Amputations in patients with diabetic foot ulcer: a retrospective study from a single centre in the Northern Territory of Australia.

BACKGROUND: Lower extremity amputations (LEAs) in diabetic patients are common in the indigenous population. There is no published data from the Northern Territory. METHODS: All patients with diabetic foot ulcer, presenting for the first time to the multi-disciplinary foot clinic at Royal Darwin Hospital, between January 2003 and June 2015, were included. These patients were followed until 2017, or death. LEA rates over the follow-up period and the risk factors were studied. RESULTS: Of the 513 included patients, 62.8% were males and 48.2% were indigenous. The majority (93.6%) had type 2 diabetes with median diabetes duration of 7.0 years (interquartile range 3-12). During the follow-up period of 5.8 years (interquartile range 3.1-9.8), a total of 435 LEAs (16.6% major; 34.7% minor) occurred in 263 patients (mean age 57.0 ± 11.8 years). In multivariate analysis, the following variables were associated with LEAs (adjusted odds ratio (95% confidence interval)): prior LEA (4.49 (1.69-11.9)); peripheral vascular disease (2.67 (1.27-5.59)); forefoot ulcer (7.72 (2.61-22.7)); Wagner grade 2 (3.71 (1.87-7.36)); and Wagner grade 3 (17.02 (3.77-76.72)). Indigenous patients were 1.8 times more likely to have LEAs than non-indigenous patients. Indigenous amputees were approximately 9 years younger than their non-indigenous counterparts. CONCLUSION: Half of patients presenting with diabetic foot ulcer had LEA during follow-up. Prior LEAs, peripheral vascular disease, forefoot ulcers and higher Wagner grades were independent risk factors for LEA. Indigenous patients were at higher risk for LEAs and were younger at the time of amputation.

Mortality in patients with diabetic foot ulcer: a retrospective study of 513 cases from a single Centre in the Northern Territory of Australia.

BACKGROUND: Diabetic foot ulcers (DFU) are a common problem in longstanding diabetes. However, mortality outcomes in Australian patients with DFU are still unclear. METHODS: All patients with DFU presenting for the first time to the Multi-Disciplinary Foot Clinic (MDFC) at Royal Darwin Hospital, Northern Territory Australia, between January 2003 and June 2015 were included in this study. These patients were followed until 2017, or death. Individual patient data was extracted from hospital and primary care information systems. Kaplan-Meier survival curves were developed. The association between various risk factors and mortality was analysed using Cox regression. RESULTS: In total 666 subjects were screened, and 513 were included in the final analysis. Of these subjects, 247 were Indigenous and 266 were non-Indigenous. The median follow-up period was 5.8 years (IQR, 3.1-9.8). The mean age at inclusion was 59.9 ± 12.3 years and 62.8% were males. The majority (93.6%) had type 2 diabetes and the median diabetes duration was 7 years (IQR, 3-12). There were 199 deaths, with a 5-year-mortality rate of 24.6%, and a 10-year-mortality rate of 45.4%. The mean age at death was 64.6 ± 11.8 years. In a multivariate analysis, the following variables were associated with mortality (adjusted HR, 95% CI): age 1.04 (1.02-1.05, P < 0.001); chronic kidney disease 1.22 (1.11-1.33, P < 0.001), and plasma albumin 0.96 (0.94-0.99, P < 0.05). The most common causes of death were chronic kidney disease (24.6%), cardiovascular events (19.6%), sepsis (15.6%), respiratory failure (10.0%), malignancy (9.5%) and multi-organ failure (5.0%). CONCLUSION: Patients with DFU have high mortality. Age, chronic kidney disease, and low albumin levels increase the risk of mortality. Strategies should focus on ulcer prevention and aggressive risk factor reduction.

Severe bilateral renal artery stenosis after transluminal radiofrequency ablation of renal sympathetic nerve plexus.

Percutaneous renal sympathetic denervation is an evolving therapy for resistant hypertension. Evidence to date demonstrates a reduction of blood pressure in the short term to medium term. Reported complications relate to problems with vascular access vessels and dissection of the renal artery. Renal artery stenosis has not been described in the literature. We present a patient with hypertensive crisis, flash pulmonary edema, and deterioration of renal function, secondary to bilateral renal artery stenosis, 9 months after renal sympathetic radiofrequency ablation denervation.

