Mechanisms underlying induction of allergic sensitization by Pru p 3.

BACKGROUND: Recently, the nature of the lipid-ligand of Pru p 3, one of the most common plant food allergens in southern Europe, has been identified as a derivative of the alkaloid camptothecin bound to phytosphingosine. However, the origin of its immunological activity is still unknown. OBJECTIVE: We sought to evaluate the role of the Pru p 3 lipid-ligand in the immunogenic activity of Pru p 3. METHODS: In vitro cultures of different cell types (monocyte-derived dendritic cells [moDCs], PBMCs [peripheral blood mononuclear cells] and epithelial and iNKT-hybridoma cell lines) have been used to determine the immunological capacity of the ligand, by measuring cell proliferation, maturation markers and cytokine production. To study the capacity of the lipid-ligand to promote sensitization to Pru p 3 in vivo, a mouse model of anaphylaxis to peach has been produced and changes in the humoral and basophil responses have been analysed. RESULTS: The lipid-ligand of Pru p 3 induced maturation of moDCsc and proliferation of PBMCs. Its immunological activity resided in the phytosphingosine tail of the ligand. The adjuvant activity of the ligand was also confirmed in vivo, where the complex of Pru p 3-ligand induced higher levels of IgE than Pru p 3 alone. The immunological capacity of the Pru p 3 ligand was mediated by CD1d, as maturation of moDCs was inhibited by anti-CD1d antibodies and Pru p 3-ligand co-localized with CD1d on epithelial cells. Finally, Pru p 3-ligand presented by CD1d was able to interact with iNKTs. CONCLUSIONS AND CLINICAL RELEVANCE: The Pru p 3 lipid-ligand could act as an adjuvant to promote sensitization to Pru p 3, through its recognition by CD1d receptors. This intrinsic adjuvant activity of the accompanying lipid cargo could be a general essential feature of the mechanism underlying the phenomenon of allergenicity.

Bronchial Challenge With Tri a 14 as an Alternative Diagnostic Test for Baker's Asthma.

BACKGROUND: Baker's asthma (BA) is the most prevalent occupational respiratory disease in developed countries. It is caused by inhalation of wheat dust in the working environment and affects 1%-10% of workers in the baking industry. Diagnosis of BA is based on bronchial challenge with wheat, a technique that carries a high risk for patients. The wheat lipid transfer protein Tri a 14 is a major allergen in BA. OBJECTIVE: The aim of our study was to characterize Tri a 14 as a marker of BA in order to prevent patients from having to undergo bronchial challenge with wheat. METHODS: The study population comprised 55 patients selected at the Rio Hortega Hospital, Valladolid, Spain. Patients with BA were diagnosed using a skin prick test (SPT) with wheat and Tri a 14 and bronchial challenge test (BCT) with wheat. Patients with food allergy had a clear clinical history of allergy to peach confirmed by positive SPT to peach extract and Pru p 3. RESULTS: All patients in the BA group had a positive SPT result with wheat (100%), and most had positive results with Tri a 14 (95%). A positive BCT result with Tri a 14 was also observed in 22 of 27 of the patients with BA (82%). The response to Tri a 14 was specifically associated with BA. CONCLUSION: Tri a 14 is a good marker of BA and can be used in SPT and BCT as an alternative diagnostic method, thus avoiding bronchial challenge with wheat and reducing the risk associated with this technique.

Bronchial challenge with Tri a 14 as an alternative diagnostic test for Baker’s asthma

Background: Baker's asthma (BA) is the most prevalent occupational respiratory disease in developed countries. It is caused by inhalation of wheat dust in the working environment and affects 1%-10% of workers in the baking industry. Diagnosis of BA is based on bronchial challenge with wheat, a technique that carries a high risk for patients. The wheat lipid transfer protein Tri a 14 is a major allergen in BA. Objective: The aim of our study was to characterize Tri a 14 as a marker of BA in order to prevent patients from having to undergo bronchial challenge with wheat. Methods: The study population comprised 55 patients selected at the Rio Hortega Hospital, Valladolid, Spain. Patients with BA were diagnosed using a skin prick test (SPT) with wheat and Tri a 14 and bronchial challenge test (BCT) with wheat. Patients with food allergy had a clear clinical history of allergy to peach confirmed by positive SPT to peach extract and Pru p 3. Results: All patients in the BA group had a positive SPT result with wheat (100%), and most had positive results with Tri a 14 (95%). A positive BCT result with Tri a 14 was also observed in 22 of 27 of the patients with BA (82%). The response to Tri a 14 was specifically associated with BA. Conclusion: Tri a 14 is a good marker of BA and can be used in SPT and BCT as an alternative diagnostic method, thus avoiding bronchial challenge with wheat and reducing the risk associated with this technique (AU)

