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I de Dios, AC Garcia-Montero, A Orfao and MA Manso

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.

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A C Garcia-Montero, I De Dios, A I Rodriguez, A Orfao and M A Manso


The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90° light scatter (SSC) parameter) and amylase content were evaluated. Amylase content/DNA ratio in pancreatic homogenates was also analyzed. The zymogen granules of the control rats were found to be distributed in two populations: a major one – R1 (95·45 ± 1·21%) – containing zymogen granules with a smaller mean size and complexity, and a minor population - R2 (4·45 ± 0·24%) – the granules of which had a mean size which was larger and more complex. At day +1 after adrenalectomy the zymogen granules were significantly (P<0·05) smaller than those of control animals. The R2 zymogen granules were similar to those from R1 as regards their size, but were more complex, suggesting that the immediate effect of glucocorticoid deprivation is to induce a depletion of the larger granules presumably belonging to the R2 population. The amount of amylase per granule did not vary at day +1 after adrenalectomy, although the amylase content/size ratio per granule was significantly (P<0·001) increased. This mechanism could be explained in terms of the existence of a bypass defined in the adrenalectomized animals between the granular content and cytosolic enzymes. Prolongation of the adrenalectomy period to 3 and 7 days resulted in a progressive increase in zymogen granule size and complexity, both parameters showing similar characteristics to those of the controls at day +7 after adrenalectomy. However, the percentage of zymogen granules within the R1 and R2 populations was clearly different from that of controls since the R2 population was much more numerous (11·25 ± 0·75% and 15·25 ± 1·15% (adrenalectomized rats at days +3 and +7 respectively) versus 4·45 ± 0·24% (controls)). An increase in the content of amylase per DNA was observed in adrenalectomized rats at day +1 although this transient effect cannot be related to glucocorticoid deprivation because it was also observed in sham-operated rats (day +1). However, a significant reduction, nearly 64%, in the amylase content/DNA ratio is produced by the absence of glucocorticoids 7 days after adrenalectomy and this is associated with a reduction in the content of amylase in each individual zymogen granule which reaches a minimum 3 days after adrenalectomy. It should be noted that, despite this, the enzyme concentration in each granule remains constant as there is a parallel decrease in the zymogen granule amylase content and size.

Journal of Endocrinology (1995) 147, 431–440

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JJ Corrales, A Lopez, J Ciudad, MT Mories, JM Miralles and A Orfao

At present, the in vivo response of T, B and natural killer (NK) cells to antithyroid drug therapy remains largely unknown. In the present study, we have prospectively analyzed the in vivo effects of methimazole treatment on a large number of circulating T and NK cell subsets, some of them expressing cell surface activation antigens involved in the very early phase of the immune response, in a group of 17 hyperthyroid, untreated patients with Graves' disease (GD). As one of the first events during T cell activation is the expression of interleukin (IL) receptors, we also studied the binding of IL-2 and IL-6 to T cells. Patients with Graves' disease were sequentially studied at diagnosis/before treatment (day 0) and 7, 14, 30, 90 and 180 days after methimazole therapy. The results were compared with both a group of 19 age- and sex-matched control volunteers and a group of 20 untreated/euthyroid patients with Graves' disease in long-term remission. The combination of flow cytometry and three-color immunofluorescence revealed a clear (P < 0.01) decrease in the percentage of NK cells before and during the whole course of therapy with respect to both controls and patients with Graves' disease who were in long-term remission. Before therapy, a marked increase (P < 0.001) in the ratio of B to NK cells was also observed; thereafter, a slight decrease in this ratio was observed, although normal values were detected only in patients in long-term remission. Expression of the CD69 early activation antigen in the hyperthyroid untreated patients with Graves' disease was clearly increased (P < 0.01) with respect to both controls and patients with Graves' disease who were in long-term remission. This abnormal CD69 expression was found to be significantly reduced (P < 0.001) by methimazole therapy, and this represents a new effect of the drug. Expression of the low-affinity receptor for IL-2 (CD25)--another early T cell activation marker--was not altered in Graves' disease, but the binding of IL-2 and IL-6 to T cells exhibited a progressive and parallel increase during the first 30 days of therapy, decreasing thereafter. Our results show that methimazole therapy downregulates the abnormally high expression of the CD69 early activation antigen on T cells, being less effective on inducing changes in other T cell activation markers and in NK cells.

