Detection of Neuroinflammation Induced by Typhoid Vaccine Using Quantitative Magnetization Transfer MR: A Randomized Crossover Study.

BACKGROUND: The role of neuroinflammation in psychiatric disorders is not well-elucidated. A noninvasive technique sensitive to low-level neuroinflammation may improve understanding of the pathophysiology of these conditions. PURPOSE: To test the ability of quantitative magnetization transfer (QMT) MR at 3 T for detection of low-level neuroinflammation induced by typhoid vaccine within a clinically reasonable scan time. STUDY TYPE: Randomized, crossover, placebo-controlled. SUBJECTS: Twenty healthy volunteers (10 males; median age 34 years). FIELD STRENGTH/SEQUENCE: Magnetization prepared rapid gradient-echo and MT-weighted 3D fast low-angle shot sequences at 3 T. ASSESSMENT: Participants were randomized to either vaccine or placebo first with imaging, then after a washout period received the converse with a second set of imaging. MT imaging, scan time, and blood-based inflammatory marker concentrations were assessed pre- and post-vaccine and placebo. Mood was assessed hourly using the Profile of Mood States questionnaire. QMT parameter maps, including the exchange rate from bound to free pool (kba) were generated using a two-pool model and then segmented into tissue type. STATISTICAL TESTS: Voxel-wise permutation-based analysis examined inflammatory-related alterations of QMT parameters. The threshold-free cluster enhancement method with family-wise error was used to correct voxel-wise results for multiple comparisons. Region of interest averages were fed into mixed models and Bonferroni corrected. Spearman correlations assessed the relationship between mood scores and QMT parameters. Results were considered significant if corrected P < 0.05. RESULTS: Scan time for the MT-weighted acquisition was approximately 11 minutes. Blood-based analysis showed higher IL-6 concentrations post-vaccine compared to post-placebo. Voxel-wise analysis found three clusters indicating an inflammatory-mediated increase in kba in cerebellar white matter. Cerebellar kba for white matter was negatively associated with vigor post-vaccine but not post-placebo. DATA CONCLUSION: This study suggested that QMT at 3 T may show some sensitivity to low-level neuroinflammation. Further studies are needed to assess the viability of QMT for use in inflammatory-based disorders. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 2.

FIB-4 Reliability in Patients With Severe Obesity: Lower Cutoffs Needed?

BACKGROUND: Liver biopsy is the gold standard to evaluate hepatic fibrosis; however, it has many drawbacks, especially in patients with severe obesity. Noninvasive testing such as the FIB-4 score is increasingly being used as the initial screening tool to identify patients at risk for advanced fibrosis. The broader applicability of FIB-4 and the precision of its cutoff values remain uncertain in metabolic dysfunction-associated steatotic liver disease and patients with severe obesity. Our study explored the correlation between FIB-4 scores and intraoperative liver biopsy in patients with severe obesity undergoing bariatric surgery. METHODS: A total of 632 patients with severe obesity underwent preoperative vibration-controlled transient elastography and intraoperative liver biopsy during bariatric surgery from January 2020 to August 2021. Variables collected included patient demographics, laboratory values, abdominal ultrasound, vibration-controlled transient elastography, and liver biopsy results. ANOVA 1-way test, χ2 tests, and Fisher exact tests were used for quantitative and qualitative variables, respectively. The 95% CIs for the mean FIB-4 scores were used to generate surrogate cutoff values. The proposed FIB-4 cutoffs for F0-1, F2, F3, and F4 were 0.62 (CI: 0.59, 0.64), 0.88 (0.74, 1.01), 1.24 (0.94, 1.54), and 1.53 (0.82, 2.24), respectively. Area under the curve (AUC) methods were used to compare traditional to proposed cutoff values. RESULTS: Applying the traditional FIB-4 cutoffs to approximate advanced fibrosis yielded an AUC of 0.5748. Use of the proposed FIB-4 cutoffs increased the AUC to 0.6899. The proposed FIB-4 cutoffs correctly identified 40 patients with biopsy-proven advanced fibrosis (F3-F4), all of which would have been missed using traditional cutoffs. CONCLUSION: Our study revealed that the use of the currently accepted FIB-4 cutoffs as the screening modality for identifying patients with advanced fibrosis due to metabolic dysfunction-associated steatotic liver disease is insufficient and will result in missing patients with histologically confirmed advanced fibrosis. Use of the revised FIB-4 scores should be considered to diagnose patients with severe obesity at high risk of liver disease progression.

