Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.

Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.

Isolation and characterization of a VHH targeting the Acinetobacter baumannii cell surface protein CsuA/B.

Acinetobacter baumannii is a Gram-negative bacterial pathogen that exhibits high intrinsic resistance to antimicrobials, with treatment often requiring the use of last-resort antibiotics. Antibiotic-resistant strains have become increasingly prevalent, underscoring a need for new therapeutic interventions. The aim of this study was to use A. baumannii outer membrane vesicles as immunogens to generate single-domain antibodies (VHHs) against bacterial cell surface targets. Llama immunization with the outer membrane vesicle preparations from four A. baumannii strains (ATCC 19606, ATCC 17961, ATCC 17975, and LAC-4) elicited a strong heavy-chain IgG response, and VHHs were selected against cell surface and/or extracellular targets. For one VHH, OMV81, the target antigen was identified using a combination of gel electrophoresis, mass spectrometry, and binding studies. Using these techniques, OMV81 was shown to specifically recognize CsuA/B, a protein subunit of the Csu pilus, with an equilibrium dissociation constant of 17 nM. OMV81 specifically bound to intact A. baumannii cells, highlighting its potential use as a targeting agent. We anticipate the ability to generate antigen-specific antibodies against cell surface A. baumannii targets could provide tools for further study and treatment of this pathogen. KEY POINTS: •Llama immunization with bacterial OMV preparations for VHH generation •A. baumannii CsuA/B, a pilus subunit, identified by mass spectrometry as VHH target •High-affinity and specific VHH binding to CsuA/B and A. baumannii cells.

Soil Microbiomes Associated with a Novel Perennial Grain Cultivated under Temperate Agricultural Conditions.

A perennial wheatgrass called Kernza perennial grains was developed by the Land Institute to harness the benefits of perenniality on soil health in a commercial farming system. This study compared bacterial and fungal soil microbiomes surrounding 1-year-old Kernza, 4-year-old Kernza, and 6-week-old winter wheat in Hudson Valley, New York.

High-definition transcranial direct current stimulation enhances network segregation during spatial navigation in mild cognitive impairment.

Spatial navigation is essential for everyday life and relies on complex network-level interactions. Recent evidence suggests that transcranial direct current stimulation (tDCS) can influence the activity of large-scale functional brain networks. We characterized brain-wide changes in functional network segregation (i.e. the balance of within vs. between-network connectivity strength) induced by high-definition (HD) tDCS in older adults with mild cognitive impairment (MCI) during virtual spatial navigation. Twenty patients with MCI and 22 cognitively intact older adults (healthy controls-HC) underwent functional magnetic resonance imaging following two counterbalanced HD-tDCS sessions (one active, one sham) that targeted the right parietal cortex (center anode at P2) and delivered 2 mA for 20 min. Compared to HC, MCI patients showed lower brain-wide network segregation following sham HD-tDCS. However, following active HD-tDCS, MCI patients' network segregation increased to levels similar to those in HC, suggesting functional normalization. Follow-up analyses indicated that the increase in network segregation for MCI patients was driven by HD-tDCS effects on the "high-level"/association brain networks, in particular the dorsal-attention and default-mode networks. HD-tDCS over the right parietal cortex may normalize the segregation/integration balance of association networks during spatial navigation in MCI patients, highlighting its potential to restore brain activity in Alzheimer's disease.

Facile Affinity Maturation of Single-Domain Antibodies Using Next-Generation DNA Sequencing.

Binding affinity is one of the primary determinants of antibody function. Here, we provide a protocol for simple and rapid affinity maturation of single-domain antibodies (sdAbs) using tandem phage display selection and next-generation DNA sequencing. The sequence of a model camelid sdAb directed against Clostridioides difficile toxin A (A26.8) was diversified using either random or site-saturation mutagenesis and cloned into a phagemid vector upstream of gene 3. The resulting phage-displayed sdAb libraries were panned against C. difficile toxin A and the panning outputs interrogated using Illumina MiSeq sequencing. Through bioinformatic analyses, we were able to identify individual affinity-enhancing amino acid substitutions in the sdAb complementarity-determining regions that, when combined, resulted in affinity improvements of approximately 10-fold. The advantages of this method are that it does not require extensive screening and characterization of individual clones, nor structural information on the mechanism of the sdAb:antigen interaction.

