Pancreas Tissue Samples Research Articles

Tissue expression of angiopoietin-like protein 4 in cattle1

Angiopoietin-like protein 4 (ANGPTL4; also known as fasting-induced adipose factor) is a plasma protein that stimulates oxidation of fatty acids and inhibits fat accumulation. The gastrointestinal tract appears to play an important role in regulating plasma ANGPTL4 concentration in some situations and may be influenced by microbes within the gastrointestinal tract. Our aim was to determine which tissues express ANGPTL4 in the bovine. Rumen, omasum, abomasum, duodenum, jejunum, ileum, colon, pancreas, liver, and subcutaneous adipose tissue samples were collected postmortem from 2 steers. Abundance of ANGPTL4 messenger RNA was quantified by quantitative real-time PCR, and was most abundant in liver and adipose tissue (P < 0.05). We also detected ANGPTL4 messenger RNA throughout the gastrointestinal tract, although its abundance was approximately 10% of that found in liver and adipose tissue. Western blot analysis revealed that ANGPTL4 protein was most abundant in liver and adipose tissue (P < 0.05), but omasal, abomasal, and ileal samples contained at least 60% as much ANGPTL4 protein as the liver and adipose tissue samples, and the protein was detected in all tissues. Finally, cross-sections of the liver, pancreas, and rumen wall were used for indirect immunofluorescent detection of ANGPTL4. Despite the low abundance of ANGPTL4 measured by quantitative real-time PCR and Western blot in ruminal tissue, immunofluorescence demonstrated that expression of ANGPTL4 in ruminal epithelial cells was equivalent to or greater than that in liver hepatocytes. These findings indicate that, as in other species studied, liver and adipose tissue are key sources of ANGPTL4 in cattle. However, the protein was also highly abundant in ruminal epithelium, making it possible that commensal microbes may influence ANGPTL4 synthesis and secretion in the ruminant gastrointestinal tract.

The protective effect of erdosteine on vancomycin-induced pancreatic damage in rats

The goal of this study was to investigate whether vancomycin (VCM) has a negative effect on pancreatic tissue and to elucidate the role of erdosteine (ERD), an expectorant and an antioxidant agent, on possible VCM-induced pancreas impairment in rats. A total of 21 male Wistar albino rats were included in this study. All animals were equally divided into three groups as follows: Controls (n = 7), VCM treated group (200 mg/kg twice daily for 7 days intraperitoneally, n = 7) and VCM (200 mg/kg) + ERD treated group (10 mg/kg day orally ERD, n = 7). The first dose of ERD administration was performed 24 h prior to VCM injection and the study was continued for 7 days. At the end of the study, all animals were sacrificed. Blood and pancreas tissue samples were collected. For biochemical analysis, serum amylase, lipase, alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT) activities were measured. For histopathological examination, pancreas tissue samples were investigated under the light microscope. VCM administration has significantly increased the serum amylase, lipase, ALP, and GGT activities, when compared with the controls. VCM + ERD administration significantly decreased the serum lipase, amylase, and GGT activities. There was no statistically significant difference between the VCM + ERD treated group and only VCM treated group by means of serum ALP levels. It has been observed that there was a prominent pancreatic tissue damage in only VCM given group. However, ERD exhibited structural protection against VCM-induced pancreatic damage and this effect was statistically significant. ERD has also obtained a marked reduction in the extent of pancreatic damage. Erdosteine may play an important role in the VCM-induced pancreatic damage and may reduce the pancreatic damage both in biochemical and histopathological aspects.

