We have previously shown a specific significant overexpression in the exocrine pancreatic tissue of two members of the regenerating gene multifamily: reg I and reg II in the non-obese diabetic (NOD) mouse during active diabetogenesis. To strengthen the hypothesis that the overexpression of these genes may represent a defence of the acinar cell against pancreatic endocrine agression, we studied the pancreatic expression and the localization of another member of this family: the pancreatitis-associated protein (PAP) in NOD mice under the same conditions. We found that NOD mice present significantly higher PAP mRNA levels than control IOPS-OF1 mice. There is no difference between female NOD mice which progressively develop type I diabetes between 100 and 200 days and male NOD mice which are protected. The only difference observed was in function of the age of onset of diabetes. Before 180 days, the PAP mRNA levels were similar to those found in NOD males and nondiabetic females, but above 180 days the levels of PAP mRNA increased significantly. More importantly immunohistological studies demonstrate a striking difference in the protein localization between normal or nondiabetic NOD mice and diabetic NOD mice. If the protein is mainly detected in the islet cells in the absence of diabetes, a specific and intense expression of PAP was observed in the acinar cells of diabetic NOD mice. In conclusion, our data demonstrates that the acinar cells may react to a long-lasting pancreatic endocrine aggression by an induction of PAP and underlines the existence of a symbiotic relationship between endocrine and exocrine tissue.

A feature associated frequently with the pathologic pancreas is the presence of tubular complexes produced by a phenotypic modulation of acinar cells that take on the characteristics of ductular cells. Since the type I Reg gene, an acinar cell product, is increased in the pancreas following an acinar injury, we aimed to evaluate whether the Reg I protein might be involved in this dedifferentiation process in the human pancreas. We studied duct-like structures in fixed human pathologic pancreatic tissues and human cells with a ductal phenotype obtained by culturing human exocrine preparations. Immunocytochemistry, Western blotting, and RT-PCR were applied for detection of type I Reg. Reg I was observed not only in acinar cells but also in the duct-like cells and dilated duct cells, both positive for cytokeratin 19. However, none of the other acinar markers was observed in these cells. In vitro, human acinar cells dedifferentiated, losing their acinar phenotype, but expression of Reg I remained constant throughout the culture duration. Furthermore, Reg I was not associated with proliferation. We demonstrated that Reg I expression was linked to acinar cell dedifferentiation. We postulate that Reg I might be used as a marker to understand the events leading to phenotypic changes of acinar cells to address the physiological role of Reg I in the pancreas.

Background We have studied gene transfer efficiency of glycosylated polylysines and glycosylated polyethylenimines as vectors in immortalized differentiated airway gland serous cells and primary cultures of human airway surface epithelial cells.Methods and results In both cell types, lactosylated PEI was more efficient for gene transfer than unsubstituted PEI and lactosylated polylysine which requires the presence of endosomolytic agents. However, for all the vectors tested, gene transfer efficiency was lower in differentiated cells as compared with poorly differentiated cells. The presence of membrane lectins, i.e. cell surface sugar-specific receptors, was evaluated using fluorescein-conjugated neoglycoproteins and microscopy or flow cytometry. In differentiated airway surface epithelial cells, membrane lectins were not expressed and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were not incorporated. This accounted in part for the lack of gene transfer efficiency in these cells. In contrast, in differentiated airway gland serous cells, expression of lectins and their endocytotic properties appeared to be similar to that observed in undifferentiated cells, and plasmid DNA/fluorescein-conjugated glycosylated polymer complexes were incorporated in similar amounts by cells in both differentiated states.Conclusions Glycosylated PEI appears to be a promising gene delivery system since it is more efficient than the sugar-free polymer and does not require endosomolytic agents. However, in differentiated airway gland serous cells, a low gene transfer efficiency was observed that could not be attributed to low expression of membrane lectins or low uptake of glycosylated complexes. An impaired intracellular trafficking of glycosylated complexes in differentiated airway gland serous cells is suggested. Copyright (C) 2002 John Wiley Sons, Ltd.