Antecedentes: El asma del panadero (BA) es la enfermedad respiratoria ocupacional más frecuente en los países occidentales. Está causada por la inhalación diaria de harina de trigo en el entorno de trabajo, afectando entre 1-10% de los trabajadores de la industria panadera. El diagnóstico de BA se basa en la provocación bronquial con trigo, una técnica de alto riesgo para los pacientes. La proteína de transferencia de lípidos de trigo (LTP) Tri a 14 ha sido descrita como alérgeno principal en esta patología. Objetivo: El objetivo de nuestro estudio ha sido caracterizar Tri a 14 como marcador de BA, y así evitar la provocación bronquial con trigo en estos pacientes. Métodos: Para ello, se seleccionaron cincuenta y cinco pacientes en el Hospital Río Hortega de Valladolid, España. Resultados: Los pacientes diagnosticados con BA mostraron prueba cutánea (SPT) positiva a trigo (100%) y la mayoría también a Tri a 14 (95%). Todos ellos, fueron sometidos a provocación bronquial con Tri a 14, observándose un resultado positivo en 22/27 de los sujetos evaluados (82%). Conclusiones: En base a esto, se puede concluir que Tri a 14 es un buen marcador de BA, y podría ser utilizado en SPT y provocación bronquial como método diagnóstico, reduciendo el uso de la provocación bronquial con trigo y el riesgo asociado a esta técnica (AU)

[Glioblastomas: gliomagenesis, genetics, angiogenesis, and microenvironment]. / Glioblastomes : oncogenèse et bases biologiques.

BACKGROUND AND PURPOSE: Glioblastomas are the most malignant gliomas of the central nervous system. Currently, numerous studies are attempting to decipher their genetic and epigenetic modifications, to identify the cells at the origin of gliomagenesis, and to better understand the molecular bases responsible for invasion and angiogenesis processes. METHODS: This article reviews recent data on the cellular and molecular biology of gliomas delineated by several teams including ours. We and others have underlined the role played by cancer stem cells in gliomagenesis; the Cancer Genome Atlas Network has described most glioblastoma genetic alterations. RESULTS: According to many studies, glioblastomas derive from malignant transformation of stem cells and/or glial precursor cells. Moreover, the topographic microenvironment is important regarding invasion and angiogenesis processes. Finally, it is now well established, thanks to IDH1 mutation identification, that primary and secondary glioblastomas are two different clinical and genetic entities. Interestingly, IDH1 mutation seems to be a very early genomic modification in astrocytoma, oligodendroglioma, and secondary glioblastoma tumorigenic processes. CONCLUSIONS: Regarding all these data, we suggest a hypothetical model of glioma initiation, growth, and progression. Moreover, the histomolecular glioma classification has been substantially revised and new therapeutic targets have been identified.

KIAA0510, the 3'-untranslated region of the tenascin-R gene, and tenascin-R are overexpressed in pilocytic astrocytomas.

AIMS: Studying the molecules and signalling pathways regulating glioma invasiveness is a major challenge because these processes determine malignancy, progression, relapse and prognosis. We took advantage of our previous study focused on genes that were critical in tumour invasion to further study here an unknown sequence, referred to as KIAA0510, the chromosomal location of which was 1q25, described as a 5596-bp long mRNA and that we found to be significantly overexpressed in pilocytic astrocytomas compared with glioblastomas. METHODS AND RESULTS: Using in silico analysis as well as Polymerase chain reaction techniques, we decipher the full genomic characterization of the KIAA0510 sequence and demonstrate that KIAA0510 constitutes the 3'-untranslated region of tenascin-R gene. We have clearly confirmed the overexpression of tenascin-R in pilocytic astrocytomas vs. glioblastomas at mRNA and protein levels. We also analysed a large series of various brain tumours and found that in the group of astrocytic tumours, tenascin-R expression decreased with malignancy, whereas oligodendrogliomas sometimes retained a high level of tenascin-R even in high-grade tumours. Gangliogliomas strongly expressed tenascin-R too. In contrast, ependymomas and meningiomas were negative. In normal brain, tenascin-R was exclusively expressed by normal oligodendrocytes and subsets of neurones during post-natal development and in adulthood, where it could differentially affect cellular adhesiveness and/or differentiation. CONCLUSION: KIAA0510, the 3'-untranslated region of the tenascin-R gene, and tenascin-R are overexpressed in pilocytic astrocytomas. Gangliogliomas shared with pilocytic astrocytomas strong tenascin-R expression. Whether tenascin-R overexpression negatively influences brain invasion remains to be determined.

