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Background: Penicillin non-susceptible (PNSP) and multi-resistant pneumococci have been prevalent in Iceland since early nineties, mainly causing problems in treatment of acute otitis media. The 10-valent protein conjugated pneumococcal vaccine (PHiD-CV) was introduced into the childhood vaccination program in 2011. The aim of the study was to investigate the changes in antimicrobial susceptibility and serotype distribution of penicillin non-susceptible pneumococci (PNSP) in Iceland 2011-2017., Methods and Findings: All pneumococcal isolates identified at the Landspítali University Hospital in 2011-2017, excluding isolates from the nasopharynx and throat were studied. Susceptibility testing was done according to the EUCAST guidelines using disk diffusion with chloramphenicol, erythromycin, clindamycin, tetracycline, trimethoprim/sulfamethoxazole and oxacillin for PNSP screening. Penicillin and ceftriaxone minimum inhibitory concentrations (MIC) were measured for oxacillin resistant isolates using the E-test. Serotyping was done using latex agglutination and/or multiplex PCR. The total number of pneumococcal isolates that met the study criteria was 1,706, of which 516 (30.2%) were PNSP, and declining with time. PNSP isolates of PHiD-CV vaccine serotypes (VT) were 362/516 (70.2%) declining with time, 132/143 (92.3%) in 2011 and 17/54 (31.5%) in 2017. PNSP were most commonly of serotype 19F, 317/516 isolates declining with time, 124/143 in 2011 and 15/54 in 2017. Their number decreased in all age groups, but mainly in the youngest children. PNSP isolates of non PHiD-CV vaccine serotypes (NVT) were 154/516, increasing with time, 11/14, in 2011 and 37/54 in 2017. The most common emerging NVTs in 2011 and 2017 were 6C, 1/143 and 10/54 respectively., Conclusions: PNSP of VTs have virtually disappeared from children with pneumococcal diseases after the initiation of pneumococcal vaccination in Iceland and a clear herd effect was observed. This was mainly driven by a decrease of PNSP isolates belonging to a serotype 19F multi-resistant lineage. However, emerging multi-resistant NVT isolates are of concern., Competing Interests: This work is an investigator-initiated study that was supported by grants from: - The Landspítali University Hospital Research Fund (MÁH) https://www.landspitali.is - GlaxoSmithKline Biologicals SA https://www.gsk.com The authors declare that this does not alter our adherence to PLOS ONE policies on sharing data and materials. Neither GlaxoSmithKline Biologicals SA, nor Landspítali University Hospital Research Fund had any role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The introduction of pneumococcal conjugate vaccines (PCVs) into childhood vaccination programs has reduced carriage of vaccine serotypes and pneumococcal disease. The 10-valent PCV was introduced in Iceland in 2011. The aim of this study was to determine PCV impact on the prevalence of serotypes, genetic lineages, and antimicrobial-resistant pneumococci isolated from the lower respiratory tract (LRT) of adults. Pneumococci isolated between 2009 and 2017 at the Landspitali University Hospital were included ( n  = 797). The hospital serves almost three-quarters of the Icelandic population. Isolates were serotyped and tested for antimicrobial susceptibility, and the genome of every other isolate collected between 2009 and 2014 was sequenced ( n  = 275). Serotypes and multilocus sequence types (STs) were extracted from the genome data. Three study periods were defined, 2009 to 2011 (PreVac), 2012 to 2014 (PostVac-I), and 2015 to 2017 (PostVac-II). The total number of isolates and vaccine-type (VT) pneumococci decreased from PreVac to PostVac-II ( n  = 314 versus n  = 230 [ p  = 0.002] and n  = 170 versus n  = 33 [ p  < 0.001], respectively), but non-vaccine-type (NVT) pneumococci increased among adults 18 to 64 years old ( n  = 56 versus n  = 114 [ p  = 0.008]). Serotype 19F decreased in the PostVac-II period; these isolates were all multidrug resistant (MDR) and were members of the Taiwan 19F -14 PMEN lineage. Serotype 6A decreased among adults ≥65 years old in the PostVac-II period ( p  = 0.037), while serotype 6C increased ( p  = 0.021) and most serotype 6C isolates were MDR. Nonencapsulated Streptococcus pneumoniae (NESp) isolates increased among adults 18 to 64 years old in the PostVac-II period, and the majority were MDR ( p  = 0.028). An overall reduction in the number of LRT samples and pneumococcus-positive cultures and significant changes in the serotype distribution became evident within 4 years, thereby demonstrating a significant herd effect., (Copyright © 2019 Quirk et al.)

