The AIDS disease of CD4C/HIV transgenic mice shows impaired germinal centers and autoantibodies and develops in the absence of IFN-gamma and IL-6.

The mechanisms responsible for degeneration of germinal centers (GC) and follicular dendritic cell (FDC) networks during progression to AIDS remain elusive. Here, we show that CD4(+) T cells from CD4C/HIV-1 Tg mice, which develop a severe AIDS-like disease, express low levels of CD40 ligand. Accordingly, GC formation, FDC networks, and immunoglobulin isotype switching are impaired in these animals. However, Tg B cells respond to in vitro CD40 stimulation. Total serum IgG levels are reduced in Tg mice, whereas total IgM levels are increased with a significant amount showing DNA specificity. IFN-gamma- and IL-6-deficient CD4C/HIV Tg mice also develop the AIDS-like disease and produce auto-Ab. Thus, CD4C/HIV Tg mice have immune dysfunction accompanied by autoimmune responses.

Lipopolysaccharide directly affects pancreatic acinar cells: implications on acute pancreatitis pathophysiology.

We have explored whether lipopolysaccharide (LPS, endotoxin) induces pancreatic injury on pancreatic acinar cells both in vivo and in vitro. Wistar male rats were treated with four intraperitoneal injections of 10 mg/kg LPS, and AR4-2J cells were exposed to increasing doses of LPS. Expression of pancreatitis-associated-protein (PAP) mRNA was strongly induced in AR4-2J cells exposed to LPS, while amylase mRNA was reduced. LPS also induced apoptosis and expression of TNF-alpha, IL-1beta, and IL-8 mRNA in AR4-2J cells. The in vivo effect of LPS showed structural signs of cellular damage, including numerous cytoplasmic vacuoles, severe nuclear alterations, and high expression of PAP mRNA. This study demonstrated that LPS induced pancreatic damage by directly affecting the pancreatic acinar cells. The role of LPS in the pathophysiology of acute pancreatitis may be partly due to the effect LPS has on the acinar cell.

Pancreatic acinar cells submitted to stress activate TNF-alpha gene expression.

To elucidate whether pancreatic acinar cell submitted to stress is able to express TNF-alpha, we studied TNF-alpha mRNA expression by Northern blot and in situ hybridization in healthy pancreas, in tissue from caerulein-induced pancreatitis and after lipopolysaccharide (LPS) treatment. In specimens from normal pancreas, TNF-alpha mRNA expression, as judged by both Northern blot and in situ hybridization, was negative, whereas a strong but transient expression was observed in acinar cells from caerulein pancreatitis and LPS treatment. TNF-alpha mRNA appeared as rapidly as 30 min after treatment, and was maximal 6 h after. At this time, there was mild infiltration consisting mostly of polymorphonuclear leukocytes (PMNL) and no signal of TNF-alpha transcript was found in their cytoplasm. Our results strongly indicate that pancreatic acinar cell is the source of TNF-alpha early in the course of acute pancreatitis and LPS treatment, and suggest that the expression of this cytokine is a part of a general response of the acinar cell to aggression.

Expression and modulation of p42/p44 MAPKs and cell cycle regulatory proteins in rat pancreas regeneration.

Pancreatic growth occurs after CCK, CCK-induced pancreatitis, and pancreatectomy; the mechanisms involved remain unknown. This study evaluates mitogen-activated protein kinase (MAPK) activation and expression of cell cycle regulatory proteins after pancreatectomy to understand the cellular and molecular mechanisms involved in pancreas regeneration. Rats were killed 1-12 days after pancreatectomy, and p42/p44 MAPK activation, expression of the cyclins D and E, cyclin-dependent kinase (Cdk)-2 activity, retinoblastoma protein (pRb) hyperphosphorylation, and expression of the cyclin kinase inhibitors p15, p21, and p27 were examined. Pancreatic remnants exhibited sustained p42/p44 MAPK activation within 8 h. Cyclins D1 and E showed maximal expression after 2 and 6 days, coinciding with maximal hyperphosphorylation of pRb and Cdk2 activity. The expression of p15 vanished after 12 h, p27 disappeared gradually, and p21 increased early. The p27 complexed with Cdk2 dissociated after 2 days, whereas p21 associated in a reverse fashion. In conclusion, sustained activation of p42/p44 MAPKs and Cdk2 along with overexpression of cyclins D1 and E and reduction of p15 and p27 cyclin inhibitors occurred early after pancreatectomy and are active factors involved in signaling that leads to pancreas regeneration.

