Isolation of cytomegalovirus-specific cytotoxic T-lymphocytes from gut-associated lymphoid tissue (GALT) of HIV type 1-infected subjects.

Cytomegalovirus (CMV) can be an important opportunistic infection in HIV-1-infected patients, particularly when the CD4+ T-cell count drops below 50 lymphocytes/mm3. CMV-associated disease, including retinitis, pneumonitis, gastroenteritis, and encephalitis, is estimated to affect up to 40% of AIDS patients. We have studied the cellular immune response to CMV in gut-associated lymphoid tissue (GALT) of HIV-1-infected patients. Two patients with chronic diarrhea of unknown etiology were examined by flexible sigmoidoscopy and upper endoscopy. Biopsy specimens were obtained from lymphoid-associated tissue sites in rectum and duodenum. Both patients were seropositive for CMV IgG, but had not been treated with ganciclovir, and neither had clinical signs of CMV disease. Mononuclear cell cultures were established from GALT and blood and assayed for the presence of CMV-specific CD8+ T cells. CD8+ T-cell phenotype and function were assessed by MHC Class I tetramer staining, using an HLA-A*0201 tetramer complex specific for peptide 495-503 (NLVPMVATV) of CMV lower matrix protein pp65, and by a standard 51Cr release assay. CMV pp65-specific cytotoxic lymphocytes (CTL) were detected in GALT and blood MNC from both patients. These results demonstrate that HIV-1-infected subjects seropositive for CMV, but without active CMV gastrointestinal disease, harbor CMV-specific CTL in intestinal lymphoid tissue. This is the first report of isolation of CMV-specific CTL in GALT and will lead to greater understanding of the pathogenesis of CMV disease in human mucosal tissue.

Characterization of HIV-1-specific cytotoxic T lymphocytes expressing the mucosal lymphocyte integrin CD103 in rectal and duodenal lymphoid tissue of HIV-1-infected subjects.

Acute HIV-1 infection depletes CD4(+) T cells in gut-associated lymphoid tissue (GALT). The failure of containment of local viral replication, and consequent CD4(+) T cell depletion, might be due to delayed mobilization of effector CD8(+) T cells or absence of functioning HIV-1-specific CD8(+) T cell effectors within GALT. No studies have addressed human intestinal HIV-1-specific CD8(+) T cell functions. We sought to determine whether functional HIV-1-specific CTL were present in GALT and whether the repertoire differed from HIV-1-specific CTL isolated from peripheral blood mononuclear cells. From three HIV-1-infected subjects, we isolated HIV-1-specific CD8(+) T cells expressing the mucosal lymphocyte integrin CD103 from GALT. These antigen-specific effector cells could be expanded in vitro and lysed target cells in an MHC class I-restricted manner. HIV-1-specific CTL could be isolated from both duodenal and rectal GALT sites, indicating that CD8(+) effectors were widespread through GALT tissue. The breadth and antigenic specificities of GALT CTL appeared to differ from those in peripheral blood in some cases. In summary, we found HIV-1-specific CD8(+) effector T cells in GALT, despite HIV-1-induced CD4(+) T cell lymphopenia. This suggests that HIV-1-specific CTL in gut tissue can be maintained with limited CD4(+) T cell help.

Role of cholecystokinin in the development and progression of acute pancreatitis and the potential of therapeutic application of cholecystokinin receptor antagonists.

This presentation reviews the role of cholecystokinin (CCK) as a contributory factor for the development and progression of acute pancreatitis (AP) and the perspective of CCK receptor antagonists for treatment of AP. High, supraphysiological concentrations of CCK induce AP in various species including man. There is also evidence that physiological increases in plasma CCK deteriorates AP in several animal models. The latter findings support the hypothesis that CCK plays a contributory or permissive role for the development of AP. The majorities of experimental studies show that the prophylactic and therapeutic use of CCK antagonists ameliorates AP. The latter effects were clearly shown for models of biliary AP in which plasma CCK is increased due to a feedback mechanism. However, CCK antagonists also had beneficial effects in models in which plasma CCK is not increased. In animal strains which do not have a CCK-A-receptor due to a genetic abnormality AP induced by a certain noxious factor does not develop to the same severity when compared to animals with a normal CCK-A-receptor. Thus, CCK acts as a permissive or contributory factor for the development and progression of AP. There is also evidence that CCK antagonists have a potential therapeutic benefit. Clinical studies will evaluate their therapeutic potential for patients with AP.

