There is no available blood marker for the preoperative diagnosis of adrenocortical malignancy. The objective of this study was to investigate the expression of extracellular vesicle-associated microRNAs and their diagnostic potential in plasma samples of patients suffering from adrenocortical tumors. Extracellular vesicles were isolated either by using Total Exosome Isolation Kit or by differential centrifugation/ultracentrifugation. Preoperative plasma extracellular vesicle samples of 6 adrenocortical adenomas (ACA) and 6 histologically verified adrenocortical cancer (ACC) were first screened by Taqman Human Microarray A-cards. Based on the results of screening, two miRNAs were selected and validated by targeted quantitative real-time PCR. The validation cohort included 18 ACAs and 16 ACCs. Beside RNA analysis, extracellular vesicle preparations were also assessed by transmission electron microscopy, flow cytometry and dynamic light scattering. Significant overexpression of hsa-miR-101 and hsa-miR-483-5p in ACC relative to ACA samples has been validated. Receiver operator characteristics of data revealed dCT hsa-miR-483-5p normalized to cel-miR-39 to have the highest diagnostic accuracy (area under curve 0.965), the sensitivity and the specifity were 87.5 and 94.44, respectively. Extracellular vesicle-associated hsa-miR-483-5p thus appears to be a promising minimally invasive biomarker in the preoperative diagnosis of ACC but needs further validation in larger cohorts of patients.

Munkank soran az extracellularis vezikulak izolalasanak es detektalasanak szamos meghatarozo preanalitikai es analitikai parameterere hivtuk fel a figyelmet. Elsőkent mutattunk ra, hogy a mikrovezikulak es a feherje-aggregatumok biofizikai parameterei jelentős mertekben atfednek, es ez zavarhatja a mikrovezikulak mereset. Kidolgoztuk annak modszeret, hogy egyazon biologiai forrasbol szarmazo kulonboző vezikula populaciokat parhuzamosan, nagy mennyisegben, intakt formaban tudjunk izolalni. Osszehasonlito proteomikai elemzest vegeztunk thymus eredetű apoptotikus testek es mikrovezikulak eseteben. Szamos T sejt jelatvitelben es immunfolyamatokban szerepet jatszo feherjet es autoantigent azonositottunk. Igazoltuk, hogy T sejt eredetű citokinek es extracellularis vezikulak egyuttes hatasat monocitak genexpressziojara. Igazoltuk, hogy a mikrovezikulak onallo ionhaztartassal rendelkeznek. Kimutattuk, hogy thymocyta exoszomak nem tartalmaznak riboszomalis RNS-eket, azonban feldusulnak bennuk kis RNS-ek (pl. bizonyos miRNS-ek). Polymyositises betegekben emelkedett keringő mikrovezikula szamot mutattunk ki, mely korrelalt a betegseg bizonyos klinikai parametereivel. Vegul elsőkent igazoltuk, hogy egeszseges T sejt eredetű mikrovezikulak CD62P-CD161 kolcsonhatas reven specifikusan kotődnek monocitak felszinehez. | In our work we drove attention to several pre-analytical and analytical parameters affecting isolation and detection of work extracellular vesicles. We were the first to describe that microvesicles share biophysical parameters with protein aggregates which may confound microvesicle assessment by flow cytometry. We developed protocols for the isolation of large amounts of intact vesicle types secreted simultaneously by the same biological source. We carried comparative proteomic analysis of murine thymus derived apoptotic bodies and microvesicles. We identified large number of proteins involved in T cell signaling or immune functions as well as autoantigens within these structures. We provided evidence fro crosstalk between T cell derived extracellular vesicles and cytokines on the gene expression of monocytes. We have shown that microvesicles possess autonomous ion homeostasis. We found that thymocyte derived exosomes lacked the 18S and 28S ribosomal RNA molecules, while they were enriched in small RNA species (e.g. certain miRNAs). We described that patients with polyomyelitis were characterized by elevated levels of circulating microvesicle. Monocyte- and B cell-derived microvesicle numbers correlated with certain clinical parameters of the diseases. Finally, for the first time we showed that HLA-G+, trophoblast-derived microvesicles isolated from healthy pregnant blood plasma samples, bound specifically to T cells via CD62P-CD161 interaction, and induced STAT3 phosphorylation.

Release of membrane vesicles, a process conserved in both prokaryotes and eukaryotes, represents an evolutionary link, and suggests essential functions of a dynamic extracellular vesicular compartment (including exosomes, microparticles or microvesicles and apoptotic bodies). Compelling evidence supports the significance of this compartment in a broad range of physiological and pathological processes. However, classification of membrane vesicles, protocols of their isolation and detection, molecular details of vesicular release, clearance and biological functions are still under intense investigation. Here, we give a comprehensive overview of extracellular vesicles. After discussing the technical pitfalls and potential artifacts of the rapidly emerging field, we compare results from meta-analyses of published proteomic studies on membrane vesicles. We also summarize clinical implications of membrane vesicles. Lessons from this compartment challenge current paradigms concerning the mechanisms of intercellular communication and immune regulation. Furthermore, its clinical implementation may open new perspectives in translational medicine both in diagnostics and therapy.

