Fasciola hepatica: Histological changes in the reproductive structures of triclabendazole (TCBZ)-sensitive and TCBZ-resistant flukes after treatment in vivo with TCBZ and the related benzimidazole derivative, Compound Alpha

Twenty-four shed-reared lambs were each infected orally with 250 metacercariae of Fasciola hepatica, using either the triclabendazole (TCBZ)-sensitive Cullompton isolate or the TCBZ-resistant Sligo isolate. Twelve weeks after infection the lambs were treated with TCBZ (10 mg/kg) or with the experimental fasciolicide, Compound Alpha (Cpd alpha), a benzimidazole derivative of TCBZ (15 mg/kg). The lambs were euthanised 48,72 and 96 h after TCBZ treatment, or 24, 48 and 72 h after Cpd a treatment, and flukes were collected from the liver and/or gall bladder of each animal. Untreated animals harbouring 12-week infections were euthanised 24 h after administration of anthelmintic to the treatment groups, and the untreated flukes provided control material. A semi-quantitative assessment of the degree of histological change induced by the two drugs after different times of exposure was achieved by scoring the intensity of three well-defined lesions that developed in the testes and uteri of a representative sample of flukes from each lamb. In general, it was found that in those tissues where active meiosis and/or mitosis occurred (testis, ovary, and vitelline follicles), there was progressive loss of cell content due to apparent failure of cell division to keep pace with expulsion of the mature or effete products. Further, actively dividing cell types tended to become individualised, rounded and condensed, characteristic of apoptotic cell death. Protein synthetic activity was apparently inhibited in the Mehlis' secretory cells. In the uterus, where successful formation of shelled eggs represents the culmination of a complex sequence of cytokinetic, cytological and synthetic activity involving the vitelline follicles, the ovary and the Mehlis' gland, histological evidence indicating failure of ovigenesis was evident from 24 h post-treatment onwards. The development of these lesions may be related to the known antitubulin activity of the benzimidazole class of anthelmintics, to the induction of apoptosis in cells where mitosis or meiosis has aborted due to failure of spindle formation, and to drug-induced inhibition of protein synthesis. The semi-quantitative findings indicated that Cpd a is slightly less efficacious than TCBZ itself in causing histological damage to the reproductive structures of TCBZ-sensitive flukes, and that, like TCBZ, it caused no histological damage in flukes of the TCBZ-resistant isolate. This study illustrates the potential utility of histological techniques for conveniently screening representative samples of flukes in field trials designed to validate instances of drug resistance or to test the efficacy of new products against known drug-resistant and drug-susceptible fluke isolates. It also provides reference criteria for drug-induced histopathological changes in fluke reproductive structures which may aid interpretation of TEM findings. (C) 2009 Elsevier B.V. All rights reserved.

Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation

We sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/l glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/l glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two- to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/l glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/l glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation.

Development of an egg hatch assay for the diagnosis of triclabendazole resistance in Fasciola hepatica: proof of concept.

The aim of this study was to develop an Egg Hatch Assay (EHA) test for the detection of triclabendazole (TCBZ) resistance in Fasciola hepatica. A number of fluke isolates were used, of differing sensitivity to TCBZ. Eggs were exposed to solutions of triclabendazole sulphoxide (TCBZ.SO) for 14 days, then triggered to hatch. Egg development was divided into 6 distinct and easily identifiable stages: dead, empty, unembryonated, cell division, eye spot and hatched. The number of eggs reaching those stages was recorded. Initially, the discriminating dose (1% hatch) was determined for the Cullompton isolate, used as TCBZ-susceptible (TCBZ-S) standard. Once this concentration had been resolved, the response of different isolates to this concentration was examined. The hatch rate of the Fairhurst isolate was not significantly different from that of the Cullompton isolate, confirming its TCBZ-S status. The Patagonia isolate has not been exposed to TCBZ in the field and should be TCBZ-S: the results of the EHA supported this. The egg hatch response of the Oberon and Dutch isolates differed significantly from that of the Cullompton isolate; the former isolates are regarded as TCBZ-resistant (TCBZ-R) and the results confirmed this. Another isolate, the Leon isolate, was originally described as being TCBZ-R, but has since been shown to be TCBZ-S. There was no difference in its response to TCBZ.SO in the EHA from the Cullompton (and Fairhurst and Patagonia) isolate(s), further indicating its TCBZ-S status. The impact of TCBZ.SO treatment on the component stages of egg development was determined and revealed differences between the isolates. In conclusion, the results of the study have shown that it is possible to discriminate between TCBZ-S and TCBZ-R isolates of F. hepatica on the basis of the response of their eggs to an EHA and the test could be used to evaluate the TCBZ sensitivity of unknown field isolates