Codistribution of TAP and the granule membrane protein GRAMP-92 in rat caerulein-induced pancreatitis.

The pathological activation of zymogens within the pancreatic acinar cell plays a role in acute pancreatitis. To identify the processing site where activation occurs, antibodies to the trypsinogen activation peptide (TAP) were used in immunofluorescence studies using frozen sections from rat pancreas. Saline controls or animals receiving caerulein in amounts producing physiological levels of pancreatic stimulation demonstrated little or no TAP immunoreactivity. However, after caerulein hyperstimulation (5 micrograms. kg-1. h-1) for 30 min and the induction of pancreatitis, TAP immunoreactivity appeared in a vesicular, supranuclear compartment that demonstrated no overlap with zymogen granules. The number of vesicles and their size increased with time. After 60 min of hyperstimulation with caerulein, most of the TAP reactivity was localized within vacuoles >/=1 micrometer that demonstrated immunoreactivity for the granule membrane protein GRAMP-92, a marker for lysosomes and recycling endosomes. Pretreatment with the protease inhibitor FUT-175 blocked the appearance of TAP after hyperstimulation. These studies provide evidence that caerulein hyperstimulation stimulates trypsinogen processing to trypsin in distinct acinar cell compartments in a time-dependent manner.

Premature trypsinogen activation during cerulein pancreatitis in rats occurs inside pancreatic acinar cells.

Although it is widely accepted that trypsinogen activation is an initiating event in the development of acute pancreatitis, its location inside the pancreas is not known. In our studies, acute edematous pancreatitis was induced in rats by one or two intraperitoneal injections of 50 microg cerulein/kg body weight. The pancreas was removed for examination 1 or 2 h after the first and the second cerulein injection, respectively. The cleavage product of trypsinogen activation, trypsinogen activation peptide, was specifically labeled on pancreatic tissue sections by a corresponding antibody, the signal enhanced by a biotin-avidin conjugate, and the site then visualized by coupled peroxidase activity on diaminobenzidine. The sections were examined by light microscopy. Trypsinogen activation peptide, reflecting activation of the pancreatic digestive enzyme trypsinogen, was detected inside pancreatic acinar cells in this animal model of acute pancreatitis. As early as 1 h after the first injection of cerulein, protease activation was seen within the apical pole of acinar cells. Protease activation was increased 2 h after the latter of two injections of cerulein and more evenly distributed within the cells. For the first time morphologic evidence confirms that the activation originates within the acinar cell, rather than from the interstitium or the duct lumen. The location of this activation at the apical site of the acinar cell indicates its origin from subcellular compartments involving the late steps in the secretory pathway.

Trypsinogen activation and glutathione content are linked to pancreatic injury in models of biliary acute pancreatitis.

