The Development of Therapeutic Radiographers in Imaging and Adaptive Radiotherapy Through Clinical Trial Quality Assurance.

AIMS: Adaptive radiotherapy (ART) is an emerging advanced treatment option for bladder cancer patients. Therapeutic radiographers (RTTs) are central to the successful delivery of this treatment. The purpose of this work was to evaluate the image-guided radiotherapy (IGRT) and ART experience of RTTs before participating in the RAIDER trial. A plan of the day (PoD) quality assurance programme was then implemented. Finally, the post-trial experience of RTTs was evaluated, together with the impact of trial quality assurance participation on their routine practice. MATERIALS AND METHODS: A pre-trial questionnaire to assess the experience of the RTT staff group in IGRT and ART in bladder cancer was sent to each centre. Responses were grouped according to experience. The PoD quality assurance programme was implemented, and the RAIDER trial commenced. During stage 1 of the trial, RTTs reported difficulties in delivering PoD and the quality assurance programme was updated accordingly. A follow-up questionnaire was sent assessing experience in IGRT and ART post-trial. Any changes in routine practice were also recorded. RESULTS: The experience of RTTs in IGRT and ART pre-trial varied. For centres deemed to have RTTs with more experience, the initial PoD quality assurance programme was streamlined. For RTTs without ART experience, the full quality assurance programme was implemented, of which 508 RTTs completed. The quality assurance programme was updated (as the trial recruited) and it was mandated that at least one representative RTT (regardless of pre-trial experience) participated in the update in real-time. The purpose of the updated quality assurance programme was to provide further support to RTTs in delivering a complex treatment. Engagement with the updated quality assurance programme was high, with RTTs in 24/33 centres participating in the real-time online workshop. All 33 UK centres reported all RTTs reviewed the updated training offline. Post-trial, the RTTs' experience in IGRT and ART was increased. CONCLUSION: Overall, 508 RTTs undertook the PoD quality assurance programme. There was a high engagement of RTTs in the PoD quality assurance programme and trial. RTTs increased their experience in IGRT and ART and subsequently updated their practice for bladder cancer and other treatment sites.

Drug-induced pyoderma gangrenosum: a model to understand the pathogenesis of pyoderma gangrenosum.

Pyoderma gangrenosum (PG) is a rare autoinflammatory condition in which the alteration of neutrophil function and the innate immune response play key roles in its pathogenesis. Cases of PG have been reported in patients being treated with certain medications, which may help us to understand some of the possible pathways involved in the aetiology of PG. The aim of this review is to review the cases of PG triggered by certain drugs and try to thoroughly understand the pathogenesis of the disease. To accomplish this, a PubMed search was completed using the following words: pyoderma gangrenosum, neutrophilic dermatosis, pathophysiology, drug-induced pyoderma gangrenosum. In total, we found 43 cases of drug-induced PG. Most of them were caused by colony-stimulating factors and small-molecule tyrosine kinase inhibitors. We propose that drugs induce PG through various mechanisms such as dysfunctional neutrophil migration and function, dysregulated inflammatory response, promotion of keratinocyte apoptosis and alteration of epigenetic mechanisms. PG is a rare condition with complex pathophysiology and drug-induced cases are even more scarce; this is the main limitation of this review. Understanding the possible mechanisms of drug-induced PG, via abnormal neutrophil migration and function, abnormal inflammation, keratinocyte apoptosis and alteration of epigenetic mechanisms would help to better understand the pathogenesis of PG and ultimately to optimize targeted therapy.

A proteomic approach for the rapid, multi-informative and reliable identification of blood.

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes). Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate.

ASC deficiency suppresses proliferation and prevents medulloblastoma incidence.

Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is silenced by promoter methylation in many types of tumors, yet ASC's role in most cancers remains unknown. Here, we show that ASC is highly expressed in a model of medulloblastoma, the most common malignant pediatric brain cancer; ASC is also expressed in human medulloblastomas. Importantly, while ASC deficiency did not affect normal cerebellar development, ASC knockout mice on the Smoothened (ND2:SmoA1) transgenic model of medulloblastoma exhibited a profound reduction in medulloblastoma incidence and a delayed tumor onset. A similar decrease in tumorigenesis with ASC deficiency was also seen in the hGFAP-Cre:SmoM2 mouse model of medulloblastoma. Interestingly, hyperproliferation of the external granule layer (EGL) was comparable at P20 in both wild-type and ASC-deficient SmoA1 mice. However, while the apoptosis and differentiation markers remained unchanged at this age, proliferation makers were decreased, and the EGL was reduced in thickness and area by P60. This reduction in proliferation with ASC deficiency was also seen in isolated SmoA1 cerebellar granule precursor cells in vitro, indicating that the effect of ASC deletion on proliferation was cell autonomous. Interestingly, ASC-deficient SmoA1 cerebella exhibited disrupted expression of genes in the transforming growth factor-ß pathway and increased level of nuclear Smad3. Taken together, these results demonstrate an unexpected role for ASC in Sonic hedgehog-driven medulloblastoma tumorigenesis, thus identifying ASC as a promising novel target for antitumor therapy.

Gastric emptying of hexose sugars: role of osmolality, molecular structure and the CCK1 receptor.

BACKGROUND: It is widely reported that hexose sugars slow gastric emptying (GE) via osmoreceptor stimulation but this remains uncertain. We evaluated the effects of a panel of hexoses of differing molecular structure, assessing the effects of osmolality, intra-individual reproducibility and the role of the CCK(1) receptor, in the regulation of GE by hexoses. METHODS: Thirty one healthy non-obese male and female subjects were studied in a series of protocols, using a (13) C-acetate breath test to evaluate GE of varying concentrations of glucose, galactose, fructose and tagatose, with water, NaCl and lactulose as controls. GE was further evaluated following the administration of a CCK(1) receptor antagonist. Three subjects underwent repeated studies to evaluate intra-individual reproducibility. KEY RESULTS: At 250 mOsmol, a hexose-specific effect was apparent: tagatose slowed GE more potently than water, glucose and fructose (P < 0.05). Fructose (P < 0.05) also slowed GE, but with substantial inter-, but not intra-, individual differences. As osmolality increased further the hexose-specific differences were lost. At 500 mOsmol, all hexoses slowed GE compared with water (P < 0.05), whereas lactulose and saline did not. The slowing of GE by hexose sugars appeared to be CCK(1) receptor-dependent. CONCLUSIONS & INFERENCES: The effects of hexose sugars on GE appear related to their molecular structure rather than osmolality per se, and are, at least in part, CCK(1) receptor-dependent.

p38 kinase is activated in the Alzheimer's disease brain.

The p38 mitogen-activated protein kinase is a stress-activated enzyme responsible for transducing inflammatory signals and initiating apoptosis. In the Alzheimer's disease (AD) brain, increased levels of phosphorylated (active) p38 were detected relative to age-matched normal brain. Intense phospho-p38 immunoreactivity was associated with neuritic plaques, neuropil threads, and neurofibrillary tangle-bearing neurons. The antibody against phosphorylated p38 recognized many of the same structures as an antibody against aberrantly phosphorylated, paired helical filament (PHF) tau, although PHF-positive tau did not cross-react with the phospho-p38 antibody. These findings suggest a neuroinflammatory mechanism in the AD brain, in which aberrant protein phosphorylation affects signal transduction elements, including the p38 kinase cascade, as well as cytoskeletal components.

N-terminal heterogeneity of parenchymal and cerebrovascular Abeta deposits.

