Strategy for Designing In Vivo Dose-Response Comparison Studies.

In preclinical drug discovery, at the step of lead optimization of a compound, in vivo experimentation can differentiate several compounds in terms of efficacy and potency in a biological system of whole living organisms. For the lead optimization study, it may be desirable to implement a dose-response design so that compound comparisons can be made from nonlinear curves fitted to the data. A dose-response design requires more thought relative to a simpler study design, needing parameters for the number of doses, the dose values, and the sample size per dose. This tutorial illustrates how to calculate statistical power, choose doses, and determine sample size per dose for a comparison of two or more dose-response curves for a future in vivo study.

To Dilute or Not to Dilute: Nominal Titer Dosing for Genetic Medicines.

Recombinant adeno-associated virus (AAV) has become a popular platform for many gene therapy applications. The strength of AAV-based products is a critical quality attribute that affects the efficacy of the drug and is measured as the concentration of vector genomes, or physical titer. Because the dosing of patients is based on the titer measurement, it is critical for manufacturers to ensure that the measured titer of the drug product is close to the actual concentration of the batch. Historically, dosing calculations have been performed using the measured titer, which is reported on the drug product label. However, due to recent regulatory guidance, sponsors are now expected to label the drug product with nominal or "target" titer. This new expectation for gene therapy products can pose a challenge in the presence of process and analytical variability. In particular, the manufacturer must decide if a dilution of the drug substance is warranted at the drug product stage to bring the strength in line with the nominal value. In this paper, we present two straightforward statistical methods to aid the manufacturer in the dilution decision. These approaches use the understanding of process and analytical variability to compute probabilities of achieving the desired drug product titer. We also provide an approach for determining an optimal assay replication strategy for achieving the desired probability of meeting drug product release specifications.

Intertumoral Heterogeneity Based on MRI Radiomic Features Estimates Recurrence in Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) heterogeneity impacts prognosis, and imaging is a potential indicator. PURPOSE: To characterize HCC image subtypes in MRI and correlate subtypes with recurrence. STUDY TYPE: Retrospective. POPULATION: A total of 440 patients (training cohort = 213, internal test cohort = 140, external test cohort = 87) from three centers. FIELD STRENGTH/SEQUENCE: 1.5-T/3.0-T, fast/turbo spin-echo T2-weighted, spin-echo echo-planar diffusion-weighted, contrast-enhanced three-dimensional gradient-recalled-echo T1-weighted with extracellular agents (Gd-DTPA, Gd-DTPA-BMA, and Gd-BOPTA). ASSESSMENT: Three-dimensional volume-of-interest of HCC was contoured on portal venous phase, then coregistered with precontrast and late arterial phases. Subtypes were identified using non-negative matrix factorization by analyzing radiomics features from volume-of-interests, and correlated with recurrence. Clinical (demographic and laboratory data), pathological, and radiologic features were compared across subtypes. Among clinical, radiologic features and subtypes, variables with variance inflation factor above 10 were excluded. Variables (P < 0.10) in univariate Cox regression were included in stepwise multivariate analysis. Three recurrence estimation models were built: clinical-radiologic model, subtype model, hybrid model integrating clinical-radiologic characteristics, and subtypes. STATISTICAL TESTS: Mann-Whitney U test, Kruskal-Wallis H test, chi-square test, Fisher's exact test, Kaplan-Meier curves, log-rank test, concordance index (C-index). Significance level: P < 0.05. RESULTS: Two subtypes were identified across three cohorts (subtype 1:subtype 2 of 86:127, 60:80, and 36:51, respectively). Subtype 1 showed higher microvascular invasion (MVI)-positive rates (53%-57% vs. 26%-31%), and worse recurrence-free survival. Hazard ratio (HR) for the subtype is 6.10 in subtype model. Clinical-radiologic model included alpha-fetoprotein (HR: 3.01), macrovascular invasion (HR: 2.32), nonsmooth tumor margin (HR: 1.81), rim enhancement (HR: 3.13), and intratumoral artery (HR: 2.21). Hybrid model included alpha-fetoprotein (HR: 2.70), nonsmooth tumor margin (HR: 1.51), rim enhancement (HR: 3.25), and subtypes (HR: 5.34). Subtype model was comparable to clinical-radiologic model (C-index: 0.71-0.73 vs. 0.71-0.73), but hybrid model outperformed both (C-index: 0.77-0.79). CONCLUSION: MRI radiomics-based clustering identified two HCC subtypes with distinct MVI status and recurrence-free survival. Hybrid model showed superior capability to estimate recurrence. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 2.

