The M1 muscarinic receptor is present in situ as a ligand-regulated mixture of monomers and oligomeric complexes

The quaternary organization of rhodopsin-like G protein-coupled receptors in native tissues is unknown. To address this we generated mice in which the M₁ muscarinic acetylcholine receptor was replaced with a C-terminally monomeric enhanced green fluorescent protein (mEGFP)–linked variant. Fluorescence imaging of brain slices demonstrated appropriate regional distribution, and using both anti-M₁ and anti–green fluorescent protein antisera the expressed transgene was detected in both cortex and hippocampus only as the full-length polypeptide. M₁-mEGFP was expressed at levels equal to the M₁ receptor in wild-type mice and was expressed throughout cell bodies and projections in cultured neurons from these animals. Signaling and behavioral studies demonstrated M₁-mEGFP was fully active. Application of fluorescence intensity fluctuation spectrometry to regions of interest within M₁-mEGFP–expressing neurons quantified local levels of expression and showed the receptor was present as a mixture of monomers, dimers, and higher-order oligomeric complexes. Treatment with both an agonist and an antagonist ligand promoted monomerization of the M₁-mEGFP receptor. The quaternary organization of a class A G protein-coupled receptor in situ was directly quantified in neurons in this study, which answers the much-debated question of the extent and potential ligand-induced regulation of basal quaternary organization of such a receptor in native tissue when present at endogenous expression levels.

Measuring and understanding the extent and potential significance of quaternary organization of members of the class A (rhodopsin-like) family of G protein-coupled receptors (GPCRs) have both fascinated and frustrated researchers for many years (1, 2). Over time, a wide range of methods have been applied to address this question, and many different GPCRs have been examined. Outcomes have ranged from assertions that such receptors are monomeric and that results consistent with other conclusions reflect either artifacts of the method of measurement or that studies have been performed at nonphysiological levels of expression of the receptor being studied, to those that have suggested rather stable dimeric or tetrameric complexes (1). Only in the case of rhodopsin, the photon receptor expressed at very high levels (in the range of 24,000–30,000 molecules/µm²) in rod outer segments of the eye, have detailed studies been conducted in situ on a class A GPCR. In this example, various studies have shown that rhodopsin is organized as rows of dimers (3, 4). However, to our knowledge, no other GPCR is expressed natively at levels akin to rhodopsin. As such, although a substantial number of studies, generally performed in transfected cell lines or in artificial bilayer systems, have provided evidence that other GPCRs can and do form dimeric and/or higher-order quaternary complexes in a concentration-dependent manner (1, 2), how levels of expression required to observe such complexes relate to expression levels in native cells and tissues has been poorly defined, as is the stability of such complexes and whether they are regulated by ligand binding.Developments in fluorescence fluctuation analysis (FFA) have facilitated efforts to define the oligomeric status of transmembrane receptor proteins (5, 6). Unlike methods based on resonance energy transfer, only a single fluorophore-linked protein is required to be expressed to use FFA. It is, therefore, more practical to use such methods in native cells and tissues if linked to genome-editing approaches and/or the generation of transgenic “knock-in” animal models in which a receptor of interest is replaced with a fluorophore-tagged, modified form of the receptor. Moreover, the recent introduction of fluorescence intensity fluctuation (FIF) spectrometry (7–10) has overcome issues with other methods based on FFA that result in information being compressed due to averaging of oligomer-size data from interrogated regions of interest (RoIs) in which complex mixtures of oligomers of different sizes may be present (7, 8).To define whether the class A M₁ muscarinic acetylcholine receptor is present in hippocampal and cortical neurons as strict monomers or as a range of monomeric, dimeric, and, potentially, oligomeric complexes, we applied FIF spectrometry to images of such neurons isolated from a line of transgenic mice in which we replaced the M₁ receptor with a form of the receptor that includes C-terminally linked monomeric enhanced green fluorescent protein (mEGFP). We first show that both expression levels and function of the introduced M₁-mEGFP construct appear equivalent to the native M₁ receptor in wild-type (WT) mice, using a range of methods and measures ranging from [³H]ligand binding and cell signaling assays to locomotion. We then demonstrate in hippocampal and cortical neurons that in the basal state, the M₁-mEGFP construct is present as a mixture of monomers and dimeric or oligomeric complexes. We also show that the presence of either an agonist or an antagonist ligand promotes monomerization of the receptor. In these studies, we combined analysis of images of a fluorophore-modified receptor in situ with calculation of receptor oligomer complexity. The studies provide a clear and unambiguous answer to a long-standing question that has been the subject of considerable debate (11–13) but that has previously been restricted to studies performed on transfected cell lines. Moreover, these studies are a model for subsequent studies for researchers who plan to explore the topic of dimerization of rhodopsin-family GPCRs.     

