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OBJECTIVE: To estimate the frequency of binge eating and binge eating disorder and their correlates in a sample of women with a history of obesity. METHOD: A group of women who had been found in a previous community study to have a body mass index > or = 30 were studied using self-report measures (n = 74) and interview (n = 62). RESULTS: One subject met criteria for binge eating disorder, while 24% reported binging. Subjects who reported binging were significantly more likely to have a past history of depressive illness. DISCUSSION: Prevalence of binge eating, binge eating disorder, and psychopathology was broadly in keeping with that found in North America. In addition, there were nonsignificant trends towards a positive family history of obesity, of childhood obesity, of early onset of dieting, of excessive concern about weight and shape, and a recent history of weight reduction. Further study is required to elucidate the causes of binge eating in the obese. [ABSTRACT FROM AUTHOR]

During adolescence, cannabis experimentation is common, and its association with interindividual variations in brain maturation well studied. Cellular and molecular underpinnings of these system-level relationships are, however, unclear. We thus conducted a three-step study. First, we exposed adolescent male mice to Δ-9-tetrahydrocannabinol (THC) or a synthetic cannabinoid WIN 55,212-2 (WIN) and assessed differentially expressed genes (DEGs), spine numbers, and dendritic complexity in their frontal cortex. Second, in human (male) adolescents, we examined group differences in cortical thickness in 34 brain regions, using magnetic resonance imaging, between those who experimented with cannabis before age 16 ( n  = 140) and those who did not ( n  = 327). Finally, we correlated spatially these group differences with gene expression of human homologs of mouse-identified DEGs. The spatial expression of 13 THC-related human homologs of DEGs correlated with cannabis-related variations in cortical thickness, and virtual histology revealed coexpression patterns of these 13 genes with cell-specific markers of astrocytes, microglia, and a type of pyramidal cells enriched in dendrite-regulating genes. Similarly, the spatial expression of 18 WIN-related human homologs of DEGs correlated with group differences in cortical thickness and showed coexpression patterns with the same three cell types. Gene ontology analysis indicated that 37 THC-related human homologs are enriched in neuron projection development, while 33 WIN-related homologs are enriched in processes associated with learning and memory. In mice, we observed spine loss and lower dendritic complexity in pyramidal cells of THC-exposed animals (vs controls). Experimentation with cannabis during adolescence may influence cortical thickness by impacting glutamatergic synapses and dendritic arborization., Competing Interests: The authors declare no competing financial interests., (Copyright © 2024 the authors.)

Species susceptible to inbreeding depression are hypothesized to combat this problem through a number of different mechanisms, including kin recognition. For species with kin recognition, it is unknown if filial recognition is innate or due to prior juvenile experience with siblings. Here, we first test for the presence of kin recognition, and then test these two hypotheses for the development of filial recognition, in the butterfly Bicyclus anynana, a species that suffers from inbreeding depression when forcibly inbred but recovers within a few generations when allowed to breed freely. We evaluate whether the rapid recovery from inbreeding depression is associated with either innate or learned filial recognition. First, we determined whether females innately prefer unrelated males over sibling males using females reared in isolation and then given a choice between an unrelated and a sibling male. Then, we determined if females raised with siblings learned to detect and avoid mating with siblings as adults when provided a choice between an unrelated male and a sibling male. Finally, we determined if females raised with siblings could learn to detect and avoid mating with familiar siblings when given a choice between familiar and unfamiliar siblings. We found that females mated randomly in all three choice combinations. Observed male behavior also did not influence female mating outcome. Our results suggest that adult females do not innately avoid or learn to avoid siblings during mate selection, and that filial detection may not be as critical to reproductive fitness in B. anynana as previously thought., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET). To provide proof of principle we have worked with Kir3 neuronal channels producing 10 different constructs which were combined into 17 donor-acceptor BRET pairs. Among these combinations, pairs bearing the donor Nano-Luc (NLuc) at the C-terminal end of Kir3.2 subunits and the FlAsH acceptor at the N-terminal end (NT) or the interfacial helix (N70) of Kir3.1 subunits were identified as potential tools. These pairs displayed significant changes in energy transfer upon activation with direct channel ligands or via stimulation of G protein-coupled receptors. Conformational changes associated with channel activation followed similar kinetics as channel currents. Dose response curves generated by different agonists in FlAsH-BRET assays displayed similar rank order of potency as those obtained with conventional BRET readouts of G protein activation and ion flux assays. Conformational biosensors as the ones reported herein should prove a valuable complement to other methodologies currently used in channel drug discovery., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Virus-Induced Gene Silencing (VIGS) is a useful method for transient downregulation of gene expression in crop plants. The geminivirus Cotton leaf crumple virus (CLCrV) has been modified to serve as a VIGS vector for persistent gene silencing in cotton. Here the use of Green Fluorescent Protein (GFP) is described as a marker for identifying silenced tissues in reproductive tissues, a procedure that requires the use of transgenic plants. Suggestions are given for isolating and cloning combinations of target and marker sequences so that the total length of inserted foreign DNA is between 500 and 750 bp. Using this strategy, extensive silencing is achieved with only 200-400 bp of sequence homologous to an endogenous gene, reducing the possibility of off-target silencing. Cotyledons can be inoculated using either the gene gun or Agrobacterium and will continue to show silencing throughout fruit and fiber development. CLCrV is not transmitted through seed, and VIGS is limited to genes expressed in the maternally derived seed coat and fiber in the developing seed. This complicates the use of GFP as a marker for VIGS because cotton fibers must be separated from unsilenced tissue in the seed to determine if they are silenced. Nevertheless, fibers from a large number of seeds can be rapidly screened following placement into 96-well plates. Methods for quantifying the extent of silencing using semiquantitative RT-PCR are given.

