Characterization of CD34+ hematopoietic cells in systemic mastocytosis: Potential role in disease dissemination.

BACKGROUND: Recent studies show that most systemic mastocytosis (SM) patients, including indolent SM (ISM) with (ISMs+) and without skin lesions (ISMs-), carry the KIT D816V mutation in PB leukocytes. We investigated the potential association between the degree of involvement of BM hematopoiesis by the KIT D816V mutation and the distribution of different maturation-associated compartments of bone marrow (BM) and peripheral blood (PB) CD34+ hematopoietic precursors (HPC) in ISM and identified the specific PB cell compartments that carry this mutation. METHODS: The distribution of different maturation-associated subsets of BM and PB CD34+ HPC from 64 newly diagnosed (KIT-mutated) ISM patients and 14 healthy controls was analyzed by flow cytometry. In 18 patients, distinct FACS-purified PB cell compartments were also investigated for the KIT mutation. RESULTS: ISM patients showed higher percentages of both BM and PB MC-committed CD34+ HPC vs controls, particularly among ISM cases with MC-restricted KIT mutation (ISMMC ); this was associated with progressive blockade of maturation of CD34+ HPC to the neutrophil lineage from ISMMC to multilineage KIT-mutated cases (ISMML ). Regarding the frequency of KIT-mutated cases and cell populations in PB, variable patterns were observed, the percentage of KIT-mutated PB CD34+ HPC, eosinophils, neutrophils, monocytes and T cells increasing from ISMs-MC and ISMs+MC to ISMML patients. CONCLUSION: The presence of the KIT D816V mutation in PB of ISM patients is associated with (early) involvement of circulating CD34+ HPC and multiple myeloid cell subpopulations, KIT-mutated PB CD34+ HPC potentially contributing to early dissemination of the disease.

Increased IL6 plasma levels in indolent systemic mastocytosis patients are associated with high risk of disease progression.

Systemic mastocytosis (SM) is a heterogeneous disease with altered interleukin (IL)-6 and IL13 plasma levels. However, no study has simultaneously investigated the plasma levels of IL1ß, IL6, IL13, CCL23 and clusterin in SM at diagnosis and correlated them with disease outcome. Here we investigated IL1ß, IL6, IL13, CCL23 and clusterin plasma levels in 75 SM patients--66 indolent SM (ISM) and 9 aggressive SM--and analyzed their prognostic impact among ISM cases grouped according to the extent of hematopoietic involvement of the bone marrow cells by the KIT D816V mutation. Although increased IL1ß, IL6 and CCL23 levels were detected in SM patients versus healthy controls, only IL6 and CCL23 levels gradually increased with disease severity. Moreover, increased IL6 plasma levels were associated with ISM progression to more aggressive disease, in particular among ISM patients with multilineal KIT mutation (ISM-ML), these patients also showing a higher frequency of organomegalies, versus other ISM-ML patients. Of note, all ISM patients who progressed had increased IL6 plasma levels already at diagnosis. Our results indicate that SM patients display an altered plasma cytokine profile already at diagnosis, increased IL6 plasma levels emerging as an early marker for disease progression among ISM cases, in particular among high-risk ISM patients who carry multilineage KIT mutation.

KIT mutation analysis in mast cell neoplasms: recommendations of the European Competence Network on Mastocytosis.

Although acquired mutations in KIT are commonly detected in various categories of mastocytosis, the methodologies applied to detect and quantify the mutant type and allele burden in various cells and tissues are poorly defined. We here propose a consensus on methodologies used to detect KIT mutations in patients with mastocytosis at diagnosis and during follow-up with sufficient precision and sensitivity in daily practice. In addition, we provide recommendations for sampling and storage of diagnostic material as well as a robust diagnostic algorithm. Using highly sensitive assays, KIT D816V can be detected in peripheral blood leukocytes from most patients with systemic mastocytosis (SM) that is a major step forward in screening and SM diagnosis. In addition, the KIT D816V allele burden can be followed quantitatively during the natural course or during therapy. Our recommendations should greatly facilitate diagnostic and follow-up investigations in SM in daily practice as well as in clinical trials. In addition, the new tools and algorithms proposed should lead to a more effective screen, early diagnosis of SM and help to avoid unnecessary referrals.

