Tejs Ehlers Klug
email: tejsehlersklug@hotmail.com

Jens-Jacob Henriksen

Kurt Fuursted

Therese Ovesen

MD Tejs Ehlers Klug

Significant pathogens in peritonsillar abscesses

Keywords: Peritonsillar abscess, acute tonsillitis, microbiology, Fusobacterium necrophorum, Beta-haemolytic Streptococci Beta-haemolytic streptococci Coag.-neg. staphylococci: Coagulase-negative staphylococci

Purpose: Peritonsillar abscesses (PTA) are polymicrobial infections, with a diverse aerobic and anaerobic flora. The aim of the present study is to compare bacteriologic culture results from patients with PTA to those from patients undergoing elective tonsillectomy (clinically non-infected tonsils), to better elucidate the pathogenic significance of various isolates. Methods: A prospective study was conducted on 36 PTA patients undergoing acute tonsillectomy and on 80 electively tonsillectomized patients. Results: Fusobacterium necrophorum (FN) and Streptococcus Group A (GAS) were isolated significantly more frequently from the tonsillar cores of PTA patients, both from the abscessed (P=0.001 and P=0.046, respectively) and non-abscessed side (P<0.001 and P=0.046, respectively), than from the tonsillar cores of electively tonsillectomized patients.

Conclusions:

Our findings indicate that FN and GAS are the prominent pathogens in PTA. In patients with PTA the incidence of FN and GAS isolated from the abscessed tonsil was the same as from the non-abscessed contralateral side, and the growth was comparable by a semi-quantitative approach. Our findings suggest that FN is also of pathogenic importance in acute tonsillitis, and that FN growth is not a subsequent phenomenon once an abscess has formed. Our findings further suggest that other factors influence the development of PTA.

Introduction

A peritonsillar abscess (PTA) is defined as a collection of pus between the tonsillar capsule and the pharyngeal constrictor muscle. It is the most frequent complication of acute tonsillitis (AT) and the prevailing cause of acute admission to the ENT-department at Aarhus University Hospital [START_REF] Rusan | An overview of the microbiology of acute ear, nose and throat infections requiring hospitalisation[END_REF].

Adolescents and young adults are most commonly affected [START_REF] Klug | Fusobacterium necrophorum: most prevalent pathogen in peritonsillar abscess in Denmark[END_REF]. Management requires surgical drainage and antimicrobial therapy.

Several studies have looked at the bacteriology of PTA aspirates [START_REF] Jokipii | Semiquantitative culture results and pathogenic significance of obligate anaerobes in peritonsillar abscesses[END_REF][START_REF] Jousimies-Somer | Bacteriologic findings in peritonsillar abscesses in young adults[END_REF][START_REF] Brook | Aerobic and anaerobic microbiology of peritonsillar abscess[END_REF][START_REF] Mitchelmore | Microbiological features and pathogenesis of peritonsillar abscesses[END_REF][START_REF] Prior | The microbiology and antibiotic treatment of peritonsillar abscesses[END_REF][START_REF] Hoffmann | Influence of antibiotic treatment on the isolation rate of group A streptococci from peritonsillar abscesses[END_REF][START_REF] Muir | Peritonsillar infection in Christchurch 1990-2: microbiology and management[END_REF][START_REF] Savolainen | Peritonsillar abscess. Clinical and microbiologic aspects and treatment regimens[END_REF][START_REF] Gavriel | Variations in the microbiology of peritonsillar abscess[END_REF][START_REF] Sunnergren | Incidence, microbiology and clinical history of peritonsillar abscesses[END_REF][START_REF] Sakae | Microbiology of peritonsillar abscesses[END_REF][START_REF] Brook | Aerobic and anaerobic bacteriology of peritonsillar abscess in children[END_REF][START_REF] Flodström | Microbiological aspects on peritonsillar abscesses[END_REF][START_REF] Sugita | Microorganisms isolated from peritonsillar abscess and indicated chemotherapy[END_REF][START_REF] Snow | The microbiology of peritonsillar sepsis[END_REF]. A mixture of aerobic and anaerobic bacteria is commonly isolated. As the cultures are obtained from an area normally heavily colonized, the pathogenic relevance of each strain is raised.

The high incidence of Streptococcus Group A (GAS) in surface swabs from AT patients and in pus aspirates from PTA patients, as well as the detection of streptococcal antibodies in these same patients has established GAS as a key pathogen in AT and PTA [4-5, 11, 16, 18-19]. Large colonyforming beta-haemolytic streptococci Group C and G have also been recovered from patients with AT and PTA [START_REF] Jokipii | Semiquantitative culture results and pathogenic significance of obligate anaerobes in peritonsillar abscesses[END_REF][START_REF] Jousimies-Somer | Bacteriologic findings in peritonsillar abscesses in young adults[END_REF][START_REF] Brook | Aerobic and anaerobic microbiology of peritonsillar abscess[END_REF][START_REF] Cimolai | Beta-haemolytic non-group A streptococci and pharyngitis: a case-control study[END_REF][START_REF] Turner | Epidemiologic evidence for Lancefield group C beta-hemolytic streptococci as a cause of exudative pharyngitis in college students[END_REF][START_REF] Lindbaek | Clinical symptoms and signs in sore throat patients with large colony variant beta-haemolytic streptococci groups C or G versus group A[END_REF].

The importance of anaerobes in PTA formation has been suspected for decades [START_REF] Jokipii | Semiquantitative culture results and pathogenic significance of obligate anaerobes in peritonsillar abscesses[END_REF][START_REF] Jousimies-Somer | Bacteriologic findings in peritonsillar abscesses in young adults[END_REF][START_REF] Brook | Aerobic and anaerobic microbiology of peritonsillar abscess[END_REF][START_REF] Mitchelmore | Microbiological features and pathogenesis of peritonsillar abscesses[END_REF][START_REF] Prior | The microbiology and antibiotic treatment of peritonsillar abscesses[END_REF], however, few attempts have been made to explore which strains are of pathogenic importance. Immune responses to Fusobacterium nucleatum and Prevotella intermedia have been detected in patients with PTA [START_REF] Brook | Immune response to Fusobacterium nucleatum and Prevotella intermedia in patients with peritonsillar cellulitis and abscess[END_REF][START_REF] Brook | Immune response to anaerobic bacteria in patients with peritonsillar cellulitis and abscess[END_REF], as well as in patients suffering from recurrent acute tonsillitis (RT) [START_REF] Brook | Immune response to Prevotella intermedia in patients with recurrent nonstreptococcal tonsillitis[END_REF], AT [START_REF] Brook | Immune response to Fusobacterium nucleatum, Prevotella intermedia and other anaerobes in children with acute tonsillitis[END_REF], peritonsillar cellulitis [START_REF] Brook | Immune response to Fusobacterium nucleatum and Prevotella intermedia in patients with peritonsillar cellulitis and abscess[END_REF][START_REF] Brook | Immune response to anaerobic bacteria in patients with peritonsillar cellulitis and abscess[END_REF], and mononucleosis [START_REF] Brook | Immune response to Fusobacterium nucleatum and Prevotella intermedia in patients with infectious mononucleosis[END_REF]. However, Fusobacterium nucleatum and Prevotella intermedia are also commonly found in clinically non-infected tonsils [START_REF] Stjernquist-Desatnik | Tonsillar microbial flora: comparison of recurrent tonsillitis and normal tonsils[END_REF][START_REF] Brook | Changes in the core tonsillar bacteriology of recurrent tonsillitis: 1977-1993[END_REF][START_REF] Brook | Bacteriology of adenoids and tonsils in children with recurrent adenotonsillitis[END_REF].

In Denmark, PTA patients under the age of 30 years are most often treated with acute bilateral tonsillectomy. To further elucidate the pathogenic significance of various bacteria associated with PTA, the present study compares bacteriologic culture results from the tonsils of PTA patients with results from clinically non-infected tonsils from electively tonsillectomized patients. Isolates from the tonsilar surface are compared with those from the tonsillar core, and those from pus aspirates.

Moreover, the bacteriology of acutely infected tonsils, without abscess formation, is examined by comparing growth from the abscessed side to that from the contralateral non-abscessed side. Only patients between the ages of 8-30 years, without antibiotic treatment during the month preceding surgery were included in the study.

Subjects and methods

Patients

Specimen collection:

For all patients after the induction of anaesthesia, coal-coated cotton swabs were rubbed thoroughly on the surfaces of each of the tonsils and placed in transport media (Stuart´s medium, SSI Diagnostic, Hilleröd, Denmark). In PTA patients, the abscess was punctured through the peritonsillar mucosa, and pus was aspirated into a sterile syringe. The tonsils were removed by blunt dissection, and placed in sterile containers separately. None of the patients received antibiotics before collection of specimens had been completed. Tonsillar tissue, pus aspirates, and surface swabs in Stuart's media were placed in a minus 80 o C freezer within minutes of collection. Surface swabs of three group 1 patients were lost before cultures were performed.