Coupled with the advantages afforded by the model plant Arabidopsis, virus-induced gene silencing (VIGS) offers a rapid means to assess gene function. The geminivirus vector based on Cabbage leaf curl virus described here has the benefits of small insert size and persistent silencing of the target gene through the life cycle of the plant. Here, we show that genetic variation in the vast collection of Arabidopsis accessions can be leveraged to ameliorate viral symptomology that accompanies the VIGS procedure. The plasticity of phenotypes under different day lengths or temperature conditions can be exploited to achieve maximum silencing efficacy in either vegetative or inflorescence tissue, according to the question being asked. Protocols and vectors for Agro-infiltration of primary leaves, subapical pricking in older plants, and microprojectile bombardment are described.

Traditional assays that monitor cAMP inhibition by opioid receptor ligands require second-messenger accumulation over periods of 10-20 minutes. Since receptor regulation occurs within a similar time frame, such assays do not discriminate the actual signal from its modulation. Here we used bioluminescence resonance energy transfer to monitor inhibition of cAMP production by δ-opioid receptor (DOR) agonists in real time. cAMP inhibition elicited by different agonists over a period of 15 minutes was biphasic, with response buildup during the first 6 to 7 minutes, followed by a second phase of response decay or of no further increment. The rate at which the cAMP response disappeared was correlated with operational parameters describing ligand efficiency [log(τ/KA)] to promote Gαi activation, as well as with ligand ability to promote internalization during the time course of the assay. Thus, ligands that displayed low signaling efficiency and poor sequestration(SB235863 ([8R-(4bS*,8aα,8aβ,12bβ)]7,10-dimethyl-1-methoxy-11-(2-ethylpropyl)oxycarbonyl 5,6,7,8,12,12b-hexahydro-(9H)-4,8-methanobenzofuro[3,2-e]pyrrolo[2,3-g]isoquinoline hydrochloride), morphine) had minimal or no response decay. On the other hand, the decay rate was pronounced for deltorphin II, [d-Pen(2), d-pen(5)]-enkephalin, met-enkephalin, and SNC-80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide), which displayed high signaling efficiency and internalization. Moreover, inhibition of internalization by dynasore reduced or abolished response decay by internalizing ligands. Unlike acute responses, endocytic profiles were not predictive of whether an agonist would induce prolonged cAMP inhibition over sustained (30-120 minutes) DOR stimulation. Taken together, the data indicate that ligand ability to evoke G-protein activation or promote endocytosis was predictive of response duration over short, but not over sustained periods of cAMP inhibition.

The 5-HT2AR (5-hydroxytryptamine-2A receptor) is a GPCR (G-protein-coupled receptor) that is implicated in the actions of hallucinogens and represents a major target of atypical antipsychotic agents. In addition to its classical signalling though PLC (phospholipase C), the receptor can activate several other pathways, including ARF (ADP-ribosylation factor)-dependent activation of PLD (phospholipase D), which appears to be achieved through a mechanism independent of heterotrimeric G-proteins. In the present study we show that wild-type and inactive constructs of PLD1 (but not PLD2) respectively facilitate and inhibit ARF-dependent PLD signalling by the 5-HT2AR. Furthermore we demonstrate that PLD1 specifically co-immunoprecipitates with the receptor and binds to a distal site in GST (glutathione transferase) fusion protein constructs of its C-terminal tail which is distinct from the ARF-interaction site, thereby suggesting the existence of a functional ARF-PLD signalling complex directly associated with this receptor. This reveals the spatial co-ordination of an important GPCR, transducer and effector into a physical complex that is likely to reinforce the impact of receptor activation on a heterotrimeric G-protein-independent signalling pathway. Signalling of this receptor through such non-canonical pathways may be important to its role in particular disorders.

