Endocanalicular transendothelial crossing (ETC): A novel intravasation mode used by HEK-EBNA293-VEGF-D cells during the metastatic process in a xenograft model.

In cancer metastasis, intravasation of the invasive tumor cell (TCi) represents one of the most relevant events. During the last years, models regarding cancer cell intravasation have been proposed, such as the "endocanalicular transendothelial crossing" (ETC) theory. This theory describes the interplay between two adjacent endothelial cells and the TCi or a leukocyte during intravasation. Two endothelial cells create a channel with their cell membranes, in which the cell fits in without involving endothelial cell intercellular junctions, reaching the lumen through a transendothelial passage. In the present study, ten SCID mice were subcutaneously xenotransplanted with the HEK-EBNA293-VEGF-D cell line and euthanized after 35 days. Post-mortem examinations were performed and proper specimens from tumors were collected. Routine histology and immunohistochemistry for Ki-67, pAKT, pERK, ZEB-1, TWIST-1, F-actin, E-cadherin and LYVE-1 were performed followed by ultrastructural serial sections analysis. A novel experimental approach involving Computed Tomography (CT) combined with 3D digital model reconstruction was employed. The analysis of activated transcription factors supports that tumor cells at the periphery potentially underwent an epithelial-to-mesenchymal transition (EMT)-like process. Topographical analysis of LYVE-1 immunolabeled lymphatics revealed a peritumoral localisation. TEM investigations of the lymphatic vessels combined with 3D digital modelling enhanced the understanding of the endotheliocytes behavior during TCi intravasation, clarifying the ETC theory. Serial ultrastructural analysis performed within tumor periphery revealed numerous cells during the ETC process. Furthermore, this study demonstrates that ETC is an intravasation mode more frequently used by the TCi than by leukocytes during intravasation in the HEK-EBNA293-VEGF-D xenograft model and lays down the potential basis for promising future studies regarding intravasation blocking therapy.

Chronic administration of green tea extract to TRAMP mice induces the collapse of Golgi apparatus in prostate secretory cells and results in alterations of protein post-translational processing.

Considering its long latency, prostate cancer (PCa) represents an ideal target for chemoprevention strategies. Green tea extract (GTE) has been proved to be one of the most promising natural substances capable of inhibiting PCa progression in animal models (transgenic adenocarcinoma of mouse prostate), as well as in humans. However, the cellular targets of the GTE action are mostly unknown. The main objective of this work was to investigate whether the endoplasmic reticulum (ER) and the Golgi apparatus (GA), known to be actively involved in sensing stress stimuli and initiating and propagating cell death signalling, may represent the subcellular targets of GTE action. To this end, 42 TRAMP mice were divided into four experimental groups: groups II and IV, received GTE in tap water (0.3 g/100 ml solution) starting at 8 weeks of age and up to the time of sacrifice. Groups I and III were respective age-matched water-fed controls. The animals were sacrificed after 4 weeks (groups I and II) or 40 weeks of treatment (groups II and IV). We also treated TRAMP-C2 cells with GTE (20 µg/ml for 7 days) to check the expression profile of clusterin (CLU), a protein involved in prostate tumourigenesis, extensively processed through ER-GA before being secreted through the plasma membrane. In vivo we found that chronic administration of GTE in TRAMP mice results in collapse of ER and GA in prostate epithelial cells. Consistently, in vitro we found that the mature, fully processed form of CLU, sCLU, is strongly reduced by GTE treatment in TRAMP-C2 cells. Taking into account the sCLU biogenesis dependence on the ER-GA integrity and the proposed anti-apoptotic role of sCLU, the possibility for GTE to counteract PCa progression by interfering with sCLU biogenesis is suggested.

The "intraendothelial canalicular formation": the route for lymphocyte diapedesis at the level of peripheral and mucosa-associated lymphoid tissue HEVs.

How the lymphocyte crosses the blood endothelium during transendothelial migration is demonstrated through the study of serial sections of high endothelial venules (HEVs) of peripheral (mesenteric lymph nodes) and mucosa-associated lymphoid tissue (Peyer's patches, vermiform appendix) during normal lymphocyte homing and experimental intestinal inflammation. The sequence of serial ultrastructural features of lymphocytes englobed in the endothelial wall at different moments of transmigration made it possible to bring out that their transendothelial migration toward the extracellular matrix of lymphoid tissues occurs through an intraendothelial canalicular formation constituted by two adjacent endothelial cells that have closed interendothelial junctions. This intraendothelial canalicular formation, morphologically unlike the transcellular and paracellular migratory pathways, is an innovative model of migratory route for lymphocyte diapedesis that does not compromise the continuity of the endothelial wall. The increased presence of lymphocytes and intraendothelial canalicular formations during experimental inflammation and the metabolic hyperactivity of the spring/summer months compared to the lethargic fast in Chiropters underscores an influence on lymphocyte traffic through the HEVs of the peripheral and mucosa-associated lymphoid tissue.