Microbiological analysis:

Microbiological analysis was carried out at the Department of Clinical Microbiology, at Aarhus University Hospital. Samples were stored at minus 80 o C until bacteriologic investigations were performed. Specimens were processed in a class-2 laminar airflow safety cabinet by aseptic technique. Tissue samples, aspirates and swabs were cultured semi-quantitative onto 5% blood agar plates, chocolate agar plates and anaerobic plates (all from SSI Diagnostic, Hilleröd, Denmark).

Semi-quantitation of growth was done by plating on the various agar plates using the dilution streak technique. The first quadrant of the plate was streaked using the tissue or swab and each successive quadrant was streaked using a new bacteriologic loop in order to dilute the number of bacteria in each quadrant. The plates were incubated at 35 o C in either a carbon dioxide (CO 2 ) enriched atmosphere for three days or anaerobically for five days using the Concept 400 anaerobic workstation (Fisher Scientific, Denmark). Quantification was expressed as 1+, 2+, 3+, or 4+ based on the number of quadrants that demonstrated bacterial growth. Bacterial growth limited to quadrant 1 was categorized as 1+, bacterial growth limited to quadrants 1 and 2 was categorized as 2+, bacterial growth limited to quadrants 1, 2, and 3 was categorized as 3+, and bacterial growth that extended to all 4 quadrants was categorized as 4+. Speciation for microorganisms was performed by standard methods [START_REF] Hagelskjaer Kristensen | Localised Fusobacterium necrophorum infections: a prospective laboratory-based Danish study[END_REF] or by using the VITEK 2 system. Special care was taken to differentiate (small colony) beta-hemolytic group C and G streptococci (Voges-Proskauer test-negative) from Streptococcus anginosus (Voges-Proskauer test-positive). Antibiotic sensitivities were determined by a standard disc diffusion method using the protocol from WWW.SRGA.ORG on isosensitivity plates (Oxoid, Denmark). Organisms of the same species were deemed indistinguishable if they had the same colony morphology, the same basic biochemical features and an identical antibiogram.

Statistical analysis:

The Fisher exact test (2-sided) was used for between-group comparisons of bacteriologic findings.

The Kruskal-Wallis test was used for comparison of semi-quantitative growth distributions, and logistic regression analysis was used for comparison of aerobic and anaerobic detection frequencies.

Bacteriological data from the right tonsil (selected at random) of electively tonsillectomized patients were used to compare to findings from PTA patients. The statistical differences having used the left tonsil data were insignificant, as the differences between the two sides were very small. Statistical significance was defined as p<.05.

The study was approved by The Ethical Committee of Aarhus County (Number 20050034).

Informed consent was obtained from all patients, in accordance with the guidelines set by The Danish National Board of Health.

For the purpose of this study, "PTA side" refers to the tonsil in close relation to the abscess and "AT side" refers to the contralateral, acutely infected tonsil (without abscess formation).

Results

The mean and median ages of PTA patients were 18.3 and 17.0 years. Controls had mean and median ages of 19.4 and 19.0 years. Three of the PTA patients had a history of RT. Within the two years preceding admission, a history of AT was noted in eight of the PTA patients: four had one case of AT, one had two cases, and three had three cases of AT. None of the patients had bilateral PTA.

In PTA patients, three or more bacterial strains were isolated from all tonsillar surface swabs, two or more were isolated from all tonsillar core tissue specimens, and at least one from each pus aspirate.

Mixed aerobic and anaerobic flora was present in all but two surface swabs and in all but one core tissue specimen. In pus aspirates, mixed aerobes and anaerobes were detected in 29 patients, while four cultures yielded anaerobes only and three aspirates contained aerobes only.

An average of 5.1 isolates (3.7 aerobes and 1.4 anaerobes) was detected in surface swabs, 5.8 isolates (3.8 aerobes and 2.1 anaerobes) were grown in core tissues, and 3.7 isolates (2.2 aerobes and 1.5 anaerobes) were found in pus aspirates.

The aerobic bacteria most frequently isolated from both surface swabs and core tissues, obtained from PTA patients, were Viridans streptococci, Neisseria species, Corynebacterium species, and Staphylococcus aureus (Table 1). The predominant anaerobic bacteria at both the surface and in the core were Prevotella species, Fusobacterium necrophorum, and other Fusobacterium species (Table 1).

Tonsillar core isolates: PTA side vs. controls:

Compared to core tissue of clinically non-infected tonsils, FN and GAS were detected significantly more frequently in core tissue from the PTA side (P=.001 and P=.046, respectively; Fisher exact test). In contrast, Fusobacterium species and Staphylococcus aureus were isolated significantly less frequently (P<.001 and P=.028, respectively) (Table 1) in core tissue from the PTA side. A semiquantitative analysis revealed also significantly lighter growth of Staphylococcus aureus (P<.001, Kruskal-Wallis test), Prevotella species (P=.002) and Fusobacterium species (P=.023) in the cultures from the PTA side cores than from control cores.

Tonsillar core isolates: AT side vs. controls:

Similar to culture results from the PTA side core, FN and GAS were detected significantly more frequently in the core of the AT side (P<.001 and P=.046, respectively) than in the core of control tonsils. In contrast, Fusobacterium species was isolated significantly less frequently from the AT side core (P<.001; Fisher exact test) compared to the control core (Table 1). Significantly lighter growth of Staphylococcus aureus (P<.001), Prevotella species (P<.001), Fusobacterium species (P=.026), and Viridans streptococci (P=.006) was obtained in cultures from the AT side core than from control cores.

Core isolates from the PTA side vs. isolates from pus aspirates:

Nearly all bacterial strains isolated from aspirates were also grown from the PTA side core (Table 2). Three patients had pure growth of one bacterial strain from aspirated pus: FN was detected in two of these patients and in one patient GAS was isolated. The semi-quantitative analysis revealed significantly lighter growth of Neisseria species (P=.034) in pus than PTA side core.

PTA side vs. AT side:

Isolation rates from the PTA side compared to the AT side were not significantly different, neither from the core nor from the surface, for all of the detected bacterial strains (Table 1).

Eighty five percent of aerobic isolates and 84% of anaerobic isolates were common to both the PTA side core and the AT side core (Table 3). Beta-haemolytic streptococci (BHS) concordance was 87% and FN concordance was 95% (Table 3). Results were similar for PTA vs. AT side surface (Table 3).

No significant semi-quantitative differences between the PTA and AT side core were found.

Nineteen of 27 BHS recovered from cores were isolated as heavy growth (4+), seven as moderate growth (3+), and one as sparse growth (2+). Similarly, 33 of 41 FN recovered from cores were isolated as heavy growth (4+) and eight as moderate growth (3+).

Isolates from the core vs. from the surface for PTA side, AT side, and controls:

Eighty five percent of aerobes and 65% of anaerobes isolated from the PTA side core were also detected from swabs of the PTA side surface (Table 4). For BHS and FN respectively, 86% and 56% of core isolates were also detected on the surface. Similarly, 84% of aerobes and 55% of anaerobes isolated from the AT side core were detected also at the AT side surface. Aerobes were predominant on the tonsillar surface compared to anaerobes, both on the PTA side and the AT side (P=.005, logistic regression analysis).

Discussion

Within the past five years, there has been an emerging focus on Fusobacterium necrophorum (FN) as a pathogen in AT [START_REF] Jensen | Detection of Fusobacterium necrophorum subsp. Funduliforme in tonsillitis in young adults by real-time PCR[END_REF][START_REF] Aliyu | Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice[END_REF][START_REF] Amess | A six-month audit of the isolation of Fusobacterium necrophorum from patients with sore throat in a district general hospital[END_REF]. Furthermore, in a retrospective study of 847 PTA patients treated at our department from 2001 to 2006, we found FN to be the most prevalent bacterial strain in pus specimens. Patients infected with this bacterium displayed significantly higher neutrophil counts and CRP values than patients infected with other bacteria, indicating a larger immune response and suggesting that FN is of pathogenic importance in PTA [START_REF] Klug | Fusobacterium necrophorum: most prevalent pathogen in peritonsillar abscess in Denmark[END_REF].

In the current study, FN was a frequent finding in PTA. We recovered FN from 60% of all aspirates. Of note, FN was isolated significantly more frequently from the cores of abscessed tonsils than from the cores of clinically non-infected tonsils from electively tonsillectomized patients (Table 1), thus supporting a pathogenic role for FN in PTA. These findings confirm the results of our retrospective study [START_REF] Klug | Fusobacterium necrophorum: most prevalent pathogen in peritonsillar abscess in Denmark[END_REF] and emphasize that FN is a very prominent pathogen in PTAat least in Denmark.

