Should pelvic exenteration for symptomatic relief in gynaecology malignancies be offered?

OBJECTIVE: To review the outcomes of pelvic exenterative surgery done with a palliative intent and evaluate its role in relapsed gynaecology malignancies. METHOD: This is a retrospective cohort study between April 2009 and May 2012 in Oxford Gynaecological Cancer Centre. Patients were identified from the oncology surgical database. RESULTS: 18 patients were identified with a mean age 54 (26-79) years, who underwent surgery for symptomatic recurrent cancer. All except one patient had radiotherapy prior to surgery. 12 patients had cervical cancer, five had vulval cancer and one had endometrial cancer. About half of the patients had major surgical complications; however, majority was patients satisfied with the outcome. CONCLUSION: Pelvic exenteration in this context carries considerable morbidity and in this series achieved good symptom control with a mean overall survival of 11 months. Careful patient selection, adequate counselling and ongoing support are imperative of successful outcome.

Utility of peritoneal lavage cytology during laparoscopic salpingo-oophorectomy: a retrospective analysis.

OBJECTIVES: To assess the significance of peritoneal washing cytology at the time of laparoscopic salpingo-oophorectomy. DESIGN: Retrospective study. SETTING: Cambridge University Hospital. POPULATION: Four hundred and nine women who underwent laparoscopic salpingo-oophorectomy by the gynaecology oncology team between 2004 and December 2009 were included. One hundred and thirteen women had risk-reducing salpingo-oophorectomy, 103 women had salpingo-oophorectomy as part of breast cancer management, 59 had simple ovarian cysts, 111 had complex ovarian cysts and 23 had the procedure done for other reasons. METHODS: Histology and cytology results were reviewed and all hospital records were checked for subsequent malignancy. Sensitivity and specificity of peritoneal washing cytology was calculated. MAIN OUTCOME MEASURES: Malignant peritoneal cytology in the absence of cancer on histopathology. RESULTS: Eleven of the 409 women in our study had occult malignancy on histopathological examination and three of them had positive peritoneal washings. One woman had positive washings from metastatic breast cancer. Thirteen women developed different malignancies subsequently but none had primary peritoneal or ovarian cancer within a median follow-up interval of 34 months. CONCLUSIONS: Peritoneal lavage cytology did not pick up any additional malignancy in the study population. Based on the evidence presented, we suggest that peritoneal washing cytology during laparoscopic salpingo-oophorectomy is of limited value and should not be practised routinely.

Sphingolipid synthesis and membrane formation by Plasmodium.

Plasmodium falciparum is a protozoan parasite that causes the most virulent o f human malarias. The asexual blood-stage organism invades and multiplies in a vacuole in the mature erythrocyte. During intravacuolar growth, it induces the formation of a novel network o f tubovesicular membranes, the TVM, that is not present in uninfected red blood cells. Recent data suggest that sphingomyelin biosynthesis by the parasite is an essential requirement for the assembly o f the TVM. Furthermore, sphingolipid synthesis as well as the formation and function o f the TVM may provide new targets for chemotherapy against malaria parasites.

Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubovesicular network in the cytoplasm of mature erythrocytes.

This work describes two unusual features of membrane development in a eukaryotic cell. (a) The induction of an extensive network of tubovesicular membranes by the malaria parasite Plasmodium falciparum in the cytoplasm of the mature erythrocyte, and its visualization with two ceramide analogues C5-DMB-ceramide and C6-NBD-ceramide. "Sectioning" of the infected erythrocytes using laser confocal microscopy has allowed the reconstruction of detailed three-dimensional images of this novel membrane network. (b) The stage-specific export of sphingomyelin synthase, a biosynthetic activity concentrated in the Golgi of mammalian cells, to this tubovesicular network. Evidence is presented that in the extracellular merozoite stage the parasite retains sphingomyelin synthase within its plasma membrane. However, intracellular ring- and trophozoite-stage parasites export a substantial fraction (approximately 26%) of sphingomyelin synthase activity to membranes beyond their plasma membrane. Importantly we do not observe synthesis of new enzyme during these intracellular stages. Taken together these results strongly suggest that the export of this classic Golgi enzyme is developmentally regulated in Plasmodium. We discuss the significance of this export and the tubovesicular network with respect to membrane development and function in the erythrocyte cytosol.

Transport of fluorescent phospholipid analogues from the erythrocyte membrane to the parasite in Plasmodium falciparum-infected cells.

