Micro-CT scouting for transmission electron microscopy of human tissue specimens.

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. We describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 µm resolution was used to determine the location of patches of the mucous membrane in osmium-stained human nasal scraping samples. Once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation.

Children with dermatological conditions admitted to paediatric intensive care: analysis of a national clinical audit database.

There is little published literature about dermatological conditions in paediatric intensive care units (PICUs). The aim of this study was to describe the range of skin disorders in children admitted to PICUs in the UK and Ireland using data from a national audit. An analysis was conducted using data for 2002 - 2010 from the Paediatric Intensive Care Audit Network (PICANet). In total, 999 admissions of 882 children were identified, representing 0.8% of all PICU admissions. The most frequent dermatological conditions were skin infections, including cellulitis and necrotizing fasciitis, and inflammatory conditions. In 28% of cases, the dermatological diagnosis was considered the reason for PICU admission, in 35% it was a manifestation of systemic disease and in 37% it was considered incidental. Overall mortality was similar to the general PICU population, with 52 deaths (5.2%), but was greater in children with vascular/haematological conditions.

Polygenic control of autoimmune diabetes in nonobese diabetic mice.

Partial exclusion mapping of the nonobese (NOD) diabetic mouse genome has shown linkage of diabetes to at least five different chromosomes. We have now excluded almost all of the genome for the presence of susceptibility genes with fully recessive effects and have obtained evidence of linkage of ten distinct loci to diabetes or the prediabetic lesion, insulitis, indicative of a polygenic mode of inheritance. The relative importance of these loci and their interactions have been assessed using a new application of multiple polychotomous regression methods. A candidate disease gene, interleukin-2 (Il-2), which is closely linked to insulitis and diabetes, is shown to have a different sequence in NOD, including an insertion and a deletion of tandem repeat sequences which encode amino acid repeats in the mature protein.

Racial and ethnic differences in patients enrolled in the national bronchiectasis and nontuberculous mycobacteria research registry.

Demographic and socioeconomic factors are recognized to contribute to disparities in healthcare outcomes. Originally, bronchiectasis was described in a population of predominantly White ethnic group of patients in which racial disparity could not be identified. The U.S. Bronchiectasis Research Registry (BRR), a centralized database of adult patients with bronchiectasis and/or NTM from 18 clinical institutions across the U.S., was created to support the research of this condition. The aim of this study is to describe the racial and ethnic distribution of patients enrolled in the BRR and evaluate factors associated with healthcare disparities within manifestations of and/or the care delivered to this population. At the time of this study, 3600 patients with bronchiectasis and/or NTM were enrolled in the BRR. Of those, 3510 participants were included in these analyses. The population was predominantly non-HispanicWhite (n = 3143, 89.5%), followed by Hispanic or Latino (n = 149, 4.3%), Asian (n = 130, 3.7%) and non-Hispanic Black (n = 88, 2.5%) participants. Testing for cystic fibrosis, immunoglobulin deficiency, and mycobacteria was not different between races, but non-Hispanic Black patients were tested less frequently for alpha-1 antitrypsin (A1AT) deficiency compared to other groups (P = 0.01). The four groups did not differ in the proportion of Pseudomonas aeruginosa or Hemophilus influenzae. There was no statistically significant difference in use of high-frequency chest wall oscillation, pulmonary rehabilitation services, or suppressive macrolide treatment across the groups (P > 0.05). There is a disproportionately high percentage of non-Hispainc White patients compared to non-Hispanic Black patients and Hispanic or Latino patients in the BRR. However, we found an overall similarity of care of BRR patients, regardless of racial and ethnic group.

Anaphylaxis admissions to UK critical care units between 2005 and 2009.