Requirement of the MAP kinase cascade for cell cycle progression and differentiation of human intestinal cells.

The intracellular signaling pathways responsible for cell cycle arrest and establishment of differentiated cells along the gut axis remain largely unknown. In the present study, we analyzed the regulation of p42/p44 mitogen-activated protein kinase (MAPK) in the process of proliferation and differentiation of human intestinal cells. In vitro studies were done in Caco-2/15 cells, a human colon cancer cell line that spontaneously differentiates into an enterocyte phenotype. In vivo studies were performed on cryostat sections of human fetal intestinal epithelium by indirect immunofluorescence. We found that inhibition of the p42/p44 MAPK signaling by the PD-98059 compound or by ectopic expression of the MAPK phosphatase-1 strongly attenuated E2F-dependent transcriptional activity in Caco-2/15 cells. p42/p44 MAPK activities dramatically decreased as soon as Caco-2/15 cells reached confluence. However, significant levels of activated p42 MAPK were detected in differentiated Caco-2/15 cells. Addition of PD-98059 during differentiation interfered with sustained activation of p42 MAPK and sucrase-isomaltase expression. Although p42/p44 MAPKs were expressed in both the villus tip and crypt cells, their phosphorylated and active forms were detected in the undifferentiated crypt cells. Our results indicate that elevated p42/p44 MAPK activities stimulate cell proliferation of intestinal cells, whereas low sustained levels of MAPK activities correlated with G1 arrest and increased expression of sucrase-isomaltase.

Lipopolysaccharides induce p8 mRNA expression in vivo and in vitro.

Systemic LPS endotoxin is associated with acute pancreatic damage. Whether damage results from direct interaction of LPS with pancreatic cells is unknown. We addressed that question by monitoring p8 expression in reponse to LPS, in vivo and in vitro, because overexpression of the p8 protein is a sensitive marker of pancreatic agression. For in vivo studies, rats were sacrificed at different times after a single intraperitoneal injection of LPS, and pancreas, liver, kidney, lung, brain, and intestine were processed for RNA preparation. In vitro, pancreatic acinar AR4-2J cells were cultivated with 0.1, 1, or 10 micrograms/ml LPS for 6, 12, or 24 h. p8 mRNA expression was monitored by Northern blotting. In vivo, it was strongly increased in the pancreas after 12 h of treatment and remained elevated after 24 h. It was also induced in kidney and liver, with a maximum at 6 and 12 h, respectively, but not in lung, brain, or intestine. In AR4-2J cells, basal p8 mRNA expression was very low and increased in a time- and dose-dependent manner after treatment with LPS. LPS-induced overexpression of p8 mRNA in vivo confirmed the adverse effect of endotoxemia on pancreas and its overexpression in vitro demonstrated a direct interaction of LPS with pancreatic cells.

Clusterin overexpression in rat pancreas during the acute phase of pancreatitis and pancreatic development.