The Influence of Zinc Supplementation on the Pancreas of Streptozotocin-Diabetic Rats

The aim of this study was to investigate the effects of zinc supplementation on somatostatin and insulin peptide expressions and biochemical parameters. Six- to 6.5-month-old female Swiss albino rats weighing 150-200 g were used. The animals were divided into four groups: group I: control (intact) animals; group II: control animals given zinc sulfate; group III: streptozotocin (STZ)- induced diabetic animals; group IV: STZ-induced diabetic animals given zinc sulfate. Fasting blood glucose and glutathione levels were measured at 0, 1, 30 and 60 days. On day 60, the pancreas tissue and blood samples were taken from the animals. Zinc supplementation caused a decrease in hyperglycemia, as well as weight increase. Zinc sulfate treatment did not affect the number of somatostatin-immunoreactive cells in the pancreas. More insulin-immunoreactive cells were observed in the pancreatic islets of the diabetic+zinc sulfate group than in the diabetic group, although it was not statistically significant. The results show that zinc supplementation may prevent diabetes in experimental animals.

Relationship of traditional and nontraditional cardiovascular risk factors to coronary artery calcium in type 2 diabetes

Background: K-cells function as targets for insulin gene therapy. In a previous study, we constructed EBV-based plasmids expressing rat preproinsulin controlled by glucose-dependent insulinotropic polypeptide promoters. In the present study, we attempted to correct hyperglycemia in vivo using genetically engineered K-cells in a mouse model of type 1 diabetes. Methods: K-cells expressing insulin were transplanted under the kidney capsules of STZ-induced diabetic mice. The blood glucose levels and body weights of the experime ntal animals were measured daily. After four weeks, the mice were injected intra-peritoneally with 2 g/ kg glucose following a 6 hr fast. Blood glucose levels were measured immediately following glucose injections. All animals were sacrificed at the end of the glucose tolerance study, and pancreas and graft-bearing kidney tissue samples were stained with antibodies against insulin, glucagon, and C-peptide. Results: The body weights of K-cell-transplanted diabetic mice increased after transplantation, whereas those of untreated diabetic control mice continued to decline. The blood glucose levels of K-cell-transplanted diabetic mice decreased gradually during the two weeks following transplantation. After intra-peritoneal injection of glucose into K-cell-transplanted diabetic mice, blood glucose levels increased at 30 minutes, and were restored to the normal range between 60 and 90 minutes, while untreated control diabetic mice continued to experience hyperglycemia. Kidney capsules containi ng transplanted K-cells were removed, and sections were stained with anti-insulin antibodies. We detected insulin-positive cells in the kidney capsules of K-cell-transplanted diabetic mice, but not in untreated control mice. Conclusion: We detected glucose-dependent insulin secretion in genetically engineered K-cells in a mouse model of type 1 diabetes. Our results suggest that genetically modified insulin producing K-cells may act as surrogate β-cells to effectively treat type 1 diabetes. (Korean Diabetes J 33:466-474, 2009)

In this issue: Proteomics 18/2008

AbstractIn this issue of Proteomics you will find the following highlighted articles:Flying through fixing formalinThere's no free lunch – whether social or scientific. If you want the long term stability of formalin, you give up the flexibility of fresh frozen tissue. Or do you? Groseclose et al. report here on preparing tissue arrays from formalin‐fixed, paraffin embedded material for MALDI MS imaging. The key is the number of procedures that can now be performed on the embedded section. In situ digestion with proteases, including trypsin, and direct deposition of matrix on the tissue array section gave good quality data that could be used for database searching and for MS/MS sequencing. Mass spectrometer imaging data could distinguish subsets of markers that distinguish tissue of origin and indicate type and stage of carcinomas. For a reliable set of marker proteins, a much larger set of samples (&gt;10×) must be tested.Groseclose, M. R. et al., Proteomics 2008, 8, 3715–3724.MALDI‐TOF MS Imaging: worth waiting forWe've all seen movies where the clues to who committed the nasty deed seem to take forever to appear. Initial peptide imaging by MS seemed to be that way. The objective of this study by Minerva et al. was to optimize the protocol for MS imaging (MSI) of thin section samples by MALDI‐TOF. Using pancreas tissue samples, which have cells with a variety of well studied peptides (e.g. pancreas beta cells, islet cells) they looked at several parameters. Factors considered were peak resolution as a function of matrix type, representative sampling, S/N, effects of rinsing, and retention of spatial distribution. After optimization, scan speed was up (200 shot/sec), and image quality was distinctly improved by such things as omitting pre‐scan washes (peptides were too mobile to wait) and tuning the matrix sprayer. As a test of the new protocol, wt and obese (ob/ob) samples were compared.Minerva, L. et al., Proteomics 2008, 8, 3763–3774.Candida candidates: Multi‐party cell wall congressCandida albicans is an unpleasant co‐resident of our living space. This opportunistic, pathogenic yeast can grow as a blastospore or in a hyphal form with true or pseudohyphae depending on its cell wall. The cell wall is important enough that it gets almost a third of the organismal budget in dry matter. These “funds” are spent on defense, (immuno, scaffolding) and offense (attachment and invasion, logistics, carbohydrates, immunomodulators). Components are classic and unconventional membrane proteins, binding through hydrogen bonds hydrophobic interactions, or covalent links. Castillo et al. give us a thorough description of cell wall components using a combination of 2‐DE, MALDI‐MS and LC‐MS/MS technology and four different fractionation chemistries: HF‐saturated pyridine, hot SDS, tryptic digestion, and cold NaOH.Castillo, L. et al., Proteomics 2008, 8, 3871–3881.