Beyond the snare: technically accessible large en bloc colonic resection in the West: an animal study.

BACKGROUND: Endoscopic submucosal dissection (ESD) and circumferential submucosal incision endoscopic mucosal resection (CSI-EMR) are techniques for en bloc excision of large sessile colonic lesions. Our aims were to compare the efficacy, safety and learning curve of colonic hybrid knife (HK) ESD versus CSI-EMR for en bloc excision of 50 mm diameter hemi-circumferential artificial lesions in a porcine model. PATIENTS AND METHODS: Two separate 50 mm diameter areas of normal recto-sigmoid mucosa were marked out in each of ten pigs. One was excised with HK-ESD using succinylated gelatin (SG) submucosal injection. The other was isolated with CSI with the Insulated Tip Knife 2 followed by SG submucosal injection then EMR with a large snare. Euthanasia and colectomy was performed at 72 h followed by blinded histopathology assessment. RESULTS: En bloc excision rates were: HK-ESD 100% versus CSI-EMR 20% (P = 0.008). The mean number of resections per lesion was HK-ESD 1 versus CSI-EMR 3 (P = 0.001). The mean dimensions of the largest specimen per technique were HK-ESD 63 × 54 mm versus CSI-EMR 49 × 41 mm (P = 0.005). Procedure duration mean was HK-ESD 54 min versus CSI-EMR 22 min (P < 0.001). When procedure duration was adjusted for the size of the resected en bloc specimen, a statistically significant and accelerated learning effect was noted for HK-ESD (r = -0.83, P = 0.003). There were no perforations and no significant bleeding. CONCLUSIONS: HK-ESD with SG submucosal injection is superior to CSI-EMR for en bloc excision of 50 mm diameter lesions in a porcine model. The technique is rapidly learnt. This novel approach may lower the barrier to colonic ESD for Western endoscopists.

Colour duplex ultrasound accurately identifies focal stenoses in dysfunctional autogenous arteriovenous fistulae.

AIMS: The aims of this study is to correlate colour duplex ultrasonography (US) with contrast fistulography for the detection of functional stenoses in the autogenous AVF (arterio-venous fistula) circuit. METHODOLOGY: Colour duplex US scans of 93 dialysis patients with dysfunctional AVF were compared with fistulograms performed within 6 weeks of the US. The AVF circuit was divided into six zones: inflow artery; anastomosis; distal vein; mid vein; proximal vein; and central vein. Colour duplex US and fistulogram images/reports were independently re-reported for stenoses in each fistula zone by two trained clinicians blinded to the outcomes. For each fistula, only zones examined by both modalities were included in the study. Kappa analysis of the results was performed to assess the accuracy of colour duplex US in the dysfunctional AVF circuit. RESULTS: Most AVF studied were radio-cephalic (59%) or brachio-cephalic (22%). Stenoses identified within the AV circuit in order of frequency were: distal vein (41), mid vein (23), arterial (12), proximal vein (7) and anastomosis (3). The interval between US and fistulogram studies was 33 + or - 29 days. Congruence of results between US and fistulograms ranged from 85% to 96%, depending on the zone examined. Kappa analysis of this US versus fistulogram data was also moderate to good, ranging from 0.72 and 0.91. CONCLUSIONS: Colour duplex US provides an accurate diagnostic assessment of a dysfunctional autogneous AVF, and is an important planning tool for subsequent open or endovascular intervention. It is particularly accurate in the peri-anastomotic area of the fistula which harbours the majority of fistula problems.

Requirement of MyD88 for macrophage-mediated islet xenograft rejection after adoptive transfer.

BACKGROUND: Porcine antigen primed and CD4+ T-cell activated macrophages are able to migrate to and destroy porcine xenografts. However, the specific signaling mechanisms involved remain to be identified. METHODS: In this study macrophages which lack the universal toll-like receptor (TLR) adaptor MyD88 were used to investigate the role of TLR in the recognition and activation of macrophages in islet xenograft rejection. Macrophages were isolated from rejecting MyD88(-/-) and wild-type C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts, and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Both wild-type C57BL/6 and MyD88(-/-) mice rejected NPCC xenografts 8 and 10 days, respectively after transplantation, and the grafts were heavily infiltrated with CD4+ T cells and macrophages. However, graft infiltrating macrophages from rejecting MyD88(-/-) recipients demonstrated impaired up-regulation of TLR expression and impaired activation phenotype, when compared to those from rejecting C57BL/6 recipients. Transfer of NOD-SCID recipients with macrophages from rejecting C57BL/6 mice resulted in NPCC xenograft rejection along with massively infiltrated macrophages 8 days after transfer, whereas NPCC xenografts in NOD-SCID mice transferred with macrophages from rejecting MyD88(-/-) mice remained intact until the end of this study, 90 days after transfer, with insulin-positive islets and no infiltration by macrophages. CONCLUSION: This study demonstrates that deletion of MyD88 causes impaired macrophage activation after pig islet xenotransplantation. However, graft survival is not prolonged and xenografts are rejected rapidly by alternate mechanisms.