An Unusual Case of Primary Gastric Balloon Cell Melanoma.

Most cases of melanoma found in the gastrointestinal tract are the result of metastasis. Although uncommon and only described in isolated case reports, primary gastric melanoma should be considered when patients present with vague gastrointestinal symptoms and a mass is identified on esophagogastroduodenoscopy or imaging. We describe a case of primary gastric balloon cell melanoma in a 73-year-old man who presented with melena. Given the high morbidity and mortality of gastric mucosal melanoma, early diagnosis and initiation of treatment can lead to improved outcomes and survival.

Development of a long-acting relaxin analogue, LY3540378, for treatment of chronic heart failure.

BACKGROUND AND PURPOSE: Chronic heart failure, a progressive disease with limited treatment options currently available, especially in heart failure with preserved ejection fraction (HFpEF), represents an unmet medical need as well as an economic burden. The development of a novel therapeutic to slow or reverse disease progression would be highly impactful to patients and society. Relaxin-2 (relaxin) is a human hormone regulating cardiovascular, renal, and pulmonary adaptations during pregnancy. A short-acting recombinant relaxin, Serelaxin, demonstrated short-term heart failure symptom relief and biomarker improvement in acute heart failure trials. Here, we present the development of a long-acting relaxin analogue to be tested in the treatment of chronic heart failure. EXPERIMENTAL APPROACH: LY3540378 is a long-acting protein therapeutic composed of a human relaxin analogue and a serum albumin-binding VHH domain. KEY RESULTS: LY3540378 is a potent agonist of the relaxin family peptide receptor 1 (RXFP1) and maintains selectivity against RXFP2/3/4 comparable to native relaxin. The half-life of LY3540378 in preclinical species is extended through high affinity binding of the albumin-binding VHH domain to serum albumin. When tested in a single dose administration, LY3540378 elicited relaxin-mediated pharmacodynamic responses, such as reduced serum osmolality and increased renal blood flow in rats. In an isoproterenol-induced cardiac hypertrophy mouse model, treatment with LY3540378 significantly reduced cardiac hypertrophy and improved isovolumetric relaxation time. In a monkey cardiovascular safety study, there were no adverse observations from administration of LY3540378. CONCLUSION AND IMPLICATIONS: LY3540378 demonstrated to be a suitable clinical development candidate, and is progressing in clinical trials.

A tough pill to swallow: Two cases of statin-induced necrotizing autoimmune myopathy manifesting as dysphagia and transaminitis.

Although 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors are well tolerated, a small subset of patients may develop autoimmune myopathy, classified as immune-mediated necrotizing myopathy. Statin-induced immune-mediated necrotizing myopathy can present as proximal muscle weakness and in some cases as dysphagia and respiratory distress. We present two cases of patients taking statins who developed dysphagia and muscle weakness found to have statin-induced immune-mediated necrotizing myopathy. Both patients were treated with immunosuppressive therapy: one did well clinically, while the other had an aggressive form of statin-induced immune-mediated necrotizing myopathy and succumbed to the disease. Although statin-induced immune-mediated necrotizing myopathy is rare, early treatment to induce remission of this disabling condition should be initiated to prevent morbidity and mortality.

Double Trouble: A Primary Epstein-Barr Virus Infection Causing Cholestatic Hepatitis and Hemophagocytic Lymphohistiocytosis.

Primary infection with Epstein-Barr virus (EBV) is very common, often manifesting as mononucleosis syndrome with fatigue, sore throat, fever, and enlarged lymph nodes. Liver involvement occurs in many cases with mildly elevated liver enzymes. However, it is rare to see EBV infection present as cholestatic hepatitis. Another rare complication of primary EBV infection is hemophagocytic lymphohistiocytosis (HLH). We describe a patient with primary EBV infection who presented with fatigue and jaundice, subsequent rash, and reactive lymphocytosis. The patient was noted to have cholestatic hepatitis and was highly suspected to have HLH based on laboratory values, including elevated ferritin, triglyceride, and interleukin-2 levels. He showed clinical improvement with HLH treatment using dexamethasone, etoposide, and rituximab. We further review the clinical manifestations, pathogenesis, and management of EBV-associated cholestatic hepatitis and EBV-HLH. Early diagnosis of primary EBV infection is emphasized in order to properly recognize and treat potentially life-threatening complications.