CONCLUSION: In models of biliary acute pancreatitis, which might resemble the situation in humans, premature activation of trypsinogen inside the pancreas ("autodigestion") occurs and is correlated with the extent of ductal and parenchymal injury. It is accompanied by a critical spending of protease inhibitors and glutathione, compromising important acinar cell defense and maintenance mechanisms. BACKGROUND: Premature activation of pancreatic digestive enzymes and profound changes of levels of certain biochemical compounds have been implicated in the pathophysiology of acute pancreatitis. Hitherto, little information on their role in biliary acute pancreatitis has been available. METHODS: Three types of injury to the pancreaticobiliary duct system of various severity were induced in rats--ligation of the common bile-pancreatic duct, retrograde infusion of electrolyte, or retrograde infusion of taurocholate solution--and were compared to sham-operated animals. Trypsin, trypsin inhibitory capacity (TIC), reduced glutathione (GSH), and other compounds were measured in pancreatic tissue. Histopathology, as well as serum amylase, lipase, and gamma-glutamyl transferase (gamma GT) were assessed. RESULTS: Histopathology and elevated activity of gamma GT in the serum revealed increasing severity of pancreatic injury from sham operation through retrograde duct infusion with taurocholate. GSH was diminished even in macroscopically normal-appearing tissue, but significantly lower in altered (hemorrhagic)-looking sections. Conversely, tissue levels of trypsin were significantly increased. TIC was elevated only in the duct obstruction model, whereas it was reduced in the retrograde duct infusion models.

Beneficial effects of L-2-oxothiazolidine-4-carboxylate on cerulein pancreatitis in mice.

BACKGROUND & AIMS: Disturbances of the thiol metabolism of acinar cells may play a role in the pathophysiology of acute pancreatitis. Cerulein-induced pancreatitis causes depletion of glutathione. The entire pancreatic thiol status was assessed in this model. The potential benefit of augmentation of pancreatic glutathione by L-2-oxothiazolidine-4-carboxylate (OTC) for the course of pancreatitis was determined. METHODS: Mice were treated with cerulein (50 microg/kg) and with or without administration of OTC (6.5 and 20 mmol/kg, respectively). Pancreatic tissue was analyzed for reduced and oxidized glutathione, nonprotein thiol, mixed disulfide, protein thiol, and protein disulfide. Histopathology and serum amylase were also assessed. RESULTS: Levels of all thiol compounds were altered profoundly at a different rate during pancreatitis. OTC caused an increase of 60% in pancreatic glutathione. Its administration at 20 mmol/kg attenuated the decrease of pancreatic glutathione and protein thiol until 8 hours and blunted the cerulein-induced increase in amylase activity and histopathologic damage. At 6.5 mmol/kg, OTC failed to show effects on all parameters. CONCLUSIONS: OTC administered in a prophylactic protocol dose-dependently exerted beneficial effects in cerulein-induced pancreatitis in mice despite only transient influence on pancreatic thiol compounds. Thiols (e.g., reduced glutathione) and their corresponding disulfides are critically involved in the pathophysiology of cerulein-induced pancreatitis.

Energy metabolism in mouse pancreas in response to different dosages of a CCK analogue.

Stimulation of the exocrine pancreas with cholecystokinin analogues leads to a variety of intraacinar processes, many coupled to energy consumption. It was hypothesized that extensive ATP depletion could play a role in the pathophysiology of acute pancreatitis, especially in the hyperstimulation (cerulein) model. Mice received seven intraperitoneal injections of cerulein at hourly intervals, at doses ranging from physiological (0.1 micrograms/kg) to pharmacological (50 micrograms/kg). A single dose of cerulein induced a 28-33% decrease in ATP, whereas a complete course of injections led to a nadir as low as 45% of the control value. The overall pattern of ATP tissue content during the observed time course was surprisingly similar in all four groups and statistically not different at any time point. Until 12 h, ATP levels in all groups remained below the control value. In contrast, serum amylase and light microscopy reflected a degree of pancreatitis in a close dose-response pattern to the administered cerulein dose. These findings suggest that ATP depletion--although probably facilitating acinar damage--does not seem to play a causal or primary role in the pathophysiology of acute pancreatitis.

Influence of ductal pressure and infusates on activity and subcellular distribution of lysosomal enzymes in the rat pancreas.

