Sialic Acid Mediated Endothelial and Hepatic Uptake: A Mechanism based Mathematic Model Elucidating the Complex Pharmacokinetics and Pharmacodynamics of Efmarodocokin Alfa, a Variably Glycosylated Fusion Protein.

Sialic acid (SA) is crucial for protecting glycoproteins from clearance. Efmarodocokin alfa (IL-22Fc), a fusion protein agonist that links IL-22 to the crystallizable fragment (Fc) of human IgG4, contains 8 N-glycosylation sites and exhibits heterogeneous and variable terminal sialylation biodistribution. This presents a unique challenge for Pharmacokinetic (PK) and Pharmacodynamic (PD) analysis and cross-species translation. In this study, we sought to understand how varying SA levels and heterogeneous distribution contribute to IL-22Fc's complex PKPD properties. We initially used homogenous drug material with varying SA levels to examine PKPD in mice. Population PKPD analysis based on mouse data revealed that SA was a critical covariate simultaneously accounting for the substantial between subject variability (BSV) in clearance (CL), distribution clearance (CLd), and volume of distribution (Vd). In addition to the well-established mechanism by which SA inhibits ASGPR activity, we hypothesized a novel mechanism by which decrease in SA increases the drug uptake by endothelial cells. This decrease in SA, leading to more endothelial uptake, was supported by the neonatal Fc receptor (FcRn) dependent cell-based transcytosis assay. The population analysis also suggested in vivo EC50 (IL-22Fc stimulating Reg3ß) was independent on SA, while the in-vitro assay indicated a contradictory finding of SA-in vitro potency relationship. We created a mechanism based mathematical (MBM) PKPD model incorporating the decrease in SA mediated endothelial and hepatic uptake, and successfully characterized the SA influence on IL-22Fc PK, as well as the increased PK exposure being responsible for increased PD. Thereby, the MBM model supported that SA has no direct impact on EC50, aligning with the population PKPD analysis. Subsequently, using the MBM PKPD model, we employed 5 subpopulation simulations to reconstitute the heterogeneity of drug material. The simulation accurately predicted the PKPD of heterogeneously and variably sialylated drug in mouse, monkey and human. The successful prospective validation confirmed the MBM's ability to predict IL-22Fc PK across variable SA levels, homogenous to heterogeneous material, and across species (R2=0.964 for clearance prediction). Our model prediction suggests an average of 1 mol/mol SA increase leads to a 50% increase in drug exposure. This underlines the significance of controlling sialic acid levels during lot-to-lot manufacturing.

Fluorine-18 labeling PEGylated 6-boronotryptophan for PET scanning of mice for assessing the pharmacokinetics for boron neutron capture therapy of brain tumors.

Two tryptophan compound classes 5- and 6-borono PEGylated boronotryptophan derivatives have been prepared for assessing their aqueous solubility as formulation of injections for boron neutron capture therapy (BNCT). The PEGylation has improved their aqueous solubility thereby increasing their test concentration in 1 mM without suffering from toxicity. In-vitro uptake assay of PEGylated 5- and 6-boronotryptophan showed that the B-10 concentration can reach 15-50 ppm in U87 cell whereas the uptake in LN229 cell varies. Shorter PEG compound 6-boronotryptophanPEG200[18F] was obtained in 1.7 % radiochemical yield and the PET-derived radioradioactivity percentage in 18 % was taken up by U87 tumor at the limb of xenograft mouse. As high as tumor to normal uptake ratio in 170 (T/N) was obtained while an inferior radioactivity uptake of 3 % and T/N of 8 was observed in LN229 xenografted mouse.

Development of a semi-automated MHC-associated peptide proteomics (MAPPs) method using streptavidin bead-based immunoaffinity capture and nano LC-MS/MS to support immunogenicity risk assessment in drug development.