Probabilidad clínica de tromboembolismo pulmonar: beneficio diagnóstico de las escalas de predicción y de los dímeros D / Clinical suspicion of pulmonary embolism: diagnostic utility of prediction scores and D-dimer measurement

Objetivo: Determinar la precisión de las escalas de Wells, Ginebra y los dímeros-D en pacientes con sospecha clínica de tromboembolismo pulmonar (TEP). Método: Estudio de cohorte prospectivo de precisión diagnóstica de la sospecha de TEP (escalas de Wells y Ginebra y los dímeros D). Análisis de una base de datos de 637 pacientes consecutivos con sospecha de TEP ingresados en el servicio de urgencias generales de un hospital terciario. La medida de resultados muestra la sensibilidad, especificidad, valores predictivos positivo/negativo (VPP/VPN) y las razones de verosimilitud positivas y negativas (RV+/RV-). El patrón oro fue la confirmación de TEP mediante la tomografía computarizada y el seguimiento a 3 meses. Resultados: La edad media fue de 67,9 (DE: 16,3) años y el 54,6% fueron mujeres. La prevalencia global de TEP fue 15,1%. Para la escala de Wells el VPN fue 85,3% (IC 95%: 82,1-88,0), el VPP 62,5% (IC 95%: 42,7-78,8), la sensibilidad 15,6% (IC 95%: 9,7-24,2), la especificidad 98,1% (IC 95%: 96,5-99,0), la RV+ 8,3 (IC 95%: 3,7-18,4) y la RV- 0,86 (IC 95%: 0,78-0,95). Para la de Ginebra, el VPN fue 82,8% (IC 95%: 78,9-86,0), el VPP 58,8% (IC 95%: 36,0-78,4), la sensibilidad 11,8% (IC 95%: 6,5-20,3), la especificidad 98,1% (IC 95%: 96,1-99,1), la RV+ 6,2 (IC 95%: 2,42-15,74) y la RV- 0,90 (IC 95%: 0,81-1,0). Para el dímero-D, el VPN fue 99,2% (IC 95%. 95,4-99,9), el VPP 21,7% (IC 95%: 18,1-25,9), la sensibilidad 98,9% (IC 95%: 94,2-99,8), especificidad 26,4% (IC 95%: 22,6-30,7), la RV+ 1,34 (IC 95%: 1,27-1,43) y la RV- 0,04 (IC 95%: 0,01-0,29). Conclusiones: De acuerdo con los resultados obtenidos la escala de Wells y Ginebra son buenas candidatas para ser utilizadas en urgencias como escalas para establecer la sospecha de TEP; y la de Wells sería la escala de elección por su superior especificidad. Sin embargo ninguna de ellas puede ser utilizada como herramienta diagnóstica por su baja sensibilidad. Así mismo, los dímeros D son una prueba que nos permite excluir el TEP, cuando el resultado es negativo, pero éstos debieran ser aplicados conjuntamente con las escalas