Association between islet xenograft rejection mediated by activated macrophages and upregulated chemokines.

OBJECTIVE: Our previous study has shown that porcine antigen-primed and CD4+ T cells activated macrophages are capable of the Recognition and rejection of porcine xenografts but not mouse allografts, and therefore suggested the involvement of signaling between the graft and macrophages in this specific graft recognition and destruction. METHODS: NOD-SCID mice were transplanted with fetal pig pancreatic fragment (FPP) before adoptive transfer with exogenous macrophages isolated from rejecting FPP xenografts of BALB/c recipient mice. The exogenous macrophages were tracked by Ly5.1 surface antigen or via CSFE staining. Gene expression of CCR2 and CCR5 and their chemokines in transplanted FPP xenografts was evaluated by real-time PCR. RESULTS: After the adoptive transfer, recently transplanted but not established FPP xenografts were rejected by exogenous activated macrophages. In the meantime, greater level of chemokine gene expression was detected in recently-transplanted compared with the established xenografts. Furthermore, expression of both CCR2 and CCR5 genes was enhanced significantly in activated macrophages when compared with non-activated macrophages. CONCLUSION: Upregulated chemokines were associated with macrophage recruitment and destruction of islet xenografts.

Chemokine and toll-like receptor signaling in macrophage mediated islet xenograft rejection.

BACKGROUND: Adoptive transfer of antigen-primed T-cell-activated macrophages into NOD-SCID mice within 14 days of foetal porcine pancreatic fragment (FPP) or foetal porcine skin (FPS) transplantation had been shown to cause xenograft rejection. In the present study, it was proposed that signaling between the graft and macrophages promoted specific graft recognition and destruction in this setting. METHODS: Exogenous macrophages isolated from rejecting FPP xenografts were transferred to NOD-SCID FPP recipients and tracked by Ly5.1 surface antigen or via CSFE staining. Monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1alpha (MIP-1alpha), macrophage inflammatory protein-1beta (MIP-1beta), regulated upon activation, normal T-cell expressed and secreted (RANTES), chemokine (C-C motif) receptor 2 (CCR2), chemokine (C-C motif) receptor 5 (CCR5), toll-like receptors (TLRs) (1-9) and gene expression in transplanted FPP xenografts was evaluated by real-time polymerase chain reaction. Gene expression of CCR2, CCR5 and TLRs was also analyzed in pooled samples of activated and non-activated macrophages. RESULTS: Exogenous macrophages were shown to track to and reject recently transplanted but not established FPP xenografts. Gene expression for MCP-1, RANTES, MIP-1alpha and MIP-1beta was at least 3-fold greater in recently transplanted compared with established xenografts (P < 0.05), and CCR2 and CCR5 gene expression was 10-fold greater in activated compared non-activated macrophages, suggesting that graft-mediated pro-inflammatory signals were important for macrophage recruitment. Specific graft recognition by macrophages may involve TLR signaling as macrophages exposed to porcine islets had higher levels of TLR gene expression compared with those exposed to allografts regardless of the level of activation. CONCLUSION: Xenografts provide additional activation signals to macrophages that are not seen following allotransplantation. This study identifies chemokines and TLR as important signals in macrophage-mediated recognition and rejection of islet xenografts.

Involvement of CCR5 signaling in macrophage recruitment to porcine islet xenografts.