Major histocompatibility complex (MHC)-Associated Peptide Proteomics (MAPPs) is an ex vivo method used to assess the immunogenicity risk of biotherapeutics. MAPPs can identify potential T-cell epitopes within the biotherapeutic molecule. Using adalimumab treated human monocyte derived dendritic cells (DCs) and a pan anti-HLA-DR antibody (Ab), we systematically automated and optimized biotin/streptavidin (SA)-capture antibody coupling, lysate incubation with capture antibody, as well as the washing and elution steps of a MAPPs method using functionalized magnetic beads and a KingFisher Magnetic Particle processor. Automation of these steps, combined with capturing using biotinylated-Ab/SA magnetic beads rather than covalently bound antibody, improved reproducibility as measured by minimal inter-and intra-day variability, as well as minimal analyst-to-analyst variability. The semi-automated MAPPs workflow improved sensitivity, allowing for a lower number of cells per analysis. The method was assessed using five different biotherapeutics with varying immunogenicity rates ranging from 0.1 to 48% ADA incidence in the clinic. Biotherapeutics with ≥10%immunogenicity incidence consistently presented more peptides (1.8-28 fold) and clusters (10-21 fold) compared to those with <10% immunogenicity incidence. Our semi-automated MAPPs method provided two main advantages over a manual workflow- the robustness and reproducibility affords confidence in the epitopes identified from as few as 5 to 10 donors and the method workflow can be readily adapted to incorporate different capture Abs in addition to anti-HLA-DR. The incorporation of semi-automated MAPPs with biotinylated-Ab/SA bead-based capture in immunogenicity screening strategies allows the generation of more consistent and reliable data, helping to improve immunogenicity prediction capabilities in drug development. MHC associated peptide proteomics (MAPPs), Immunogenicity risk assessment, in vitro/ex vivo, biotherapeutics, Major Histocompatibility Complex Class II (MHC II), LC-MS, Immunoaffinity Capture, streptavidin magnetic beads.

An Antibody-Dependent Cellular Cytotoxicity Assay for Detecting Ocrelizumab Neutralizing Antibody.

Ocrelizumab (OCREVUS®) is a humanized anti-CD20 monoclonal antibody approved for the treatment of adult patients with relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). Here, we discuss the strategic and technical considerations needed to develop a robust antibody-dependent cellular cytotoxicity (ADCC)-based neutralizing antibody (NAb) assay to detect anti-ocrelizumab NAb in patients enrolled in the ocrelizumab registered clinical trials. The NAb detection assay consisted of a two-tier assay that included a screening assay and a confirmation assay. In the screening assay, patient samples were analyzed in the presence of ocrelizumab. Samples that tested positive in the screening assay were subsequently analyzed in the confirmatory assay where another anti-CD20 mAb, obinutuzumab, was replaced by ocrelizumab, to verify NAb specificity. Both assays utilized MEC-2 cells, a chronic B cell leukemia cell line, pre-labeled with calcein AM as the target cells, and natural killer (NK) cells engineered to stably express Fc gamma receptor IIIa_ F158 as effector cells. Both cell lines were prepared to be thaw-and-use cells. The NAb assay measures fluorescence from the calcein AM released into the assay media upon the lysis of target cells by ADCC in the presence of ocrelizumab or obinutuzumab. Our validated NAb assay showed a relative sensitivity of 743 ng/mL and can detect 1500 ng/mL of a surrogate positive control antibody in the presence of 1500 ng/mL ocrelizumab. This ADCC assay is the first reported NAb assay that directly measures target cell lysis by using thaw-and-use target and effector cells simultaneously.

Neutralizing Antibody Validation Testing and Reporting Harmonization.

Evolving immunogenicity assay performance expectations and a lack of harmonized neutralizing antibody validation testing and reporting tools have resulted in significant time spent by health authorities and sponsors on resolving filing queries. A team of experts within the American Association of Pharmaceutical Scientists' Therapeutic Product Immunogenicity Community across industry and the Food and Drug Administration addressed challenges unique to cell-based and non-cell-based neutralizing antibody assays. Harmonization of validation expectations and data reporting will facilitate filings to health authorities and are described in this manuscript. This team provides validation testing and reporting strategies and tools for the following assessments: (1) format selection; (2) cut point; (3) assay acceptance criteria; (4) control precision; (5) sensitivity including positive control selection and performance tracking; (6) negative control selection; (7) selectivity/specificity including matrix interference, hemolysis, lipemia, bilirubin, concomitant medications, and structurally similar analytes; (8) drug tolerance; (9) target tolerance; (10) sample stability; and (11) assay robustness.

Survey Outcome on Immunogenicity Risk Assessment Tools for Biotherapeutics: an Insight into Consensus on Methods, Application, and Utility in Drug Development.