Pre-operative enhanced magnetic resonance imaging combined with clinical features predict early recurrence of hepatocellular carcinoma after radical resection.

BACKGROUND: Indentifying predictive factors for postoperative recurrence of hepatocellular carcinoma (HCC) has great significance for patient prognosis. AIM: To explore the value of gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) enhanced magnetic resonance imaging (MRI) combined with clinical features in predicting early recurrence of HCC after resection. METHODS: A total of 161 patients with pathologically confirmed HCC were enrolled. The patients were divided into early recurrence and non-early recurrence group based on the follow-up results. The clinical, laboratory, pathological results and Gd-EOB-DTPA enhanced MRI imaging features were analyzed. RESULTS: Of 161 patients, 73 had early recurrence and 88 were had non-early recurrence. Univariate analysis showed that patient age, gender, serum alpha-fetoprotein level, the Barcelona Clinic Liver Cancer stage, China liver cancer (CNLC) stage, microvascular invasion (MVI), pathological satellite focus, tumor size, tumor number, tumor boundary, tumor capsule, intratumoral necrosis, portal vein tumor thrombus, large vessel invasion, nonperipheral washout, peritumoral enhancement, hepatobiliary phase (HBP)/tumor signal intensity (SI)/peritumoral SI, HBP peritumoral low signal and peritumoral delay enhancement were significantly associated with early recurrence of HCC after operation. Multivariate logistic regression analysis showed that patient age, MVI, CNLC stage, tumor boundary and large vessel invasion were independent predictive factors. External data validation indicated that the area under the curve of the combined predictors was 0.861, suggesting that multivariate logistic regression was a reasonable predictive model for early recurrence of HCC. CONCLUSION: Gd-EOB-DTPA enhanced MRI combined with clinical features would help predicting the early recurrence of HCC after operation.

Analysis of SARS-CoV-2 Emergent Variants Following AZD7442 (Tixagevimab/Cilgavimab) for Early Outpatient Treatment of COVID-19 (TACKLE Trial).

INTRODUCTION: AZD7442 (tixagevimab/cilgavimab) comprises neutralising monoclonal antibodies (mAbs) that bind to distinct non-overlapping epitopes on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Viral evolution during mAb therapy can select for variants with reduced neutralisation susceptibility. We examined treatment-emergent SARS-CoV-2 variants during TACKLE (NCT04723394), a phase 3 study of AZD7442 for early outpatient treatment of coronavirus disease 2019 (COVID-19). METHODS: Non-hospitalised adults with mild-to-moderate COVID-19 were randomised and dosed ≤ 7 days from symptom onset with AZD7442 (n = 452) or placebo (n = 451). Next-generation sequencing of the spike gene was performed on SARS-CoV-2 reverse-transcription polymerase chain reaction-positive nasopharyngeal swabs at baseline and study days 3, 6, and 15 post dosing. SARS-CoV-2 lineages were assigned using spike nucleotide sequences. Amino acid substitutions were analysed at allele fractions (AF; % of sequence reads represented by substitution) ≥ 25% and 3% to 25%. In vitro susceptibility to tixagevimab, cilgavimab, and AZD7442 was evaluated for all identified treatment-emergent variants using a pseudotyped microneutralisation assay. RESULTS: Longitudinal spike sequences were available for 461 participants (AZD7442, n = 235; placebo, n = 226) and showed that treatment-emergent variants at any time were rare, with 5 (2.1%) AZD7442 participants presenting ≥ 1 substitution in tixagevimab/cilgavimab binding sites at AF ≥ 25%. At AF 3% to 25%, treatment-emergent variants were observed in 15 (6.4%) AZD7442 and 12 (5.3%) placebo participants. All treatment-emergent variants showed in vitro susceptibility to AZD7442. CONCLUSION: These data indicate that AZD7442 creates a high genetic barrier for resistance and is a feasible option for COVID-19 treatment.

