steps-for-autologous-ex-vivo-perfused-porcine-liver-kidney

Steps for the Autologous <em>Ex vivo</em> Perfused Porcine Liver-kidney Experiment

The use of ex vivo perfused models can mimic the physiological conditions of the liver for short periods, but to maintain normal homeostasis for an ...

University Hospitals of Leicester

Apoptin, a chicken anemia virus-encoded protein, induces apoptosis in human tumor cells but not in normal cells. The tumor-specific activity of Apoptin is correlated with its nuclear localization in tumor cells. In an attempt to elucidate the molecular mechanism of Apoptin-induced apoptosis, we identified human Hippi, the protein interactor and apoptosis co-mediator of Huntingtin interacting protein 1, as one of the Apoptin-associated proteins by yeast two-hybrid screen. We also demonstrated that Hippi could interact with Apoptin both in vitro and in human cells. Furthermore, subcellular localization studies showed that Hippi and Apoptin perfectly colocalized in the cytoplasm of normal human HEL cells, whereas in cancerous HeLa cells most Apoptin and Hippi were located separately in the nucleus and cytoplasm and, thus, showed only a modest colocalization. Mapping studies indicate that Hippi binds within the self-multimerization domain of Apoptin, and Apoptin binds to the C-terminal half of Hippi, including its death effector domain-like motif. Our results suggest that the Apoptin-Hippi interaction may play a role in the suppression of apoptosis in normal cells.

The viral death protein Apoptin interacts with Hippi, the protein interactor of Huntingtin-interacting protein 1.

Heat-labile enterotoxin (Etx) produced by certain strains of Escherichia coli is a major virulence factor related to cholera toxin. Both are hexameric proteins comprising one A-subunit and five B-subunits. The pentameric B-subunit of E. coli has a high affinity for G(M1)-ganglioside receptors on gut epithelial cells and is directly responsible for toxin entry. The pentameric B-subunit (EtxB(5)) is an exceptionally stable protein, being able to maintain its quaternary structure over a wide pH range (2.0- 11.0). However, little is known about the formation of the pentameric structure (EtxB(5)) from newly synthesized B-subunit monomers (EtxB(1)). We previously described and characterized a mAb (LDS47) that was shown to be highly specific for an N-terminal decapeptide region of EtxB(1) (Amin, T., Larkins, A., James, R. F. L., and Hirst, T. R. (1995) J. Biol. Chem. 270, 20143-20150). Here we also describe a mAb (LDS16) with exquisite specificity for pentameric EtxB. In this study, we have used these two mAbs, in combination, to probe the in vitro assembly of EtxB(5) from EtxB(1). EtxB pentamers disassemble in highly acidic conditions, giving rise to monomeric B-subunits that can reassemble if placed in buffers of neutral pH. Using this in vitro assembly model, it was found that at a molar ratio of 1:1; LDS47:EtxB, 50% of reassembly was inhibited, and that this inhibition increased to 90% at a ratio of 2:1. These results infer that the N-terminal decapeptide region (APQSITELCS) defined by the LDS47 antibody is crucial for competent pentameric B-subunit assembly and stabilization.

Inhibition of Escherichia coli heat-labile enterotoxin B subunit pentamer (EtxB5) assembly in vitro using monoclonal antibodies.

Previously we have described studies on in vitro pentamer assembly of Escherichia coli (E. coli) derived heat-labile enterotoxin B subunit (EtxB) using conventional monoclonal antibodies (Amin et al., JBC 1995: 270, 20143-50 and Chung et al., JBC 2006: 281, 39465-70). To extend these studies further we have used phage-display to select single-chain Fragment variable (scFv) antibodies against different forms of the B-subunit. Two clones exhibiting strong and differential binding were chosen for detailed characterization. A comprehensive sequence analysis was performed to assign the framework and complementary-determining regions and a nonsense mutation present in one of these (scFv-B1.3.9) was corrected. Binding analysis showed that scFv-B1.3.9 bound in ELISA to both heat-denatured monomeric B-subunits (EtxB1) and also displayed cross-reactivity towards pentameric EtxB (EtxB5), although there was no reactivity towards monoganglioside (GM1) captured EtxB5. Another antibody (scFv-B5.2.14) had a different reactivity profile and, in ELISA, bound only to EtxB5 but not to EtxB1 or to EtxB5 captured via GM1. Surprisingly, in competition experiments, the assembled pentameric B-subunit inhibited binding of scFv-B5.2.14 to immobilized EtxB5 only weakly, whereas reduced, but not oxidized, monomeric EtxB1 was an efficient competitor. These results clearly demonstrate that B1.3.9 and B5.2.14 have different specificities for cryptic epitopes not accessible in the fully assembled GM1 bound pentameric form of EtxB. Taken together our results show that we were able to successfully isolate and characterize recombinant scFvs that differentially recognize diverse denatured forms or assembly intermediates of the heat-labile enterotoxin B subunit of E. coli.

Phage-display derived single-chain fragment variable (scFv) antibodies recognizing conformational epitopes of Escherichia coli heat-labile enterotoxin B-subunit.

Islet autotransplantation requires access to the portal vein or tributaries. We originally infused islets into the liver via the middle or right colic veins, but since 2005 we have used the recanalised umbilical vein. Here, we describe the technique, the advantages and the early results achieved.

Use of the recanalised umbilical vein for islet autotransplantation following total pancreatectomy.

