Neutrophil serine proteases in cystic fibrosis: role in disease pathogenesis and rationale as a therapeutic target.

Chronic airway inflammation is a central feature in the pathogenesis of bronchiectasis (BE), which can be caused by cystic fibrosis (CFBE; hereafter referred to as CF lung disease) and non-CF-related conditions (NCFBE). Inflammation in both CF lung disease and NCFBE is predominantly driven by neutrophils, which release proinflammatory cytokines and granule proteins, including neutrophil serine proteases (NSPs). NSPs include neutrophil elastase, proteinase 3 and cathepsin G. An imbalance between NSPs and their antiproteases has been observed in people with CF lung disease and people with NCFBE. While the role of the protease/antiprotease imbalance is well established in both CF lung disease and NCFBE, effective therapies targeting NSPs are lacking. In recent years, the introduction of CF transmembrane conductance regulator (CFTR) modulator therapy has immensely improved outcomes in many people with CF (pwCF). Despite this, evidence suggests that airway inflammation persists, even in pwCF treated with CFTR modulator therapy. In this review, we summarise current data on neutrophilic inflammation in CF lung disease to assess whether neutrophilic inflammation and high, uncontrolled NSP levels play similar roles in CF lung disease and in NCFBE. We discuss similarities between the neutrophilic inflammatory profiles of people with CF lung disease and NCFBE, potentially supporting a similar therapeutic approach. Additionally, we present evidence suggesting that neutrophilic inflammation persists in pwCF treated with CFTR modulator therapy, at levels similar to those in people with NCFBE. Collectively, these findings highlight the ongoing need for new treatment strategies targeting neutrophilic inflammation in CF lung disease.

Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of ERM Phosphorylation.

Astrocytes, a major glial cell type of the brain, regulate synapse numbers and function. However, whether astrocyte dysfunction can cause synaptic pathologies in neurological disorders such as Parkinson's Disease (PD) is unknown. Here, we investigated the impact of the most common PD-linked mutation in the leucine-rich repeat kinase 2 (LRRK2) gene (G2019S) on the synaptic functions of astrocytes. We found that both in human and mouse cortex, the LRRK2 G2019S mutation causes astrocyte morphology deficits and enhances the phosphorylation of the ERM proteins (Ezrin, Radixin, and Moesin), which are important components of perisynaptic astrocyte processes. Reducing ERM phosphorylation in LRRK2 G2019S mouse astrocytes restored astrocyte morphology and corrected excitatory synaptic deficits. Using an in vivo BioID proteomic approach, we found Ezrin, the most abundant astrocytic ERM protein, interacts with the Autophagy-Related 7 (Atg7), a master regulator of catabolic processes. The Ezrin/Atg7 interaction is inhibited by Ezrin phosphorylation, thus diminished in the LRRK2 G2019S astrocytes. Importantly, Atg7 function is required to maintain proper astrocyte morphology. These studies reveal an astrocytic molecular mechanism that could serve as a therapeutic target in PD.

Comparative transcriptomics of broad-spectrum and synthetic cannabidiol treated C2C12 skeletal myotubes.

Cannabidiol (CBD) is widely used in sports for recovery, pain management, and sleep improvement, yet its effects on muscle are not well understood. This study aimed to determine the transcriptional response of murine skeletal muscle myotubes to broad-spectrum CBD and synthetic CBD (sCBD). Differentiated C2C12 myotubes were treated with 10 µM CBD, sCBD, or vehicle control (DMSO) for 24 h before RNA extraction. Poly-A tail-enriched mRNA libraries were constructed and sequenced using 2 × 50 bp paired-end sequencing. CBD and sCBD treatment induced 4489 and 1979 differentially expressed genes (DEGs; p < 0.001, FDR step-up <0.05), respectively, with common upregulation of 857 genes and common downregulation of 648 genes. Common upregulated DEGs were associated with "response to unfolded protein," "cell redox homeostasis," "endoplasmic reticulum stress," "oxidative stress," and "cellular response to hypoxia." Common downregulated DEGs were linked to "sarcomere organization," "skeletal muscle tissue development," "regulation of muscle contraction," and "muscle contraction." CBD treatment induced unique DEGs compared to sCBD. The data indicate CBD may induce mild cellular stress, activating pathways associated with altered redox balance, unfolded protein response, and endoplasmic reticulum stress. We hypothesize that CBD interacts with muscle and may elicit a "mitohormetic" effect that warrants further investigation.