The goals of this study were twofold: to determine whether species differences in Abeta N-terminal heterogeneity explain the absence of neuritic plaques in the aged dog and aged bear in contrast to the human; and to compare Abeta N-terminal isoforms in parenchymal vs cerebrovascular Abeta (CVA) deposits in each of the species, and in individuals with Alzheimer disease (AD) vs nondemented individuals. N-terminal heterogeneity can affect the aggregation, toxicity, and stability of Abeta. The human, polar bear, and dog brain share an identical Abeta amino acid sequence. Tissues were immunostained using affinity-purified polyclonal antibodies specific for the L-aspartate residue of Abeta at position one (AbetaN1[D]), D-aspartate at N1 (AbetaN1[rD]), and pyroglutamate at N3 (AbetaN3[pE]) and p3, a peptide beginning with leucine at N17 (AbetaN17[L]). The results demonstrate that each Abeta N-terminal isoform can be present in diffuse plaques and CVA deposits in AD brain, nondemented human, and the examined aged animal models. Though each Abeta N-terminal isoform was present in diffuse plaques, the average amyloid burden of each isoform was highest in AD vs polar bear and dog (beagle) brain. Moreover, the ratio of AbetaN3(pE) (an isoform that is resistant to degradation by most aminopeptidases) vs AbetaN17(L)-x (the potentially nonamyloidogenic p3 fragment) was greatest in the human brain when compared with aged dog or polar bear. Neuritic plaques in AD brain typically immunostained with antibodies against AbetaN1(D) and AbetaN3(pE), but not AbetaN17(L) or AbetaN1(rD). Neuritic deposits in nondemented individuals with atherosclerotic and vascular hypertensive changes could be identified with AbetaN1(D), AbetaN3(pE), and AbetaN1(rD). The presence of AbetaN1(rD) in neuritic plaques in nondemented individuals with atherosclerosis or hypertension, but not in AD, suggests a different evolution of the plaques in the two conditions. AbetaN1(rD) was usually absent in human CVA, except in AD cases with atherosclerotic and vascular hypertensive changes. Together, the results demonstrate that diffuse plaques, neuritic plaques, and CVA deposits are each associated with distinct profiles of Abeta N-terminal isoforms.

Determination of water in forages and animal feeds by Karl Fischer titration.

Oven methods for determining moisture (volatiles) in forages and other animal feeds are empirical. The moisture concentration obtained depends upon the time and temperature the sample was dried and is influenced by the presence of other volatiles than water. A validated reference method to measure water in forages and animal feeds could be used to evaluate the appropriateness of oven methods for various types of animal feeds and forages. Karl Fischer titration is a well-established method for determining water. However, thorough extraction of water from forages and feeds is a challenge because they often contain cellular structures that release water slowly. Water was successfully extracted into methanol-formamide (50 + 50) by high-speed homogenization and then titrated directly at 50 degrees C with a one-component Karl Fischer reagent based on imidazole. The method is described in detail, results of day-to-day repeatability and laboratory-to-laboratory reproducibility are reported, and preliminary comparison data between oven methods are provided.

Interleukin-4 receptor blockade prevents airway responses induced by antigen challenge in mice.

The functional role of interleukin (IL)-4 in the development of airway hyperresponsiveness (AHR) and pulmonary eosinophilia in response to sensitization and challenge of mice with sheep red blood cells (SRBC) was examined. Control- and SRBC-sensitized A/J mice were treated with an antibody to the murine IL-4 receptor (anti-IL-4R) 3 days before intratracheal challenge with the antigen or vehicle only. Blockade of IL-4R significantly reduced antigen-induced AHR and prevented increases in goblet cells and bronchoalveolar lavage (BAL) eosinophils. Treatment with anti-IL-4R did not affect antigen-induced increases in lung mRNA and BAL protein levels of IL-5 and interferon-gamma or IL-4 mRNA but did significantly increase IL-4 protein levels. Antigen-induced AHR was not reduced by treatment with antibodies to the adhesion molecules, vascular cell adhesion molecule-1 and very late activation antigen-4. Administration of IL-4 over a 7-day period did not increase airway reactivity or induce any changes in BAL cell numbers in naive mice. These results demonstrate that IL-4 is necessary for in vivo development of antigen-induced AHR, goblet cell metaplasia, and pulmonary eosinophilia.

Carboxy terminal of beta-amyloid deposits in aged human, canine, and polar bear brains.

Immunocytochemistry, using antibodies specific for different carboxy termini of beta-amyloid. A beta 40 and A beta 42(43), was used to compare beta-amyloid deposits in aged animal models to nondemented and demented Alzheimer's disease human cases. Aged beagle dogs exhibit diffuse plaques in the absence of neurofibrillary pathology and the aged polar bear brains contain diffuse plaques and PHF-1-positive neurofibrillary tangles. The brains of nondemented human subjects displayed abundant diffuse plaques, whereas the AD cases had both diffuse and mature (cored) neuritic plaques. Diffuse plaques were positively immunostained with an antibody against A beta 42(43) in all examined species, whereas A beta 40 immunopositive mature plaques were observed only in the human brain. Anti-A beta 40 strongly immunolabeled cerebrovascular beta-amyloid deposits in each of the species examined, although some deposits in the polar bear brain were preferentially labeled with anti-A beta 42(43). beta-amyloid deposition was evident in the outer molecular layer of the dentate gyrus in the aged dog, polar bear, and human. Within this layer, A beta 42 was present as diffuse deposits, although these deposits were morphologically distinct in each of the examined animal models. In dogs, A beta 42 was cloud-like in nature; the polar bear demonstrated a more aggregated type of deposition, and the nondemented human displayed well-defined deposits. Alzheimer's disease cases were most frequently marked by neuritic plaques in this region. Taken together, the data indicate that beta-amyloid deposition in aged mammals is similar to the earliest stages observed in human brain. In each species, A beta 42(43) is the initially deposited isoform in diffuse plaques.

