Advances in membrane emulsification. Part B: recent developments in modelling and scale-up approaches.

Membrane emulsification is a promising process for formulating emulsions and particulates. It offers many advantages over conventional 'high-shear' processes with narrower size distribution products, higher batch repeatability and lower energy consumption commonly demonstrated at a small scale. Since the process was first introduced around 25 years ago, understanding of the underlying mechanisms involved during microstructure formation has advanced significantly leading to the development of modelling approaches that predict processing output; e.g. emulsion droplet size and throughput. The accuracy and ease of application of these models is important to allow for the development of design equations which can potentially facilitate scale-up of the process and meet the manufacturer's specific requirements. Part B of this review considers the advantages and disadvantages of a variety of models developed to predict droplet size, flow behaviour and other phenomena (namely droplet-droplet interactions), with presentation of the appropriate formulae where necessary. Furthermore, the advancement of the process towards an industrial scale is also highlighted with additional recommendations by the authors for future work.

Advances in membrane emulsification. Part A: recent developments in processing aspects and microstructural design approaches.

Modern emulsion processing technology is strongly influenced by the market demands for products that are microstructure-driven and possess precisely controlled properties. Novel cost-effective processing techniques, such as membrane emulsification, have been explored and customised in the search for better control over the microstructure, and subsequently the quality of the final product. Part A of this review reports on the state of the art in membrane emulsification techniques, focusing on novel membrane materials and proof of concept experimental set-ups. Engineering advantages and limitations of a range of membrane techniques are critically discussed and linked to a variety of simple and complex structures (e.g. foams, particulates, liposomes etc.) produced specifically using those techniques.

Histological changes during extracorporeal perfusions of the porcine liver: implications for temporary support during acute liver failures.

Ex vivo perfused porcine livers have been used for temporary support during acute liver failure. The aim of this study was to assess both the histological changes and temporal pattern of the changes that occur during extracorporeal liver perfusions and to correlate these with factors that may influence them. Five porcine livers were harvested, preserved in cold ice and reperfused for 6 h in an extracorporeal circuit using autologous normothermic blood. Tissue biopsies were collected hourly. The Ishak score was used to quantify hepatic necrosis, and immunohistochemistry was used to evaluate apoptosis and regeneration. Liver weight, perfusion parameters, arterial blood gases and blood samples were also collected. The Ishak score peaked immediately before and 4 h after the start of reperfusion. Scattered necrosis, microvesicular steatotic vacuolization, sinusoidal dilatation and red cell extravasation were present. Anion gap acidosis was associated with the Ishak score. An inverse correlation was present between liver regeneration and necrosis, and between liver weight and regeneration. No changes were observed for apoptosis. Among the inflammatory cytokines evaluated, interleukin-6 and -8 levels increased significantly during the perfusions. Hepatic necrosis was always present during the extracorporeal perfusions, followed a definite pattern and was inversely correlated with regeneration. Apoptosis did not increase over baseline levels. The meaning of these findings and their correlation with clinical outcomes during acute hepatic failures deserve further investigation.

Heat shock protein 70 expression following hepatic radiofrequency ablation is affected by adjacent vasculature.

BACKGROUND: Heat shock proteins are a highly conserved family of stress response proteins. Members of the heat shock protein 70 (Hsp70) family prevent protein misfolding and aggregation. Following radiofrequency ablation of unresectable liver tumors an interface appears between the irreversibly damaged and normal liver. The fate of this transition zone is critical and is believed to be responsible for local recurrences. Hsp70 is expressed in response to thermal stress and may influence the fate of cells in this transitional zone. It is also recognized that the presence of large vessels or a perivascular location of tumors also influences the recurrence rate. The aim of this study is to examine the transition zone and observe the effect of local blood flow on ablation morphology and Hsp70 expression. METHODS: Radiofrequency ablation was performed in 25 rats at various distances from the liver hilum. Tissue was retrieved and analysed at time points 0, 4, 24, 48 h, and 2 wk following treatment. Tissue was analyzed histologically with hematoxylin and eosin staining (H and E,) and immunohistochemically for Hsp70 expression. RESULTS: All rats survived the procedure. H and E staining revealed previously unreported foci of apoptosis at the ablation edge and deep in the normal hepatic parenchyma. Hsp70 was expressed in the transition zone at 4 h and peaked at 24 h. The degree of Hsp70 expression was significantly influenced by the distance from surrounding vasculature. CONCLUSIONS: This study reports several previously unreported findings. There is increased apoptosis distal to the ablated zone suggests leakage of radiofrequency (RF) current down blood vessels originating in the ablation zone. The degree of Hsp70 expression in the transition zone correlates with time after treatment and the size and location of any adjacent vasculature. These findings suggest that heat shock proteins may play a role in the ability of damaged cells to recover and survive at the periphery of an ablation zone.

