Richard Downing

Assessment of the reproducibility of alginate encapsulation of pancreatic islets using the MTT colorimetric assay.

Radioisotope diffusion experiments demonstrate that alginate/polyaminoacid encapsulation can prevent antibody and cytotoxic cell contact in vitro. The unpredictable outcome of xenotransplantation of encapsulated islets may reflect incomplete encapsulation. We have assessed a cytotoxic/MTT (tetrazolium) assay to test antibody permeability of capsules. Samples of free porcine islet tissue, and islet tissue encapsulated in alginate/poly-l-lysine/alginate microspheres were incubated with fresh autologous pig serum or normal human serum overnight. Cell metabolism was assessed by the MTT assay. Data from eight experiments (10 replicates/experiment) were analyzed using the Mann-Whitney U-test. Values were deemed significant when p < 0.05. Free islets cultured in human serum showed a significant reduction in metabolism when compared with islets cultured in pig serum: percentage reduction 52 ± 23% (mean ± SD). The differences in formazan production were significant in all experiments (p < 0.05). Alginate encapsulation of islets or removal of xeno-reactive antibodies in human serum by adsorption was shown to prevent the effects of complement-mediated lysis. However, in one of the eight experiments, there was a significant reduction in islet metabolism after exposure of encapsulated porcine islets to human serum. In conclusion, it has been shown that alginate encapsulation can prevent complement-mediated lysis. However, the encapsulation process employed was imperfect and did not prevent complement-mediated lysis of porcine islets in all experiments. The cytotoxicity/MTT assay allows investigation of the permeability of capsules to serum antibodies and could be performed to determine the viability of the islets and the integrity of microcapsules prior to transplantation.

Human islet isolation: semi-automated and manual methods

Large yields of high-viability human islets are necessary to service the expanding programmes of islet transplantation worldwide; similarly, there is an increasing demand from diabetes researchers for a reliable and cost-effective supply of human islets. The two main isolation methods are ‘semi-automated’ and ‘manual’. Both methods rely on prompt and careful removal and transfer of the donor pancreas to allow isolation to commence, preferably within eight hours. Each method involves exocrine digestion with high-activity collagenase (Liberase). The semi-automated method is standardised, generally provides higher islet yields and is used for clinical transplant purposes, although it is not suitable for all donor pancreata. The manual method is less expensive and more adaptable and enables islets to be isolated for research from most donor pancreata.

Rotational co-culture of clonal β-cells with endothelial cells: effect of PPAR-γ agonism in vitro on insulin and VEGF secretion

Aim: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co‐culture of insulin secreting β‐cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator‐activated receptor gamma (PPAR‐γ) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function.

Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro: implications for pancreatic islet transplantation.

Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p < 0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 μg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of β-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close proximity to human AEC. Such an approach may reduce the need for chronic systemic immunosuppression, thus making islet transplantation a more attractive treatment option for the management of insulin-dependent diabetes.

Low gravity rotational culture and the integration of immunomodulatory stem cells reduce human islet allo-reactivity.

Modification of human islets prior to transplantation may improve long‐term clinical outcome in terms of diabetes management, by supporting graft function and reducing the potential for allo‐rejection. Intragraft incorporation of stem cells secreting beta (β)‐cell trophic and immunomodulatory factors represents a credible approach, but requires suitable culture methods to facilitate islet alteration without compromising integrity. This study employed a three‐dimensional rotational cell culture system (RCCS) to achieve modification, preserve function, and ultimately influence immune cell responsiveness to human islets. Islets underwent intentional dispersal and rotational culture‐assisted aggregation with amniotic epithelial cells (AEC) exhibiting intrinsic immunomodulatory potential. Reassembled islet constructs were assessed for functional integrity, and their ability to induce an allo‐response in discrete T‐cell subsets determined using mixed islet:lymphocyte reaction assays. RCCS supported the formation of islet:AEC aggregates with improved insulin secretory capacity compared to unmodified islets. Further, the allo‐response of peripheral blood mononuclear cell (PBMC) and purified CD4+ and CD8+ T‐cell subsets to AEC‐bearing grafts was significantly (p < 0.05) attenuated. Rotational culture enables pre‐transplant islet modification involving their integration with immunomodulatory stem cells capable of subduing the allo‐reactivity of T cells relevant to islet rejection. The approach may play a role in achieving acute and long‐term graft survival in islet transplantation.