Anaphylaxis is a life-threatening emergency that may necessitate admission to a critical care unit. There are no reports of the frequency of admission to critical care units for patients with anaphylaxis or indeed any description of their demographic characteristics or outcomes. We analysed all physician-diagnosed cases of anaphylaxis over a 5-year period in national audit data from critical care units across the UK. Over the period 2005-2009, there were 81 paediatric and 1269 adult admissions with anaphylaxis admitted to UK critical care units (0.1% of admissions to paediatric units and 0.3% of admissions to adult units). Absolute numbers in both children and adults rose year on year. There were comparable proportions of admissions in female and male children (female = 47% and male = 53%; rate ratios (RR) = 0.88, 95% CI 0.64-1.20), but a greater proportion of adult female admissions (female = 65% and male = 35%; RR = 1.83, 95% CI 1.68-1.99). Survival to unit discharge was 95% (77/81) for children, and survival to hospital discharge was 92% (1166/1269) for adults. Each UK critical care unit is likely to see at least one anaphylaxis case per year. The number of admissions has risen in both children and adults. Although admission ratios between the sexes were comparable in children, there was a female preponderance in adult life. Survival rates were high at over 90%.

The use of a measure of acute irritation to predict the outcome of repeated usage of hand soap products.

BACKGROUND: Healthcare-associated infection is an important worldwide problem that could be reduced by better hand hygiene practice. However, an increasing number of healthcare workers are experiencing irritant contact dermatitis of the hands as a result of repeated hand washing. This may lead to a reduced level of compliance with regard to hand hygiene. OBJECTIVES: To assess whether a measure of acute irritation by hand soaps could predict the effects of repeated usage over a 2-week period. METHODS: In a double-blind, randomized comparison study, the comparative irritation potential of four different hand soaps was assessed over a 24-h treatment period. The effect of repeated hand washing with the hand soap products over a 2-week period in healthy adult volunteers on skin barrier function was then determined by assessment of transepidermal water loss (TEWL), epidermal hydration and a visual assessment using the Hand Eczema Severity Index (HECSI) at days 0, 7 and 14. RESULTS: A total of 121 subjects from the 123 recruited completed phase 1 of the study. All four products were seen to be significantly different from each other in terms of the irritant reaction observed and all products resulted in a significantly higher irritation compared with the no-treatment control. Seventy-nine of the initial 121 subjects were then enrolled into the repeated usage study. A statistically significant worsening of the clinical condition of the skin as measured by HECSI was seen from baseline to day 14 in those subjects repeatedly washing their hands with two of the four soap products (products C and D) with P-values of 0·02 and 0·01, respectively. Subclinical assessment of the skin barrier function by measuring epidermal hydration was significantly increased from baseline to day 7 after repeated hand washing with products A, B and D but overall no significant change was seen in all four products tested by day 14. A statistically significant increase in TEWL at day 14 was seen for product A (P = 0·02) indicating a worsening of skin barrier function. This effect was also seen initially for product D at day 7 although this was then lost at day 14. Further regression analysis was then performed to see if the acute irritant test data for each product correlated with the skin barrier data from the repeated usage component of the study. This showed that the results of acute irritant testing of the individual products did not predict the results of chronic use of hand soaps. CONCLUSIONS: The results from phase 2 of our study confirm the work of previous studies that show that regular exposure to irritants in daily life leads to stratum corneum damage and impairment of the skin barrier. Although significant differences were seen between the products in phase 1 of the study, regression analysis showed that the results of patch testing of the individual products did not predict the results of chronic use of hand soaps. When designing a study to assess the effects of cumulative use of a product on the skin, the study should mirror the use conditions of the product as closely as possible.

The specificity of rejection and the absence of susceptibility of pancreatic islet beta cells to nonspecific immune destruction in mixed strain islets grafted beneath the renal capsule in the rat.

The specificity of rejection of isolated pancreatic islets was examined in the rat using a quantitative model in which syngeneic (DA) or a mixture of syngeneic and allogeneic (DA and LEW or PVG) islets were implanted beneath the capsule of the kidney of nondiabetic normal rats (DA). 3 wk after transplantation total insulin extraction assays of the kidney with its islet implant together with immunohistological examination of the site of transplantation for evidence of syngeneic or allogeneic tissue demonstrated the total destruction of allogeneic islets without any evidence of damage to syngeneic islets either distant or in immediate proximity to allogeneic islets. Pancreatic islets, and especially beta cells, appear to be particularly vulnerable to the effector arm of both autoimmune and alloimmune responses, a vulnerability that has been attributed to the cytotoxic effects of lymphokines, notably IL-1, released in both autoimmune and alloimmune responses. The experiments reported here demonstrate not only the exquisite specificity of the allograft reaction but are not compatible with a hypothesis that B cells within an intact islet are nonspecifically susceptible to destruction by lymphokines.

