Discovery of PLD4 modulators by high-throughput screening and kinetic analysis.

Phospholipase D3 (PLD3) and D4 (PLD4) are endolysosomal exonucleases of ssDNA and ssRNA that regulate innate immunity. Polymorphisms of these enzymes are correlated with numerous human diseases, including Alzheimer's, rheumatoid arthritis, and systemic sclerosis. Pharmacological modulation of these immunoregulatory proteins may yield novel immunotherapies and adjuvants. A previous study reported a high-throughput screen (N = 17,952) that discovered a PLD3-selective activator and inhibitor, as well as a nonselective inhibitor, but failed to identify selective modulators of PLD4. However, modulators selective for PLD4 are therapeutically pertinent, since recent reports have shown that regulating this protein has direct implications in cancer and autoimmune diseases. Furthermore, the high expression of PLD4 in dendritic and myeloid cells, in comparison to the broader expression of PLD3, presents the opportunity for a cell-targeted immunotherapy. Here, we describe screening of an expended diversity library (N = 45,760) with an improved platform and report the discovery of one inhibitor and three activators selective for PLD4. Furthermore, kinetic modeling and structural analysis suggest mechanistic differences in the modulation of these hits. These findings further establish the utility of this screening platform and provide a set of chemical scaffolds to guide future small-molecule development for this novel immunoregulator target.

Plasma Myokine Profiles in Patients With AChR- and MuSK-Ab-Positive Myasthenia Gravis.

BACKGROUND AND PURPOSE: Myokines include cytokines secreted by muscle fibers, which are the final targets of myasthenia gravis (MG). This pilot study investigated whether myokine plasma concentrations are altered in patients with MG and assessed the association between the concentration of each myokine and disease severity. METHODS: We compared the plasma concentrations of 15 myokines in 63 patients with acetylcholine receptor antibody (Ab)-positive MG and 14 with muscle-specific tyrosine kinase Ab-positive MG (MuSK MG) with those in 15 healthy controls. Plasma myokine concentrations were measured using a Luminex multiplex assay kit with magnetic beads that contained Abs for 15 myokines. Correlations between myokine concentration and clinical scale results were analyzed. RESULTS: The concentration of fractalkine in plasma was higher in MG (median [interquartile range]=419.6 [38.7-732.5] pg/mL) than in controls (158.5 [0.0-313.2] pg/mL, p=0.034). The leukemia inhibitory factor concentration was also found to be higher in MuSK MG (29.9 [8.7-40.1] pg/mL) than in healthy controls (7.6 [0.0-15.6] pg/mL, p=0.013). Fatty-acid-binding protein 3 (FABP3) concentrations in plasma were positively associated with clinical parameters for MG severity, including scores on the Quantitative Myasthenia Gravis score (p=0.008), Myasthenia Gravis Activities of Daily Living (p=0.003), and Myasthenia Gravis Composite (p=0.024) scales. FABP3 concentration in plasma tended to decrease after treatment in patients without additional relapse but increased in those with further relapse. CONCLUSIONS: The plasma myokine profile was significantly altered in patients with MG. FABP3 concentration may be useful in assessing disease severity and predicting the treatment response.

Pure uric acid stone prediction model using the variant coefficient of stone density measured by thresholding 3D segmentation-based methods: A multicenter study.