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos,Peru

Over half the world’s population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito–human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = −9.11) and 12.4% (95% CI 4.2%, ∞); z = −2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Aedes-borne viral diseases (ABVDs) [e.g., dengue (DENV), chikungunya, Zika (ZIKV), and yellow fever] are devastating, expanding global public health threats that disproportionally affect low- and middle-income countries. DENV, one of the most rapidly increasing vector-borne infectious diseases, results in ∼400 million infections each year (1, 2), with 4 billion people at risk for infection annually (3). Currently, the primary means for ABVD prevention is controlling the primary mosquito vector, Aedes aegypti. Existing vector control interventions, however, have failed to prevent ABV transmission and epidemics (4–6).There is an urgent need to develop evidence-based guidance for the use of new and existing ABV vector control tools. The evidence base for vector control against ABVs is weak, despite considerable government investments in World Health Organization (WHO)-recommended control of larval habitats (larviciding, container removal) and ultra-low-volume insecticide spraying (4, 5, 7–9). These strategies continue to be implemented despite the lack of rigorously generated data from controlled clinical trials demonstrating they reduce ABV infection or disease (6). The only ABV intervention with a proven epidemiological impact in a cluster-randomized control trial (cRCT) assessed community mobilization to reduce mosquito larval habitats (10). A recent test-negative trial with Wolbachia-infected mosquitoes reported a significant reduction of DENV illness in Indonesia (11).Spatial repellents (SRs) are devices that contain volatile active ingredients that disperse in air. The active ingredients can repel mosquitoes from entering a treated space, inhibit attraction to human host cues, or disrupt mosquito biting and blood-feeding behavior and, thus, interfere with mosquito–human contact (12–14). Any of these outcomes reduce the probability of pathogen transmission. Pyrethroid-based SRs have shown efficacy in reducing malaria infections in China (15) and Indonesia (16). There have, however, been no clinical trials evaluating the protective efficacy (PE) of SRs against ABV infection or disease.To generate evidence for public health consideration, we conducted a double-blinded, parallel cRCT to demonstrate and quantify the PE of a transfluthrin-based SR to reduce ABV infection incidence over 2 y in a human cohort in Iquitos, Peru.     

The Unrealized Potential of Cervical Smears

We have a simple, efficient method for early detection of cervical cancer, yet there has been little change in mortality of this disease in 10 years. We need some new approach if we are to reach more women with this screening method, including prenatal patients under 25.     

Getting To Outcomes: a community-based participatory approach to preventive interventions

Bouget, D. (1998). The Juppé Plan and the future of the French social welfare system. Journal of European Social Policy, 8(2) 154-172.     

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c- derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid- specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.     

In vivo and in vitro studies into the immunological changes following iodine 131 therapy for Graves' disease

Radio-iodine therapy for Graves' disease is followed by immunological changes in addition to effects on thyroid hormone production. The present study examined these changes and the mechanisms responsible for them. Of the 15 patients enrolled in the study, 10 became hypothyroid in the first year after iodine 131 therapy. Patients who became hypothyroid had a tendency to show a rise in serum thyrotropin receptor antibody levels (30 ± 14 to 40 ± 9 units; NS) and a significant rise in immunoglobulin production (324 ± 153 to 740 ±200 ng/ml; P < 0.0005) from mitogen-stimulated peripheral blood lymphocytes (a measure of B-cell activity) 2 months after iodine 131 therapy. The increases were not seen in the patients who remained euthyroid at 1 year. In vitro studies suggested that the rise in B-cell activity is due to a fall in suppressor T cell numbers, a change shown to occur following iodine 131 therapy in previous studies. Our results indicate that immunological changes do arise after iodine 131 therapy for Graves' disease but appear to be confined to patients who subsequently became hypothyroid. It is not possible from this study to determine whether the immunological changes appear as a consequence of thyroidal destruction leading to hypothyroidism or whether they contribute directly to it. Offprint requests to: R. Wilson     

Acute haemodynamic and metabolic effects of dopexamine, a new dopaminergic receptor agonist, in patients with chronic heart failure.