A survey conducted by the Therapeutic Product Immunogenicity (TPI) community within the American Association of Pharmaceutical Scientists (AAPS) posed questions to the participants on their immunogenicity risk assessment strategies prior to clinical development. The survey was conducted in 2 phases spanning 5 years, and queried information about in silico algorithms and in vitro assay formats for immunogenicity risk assessments and how the data were used to inform early developability effort in discovery, chemistry, manufacturing and control (CMC), and non-clinical stages of development. The key findings representing the trends from a majority of the participants included the use of high throughput in silico algorithms, human immune cell-based assays, and proteomics based outputs, as well as specialized assays when therapeutic mechanism of action could impact risk assessment. Additional insights into the CMC-related risks could also be gathered with the same tools to inform future process development and de-risk critical quality attributes with uncertain and unknown risks. The use of the outputs beyond supporting early development activities was also noted with participants utilizing the risk assessments to drive their clinical strategy and streamline bioanalysis.

Status of resource recycling stations in Taiwan and recycling work-related health effects.

Resource recycling has become an integral part of environmental protection efforts. At present, the development of Taiwan's resource recovery and related works are quite mature. However, laborers or volunteers working in resource recycling stations may be exposed to different types of hazards during the recycling process. These hazards can be divided into biological, chemical, and musculoskeletal problems. These hazards are usually related to the work environment and work habits; therefore, a related control strategy is needed. Tzu Chi's recycling business has been running for over 30 years. In addition to leading the trend of resource recycling in Taiwan, many elderly people have also participated in Tzu Chi recycling stations as volunteers. These older volunteers may be more sensitive to exposure to hazards, and thus the focus of this review is to illustrate the possible hazards and health impacts of resource recovery work and to recommend relevant interventions to improve occupational health during resource recovery work.

The efficacy and safety of cupping as complementary and alternative therapy for metabolic syndrome: A systematic review and meta-analysis.

INTRODUCTION: This systematic review and meta-analysis aimed to assess the efficacy and safety of cupping therapy in patients with metabolic syndrome (MetS). METHODS: This systematic review focused on patients with MetS and included randomized controlled trials (RCTs) that compared the effects of cupping therapy with control groups. A total of 12 electronic databases were searched from inception until February 03, 2023. The main outcome after the meta-analysis was waist circumference; the others included anthropometric variables, blood pressure, lipid profile, fasting blood glucose level, and high-sensitivity C-reactive protein level. The incidence of adverse events and the follow-up courses were also evaluated. Risk of bias (ROB) was evaluated using ROB 2.0 from the Cochrane Handbook. RESULTS: This systematic review included five studies involving 489 patients. Some risks of bias were also identified. The meta-analysis revealed a statistically significance in waist circumference (MD = -6.07, 95% CI: -8.44 to -3.71, P < .001, I2 = 61%, τ2 = 3.4), body weight (MD = -2.46, 95% CI: -4.25 to -0.68, P = .007, I2 = 0%, τ2 = 0) and body mass index (MD = -1.26, 95% CI: -2.11 to -0.40, P = .004, I2 = 0%, τ2 = 0) between the cupping therapy and control groups. However, there were no significant results in total fat percentage and blood pressure values. Regarding biochemical markers, cupping significantly lowered the concentration of low-density lipoprotein cholesterol (MD = -3.98, 95% CI: -6.99 to -0.96, P = .010, I2 = 0%, τ2 = 0) but had no significant effect on total cholesterol, triglyceride, high-density lipoprotein cholesterol, fasting blood glucose, and high-sensitivity C-reactive protein. 3 RCTs reported no adverse events. CONCLUSIONS: Despite some ROB and low to substantial heterogeneity of the included studies, cupping therapy can be considered a safe and effective complementary intervention for reducing waist circumference, body weight, body mass index, and low-density lipoprotein cholesterol in patients with MetS. In the future, well-designed, high-quality, rigorous methodology, and long-term RCTs in this population are required to assess the efficacy and safety of cupping therapy.

Recent Development of Radiofluorination of Boron Agents for Boron Neutron Capture Therapy of Tumor: Creation of 18F-Labeled C-F and B-F Linkages.