BACKGROUND & AIMS: Subcellular redistribution of lysosomal enzymes into the zymogen-enriched fraction (cosedimentation) in pancreatic homogenates occurs after different pancreatic injuries and has been proposed to be the trigger event for acute pancreatitis. This phenomenon is now studied in models of biliary pancreatitis. METHODS: The bile-pancreatic duct in rats was either obstructed or retrogradely infused at different degrees of pressure and with solutions of various injurious potential. Controls were untreated or sham operated. Six hours later, the pancreas was analyzed for the total activity of cathepsin B and beta-galactosidase and their distribution among subcellular fractions. RESULTS: In control animals, 17% and 29%, respectively, of these lysosomal enzymes were found in the zymogen fraction. Redistribution occurred after all duct manipulations, including obstruction. In contrast to sham operation and duct obstruction, all modes of duct infusion resulted in marked increases in the total activity of lysosomal enzymes. CONCLUSIONS: Increased lysosomal activity in models of biliary pancreatitis might contribute to acinar injury or represent a cellular repair mechanism. Cosedimentation at a certain extent is a physiological event. Redistribution reflects a uniform response to a range of perturbations, some of which do not cause pancreatitis. Thus, it seems unlikely that redistribution is the trigger event for acute pancreatitis.

Consensus statement: octreotide dose titration in secretory diarrhea. Diarrhea Management Consensus Development Panel.

Octreotide is an effective therapeutic option in controlling secretory diarrhea of varied etiology. However, marked patient-to-patient differences in the antidiarrheal effects necessitate titration of octreotide dose in individual patients to achieve optimal symptom control. A consensus development panel established guidelines for octreotide dose titration in patients with secretory diarrhea. Overall, the panel recommended an aggressive approach in selecting the initial octreotide dose and in making subsequent dose escalations in patients with secretory diarrhea due to gastrointestinal tumors (eg, carcinoids, VIPomas), AIDS, dumping syndrome, short bowel syndrome, radiotherapy, or chemotherapy. To avoid hypoglycemia in patients with diabetes mellitus-associated secretory diarrhea, the panel recommended a low initial octreotide dose and a conservative titration regimen with close monitoring a blood glucose levels. The end point of therapy should focus on a reduction in diarrhea (frequency of bowel movements or stool volume) rather than normalization of hormonal profile. Overall, octreotide is well tolerated; principal side effects are transient injection site pain and gastrointestinal discomfort. For many patients with secretory diarrhea, octreotide therapy is expected to improve the overall health and quality of life and in the long run will lessen health care costs.

Intrapancreatic zymogen activation and levels of ATP and glutathione during caerulein pancreatitis in rats.

Studies in acutely inflamed pancreatic tissue in humans and animals suggest that premature activation of proteases within the gland plays a key role in its pathophysiology. The present study aimed to detect such protease activation in relation to protease inhibition and to changes in the concentrations of the vital cellular compounds ATP and glutathione in pancreatic tissue during caerulein-induced pancreatitis in rats. Within 1 h after supramaximal stimulation by intraperitoneal caerulein injection, pancreatic tissue activities of enzymatically active trypsin and elastase showed significant increases, accompanied by a twofold increase in trypsin inhibitory capacity. Over the same time course pancreatic ATP and glutathione concentrations dropped to 38% and 47%, respectively, after 1 h and reached a nadir of 22% and 28%, respectively, after 4-8 h. Intrapancreatic trypsin activation in this model, despite increasing trypsin inhibitory capacity, indicates concealed liberation of even more protease or enzyme-inhibitor complex instability. It is hypothesized that early acinar glutathione depletion, in part due to diminished ATP, could play a role in the premature activation of digestive enzymes by impairment of the integrity of the cytoskeleton and cell organelles or lowered defense capabilities against oxidant stress, finally leading to acute pancreatitis.

Endoscopy training in a three-year curriculum.

Endoscopic training has become an increasingly important part of training in gastroenterology in recent years. As plans are developed to require 3 years of training in gastroenterology for board eligibility, the outline of a 3-year curriculum is proposed that would incorporate both "basic" training and "advanced" training (where offered) in endoscopy as integral components of a flexible plan designed to suit the needs and capabilities of both trainees and programs.

Hydrocolonic ultrasonography in the detection of colonic polyps and tumors.