Real world analysis of treatment change and response in adults with attention-deficit/hyperactivity disorder (ADHD) alone and with concomitant psychiatric comorbidities: results from an electronic health record database study is the United States.

BACKGROUND: The objectives of this study were to examine the association of psychiatric comorbidities and patient characteristics with treatment change and response as well as to assess the association between treatment change and healthcare resource utilization (HCRU) among adult patients with attention-deficit/hyperactivity disorder (ADHD) and psychiatric comorbidities. METHODS: De-identified electronic health records from the NeuroBlu Database (2002-2021) were used to select patients ≥ 18 years with ADHD who were prescribed ADHD-specific medication. The index date was set as the first prescription of ADHD medication. The outcomes were treatment change (discontinuation, switch, add-on, or drop) and HCRU (inpatient, outpatient, composite) within 12 months of follow-up. Cox proportional-hazard model was used to assess the association between clinical and demographic patient characteristics and treatment change, while generalized linear model with negative binomial distribution and log link function was used to assess the association between key risk factors linked to treatment change and HCRU rates. RESULTS: A total of 3,387 patients with ADHD were included (ADHD only: 1,261; ADHD + major depressive disorder (MDD): 755; ADHD + anxiety disorder: 467; ADHD + mood disorder: 164). Nearly half (44.8%) of the study cohort experienced a treatment change within the 12-month follow-up period. Treatment switch and add-on were more common in patients with ADHD and comorbid MDD and anxiety disorder (switch: 18.9%; add-on: 20.5%) compared to other cohorts (range for switch: 8.5-13.6%; range for add-on: 8.9-12.1%) Survival analysis demonstrated that the probability of treatment change within 12 months from treatment initiation in the study cohort was estimated to be 42.4%. Outpatient visit rates statistically significantly increased from baseline (mean [SD] 1.03 [1.84] visits/month) to 3 months post-index (mean [SD] 1.62 [1.91] visits/month; p < 0.001), followed by a gradual decline up to 12 months post-index. Being prescribed both a stimulant and a non-stimulant at index date was statistically significantly associated with increased risk of treatment change (adjusted hazard ratio: 1.64; 95% CI: 1.13, 2.38; p = 0.01). CONCLUSIONS: This real-world study found that treatment change was common among patients with ADHD and psychiatric comorbidities. These findings support the need for future studies to examine the unmet medical and treatment needs of this complex patient population.

Omalizumab Implementation in Practice: Lessons Learned from the OUtMATCH Study.

In February 2024, omalizumab was approved by the Food and Drug Administration for the treatment of food allergy, based on data from the landmark Phase 3 clinical trial "Omalizumab as Monotherapy and as Adjunct Therapy in Children and Adults (OUtMATCH)." In this Rostrum, OUtMATCH investigators share their perspectives on the trial results, the implications for translation into daily practice, and on remaining gaps in the field. The study met its primary and key secondary endpoints, demonstrating a large effect size in multi-allergen desensitization compared to placebo; yet there were some participants who did not respond, and the percentage of responders tolerating all 3 food allergens was lower than that for single foods. . Clinicians are likely to have many questions about appropriate patient selection, monitoring for treatment responsiveness, and how to manage off-label considerations such as dietary incorporation or co-treatment with oral immunotherapy. Additional research is needed to answer these remaining questions and ensure that the translation of omalizumab in real-world practice leads to high-quality outcomes.

The Role of Food Allergy in Atopic Dermatitis.