When FN was present at the abscessed site we were able to recover it in nearly all cases from both the abscess aspirate, as well as the tonsillar core. However, swabs of the abscessed tonsil yielded FN much less frequently (56% incidence in cores of abscessed tonsils vs. 27% in surface swabs from the same tonsils) indicating that swabs are not a reliable clinical tool for detecting the organism, or that our method of collection of the swabs was not optimal for culturing FN downstream.

FN, an obligate, anaerobic, Gram-negative rod, is most commonly associated with Lemierre´s Syndrome. However, unlike in Lemierre´s Syndrome, most FN infections remain localised [START_REF] Hagelskjaer Kristensen | Localised Fusobacterium necrophorum infections: a prospective laboratory-based Danish study[END_REF]. In contrast to FN, Fusobacterium species are not considered of pathogenic importance in ENT infections.

In Finland, Jousimies-Somer at al. detected FN in 38% of PTA aspirates and found an association to previous tonsillar / peritonsillar infections and previous use of antimicrobial therapy [START_REF] Jousimies-Somer | Bacteriologic findings in peritonsillar abscesses in young adults[END_REF]. We did not find an association between incidence of FN and previous events of AT. Brook [START_REF] Brook | Aerobic and anaerobic bacteriology of peritonsillar abscess in children[END_REF] and Jokipii et al [START_REF] Jokipii | Semiquantitative culture results and pathogenic significance of obligate anaerobes in peritonsillar abscesses[END_REF] also detected FN in 13% and 7% of PTA aspirates, respectively.

As expected, GAS was also detected significantly more frequently from the cores of the PTA side than from the control cores confirming the well-documented role of GAS in PTA. In our study, GAS was isolated in 19% of PTA patients, which is less than reported by some studies [START_REF] Jousimies-Somer | Bacteriologic findings in peritonsillar abscesses in young adults[END_REF][START_REF] Savolainen | Peritonsillar abscess. Clinical and microbiologic aspects and treatment regimens[END_REF][START_REF] Flodström | Microbiological aspects on peritonsillar abscesses[END_REF] and similar to the findings of other studies [START_REF] Jokipii | Semiquantitative culture results and pathogenic significance of obligate anaerobes in peritonsillar abscesses[END_REF][START_REF] Hoffmann | Influence of antibiotic treatment on the isolation rate of group A streptococci from peritonsillar abscesses[END_REF][START_REF] Muir | Peritonsillar infection in Christchurch 1990-2: microbiology and management[END_REF][START_REF] Gavriel | Variations in the microbiology of peritonsillar abscess[END_REF][START_REF] Sunnergren | Incidence, microbiology and clinical history of peritonsillar abscesses[END_REF][START_REF] Sakae | Microbiology of peritonsillar abscesses[END_REF][START_REF] Brook | Aerobic and anaerobic bacteriology of peritonsillar abscess in children[END_REF].

Fusobacterium species and Staphylococcus aureus were isolated significantly less frequently and in significantly lighter growth from PTA side cores than control cores. We interpret these findings as signs of overgrowth by the pathogens described above. It stresses the belief that Staphylococcus aureus does not exert a pathogenic role in PTA.

By comparing the bacteriology from the PTA side and the non-abscessed, but acutely inflamed, contralateral tonsil, we conclude that the core bacteriology was almost identical between the PTA side and AT side (Table 3). This suggests that other factors are of major importance in the development of PTA. It also indicates that growth of FN is not a subsequent overgrowth phenomenon once an abscess is formed, but that FN is a primary pathogen with importance not only in the pathogenesis of PTA, but also in severe, non-abscessed AT. This is in agreement with recent studies suggesting FN could be involved in AT [START_REF] Jensen | Detection of Fusobacterium necrophorum subsp. Funduliforme in tonsillitis in young adults by real-time PCR[END_REF][START_REF] Aliyu | Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice[END_REF][START_REF] Amess | A six-month audit of the isolation of Fusobacterium necrophorum from patients with sore throat in a district general hospital[END_REF][START_REF] Batty | Prevalence of Fusobacterium necrophorum and other upper respiratory tract pathogens isolated from throat swabs[END_REF]. Batty et al. found FN in 10% of all throat swabs and a clinical diagnosis of tonsillitis was equally likely to be associated with GAS or FN infection [START_REF] Batty | Prevalence of Fusobacterium necrophorum and other upper respiratory tract pathogens isolated from throat swabs[END_REF]. In a polymerase chain reaction (PCR)-based study, conducted in Denmark, FN was detected in 15% of throat swabs from tonsillitis patients aged 18 to 32 years, and the investigators concluded that FN could be a cause of AT and may account for some of the cases previously assumed to be of viral aetiology [START_REF] Jensen | Detection of Fusobacterium necrophorum subsp. Funduliforme in tonsillitis in young adults by real-time PCR[END_REF]. Also using PCR, Aliyu et al. identified FN in 10 % of throat swabs from patients presenting to general practitioners with pharyngitis, but they were unable to recover FN from the throats of healthy control subjects [START_REF] Aliyu | Real-time PCR investigation into the importance of Fusobacterium necrophorum as a cause of acute pharyngitis in general practice[END_REF].

We found that the probability of detecting the pathogens isolated from pus and PTA side core was the same whether swabbing the surface of PTA side or AT side tonsil (Table 3). This finding confirmed the high concordances between the AT and PTA side and likely reflects the high concentrations of bacteria in the acutely infected tonsils. No former studies have made these comparisons, but Brook et al have shown the necessity of swabbing both tonsils in patients with AT in order to detect all cases of acute GAS tonsillitis [START_REF] Brook | Discrepancies in the recovery of group A beta-hemolytic streptococci from both tonsillar surfaces[END_REF]. Our results do not confirm the relevance of swabbing both tonsils, but it might reflect the fact that surface swabs were obtained under optimal circumstances in anaesthetized patients and using mouth gag.

We found that surface swabs are not reliable at detecting anaerobes, such as FN (Table 4). In contrast, surface swabs were better at detecting aerobes, for example 86% of BHS isolated from cores was detected. We found that bacterial detection rates from surface swabs were higher in acutely infected tonsils than in non-infected tonsils, but consistent with the trend of detecting aerobes more frequently than anaerobes in surface swabs. These findings have important clinical implications as cases of AT caused by FN, would be missed by a surface swab of the infected tonsils. Whether PCR and other more sensitive detection methods would be helpful in making the diagnosis remains unknown.

In part the lack of consistency with regards to microbiologic findings in former PTA studies may be attributable to differences in culture methods, in specimen collection and handling, and in the patient population (e.g. age, geography). Interpretation of bacteriologic findings in former studies is made even more obscure by the fact that in many studies patients with and without prior antibiotic treatment are pooled together. Some studies have found quantitative and qualitative differences in bacteriologic culture results after antibiotic treatment [START_REF] Jokipii | Semiquantitative culture results and pathogenic significance of obligate anaerobes in peritonsillar abscesses[END_REF][START_REF] Mitchelmore | Microbiological features and pathogenesis of peritonsillar abscesses[END_REF][START_REF] Hoffmann | Influence of antibiotic treatment on the isolation rate of group A streptococci from peritonsillar abscesses[END_REF][START_REF] Sakae | Microbiology of peritonsillar abscesses[END_REF] while others have not [START_REF] Jousimies-Somer | Bacteriologic findings in peritonsillar abscesses in young adults[END_REF][START_REF] Brook | Aerobic and anaerobic microbiology of peritonsillar abscess[END_REF]. Only Flodström et al [START_REF] Flodström | Microbiological aspects on peritonsillar abscesses[END_REF], in 1976, have studied the microbiologic flora exclusively in PTA patients with no prior antibiotic treatment. None of the former PTA studies have explored the significance of bacteria isolated from PTA patients by comparing the findings with the bacterial flora of non-acutely infected tonsils.

To make such a comparison, comparable materials must be obtained and tonsillar core tissues are therefore needed as culture specimens. We hypothesized that potential pathogens isolated from aspirated pus would also be present in PTA side core tissue. However, even more potential pathogens were isolated from PTA side cores than aspirated pus, and only very few pathogens were isolated in pus only (Table 4). We therefore believe that tonsillar core tissues formed appropriate basis of comparison to the flora found in clinically non-infected tonsils of electively tonsillectomized patients.