CF-KM4 (cystic fibrosis transmebrane conductance regulator-deficient) and MM-39 (healthy) cells, two serous cell lines from submucosal tracheal glands, were found to be poorly susceptible to adenovirus (Ad)5 infection and Ad5-mediated gene transduction. The major limiting steps apparently resided in the primary events of Ad5 interaction, i.e., cell attachment and entry. Both CF-KM4 and MM-39 cells failed to express the Coxsackie-Ad receptor (CAR), and experimental data suggested that alpha[2-->6]-linked sialic acid residues of sialoglycoproteins (SAGP) in CF-KM4 cells, and heparan sulfate glycosaminoglycans (HS-GAG) in MM-39, were used as receptors by Ad5 virions. Ad5 attached to SAGP and HS-GAG receptors via its fiber knob domain, but entered the cells via a penton base- and Arg-Gly-Asp (RGD)-integrin-independent pathway. The block to Ad5-mediated gene transfer in MM-39 and KM4 cells could be overcome by conferring to the vector a novel cell-binding specificity. Thus, Ad5 vectors carrying a stretch of 7-lysine residues genetically inserted at the C-terminus of the fiber knob were found to transduce MM-39 cells with a 10- to 20-fold higher efficiency than the original vectors, but the transduction of CF-KM4 was not significantly improved. Retargeting Ad5 to integrin receptors via RGD peptide ligands, inserted at the extremity of the fiber shaft, resulted in a transducing efficiency of 20- and 50-fold higher in MM-39 and KM4 cells, respectively, compared with Ad5 vectors carrying fibers terminated by their natural knob domain.

In human pancreas two genes, reg I alpha and reg I beta, have been characterized but only the reg I alpha protein has been isolated from human pancreatic secretion. To examine their respective physiological roles in fetal and adult pancreas we have compared the patterns of gene expression using a specific RT-PCR method. No progressive evolution in the two mRNAs levels was observed during fetal development (16--41 weeks). A discoordinate expression of the two genes was found with a higher level of reg I alpha mRNA in fetus and a higher level of regI beta in adult. In addition, if reg I alpha mRNA level was correlated with the expression of genes encoding exocrine proteins in adults, reg I beta mRNA level presented no correlation with any ductular, endocrine, or exocrine gene expression. In human pancreatic cell lines we showed the only expression of reg I beta gene and protein. All these data suggest that the two reg genes and proteins could play different roles in the pancreas.

Human tracheal glands cells (HTGC) in culture are able to respond to adrenergic, cholinergic and purinergic agonists by increasing their serous and mucin secretions. These secretagogues are also able to maintain an optimal responsiveness of serous cells to stimulation when they are regularly and briefly delivered to the cells, making the HTGC a suitable model to study the serous secretion (Merten, in press). Our interest has been focused on the effects of cholinergic and purinergic secretagogues associated to histamine, on the mucous function of the transformed human tracheal gland cell line MM-39, which has a mixed, both serous and mucous, phenotype. When the cells were exposed to short stimulation every 2 days for 3 weeks with 10 or 100 μM carbachol, UTP and histamine, modifications of their mucous phenotype were observed. The expression of MUC genes appeared dependent on the culture conditions. Transcripts of MUC1, MUC4, and MUC5B genes were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 10 μM, in contrast to the unstimulated expression of MUC1 and MUC4 in control cells. MUC1, MUC4, MUC7, MUC6 and MUC11 transcripts were observed when the cells were regularly exposed to the mixture of secretagogues at a concentration of 100 μM. These culture conditions were also able to induce an α1,2-fucosyltransferase activity absent in the MM-39 cells cultivated with standard conditions. There was no marked effect on the α2,3-sialyltransferase activity although the expression pattern of the sialyltransferase genes was reduced to the unique presence of ST3Gal III. In conclusion, MM-39 cells exposed to repeated stimulation by secretagogues at different concentrations express different sero-mucous phenotypes.