Objective: To assess the utility of the Wells and Geneva scoring systems and D-dimer measurements when pulmonary embolism (PE) is suspected. Methods: Prospective cohort study of the diagnostic performance of the Wells and Geneva scores and D-dimer measurement. We analyzed data for 637 consecutive patients suspected of having PE; the patients were being treated in the emergency department of a tertiary care hospital. The following performance measures were assessed: sensitivity, specificity, positive and negative predictive values (PPV and NPV), and the positive and negative likelihood ratios (LR+ and LR-). PE was confirmed by computed tomography and the patients were followed for the recommended 3 months. Results: The mean (SD) age was 67.9 (16.3) years and 54.6% of the patients were women. The prevalence of PE in the cohort was 15.1%. For the Wells score, the NPV was 85.3% (95% CI, 82.1%-88.0%), and the PPV was 62.5% (95% CI, 42.7%-78.8%). Sensitivity was 15.6% (95% CI, 9.7%-24.2%) and specificity 98.1% (95% CI, 96.5%-99.0%). The LR+ was 8.3 (95% CI, 3.7-18.4), and the LR- was 0.86 (95% CI, 0.78-0.95). For the Geneva score, the NPV was 82.8% (95% CI, 78.9%-86.0%), and the PPV was 58.8% (95% CI, 36.0%-78.4%). Sensitivity was 11.8% (95% CI, 6.5%-20.3%) and specificity 98.1% (95% CI, 96.1%-99.1%). The LR+ was 6.2 (95% CI, 2.42-15.74), and the LR- was 0.90 (95% CI, 0.81-1.0). For D-dimer level, the NPV was 99.2% (95% CI, 95.4%-99.9%), and the PPV was 21.7% (95% CI, 18.1%-25.9%). Sensitivity was 98.9% (95% CI, 94.2%-99.8%) and specificity 26.4% (95%, 22.6%-30.7%). The LR+ was 1.34 (95% CI, 1.27-1.43), and the LR- was 0.04 (95% CI, 0.01-0.29). Conclusions: The Wells and Geneva scoring systems are good useful in the emergency department for establishing a preliminary clinical diagnosis of suspected PE. The Wells score has better specificity. However, diagnosis cannot be based on any of these tools because of their low sensitivity. Similarly, D-dimer measurement can allow PE to be ruled out when the result is negative, but this criterion must be used in conjunction with the scoring systems

Current state of biology and diagnosis of clonal mast cell diseases in adults.

Mastocytosis comprises a heterogeneous group of disorders characterized by the presence of clonal mast cells (MC) in organs such as skin, bone marrow (BM), and gastrointestinal tract, among other tissues. The clonal nature of the disease can be established in most adult patients by the demonstration of activating KIT mutations in their BM MC. When highly sensitive techniques capable of identifying cells present at very low frequencies in a sample are applied, BM MC from virtually all systemic mastocytosis patients display unique immunophenotypical features, particularly the aberrant expression of CD25. By contrast, large, multifocal BM MC aggregates (the only World Health Organization major criterion for systemic mastocytosis) are absent in a significant proportion of patients fulfilling at least three minor criteria for systemic mastocytosis, particularly in subjects studied at early stages of the disease with very low MC burden. Moreover, recent molecular and immunophenotypical investigations of BM MC from patients with indolent systemic mastocytosis have revealed a close association of some biological features (e.g., multilineage involvement of hematopoiesis by the KIT mutation and an immature mast cell immunophenotype) with an increased risk for disease progression. These observations support the fact that, although the current consensus diagnostic criteria for systemic mastocytosis have been a major advance for the diagnosis and classification of the disease, rationale usage of the most sensitive diagnostic techniques available nowadays is needed to improve the diagnosis, refine the classification, and reach objective prognostic stratification of adult mastocytosis.

Validation of the REMA score for predicting mast cell clonality and systemic mastocytosis in patients with systemic mast cell activation symptoms.

