Nonapical and Cytoplasmic Expression of Interleukin-8, CXCR1, and CXCR2 Correlates with Cell Proliferation and Microvessel Density in Prostate Cancer

Purpose: We characterized interleukin-8 (IL-8) and IL-8 receptor expression (CXCR1 and CXCR2) in prostate cancer to address their significance to this disease. Experimental Design: Immunohistochemistry was conducted on 40 cases of human prostate biopsy containing histologically normal and neoplastic tissue, excised from patients with locally confined or invasive androgen-dependent prostate cancer, and 10 cases of transurethral resection of the prostate material from patients with androgen-independent disease. Results: Weak to moderate IL-8 expression was strictly localized to the apical membrane of normal prostate epithelium. In contrast, membranous expression of IL-8, CXCR1, and CXCR2 was nonapical in cancer cells of Gleason pattern 3 and 4, whereas circumferential expression was present in Gleason pattern 5 and androgen-independent prostate cancer. Each of IL-8, CXCR1, and CXCR2 were also increasingly localized to the cytoplasm of cancer cells in correlation with advancing stage of disease. Cytoplasmic expression (but not apical membrane expression) of IL-8 in Gleason pattern 3 and 4 cancer correlated with Ki-67 expression (R = 0.79; P <0.001), cyclin D1 expression (R = 0.79; P <0.001), and microvessel density (R = 0.81; P <0.001). In vitro studies on androgen-independent PC3 cells confirmed the mitogenic activity of IL-8, increasing the rate of cell proliferation through activation of both CXCR1 and CXCR2 receptors. Conclusions: We propose that the concurrent increase in IL-8 and IL-8 receptor expression in human prostate cancer induces autocrine signaling that may be functionally significant in initiating and promoting the progression of prostate cancer by underpinning cell proliferation and angiogenesis.

Distribution of the Receptor for Advanced Glycation End Products (RAGE) in the Human Male Reproductive Tract. Prevalence in Men with Diabetes Mellitus.

BACKGROUND: Diabetics have a significantly higher percentage of sperm with nuclear DNA (nDNA) fragmentation and increased levels of advanced glycation end products (AGEs), in their testis, epididymis and sperm. As the receptor for AGEs (RAGE) is important to oxidative stress and cell dysfunction, we hypothesise, that it may be involved in sperm nDNA damage. METHODS: Immunohistochemistry was performed to determine the presence of RAGE in the human testis and epididymis. A comparison of the receptor's incidence and localisation on sperm from 10 diabetic and 11 non-diabetic men was conducted by blind semi-quantitative assessment of the immunostaining. ELISA analysis ascertained RAGE levels in seminal plasma and sperm from 21 diabetic and 31 non-diabetic subjects. Dual labelling immunolocalisation was employed to evaluate RAGE's precise location on the sperm head. RESULTS: RAGE was found throughout the testis, caput epididymis, particularly the principle cells apical region, and on sperm acrosomes. The number of sperm displaying RAGE and the overall protein amount found in sperm and seminal plasma were significantly higher in samples from diabetic men (p

Characterization of a spontaneous mouse retinal pigment epithelial cell line B6-RPE07.

PURPOSE. A spontaneously arising retinal pigment epithelial (RPE) cell line (B6-RPE07) was cloned from a primary culture of mouse RPE cells and maintained in culture for more than 18 months. Morphologic and functional properties of this cell line have been characterized.

METHODS. The morphology of the B6-RPE07 cells was examined by phase-contrast light microscopy, electron microscopy, and confocal microscopy. Barrier properties were measured by the flux of fluorescence from the apical to the basolateral compartment of culture chambers. The abilities of the cells to bind/phagocytose photoreceptor outer segments (POS) were determined by confocal microscopy, electron microscopy, and flow cytometry. Cytokine/chemokine secretion was measured by cytometric bead array. The expression of visual cycle proteins was determined by RT-PCR and Western blotting.