Familial influence on plaque formation in the beagle brain.

Aged canines exhibit central neuropathological changes strikingly similar to those seen in patients with Alzheimer's disease. In this study, brain tissue from pure bred beagles raised in a controlled environment were examined for Alzheimer-like pathology. The mean age of the animals was 15.6 years. The incidence of plaques among these 29 dogs was 65.5%. Of the 19 samples that demonstrated Alzheimer-like pathology, 18 were characterized as diffuse and one as neuritic. Plaque density was found to be independent of age. Plaque numbers were highest in the perirhinal cortex and the adjacent temporal cortex. Familial influence on plaque development is supported by congruence within 15 of the 16 litters examined (p < 0.001). In this environmentally controlled group the diffuse plaques were rarely converted to the dense neuritic plaques found in Alzheimer's disease.

Bile acid profiles in peroxisomal 3-oxoacyl-coenzyme A thiolase deficiency.

Fast atom bombardment mass spectrometry and gas chromatography-mass spectrometry were used to analyze bile acids in the body fluids of an infant (L.C.) whose liver contained no immunoreactive peroxisomal 3-oxoacyl-CoA thiolase. The profiles were compared with those of six patients with undetectable peroxisomes (Zellweger syndrome) and two siblings (N.B. and I.B.) whose defect of peroxisomal beta-oxidation could not be localized by morphological studies of peroxisomes or by immunoblotting of peroxisomal beta-oxidation proteins. 3 alpha, 7 alpha, 12 alpha-Trihydroxy-5 beta-cholestan-26-oic acid (THCA) was present in bile and plasma of all patients. However, bile from L.C., N.B. and I.B. contained unconjugated varanic acid (3 alpha, 7 alpha, 12 alpha, 24-tetrahydroxy-5 beta-cholestan-26-oic acid) as the major C27 bile acid, whereas bile from Zellweger patients contained only small amounts of varanic acid. In the bile from L.C. two isomers of varanic acid were present; in the bile from N.B. and I.B. a single isomer predominated. L.C., N.B., and I.B. all produced bile containing small amounts of (24E)-3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-24-en-26-oic acid [( 24E]-delta 24-THCA), its [24Z]- isomer, 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholest-23-en-26-oic acid and 3 alpha, 7 alpha, 12 alpha-trihydroxy-27-nor-5 beta-cholestan-24-one. The results provide evidence for peroxisomal pathways for cholic acid synthesis in man via THCA, delta 24-THCA and varanic acid and show that bile acid analyses can be used to diagnose peroxisomal thiolase deficiency.

Rat gastrointestinal transglutaminase: demonstration of enzyme activity and cell and tissue distributions.

The properties, tissue and cellular distribution of intestinal transglutaminase have been investigated. Transglutaminase was assayed with dimethylcasein and [14C]putrescine as substrates. The enzyme has maximum activity at pH 10, although more reliable assays are made at pH 9. Transglutaminase showed an absolute requirement for Ca2+ and exhibited linear assay kinetics. The Km for putrescine was approx. 0.15 mmol/l. Tissue distribution studies suggest transglutaminase is more active in the more muscular segments of the gut. The cellular localization in jejunum was investigated by sequential cell release techniques. Approximately 2 per cent of the total activity was found in the enterocytes and crypt cells. Most of the activity was in the submucosa and serosa suggesting an interstitial cell localization. Acute hypoplastic enteropathy induced by methotrexate was accompanied by a striking decrease in mucosal transglutaminase but the activity returned to control values by 72 h. There was no significant increase in activity during the period of intense crypt cell hyperplasia and it is concluded that intestinal transglutaminase is not implicated in crypt cell proliferation.