Understanding the structure of a bacterial population is essential in order to understand bacterial evolution. Estimating the core genome (those genes common to all, or nearly all, strains of a species) is a key component of such analyses. The size and composition of the core genome varies by dataset, but we hypothesized that the variation between different collections of the same bacterial species would be minimal. To investigate this, we analyzed the genome sequences of 3,118 pneumococci recovered from healthy individuals in Reykjavik (Iceland), Southampton (United Kingdom), Boston (United States), and Maela (Thailand). The analyses revealed a "supercore" genome (genes shared by all 3,118 pneumococci) of 558 genes, although an additional 354 core genes were shared by pneumococci from Reykjavik, Southampton, and Boston. Overall, the size and composition of the core and pan-genomes among pneumococci recovered in Reykjavik, Southampton, and Boston were similar. Maela pneumococci were distinctly different in that they had a smaller core genome and larger pan-genome. The pan-genome of Maela pneumococci contained several >25 Kb sequence regions (flanked by pneumococcal genes) that were homologous to genomic regions found in other bacterial species. Overall, our work revealed that some subsets of the global pneumococcal population are highly heterogeneous, and our hypothesis was rejected. This is an important finding in terms of understanding genetic variation among pneumococci and is also an essential point of consideration before generalizing the findings from a single dataset to the wider pneumococcal population.

Vaccination with pneumococcal conjugate vaccines (PCVs) disrupts the pneumococcal population. Our aim was to determine the impact of the 10-valent PCV on the serotypes, genetic lineages, and antimicrobial susceptibility of pneumococci isolated from children in Iceland. Pneumococci were collected between 2009 and 2017 from the nasopharynges of healthy children attending 15 day care centers and from the middle ears (MEs) of children with acute otitis media from the greater Reykjavik capital area. Isolates were serotyped and tested for antimicrobial susceptibility. Whole-genome sequencing (WGS) was performed on alternate isolates from 2009 to 2014, and serotypes and multilocus sequence types (STs) were extracted from the WGS data. Two study periods were defined: 2009 to 2011 (PreVac) and 2012 to 2017 (PostVac). The overall nasopharyngeal carriage rate was similar between the two periods (67.3% PreVac and 61.5% PostVac, P = 0.090). Vaccine-type (VT) pneumococci decreased and nonvaccine-type (NVT) pneumococci (serotypes 6C, 15A, 15B/C, 21, 22F, 23A, 23B, 35F, and 35B) significantly increased in different age strata post-PCV introduction. The total number of pneumococci recovered from ME samples significantly decreased as did the proportion that were VTs, although NVT pneumococci (6C, 15B/C, 23A, and 23B) increased significantly. Most serotype 6C pneumococci were multidrug resistant (MDR). Serotype 19F was the predominant serotype associated with MEs, and it significantly decreased post-PCV introduction: these isolates were predominantly MDR and of the Taiwan 19F -14 PMEN lineage. Overall, the nasopharyngeal carriage rate remained constant and the number of ME-associated pneumococci decreased significantly post-PCV introduction; however, there was a concomitant and statistically significant shift from VTs to NVTs in both collections of pneumococci., (Copyright © 2018 Quirk et al.)

