Expression of the enteroviral capsid protein VP1 in the islet cells of patients with type 1 diabetes is associated with induction of protein kinase R and downregulation of Mcl-1.

AIMS/HYPOTHESIS: Immunohistochemical staining reveals that the enteroviral capsid protein VP1 is present at higher frequency in the insulin-containing islets of patients with recent-onset type 1 diabetes than in controls. This is consistent with epidemiological evidence suggesting that enteroviral infection may contribute to the autoimmune response in type 1 diabetes. However, immunostaining of VP1 is not definitive since the antibody widely used to detect the protein (Clone 5D8/1) might also cross-react with additional proteins under some conditions. Therefore, we sought to verify that VP1 immunopositivity correlates with additional markers of viral infection. METHODS: Antigen immunoreactivity was examined in formalin-fixed, paraffin-embedded, pancreases from two different collections of type 1 diabetes and control cases: a historical collection from the UK and the nPOD (network of Pancreatic Organ donors with Diabetes) cohort from the USA. RESULTS: VP1 immunoreactivity was present in ~20% of insulin-containing islets of both cohorts under stringent conditions but was absent from insulin-deficient islets. The presence of VP1 was restricted to beta cells but only a minority of these contained the antigen. The innate viral sensor, protein kinase R (PKR) was upregulated selectively in beta cells that were immunopositive for VP1. The anti-apoptotic protein myeloid cell leukaemia sequence-1 (Mcl-1) was abundant in beta cells that were immunonegative for VP1 but Mcl-1 was depleted in cells containing VP1. CONCLUSIONS/INTERPRETATION: The presence of immunoreactive VP1 within beta cells in type 1 diabetes is associated with a cellular phenotype consistent with the activation of antiviral response pathways and enhanced sensitivity to apoptosis. However, definitive studies confirming whether viral infections are causal to beta cell loss in human diabetes are still awaited.

Prognostic value of tumour necrosis and host inflammatory responses in colorectal cancer.

BACKGROUND: Tumour necrosis is a marker of poor prognosis in some tumours but the mechanism is unclear. This study examined the prognostic value of tumour necrosis and host inflammatory responses in colorectal cancer. METHODS: This was a retrospective study of patients undergoing potentially curative resection of colorectal cancer at a single surgical institution over a 10-year period. Patients who underwent preoperative radiotherapy were excluded. The systemic and local inflammatory responses were assessed using the modified Glasgow Prognostic Score and Klintrup-Makinen criteria respectively. Original tumour sections were retrieved and necrosis graded as absent, focal, moderate or extensive. Associations between necrosis and clinicopathological variables were examined, and multivariable survival analyses carried out. RESULTS: A total of 343 patients were included between 1997 and 2007. Tumour necrosis was graded as absent in 32 (9·3 per cent), focal in 166 (48·4 per cent), moderate in 101 (29·4 per cent) and extensive in 44 (12·8 per cent). There were significant associations between tumour necrosis and anaemia (P = 0·022), white cell count (P = 0·006), systemic inflammatory response (P < 0·001), local inflammatory cell infiltrate (P = 0·004), tumour node metastasis (TNM) stage (P = 0·015) and Petersen Index (P = 0·003). On univariable survival analysis, tumour necrosis was associated with cancer-specific survival (P < 0·001). On multivariable survival analysis, age (hazard ratio (HR) 1·29, 95 per cent confidence interval 1·00 to 1·66), systemic inflammatory response (HR 1·74, 1·27 to 2·39), low-grade local inflammatory cell infiltrate (HR 2·65, 1·52 to 4·63), TNM stage (HR 1·55, 1·02 to 2·35) and high-risk Petersen Index (HR 3·50, 2·21 to 5·55) were associated with reduced cancer-specific survival. CONCLUSION: The impact of tumour necrosis on colorectal cancer survival may be due to close associations with the host systemic and local inflammatory responses.

Determinants of short- and long-term outcome in patients undergoing simultaneous resection of colorectal cancer and synchronous colorectal liver metastases.