A randomized, double-blind, placebo-controlled, hybrid parallel-arm study of low-dose naltrexone as an adjunctive anti-inflammatory treatment for major depressive disorder.

BACKGROUND: Major depressive disorder (MDD) is a leading cause of disability worldwide. The current treatments are ineffective in approximately one-third of patients, resulting in a large economic burden and reduced quality of life for a significant proportion of the global population. There is considerable evidence that increased inflammation may distinguish a sub-type of MDD, and there are no validated diagnostic tools or treatments for neuroinflammation in MDD patients. The current study aims to explore the potential role of low-dose naltrexone (LDN), a drug with purported anti-inflammatory properties in the central nervous system, as an adjunctive treatment in patients with MDD. METHODS/DESIGN: This double-blind placebo-controlled hybrid parallel arm study enables the exploration of peripheral and central inflammatory markers with LDN as an approach to investigate inflammation as a pathophysiological contributor to MDD. Eligible participants with MDD (n = 48) will be stratified into the high and low inflammatory groups according to the levels of high-sensitivity C-reactive protein (hs-CRP) and then randomized to receive LDN or placebo for an initial 12 weeks, followed by a further 12 weeks during which all participants will receive LDN. The primary outcome measure will be the Montgomery-Åsberg Depression Rating Scale (MADRS) administered at baseline, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 14 weeks, 16 weeks, 20 weeks, and 24 weeks, to assess the effectiveness of the anti-depressant response. The secondary outcomes include the use of MRI techniques including quantitative magnetization transfer (qMT), echo-planar spectroscopic imaging (EPSI), and diffusion-weighted imaging (DWI) to help to elucidate the neurobiological mechanism of LDN, and the inflammatory mechanisms in action in MDD. Electroencephalography, blood samples, cognitive tasks, and additional questionnaires will also be used to determine if there is a specific profile of symptoms in individuals with inflammatory MDD. Healthy participants (n = 24) will be recruited for baseline outcome measures only, to enable comparison with patients with MDD. DISCUSSION: This trial contributes to the literature on inflammation in MDD, including the understanding of the pathophysiology and efficacy of anti-inflammatory treatments. The investigation of inflammatory mechanisms in MDD is an important first step in the development of biomarkers to classify patient sub-groups, increase the accuracy of diagnosis, and tailor the approach to patients in clinical practice. This study may provide evidence of the benefit of LDN for the groups in whom conventional anti-depressants are ineffective and lead the way for translation into clinical practice. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN12622000881730 . Registered on 21 June 2022.

Active conductive head cooling of normal and infarcted brain: A magnetic resonance spectroscopy imaging study.

Active conductive head cooling is a simple and non-invasive intervention that may slow infarct growth in ischemic stroke. We investigated the effect of active conductive head cooling on brain temperature using whole brain echo-planar spectroscopic imaging. A cooling cap (WElkins Temperature Regulation System, 2nd Gen) was used to administer cooling for 80 minutes to healthy volunteers and chronic stroke patients. Whole brain echo-planar spectroscopic imaging scans were obtained before and after cooling. Brain temperature was estimated using the Metabolite Imaging and Data Analysis System software package, which allows voxel-level temperature calculations using the chemical shift difference between metabolite (N-acetylaspartate, creatine, choline) and water resonances. Eleven participants (six healthy volunteers, five post-stroke) underwent 80 ± 5 minutes of cooling. The average temperature of the coolant was 1.3 ± 0.5°C below zero. Significant reductions in brain temperature (ΔT = -0.9 ± 0.7°C, P = 0.002), and to a lesser extent, rectal temperature (ΔT = -0.3 ± 0.1°C, P = 0.03) were observed. Exploratory analysis showed that the occipital lobes had the greatest reduction in temperature (ΔT = -1.5 ± 1.2°C, P = 0.002). Regions of infarction had similar temperature reductions to the contralateral normal brain. Future research could investigate the feasibility of head cooling as a potential neuroprotective strategy in patients being considered for acute stroke therapies.