The proportion of tumour cells is an independent predictor for survival in colorectal cancer patients.

BACKGROUND: The proportion of epithelial and stromal cells in tumours is thought to have an important role in the progression of epithelial malignancy. We aimed to determine whether the relative proportion of tumour (PoT) was related to survival in colorectal cancer. METHODS: The PoT at the luminal surface was measured by point counting using virtual tissue sections in a series of 145 colorectal cancer cases. The relationship of PoT to clinicopathological parameters including cancer-specific survival was analysed. Modified receiver operating characteristic curves were used to determine the optimum cut off points to dichotomise the data for survival analyses. RESULTS: Tumours with PoT-low (

A double-blind, randomized study to assess the effectiveness of different moisturizers in preventing dermatitis induced by hand washing to simulate healthcare use.

BACKGROUND: Healthcare-associated infection is an important worldwide problem that could be reduced by better hand hygiene practice. However, irritant contact dermatitis of the hands as a result of repeated hand washing is a potential complication that may be preventable by the regular use of an emollient. OBJECTIVES: To assess the effect of moisturizer application after repeated hand washing (15 times daily) vs. soap alone. METHODS: In a double-blind, randomized study, the effect of five different moisturizers on skin barrier function was determined by assessment after repeated hand washing over a 2-week period in healthy adult volunteers. Assessments of transepidermal water loss (TEWL), epidermal hydration and a visual assessment using the Hand Eczema Severity Index (HECSI) were made at days 0, 7 and 14. RESULTS: In total, 132 patients were enrolled into the study. A statistically significant worsening of the clinical condition of the skin as measured by HECSI was seen from baseline to day 14 (P = 0.003) in those subjects repeatedly washing their hands with soap without subsequent application of moisturizer. No change was seen in the groups using moisturizer. Subclinical assessment of epidermal hydration as a measure of skin barrier function showed significant increases from baseline to day 14 after the use of three of the five moisturizing products (P = 0.041, 0.001 and 0.009). Three of the five moisturizers tested led to a statistically significant decrease in TEWL at day 7 of repeated hand washing. This effect was sustained for one moisturizing product at day 14 of hand washing (P = 0.044). CONCLUSIONS: These results support the view that the regular application of moisturizers to normal skin offers a protective effect against repeated exposure to irritants, with no evidence of a reduction in barrier efficiency allowing the easier permeation of irritant substances into the skin as has been suggested by other studies. Regular use of emollient in the healthcare environment may prevent the development of dermatitis.

Predictors of return to work following carotid endarterectomy.

BACKGROUND: Carotid endarterectomy (CEA) is an important part of secondary prevention in selected patients following a transient ischaemic attack or stroke. A key marker of success, return to work following surgery, was assessed in a retrospective cohort study. METHODS: Patients from the UK aged less than 65 years at operation were sent a questionnaire concerning return to work after CEA. Data were analysed using univariable tests and logistic regression. RESULTS: Some 174 (64.4 per cent) of 270 patients responded; their median age was 60 (range 35-64) years and 124 were men. Seventy-five per cent of respondents employed preoperatively returned to work following CEA. Newly retiring patients were older (62 versus 58 years; P < 0.001). Univariable analysis confirmed that age and preoperative stroke influenced return to work. The adjusted odds ratio for patients with versus without a preoperative stroke was 0.46 (95 per cent confidence interval 0.22 to 0.97) (P = 0.040). Median convalescence was 4 weeks, but was shorter in the self-employed (P = 0.039) and prolonged in patients with symptomatic cardiovascular disease (P = 0.023) and those who required postoperative critical care (P = 0.039). CONCLUSION: Return to work following CEA was influenced by age and preoperative stroke.