PURPOSE: The optimal surgical strategy for patients presenting with colorectal liver metastases has yet to be determined. Short- and long-term outcomes must be considered if simultaneous resection of primary and liver metastases is to gain acceptance. We examine the prognostic value of patient and tumour characteristics in predicting short- and long-term outcomes following simultaneous resection for synchronous disease. METHODS: Forty-six patients undergoing simultaneous resection between April 2002 and June 2010 in a single institution were included. Patient characteristics included preoperative ASA grade and POSSUM. Tumour characteristics included TNM stage, Petersen Index and the Clinical Risk Score. RESULTS: There were no postoperative deaths. The most common complications were atrial fibrillation (seven patients) and pneumonia (seven patients). Mean hospital stay with an uncomplicated postoperative recovery was 11 days versus 17 days with complicated recovery. Age (p = 0.015), ASA grade (p = 0.010) and POSSUM score (p = 0.032) were associated with postoperative complications. No pathological characteristics of the primary or secondary tumours related to surgical morbidity. Median follow-up was 37 months (5-87) during which 24 patients died, 23 from cancer. Twenty-seven had disease recurrence. N stage of the primary (p = 0.035), high-risk Petersen Index of the primary (p = 0.010) and Clinical Risk Score ≥ 3 (p = 0.005) were associated with poorer recurrence-free and cancer-specific survival. CONCLUSIONS: Post operative morbidity was determined by patient factors rather than operative or tumour characteristics. In addition to the Clinical Risk Score, pathological characteristics of the primary are important determinants of long-term outcome following simultaneous resection for synchronous disease.

Immunohistochemical analysis of the relationship between islet cell proliferation and the production of the enteroviral capsid protein, VP1, in the islets of patients with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: The enteroviral capsid protein, VP1, was recently shown to be present in some beta cells in more than 60% of patients with recent-onset type 1 diabetes but in very few age-matched controls. The rate of proliferation of islet cells was also markedly increased in the type 1 diabetic patients. As it has been suggested that enteroviruses replicate most efficiently in proliferating cells, we have investigated whether VP1 is preferentially present in proliferating beta cells in type 1 diabetes. METHODS: Combined immunoperoxidase and immunofluorescence staining was used to record the presence of enteroviral VP1, insulin and Ki67 in the islets of recent-onset type 1 diabetic patients. RESULTS: From a total of 1,175 islets, 359 (30.5%) contained insulin. VP1-producing endocrine cells were found in 72 islets (6.1% of total), all of which retained insulin. Ki67(+) endocrine cells were present in 52 (4.4%) islets, with 44 (84.6%) of these being insulin-positive. Overall, 28 of 1,175 (2.4%) islets contained both Ki67(+) cells and VP1(+) cells. Dual positivity of these markers accounted for 38.9% of the total VP1(+) islets and 53.8% of the total Ki67(+) islets. No individual islet cells were dual-positive for Ki67 and VP1. CONCLUSIONS/INTERPRETATION: Ki67(+) cells were frequently observed in islets that also contained VP1(+) cells, suggesting that the factors facilitating viral replication may also drive islet cell proliferation. However, in an individual cell, VP1 production does not require concurrent beta cell proliferation.

An inflammation-based prognostic score (mGPS) predicts cancer survival independent of tumour site: a Glasgow Inflammation Outcome Study.

