In vitro induction of Entamoeba gingivalis cyst-like structures from trophozoites in response to antibiotic treatment.

Background: Entamoeba gingivalis (E. gingivalis) is an anaerobic protozoan that is strongly associated with inflamed periodontal pockets. It is able to invade the mucosal epithelium of the human host, where it can feed on epithelial cells and elicit a severe innate immune response. Unlike other Entamoeba species, it is considered that E. gingivalis cannot form cysts, because it is a non-infectious protozoan. The lack of encystation capability would make it susceptible to periodontal treatment. However, it is not clear how the human host becomes infected with E. gingivalis trophozoites. We investigated the ability of E. gingivalis to encapsulate in response to an unfavorable environment in vitro. Methods: Different strains of E. gingivalis, isolated from inflamed periodontal pocket samples, were cultured for 8 days in the presence or absence of the antimicrobials amoxycillin and metronidazole. To reveal cyst formation, we investigated the morphology and ultrastructure of the amoeba by light, fluorescence, transmission and scanning electron microscopy. We also used the fluorescent dye calcofluor white M2R to demonstrate chitin present in the cyst wall. Results: We observed exocysts and an intra-cystic space separating the encapsulated trophozoite from the environment. Remarkably, cysts showed a smooth surface, polygonal edges and smaller size compared to free-living trophozoites. In addition, encapsulated trophozoites that detached from the cyst wall had a dense cytoplasma without phagocytic vesicles. The cyst walls consisted of chitin as in other Entamoba species. The encapsulated trophozoids were mononuclear after antibioticinduced encapsulation. Discussion: We conclude that E. gingivalis cyst formation has significant implications for dissemination and infection and may explain why established treatment approaches often fail to halt periodontal tissue destruction during periodontitis and peri-implantitis.

Multicystic peritoneal tumor in two layer hens.

In chicken, peritoneal cystic lesions have not been clearly categorized. In this study, diffuse peritoneal multiple cysts were observed in two layer hens. The cysts in the serosa were lined with single layers of squamous or cuboidal cells. The papillary proliferations of columnar cells were also observed in one case. The smooth muscle layer or mass were observed around the cysts in both cases. The cystic lining cells were positive for pan-cytokeratin, vimentin, S100 and Wilms tumor 1. Ultrastructurally, they had sparsely microvilli on the luminal surface. The histological results indicated the present cases were multicystic mesothelioma, but also had characteristics of Mullerian epithelium. This is the first report describing the detailed pathological feature of unique multicystic tumor in chicken.

Verruciform xanthoma accompanying a cystic growth: A case report.

Verruciform xanthoma, an uncommon, benign lesion with characteristic histopathological features, usually develops on the oral mucosa or genital area. We present an unusual case of verruciform xanthoma observed on the inguinal skin of a 52-year-old healthy man along with an underlying cystic component. The superficial lesion was a pedunculated nodule with a fissured surface and an 18-mm mound-like pigmented tumor underneath it. The histopathologically deep lesion was continuously attached to the superficial lesion. It was composed of fistula or sinus-like spaces and covered with acanthotic epithelium. The epidermis and upper dermis of both lesions showed identical histopathological findings: varying degrees of acanthosis, elongation of rete ridges, eosinophilic parakeratotic layer extending toward the dermis, and densely infiltrating foam cells confined to the papillary layer of the dermis. This finding of a cystic component in the deep dermis expands the histopathologic features of verruciform xanthoma.

Demonstration and Characterization of Cyst-Like Structures in the Life Cycle of Trichomonas vaginalis.