Atopic dermatitis (AD) and food allergies are 2 atopic conditions that tend to develop early in life. Their interrelationship has been a topic of controversy and many studies. The presence of atopic dermatitis in infancy and early childhood, particularly if severe, is a risk factor for the development of immunoglobulin E (IgE) -mediated food allergies. While it is common for children with AD to demonstrate extensive sensitization to foods, serum IgE testing is not always indicative of clinical allergy.

Timing of Food Introduction and Allergy Prevention: An Update.

Recommendations about allergy prevention through diet are rapidly changing. In just the past several years, multiple organizations have provided updated guidance and recommendations about infant feeding based on recent studies and meta-analyses. In addition to the increased number of studies supporting the benefit of early introduction of allergenic foods, in particular peanut and egg, recent studies demonstrate that infant and maternal diet diversity may also reduce risk of food allergy and atopy. Skin emollients have not been found to be helpful in prevention of food allergy, and more evidence is needed to determine if emollients play a role in prevention of atopic dermatitis.

In silico mediated workflow for rapid development of downstream processing: Orthogonal product-related impurity removal for a Fc-containing therapeutic.

Therapeutic formats derived from the monoclonal antibody structure have been gaining significant traction in the biopharmaceutical market. Being structurally similar to mAbs, most Fc-containing therapeutics exhibit product-related impurities in the form of aggregates, charge variants, fragments, and glycoforms, which are inherently challenging to remove. In this work, we developed a workflow that employed rapid resin screening in conjunction with an in silico tool to identify and rank orthogonally selective processes for the removal of product-related impurities from a Fc-containing therapeutic product. Linear salt gradient screens were performed at various pH conditions on a set of ion-exchange, multimodal ion-exchange, and hydrophobic interaction resins. Select fractions from the screening experiments were analyzed by three different analytical techniques to characterize aggregates, charge variants, fragments, and glycoforms. The retention database generated by the resin screens and subsequent impurity characterization were then processed by an in silico tool that generated and ranked all possible two-step resin sequences for the removal of product-related impurities. A highly-ranked process was then evaluated and refined at the bench-scale to develop a completely flowthrough two-step polishing process which resulted in complete removal of the Man5 glycoform and aggregate impurities with a 73% overall yield. The successful implementation of the in silico mediated workflow suggests the possibility of a platformable workflow that could facilitate polishing process development for a wide variety of mAb-based therapeutics.

HYPERACUTE CUTIBACTERIUM ACNES ENDOPHTHALMITIS AFTER CATARACT SURGERY.

PURPOSE: Postoperative endophthalmitis is a relatively uncommon, but potentially visually devastating, complication associated with cataract surgery. Specific microbial causes of endophthalmitis are characteristically associated with particular disease time courses. Although Cutibacterium acnes is typically associated with an indolent course of inflammation, we report a case of C. acnes endophthalmitis with onset on postoperative day (POD) 1 and a positive culture from POD 2. METHODS: This is a case report. RESULTS: A 56-year-old man underwent cataract extraction and posterior chamber intraocular lens placement in his left eye. On POD 1, he presented with severe discomfort, reduced visual acuity, and significant inflammation. On POD 2, his anterior chamber was tapped and injected with broad-spectrum antibiotics and steroids. The inflammation ultimately resolved, and his visual acuity improved to 20/20. CONCLUSION: C. acnes is a rare cause of hyperacute-onset postoperative endophthalmitis. Maintaining a high clinical suspicion and initiating prompt treatment can help to optimize long-term visual outcomes.

Behavioral characteristics of toddlers later identified with an autism diagnosis, ADHD symptoms, or combined autism and ADHD symptoms.

