Cell-mediated immunity in patients with cystic fibrosis.

Leucocytes from 26 patients with cystic fibrosis (CF) and 18 healthy controls were investigated by migration inhibition induced by a variety of antigens. In patients with CF cell-mediated immunity was found to human lung and pancreatic tissue extracts as well as to Aspergillus fumigatus, Pseudomonas aeruginosa, and food antigens but not to brain, heart, or kidney. Those patients with the severest form of the disease had the greatest impairment of cell-mediated immunity, but this impairment could be reversed by steroid treatment. Cell-mediated cytotoxicity may also be concerned in the pathogenesis of CF.     

吗啡成瘾大鼠胰腺外分泌的变化

本实验采用急性吗啡成瘾法,观察了吗啡成瘾大鼠在整体胰导管引流和离体胰片灌流条件下,胰腺对ＣＣＫ８刺激的反应。结果如下：（１）吗啡成瘾大鼠对ＣＣＫ８诱导的淀粉酶分泌反应降低;（２）吗啡成瘾大鼠胰腺组织中淀粉酶含量降低。提示吗啡成瘾大鼠胰淀粉酶合成受到抑制。     

In vitro and in vivo protective effect of Ganoderma lucidum polysaccharides on alloxan-induced pancreatic islets damage

This study was undertaken to investigate the protective effect against alloxan-induced pancreatic islets damage by Ganoderma lucidum Polysaccharides (Gl-PS) isolated from the fruiting body of Ganoderma lucidum (Leyss. ex Fr.) Karst. In vitro, alloxan caused dose-dependent toxicity on the isolated pancreatic islets. Pre-treatment of islets with Gl-PS for 12 h and 24 h significantly reversed alloxan-induced islets viability loss. Gl-PS was also found to inhibit the free radicals production induced by alloxan in the isolated pancreatic islets using confocal microscopy. Gl-PS dose-dependently increased serum insulin and reduced serum glucose levels when pretreated intragastrically for 10 days in alloxan-induced diabetic mice. It was found that the pancreas homogenates had higher lipid peroxidation products in alloxan-treated mice than in the Gl-PS-treated animals. Aldehyde fuchsin staining revealed that alloxan caused nearly all the beta cells disappearing from the pancreatic islets, while Gl-PS partly protected the beta cells from necrosis. Alloxan (60 mg/kg) induced NF-kappa B activation in the pancreas at 30 min after injection, pretreatment with Gl-PS inhibited alloxan-induced activation of NF-kappa B. These results suggest that Gl-PS was useful in protecting against alloxan-induced pancreatic islets damage in vitro and in vivo; one of the mechanisms is through its scavenging ability to protect the pancreatic islets from free radicals-damage induced by alloxan.     

Non-essential amino acids play an important role in adaptation of the rat exocrine pancreas to high nitrogen feeding

We have previously demonstrated that feeding a diet with a high amino acid (60% AA diet) content, as a mixture simulating casein, induced pancreatic growth and pancreatic protease production in rats. In the present study, we examined the effects of an increasing dietary content of essential amino acids (EAA, x1 - x3 in exp. 1 and x1 - x3.3 in exp. 2) and non-essential amino acids (NEAA, x1 - x3 in exp. 1 and x1 - x5.2 in exp. 2) on pancreatic growth, amylase and protease adaptation using casein-type amino acid mixtures (exp. 1, basal diet; 20% AA diet) and egg white-type amino acid mixtures (exp. 2, basal diet; 12% AA diet). Pancreatic growth and trypsin activity were induced as the dietary content of NEAA was increased in experiments 1 and 2. Amylase activity in the pancreas was also induced as the dietary content of NEAA was increased, even with the decrease in dietary carbohydrate in experiment 2. The values of all pancreatic variables decreased with the increase in dietary EAA (x2 and x3) without an increase in NEAA. The changes in the pancreas were coincident with increases in plasma arginine and lysine concentrations and a decrease in the plasma alanine concentration. In rats fed a 60% AA diet (EAA and NEAA x3), in the case of which the EAA content was balanced with the NEAA content, pancreatic growth and protease production increased and reached maximum levels as the plasma amino acid concentrations decreased, except for alanine. These results show that NEAA, not EAA, are associated with induction of pancreatic growth and protease production upon feeding a diet with a high AA content, and that some metabolites may be involved in the induction process. The suppression of pancreatic growth and protease production in rats fed the high EAA diets without balanced NEAA may be associated with impairment of amino acid metabolism rather than the increments in the concentration of one or more essential amino acids. Our results also suggest that there is an unknown mechanism or unknown factors involved in regulating pancreatic amylase.     

