Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation.

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41-59, BMI 26-38, cold ischemic time < 10 h). Collagen IV, pan-laminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan-laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan-laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.

Laparoscopic intussuscepting pancreaticojejunostomy.

Perhaps the greatest barrier to adoption of laparoscopic pancreaticoduodenectomy by experienced pancreatic surgeons is the technical challenge of constructing the pancreaticojejunostomy (PJ). The authors present a less demanding PJ technique they have developed that creates an end-to-end intussuscepting anastomosis using a running monofilament suture. This method reduces technical complexity and operative time while producing acceptably comparable outcomes.

Longevity of human islet α- and ß-cells.

Pancreatic islet cell regeneration is considered to be important in the onset and progression of diabetes and as a potential cell therapy. Current hypotheses, largely based on rodent studies, indicate continuous turnover and plasticity of α- and ß-cells in adults; cell populations in rodents respond to increased secretory demand in obesity (30-fold ß-cell increase) and pregnancy. Turnover and plasticity of islet cells decrease in mice within >1 year. In man, morphometric observations on postmortem pancreas have indicated that the cellular expansion is much smaller than the increased insulin secretion that accompanies obesity. Longevity of ß-cells in humans >20-30 years has been shown by (14) C measurements and bromo-deoxyuridine (BrdU) incorporation and there is an age-related decline in the expression of proteins associated with cell division and regeneration including cyclin D3 and PDX-1. Quantitative estimation and mathematical modelling of the longevity marker, cellular lipofuscin body content, in islets of subjects aged 1-84 years indicated an age-related increase and that 97% of the human ß-cell population was established by the age of 20. New data show that human α-cell lipofuscin content is less than that seen in ß-cells, but the age-related accumulation is similar; lipofuscin-positive (aged) cells form ≥ 95% of the population after 20 years. Increased turnover of cellular organelles such as mitochondria and endoplasmic reticulum could contribute to lipofuscin accumulation with age in long-lived cells. Induction of regeneration of human islet cells will require understanding of the mechanisms associated with age-related senescence.

Measurements of electron transport in foils irradiated with a picosecond time scale laser pulse.

The heating of solid foils by a picosecond time scale laser pulse has been studied by using x-ray emission spectroscopy. The target material was plastic foil with a buried layer of a spectroscopic tracer material. The laser pulse length was either 0.5 or 2 ps, which resulted in a laser irradiance that varied over the range 10(16)-10(19) W/cm(2). Time-resolved measurements of the buried layer emission spectra using an ultrafast x-ray streak camera were used to infer the density and temperature conditions as a function of laser parameters and depth of the buried layer. Comparison of the data to different models of electron transport showed that they are consistent with a model of electron transport that predicts the bulk of the target heating is due to return currents.

The long lifespan and low turnover of human islet beta cells estimated by mathematical modelling of lipofuscin accumulation.

AIMS/HYPOTHESIS: Defects in pancreatic beta cell turnover are implicated in the pathogenesis of type 2 diabetes by genetic markers for diabetes. Decreased beta cell neogenesis could contribute to diabetes. The longevity and turnover of human beta cells is unknown; in rodents <1 year old, a half-life of 30 days is estimated. Intracellular lipofuscin body (LB) accumulation is a hallmark of ageing in neurons. To estimate the lifespan of human beta cells, we measured beta cell LB accumulation in individuals aged 1-81 years. METHODS: LB content was determined by electron microscopical morphometry in sections of beta cells from human (non-diabetic, n = 45; type 2 diabetic, n = 10) and non-human primates (n = 10; 5-30 years) and from 15 mice aged 10-99 weeks. Total cellular LB content was estimated by three-dimensional (3D) mathematical modelling. RESULTS: LB area proportion was significantly correlated with age in human and non-human primates. The proportion of human LB-positive beta cells was significantly related to age, with no apparent differences in type 2 diabetes or obesity. LB content was low in human insulinomas (n = 5) and alpha cells and in mouse beta cells (LB content in mouse <10% human). Using 3D electron microscopy and 3D mathematical modelling, the LB-positive human beta cells (representing aged cells) increased from >or=90% (<10 years) to >or=97% (>20 years) and remained constant thereafter. CONCLUSIONS/INTERPRETATION: Human beta cells, unlike those of young rodents, are long-lived. LB proportions in type 2 diabetes and obesity suggest that little adaptive change occurs in the adult human beta cell population, which is largely established by age 20 years.

Heteroconium and Pirozynskiella n. gen., with comments on conidium transseptation.

