Ivana Bočina

Photobacterium damselae subsp. piscicida Outbreak in Cage-Reared Atlantic Bluefin Tuna Thunnus thynnus

Abstract High mortalities of the bluefin tuna Thunnus thynnus reared in Adriatic Sea cages occurred during spring 2004. Outbreaks had been previously reported at the same facility in similar conditions in the tuna population harvested in winter 2003. Fish displayed no external clinical signs of disease except for changed coloration and atypical swimming, and gross pathology was limited to general signs of septicemia. Histological and bacteriological findings suggested an epizootic outbreak of pasteurellosis. The pathology suggested an acute infection with the changes characteristic of septicemia. Most fish died in 1–2 d without gross pathology, possibly because of the virulence of the bacterial strain or reduced resistance of the fish. A low percentage of fish developed a chronic form of infection with disseminated granulomas in kidneys but also showed no gross pathology. Tuna parasitofauna was evaluated and did not appear to affect the development and the course of the disease.

Tubulogenesis in a simple cell cord requires the formation of bi-apical cells through two discrete Par domains

Apico-basal polarization is a crucial step in the de novo formation of biological tubes. In Ciona notochord, tubulogenesis occurs in a single file of cells in the absence of cell proliferation. This configuration presents a unique challenge for the formation of a continuous lumen. Here, we show that this geometric configuration is associated with a novel polarization strategy: the generation of bipolar epithelial cells possessing two apical/luminal domains instead of one, as in the conventional epithelium. At the molecular level, cells establish two discrete Par3/Par6/aPKC patches, and form two sets of tight junctions, in opposite points of the cells. The key molecule controlling the formation of both domains is Par3. Changing the position of the cells within the organ fundamentally changes their polarity and the number of apical domains they develop. These results reveal a new mechanism for tubulogenesis from the simplest cell arrangement, which occurs in other developmental contexts, including vertebrate vascular anastomosis.

Anion translocation through an Slc26 transporter mediates lumen expansion during tubulogenesis

Lumen formation is a critical event in biological tube formation, yet its molecular mechanisms remain poorly understood. Specifically, how lumen expansion is coordinated with other processes of tubulogenesis is not well known, and the role of membrane transporters in tubulogenesis during development has not been adequately addressed. Here we identify a solute carrier 26 (Slc26) family protein as an essential regulator of tubulogenesis using the notochord of the invertebrate chordate Ciona intestinalis as a model. Ci-Slc26aα is indispensable for lumen formation and expansion, but not for apical/luminal membrane formation and lumen connection. Ci-Slc26aα acts as an anion transporter, mediating the electrogenic exchange of sulfate or oxalate for chloride or bicarbonate and electroneutral chloride:bicarbonate exchange. Mutant rescue assays show that this transport activity is essential for Ci-Slc26aα’s in vivo function. Our work reveals the consequences and relationships of several key processes in lumen formation, and establishes an in vivo assay for studying the molecular basis of the transport properties of SLC26 family transporters and their related diseases.

Expression analysis of the Atlantic bluefin tuna (Thunnus thynnus) pro-inflammatory cytokines, IL-1β, TNFα1 and TNFα2 in response to parasites Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea)

Pro-inflammatory cytokines play an important role in teleost defence against numerous types of pathogens, therefore are often used as biomarkers during various infections. In order to evaluate Atlantic bluefin tuna IL-1β, TNFα1 and TNFα2 induction by PAMPs, we quantified their expression during in vitro stimulation of peripheral blood leukocytes by LPS and Poly I:C. Furthermore, their role in acute and chronic parasitic infection was examined during natural infection of Pseudocycnus appendiculatus (Copepoda) and Didymosulcus katsuwonicola (Digenea), as well as during leukocyte exposure to total protein extracts isolated from two parasite species. Induction of ABT IL-1β and TNFα2 by PAMPs and protein extracts from D. katsuwonicola and P. appendiculatus, as well as during natural infection with two parasites, suggests these cytokines play an important role in inflammation, being engaged in controlling parasite infections, in contrast to ABT TNFα1. Cellular innate response to the digenean D. katsuwonicola showed rather chronic character, resulting with parasite encapsulation in connective tissue. Mast cells, eosinophils, goblet cells, and occasional rodlet cells found at the site of infection, along with the induction of TNFα2, suggest the presence of a moderate inflammatory reaction that fails to seriously endanger digenean existence. In contrast, copepod P. appendiculatus, attached to the gill epithelium by clamping, caused direct tissue disruption with undergoing necrotic or apoptotic processes, and extensive proliferation of rodlet and goblet cells. Differential expression patterns of target cytokines in tissue surrounding two parasites and in vitro PBL model suggest that quality and quantity of tuna immune response is conditioned by parasite adaptive mechanisms and pathogenicity.