Boron neutron capture therapy (BNCT) is a binary therapeutic technique employing a boron agent to be delivered to the tumor site followed by the irradiation of neutrons. Biofunctional molecules/nanoparticles labeled with F-18 can provide an initial pharmacokinetic profile of patients to guide the subsequent treatment planning procedure of BNCT. Borono phenylalanine (BPA), recognized by the l-type amino acid transporter, can cross the blood-brain barrier and be accumulated in gliomas. The radiofluoro BNCT agents are reviewed by considering (1) less cytotoxicity, (2) diagnosing and therapeutic purposes, (3) aqueous solubility and extraction route, as well as (4), the trifluoroborate effect. A trifluoroborate-containing amino acid such as fluoroboronotyrosine (FBY) represents an example with both functionalities of imaging and therapeutics. Comparing with the insignificant cytotoxicity of clinical BPA with IC50 > 500 µM, FBY also shows minute toxicity with IC50 > 500 µM. [18F]FBY is a potential diagnostic agent for its tumor to normal accumulation (T/N) ratio, which ranges from 2.3 to 24.5 from positron emission tomography, whereas the T/N ratio of FBPA is greater than 2.5. Additionally, in serving as a BNCT therapeutic agent, the boron concentration of FBY accumulated in gliomas remains uncertain. The solubility of 3-BPA is better than that of BPA, as evidenced by the cerebral dose of 3.4%ID/g vs. 2.2%ID/g, respectively. While the extraction route of d-BPA differs from that of BPA, an impressive T/N ratio of 6.9 vs. 1.5 is noted. [18F]FBPA, the most common clinical boron agent, facilitates the application of BPA in clinical BNCT. In addition to [18F]FBY, [18F] trifluoroborated nucleoside analog obtained through 1,3-dipolar cycloaddition shows marked tumoral uptake of 1.5%ID/g. Other examples using electrophilic and nucleophilic fluorination on the boron compounds are also reviewed, including diboronopinacolone phenylalanine and nonsteroidal anti-inflammatory agents.

Endoscopic ultrasound-guided balloon-occluded gastrojejunostomy bypass, duodenal stent or laparoscopic gastrojejunostomy for unresectable malignant gastric outlet obstruction.

OBJECTIVES: Malignant gastric outlet obstruction (GOO) can be relieved by either laparoscopic gastrojejunostomy (LGJ), endoscopic stenting (SEMS) or endoscopic ultrasound-guided gastrojejunostomy (endoscopic ultrasound-guided balloon-occluded gastrojejunostomy bypass; EPASS). This study aimed to compare the outcomes of the three treatment methods. METHODS: This was a retrospective study of patients who suffered from malignant GOO between January 2012 to November 2020 that received either EPASS, LGJ or SEMS. The outcomes included the technical and clinical success, 30-day adverse events and mortality, pre and post stenting GOO scores (GOOSs), stent patency and causes of stent dysfunction. RESULTS: One hundred and fourteen patients were included (30 EPASS, 35 LGJ, 49 SEMS). The technical success of EPASS, LGJ and SEMS were 93.3%, 100%, 100% (P = 0.058) and clinical success rates were 93.3%, 80%, 87.8% (P = 0.276), respectively. Procedural time was longest for the LGJ group (P < 0.001). The EPASS group had the shortest hospital stay (EPASS 1.5 [1-17], LGJ 7 [2-44], SEMS 5 [2-46] days, P < 0.001). EPASS group also had the lowest rates of recurrent obstruction (EPASS 3.3%, LGJ 17.1%, SEMS 36.7%, P = 0.002) and re-intervention (EPASS 3.3%, LGJ 17.1%, SEMS 26.5%, P = 0.031). The 1-month GOOS was highest in the EPASS group (EPASS 3 [1-3], LGJ 3 [0-3], SEMS 2 [0-3], P = 0.028). CONCLUSION: Endoscopic ultrasound-guided gastrojejunostomy was associated with better clinical outcomes then the other two procedures. The procedure may be the best option provided that the expertise is available.

Preclinical development of ZED8, an 89Zr immuno-PET reagent for monitoring tumor CD8 status in patients undergoing cancer immunotherapy.

BACKGROUND: ZED8 is a novel monovalent antibody labeled with zirconium-89 for the molecular imaging of CD8. This work describes nonclinical studies performed in part to provide rationale for and to inform expectations in the early clinical development of ZED8, such as in the studies outlined in clinical trial registry NCT04029181 [1]. METHODS: Surface plasmon resonance, X-ray crystallography, and flow cytometry were used to characterize the ZED8-CD8 binding interaction, its specificity, and its impact on T cell function. Immuno-PET with ZED8 was assessed in huCD8+ tumor-bearing mice and in non-human primates. Plasma antibody levels were measured by ELISA to determine pharmacokinetic parameters, and OLINDA 1.0 was used to estimate radiation dosimetry from image-derived biodistribution data. RESULTS: ZED8 selectively binds to human CD8α at a binding site approximately 9 Å from that of MHCI making mutual interference unlikely. The equilibrium dissociation constant (KD) is 5 nM. ZED8 binds to cynomolgus CD8 with reduced affinity (66 nM) but it has no measurable affinity for rat or mouse CD8. In a series of lymphoma xenografts, ZED8 imaging was able to identify different CD8 levels concordant with flow cytometry. In cynomolgus monkeys with tool compound 89Zr-aCD8v17, lymph nodes were conspicuous by imaging 24 h post-injection, and the pharmacokinetics suggested a flat-fixed first-in-human dose of 4 mg per subject. The whole-body effective dose for an adult human was estimated to be 0.48 mSv/MBq, comparable to existing 89Zr immuno-PET reagents. CONCLUSION: 89Zr immuno-PET with ZED8 appears to be a promising biomarker of tissue CD8 levels suitable for clinical evaluation in cancer patients eligible for immunotherapy.