Trichomonas vaginalis is the parasitic protozoan residing in human urogenital tract causing trichomoniasis, which is the leading non-viral sexually transmitted disease. It has cosmopolitan distribution throughout the globe and affects both men and women. Lifecycle of the parasite has been traditionally described as consisting of motile and symptom-causing trophozoites. Chemical and temperature perturbations in trophozoites have been shown to aid conversion to pseudocysts, which is poorly investigated. In the current study, we show the formation of viable cyst-like structures (CLS) in stationary phase of T. vaginalis axenic culture. We used a fluorescent stain called calcofluor white, which specifically binds to chitin and cellulose-containing structures, to score for T. vaginalis CLS. Using flow cytometry, we demonstrated and quantitated the processes of encystation as well as excystation; thus, completing the parasite's lifecycle in vitro without any chemical/temperature alterations. Like cysts from other protozoan parasites such as Entamoeba histolytica and Giardia lamblia, T. vaginalis CLS appeared spherical, immotile, and resistant to osmotic lysis and detergent treatments. Ultrastructure of CLS demonstrated by Transmission Electron Microscopy showed a thick electron-dense deposition along its outer membrane. To probe the physiological role of CLS, we exposed parasites to vaginal pH and observed that trophozoites took this as a cue to convert to CLS. Further, upon co- culturing with cells of cervical origin, CLS rapidly excysted to form trophozoites which abrogated the cervical cell monolayer in a dose-dependent manner. To further corroborate the presence of two distinct forms in T. vaginalis, we performed two-dimensional gel electrophoresis and global, untargeted mass spectrometry to highlight differences in the proteome with trophozoites. Interestingly, CLS remained viable in chlorinated swimming pool water implicating the possibility of its role as environmentally resistant structures involved in non-sexual mode of parasite transmission. Finally, we showed that symptomatic human patient vaginal swabs had both T. vaginalis trophozoites and CLS; thus, highlighting its importance in clinical infections. Overall, our study highlights the plasticity of the pathogen and its rapid adaption when subjected to stressful environmental cues and suggests an important role of CLS in the parasite's life cycle, pathogenesis and transmission.

Aberrant endocytosis leads to the loss of normal mitotic spindle orientation during epithelial glandular morphogenesis.

Epithelial cells form tissues with many functions, including secretion and environmental separation and protection. Glandular epithelial tissues comprise cysts and tubules that are formed from a polarized, single-epithelial cell layer surrounding a central, fluid-filled lumen. The pathways regulating key processes in epithelial tissue morphogenesis such as mitotic spindle formation are incompletely understood, but are important to investigate, as their dysregulation is a signature of epithelial tumors. Here, we describe a signaling axis that manifests in a defect in mitotic spindle orientation during epithelial growth and cystogenesis. We found that activation of the small GTPase ADP-ribosylation factor 6 (ARF6) results in the sustained internalization of cell-surface components such as the cMet receptor and the cell-adhesion molecule E-cadherin. The spindle orientation defect arising from elevated levels of ARF6-GTP required an increase in cMet endocytosis, but was independent of E-cadherin internalization or elevated extracellular signal-regulated kinase (ERK) activity resulting from internalized receptor signaling on endosomes. Misorientation of the mitotic spindle resulted in the development of epithelial cysts with structural abnormalities, the most conspicuous of which was the presence of multiple intercellular lumens. Abnormal mitotic spindle orientation was necessary but insufficient to disrupt glandular development, as blocking the strong prosurvival signal resulting from ERK hyperactivation yielded structurally normal cysts despite continued manifestation of spindle orientation defects. Our findings highlight a previously unknown link between ARF6 activation, cMet receptor internalization, and mitotic spindle orientation during epithelial glandular morphogenesis.

α5ß1 integrin mediates pulmonary epithelial cyst formation.

BACKGROUND: Formation of the epithelial cyst involves the establishment of apical-basolateral polarity through a series of cellular interactions that are in part mediated by the extracellular matrix (ECM). We report that in a three-dimensional multi-cellular self-assembly model of lung development, α5 integrin regulates epithelial cyst formation through organization of soluble fibronectin matrix into insoluble fibrils through a process called fibrillogenesis. RESULTS: Dissociated murine embryonic lung cells self-assemble into three-dimensional pulmonary bodies that are dependent on α5ß1 integrin mediated fibrillogenesis for cell-cell mediated self-assembly: compaction and epithelial cyst formation. Knockdown of α5 integrin resulted in a significant increase in another mediator of fibrillogenesis, αV integrin. Compensatory increased expression of another mediator of fibrillogenesis, αV integrin, was not sufficient to normalize epithelial cyst formation. Loss of α5 integrin-mediated fibrillogenesis perturbed the ability of clustered epithelial cells to establish clear polarity, loss of epithelial cell pyramidal shape, and disrupted apical F-actin-rich deposition. Lack of rich central epithelial localization of F-actin cytoskeleton and Podocalyxin suggests that loss of α5 integrin-mediated fibrillogenesis interferes with the normal cytoskeleton organization that facilitates epithelial cysts polarization. CONCLUSIONS: We conclude that lung epithelial cyst formation in development is mediated in part by α5ß1 integrin dependent fibrillogenesis. Developmental Dynamics 246:475-484, 2016. © 2017 Wiley Periodicals, Inc.