BACKGROUND: A variable percentage of patients with systemic mast cell (MC) activation symptoms meet criteria for systemic mastocytosis (SM). We prospectively evaluated the clinical utility of the REMA score versus serum baseline tryptase (sBt) levels for predicting MC clonality and SM in 158 patients with systemic MC activation symptoms in the absence of mastocytosis in the skin (MIS). METHODS: World Health Organization criteria for SM were applied in all cases. MC clonality was defined as the presence of KIT-mutated MC or by a clonal HUMARA test. The REMA score consisted of the assignment of positive or negative points as follows: male (+1), female (-1), sBt <15 µg/l (-1) or >25 µg/l (+2), presence (-2) or absence (+1) of pruritus, hives or angioedema and presence (+3) of presyncope or syncope. Efficiency of the REMA score for predicting MC clonality and SM was assessed by receiver operating characteristic (ROC) curve analyses and compared to those obtained by means of sBt levels alone. RESULTS: Molecular studies revealed the presence of clonal MC in 68/80 SM cases and in 11/78 patients who did not meet the criteria for SM. ROC curve analyses confirmed the greater sensitivity and a similar specificity of the REMA score versus sBt levels (84 vs. 59% and 74 vs. 70% for MC clonality and 87 vs. 62% and 73 vs. 71% for SM, respectively). CONCLUSIONS: Our results confirm the clinical utility of the REMA score to predict MC clonality and SM in patients suffering from systemic MC activation symptoms without MIS.

An immature immunophenotype of bone marrow mast cells predicts for multilineage D816V KIT mutation in systemic mastocytosis.

D816V KIT mutation of bone marrow (BM) mast cells (MC) is a common feature to systemic mastocytosis (SM) patients. Nevertheless, occurrence of the KIT mutation in BM cell compartments other than MC is associated with progression to more aggressive forms of the disease and poor outcome in indolent SM (ISM). Here, we assessed the potential association between the immunophenotype of MC and multilineage KIT mutation in the BM of SM patients through the investigation of the flow cytometric protein expression profile (PEP) of bone marrow mast cells (BMMC) from 70 control individuals and 206 SM patients, classified according to the WHO (World Health Organization), and the degree of involvement of BM hematopoiesis by the D816V KIT mutation; additionally, we developed a score-based class prediction algorithm for the detection of SM cases with multilineage mutation. Our results show that aberrant expression of CD25 with a FcɛRI(lo), FSC(lo), SSC(lo) and CD45(lo) immature phenotype of BMMC, in the absence of coexisting normal MC in the BM, was associated with multilineage involvement by the D816V KIT mutation, regardless of the diagnostic subtype of the disease (for example, indolent vs aggressive SM), which supports the utility of the immunophenotype of BMMC as a surrogate marker to screen for multilineage KIT mutation in ISM.

Redox-sensitive modulation of CD45 expression in pancreatic acinar cells during acute pancreatitis.

CD45, a transmembrane protein tyrosine phosphatase required for signal transduction in leukocytes, has recently been found in pancreatic acinar cells. We have investigated the relationship between kinetic expression of CD45 on acinar cells during acute pancreatitis (AP) and the ability of these cells to produce tumour necrosis factor-alpha (TNF-alpha) through mechanisms sensitive to the cellular redox state. Flow cytometric analysis showed a significant decrease in the constitutive expression of CD45 in acinar cells from six hours onwards after inducing AP by bile-pancreatic duct obstruction (BPDO) in parallel with a significant increase in acinar TNF-alpha production. Changes in protein expression on the acinar cell surface preceded CD45 mRNA down-regulation, which was not found until 12 hours after BPDO. N-Acetylcysteine treatment delayed and reduced the down-regulation of CD45 expression induced by AP and prevented acinar cells from producing TNF-alpha. Our results show that CD45 expression is down-regulated in acinar cells during acute pancreatitis by redox-sensitive mechanisms, and they support the notion that CD45 negatively controls the production of cytokines in pancreatic acinar cells.

Transforming growth factor beta-1 enhances Smad transcriptional activity through activation of p8 gene expression.