BACKGROUND: Autism commonly co-occurs with attention-deficit/hyperactivity disorder (ADHD), but less is known regarding how ADHD symptoms impact the early presentation of autism. This study examined early behavioral characteristics of a community sample of toddlers later identified with autism diagnosis, ADHD symptoms, combined autism and ADHD symptoms, or neither condition. METHODS: Participants were 506 toddlers who were part of a longitudinal study of children's behavioral development. Parents completed questionnaires about their children's behavior at two time points. Four groups were identified based on study measures or medical record: autism diagnosis (n = 45), elevated ADHD symptoms (n = 70), autism and ADHD symptoms (n = 30), or neurotypical development (n = 361). Relationships between early parent report of autism- and ADHD-related behaviors, social-emotional and behavioral functioning, and caregiver experience and subsequent group designation were evaluated with adjusted linear regression models controlling for sex. RESULTS: Significant group differences were found in measures of autism-related behaviors, ADHD-related behaviors, externalizing and internalizing behaviors, and parent support needs (p < .0001). Pairwise comparisons indicated toddlers later identified with combined autism diagnosis and ADHD symptoms had higher levels of autism-related behaviors, externalizing and internalizing behaviors, and autism-related parent support needs compared to the other groups. Toddlers with subsequent elevated ADHD symptoms or combined autism diagnosis and ADHD symptoms exhibited similar levels of ADHD-related behaviors, while both groups displayed more ADHD-related behaviors than toddlers subsequently identified with autism or those with neither condition. CONCLUSIONS: In this community sample, toddlers for whom combined autism diagnosis and ADHD symptoms were subsequently identified showed a distinct presentation characterized by higher early autism-related behaviors, broader behavioral concerns, and higher parent support needs. Presence of ADHD symptoms (alone or in combination with autism) was associated with higher parent-reported ADHD-related behaviors during toddlerhood. Results indicate that ADHD-related behaviors are manifest by toddlerhood, supporting screening for both autism and ADHD during early childhood.

Proximity analysis of native proteomes reveals phenotypic modifiers in a mouse model of autism and related neurodevelopmental conditions.

One of the main drivers of autism spectrum disorder is risk alleles within hundreds of genes, which may interact within shared but unknown protein complexes. Here we develop a scalable genome-editing-mediated approach to target 14 high-confidence autism risk genes within the mouse brain for proximity-based endogenous proteomics, achieving the identification of high-specificity spatial proteomes. The resulting native proximity proteomes are enriched for human genes dysregulated in the brain of autistic individuals, and reveal proximity interactions between proteins from high-confidence risk genes with those of lower-confidence that may provide new avenues to prioritize genetic risk. Importantly, the datasets are enriched for shared cellular functions and genetic interactions that may underlie the condition. We test this notion by spatial proteomics and CRISPR-based regulation of expression in two autism models, demonstrating functional interactions that modulate mechanisms of their dysregulation. Together, these results reveal native proteome networks in vivo relevant to autism, providing new inroads for understanding and manipulating the cellular drivers underpinning its etiology.

Outcomes of Deep Hypothermic Circulatory Arrest for Descending and Thoracoabdominal Aneurysm Repair.

BACKGROUND: Deep hypothermic circulatory arrest (DHCA) in patients undergoing descending thoracic (DTAA) or thoracoabdominal aortic aneurysm (TAAA) repair is associated with increased morbidity and mortality. We present our outcomes after open DTAA and TAAA repair with and without DHCA. METHODS: From 1999 to 2022, 81 (38.8%) patients undergoing DTAA or TAAA repair required DHCA because proximal cross-clamping was not feasible or aneurysmal pathology extended into the arch and 128 (61.2%) patients required only distal bypass. Because of intrinsic pathological differences in patients requiring DHCA, confidence intervals (CIs) were used to compare groups in lieu of formal hypothesis tests. RESULTS: DHCA patients had more chronic dissections (64.2% vs. 43.8%, 95% CI for difference: 6-35%) and higher body mass indices (29.5 ± 6.8 vs. 27.2 ± 6.6, CI: 26-421%). More non-DHCA patients had medial degeneration (9.9% vs. 31.3%, CI: -33 to -7%). There were 10 (12.4%) in-hospital deaths for the DHCA and 10 (7.8%) for the non-DHCA group (CI: -5 to 14%). Survival at 10 years was 52.6% (CI: 42.1-65.7%) for the non-DHCA group and 48.3% (CI: 40.3-57.9%) for the DHCA group. The only meaningful differences in postoperative outcomes were intensive care unit (5.5 days vs. 6 days, CI: 12-410%) and hospital stay (19 days vs. 12 days, CI: 74-470%), which were longer in the DHCA group. CONCLUSIONS: Despite longer intensive care unit and hospital length of stays, selective use of DHCA is safe and effective with comparable morbidity and mortality to non-DHCA in open DTAA and TAAA repair.