The asexual development of the human malaria parasite Plasmodium falciparum is largely intraerythrocytic. When 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazole-4-yl)amino]caproyl] phosphatidylcholine (NBD-PC) was incorporated into infected and uninfected erythrocyte membranes at 0 degrees C, it remained at the cell surface. At 10 degrees C, the lipid was rapidly internalized in infected erythrocytes at all stages of parasite growth. Our results indicate that the internalization of NDB-PC was not because of endocytosis but rapid transbilayer lipid flip-flop at the infected erythrocyte membrane, followed by monomer diffusion to the parasite. Internalization of the lipid was inhibited by (a) depleting cellular ATP levels; (b) pretreating the cells with N-ethyl maleimide or diethylpyrocarbonate; and (c) 10 mM L-alpha-glycerophosphorylcholine. The evidence suggests protein-mediated and energy dependent transmembrane movement of the PC analogue. The conditions for the internalization of another phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) were distinct from that of NBD-PC and suggest the presence of additional mechanism(s) of parasite-mediated lipid transport in the infected host membrane. In spite of the lack of bulk, constitutive endocytosis at the red cell membrane, the uptake of Lucifer yellow by mature infected cells suggests that microdomains of pinocytotic activity are induced by the intracellular parasite. The results indicate the presence of parasite-induced mechanisms of lipid transport in infected erythrocyte membranes that modify host membrane properties and may have important implications on phospholipid asymmetry in these membranes.

Partitioning of the Golgi apparatus during mitosis in living HeLa cells.

The Golgi apparatus of HeLa cells was fluorescently tagged with a green fluorescent protein (GFP), localized by attachment to the NH2-terminal retention signal of N-acetylglucosaminyltransferase I (NAGT I). The location was confirmed by immunogold and immunofluorescence microscopy using a variety of Golgi markers. The behavior of the fluorescent Golgi marker was observed in fixed and living mitotic cells using confocal microscopy. By metaphase, cells contained a constant number of Golgi fragments dispersed throughout the cytoplasm. Conventional and cryoimmunoelectron microscopy showed that the NAGT I-GFP chimera (NAGFP)-positive fragments were tubulo-vesicular mitotic Golgi clusters. Mitotic conversion of Golgi stacks into mitotic clusters had surprisingly little effect on the polarity of Golgi membrane markers at the level of fluorescence microscopy. In living cells, there was little self-directed movement of the clusters in the period from metaphase to early telophase. In late telophase, the Golgi ribbon began to be reformed by a dynamic process of congregation and tubulation of the newly inherited Golgi fragments. The accuracy of partitioning the NAGFP-tagged Golgi was found to exceed that expected for a stochastic partitioning process. The results provide direct evidence for mitotic clusters as the unit of partitioning and suggest that precise regulation of the number, position, and compartmentation of mitotic membranes is a critical feature for the ordered inheritance of the Golgi apparatus.

A membrane network for nutrient import in red cells infected with the malaria parasite.

The human malaria parasite Plasmodium falciparum exports an interconnected network of tubovesicular membranes (the TVM) that extends from the parasite's vacuolar membrane to the periphery of the red cell. Here it is shown that extracellular solutes such as Lucifer yellow enter the TVM and are delivered to the parasite. Blocking the assembly of the network blocked the delivery of exogenous Lucifer yellow, nucleosides, and amino acids to the parasite without inhibiting secretion of plasmodial proteins. These data suggest that the TVM is a transport network that allows nutrients efficient access to the parasite and could be used to deliver antimalarial drugs directly into the parasite.

Intracellular trafficking in Plasmodium-infected erythrocytes.

The past few years have witnessed considerable progress in molecular and biochemical studies of intracellular trafficking in malaria-infected red cells. Highlights include the identification of solute channels in the vacuolar membrane and the red blood cell membrane, a tubovesicular membrane network that delivers exogenous nutrients and drugs to the parasite, and parasite gene families that mediate adherence to endothelial cells and red cells.

The movement of fluorescent endocytic tracers in Plasmodium falciparum infected erythrocytes.