Circuit-specific gene therapy reverses core symptoms in a primate Parkinson's disease model.

Parkinson's disease (PD) is a debilitating neurodegenerative disorder. Its symptoms are typically treated with levodopa or dopamine receptor agonists, but its action lacks specificity due to the wide distribution of dopamine receptors in the central nervous system and periphery. Here, we report the development of a gene therapy strategy to selectively manipulate PD-affected circuitry. Targeting striatal D1 medium spiny neurons (MSNs), whose activity is chronically suppressed in PD, we engineered a therapeutic strategy comprised of a highly efficient retrograde adeno-associated virus (AAV), promoter elements with strong D1-MSN activity, and a chemogenetic effector to enable precise D1-MSN activation after systemic ligand administration. Application of this therapeutic approach rescues locomotion, tremor, and motor skill defects in both mouse and primate models of PD, supporting the feasibility of targeted circuit modulation tools for the treatment of PD in humans.

Fc-mediated functions of nirsevimab complement direct respiratory syncytial virus neutralization but are not required for optimal prophylactic protection.

Introduction: Nirsevimab is an extended half-life (M252Y/S254T/T256E [YTE]-modified) monoclonal antibody to the pre-fusion conformation of the respiratory syncytial virus (RSV) Fusion protein, with established efficacy in preventing RSV-associated lower respiratory tract infection in infants for the duration of a typical RSV season. Previous studies suggest that nirsevimab confers protection via direct virus neutralization. Here we use preclinical models to explore whether fragment crystallizable (Fc)-mediated effector functions contribute to nirsevimab-mediated protection. Methods: Nirsevimab, MEDI8897* (i.e., nirsevimab without the YTE modification), and MEDI8897*-TM (i.e., MEDI8897* without Fc effector functions) binding to Fc γ receptors (FcγRs) was evaluated using surface plasmon resonance. Antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent cellular phagocytosis (ADCP), antibody-dependent complement deposition (ADCD), and antibody-dependent cellular cytotoxicity (ADCC) were assessed through in vitro and ex vivo serological analyses. A cotton rat challenge study was performed with MEDI8897* and MEDI8897*-TM to explore whether Fc effector functions contribute to protection from RSV. Results: Nirsevimab and MEDI8897* exhibited binding to a range of FcγRs, with expected reductions in FcγR binding affinities observed for MEDI8897*-TM. Nirsevimab exhibited in vitro ADNP, ADCP, ADCD, and ADCC activity above background levels, and similar ADNP, ADCP, and ADCD activity to palivizumab. Nirsevimab administration increased ex vivo ADNP, ADCP, and ADCD activity in participant serum from the MELODY study (NCT03979313). However, ADCC levels remained similar between nirsevimab and placebo. MEDI8897* and MEDI8897*-TM exhibited similar dose-dependent reduction in lung and nasal turbinate RSV titers in the cotton rat model. Conclusion: Nirsevimab possesses Fc effector activity comparable with the current standard of care, palivizumab. However, despite possessing the capacity for Fc effector activity, data from RSV challenge experiments illustrate that nirsevimab-mediated protection is primarily dependent on direct virus neutralization.

Whole-cage randomization for animal studies with unequal cage or group sizes.