The retinoblastoma-related protein (RBR) is required for cell cycle control and differentiation and is expressed throughout the life of plants and animals. In this study, the tomato golden mosaic virus (TGMV) geminivirus vector was used to silence NbRBR1 in Nicotiana benthamiana by microprojectile bombardment into fully developed leaves. Similar to previous results using agroinoculation of a tobacco rattle virus silencing vector [Park et al. (Plant J 42:153, 2005)], developmental defects caused by disruptions in cell size and number were seen in new growth. Leaf midvein cross-sections showed tissue-specific differences in size, cell number, and cell morphology. While cortical cell numbers decreased, size increased to maintain overall shape. In contrast, xylem parenchyma cells increased approximately three fold but remained small. Normally straight flowers often curved up to 360 degrees without a significant change in size. However, the most striking phenotype was cell death in mature cells after a delay of 3-4 weeks. Trypan blue staining confirmed cell death and demonstrated that cell death was absent in similarly treated leaves of wild type TGMV-inoculated plants. Quantitative RT-PCR confirmed that the mature TGMV:RBR-inoculated leaves still maintained reduced accumulation of RBR transcript at 4 weeks compared to controls. The results suggest that either inappropriate activation of the cell cycle is lethal in plants or that RBR has other functions, unrelated to the cell cycle. The results also demonstrate that continual transcription of RBR is necessary for cell survival.

In this study we have shown that N376 to D mutation in the conserved NPxxY motif within the carboxy terminal tail domain (CT) of the 5-HT2A receptor alters the binding preference of GST-fusion protein constructs of the CT domain from ARF1 to an alternative isoform, ARF6. These findings were corroborated by experiments investigating co-immunoprecipitation of the wild type (WT) and N376D mutant of the 5-HT2A receptor with ARF1 or 6 or dominant negative ARF1/6 constructs co-expressed in COS7 cells. In functional assays of 5-HT-induced phospholipase D (PLD) activation responses of the WT receptor were inhibited by a dominant negative mutant of ARF1 but not ARF6, whereas responses of the N376D mutant were strongly inhibited by negative mutant ARF6. No equivalent effect of the ARF mutants was seen on phospholipase C activation. In experiments assaying 5-HT-induced increases in [35S]GTPgammaS binding to ARF 1/6 immunoprecipitates as a measure of ARF activation, increased ARF6 activation was seen only with the mutant receptor. When cellular PLD responses of other NPxxY- or a DPxxY-containing GPCRs were measured in the presence of dominant negative ARF1/6 constructs, the majority, but not all, fitted the pattern exemplified by the 5-HT2A receptor and its N376D mutant. These data suggest that the presence of the N or a D in this highly conserved motif is an important, but not exclusive, determinant of which ARF isoform interacts with the GPCR.

Investigations were undertaken to determine the cause of decreases over time in the release rate from levonorgestrel (LNG) implants consisting of silicone elastomer tubing filled with crystalline steroid. Emptying and refilling with the same steroid partially restored release rate. Surprisingly, a further increment in release rates was attained if the tubing was briefly irrigated with methanol before refill. Fractional crystallization showed that release-modulating factors could be concentrated in mother liquors and were initially present as impurities. Boiling LNG in ethanol or methanol produced a number of release-modulating factors of which the most prominent was also found in one production lot of LNG. It was identified as 6beta-hydroxy-levonorgestrel (6beta-OH-LNG). Added to LNG at the 2% level, 6beta-OH-LNG decreased the release rate by 27%., (Copyright 2004 Wiley-Liss, Inc. and the American Pharmacists Association)

The 5-hydroxytryptamine 2A receptor (5-HT2AR) is a member of the class I family of rhodopsin-related G protein-coupled receptors. The receptor is known to activate phospholipase C via the heterotrimeric G proteins Gq/11, but we showed previously that it can also signal through the phospholipase D (PLD) pathway in an ADP-ribosylation factor (ARF)-dependent manner that seems to be independent of Gq/11 (Mitchell et al., 1998). Both coimmunoprecipitation experiments and the effects of negative mutant ARF constructs on 5-HT2AR-induced PLD activation here suggested that ARF1 may play a greater role than ARF6 in the function of this receptor. Furthermore, we demonstrated using glutathione S-transferase (GST)-fusion proteins of receptor domains that ARF1 and ARF6 bind to the third intracellular loop (i3) and the carboxy terminal tail (ct) of the 5-HT2AR. The association of ARF1 with the ct domain of the receptor was stronger than its interaction with i3, or the interactions of ARF6 with either construct. Experiments using ARF mutants that are deficient in GTP loading, and the in vitro addition of GTPgammaS suggested that GTP loading enhances ARF1 binding to the receptor. The N376PxxY motif in the transmembrane 7 domain of the receptor (rather than a N376DPxxY mutant form) was shown to be essential for ARF-dependent PLD signaling and ARF1 coimmunoprecipitation. In GST-fusion proteins of the 5-HT2AR ct domain, mutation of Asn376 to Asp also markedly reduced ARF1-HA binding, although additional motifs in the Asn376-Asn384 sequence and to a lesser extent elsewhere, seem also to contribute to the interaction.