Heteroconium citharexyli, the type species of this genus, is illustrated and redescribed as a sooty mold bearing acropetal chains of conidia showing a basifugal sequence of septation. Heteroconium neriifoliae, H. glutinosa and the Heteroconium synanamorph of Antennulariella concinna are congeneric. The latter species is neotypified, illustrated and described. Pirozynskiella new genus, typified by P. solaninum comb. nov. (-Helminthosporium solaninum), differs from Heteroconium in having an obligate association with asterinaceous fungi and in the centrifugal sequence of conidium transseptation after the initial median septum. Pirozynskiella costaricensis comb. nov. (-Dendryphion costaricensis) is illustrated and described. Heteroconium tetracoilum and H. chaetospira are fungicoles of wood and bark; the former has basifugal conidium septation and the latter has a centrifugal sequence. The two latter species can be excluded from the Heteroconium. Basifugal and centrifugal septation of conidia is discussed with reference to several sooty molds, to some foliicolous anamorphs with subcuticular hyphae (Heterosporiopsis) and to some wood and bark hyphomycetes (Septonema, Taeniolella). Ten other species included in Heteroconium are known to me only from their original descriptions; only one (H. asiaticum) is probably a sooty mold.

Taxonomy, nomenclature and phylogeny of three cladosporium-like hyphomycetes, Sorocybe resinae, Seifertia azaleae and the Hormoconis anamorph of Amorphotheca resinae.

Using morphological characters, cultural characters, large subunit and internal transcribed spacer rDNA (ITS) sequences, and provisions of the International Code of Botanical Nomenclature, this paper attempts to resolve the taxonomic and nomenclatural confusion surrounding three species of cladosporium-like hyphomycetes. The type specimen of Hormodendrum resinae, the basis for the use of the epithet resinae for the creosote fungus {either as Hormoconis resinae or Cladosporium resinae) represents the mononematous synanamorph of the synnematous, resinicolous fungus Sorocybe resinae. The phylogenetic relationships of the creosote fungus, which is the anamorph of Amorphotheca resinae, are with the family Myxotrichaceae, whereas S. resinae is related to Capronia (Chaetothyriales, Herpotrichiellaceae). Our data support the segregation of Pycnostysanus azaleae, the cause of bud blast of rhododendrons, in the recently described anamorph genus Seifertia, distinct from Sorocybe; this species is related to the Dothideomycetes but its exact phylogenetic placement is uncertain. To formally stabilize the name of the anamorph of the creosote fungus, conservation of Hormodendrum resinae with a new holotype should be considered. The paraphyly of the family Myxotrichaceae with the Amorphothecaceae suggested by ITS sequences should be confirmed with additional genes.

The battle for Goose Green--the RMO's view.

By virtue of the Battalion I serve with, I was the first Task Force Doctor on to the Falklands. On Friday the 21st May, 2 Para made an assault beach landing, thankfully unopposed, on San Carlos beach, the RAP was with them.

Descending necrotising mediastinitis: a safe treatment algorithm.

Descending necrotising mediastinitis can complicate oropharyngeal infection and has a high associated mortality. We present three cases treated in our department and propose a treatment algorithm based on our experience and literature review. The primary oropharyngeal infection was peritonsillar abscess in two cases and odontogenic abscess in one. Two patients underwent cervicotomy and later thoracotomy. The third underwent cervicotomy with transcervical mediastinal drainage and later required pericardial drainage via a subxiphoid incision. All recovered fully and were discharged within 6 weeks. To enable successful treatment, diagnosis needs to be prompt and surgical drainage adequate. Thoracic management of the chest is essential.

A new name for Torula glutinosa in Heteroconium.

Torula glutinosa, a sooty mold on living leaves and stems of Eriodictyon spp. from California is illustrated and described. It shares, with the type species of Heteroconium, H. citharexyli, acropetal conidiogenesis of chains of conidia of variable length and acropetal transseptation. An unnamed synanamorph is recognized and described.

Comparison of the collagen VI content within the islet-exocrine interface of the head, body, and tail regions of the human pancreas.

Efficient islet isolation depends on the use of collagenase to digest the extracellular matrix within the islet-exocrine interface, the molecular structure of which is poorly understood. Recently it has been reported that transplantable yields of islets can be isolated from the tail segment of the pancreas alone. This study aimed to quantify and compare the amount of collagenase-resistant collagen VI within the islet-exocrine interface of the head, body, and tail of the human pancreas. Human adult pancreata (n = 5) were retrieved from heart-beating donors (age range, 40-62 years; cold ischemia times <10 hours). Tissue blocks from the head, body, and tail region of each pancreas were fixed in formalin and processed for immuno-labelling of collagen VI, which was quantified in the islet-exocrine interface using a Zeiss KS-400 image analysis system. Data were expressed as area of collagen at the interface relative to the islet area. Statistical analysis was done using paired t test. The mean islet areas in the head, body, and tail regions were not significantly different. Collagen VI was uniformly present within the islet-exocrine interface of all regions of the pancreas and was 0.326 +/- 0.064, 0.324 +/- 0.060, and 0.334 +/- 0.052 microm(2)/islet area (P = .441) in the head, body, and tail, respectively. The content of collagen VI within the islet-exocrine interface was uniform throughout all parts of the adult pancreas. Targeting this collagen subtype with novel collagenase blends may result in consistently improved islet yields and enable a wider number of available donor pancreata to be used.