Urinary stones are common urological diseases with increasing prevalence and incidence worldwide. Among the various types of stones, uric acid stones can be dissolved by oral chemolysis without any surgical procedure. Therefore, our study demonstrates that variant coefficient of stone density measured by thresholding a three-dimensional segmentation-based method from noncontrast computed tomography images can be used to identify pure uric acid stones from non-pure uric acid stones. This study provides a preoperative pure uric acid stone prediction model that could reduce invasive procedural treatments. The pure uric acid stone prediction model may offer optimized clinical decision-making for patients with urinary stones. BACKGROUND AND OBJECTIVES: While most urinary stones are managed with interventional therapy, uric acid (UA) stones can be dissolved by oral chemolysis without invasive procedures. This study aimed to develop and validate a pure UA (pUA) stone prediction model using a variant coefficient of stone density (VCSD) measured by thresholding a three-dimensional (3D) segmentation-based method. METHODS: Patients with urolithiasis treated at Keimyung University Dongsan Hospital between January 2017 and December 2020 were divided into training and internal validation sets, and patients from Kyungpook National University Hospital between January 2017 and December 2018 were used as an external validation set. Each stone was segmented by a thresholding 3D segmentation-based method using an attenuation threshold of 130 Hounsfield units. VCSD was calculated as the stone heterogeneity index divided by the mean stone density. RESULTS: A total of 1175 urinary stone cases in 1023 patients were enrolled in this study. Of these, 224 (19.1%) were pUA stone cases. Among the potential predictors, thresholding 3D segmentation-based VCSD, age, sex, radio-opacity, hypertension, diabetes, and urine pH were identified as independent pUA stone predictors, and VCSD was the most powerful indicator. The pUA stone prediction model showed good discrimination, yielding area under the receiver operating characteristic curve of 0.960 (95% confidence interval (CI): 0.940-0.979, P < 0.001), 0.931 (95% CI: 0.875-0.987, P < 0.001), and 0.938 (95% CI: 0.912-0.965, P < 0.001) in the training, internal validation, and external validation sets, respectively. CONCLUSIONS: VCSD measured using 3D segmentation was a decisive independent predictive factor for pUA stones. Furthermore, the established prediction model with VCSD can serve as a noninvasive preoperative tool to identify pUA stones.

Korean Red Ginseng slows coreceptor switch in HIV-1 infected patients.

Background: Human immunodeficiency virus-1 (HIV-1) that binds to the coreceptor CCR5 (R5 viruses) can evolve into viruses that bind to the coreceptor CXCR4 (X4 viruses), with high viral replication rates governing this coreceptor switch. Korean Red Ginseng (KRG) treatment of HIV-1 infected patients has been found to slow the depletion of CD4+ T cells. This study assessed whether the KRG-associated slow depletion of CD4+ T cells was associated with coreceptor switching. Methods: This study included 146 HIV-1-infected patients naïve to antiretroviral therapy (ART) and seven patients receiving ART. A total of 540 blood samples were obtained from these patients over 122 ± 129 months. Their env genes were amplified by nested PCR or RT-PCR and subjected to direct sequencing. Tropism was determined with a 10% false positive rate (FPR) cutoff. Results: Of the 146 patients naïve to ART, 102 were KRG-naïve, and 44 had been treated with KRG. Evaluation of initial samples showed that coreceptor switch had occurred in 19 patients, later occurring in 38 additional patients. There was a significant correlation between the amount of KRG and FPR. Based on initial samples, the R5 maintenance period was extended 2.35-fold, with the coreceptor switch being delayed 2.42-fold in KRG-treated compared with KRG-naïve patients. The coreceptor switch occurred in 85% of a homogeneous cohort. The proportion of patients who maintained R5 for ≥10 years was significantly higher in long-term slow progressors than in typical progressors. Conclusion: KRG therapy extends R5 maintenance period by increasing FPR, thereby slowing the coreceptor switch.

Impact of HIV-1 subtypes on gross deletion in the nef gene after Korean Red Ginseng treatment.