Molecular mechanisms associated with apoptosis in pancreas remain largely unknown. Clusterin mRNA is induced in several tissues in response to most apoptotic stimuli. In these tissues, clusterin has an antiapoptotic activity. The aim of this work was to test whether clusterin, which is not expressed in normal pancreas, was induced in pancreas during pancreatitis and pancreatic development. Clusterin mRNA levels were strongly increased 6 h after pancreatitis induction. Maximal expression happened between 24-48 h and decreased progressively to undetectable levels at day 5. Clusterin mRNA was expressed with similar intensity in oedematous caerulein-induced pancreatitis and in response to various degrees of necrohaemorrhagic taurocholate-induced pancreatitis, indicating a maximal gene activity in all types of pancreatitis; in situ hybridization showed that the acinar cells and some ducts expressed clusterin mRNA. A single band of about 35-38 kDa was detected by western blot in pancreatic homogenates and in pancreatic juice from rats with acute pancreatitis, but not from control rats. Clusterin mRNA expression was strong in late fetal life and remains high until day 11 post-partum, then decreased progressively with a minimum from 35 to 90 days post-partum. Clusterin mRNA levels were strongly induced in pancreatic acinar AR4-2J cells in response to various apoptotic stimuli (i.e., cycloheximide, staurosporine, ceramide and H2O2) but not with interleukin (IL)-1, IL-4 or IL-6 or heat shock, which do not induce apoptosis in AR4-2J cells. In conclusion, we demonstrated that clusterin is synthesized and released by the pancreas. Its strong expression during acute pancreatitis suggests its involvement in the pancreatic response to injury. Clusterin is also induced during pancreatic development. Because these situations are associated with apoptosis and clusterin was shown to protect against apoptosis, we speculate that clusterin could be involved in the control of acinar cell apoptosis.

Pancreatic GAPDH gene expression during ontogeny and acute pancreatitis induced by caerulein.

Recent studies indicated that expression of the housekeeping gene GAPDH is highly regulated during proliferation and differentiation. The objective of this study was to characterize by Northern blot the GAPDH mRNA expression in rat pancreas development and regeneration following acute pancreatitis induction by caerulein. Pancreatic GAPDH mRNA levels were the highest between fetal day 19 and the 11 postnatal day; they decreased to their lowest level after weaning on day 26. In acute pancreatitis, GAPDH mRNA levels were clearly increased 18 h after its initiation, were maximal during the first two days of induction and then decreased to control values after 9 days. These data demonstrate that overexpression of GAPDH may be implicated in pancreatic development, maturation and pancreas regeneration after acute pancreatitis.

Downregulation of IGF-I mRNA expression during postnatal pancreatic development and overexpression after subtotal pancreatectomy and acute pancreatitis in the rat pancreas.

Insulin-like growth factors (IGFs) are important peptides involved in the regulation of cell growth and differentiation in many tissues. The ontogeny of IGF-I was examined in pancreata from 19-day rat fetuses, newborns and 5-, 11-, 26- and 70-day-old rats. For the regeneration studies two models were used: (i) 90% pancreatectomy was carried out and the rats were killed at 1, 2, 3 and 6 days after resection; (ii) acute pancreatitis was induced with caerulein (12 micrograms/body weight three times a day every 8 h for 2 days) and the rats were killed at 1, 2, 5, 7 and 9 days after the first injection. Total RNA was extracted by the guanidinium isothiocyanate method and Northern blots were performed using total RNA and labeled cRNA probes. Abundance of the different mRNA transcripts was estimated by densitometric scanning and normalized to the abundance of 18 S rRNA for each time point. Northern blot analysis during ontogeny showed four (0.8-1.2, 1.9, 4.7 and 7.5 kb) major transcripts in the rat pancreas and liver. Total IGF-I mRNA was 40-fold higher in the adult liver than in the adult pancreas. Moreover, in the liver, IGF-I mRNA levels were higher in the adult than in the fetus, whereas in the pancreas, the highest levels were observed around birth. During the first 3 days after pancreatectomy, a peak of maximal expression was observed after the second day. Densitometric analysis of each IGF-I mRNA species showed concomitant increases in all transcripts. After 6 days, all transcripts had returned to near-control values. IGF-I mRNA expression 2 days after pancreatectomy was 3.5-fold higher than in the newborn. During the first 2 days of acute pancreatitis induction, overexpression of IGF-I mRNA was observed. However, soon after the second day of caerulein treatment, the 7.5 kb transcripts remained elevated whereas those of the others regressed toward control values. Our results show that IGF-I mRNA is overexpressed in both models of pancreatic regeneration but downregulated in the normal adult pancreas.