The addition of a kidney to the ex vivo liver perfused model may facilitate the circuit homeostatic balance of important biochemical parameters (i.e. pH changes, urea and creatinine, or glucose levels) but might also increase the inflammatory reaction produced. In this study, we compared the production of various cytokines between liver-kidney and liver-alone circuits. Seven livers were harvested from female pigs and perfused for 6 h. In five additional experiments, a kidney was also harvested and connected in parallel. Blood samples for interleukins (IL) 1, 2, 4, 6, 8, 10, and 12, interferon (IFN)-γ and tumor necrosis factor (TNF)-α were collected before perfusion and at hours 1, 2, 4 and 6 postperfusion. In the combined liver-kidney circuit, a significant increase was present only for IL-6 and IL-8, but this did not differ significantly from those recorded in the liver-alone circuit. All other cytokines were not modified from baseline levels. The addition of a kidney to the perfusion circuit does not stimulate a greater inflammatory reaction than that of the liver alone and therefore further confirms the safety of the experimental setups in view of more delicate experiments requiring strict homeostatic conditions.

Addition of a kidney to the normothermic ex vivo perfused porcine liver model does not increase cytokine response.

Microbubbles have improved ultrasonography imaging techniques over the past 2 decades. Their safety, versatility, and easiness of use have rendered them equal or even superior in some instances to other imaging modalities such as computed tomography and magnetic resonance imaging. Herein, we conducted a literature review to present their types, general behavior in tissues, and current and potential use in clinical practice.

Targeted microbubbles in the experimental and clinical setting.

The ex vivo porcine liver perfused model isolates the organ from extrinsic regulatory mechanisms, facilitating an improved understanding of the organ physiology and reaction to various conditions. We have assessed the influence of the addition of a porcine kidney to the circuit.

The autologous normothermic ex vivo perfused porcine liver-kidney model: improving the circuit's biochemical and acid-base environment.

We already developed an ex vivo liver-kidney model perfused for 6 h in which the kidney acted as a homeostatic organ to improve the circuit milieu compared to liver alone. In the current study, we extended the multiorgan perfusions to 24 h to evaluate the results and eventual pitfalls manifesting with longer durations. Five livers and kidneys were harvested from female pigs and perfused over 24 h. The extracorporeal circuit included a centrifugal pump, heat exchanger, and oxygenator. The primary end point of the study was the evaluation of the organ functions as gathered from biochemical and acid-base parameters. In the combined liver-kidney circuit, the organs survived and maintained an acceptable homeostasis for different lengths of time, longer for the liver (up to 19-23 h of perfusions) than the kidney (9-13 h of perfusions). Furthermore, glucose and creatinine values decreased significantly over time (from the 5th and 9th hour of perfusion onward). The addition of a kidney to the perfusion circuit improved the biochemical environment by removing excess products from ongoing metabolic processes. The consequence is a more physiological milieu that could improve results from future experimental studies. However, it is likely that long perfusions require some nutritional support over the hours to maintain the organ's vitality and functionality throughout the experiments.

The development of a multiorgan ex vivo perfused model: results with the porcine liver-kidney circuit over 24 hours.

The use of contrast agents (CA) with liver ultrasound (US) has gained recently an established role for the diagnosis of various hepatic diseases due to their safety, high versatility and low costs (contrast-enhanced ultrasound: CEUS). The purpose of this review is to provide a state-of-the-art summary of the available evidence for their use in the characterization of focal liver lesions. A published work search was conducted for all preclinical and clinical studies involving CA on hepatic US imaging. CEUS increases the sensitivity for lesion detection and the specificity to differentiate between benign and malignant diseases due to the enhanced visualization of the tumor microcirculation. Results achieved seem at least equivalent to those of spiral computed tomography or magnetic resonance imaging. The association of CA with intraoperative ultrasound has changed the surgical approach in 25% of patients and guarantees complete ablations by a single session in most of them. CEUS provides detailed information about tumor vasculature, improves the preoperative characterization and therefore the therapeutic strategy, and can evaluate the intraoperative completeness of the ablation.

Contrast-enhanced ultrasound in the preoperative, intraoperative and postoperative assessment of liver lesions.

Following transplantation, areas of hypoperfusion can be associated with metabolic changes and poor organ recovery. Our study evaluated contrast-enhanced ultrasound (CEUS) agents for the detection of such areas. Livers were collected from ten pigs, connected to extracorporeal circuits and perfused using autologous blood. After 1 and 4 h livers were scanned with an ultrasound machine following the administration of CEUS agents. Biopsies from perfused and non-perfused areas were collected. The entire parenchyma enhanced strongly on non-contrast ultrasound at 1 h with no perfusion defects. Four hours later multiple perfusion defects manifested not evident with non-contrast ultrasound. Histology confirmed non-perfused areas corresponded to ischemic zones. In our model the addition of CEUS revealed perfusion defects after 4 h. This might facilitate detection and characterization of perfusion defects in transplanted livers.

Wen Yuan Chung

Department of Hepatobiliary and Pancreatic Surgery

University Hospitals of Leicester

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Wen Yuan Chung

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Hepatobiliary and Pancreatic Surgery

University Hospitals of Leicester

Steps for the Autologous Ex vivo Perfused Porcine Liver-kidney Experiment

Department of Hepatobiliary and Pancreatic Surgery