RESULTS. In standard culture conditions, B6-RPE07 cells display cobblestone morphology. When cultured on three-dimensional (3D) collagen gel–coated membranes, B6-RPE07 cells exhibit a monolayer epithelial polarization with apical surface microvilli. Immunohistochemistry of B6-RPE07 cultures revealed a high expression of pan-cytokeratin. B6-RPE07 cells also expressed the retinal pigment epithelium-specific marker CRALBP, but not RPE65. Cell junction proteins ZO-1 and ß-catenin, but not claudin-1/3 or occludin-1, were observed in B6-RPE07 cells. B6-RPE07 cells are able to bind, phagocytose, and digest POS. Finally, B6-RPE07 cells produce high levels of IL-6 and CCL2.

CONCLUSIONS. This is the first report of a mouse RPE cell line with morphology, phenotype, and function similar to those of in vivo mouse RPE cells. This cell line will be a valuable resource for future RPE studies, in particular for in vivo gene modification and transplantation studies.

A 3-D well-differentiated model of pediatric bronchial epithelium demonstrates unstimulated morphological differences between asthmatic and nonasthmatic cells

There is a need for reproducible and effective models of pediatric bronchial epithelium to study disease states such as asthma. We aimed to develop, characterize, and differentiate an effective, an efficient, and a reliable three-dimensional model of pediatric bronchial epithelium to test the hypothesis that children with asthma differ in their epithelial morphologic phenotype when compared with nonasthmatic children. Primary cell cultures from both asthmatic and nonasthmatic children were grown and differentiated at the air-liquid interface for 28 d. Tight junction formation, MUC5AC secretion, IL-8, IL-6, prostaglandin E2 production, and the percentage of goblet and ciliated cells in culture were assessed. Well-differentiated, multilayered, columnar epithelium containing both ciliated and goblet cells from asthmatic and nonasthmatic subjects were generated. All cultures demonstrated tight junction formation at the apical surface and exhibited mucus production and secretion. Asthmatic and nonasthmatic cultures secreted similar quantities of IL-8, IL-6, and prostaglandin E2. Cultures developed from asthmatic children contained considerably more goblet cells and fewer ciliated cells compared with those from nonasthmatic children. A well-differentiated model of pediatric epithelium has been developed that will be useful for more in vivo like study of the mechanisms at play during asthma.

Cytopathogenesis of Sendai virus in well-differentiated primary pediatric bronchial epithelial cells

Sendai virus (SeV) is a murine respiratory virus of considerable interest as a gene therapy or vaccine vector, as it is considered nonpathogenic in humans. However, little is known about its interaction with the human respiratory tract. To address this, we developed a model of respiratory virus infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs). These physiologically authentic cultures are comprised of polarized pseudostratified multilayered epithelium containing ciliated, goblet, and basal cells and intact tight junctions. To facilitate our studies, we rescued a replication-competent recombinant SeV expressing enhanced green fluorescent protein (rSeV/eGFP). rSeV/eGFP infected WD-PBECs efficiently and progressively and was restricted to ciliated and nonciliated cells, not goblet cells, on the apical surface. Considerable cytopathology was evident in the rSeV/eGFP-infected cultures postinfection. This manifested itself by ciliostasis, cell sloughing, apoptosis, and extensive degeneration of WD-PBEC cultures. Syncytia were also evident, along with significant basolateral secretion of proinflammatory chemokines, including IP-10, RANTES, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), interleukin 6 (IL-6), and IL-8. Such deleterious responses are difficult to reconcile with a lack of pathogenesis in humans and suggest that caution may be required in exploiting replication-competent SeV as a vaccine vector. Alternatively, such robust responses might constitute appropriate normal host responses to viral infection and be a prerequisite for the induction of efficient immune responses.

Piperonyl butoxide enhances triclabendazole action against triclabendazole-resistant Fasciola hepatica.