Background: The number of primary human immunodeficiency virus (HIV)-1 non-B subtype infections (non-B) and that of reports regarding the differences in the pathogenesis of subtype B and non-B infections are increasing. However, to the best of our knowledge, there have been no reports on gross deletion in the nef gene (gΔnef) in non-B infections. Methods: To determine whether there is a difference in the change in CD4+ T cells after treatment with Korean Red Ginseng (KRG) between patients with subtype B and non-B infections, we retrospectively analyzed and compared the annual decrease in CD4+ T cells (AD) and the proportion of gΔnef in 77 patients who were followed for more than 10 years in the absence of combination antiretroviral therapy. Results: Overall, AD was significantly faster in patients with non-B infections than in those with subtype B infections. Survival analysis showed that the survival probability was significantly higher in subtype B than in non B-infected patients. These differences mainly resulted from significant differences in the amount of KRG and age. In the patients treated with KRG, there was a significant correlation between the amount of KRG and the AD in both subtypes. Interestingly, there was a significant correlation between the amount of KRG and the proportion of gΔnef in patients infected with subtype B, but not in those infected with non-B. The same phenomenon was observed when the KRG dose was adjusted. Conclusion: Our results suggest that non-B may be biologically more stable than subtype B.

Development of an Effective Monoclonal Antibody against Heroin and Its Metabolites Reveals Therapies Have Mistargeted 6-Monoacetylmorphine and Morphine over Heroin.

The opioid epidemic is a global public health crisis that has failed to abate with current pharmaceutical treatments. Moreover, these FDA-approved drugs possess numerous problems such as adverse side effects, short half-lives, abuse potential, and recidivism after discontinued use. An alternative treatment model for opioid use disorders is immunopharmacotherapy, where antibodies are produced to inhibit illicit substances by sequestering the drug in the periphery. Immunopharmacotherapeutics against heroin have engaged both active and passive vaccines targeting heroin's metabolites, 6-monoacetylmorphine (6-AM) and morphine, since decades of research have stated that heroin's psychoactive and lethal effects are mainly attributed to these compounds. However, concerted efforts to develop effective immunopharmacotherapies against heroin abuse have faced little clinical advancement, suggesting a need for reassessing drug target selection. To address this issue, four unique monoclonal antibodies were procured with distinct affinity to either heroin, 6-AM, or morphine. Examination of these antibodies through in vitro and in vivo tests revealed monoclonal antibody 11D12 as the optimal therapeutic and provided crucial insights into the key chemical species to target for blunting heroin's psychoactive and lethal effects. These findings offer clarification into the problematic attempts of therapeutics targeting heroin's metabolites and provide a path forward for future heroin immunopharmacotherapy development.

A Vaccine against Benzimidazole-Derived New Psychoactive Substances That Are More Potent Than Fentanyl.

New psychoactive substance (NPS) opioids have proliferated within the international drug market. While synthetic opioids are traditionally composed of fentanyl analogues, benzimidazole-derived isotonitazene and its derivatives are the current NPS opioids of concern. Hence, in this study, we implement immunopharmacotherapy wherein antibodies are produced with high titers and nanomolar affinity to multiple benzimidazole-derived NPS opioids (BNO). Notably, these antibodies blunt psychoactive and physiological repercussions from BNO exposure, which was observed through antinociception, whole-body plethysmography, and blood-brain biodistribution studies. Moreover, we detail previously unreported pharmacokinetics of these drugs, which explains the struggle of traditional pharmaceutical opioid antagonists against BNO substances. These findings provide further insight into the in vivo effects of BNO drugs and the development of effective broad-spectrum therapeutics against NPS opioids.

Development of effective therapeutics for polysubstance use disorders.

Traditional pharmacotherapies for substance use disorders have focused on mono-substance abuse. However, recent epidemiological studies have found polysubstance use disorders (PUD) are becoming more prevalent and the abuse of adulterated drugs has led to increasing unintentional overdose deaths. Unfortunately, there are no approved pharmacological agents for PUD. Hence, a therapeutic model of interest to address this growing epidemic is immunopharmacotherapy, where individuals are inoculated with conjugate vaccines formulated with haptens that mimic the drug of abuse. These conjugate vaccines have demonstrated significant therapeutic potential against mono-substance abuse, thus recent studies have applied this model to address PUD. This review presents immunopharmacotherapeutic advancements against polysubstance abuse and discusses necessary developments for conjugate vaccines in order to effectively treat this unaddressed epidemic.

Broadly Neutralizing Synthetic Cannabinoid Vaccines.