Relevance of combinatorial profiles of intermediate filaments and transcription factors for glioma histogenesis.

In order to define specific markers for histogenesis of three well-characterized subgroups of human gliomas (pilocytic astrocytomas, glioblastoma multiforme and oligodendrogliomas), we studied the expression of relevant markers that characterize gliomagenesis, by immunohistochemistry and in situ hybridization. They include the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin and nestin, the transcription factors Olig2, Nkx2.2 and Sox10, and the proteolipid protein transcripts plp/dm20. We show that the three major categories of human gliomas express a combinatorial profile of markers that gives new insights to their histogenesis and may help diagnosis. Pilocytic astrocytomas strongly express GFAP, vimentin, Olig2, Nkx2.2 and Sox10 but not nestin. In contrast, glioblastomas strongly express GFAP, vimentin and nestin but these tumours are heterogeneous regarding the expression of the transcription factors studied. Finally, in oligodendrogliomas, intermediate filament proteins are generally not observed whereas Olig2 was found in almost all tumour cells nuclei while only a subpopulation of tumour cells expressed Nkx2.2 and Sox10.

Duloxetina versus placebo en el tratamiento de la incontinencia urinaria de esfuerzo: ensayo clínico controlado en mujeres de la III Región de Atacama / Duloxetine vs. placebo in the stress incontinence treatment: controled clinical assay in women of Atacama area

Actualmente para la IOE los tratamientos aceptados son el entrenamiento muscular pélvico, intervenciones de hábitos y perfil biométrico y la cirugía, no estando plenamente establecido el tratamiento farmacológico para esta patología. La efectividad de la Duloxetina está relacionada con su capacidad de inhibición de la recaptación pre sináptica neuronal de serotonina y norepinefrina en el espacio sináptico del sistema nervioso central, promoviendo un tono esfinteriano uretral más potente durante el estrés físico y llenado vesical. En la actualidad no se han reportado experiencias nacionales con este fármaco para el tratamiento de la IOE en mujeres. Objetivos: Determinar la eficacia y tolerancia de la Duloxetina en el tratamiento médico de la IOE, evaluando la mejoría en la calidad de vida, la disminución de la frecuencia de escapes urinarios y los efectos adversos. Pacientes y método: Ensayo prospectivo randomizado doble ciego que incluyó 64 mujeres no embarazadas de 23 a 73 años (promedio 54 años), con síntomas predominantes de IOE de más de 3 meses de evolución. Los criterios de inclusión fueron: frecuencia de episodios de incontinencia (FEI) >= 4 a la semana, ausencia de síntomas predominantes de urge-incontinencia, frecuencia miccional (FM) normal (<= a 7/2), capacidad vesical >= a 400 ml. medida por infusión supina y prueba de tos y pañal positivas. Se realizó estudio urodinámico a un grupo aleatorio de 30 pacientes. Las pacientes fueron randomizadas recibiendo Duloxetina (40 mg c/12 horas vo) o placebo 2 veces al día, por 3 meses. Todas ellas fueron controladas mensualmente, evaluando la FEI, FM, cuestionario de calidad de vida por incontinencia (I-QOL), efectos adversos y adhesión al tratamiento. Se utilizó el software SPSS(r) para el análisis de resultados incluyendo test de Wilcoxon y Ancova. Resultados: El promedio de la FEI disminuyó significativamente con Duloxetina versus placebo (59 por ciento vs 28 por ciento, p<0,01), siendo mayor aún en el gru...