G protein-coupled receptors can potentially activate phospholipase D (PLD) by a number of routes. We show here that the native M3 muscarinic receptor in 1321N1 cells and an epitope-tagged M3 receptor expressed in COS7 cells substantially utilize an ADP-ribosylation factor (ARF)-dependent route of PLD activation. This pathway is activated at the plasma membrane but appears to be largely independent of G, phospholipase C, Ca2+ q/11, protein kinase C, tyrosine kinases, and phosphatidyl inositol 3-kinase. We report instead that it involves physical association of ARF with the M3 receptor as demonstrated by co-immunoprecipitation and by in vitro interaction with a glutathione S-transferase fusion protein of the receptor's third intracellular loop domain. Experiments with mutant constructs of ARF1/6 and PLD1/2 indicate that the M3 receptor displays a major ARF1-dependent route of PLD1 activation with an additional ARF6-dependent pathway to PLD1 or PLD2. Examples of other G protein-coupled receptors assessed in comparison display alternative pathways of protein kinase C- or ARF6-dependent activation of PLD2.

The PAC(1), VPAC(1) and VPAC(2) receptors are members of the secretin (Group II) family of G protein-coupled receptors. All members of this family activate adenylate cyclase and several have also been shown to activate phospholipase C. We have recently reported that the rat VPAC(1), VPAC(2) and PAC(1) receptors activate phospholipase D and that distinct pathways are utilised by two intracellular loop 3 splice variants of PAC(1), one of which is ARF-dependent. Phospholipase D activation by the hop1, but not the null (short), form of the PAC(1) receptor is sensitive to brefeldin A, an inhibitor of GTP exchange at ARF. We have expressed the null and hop1 intracellular loop 3 domains of the human PAC(1) receptor in bacteria as GST-fusion proteins and used them as peptide affinity matrices to determine whether a functional interaction exists between these domains and ARF. Using this GST pull-down assay, we have shown binding of the small G protein ARF6 to the hop1 but not the null domain of this receptor.

The receptors for the neuropeptides vasoactive intestinal polypeptide and pituitary adenylate cyclase-activating polypeptide are strong activators of adenylate cyclase, but recent evidence suggests that they can elicit a number of additional intracellular signals. Some of these are likely to be downstream of the conventional adenylate cyclase pathway, but it is now clear that others reflect novel primary coupling events of the receptors.

The VPAC(1) and VPAC(2) receptors for vasoactive intestinal polypeptide and the PAC(1) receptor for pituitary adenylate cyclase-activating polypeptide are members of a subfamily of G protein-coupled receptors (GPCRs). We recently reported that phospholipase D (PLD) activation by members of the rhodopsin group of GPCRs occurs by at least two routes, one of which seems to involve the small G protein ADP-ribosylation factor (ARF) and its physical association with GPCRs. Here we report that rat VPAC and PAC(1) receptors can also stimulate PLD (albeit less potently than adenylate cyclase) in transfected cells and also in cells where they are natively expressed. PLD responses of the VPAC receptors and the hop1 spice variant of the PAC(1) receptor but not its null form are sensitive to brefeldin A (BFA), an inhibitor of GTP exchange at ARF. The presence of the hop1 cassette in the rat PAC(1) receptor facilitates PLD activation in the absence of marked changes in ligand binding, receptor internalization, and adenylate cyclase activation, with some reduction in phospholipase C activation. Both VPAC(2) and PAC(1-hop1) (but not PAC(1-null)) receptors were shown to associate with immunoprecipitates directed against native or epitope-tagged ARF. A chimeric construct of the VPAC(2) receptor body with intracellular loop 3 (i3) of the PAC(1-null) receptor mediated BFA-insensitive activation of PLD, whereas the response of the corresponding PAC(1-hop1) construct was BFA-sensitive. Motifs in i3 of the PAC(1-hop1) receptor may act as critical determinants of coupling to ARF-dependent PLD activation by contributing to the GPCR:ARF interface.

The vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating polypeptide type 2 (VPAC(2)) receptor was shown to induce both [(3)H]inositol phosphate ([(3)H]InsP)and cAMP production in transfected COS7 cells and in GH(3) cells where it is natively expressed. Neither cholera toxin nor forskolin could elicit an equivalent [(3)H]InsP response, suggesting independent coupling of the two pathways. The VPAC(2) receptor-mediated [(3)H]InsP response was partially inhibited by pertussis toxin (Ptx) and by the G beta gamma-sequestering C-terminal fragment of GRK2 (GRK2-ct) in COS7 and GH(3) cells, whereas responses of control receptors were unaffected. Blockers of receptor-activated Ca(2+) influx pathways (Co(2+) and SKF 96365) also partially inhibited VPAC(2) receptor-mediated [(3)H]InsP responses. This inhibition was not present in the component of the response remaining after Ptx treatment. A range of blockers of voltage-sensitive Ca(2+) channels were ineffective, consistent with the reported lack of these channels in COS7 cells. The data suggest that the VPAC(2) receptor may couple to phospholipase C through both Ptx-insensitive and Ptx-sensitive G proteins (G(q/11) and G(i/o), respectively) to generate [(3)H]InsP. In addition to G beta gamma, G(i/o) activation appears to require receptor-activated Ca(2+) entry. This is consistent with the possibility that not only G alpha(q/11)-responsive and G beta gamma-responsive isoforms of phospholipase C but also Ca(2+)-responsive forms may contribute to the overall [(3)H]InsP response.