The "mode" of lymphocyte extravasation through HEV of Peyer's patches and its role in normal homing and inflammation.

The mode of lymphocyte transendothelial migration in the postcapillary high endothelial venules (HEVs) of Peyer's patches during normal homing and acute inflammation in the guinea pig was studied. It is common opinion that the lymphocyte transendothelial passage from the blood stream into the extravasal lymphoid tissue calls for a multistep process of endothelial and lymphocyte molecules favoring tethering, rolling, activation, arrest and its firm adhesion to the endothelial luminal surface. Ultrastructural serial pictures and the three-dimensional reconstruction of HEVs with lymphocytes during different moments of their transmigration through the endothelial wall enabled us to demonstrate in vivo the morphological modality of their extravasation in lymphoid tissue. The latter is accomplished by means of an intraendothelial canalicular formation (6.8-7.2 microm long and 2.1-2.2 microm in diameter), whose creation depends on the particular behavior of adjacent endothelial cells, without compromising the interendothelial contacts. This new canalicular pathway of lymphocyte extravasation, particularly selective for the B cell, does not permit confirmation of the dogmas of the transcellular and paracellular (open interendothelial junctions) modes that have prevailed in recent decades. The lack of knowledge regarding the molecular bases that would induce constitution of this intraendothelial canalicular formation is a critical point for stimulating future interdisciplinary research aimed at developing strategies for modulating normal lymphocyte homing and in inflammation.

Tumor cell transendothelial passage in the absorbing lymphatic vessel of transgenic adenocarcinoma mouse prostate.

The distribution and fine structure of the tumor-associated absorbing lymphatic vessel in the tumor mass of prostate adenocarcinoma and of seminal vesicle metastasis in transgenic mice was studied for the purpose of understanding the modality of tumor cell transendothelial passage from the extravasal matrix into the lymphatic vessel. In the tumor mass, two main cell populations were identified: stromal tumor cells and the invasive phenotype tumor (IPT) cells, having characteristics such as a highly electron-dense matrix rich in small granules lacking a dense core and massed nuclear chromatin, which is positive to immunostaining with anti-SV40 large T antigen antibody. Based on the ultrastructural pictures of different moments of the IPT cell transendothelial passage by ultrathin serial sections of the tumor-associated absorbing lymphatic vessel, the manner of its transendothelial passage through the intraendothelial channel, without involving intercellular contacts, was demonstrated. The presence of IPT cells in the parenchyma of satellite lymph node highlights its significant role in metastatic diffusion. The intraendothelial channel is the reply to the lack of knowledge regarding the intravasation of the tumor cell into the lymphatic circulation. The lymphatic endothelium would organize this channel on the basis of tumor cell-endothelial cell-extravasal matrix molecular interactions, which are as yet unidentified.

On the transendothelial passage of tumor cell from extravasal matrix into the lumen of absorbing lymphatic vessel.

The aim of the research is the study of ultrastructural characteristics of the absorbing lymphatic vessel and of tumor cell passage through the endothelial lymphatic wall in (a) subcutaneous xenografts of T84 colon adenocarcinoma and B16 melanoma cell lines in nude mice and (b) human colorectal cancer. It was found that the tumor-associated absorbing lymphatic (TAAL) vessel has the same ultrastructural characteristics as the absorbing lymphatic vessel in normal organs, and it is provided with an endothelial wall wholly lacking a continuous basement membrane, pores, fenestrations, and open junctions. The TAAL vessel is always missing in the studied tumor masses as far as the central stroma is concerned, whereas it is always present in the peripheral area of the tumor and in the peritumoral connective tissue. The factors of extravasal matrix that play an active role in migration process of invasive phenotype tumor (IPT) cell after its detachment from tumor mass, as well as the role of cytoplasmic protrusions (pseudopod-like) in lymphatic recognition, were considered. For the first time, this study demonstrated the transendothelial passage of IPT cell inside the TAAL vessel lumen, which takes place by means of the intraendothelial channel (approximately 1.8-2.1 mum in diameter and 6.8-7.2 microm in length). This channel is to be considered a transient morphological entity organized by TAAL vessel endothelium by means of still unidentified molecular mechanisms. Therefore, it appears to be ascertained that the intraendothelial channel represents a step forward in the knowledge of the drainage into lymphatic circulation of interstitial fluid and the answer to the lack of knowledge expressed till today by researchers concerning the modality of passage of the tumor cell through the endothelial wall of the TAAL vessel.