A study has been carried out to determine whether the action of triclabendazole (TCBZ) against the liver fluke, Fasciola hepatica is altered by inhibition of the cytochrome P450 (CYP 450)-mediated drug metabolism pathway. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for these experiments, the basic design of which is given in the paper by Devine et al. (2010a). Piperonyl butoxide (PB) was the CYP P450 inhibitor used. Morphological changes resulting from drug treatment and following metabolic inhibition were assessed by means of transmission electron microscopy. After treatment with either TCBZ or TCBZ.SO on their own, there was greater disruption to the TCBZ-susceptible than TCBZ-resistant isolate. However, co-incubation with PB+TCBZ, but more particularly PB+TCBZ.SO, led to greater changes to the TCBZ-resistant isolate than with each drug on its own, with blebbing of the apical plasma membrane, severe swelling of the basal infolds and their associated mucopolysaccharide masses in the syncytium and flooding in the internal tissues. Golgi complexes were greatly reduced or absent in the tegumental cells and the synthesis and production of secretory bodies were badly disrupted. The mitochondria were swollen throughout the tegumental system and the somatic muscle blocks were disrupted. With the TCBZ-susceptible Cullompton isolate, there was a limited increase in drug action following co-incubation with PB. The results provide evidence that the condition of a TCBZ-resistant fluke can be altered by inhibition of drug metabolism. Moreover, they support the concept that altered drug metabolism contributes to the mechanism of resistance to TCBZ

In vitro modeling of respiratory syncytial virus infection of pediatric bronchial epithelium, the primary target of infection in vivo

Respiratory syncytial virus (RSV) is the major viral cause of severe pulmonary disease in young infants worldwide. However, the mechanisms by which RSV causes disease in humans remain poorly understood. To help bridge this gap, we developed an ex vivo/in vitro model of RSV infection based on well-differentiated primary pediatric bronchial epithelial cells (WD-PBECs), the primary targets of RSV infection in vivo. Our RSV/WD-PBEC model demonstrated remarkable similarities to hallmarks of RSV infection in infant lungs. These hallmarks included restriction of infection to noncontiguous or small clumps of apical ciliated and occasional nonciliated epithelial cells, apoptosis and sloughing of apical epithelial cells, occasional syncytium formation, goblet cell hyperplasia/metaplasia, and mucus hypersecretion. RSV was shed exclusively from the apical surface at titers consistent with those in airway aspirates from hospitalized infants. Furthermore, secretion of proinflammatory chemokines such as CXCL10, CCL5, IL-6, and CXCL8 reflected those chemokines present in airway aspirates. Interestingly, a recent RSV clinical isolate induced more cytopathogenesis than the prototypic A2 strain. Our findings indicate that this RSV/WD-PBEC model provides an authentic surrogate for RSV infection of airway epithelium in vivo. As such, this model may provide insights into RSV pathogenesis in humans that ultimately lead to successful RSV vaccines or therapeutics.

Ultrastructural observations on rhabdite formation in the planarian, Artioposthia triangulata

The epidermis of the predatory terrestrial flatworm. Artioposthia triangulata has been examined by transmission electron microscopy for the presence of rhabdiform secretions. Two types of secretion are present: epidermal rhabdoids, produced by a special type of epidermal cell and true adenal rhabdites produced by gland cells beneath the epidermis. The epidermal rhabdoids are formed from Golgi-derived vesicles, which Fuse together to form the developing rhabdoid. Within the latter is a filamentous network on which granular material is deposited and coalesces to form a rod-shaped inclusion. The rhabdoids accumulate in the apical region of the cell and release their contents from the apical surface. The adenal rhabdites are formed by Golgi-derived vesicles. which become more elongated and their contents more electron-dense as they mature. The vesicles Fuse together to form the primordial rhabdite, which continues to lengthen with the addition of further vesicles. The neck of the rhabdite-forming cell passes between the muscle layers and through the basement membrane to open into the base of the epidermal cell. The rhabdites move from the cell body through the neck into the cytoplasm of the epidermal cell and make their way to the apical surface where they are released to the exterior.