We report that exposure of mouse embryonic fibroblasts to transforming growth factor beta-1 (TGFbeta-1) (5 ng/ml) results in a strong activation of p8 mRNA expression that precedes the induction of cell growth. Involvement of the p8 promoter in the regulation was demonstrated by using a p8-chloramphenicol acetyltransferase construct. We therefore speculated that p8 might be a mediator of TGFbeta-1 in these cells. The incorporation of [(3)H]thymidine on treatment with TGFbeta-1 was indeed significantly higher in p8(+/+) fibroblasts than in p8(-/-) fibroblasts. Smad transcriptional activity was used as marker of the TGFbeta-1 signalling pathway, to probe the lower p8(-/-) response to TGFbeta-1. Two Smad-binding elements (SBEs)-luciferase constructs were transfected into p8(-/-) and p8(+/+) embryonic fibroblasts before treatment with TGFbeta-1. A lower level of Smad transactivation was observed in p8(-/-) embryonic fibroblasts, under basal conditions and after stimulation with TGFbeta-1. To test whether Smad underexpression in p8(-/-) cells was actually due to p8 depletion, p8(-/-) embryonic fibroblasts were transfected with a human p8 expression plasmid together with an SBE-luciferase construct. The expression of p8 restored Smad transactivation in unstimulated and TGFbeta-1-treated cells to the level found in p8(+/+) cells. We concluded that TGFbeta-1 activates p8 expression, which in turn enhances the Smad-transactivating function responsible for TGFbeta-1 activity.

Expression of the stress-induced p8 mRNA is transiently activated after culture medium change.

We report here that the mere fact of changing culture medium for fresh medium induced in several cell lines the expression of stress-activated genes including protein kinases p38, JNK and ERK1/2 and the transcription factor C/EBPbeta. As a consequence, p8, a gene induced by stress in several tissues, was strongly up-regulated. Induction did not occur after change for cell-conditioned medium. Induction was however transient, with a peak at 60 min for p38, at 15-30 min for JNK and at 15 min for ERK1/2, at 2-3 hours for C/EBPbeta and at 4-6 hours for p8. Repression of the induction was due to the secretion of thermolabile molecule(s) that progressively conditioned the medium. As low as 25% of conditioned medium added to fresh culture medium was sufficient to abolish the stress response. Taken together, our data indicate that the renewal of culture medium induces a transient cellular stress that may be a source of artifacts in experiments performed shortly after a change of culture medium.

The recovery of acute pancreatitis depends on the enzyme amount stored in zymogen granules at early stages.

Little is known about the changes in pancreatic enzyme storage in acute pancreatitis. We have performed flow cytometric studies of zymogen granules from rats with acute pancreatitis induced by hyperstimulation with caerulein. A comparison was made with rats treated with hydrocortisone (10 mg/kg/day) over 7 days before inducing pancreatitis in order to find out whether the amount of enzymes stored in the pancreas plays a key role in the development of pancreatitis. The potentially therapeutic effect of L-364,718 (0.1 mg/kg/day, for 7 days), a CCK receptor antagonist, was assayed in the rats with caerulein-induced pancreatitis which had previously received the hydrocortisone treatment. A significant increase in the intragranular enzyme content was observed 5 h after hyperstimulation with caerulein. The highest values were reached in the rats previously treated with hydrocortisone. The greatest pancreatic enzyme load was parallel to the highest values in plasma amylase, edema and haematocrit observed. Acute pancreatitis was reversed seven days later. At this stage smaller granules appeared in the pancreas whose enzyme content was similar to that of controls when no treatment was applied after pancreatitis. In contrast, L-364,718 administration prevented the favourable evolution of pancreatitis since the antagonism exerted on CCK receptors induced a blockade of secretion of the large amounts of enzymes stored in the pancreas. Moreover, the enzyme content in zymogen granules was below normal values since the stimulatory CCK action on enzyme synthesis can be inhibited by L-364,718. Our results suggest that the efficiency of CCK antagonists, as potential therapy, would also depend on the load of enzymes in the pancreas when acute pancreatitis is produced.

Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats.

Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.