FASCIOLA-HEPATICA - DISRUPTION OF SPERMATOGENESIS BY THE MICROFILAMENT INHIBITOR CYTOCHALASIN-B

The distribution of actin filaments in the spermatogenic cells of Fasciola hepatica was determined using a fluorescent derivative of phalloidin. Actin was localised primarily in the region of separation of a secondary spermatogonium from a primary spermatogonium, in the inner faces at the centre of four-cell clusters of tertiary spermatogonia and in the cytophore region of spermatocyte and spermatid rosettes. The effect of the microfilament inhibitor cytochalasin B (100-mu-g/ml) on the ultrastructure of the spermatogenic cells was determined in vitro by transmission electron microscopy using tissue-slice material. Cytochalasin B treatment led to the formation of bi- and multinucleate cells, whose frequency increased with progressively longer incubation periods. Few typical rosettes of spermatocyte and spermatid cells were evident from 6 h onwards, being replaced by syncytial masses of cells. Spermatozoon formation became abnormal in the longer treatment periods, the spermatozoa containing variable numbers of axonemes and an altered distribution of cortical microtubules. Multiple axonemes were observed in the cytoplasm of spermatid cells. The results are discussed in relation to the established role of actin in the cytokinesis phase of cell division and to the effects of cytochalasin B on other tissues and organ systems within the fluke.

Rescue of glandular dysmorphogenesis in PTEN-deficient colorectal cancer epithelium by PPARγ-targeted therapy

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-? (PPAR?), cultures were treated with the PPAR? ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPAR? antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPAR? ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.

Genomic instability in Chinese hamster cells after exposure to X rays or alpha particles of different mean linear energy transfer

Evidence has accumulated that radiation induces a transmissible persistent destabilization of the genome, which mag. result in effects arising in the progeny of irradiated but surviving cells. An enhanced death rate among the progeny of cells surviving irradiation persists for many generations in the form of a reduced plating efficiency. Such delayed reproductive death is correlated with an increased occurrence of micronuclei. Since it has been suggested that radiation-induced chromosomal instability might depend on the radiation quality, we investigated the effects of alpha particles of different LET by looking at the frequency of delayed micronuclei in Chinese hamster V79 cells after cytochalasin-induced block of cell division, A dose-dependent increase in the frequency of micronuclei was found in cells assayed 1 week postirradiation or later. Also, there was a persistent increase in the frequency of dicentrics in surviving irradiated cells, Moreover, we found an increased micronucleus frequency in all of the 30 clones isolated from individual cells which had been irradiated with doses equivalent to either one, two or three alpha-particle traversals per cell nucleus, We conclude that the target for genomic instability in Chinese hamster cells must be larger than the cell nucleus. (C) 1997 by Radiation Research Society

Reappraisal of the type species of Polysiphonia (Rhodomelaceae, Rhodophyta)

The genus Polysiphonia Greville, nom. cons., has had a long and confused nomenclatural history. At present, Polysiphonia has a wide circumscription, including at least 200 species, but it is heterogeneous in many vegetative and reproductive developmental features. Central to any re-evaluation of the genus is a detailed examination of the type species of Polysiphonia, P. urceolata (Lightfoot ex Dillwyn) Greville, which is conspecific with P. stricta (Dillwyn) Greville. We here report on the vegetative and reproductive morphology of P. stricta, including P, urceolata, based on type and other material from the British Isles. Thalli consist of prostrate and erect ecorticate axes with four pericentral cells, attached by unicellular rhizoids remaining in open connection with pericentral cells. Prostrate axes lack vegetative trichoblasts; trichoblasts occur seasonally on erect axes. Branch initials are cut off from the subapical cell at intervals of four or five segments in dichotomous and alternating pairs rather than being formed horn each axial cell in the spiral pattern typical of most species of Polysiphonia. Spermatangial branch initials, which are trichoblast homologues, are produced directly from each axial cell at the tips of erect branches, not subtended by trichoblasts, and have two- to five-celled sterile tips when mature. The mature carpogonial branch is four-celled with a two-celled first sterile group and a one-celled second sterile group. Following presumed fertilization, direct fusion apparently takes place between carpogonium and auxiliary tell; mature cystocarps are usually urceolate. Tetrasporangia are formed from the third pericentral cell, in straight series, and have two pre-sporangial cover cells. Previous accounts of a third, post-sporangial cover cell could not be substantiated. P. stricta and a small group of other Polysiphonia species differ in several important respects from most members of the genus, which have rhizoids cut off from pericentral cells by a cell division, abundant trichoblasts, spirally arranged tetrasporangia and a post-sporangial cover cell. The branching pattern of P. stricta highlights the difficulties of distinguishing between the tribes Polysiphonieae and Pterosiphonieae.