Effects of High Definition-Transcranial Direct Current Stimulation on Local GABA and Glutamate Levels Among Older Adults with and without Mild Cognitive Impairment: An Exploratory Study.

BACKGROUND: Prior research, primarily with young adults, suggests transcranial direct current stimulation (tDCS) effects are driven by the primary excitatory and/or inhibitory neurotransmitters, glutamate, and gamma-aminobutyric acid (GABA), respectively. OBJECTIVE: We examined the neurometabolic mechanisms of tDCS in older adults with and without mild cognitive impairment (MCI). METHODS: We used data from a double-blind, cross-over, randomized controlled trial (NCT01958437) in 32 older adults to evaluate high definition (HD)-tDCS-induced changes in glutamate and GABA via magnetic resonance spectroscopy (MRS). Participants underwent MRS following two counterbalanced HD-tDCS sessions (one active, one sham) that targeted the right superior parietal cortex (center anode at P2) and delivered 2mA for 20 minutes. RESULTS: Relative to sham, and when co-varying for MRS voxel overlap and right superior parietal volume, active HD-tDCS significantly increased GABA and decreased the ratio of glutamate to GABA. No changes were observed in a left prefrontal control MRS voxel. Although we did not find a significant correlation between strength of delivered current (measured via MRI-based computational modeling) and neurometabolite change, there was a robust positive relationship between the volume of right superior parietal cortex and neurometabolite change. CONCLUSION: Our preliminary findings of increased GABA and reduced glutamate/GABA ratio raise the possibility that (HD-)tDCS effects differ by age. Moreover, age- and disease-related regional brain volume loss may be especially important to consider when planning future studies. Replication would emphasize the importance of developing population-specific tDCS parameters that consider structural and physiologic changes associated with "normal" and pathological aging.

Incorporation of a Novel CD16-Specific Single-Domain Antibody into Multispecific Natural Killer Cell Engagers With Potent ADCC.

Multispecific antibodies that bridge immune effector and tumor cells have shown promising preclinical and clinical efficacies. Here, we isolated and characterized novel llama single-domain antibodies (sdAbs) against CD16. One sdAb, NRC-sdAb048, bound recombinant human and cynomolgus monkey CD16 ectodomains with equivalent affinity (KD: 1 nM) but did not recognize murine CD16. Binding was similar for human CD16a expressed on NK cells and CD16b (NA2) expressed on neutrophils but dramatically weaker (KD: ∼6 µM) for the CD16b (NA1) allotype. The sdAb stained primary human peripheral blood NK cells. Irrespective of fusion orientation and linker length, bispecific sdAb-sdAb and sdAb-scFv dimers (anti-CD16/EGFR, anti-CD16/HER2, and anti-CD16/CD19) retained full binding affinity for each target, coengaged both antigens simultaneously, elicited ADCC against target antigen-expressing tumor cells in a reporter bioassay, and triggered target-specific activation and degranulation of primary NK cells as measured via interferon-γ and CD107a expression. These molecules may have applications in cancer immunotherapy.

Serum albumin-binding VH Hs with variable pH sensitivities enable tailored half-life extension of biologics.