Transitory cystic cavities in the developing mammalian brain - normal or anomalous?

Transitory cavities associated with the ventricular system represent probably one of the most unique features in the developing mammalian brain. In rodents, the cavities exist transiently in the developing brain and do not appear to be associated with any pathological events. Among the various cavities, the pyramidal-shaped cavum septum pellucidum (CSP) located beneath the corpus callosum and between the lateral ventricles is most well defined. In addition to the CSP are the bilateral subependymal cysts that are consistently associated with the third and fourth ventricles as well as the aqueduct. The cavities/cysts contain a large number of amoeboid microglia expressing surface receptors and hydrolytic enzymes common to tissue macrophages. The significance of these cavities in the developing brain remains a conjecture. Firstly, the cavity walls are free of an apparent epithelial lining; hence, it is speculated that the cavities that appear to communicate with the widened neighboring interstitial tissue spaces may have resulted from physical traction due to the rapid growth of the perinatal brain. Secondly, the cavities contain prominent clusters of amoeboid microglia that may be involved in clearing the debris of degenerating axons and cells resulting from the early brain tissue remodeling. With the increase in brain tissue compactness following the beginning of myelination in the second postnatal week, all cavities are obliterated; concomitantly, the number of amoeboid microglia in them diminishes and all this might signal further maturation of the brain.

Morphological and Genetic Diversity of Opisthosporidia: New Aphelid Paraphelidium tribonemae gen. et sp. nov.

Aphelids are a poorly known group of parasitoids of algae that have raised considerable interest due to their pivotal phylogenetic position. Together with Cryptomycota and the highly derived Microsporidia, they have been recently re-classified as the Opisthosporidia, which constitute the sister group to the fungi within the Holomycota. Despite their huge diversity, as revealed by molecular environmental studies, and their phylogenetic interest, only three genera have been described (Aphelidium, Amoeboaphelidium, and Pseudaphelidium), from which 18S rRNA gene sequences exist only for Amoeboaphelidium and Aphelidium species. Here, we describe the life cycle and ultrastructure of a new representative of Aphelida, Paraphelidium tribonemae gen. et sp. nov., and provide the first 18S rRNA gene sequence obtained for this genus. Molecular phylogenetic analysis indicates that Paraphelidium is distantly related to both Aphelidium and Amoebaphelidium, highlighting the wide genetic diversity of aphelids. Paraphelidium tribonemae has amoeboflagellate zoospores containing a lipid-microbody complex, dictyosomes, and mitochondria with rhomboid cristae, which are also present in trophonts and plasmodia. The amoeboid trophont uses pseudopodia to feed from the host cytoplasm. Although genetically distinct, the genus Paraphelidium is morphologically indistinguishable from other aphelid genera and has zoospores able to produce lamellipodia with subfilopodia like those of Amoeboaphelidium.

The first record of calcareous dinoflagellate cysts from the Upper Cretaceous of the Volga River Region.

The taxonomic position of the microproblematics previously considered to be Foraminifera or Calcisphaerulidae is established. The first record from Russia of calcareous cysts of Dinoflagellata is presented. The microfossils originate from the Maastrichtian siliceous clay of the Volga River Region.

Entamoeba marina n. sp.; a New Species of Entamoeba Isolated from Tidal Flat Sediment of Iriomote Island, Okinawa, Japan.