The fluorescent lipophilic probe 1,1'-dihexadecyl-3-3'-3-3'- tetramethylindocarbocyanine (diIC16) inserted in the red cell surface, functioned as a non-exchangeable lipid marker which was not metabolised or toxic in plasmodial cultures. Invasion by Plasmodium falciparum resulted in the internalisation of the lipid, suggesting the uptake of red cell membrane components during parasite entry. The fluorescent lipid was not transported from red cell to parasite membranes at subsequent stages of development, but label in the erythrocyte-derived parasitophorous vacuole was eventually detected in daughter merozoites. Fluorescent dextrans of 10 kDa in the extracellular medium were also not internalised during intraerythrocytic parasite growth. The results support that with the exception of the invasion step, plasmodial infection does not induce endocytosis in the erythrocyte membrane. Despite the lack of endocytosis, both D and L stereoisomers of the head group blocked phospholipid analogue N-4-nitrobenzo-2-oxa-1,3-diazoledipalmitoyl phosphatidylethanolamine (N-NBD-PE) inserted in the erythrocyte membrane, were internalised by mature infected erythrocytes. Lipid internalisation occurred by a non head group dependent parasite mechanism, which could account for the stage-specific uptake of phospholipids observed in mature infected erythrocytes. We were unable to detect the transport of carbocyanine dyes and N-NBD-PE from intracellular parasites back to the erythrocyte membrane. Additionally, the carbocyanine dyes were not transferred between adjacent organisms in a double infected red cell. The data argue for the absence of bulk membrane lipid transport between individual parasites and their host cell bilayer in an infected erythrocyte.

Brefeldin A inhibits protein secretion and parasite maturation in the ring stage of Plasmodium falciparum.

The release of all newly synthesized soluble proteins from the ring stage of Plasmodium falciparum-infected erythrocytes was reversibly blocked by brefeldin A, indicating the presence of a conserved step of classical eukaryotic secretory export within the parasite. This implies that proteins exported to the erythrocyte cytosol undergo secretory release at the parasite plasma membrane and subsequent translocation across the vacuolar membrane. Along with inhibiting protein export brefeldin arrested parasite maturation, but the cells remained viable even after 24 h in the presence of the drug. The results suggest that secretory export may be important for development, but not for immediate survival, at the ring stage.

A transferrin-independent iron uptake activity in Plasmodium falciparum-infected and uninfected erythrocytes.

Non-heme iron is essential for the asexual growth of the human malaria parasite Plasmodium falciparum in mature erythrocytes. Utilization of iron bound to serum transferrin by the parasitized cells has been postulated, but direct evidence for its specific delivery has not been reported. Here we demonstrate that normal levels of transferrin in human serum are not required for intraerythrocytic P. falciparum growth: culture medium immunodepleted 500-1000 fold in human transferrin was capable of supporting parasitemias and rates of invasion comparable to those observed in non-depleted medium. 55Fe bound to transferrin was not taken up by infected cells. A transferrin-independent non-heme iron uptake activity was, however, detected in both infected and uninfected erythrocytes when iron was presented to the cells as 55Fe-NTA or 55Fe-citrate. Although the uptake activity was not parasite specific, the radiolabel was found in association with parasites mechanically released from the infected erythrocytes, indicating that it is delivered to the intracellular organism. Evidence is presented that the transferrin-independent iron uptake activity is time-, temperature- and concentration-dependent, but apparently not energy-dependent.

Synthesis and secretion of proteins by released malarial parasites.

Controlled mechanical homogenization of Plasmodium falciparum-infected erythrocytes releases parasites of a quality sufficient for studying the export of newly synthesized plasmodial proteins. Protein synthesis occurs within intact released parasites as defined by resistance of acid-insoluble incorporation of radiolabel to high levels of exogenously added EDTA, hexokinase, and RNaseA. While exogenously added ATP and erythrocyte cytosol were not essential for biosynthetic activity at levels comparable to that seen in infected erythrocytes, the addition of an extracellular ATP regenerating system (ARS) stimulated the synthesis of parasite proteins. Conversely, parasite viability and biosynthetic activity are decreased by the addition of a non-hydrolyzable ATP analogue (ATP gamma S), ADP, or ATP in the absence of a regenerating system. These data suggest a metabolic interdependence between extracellular energy metabolism and biosynthetic functions within the parasite. The export of a predominant subset of proteins was retarded in the presence of Brefeldin A, indicating the existence of a classical secretory pathway characteristic of that seen in higher eukaryotic cells. Interestingly, a Brefeldin A-insensitive component of export was also consistently observed; this may suggest the existence of an additional alternative secretory mechanism in malaria.

Artemisinin and its derivatives are transported by a vacuolar-network of Plasmodium falciparum and their anti-malarial activities are additive with toxic sphingolipid analogues that block the network.