Prolonged serum half-life is required for the efficacy of most protein therapeutics. One strategy for half-life extension is to exploit the long circulating half-life of serum albumin by incorporating a binding moiety that recognizes albumin. Here, we describe camelid single-domain antibodies (VH Hs) that bind the serum albumins of multiple species with moderate to high affinity at both neutral and endosomal pH and significantly extend the serum half-lives of multiple proteins in rats from minutes to days. We serendipitously identified an additional VH H (M75) that is naturally pH-sensitive: at endosomal pH, binding affinity for human serum albumin (HSA) was dramatically weakened and binding to rat serum albumin (RSA) was undetectable. Domain mapping revealed that M75 bound to HSA domain 1 and 2. Moreover, alanine scanning of HSA His residues suggested a critical role for His247, located in HSA domain 2, in M75 binding and its pH dependence. Isothermal titration calorimetry experiments were suggestive of proton-linked binding of M75 to HSA, with differing binding enthalpies observed for full-length HSA and an HSA domain 1-domain 2 fusion protein in which surface-exposed His residues were substituted with Ala. M75 conferred moderate half-life extension in rats, from minutes to hours, likely due to rapid dissociation from RSA during FcRn-mediated endosomal recycling in tandem with albumin conformational changes induced by M75 binding that prevented interaction with FcRn. Humanized VH Hs maintained in vivo half-life extension capabilities. These VH Hs represent a new set of tools for extending protein therapeutic half-life and one (M75) demonstrates a unique pH-sensitive binding interaction that can be exploited to achieve modest in vivo half-life.

The TRANScending Love Arts-Based Workshop to Address Self-Acceptance and Intersectional Stigma Among Transgender Women of Color in Toronto, Canada: Findings from a Qualitative Implementation Science Study.

Purpose: Transgender (trans) women of color's HIV vulnerabilities are shaped by social exclusion and intersectional stigma. There is a dearth of tailored HIV prevention interventions with trans women of color in Canada. The objective of the study was to explore trans women of color's HIV prevention priorities and to pilot test an intervention developed from these priorities. Methods: We conducted a qualitative implementation science study to develop HIV intervention strategies with trans women of color in Toronto, Canada. First, we conducted a focus group with trans women of color (n=8) to explore HIV prevention priorities. Second, we held a consultation with trans women of color community leaders (n=2). Findings informed the development of the TRANScending Love (T-Love) arts-based workshop that we pilot tested with three groups of trans women of color (n=18). Workshops were directly followed by focus groups to examine T-Love products and processes. Results: Focus group participants called for researchers to shift the focus away from trans women's bodies and HIV risks to address low self-acceptance produced by intersecting forms of stigma. The community leader consultation articulated the potential for strengths-focused arts-based approaches to address self-worth. T-Love participants described how workshops fostered self-acceptance and built connections between trans women of color. Conclusions: Findings demonstrate the feasibility and acceptability of an arts-based strategy with trans women of color to elicit group-based sharing of journeys to self-acceptance, fostering feelings of solidarity and connection. Providing opportunities for dialogue and reflection about individual and collective strengths may reduce internalized stigma among trans women of color.

Neutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains.

An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C. difficile immunotherapeutics, we previously isolated several single-domain antibodies (VHHs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing VHHs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 VHHs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as KD = 70 pM, none of the VHHs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with VHH-VHH dimers and to a much greater extent with VHH-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several VHH-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other VHH-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the VHHs described here are effective at toxin B neutralization when formatted as bivalent VHH-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site.

Pathways from Resilient Coping to Safer Sex Communication Among African, Caribbean, and Black Women in Toronto, Canada: Results from a Cross-sectional Survey.

PURPOSE: African, Caribbean, and Black (ACB) women in Canada are disproportionately impacted by HIV and other sexually transmitted infections. Although there is reported suboptimal consistent condom use with ACB women, limited research has explored safer sex communication among this population. Coping frameworks highlight the role that resilient coping and condom use self-efficacy may play in facilitating safer sex communication. Structural perspectives stress the need to explore associations between HIV vulnerabilities and food insecurity. We examined pathways from resilient coping to safer sex communication through the mediator of condom use self-efficacy among ACB women in Toronto. METHOD: We conducted a cross-sectional survey with a purposive sample of ACB women aged 16 and older across Toronto, Canada. We conducted path analysis to test the direct effects of resilient coping on safer sex communication, and indirect pathways through the mediator (condom use self-efficacy) while controlling for food insecurity. RESULTS: Participant (n = 80; mean age 27, SD 7.93) ethnicities included African (58.8%, n = 47), Caribbean (30%, n = 24), and others (11.3%, n = 9). Participants with food security reported significantly higher safer sex communication. We found no direct effect of resilient coping on safer sex communication. Findings support the hypothesized mediation process; resilient coping was associated with condom use self-efficacy, which in turn was associated with safer sex communication. CONCLUSION: Findings that condom use self-efficacy mediated the association between resilient coping and safer sex communication align with theoretical assertions of the protective role of adaptive coping strategies. Findings can inform tailored HIV and STI preventive interventions with ACB women.