Reaction of the mussel Mytilus galloprovincialis (Bivalvia) to Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria) concurrent infestation

In total 480 individuals of Mytilus galloprovincialis were sampled monthly from October 2009 to September 2010, at the shellfish farm in the Mali Ston Bay, south Adriatic Sea (Croatia) in order to assess the extent of pathology imposed by two parasites, Eugymnanthea inquilina (Cnidaria) and Urastoma cyprinae (Turbellaria). Although a deteriorating impact on host reproduction or condition index was lacking, we evidenced ultrastructural and functional alteration in host cells at the attachment site. Ultrastructural changes included hemocytic encapsulation of the turbellarian and cell desquamation in medusoid infestation. Caspase positive reaction inferred by immunohistochemistry (IHC) was triggered in cases of turbellarian infestation, in contrast with hydroids, suggesting that the former exhibits more complex host-parasite interaction, reflected in the persistent attempts of the parasite to survive bivalve reaction. We have evidenced that both organisms trigger specific host reaction that although not costly in terms of host reproductive cycle or growth, results in mild tissue destruction and hemocyte activation. A lower degree of tissue reaction was observed in cases of hydroid infestation, compared to turbellarian.

Immunohistochemical and electronmicroscopic features of mesenchymal-to-epithelial transition in human developing, postnatal and nephrotic podocytes

Differentiation of human podocytes starts with mesenchymal-to-epithelial transition (MET) of the metanephric mesenchyme into the S-shaped nephrons. During further development, differentiating podocytes regain mesenchyme-like cell characteristics by epithelial-to-mesenchymal transition (EMT), leading to formation of the terminally differentiated, non-dividing cell. Both MET and EMT processes involve changes in content and organization of cytoskeletal and actin filaments, accompanied by the increased glomerular vascularization. Here, we analyze and compare normal human developing, postnatal and nephrotic podocytes and glomeruli, using immunohistochemical and double immunofluorescent methods for detection of markers of cytoskeletal filaments (nestin, cytokeratin 10—CK10, vimentin and α-SMA), vasculogenesis (CD31 and VEGF) and podocyte function (receptor for advanced glycation end products, RAGE). In addition, electron microscopy is used to detect ultrastructural changes of the podocytes. Early metanephric cup mesenchyme expresses all investigated markers except α-SMA, which characterizes only surface mesenchymal cells. In differentiating podocytes and cells of Bowman’s capsule (parietal podocytes) nestin decreases, vimentin increases, while CK10 gradually disappears. Increase in α-SMA is associated with blood vessels development, appearance of podocyte pedicles and slit diaphragm and loss of intercellular connections (zonulae adherentes). Increase in CD31 characterizes vascular glomerular tufts development, while decrease in RAGE expression accompanies normal podocyte differentiation. In congenital nephrotic syndrome of the Finnish type, dedifferentiated podocytes display changes in cytoskeletal filaments and depletion of podocyte pedicles, while glomerular vascular supply is diminished. Our data also suggest high potential of metanephric mesenchyme and parietal podocytes in possible regeneration of the damaged podocytes.

Expression of epithelial and mesenchymal differentiation markers in the early human gonadal development

Expressions of cytokeratin 8 (CK8), vimentin, nestin, and alpha‐smooth‐muscle‐actin (alpha‐SMA) were analyzed in the developing gonads of 12, 5–9 week old (W) human conceptuses by immunohistochemistry and immunofluorescence. During the investigated period, the number of CK8 positive cells increased from 56% to 92% in the gonadal surface epithelium, from 50% to 60% in the stroma, and from 23% to 42% in the medulla. In the early fetal period, the cell expression of CK8 increased in all gonadal parts, whereas primordial germ cells (PGC) remained negative. The expression of vimentin increased in the gonad stroma (gs) from 73% to 88%, and in the surface epithelium from 18% to 97% until ninth W. The medulla had the highest expression of vimentin in the seventh to eighth W (93%). Vimentin and CK8 colocalized in the somatic cells, while some PGCs showed vimentin expression only. Initially, nestin was positive in the gonad surface epithelium (8%) and stroma (52%), however during further development it decreased to 1% and 33%, respectively. In the early fetal period, the nestin positive cells decreased from 44% to 31% in the gonad medulla. Alpha‐SMA was positive only in the blood vessels and mesonephros. The described pattern of expression of intermediate filaments (IF) in developing human gonads suggests their role in the control of PGC apoptosis, early differentiation of gs cells and cell migration. Both epithelial and mesenchymal origins of follicular cells and possible epithelial‐to‐mesenchymal transition of somatic cells is proposed. Lastly, IF intensity expression varies depending on the cell type and developmental period analyzed. Anat Rec, 300:1315–1326, 2017.