Ideally our bacteriologic findings in patients with PTA would be compared to the flora of tonsillar tissue of healthy subjects (without a history of prior tonsillar disease). Unfortunately, for ethical reasons, such specimens were unobtainable in the present study. Instead, tonsils from patients undergoing elective tonsillectomy were used, thus isolates may not represent "normal" tonsillar flora. A few studies have been conducted comparing core tissue from normal tonsils with that from patients suffering from RT and tonsillar hypertrophy (TH) [START_REF] Stjernquist-Desatnik | Tonsillar microbial flora: comparison of recurrent tonsillitis and normal tonsils[END_REF][START_REF] Brook | Microbiology of "normal" tonsils[END_REF][START_REF] Stjernquist-Desatnik | High recovery of Haemophilus influenzae and group A streptococci in recurrent tonsillar infection or hypertrophy as compared with normal tonsils[END_REF]. Brook et al., in a study of eight children, found similar organisms in normal and recurrently inflamed tonsils, but the concentration of all BHS, all Bacteroides species, and all Peptostreptococcus species was higher in recurrently inflamed tonsils [START_REF] Brook | Microbiology of "normal" tonsils[END_REF]. A study by Stjernquist-Desatnik et al. detected significantly fewer BHS, in particular GAS, as well as Haemophilus influenzae, in control patients with sleep apnea compared to patients with TH and RT [START_REF] Stjernquist-Desatnik | High recovery of Haemophilus influenzae and group A streptococci in recurrent tonsillar infection or hypertrophy as compared with normal tonsils[END_REF]. However, further studies by Stjernquist-Desatnik et al., using a semi-quantitative method to compare the culture results from the above patient group, found no significant differences between groups with regards to frequency and quantity of aerobic and anerobic bacteria [START_REF] Stjernquist-Desatnik | Tonsillar microbial flora: comparison of recurrent tonsillitis and normal tonsils[END_REF]. In the present study, the culture results from the four subgroups of electively tonsillectomized patients were comparable to those found in the tonsillar cores of healthy control patients in the studies discussed above. Hence, we believe that tonsillar tissue from patients undergoing elective tonsillectomy can serve as a control in our study.

Due to the amount of specimens, some of which were obtained at night, we kept the specimens at minus 80 o C until cultures were made. Studies of the effect of freezing specimens at minus 80 o C do not seem to alter the ability to isolate organisms [START_REF] Green | Impact of freezing on the future utility of archived surveillance culture specimens[END_REF][START_REF] Dan | Comparison of preservation media and freezing conditions for storage of specimens of faeces[END_REF][START_REF] Siberry | Comparison of different culture media and storage temperatures for the long-term preservation of Streptococcus pneumoniae in the tropics[END_REF]. A weakness of the study is that we did not test the effect of freezing on recovery. Some bacteria sensitive to freezing or in low numbers might not have been detected. However, as bacteria were commonly found in high numbers and control specimens were treated in the same way, the risk of bias of results seems limited.

In summary, the present study is the first to compare bacteriological data from PTA patients with non-clinically infected tonsils. Patients were from the same geographic area, over the time period from November, 2005 to February, 2009, were well age matched between the PTA and the control group, and had not taken antibiotics in the month preceding the tonsillectomy. FN and GAS were isolated significantly more frequently from tonsillar cores of PTA patients, both at the side of the abscess and the contralateral side, than from tonsillar cores of electively tonsillectomized patients.

Our findings add to the growing body of evidence that FN is a prominent pathogen in PTA and AT.

As FN and GAS were isolated as frequently from the PTA side as from the AT side, and as growth in culture from the two sides seemed comparable, it appears that other factors play an important role in the development of PTA. Further studies to investigate the viral composition of the tonsils and immunologic studies are warranted.

Tables

:

  Patients were enrolled in the study between November, 2005 and February, 2009 at three ENT Departments in Denmark. The study consisted of two patient groups: (1) 36 patients with unilateral PTA undergoing acute bilateral tonsillectomy and (2) 80 patients admitted for elective tonsillectomy (controls). The control patients were categorized into four subgroups according to their indication for tonsillectomy: (a) recurrent tonsillitis (RT, more than five episodes within two years) (30 patients), (b) tonsillar hypertrophy (TH, with a history of airway obstruction) (20 patients), (c) both RT and TH (20 patients), and (d) halitosis or persistant sore throat syndrome (PSTS)(10 patients).

Table 1 .

 1 Isolation rates of organisms from tonsillar surfaces and cores from PTA side and AT side of PTA patients and right sided surfaces and cores of electively tonsillectomized patients (controls)

	Surface	Core

Table 2 .

 2 Number of isolates from PTA aspirates and PTA side core

	tissues
		No. isolates
	Organism	Aspirate	Core	Aspirate
		only	only	& Core
	Aerobic
	Beta-haemolytic streptococci
	Group A	0	0	7
	Group C	0	1	2
	Group G	1	2	1
	Not grouped	1	1	1
	Total	2	4	11
	Streptococus group B	0	1	0
	Streptococcus pneumoniae	0	1	0
	Viridans streptococci	2	18	38
	Staphylococcus aureus	0	10	2
	Coagulase-negative staphylococci	2	0	1
	Eikenella corrodens	1	2	0
	Neisseria species	0	20	5
	Corynebacterium species	1	16	2
	Anaerobic
	Fusobacterium necrophorum	2	1	19
	Fusobacterium species	0	4	6
	Prevotella species	0	17	27
	Other anaerobes	0	1	0
	Yeast	0	2	0

Table 3 .

 3 Bacterial concordances between PTA side and AT side surface swabs and core tissues

	Surface	Core

Table 4 .

 4 Number of isolates from PTA side surface swabs and core tissues

	No. isolates

Coag.-neg. staphylococci: Coagulase-negative staphylococci

Acknowledgments

The authors wish to acknowledge Department of Biostatistics, Aarhus University, for advices concerning statistical analyses, and Dr Maria Rusan for helpful advice on drafting the manuscript.

The authors declare that they have no conflict of interest.

Sources of financial support: None.

Meetings where the information has previously been presented: None.


Nicola J Smith
email: nicola.smith@bio.gla.ac.uk

Kirstie A Bennett

Graeme Milligan

When simple agonism is not enough: emerging modalities of GPCR ligands

Keywords: G protein-coupled receptor, Functional selectivity, Allosterism, Ago-allosterism, Dualsteric ligand, Super-agonism

Recent advances in G protein-coupled receptors have challenged traditional definitions of agonism, antagonism, affinity and efficacy. The discovery of agonist functional selectivity and receptor allosterism has meant researchers have an expanded canvas for designing and discovering novel drugs. Here we describe modes of agonism emerging from the discovery of functional selectivity and allosterism. We discuss the concept of ago-allosterism, where ligands can initiate signaling by themselves and influence the actions of another ligand at the same receptor. We introduce the concept of dualsteric ligands that consist of distinct elements which bind to each of the orthosteric and an allosteric domain on a single receptor to enhance subtype selectivity. Finally, the concept that efficacy should be defined by the activity of an endogenous ligand will be challenged by the discovery that some ligands act as 'super-agonists' in specific pathways or at certain receptor mutations.

M a n u s c r i p t 3 For decades now, the seven transmembrane-spanning (7TM) family of G proteincoupled receptors (GPCRs) has been a fruitful source of drug targets for the pharmaceutical industry, with blockbuster drugs acting at numerous GPCRs, most notably the monoaminergic (such as histamine, serotonin and the catecholamines) and angiotensin receptor families, constituting over 26% of all FDA-approved drugs [START_REF] Overington | How many drug targets are there?[END_REF]. However, few recently approved GPCR-directed medicines regulate previously untapped pharmacological targets, in spite of concerted drug discovery programs within the pharmaceutical industry.

Failure to substantially expand the number of truly 'druggable' GPCR targets may be due to a number of reasons. Until very recently, little structural information has existed for GPCRs, meaning that rational drug design has had to rely upon homology modeling using the low homology template of rhodopsin [START_REF] Michino | Community-wide assessment of GPCR structure modelling and ligand docking: GPCR Dock 2008[END_REF][START_REF] Mobarec | Effects of an oral ghrelin mimetic on body composition and clinical outcomes in healthy older adults: a randomized trial[END_REF]. The recent publication of three high resolution crystal structures of clinically important GPCRs has therefore significantly expanded our understanding of how a ligand may gain access to and bind within the receptor binding pocket [START_REF] Cherezov | High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor[END_REF][START_REF] Jaakola | The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist[END_REF][START_REF] Kobilka | New G-protein-coupled receptor crystal structures: insights and limitations[END_REF], Rasmussen et al., 2007, Rosenbaum et al., 2007[START_REF] Rosenbaum | The structure and function of G-protein-coupled receptors[END_REF].