Background: Since the introduction of pneumococcal conjugate vaccines, vaccine type pneumococcal carriage and disease has decreased world-wide. The aim was to monitor changes in the nasopharyngeal carriage of pneumococci, the distribution of serotypes and antimicrobial resistance in children before and after initiation of the 10-valent pneumococcal vaccination in 2011, in a previously unvaccinated population., Methods: Repeated cross-sectional study at 15day-care centres in greater Reykjavik area. Nasopharyngeal swabs were collected yearly in March from 2009 to 2015. The swabs were selectively cultured for pneumococci, which were serotyped using latex agglutination and/or PCR and antimicrobial susceptibility determined. Two independent studies were conducted. In study 1, on total impact, isolates from children aged <4years were included. The vaccine-eligible-cohort (birth-years: 2011-2013, sampled in 2013-2015) was compared with children at the same age born in 2005-2010 and sampled in 2009-2012. In study 2 on herd effect, isolates from older non-vaccine-eligible children (3.5-6.3years) were compared for the periods before and after the vaccination (2009-2011 vs 2013-2015. Vaccine impact was determined using 1-odds-ratio., Results: Following vaccination, the vaccine impact on vaccine type acquisition was 94% (95% CI: 91-96%) in study 1 and 56% (95% CI: 44-65%) in study 2. The impact on serotype 6A was 33% (95% CI: -9%; 59%) in study 1 and 42% (95% CI: 10-63%) in study 2 with minimal effect on 19A. The non-vaccine serotypes/groups 6C, 11, 15 and 23B were the most common serotypes/groups after vaccination. Isolates from the vaccine-eligible-cohort had lower penicillin MICs, less resistance to erythromycin and co-trimoxazole and less multi resistance than isolates from the control-group., Conclusions: The efficacy of the vaccination on vaccine serotypes was high, and a milder effect on vaccine-associated-serotype 6A was observed for the vaccine-eligible-cohort. There was a significant herd effect on vaccine types in older non-vaccine-eligible children. Overall antimicrobial non-susceptibility was reduced., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Information on pneumococcal serotype distribution before vaccination is a prerequisite for evaluation of vaccine effect. The aim was to investigate the prevalence of pneumococcal serotypes isolated from middle ear (ME), lower respiratory tract (LRT) and from invasive disease (IPD) in Iceland prior to implementation of ten-valent pneumococcal Haemophilus influenzae protein D conjugate vaccine (PHiD-CV-10) into the infant vaccination program (April 2011)., Methods and Findings: All isolates cultured 2007-2011 from ME, LRT and IPD identified as pneumococci were serotyped and tested for susceptibility at the Clinical Microbiology Department, Landspitali University Hospital that serves approximately 85% of the Icelandic population. Pneumococcal isolates were 1711 and 1616 (94.4%) were available for serotyping and included. Isolates belonging to PHiD-CV10 serotypes (VTs) were 1052 (65.1%). Isolates from ME were 879 (54.4%), with 639 (72.7%) from 0-1 year old patients and 651 of VTs (74%). Isolates from LRT were 564 (34.9%), with 292 (51.8%) from ≥65 years old patients, and 300 (53.2%) of VTs. IPD isolates were 173 (10.7%), although more evenly distributed according to age than isolates from the other sites most were from adults and the youngest age group,101 (58.4%) isolates were of VTs. The most common serotype was 19F, 583 (36.1%). Its prevalence was highest in ME, 400 (45.5%), 172 (30.5%) in LRT and 11 isolates (6.4%), in IPD. Penicillin non-susceptible isolates were 651 (40.3%), mainly belonging to VTs, 611 (93.9%), including 535 (82.2%) of 19F., Conclusions: Multiresistant isolates of serotype 19F were highly prevalent, especially from ME of young children but also from LRT of adults. Serotype 14 was the most common serotype in IPD. The rate of VTs was high and almost all PNSP were of VTs. There was great difference in vaccine coverage between sampling sites, also reflecting difference in vaccine coverage by age groups., Competing Interests: Competing Interests: Some of the authors (HE, ÁH and KGK) have received a research grant from GSK, to study the effectiveness of pneumococcal vaccination in Iceland. That study is an investigator initiated study with no conflicting interests related to the current study. GSK did not fund this specific study. There are no patents, products in development or marketed products to declare. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

The pneumococcus is a leading global pathogen and a key virulence factor possessed by the majority of pneumococci is an antigenic polysaccharide capsule ('serotype'), which is encoded by the capsular ( cps ) locus. Approximately 100 different serotypes are known, but the extent of sequence diversity within the cps loci of individual serotypes is not well understood. Investigating serotype-specific sequence variation is crucial to the design of sequence-based serotyping methodology, understanding pneumococcal conjugate vaccine (PCV) effectiveness and the design of future PCVs. The availability of large genome datasets makes it possible to assess population-level variation among pneumococcal serotypes and in this study 5405 pneumococcal genomes were used to investigate cps locus diversity among 49 different serotypes. Pneumococci had been recovered between 1916 and 2014 from people of all ages living in 51 countries. Serotypes were deduced bioinformatically, cps locus sequences were extracted and variation was assessed within the cps locus, in the context of pneumococcal genetic lineages. Overall, cps locus sequence diversity varied markedly: low to moderate diversity was revealed among serogroups/types 1, 3, 7, 9, 11 and 22; whereas serogroups/types 6, 19, 23, 14, 15, 18, 33 and 35 displayed high diversity. Putative novel and/or hybrid cps loci were identified among all serogroups/types apart from 1, 3 and 9. This study demonstrated that cps locus sequence diversity varied widely between serogroups/types. Investigation of the biochemical structure of the polysaccharide capsule of major variants, particularly PCV-related serotypes and those that appear to be novel or hybrids, is warranted.