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

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.

The porcine hepatic arterial supply, its variations and their influence on the extracorporeal perfusion of the liver.

BACKGROUND: Variations in the porcine hepatic vasculature may contribute to the outcome of experiments based on liver perfusion models. We studied the hepatic artery (HA), variations of its branches, and the correlation with the resultant perfusion. MATERIALS AND METHODS: Nineteen animals were used. After 6 h of perfusion, dissection of the HA and its branches was conducted up to the insertion in the liver parenchyma. Data about the macroscopic appearance of lobes and the pattern of branching were recorded. RESULTS: In all cases, the HA bifurcated into two constant branches, one for the LL/LM lobes (further divided in two vessels for each lobe) and one for the RM lobe. Five main patterns were identified involving vessels for the RL and the CL lobes. Two variations produced complete and uniform perfusion of the entire liver in all cases, while in the remaining cases, only part of them were completely perfused. CONCLUSIONS: Some variants of the porcine HA and its branches are associated with the risk of incomplete perfusion if the perfusion cannula placement does not take the individual hepatic arterial anatomy into account. Understanding of the hepatic arteries branching patterns is essential when ex vivo perfusion models are being established.

International multicentre prospective study on microwave ablation of liver tumours: preliminary results.

BACKGROUND: Microwave ablation (MWA) is increasingly utilized in the treatment of hepatic tumours. Promising single-centre reports have demonstrated its safety and efficacy, but this modality has not been studied in a prospective, multicentre study. METHODS: Eighteen international centres recorded operative and perioperative data for patients undergoing MWA for tumours of any origin in a voluntary Internet-based database. All patients underwent operative MWA using a 2.45-GHz generator with a 5-mm antenna. RESULTS: Of the 140 patients, 114 (81.4%) were treated with MWA alone and 26 (18.6%) were treated with MWA combined with resection. Multiple tumours were treated with MWA in 40.0% of patients. A total of 299 tumours were treated in these 140 patients. The median size of ablated lesions was 2.5 cm (range: 0.5-9.5 cm). Tumours were treated with a median of one application (range: 1-6 applications) for a median of 4 min (range: 0.5-30.0 min). A power setting of 100 W was used in 78.9% of cases. Major morbidity was 8.3% and in-hospital mortality was 1.9%. CONCLUSIONS: These multi-institution data demonstrate rapid ablation time and low morbidity and mortality rates in patients undergoing operative MWA with a high rate of multiple ablations and concomitant hepatic resection. Longterm follow-up will be required to determine the efficacy of MWA relative to other forms of ablative therapy.

Renal effects of microwave ablation compared with radiofrequency, cryotherapy and surgical resection at different volumes of the liver treated.

BACKGROUND: Renal changes after microwave tissue ablation (MTA) were compared with those following hepatic resection, cryotherapy (CRYO), and radiofrequency ablation (RFA). Structural damage producing renal impairment has been assessed directly by examining tissue specimens and by serum analysis for two sensitive biomarkers, retinol binding protein (RBP) and the heat shock protein 70 (HSP-70) for each modality at different ablation volumes. METHODS: Live rats underwent MTA, surgical resection, CRYO or RFA of 15, 33 or 66% of total hepatic volume. Urine and tissue samples were collected at the time of death. Percentage of tubules with casts and glomerular damage, tissue expression of HSP-70 and urine RBP were evaluated and compared. Behaviour of the animals was also assessed by means of five different parameters and combined to produce a response score. RESULTS: All RFA and CRYO rats undergoing 66% died and these animals had >60% of damaged tubuli and 8% of altered glomeruli. No animals treated by MTA or surgical resection died. Cut-off values (those predicting fatal treatments) could be identified for levels of HSP-70 and RBP. CONCLUSIONS: Large volume MTA is associated with a significant reduced renal damage and is well tolerated compared with RFA and CRYO.