Following good statistical practice, in vivo study investigators allocate animals into two or more treatment groups using a randomization routine to eliminate selection bias and balance known and unknown confounding factors. For some studies, however, randomization at the individual animal level cannot be implemented. For example, for studies that involve co-housed male mice, an animal-level randomization can place unfamiliar mice together in the same cage, which can trigger fighting. To meet the ethical obligations to enhance the welfare of an animal used in science, the experimental procedures are, therefore, often modified, and male mice, possibly from the same brood, may be housed together. It follows that animal allocation into groups must proceed at the whole-cage level. Given the small sample sizes in animal studies, controlling baseline variables can be quite challenging. The difficulty greatly increases with a whole-cage randomization restriction. When the number of animals per cage or the treatment group sizes are unequal, there is no algorithm in the literature to perform the task. We propose a novel, fast, and reliable algorithm to provide a whole-cage randomization that balances one or more baseline variables across groups. The algorithm was applied to a realistic example dataset.

Latest advances: Improving the anti-inflammatory and immunomodulatory properties of PEEK materials.

Excellent biocompatibility, mechanical properties, chemical stability, and elastic modulus close to bone tissue make polyetheretherketone (PEEK) a promising orthopedic implant material. However, biological inertness has hindered the clinical applications of PEEK. The immune responses and inflammatory reactions after implantation would interfere with the osteogenic process. Eventually, the proliferation of fibrous tissue and the formation of fibrous capsules would result in a loose connection between PEEK and bone, leading to implantation failure. Previous studies focused on improving the osteogenic properties and antibacterial ability of PEEK with various modification techniques. However, few studies have been conducted on the immunomodulatory capacity of PEEK. New clinical applications and advances in processing technology, research, and reports on the immunomodulatory capacity of PEEK have received increasing attention in recent years. Researchers have designed numerous modification techniques, including drug delivery systems, surface chemical modifications, and surface porous treatments, to modulate the post-implantation immune response to address the regulatory factors of the mechanism. These studies provide essential ideas and technical preconditions for the development and research of the next generation of PEEK biological implant materials. This paper summarizes the mechanism by which the immune response after PEEK implantation leads to fibrous capsule formation; it also focuses on modification techniques to improve the anti-inflammatory and immunomodulatory abilities of PEEK. We also discuss the limitations of the existing modification techniques and present the corresponding future perspectives.

Breakthrough SARS-CoV-2 Infections in the PROVENT Prevention Trial Were Not Associated With AZD7442 (Tixagevimab/Cilgavimab) Resistant Variants.

BACKGROUND: We report spike protein-based lineage and AZD7442 (tixagevimab/cilgavimab) neutralizing activity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants identified from breakthrough infections in the PROVENT preexposure prophylaxis trial. METHODS: Variants identified from PROVENT participants with reverse-transcription polymerase chain reaction-positive symptomatic illness were phenotypically assessed to determine neutralization susceptibility of variant-specific pseudotyped virus-like particles. RESULTS: At completion of 6 months' follow-up, no AZD7442-resistant variants were observed in breakthrough coronavirus disease 2019 (COVID-19) cases. SARS-CoV-2 neutralizing antibody titers were similar in breakthrough and nonbreakthrough cases. CONCLUSIONS: Symptomatic COVID-19 breakthrough cases in PROVENT were not due to resistance-associated substitutions in AZD7442 binding sites or lack of AZD7442 exposure. CLINICAL TRIALS REGISTRATION: NCT04625725.

Durability of neutralizing RSV antibodies following nirsevimab administration and elicitation of the natural immune response to RSV infection in infants.