Synthetic cannabinoids (SCs) constitute a significant portion of psychoactive substances forming a major public health risk. Due to the wide variety of SCs, broadly neutralizing antibodies generated by active immunization present an intriguing pathway to combat cannabinoid use disorder. Here, we probed hapten design for antibody affinity and cross reactivity against two classes of SCs. Of the 10 haptens screened, 3 vaccine groups revealed submicromolar IC50, each targeting 5-6 compounds in our panel of 22 drugs. Moreover, SCs were successfully sequestered when administered by vaping or intraperitoneal injection, which was confirmed within animal models by observing locomotion, body temperature, and pharmacokinetics. We also discovered synergistic effects to simultaneously blunt two drug classes through an admixture vaccine approach. Collectively, our study provides a comprehensive foundation for the development of vaccines against SCs.

Immunopharmacotherapeutic advancements in addressing methamphetamine abuse.

Methamphetamine (METH) is an illicit psychostimulant that is known to account for substance abuse disorders globally, second only to opioids, yet has no approved pharmacotherapies. Traditional therapies employ small molecule agonists or antagonists for substance use disorders or overdose reversal by targeting drug-specific receptors in the brain. However, the comprehensive mechanism of METH on multiple sites within the central nervous system (CNS) implies its receptors lack the high affinity and specificity required for an "ideal" drug target. The alternative to pharmacotherapies is to sequester abused drugs in the periphery, effectively eliminating the effects from CNS receptor occupation through pharmacokinetic antagonism. This review presents updates on immunopharmacotherapeutic advancements in addressing methamphetamine abuse by focusing on the cultivation of research optimization strategies regarding hapten chemistry, carrier proteins, and adjuvants implemented in active immunization. Furthermore, we discuss necessary developments for each component of active immunopharmacotherapies and the future of active vaccines in treating METH use disorder.

Improvements on a chemically contiguous hapten for a vaccine to address fentanyl-contaminated heroin.

Unintentional overdose deaths related to opioids and psychostimulants have increased in prevalence due to the adulteration of these drugs with fentanyl. Synergistic effects between illicit compounds and fentanyl cause aggravated respiratory depression, leading to inadvertent fatalities. Traditional small-molecule therapies implemented in the expanding opioid epidemic present numerous problems since they interact with the same opioid receptors in the brain as the abused drugs. In this study, we report an optimized dual hapten for use as an immunopharmacotherapeutic tool in order to develop antibodies capable of binding to fentanyl-contaminated heroin in the periphery, thus impeding the drugs' psychoactive effects on the central nervous system. This vaccine produced antibodies with nanomolar affinities and effectively blocked opioid analgesic effects elicited by adulterated heroin. These findings provide further insight into the development of chemically contiguous haptens for broad-spectrum immunopharmacotherapies against opioid use disorders.

Discovery of Chemicals to Either Clear or Indicate Amyloid Aggregates by Targeting Memory-Impairing Anti-Parallel Aß Dimers.

Amyloid-ß (Aß) oligomers are implicated in Alzheimer disease (AD). However, their unstable nature and heterogeneous state disrupts elucidation of their explicit role in AD progression, impeding the development of tools targeting soluble Aß oligomers. Herein parallel and anti-parallel variants of Aß(1-40) dimers were designed and synthesized, and their pathogenic properties in AD models characterized. Anti-parallel dimers induced cognitive impairments with increased amyloidogenesis and cytotoxicity, and this dimer was then used in a screening platform. Through screening, two FDA-approved drugs, Oxytetracycline and Sunitinib, were identified to dissociate Aß oligomers and plaques to monomers in 5XFAD transgenic mice. In addition, fluorescent Astrophloxine was shown to detect aggregated Aß in brain tissue and cerebrospinal fluid samples of AD mice. This screening platform provides a stable and homogeneous environment for observing Aß interactions with dimer-specific molecules.

Left atrial imaging and registration of fibrosis with conduction voltages using LGE-MRI and electroanatomical mapping.