Effects of hypoxia due to isovolemic hemodilution on an ex vivo normothermic perfused liver model.

BACKGROUND: The liver has a complex hormonal and nervous control mechanism leading to difficulty in the interpretations of its responses to chronic hypoxia. Theoretically an ex vivo perfused model of the liver should, by dissociating the organ from the extrinsic regulatory mechanisms, allow a better and unequivocal analysis of changes obtained. MATERIALS AND METHODS: Twelve livers were harvested from female pigs and perfused for 6 h. Hypoxia was produced by means of isovolemic hemodilution with hemoglobin and hematocrit reduced to 40% of the baseline value. The extracorporeal circuit included a centrifugal pump, heat exchanger, and oxygenator. Every hour, physiological parameters (arterial/portal venous pressures and flows) were measured and blood samples were collected for the analysis of hemoglobin, red blood cells, hematocrit, lactate, glucose, albumin, alanine aminotransferase, alkaline phosphatase, and total bilirubin, arterial and venous blood gases. The arterio-venous oxygen and carbon dioxide differences, and the hepatic metabolic rate for oxygen, were also calculated. Primary endpoint of the study was the glucose response of the liver to acute hypoxia. Secondary endpoints were eventual changes of markers for hepatic viability and functionality. RESULTS: Most parameters showed significant variability during the first h of perfusion but subsequently normalized and remained stable at baseline values for the following 5 h. A strong and significant hyperglycemic response was present throughout the experiment (P < 0.001). Lactate rose steadily throughout the study period and after 6 h of perfusion there was a significant deviation from initial values (P < 0.05). Albumin did not change significantly throughout the study although a trend towards decreasing values was observed (Friedman test, P = NS). After an initial rise in levels of alanine transaminase and alkaline phosphatase following perfusion (P < 0.01), values remained constant without any further increase. CONCLUSIONS: Following reperfusion in an ex vivo model, the liver reacts to low oxygen concentrations mobilizing glycogen deposits. This mechanism depends on an intrinsic sensibility of hepatocytes to hypoxia, as demonstrated by the ex vivo liver perfusion. These findings improve our knowledge in organ preservation for liver transplantation.

Bile changes after liver surgery: experimental and clinical lessons for future applications.

INTRODUCTION: The aim of this review is to summarize the available evidence for changes in bile composition following liver surgery and assess their use in predicting post-operative complications. MATERIALS AND METHODS: A literature search was undertaken for all studies focusing on bile composition, bile volume and analysis. Articles were selected from MEDLINE, Embase and the Cochrane Central Register of Controlled Trials databases up to May 2009. RESULTS: Low values of pre-operative bilirubin diglucuronide predict reduced post-operative liver function and the occurrence of jaundice. Low concentrations of hepatocyte growth factor and interleukin-6 in bile following surgery are associated with the subsequent development of liver failure and are probably surrogate markers for situations where the resultant hepatic regeneration is inadequate. CONCLUSIONS: Analysis of the composition and quality of bile is probably underused as a tool for the pre-operative screening and early post-operative monitoring of patients at high risk of developing liver failure following major hepatobiliary procedures.

Immunoregulatory effects of liver ablation therapies for the treatment of primary and metastatic liver malignancies.

Ablation of liver tissue produces in situ tumoural antigens and elicits specific immune responses. The aim of this review is to examine the available data about the local and systemic responses produced and to compare differences between the methods available. A literature search was undertaken for all papers focusing on immune responses following ablative therapy of the liver, including experimental and clinical studies. Following ablative procedures, the cellular response is elicited by the presentation of antigens by dendritic cells to specific CD4(+) T cells, which in turn stimulate natural killer or CD8(+) cytotoxic cells. The local release of intracellular debris activates Kupffer cells to produce cytokines, which, in the immediate vicinity, activate monocytes/macrophages or specific T cells that respond and produce systemic reactions such as fever, thrombocytopaenia or shock. The immune responses elicited by cryotherapy, both cellular and cytokine, seem far greater than those produced by radiofrequency or microwave ablation, probably as a consequence of the peculiar mechanism of cell death of the former (disruptive necrosis). This mechanism is considered central to the pathogenesis of cryoshock. Ablative techniques stimulate the immune system and provide an easy way to achieve in vivo vaccination against tumoural antigens. Immunomodulatory approaches have the potential to augment the initial immune stimulation and this combined approach could pave the way to a more selective and specific method of treating liver tumours.