Adapting and pilot testing the Healthy Love HIV and sexually transmitted infection prevention intervention with African, Caribbean and Black women in community-based settings in Toronto, Canada.

We adapted the Healthy Love Workshop (HLW), an HIV prevention workshop for African American women in the United States, for African, Caribbean and Black (ACB) women in Toronto, Canada. We conducted a pilot study with ACB women ( n = 80) in ten community-based settings with pre-test (T1), post-test (T2) and three-month follow-up (T3) surveys. Mixed-effect regression results indicated significant increases in condom use self-efficacy and sexually transmitted infection (STI) knowledge scores from T1 to T3. Qualitative feedback revealed increased STI knowledge, confidence using condoms and suggestions for future HLWs. Findings highlight the promise of the adapted HLW for HIV/STI prevention with ACB women in Canada.

Application of Assisted Design of Antibody and Protein Therapeutics (ADAPT) improves efficacy of a Clostridium difficile toxin A single-domain antibody.

Assisted Design of Antibody and Protein Therapeutics (ADAPT) is an affinity maturation platform interleaving predictions and testing that was previously validated on monoclonal antibodies (mAbs). This study expands the applicability of ADAPT to single-domain antibodies (sdAbs), a promising class of recombinant antibody-based biologics. As a test case, we used the camelid sdAb A26.8, a VHH that binds Clostridium difficile toxin A (TcdA) relatively weakly but displays a reasonable level of TcdA neutralization. ADAPT-guided A26.8 affinity maturation resulted in an improvement of one order of magnitude by point mutations only, reaching an equilibrium dissociation constant (KD) of 2 nM, with the best binding mutants having similar or improved stabilities relative to the parent sdAb. This affinity improvement generated a 6-fold enhancement of efficacy at the cellular level; the A26.8 double-mutant T56R,T103R neutralizes TcdA cytotoxicity with an IC50 of 12 nM. The designed mutants with increased affinities are predicted to establish novel electrostatic interactions with the antigen. Almost full additivity of mutation effects is observed, except for positively charged residues introduced at adjacent positions. Furthermore, analysis of false-positive predictions points to general directions for improving the ADAPT platform. ADAPT guided the efficacy enhancement of an anti-toxin sdAb, an alternative therapeutic modality for C. difficile.

"It's for us -newcomers, LGBTQ persons, and HIV-positive persons. You feel free to be": a qualitative study exploring social support group participation among African and Caribbean lesbian, gay, bisexual and transgender newcomers and refugees in Toronto, Canada.

BACKGROUND: Stigma and discrimination harm the wellbeing of lesbian, gay, bisexual and transgender (LGBT) people and contribute to migration from contexts of sexual persecution and criminalization. Yet LGBT newcomers and refugees often face marginalization and struggles meeting the social determinants of health (SDOH) following immigration to countries such as Canada. Social isolation is a key social determinant of health that may play a significant role in shaping health disparities among LGBT newcomers and refugees. Social support may moderate the effect of stressors on mental health, reduce social isolation, and build social networks. Scant research, however, has examined social support groups targeting LGBT newcomers and refugees. The purpose of this qualitative study was to explore experiences of social support group participation among LGBT African and Caribbean newcomers and refugees in an urban Canadian city. METHODS: We conducted 3 focus groups with a venue-based sample of LGBT African and Caribbean newcomers and refugees (n = 29) who attended social support groups at an ethno-specific AIDS Service Organization. Focus groups followed a semi-structured interview guide and were analyzed using narrative thematic techniques. RESULTS: Participant narratives highlighted immigration stressors, social isolation, mental health issues, and challenges meeting the SDOH. Findings reveal multi-level benefits of social support group participation at intrapersonal (self-acceptance, improved mental health), interpersonal (reduced isolation, friendships), community (reciprocity, reduced stigma and discrimination), and structural (housing, employment, immigration, health care) levels. CONCLUSIONS: Findings suggest that social support groups tailored for LGBT African and Caribbean newcomers and refugees can address social isolation, community resilience, and enhance resource access. Health care providers can provide support groups, culturally and LGBT competent health services, and resource access to promote LGBT newcomers and refugees' health and wellbeing.