An improved method of fluorescent dual insulin and endothelial staining allows visualisation of the revascularisation of intraportally transplanted islets.

BACKGROUND: Intraportally transplanted islets are avascular at the time of transplantation and take up to 14 days to fully revascularize, during which time up to 60% of islet mass may be lost. To investigate and improve islet revascularization, a robust method for the visualization and quantification of this process is required. METHODS: Islets isolated from Lewis rats were transplanted intraportally into the liver of diabetic syngeneic Lewis recipients. The animals were humanely killed either on the day of transplant or at 3, 5, 7, or 14 days posttransplant. The harvested livers were sectioned and stained with Bandeiraea simplicifolia lectin (for endothelial cells) and anti-insulin antibody and counterstained with DAPI. The slides were visualized with a fluorescent microscope. RESULTS: Islets were visualized over the whole time course. Insulin and endothelial staining was clearly visualized on the day of transplantation, but by day 3 endothelial staining was scarce within the islet. By day 5, early vessel formation could be seen within the islet, but insulin staining was patchy and associated with apoptotic nuclei. By day 7, vessels could be seen throughout the islet and insulin staining had returned. Day 14 sections showed a fully revascularized islet. CONCLUSIONS: The staining provided good delineation of islet endothelium and beta-cell location, with clear observation of the revascularization process. This technique also suggests that days 3 through 5 may be a critical period for islet survival and provides a good model for studying the effects of manipulating the revascularization process.

Student attendance during college-based lectures: a pilot study.

AIM: To identify the factors that influence attendance and absenteeism among a group of second-year nursing students during the theory component of the Fitness for Practice (FFP) curriculum. METHOD: In 2004, a non-randomised questionnaire was used to elicit information about the factors surrounding absenteeism from 75 adult branch nursing students within the first FFP cohort. The questionnaire consisted of 48 questions and was designed to generate a mixture of quantitative and qualitative data. Absence was recorded for the first 91 weeks of the programme. RESULTS: The main reasons identified for absence included: illness, family commitments, dental and medical appointments, and impending assignment submissions. Other factors that might influence college attendance included a dislike of certain subjects, with ethics, law and social policy identified as the least popular subjects. Students also admitted to an increase in absence around the time when assignments are due for submission and occasionally pretended to be ill. CONCLUSION: Further studies should be undertaken with other pre-registration nursing student cohorts to compare the results with this research. There should be: an increase in self-directed learning; a 'family-friendly' approach to the curriculum by allocating self-directed study during school holidays; a reduction in the number of theory hours to coincide with students' external commitments and to assist them with the demands of studying; and time for private study before the submission of theoretical assignments.

Substrates modified by advanced glycation end-products cause dysfunction and death in retinal pericytes by reducing survival signals mediated by platelet-derived growth factor.

AIMS/HYPOTHESIS: Premature death of retinal pericytes is a pathophysiological hallmark of diabetic retinopathy. Among the mechanisms proposed for pericyte death is exposure to AGE, which accumulate during diabetes. The current study used an in vitro model, whereby retinal pericytes were exposed to AGE-modified substrate and the mechanisms underlying pericyte death explored. METHODS: Pericytes were isolated from bovine retinal capillaries and propagated on AGE-modified basement membrane (BM) extract or non-modified native BM. The extent of AGE modification was analysed. Proliferative responses of retinal pericytes propagated on AGE-modified BM were investigated using a 5-bromo-2-deoxy-uridine-based assay. The effect of extrinsically added platelet-derived growth factor (PDGF) isoforms on these proliferative responses was also analysed alongside mRNA expression of the PDGF receptors. Apoptotic death of retinal pericytes grown on AGE-modified BM was investigated using terminal deoxynucleotidyl transferase-mediated dUTP nick end-labelling labelling, mitochondrial membrane depolarisation and by morphological assessment. We also measured both the ability of PDGF to reverse Akt dephosphorylation that was mediated by AGE-modified BM, and increased pericyte apoptosis. RESULTS: Retinal pericytes exposed to AGE-modified BM showed reduced proliferative responses in comparison to controls (p<0.05-0.01), although this effect was reversed at low-AGE modifications. PDGF mRNA levels were differentially altered by exposure to low and high AGE levels, and AGE-modified BM caused significantly increased apoptosis in retinal pericytes. Pre-treatment of AGE-modified BM with PDGF-AA and -BB reversed the apoptosis (p<0.05-0.001) and restored Akt phosphorylation in retinal pericytes. CONCLUSIONS/INTERPRETATION: Evidence suggests that substrate-derived AGE such as those that occur during diabetes could have a major influence on retinal pericyte survival. During diabetic retinopathy, AGE modification of vascular BM may reduce bioavailability of pro-survival factors for retinal pericytes.