Double Balloon Enteroscopy in Recipients of Simultaneous Enteric-Drainaged Pancreas-Kidney Transplantation - Initial Experience

Simultaneous enteric drainaged pancreas-kidney (SPK) transplantation is accepted treatment modality for patients with type 1 diabetes and end-stage renal insufficiency. The most frequent reason for loosing the pancreatic graft is rejection process. The histological examinations of donor pancreas or duodenum tissue sample are the only methods to diagnose the pancreatic rejection in transplant recipients. Different techniques to obtain pancreas tissue samples were described, however till now duodenal sampling were reported only under special circumstances (as temporal enterostomy).

Tissue Expression of Fasting‐Induced Adipocyte Factor in Cattle

Fasting‐induced adipocyte factor (FIAF; also known as angiopoietin‐like 4) is a plasma protein that stimulates the oxidation of fatty acids and inhibits fat accumulation. The gastrointestinal tract appears to play an important role in regulating plasma FIAF concentration in some situations. Bovine FIAF shares 87% homology with the mouse protein. Our aim was to determine tissue expression of FAIF in the bovine. Rumen, omasum, abomasum, duodenum, jejunum, ileum, colon, pancreas, liver and adipose tissue samples were collected postmortem from 2 steers. FIAF mRNA was quantified by qPCR, and was most abundant in liver (420 arbitrary units; AU) and adipose tissue (320 AU). We detected FIAF mRNA throughout the gastrointestinal tract, with values ranging from 10 AU in the abomasum to 28 AU in the abomasum. By Western blot, FIAF protein was most abundant in liver and adipose tissue. However, FIAF protein was barely detectable in the rumen or duodenum, but was highly abundant in the omasum. Finally, cross‐sections of the rumen wall were used for immunohistochemistry staining, and strong signals for FIAF were detected in the epithelial layer. These findings suggest host/microbe interactions may influence FIAF expression in the ruminant gastrointestinal tract.

Protective effect of vanadyl sulfate on the pancreas of streptozotocin-induced diabetic rats