NADPH oxidase 2 plays a critical role in dysfunction and apoptosis of pancreatic β-cells induced by very low-density lipoprotein

In type 2 diabetes, pancreatic β-cells cannot secret enough insulin compensate for insulin resistance, which are often accompanied by abnormality in lipid metabolism such as hypertriglyceridemia. It is reported that oxidative stress is involved in pancreatic β-cell dysfunction. However, molecular mechanisms linking between excessive generations of reactive oxygen species (ROS) and β-cell dysfunction and apoptosis induced by high levels of very low-density lipoprotein (VLDL) are poorly understood. In this study, we test the hypothesis that NADPH oxidase 2 (NOX2)-derived ROS may play a key role in dysfunction and apoptosis of pancreatic β-cell induced by VLDL. Our results show that the ApoCIII transgenic mice displayed increased serum TG levels, enhanced generation of ROS and impaired insulin content in pancreatic β-cells. In vitro, the treatment of pancreatic NIT-1 cells with 1?mg/ml VLDL for 12?h stimulated NOX2-derived ROS generation, decreased expression and secretion of insulin. Furthermore, we found that VLDL induced dysfunction and apoptosis of pancreatic β-cells through JNK and p53 pathways, which were rescued by siRNA-mediated NOX2 reduction. In conclusion, our data demonstrate a critical role of NOX2-derived ROS in dysfunction and apoptosis through JNK and p53 pathways in pancreatic β-cells induced by VLDL.     

Catharanthine: A novel stimulator of pancreatic enzyme release

Summary The plant alkaloid, catharanthine, was shown to stimulate release of amylase from pancreatic fragments and to cause extensive degranulation of pancreatic acinar cells with accumulation of membrane material in the Golgi region. The extent and time course of maximal catharanthine stimulation was comparable to that induced by the cholinergic analog bethanechol. Antimycin inhibited the action of catharanthine while atropine did not. Removal of Ca²⁺ from the incubation medium inhibited amylase release induced by catharanthine but did not affect release induced by bethanechol. Catharanthine induced a delayed release of ⁴⁵Ca²⁺ from prelabeled pancreatic fragments as compared to bethanechol. It is suggested therefore that catharanthine activates the physiological pathway controlling amylase release by causing a rise in cytoplasmic Ca²⁺ but the mechanism by which this occurs is different from that caused by physiological secretagogues.Supported by a grant from the NIH (GM-19998)I am indebted for technical assistance to E. Roach and S. Bennett     

Coxsackie B4 virus induces short-term changes in the metabolic functions of mouse pancreatic islets in vitro

Mouse pancreatic islets cultured in vitro were infected with a tissue culture-adapted or a mouse pancreas-adapted strain of Coxsackie B4 (CB4) virus. The effects of the viruses on the islets were assessed by examination of their biochemical functions. It was found that the mouse pancreas-adapted strain of CB4 induced a 'leakage' of insulin from islets incubated at a basal (2 mmol l-1) glucose concentration, both at two and four days following infection. However, at a stimulatory concentration of glucose (20 mmol l-1) the rate of insulin secretion appeared to be normal in these islets. At two days the rate of total protein synthesis in islets infected with mouse pancreas-adapted CB4, incubated at high glucose concentration, was reduced; at four days the degree of inhibition was more severe, the rate at basal glucose concentration falling to half that of the control islets and at the stimulatory glucose concentration to a quarter of the control islets. (Pro)insulin biosynthesis was also inhibited, the rate being reduced to less than half the mean control value in islets infected with mouse pancreas-adapted CB4 virus at 20 mmol l-1 glucose at two days; at four days the rate was greatly reduced at both 2 and 20 mmol l-1 glucose. It is concluded from this study that only certain strains of CB4 virus can infect mouse pancreatic islets in vitro and that infection with strains of virus tropic for the islets leads to an impairment of metabolic functions of the B-cells, and is not necessarily lytic.     

