Pancreas Acinar Cell Regeneration: XII. Protein Metabolism as Determined by 3H-Leucine Autoradiography

Protein metabolism was studied in the regenerating rat pancreas acinar cell by ³H-leucine autoradiography. Rats were placed for 10 days on a protein-free ethionine (PFE) regimen which caused necrosis of most of the acinar cells. Withdrawing the PFE regimen and instituting a stock diet (SD) on day 11 was followed by good restitution of acinar cell morphology. DNA synthesis peaked at day 15. Intraperitoneal injection of ³H-leucine and sacrifice at 5, 10 and 30 minutes and at 1, 4 and 24 hours after injection at PFE-SD days 10, 12, 15 and 29 and at the same time periods in control SD animals provided tissue for autoradiography. At PFE-SD days 10, 12 and 15, there were great increases in the concentration of autoradiographic grains over the nucleolus, nucleus and cytoplasm of acinar cells as compared to similar compartments in SD animals. There also were more total grains per nucleus and nucleolus in the PFE-SD animals at these days. The grains per unit area of nucleus and cytoplasm of PFE-SD acinar cells at days 10, 12 and 15 increased to about the same degree, but the increase in concentration of grains per unit area of nucleolus was much higher than in the two other compartments. The increase in total number of grains in the nucleolus was much higher than that per nucleus in PFE-SD animals on days 12 and 15. Some of the disproportionately high concentration and amount of newly synthesized labeled protein in the nucleolus at PFE-SD days 12 and 15 may have been involved in genomic activity, giving rise to the peak of DNA synthesis at day 15.     

Formation and fate of ethionine-induced cytoplasmic crystalloids in rat parotid acinar cells

The formation and fate of cytoplasmic crystalloids in rat parotid acinar cells were investigated during ethionine intoxication and recovery. By day 3 of ethionine treatment, acinar cells had numerous autophagic vacuoles containing recognizable secretory granules and fragments of rough endoplasmic reticulum. By day 5, immature crystalloids were present in many of the autophagic vacuoles, and as the crystalloids matured, a 7-nm periodicity became apparent. Crystalloids were never observed in the Golgi saccules or in any other organelle associated with secretory granule formation. When ethionine treatment was stopped, the acinar cells rapidly returned to their normal morphology. The majority of the crystalloids and autophagic vacuoles were lost from the cells during the first two to three days of recovery. At this time annulate lamellae were present intracellularly, and macrophages, many contaning crystalloids, were associated with the basal surface of the acinar cells. These results indicate that the cytoplasmic crystalloids are formed in autophagic vacuoles, and do not represent an abnormal secretory product. Additionally, during recovery crystalloids may be removed from the acinar cells by interaction with macrophages. The sequence of autophagic vacuole formation, development of crystalloids, macrophage infiltration and phagocytosis of acinar cell debris appears to be a non-specific response of the rat parotid gland to cellular injury occurring in a variety of experimental and pathological conditions.     

Sequential ultrastructural changes of the pancreas in zinc toxicosis in ducklings.

The sequential ultrastructural alterations of the pancreas in zinc toxicosis were examined in ducklings fed 2500 ppm Zn (as ZnSO4) for 56 days. From days 3 to 17, acinar cells had cytoplasmic vacuoles that contained electron-dense, zymogen-like material and increased autophagocytosis. Other changes were swollen mitochondria and dilatation, vesiculation, degranulation and intracisternal sequestration of rough endoplasmic reticulum. Apoptosis was the predominant form of cell deletion. By day 10, acinar cellular atrophy and interstitial fibrosis were noted. Islets appeared normal. After day 19, the pancreas consisted of ductlike structures embedded in fibrous connective tissue with a minimal inflammatory cell response. These ductlike structures were lined by attenuated to cuboidal, atrophic acinar cells. Many cells contained granular, electron-dense cytoplasmic debris that served as a marker of previous cell damage. This ultrastructural study provides support for a previously proposed theory that ductlike structures (tubular complexes) arise by atrophy and dedifferentiation of acinar cells.     

Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline-deficient diet.

Female, albino mice were fed a choline-deficient diet containing 0.5% DL-ethionine. All animals died within 5 days due to the development of an acute hemorrhagic pancreatis with fat necrosis throughout the peritoneal cavity. The apancreatitis was characterized by a massive necrosis of the exocrine parenchyma with intense hemorrhage and inflammatory reaction of the stroma. The sequence of histologic and ultrastructural alterations occurring in the acinar cells of the pancreas were studied in mice fed the diet for 1, 2, and 3 days. Major findings consited of accumulation of zymogen granules, vacuolation due to foci of cytoplasmic degradation, and alterations in the morphology of the zymogen granules. The pancreatitis appears to be due to intraparenchymal activation of zymogens, resulting from a synergistic action of choline deficiency with the basic toxicity of ethionine toward the acinar cells of the pancreas. The experimental model simulates closely the acute hemorrhagic pancreatitis with fat necrosis occurring in humans and may prove useful for exploring the pathogenesis of this condition.     

Effect on cellular and humoral immune responses of the AS03 adjuvant system in an A/H1N1/2009 influenza virus vaccine administered to adults during two randomized controlled trials

The influence of AS03_A, a tocopherol oil-in-water emulsion-based adjuvant system, on humoral and cell-mediated responses to A/California/7/2009 H1N1 pandemic vaccine was investigated. In two observer-blind studies, a total of 261 healthy adults aged 18 to 60 years were randomized to receive either AS03_A-adjuvanted H1N1 vaccine containing 3.75 μg hemagglutinin (HA) or nonadjuvanted H1N1 vaccine containing 15 or 3.75 μg HA on days 0 and 21. Hemagglutination inhibition (HI) antibody and T-cell responses were analyzed up to day 42. A first dose of AS03_A-adjuvanted vaccine (3.75 μg HA) or nonadjuvanted vaccine (15 μg HA) induced HI responses of similar magnitudes that exceeded licensure criteria (e.g., 94 to 100% with titers of ≥40). A lower response following 3.75 μg HA without adjuvant was observed (73% with titers of ≥40). Following a second dose, geometric mean HI titers at day 42 were higher for AS03_A-adjuvanted vaccine (636 and 637) relative to nonadjuvanted vaccine (341 for 15 μg HA and 150 for 3.75 μg HA). Over the 42-day period, the increase in frequency of A/H1N1/2009-specific CD4⁺ T cells was significantly higher in the adjuvanted group than in the nonadjuvanted group. There was no evidence of correlation between baseline CD4⁺ T-cell frequencies and day 21 HI antibody titers, while there was some correlation (R = 0.35) between day 21 CD4⁺ T-cell frequencies and day 42 HI titers. AS03_A adjuvant enhanced the humoral and CD4⁺ T-cell-mediated responses to A/H1N1/2009 vaccine. Baseline A/H1N1/2009-specific CD4⁺ T-cell frequencies did not predict post-dose 1 antibody responses, but there was some correlation between post-dose 1 CD4⁺ T-cell frequencies and post-dose 2 antibody responses.     