Brain temperature as an indicator of neuroinflammation induced by typhoid vaccine: Assessment using whole-brain magnetic resonance spectroscopy in a randomised crossover study.

Prior studies indicate a pathogenic role of neuroinflammation in psychiatric disorders; however, there are no accepted methods that can reliably measure low-level neuroinflammation non-invasively in these individuals. Magnetic resonance spectroscopic imaging (MRSI) is a versatile, non-invasive neuroimaging technique that demonstrates sensitivity to brain inflammation. MRSI in conjunction with echo-planar spectroscopic imaging (EPSI) measures brain metabolites to derive whole-brain and regional brain temperatures, which may increase in neuroinflammation. The validity of MRSI/EPSI for measurement of low level neuroinflammation was tested using a safe experimental model of human brain inflammation - intramuscular administration of typhoid vaccine. Twenty healthy volunteers participated in a double-blind, placebo-controlled crossover study including MRSI/EPSI scans before and 3 h after vaccine/placebo administration. Body temperature and mood, assessed using the Profile of Mood States, were measured every hour up to four hours post-treatment administration. A mixed model analysis of variance was used to test for treatment effects. A significant proportion of brain regions (44/47) increased in temperature post-vaccine compared to post-placebo (p < 0.0001). For temperature change in the brain as a whole, there was no significant treatment effect. Significant associations were seen between mood scores assessed at 4 h and whole brain and regional temperatures post-treatment. Findings indicate that regional brain temperature may be a more sensitive measure of low-level neuroinflammation than whole-brain temperature. Future work where these measurement techniques are applied to populations with psychiatric disorders would be of clinical interest.

A national study of the mental health literacy of community pharmacists.

BACKGROUND: Community pharmacists are in a prime position to communicate with and assist those with mental health needs. However, mental health literacy, which includes beliefs and knowledge of mental health conditions, can impact the provision of pharmacy services. The mental health literacy of community pharmacists in New Zealand is currently unknown. OBJECTIVES: To assess the mental health literacy of community pharmacists in New Zealand. METHODS: We employed a national cross-sectional online survey, evaluating attitudes towards mental illness, ability to recognise depression using a vignette and followed by questions related to the helpfulness of various interventions, and willingness to provide pharmacy services for people with mental illness in comparison to cardiovascular diseases. Additionally, opportunities for mental health training were explored. Participants were community pharmacists working in New Zealand contacted via mailing lists of professional bodies. RESULTS: We received responses from 346 participants. The majority of participants showed positive attitudes towards mental illness and correctly identified depression in the vignette (87%). Participants rated counsellors (84%) and physical activity (92%) as the most helpful professionals and intervention respectively while only 43% considered antidepressants as helpful for depression. When compared to other people in the community, long-term functioning of the individual described in the vignette was rated poorly, especially in terms of increased likelihood to attempt suicide (85%) and reduced likelihood to be a productive worker (64%). Approximately 30% of participants reported reduced confidence/comfort while approximately half of participants reported greater interest in providing mental health-related care compared to cardiovascular disease. The participants also highlighted several areas for future mental health training they wished to undertake. CONCLUSIONS: We have identified positive attitudes towards mental illness in our study. Participants correctly identified and supported evidence-based interventions for mild to moderate depression. However, we highlighted the need for ongoing mental health training to address knowledge gaps and enhance the confidence in providing mental health-related care.

Investigating whole-brain metabolite abnormalities in the chronic stages of moderate or severe traumatic brain injury.