The aim of this study is to examine from a biochemical and histological perspective, whether vanadium has a protective effect on the pancreas of diabetic rats. Male, 6–6.5 months old, Swiss albino rats were divided into four groups. Group I: control (intact) animals ( n = 13). Group II: control rats given vanadyl sulfate ( n = 5). Group III: streptozotocin-induced diabetic animals ( n = 11). Group IV: streptozotocin-induced diabetic animals given vanadyl sulfate ( n = 11). Vanadyl sulfate was given by gavage technique to rats in a dose of 100 mg/kg daily for 60 days, after experimental animals were made diabetic. On day 60, the pancreas tissue and blood samples were taken from the animals. In the streptozotocin-induced diabetic group, blood glucose levels significantly increased in contrast to the loss of body weight, but vanadyl sulfate in streptozotocin-diabetic rats reduced blood glucose levels and increased both blood glutathione levels and body weight. Tissue sections were immunostained using an insulin antibody. The control group given vanadyl sulfate was no different from the other intact control group considering the insulin immunoreactivity in B cells. In pancreatic islets of the diabetic group, a decrease in the number of immunoreactive B cells was observed in comparison to the control group. On the other hand, pancreatic islets of the diabetic group given vanadyl sulfate showed a higher number of immunoreactive B cells in comparison to the diabetic group. According to the immunohistochemical and biochemical results obtained, it was concluded that vanadyl sulfate can regenerate B cells of endocrine pancreas in experimental diabetes.

Cryo-fracturing and cryo-planing for in-lens cryo-SEM, using a newly designed diamond knife.

High-pressure frozen rat pancreas tissue samples were cryo-fractured and cryo-sectioned with a diamond knife in the microtome of a freeze-etching device. The bulk fracture faces and block faces were investigated in the frozen-hydrated state by use of a cryo-stage in an in-lens SEM. With this combination, relevant biological structures can be investigated with a few nanometers resolution in a near lifelike state, preventing the artifacts of conventional fixation techniques. Compared to TEM replica techniques, the presented method is more versatile since no replica cleaning is necessary. Additional structures can be made visible by controlled sublimation of ice, leading to a better understanding of the three-dimensional organization of organelles, such as the endoplasmic reticulum.

An experimental study on the pharmacokinetics of 5-fluorouracil regional chemotherapy for pancreatic cancer.

Recently a few centers reported promising results of regional intra-arterial chemotherapy for pancreatic cancer. However, the detailed pharmacokinetics and the side effects of anticancer agents remain unclear. Catheters were introduced into the gastroduodenal artery and the splenic artery of dogs. In group I, arterial infusion of 5-fluorouracil (5-FU) was performed over 10 minutes. In group II, 5-FU was infused systemically. In group III, an intra-arterial infusion was repeated weekly three times. Blood samples and liver and pancreas tissue samples were obtained to determine 5-FU levels. In a subset of each group, the pancreas, duodenum, and liver were excised for histological analyses. Immediately after the infusion of 5-FU, the portal level in group I was higher than that in group II. However, the mean systemic level in group I was lower than in group II. The mean tissue concentration in the pancreas in group I was significantly higher than that of group II. Histological examination revealed no microscopic alterations after treatment in all groups, including group III. This fundamental study suggested that intra-arterial chemotherapy of 5-FU for pancreatic cancer allows higher regional drug delivery without adverse effects on normal regions of the pancreas, the duodenum, and the liver.

Up-regulation of p75 neurotrophin receptor (p75NTR) is associated with apoptosis in chronic pancreatitis.

Activation of apoptosis in chronic pancreatitis has been demonstrated. The low-affinity neurotrophin receptor p75 (p75NTR) mediates apoptosis in many cell types in vivo and in vitro. The aim of this study was to examine whether p75NTR is involved in the apoptotic process in chronic pancreatitis. The quantity and localization of the receptor was evaluated using northern blot analysis, in situ hybridization, immunohistochemistry, and western blot analysis. Apoptosis was determined by TUNEL assay. By northern blot analysis, p75NTR mRNA levels were increased 40-fold in chronic pancreatitis compared with normal pancreas (P < 0.01). In situ hybridization revealed weak p75NTR mRNA expression in some ductal cells in the normal pancreas. In contrast in chronic pancreatitis moderate p75NTR expression was present in acinar cells next to fibrosis, ductal cells, and cells of ductular structures as well as in some islet cells. Immunostaining of p75NTR in normal pancreas and chronic pancreatitis tissue samples showed a similar intensity and distribution pattern as found by in situ hybridization. Higher p75NTR protein levels could be confirmed by western blot analysis, which revealed an 8.6-fold increase of p75NTR in chronic pancreatitis. TUNEL staining showed, in chronic pancreatitis samples, positivity in some acinar cells next to fibrosis, some ductal cells, and cells of ductular structures. Also some islet cells were positive by TUNEL staining. The presence of p75NTR immunoreactivity was positively correlated (P < 0.05) with the apoptotic index in the exocrine and endocrine pancreas. In conclusion, p75NTR, the low-affinity receptor of neurotrophins which mediates apoptosis, is up-regulated in CP and is involved in the apoptotic process of the exocrine and endocrine pancreas.