Immunoglobulin peptide chain synthesis in the newborn rabbit

Lymphoid tissue from newborn rabbits was examined using fluorochrome conjugated goat antisera to rabbit γ, μ and α heavy chain and mixed light chain. Unimmunized newborn rabbits examined at intervals up to 20 days of age revealed a very low background of approximately one in 10⁵ cells which stained with either the anti-μ, anti-γ or anti-L chain reagents. Newborn rabbits, immunized intraperitoneally on the day of birth with S. paratyphi B (4, 5, 12:b), showed a 1000-fold increase in the number of stained cells; μ heavy chain containing cells were found 16 hours after immunization, and γ heavy chain containing cells were found 20 hours after immunization in the rabbit spleen tissue. The numbers of μ containing cells and γ containing cells were approximately equal at 5 days of age in the immunized groups. No α heavy chain containing cells were found in any of the rabbits studied. All cells which stained with the anti-μ or γ heavy chain reagent were also stained with the anti-light chain reagent.The cellular response of the immunized newborn rabbits could be inhibited by passive antibody to S. paratyphi B, either given at the time of immunization, or passively acquired from the doe. Specificity of inhibition was indicated by the failure of sheep red blood cells (SRBC) stroma to be inhibited in animals so suppressed.The range of cellular morphology of the γ chain containing and the μ chain containing cells was indistinguishable. The earliest cell to be stained with either anti-γ or anti-μ was a large blast-like cell with a thin rim of cytoplasm and a large nucleus. A full range of forms between this and typical plasma cells appeared later in each case.These findings present a paradox since γG antibody to this antigen does not appear in the serum of such animals following γM class in the usual sequence characteristic of adult animals. Possible explanations of this paradox are explored.     

Transient and ectopic expression of lumican by acinar cells in L-arginine-induced acute pancreatitis

Lumican is a member of a small leucine-rich proteoglycan family. We previously found that lumican mRNA and its protein were ectopically and highly expressed in acinar cells in chronic pancreatitis (CP)-like lesions close to pancreatic cancer cells. CP-like lesions are characterized by acinar and ductal-ductular cell proliferation with expanding fibrosis. This finding suggests that lumican is ectopically synthesized by acinar cells under chronic inflammatory conditions and plays a role in fibrosis of the pancreas. However, the expression and role of lumican in acute inflammatory changes of the pancreas are not completely elucidated. In the present study, we aim to clarify whether lumican mRNA and its protein are expressed in exocrine or endocrine components in acute pancreatitis (AP). For experimental AP, Wistar rats received an intraperitoneal injection of L-arginine. Western blot analysis showed an intense 50-kDa band corresponding to the lumican protein in normal and L-arginine-treated rat pancreas. After L-arginine injection, three intense bands at 42, 57, and 92 kDa were detected on day 1. Immunohistochemically, the lumican protein was localized in ductal and a few centroacinar cells in the normal pancreas. After L-arginine injection, an immature fibrosis with fragmented and loose collagen fibers was observed in AP on day 4 and lumican immunoreactivity was detected in the collagen fibers. Lumican mRNA was faintly detected in islet cells in the normal pancreas, but it was strongly expressed in acinar and islet cells on day 1. Furthermore, lumican mRNA was expressed in many proliferating fibroblasts on day 4 by in situ hybridization. These findings indicate that lumican is transiently synthesized by acinar cells and fibroblasts in AP. Lumican proteins synthesized by acinar cells, islet cells, and fibroblasts may contribute to immature and transient fibrosis of AP.     

Effect of d,l-ethionine administration on the histomorphology of canine pancreatic acinar and β-cells

d,l-Ethionine produces pancreatic exocrine necrosis and islet proliferation in hamsters and dogs. As a first step in examining whether induction of islet proliferation has therapeutic applications in animals with exhausted or destroyed insulin-producing beta-cells, we studied pancreatic pathological alterations after intravenous administration of d,l-ethionine in normal dogs. Histomorphological changes in pancreatic acinar cells and beta-cells were assessed in three groups of six clinically normal crossbred dogs administered d,l-ethionine (100 mg/ kg) intravenously three times a week for two weeks. Six additional dogs served as untreated controls. Group I was euthanased and necropsied on day 15 (72 hours after the final dose of ethionine). Groups II and III were euthanased on days 29 and 43 respectively. Severe acinar destruction occurred resulting in significant (p < 0.001) shrinkage of the pancreas in all groups. Although there was variability in histomorphology within groups, pancreases of group I generally exhibited widespread loss of pancreatic acinar structure. Remaining acinar cells were difficult to discern from other cell types within lobules and were surrounded by infiltrates, predominantly of lymphocytes. Partial acinar cellular regeneration had occurred by day 29, but was still incomplete at day 43. Immunohistochemistry suggested that the effect of d,l-ethionine administration on the histomorphology of beta-cells in the left lobe was minimal; however, morphometry demonstrated a significant (p < 0.05) increase in the number of individual beta-cells in groups II and III, and clusters of 2-10, 10-20 and 20+ cells in Group II. It is probable that the apparent increase in the number of individual and other beta-cell arrangements observed in some groups resulted primarily from alterations in the exocrine tissue, although some beta-cell hyperplasia cannot be excluded completely.     