Evaluation of Monteggia Fracture Outcomes: Acute to Chronic.

INTRODUCTION: Our study aims to characterize the results of Monteggia fractures treated in our practice and to determine factors associated with good or poor outcomes. METHODS: A retrospective review of children aged 17 and under with acute, subacute, or chronic Monteggia fractures who were treated at our institution was performed. The primary outcomes were initial reduction and maintenance of joint reduction, while the secondary outcomes were elbow flexion/extension and forearm supination/pronation. RESULTS: Seventeen patients with Monteggia fractures were identified. Two patients were excluded: 1 was lost to follow-up and 1 had congenital absence of the elbow flexors. Thus, our final cohort was 15 patients (acute n = 3, subacute n = 4, chronic group n = 8). Median final follow-up was 1.9 years (range = 34 days-8 years). CONCLUSION: Preoperative range of motion (ROM) was the most important factor in determining postoperative ROM in this cohort of patients with chronic Monteggia fractures. All patients who presented with excellent preoperative ROM, regardless of their timing category, had an excellent ROM outcome. Time from initial injury also played an important role. All patients in the acute and subacute categories had good or excellent postoperative ROM. Patients who were further from the initial injury were more likely to present with worse preoperative ROM and, in turn, had worse outcomes with postoperative ROM.

Predicting the Hallucinogenic Potential of Molecules Using Artificial Intelligence.

The development of new drugs addressing serious mental health and other disorders should avoid the psychedelic experience. Analogs of psychedelic drugs can have clinical utility and are termed "psychoplastogens". These represent promising candidates for treating opioid use disorder to reduce drug dependence, with rarely reported serious adverse effects. This drug abuse cessation is linked to the induction of neuritogenesis and increased neuroplasticity, a hallmark of psychedelic molecules, such as lysergic acid diethylamine. Some, but not all psychoplastogens may act through the G-protein coupled receptor (GPCR) 5HT2A whereas others may display very different polypharmacology making prediction of hallucinogenic potential challenging. In the process of developing tools to help design new psychoplastogens, we have used artificial intelligence in the form of machine learning classification models for predicting psychedelic effects using a published in vitro data set from PsychLight (support vector classification (SVC), area under the curve (AUC) 0.74) and in vivo human data derived from books from Shulgin and Shulgin (SVC, AUC, 0.72) with nested five-fold cross validation. We have also explored conformal predictors with ECFP6 and electrostatic descriptors in an effort to optimize them. These models have been used to predict known 5HT2A agonists to assess their potential to act as psychedelics and induce hallucinations for PsychLight (SVC, AUC 0.97) and Shulgin and Shulgin (random forest, AUC 0.71). We have tested these models with head twitch data from the mouse. This predictive capability is desirable to reliably design new psychoplastogens that lack in vivo hallucinogenic potential and help assess existing and future molecules for this potential. These efforts also provide useful insights into understanding the psychedelic structure activity relationship.

Longitudinal Monitoring of the Effects of Anti-Adenoviral Treatment Regimens in a Permissive In Vivo Model.

Adenovirus infections of immunocompromised patients can cause life-threatening disseminated disease. While there are presently no drugs specifically approved to treat these infections, there are several compounds that showed efficacy against adenovirus in preclinical studies. For any such compound, low toxicity is an essential requirement. As cumulative drug effects can accentuate pathology, especially in patients with other morbidities, it is important to limit antiviral exposure to what is absolutely necessary. This is achievable by monitoring the virus burden of the patients and administering antivirals to suppress virus replication to a non-pathogenic level. We modeled such a system using Syrian hamsters infected with a replication-competent adenovirus vector, in which luciferase expression is coupled to virus replication. We found that virus replication could be followed in vivo in the same animal by repeated measurement of luciferase expression. To test the utility of an interrupted treatment regimen, we used NPP-669 and valganciclovir, two antiviral compounds with high and moderate anti-adenoviral efficacy, respectively. We found that short-term treatment of adenovirus-infected hamsters at times of peak virus replication can prevent virus-associated pathology. Thus, we believe that this animal model can be used to model different treatment regimens for anti-adenoviral compounds.