Connective Tissue Growth Factor is Involved in Pancreatic Repair and Tissue Remodeling in Human and Rat Acute Necrotizing Pancreatitis

To analyze the involvement of connective tissue growth factor (CTGF) in the transforming growth factor-beta (TGF-beta) pathway during acute necrotizing pancreatitis (ANP) in humans and rats. Connective tissue growth factor is involved in several fibrotic diseases and has a critical role in fibrogenesis and tissue remodeling after injury. Normal human pancreas tissue samples were obtained through an organ donor program from five individuals without a history of pancreatic disease. Human ANP tissues were obtained from eight persons undergoing surgery for this disease. In rats, ANP was induced by intraductal infusion of taurocholate. The expression of CTGF was studied by Northern blot analysis, in situ hybridization, and immunohistochemistry in both human and rat pancreatic tissue samples. Northern blot analysis revealed enhanced CTGF mRNA expression in human ANP tissue samples compared with normal controls. In addition, a concomitant increase in TGF-beta1 was present. By in situ hybridization, CTGF mRNA was localized in the remaining acinar and ductal cells and in fibroblasts. In regions of intense damage adjacent to areas of necrosis, CTGF mRNA signals were most intense. Inflammatory cells were devoid of any CTGF mRNA signals. By immunohistochemistry, CTGF protein was localized at high levels in the same cell types as CTGF mRNA. In ANP in rats, concomitantly enhanced mRNA levels of CTGF, TGF-beta1, and collagen type 1 were present, with a biphasic peak pattern on days 2 to 3 and day 7 after induction of ANP. These data indicate that CTGF participates in tissue remodeling in ANP. The expression of CTGF predominantly in the remaining acinar and ductal cells indicates that extracellular matrix synthesis after necrosis is at least partly regulated by the remaining pancreatic parenchyma and only to a minor extent by inflammatory cells. Blockage of CTGF, a downstream mediator of TGF-beta in fibrogenesis, might be useful as a target to influence and reduce fibrogenesis in this disorder.

Novel mutations in the polyadenine tract of the transforming growth factor β type II receptor gene are found in a subpopulation of human pancreatic adenocarcinomas

In this study, we determined the incidence of microsatellite instability (MIN) in pancreatic adenocarcinoma and determined whether MIN might target, for mutations, the simple nucleotide repeats of the transforming growth factor beta type II receptor (TGFBR2) gene. Forty-eight surgically resected pancreatic tumor tissue samples and two normal pancreas tissue samples were analyzed in this study. Microsatellite analysis was performed for six loci in 14 of the 48 tumor specimens for which we had matching normal genomic DNA. Only four of the 14 tumors (29%) were MIN-positive as determined by the presence of microsatellite variations in more than one locus. Interestingly, eight of the 14 specimens (57%) showed microsatellite variations or loss of heterozygosity at D18S34, suggesting that this locus may be a critical region of genetic instability in pancreatic tumorigenesis. Of the 48 tumors, only two (4%) showed mutations in the polyA region, one of the MIN-targeted sites of the TGFBR2 gene. DNA sequence analysis of these two specimens showed the presence of a two-base deletion in one tumor specimen and the other tumor specimen showed a base substitution in the polyA tract at codon 128 of the TGFBR2 gene. The fact that these mutations occurred in the polyA tract of some pancreatic tumors suggests that a subpopulation of these tumors may be susceptible to MIN-targeted mutations. The incidence of these mutations are low and similar to that reported for nonhereditary, sporadic colon cancers.