BACKGROUND: Evidence suggests that neurometabolic abnormalities can persist after traumatic brain injury (TBI) and drive clinical symptoms such as fatigue and cognitive disruption. Magnetic resonance spectroscopy has been used to investigate metabolite abnormalities following TBI, but few studies have obtained data beyond the subacute stage or over large brain regions. OBJECTIVE: To measure whole-brain metabolites in chronic stages of TBI. DESIGN: Observational study. SETTING: University. PARTICIPANTS: Eleven men with a moderate or severe TBI more than 12 months prior and 10 age-matched healthy controls completed whole-brain spectroscopic imaging. MAIN MEASURES: Ratios of N-acetylaspartate (NAA), choline (CHO), and myo-inositol (MI) to creatine (CR) were measured in whole-brain gray and white matter as well as 64 brain regions of interest. Arterial spin labeling (ASL) data were also collected to investigate whether metabolite abnormalities were accompanied by differences in cerebral perfusion. RESULTS: There were no differences in metabolite ratios within whole-brain gray and white matter regions of interest (ROIs). Linear regression showed lower NAA/CR in the white matter of the left occipital lobe but higher NAA/CR in the gray matter of the left parietal lobe. Metabolite abnormalities were observed in several brain regions in the TBI group including the corpus callosum, putamen, and posterior cingulate. However, none of the findings survived correction for multiple comparison. There were no differences in cerebral blood flow between patients and controls. CONCLUSION: Higher MI/CR may indicate ongoing gliosis, and it has been suggested that low CHO/CR at chronic time points may indicate cell death or lack of healthy turnover and repair. However, with the small sample size of this study, we caution against the over interpretation of our results. None of the findings within ROIs survived correction for multiple comparison. Thus, they may be considered possible avenues for future research in this area.

Kidney Autotransplantation for Treatment of Ureteric Obstruction: A Case Report and Brief Review of the Literature.

Autologous kidney transplantation is a relatively rare procedure that has been used as an alternative treatment for a variety of complex genitourinary problems, in particular for the treatment of complex proximal ureteral strictures. In this case report, a 47-year-old male, who had undergone a living donor nephrectomy 14 years earlier, presented with episodes of acute kidney injury on chronic kidney disease. He was found to have a complex proximal ureter stricture of his solitary right kidney. He underwent nephrectomy with subsequent autotransplantation of the kidney into the right iliac fossa. His renal function improved significantly after surgery. Renal autotransplantation may be considered for the management of proximal ureteral obstruction when alternative options are contraindicated.

Evaluating the optimization of the use of sedating antihistamines in a community pharmacy: a retrospective quality improvement study in Singapore.

OBJECTIVES: This study aimed to retrospectively assess whether community pharmacy customers accepted the pharmacist's recommendations for the selection of an antihistamine based on medicine optimization guidelines. METHODS: A retrospective study was conducted on the implementation of an antihistamine use optimization guide for patients who were seeking first-generation antihistamines between July and December 2019 across forty-five community pharmacies in Singapore. The primary outcome measure was the acceptance rate of ceasing or substituting first-generation antihistamine with a second-generation antihistamine. Secondary measures included the reduction in types of first-generation antihistamines used, adverse drug-related events reported, intended use of antihistamines and the types of recommendations. KEY FINDINGS: During the study period, 2328 patients fulfilled the inclusion criteria, out of which 523 patients agreed to optimize their use of sedating first-generation antihistamines. Chlorpheniramine (95.2%) was the most widely accepted first-generation antihistamine for optimization, with 59.6% of the users experiencing adverse events, the most common being drowsiness (53.2%). The main indication of use was allergic conditions (allergic rhinitis or atopic dermatitis) (70.3%). After implementation of the guide, most interventions were direct substitution (72.8%) with a less-sedating antihistamine, followed by gradual tapering (22.6%). Loratadine, a second-generation antihistamine, was most frequently (51.4%) used to substitute first-generation antihistamines. The optimization guide can potentially reduce adverse effects in 59.6% (297 patients) of chlorpheniramine users, which were mainly drowsiness (265 patients; 53.2%) and dry mouth (14 patients; 2.8%). CONCLUSIONS: This study highlighted the importance of assessing and reducing potentially inappropriate first-generation antihistamine self-use and that a guided approach and substitution with less-sedating antihistamines can be employed in the community pharmacy setting.

A structure-based engineering approach to abrogate pre-existing antibody binding to biotherapeutics.