GH3 cells were stably transfected with the wild-type murine GnRH receptor and a clonal cell line selected on the basis of inositol phosphate production and PRL/GH release in response to GnRH. This cell line (wt28) was characterized by [125I]GnRH analog binding, [3H]inositol phosphate response to GnRH, and hormone secretion. We examined the activation of the mitogen-activated protein kinase isoforms, extracellular signal-regulated kinase 1/2 (ERK1/2) and tyrosine kinases in wt28 cells and alphaT3-1 cells (which express a native GnRH) using specific phospho-ERK1/2 and phosphotyrosine antibodies. Concentration-response and time-course data revealed that a sustained ERK1/2 response was seen only in aT3-1 cells. Furthermore, GnRH-induced tyrosine phosphorylation was detectable in alphaT3-1 cells, but not in wt28 cells. Activators for several different signaling pathways revealed distinct differences between the cell types. Protein kinase C activation by phorbol 12,13-dibutyrate was very effective in alphaT3-1 cells at phosphorylation of both ERK1/2 and tyrosine, whereas raising cAMP levels using forskolin also strongly increased wt28 cell ERK1/2 phosphorylation. Only the tyrosine phosphatase inhibitor pervanadate increased tyrosine phosphorylation in wt28 cells. The lack of sustained ERK1/2 phosphorylation in wt28 cells could be the result of minimal tyrosine kinase activation by GnRH compounded by a different pathway profile for ERK1/2 activation. When pervanadate and GnRH were combined, ERK1/2 phosphorylation was synergistic and sustained in wt28 cells, whereas the response was additive in alphaT3-1 cells. In sum, the intracellular pathways leading to ERK1/2 and tyrosine phosphorylation in alphaT3-1 and wt28 cells are distinct; thus, activating GnRH receptors in each of the two cell types leads to different sequelae of events regarding ERK1/2 activation.

Norplant subdermal implants containing levonorgestrel were used for contraception and compared with the Copper T, Model T Cu 200, during 42 months of use in an open study in Chile, the Dominican Republic, and Finland. Among 324 women enrolled for the implant regimen, there were no pregnancies in the 1st 2.5 years, and 2 by the end of 42 months. 1st segment net and gross cumulative pregnancy rates were 0.7 and 1.2/100 respectively, at 3.5 years. Cumulative pregnancy rates for the T Cu 200 group were 2.9, net and 3.5/100, gross at 42 months. More than 1/2 of the implant acceptors, 51.6/100 were continuing use at 3.5 years, somewhat above the continuation rate of the T Cu acceptors, 43.5/100, but not significantly so.

Polydimethylsiloxane (Silastic) capsules containing megestrol acetate have been implanted in subcutaneous tissue as a method of long-term contraception. Histological studies revealed a granulomatous foreign body reaction around these capsules with birefringent crystals in multinucleated giant cells. Although several authors had interpreted these crystals as steroids, this seemed unlikely since the tissue had been processed with organic solvents. Analysis of these crystals by polarization microscopy, scanning electron microscopy, and X-ray microanalysis demonstrated that the material was talc. The talc was probably introduced as a contaminant from gloves during the implantation. Further analysis showed that capsule fragmentation could not have produced the material since Silastic particles could not be detected.

NORPLANTTM (Laboratorios Gutfol, S.A., Mexico City, Mexico) subdermal implants containing levonorgestrel were accepted by 816 women in a two-phase study initiated in 1975 and augmented in 1978. Through 3 years of first-segment use, acceptors experienced five pregnancies, a cumulative net rate of 0.7 per 100, and a gross pregnancy rate of 1.1 per 100. First-segment continuation at 3 years was at the rate of 44.6 per 100, an annual average of 76 per 100. NORPLANT users experienced irregular menstruation, with high interindividual variation, but with normal average numbers of bleeding days and of bleeding episodes. Hemoglobin levels increased moderately during implant use. The incidence of systolic blood pressure readings above 145 mm Hg or diastolic readings above 95 mm Hg was similar to that observed among users of the TCu 200 intrauterine device enrolled under the same selection criteria in the same clinics between the two phases of the NORPLANT study. Comparative data on pregnancy and other rates are also given for these intrauterine device acceptors.