FASCIOLA-HEPATICA - ULTRASTRUCTURAL-CHANGES TO THE TEGUMENT OF JUVENILE FLUKES FOLLOWING INCUBATION IN-VITRO WITH THE DEACETYLATED (AMINE) METABOLITE OF DIAMPHENETHIDE

Ultrastructural changes to the tegument of 5-week-old, 3-week-old and freshly-excysted Fasciola hepatica following in vitro incubation with the deacetylated (amine) metabolite of diamphenethide (DAMD, 10 mu gml(-1)) were examined by transmission electron microscopy, A similar sequence of tegumental changes occurred in all three age groups of fluke, although, with increasing fluke age, the time before onset increased and the damage became more extensive. The 5-week-old flukes showed an initial stress response after 3 h, typified by blebbing of the apical plasma membrane, formation of microvilli and an accumulation and accelerated release of secretory bodies at the tegumental apex, as well as swelling of the basal infolds, The swelling increased in extent with progressively longer periods of incubation in DAMD, leading to extreme edema and sloughing of the tegument after 9 h. The 3-week-old flukes showed a stress response and swelling of the basal infolds after only 1.5 h, although sloughing of the tegument did not occur until after 9 h. In the freshly-excysted metacercaria, a stress response and some sloughing of the tegument were evident after only 0.5 h. At all stages of development, the ventral tegument was more severely affected than the dorsal, Changes also occurred to the tegumental cells which were indicative of a disruption in the synthesis and release of tegumental secretory bodies: the amount of GER became reduced, the cisternae became swollen and their ribosomal covering decreased, the Golgi complexes disappeared from the cells and the numbers of secretory bodies in the cells also decreased, The heterochromatin content of the nuclei increased and eventually the tegumental cells began to break down, Again, the changes became apparent more rapidly at the earlier stages of development. The ultrastructural changes to the tegument are linked to a possible mode of action for diamphenethide as an inhibitor of protein synthesis. In turn, the results may help to explain the drug's high efficacy against juvenile stages of F. hepatica.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROFILAMENT INHIBITOR CYTOCHALASIN-B ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microfilament inhibitor cytochalasin B (10 and 100-mu-g/ml) on the ultrastructure of adult Fasciola hepatica was determined in vitro by scanning and transmission electron microscopy (SEM, TEM) using both intact flukes and tissue-slice material. SEM revealed that initial swelling of the tegument led to surface blebbing and limited areas of sloughing after 24 h treatment at 100-mu-g/ml. In the tegumental syncytium, basal accumulations of secretory bodies (especially T2s) were evident in the earlier time periods but declined with longer incubations, until few secretory bodies remained in the syncytium overall. Blebbing of the apical plasma membrane and occasional areas of breakdown and sloughing of the tegument were observed over longer periods of treatment at 100-mu-g/ml. In the tegumental cell bodies, the Golgi complexes gradually decreased in size and activity, and few secretory bodies were produced. In the later time periods, the cells assumed abnormal shapes, the cytoplasm shrinking in towards the nucleus. In the vitelline follicles, a random dispersion of shell protein globules was evident within the intermediate-type cells, rather than their being organized into distinct shell globule clusters. Disruption of this process was more severe at the higher concentration of 100-mu-g/ml and again was more evident in tissue-slice material. In the latter, after prolonged (12 h) exposure to cytochalasin B, the intermediate and mature vitelline cells were filled with loosely packed and expanded shell globule clusters, containing few shell protein globules. The mature vitelline cells continued to lay down "yolk" globules and glycogen deposits. Disruption of the network of processes from the nurse cells was evident at the higher concentration of cytochalasin. Spaces began to appear between the vitelline cells and grew larger with progressively longer incubation periods, and the cells themselves assumed abnormal shapes. A number of binucleate stem cells were observed in tissue-slice material at the longest incubation period (12 h).

Taxonomy and phylogeny of Osmundea (Rhodomelaceae, Rhodophyta) in Atlantic Europe