Microvesicles are membrane-covered cell fragments whose size varies between 30 and 1,000 nm. They are generated by all cell types, constituvely and in response to activation signals. Their importance in intercellular communication has been only recently discovered. They seem to enhance the potential of information transfer between cells, displaying a large number of proteins and lipids as membrane constituents and as components of the inner vesicular content. The content reflects the phenotype of the donor cell and allows the identification of the microvesicular origine as well. Complex "packets" of molecules are transmitted to the target cells this way, modifying their cellular physiology. Additionally, epigenetic changes may be induced by transmitted DNA and RNAs, that have also been identified in these vesicles. The vesicles can act in close and far distances as well. Microvesicles have been implicated in several physiological and pathological processes. There is an increasing evidence, that they play a pivotal role in tumorigenesis. Vesicles shedding from tumor cells reflect the special potential of the tumor for survival and expansion, independently from cell-to-cell contact. Tumor derived vesicles are fully equipped to facilitate the escape of tumor cells from immune surveillance through their protein and RNA content, at the same time they are involved in the establishment of an optimal environment for newly formed and metastatic tumor cells, influencing angiogenesis and the reorganization of the extracellular matrix. As immune cells, endothels, platelets and stem cells also release microvesicles, a multilevel communication network draws up, allowing a complex interplay between the cells. The concentration of tumor derived vesicles increases in blood plasma and other body fluids with the progression of the disease; therefor they may serve as prognostic markers. The microvesicular approach can offer new perspectives: interfering with the formation, release and propagation of these vesicles, they can be considered as new targets in tumor therapy.

Microvesicles (MVs) are membrane-covered cell fragments released by most cell types during apoptosis or activation. They are increasingly considered to play a pivotal role in information transfer between cells. Their presence and role have been proven in several physiological and pathological processes, such as immune modulation in inflammation and pregnancy, or blood coagulation and cancer. MVs represent a newly recognized system of intercellular communications. They not only may serve as prognostic markers in different diseases, but could also hold the potential to be new therapeutic targets or drug delivery systems.The present overview aims to highlight some aspects of this new means of cellular communication: "microvesicular communication".

Microvesicles (MVs) can derive from several cell types and their membranes contain cell surface elements. Their role is increasingly recognized in cell-to-cell communication, as they act as both paracrine and remote messengers, occurring in circulating form as well as in plasma. Successful pregnancy requires a series of interactions between the maternal immune system and the implanted fetus, such that the semi-allograft will not be rejected. These interactions occur at the materno-placental interface and/or at a systemic level. In the present study we identified for the first time the in vivo plasma pattern of the MVs of third-trimester, healthy pregnant women, their cellular origin, and their target cells using flow cytometry and confocal laser microscopy. We searched for the cellular target molecules of thrombocyte-derived MVs with the help of neutralizing antibodies. We examined the in vitro effects of MVs on STAT3 phosphorylation of primary lymphocytes and Jurkat cells. We found that both placental trophoblast-derived and maternal thrombocyte-derived MVs bind to circulating peripheral T lymphocytes, but not to B lymphocytes or NK cells. We were able to show that the P-selectin (CD62P)–PSGL-1 (CD162) interaction is one mechanism binding platelet-derived MVs to T cells. We were also able to demonstrate that MV–lymphocyte interactions induce STAT3 phosphorylation in T cells. Our findings indicate that both thrombocyte- and trophoblast-derived MVs may play an important role in the immunomodulation of pregnancy. We suggest that the transfer of different signals via MVs represents a novel form of communication between the placenta and the maternal immune system, and that MVs contribute to the establishment of stable immune tolerance to the semi-allograft fetus.

The present study focuses on histaminergic regulation of Leydig cell physiology, since limited information is available so far. To evaluate the dependency of Leydig cells on histamine (HA), we performed experiments using highly purified Leydig cells in culture, isolated from wild type (WT) and histidine decarboxylase (Hdc) gene knockout (HDC KO)-so HA-deprived-mice. HDC KO Leydig cells showed lower basal and human choriogonadotropin (hCG)-induced testosterone production compared to WT Leydig cells, presumably due to altered P450scc gene (Cyp11a1) expression levels. Moreover, in HDC KO cells, hCG did not increase basal expression levels of HA H1 and H2 receptor genes, while the hormone showed a significant inducing effect in WT cells. Based on these findings, we propose that prolonged HA deficiency in HDC KO mice affects various aspects of Leydig cell physiology, most importantly the response to hCG, providing definite evidence that HA plays a role as direct modulator of Leydig cell function and steroid synthesis in the testis. Also, the results presented herein constitute the first molecular evidence for the expression of HA H1 and H2 receptor subtypes in isolated Leydig cells.