Introduction: The stress urinary incontinence (SUI) is the most common of the urinary incontinences and it has become a relevant urological and public health problem all over the world. Currently the most accepted forms of therapy for SUI are pelvic floor muscle training, behavioural and biometrical profile interventions and surgery, pharmacological treatment is not fully established for this pathology. The entrenaeffectiveness of duloxetine is related with its ability of inhibit serotonin and norepinephrine reuptake in the synaptic space of the central nervous system, increasing the urethral sphincter tone during the physical stress and bladder filling. Currently, no national experiences have been reported with this drug for the medical treatment of the SIU in women. Objectives: To assess the efficacy and safety of duloxetine in the medical treatment of SIU, valuating improvements in life quality, the diminishing of urinary leakage frecuency and adverse events. Patients and methods: Prospective double-blind randomized study which enrolled 64 nonpregnant women aged 23 to 73 years (54 years average), with a predominant symptom of SIU of more than 3 months of evolution. The inclusion criteria were: weekly incontinence episode frecuency (IEF) of 4 or greater, the absence of predominant symptoms of urge incontinence, normal diurnal and nocturnal frecuency (≤ a 7/2), a bladder capacity of 400 ml or greater measured with supine infusion, and a positive cough stress test and stress pad test. Urodynamic study was assessed to a random group of 30 patients. Subjects were randomized to duloxetine (40 mg BID) or placebo BID, for 3 months. All of them were controlled monthly, evaluating the IEF, voiding frecuency, Incontinence Quality of Life (I-QOL) questionnaire, adverse events and adhesion to treatment. SPSS® was used to analize the results including the Wilcoxon and Ancova tests. Results: There was a significant decrease in IEF with duloxetine compared with placebo...

In vitro identification and functional characterization of glial precursor cells in human gliomas.

Human gliomas including astrocytomas and oligodendrogliomas are defined as being composed of neoplastic astrocytes and oligodendrocytes respectively. Here, on the basis of in vitro functional assays, we show that gliomas contain a mixture of glial progenitor cells and their progeny. We have set up explant cultures from pilocytic astrocytomas, glioblastomas and oligodendrogliomas and studied antigens that characterize glial lineage, from the precursor cells (glial restricted precursors and oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells expressing the A2B5 ganglioside) to the differentiated cells (oligodendrocyte and type-1 and type-2 astrocytes). All tumoral explants contain A2B5+ cells and can generate migrating cells with distinctive functional properties according to glioma subtypes. In pilocytic astrocytomas, very few migrating cells are dividing and can differentiate in type-2 astrocytes or towards the oligodendrocyte lineage. In glioblastomas, most migrating cells are dividing, express A2B5 or glial fibrillary acid protein (GFAP) and can generate oligodendrocytes and type-1 and type-2 astrocytes in appropriate medium. Oligodendroglioma explants are made by actively dividing glial precursor cells expressing A2B5 or PSA-NCAM. Only few cells can migrate and differentiation towards oligodendrocyte lineage does not occur. Isolated A2B5+ cells from both glioblastomas and oligodendrogliomas showed similar genetic alterations as the whole tumour. Therefore, pilocytic astrocytomas contain slowly dividing oligodendrocyte-type2-astrocyte/oligodendrocyte precursor cells in keeping with their benign behaviour whereas both glioblastomas and oligodendrogliomas contain neoplastic glial restricted precursor cells. In oligodendrogliomas, these cells are trapped in undifferentiated and proliferating state. The precursor cells properties present in gliomas give new insight into their histogenesis and open up new avenues for research in the field of gliomagenesis.

Identification of genes differentially expressed in glioblastoma versus pilocytic astrocytoma using Suppression Subtractive Hybridization.

Glioblastoma (GBM) is a highly malignant glioma, which has the propensity to infiltrate throughout the brain in contrast to pilocytic astrocytoma (PA) of the posterior fossa, which does not spread and can be cured by surgery. We have used Suppression Subtractive Hybridization to define markers that better delineate the molecular basis of brain invasion and distinguish these tumor groups. We have identified 106 genes expressed in PA versus GBM and 80 genes expressed in GBM versus PA. Subsequent analysis identified a subset of 20 transcripts showing a common differential expression pattern for the two groups. GBM differs from PA by the expression of five genes involved in invasion and angiogenesis: fibronectin, osteopontin, chitinase-3-like-1 (YKL-40), keratoepithelin and fibromodulin. PA differs from GBM by the expression of genes related to metabolism (apolipoprotein D), proteolysis (protease-serine-11), receptor and signal transduction (PLEKHB1 for Pleckstrin-Homology-domain-containing-protein-family-B-member-1), transcription/translation (eukaryotic-translation-elongation-factor-1-alpha1) processes and cell adhesion (SPOCK1 for SPARC/Osteonectin-CWCV-kazal-like-domains-proteoglycan). The expression of these genes was confirmed by real-time quantitative RT-PCR and immunohistochemistry. This study highlights the crucial role of brain invasion in GBM and identifies specific molecules involved in this process. In addition, it offers a restricted list of markers that accurately distinguish PA from GBM.