Most successful GPCR drugs on the market have been developed from the knowledge of and structure-activity relationships around an existing ligand template, such as adrenaline [START_REF] Griffith | Adrenergic receptors and drugs affecting adrenergic neurotransmission[END_REF]. However, roughly 100 non-A c c e p t e d M a n u s c r i p t 4 chemosensory GPCRs remain to be 'deorphanized', in that they are yet to be matched to an endogenous ligand partner [START_REF] Chung | Orphan GPCR research[END_REF]. Of those recently deorphanized, many of the identified ligands have poor potency and undefined affinity at the receptor (for example, the free fatty acid receptor family [START_REF] Stoddart | International Union of Pharmacology. LXXI. Free fatty acid receptors FFA1, -2, and -3: pharmacology and pathophysiological functions[END_REF], GPR35 (Wang et al., 2006a), GPR109A and GPR109B [START_REF] Tunaru | PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect[END_REF], Wise et al., 2003), GPR120 [START_REF] Hirasawa | Free fatty acids regulate gut incretin glucagon-like peptide-1 secretion through GPR120[END_REF] and GPR84 (Wang et al., 2006b)) and are therefore a challenge for rational drug design.

Probably the biggest contributor to the lack of new ligand classes is the way in which chemical libraries have traditionally been screened (Kenakin, 2009b, Langmead andChristopoulos, 2006); high throughput screening of libraries has often been limited to a single readout, be it receptor binding, G protein activation or second messenger generation. In some cases, targets are even modified such that receptor signaling is directed towards a non-native readout, for example via the co-expression of chimeric or promiscuous G proteins [START_REF] Milligan | Heterotrimeric G-proteins: a short history[END_REF]. Such a streamlined approach to drug discovery has undoubtedly advanced GPCR research efforts but two key ligand actions were overlooked by such approaches, namely allosterism and functional selectivity. In the present review, we will briefly introduce the concepts of allosterism and functional selectivity and highlight the exciting prospects for novel agonist medicines to emerge from a more detailed understanding of these phenomena. An appreciation of the newer modalities of agonism should contribute to the

Functional Selectivity and Allosterism at GPCRs

Functional Selectivity

The notion of functional selectivity [also known as or encompassed by the terms biased agonism, agonist-directed trafficking of receptor stimulus, pleiotropy, or pluridimensional efficacy [START_REF] Galandrin | The evasive nature of drug efficacy: implications for drug discovery[END_REF][START_REF] Kenakin | Functional selectivity through protean and biased agonism: who steers the ship?[END_REF][START_REF] Kenakin | What systems can and can't do[END_REF][START_REF] Urban | Functional selectivity and classical concepts of quantitative pharmacology[END_REF]] has arisen from the accumulation of experimental evidence showing that certain ligands, or certain biological systems, favor activation of one signaling pathway over another and is most likely a common phenomenon across many receptor types. As such, while an endogenous full agonist is theoretically capable of activating all of the possible pathways that its cognate receptor can couple to (provided it is in the appropriate cellular background), another ligand may selectively activate only a subset of endpoints, presumably because only a subset of receptor conformations are stabilized by the interaction.

Functional selectivity is commonly determined through comparison of the activities of two or more ligands across multiple signaling pathways, thus functional selectivity is usually manifested by changes in rank order of efficacy, i.e. the extent of responsiveness of the system, and/or by changes in rank order of potency of different ligands acting at the same receptor. Changes in efficacy have been demonstrated, for example, at the pituitary adenylate cyclaseactivating peptide (PACAP) receptor, where the agonists PACAP 1-27 and A c c e p t e d M a n u s c r i p t 6 PACAP 1-38 stimulate PACAP-receptor mediated adenylate cyclase activation with equal potencies, but only PACAP 1-38 could evoke an increase in inositol phosphate levels through activation of phospholipase Cβ [START_REF] Spengler | Differential signal transduction by five splice variants of the PACAP receptor[END_REF].

For the Gonadotropin Releasing Hormone (GnRH) receptor, the two endogenous agonists, GnRH I and GnRH II, display functional selectivity as measured by differences in rank order of potency at more distal endpoints [START_REF] Millar | Diversity of actions of GnRHs mediated by ligand-induced selective signaling[END_REF]) -GnRH I is more potent than GnRH II at stimulating gonadotrophin release, while the rank order of potency is reversed for inhibition of cell growth [START_REF] Maudsley | Gonadotropin-releasing hormone (GnRH) antagonists promote proapoptotic signaling in peripheral reproductive tumor cells by activating a Galphai-coupling state of the type I GnRH receptor[END_REF]. Both changes in the rank order of potency and efficacy of compounds have been observed at the human D 2L receptor expressed in CHO cells. For example, the compound S(+)-propylnorapomorphine (SNPA) acts as a full agonist with efficacy equal to quinpirole in inhibiting forskolin-stimulated cAMP accumulation, but acts with lower efficacy and potency than quinpirole in activating MAP kinase phosphorylation [START_REF] Gay | Functional selectivity of D2 receptor ligands in a Chinese hamster ovary hD2L cell line: evidence for induction of ligand-specific receptor states[END_REF].

While alterations in potency can sometimes be explained by receptor reserve, the general phenomenon of functional selectivity is distinct to and not an artifact of receptor reserve (receptor reserve is unique to each agonist in the system).

For example, for the dopamine receptor it is known that there is a higher presynaptic D 2 receptor reserve than post-synaptic receptor reserve (in a situation where there is high receptor reserve a partial agonist may have increased potency and efficacy), however, the agonist dihydrexidine has greater functional activity at post-synaptic D 2 receptors than pre-synaptic [START_REF] Mailman | GPCR functional selectivity has therapeutic impact[END_REF]. Instead, for example, Kobilka and colleagues (Ghanouni et al., 2001a, Ghanouni et al., 2001b[START_REF] Swaminath | Sequential binding of agonists to the beta2 adrenoceptor. Kinetic evidence for intermediate conformational states[END_REF] have used an environmentally-sensitive fluorophore to demonstrate two distinct fluorescence lifetimes upon full agonist stimulation with isoproterenol, while the partial agonist dobutamine only stabilized one of the observed conformations. These conformations were later found to correspond to specific ligand-mediated outcomes [START_REF] Swaminath | Probing the beta2 adrenoceptor binding site with catechol reveals differences in binding and activation by agonists and partial agonists[END_REF]. More recently, multiple conformations of the purified and reconstituted beta2adrenoceptor have been identified based upon their propensity to couple to purified Gα s in the absence or presence of guanine nucleotides (Yao et al., 2009). Meanwhile, in a very recent study, Bokoch and colleagues (2010) used solid-state NMR to identify changes within the extracellular surface of the receptor in response to ligand binding. Interestingly, they found that ligands with different efficacies that bound within the transmembrane section of the receptor were able to induce conformational changes in the extracellular space (monitored at a salt bridge formed between extracellular loops 2 and 3) to different degrees [START_REF] Bokoch | Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor[END_REF]. In support of the beta2-adrenoceptor studies, molecular modeling in combination with site-directed mutagenesis has been used for the GnRH receptor to support the hypothesis that GnRH I and GnRH II stabilize distinct conformations of the receptor by making a series of different contacts A c c e p t e d M a n u s c r i p t 8 within the binding pocket in addition to their shared binding interactions [START_REF] Millar | Diversity of actions of GnRHs mediated by ligand-induced selective signaling[END_REF].

Allosteric modulators

Conventional agonists target the same binding site on a receptor as the endogenous ligand, termed the orthosteric site; however ligands may also bind to a topographically distinct site on the receptor, referred to as an allosteric site.

Binding of an allosteric ligand to a receptor can modulate the potency/affinity and/or the efficacy of the orthosteric ligand by altering the global conformation of the receptor compared to binding of the orthosteric ligand alone. Although by definition the binding site of an allosteric modulator is distinct from the orthosteric site [START_REF] Nawaratne | Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms[END_REF], in general, binding sites for allosteric modulators are not well defined but can be located close to [START_REF] Christopoulos | G protein-coupled receptor allosterism and complexing[END_REF] (e.g. the muscarinic acetylcholine receptor family [START_REF] Birdsall | Allosterism at muscarinic receptors: ligands and mechanisms[END_REF]; and the free fatty acid 2 receptor [START_REF] Lee | Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2[END_REF], Milligan et al., 2009)) or far from the orthosteric site (e.g. the metabolic glutamate receptors [START_REF] Knoflach | Positive allosteric modulators of metabotropic glutamate 1 receptor: characterization, mechanism of action, and binding site[END_REF]). Although this review is focused on ligands as allosteric modulators, the most common allosteric modulators of G protein-coupled receptors are the G proteins themselves [START_REF] Milligan | The impact of G-proteins on constitutive GPCR activity[END_REF]; the role of these and other interacting proteins, such as receptor activity modifying proteins, have been reviewed extensively elsewhere [START_REF] Kenakin | What systems can and can't do[END_REF], Kenakin, 2009a).