There is great need to identify and characterize drug targets and chemotherapeutic strategies against malaria. Here we show that a vacuolar-network induced by the human malaria parasite Plasmodium falciparum, is a major import pathway for artemisinin, a leading, new anti-malarial that is known to be effective against drug resistant strains. We also show that artemisinin-treatment induces aberrant, budding of a vacuolar-network membrane protein and its antimalarial activity is additive with toxic sphingolipid analogues that block the network. The data suggest that artemisinin alters membrane protein export from the vacuolar-network and combinations with anti-network reagents have the potential to provide powerful new chemotherapy for drug resistant malaria.

The human malaria parasite Plasmodium falciparum exports the ATP-binding cassette protein PFGCN20 to membrane structures in the host red blood cell.

PFGCN20 is a member of the ATP-binding cassette family of proteins that is closely related to the yeast translational regulator Gcn20p. We have generated a polyclonal antibody against the N-terminal region of PFGCN20 and studied the cellular localization of PFGCN20 throughout the erythrocytic life cycle of Plasmodium falciparum. PFGCN20 was found to be present at all stages and a pronounced export of PFGCN20 into the erythrocyte was observed in the trophozoite and schizont stages. In the indirect immunofluorescence assay, PFGCN20 was found to display significant colocalization with antigens detected by the monoclonal antibody 41E11. In contrast, there was only a minimal overlap of PFGCN20 localization with EMP2 and HRP2. Immunoelectron microscopy demonstrated a pronounced accumulation of PFGCN20 in the lumen of the parasitophorous vacuole and deconvolution fluorescence microscopy showed membrane association with selective regions of a tubovesicular network in the red cell. We also observed a concentration of PFGCN20 in electron-dense plaques just underneath the parasite's plasma membrane and an association of PFGCN20 with cytoplasmic vesicular structures within the parasite. The observed export of PFGCN20 and its association with the tubovesicular network in host red cells, may be indicative of the fact that PFGCN20 functions as ATP-binding subunit of an unknown multimeric ABC-transporter. The cytoplasmic localization of PFGCN20 in the parasite, however, suggests that the involvement of PFGCN20 in translational regulation or other cytoplasmic biological functions cannot be ruled out.

Stage-specific expression of plasmodial proteins containing an antigenic marker of the intraerythrocytic cisternae.

A monoclonal antibody, LWLI, recognized 3 proteins of 45, 50 and 102 kDa in Plasmodium falciparum-infected erythrocytes. The 45- and 50-kDa proteins were parasite-encoded and displayed markedly different peptide maps, indicating that they were distinct plasmodial polypeptides with a common antigenic epitope rather than differentially processed forms of a primary translational product. The 45-kDa protein was present throughout intraerythrocytic growth, while the 50-kDa molecule was not detected earlier than 11 h in the life cycle. The 102-kDa protein was only expressed in trophozoite- and schizont-infected red cells: its structural relationship to the 45- and 50-kDa proteins, if any, remains undefined. By indirect immunofluorescence and immunoelectron microscopy, LWLI bound to flattened intraerythrocytic cisternae exported into the erythrocyte cytoplasm. The results support the theory that proteins recognized by the antibody were concentrated in these compartments and their common antigenic epitope may serve as a marker for the cisternae. Stage-specific expression of LWLI reactive proteins implicates developmental regulation of cisternal functions during asexual parasite development.

The accumulation and metabolism of a fluorescent ceramide derivative in Plasmodium falciparum-infected erythrocytes.

We have examined the accumulation and metabolism of N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]aminocaproyl sphingosine (C6-NBD-cer) in Plasmodium falciparum FCR-3/A2-infected erythrocytes. C6-NBD-cer transferred to live infected erythrocytes at 2 degrees C to label the infected red cell surface and intracellular parasite membranes. Subsequent incubation for 30 min at 2 degrees C, resulted in a depletion of the ceramide label from the red cell membrane and an accumulation of fluorescence in parasite membranes, by an energy independent process. When the cells were subsequently warmed to 37 degrees C for 30 min, virtually all of the ceramide was converted to N-[7-(4-nitrobenzo-2-oxa-1,3- diazole)]aminocaproyl sphingosine-1-phosphocholine (C6-NBD-Sm). Uninfected erythrocytes were incapble of sphingomyelin synthesis. By fluorescence microscopy, sphingomyelin synthesis in infected erythrocytes occurred in compartments morphologically similar to those accumulating ceramide. To examine the intracellular sites of ceramide accumulation glutaraldehyde fixed cells were labeled with C6-NBD-ceramide and subsequently back extracted to remove excess probe. This resulted in a depletion of label at the red cell membrane but prominent fluorescence remained associated with the parasite. Photobleaching in the presence of diaminobenzidine resulted in precipitates in intraerythrocytic cisternae and the vacuolar membrane surrounding the parasite, rather than a perinuclear Golgi apparatus within the organism. The results support a novel organisation of plasmodial membranes regulating the accumulation and metabolism of C6-NBD-cer in infected erythrocytes.