Antiinflammation Derived Suzuki-Coupled Fenbufens as COX-2 Inhibitors: Minilibrary Construction and Bioassay.

A small fenbufen library comprising 18 compounds was prepared via Suzuki Miyara coupling. The five-step preparations deliver 9-17% biphenyl compounds in total yield. These fenbufen analogs exert insignificant activity against the IL-1 release as well as inhibiting cyclooxygenase 2 considerably. Both the para-amino and para-hydroxy mono substituents display the most substantial COX-2 inhibition, particularly the latter one showing a comparable activity as celecoxib. The most COX-2 selective and bioactive disubstituted compound encompasses one electron-withdrawing methyl and one electron-donating fluoro groups in one arene. COX-2 is selective but not COX-2 to bioactive compounds that contain both two electron-withdrawing groups; disubstituted analogs with both resonance-formable electron-donating dihydroxy groups display high COX-2 activity but inferior COX-2 selectivity. In silico simulation and modeling for three COX-2 active-p-fluoro, p-hydroxy and p-amino-fenbufens show a preferable docking to COX-2 than COX-1. The most stabilization by the p-hydroxy fenbufen with COX-2 predicted by theoretical simulation is consistent with its prominent COX-2 inhibition resulting from experiments.

Introducing dendritic cell antibody internalization as an immunogenicity risk assessment tool.

Aim: Immunogenicity risk assessment assays are powerful tools that assess the relative immunogenicity of potential biotherapeutics. We detail here the development of a novel assay that measures the degree of antibody internalization by antigen-presenting cells as a predictor of immunogenicity. Results & methodology: The assay uses the fluorescence signal from the antibody bound to the outside of the cell as well as inside the cell to determine internalization. To calculate the amount of internalized antibody, the fluorescent signal from the outside was subtracted from the fluorescent signal from the inside, which is referred to as the internalization index. Conclusion: This assay format demonstrated that antibody-based biotherapeutics with higher clinical immunogenicity internalized to a higher degree than therapeutic antibodies with lower clinical immunogenicity.

Efficacy of Acupuncture Treatment for Incidence of Poststroke Comorbidities: A Systematic Review and Meta-Analysis of Nationalized Cohort Studies.

Acupuncture has been applied as a complementary therapy in stroke survivors worldwide and approved to be beneficial to stroke recovery. However, there is little medical evidence regarding the association between acupuncture and the risk of poststroke comorbidities. We reviewed big data studies from the Taiwan National Health Insurance Research Database to investigate the risk of poststroke comorbidities after acupuncture treatment in a real-world situation. Ten English (PubMed, Embase, Medline, Cochrane, Alt HealthWatch, CINAHL, Health Source, PsycINFO, PsycARTICLES, and Psychology and Behavioral Sciences Collection) and two Chinese (AiritiLibray and Visualizing Health Data) electronic databases were searched from inception until December 2020 for nationalized cohort studies comparing the effects of acupuncture treatment with a nonacupuncture control group among stroke patients. Eight nationalized cohort studies were included. Six of eight studies showed a moderate overall risk of bias, while two studies showed a serious overall risk of bias. Included studies have investigated the effect of acupuncture in reducing the risk of seven medical conditions after stroke, including stroke recurrence, new-onset acute myocardial infarction (AMI), pneumonia, dementia, epilepsy, urinary tract infection (UTI), and depression. The meta-analysis showed clinically significant reductions in the risk of poststroke comorbidities in the acupuncture group compared to the nonacupuncture group (HR, 0.776; 95% CI, 0.719-0.838; p < 0.0001). In this systematic review and meta-analysis of nationalized cohort studies, acupuncture showed clinically relevant benefits in reducing the incidence of poststroke comorbidities, such as stroke recurrence, new-onset acute myocardial infarction (AMI), pneumonia, dementia, epilepsy, and UTI.