A c c e p t e d M a n u s c r i p t 9 Allosteric modulators are well-established therapeutic agents of ion channels but until recently have not been a traditional focus of drug discovery efforts at GPCRs [START_REF] Conn | Allosteric modulators of GPCRs: a novel approach for the treatment of CNS disorders[END_REF]. However in the last few years two allosteric modulators have entered the market as therapeutic targets of GPCRs: cinacalcet, a modulator of the calcium-sensing receptor [START_REF] Harrington | Calcium sensing receptor activators: calcimimetics[END_REF], and maraviroc, a modulator of the chemokine receptor CCR5 [START_REF] Dorr | Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity[END_REF], with the allosteric modulator LY2033298 acting at the muscarinic M 4 receptor showing potential as an anti-psychotic agent [START_REF] Chan | Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia[END_REF].

Emerging Agonistic Modalities

Allosteric agonists and ago-allosteric modulation

As introduced above, allosteric modulators can act to modify an orthosteric ligand's affinity for a receptor and/or modulate its efficacy, events which are both saturable and probe-dependent [i.e. depend upon the ligands being studied [START_REF] Leach | Allosteric GPCR modulators: taking advantage of permissive receptor pharmacology[END_REF], May et al., 2007b)]. Additionally, a ligand binding at an allosteric site can have efficacy in its own right; this is termed an allosteric agonist or ago-allosteric modulator and is accounted for by the Allosteric Two-State Model (Langmead and Christopoulos, 2006, May et al., 2007b, Schwartz and Holst, 2006).

Ago-allosteric modulation is not a new phenomenon; it was first described in 1990 at the adenosine A 1 receptor where the allosteric modulator PD81723 was shown to not only activate the Gα i/o pathway on its own but also to enhance the were present [START_REF] Bruns | Allosteric enhancement of adenosine A1 receptor binding and function by 2-amino-3-benzoylthiophenes[END_REF]. Indeed, ago-allosteric modulation has also been described at a wide variety of receptors including the GABA B receptor [START_REF] Binet | The heptahelical domain of GABA(B2) is activated directly by CGP7930, a positive allosteric modulator of the GABA(B) receptor[END_REF], the M 1 and M 2 muscarinic acetylcholine receptors (Langmead et al., 2006, May et al., 2007a), the free fatty acid 2 receptor [START_REF] Lee | Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2[END_REF], Milligan et al., 2009), and the glucagon-like peptide 1 receptor [START_REF] Teng | Small molecule agoallosteric modulators of the human glucagon-like peptide-1 (hGLP-1) receptor[END_REF]. Synthetic ago-allosteric modulators were also described for the ghrelin receptor [START_REF] Holst | Nonpeptide and peptide growth hormone secretagogues act both as ghrelin receptor agonist and as positive or negative allosteric modulators of ghrelin signaling[END_REF] although further studies have suggested that these compounds act as simple orthosteric agonists (see section below on superagonists).

In addition to their modulatory and agonistic qualities, some ago-allosteric modulators have also been suggested to display functional selectivity. For example, [START_REF] Thomas | G protein coupling and signaling pathway activation by m1 muscarinic acetylcholine receptor orthosteric and allosteric agonists[END_REF] demonstrated differential G protein coupling depending upon whether the ligand was an allosteric modulator or allosteric agonist at the M 1 mACh receptor [START_REF] Thomas | G protein coupling and signaling pathway activation by m1 muscarinic acetylcholine receptor orthosteric and allosteric agonists[END_REF]. More recently, [START_REF] Thomas | Contrasting effects of allosteric and orthosteric agonists on m1 muscarinic acetylcholine receptor internalization and down-regulation[END_REF] extended these studies to demonstrate that the ago-allosteric modulator, AC42, did not initiate receptor internalization and down-regulation, unlike traditional orthosteric ligands [START_REF] Thomas | Contrasting effects of allosteric and orthosteric agonists on m1 muscarinic acetylcholine receptor internalization and down-regulation[END_REF]. For the M 4 mACh receptor, the ago-allosteric modulator LY2033298 was found to modulate signaling via [ 35 S]GTPγS, extracellular signal-regulated kinases 1 and 2 or glycogen synthase kinase 3β to different degrees [START_REF] Leach | Molecular mechanisms of action and in vivo validation of an M4 muscarinic acetylcholine receptor allosteric modulator with potential antipsychotic properties[END_REF]. In an elegant follow up to this study, [START_REF] Nawaratne | Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms[END_REF] have used site-directed A c c e p t e d M a n u s c r i p t 11 mutagenesis to identify key contacts or domains within the M 4 mACh receptor for the ago-allosteric properties of LY2033298. Intriguingly, they were able to dissect out individual regions responsible for each of allosteric binding, allosteric agonism and the communication between orthosteric and allosteric sites resulting in modulation of either binding or function [START_REF] Nawaratne | Structural determinants of allosteric agonism and modulation at the M4 muscarinic acetylcholine receptor: identification of ligand-specific and global activation mechanisms[END_REF]. Clearly, agoallosteric modulators may prove to be useful tools for regulating distinct receptor signals.

Allosterism across a hetero-dimer

Given the evidence that GPCRs can form dimers, or higher oligomer structures, it is not inconceivable that allosteric modulation may occur across the dimer interface [START_REF] Milligan | Allosteric modulation of heterodimeric Gprotein-coupled receptors[END_REF]. Indeed, evidence to support allosteric modulation in receptor dimers has been supplied by studies into allosteric regulation of the aminobutyric acid (GABA) B receptor. The GABA B receptor is an obligatory hetero-dimeric receptor composed of two subunits; GABA B1 and GABA B2 [START_REF] Jones | GABA(B) receptors function as a heteromeric assembly of the subunits GABA(B)R1 and GABA(B)R2[END_REF][START_REF] Kaupmann | GABA(B)-receptor subtypes assemble into functional heteromeric complexes[END_REF], White et al., 1998).

Interestingly, Binet and colleagues demonstrated that the positive allosteric modulator CGP7930 activated a mutant receptor that consisted solely of the GABA B2 subunit. As the endogenous ligand GABA binds solely to the GABA B1 subunit, this data suggests CGP7930 could be imparting its allosteric effect across the dimer [START_REF] Binet | The heptahelical domain of GABA(B2) is activated directly by CGP7930, a positive allosteric modulator of the GABA(B) receptor[END_REF].

A c c e p t e d M a n u s c r i p t 12

The emergence of ago-allosterism has a number of implications for drug discovery. Firstly, allosteric binding sites are considered to have been under less evolutionary pressure for their maintenance than the orthosteric binding site of a GPCR and, therefore, are likely to display heterogeneity across a receptor family.

Thus, ago-allosterism provides an opportunity for more selective agonism.

Furthermore, allosteric modulators have been heralded as a safer alternative to orthosteric ligands as they exhibit a maximal 'ceiling effect', that is, their effect on affinity or efficacy of an orthosteric ligand is saturable. However, a number of ago-allosteric modulators appear to be intrinsic full agonists with efficacy equivalent to that of the endogenous ligand, despite binding to a distinct sitethis would appear to negate the clear benefit of positive allosteric ligands in overdose, and also makes determination of experimental parameters of allosterism difficult. In light of increased appreciation of functional selectivity, it is becoming more important to measure many functional endpoints. This becomes an onerous task once the additional complication of probe-dependence of allosterism is consideredwhich ligands should be used as orthosteric probes to assess the allosteric effects of an ago-allosteric modulator? To extend the problem further, which animal models would be suitable to measure physiological and pathological endpoints of a pre-clinical ago-allosteric modulator? There are many examples in which orthosteric ligands identified initially at a human GPCR expressed in a heterologous cell line display significantly different potency and/or efficacy at species orthologues of the GPCR. This poses major challenges for translation of basic pharmacological information into animal models, and if A c c e p t e d M a n u s c r i p t 13 allosteric sites are indeed less well conserved throughout mammalian species than orthosteric binding sites, these issues of translation will undoubtedly generate even more substantial challenges.

Efficacy and super agonism

The term efficacy was coined by Stephenson [START_REF] Stephenson | A modification of receptor theory[END_REF]) and refers to the ability of a ligand, once bound to a receptor, to elicit a physiological or pharmacological response. The parameter most frequently used to assess efficacy is the maximal agonist effect (E max ), which is the maximal asymptote of a concentration-response curve fitted to experimental data [START_REF] Strange | Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors[END_REF].