Identification and localization of rab6, separation of rab6 from ERD2 and implications for an 'unstacked' Golgi, in Plasmodium falciparum.

The rab6 gene product in mammalian cells and yeast is localized to and regulates protein transport in the medial and trans Golgi cisternae, as well as the trans Golgi network. We have identified a homologue in the malaria parasite Plasmodium falciparum which displays a rab-like sequence that is 62.4% identical to mammalian rab6. In addition the parasite gene (Pfrab6 gene) contains an N-terminal hydrophobic domain, unique to P. falciparum. Antibodies developed to Pfrab6 localize protein in 4-7 well-resolved sites in a ring-stage parasite, as detected by high resolution fluorescence microscopy. This suggests that there are multiple, distinct foci of medial/trans Golgi membranes in a ring. ERD2 is a cis Golgi marker in mammalian cells. The plasmodial homologue of ERD2 (PfERD2) is concentrated in a single perinuclear region in a ring-stage parasite. This site is distinct from the Pfrab6 membranes, indicating that early and late Golgi markers can be segregated in P. falciparum. Mammalian cells contain a single Golgi complex where cis medial and trans markers are tightly stacked in closely apposed cisternae. In P. falciparum-rings however, rab6-associated membranes are not invariably 'stacked' with an ERD2 structure. In immunoelectron microscopy studies, both the PfERD2- and Pfrab6-associated membranes appear tubulovesicular in nature, devoid of cisternal morphology. Hence the Golgi of ring stage parasites may comprise of multiple, 'unstacked' tubulovesicular clusters, suggesting a primitive organization of the organelle in Plasmodia.

Rapid recombination among transfected plasmids, chimeric episome formation and trans gene expression in Plasmodium falciparum.

Although recombination is known to be important to generating diversity in the human malaria parasite P. falciparum, the low efficiencies of transfection and the fact that integration of transfected DNA into chromosomes is observed only after long periods (typically 12 weeks or more) have made it difficult to genetically manipulate the blood stages of this major human pathogen. Here we show that co-transfection of a P. falciparum line with two plasmids, one expressing a green fluorescent protein (gfp) reporter and the other expressing a drug resistance marker (Tgdhfr-ts M23), allowed selection of a population in which about approximately 30% of the parasites produce GFP. In these GFP-producing parasites, the transfected plasmids had recombined into chimeric episomes as large as 20 kb and could be maintained under drug pressure for at least 16 weeks. Our data suggest that chimera formation occurs early (detected by 7--14 days) and that it involves homologous recombination favored by presence of the same P. falciparum 5'hrp3 UTR promoting transcription from each plasmid. This indicates the presence of high levels of homologous recombination activity in blood stage parasites that can be used to drive rapid recombination of newly introduced DNA, study mechanisms of recombination, and introduce genes for trans expression in P. falciparum.

Transport mechanisms in Plasmodium-infected erythrocytes: lipid rafts and a tubovesicular network.

The mature human erythrocyte is a simple cell that is devoid of intracellular organelles and does not show endocytic or phagocytic activity at the plasma membrane. However, following infection by Plasmodium, the erythrocyte undergoes several morphological and functional changes. Parasite-derived proteins are exported into the erythrocyte cytoplasm and to the membrane, while several proteins are localised to the parasitophorous vacuolar membrane and to the tubovesicular membranous network structures surrounding the parasite. Recent evidence indicates that multiple host proteins, independent of the type of their membrane anchor, that exist in detergent-resistant membrane (DRM) rafts or microdomains enter this apicomplexan vacuole. The internalised host components along with the parasite-encoded transmembrane protein PfEXP1 can be detected as DRM rafts in the vacuole. It appears that in Plasmodium-infected erythrocytes lipid rafts may play a role in endovacuolation and macromolecular transport.