Mechanism of induction of immunological tolerance. IV. The effects of ultra-low doses of flagellin

Minute amounts of Salmonella adelaide flagellin were shown to be capable of inducing tolerance in newborn Wistar rats. With the range of doses, 10^-9–10^-1 μg, the following results were obtained:(1) Tolerance could be induced in two zones of dosage by daily injections of flagellin during the first 2 weeks of life. The low zone corresponded to a dose of 10^-7 μg/g body weight and the high zone to 10^-3 μg/g body weight. Doses between these evoked an immune response.(2) Daily injections of flagellin for 6 weeks resulted in a shift in the dosage required for low zone tolerance from 10^-7 μg/g body weight to 10^-5 μg/g body weight.(3) The events of low zone tolerance occurred at an antigen concentration, which, in an organ such as the spleen of a newborn rat, never exceeded about 10^-14 M. The high zone of tolerance corresponded to an antigen concentration of about 10^-10 M.     

Prevalence of occult adrenal insufficiency and the prognostic value of a short corticotropin stimulation test in patients with septic shock

Background:

Corticosteroid insufficiency in acute illness can be difficult to discern clinically. Occult adrenal insufficiency (i.e., Δmax ≤9 μg/dL) after corticotropin may be associated with a high mortality rate.

Objective:

To assess the prevalence of occult adrenal insufficiency and the prognostic value of short corticotropin stimulation test in patients with septic shock.

Materials and Methods:

A total of 30 consecutive patients admitted in the adult intensive care unit of the Sheri Kashmir Institute of Medical Sciences who met the clinical criteria for septic shock were prospectively enrolled in the study. A low dose (1 μg) short corticotropin stimulation test was performed; blood samples were taken before the injection (T0) and 30 (T30) and 60 (T60) minutes afterward.

Results:

The prevalence of occult adrenal insufficiency was 57%. The 28-day mortality rate was 60% and the median time to death was 12 days. The following seven variables remained independently associated with death: organ system failure scores, simplified acute physiology score II score, mean arterial pressure, low platelet count, PaO₂:FIO₂, random baseline cortisol (T0) >34 μg/dL, and maximum variation after test (Δmax) of ≤9 μg/dL. Three different mortality patterns were observed: (I) low (T0 ≤34 μg/dL and Δmax >9 μg/dL; a 28-day mortality rate of 33%),(II) intermediate (T0 >34 μg/dL and Δmax >9 μg/dL or T0 ≤34 μg/dL and Δmax ≤9 μg/dL; a 28-day mortality rate of 71%), and (III) high (T0 >34 μg/dL and Δmax ≤9 μg/dL; a 28-day mortality rate of 82%).

Conclusion:

A short corticotropin test using low-dose corticotropin (1 μg) has a good prognostic value. High basal cortisol and a low increase in cortisol on corticotropin stimulation test are predictors of a poor outcome in patients with septic shock.     