N-acetylcysteine prevents intra-acinar oxygen free radical production in pancreatic duct obstruction-induced acute pancreatitis

Although oxygen free radicals (OFR) are considered to be one of the pathophysiological mechanisms involved in acute pancreatitis (AP), the contribution of acinar cells to their production is not well established. The aim of the present study was to determine the effect of N-acetylcysteine (NAC) in the course of AP induced by pancreatic duct obstruction (PDO) in rats, directly analysing by flow cytometry the quantity of OFR generated in acinar cells. NAC (50 mg/kg) was administered 1 h before and 1 h after PDO. Measurements by flow cytometry of OFR generated in acinar cells were taken at different PDO times over 24 h, using dihydrorhodamine-123 as fluorescent dye. Histological studies of pancreas and measurements of neutrophil infiltration in the pancreas, pancreatic glutathione (GSH), malondialdehyde (MDA) levels, plasma amylase activity and hemoconcentration were carried out in order to assess the severity of AP at different stages. NAC effectively blunted GSH depletion at early AP stages and prevented OFR generation found in acinar cells as a consequence of AP induced by PDO. This attenuation of the redox state impairment reduced cellular oxidative damage, as reflected by less severe pancreatic lesions, normal pancreatic MDA levels, as well as diminished neutrophil infiltration in pancreas. Hyperamylasemia and hemoconcentration following AP induction were ameliorated by NAC administration at early stages, when oxidative stress seems to be critical in the development of pancreatitis. In conclusion, NAC reinforces the antioxidant defences in acinar cells, preventing OFR generation therefore attenuating oxidative damage and subsequently reducing the severity of PDO-induced AP at early stages of the disease.     

Reversal of alpha-difluoromethylornithine inhibition of caerulein-induced pancreatic growth by putrescine

The role of ornithine decarboxylase and of polyamines was investigated on caerulein-induced pancreatic growth through the use of alpha-difluoromethylornithine (DFMO) and putrescine. Caerulein, the cholecystokinin analog, given at a dose of 1 microgram . kg-1 three times a day was associated with pancreatic hyperplasia and hypertrophy after 2 and 4 days of treatment. The present study shows that putrescine, given once daily i.p. at a dose of 300 mumol . kg-1, can reverse the previously observed DFMO inhibition on pancreatic DNA content increments stimulated by caerulein. It was also observed that putrescine inhibits severely the 2-day caerulein-induced pancreatic hypertrophy, yet interferes only moderately with 4 days of caerulein treatment. These data lend further support to the involvement of ornithine decarboxylase and polyamines in induced pancreatic growth.     

Streptozotocin-induced suppression of FAD-linked glycerophosphate dehydrogenase in pancreatic islets of adult rats.

Injection of streptozotocin (30-40 mg/kg body weight) to adult rats caused within 4-6 days a sizeable decrease in the activity of FAD-linked glycerophosphate dehydrogenase in pancreatic islets, with little change in either glutamate dehydrogenase or 2-oxoglutarate dehydrogenase activity. The severity of the enzymatic defect was related to that of the diabetic state, although a decreased enzymic activity was also observed in islets from virtually normoglycemic animals examined 2-3 weeks after streptozotocin injection. The administration of nicotinamide prior to that of streptozotocin prevented the change in enzymic activity. It is proposed that the enzymatic defect, rather than being attributable to a genomic effect of streptozotocin, may reflect the preferential impairment of a subpopulation of pancreatic B-cells.     

Involvement of mitochondrial dysfunction in human islet amyloid polypeptide-induced apoptosis in INS-1E pancreatic beta cells: An effect attenuated by phycocyanin

Misfolded human islet amyloid polypeptide (hIAPP) in pancreatic islets is associated with the loss of insulin-secreting beta cells in type 2 diabetes. Insulin secretion impairment and cell apoptosis can be due to mitochondrial dysfunction in pancreatic beta cells. Currently, there is little information about the effect of hIAPP on mitochondrial function. In this study, we used INS-1E rat insulinoma beta cells as a model to investigate the role of mitochondria in hIAPP-induced apoptosis and the protective effects of phycocyanin (PC). We demonstrated that hIAPP induced apoptosis in INS-1E cells was associated with the disruption of mitochondrial function, as evidenced by ATP depletion, mitochondrial mass reduction, mitochondrial fragmentation and loss of mitochondrial membrane potential (ΔΨ(m)). Further molecular analysis showed that hIAPP induced changes in the expression of Bcl-2 family members, release of cytochrome c and apoptosis-inducing factor (AIF) from mitochondria into cytosol, activation of caspases and cleavage of poly (ADP-ribose) polymerase. Interestingly, the hIAPP-induced mitochondrial dysfunction in INS-1E cells was effectively restored by co-treatment of PC. Moreover, there was crosstalk between the extrinsic and intrinsic apoptotic pathways as demonstrated by cleavage of Bid by caspase-8 in the apoptotic process triggered by hIAPP. Taken together, we demonstrated for the first time the involvement of mitochondrial dysfunction in hIAPP-induced INS-1E cell apoptosis. Attenuation of mitochondrial dysfunction provides a mechanism for the protective effects of PC.     