Impact of tendon transfer on scapulothoracic and glenohumeral motion in children with brachial plexus birth injuries.

BACKGROUND: Tendon transfers are often utilized to improve shoulder external rotation and abduction in children with brachial plexus birth injuries and are designed to improve glenohumeral (GH) joint motion. However, changes in scapulothoracic (ST) and glenohumeral (GH) joint function after tendon transfer are not well defined. The purpose of this study was to quantify changes in GH, ST, and humerothoracic (HT) joint function before and after tendon transfer, and we hypothesized that tendon transfers would reorient the arc of motion into more external rotation and abduction, but not increase GH motion. METHODS: Motion analysis was performed in 15 children (ages 3-16) before and after transfer of teres major and/or latissimus dorsi. Scapulothoracic, GH, and HT joint angles were measured in a neutral, resting position and each of the modified Mallet positions. Joint angular displacements from the neutral position and the total arc of internal-external rotation for each joint were also calculated. Relevant joint angles, joint angular displacements, and internal-external rotation arcs were compared using multivariate analyses of variance with repeated measures and univariate post-hoc analyses. RESULTS: Glenohumeral and HT external rotation were significantly increased in all positions postoperatively. The arc of GH internal-external rotation was unchanged, but oriented in more external rotation after surgery. Only 6 patients gained active external rotation. Glenohumeral and HT internal rotation were significantly decreased after surgery, but ST internal rotation was significantly increased. Two patients had loss of midline function. In the abduction position, GH elevation joint angles were unchanged, but ST and HT elevation increased. DISCUSSION: Only four patients gained active GH external rotation and maintained their internal rotation. Each of those patients underwent isolated tendon transfer without concomitant joint release. Seven patients maintained their preoperative internal rotation, which was attributed to increased ST internal rotation. The other half of patients lost internal rotation and gained external rotation through reorientation of the arc of rotation. Nine patients gained HT elevation, with three attributed to increased ST upward rotation, five attributed to a combination of increased ST upward rotation and increased GH elevation, and one attributed to increased GH abduction contracture. These findings challenge the dogma that teres major/latissimus dorsi tendon transfers augment GH motion and highlight the importance of ST function for outcome determination.

De Novo Design of Peptide Binders to Conformationally Diverse Targets with Contrastive Language Modeling.

Designing binders to target undruggable proteins presents a formidable challenge in drug discovery, requiring innovative approaches to overcome the lack of putative binding sites. Recently, generative models have been trained to design binding proteins via three-dimensional structures of target proteins, but as a result, struggle to design binders to disordered or conformationally unstable targets. In this work, we provide a generalizable algorithmic framework to design short, target-binding linear peptides, requiring only the amino acid sequence of the target protein. To do this, we propose a process to generate naturalistic peptide candidates through Gaussian perturbation of the peptidic latent space of the ESM-2 protein language model, and subsequently screen these novel linear sequences for target-selective interaction activity via a CLIP-based contrastive learning architecture. By integrating these generative and discriminative steps, we create a Peptide Prioritization via CLIP (PepPrCLIP) pipeline and validate highly-ranked, target-specific peptides experimentally, both as inhibitory peptides and as fusions to E3 ubiquitin ligase domains, demonstrating functionally potent binding and degradation of conformationally diverse protein targets in vitro. Overall, our design strategy provides a modular toolkit for designing short binding linear peptides to any target protein without the reliance on stable and ordered tertiary structure, enabling generation of programmable modulators to undruggable and disordered proteins such as transcription factors and fusion oncoproteins.