Pancreatitis-associated protein (HIP/PAP) gene expression is upregulated in NOD mice pancreas and localized in exocrine tissue during diabetes.

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.

Attenuation of FGF signalling in mouse beta-cells leads to diabetes.

Fibroblast growth factor (FGF) signalling has been implicated in patterning, proliferation and cell differentiation in many organs, including the developing pancreas. Here we show that the FGF receptors (FGFRs) 1 and 2, together with the ligands FGF1, FGF2, FGF4, FGF5, FGF7 and FGF10, are expressed in adult mouse beta-cells, indicating that FGF signalling may have a role in differentiated beta-cells. When we perturbed signalling by expressing dominant-negative forms of the receptors, FGFR1c and FGFR2b, in the pancreas, we found that that mice with attenuated FGFR1c signalling, but not those with reduced FGFR2b signalling, develop diabetes with age and exhibit a decreased number of beta-cells, impaired expression of glucose transporter 2 and increased proinsulin content in beta-cells owing to impaired expression of prohormone convertases 1/3 and 2. These defects are all characteristic of patients with type-2 diabetes. Mutations in the homeobox gene Ipf1/Pdx1 are linked to diabetes in both mouse and human. We also show that Ipf1/Pdx1 is required for the expression of FGFR1 signalling components in beta-cells, indicating that Ipf1/Pdx1 acts upstream of FGFR1 signalling in beta-cells to maintain proper glucose sensing, insulin processing and glucose homeostasis.

Sexual dimorphism of pancreatic gene expression in normal mice.

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.

Overexpression of the reg gene in non-obese diabetic mouse pancreas during active diabetogenesis is restricted to exocrine tissue.

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.

Serum reg protein level is not related to the beta cell destruction/regeneration process during early phases of diabetogenesis in type I diabetes.

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.

Expression of peptide-23/pancreatitis-associated protein and Reg genes in human pituitary and adenomas: comparison with other fetal and adult human tissues.

Peptide 23, the rat homolog of the human pancreatitis-associated protein (PAP)/hepatocarcinoma-intestine-pancreas (HIP) protein, has been identified in primary culture of rat pituitary cells. Its secretion was shown to be stimulated by GH-releasing factor and inhibited by somatostatin in a similar fashion to GH. This observation led the researchers to speculate that peptide 23 does have a physiological hormonal role. We tested this hypothesis by screening by RT-PCR reactions the expression of the PAP/HIP gene in several human pituitary adenomas, especially GH-producing adenomas. Our results show a weak expression of the PAP/HIP gene in the pituitary gland and in most of the tumors, but independent of their origin. The significant homology of the PAP/HIP gene to the Reg gene family prompted us to study in the same pituitary adenomas the presence of the related Reg genes. Reg expression was never observed in the adenomas tested or in the pituitary gland. In contrast, the RegL transcript was observed in pituitary gland and in some subtypes of adenomas. We then extended our work to normal adults and developing human tissues to compare the expression patterns of the PAP/Reg gene family. We observed the presence of the PAP/HIP transcript in each tissue tested. In contrast, the Reg gene was expressed only in fetal pancreas and in some adult tissues, whereas the RegL gene was expressed not only in fetal pancreas but also in fetal colon and brain as well as some adult tissues. In conclusion, our results show that all of the human fetal and adult tissues examined express at least one of the different transcripts of the PAP/Reg family, suggesting that the regulation of these homologous genes is coordinately controlled.

Specific reg II gene overexpression in the non-obese diabetic mouse pancreas during active diabetogenesis.