In order to investigate the influence of inflammation on the peripheral glycosylation of airway mucins, a human respiratory glandular cell line (MM-39) was treated by TNFα. The expression and the activity of sialyl- and fucosyl-transferases, involved in the biosynthesis of peripheral carbohydrate determinants like sialyl-Lewis x, were investigated by RT-PCR and by HPAEC respectively. The mRNA steady-state level of sialyl- (ST3Gal III) and of fucosyl- (FUT3) transferases was moderately up-regulated by TNFα; a 52% increase of α2,3-sialyltransferase activity was also observed in TNFα-stimulated MM-39 cells. After metabolic radio-labelling with [3H]glucosamine and [3H]fucose, the mucins released in␣the culture supernatant were purified by Sepharose CL-4B, density-gradient centrifugation and treatment with glycosaminoglycans-degrading enzymes. The mucins, released in the culture supernatant from control MM-39 cells, were constituted by two populations of molecules having the same 1.39–1.44 mg/ml density but carrying either high or low amounts of sialic acid residues at their periphery. TNFα was able to increase the sialylation of the weakly sialylated mucins. This effect and the enhancement of the α2,3-sialyltransferase activity by TNFα argue in favour of a regulation of the mucin sialylation by this pro-inflammatory cytokine. Despite the moderate overexpression of FUT3, no fucosylation of mucins produced by MM-39 cells was induced by TNFα. In conclusion, the influence of TNFα on the sialylation of mucins could explain why the mucins from infected patients suffering either from cystic fibrosis or from chronic bronchitis are more sialylated.

Background We recently designed a cationic polymer called histidylated polylysine made of polylysine partially substituted with histidyl residues which become protonated at slightly acidic pH. This polymer is thought to induce the leakage of acidic vesicles containing plasmid/histidylated polylysine complexes.Methods and results Here, we have analyzed the ability of histidylated polylysine to transfer reporter or CFTR genes into immortalized cystic fibrosis airway surface epithelial cells (Sigma CFTE29o- cells) and airway gland serous cells (CF-KM4 cells) which are both important targets far cystic fibrosis gene therapy. The luciferase reporter gene expression measured after gene transfer with histidylated polylysine into both cell lines was quite high and similar to that obtained with commercially available vectors. In addition, the level of expression was not dependent on the presence of a membrane disrupting agent such as chloroquine. Histidylated complexes were present in slightly acidic non-lysosomal cellular compartments as shown by a cytological approach using biotinylated plasmid, lysosome-specific antibodies and confocal microscopy. Histidylated complexes appeared to be of small size when prepared at low ionic strength and formed aggregates upon increasing the ionic strength. However, aggregate formation was prevented by the addition of 10% fetal bovine serum. Gene transfer efficiency varied with the size of the complexes and decreased when small particles were used.Conclusions These results suggest that histidylated polylysine may be an efficient non-viral vector for gene transfer into cystic fibrosis airway surface epithelial cells and airway gland serous cells. Copyright (C) 2000 John Wiley & Sons, Ltd.

We demonstrated pancreatic reg gene overexpression in non-obese diabetic (NOD) mice during active diabetogenesis. The aim of this study was to determine in which part of the pancreas (endocrine and/or exocrine) the gene(s) and the protein(s) were expressed and if their localization changed with progression of the disease. In situ hybridization analysis and immunocytochemical studies were carried out on pancreas of female and male NOD mice. Both develop insulitis but diabetes develops only in females and in males only when treated by cyclophosphamide. Our results show that whatever the age, sex, and presence of insulitis and/or diabetes, the expression of reg mRNAs and of the corresponding protein(s) was restricted to exocrine tissue. Moreover, reg remains localized in acinar cells in the two opposite situations of (a) cyclophosphamide-treated males in a prediabetic stage presenting a high level of both insulin and reg mRNAs, and (b) the overtly diabetic females with no insulin but a high level of reg mRNA. These findings suggest that overexpression of the reg gene(s) might represent a defense of the acinar cell against pancreatic aggression.

The lipase gene family contains a large number of members. Among the most closely related are pancreatic triglyceride lipase (PTL) and two pancreatic lipase-related proteins (PLRP1 and PLRP2). Previous studies in rodents demonstrated divergent temporal expression of the genes encoding these proteins. PLRP1 and PLRP2 were expressed in fetal pancreas, whereas PTL was not expressed until pups were several weeks old. To determine whether the human pancreas has a similar expression pattern for these genes, we determined the levels of each mRNA in fetal pancreas at various ages. A reverse transcriptase-PCR method was developed and used to quantify the mRNA levels for the three species normalized to the mRNA encoding cyclophillin. The mRNA encoding PLRP1 and PLRP2 was present by 16 wk in the fetal pancreas. In contrast, the mRNA encoding PTL was not present in the fetal pancreas. This pattern of expression suggests that the genes encoding theses proteins have different regulatory elements controlling temporal expression and provides another example of nonparallel expression of genes encoding pancreatic exocrine proteins.

Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally accepted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistandard RT-PCR method. This was based on the observation that the CFTR and ribosomal phosphoprotein PO (PR-PO) genes have retained important sequence homologies between rat and human species, allowing the use of rat RNA as an internal standard. A mixture of rat and human RNAs is simultaneously reverse-transcribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by restriction analysis. PR-PO is analyzed similarly and serves as a control of template loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) incubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. This method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cystic fibrosis patients.

A differential pancreatic behavior observed between male and female mice in diabetes and pancreatitis led us to study the gene and protein expressions of endocrine and exocrine pancreatic proteins in normal mice. We compared the levels of expression of six pancreatic genes and of four of the corresponding proteins in male and female mice OF1. Amylase gene expression was found to be significantly higher in females than in males, whereas trypsinogen and lipase gene expression were significantly lower. For chymotrypsinogen, reg, and insulin the differences were not significant. This sexual dimorphism did not exist in rat pancreas, where no gender difference was observed. After characterization of mice enzymes by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and antibodies directed to the closely related human pancreatic enzymes, we have compared the levels of these proteins in mice pancreatic homogenates. No significant difference was observed between males and females at the level of protein expression. These data suggest a hormonal sexual difference in the regulation of pancreatic protein synthesis at the pre- and posttranscriptional levels in normal mice, which may play a role in the development of mice pancreatic diseases.

ABSTRACT ATP and UTP have been proposed for use as therapeutic treatment of the abnormal ion transport in the airway epithelium in cystic fibrosis (CF), the most characteristic feature of which is permanent infection by Pseudomonas aeruginosa . As for diverse gram-negative bacteria, this pathogenic bacterium accumulates diffusible N -acylhomoserine lactone (AHL) signal molecules, and when a threshold concentration is reached, virulence factor genes are activated. Human submucosal tracheal gland serous (HTGS) cells are believed to play a major role in the physiopathology of CF. Since ATP and UTP stimulate CF epithelial cells through P2Y receptors, we sought to determine whether CF HTGS cells are capable of responding to the AHLs N -butanoyl- l -homoserine lactone (BHL), N -hexanoyl- l -homoserine lactone (HHL), N -(3-oxododecanoyl)- l -homoserine lactone (OdDHL), and N -(3-oxohexanoyl)- l -homoserine lactone (OHHL), with special reference to P2Y receptors. All AHLs inhibited ATP- and UTP-induced secretion by CF HTGS cells. The 50% inhibitory concentrations were as high as 10 and 5 μM for BHL and HHL, respectively, but were only 0.3 and 0.4 pM for OdDHL and OHHL, respectively. Furthermore, all AHLs down-regulated the expression of the P2Y2 and P2Y4 receptors. Ibuprofen and nordihydroguaiaretic acid were able to prevent AHL inhibition of the responses to nucleotides, but neither dexamethasone nor indomethacin was able to do this. These data indicate that AHLs may alter responsiveness to ATP and UTP by CF HTGS cells and suggest that, in addition to ATP and/or UTP analogues, ibuprofen may be of use for a combinational pharmacological therapy for CF.