In vitro and in vivo studies of RNAse of Bacillus intermedius

Cytotoxicity of RNAase from Bacillus intermedius was studied in vitro and in vivo. It was shown that the enzyme had slightly pronounced cytotoxicity according to the tests with inhibition of cell proliferation and biosynthesis of cell nucleic acids. The RNAase was also shown to impair the vital staining by neutral red. The efficiency of the impairment much more depended on the enzyme catalytic activity than on the proliferation and biosynthesis of nucleic acids. In vivo toxicity of RNAase from B. intermedius was 3-5 times higher than that of pancreatic RNAase. Possible mechanisms of the different toxicity of the enzymes are discussed.     

Elevation of plasma CCK concentration after intestinal administration of a pancreatic enzyme secretion-stimulating peptide purified from rat bile-pancreatic juice: analysis with N-terminal region specific radioimmunoassay

The rat plasma cholecystokinin (CCK) concentration was measured after intestinal administration of a peptide purified from rat bile-pancreatic juice, which has a stimulatory effect on pancreatic enzyme secretion. The plasma CCK concentration was measured by means of a radioimmunoassay using CCK-8 N-terminal specific antibody, OAL-656. In experimental rats with protease-free intestines, intraduodenal infusion of 10 micrograms of the purified peptide, which stimulates pancreatic enzyme secretion 2.0-2.5 fold, induced a significant increase in the plasma CCK level. Furthermore, after removal of CCK from the plasma by immunoabsorption with an OAL-656-bound Sepharose 4B column, the stimulatory effect of the plasma on pancreatic enzyme secretion was abolished when it was injected intravenously into recipient rats. It was concluded that this peptide stimulates the release of CCK in the intestine and that this is responsible at least in part for the pancreatic enzyme secretion-stimulating activity of the peptide.     

无Ca^2＋外淋巴灌流对短时噪声听损伤的作用

已有报道Ｃａ２＋在噪声所致耳蜗病理损伤中起重要作用。本实验比较采用不含Ｃａ２＋人工外淋巴鼓阶灌流和对照灌流耳蜗在接受到１１５ｄＢＳＰＬ白噪声暴露１ｈ后耳蜗各种生物电反应的差异,以探索降低外淋巴Ｃａ２＋浓度是否能减轻噪声所致的听觉损伤。结果提示,用无Ｃａ２＋外淋巴降低鼓阶Ｃａ２＋浓度可部分压抑耳蜗声电响应,但强噪声所致的耳蜗功能损伤将减轻。本文在多种电生理指标的综合分析中对此种低Ｃａ２＋保护的机制做了初步探讨。     

Effects of some gastrointestinal peptides on pancreatic growth in rats (preliminary results)

The purpose of this study was to estimate the effects of cholecystokinin (CCK), somatostatin (SS) pancreatic polypeptide (PP) and their interaction with each other, given them in single doses, on pancreatic secretion and pancreatic growth after long-term treatment in rats. The acute secretory effects of the above mentioned peptides were studied on conscious rats supplied with pancreatic, gastric and jugular vein cannulae. The pancreatic growth was characterized by measurements of pancreatic weight, desoxyribonucleic acid (DNA), protein, trypsin and amylase content after 5 days treatment. Amylase output was increased by caerulein alone, and given it in combination with somatostatin (SS), while its value decreased by SS alone. After 5 days treatment, the pancreatic weight, trypsin and amylase activity (hypertrophy) was increased by caerulein, and these values were not altered by S alone. In combinative administration of caerulein with somatostatin, the stimulatory effect by caerulein was decreased. PP given alone or in combination with caerulein decreased both the basal and stimulated amylase output. PP given for 5 days decreased pancreatic trypsin and amylase contents and counteracted the stimulatory effect by caerulein to these enzymes' contents. It has been concluded that: 1. caerulein stimulates both pancreatic enzyme secretion and pancreatic growth; 2. somatostatin inhibits the pancreatic secretion and caerulein induced pancreatic growth, but it does not affect the spontaneous growth of pancreas; 3. pancreatic polypeptide inhibits the pancreatic secretion and decreases pancreatic trypsin and amylase contents.     

Biochemical reactions involved in pancreatic enzyme secretion. 4. Effects of cytochalasin B on functions of the exocrine pancreas.

These experiments were performed to evaluate the effects of cytochalasin B on pancreatic enzyme secretion and thus perhaps establish a role for microfilaments in the exocytosis process. The alkaloid at a concentration of 1 microgram/ml (2micron) inhibits amylase secretion induced by urecholine or cholecystokinin-pancreozymin (CCK-PZ) but does not modify that induced by dibutyryl cyclic AMP. The inhibitory effect of the drug is reversible after a 30-min washing out period. It does not affect O2 consumption, basal calcium efflux, or efflux caused by CCK-PZ. Amino acid accumulation in the tissue and their incorporation into proteins are not modified. It is suggested that cytochalasin B inhibits pancreatic enzyme secretion, probably through an effect on the microfilament system.     