Effect of donor treatment with heparinoids on graft function after intraportal transplantation of a marginal islet mass: an experimental study.

Heparinoids interact with factors that are involved in ischemia-reperfusion injury and thus may prevent organ injury. We therefore studied the effects on subsequent intraportal islet transplantation of systemic administration of unfractionated and N-desulphated heparin to donors prior to pancreatectomy. Donor rats were given an intravenous injection of either heparin (1.3 mg/kg or 13.3 mg/kg; 200 U/kg or 2000 U/kg, respectively) or N-desulphated heparin (50 mg/kg; approximately 5 U/kg) at 5 to 10 minutes prior to pancreas procurement. Five hundred freshly isolated islets were injected intraportally into syngeneic male Lewis recipients that had developed streptozotocin-induced diabetes. Blood glucose and body weight were monitored for 5 weeks thereafter. Rats transplanted with islets from donors given high dose heparin showed a fall in blood glucose from 25.1 +/- 1.4 to 11.0 +/- 2.7 mmol/L (P <.01) with 60% of animals euglycemic within the first week. In contrast, the controls, did not show a fall in glucose levels at 1 week and none had become euglycaemic. Normalization of glycemia was slower in recipients of islets from animals treated with the lower dose of heparin. Results were intermediate with islets from donors given N-desulphated heparin. Nevertheless, all heparinoids used in this study caused more than a doubling of the number of animals achieving normoglycemia by 3 to 4 weeks. We hypothesize that pretreatment of the donor with heparin protects islet integrity during procurement and isolation and hence accelerates islet engraftment and remodelling. Since the effect was seen with N-desulphated heparin, which has negligible anticoagulant properties, we believe the mechanism to be independent of the anticoagulant activity.

Surviving native beta-cells determine outcome of syngeneic intraportal islet transplantation.

In moderately diabetic rats (plasma glucose 20-30 mmol/L), where there is some residual pancreatic islet function, normoglycemia can be restored by transplantation of pancreatic islets into the liver via the portal vein. To examine whether normoglycemia can also be achieved in more severely diabetic animals (which more closely resemble human type I diabetes), we have compared the effect of transplanting 1000 islets intraportally in Lewis rats made moderately diabetic (55 mg/kg streptozotocin injected IP while nonfasting) or severely diabetic (65 mg/kg streptozotocin injected IP while fasting). In the moderately diabetic rats in which residual pancreatic insulin was 128 +/- 40 mU insulin (2.0% of control), plasma glucose stabilized (32 +/- 2.8 mmol/L at 1 week, 34 +/- 2 mmol/L at 3 weeks) as did body weight (falling from 290 +/- 5 to 265 +/- 5 g at 1 week and 253 +/- 6 g at 3 weeks). In contrast, in severely diabetic rats in which residual pancreatic insulin was only 13.5 +/- 4.2 mU insulin (0.21% of control), there was a progressive rise in plasma glucose (30 +/- 1.3 mmol/L at 1 week, 49 +/- 4 mmol/L at 2 weeks, and 67 +/- 7 mmol/L at 3 weeks) and a progressive fall in body weight (from 304 +/- 10 to 260 +/- 5 g by week 1 and to 209 +/- 6 g by week 3). Following islet transplantation, nonfasting plasma glucose normalized in moderately diabetic rats (10.5 +/- 0.6 vs. 9.1 +/- 0.6 mmol/L in nondiabetic controls, NS) after 23 +/- 5 days. In contrast, in the severely diabetic rats plasma glucose stabilized at 32 +/- 5 mmol/L (p < 0.05 compared to moderately diabetic group) but did not normalize. This difference was not attributable to different plasma glucose levels at the time of transplantation (35.1 +/- 1.8 in moderately diabetic vs. 32.5 +/- 2.5 mmol/L in severely diabetic rats). These observations demonstrate that residual native beta-cells (equivalent to only 60-80 islets) contribute to the survival or function of intraportally transplanted islets.