Safety and Immunogenicity of a Single Oral Dose of Recombinant Double Mutant Heat-Labile Toxin Derived from Enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a primary cause of traveler''s diarrhea for which there is no licensed vaccine. This phase 1 trial determined the safety and immunogenicity of a recombinantly produced double mutant heat-labile enterotoxin (dmLT) of ETEC. It was administered as a single oral dose of dmLT in escalating doses of 5 μg, 25 μg, 50 μg, and 100 μg, followed by a 72-h inpatient observation, outpatient visits at 8, 14, and 28 days, and telephone calls at 2 and 6 months postvaccination. Safety was assessed by frequency of adverse events, and immune responses determined after immunization included dmLT-specific serum IgA and IgG, fecal IgA, antibody-secreting cells (ASC), and antibodies in lymphocyte supernatant (ALS) responses. All doses were well tolerated by the 36 healthy adults enrolled. Immune responses were limited in the 5- and 25-μg dose recipients. The 50-μg dose recipients trended toward stronger responses than the 100-μg dose recipients by serum IgA (67% versus 33%, P = 0.22), serum IgG (58% versus 33%, P = 0.41), and fecal IgA (58% versus 33%, P = 0.41). By day 14 postvaccination, there were significantly more positive responders (≥4-fold increase from baseline) among the 50- versus 100-μg dose recipients for serum IgA (P = 0.036) but not serum IgG (P = 0.21). In conclusion, a single oral dose of dmLT was well tolerated and immunogenic, with immune responses plateauing at the 50-μg dose. (This clinical trial is registered at www.clinicaltrials.gov, registration number {"type":"clinical-trial","attrs":{"text":"NCT01147445","term_id":"NCT01147445"}}NCT01147445.)     

Studies on Acute Methionine Toxicity: I. Nucleolar Disaggregation in Guinea Pig Hepatic Cells with Methionine or Ethionine and Its Reversal with Adenine

The effects of methionine and ethionine on the fine structure of hepatic cell nucleoli of guinea pigs and rats were investigated. A single intraperitoneal injection of methionine into guinea pigs results in the disruption of nucleolonema as early as 2 hours after the injection. By 4 hours, nucleoli show complete fragmentation consisting of many small fragments and small remnants of nucleoli. Large aggregates of interchromatinic granules and condensation of chromatin appear in the nucleoplasm. These changes are remarkably similar to the lesions induced by ethionine in the liver of the rat or the guinea pig. The methionine-induced nuclear and nucleolar lesions persist up to 10 hours after the injection. The administration of adenine 4 hours after the methionine injection reverses the nucleolar lesions by 8 hours. The appearance of incompletely reconstructed nucleoli with twisted ropelike structures suggests a pattern of recovery very similar to the adenine-induced nucleolar reformation in ethionine-treated rats. Injecting methionine into rats induced no nucleolar abnormalities. It is suggested that the mechanism of nucleolar fragmentation induced by methionine or ethionine is related to the accumulation of S-adenosyl compounds with concomitant ATP deficiency in the liver.     

Regional differences in the cellular proliferation activity of the regenerating rat pancreas after partial pancreatectomy

The proliferation activity of component cells and its regional differences in the regenerating rat pancreas after 90% pancreatectomy were examined by bromodeoxyuridine (BrdU) immunohistochemistry. Cells of the ductal system and the centroacinar cells showed a rapid increase in labeling indices at day 2 after pancreatectomy, followed by a second peak of a mild increase at days 5 to 7. No regional difference in the labeling index was recognized in the ductal elements. In contrast, the labeling index of acinar cells started to increase at day 3, reaching a definite peak at day 5. Furthermore, acinar cells in the region close to the duodenum had labeling indices more than 2 times higher than those in the portions further away from the duodenum. Acinar cells increased in number as early as from day 3 after surgery. These result suggested that the parental cells of regeneration were located in the ductal epithelium. It is highly probable that the proliferation of acinar cells is controlled by some unknown trophic factor(s) which is released locally from the duodenum, but does not involve a neural or a circulatory route. The phenomenon may be closely linked to the known fact that the incidence of pancreatic cancer is highest in the head region.     

Multiple atypical acinar cell nodules of the pancreas

Although acinar cell nodules of the pancreas have been described in rats given carcinogenic chemicals, similar nodules have not been reported in humans. This report describes two cases of nodular acinar cell lesions in the pancreas in patients who died of insulin secreting islet cell adenoma and of bronchogenic carcinoma. The lesions consisted of multiple nodules that were well demarcated from the surrounding acinar tissue and were composed of zymogen granule containing acinar cells with a pale to pink cytoplasm. The significance of these atypical acinar cell nodules in regard to their being possible precursor lesions of acinar cell carcinoma of the pancreas is discussed.     