Antihyperglycemic, antistress and nootropic activity of roots of Rubia cordifolia Linn

Effect of alcoholic extract of roots of Rubia cordifolia was studied on elevated blood glucose level in alloxan treated animals. The extract reduced the blood sugar level raised by alloxan. Effect of alcoholic extract was also investigated on cold restraint induced stress and on scopolamine-induced memory impairment. Alcoholic extract enhanced brain gamma-amino-n-butyric acid (GABA) levels and decreased brain dopamine and plasma corticosterone levels. Acidity and ulcers caused due to cold restraint stress were inhibited by alcoholic extract. Animals treated with alcoholic extract spent more time in open arm in elevated plus maze model. It also antagonized scopolamine induced learning and memory impairment. Baclofen induced catatonia was potentiated by alcoholic extract.     

Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs

Incubation of guinea pig pancreatic lobules in Ca++-free Krebs-Ringer bicarbonate solution (KRB) containing 0.5 mM ethylene glycol-bis(beta- aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) results in the progressive fragmentation of the occluding zonulae (ZO) with formation of multiple discrete junctions (fasciae occludentes) localized in the lateral and lumenal plasmalemma. After 1--2 h of such incubation, most ZO appear completely disassembled. This results in the disappearance of the heterogeneity in density of intramembrane particles on the P- fracture faces of the basolateral and lumenal plasmalemma. If Ca++ ions are reintroduced into the incubation fluid at this point, continous zonulae reform around the apices of the cells; in contrast, the density of intramembrane particles (imp) at the lumenal plasmalemma remains the same as in the basolateral region, at least for 3 h after Ca++ reintroduction. When added to the incubation fluid, cycloheximide (at a dose known to inhibit protein synthesis greater than 95%) and cytochalasin B (at doses which disrupt microfilaments and modify the cell shape) had no effect on the organization of ZO, on their disassembly in Ca++-free, EGTA medium, or on their Ca++-dependent reformation. Likewise, the organization and disassembly of ZO were unaffected by colchicine; however, after treatment with the latter drug the reassembly was defective, with formation of strand networks on the lateral surface and incomplete segregation of the lumenal region. Antimycin A, on the other hand, when added to the Ca++-EGTA medium, induced a large proliferation of long, infrequently anastomosed junctional strands, usually arranged to form ribbons, festoons, and other bizarre arrays. The possible relationship of these in vitro findings to the in vivo biogenesis and turnover of occluding junctions is discussed. It is suggested that the impairment of reassembly of zonulae by colchicine might be correlated with the disorder induced by the drug on the general organization of pancreatic exocrine cells. Moreover, antimycin A could act by promoting the aggregation of a pool of free junctional strand components (or precursors) that might exist normally in pancreatic exocrine cells.     

SREBP‐1c,Pdx‐1, and GLP‐1R involved in palmitate–EPA regulated glucose‐stimulated insulin secretion in INS‐1 cells

Impairment of glucose‐stimulated insulin secretion (GSIS) caused by glucolipotoxicity is an essential feature in type 2 diabetes mellitus (T2DM). Palmitate and eicosapentaenoate (EPA), because of their lipotoxicity and protection effect, were found to impair or restore the GSIS in beta cells. Furthermore, palmitate was found to up‐regulate the expression level of sterol regulatory element‐binding protein (SREBP)‐1c and down‐regulate the levels of pancreatic and duodenal homeobox (Pdx)‐1 and glucagon‐like peptide (GLP)‐1 receptor (GLP‐1R) in INS‐1 cells. To investigate the underlying mechanism, the lentiviral system was used to knock‐down or over‐express SREBP‐1c and Pdx‐1, respectively. It was found that palmitate failed to suppress the expression of Pdx‐1 and GLP‐1R in SREBP‐1c‐deficient INS‐1 cells. Moreover, down‐regulation of Pdx‐1 could cause the low expression of GLP‐1R with/without palmitate treatment. Additionally, either SREBP‐1c down‐regulation or Pdx‐1 over‐expression could partially alleviate palmitate‐induced GSIS impairment. These results suggested that sequent SREBP‐1c‐Pdx‐1‐GLP‐1R signal pathway was involved in the palmitate‐caused GSIS impairment in beta cells. J. Cell. Biochem. 111: 634–642, 2010. © 2010 Wiley‐Liss, Inc.