Interleukin-4 -590 (C>T), toll-like receptor-2 +2258 (G>A) and matrix metalloproteinase-9 -1562 (C>T) polymorphisms in pre-eclampsia.

Functional single nucleotide polymorphisms (SNPs) of interleukin (IL)-4 -590 (C>T), toll-like receptor (TLR)-2 +2258 (G>A) and matrix metalloproteinase (MMP)-9 -1562 (C>T) were examined by polymerase chain reaction-restriction fragment length polymorphism to identify their merit as genetic markers for pre-eclampsia. One hundred and seventeen pre-eclamptic women and 146 control subjects with uncomplicated singleton pregnancies participated in this study, conducted at Leeds General Infirmary and St James's University Hospital. While the TLR-2 +2258 (G>A) and MMP-9 -1562 (C>T) SNPs failed to present any significant association with pre-eclampsia, there was a marked trend for an association between the IL-4 -590 (C>T) SNP and pre-eclampsia (chi(2)= 5.87, P = 0.055), with a prevalence of TT homozygous women in this group (OR 4.455, 95% CI 1.286-15.350).

Protease activity in pancreatic islet isolation by enzymatic digestion.

Commercial Collagenase* prepared from Clostridium histolyticum is widely used in isolation of pancreatic islets. It is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches. Our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized. Comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that they had very similar activity against collagen (measured by digestion of insoluble collagen followed by assay of soluble products with ninhydrin) but substantially different activities against azocasein as measured by optical density increase (measured by release of dye). Eighteen batches of Collagenase were examined for efficiency in islet isolation, and the yields obtained correlated with manufacturer's data of activity against casein. The data show that low caseinase activity is associated with performance in islet isolation (r = .5 after adjusting for collagenase activity). The effect of supplementing a batch of collagenase, known to be poor in isolating islets, with proteolytic enzymes was investigated. Trypsin and papain had apparently no effect, but dispase significantly increased yield. Dispase alone failed to digest pancreas. Size-exclusion high-performance liquid chromatography identified a peak associated with high protease activity and efficiency in islet isolation, having an Mr of approximately 30,000, compared to 78,000 for collagenase. The protease, like collagenase, is inhibited by EDTA. Increased Ca2+ and Mg2+ (up to 10 mM) did not affect activity. Both the protease and collagenase are stable under normal use but are inactivated by heating at 56 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for isolation of islets of Langerhans from the human pancreas.

A method has been developed for the isolation of islets of Langerhans from the human pancreas. The average number of islets isolated was 1011 islets per gram of pancreas (SD 475, range 752-2111), and the purity of the preparation as defined by histologic examination and specific staining for insulin varied from 10% to 40%. Islet structure was well preserved and the islets were shown to be viable by supravital staining, demonstration of insulin response to glucose, and by transplantation of isolated islets beneath the renal capsule of nude mice. The essential features of this technique for isolation of human islets include injection of a high concentration of collagenase (6 mg/ml) into the pancreatic duct under pressure, followed by a short incubation (23 min) at 39 degrees C. The gland is then dispersed by a process of teasing and shaking, and the islets are separated by a two-stage process of filtration on a nylon mesh to remove the larger islets and centrifugation on a preformed Ficoll density gradient to separate the small islets.

Metabolic function of intraportal and intrasplenic islet autografts in cynomolgus monkeys.

Intraportal islet autografting can restore near-normal glucose homeostasis in large diabetic animals, but the long-term failure rate of such grafts remains high. To assess the effect of the site of transplantation, we compared the hormonal responses to glucose (500 mg/kg i.v.) of intraportal (IP) and intrasplenic (IS) islet autografts in the cynomolgus monkey previously rendered diabetic by total pancreatectomy. Intravenous glucose tolerance tests (IVGTTs) 6 wk after IP grafting (n = 10) demonstrated nearly normal plasma glucose changes, with qualitatively normal but quantitatively reduced insulin and glucagon responses; only two animals have maintained these responses for greater than 2 yr. IVGTTs 6 wk after IS grafting (n = 4) demonstrated more abnormal plasma glucose changes, with qualitatively normal but weak insulin responses and glucagon levels that did not fall in response to hyperglycemia; only one animal has maintained fasting normoglycemia for greater than 9 mo. These results suggest that IS transplantation confers no benefit over IP transplantation in this model.