Vagus nerve stimulation mitigates intrinsic cardiac neuronal and adverse myocyte remodeling postmyocardial infarction.

This paper aims to determine whether chronic vagus nerve stimulation (VNS) mitigates myocardial infarction (MI)-induced remodeling of the intrinsic cardiac nervous system (ICNS), along with the cardiac tissue it regulates. Guinea pigs underwent VNS implantation on the right cervical vagus. Two weeks later, MI was produced by ligating the ventral descending coronary artery. VNS stimulation started 7 days post-MI (20 Hz, 0.9 ± 0.2 mA, 14 s on, 48 s off; VNS-MI, n = 7) and was compared with time-matched MI animals with sham VNS (MI n = 7) vs. untreated controls (n = 8). Echocardiograms were performed before and at 90 days post-MI. At termination, IC neuronal intracellular voltage recordings were obtained from whole-mount neuronal plexuses. MI increased left ventricular end systolic volume (LVESV) 30% (P = 0.027) and reduced LV ejection fraction (LVEF) 6.5% (P < 0.001) at 90 days post-MI compared with baseline. In the VNS-MI group, LVESV and LVEF did not differ from baseline. IC neurons showed depolarization of resting membrane potentials and increased input resistance in MI compared with VNS-MI and sham controls (P < 0.05). Neuronal excitability and sensitivity to norepinephrine increased in MI and VNS-MI groups compared with controls (P < 0.05). Synaptic efficacy, as determined by evoked responses to stimulating input axons, was reduced in VNS-MI compared with MI or controls (P < 0.05). VNS induced changes in myocytes, consistent with enhanced glycogenolysis, and blunted the MI-induced increase in the proapoptotic Bcl-2-associated X protein (P < 0.05). VNS mitigates MI-induced remodeling of the ICNS, correspondingly preserving ventricular function via both neural and cardiomyocyte-dependent actions.

Angiotensin receptors alter myocardial infarction-induced remodeling of the guinea pig cardiac plexus.

Neurohumoral remodeling is fundamental to the evolution of heart disease. This study examined the effects of chronic treatment with an ACE inhibitor (captopril, 3 mg·kg(-1)·day(-1)), AT1 receptor antagonist (losartan, 3 mg·kg(-1)·day(-1)), or AT2 receptor agonist (CGP42112A, 0.14 mg·kg(-1)·day(-1)) on remodeling of the guinea pig intrinsic cardiac plexus following chronic myocardial infarction (MI). MI was surgically induced and animals recovered for 6 or 7 wk, with or without drug treatment. Intracellular voltage recordings from whole mounts of the cardiac plexus were used to monitor changes in neuronal responses to norepinephrine (NE), muscarinic agonists (bethanechol), or ANG II. MI produced an increase in neuronal excitability with NE and a loss of sensitivity to ANG II. MI animals treated with captopril exhibited increased neuronal excitability with NE application, while MI animals treated with CGP42112A did not. Losartan treatment of MI animals did not alter excitability with NE compared with untreated MIs, but these animals did show an enhanced synaptic efficacy. This effect on synaptic function was likely due to presynaptic AT1 receptors, since ANG II was able to reduce output to nerve fiber stimulation in control animals, and this effect was prevented by inclusion of losartan in the bath solution. Analysis of AT receptor expression by Western blot showed a decrease in both AT1 and AT2 receptors with MI that was reversed by all three drug treatments. These data indicate that neuronal remodeling of the guinea pig cardiac plexus following MI is mediated, in part, by activation of both AT1 and AT2 receptors.