Listeria Infected Pseudoaneurysm of the Superficial Femoral Artery.

We present a unique case of listeria-infected pseudoaneurysm of the superficial femoral artery. Listeria monocytogenes, a gram positive, intracellular bacterium, is widespread in the environment. The manifestation of infection in humans ranges from mild gastroenteritis to life-threatening listeriosis. The incidence in the healthy population is low (0.7 per 100,000) but rises sharply in pregnancy, the immunocompromised and patients with malignancy. Listeria rarely causes mycotic aneurysm formation, with fewer than 40 cases reported in the literature. Further investigation revealed the patient to be diagnosed with a hepatocellular carcinoma. It is likely that the two pathologies were linked. Hence, we would advocate investigation for underlying malignancy in patients presenting with a mycotic aneurysm, particularly those resulting from Listeria infection.

Accelerated development of mesenteric and renal artery calcific atherosclerosis following radiotherapy for testicular cancer.

We present a case of a 61-year-old male presenting with post-prandial epigastric pain and marked weight loss. Investigation revealed calcific atherosclerosis of the abdominal aorta, coeliac axis, superior mesenteric (SMA) and renal arteries. He had undergone radiotherapy for testicular teratoma 34 years previously. Percutaneous mesenteric revascularization by primary stenting of the SMA proved successful. Radiotherapy for intra-abdominal malignancy has the potential to induce both acute and chronic enteritis and an accelerated atherosclerotic process in the arteries within the field of beam.

Isolated Dissecting Aneurysms of the Abdominal Aorta and the Superior Mesenteric Artery. A Case Report and Literature Review

Supracoeliac abdominal aortic dissections are rare and require complex interventions for repair. Superior mesenteric artery (SMA) dissections are also rare and even less frequently reported to involve aneurysmal change. We present the case of a 65-year-old man with a dissecting supracoeliac aortoiliac aneurysm and a separate dissecting aneurysm of the SMA The surgical intervention performed and a review of the literature on the management of SMA dissection in the endovascular era are presented.

Pre-transplant signal induction for vascularisation in human islets

Human islet transplant success is partially impaired by slow revascularisation. Our study investigated the potential for rotational cell culture (RC) of human islets combined with thiazolidinedione (TZD) stimulation of peroxisome proliferator-activated receptor gamma (PPARγ) to upregulate vascular endothelial growth factor (VEGF) expression in the islets. Four groups of human islets were studied: static culture (SC) with and without 25 mmol/L TZD and RC with and without 25 mmol/L TZD. These were assessed for insulin secretion and soluble VEGF-A release. Both proteins were quantified by enzyme-linked immunosorbent assay (ELISA), supported with qualitative immunofluorescence staining. RC + TZD increased insulin secretion by >20% (p < 0.05–0.001) in response to 16.7 mmol/L glucose and 16.7 mmol/L glucose + 10 mmol/L theophylline (G + T). This effect was seen at all time intervals compared with SC and without addition of TZD. Soluble VEGF-A release was significantly augmented by RC and TZD exposure with an increased effect of >30% (p < 0.001) at 72 h under both SC and RC conditions. RC supplemented with a TZD enhances and prolongs the release of insulin and soluble VEGF-A by isolated human islets.

Human islet transplantation: expectation versus reality

Although experimental islet isolation and transplantation has continued for over 25 years, the results of human islet transplantation in patients with type 1 diabetes were disappointing until June 2000 when the University of Alberta, Edmonton, Canada reported 100% insulin independence in a cohort of seven patients. The study introduced several innovations now under evaluation worldwide. In the United Kingdom, an Islet Transplant Consortium has been established to co-ordinate clinical trials in several centres. The Canadian results have renewed hope of establishing islet transplantation as a treatment for diabetes while highlighting the need to identify plentiful sources of insulin secreting tissue and alternatives to current immunosuppressive therapies.