Two species of Osmundea Stackhouse (Rhodomelaceae, Rhodophyta) that occur in Atlantic Europe have been confused under the names Osmundea ramosissima (Oeder) Athanasiadis and Osmundea truncata (Kutzing) Nam et Maggs, regarded until now as a synonym of O. ramosissima, An epitype from its type locality (Stavanger, Norway) is selected for Osmundea ramosissima Athanasiadis, recognized here as a valid name for Fucus ramosissimus Oeder, nom. illeg. Details of vegetative and reproductive morphology of O. ramosissima are reported, based on material from France, the British Isles, and Helgoland. Osmundea ramosissima resembles other species of Osmundea in its vegetative axial segments with two pericentral cells and one trichoblast, spermatangial development from apical and epidermal cells (filament type), the formation of five pericentral cells in the procarp-bearing segment of the female trichoblast, and tetrasporangial production from random epidermal cells. Among the species of Osmundea, O. ramosissima is most similar to O. truncata. Both species have discoid holdfasts, secondary pit connections between epidermal cells, and cup-shaped spermatangial pits. They differ in that: (a) O. ramosissima lacks lenticular wail thickenings and refractive needle-like inclusions in medullary cells, both of which are present in O. truncata; (b) O. ramosissima has branched spermatangial filaments that terminate in a cluster of several cells, whereas in O. truncata the unbranched spermatangial filaments have a single large terminal sterile cell; and (c) cystocarps of O. ramosissima lack protuberant ostioles but ostioles are remarkably protuberant in o. truncata. Phylogenetic analyses of rbcL sequences of Laurencia obtusa (Hudson) Lamouroux and all five Atlantic European species of Osmundea, including the type species, strongly support the generic status of Osmundea. Osmundea ramosissima and O. truncata are closely related (5.2% sequence divergence) and form a well-supported clade sister to a clade consisting of O. pinnatifida (Hudson) Stack-house, O. osmunda Stackhouse and O. hybrida (A. P. de Candolle) Nam. The formation of secondary pit connections between epidermal cells is a synapomorphy for the O. ramosissima + O. truncata clade. The close relationship between species with cup-shaped spermatangial pits (Osmundea hybrida) and urn-shaped pits (Osmundea pinnatifida and Osmundea osmunda) shows that spermatangial pit shape is not an important phylogenetic character. Parsimony analysis of a morphological data set also supports the genus Osmundea but conflicts with the molecular trees in infrageneric relationships, placing O. hybrida basal within the Osmundea clade and grouping O. osmunda and O. pinnatifida but not O. truncata and O. ramosissima. A key to Osmundea species is presented.

RESURRECTION OF THE GENUS OSMUNDEA WITH AN EMENDATION OF THE GENERIC DELINEATION OF LAURENCIA (CERAMIALES, RHODOPHYTA)

Vegetative and reproductive development of some European and Californian species of Laurencia Lamouroux (Ceramiales, Rhodophyta), L. obtusa (Hudson) Lamouroux, L. spectabilis Postels et Ruprecht, L. crispa Hollenberg, L. osmunda (S.G. Gmelin) Maggs et Hommersand, L. pinnatifida (Hudson) Lamouroux and L. truncata Kutzing, is investigated on the basis of liquid-preserved and herbarium specimens. The latter five species share several features, but they differ distinctly from L. obtusa, the lectotype of the genus, in essential anatomical characters of vegetative and male reproductive structures and tetrasporangial development. In these five species each vegetative axial segment produces two rather than four pericentral cells, and spermatangial branches (filaments) are produced in apical pits of branchlets from apical and epidermal cells rather than from trichoblasts arising from axial cells. The spermatangial branches are usually branched alternately and usually terminate in a cluster of several large sterile vesicular cells, rather than being branched dichotomously and terminating in a single, or occasionally a row of two, large sterile vesicular cells as in L. obtusa. Apical spermatangial pits of fertile male branchlets (except for those in L. truncata) are pocket- (or urn)-shaped, with an ostiole-like upper opening, rather than cup- (or bowl)-shaped. In these five species tetrasporangia are produced laterally from random epidermal cells rather than abaxially from particular pericentral cells (the third and fourth ones) as in L. obtusa, and the two presporangial cover cells are aligned parallel rather than transverse to the stichidial axis in surface view. These important differences strongly suggest that L. spectabilis, L. crispa, L. osmunda, L. pinnatifida and L. truncata occupy a phylogenetically different position from L. obtusa, and lead to the conclusion that the genus Osmundea Stackhouse, which was based on 0. expansa Stackhouse, nom. illeg. (= Laurencia osmunda) and which has been a nomen rejiciendum as an earlier facultative synonym of Laurencia, should be resurrected. Emendations of the generic criteria of Laurencia and Osmundea are proposed here, and relevant nomenclatural changes for several Laurencia species are also included.