Successful pregnancy is closely related to polarization toward a Th2 type immune response. As histamine is known to initiate Th2 dominance during inflammatory processes we raised the question whether histamine has any effect on the actual tuning of proper cytokine balance for the proceeding of the gestation. Histamine has multiple functions in the process of pregnancy, different studies have shown the direct and/or indirect presence of histamine action in the placenta as well. As HDC is the unique histamine producing enzyme in eukaryotes, we used HDC (so endogenous histamine)-deficient knockout mice as reliable model for studying histamine-related processes in vivo. We examined the placental histamine content and the expression of histamine receptors and Th1/Th2/Th3 type cytokines in the placenta. We showed for the first time the influence of histamine on the orchestrated regulation of placental cytokine expression. In the absence of local histamine the cytokine balance is shifted toward Th1 types at the maternal–placental interface, threatening pregnancy. We also measured splenic lymphocyte subpopulation ratios in pregnant and non-pregnant mice and found that in pregnancy they are independent of the presence of histamine.

PROBLEM:Histamine induces a Th2 shift. As successful allopregnancy is characterized by a peripheral Th2 dominance, we investigated the role of histamine in reproduction.METHOD OF STUDY:HDC knockout (HDC-/-) or wild-type (HDC+/+) mice kept on histamine-deficient or normal diet were mated. Appearance of vaginal plugs indicated day 0.5 of pregnancy. On day 10.5 uteri were inspected. Splenic IFN-gamma production and cytotoxic activity were determined.RESULTS:In HDC+/+ or HDC-/- females on normal diet, plugs appeared between 3 and 6 days. In 80% of the (HDC-/-)/(HDC-/-) matings on histamine-deficient diet, no vaginal plugs were observed for more than 1 month. After replacing males with the wild type, plugs appeared within 3 days. In HDC-/- mice, litter size was lower than in HDC+/+ animals. Cytotoxicity and IFN-gamma production were significantly increased in non-pregnant histamine-deficient mice, but not in pregnant mice.CONCLUSION:Histamine affects male mating behavior, but is not indispensable for successful pregnancy.

Histamine is synthesized in cells by histidine decarboxylase (HDC). HDC-deficient knockout (KO) mice lack functional HDC and histamine in the tissues. In the present study we used this in vivo model for studying the role of HDC deficiency in the regulation of male steroid hormone metabolism. In agreement with earlier studies showing the lack of effects of central histamine on the basal secretion of gonadotrope hormones, we found no difference with in situ hybridization in the expression of GnRH in the hypothalamus of wild type and KO mice. The tissue concentrations of testosterone and several androgenic steroids were significantly elevated in the testes but not in the adrenal glands of HDC-KO mice. In contrast, serum estradiol levels failed to show a significant difference between the two groups. The weight of the testes was significantly smaller in both 7-day-old and adult KO mice. The ultrastructure of the adult testis indicated elevated steroid synthesis with more tightly coiled membranous whorls in Leydig cells. The present results suggest that changes in reproductive functions and sex steroid secretion in male HDC-KO mice are not due to altered hypothalamic GnRH expression but are probably related to definite modifications during fetal development of KO mice reinforced later by the lack of the effect of peripheral histamine. This may provide in vivo evidence that peripheral histamine is an important regulatory factor of male gonadal development during embryogenesis and of sex steroid metabolism later in adulthood.

Alterations of steroid hormone biosynthesis and metabolism are suspected to be involved in the pathogenesis of several diseases. Several polymorphisms of the enzymes involved in these processes have already been described and some could be associated with certain diseases. We attempted to examine the sequence variants of these genes in order to find novel variants by an in silico analysis. We analyzed the known human nucleotide sequences of the enzymes p450 side-chain cleavage enzyme, steroid 17-alpha-hydroxylase/17,20-lyase, 3-beta-hydroxysteroid dehydrogenase types 1 and 2, 21-hydroxylase, 11-beta-hydroxylase, aldosterone synthase, aromatase, 11-beta-hydroxysteroid dehydrogenase types 1 and 2, steroid 5-alpha-reductase types 1 and 2, steroid 5-beta-reductase, dehydroepiandrosterone sulfotransferase, 17-beta-hydroxysteroid dehydrogenase types 1-3. The analysis was performed using the National Center for Biotechnology Information Database by the search tool blastn. We found numerous sequence variants in both coding and non-coding sequences. The majority of these sequence variants have already been described, nevertheless, some appear as novel variants. Some of these may also have functional significance. We hypothesize over the possible significance of these findings and briefly review the available literature.

In the rat, the peroxysome proliferator activated receptor (PPAR) inducer clofibrate can moderately influence the hormone (testosterone) level and, after single perinatal treatment, irreversibly affects sexual behavior through the mechanism of hormonal imprinting. The thymic glucocorticoid and estrogen receptors weren't significantly influenced. The experiments call the attention to the universality of false imprinting by molecules able to bind to the steroid/thyroid receptor superfamily, and point to the different sensitivity to different ligands.

Serum testosterone concentration was significantly elevated in adult male rats by a single perinatal allylestrenol administration. One week after a second allylestrenol treatment in adulthood the hormone concentration dropped below the control values. Serum progesterone concentration was significantly lowered in adult female rats by a single perinatal allylestrenol administration. Following a second allylestrenol treatment in adulthood the hormone concentration reached the control values. These experiments demonstrate that the hormonal imprinting caused by allylestrenol (a steroid used in the treatment of endangered pregnancy) not only acts at receptor level and produces changes in sexual behaviour, but also induces modifications in serum hormone concentrations.