Nirsevimab is an extended half-life monoclonal antibody specific for the prefusion conformation of the respiratory syncytial virus (RSV) F protein, which has been studied in preterm and full-term infants in the phase 2b and phase 3 MELODY trials. We analyzed serum samples collected from 2,143 infants during these studies to characterize baseline levels of RSV-specific immunoglobulin G antibodies and neutralizing antibodies (NAbs), duration of RSV NAb levels following nirsevimab administration, the risk of RSV exposure during the first year of life and the infant's adaptive immune response to RSV following nirsevimab administration. Baseline RSV antibody levels varied widely; consistent with reports that maternal antibodies are transferred late in the third trimester, preterm infants had lower baseline RSV antibody levels than full-term infants. Nirsevimab recipients had RSV NAb levels >140-fold higher than baseline at day 31 and remained >50-fold higher at day 151 and >7-fold higher at day 361. Similar seroresponse rates to the postfusion form of RSV F protein in nirsevimab recipients (68-69%) compared with placebo recipients (63-70%; not statistically significant) suggest that while nirsevimab protects from RSV disease, it still allows an active immune response. In summary, nirsevimab provided sustained, high levels of NAb throughout an infant's first RSV season and prevented RSV disease while allowing the development of an immune response to RSV.

Molecular Characterization of AZD7442 (Tixagevimab-Cilgavimab) Neutralization of SARS-CoV-2 Omicron Subvariants.

Therapeutic anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (MAbs) provide immunosuppressed and vulnerable populations with prophylactic and treatment interventions against coronavirus disease 2019 (COVID-19). AZD7442 (tixagevimab-cilgavimab) is a combination of extended-half-life neutralizing MAbs that bind to distinct epitopes on the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. The Omicron variant of concern carries mutations at >35 positions in the spike protein and has undergone further genetic diversification since its emergence in November 2021. Here, we characterize the in vitro neutralization activity of AZD7442 toward major viral subvariants circulating worldwide during the first 9 months of the Omicron wave. BA.2 and its derived subvariants showed the highest susceptibility to AZD7442, while BA.1 and BA.1.1 showed a lower susceptibility. BA.4/BA.5 had a susceptibility level intermediate between BA.1 and BA.2. Mutagenesis of parental Omicron subvariant spike proteins was performed to establish a molecular model to describe the underlying determinants of neutralization by AZD7442 and its component MAbs. The concurrent mutation of residues at positions 446 and 493, located in the tixagevimab and cilgavimab binding sites, was sufficient to enhance in vitro susceptibility of BA.1 to AZD7442 and its component MAbs to levels similar to the Wuhan-Hu-1+D614G virus. AZD7442 maintained neutralization activity against all Omicron subvariants tested up to and including BA.5. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time molecular surveillance and assessment of in vitro activity of MAbs used in prophylaxis against and the treatment of COVID-19. IMPORTANCE MAbs are key therapeutic options for COVID-19 prophylaxis and treatment in immunosuppressed and vulnerable populations. Due to the emergence of SARS-CoV-2 variants, including Omicron, it is vital to ensure that neutralization is maintained for MAb-based interventions. We studied the in vitro neutralization of AZD7442 (tixagevimab-cilgavimab), a cocktail of two long-acting MAbs targeting the SARS-CoV-2 spike protein, toward Omicron subvariants circulating from November 2021 to July 2022. AZD7442 neutralized major Omicron subvariants up to and including BA.5. The mechanism of action responsible for the lower in vitro susceptibility of BA.1 to AZD7442 was investigated using in vitro mutagenesis and molecular modeling. A combination of mutations at two spike protein positions, namely, 446 and 493, was sufficient to enhance BA.1 susceptibility to AZD7442 to levels similar to the Wuhan-Hu-1+D614G ancestral virus. The evolving nature of the SARS-CoV-2 pandemic warrants continuing real-time global molecular surveillance and mechanistic studies of therapeutic MAbs for COVID-19.

Tree-based machine learning model for visualizing complex relationships between biochar properties and anaerobic digestion.