Cloning and expression of the rat p8 cDNA, a new gene activated in pancreas during the acute phase of pancreatitis, pancreatic development, and regeneration, and which promotes cellular growth.

To characterize at the molecular level the pancreatic emergency program set up by the pancreatic cells in response to pancreatitis, we have developed a strategy in which the phenotype of the pancreatitis affected pancreas is established by characterization of a large number of its transcripts. Herein, we describe the cloning, sequence, and expression of a new gene, named p8, which is strongly activated in pancreatic acinar cells during the acute phase of pancreatitis, in developing pancreas and during pancreatic regeneration. In acinar cells, p8 mRNA is expressed rapidly and specifically in response to cellular pancreatitis-induced injury; its induction occurred almost similarly in edematous and necrohemorrhagic pancreatitis, indicating that p8 mRNA is maximally activated even in response to a mild pancreatic injury. Furthermore, in vitro studies suggest that p8 mRNA is induced in pancreatic and non-pancreatic cells in response to some apoptotic stimuli. p8 acts as a promoter of cellular growth factor when its cDNA is transfected into COS-7 and AR4-2J cells. Although we failed to identify p8-related sequences, analysis of its primary and secondary structure suggests that p8 is a basic helix-turn-helix-containing gene with slight homology to several homeotic genes and sufficient signal to be targeted to the nucleus. We therefore propose p8 as a putative transcriptional factor which can regulate pancreatic growth.

Ontogeny of hepatocyte growth factor and c-met/hgf receptor in rat pancreas.

Hepatocyte growth factor has several activities in epithelial cells, including mitogenesis, dissociation of epithelial sheets, stimulation of motility, and promotion of matrix invasion. HGF is the ligand of a dimeric transmembrane tyrosine kinase receptor encoded by the met proto-oncogene. The objective of this study was to characterize by Northern blot the HGF and c-met mRNA expression in rat pancreas during development. Our findings show that simultaneous expression of both messengers occurs during the early development of the rat pancreas. Pancreatic HGF and c-met mRNA levels were higher between late fetal and 11 post-natal days and then decreased to a very low level at 26 days. This low level was maintained until the adult age. We conclude that HGF gene expression and c-met pancreatic gene expression are age dependent with the highest expression before weaning. These new data demonstrate that HGF and its receptor may be implicated in normal pancreas development and maturation.

Effect of ethanol intake on pancreatic exocrine secretion in mice.

Swiss mice were fed conventional lab chow and 10% ethanol or water as drinking fluid for 2 weeks. Pancreatic juice was obtained by cannulation of the bile pancreatic common duct of mice anesthetized with urethane. Isolated pancreatic lobules were also obtained. The flow rate and the amylase output were determined in pure pancreatic juice. The release of amylase was measured in pancreatic lobule preparations. The basal pancreatic juice flow rate and the amylase output were significantly increased by ethanol consumption. The magnitude of the pancreatic juice flow rate and the amylase output responses to increasing doses of bethanechol, a cholinergic agent, was significantly decreased in ethanol-fed mice. The amount of spontaneously released amylase was higher in pancreatic lobule preparations from ethanol-fed animals than that from control mice, and the difference was abolished by addition of atropine to the incubation media. The amylase release rate in response to increasing doses of bethanechol was significantly reduced in lobule preparations from the ethanol-fed group. These data indicate that ethanol intake in mice has a stimulating effect on the spontaneous pancreatic secretion and lends support to the hypothesis that ethanol consumption increases the intrapancreatic cholinergic tone.

Transcriptional regulation by cholecystokinin-pancreozymin in rat pancreas.