Development of biotherapeutics is hampered by the inherent risk of immunogenicity, which requires extensive clinical assessment and possible re-engineering efforts for mitigation. The focus in the pre-clinical phase is to determine the likelihood of developing treatment-emergent anti-drug antibodies (TE-ADA) and presence of pre-existing ADA in drug-naïve individuals as risk-profiling strategies. Pre-existing ADAs are routinely identified during clinical immunogenicity assessment, but their origin and impact on drug safety and efficacy have not been fully elucidated. One specific class of pre-existing ADAs has been described, which targets neoepitopes of antibody fragments, including Fabs, VH, or VHH domains in isolation from their IgG context. With the increasing number of antibody fragments and other small binding scaffolds entering the clinic, a widely applicable method to mitigate pre-existing reactivity against these molecules is desirable. Here is described a structure-based engineering approach to abrogate pre-existing ADA reactivity to the C-terminal neoepitope of VH(H)s. On the basis of 3D structures, small modifications applicable to any VH(H) are devised that would not impact developability or antigen binding. In-silico B cell epitope mapping algorithms were used to rank the modified VHH variants by antigenicity; however, the limited discriminating capacity of the computational methods prompted an experimental evaluation of the engineered molecules. The results identified numerous modifications capable of reducing pre-existing ADA binding. The most efficient consisted of the addition of two proline residues at the VHH C-terminus, which led to no detectable pre-existing ADA reactivity while maintaining favorable developability characteristics. The method described, and the modifications identified thereby, may provide a broadly applicable solution to mitigate immunogenicity risk of antibody-fragments in the clinic and increase safety and efficacy of this promising new class of biotherapeutics.

A Placebo-Controlled, Pseudo-Randomized, Crossover Trial of Botanical Agents for Gulf War Illness: Curcumin (Curcuma longa), Boswellia (Boswellia serrata), and French Maritime Pine Bark (Pinus pinaster).

This report is part of a larger study designed to rapidly and efficiently screen potential treatments for Gulf War Illness (GWI) by testing nine different botanicals. In this placebo-controlled, pseudo-randomized, crossover clinical trial of 20 men with GWI, we tested three botanical agents with putative peripheral and central anti-inflammatory actions: curcumin (Curcuma longa), boswellia (Boswellia serrata), and French maritime pine bark extract (Pinus pinaster). Participants completed 30 +/- 3 days of baseline symptom reports, followed by 30 +/- 3 days of placebo, 30 +/- 3 days of lower-dose botanical, and 30 +/- 3 days of higher-dose botanical. Participants then repeated the process with a new botanical until completing up to three botanical cycles. Data were analyzed using linear mixed models. Curcumin reduced GWI symptom severity significantly more than placebo at both the lower (p < 0.0001) and higher (p = 0.0003) dosages. Boswellia was not more effective than placebo at reducing GWI symptoms at either the lower (p = 0.726) or higher (p = 0.869) dosages. Maritime pine was not more effective than placebo at the lower dosage (p = 0.954) but was more effective than placebo at the higher dosage (p = 0.006). This study provides preliminary evidence that curcumin and maritime pine may help alleviate symptoms of GWI. As a screening study, a final determination of the efficacy of these compounds for all individuals with GWI cannot be made, and further studies will need to be conducted to determine strength and durability of effects, as well as optimal dosage. These results suggest that GWI may, at least in part, involve systemic inflammatory processes. This trial was registered on ClinicalTrials.gov (NCT02909686) on 13 September 2016.

A Placebo-Controlled, Pseudo-Randomized, Crossover Trial of Botanical Agents for Gulf War Illness: Resveratrol (Polygonum cuspidatum), Luteolin, and Fisetin (Rhus succedanea).