Nucleolar segregation as an early marker for DNA damage; an experimental study in rats treated with 4-hydroxyaminoquinoline 1-oxide

Male 6-week-old Sprague Dawley rats were given a single intravenous injection of 4-hydroxyamino-quinoline 1-oxide (4HAQO) at a dose of 20 mg/kg in order to produce ultrastructural changes as possible morphological biomarkers for toxicity. Immunohistochemically demonstrated formation of 4HAQO-DNA adduct was correlated with the changes found. Nucleolar alteration, demonstrable by electron microscopy as segregation of nucleolar components into granular and fibrillar compartments, was evident in cells of the target organs, exocrine pancreas and adrenocortex, but not of the non-target liver parenchyma. Sequential observation clarified that such alteration was highest in frequency 6 h and 4 h after 4HAQO administration in pancreatic acinar cells and adrenocortical cells respectively. Electron microscopically, apoptotic changes of acinar cells were evident 2 h after injection of 4HAQO. DNA adduct formation was consistently demonstrated in the same target organs showing nucleolar segregation, the highest frequency being noted 4 h after 4HAQO treatment in both pancreatic acinar cells and adrenocortical cells. Our results thus indicate an identity of the target cells for nucleolar segregation and 4HAQO-DNA adduct formation which correlates with 4HAQO-toxicity. We suggest that nucleolar segregation occurs subsequent to the generation of DNA damage.     

Norepinephrine supplemented with dobutamine or epinephrine for the cardiovascular support of patients with septic shock

Background and Aims:

Sepsis management remains a great challenge for intensive care medicine. The aim of this study was to evaluate the effect of adding dobutamine versus epinephrine to norepinephrine in treating septic shock patients refractory to fluid therapy.

Materials and Methods:

Sixty adult patients with the diagnosis of septic shock were included in this study. Norepinephrine infusion was started at a dose of 0.05 μg/kg/min, and increased gradually up to 0.1 μg/kg/min. Upon reaching this dose, patients with mean arterial pressure <70 mmHg were further divided randomly into two equal groups. In group I: the patients continued on norepinephrine and dobutamine was added at a starting dose of 3 μg/kg/min and increased in increments of 2 μg/kg/min up to 20 μg/kg/min. In group II: the patients continued on norepinephrine and epinephrine was added in a starting dose of 0.05 μg/kg/ min and increased in increments of 0.03 μg/kg/min up to 0.3 μg/kg/min.

Results:

Group II patients developed significantly better cardiovascular parameters, lower arterial pH and higher serum lactate and urine output; however, the 28-day mortality and major adverse effects were comparable in both groups.

Conclusions:

The addition of epinephrine to norepinephrine has positive effects on the cardiovascular parameters but negative results on the serum lactate concentration and systemic pH compared with the addition of dobutamine to norepinephrine.     

In Vitro Activity of Amikacin against Isolates of Mycobacterium avium Complex with Proposed MIC Breakpoints and Finding of a 16S rRNA Gene Mutation in Treated Isolates

