence staining showed a remarkable degree of similarity in the distribution of the StAR protein and mitochondria, indicating mitochondrial localization of StAR.

Localization of cytochrome P450 aromatase and estrogen receptors alpha and beta in testicular cells--an immunohistochemical study of the bank vole.

Age- and light-dark cycle-induced changes in immunoexpression of aromatase and estrogen receptors alpha and beta were studied in testes of a seasonally breeding rodent, the bank vole. Seasonal breeding can be mimicked by exposure to different light cycle regimes. In testes of animals that were exposed to long light cycles of 18 h light and 6 h darkness aromatase was in Leydig cells and seminiferous tubules, mainly in spermatocytes, whereas in animals exposed to short light cycles (6 h light and 18 h darkness), only Leydig cells exhibited positive immunostaining for aromatase. Whatever the age of animals, immunostaining for estrogen receptor alpha was restricted to Leydig cells, whereas estrogen receptor beta immunoreactivity was mainly confined to Sertoli cells of both of immature and adult animals, independently of the regimes of light. Additionally, in testes of animals that were exposed to long light cycles, estrogen receptor beta immunoreactivity was observed in seminiferous tubules. Nuclei of germ cells, predominantly spermatocytes and elongated spermatids, were strongly positive which correlated well with aromatase immunoreactivity. As far as we know, the present study is the first study that describes immunoexpression of aromatase and both estrogen receptors alpha and beta in testis of the bank vole. We provide strong evidence that estrogens are not only produced in Leydig cells but also in germ cells in this rodent. These female hormones may play a physiological role in testis, likely in the development of germ cells during spermatogenesis.

[Radiological evaluation of pineal pathology and its regions]. / Diagnostyka radiologiczna patologii szyszynki i jej okolicy.

Contemporary neuroimaging procedures facilitate the differential diagnosis of pineal region pathology. They are of particular importance in malignant pineal tumours which may have non characteristic clinical presentation. Computed tomography and magnetic resonance imaging with intravenous infusion of paramagnetic contrast allow to determine tumour size and localization as well as its morphologic features and growth dynamics. The diagnosis obtained is a valid basis for treatment strategies.

[The battle against acute infections in Poland between the two world wars of the twentieth century, 1918-1939]. / Zwalczanie ostrych chorób zakaznych w Polsce w dwudziestoleciu miedzywojennym, 1918-1939.

In the health policy of the Polish State during the period between the two World Wars, the fight against acute and chronic infectious diseases was considered as a great priority. Besides legislative activities, the most important task in this policy, was the organization of the institutional forms of medical care and permanent preventive activities, as well as the formation of the people's health awareness and responsibility. Those activities resulted in, among others, the reduction of morbidity and mortality due to acute infections and social diseases. The progress in the health care was the result of a consistent cooperation between all medical care sectors, medical associations, social and scientific institutions and the essential financial support from the international organizations.

[Establishment and development of the self-governed health case system in the Austrian sector of Poland]. / Geneza i rozwój samorzadowego lecznictwa na ziemiach polskich w zaborze austriackim.

Transformation and development of organisational matters, characteristic for the 18th and 19th centuries, included the issue of public health care. The development of economy, social and medical sciences changed the understanding of joint responsibility of the state for public health. The Teresian-Josephine reforms in Austria, concerning public issues, including the sanitary-epidemiological matters, were enforced in the areas of Polish annexed territory in 1772 and 1795. The normative regulations for Galicia and Lodomeria from 1773, 1861, 1891 established the responsibilities and rights for medical practice, activities of sanitary institutions and general medical care for the society. Austrian sanitary legislation created central institutions and self-governed local structures, responsible for public health. Introduction of modern local administration had a deciding effect on the development of self-governed institutions in closed and open health care system. Apart from sanitary national administration, public health care matters were governed by local-governed authorities of the insurance sector and private subjects. This advanced decentralisation and handing of health care matters to local self-governments shaped the new system of public health care.

[Public health matters in the early-Capitalistic period (XVII-XIX century]. / Zagadnienie zdrowia publicznego w okresie wczesno-Kapitalistycznym (XVII-XIX wiek).

Modern opinion about people community formulated by philosophers and economists has shown essence of the public health care problem. Together with development of medical knowledge about man and his social role, sense of understanding joint responsibility of the government for health matters of the nation has been changing. Political and economical transformations in the era of early industrialisation tiggered in people need for making their material and health state better. This base was strenghtened by the French Revolution in proclaimed rights to freedom and individual life in the society. Modern adjusted to the epoch inner structures of undergoing modernization European countries, considered issues of the public health care in wider range. Sanitary, hygienic and epidemic problems as well as new health- and life-threatening factors in working population and pathology.

[Developmental tendencies of public health services on Polish lands before and after 1918]. / Tendencje rozwojowe publicznej sluzby zdrowia na ziemiach polskich przed i po 1918 roku.

On the basis of the up to now historic-medical researches we can state that sanitarian legislation of the repacious states (Austria, Prussia, Russia) had a decisive influence on public health services development on Polish lands in XIX century. In the elaboration of the polish services project, actively participated the medical Association of Cracow and Warsaw. The presented approaches realizes in the liberated polish State, considered the desire for health services administration union, as well as underlined the decisive role of the urban and district self-government in the organization of health care services. In the multisectorial structure of medical care, public health services-state and self-governed, were rendering services for the whole of population. Some of the important achievements were: accessibility to the public of numerous medical care units, declination of mortality coefficient because of acute and social infective diseases, wide range of preventive activities, etc.

Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs.

We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar VL amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL genes were initially coupled to a synthetic C kappa gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C kappa domains with a C lambda 1 domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C kappa and C lambda 1 forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

[Public health care in Poland in the Prussian Sector]. / Ochrona zdrowia publicznego na ziemiach polskich w zaborze pruskim.

Modernization processes in modern European countries, characterised by the development of contemporary organisational structures, have led to a wider range of public health and hygiene activities. New, developing ideas of public health care demonstrated the need for competent institutions, operating within national government and administration structures, capable of preventing mass epidemics and of eliminating factors from the external environment which area danger to health. These attempts were fulfilled in the beginning of the 19th century. The Prussian state and then united German empire was, at that time, a classical example for the effective organisation of self-governed public health care organisations. In the Prussian sector, public health care matters were governed by the boards of provincial, regional and district authorities. In this sector public health care was also the responsibility of sanitary commissions, which were a continuation of the earlier, so called, medical police forces. The studies devoted to the issue formulated in the title show that within the three-sector scale in partitioned Poland, the Prussian sector could serve as an example of optimal systemic solutions, not only for self-governed health care, but also for a health insurance system. These concepts were the basis for the model of public health care after 1918, in the Public state liberated from its oppressors.