BACKGROUND: Hydrocolonic ultrasonography--abdominal ultrasonography in conjunction with the retrograde instillation of water into the colon--has been advocated as an alternative to colonoscopy for detecting colorectal polyps and cancer. We conducted a prospective, blinded trial to evaluate the procedure further. METHODS: Fifty-two consecutive patients (50 men and 2 women; average age, 62 years) who were referred for colonoscopy underwent hydrocolonic ultrasonography followed by colonoscopy. The physicians performing colonoscopy were blinded to the ultrasound results. Patients who had a history of colonic polyps or tumors or who had previously undergone flexible sigmoidoscopy or colonoscopy were excluded. RESULTS: Twenty-two patients had normal results on colonoscopy, 26 had polyps, 3 had cancer and polyps, and 1 had cancer alone. Twenty patients had polyps less than 7 mm in diameter, eight had polyps 7 mm or more in diameter, and one had a polyp of unknown size. Hydrocolonic ultrasonography did not detect any cancers and detected only one polyp > or = 7 mm and one polyp < 7 mm in diameter. The overall sensitivity of ultrasonography for identifying any polyp was 6.9 percent, and for identifying a polyp > or = 7 mm, it was 12.5 percent. Ultrasonography suggested the presence of five masses and five polyps that were not confirmed by colonoscopy. Six patients had incomplete ultrasound studies because of discomfort or the inability to retain water. There were two complications: one patient had two vasovagal episodes, and another had diaphoresis. CONCLUSIONS: Hydrocolonic ultrasonography was less useful than colonoscopy for detecting colorectal polyps and cancers. The usefulness of the technique in screening for colonic polyps and tumors appears to be limited.

The level of the zymogen granule protein GP2 is elevated in a rat model for acute pancreatitis.

BACKGROUND/AIMS: GP2 is the major membrane protein in pancreatic zymogen granules. It is linked to the membrane via a glycosyl-phosphatidylinositol linkage. After cleavage, a significant fraction of GP2 becomes soluble. The present study assessed whether GP2 is a useful serum marker for acute pancreatitis. METHODS: Using an anti-GP2 monoclonal antibody, an enzyme-linked immunosorbent assay was developed to measure the serum levels of GP2 in rats with cerulein-induced acute pancreatitis. RESULTS: The anti-GP2 antibody was specific because it did not cross-react with uromodulin, a structurally similar protein to GP2, or to protein extracts from nonpancreatic tissues. Eight hours after the induction of pancreatitis, the serum levels of amylase, lipase, and GP2 peaked. Peak GP2 levels were 4.2 times higher than those of controls. At 24 hours, GP2 was still 70% of the peak level, whereas amylase and lipase were 5.5% and 0.5%, respectively, of their peak levels. CONCLUSIONS: GP2 may serve as a potentially valuable marker for clinical acute pancreatitis.

The effect of L-buthionine-[S,R]-sulfoximine on the pancreas in mice. A model of weakening glutathione-based defense mechanisms.

L-Buthionine-[S,R]-Sulfoximine (BSO) decreases glutathione levels in various organs by inhibition of gamma-glutamylcysteine synthetase. We have examined the levels of total glutathione and oxidized glutathione in the pancreas of mice, as well as serum amylase and pancreatic histology, after BSO administration in two different ways. The injection of a single dose of BSO (5 mmol/kg body wt) decreased total glutathione to 10% of the control value. A similar depletion was observed after 24 h of oral administration of a 10 mM BSO solution, without changes in the levels of oxidized glutathione. BSO-induced pancreatic glutathione depletion--even if maintained for up to 14 d--did not cause morphological alterations of the pancreas or hyperamylasemia. Thus pancreatic glutathione depletion in itself does not lead to pancreatitis, although during development of experimental acute pancreatitis, glutathione depletion has been described. BSO might be used in animal models to weaken the glutathione-based acinar defense mechanisms against oxidant stress or to alter other physiologic processes in which glutathione is involved.