The pneumococcus is a leading pathogen infecting children and adults. Safe, effective vaccines exist, and they work by inducing antibodies to the polysaccharide capsule (unique for each serotype) that surrounds the cell; however, current vaccines are limited by the fact that only a few of the nearly 100 antigenically distinct serotypes are included in the formulations. Within the serotypes, serogroup 6 pneumococci are a frequent cause of serious disease and common colonizers of the nasopharynx in children. Serotype 6E was first reported in 2004 but was thought to be rare; however, we and others have detected serotype 6E among recent pneumococcal collections. Therefore, we analyzed a diverse data set of ∼1,000 serogroup 6 genomes, assessed the prevalence and distribution of serotype 6E, analyzed the genetic diversity among serogroup 6 pneumococci, and investigated whether pneumococcal conjugate vaccine-induced serotype 6A and 6B antibodies mediate the killing of serotype 6E pneumococci. We found that 43% of all genomes were of serotype 6E, and they were recovered worldwide from healthy children and patients of all ages with pneumococcal disease. Four genetic lineages, three of which were multidrug resistant, described ∼90% of the serotype 6E pneumococci. Serological assays demonstrated that vaccine-induced serotype 6B antibodies were able to elicit killing of serotype 6E pneumococci. We also revealed three major genetic clusters of serotype 6A capsular sequences, discovered a new hybrid 6C/6E serotype, and identified 44 examples of serotype switching. Therefore, while vaccines appear to offer protection against serotype 6E, genetic variants may reduce vaccine efficacy in the longer term because of the emergence of serotypes that can evade vaccine-induced immunity., (Copyright © 2015, van Tonder et al.)

Both adipose and epithelial cells isolated from the mammary glands of pregnant and lactating rats show 11 beta-hydroxysteroid dehydrogenase (11-HSD) activity, as measured by conversion of corticosterone to 11-dehydrocorticosterone. Activity in adipose cells from pregnant rats is 3-fold higher than in lactating rats. Epithelial cells from pregnant rats show one-twentieth of the activity of adipose cells, and activity is lower still in epithelial cells from lactating rats. Explants incubated for 48 h extensively metabolized corticosterone to 11-dehydrocorticosterone, and to a much lesser extent to a second unknown metabolite which is found in tissue extracts but not conditioned medium. Mammary gland 11-HSD may thus constitute one of the physiological mechanisms preventing premature milk production in response to glucocorticoids.

Levels of 11 beta-hydroxysteroid dehydrogenase activity in mammary gland homogenates from pregnant and lactating Sprague-Dawley rats were determined by incubation with [3H]corticosterone under standard conditions, followed by thin-layer chromatography of incubated media. Enzyme activity was high in virgin and pregnant rats, but fell soon after parturition, suggesting a possible role for this enzyme in the co-ordinate regulation of glucocorticoid effects on milk protein synthesis.

The granulosa luteal cells of the sheep corpus luteum secrete their hormonal products by exocytosis of granules. Electron micrographs of randomly selected granulosa cells from nine corpora lutea at day 10 of the oestrous cycle were examined to obtain the cellular density of these granules. From the area of the cell, calculated using an x-y digitiser, and the number of granules observed, the number of intracellular secretory granules per microns 3 of luteal cell cytoplasm was calculated. There was a large variation in the number of granules per cell within the same corpus luteum and between corpora lutea taken at the same stage of the cycle. The number of intracellular granules in nine corpora lutea varied from 2.12 +/- 1.05 granules per microns 3 (mean, SD, n = 30) cells to 0.36 +/- 0.18 granules per microns 3 (mean, SD, n = 26). These morphological data suggest that the variation in granule synthesis in individual cells may contribute to the variation in hormone content of corpora lutea at the same stage of the cycle, and the episodic release of hormone into the plasma.