Transendothelial transport and migration in vessels of the apparatus lymphaticus periphericus absorbens (ALPA).

The vessel of the apparatus lymphaticus periphericus absorbens (ALPA) represents the sector with high absorption capacity of the canalization of the lymphatic vascular system. It plays a basic role in preserving tissue homeostasis and in directing interstitial capillary filtrate back to the bloodstream. ALPA lymphatic endothelium differs from the endothelia of conduction and flowing vessels (precollectors, prelymph nodal and postlymph nodal collectors, main trunks), since it presents a discontinuous basement membrane, which is often absent, and lacks pores and fenestrations. The mesenchymal origin of the ALPA lymphatic vessel, morphological and ultrastructural aspects, intrinsic contractile properties, the presence of valves, innervation, and specific lymphatic markers that reliably distinguish it from blood capillaries are studied. Furthermore, its role in lymph formation through different mechanisms (hydrostatic pressure and colloidal osmotic-reticular mechanisms, vesicular pathway, and intraendothelial channel) is investigated. We have studied morphological and biomolecular mechanisms that control the transendothelial migration, from the extracellular interstitial matrix into the lumen of the lymphatic vessel, of cells involved in immune response and resistance (lymphocyte recirculation, etc.) and in the tumoral metastatic process via the lymphatic system. Finally, future research prospects, clinical implications, and therapeutic strategies are considered.

Structure, lymphatic vascularization and lymphocyte migration in mucosa-associated lymphoid tissue.

In this review, we consider the morphological aspects and topographical arrangement of gut-associated lymphoid tissue (GALT) (solitary and aggregate lymph nodules or Peyer's patches) and of vermiform appendix in the human child and in some mammals. The spatial arrangement of the vessels belonging to apparatus lymphaticus periphericus absorbens (ALPA) and of blood vessels within each lymphoid follicle as well as the ultrastructural characteristics of the lymphatic endothelium with high absorption capacity are considered. Particular attention is also paid to the morphological and biomolecular mechanisms inducing lymphocyte transendothelial migration to the bloodstream by means of lymphatic vessels as well as their passage from blood into lymphoid tissue through the high endothelial venules (HEVs). The preferential transendothelial passage of lymphocytes and polymorphonuclear neutrophils within ALPA vessels of the interfollicular area does not occur following the opening of intercellular contacts, but rather it occurs by means of 'intraendothelial channels'. In HEVs, on the contrary, the hypothesis is plausible that lymphocyte transendothelial migration into lymphoid tissue occurs through a channel-shaped endothelial invagination entirely independent of interendothelial contacts. The lymph of ALPA vessels of the single Peyer's patch is conveyed into precollector lymphatic vessels and into prelymph nodal collectors, totally independent of the ALPA vessels of the gut segments devoid of lymphoid tissue. The quantitative distribution of T lymphocytes in the lymph of mucosal ALPA vessels suggests a prevalent function of fluid uptake, whereas a reservoir and supply function is implicated for the vessels of interfollicular area. The precollector lymphatic vessels and prelymph nodal collectors are considered to be vessels with low absorption capacity, whose main function is lymph conduction and flow.

Ultrastructural aspects of the follicular cells of the pars tuberalis in bats related to the seasonal cycle.