The correlations of plasma levels of levonorgestrel, megestrol acetate and norgestrienone with the doses delivered from subdermal implants and contraceptive rings have been examined. Differences in plasma levels as large as three-fold between different subjects receiving nearly identical doses were observed with all three steroids. The relative plasma levels seen from one subject to another showed high consistency over time. Part of the explanation may lie in differing levels of sex hormone binding globulin, but additional factors must be operative since megestrol acetate binds only weakly with this carrier.

Silastic implants containing the progestin, levonorgestrel, were tested as long-term contraceptives in 101 women. After three full years of exposure and 2,998 woman-months of use, no pregnancies had occurred. The continuation rates were 87% at 12 months, 79% at 24 months and 66% at 36 months. The most important side effect was excessive or irregular bleeding during the first year. No treatment was offered for this side effect other than vitamins and iron or change of method, with the exception of 3 cases where ethinyl estradiol was used one time for 2 weeks each. Bleeding disturbances led 8 patients to ask for removal of implants. Other side effects were headache, acne and lower abdominal pain. Blood and urine analysis tested 17 different parameters and all but plasma cortisol remained within normal limits throughout the study. A general tendency toward lowered cortisol values was observed and two subjects had more than one value below the normal limit for the population during the study. Glucose tolerance tests during the second year were abnormal in two women with familial diabetes but they returned to normal values spontaneously at the next test. It is concluded that levonorgestrel implants offer effective protection against pregnancy during the first three years of continuous use. Their acceptability and few side effects justify larger trials, especially if treatment of bleeding irregularities is introduced.

1. Intravenous administration of platelet activating factor, Paf (600 ng kg-1 h-1) to ventilated anaesthetised guinea-pigs induced bronchial hyperresponsiveness to i.v. acetylcholine. 2. Pretreatment of ventilated, anaesthetised guinea-pigs with the beta-adrenoceptor antagonist, propranolol, or the non-steroidal anti-inflammatory drug, indomethacin, induced bronchial hyperresponsiveness to i.v. histamine. 3. Paf-induced bronchial hyperresponsiveness was significantly attenuated by pretreatment with three different Paf antagonists, CV-3988, BN 52021 and WEB 2086. 4. Pretreatment of guinea-pigs with CV-3988, BN 52021 or WEB 2086 at doses inhibiting Paf-induced bronchial hyperresponsiveness, had no significant effect on propranolol or indomethacin-induced bronchial hyperresponsiveness. 5. It is suggested that bronchial hyperresponsiveness induced by propranolol or indomethacin is not secondary to Paf release in the guinea-pig.

Three forms of subdermal implants containing levonorgestrel are described. These are: capsules, in which the powdered drug is sealed inside of lengths of Medical Grade Silastic tubing; homogeneous rods, in which the drug is uniformly dispersed in Silastic 382 Medical Grade Elastomer; and covered rods, in which a core rod of drug and filler-free polydimethylsiloxane polymer (50:50, Wt:Wt) are sealed inside thin-walled Silastic tubing. Long-term in vivo release rates from human subjects are presented; 6.5 years for capsules, 3.6 years for homogeneous rods and 4 years for covered rods. Sets of six capsules release a decreasing amount of drug through the first few hundred days in situ and after 500 days a fairly constant rate of about 35 micrograms per day is released (2 micrograms/cm). Homogeneous rods deliver a continuously declining amount of drug during the entire time studied. In the first 100 days the release averages 136 micrograms per day from a set of three 3-cm rods (15 micrograms/cm), gradually declining to 30 micrograms per day (3.3 micrograms/cm) from day 800 to day 1300. The covered rods deliver at a constant rate of 17.5 micrograms per day for a 3-cm rod (5.83 micrograms/cm) through 4 years.

Platelet activating factor (PAF-acether) was shown to cause a fall in the circulating platelet count and blood pressure (BP) in anaesthetised rabbits. CV3988 caused a dose-dependent inhibition of these responses to a low dose of PAF-acether (150 ng X kg-1). CV3988 itself was found to cause a fall in both BP and platelet count in vivo. Collagen (40 micrograms X kg-1) i.v. caused sudden death in rabbits and pretreatment with CV3988 (5 mg X kg-1) caused a 62% inhibition of the platelet count response to collagen without significantly increasing the survival rate. In the rat, collagen (40 micrograms X kg-1) was not lethal and CV3988 caused a dose-dependent inhibition of the fall in platelet count due to collagen, but this action was short-lived. CV3988 also caused agonist actions in the rat. These results show that CV3988 inhibits the effects of PAF-acether in vivo and suggests that PAF-acether may play a role in mediating collagen-induced platelet aggregation in vivo.