We have identified and characterized aldosterone-binding sites in the mammary gland of pregnant and lactating rats by using the highly specific glucocorticoid RU26988 (11 beta, 17 beta-dihydroxy-17 alpha-propynyl-androsta-1,4,6-triene-3-one) to eliminate aldosterone binding to classical glucocorticoid (type II)-binding sites. The hierarchy of affinity of steroids competing for aldosterone in the presence of RU26988 was similar in mammary glands of pregnant and lactating rats and kidneys of lactating rats: deoxycorticosterone greater than aldosterone greater than or equal to corticosterone greater than dexamethasone; this pattern is similar but not identical to that for the mineralocorticoid (type I) receptor of a classical mineralocorticoid target organ, the kidney of the virgin female rat, where aldosterone has a higher affinity than corticosterone. This pattern (aldosterone greater than corticosterone) was also found in renal cytosols of pregnant rats. To define these aldosterone-binding sites in terms of dissociation constant and number of sites, it was necessary to pretreat cytosols with charcoal. Without this treatment, curvilinear Scatchard plots were obtained. After pretreatment with charcoal, subsequent Scatchard analysis indicated a single class of site with low capacity (3-33 fmol/mg protein) and dissociation constants of about 1-3 nM in kidneys and mammary glands of pregnant and lactating rats. The occupancy of this site in vivo may depend on the relative tissue concentrations of aldosterone or corticosterone, which, in turn, are modulated by the levels of extravascular transcortin.

The characteristics of pregnant rat mammary gland progesteronee receptors have been studied by the use of two synthetic probes, [3H]R5020 (3H-labeled 17 alpha, 21-dimethyl-19-nor-pregn-4,9-diene-3,20-dione) amd [3H]ORG 2058 (3H-labeled 16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20-dione). [3H] ORG 2058 bound with high affinity (Kd at 4 C, approximately 0.5 nM) to an apparent single class of displaceable sites, with a hierarchy of competition as follows: ORG 2058 greater than R5020 greater than or equal to progesterone greater than dexamethasone. In contrast, [3H]R5020 showed binding in mammary gland cytosols which was nonlinear with protein concentration, of intermediate affinity (Kd at 4 C, approximately 5 nM), and poorly displaced by nonradioactive R5020; the hierarchy of competitors was R5020 greater than progesterone greater than dexamethasone = ORG 2058. Parallel studies in rat uterine cytosols showed both [3H]ORG 2058 and [3H]R5020 to bind with high affinity to an apparently identical class of sites; the hierarchy of competition for both was ORG 2058 greater than or equal to R5020 greater than progesteron greater than dexamethasone. We conclude that in pregnant rat mammary gland, as opposed to rat uterus and other tissues, R5020 is unsuitable as a progesterone receptor probe; however, the affinity, capacity, ad specificity characteristics of progesterone receptors in this tissue can be described by the use of ORG 2058.

Displaceable binding of the synthetic progestin [3H]ORG 2058 (16 alpha-ethyl-21-hydroxy-19-nor-pregn-4-ene-3,20 dione) was found in ammonium sulphate treated cytosols from uterus and mammary gland from lactating rats. Specificity studies, however, showed that in mammary gland but not uterus this binding was exclusively to glucocorticoid receptors, and that specific progesterone receptors were undetectable. In contrast, after in vivo administration of oestradiol benzoate in oil (4 micrograms at -48, -24h), [3H]ORG 2058 bound predominantly to specific, high affinity progesterone receptors in lactating mammary gland. Thus though in lactation progesterone receptors are normally absent from the mammary gland, they can be induced by oestrogen administration.

Explants of mammary glands from pregnant or lactating rats were shown to differ markedly in their response to glucocorticoid and progestin in terms of alpha-lactalbumin production. Tissue from day 14 pregnant rats had low initial tissue levels and basal production of alpha-lactalbumin, and showed a dose-biphasic response to glucocorticoids, with significantly higher production at 3 nM than 300 nM RU26988; the progestin ORG 2058 inhibited production. In explants from lactating rats (day 1 and day 5) tissue content and basal production of alpha-lactalbumin were markedly increased compared with day 14 pregnant, but were unaltered by glucocorticoid or progestin. Animals near parturition (day 19, day 20) showed a transitional pattern, with considerable variation between animals. Basal production and tissue content correlated closely; as levels rose, the tissues became progressively less responsive to the effects of both classes of steroid. We interpret these data as evidence for a switch, in terms of glucocorticoid control of alpha-lactalbumin gene expression pre-partum; the mechanism(s) underlying this switch remain to be explored.