Structural and functional characterization of the mouse p8 gene: promotion of transcription by the CAAT-enhancer binding protein alpha (C/EBPalpha) and C/EBPbeta trans-acting factors involves a C/EBP cis-acting element and other regions of the promoter.

Rat p8 mRNA was discovered because of its strong activation in pancreas during the acute phase of pancreatitis. We report here structural and functional data on the mouse p8 gene. The mouse p8 polypeptide is 80 amino acids long and shows 91% and 75% identity with its rat and human counterparts respectively. The p8 gene is organized into three exons interrupted by two introns. Promoter regions involved in the regulation of p8 gene expression in NIH 3T3 cells were analysed. Chloramphenicol acetyltransferase (CAT) reporter assays with progressive deletions of the 5' flanking region of the mouse p8 gene revealed four silencer elements located from nucleotides -5000 to -1472, -1471 to -671, -670 to -473, and -239 to -117 respectively. One positive element was identified between nucleotides -117 and -72. We identified a CAAT-enhancer binding protein (C/EBP) cis-acting element at position -111. Site-directed mutagenesis of this consensus site decreased promoter activity to 5% of that of the wild-type. An electrophoretic mobility-shift assay, using an oligonucleotide probe corresponding to the C/EBP consensus and nuclear extracts of NIH 3T3 cells transfected with C/EBPalpha or C/EBPbeta expression vectors, generated specific DNA-protein complexes that were supershifted with specific antibodies against C/EBPalpha and C/EBPbeta. Co-transfection with C/EBPalpha or C/EBPbeta expression vectors and the p-116/+36p8-CAT construct increased the reporter gene activity in a dose-dependent fashion. Surprisingly, overexpression of C/EBPalpha or C/EBPbeta still increased the promoter activity of both pC/EBPmut-116/+36p8-CAT (which contains the C/EBP mutated site) and the p-71/+36-CAT construct (which does not contain the C/EBP site). Collectively, these results show that C/EBPalpha and C/EBPbeta trans-acting factors can promote transcription of the mouse p8 gene (i) by direct binding to the C/EBP consensus site, and (ii) by enhancing the activity of other trans-acting factors interacting with the p8 promoter.

Zymogen granule alterations in caerulein-induced pancreatitis developed during continuous hydrocortisone administration.

BACKGROUND: The amount of enzymes stored in individual zymogen granules and the glycosylation of their membrane have been analysed in rats with acute pancreatitis induced by caerulein after hydrocortisone treatment. The consequences of prolonging hydrocortisone administration after pancreatitis and the use of the cholecystokinin (CCK) receptor antagonist, L-364,718, have also been evaluated. MATERIALS AND METHODS: Analysis was performed using flow cytometry. RESULTS: Caerulein-induced pancreatitis in rats previously treated for 7 days with hydrocortisone (10 mg kg-1 per day) revealed alterations in enzyme storage in the pancreas. Significant increases in amylase and trypsinogen contents in zymogen granules were observed, an effect associated with a reduction in L-fucose glycoconjugates. Pancreatitis persists 7 days later if hydrocortisone treatment is prolonged. At this stage, a reduced granule fucosylation was still observed, and a significant decrease in the amount of trypsinogen stored in the granules was found. However, hydrocortisone administration led to an increase in intragranular amylase quantities up to normal values, even when L-364,718 was simultaneously administered, but it reverted to plasma as a consequence of pancreatitis. The amount of N-acetyl D-glucosamine in the zymogen granule membrane was not altered by caerulein acute pancreatitis induced under continuous hydrocortisone treatment, but it was decreased by the administration of L-364,718 over 7 days after pancreatitis induction. CONCLUSIONS: The administration of hydrocortisone after the development of pancreatitis prevented recurrence of the disease. L-364,718 proved to be detrimental, not only failing to reduce the symptoms of pancreatitis but also altering the glycoproteins of zymogen granule membrane.

Effect of cholecystokinin blockade on the recovery of alterations induced by acute pancreatitis in glycoconjugates of rat zymogen granules.