Chloride channels play an important role in the physiology and pathophysiology of epithelia, but their pharmacology is still poorly developed. We have chemically synthesized a series of substituted benzo[c]quinolizinium (MPB) compounds. Among them, 6-hydroxy-7-chlorobenzo[c]quinolizinium (MPB-27) and 6-hydroxy-10-chlorobenzo[c]quinolizinium (MPB-07), which we show to be potent and selective activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. We examined the effect of MPB compounds on the activity of CFTR channels in a variety of established epithelial and nonepithelial cell systems. Using the iodide efflux technique, we show that MPB compounds activate CFTR chloride channels in Chinese hamster ovary (CHO) cells stably expressing CFTR but not in CHO cells lacking CFTR. Single and whole cell patch clamp recordings from CHO cells confirm that CFTR is the only channel activated by the drugs. Ussing chamber experiments reveal that the apical addition of MPB to human nasal epithelial cells produces a large increase of the short circuit current. This current can be totally inhibited by glibenclamide. Whole cell experiments performed on native respiratory cells isolated from wild type and CF null mice also show that MPB compounds specifically activate CFTR channels. The activation of CFTR by MPB compounds was glibenclamide-sensitive and 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid-insensitive. In the human tracheal gland cell line MM39, MPB drugs activate CFTR channels and stimulate the secretion of the antibacterial secretory leukoproteinase inhibitor. In submandibular acinar cells, MPB compounds slightly stimulate CFTR-mediated submandibular mucin secretion without changing intracellular cAMP and ATP levels. Similarly, in CHO cells MPB compounds have no effect on the intracellular levels of cAMP and ATP or on the activity of various protein phosphatases (PP1, PP2A, PP2C, or alkaline phosphatase). Our results provide evidence that substituted benzo[c]quinolizinium compounds are a novel family of activators of CFTR and of CFTR-mediated protein secretion and therefore represent a new tool to study CFTR-mediated chloride and secretory functions in epithelial tissues.

OBJECTIVE: In type I diabetes mellitus, early markers of beta cell damage are needed in order to detect the infraclinical development of the disease. The reg protein may be a good candidate, as the reg gene has been proposed to play a role in the pancreatic beta cell destruction/regeneration process during diabetogenesis in animal models of autoimmune diabetes. The aim of this study was to test the hypothesis whether serum reg protein level could be representative of either the destructive or regenerative process at the beta cell level during the early phases of type I diabetes in humans. DESIGN AND METHODS: We used a highly specific immunoassay to measure serum reg protein level in controls and in three groups of either diabetes prone or diabetic subjects: recently diagnosed diabetic patients, long-standing diabetic patients and islet cell antibody-positive non-diabetic subjects. RESULTS: We found no significant difference between the values observed in these three groups in comparison with control group (90.7+/-18.1ng/ml, 83.1+/-5.6ng/ml, 98.7+/-24.5ng/ml vs 85.5+/- 5.6ng/ml respectively). Moreover, when the insulin reserve was evaluated at 6 months in the recently diagnosed group, serum reg protein levels were not different between patients with or without residual insulin secretion (at onset: 103+/-42 vs 70.3+/-8. 5ng/ml respectively; at 6 months: 79.7+/-25.8ng/ml vs 81.6+/-15ng/ml respectively). In contrast, trypsin levels were significantly lower in every group of diabetic patients. Results were expressed as means +/- S.E.M. and groups compared by Student's t-test (P<0.05). CONCLUSIONS: We conclude that serum reg protein level cannot be used as a marker for the progression of the diabetogenic process in type I diabetes.

Human tracheal gland serous (HTGS) cells are now considered one principal pulmonary target for the gene therapy of cystic fibrosis (CF). We developed a CF tracheal gland serous cell line, CF-KM4, obtained by the transformation of primary cultures of CF tracheal gland serous cells homozygous for the DeltaF508 mutation by using the wild-type SV40 virus. This cell line retained epithelial and secretory features of the native CF-HTGS cells in primary culture, namely, presence of cytokeratin, constitutive secretion of secretory leukocyte proteinase inhibitor, absence of responsiveness to carbachol and isoproterenol, and defective cyclic adenosine monophosphate-dependent chloride channel activity. Adenovirus-mediated CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells corrected the defective chloride channel activity as well as the responsiveness to adrenergic and cholinergic agonists. In contrast, control transfection using adenovirus-mediated beta-galactosidase gene transfer was totally ineffective. In conclusion, these results present a stable CF tracheal gland cell line that has retained its epithelial and CF-specific defective secretory characteristics which are corrected after CFTR gene transfer. This cell line therefore appears to be a useful tool for large-scale molecular and cellular pharmacologic investigations designed to test potential therapies of the disease CF.