BACKGROUND: Porcine antigen primed and CD4+ T-cell-activated macrophages are capable of both recognition and rejection of porcine xenografts. However, the specific signaling mechanisms involved remains to be addressed. The aim of this study was to examine the role of chemokine receptor and CD40 signaling in macrophage recruitment and graft destruction. METHODS: Macrophages were isolated from rejecting CCR2, CCR5, CD40 and control C57BL/6 mice that were recipients of neonatal porcine pancreatic cell cluster (NPCC) xenografts and were transferred to NPCC recipient NOD-SCID mice. RESULTS: Macrophages isolated from rejecting NPCC xenografts in CD40 and wildtype C57BL/6 mice demonstrated upregulated expression of macrophage activation markers as well as CCR5 and CCR2 genes, and caused pig islet xenograft destruction 8 days after transfer to NOD-SCID recipients. Graft infiltrating macrophages from rejecting CCR2 mice showed a similar activation phenotype and destroyed NPCC xenografts 10 days after transfer to NOD-SCID mice. Blockade of MCP-1 by anti-MCP-1 mAb did not prolong graft survival in CD4+ T cell reconstituted NPCC recipient NOD-SCID mice. By contrast, the graft infiltrating macrophages from rejecting CCR5 recipients showed impaired macrophage activation when compared to control C57BL/6 recipients, and transfer of these macrophages did not result in xenograft destruction in NOD-SCID recipients until day 16 after transfer. Analysis of graft infiltrating macrophages from these rejecting NOD-SCID mice showed an impaired activation phenotype. CONCLUSION: These results demonstrate that CCR5 is involved in both the activation and recruitment of macrophages to rejecting islet xenografts but other pathways are involved.

Overexpression of glutathione peroxidase with two isoforms of superoxide dismutase protects mouse islets from oxidative injury and improves islet graft function.

Primary nonfunction of transplanted islets results in part from their sensitivity to reactive oxygen species (ROS) generated during the isolation and transplantation process. Our aim was to examine whether coexpression of antioxidant enzymes to detoxify multiple ROS increased the resistance of mouse islets to oxidative stress and improved the initial function of islet grafts. Islets from transgenic mice expressing combinations of human copper/zinc superoxide dismutase (SOD), extracellular SOD, and cellular glutathione peroxidase (Gpx-1) were subjected to oxidative stress in vitro. Relative viability after hypoxanthine/xanthine oxidase treatment was as follows: extracellular SOD + Gpx-1 + Cu/Zn SOD > extracellular SOD + Gpx-1 > extracellular SOD > wild type. Expression of all three enzymes was the only combination protective against hypoxia/reoxygenation. Islets from transgenic or control wild-type mice were then transplanted into streptozotocin-induced diabetic recipients in a syngeneic marginal islet mass model, and blood glucose levels were monitored for 7 days. In contrast to single- and double-transgenic grafts, triple-transgenic grafts significantly improved control of blood glucose compared with wild type. Our results indicate that coexpression of antioxidant enzymes has a complementary beneficial effect and may be a useful approach to reduce primary nonfunction of islet grafts.

Fate of alphaGal +/+ pancreatic islet grafts after transplantation into alphaGal knockout mice.

BACKGROUND: Important phylogenetic differences between pig and human tissues prevent xenotransplantation from becoming a clinically feasible option. Humans lack the galactose-alpha1,3-galactose (alphaGal) epitope on endothelial cell surfaces and therefore have preformed anti-alphaGal antibodies. The role of these antibodies in rejection of non-vascular xenografts remains controversial. This study investigated the role of anti-alphaGal antibodies in rejection of non-vascularized alphaGal+/+ grafts in alphaGal -/- mice. METHODS: alphaGal +/+ and alphaGal -/- pancreatic islets were transplanted under the renal capsule of streptozotocin-induced diabetic (1) alphaGal -/- mice and (2) alphaGal +/+ mice. alphaGal -/- recepients were immunized with rabbit red blood cell membranes (RRBCs) to produce elevated anti-alphaGal antibody levels. RESULTS: Six of the 18 alphaGal -/- mice rejected the alphaGal +/+ grafts within 68 days whereas indefinite graft survival was achieved in the control groups. Animals with surviving islet grafts were challenged with alphaGal +/+ skin grafts. Although all alphaGal +/+ skin grafts were rejected within 58 days, the islet grafts remained intact. This observation correlated with the level of alphaGal expression (which was very low on islets compared to skin) rather than the actual titre of anti-alphaGal antibody. DISCUSSION: The results suggest that the level of alphaGal expression plays an important role in graft survival. Therefore, its removal is important in the development of a pig islet donor for future clinical therapy.