The highly selective Type II glucocorticoid ligand RU28362 showed a clear biphasic effect on alpha-lactalbumin (alpha-LA) production in rat mammary gland explants, with a peak at 1 nM and a return to basal levels at 30-300 nM; dexamethasone showed a similar profile. Corticosterone, which has a higher affinity for Type I than Type II receptors, produced a variable response. In six out of eleven studies this was biphasic, with a maximum at 300 nM; in five no increase above baseline was seen. Classical Type I receptor ligands--aldosterone and deoxycorticosterone--showed responses parallel to their Type II agonist activity. We interpret these data as follows occupancy of Type I receptors does not increase alpha-LA production the response to selective Type II receptor ligands is truly biphasic and one explanation of this pattern may be the existence of both "turn-on" and "turn-off" acceptor sites in the nucleus.

RU26988 [11 beta, 17 beta-dihydroxy-17 alpha-(1-propynyl)androsta-1,4,6-trien-3-one], a specific type II (glucocorticoid) ligand with negligible affinity for type I (mineralocorticoid) receptors, increased alpha-lactalbumin production in mammary gland explants from midpregnant rats when cultured in vitro in the presence of insulin (5 micrograms/ml) and rat PRL (1 microgram/ml). The dose-response curve was biphasic, over a 3-300 nM dose range, with maximum at 3-10 nM, followed by a progressive decline toward or to control levels at 300 nM. A similar dose-response curve was obtained with another type II-specific ligand, RU28362 [11 beta, 17 beta-dihydroxy-6-methyl-17 alpha-(1-propynyl)androsta-1,4,6-trien-3-one], and with dexamethasone. This biphasic response had been previously reported for mouse mammary gland explants using cortisol, which binds to both type I and type II receptors. Our experiments, therefore, show that occupancy of type II receptors alone can be responsible for the stimulation at low steroid concentrations, followed by the decrease from peak levels observed at higher steroid concentrations. On the basis of these data, we propose a model to account for these findings, based on ligand binding to a single class of receptors and the putative existence of both turn-on and turn-off glucocorticoid regulatory elements in the nucleus.

Whey acidic protein (WAP) has been measured by radioimmunoassay in the mammary gland of rats over pregnancy and lactation, and in mammary gland explants incubated with glucocorticoid or progestin in vitro. The radioimmunoassay used a novel fusion protein, glutathione transferase-WAP (GT-WAP), which can be iodinated with ease, unlike the native protein. Mammary gland WAP levels were low (less than 100 ng/mg tissue) until 2-3 days before parturition, but rose to 3 micrograms/mg tissue at day 21 of pregnancy. Immediately after parturition WAP content decreased to 1 micrograms/mg tissue, and then increased to greater than 5 micrograms/mg tissue during mid-lactation. Similarly, alpha-lactalbumin content was low throughout pregnancy (less than 10 ng/mg tissue) until day 20. Thereafter, values rose on the last day of pregnancy and the first day of lactation, fell briefly in early lactation like WAP, and rose to plateau levels by day 11 of lactation. In vitro explants prepared from mid-pregnant rats (day 14) synthesized WAP in the presence of insulin and prolactin. The synthetic glucocorticoid RU26988 (11 beta,17 beta-hydroxy-17 alpha-(1-propynyl)-androsta-1,4,6-trien-3-one) progressively increased WAP production to a maximum of greater than 10-fold basal (in the presence of insulin and prolactin) at 300 nM, in contrast with alpha-lactalbumin which showed a biphasic dose-response curve in explants from mid-pregnant rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Mammary gland explants from pregnant (day 12-15) rats were cultured with insulin and prolactin, and their content and secretion of alpha-lactalbumin determined after exposure to a wide range of doses (0.01-300 nM) of the specific synthetic progestin (ORG2058), alone or with a maximally stimulatory dose of the highly specific glucocorticoid RU26988. ORG2058 alone suppressed alpha-lactalbumin synthesis below baseline, with a half-maximal effect at a concentration of less than 0.1 nM; RU26988-stimulated secretion was similarly abrogated by ORG2058, similarly with a half maximally effective dose of less than 0.1 nM. We interpret these data as suggesting that (i) given the specificity and doses of the steroids used the effect of progestins on alpha lactalbumin synthesis is directly via progesterone receptor occupancy, and not by competing with glucocorticoids for glucocorticoid receptors and (ii) given the shift to the left in the alpha-lactalbumin response (half maximal less than 0.1 nM ORG2058) compared with receptor binding (Kd (37 degrees C) greater than 1 nM), one possible model for such sensitivity is that of multiple, independent regulatory elements on the chromatin controlling alpha-lactalbumin gene expression, occupancy of any one of which by an activated progesterone receptor is sufficient to abrogate transcription.