Glutathione and ATP levels, subcellular distribution of enzymes, and permeability of duct system in rabbit pancreas following intravenous administration of alcohol and cerulein.

In order to reproduce what might occur during the initial phase in some cases of acute alcohol-induced pancreatitis, rabbits were infused with diluted ethanol and low-dose cerulein. The duct permeability was assessed by recovery of fluoresceinated dextran (molecular weight 19,500) in central venous blood following orthograde duct perfusion with this substance in the anesthetized animal. Serum ethanol, lipase, and amylase were measured; pancreatic duct morphology was examined by light microscopy and electron microscopy. ATP and glutathione were measured, as were amylase, trypsinogen/trypsin, cathepsin B, and DNA levels in differential centrifugates. As expected, acinar amylase and trypsinogen showed a significant decrease in the experimental group; cathepsin B activity was similarly diminished. Compared with the control group, the activity of serum amylase and lipase in the experimental group demonstrated a significant increase. However, no differences between saline-infused control animals and the treated group regarding pancreatic duct permeability, continuity of lumen-lining epithelium, ATP and glutathione levels, and the relative subcellular distribution of pancreatic digestive and lysosomal enzymes were observed. Thus, our findings do not support the relevance of some of the most common hypotheses on the pathophysiology of acute pancreatitis in its early stage for at least a certain subgroup of patients with acute alcohol-induced pancreatitis.

Effects of bile and pancreatic digestive enzymes on permeability of the pancreatic duct system in rabbits.

In order to reproduce what may occur during the initial phase of biliary acute pancreatitis, the rabbit pancreatic duct was perfused with preincubated mixtures of bile and different digestive enzymes at low physiologic pressure. Permeability of the pancreatic duct system, serum amylase, and histological appearance of pancreatic tissue were studied after orthograde duct perfusion in the anesthetized animal. The ductal permeability was estimated by recovery of fluoresceinated dextran (molecular weight 17,200) in central venous blood following duct perfusion with this substance. Perfusion with preincubated bile failed to increase permeability significantly (11.10 +/- 3.04 nmol/L compared to 5.80 +/- 2.71 nmol/L in the control group), whereas mixtures of bile and trypsin (27.19 +/- 5.21 nmol/L), bile and lipase (16.68 +/- 3.75 nmol/L), and bile and pancreatic juice (13.92 +/- 0.48 nmol/L) caused significant increases (p < 0.05). Similar observations were made regarding serum amylase and histology. Thus, the presence of mixtures of bile with pancreatic enzymes (following their prolonged common incubation) in the absence of elevated pressure, results in an increase in duct permeability for molecules up to the size range of pancreatic enzymes and thereby may contribute to the initiation of acute pancreatitis.

Receptor strategies in pancreatitis.

A variety of receptors on pancreatic acinar and duct cells regulate both pancreatic exocrine secretion and intracellular processes. These receptors are potential sites of action for therapeutic agents in the treatment of pancreatitis. Cholecystokinin (CCK) receptor antagonists, which may reduce the level of metabolic "stress" on acinar cells, have been shown to mitigate the severity of acute pancreatitis in a number of models. Not all studies have shown a benefit, however, and differences may exist between different structural classes of antagonists. Because increased pancreatic stimulation due to loss of feedback inhibition of CCK has been proposed to contribute to the pain of some patients with chronic pancreatitis, CCK receptor antagonists could also be of benefit in this setting. Somatostatin and its analogs diminish pancreatic secretion of water and electrolytes and have been effective in treating pancreatic fistulas and pseudocysts. These agents are also being evaluated for their ability to reduce pain in chronic pancreatitis (perhaps by reducing ductal pressure by diminishing secretory volume) and mitigating the severity of acute pancreatitis (possibly by reducing the metabolic load on acinar cells). Recently described secretin receptor antagonists may also have therapeutic value as a means of selectively inhibiting pancreatic secretion of water and electrolytes.