INTRODUCTION: A selective combination of C-reactive protein and albumin (termed the modified Glasgow Prognostic Score, mGPS) has been shown to have prognostic value, independent of tumour stage, in lung, gastrointestinal and renal cancers. It is also of interest that liver function tests such as bilirubin, alkaline phosphatase and γ-glutamyl transferase, as well as serum calcium, have also been reported to predict cancer survival. The aim of the present study was to examine the relationship between an inflammation-based prognostic score (mGPS), biochemical parameters, tumour site and survival in a large cohort of patients with cancer. METHODS: Patients (n=21,669) who had an incidental blood sample taken between 2000 and 2006 for C-reactive protein, albumin and calcium (and liver function tests where available) and a diagnosis of cancer were identified. Of this group 9608 patients who had an ongoing malignant process were studied (sampled within 2 years before diagnosis). Also a subgroup of 5397 sampled at the time of diagnosis (sampled within 2 months prior to diagnosis) were examined. Cancers were grouped by tumour site in accordance with International Classification of Diseases 10 (ICD 10). RESULTS: On follow up, there were 6005 (63%) deaths of which 5122 (53%) were cancer deaths. The median time from blood sampling to diagnosis was 1.4 months. Increasing age, male gender and increasing deprivation was associated with a reduced 5-year overall and cancer-specific survival (all P<0.001). An elevated mGPS, adjusted calcium, bilirubin, alkaline phosphatase, aspartate transaminase, alanine transaminase and γ-glutamyl transferase were associated with a reduced 5-year overall and cancer-specific survival (independent of age, sex and deprivation in all patients sampled), as well as within the time of diagnosis subgroup (all P<0.001). An increasing mGPS was predictive of a reduced cancer-specific survival in all cancers (all P<0.001). CONCLUSION: The results of the present study indicate that the mGPS is a powerful prognostic factor when compared with other biochemical parameters and independent of tumour site in patients with cancer.

Evidence of increased islet cell proliferation in patients with recent-onset type 1 diabetes.

AIMS/HYPOTHESIS: In adults, the rate of beta cell replication is normally very low, but recent evidence suggests that it may increase during insulitis. We therefore studied tissue from donors with recent-onset type 1 diabetes to establish whether islet cell proliferation is increased during the disease process. METHODS: Paraffin-embedded pancreatic sections from ten donors with recent-onset type 1 diabetes and a range of relevant controls were stained by immunohistochemical techniques with antibodies against the proliferation markers Ki67 and minichromosome maintenance protein-2 (MCM-2). A combination staining technique involving immunoperoxidase and immunofluorescence methods was developed to quantify the numbers of alpha and beta cells with Ki67-positive nuclei and to investigate the relationship between insulitis and islet cell proliferation. RESULTS: In non-diabetic control donors, only 1.1 +/- 0.3% (mean +/- SEM) of islets contained one or more Ki67(+) islet cells, whereas this proportion was increased markedly in recent-onset type 1 diabetes (10.88 +/- 2.5%; p < 0.005). An equivalent increase in Ki67(+) staining occurred in alpha and beta cells and was correlated positively with the presence of insulitis. A significant increase in the labelling of islet cells from type 1 diabetic donors was also seen when MCM-2 staining was employed. Increased islet cell proliferation was not evident in three donors with longer duration type 1 diabetes or in ten type 2 diabetic donors. CONCLUSIONS/INTERPRETATION: Alpha and beta cells undergo a marked increase in proliferation during the progression of type 1 diabetes in humans. The results imply that islet cell proliferation is re-initiated in response to the autoimmune attack associated with type 1 diabetes.

The relationship between the presence and site of cancer, an inflammation-based prognostic score and biochemical parameters. Initial results of the Glasgow Inflammation Outcome Study.