Dopexamine, a new compound with postjunctional dopamine receptor activating and beta adrenoceptor agonist properties, was given to 10 patients with chronic heart failure at diagnostic cardiac catheterisation to investigate its acute haemodynamic and metabolic effects. The drug was administered by intravenous infusion in three incremental doses and produced significant dose related increases in cardiac index, stroke volume index, and heart rate and falls in systemic vascular resistance and left ventricular end diastolic pressure; aortic and pulmonary artery pressures were unchanged. Isovolumic phase (max dP/dt and KVmax) and ejection phase (peak aortic blood velocity, maximum acceleration of blood, and maximum rate of change of power with time during ejection) indices of myocardial contractility were all increased by dopexamine but these changes were hard to interpret in the presence of an increase in heart rate. Myocardial efficiency and ejection fraction were both increased and left ventricular end diastolic and end systolic volumes fell. These largely beneficial changes were achieved without a statistically significant increase in myocardial oxygen consumption or disturbance of myocardial metabolic function. Dopexamine was well tolerated but tremor was reported by two patients at the intermediate dose and mild chest pain by two patients at the high dose.     

Efficacy of a simplified lower gastrointestinal flexible endoscope cleaning method

Published guidelines from the Center for Disease Control (CDC) “strongly recommended” gas sterilization or 30 minutes of high-level disinfection with either 2 percent glutaradehyde or 6 percent hydrogen peroxide following each flexible endoscope cleansing for proper care. The guidelines were proposed on the basis of previous CDC studies performed on glutaraldehyde disinfection of respiratory equipment. A prospective study was performed culturing flexible endoscopes following cannulation of the lower gastroinstestinal tract and cleansing. A uniform endoscope cleansing method without gas sterilization or high-level disinfection was used between patients. Thirty aerobic and 30 anaerobic RODAC bacterial culturings revealed no obligate anaerobic organism growth and only sparse, aerobic, environmental and cutaneous organism growth. These were no instances of documented or suspected postendoscopy infectious complications. Our results indicate that high-level disinfection and gas sterilization of flexible endoscopes are not necessary to prevent bacterial disease transmission from patient to patient. Read at the joint meeting of the American Society of Colon and Rectal Surgeons with the Section of Colo-Proctology, Royal Society of Medicine, and the Section of Colonic and Rectal Surgery, Royal Australasian College of Surgeons, New Orleans, Louisiana, May 6 to 11, 1984.     

Effects of intracoronary and intravenous amrinone infusions in patients with cardiac failure and patients with near normal cardiac function

The effects of intracoronary and intravenous infusions of amrinone were studied to distinguish the drug's direct cardiac actions from its peripheral vascular and neuroendocrine properties. Intracoronary infusions of amrinone were found to have no haemodynamic effect other than producing a slight reduction in the left ventricular ejection fraction and some suggestion of coronary vasodilatation in patients with impaired left ventricular function. They did not improve contractility, cardiac output, or filling pressures and had no significant effect on myocardial metabolism, although therapeutic concentrations of the drug were detected in coronary sinus blood. Intravenously administered amrinone reduced filling pressures and improved the cardiac index in all patients, but haemodynamic improvements were most pronounced in the patients with the worst cardiac function. These changes were accompanied by improvements in the indices of contractility only in patients in whom alterations in concentrations of free fatty acid, glycerol, and glucose suggested peripheral catecholamine release. In the patients with the best basal cardiac function intravenously administered amrinone produced a reduction in myocardial work and evidence of myocardial ischaemia, as a result of excessive reduction of coronary perfusion pressure and increased heart rate, without any appreciable increase in cardiac index. It is concluded that, at the concentrations of the drug that can be achieved in man without adverse effects, amrinone has no direct positive inotropic effect. Haemodynamic changes are predominantly the result of vasodilatation, although catecholamines may be released in some patients.