The reg gene, previously described in islets of 90% pancreatectomized and nicotinamide-treated rats, has been shown to be expressed in many pharmacological or surgical animal models of beta cell regeneration. We have studied the non-obese diabetic (NOD) mouse, which represents a good model of spontaneous autoimmune diabetes in which regenerative processes have recently been demonstrated. Two reg genes have been described in the mouse genome, both recognized by the human reg cDNA. In a previous work, using the human probe, we have demonstrated a strong correlation between pancreatic reg gene expression and the likelihood of developing diabetes. In the present study, we have examined the respective levels of both mouse reg I and reg II mRNA in the NOD mouse pancreas using their specific cDNA probes. We found that reg II expression was specifically prevalent compared to reg I, irrespective of sex or state of the disease. Reg II mRNA was particularly increased in overtly diabetic female mice and in cyclophosphamide-treated male mice. These data underline the need to study separately the reg genes using specific probes and show that both reg genes are subjected to various regulations, strongly suggesting that their physiological functions may be different.

Pseudomonas aeruginosa lipopolysaccharide induces CF-like alteration of protein secretion by human tracheal gland cells.

Human tracheal gland (HTG) serous cells are now believed to play a major role in the physiopathology of cystic fibrosis. Because of the persistent inflammation and the specific infection by Pseudomonas aeruginosa in the lung, we looked for the action of the lipopolysaccharide (LPS) of this bacteria on human tracheal gland cells in culture by studying the secretion of the secretory leukocyte proteinase inhibitor (SLPI) which is a specific serous secretory marker of these cells. Treatment with Pseudomonas aeruginosa LPS resulted in a significant dose-dependent increase in the basal production of SLPI (+ 250 +/- 25%) whilst the SLPI transcript mRNA levels remained unchanged. This LPS-induced increase in secretion was inhibited by glucocorticoides. Furthermore, LPS treatment of HTG cells induces a loss of responsiveness to carbachol and isoproterenol but not to adenosine triphosphate. These findings indicate that HTG cells treated by Pseudomonas aeruginosa LPS have the same behavior as those previously observed with CF-HTG cells. Exploration by using reverse transcriptase polymerase chain reaction amplification showed that LPS downregulated cystic fibrosis transmembrane conductance regulator (CFTR) mRNA expression in HTG cells indicative of a link between CFTR function and consequent CF-like alteration in protein secretory process.

Reg protein: a potential beta-cell-specific growth factor?

Beta-cell regeneration in adult pancreas is usually considered to be limited. However, various animal models suggest that this tissue is still capable of regeneration under certain conditions. Reg protein could be responsible for this replicative process. The reg gene codes for a 166 amino-acid protein usually synthesized and secreted by pancreatic acinar cells but expressed in islet beta cells during experimental regenerative processes in animals (90% pancreatectomy + nicotinamide, or insulinoma tumor removal in rats, or the "wrapping pancreas model" in the hamster). In addition, recombinant rat reg protein can stimulate beta-cell replication in vivo and in vitro. In animal models of Type 1 diabetes mellitus, reg gene overexpression occurs during active phases of diabetogenesis and could be a defence mechanism. During human pancreatic development, reg gene is expressed at an early stage but is not associated with the expression of other pancreatic genes. Conversely, gene expression for reg and insulin are correlated in adult pancreas. Accordingly, reg protein could be a beta-cell-specific growth factor implicated in the maintenance of beta-cell mass, especially in adult pancreas.

Pancreatic regenerating gene overexpression in the nonobese diabetic mouse during active diabetogenesis.

The reg gene has previously been shown to be associated with regeneration of pancreatic islets. Strategies for influencing the replication and the growth of the beta-cell mass may be important for prevention and/or treatment of type I diabetes. In this study, we have examined the level of reg gene expression at various degrees of diabetogenesis in the pancreas of the NOD mouse (male, female, and cyclophosphamide-treated male) using both human reg cDNA as the probe and dot blot analysis. The expression of the reg gene was found to be significantly increased in female mice compared with male mice, and in both cases, the expression level was not influenced by age. Nondiabetic female mice have a significantly higher expression of the gene than diabetic female mice, and there was a positive correlation between the age of diabetes onset and the reg mRNA level. In addition, overexpression of the reg gene was found in male mice treated by cyclophosphamide, an agent known to be a potent inducer of diabetes in male NOD mice. None of these results were found in the diabetes-resistant control OF1 mice, in which pancreatic reg gene expression did not differ between female and male mice treated or untreated with cyclophosphamide. All of these data suggest that there is a strong correlation between reg gene expression in the pancreas of the NOD mouse and the likelihood of developing diabetes.