Amikacin is a major drug used for the treatment of Mycobacterium avium complex (MAC) disease, but standard laboratory guidelines for susceptibility testing are not available. This study presents in vitro amikacin MICs for 462 consecutive clinical isolates of the MAC using a broth microdilution assay. Approximately 50% of isolates had amikacin MICs of 8 μg/ml, and 86% had MICs of ≤16 μg/ml. Of the eight isolates (1.7%) with MICs of 64 μg/ml, five had an MIC of 32 μg/ml on repeat testing. Ten isolates (2.1%) had an initial amikacin MIC of >64 μg/ml, of which seven (1.5%) had MICs of >64 μg/ml on repeat testing. These seven isolates had a 16S rRNA gene A1408G mutation and included M. avium, Mycobacterium intracellulare, and Mycobacterium chimaera. Clinical data were available for five of these seven isolates, all of which had received prolonged (>6 months) prior therapy, with four that were known to be treated with amikacin. The 16S mutation was not detected in isolates with MICs of ≤64 μg/ml. We recommend primary testing of amikacin against isolates of the MAC and propose MIC guidelines for breakpoints that are identical to the CLSI guidelines for Mycobacterium abscessus: ≤16 μg/ml for susceptible, 32 μg/ml for intermediate, and ≥64 μg/ml for resistant. If considered and approved by the CLSI, this will be only the second drug recommended for primary susceptibility testing against the MAC and should facilitate its use for both intravenous and inhaled drug therapies.     

Increase in zymogen granule volume accounts for increase in volume density during prenatal development of pancreas

The sudden increase in volume density of zymogen granules in acinar cells of the fetal rat pancreas was examined with particular attention to the respective roles of granule size and number in this event. Volume density increased some twelvefold, from about 3% of cytoplasmic volume at 17 days to about 45% at 20 days, following a sigmoidal pattern in which the greatest rate of increase occurred during day 18. This increase in volume density was primarily the result of an increase in granule volume. Zymogen granule diameter increased from 0.55 μm at 17 days to 1.20 μm at 20 days, an order of magnitude increase in average granule volume. The total number of granules in the tissue increased in proportion to the increase in organ weight (cell number and size), but changes in the number of granules per unit cytoplasmic volume were minor (+ 40%) in comparison to the increase in volume density. The distribution of granule diameter was roughly normal and unimodal at each time interval, and the increase in average diameter over time was marked by an increase in the upper limit of the size distribution and an increased percentage of large granules. The size of condensing vacuoles also increased during this period, and their distributions were roughly coextensive with those seen for zymogen granules at the same time. The potential origins of changes in granule size are discussed, as well as the important effect that size has on the number of granules observed in “two-dimensional” tissue sections viewed in the electron microscope. If size is not considered in our estimates, then we underestimate the numerical density in cells with small granules compared to those with large granules. The results indicate the central role of granule size, as opposed to number, in determining granule volume density in the embryonic pancreas.     

Clinical evaluation, dose-finding and acceptability of AERODIOL, the pulsed estrogen therapy for treatment of climacteric symptoms

S21400 (AERODIOL^®) is a new intranasal formulation of 17β-estradiol. It provides a pulsed estrogen therapy that ensures sufficient estrogenisation of tissues to treat estrogen deficiency symptoms, particularly those of the menopause. This multicentric study was designed to determine dose-range, efficacy and acceptability of S21400. One hundred and thirty four women were allocated a daily dose of 100–900 μg for 12 weeks. The doses of 100, 600 and 900 μg were given in two daily administrations, the doses of 200, 300 and 450 μg were given in one and two daily administrations. Oral progestogen was added the last 10–14 days of each cycle of estrogen therapy in all non-hysterectornized women. S21400 showed a dose-effect relationship and provided adequate estrogenisation in more than 80% of patients receiving a dose ranging from 200 to 600 μg daily. Hormonal impregnation was judged sufficient in 23% of women receiving the lowest dose (100 μg). It was often considered excessive for daily doses of 900 μg (36%). After 12 weeks of treatment, efficacy was similar whether the total daily dose was given in one or two administrations. Treatment was well tolerated and accepted, with only minor nasal events (prickling, sneezing). It was perceived by 92% of patients as good or excellent and 81% chose to continue the nasal treatment when it was offered to them. An initial dose of 300 μg per day provides an optimal efficacy/tolerability ratio.

In summary, the pulsed estrogen therapy with AERODIOL^® in one daily administration offers a safe, well accepted and highly effective treatment to alleviate climacteric symptoms. It can be adapted easily to ensure optimal clinical efficacy.