A chronic multi-symptom illness of unknown etiology, Gulf War Illness (GWI) affects 175,000 to 250,000 veterans of the Gulf War. Because inflammation has suspected involvement in the pathophysiology of GWI, botanical treatments that target inflammation may be beneficial in reducing symptoms. No FDA-approved treatments currently exist for GWI, and rapid prioritization of agents for future efficacy testing is important. This study is part of a larger project that screened nine different botanical compounds with purported anti-inflammatory properties for potential treatment of GWI. We tested three botanicals (resveratrol [Polygonum cuspidatum], luteolin, and fisetin [Rhus succedanea]) on symptom severity of GWI in this placebo-controlled, pseudo-randomized clinical trial. Twenty-one male veterans with GWI completed the study protocol, which consisted of 1 month (30 days ± 3) of baseline symptom reports, 1 month of placebo, 1 month of lower-dose botanical, and 1 month of higher-dose botanical. Participants completed up to 3 different botanicals, repeating the placebo, lower-dose, and higher-dose cycle for each botanical assigned. Linear mixed models were used for analyses. Resveratrol reduced GWI symptom severity significantly more than placebo at both the lower (p = 0.035) and higher (p = 0.004) dosages. Luteolin did not decrease symptom severity more than placebo at either the lower (p = 0.718) or higher dosages (p = 0.492). Similarly, fisetin did not reduce symptom severity at either the lower (p = 0.504) or higher (p = 0.616) dosages. Preliminary findings from this screening study suggest that resveratrol may be beneficial in reducing symptoms of GWI and should be prioritized for future testing. Larger trials are required to determine efficacy, response rates, durability of effects, safety, and optimal dosage. This trial was registered on ClinicalTrials.gov (NCT02909686) on 13 September 2016.

Binding affinity determination of therapeutic antibodies to membrane protein targets: Kinetic Exclusion Assay using cellular membranes for anti-CD20 antibody.

Antibody-based therapeutics targeting membrane proteins have evolved as a major modality for the treatment of cancer, inflammation and autoimmune diseases. There are numerous challenges, ranging from desired epitope expression to reliable binding/functional assays which are associated with developing antibodies for this target class. Specifically, having a robust methodology for characterizing antibody interaction with a membrane protein target is essential for providing guidance on dosing, potency and thus expected efficacy. Fluorescence-activated cell sorting (FACS) has been commonly used to characterize antibodies binding to membrane protein targets. FACS provides information about the antibody-receptor complex (antibody bound to cells) and the apparent equilibrium dissociation constant (KD') is elucidated by fitting the antibody-receptor binding isotherm as a function of total antibody concentration to a nonlinear regression model. Conversely, Kinetic Exclusion Assay (KinExA) has been used to measure solution-based equilibrium dissociation constant (KD) of antibodies. Here, KD is determined by measuring the free antibody concentration at equilibrium in a series of solutions in which the antibody is at constant concentration and the receptor (either in the membrane or the cell) is titrated. We measured the binding affinity of the anti-CD20 antibody, Rituximab, using both FACS and KinExA. There was ~25-fold difference in the binding affinity measured by these two techniques. We have explored this discrepancy through additional experiments around the mathematical framework involved in the analysis of these two different binding assays. Finally, our study concluded that KinExA enables accurate measurement of the KD for strong protein-protein interactions (sub-nanomolar values) compared to FACS.

Development of a FRET-Based Assay for Analysis of mAbs Internalization and Processing by Dendritic Cells in Preclinical Immunogenicity Risk Assessment.

Treatment-emergent antidrug antibodies (TE-ADA) pose a major challenge to the development of biotherapeutics. The antidrug antibody responses are highly orchestrated and involve many types of immune cells and biological processes. Biological drug internalization and processing by antigen-presenting cells (APCs) are two initial and critical steps in the cascade of events leading to T cell-dependent ADA production. The assays thus far described in literature to evaluate immunogenicity potential/risk as a function of APC activity mainly focus on internalization of labeled drug candidates in vitro. Herein, we describe a high-throughput Förster Resonance Energy Transfer (FRET)-based assay for assessing both internalization and processing using CD14+ monocyte-derived dendritic cells (DCs) as APCs. Antigen-binding fragment F(ab')2 against IgG fragment crystallizable gamma (Fcγ) was labeled with the activatable FRET pair TAMRA-QSY7 as a universal probe for antibodies and proteins with a fragment crystallizable (Fc) domain. The assay was qualified using six mAbs of known clinical immunogenicity and one IgG1 isotype antibody using Design of Experiment (DoE). Correlation analysis of internalization and clinical immunogenicity data showed that this FRET-based internalization assay was able to detect clinically immunogenic antibodies. This method provides a tool for analyzing/screening the immunogenicity risk of biological candidates by assessing one of the critical components of the ADA formation process within the broader context of an immunogenicity risk assessment strategy.