The genus Entamoeba includes anaerobic lobose amoebae, most of which are parasites of various vertebrates and invertebrates. We report a new Entamoeba species, E. marina n. sp. that was isolated from a sample of tidal flat sediment collected at Iriomote Island, Okinawa, Japan. Trophozoites of E. marina were 12.8-32.1 µm in length and 6.8-15.9 µm in width, whereas the cysts were 8.9-15.8 µm in diam. and contained four nuclei. The E. marina cells contained a rounded nucleus with a small centric karyosome and uniformly arranged peripheral chromatin. Although E. marina is morphologically indistinguishable from other tetranucleated cyst-forming Entamoeba species, E. marina can be distinguished from them based on the combination of molecular phylogenetic analyses using SSU rDNA gene and the difference of collection sites. Therefore, we propose E. marina as a new species of the genus Entamoeba.

The sperm ultrastructure and spermiogenesis of Tribolium castaneum (Coleoptera: Tenebrionidae) with evidence of cyst degeneration.

Previous studies on the spermatogenesis of tenebrionid beetles showed the unusual formation of two antiparallel sperm bundles per cyst. In this work we reported this feature also in Tribolium castaneum using light and transmission electron microscopy. The sperm structure of T. castaneum, similar to other tenebrionids, consists of a three-layered acrosome, an elongated nucleus and a flagellum with a 9+9+2 axoneme, two accessory bodies and two asymmetric mitochondrial derivatives. The presence of two antiparallel sperm bundles per cyst also in Meloidae and Rhipiphoridae suggests that it is a strong trait synapomorphic for Tenebrionoidea. The huge degeneration of whole sperm cells in several cysts of testes during spermiogenesis is also described.

Preventive prospective of triclosan and triclosan-liposomal nanoparticles against experimental infection with a cystogenic ME49 strain of Toxoplasma gondii.

The preventative effect of triclosan (TS) and TS liposomal nanoparticles was studied on the early establishment of chronic infection with Toxoplasma gondii (T. gondii). Swiss albino mice were orally infected with 10 cysts of avirulent ME49 strain of T. gondii, and 2 weeks later they were orally treated with dual daily doses of 200mg/kg and 120 mg/kg TS and TS liposomes for 30 days; respectively. Effect of TS and TS liposomes was parasitologically and ultrastructurally evaluated, versus infected non-treated control. Their safety was biochemically assessed. Parasitologically, both TS and TS liposomes induced significant reduction in mice mortality, brain parasite burden and infectivity of cysts obtained from the brains of treated mice. Ultrastructurally, scanning electron microscopy of cysts obtained from infected mice treated with either TS or TS liposomes showed surface irregularities, protrusions and depressions. Transmission electron microscopy revealed disintegration of the cyst wall and vacuolation of the bradyzoites with disintegration of plasma membranes of both cysts and bradyzoites whether treated with TS or TS liposomes. Biochemical study reflected the safety of the TS and TS liposomes. Therefore, TS proved an effective, promising and safe preventive drug against early establishment of chronic toxoplasmosis. Loading TS on liposomes marginally enhanced its efficacy against T. gondii cysts yet allowed its use in a lower dose.

Centrosomal abnormalities characterize human and rodent cystic cholangiocytes and are associated with Cdc25A overexpression.

Hepatic cystogenesis in polycystic liver diseases is associated with abnormalities of cholangiocyte cilia. Given the crucial association between cilia and centrosomes, we tested the hypothesis that centrosomal defects occur in cystic cholangiocytes of rodents (Pkd2(WS25/-) mice and PCK rats) and of patients with polycystic liver diseases, contributing to disturbed ciliogenesis and cyst formation. We examined centrosomal cytoarchitecture in control and cystic cholangiocytes, the effects of centrosomal abnormalities on ciliogenesis, and the role of the cell-cycle regulator Cdc25A in centrosomal defects by depleting cholangiocytes of Cdc25A in vitro and in vivo and evaluating centrosome morphology, cell-cycle progression, proliferation, ciliogenesis, and cystogenesis. The cystic cholangiocytes had atypical centrosome positioning, supernumerary centrosomes, multipolar spindles, and extra cilia. Structurally aberrant cilia were present in cystic cholangiocytes during ciliogenesis. Depletion of Cdc25A resulted in i) a decreased number of centrosomes and multiciliated cholangiocytes, ii) an increased fraction of ciliated cholangiocytes with longer cilia, iii) a decreased proportion of cholangiocytes in G1/G0 and S phases of the cell cycle, iv) decreased cell proliferation, and v) reduced cyst growth in vitro and in vivo. Our data support the hypothesis that centrosomal abnormalities in cholangiocytes are associated with aberrant ciliogenesis and that accelerated cystogenesis is likely due to overexpression of Cdc25A, providing additional evidence that pharmacological targeting of Cdc25A has therapeutic potential in polycystic liver diseases.