Fasciola hepatica:Histological demonstration of apoptosis in the reproductive organs of flukes of triclabendazole-sensitive and triclabendazole-resistant isolates, and in field-derived flukes from triclabendazole-treated hosts, using in situ hybridisation to visualise endonuclease-generated DNA strand breaks

Investigation of the triclabendazole (TCBZ) resistance status of populations of Fasciola hepatica in field cases of fasciolosis, where treatment failure has been reported, can be supported by histological examination of flukes collected from recently treated hosts. In TCBZ-sensitive flukes (TCBZ-S) exposed to TCBZ metabolites for 1-4. days in vivo, but not in TCBZ-resistant flukes (TCBZ-R), morphological changes suggestive of apoptosis occur in cells undergoing meiosis or mitosis in the testis, ovary and vitelline follicles. In order to verify or refute the contention that efficacy of TCBZ treatment is associated with apoptosis in the reproductive organs of flukes, histological sections of TCBZ-S (Cullompton isolate) flukes and TCBZ-R (Sligo isolate) flukes were subjected to the TdT-mediated dUDP nick end labelling (TUNEL) in situ hybridisation method, a commercially available test specifically designed to label endonuclease-induced DNA strand breaks associated with apoptosis. Additionally, sections of in vivo-treated and untreated flukes originating from field outbreaks of suspected TCBZ-S and TCBZ-R fasciolosis were labelled by the TUNEL method. It was found that in treated TCBZ-S flukes, strong positive labelling indicating apoptosis was associated with morphologically abnormal cells undergoing mitosis or meiosis in the testis, ovary and vitelline follicles. Background labelling in the positive testis sections was attributed to heterophagy of cell debris by the sustentacular tissue. The triggering of apoptosis was probably related to failure of spindle formation at cell division, supporting the contention that TCBZ inhibits microtubule formation. In treated TCBZ-R (Sligo Type 1) flukes, and in treated flukes from field outbreaks of suspected TCBZ-R fasciolosis, no significant labelling was observed, while sections of fluke derived from a field case of fasciolosis where TCBZ resistance was not suspected were heavily labelled. Light labelling was associated with the testis of untreated Cullompton (TCBZ-S) and Sligo Type 2 (TCBZ-R) flukes, which exhibit abnormal spermatogenesis and spermiogenesis, respectively. This was attributed to apoptosis and to heterophagy of effete germ line cells by the sustentacular tissue. It is concluded that demonstration of apoptosis by in situ hybridisation using the TUNEL method on sections of 1-4. days in vivo TCBZ-treated F. hepatica can contribute to the diagnosis of TCBZ resistance in field outbreaks of fasciolosis. © 2012 Elsevier B.V.

Dynamic oscillation of translation and stress granule formation mark the cellular response to virus infection

Virus infection-induced global protein synthesis suppression is linked to assembly of stress granules (SGs), cytosolic aggregates of stalled translation preinitiation complexes. To study long-term stress responses, we developed an imaging approach for extended observation and analysis of SG dynamics during persistent hepatitis C virus (HCV) infection. In combination with type 1 interferon, HCV infection induces highly dynamic assembly/disassembly of cytoplasmic SGs, concomitant with phases of active and stalled translation, delayed cell division, and prolonged cell survival. Double-stranded RNA (dsRNA), independent of viral replication, is sufficient to trigger these oscillations. Translation initiation factor eIF2a phosphorylation by protein kinase R mediates SG formation and translation arrest. This is antagonized by the upregulation of GADD34, the regulatory subunit of protein phosphatase 1 dephosphorylating eIF2a. Stress response oscillation is a general mechanism to prevent long-lasting translation repression and a conserved host cell reaction to multiple RNA viruses, which HCV may exploit to establish persistence.