Lectin-binding studies have been performed on rat zymogen granules to investigate alterations in the carbohydrate membrane composition that occur in acute pancreatitis induced by caerulein. The influence of treatment with hydrocortisone for seven days before inducing pancreatitis was also studied. Lectin labeling on zymogen granules was also analyzed seven days after inducing pancreatitis in rats that had previously received a hydrocortisone treatment. During this period L 364,718 (0.1 mg/kg)--specific cholecystokinin (CCK) receptor antagonist--was administered daily to some of the rats, and no treatment was applied to others. Using fluorescein-labelled T. purpureus (TP)lectin, a significant decrease in the amount of L-fucose in the granule membrane was observed in rats with caerulein-induced pancreatitis. This effect was directly caused by the pancreatitis and was not influenced by previous hydrocortisone treatment. Seven days later, the density of TP receptors in the granule membrane was similar to the controls both in L-364,718-treated and untreated rats. Therefore, we suggest that endogenous CCK is not an essential factor in the recovery of L-fucose containing glycoconjugates the granule membrane after pancreatitis. Acute pancreatitis did not alter the expression of wheat germ agglutinin (WGA) receptors in the zymogen granule membrane. WGA specifically binds N-acetyl glucosamine and sialic acids. L 364,718 administered for seven days after inducing pancreatitis significantly reduced WGA binding, untreated rats showed a normal zymogen granule membrane. Therefore, the blockade of CCK-induced alterations in membrane glycoconjugates enriched in N-acetyl glucosamine and sialic acid of newly formed granules after pancreatitis, a finding that could explain the delay in the regression of the disease.

Glucocorticoids regulate L-fucose glycoconjugates in rat pancreatic zymogen granules.

Lectin-binding studies were performed on rat pancreatic zymogen granules to investigate the influence of glucocorticoid levels on saccharide membrane composition. The following animal groups were used: (1) control rats; (2) rats treated with hydrocortisone (1, 10 and 25 mg/kg/day) for 1, 3 and 8 days; (3) postadrenalectomized rats at days +1, +3 and +8; and (4) adrenalectomized rats receiving hydrocortisone therapy (10 mg/kg/day) for 8 days. By flow cytometry, fluoresceinated (FITC) lectins were used to measure the amount of Concanavalin A (Con A) (specific for D-mannose), wheat germ agglutinin (WGA) (specific for N-acetyl-D-glucosamine) and sialic acids and Tetragonolobus purpureus (TP) (specific for L-fucose) bound to individual zymogen granules from two subpopulations, Z1 and Z2, identified on the basis of their forward and side scatter properties. The molar ratio of the different FITC-lectins revealed significant differences in the glycoconjugate composition of Z1 and Z2 granules, the Z1 granules showing higher ratios of N-acetyl-D-glucosamine:L-fucose and N-acetyl-D-glucosamine:D-mannose, both in control, adrenalectomized and hydrocortisone-treated rats. It was also observed that N-acetyl-D-glucosamine and/or sialic acids were more abundant than L-fucose and D-mannose in the zymogen granule membrane. Z1 and Z2 granules had different glycosylation patterns. Neither adrenalectomy nor hydrocortisone treatments varied the Con A binding to zymogen granules. An increase in WGA binding was only induced by administration of very high doses of hydrocortisone (25 mg/kg/day) for 8 days, an effect not directly related to glucocorticoids. In contrast, a correlation between the FITC-TP labelling and glucocorticoid levels can be established, so that, in a time-dose dependent way, an increase was observed in zymogen granules of rats treated with hydrocortisone while a decreased TP binding was found in adrenalectomized rats-an effect which was reversed with hydrocortisone therapy. Therefore, glucocorticoids exert a direct influence on the saccharide composition of rat pancreatic zymogen granules, regulating the amount of L-fucose glycoconjugates, with Z2 granules more sensitive than Z1 ones.