The ideal conditions for anaerobic digestion experiments with biochar addition are challenging to thoroughly study due to different experimental purposes. Therefore, three tree-based machine learning models were developed to depict the intricate connection between biochar properties and anaerobic digestion. For the methane yield and maximum methane production rate, the gradient boosting decision tree produced R2 values of 0.84 and 0.69, respectively. According to feature analysis, digestion time and particle size had a substantial impact on the methane yield and production rate, respectively. When particle sizes were in the range of 0.3-0.5 mm and the specific surface area was approximately 290 m2/g, corresponding to a range of O content (>31%) and biochar addition (>20 g/L), the maximum promotion of methane yield and maximum methane production rate were attained. Therefore, this study presents new insights into the effects of biochar on anaerobic digestion through tree-based machine learning.

Recent advances in nanomaterials for the treatment of spinal cord injury.

Spinal cord injuries (SCIs) are devastating. In SCIs, a powerful traumatic force impacting the spinal cord results in the permanent loss of nerve function below the injury level, leaving the patient paralyzed and wheelchair-bound for the remainder of his/her life. Unfortunately, clinical treatment that depends on surgical decompression appears to be unable to handle damaged nerves, and high-dose methylprednisolone-based therapy is also associated with problems, such as infection, gastrointestinal bleeding, femoral head necrosis, obesity, and hyperglycemia. Nanomaterials have opened new avenues for SCI treatment. Among them, performance-based nanomaterials derived from a variety of materials facilitate improvements in the microenvironment of traumatic injury and, in some cases, promote neuron regeneration. Nanoparticulate drug delivery systems enable the optimization of drug effects and drug bioavailability, thus contributing to the development of novel treatments. The improved efficiency and accuracy of gene delivery will also benefit the exploration of SCI mechanisms and the understanding of key genes and signaling pathways. Herein, we reviewed different types of nanomaterials applied to the treatment of SCI and summarized their functions and advantages to provide new perspectives for future clinical therapies.

ROS-responsive magnesium-containing microspheres for antioxidative treatment of intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a degenerative disease characterized by lower-back pain, causing disability globally. Antioxidant therapy is currently considered one of the most promising strategies for IVDD treatment, given the crucial role of reactive oxygen species (ROS) in IVDD pathogenesis. Herein, a ROS-responsive magnesium-containing microsphere (Mg@PLPE MS) was constructed for the antioxidative treatment of IVDD. The Mg@PLPE MS has a core-shell structure comprising poly(lactic-co-glycolic acid) (PLGA) and ROS-responsive polymer poly(PBT-co-EGDM) as the shell and a magnesium microparticle as the core. The poly(PBT-co-EGDM) can be destroyed by H2O2 through the H2O2-triggered hydrophobic-to-hydrophilic transition, subsequently promoting an Mg-water reaction to produce H2. Thus, Mg@PLPE MS provides a valuable platform for H2O2 elimination and controlled H2 release. The generated H2 scavenge for ROS by reacting with noxious •OH. Notably, the Mg@PLPE MS exerted significant antioxidative and anti-inflammatory effects in a disc degeneration rat model and alleviated extracellular matrix degradation and disc cells apoptosis, thereby underlining its efficacy in IVDD treatment. The Mg@PLPE MS also exhibited robust biocompatibility and negligible toxicity, presenting the promise for the antioxidative treatment of IVDD in vivo. STATEMENT OF SIGNIFICANCE: Antioxidant therapy is currently considered one of the most promising strategies for intervertebral disc degeneration (IVDD) treatment, given the crucial role of reactive oxygen species (ROS) in IVDD pathogenesis. Here, ROS-responsive magnesium-containing microspheres (Mg@PLPE MSs) were constructed to alleviate IVDD through controlled release of hydrogen gas. The Mg@PLPE MSs can effectively scavenge overproduced ROS by simultaneously reacting with H2O2 and •OH, thus creating a suitable microenvironment for inhibition of ECM degradation. As a result, Mg@PLPE MSs treated IVDD rats exhibit minimal nucleus pulposus decrease, less extracellular matrix degradation, minimal radial fissure of fibrous rings, and higher disc height index. Therefore, the as-prepared Mg@PLPE MSs may shed a new light on clinical treatment of IVDD.