Subcutaneously placed Silastic capsules containing levonorgestrel are effective for 5 years and have a higher continuation rate than other methods of reversible contraception. Six 3 cm capsules are required to achieve satisfactory circulating levels of levonorgestrel. Two 4 cm covered Silastic rods containing levonorgestrel, which are easier to manufacture, insert, and remove than the capsules, produce similar in vitro release rates. This study compared clinical and metabolic effects as well as bleeding patterns in 23 women using either six capsules (n = 11) or two covered rods (n = 12). Serum levels of levonorgestrel, lipids, and lipoproteins as well as frequency of elevated progesterone levels were compared in serum samples obtained before treatment and 1, 6, 12, 18, and 24 months after insertion with the two systems. While bleeding patterns were similar for users of the two systems, rod users had slightly higher serum levels of levonorgestrel and a lower incidence of cycles with elevated progesterone levels. Therefore, rods could replace capsules as a long-term, reversible contraceptive method.

Two models of levonorgestrel-releasing intrauterine devices were studied. Model A was designed to deliver 20 micrograms/day, and model B was designed to deliver 30 micrograms/day. Plasma concentrations of levonorgestrel were determined in blood samples taken from women who used the levonorgestrel-releasing intrauterine devices (IUDs) and who participated in a clinical trial for more than 5 years. During clinical studies IUDs have been removed for various reasons between 8 and 40 months after insertion. The rate of release of levonorgestrel from the IUDs was calculated by determining the remaining amount of levonorgestrel. Plasma concentrations of levonorgestrel ranged between a mean +/- standard deviation (SD) concentration of 166 +/- 75 pg/ml and 131 +/- 32 pg/ml for the first 18 months of use and between 101 +/- 37 pg/ml and 74 +/- 15 pg/ml at 24 and 60 months after insertion of the IUD. The plasma concentrations from 24 months through 60 months were significantly lower than concentrations measured during initial months of IUD use, but not between the two devices. There was a strong correlation between the time of use and the amount of levonorgestrel lost from the IUDs. The calculated mean daily release of levonorgestrel was 17.6 micrograms for model A and 22.2 micrograms for model B. This gives a calculated lifetime of more than 6 years for a levonorgestrel-releasing IUD.

Ten healthy, normally menstruating women attending a family planning clinic in Santo Domingo Participated in a study to determine the effects on plasma lipid levels of levonorgestrel and estradiol released from a contraceptive ring. A schedule of 21 days of use followed by 7 days of non-use was followed for 6 cycles. During the first two cycles of use, concentrations of cholesterol, HDL cholesterol, triglycerides and LDL cholesterol declined significantly from control levels, up to 25% for cholesterol, 28% for HDL cholesterol, 45% for friglycerides and 24% for LDL cholesterol. There were no subsequent changes with continued use. These declines are similar in direction but of lesser magnitude than those reported from clinics in other countries where pretreatment plasma levels of the same lipids are considerably higher. There was no significant change in the total cholesterol to HDL cholesterol ratio during treatment.

Norgestrienone implants delivering approximately 225 microgram/ day were tested clinically for contraceptive effectiveness and acceptability in 145 women. Five pregnancies occurred in 2259 woman-months of use, one in the 11th month, one in the 15th and three in the 16th month of use. Continuation rate at 12 months was 86.7. The number of bleeding runs and bleeding days was increased in approximately 12% of the subjects. Ten percent of the patients had no bleeding in the first 90 days of treatment. Changes in bleeding pattern led to closures in four cases. Headache and signs of mild androgenicity were among the leading side effects. Blood and urine analysis throughout the study showed normal values of 17 different parameters, but a tendency to lower cholesterolemia not associated with changes in thyroid hormone levels, was observed in several patients. Cortisol was found slightly under the lower normal range in one subject without clinical manifestations of hypoadrenalism. It is concluded that norgestrienone implants should be replaced every twelve months for maximal contraceptive effect and because of their efficacy and good acceptability, evaluation of their long term use is warranted.

1 Intravenous infusion of platelet activating factor (Paf), adenosine diphosphate (ADP), collagen and the thromboxane-mimetic U46619 induced a dose-related accumulation of 111indium-labelled platelets into the thoracic region of anaesthetized guinea-pigs. 2 Intravenous infusion of Paf increased the reactivity of the airways to the spasmogen histamine. Such changes were not observed following treatment with ADP, collagen or U46619. 3 Paf-induced bronchial hyperreactivity is not secondary to pulmonary platelet recruitment, changes in basal lung function or cardiovascular changes. 4 Paf-induced bronchial hyperreactivity is not dependent upon the endogenous generation of ADP or thromboxane.

Silastic implants containing the progestin, levonorgestrel, were tested as a long-term contraceptive system in 101 women. During five full years of use, no pregnancies occurred. The 5-year continuation rate was 54%. Menstrual irregularities were the most frequent reason for termination of use but only during the first year. More than half of the terminations for this reason were in the first year. Some of the subjects elected to continue use of the implants beyond 5 years, allowing release rate data to be obtained through 6 years. From the second through the sixth year of use, the implants delivered a constant 30 micrograms per day of levonorgestrel to the subjects, and even after six years 57% of the original steroid content remained in the capsules. Return of fertility following removal was essentially immediate and not related to time of use. Medical reasons for removal were infrequent and no pattern was discernible.