We have isolated epithelial cell clusters from mammary glands of pregnant and lactating rats by collagenase-hyaluronidase-deoxyribonuclease digestion, followed by Ficoll density-gradient centrifugation. Clusters of greater than 90% viable cells were identified by light microscopy as essentially devoid of other cell types; the integrity of their subcellular organelles verified by electron microscopy. Binding characteristics of the synthetic glucocorticoid [3H]dexamethasone were studied in cytosols prepared from isolated cell clusters. Cytosols from both pregnant and lactating rats bound [3H]dexamethasone with high affinity to a single class of low capacity binding sites. In both types of cytosol the dissociation constant (Kd 4 degrees C approximately/nM) of the binding was similar; the number of sites per cell in lactating rats was approximately double that in pregnant rats. The specificity of binding was typical of a classical glucocorticoid receptor, with a hierarchy of affinity by competition studies dexamethasone greater than progesterone greater than aldosterone much much greater than testosterone = estradiol. In particular, no difference in progesterone affinity for these glucocorticoid receptors was seen between pregnancy and lactation. This suggests that reported differences in inhibitory action of progesterone, pregnancy versus post-partum, are not glucocorticoid-receptor mediated.

Incubation of hen erythrocytes with Ca2+ and the bivalent-cation ionophore A23187 induced slight cell fusion in 1 h at 37 degrees C, and extensive fusion during a subsequent 15 min at 47 degrees C. Redistributions of intramembranous particles were observed, possibly involving interactions between Ca2+ and phospholipids, which are discussed in relation to molecular mechanimss of cell fusion.

PMC42 is a new human breast carcinoma cell line. In this report the content of estrogen, progesterone, and glucocorticoid receptors in PMC42 has been determined. The estrogen receptor content (1,750 cytoplasmic sites and 350 nuclear sites) was lower than that described for MCF7 and T47D. The cells would not proliferate in serum-free medium without the addition of beta-estradiol (optimum concentration 10(-8) M) or progesterone (5 X 10(-8) M). The addition of both hormones induced a more than additive increase in proliferation (P less than .005, n = 18). Similarly, addition of insulin or hydrocortisone induced proliferation; however, in this case, the effect of the hormones together was only additive. The addition of tamoxifen (10(-6) M) led to a significant decrease in cell numbers and inhibited the stimulatory effects of 10(-8) M beta-estradiol.

Though adrenal steroids are required for the production of alpha-lactalbumin by mammary gland explants, the physiological class of steroid activity (mineralocorticoid, glucocorticoid) remains to be established. alpha-Lactalbumin production by mammary gland explants from mid-pregnant rats has been shown in previous studies to be increased by high doses of aldosterone, but not by deoxycorticosterone; in six of 11 experiments corticosterone, the physiological glucocorticoid in the rat, elevated alpha-lactalbumin; in five other studies corticosterone had no effect. In the present studies the mineralocorticoid antagonist, spirolactone, at very high doses (3-10 mumol/l) blocked the stimulatory effect on alpha-lactalbumin levels of both 30 nmol/l corticosterone and 3 nmol/l RU 26988, a pure synthetic glucocorticoid (Type II) receptor agonist. Receptor studies, however, indicated that this antagonism is consistent with Type II, glucocorticoid receptor occupancy by spirolactone. Since deoxycorticosterone is without agonist effect, and only very high doses of spirolactone affect alpha-lactalbumin synthesis, we conclude that the effect of adrenal steroids on alpha-lactalbumin production is a manifestation of glucocorticoid and not mineralocorticoid activity.