Protease-resistant single-domain antibodies inhibit Campylobacter jejuni motility.

Camelid heavy-chain antibody variable domains (VHHs) are emerging as potential antimicrobial reagents. We have engineered a previously isolated VHH (FlagV1M), which binds Campylobacter jejuni flagella, for greater thermal and proteolytic stability. Mutants of FlagV1M were obtained from an error-prone polymerase chain reaction library that was panned in the presence of gastrointestinal (GI) proteases. Additional FlagV1M mutants were obtained through disulfide-bond engineering. Each approach produced VHHs with enhanced thermal stability and protease resistance. When the beneficial mutations from both approaches were combined, a hyperstabilized VHH was created with superior stability. The hyperstabilized VHH bound C. jejuni flagella with wild-type affinity and was capable of potently inhibiting C. jejuni motility in assays performed after sequential digestion with three major GI proteases, demonstrating the remarkable stability imparted to the VHH by combining our engineering approaches.

Dynamic remodeling of the guinea pig intrinsic cardiac plexus induced by chronic myocardial infarction.

Myocardial infarction (MI) is associated with remodeling of the heart and neurohumoral control systems. The objective of this study was to define time-dependent changes in intrinsic cardiac (IC) neuronal excitability, synaptic efficacy, and neurochemical modulation following MI. MI was produced in guinea pigs by ligation of the coronary artery and associated vein on the dorsal surface of the heart. Animals were recovered for 4, 7, 14, or 50 days. Intracellular voltage recordings were obtained in whole mounts of the cardiac neuronal plexus to determine passive and active neuronal properties of IC neurons. Immunohistochemical analysis demonstrated an immediate and persistent increase in the percentage of IC neurons immunoreactive for neuronal nitric oxide synthase. Examination of individual neuronal properties demonstrated that after hyperpolarizing potentials were significantly decreased in both amplitude and time course of recovery at 7 days post-MI. These parameters returned to control values by 50 days post-MI. Synaptic efficacy, as determined by the stimulation of axonal inputs, was enhanced at 7 days post-MI only. Neuronal excitability in absence of agonist challenge was unchanged following MI. Norepinephrine increased IC excitability to intracellular current injections, a response that was augmented post-MI. Angiotensin II potentiation of norepinephrine and bethanechol-induced excitability, evident in controls, was abolished post-MI. This study demonstrates that MI induces both persistent and transient changes in IC neuronal functions immediately following injury. Alterations in the IC neuronal network, which persist for weeks after the initial insult, may lead to alterations in autonomic signaling and cardiac control.

Does an Acute Surgical Model increase the rate of negative appendicectomy or perforated appendicitis?

INTRODUCTION: The clinical outcomes from suspected appendicitis depend on balancing the rate of negative appendicectomy (NA) with perforated appendicitis (PA). An Acute Surgical Model (ASM) was introduced at Geelong Hospital (GH) in 2011 involving a dedicated emergency general surgery theatre list every business day giving greater access to theatre for general surgeons. The aim of this study was to evaluate the effect of the ASM at GH on the management of appendicitis, in particular the NA and PA rates. METHODS: Data for 357 patients undergoing emergency appendicectomy was collected prospectively over 1 year (2011) and compared with a historical control group of 351 patients (2010). The data was analysed for patient demographics, preoperative diagnostic radiology and outcomes including NA and PA rates and complications. The negative appendicectomy rates were compared with contemporary studies. RESULTS: There was no difference between the two groups in rates of negative appendicectomy 21% (ASM; 73/357) versus 21% (Control; 73/351) P = 0.98, or perforated appendicitis 17% (ASM; 61/357) versus 13% (Control; 47/351) P = 0.18. The introduction of the ASM corresponded to a significantly lower proportion of emergency appendicectomies overnight (4% [16/357] versus 12% [44/351] P = 0.005). There was no significant difference in the use of preoperative diagnostic radiology or complications. Matched contemporary studies had a NA rate of 26%. CONCLUSION: The introduction of the ASM at GH has not significantly altered the rate of NA or PA. The NA rate at GH is comparable to other published UK and Australian series.