BACKGROUND AND PURPOSE: Abnormal electrical conduction and excitability associated with fibrosis in the left atrium (LA) may serve as a substrate for atrial fibrillation (AF). Electroanatomical voltage mapping systems (EAMs) have become a dominant facilitator to treat AF with catheter ablation assisted by additional diagnostic imaging modalities. Importantly, AF has been associated with structural changes to the extracellular matrix of the myocardium, including increased collagen deposition-a process known as fibrosis. Late gadolinium enhancement-magnetic resonance imaging (LGE-MRI) may aid in guiding AF cardiac ablation therapy by determination of location of fibrosis in the LA. To locate fibrosis for cardiac ablation, however, accurate registration between EAMs and LGE-MRI data is crucial. The purpose of this work was to develop a method for registering EAMs with late gadolinium enhancement-magnetic resonance (LGE-MR) images of fibrosis. METHODS: Twenty patients with persistent AF, who underwent magnetic resonance imaging scanning and EAMs prior to first-time catheter ablation, participated in the study. In our registration pipeline, LGE-MR images were registered to the left atrial surface on EAMs using manual alignment followed by iterative closest point (ICP), and non-rigid ICP (NICP) algorithm. RESULTS AND CONCLUSIONS: The results demonstrate that NICP provided a substantial reduction in registration error when compared to the use of affine ICP alone. Regions of fibrosis on LGE-MR images identified using the signal threshold to reference mean threshold demonstrated the most regional overlap with low bipolar voltage points on EAMs. Successful co-registration of LGE-MR images to EAMs may assist electro-physiologists in selecting candidate targets for ablation and ultimately, reduce the rate of AF recurrence for patients.

Diagnosis of Alzheimer's disease utilizing amyloid and tau as fluid biomarkers.

Current technological advancements in clinical and research settings have permitted a more intensive and comprehensive understanding of Alzheimer's disease (AD). This development in knowledge regarding AD pathogenesis has been implemented to produce disease-modifying drugs. The potential for accessible and effective therapeutic methods has generated a need for detecting this neurodegenerative disorder during early stages of progression because such remedial effects are more profound when implemented during the initial, prolonged prodromal stages of pathogenesis. The aggregation of amyloid-ß (Aß) and tau isoforms are characteristic of AD; thus, they are considered core candidate biomarkers. However, research attempting to establish the reliability of Aß and tau as biomarkers has culminated in an amalgamation of contradictory results and theories regarding the biomarker concentrations necessary for an accurate diagnosis. In this review, we consider the capabilities and limitations of fluid biomarkers collected from cerebrospinal fluid, blood, and oral, ocular, and olfactory secretions as diagnostic tools for AD, along with the impact of the integration of these biomarkers in clinical settings. Furthermore, the evolution of diagnostic criteria and novel research findings are discussed. This review is a summary and reflection of the ongoing concerted efforts to establish fluid biomarkers as a diagnostic tool and implement them in diagnostic procedures.

Comparative analyses of plasma amyloid-ß levels in heterogeneous and monomerized states by interdigitated microelectrode sensor system.

Detection of amyloid-ß (Aß) aggregates contributes to the diagnosis of Alzheimer disease (AD). Plasma Aß is deemed a less invasive and more accessible hallmark of AD, as Aß can penetrate blood-brain barriers. However, correlations between biofluidic Aß concentrations and AD progression has been tenuous. Here, we introduce a diagnostic technique that compares the heterogeneous and the monomerized states of Aß in plasma. We used a small molecule, EPPS [4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid], to dissociate aggregated Aß into monomers to enhance quantification accuracy. Subsequently, Aß levels of EPPS-treated plasma were compared to those of untreated samples to minimize inter- and intraindividual variations. The interdigitated microelectrode sensor system was used to measure plasma Aß levels on a scale of 0.1 pg/ml. The implementation of this self-standard blood test resulted in substantial distinctions between patients with AD and individuals with normal cognition (NC), with selectivity and sensitivity over 90%.