1. The fusion of human erythrocytes into multicellular bodies that is induced by microdroplets of oleoylglycerol was investigated by optical and electron microscopy, and by gel electrophoresis of membrane proteins. 2. At the highest concentrations of oleoylglycerol and Ca(2+) used, at least 80% of the cells fused after 30min at 37 degrees C and only about 5% of the cells had completely lysed; the shapes of fused multicellular bodies were usually retained in ;ghosts' prepared by hypo-osmotic lysis. 3. The rate of cell fusion was related to the concentration of Ca(2+), although some cells fused when no exogenous Ca(2+) was present. 4. Interactions of microdroplets of oleoylglycerol with the cells led to abnormalities in the structural appearance of the erythrocyte membrane; subsequent membrane fusion occurred, at least in some instances, at the sites of the microdroplets. 5. The intramembranous particles on the P-fracture face of the treated cells were more randomly distributed, but not significantly increased in number by comparison with the control cells. 6. Gel electrophoresis of the proteins of ;ghosts' prepared from fused human erythrocytes showed a production of material of very high molecular weight, the development of a new component in the band-3 region, an increased staining of bands 4.3 and 4.5, and a new component moving slightly faster than band 6. 7. Bands 2.1-2.3 were altered, band 3 was decreased and band 4.1 was lost. 8. Most, but not all, of the changes in the membrane proteins appeared to result from the entry of Ca(2+) into the cell. 9. 1-Chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one partially inhibited both cell fusion and the associated decrease in band-3 protein. 10. The possibility that proteolytic degradation of membrane proteins may be involved in cell fusion induced by oleoylglycerol is considered, and some implications of this possibility are discussed.

In most steroid-modulated systems, responses are closely coupled to receptor occupancy, with half-maximal responses at Kd, and near-maximal at greater than or equal to 10 Kd. Though glucocorticoids have been reported to increase, and progesterone to lower, levels of various milk proteins, clear differences in glucocorticoid dose-response profiles have been previously described. In mouse mammary gland explants in vitro, Ono and Oka showed that casein synthesis was stimulated by cortisol to plateau levels with progressively higher doses; in contrast, the dose-response curve for alpha-lactalbumin (alpha-LA) was biphasic, with stimulation at low doses and a return to baseline at higher cortisol levels. Since cortisol is both non-physiologic in the mouse, and has higher affinity for Type I (mineralocorticoid-like) than Type II (glucocorticoid) receptors, this might reflect an induction of alpha-LA synthesis via Type I receptor occupancy, and a suppression via Type II receptor occupancy. To examine this possibility we have used highly selective Type II receptor ligands, and in addition have defined the role of progestins as candidate inhibitors of alpha-LA synthesis. We have thus incubated mid-pregnant rat mammary gland explants in medium including insulin and prolactin, with increasing concentrations of highly specific Type II ligand (RU26988 or RU28362) and/or the selective progestin ORG 2058, and determined tissue and medium content of alpha-LA 48 h later. RU26988/RU28362 increased alpha-LA to a maximum of 3-6-fold basal at approximately 3 nM, well below the Kd (37 degrees C) for Type II receptors (8-28 nM); levels fell progressively with increasing concentration of ligand, returning to baseline by 30-300 nM. ORG 2058 lowered both basal and RU26988-stimulated levels, with the half-maximally effective dose being approximately 10(-10) M, again well below Kd for receptor binding. To accommodate the observed biphasic dose-response curve for highly specific glucocorticoids, and the shift-to-the-left for both classes of effector, we have proposed a model of a single "turn-on" nuclear acceptor site for glucocorticoid receptors (glucocorticoid regulatory element: GRE) and multiple, pre-emptive "turn-off" GREs, occupancy of any one of which will abrogate transcription; for progestins, we propose that all the GRE are alternate "turn-off" sites. Consistent with such a hypothesis, Qasba and Safaya have shown that the alpha-LA genome contains six core consensus sequences (TGT TCCT) which are putative GRE, and von der Ahe et al. that both progesterone receptors and glucocorticoid receptors may bind to GRE sites.

1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg(2+)-dependent and Na(+),K(+)-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.

The kinetics of incorporation of [(3)H]lysine and [(14)C]glucosamine into kidney brush borders were studied in vivo. The patterns of incorporation and loss of radioactivity from the brush borders were similar for both radioactively labelled precursors. Maximal labelling occurred 15-20h after injection of the precursors, and then the radioactivity declined rapidly until 50h. The radioactivity of brush borders then remained constant until 120h and thereafter declined slowly. These results are interpreted as indicating that two processes contribute to the turnover of brush-border components, pinocytosis and a slower turnover of the components of the microvilli. Studies of the distribution of radioactivity among glycoprotein components of the brush borders, separated by polyacrylamide-gel electrophoresis, indicated that the membrane components turned over in unison.