Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles.

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.

The role of microvesicles in malignancies.

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.

Prolonged histamine deficiency in histidine decarboxylase gene knockout mice affects Leydig cell function.

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.

Evaluation and diagnostic potential of circulating extracellular vesicle-associated microRNAs in adrenocortical tumors.

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.

Genomics of steroid hormones: in silico analysis of nucleotide sequence variants (polymorphisms) of the enzymes involved in the biosynthesis and metabolism of steroid hormones.

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.

The role of membrane vesicles in tumorigenesis.

Membrane vesicles are membrane-covered cell fragments generated by all cell types. They comprise a recently recognized new system of intercellular communication, believed to play a pivotal role in information transfer between cells, as they display a large number of biomolecules enclosed within the membrane as well as in the membrane proper. The phenotype of the donor cell is reflected in the vesicular protein content, which also allows the identification of the original cell. Membrane vesicles have been implicated in several physiological and pathological processes, most notably in tumorigenesis. Tumor-derived vesicles may serve as prognostic markers, they were detected in blood plasma and in other body fluids. Their size varies between 30 and 1000 nm. All of them reflect the special potential of tumor cells for survival and for the expansion of the tumor, independently from cell-to-cell contact. Tumor-derived vesicles have the potential 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 a beneficial environment for newly formed and migrating tumor cells, influencing angiogenesis and the reorganization of the extracellular matrix. Elucidating the properties of tumor-derived vesicles should increase our understanding in tumor biology and open new perspectives in cancer treatment. Tumor-derived vesicles are involved in tumorigenesis at multiple level and drugs themselves can be expulsed from tumor cells via vesicles. Consequently, interfering with the formation, release and propagation of these vesicles can be a novel and alternative issue in cancer therapy. The present review is an overview of the roles of membrane vesicles in tumorigenesis showing also the potential to consider them as new targets in tumor therapy.

Impaired reproduction of histamine deficient (histidine-decarboxylase knockout) mice is caused predominantly by a decreased male mating behavior.

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.