The topography and structure of the follicular cells and the follicular cavity of the hypophyseal pars tuberalis (PT) were studied in adult hibernating bats (Pipistrellus pipistrellus and Rhinolophus ferrumequinum) of both sexes, during the annual seasonal cycle and the reproductive cycle. The follicular cells were found to be organized around a central cavity. They showed a polyhedral shape and apical microvilli protruding into central cavities. During hibernation, the follicular cells showed active cytoplasmic organelles, clusters of glycogen particles, and lipid droplets. In the supranuclear cytoplasm, 9+2 type cilia, some dense bodies, microvesicular vacuoles, and thin actin-like filaments (rather scarce during autumn) were detected. The contents of the follicular cavity showed well-defined ultrastructural seasonal characteristics, with a colloid-like aspect during awakening and a strongly granular aspect during autumn oestrus and mating. Positive staining for PAS and paraldehyde fuchsin, and a marked reaction to lectins PHA-L4, MAM, and RCA 60 suggested the presence of sialo-glycoproteins in the follicular cavities. Both follicular and endocrine PT-specific cells appeared to mark the boundary of follicular cavities. This finding suggests that the follicular cavity contents are comprised of both types of cells, rather than by cell fragmentation or degeneration products.

Fine structure and photoperiodical seasonal changes in Pars tuberalis of hibernating bats.

In Pars tuberalis (PT) of pituitary gland of hibernating bats, extending cranially along the ventral face of the hypothalamic median eminence, around the hypophyseal stalk, and caudally continuing in the Pars distalis (PD), pt specific cells, follicular cells and gonadotropic cells were distinguished. Pt specific cells contain peculiar secretory granules positive to lectin WGA and negative to lectins LFA and PNA, positive to S-100 protein labeling and to PD hormones antisera. During hibernation they present a low numerical density of both secretory granules and melatonin binding sites. After light exposure, on the other hand, the latter increase in density and are associated with marked secretion synthetic activity and exocytosis. These aspects result to be more balanced in animals sacrificed during summer. These changes seem to support the hypothesis of marked annual changes even in animal species with seasonal rhythmicity of metabolisms and gonads. Follicular cells, organized in closed follicles, have slightly developed RER and Golgi apparatus during hibernation, whereas they seem to show an increased secretory activity after light exposure and during summer. In perinuclear and supranuclear cytoplasm, glycogen particles clusters (peculiar of hibernation), cilia 9+2 and multivesicular bodies were identified. Concerning FSH cells, a reduced numerical density during hibernation, the secretory granules morphological characteristics and their probable involvement in photoperiod-linked reproductive functions are investigated.

Ultrastructure of absorbing peripheral lymphatic vessel (ALPA) in guinea pig Peyer's patches.

Corrosion casts from neoprene direct injection of lymphatic and blood vessels in guinea pig gut-associated lymphoid tissue, that is, solitary lymphoid follicles and Peyer's patches, have shown both their numerical density and their topographical arrangement in physiological conditions, after starvation and lymphatic stasis. The absorbing peripheral lymphatic vessel (ALPA) begins with the lacteal vessel, which continues in the mucosal lymphatic network. The latter is formed by subepithelial and interfollicular vessels wrapping single lymphoid follicles like a basket. Interfollicular vessels drain in the submucosal network, which flows into muscular tunica vessels with nonsegmentary bicuspid valves. They in turn drain lymph in subserosal precollectors and then in prelymphonodal collectors with conduction function. The follicles' germinal center and dome are completely devoid of ALPA vessels, while they are rich in blood vessels. Ultrastructurally, the ALPA vessel wall consists of a monolayer of endothelial cells devoid of pores, fenestrations, and open junctions and lacking a continuous basal lamina. Endothelial cells are joined by overlapping and interdigitating intercellular contacts, while end-to-end contacts are rare. They have a sizeable cell body, containing the nucleus and the common endocytoplasmic organelles, and a peripheral cytoplasm with actin-like filament bundles, free microvesicles or forming channels and a few rough-surfaced encloplasmic reticulum (RER) canaliculi. The presence of intraendothelial channels crossed by lymphocytes can often be detected within the endothelial wall during the different phases of cell transendothelial migration from lymphoid tissue to lymphatic vessel lumen. These channels undergo a numerical increase during starvation, while they are scarce during lymphatic stasis. We have quantitatively evaluated the prevalence of T lymphocytes in the lymph of interfollicular ALPA vessels and of prelymphonodal collectors draining the small intestine tract with or without Peyer's patches, under physiological and experimental conditions (starvation, lymphatic stasis).

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells.

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)-2, IL-12, and IL-15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus-infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL-2, IL-12, and IL-15 on PKCalpha and PKCepsilon--a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCalpha and PKCepsilon activities; 2) whereas PKCepsilon activity is induced by cytokine stimulation, PKCalpha activity is inhibited; and 3) both the induction of PKCepsilon and the inhibition of PKCalpha functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine-induced NK cell stimulation.