BACKGROUND: Cancer incidence is increasing in the United Kingdom, as well as on a global basis. Biochemical parameters, such as C-reactive protein and albumin (combined to form the modified Glasgow Prognostic Score, mGPS), alkaline phosphatase (Alk phos), gamma-glutamyl transferase (GGT) and serum calcium have been reported to be associated with cancer and non-cancer mortality. Therefore, to definitively examine the interrelationships between the above biochemical parameters, the mGPS and the presence of cancer, the Glasgow Inflammation Outcome Study was undertaken. The aim of this initial study was to examine the effect of cancer on markers of systemic inflammation induced by the liver (mGPS) and on levels of routine biochemical parameters. METHODS: Patients (n=223 303) who had a single incidental sample taken for C-reactive protein, albumin, calcium and serum liver function tests where available, between 2000 and 2008 were studied. Those with a pathological diagnosis of cancer (n=22 715) were identified. The mGPS was constructed and liver function tests classified in accordance with the local reference ranges. RESULTS: Patients with cancer had higher C-reactive protein and lower albumin levels (and thus a higher mGPS), higher adjusted calcium, Alk phos and GGT levels, but lower aspartate transaminase (AST) and alanine transaminase (ALT) levels (all P<0.001). The strongest associations (Spearman's correlation > or =0.3) in both the non-cancer and cancer groups were found between albumin, C-reactive protein and Alk phos, AST and ALT, AST and GGT and ALT and GGT (all P<0.001). On multivariate analysis, the associations with the presence of cancer remained with age, deprivation, C-reactive protein, albumin, adjusted calcium, Alk phos and GGT (all P<0.01). Patients following a diagnosis of cancer had lower albumin levels and thus higher mGPS (all P<0.001). Also, post-diagnosis patients were more likely to have lower adjusted calcium, bilirubin, Alk Phos, AST, ALT and GGT levels (all P<0.05). When the cancer diagnoses were ranked from those with the lowest proportion of mGPS 1 or 2 to those with the highest, the percentage of cases with a mGPS of 1 or 2 ranged from 21% in breast cancer to 46% in prostate cancer and to 68% in pulmonary cancer. Compared with breast cancer the mGPS was significantly higher in those diagnosed with dermatological, bladder, endocrinological, gynaecological, prostate, musculoskeletal, gastroesophageal, haematological, renal, colorectal, head and neck, pancreaticobiliary and pulmonary cancers (all P<0.001). CONCLUSION: The results of the present study indicate that the systemic inflammatory response is common in a large patient cohort, increased by the presence of cancer and associated with the perturbation of a number of biochemical parameters previously reported to be associated with mortality. There is a striking parallel between the proportions of cases with a mGPS of 1 or 2 and reported survival rates in these tumours.

The prevalence of enteroviral capsid protein vp1 immunostaining in pancreatic islets in human type 1 diabetes.

AIMS/HYPOTHESIS: Evidence that the beta cells of human patients with type 1 diabetes can be infected with enterovirus is accumulating, but it remains unclear whether such infections occur at high frequency and are important in the disease process. We have now assessed the prevalence of enteroviral capsid protein vp1 (vp1) staining in a large cohort of autopsy pancreases of recent-onset type 1 diabetic patients and a range of controls. METHODS: Serial sections of paraffin-embedded pancreatic autopsy samples from 72 recent-onset type 1 diabetes patients and up to 161 controls were immunostained for insulin, glucagon, vp1, double-stranded RNA activated protein kinase R (PKR) and MHC class I. RESULTS: vp1-immunopositive cells were detected in multiple islets of 44 out of 72 young recent-onset type 1 diabetic patients, compared with a total of only three islets in three out of 50 neonatal and paediatric normal controls. vp1 staining was restricted to insulin-containing beta cells. Among the control pancreases, vp1 immunopositivity was also observed in some islets from ten out of 25 type 2 diabetic patients. A strong correlation was established between islet cell vp1 positivity and PKR production in insulin-containing islets of both type 1 and type 2 diabetic patients, consistent with a persistent viral infection of the islets. CONCLUSIONS/INTERPRETATION: Immunoreactive vp1 is commonly found in the islets of recent-onset type 1 diabetes patients, but only rarely in normal paediatric controls. vp1 immunostaining was also observed in some islets of type 2 diabetes patients, suggesting that the phenomenon is not restricted to type 1 diabetes patients.

Comparison of tumour-based (Petersen Index) and inflammation-based (Glasgow Prognostic Score) scoring systems in patients undergoing curative resection for colon cancer.

After resection, it is important to identify colon cancer patients, who are at a high risk of recurrence and who may benefit from adjuvant treatment. The Petersen Index (PI), a prognostic model based on pathological criteria is validated in Dukes' B and C disease. Similarly, the modified Glasgow Prognostic Score (mGPS) based on biochemical criteria has also been validated. This study compares both the scores in patients undergoing curative resection of colon cancer. A total of 244 patients underwent elective resection between 1997 and 2005. The PI was constructed from pathological reports; the mGPS was measured pre-operatively. The median follow-up was 67 months (minimum 36 months) during which 109 patients died; 68 of them from cancer. On multivariate analysis of age, Dukes' stage, PI and mGPS, age (hazard ratio, HR, 1.74, P=0.001), Dukes' stage (HR, 3.63, P<0.001), PI (HR, 2.05, P=0.010) and mGPS (HR, 2.34, P<0.001) were associated independently with cancer-specific survival. Three-year cancer-specific survival rates for Dukes' B patients with the low-risk PI were 98, 92 and 82% for the mGPS of 0, 1 and 2, respectively (P<0.05). The high-risk PI population is small, in particular for Dukes' B disease (9%). The mGPS further stratifies those patients classified as low risk by the PI. Combining both the scoring systems could identify patients who have undergone curative surgery but are at high-risk of cancer-related death, therefore guiding management and trial stratification.