Hepatic microwave ablation: a review of the histological changes following thermal damage.

Microwave (MW) ablation therapy is a local treatment by which tumours are destroyed by coagulation from the passage of MWs into cells. The aim of this review is to examine histological results obtained from preclinical and clinical studies. A literature search was undertaken for all studies focusing on MW therapy and in which lesions were excised for a complete histopathological examination after treatment. Two main zones were described after ablative therapy (central and transitional). Both corresponded to specific microscopic characteristics and evolved over time in a precise manner. No viable cells even up to 6 cm in diameter were demonstrated in 93% of lesions after treatment. Microwave therapy is a reliable technique under a variety of clinical situations. Future investigations are needed to compare MW with other ablative techniques to identify factors that influence the effectiveness of the various techniques and to determine specific indications.

Adherence to vaccination guidelines post splenectomy: A five year follow up study.

Following a splenectomy patients are at increased risk of significant infections. In its most severe form, overwhelming post-splenectomy infection (OPSI) has a mortality rate of up to 80%. In this study we aim to establish the adherence to vaccination and antibiotic national guidelines in splenectomised patients. A retrospective study of 100 patients who underwent splenectomy (21 emergency, 79 elective), in two teaching hospitals was undertaken over a five-year period. Patients were followed up for five years. Hospital and GP records were reviewed for adherence to pre, intra and postoperative vaccination, thromboprophylaxis and antibiotic guidance. Eighty-six eligible patients (91.5%) received their Haemophilus influenzae B, meningococcal C and pneumococcus vaccinations peri-operatively. Eighty-one (86%) received post-operative antibiotics. Ninety-nine percent of patients received thromboprophylaxis treatment. Eighty-nine (95%) were treated with long-term antibiotic prophylaxis. Only 20 patients (23%) had an emergency supply of antibiotics. Ninety-five percent of patients were administered an annual influenza vaccination and 84% of eligible patients received a five-year pneumococcal booster vaccination. Improvement in the management of this patient cohort can be achieved by a multidisciplinary approach involving adherence to national guidelines, standardised trust protocols, patient information leaflets and advice detailing risk of infection, standardised GP letters and a splenectomy register to monitor and manage this vulnerable group of patients.

Expression of thrombospondin-1 in resected colorectal liver metastases predicts poor prognosis.

PURPOSE: The aim of this study was to examine the expression and prognostic relevance of thrombospondin-1 (TSP-1) in tumor biopsies taken from a consecutive series of liver resections done at the University Hospitals of Leicester and the Royal Liverpool Hospital. EXPERIMENTAL DESIGN: Patients having undergone a liver resection for colorectal liver metastases at our institutions between 1993 and 1999 inclusive were eligible. Inclusion criteria were curative intent, sufficient tumor biopsy, and patient follow-up data. One hundred eighty-two patients were considered in this study. Standard immunohistochemical techniques were used to study the expression of TSP-1 in 5-microm tumor sections from paraffin-embedded tissue blocks. TSP-1 was correlated with survival using the Kaplan-Meier method and log-rank test for univariate analysis and the Cox proportional hazard model for multivariate analysis. RESULTS: One hundred eighty-two patients (male, n = 122 and female, n = 60) ages between 25 and 81 years (mean, 61 years) were included. TSP-1 was expressed around blood vessels (n = 45, 25%) or in the stroma (n = 59, 33%). No expression was detected in the remaining tumors. TSP-1 significantly correlated with poor survival on univariate (P = 0.01 for perivascular expression and P = 0.03 for stromal expression) and multivariate analysis (P = 0.01 for perivascular expression). CONCLUSION: TSP-1 is a negatively prognostic factor for survival in resected colorectal liver metastases.

Laparoscopic ligation of splenic artery aneurysm.

Splenic artery aneurysms are rare and often asymptomatic; however, rupture may prove fatal. Although many can be treated with percutaneous embolization, tortuosity of the artery may render this approach impossible. Presented is a case report describing laparoscopic ligation of a splenic artery aneurysm after failed attempt at embolization.

Rapid microwave ablation of large hepatocellular carcinoma in a high-risk patient.