A comparison of statistical methods for animal oncology studies.

In pre-clinical oncology studies, tumor-bearing animals are treated and observed over a period of time in order to measure and compare the efficacy of one or more cancer-intervention therapies along with a placebo/standard of care group. A data analysis is typically carried out by modeling and comparing tumor volumes, functions of tumor volumes, or survival. Data analysis on tumor volumes is complicated because animals under observation may be euthanized prior to the end of the study for one or more reasons, such as when an animal's tumor volume exceeds an upper threshold. In such a case, the tumor volume is missing not-at-random for the time remaining in the study. To work around the non-random missingness issue, several statistical methods have been proposed in the literature, including the rate of change in log tumor volume and partial area under the curve. In this work, an examination and comparison of the test size and statistical power of these and other popular methods for the analysis of tumor volume data is performed through realistic Monte Carlo computer simulations. The performance, advantages, and drawbacks of popular statistical methods for animal oncology studies are reported. The recommended methods are applied to a real data set.

Unraveling the mechanisms of intervertebral disc degeneration: an exploration of the p38 MAPK signaling pathway.

Intervertebral disc (IVD) degeneration (IDD) is a worldwide spinal degenerative disease. Low back pain (LBP) is frequently caused by a variety of conditions brought on by IDD, including IVD herniation and spinal stenosis, etc. These conditions bring substantial physical and psychological pressure and economic burden to patients. IDD is closely tied with the structural or functional changes of the IVD tissue and can be caused by various complex factors like senescence, genetics, and trauma. The IVD dysfunction and structural changes can result from extracellular matrix (ECM) degradation, differentiation, inflammation, oxidative stress, mechanical stress, and senescence of IVD cells. At present, the treatment of IDD is basically to alleviate the symptoms, but not from the pathophysiological changes of IVD. Interestingly, the p38 mitogen-activated protein kinase (p38 MAPK) signaling pathway is involved in many processes of IDD, including inflammation, ECM degradation, apoptosis, senescence, proliferation, oxidative stress, and autophagy. These activities in degenerated IVD tissue are closely relevant to the development trend of IDD. Hence, the p38 MAPK signaling pathway may be a fitting curative target for IDD. In order to better understand the pathophysiological alterations of the intervertebral disc tissue during IDD and offer potential paths for targeted treatments for intervertebral disc degeneration, this article reviews the purpose of the p38 MAPK signaling pathway in IDD.

Qualification of a Biolayer Interferometry Assay to Support AZD7442 Resistance Monitoring.