Mechanism of cystogenesis in nephrotic kidneys: a histopathological study

BackgroundNephrotic syndrome (NS) is pathological condition characterized by heavy proteinuria. Our study investigates hypothesis that change in cell proliferation of proximal tubules influences primary cilia structure and function and promotes cystogenesis in congenital nephrotic syndrome of the Finnish type (CNF) and focal segmental glomerulosclerosis (FSGS).MethodsCNF kidneys were analyzed genetically. Proliferation (Ki-67), apoptosis (caspase-3), and primary cilia (α-tubulin) length and structure were analyzed immunohistochemically and ultrastructurally in healthy, CNF and FSGS kidneys. Cyst diameters were measured and correlated with proliferation index.ResultsProximal tubules cells of healthy kidneys did not proliferate. In nephrotic kidneys, tubules with apparently normal diameter covered by cuboidal/columnar epithelium (PTNC) contained 81.54% of proliferating cells in CNF and 36.18% in FSGS, while cysts covered with columnar epithelium (CC) contained 37.52% of proliferating cells in CNF and 45.23% in FSGS. The largest cysts, covered with squamous epithelium (CS) had 11.54% of proliferating cells in CNF and 13.76% in FSGS. Increase in cysts diameter correlated with changes in proliferation index, tubular cells shape, primary cilia formation and appearance of apoptotic cells.ConclusionsWe present a novel histopathological data on the structure and possible changes in function of tubular cell in NS kidneys during cystogenesis. We suggest existence of common principles of cystogenesis in CNF and FSGS kidneys, including serious disturbances of tubular cells proliferation and apoptosis, and faulty primary cilia signaling leading to deterioration of proteinuria in NS kidneys.

Cilia-like structures anchor the amphioxus notochord to its sheath.

Body stiffness is important during undulatory locomotion in fish. In amphioxus, the myosepta play an important role in transmission of muscular forces to the notochord. In order to define the specific supporting role of the notochord in amphioxus during locomotion, the ultrastructure of 10 adult amphioxus specimens was analyzed using transmission electron microscopy. Numerous cilia-like structures were found on the surface of each notochordal cell at the sites of their attachment to the notochordal sheath. Ultrastructurally, these structures consisted of the characteristic arrangement of peripheral and central microtubular doublets and were anchored to the inner layer of the notochordal sheath. Immunohistochemically, a positive reaction to applied dynein and β-tubulin antibodies characterized the area of the cilia-like structures. We propose that reduced back-and-forth movements of the cilia-like structures might contribute to the flow of the fluid content inside the notochord, thus modulating the stiffness of the amphioxus body during its undulatory locomotion.

Redescription of Myxidium sphaericum Thélohan, 1895 and Ceratomyxa beloneae Lubat et al., 1989 from the gall bladder of the garpike, Belone belone in the Adriatic Sea

The garpike Belone belone (L.) is an epipelagic fish abundant in the Mediterranean, parasitized by two coelozoic myxosporidians in the gall bladder — Myxidium sphaericum Thélohan, 1895 and Ceratomyxa beloneae Lubat, Radujković, Marques et Bouix, 1989. In order to redescribe these intriguing and taxonomically complex species, whose original descriptions are limited only to line drawings based on light microscopy, we studied the morphology and ultrastructure of their vegetative and sporogenic stages. M. sphaericum has an oval to sigmoid or mild crescent shape in the frontal view, large conical polar capsules, opening at the spore end, in opposite direction of the spore length, with 8–9 coils of polar filament. C. beloneae has elongated and slightly arched valves; symmetrical and smooth, with the posterior surface almost straight and anterior surface mildly inclinated, having 5 coils of the polar filament.