Cholecystokinin-pancreozymin (CCK-PZ) is involved in the regulation of pancreatic protein synthesis and secretion. We demonstrate here that CCK-PZ also stimulates RNA synthesis. Rats were killed 0, 30, 60, 120, 240 or 480 min after intraperitoneal injection of CCK-PZ (8 U/kg). Nuclei were prepared from pancreata and used for in vitro RNA synthesis ('run-on' experiments) in the presence of [alpha-32P]UTP. Total RNA synthesis increased after CCK-PZ with maximum UTP incorporation at 60 min. Contributions of RNA polymerase II, responsible for mRNA synthesis, and RNA polymerases I and III could be separately estimated by using alpha-amanitin. RNA polymerase I and III activities increased by 68% after CCK-PZ, whereas RNA polymerase II activity increased by 113%. Rates of synthesis of amylase, chymotrypsinogen B, trypsinogen I and actin mRNAs were estimated by quantitative hybridization of newly synthesized transcripts to specific cDNA clones. Synthesis of the four RNAs increased with a maximum between 60 and 120 min after CCK-PZ, stimulation being more important for the serine proteinases than for amylase. It was concluded that, in rat pancreas, CCK-PZ controls gene expression at the transcriptional level.

Changes in gene expression during pancreatic regeneration: activation of c-myc and H-ras oncogenes in the rat pancreas.

Expression of the c-myc and H-ras oncogenes, and of several genes specifically expressed in adult rat pancreas was investigated by monitoring changes in corresponding mRNA concentrations, by dot-blot hybridization, during the early phase of regeneration following subtotal pancreatectomy. The oncogenes c-myc and H-ras were overexpressed after 12-24 and 48 h, respectively, then returned to basal levels. The concentrations of mRNAs encoding amylase, chymotrypsinogen B, and trypsinogen I decreased during the regeneration time. By contrast, proinsulin I mRNA concentration was increased at 12-48 h after surgical resection, and actin mRNA concentration was increased at 12-48 h after surgical resection, and actin mRNA concentration was increased at 12 h after subtotal pancreatectomy and remained elevated thereafter. We concluded that regeneration after subtotal pancreatectomy is accompanied by repression of certain genes that are expressed in differentiated pancreatic tissue, and that derepression of other genes may be necessary for starting and/or maintaining the process of pancreatic regeneration.

Changes in pancreatic trophism and gene expression during a prolonged fasting period in rats.

To examine the effects of fasting on trophism and gene expression in pancreas, adult male rats were deprived of food from 0-6 d. Total DNA, RNA, and proteins, and specific mRNAs for rat amylase, chymotrypsinogen B, trypsinogen I, proinsulin I, and actin (assessed by employing cloned cDNAs and dot-blot hybridization) were quantitated in pancreas. Body and pancreatic wt diminished progressively to reach 65 and 75% of initial values at the 6th d of fasting. Protein/DNA and total RNA/DNA ratios decreased 2.04 and 2.31-fold, respectively, during 6 d of fasting. The concentration of amylase, chymotrypsinogen B, trypsinogen I, and actin mRNA, expressed as cpm/microgram RNA, decreased significantly throughout the study period, whereas the decrease observed in Proinsulin I mRNA concentration was not significantly different. When mRNA concentrations were refereed to the total content of DNA, however, the decrease was significant for all messengers tested. It is concluded that the prolonged absence of nutrients in the digestive tract exerts negative trophic influence on pancreas and triggers differential changes in pancreatic gene expression. These changes are gradual, asynchronic, and nonparallel.

Changes in growth and pancreatic mRNA concentrations during postnatal development of rat pancreas.

Changes in pancreatic growth and in mRNA concentrations in rat pancreas were monitored by dot-blot hybridization with cloned cDNAs of rat amylase, chymotrypsinogen B, proinsulin I, and actin during the pre- and postnatal period in the rat. Wistar rats were killed at the 18th day of gestation and at the 1st, 10th, 20th, 35th, and 87th day of postnatal life. It was concluded from the ratio of pancreatic weight/body weight that pancreatic growth preceded body growth. Pancreatic protein and total RNA concentration increased 2.9 times during the period studies. All studied mRNAs increased in concentration during the postnatal development period. Messenger RNA for chymotrypsinogen B and proinsulin I exhibited a significant increase after birth, decreased by the 10th day of life, and increased thereafter. For amylase mRNA, no significant changes were observed around birth, a progressive increase occurring thereafter up to the 87th day of life. The mRNA for actin showed a progressive increase between the 18th day of gestation and the 20th postnatal day, after which it remained stable. We concluded that each mRNA showed a singular profile of increase during postnatal development.