AZD7442, a combination of two long-acting monoclonal antibodies (tixagevimab [AZD8895] and cilgavimab [AZD1061]), has been authorized for the prevention and treatment of coronavirus disease 2019 (COVID-19). The rapid emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants requires methods capable of quickly characterizing resistance to AZD7442. To support AZD7442 resistance monitoring, a biolayer interferometry (BLI) assay was developed to screen the binding of tixagevimab and cilgavimab to SARS-CoV-2 spike proteins to reduce the number of viral variants for neutralization susceptibility verification. Six spike variants were chosen to assess the assay's performance: four with decreased affinity for tixagevimab (F486S:D614G and F486W:D614G proteins) or cilgavimab (S494L:D614G and K444R:D614G proteins) and two reference proteins (wild-type HexaPro and D614G protein). Equilibrium dissociation constant (KD) values from each spike protein were used to determine shifts in binding affinity. The assay's precision, range, linearity, and limits of quantitation were established. Qualification acceptance criteria determined whether the assay was fit for purpose. By bypassing protein purification, the BLI assay provided increased screening throughput. Although limited correlation between pseudotype neutralization and BLI data (50% inhibitory concentration versus KD) was observed for full immunoglobulins (IgGs), the correlations for antibody fragments (Fabs) were stronger and reflected a better comparison of antibody binding kinetics with neutralization potency. Therefore, despite strong assay performance characteristics, the use of full IgGs limited the screening utility of the assay; however, the Fab approach warrants further exploration as a rapid, high-throughput variant-screening method for future resistance-monitoring programs. IMPORTANCE SARS-CoV-2 variants harbor multiple substitutions in their spike trimers, potentially leading to breakthrough infections and clinical resistance to immune therapies. For this reason, a BLI assay was developed and qualified to evaluate the reliability of screening SARS-CoV-2 spike trimer variants against anti-SARS-CoV-2 monoclonal antibodies (MAbs) tixagevimab and cilgavimab, the components of AZD7442, prior to in vitro pseudovirus neutralization susceptibility verification testing. The assay bypasses protein purification with rapid assessment of the binding affinity of each MAb for each recombinant protein, potentially providing an efficient preliminary selection step, thus allowing a reduced testing burden in the more technically complex viral neutralization assays. Despite precise and specific measures, an avidity effect associated with MAb binding to the trimer confounded correlation with neutralization potency, negating the assay's utility as a surrogate for neutralizing antibody potency. Improved correlation with Fabs suggests that assay optimization could overcome any avidity limitation, warranting further exploration to support future resistance-monitoring programs.

Neutralizing Staphylococcus aureus Virulence with AZD6389, a Three mAb Combination, Accelerates Closure of a Diabetic Polymicrobial Wound.

Nonhealing diabetic foot ulcers (DFU), a major complication of diabetes, are associated with high morbidity and mortality despite current standard of care. Since Staphylococcus aureus is the most common pathogen isolated from nonhealing and infected DFU, we hypothesized that S. aureus virulence factors would damage tissue, promote immune evasion and alter the microbiome, leading to bacterial persistence and delayed wound healing. In a diabetic mouse polymicrobial wound model with S. aureus, Pseudomonas aeruginosa, and Streptococcus pyogenes, we report a rapid bacterial proliferation, prolonged pro-inflammatory response and large necrotic lesions unclosed for up to 40 days. Treatment with AZD6389, a three-monoclonal antibody combination targeting S. aureus alpha toxin, 4 secreted leukotoxins, and fibrinogen binding cell-surface adhesin clumping factor A resulted in full skin re-epithelization 21 days after inoculation. By neutralizing multiple virulence factors, AZD6389 effectively blocked bacterial agglutination and S. aureus-mediated cell killing, abrogated S. aureus-mediated immune evasion and targeted the bacteria for opsonophagocytic killing. Neutralizing S. aureus virulence not only facilitated S. aureus clearance in lesions, but also reduced S. pyogenes and P. aeruginosa numbers, damaging inflammatory mediators and markers for neutrophil extracellular trap formation 14 days post initiation. Collectively, our data suggest that AZD6389 holds promise as an immunotherapeutic approach against DFU complications. IMPORTANCE Diabetic foot ulcers (DFU) represent a major complication of diabetes and are associated with poor quality of life and increased morbidity and mortality despite standard of care. They have a complex pathogenesis starting with superficial skin lesions, which often progress to deeper tissue structures up to the bone and ultimately require limb amputation. The skin microbiome of diabetic patients has emerged as having an impact on DFU occurrence and chronicity. DFU are mostly polymicrobial, and the Gram-positive bacterium Staphylococcus aureus detected in more than 95% of cases. S. aureus possess a collection of virulence factors which participate in disease progression and may facilitate growth of other pathogens. Here we show in a diabetic mouse wound model that targeting some specific S. aureus virulence factors with a multimechanistic antibody combination accelerated wound closure and promoted full skin re-epithelization. This work opens promising new avenues for the treatment of DFU.