Analysis of islet inflammation in human type 1 diabetes.

The immunopathology of type 1 diabetes (T1D) has proved difficult to study in man because of the limited availability of appropriate samples, but we now report a detailed study charting the evolution of insulitis in human T1D. Pancreas samples removed post-mortem from 29 patients (mean age 11.7 years) with recent-onset T1D were analysed by immunohistochemistry. The cell types constituting the inflammatory infiltrate within islets (insulitis) were determined in parallel with islet insulin content. CD8(+) cytotoxic T cells were the most abundant population during insulitis. Macrophages (CD68(+)) were also present during both early and later insulitis, although in fewer numbers. CD20(+) cells were present in only small numbers in early insulitis but were recruited to islets as beta cell death progressed. CD138(+) plasma cells were infrequent at all stages of insulitis. CD4(+) cells were present in the islet infiltrate in all patients but were less abundant than CD8(+) or CD68(+) cells. Forkhead box protein P3(+) regulatory T cells were detected in the islets of only a single patient. Natural killer cells were detected rarely, even in heavily inflamed islets. The results suggest a defined sequence of immune cell recruitment in human T1D. They imply that both CD8(+) cytotoxic cells and macrophages may contribute to beta cell death during early insulitis. CD20(+) cells are recruited in greatest numbers during late insulitis, suggesting an increasing role for these cells as insulitis develops. Natural killer cells and forkhead box protein P3(+) T cells do not appear to be required for beta cell death.

Aberrant expression of HLA-DR antigens by insulin-containing beta-cells in recent-onset type I diabetes mellitus.

The pancreases of 14 children who died of type I diabetes were studied immunohistochemically for aberrant expression of HLA-DR antigens on islet endocrine cells. Two cases in which no residual insulin-secreting beta-cells were present had no evidence of HLA-DR expression on endocrine cells. Insulin-containing islets were present in the remainder, and HLA-DR-positive endocrine cells were demonstrable in all of them. Endocrine cells expressing HLA-DR were present in 171 of 630 insulin-containing islets from all the cases. However, HLA-DR-positive endocrine cells were not seen in 2060 insulin-deficient islets, providing evidence that of the four hormone-producing cells in the pancreas only the beta-cells expressed HLA-DR. Sections double stained for HLA-DR and the pancreatic hormones confirmed this view. Most islets in which HLA-DR-positive endocrine cells were seen had no evidence of insulitis, suggesting that within an individual islet, aberrant expression of HLA-DR on beta-cells may precede the inflammatory infiltrate.

Accuracy of thin section magnetic resonance using phased-array pelvic coil in predicting the T-staging of rectal cancer.

Magnetic resonance (MR) imaging may contribute to staging rectal cancer and inform the decision regarding administration of pre-operative radiotherapy. The accuracy of MR has been debated. The aim of the present study was to determine the accuracy of thin section T2-weighted MR images in rectal cancer patients. MR results were compared with histological assessment of resection specimens. Over a 2-year period, 42 patients were studied. Histological staging was pT2 n = 13, pT3 n = 25 and pT4 n = 4. MR diagnostic accuracy was 74%. MR sensitivity and specificity was 62% and 79% for pT2 lesions, 84% and 59% for pT3 lesions and 50% and 76% for pT4 lesions. Estimation of tumour penetration by thin section MR imaging of rectal cancers using pelvic phased-array coil has moderate diagnostic accuracy. The limitations of MR should be acknowledged when selecting rectal cancer patients for pre-operative radiotherapy.

Pancreatic beta-cell damage. In search of novel pathogenetic factors.