A 74-year-old male with an inoperable, large (6 cm in diameter) primary hepatocellular carcinoma of the liver was successfully treated using a novel microwave ablating system. Using a single applicator, the tumour was treated at 150 W for 4 minutes. An ablation zone 8 cm in diameter was achieved, which gradually shrunk to form scar tissue that remained unchanged without tumour recurrence for 2 years.

Characterization and propagation of tumor initiating cells derived from colorectal liver metastases: trials, tribulations and a cautionary note.

Tumor initiating cells (TIC) are increasingly being put forward as a potential target for intervention within colorectal cancer. Whilst characterisation and outgrowth of these cells has been extensively undertaken in primary colorectal cancers, few data are available describing characteristics within the metastatic setting. Tissue was obtained from patients undergoing surgical resection for colorectal liver metastases, and processed into single cell suspension for assessment. Tumor initiating cells from liver metastases were characterised using combinations of EPCAM, Aldehyde dehydrogenase activity, CD133 and CD26. CD133 expression was significantly lower in patients who had received chemotherapy, but this was accounted for by a decrease observed in the male patient cohort only. ALDHhigh populations were rare (0.4 and 0.3% for EPCAM+/ALDHhigh/CD133- and EPCAM+/ALDHhigh/CD133+ populations respectively) and below the limits of detection in 28% of samples. Spheroid outgrowth of metastatic tumor cells across all samples could not be readily achieved using standard spheroid-formation techniques, thus requiring further method validation to reliably propagate cells from the majority of tissues. Spheroid formation was not enhanced using additional growth factors or fibroblast co-culture, but once cells were passaged through NOD-SCID mice, spheroid formation was observed in 82% samples, accompanied by a significant increase in CD26. Order of spheroid forming ability was ALDHhigh>CD133>CD26. Samples sorted by these markers each had the ability to reform ALDHhigh, CD133 and CD26 positive populations to a similar extent, suggestive of a high degree of plasticity for each population. Ex vivo TIC models are increasingly being utilised to assess efficacy of therapeutic interventions. It is therefore essential that such investigations use well-characterised models that are able to sustain TIC populations across a large patient cohort in order that the inherent heterogeneity observed in cancer populations is maintained.

Curcumin inhibits cancer stem cell phenotypes in ex vivo models of colorectal liver metastases, and is clinically safe and tolerable in combination with FOLFOX chemotherapy.

In vitro and pre-clinical studies have suggested that addition of the diet-derived agent curcumin may provide a suitable adjunct to enhance efficacy of chemotherapy in models of colorectal cancer. However, the majority of evidence for this currently derives from established cell lines. Here, we utilised patient-derived colorectal liver metastases (CRLM) to assess whether curcumin may provide added benefit over 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) in cancer stem cell (CSC) models. Combination of curcumin with FOLFOX chemotherapy was then assessed clinically in a phase I dose escalation study. Curcumin alone and in combination significantly reduced spheroid number in CRLM CSC models, and decreased the number of cells with high aldehyde dehydrogenase activity (ALDH(high)/CD133(-)). Addition of curcumin to oxaliplatin/5-FU enhanced anti-proliferative and pro-apoptotic effects in a proportion of patient-derived explants, whilst reducing expression of stem cell-associated markers ALDH and CD133. The phase I dose escalation study revealed curcumin to be a safe and tolerable adjunct to FOLFOX chemotherapy in patients with CRLM (n = 12) at doses up to 2 grams daily. Curcumin may provide added benefit in subsets of patients when administered with FOLFOX, and is a well-tolerated chemotherapy adjunct.

Post-traumatic hepatic artery aneurysm--surgical treatment: report of a case.

Hepatic artery aneurysms (HAAs) are uncommon. We present the diagnosis and surgical treatment of a post-traumatic, extrahepatic artery aneurysm.

Laparoscopic distal pancreatectomy for lymphoepithelial cyst of the pancreas.

Lymphoepithelial cysts are rare pancreatic lesions. This case report describes the first excision of such a lesion by laparoscopic distal pancreatectomy which is a recognized procedure for treatment of cystic pancreatic neoplasms. Our patient underwent complete excision of the lesion and has enjoyed complete resolution of his symptoms. Laparoscopic distal pancreatectomy may be a suitable choice for first-line therapy for such lesions.