Efficacy is a relative phenomenon and so is usually assessed by comparing the E max of the test ligand to that of another ligand, most commonly the endogenous agonist (although this is clearly not practical for an orphan receptor). This allows ligands to be characterized either as full agonists (ligands that produce the 'maximal' response i.e. the maximum possible response produced by a ligand in the particular cell/tissue of study), partial agonists (ligands that produce a submaximal response) or antagonists (ligands that bind to receptor but do not possess efficacy). The relative nature of efficacy, however, means that each time a new ligand is identified as having the maximal response in a system, ligands that were previously considered to be full agonists are subsequently re-classified as partial agonists. Whilst pharmacologically correct, such an approach can be cumbersome and draw focus away from the endogenous agonist. This has led to some commentators to coin the term 'super agonism' to describe situations A c c e p t e d M a n u s c r i p t 14 where synthetic orthosteric ligands that share an overlapping binding site with the endogenous ligand(s) have been demonstrated to activate the receptor with higher efficacy than the endogenous agonist [START_REF] Niemczyk | A super-agonist of growth hormone-releasing hormone causes rapid improvement of nutritional status in patients with chronic kidney disease[END_REF][START_REF] Tan | Heterozygous alpha 2A-adrenergic receptor mice unveil unique therapeutic benefits of partial agonists[END_REF].

Super agonists may prove to be useful therapeutic ligands. The non-peptide growth hormone secretagogue MK-677 is currently undergoing clinical trials as its growth hormone-releasing properties may be clinically useful for the treatment of idiopathic short stature or ageing (in which there is a natural decline in GH release). In one short (2 year) clinical trial healthy older adults who received MK-677 showed sustained increases in amplitude of pulsatile growth hormone secretion to levels observed in young adults [START_REF] Mobarec | Effects of an oral ghrelin mimetic on body composition and clinical outcomes in healthy older adults: a randomized trial[END_REF]. Interestingly, MK-677, (along with two other synthetic agonists, growth hormone releasing peptide-6 and L-692,585) has been described as a super agonist of the ghrelin receptor in the G αo1 pathway [START_REF] Bennett | Growth hormone secretagogues and growth hormone releasing peptides act as orthosteric super-agonists but not allosteric regulators for activation of the G protein Galpha(o1) by the Ghrelin receptor[END_REF]. In a separate study, MK-677 was also described as a super agonist of the ghrelin receptor in β-arrestin mobilization studies and in serum-responsive element mediated transcription assays [START_REF] Holst | Ghrelin receptor inverse agonists: identification of an active peptide core and its interaction epitopes on the receptor[END_REF]. Although the benefits of MK-677 in the treatment of growth hormone disorders has not been linked to its enhanced efficacy per se, MK-677 has been shown to act as a super agonist and may well become a clinically useful drug.

The study into activation of the ghrelin receptor by [START_REF] Holst | Nonpeptide and peptide growth hormone secretagogues act both as ghrelin receptor agonist and as positive or negative allosteric modulators of ghrelin signaling[END_REF] also demonstrates an important point -that efficacy varies depending on the assay A c c e p t e d M a n u s c r i p t 15 used to measure it, with MK-677 shown to act with efficacy equal to that of the endogenous ligand, ghrelin, in calcium mobilization and inositol phosphate accumulation assays [START_REF] Holst | Nonpeptide and peptide growth hormone secretagogues act both as ghrelin receptor agonist and as positive or negative allosteric modulators of ghrelin signaling[END_REF]. It has long been known that efficacy of a ligand may vary depending on the assay used. Thus, measuring efficacy in downstream assays where a degree of response amplification occurs can result in a masking of differences in ligand efficacies. Measurement of efficacy in downstream systems also means that feedback, cross regulation and even convergence of different effector systems may also be integrated into the measured signal, complicating experimental results [START_REF] Wess | Molecular basis of receptor/G-protein-coupling selectivity[END_REF]. Thus it is best to measure maximum efficacy in response systems close to the receptor where few compounds will reach the maximal system response [START_REF] Strange | Agonist binding, agonist affinity and agonist efficacy at G protein-coupled receptors[END_REF]. Such an approach may reveal hitherto unrecognized super agonists and may highlight functional selectivity. It could be argued that super agonism is purely an artifact of, for example, alterations in receptor desensitization/recycling rates or alterations in ligand affinity. However, a study by [START_REF] Engel | Low affinity analogs of thyrotropinreleasing hormone are super-agonists[END_REF] demonstrated, for the thyrotropin-releasing hormone 1 receptor, that the thyrotropin-releasing hormone (TRH) analogue R-desaza TRH ((1R)-(3oxocyclopentyl-His-ProNH 2 )) exhibited a lower potency but a higher efficacy than TRH at similar occupancy of the receptor. By using ligands that affect receptor recycling or inhibit specific protein kinases, such as the selective protein kinase C inhibitor, Ro-31-8425, Engel et al. suggested that R-desaza TRH still acted as a super agonist with respect to TRH signaling [START_REF] Engel | Low affinity analogs of thyrotropinreleasing hormone are super-agonists[END_REF]. Although the effects of various G protein-coupled receptor kinases (GRKs) or regulators of G A c c e p t e d M a n u s c r i p t 16 protein signaling (RGS) proteins should be investigated before firm conclusions are drawn, it is possible that super agonism may not be an artifact of altered receptor number at the cell surface but is indeed a result of altered receptor-G protein interactions.

Dualsteric ligand design

The concept of a ligand binding simultaneously to more than one site on a GPCR and resulting in enhanced binding, efficacy or duration of signal is not new; for example, the beta2-adrenoceptor agonist, salmeterol, is thought to be long-acting because the extended aliphatic chain is tethered to a site on the beta2adrenoceptor that is distinct from the orthosteric site (although there is no suggestion that this interaction is allosteric; [START_REF] Griffith | Adrenergic receptors and drugs affecting adrenergic neurotransmission[END_REF]). However, the rational design of orthosteric agonists that possess enhanced receptor subtype selectivity due to additional binding at an allosteric site is a new and exciting approach to drug discovery. For the purpose of this review, we will define such chemicals as dualsteric ligands (rather than the terms bitopic or hybrid ligands, that are also in current usage) to reflect the presence of both orthosteric and allosteric moieties and to distinguish them from heterobivalent ligands spanning across a homo-or hetero-dimer (e.g. KDN-21, which spans delta and kappa opioid receptors, [START_REF] Bhushan | A bivalent ligand (KDN-21) reveals spinal delta and kappa opioid receptors are organized as heterodimers that give rise to delta(1) and kappa(2) phenotypes. Selective targeting of delta-kappa heterodimers[END_REF]) or binding to two separate GPCRs (e.g. a peptide coagonist at glucagon and glucagon-like peptide 1 receptors, [START_REF] Day | A new glucagon and GLP-1 co-agonist eliminates obesity in rodents[END_REF]).

A c c e p t e d M a n u s c r i p t 17

It is no coincidence that the majority of studies to date examining dualsteric ligands have focused on the muscarinic family of acetylcholine receptors (mAChRs). Consisting of five subtypes, M 1 -M 5 , the highly conserved nature of the orthosteric binding pocket has meant that few selective agonists have progressed to clinical trials, despite a high degree of confidence that these would be appropriate therapeutic targets [START_REF] Hulme | Muscarinic receptor subtypes[END_REF]). In the case of M 1 agonists for the treatment of the early stages of Alzheimer's disease, at least five compounds have failed to progress from early trials to the clinic owing to poor selectivity resulting in cardiovascular side effects via M 2 and M 3 mAChRs [START_REF] Langmead | Muscarinic acetylcholine receptors as CNS drug targets[END_REF]. While the orthosteric binding pockets of the mAChRs are poorly conserved, considerable divergence is found in the allosteric sites of this family of GPCRs [START_REF] Christopoulos | G protein-coupled receptor allosterism and complexing[END_REF]. Thus, the combination of potent orthosteric moieties with near-neutral allosteric compounds into a single hybrid ligand would provide an ideal mechanism for conferring increased subtype selectivity. Indeed, close examination of one existing subtype-selective ligand,