Actin-binding proteins in the Arabidopsis genome database: properties of functionally distinct plant actin-depolymerizing factors/cofilins

The plant actin cytoskeleton is a highly dynamic, fibrous structure essential in many cellular processes including cell division and cytoplasmic streaming. This structure is stimulus responsive, being affected by internal stimuli, by biotic and abiotic stresses mediated in signal transduction pathways by actin-binding proteins. The completion of the Arabidopsis genome sequence has allowed a comparative identification of many actin-binding proteins. However, not all are conserved in plants, which possibly reflects the differences in the processes involved in morphogenesis between plant and other cells. Here we have searched for the Arabidopsis equivalents of 67 animal/fungal actin-binding proteins and show that 36 are not conserved in plants. One protein that is conserved across phylogeny is actin-depolymerizing factor or cofilin and we describe our work on the activity of vegetative tissue and pollen-specific isoforms of this protein in plant cells, concluding that they are functionally distinct.

The ASK1 and ASK2 genes are essential for Arabidopsis early development.

The requirement of CUL1 for Arabidopsis embryogenesis suggests that Skp1-CUL1-F-box protein (SCF) complexes play important roles during embryo development. Among the 21 Arabidopsis Skp1-like genes (ASKs), it is unknown which ASK gene(s) is essential for embryo development. In this study, we demonstrate a vital role for ASK1 and ASK2 in Arabidopsis embryogenesis and postembryonic development through analysis of the ask1 ask2 double mutant. Our detailed analysis indicates that the double mutations in both ASK1 and ASK2 affect cell division and cell expansion/elongation and cause a developmental delay during embryogenesis and lethality in seedling growth. The expression patterns of ASK1 and ASK2 were examined further and found to be consistent with their roles in embryogenesis and seedling development. We propose that mutations in ASK1 and ASK2 abolish all of the ASK1- and ASK2-based SCF and non-SCF complexes, resulting in alteration of gene expression and leading to defects in growth and development.

The effect of endothelin 1 on the retinal microvascular pericyte

The effect of the highly vasoactive peptide endothelin 1 (ET1) was tested on bovine retinal microvascular pericytes propagated in vitro. Specific binding of 125I-ET1 to retinal pericytes was documented by autoradiography. ET1 caused contraction of pericytes at a concentration of 0.1 nM which was accompanied by increases in inositol phosphates. Exposure of pericytes to 10 nM ET1 resulted in the aggregation and realignment of muscle-specific actins into bundles which were oriented parallel to the long axis of the cell, and ET1 was also mitogenic to pericytes in the presence of low levels of fetal calf serum. These observations suggest that ET1 may play an important role in endothelial cell-pericyte interactions within the microvasculature of the retina and that it may be involved in the autoregulation of retinal blood flow.

In vitro viability of choroidal melanomata following pre-enucleation irradiation

In a group of eighteen patients with uveal melanomas, seven underwent low-dose pre-enucleation irradiation of approximately 2000 cGy. All the tumours were propagated in tissue culture and the growth characteristics of tumour cells from irradiated eyes were compared with tumour cells from non-irradiated eyes. Cultures were observed with phase-contrast microscopy, and radioactive thymidine labelling was used to study cell turnover. Although tissue samples from peripheral areas of irradiated tumours produced a mixture of viable and non-viable cells, with reduced ability to attach to substrate, central regions of irradiated tumours contained viable cells which propagated freely in tissue culture.

Focal irradiation of perforating eye injuries

Plaques constructed with 125I were used to irradiate the sites of perforating ocular injuries in rabbits. An approximate dose of 16Gy given over a period of 6 days was shown to significantly reduce intraocular cellular proliferation when irradiation was commenced within 24 hours after injury. If irradiation was delayed until day 5, this reduction in cellular proliferation and intraocular membrane formation did not occur. Smaller radiation doses of approximately 6Gy given within 24 hours post-injury and administered over 6 days also reduced the extent of cellular proliferation but was not as effective as the 16Gy dose.

A light microscopic and autoradiographic study of non-irradiated and irradiated ocular wounds

Focal gamma irradiation was used to limit the intraocular extension of scar tissue which typically occurs after posterior perforating injury to the eye. Standard posterior perforating injuries were created in the right eye of forty-eight rabbits, half of which had the site of perforation focally irradiated using a Cobalt 60 ophthalmic plaque. Non-irradiated wounds healed with profuse formation of highly cellular and vascularised granulation tissue which invaded the vitreous to form contractile vitreo-retinal membranes. In irradiated eyes vitreo-retinal membrane formation was infrequent; the wounds showing only sparse granulation tissue with little or no extension into the vitreous cavity. Autoradiographic studies carried out in a second group of 40 animals showed that the episclera was the main source of the proliferating fibroblasts, and cell counts confirmed that the inflammatory and repair responses in irradiated wounds were both delayed and attenuated.