Changes in pancreatic exocrine secretion after repeated haloperidol administration.

The effect of repeated administration of haloperidol on the pancreatic secretion was studied in urethane-anesthetized Swiss mice. Haloperidol (2 mg/kg) injected daily i.p. for 7 days, increase the volume and protein content of the basal pancreatic juice significantly. This secretory activity was partially blocked by i.p. injection of atropine (5 mg/kg), both in control and treated animals. The volume of the secretory response to bethanechol, a cholinergic agonist, was decreased by haloperidol without any change in amylase release. From these findings it is concluded that repeated haloperidol treatment produces an increase of basal pancreatic secretion, which is probably the result of changes in the sensitivity of dopamine receptors of the gland.

Synthesis and contents of pancreatic exportable enzymes. Effect of oleic acid intraduodenal administration.

The effect of intraduodenal oleic acid administration on protein synthesis and enzymatic levels in rat pancreas was investigated. Sprague-Dawley rats were sacrificed at 20, 40, 60, and 80 min after intraduodenal oleic acid administration. Ten minutes before sacrifice, the rats were injected with 50 microCi 3H-Phenylalanine intraperitoneally. Amylase (Am), chymotrypsinogen (Chtg), trypsinogen (Tg) and lipase (Li) activities, and 3H-Phenylalanine incorporation to total secretory proteins were determined in pancreas homogenates. Forty minutes after oleic acid administration, the activities of Chtg, Tg and Li were significantly increased (45, 38 and 23%, respectively) above those from control rats. Amylase levels were not modified. Enzyme activities decreased below baseline levels by 60 and 80 min after oleic acid administration. The 3H-phenylalanine incorporation pattern exhibited a peak at 40 min. We conclude that intraduodenal oleic acid administration stimulates intrapancreatic enzyme content in a non-parallel fashion, before enzyme activities decreased below those from control rats. Protein synthesis was similarly affected by intraduodenal oleic acid.

Synthesis and contents of pancreatic exportable enzymes. Effect of oleic acid intraduodenal administration.

The effect of intraduodenal oleic acid administration on protein synthesis and enzymatic levels in rat pancreas was investigated. Sprague-Dawley rats were sacrificed at 20, 40, 60, and 80 min after intraduodenal oleic acid administration. Ten minutes before sacrifice, the rats were injected with 50 microCi 3H-Phenylalanine intraperitoneally. Amylase (Am), chymotrypsinogen (Chtg), trypsinogen (Tg) and lipase (Li) activities, and 3H-Phenylalanine incorporation to total secretory proteins were determined in pancreas homogenates. Forty minutes after oleic acid administration, the activities of Chtg, Tg and Li were significantly increased (45, 38 and 23

, respectively) above those from control rats. Amylase levels were not modified. Enzyme activities decreased below baseline levels by 60 and 80 min after oleic acid administration. The 3H-phenylalanine incorporation pattern exhibited a peak at 40 min. We conclude that intraduodenal oleic acid administration stimulates intrapancreatic enzyme content in a non-parallel fashion, before enzyme activities decreased below those from control rats. Protein synthesis was similarly affected by intraduodenal oleic acid.

Bethanechol-induced restricted stimulation of pancreatic juice secretion in mice.

Flow and enzymatic output from mouse pancreas were studied "in vivo" under bethanechol stimulation. A biphasic dose response curve was found in both, enzymatic output and juice flow, implicating that exocrine pancreatic juice secretion was also involved in restricted stimulation phenomenon.