Effects of multipurpose solutions on the viability and encystment of acanthamoeba determined by flow cytometry.

PURPOSE: To evaluate simultaneously the effects of multipurpose contact lens care solution (MPS) on the viability and encystment of Acanthamoeba using flow cytometry. METHODS: Viability and encystment rate were evaluated using Acanthamoeba castellanii (ATCC 50514 and ATCC 50370) and three clinical strains of Acanthamoeba spp. isolated from patients with Acanthamoeba keratitis. Acanthamoeba trophozoites (1.0 × 10(5) cells/mL) were exposed to four kinds of commercially available MPSs for 24 hours. After dispensing the cell suspension into two portions, one portion was stained with 0.004% Congo Red (CR), a fluorescence dye to stain the inner cell wall of cysts, and the other portion was stained with a mixture of Congo Red and 3% sarkosyl (CRS), a detergent to lyse the trophozoites and pseudocysts. Flow cytometric analysis of the treated portions was then carried out on an EPICS ALTRA flow cytometer. The encystment rate and disinfecting efficacies (percentage of rounded trophozoites, "pseudocyst") were calculated by the rates of CR-stained, CR-nonstained, and CRS-stained populations, respectively. Ultrastructural features of resistant (mature or immature) cysts and pseudocysts were observed by transmission electron microscopy. RESULTS: Resistant cysts and rounded trophozoites (pseudocysts) were stained with CR, whereas native (unrounded) trophozoites were not. Resistant cysts were also stained with CRS unlike pseudocysts. Three clinical isolates showed higher resistance and higher encystment rates than two ATCC strains when treated with encystment-positive control solution. Disinfecting efficacy of each MPS was not directly related to each encystment rate. Transmission electron microscopy observations showed basic differences in the ultrastructure of pseudocysts produced by MPSs and resistant cysts. CONCLUSIONS: These results suggest that viability and encystment of Acanthamoeba are independent phenomena, and therefore disinfecting efficacy of MPS and encystment rates of Acanthamoeba should be evaluated, respectively. Thus, it is important to evaluate simultaneously the disinfecting efficacies and encystment rates of newly developed premarket MPS using the authors' novel flow cytometric methods.

Pathological features of salivary gland cysts in a Shiba dog with GM1 gangliosidosis: a possible misdiagnosis as malignancy.

Salivary gland cysts are often concurrent with GM1 gangliosidosis in Shiba dogs. Although the etiology is unknown, these cysts may be misdiagnosed as malignant due to the accumulation of foamy cells. The present study investigated the cytological, histopathological, immunohistochemical and electron microscopic characteristics of salivary gland cysts in a Shiba dog affected with GM1 gangliosidosis. The salivary gland masses were surgically enucleated and examined clinicopathologically and pathologically in a 7-month-old Shiba dog with GM1 gangliosidosis. Many large cells with rich cytoplasm including vacuoles of various sizes, i.e., foamy cells, were observed in stamp smears from the cut-surface of the masses and histopathologically in major parts of the cyst wall. Some of these foamy cells presented features similar to a spider-web appearance. The foamy cells were confirmed to have originated from macrophages based on marked immunohistochemical expression of vimentin, HLA-DR, lysozyme and Iba1. An ultrastructural study demonstrated electron-dense vesicular structures in the vacuolated cells. Therefore, the masses were diagnosed pathologically as benign salivary gland cysts with accumulation of foamy cells. In conclusion, the histopathological features of the salivary gland cysts in this Shiba dog were similar to those of lipoma and/or liposarcoma. In such cases, immunohistochemical and ultrastructural examinations were useful in the differential diagnosis. Practitioners, clinical pathologists and pathologists should take GM1 gangliosidosis into consideration when they encounter salivary gland cysts in Shiba dogs.