Long-term blockade of cholecystokinin (CCK): effects of L 364,718 (a CCK receptor antagonist) on pancreatic enzyme storage and secretion.

To determine the effect of long-term blockade of cholecystokinin (CCK) on both pancreatic storage and secretion processes, L 364,718 (a CCK receptor antagonist) was administered to rats at 0.1 mg/kg/day for 3, 7, and 15 days. Zymogen granules were analyzed by flow cytometry to determine their light scattering properties-forward scatter and side scatter-as well as their amylase content measured by a specific antiserum. The mean number of zymogen granules per cell was counted on pancreatic sections using electron microscopy. DNA content, pancreatic weight, and enzyme secretion were also studied under both basal conditions and CCK infusion at a dose of 1.25 micrograms/kg/h, which is able to displace the CCK receptor antagonist. Two subpopulations of zymogen granules (Z1 and Z2) were identified on the basis of their light scattering parameters, in both control and L 364,718-treated rats. L 364,718 administered for 3, 7, and 15 days induced a significant reduction in the amylase content of individual zymogen granules, for both Z1 and Z2 zymogen granule subsets. In contrast, the number of zymogen granules per cell increased from day +3 of treatment onward, the highest values being detected at day +7. Hyperplasia was observed only at day +15. Basal enzyme secretion decreased significantly in rats treated with L 364,718 for 3 and 7 days but recovered to control values after 15 days of treatment. No significant differences in CCK-stimulated amylase secretion were observed between control and L 364,718-treated rats. At day +15 of L 364,718 treatment a significant increase in enzyme secretion was observed with respect to shorter treatment periods; this was associated with a significant increase in both the number of cells and the number of zymogen granules per cell. Our results indicate that chronic administration of L 364,718 induces a biphasic effect on pancreatic function. Interestingly, although enzyme secretion reached recovery after long-term treatment (15 days), the storage process is altered since the mean enzyme content in each individual zymogen granule remains significantly reduced.

Hydrocortisone induces an increase of amylase content in individual zymogen granules from rat pancreas.

This study was performed to evaluate the effects of different doses of hydrocortisone (1, 10 and 25 mg/kg/day) administered for 1, 3 and 8 days on pancreatic enzyme storage in rats. The enzyme content in both pancreas homogenates and in individual isolated zymogen granules (ZGs) was measured using standard biochemical assays and flow cytometry, respectively. Hydrocortisone did not alter the total amount of pancreatic DNA but increased the pancreas enzyme content in a time-dose-dependent way. Amylase activity was significantly increased after hydrocortisone administration at day +8 when 10 mg/kg/day was used, and from the first day of treatment when 25 mg/kg/day was administered. A significant increase in trypsin activity was also observed in response to 25 mg/kg/day of hydrocortisone but only from the third day of treatment onwards. As compared with control rats, chronic administration of either 1 or 10 mg/kg/day of hydrocortisone did not alter significantly either the size or the percentage of the two ZG subpopulations (Z1 and Z2) identified in the pancreas by flow cytometry; in addition, no significant changes were observed in the mean amylase content per individual granule, although its mean concentration increased in rats treated with 10 mg/kg/day for 3 and 8 days. Nevertheless, when 25 mg/kg/day of hydrocortisone were administered for 1 and 3 days, a significant increase in the proportion of Z1 ZGs was observed, which may be related to the formation of new and smaller ZGs. When a very high dose of hydrocortisone (25 mg/kg/day) was used, an overall increase in the pancreatic enzyme content related to an increase in the mean amylase content per individual ZG was observed; this effect was apparent from the first day of treatment in the Z1 subset of ZGs and from day +3 in the Z2 subpopulation. Only a high concentration of hydrocortisone was able to alter the enzyme storage process in individual zymogen granules, but they maintain a normal enzyme load at lower hydrocortisone doses.