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.

Observational study of children admitted to United Kingdom and Republic of Ireland Paediatric Intensive Care Units after out-of-hospital cardiac arrest.

AIMS: To estimate the prevalence of children admitted after out-of-hospital cardiac arrest (OHCA) to UK and Republic of Ireland (RoI) Paediatric Intensive Care Units (PICUs) and factors associated with mortality to inform future clinical trial feasibility. METHOD: Observational study using a prospectively collected dataset of the Paediatric Intensive Care Audit Network (PICANet) of 33 UK and RoI PICUs (January 2003 to June 2010). Cases (0 to <16 years), with documented OHCA surviving to PICU admission and requiring mechanical ventilation were included. Main outcomes were prevalence for admission and death within PICU. Factors associated with mortality were examined with multiple logistic regression analysis. RESULTS: 827 of 111,170 admissions (0.73%; 95% CI [0.48 to 0.98%]) were identified as children admitted following OHCA. PICU mortality for OHCA was 50.5% (418/827). Recruitment into an adequately sized clinical trial would not be feasible with the current prevalence rate. Characteristics at PICU admission associated with increased risk of death included; bilateral unreactive pupils, genetically inherited condition, inter-hospital transfer to PICU, requirement for vasoactive drugs and greater base deficit. Factors associated with reduced risk of death were submersion or a respiratory aetiology and pre-existing respiratory or cardiac conditions. CONCLUSIONS: Less than 120 children a year are admitted to PICUs in the UK and RoI after OHCA, limiting options for conducting UK intervention trials. The risk factors associated with mortality identified in this study will allow risk stratification in future studies.

The effect of hyperglycemia on isolated rodent islets transplanted to the kidney capsule site.

The effect of hyperglycemia on transplanted rat pancreatic islets was studied using a new technique for transplanting a defined number of islets in a blood clot. Normal or streptozotocin-diabetic DA rats were given 400 DA islets under the left kidney capsule (a number shown to be insufficient to reverse diabetes). After 2 weeks the diabetic rats were given a further 1000 islets under the right kidney capsule to reverse diabetes. Kidneys from both groups were examined at 2 weeks after the initial islet implant and at 3 months for both gross and histological appearance and for insulin content. After 2 weeks left kidneys from nondiabetic rats showed abundant islet tissue, with an insulin content of 116 (+/- 14 SEM) milliunits, compared to 1 +/- 0.5 milliunits in the right kidney. Kidneys from diabetic rats showed no islets recognizable grossly. Histological examination showed vacuolated tissue scarcely recognizable as islet tissue, and the insulin content of the left kidney was reduced to 18 +/- 5 milliunits. However, 3 months after reversal of diabetes by transplantation of 1000 islets to the right kidney, histologically "normal" islet tissue was again visible on the left kidney, and the insulin content was 160 +/- 36 milliunits. Islets left in normal animals for 3 months contained 195 +/- 50 milliunits. These experiments show that islets implanted beneath the kidney capsule in diabetic rats are not destroyed by two weeks hyperglycemia. This suggests that protection of islets after implantation by insulin treatment is unnecessary, even in the presence of a persistently raised blood sugar.

A technique for transplanting pancreatic islets as a vascularized graft and prevention of rejection with cyclosporin A.

Transplants of isolated allogeneic pancreatic islets behave as if extremely immunogenic but vascularized segmental pancreatic allografts do not evoke such a rapid rejection of islet tissue. We have transplanted rat pancreatic islets as a vascularized graft by implanting isolated islets under the renal capsule of a syngeneic diabetic rat and, after successful reversal of diabetes, a composite graft of kidney and islets was transplanted into an allogeneic diabetic host. Cyclosporin A was used to suppress rejection of the allografts in doses that have previously been shown to completely suppress rejection of renal allografts and delay rejection of vascularized segmental pancreatic allografts. This strategy produced indefinite survival of the allogeneic isolated pancreatic islets.