Plasma concentrations of cholesterol, HDL-cholesterol, and levonorgestrel were determined in two groups of women using levonorgestrel-releasing subdermal implants. One group used six capsules (NORPLANT)*; the other six covered rods. Plasma concentrations of levonorgestrel among NORPLANT users averaged 700 pg/ml in the first two weeks of use, decreased to 300 pg/ml at 8 weeks, and to about 230 pg/ml by 50 weeks. Concentrations among covered rod users were 1.4 to 1.7 times higher at comparable time periods. Total serum cholesterol and HDL-cholesterol were decreased as compared with controls at all sampling intervals during the 114 weeks of the trial, although the differences did not meet tests of significance at all time periods. Decreases during the test period were of the order of 10 percent, except for total cholesterol among covered rod users where the decrease was less. Cholesterol to HDL-cholesterol ratios did not differ significantly from control values at any sampling period.

An intravenous infusion of platelet activating factor (Paf) in the guinea-pig elicits an increase in bronchial responsiveness to the spasmogens, histamine and bombesin. Airways obstruction induced by bombesin in Paf-treated animals is poorly reversed by isoprenaline compared to comparable airways obstruction induced by bombesin in vehicle-treated animals. Isoprenaline induced a comparable dose-related relaxation in vitro of tracheal smooth muscle isolated from Paf- and vehicle-treated animals. No change in beta-adrenoceptor numbers or binding affinity was observed in lungs removed from Paf-treated animals in comparison with those from vehicle-treated animals, or after direct incubation with Paf in vitro. The reduced bronchodilator responsiveness to isoprenaline in Paf-treated animals is not related to changes in pulmonary beta-adrenoceptor function. These results suggest that non-spasmogenic elements may contribute to airways obstruction induced in hyper-responsive animals.

Two groups of implant users and two groups of IUD users participated in the study at different times. In the first groups, fractionation of lipoproteins was performed on serum samples from 28 subjects who had used the implants for 37 months and from 26 subjects who had used the Copper TCu 200 for 30 months. Users of the implants had significantly lower levels of total cholesterol, triglycerides and LDL-cholesterol than users of the IUDs. HDL-cholesterol levels were not different between the groups. In the other groups of women, general chemistries and selected hormone assays were carried out on samples from 30 subjects who had used implants for 51 months and from 24 subjects who had used the TCu 200 IUDs for 43 months. The parameters studied were the SM-12 chemistry group profile, estradiol, cortisol, testosterone, triiodothyronine, thyroxine and thyroid stimulating hormone. In the implant group, mean serum glucose levels were statistically significantly elevated and inorganic phosphorus levels were significantly reduced as compared to the IUD group. In both cases all individual measurements were within the normal range for the population. Testosterone and triiodothyronine levels in the implant group were significantly lower than in the IUD group, but no individual values were outside the normal range for the population. Mean values for all other parameters were not significantly different between the groups.

A total of 189 women volunteered to accept subdermal implants for contraception. The implants were "covered rods", consisting of a core rod containing equal parts by weight of levonorgestrel and polydimethylsiloxane and sealed inside a thin-walled tube of Silastic tubing with medical adhesive. In one study 78 women used 4 3cm rods (study 07) and in the other 111 women used 6 3cm rods. In 5 years of use there were no pregnancies in either group. Terminations because of menstrual problems were twice as frequent among the 4-rod users than among users of the 6 rods. Menstrual pattern analysis is presented for the two rod regimens and compared with the previously reported patterns for the 6-capsule regimen (NORPLANT). Long--term in vivo release rates are also presented.

An effort was made to achieve different levels of plasma levonorgestrel (13-ethyl-17 alpha-ethynyl-17-hydroxy-gon-4-en-3-one) (Ng) concentrations by the intra-uterine admininistration of levonorgestrel-releasing intra-uterine devices (IUD). Three different models were designed to release different doses of Ng daily. Sixteen women volunteered for the study and had a Ng IUD inserted post-menstrually. The effect of the IUDs on the ovulatory pattern was studied by measuring plasma concentration of oestradiol and progesterone by radioimmunoassay. Ng plasma concentrations were also measured by radioimmunoassay. In vitro experiments to measure the release of Ng from each type of device were carried out. Although some overlapping of mean plasma levels of Ng occurred during use of the three models of the IUDs, suppression of ovulation was clearly dose-dependent. Wide variations in plasma levels of Ng were seen between individuals but quite uniform plasma levels were seen in each subject for up to one year. IUDs where in vitro release rates were equivalent to each other also resulted in wide individual variations in plasma concentrations of Ng.