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J J Corrales, A Orfao, A López, J Ciudad and M T Mories


The immunosuppressive effects of antithyroid drug therapy are well recognized; however, the cellular mechanisms underlying their action remain largely unknown. In the present paper we have prospectively analyzed the in vivo effects of methimazole treatment on a large number of circulating B cell subsets, involved in the effector phase of the immune response, in a group of 18 hyperthyroid patients with Graves' disease (GD). The patients were sequentially studied before (day 0) and 7, 14, 30, 90 and 180 days after methimazole therapy. The results were compared with both a group of 19 age- and sex-matched healthy controls and a group of 20 untreated/euthyroid GD patients in long-term remission. The combination of flow cytometry and three colour immunofluorescence revealed a clear increase (P<0·001) in the numbers of circulating total B cells (CD19+) due to a significant increase (P<0·001) in the CD5+, FMC7+, CD5+/FMC7+ and CD23+ B cell subsets in hyperthyroid GD patients with respect to both healthy individuals and to GD patients in long-term remission. The absolute numbers of all these B cell subsets analyzed before treatment, although abnormal, were not statistically different from those observed during the whole period of therapy. When comparing the percentages of these B cell subsets during treatment, significant changes (P<0·001) were only observed in the proportion of CD5+, CD5+/FMC7+ and CD5 −B cells at the end of the follow-up period with respect to those found both before and during the first month of therapy. Whereas CD5+ and CD5+/FMC7+ B cells decreased (P<0·001) after 3 months of therapy, CD5 − B cells showed a significant increase (P<0·001) at the end of therapy. It is remarkable that the percentage of CD5+, CD5+/FMC7+, CD5 − and CD23+ B cell subsets were abnormal during the whole period of treatment and that they never reached normal values. These results show that, in vivo, GD patients treated with methimazole exhibited an abnormal but rather stable pattern of B cell distribution, similar to that present in hyperthyroid untreated GD patients, except for the CD5+ and CD5 − B cell populations. Our findings suggest that in vivo methimazole therapy would not directly have an important influence on circulating B cell subsets.

Journal of Endocrinology (1996) 151, 231–240

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E. Carbajo-Pérez, S. Carbajo, A. Orfao, J. L. Vicente-Villardón and R. Vázquez


Flow cytometric analysis of nuclei stained with propidium iodide (PI) has been used to study the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male Sprague–Dawley rats at different times of the day. According to PI fluorescence intensity the relative numbers of cells in S phase (cells with a DNA content between that of somatic cells in interphase (2n) and that of somatic cells after duplication of the DNA prior to cell division (4n)) and G2/M phase (4n) were calculated. A significant circadian rhythm was found for cells in both the S phase (P < 0·05) and the G2/M phase (P < 0·01). The wave of cells in S phase with a peak at the middle of the light period (14.00 h) precedes by about 6 h the wave of cells in G2/M phase (peak at 20.00 h). Most of the DNA-replicating cells were found during the early S phase at 11.00 h, advancing further up to the middle of this phase at 14.00 h. Cells were distributed homogeneously throughout the S phase at 17.00 h. These data strongly suggest that the beginning of the light period triggers a wave of cells to leave G0/G1 into S phase.

Journal of Endocrinology (1991) 129, 329–333

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J J Corrales, M Almeida, R Burgo, M T Mories, J M Miralles and A Orfao

Androgens are considered to have immunomodulatory effects but their cellular mechanisms of action remain largely unknown. In the present study we prospectively analyzed the serial effects of androgen-replacement therapy on both the distribution of peripheral blood lymphocytes, monocytes and dendritic cells as well as on the production of interleukin (IL)-1β, IL-6 and tumor necrosis factor α (TNFα) inflammatory cytokines by circulating monocytes and CD33 myeloid, CD16 and plasmacytoid dendritic cell subsets, the most potent antigen-presenting cells (APCs) in type-2 diabetic men with partial androgen deficiency. Analyses were performed before therapy and at 1, 3, 6 and 12 months after treatment with 150 mg testosterone enanthate every 2 weeks in a group of 13 type-2 diabetic men. Our results show for the first time that testosterone-replacement therapy is associated with a reduction or complete abrogation of spontaneous ex vivo production of IL-1β, IL-6 and TNFα by APCs. Meanwhile, the in vitro production of inflammatory cytokines by these cells after stimulation with lipopolysaccharide plus recombinant human interferon-γ remained unchanged, suggesting that APCs preserve their constitutive machinery to produce inflammatory cytokines under androgen treatment. These results confirm and extend previous observations about the anti-inflammatory effects of androgen therapy on APCs in a new, previously unexplored model of androgen deficiency; namely, aging type-2 diabetic men. A decreased production of inflammatory cytokines by APCs might have important consequences for sex differences in susceptibility to autoimmune diseases, inflammatory response to injury and atheromatosis.