In-vivo Tissue Uptake and Retention of Sn-117m(4+)DTPA in a Human Subject with Metastatic Bone Pain and in Normal Mice

Organ and tissue uptake and retention of Sn-117m(4+)DTPA were studied in a human subject treated for metastatic bone pain, and the results were compared with the biodistribution studies in five normal mice. The explanted organs from a patient who received a therapy dose of 18.6 mCi (688.2 MBq) Sn-117m(4+)DTPA and who died 47 days later were imaged with a γ-camera, and tissue samples were counted and also autoradiographed. Bone, muscle, liver, fat, lungs, kidneys, spleen, heart and pancreas tissue samples were assayed in a well counter for radioactivity. Regions of interest were drawn over bone and major organs to calculate and quantify clearance times using three in vivo Sn-117m(4+)DTPA whole-body scintigrams acquired at 1, 24 and 168 h after injection. Five normal mice injected with the same batch of Sn-117m(4+)DTPA as used for the human subject were sacrificed at 24 h, and tissue samples were collected and assayed for radioactivity for comparison with the human data. For the human subject, whole-body retention at 47 days postinjection was 81% of the injected dose, and the rest (19%) was excreted in urine. Of the whole-body retained activity at 47 days, 82.4% was in bone, 7.8% in the muscle and 1.5% in the liver, and the rest was distributed among other tissues. γ-Ray scintigrams and electron autoradiographs of coronal slices of the thoracolumbar vertebral body showed heterogenous metastatic involvement with normal bone between metastatic lesions. There was nonuniform distribution of radioactivity even within a single vertebral body, indicating normal bone between metastatic lesions. Lesion-to-nonlesion ratios ranged from 3 to 5. However, the osteoid-to-marrow cavity deposition ratio, from the microautoradiographs, was 11:1. The peak uptake in the human bone was seen at 137 h with no biological clearance. Soft tissues showed peak uptake at 1 h and exhibited three compartmental clearance components. Whole-body retention in normal mice was 38.7% of the injected dose at 24 h and the rest was excreted. At 24 h postinjection, bone in mice showed 84.2% of the whole-body retention, muscle 1.7% and liver 1.4%, and the rest was distributed in other soft tissues. Percent distribution of the retained dose among bone, muscle, liver and other soft tissues is very similar between mice and a human subject. To calculate precise radiation absorbed doses from bone pain palliation radionuclides, it is necessary to take into account soft-tissue uptake and retention that may not be readily evident from routine external γ-scintigraphy.

Prostaglandin D2, F2 alpha, E2, and E1 in early phase of experimental acute necrohemorrhagic pancreatitis in rats.

Changes in endogenous pancreas production of prostaglandins D2, F2 alpha, E2, and E1 in early stages of acute necrotizing pancreatitis induced by intraductal administration of 3.5% sodium taurocholate have been determined by radioimmunoassay of chromatographically purified tissue extracts. For this purpose 18 male Wistar rats were randomized in three groups: control, pancreatitis, and pancreatitis plus indomethacin. Pancreas tissue samples were obtained 5 min after pancreatitis induction. In the pancreatitis-induced group, prostaglandins D2, F2 alpha, and E2 show significantly increased tissue levels relative to the controls whereas prostaglandin E1 remains unmodified. These results suggest a role for series 2 prostaglandins in the earlier stages of pancreatitis.

32P-postlabelling analysis of DNA from human tissues

DNA extracted from human lung, bladder, liver, pancreas, cervix and breast tissue samples taken at autopsy (37 sample sets) was analysed by the nuclease P1 enhancement modification of the 32P-postlabelling assay for levels of aromatic carcinogen DNA adducts. Results were combined with those from a previous study for statistical analysis of 56 sample set (32 male + 24 female). A strong trend was seen for increased adduct levels in the lung DNA of smokers and a weak association for the bladder DNA of smokers compared to non-smokers. Aromatic adducts were also detected in other tissues.