A model for the possible pathogenesis of insulin-dependent diabetes mellitus (IDDM) is presented. It is partly based on studies of autoimmune thyroiditis and founded on the idea that inappropriate class II molecule expression in insulin-producing beta-cells of pancreatic islets could cause an organ-specific autoimmune disorder. The model is supported by results of several IDDM case studies as well as by the critical evaluation of the known effects of various lymphokines and lymphotoxins on endocrine target tissues. The possible roles of islet capillary endothelial cells and islet cell antibodies in the pathogenesis of IDDM are discussed.

Localization of alpha-interferon in the human feto-placental unit.

The distribution of alpha-interferon in human placental tissue was investigated by immunocytochemical study of paraffin wax-embedded tissue sections using a sheep alpha-interferon antiserum. Fifty-eight placentas of gestational ages from 8 to 40 weeks were examined. alpha-Interferon was present in the syncytiotrophoblast of the chorionic villi of all placentas and was also in macrophages in 28 cases. The appearances suggest production of interferon in human placental trophoblast and, in view of its diverse biological effects, support the concept of a role for alpha-interferon in the complex series of events required for successful gestation.

The pathology of the endocrine pancreas in type 1 (insulin-dependent) diabetes mellitus.

Type 1 diabetes is an organ-specific autoimmune disease in which the insulin-secreting B cell is destroyed. Both genetic factors (linked to class II MHC genes) and environmental agents (viruses, diet) appear to be involved in the aetiology. Study of autopsy pancreases of children who die at presentation of their disease has proved elucidating. In such pancreases islets before, during and after B cell destruction, are all visible. The earliest defined immunological event in the disease process appears to be expression of interferon-alpha by insulin-containing B cells. Secretion of this cytokine is associated with hyperexpression of class I MHC by all the endocrine cells within insulin-containing islets. Another immunological phenomenon which is unique to type I diabetes is the presence of aberrant class II MHC molecule expression by B cells. This may induce autoimmunity by allowing antigen presentation of B cell specific antigens. If the onset of the disease process is marked by interferon-alpha expression by B cells then a search for the presence of a continuing viral infection in these cells may prove profitable, although no viruses have been found in them to date.

Morphological study of the relation between accidental hypothermia and acute pancreatitis.

There is a recognised but poorly understood association between hypothermia and acute pancreatitis. A histological study of the pancreas was made in eight patients with accidental hypothermia who had evidence of pancreatitis at necropsy. From an analysis of the patterns of parenchymal necrosis in the pancreas it was thought that there were at least three possible mechanisms for the relation between hypothermia and pancreatitis. Firstly, that ischaemic pancreatitis may result from the "microcirculatory shock" of hypothermia. Secondly, that both hypothermia and pancreatitis may be secondary to alcohol abuse: and finally, that severe pancreatitis may be the primary disease and that hypothermia results from the patients' social circumstances.

Histological evidence of initiating factors in acute necrotising pancreatitis in man.

In this necropsy review of 37 cases of acute pancreatitis, evidence is presented that there are at least two different initiating mechanisms in acute necrotising pancreatitis in man. Firstly, there is primary duct inflammation, with subsequent inflammation and necrosis of the pancreatic parenchyma surrounding the excretory ducts. Included in this group are cases secondary to alcohol abuse and cholelithiasis. Secondly, there is necrosis confined to the microcirculatory periphery of the pancreatic lobule. These cases are usually secondary to some form of shock (septic or cardiogenic) and are thought to represent ischaemic pancreatitis. These patterns of necrosis are discussed in relation to the pancreatic anatomy and clinical findings.

Use of microwave oven improves morphology and staining of cryostat sections.

The quality of microscopic image of cryostat sections that had been subjected to microwave assisted fixation was compared with that resulting from conventional air drying of the sections. The role of microwaves in producing rapid special stains on cryostat sections was also assessed. Methods used permitted stains such as periodic acid Schiff, alcian blue, Gordon and Sweets's reticulin, Masson Fontana, Elastica, Prussian blue and Van Gieson to be performed within three minutes of cutting a cryostat section. The cytological detail of nuclei was much clearer using the microwave technique, allowing more accurate determination of cell type. The microwave oven seems to have major potential in improving the diagnostic accuracy of surgical frozen sections.