McN-A-343, has revealed its mode of binding to the M 2 mACh receptor to be bitopic [START_REF] Valant | A novel mechanism of G protein-coupled receptor functional selectivity. Muscarinic partial agonist McN-A-343 as a bitopic orthosteric/allosteric ligand[END_REF][START_REF] Valant | Orthosteric/allosteric bitopic ligands: going hybrid at GPCRs[END_REF] and it is conceivable that other ligands with clear selectivity interact with their preferred GPCR in the same manner. Holzgrabe and colleagues [START_REF] Disingrini | Design, synthesis, and action of oxotremorine-related hybrid-type allosteric modulators of muscarinic acetylcholine receptors[END_REF] employed a rational approach to the design of bitopic ligands and synthesized a panel of hybrids incorporating the potent yet non-selective agonists, oxotremorine, oxotremorine-M and analogues, and the M 2 mAChR-selective negative allosteric modulators W84 or naphmethonium. Although an agonist backbone was used to target the A c c e p t e d M a n u s c r i p t 19 Not only can subtype selectivity be enhanced by incorporating allosteric and orthosteric moieties into a dualsteric compound, but functional selectivity can be achieved, highlighting the promise for this approach in disease states where activation of only a subset of pathways from a receptor is desirable (e.g., beta-arrestin2-mediated inotropy and lusitropy [START_REF] Rajagopal | Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes[END_REF] without G protein-mediated cardiac hypertrophy for angiotensin receptors [reviewed in [START_REF] Smith | Signal switching, crosstalk, and arrestin scaffolds: novel G protein-coupled receptor signaling in cardiovascular disease[END_REF]] or β-arrestin-dependent cardioprotection [START_REF] Noma | Beta-arrestin-mediated beta1-adrenergic receptor transactivation of the EGFR confers cardioprotection[END_REF] without cardiac hypertrophy or progression to heart failure [START_REF] Patel | Physiologic and cardiac roles of beta-arrestins[END_REF]). When investigating the nature of agonism to result from the dualsteric ligands hybrid 1 and hybrid 2, whole cell biophysical experiments in the presence of the Gα i/o inhibitor, pertussis toxin, demonstrated that while the endogenous agonist, acetylcholine, and the original orthosteric template, iperoxo, were still able to cause a cellular response, the dualsteric agonists were rendered inactive [START_REF] Antony | Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity[END_REF], Kebig et al., 2009). Thus, the dualsteric ligands were functionally selective for the Gα i/o pathway.

So what distinguishes dualsteric ligands from ago-allosteric modulators? Agoallosteric ligands both bind and activate the receptor by interacting with the allosteric binding site, meaning that an orthosteric ligand is able to co-bind to the receptor and that any observed agonism would be the resulting combination of orthosteric agonism, allosteric-site agonism and any co-operativity that occurred between the two sites. In contrast, dualsteric agonists bind simultaneously to the the interaction between the allosteric modulator and M 2 mAChR hybrid 1 ligand was found to be competitive (indicating that the dualsteric ligand bound at the allosteric site of the receptor) as was, at lower concentrations of antagonist, the interaction between hybrid 1 and the orthosteric antagonist atropine [START_REF] Antony | Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity[END_REF]. Interestingly, at higher concentrations of atropine, hybrid 1 was no longer able to competitively overcome atropine binding and the rightward shift appeared to be saturating (negative allosteric modulation)the authors concluded that this reflected the displacement of the agonistic fragment of hybrid 1 from the orthosteric binding site by atropine (hence the reduction in signal) but the retention of allosteric fragment binding at the allosteric site [START_REF] Antony | Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity[END_REF]. Thus, although dualsteric ligands appear to exert their agonistic effects via the orthosteric site, their pharmacology has the potential to be equally if not more complicated than an ago-allosteric modulator because the ligand can act as an agonist at the orthosteric site, an allosteric modulator if the orthosteric site is bound by another ligand and an antagonist for either allosteric or orthosteric ligands. The capacity for the allosteric moiety of a dualsteric ligand to bind to the receptor without the agonistic fragment occupying the receptor means that if the rationale for designing a dualsteric ligand was to obtain receptor subtype selectivity, the allosteric fragment should ideally have little to no co-operativity.

Otherwise, orthosteric agonist efficacy may be affected (May et al., 2007b). For decades, the traditional model of receptor activation involves the interaction of a ligand at the binding site of a receptor somewhat like a key fitting into a lock (illustrated diagrammatically in A). In the case of agonism, binding of an orthosteric agonist induces conformational changes in the receptor that result in a functional response; thus the ligand possesses efficacy. As such, an inverse agonist at the orthosteric site will act to reduce any existing function of the receptor while a neutral antagonist will occupy the binding site but will not possess efficacy. More recently, it has become apparent that compounds are able to bind and interact at a site distinct from the orthosteric sitethis is referred to as the allosteric site and is represented by the rectangular ligand in diagrams (B-E). An allosteric modulator can bind to a G protein-coupled receptor Another possible interaction predicted by the Allosteric Two-State Model (May et al., 2007) of receptor function is allosteric agonism (F), in which case a ligand binds to an allosteric site on the receptor and causes a functional response. The allosteric agonist may also allosterically modulate the affinity and/or efficacy (as per C-E) of a ligand at the orthosteric site, in which case it is referred to as an ago-allosteric modulator (G). Less frequently, an agonist may possess efficacy above that of the endogenous ligand (so-called super-agonism; although represented here at the orthosteric site there is no reason why super-agonism could not occur via allosteric agonism or ago-allosterism). Finally, subtype selectivity can be enhanced by the design of dualsteric ligands in which a potent but non-selective orthosteric ligand is fused to a more selective moiety that interacts at the allosteric binding site (I). However, there is also the possibility that the dualsteric ligand will bind only at the allosteric site, allowing access for another ligand to bind orthostericallythe affinity and/or efficacy of the orthosteric interaction may be modulated by the allosteric moiety of the dualsteric ligand, depending upon its allosteric properties. Ideally, if the purpose of designing a dualsteric ligand is to generate an agonist with enhanced subtype specificity, then the allosteric moiety should have near-neutral co-operativity.

  potent and selective agonists at hitherto untapped drug targets.

  thought to derive from ligands stabilizing GPCRs in specific conformations that favor activation of a particular downstream effector. A number of elegant examples of distinct receptor conformations stabilized by ligands with different efficacies have been provided for the beta2-adrenoceptor;

  A c c e p t e d M a n u s c r i p t 10 binding of the orthosteric radioligand [ 3 H]-cyclohexyladenosine when both ligands

  sites and activate the receptor via the orthosteric site: this was demonstrated by competition experiments in [ 35 S]GTPγS assays, where

  Understanding of the mode of action of synthetic ligands that target members of the G protein-coupled family of receptors has recently been extended greatly by detailed analysis, often in parallel, of multiple elements of their ability to bind and/or activate receptors. Some of the emerging modalities offer distinctly novel avenues to drug discovery that are likely to revitalize programs of work on receptors that had appeared intractable or where receptor subtype side effects appeared to eliminate the use of conventional ligands.

  Figure Legend

  of agonist (B) without effect. However, in the presence of an orthosteric ligand (for the purposes of this review, an orthosteric agonist), an allosteric modulator can positively or negatively affect either the affinity (C), efficacy (D) or both the affinity and efficacy (E) of the orthosteric ligand.

  here, it is important to remember that each of the examples of efficacy above may represent full, partial or inverse agonism or even activation of subsets of the full complement of signaling pathways (functional selectivity).

Acknowledgments

NJS is a National Health and Medical Research Council/National Heart Foundation of Australia C.J. Martin Overseas Research Fellow. KAB was supported by a CASE studentship from the Biotechnology and Biological Sciences Research Council (BBSRC).

A c c e p t e d M a n u s c r i p t 18 orthosteric binding site, the dualsteric ligands generally displayed antagonism in a range of organ preparations and intrinsic efficacy was only revealed in a [ 35 S]-GTPγS measure of G protein activation in membranes from a heterologous cell culture system. Little subtype selectivity was observed. In contrast, reasonable subtype selectivity for the M 2 mAChR was obtained by [START_REF] Steinfeld | A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor[END_REF], who generated a high affinity antagonist, THRX-160209, comprising a benzhydryl group linked to a 4-aminopiperidine motif by a C7 polymethylene chain [START_REF] Steinfeld | A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor[END_REF].

A very recent example of the promise of dualsteric ligands comes from Mohr and colleagues ( 2009), who extended the approach of Disingrini et al. above (2006) by again using high affinity M 2 mAChR-selective allosteric fragments but this time fused to the potent orthosteric muscarinic agonist, iperoxo [START_REF] Antony | Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity[END_REF].

The authors successfully created dualsteric ligands that were able to act as full agonists with good selectivity for the M 2 mAChR both in vitro and in whole tissue pharmacology experiments. Unlike THRX-160209, which displayed markedly enhanced affinity for the receptor compared to its individual components [START_REF] Steinfeld | A novel multivalent ligand that bridges the allosteric and orthosteric binding sites of the M2 muscarinic receptor[END_REF], neither hybrid 1 nor hybrid 2 displayed affinity or potency greater than iperoxo, the orthosteric building block [START_REF] Antony | Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity[END_REF]. Thus, despite the expectation that fusing two separate binding partners into a single molecule would be energetically favorable and confer enhanced affinity upon the resultant dualsteric compound, it is clear that the nature of the ligand fragment influences the measured receptor binding or function (May et al., 2007b).