The Potential Win-win Strategy for Healthy Aging and Environmental Protection: Environmental Volunteering.

PURPOSE: To explore the physical activity level of community environmental volunteering (CEV) participants and the differences in physical functions and daily activity patterns between the older adults who engaged in intensive CEV (≥15 hours/week) and non-intensive CEV (<15 hours/week) groups. DESIGN: Cross-sectional study. SETTING: Three recycling stations in Taiwan. SAMPLE: In total, 113 community-dwelling older adults who regularly participated in CEV. The response rate was 53%. MEASURES: The ActiGraph wGT3x-BT accelerometer for the percentage of sedentary, light, and moderate to vigorous physical activity (MVPA) of CEV time and awaken time; the Jamar hand dynamometer for grip strength; and the MicroFET3 muscle testing dynamometer for knee extension strength. ANALYSIS: Analysis of covariance with the baseline characteristics as covariates. RESULTS: Overall, MVPA, light, and sedentary activities accounted for 53.73%, 41.10%, and 5.23% of CEV time, respectively. The intensive group (n = 61) displayed greater dominant handgrip strength (P = .004) and higher MVPA percentage in daily life (P = .044) than the non-intensive group (n = 52). CONCLUSION: CEV provides sufficient opportunities for older adults to perform physical activity. Intensive CEV is related to greater handgrip strength but not lower limb strength. Further study is needed to establish the causal relationship between CEV and health variates.

Methods for Functional Characterization of FcRn Interactions with Therapeutic Antibodies and Fc-Fusion Proteins.

The neonatal Fc receptor (FcRn) plays a key role in determining the pharmacokinetic behavior of therapeutic monoclonal antibodies (mAbs). FcRn-mediated intracellular trafficking mechanisms extend the half-lives of mAbs by rescuing them from lysosomal degradation and contribute to their transportation from the vascular space to tissue compartments such as placenta and mucosal surfaces. It is important to characterize the FcRn interactions of therapeutic mAbs and Fc-fusion proteins due to its potential impact on their in vivo pharmacokinetic properties such as clearance and half-life. In this chapter, we describe protocols for two cell-based assays that measure the total function of FcRn which involves pH-dependent association and dissociation with IgG-Fc, as well as FcRn-mediated intracellular trafficking parameters. These assays are suitable for characterization of FcRn interactions with therapeutic mAbs and Fc-fusion proteins for the purpose of assessing lot-to-lot consistency and the structural and functional integrity of the Fc domain. In addition, they may serve as cost-effective screening tools for the evaluation of mAb-based drug candidates during lead selection and optimization for desired pharmacokinetic properties.

Method for Measurement of Antibody-Dependent Cellular Phagocytosis.

Antibody-based therapeutics are powerful tools to treat disease. While their mechanism of action (MOA) always involves binding to a specific target via the antibody-binding fragment (Fab) region of the antibody, the induction of immune-mediated effector functions through the fragment crystallizable (Fc) region is a vital aspect of antibody therapeutics targeting tumor cells. Cross-linking of the Fc gamma receptors (FcγRs) via cell-bound antibodies activate immune effector cells, leading to antibody-dependent cellular cytotoxicity via natural killer (NK) cells. Linking of FcγRs on macrophages triggers the process of antibody-dependent cellular phagocytosis (ADCP), where antibody-opsonized target cells are internalized in phagosomes and degraded through the process of phagosome maturation and acidification. ADCP activity can be challenging to measure accurately due to the difficulty in differentiating target cells that are bound to a macrophage versus those that are internalized within phagosomes. In this chapter, we describe a protocol that measures ADCP activity by labeling target cells with a pH-sensitive dye that fluoresces brightly in mature phagosomes. The ADCP activity of therapeutics is then measured via flow cytometry. This assay is capable of detecting glycosylation differences arising from manufacturing processes and is suitable for evaluation of ADCP activity of monoclonal antibodies (mAb) to support in vitro biological characterization of drug candidates and lead candidate selection for desirable effector functions.

Hydroxychloroquine inhibits the mitochondrial antioxidant system in activated T cells.

Although hydroxychloroquine (HCQ) has long been used to treat autoimmune diseases, its mechanism of action remains poorly understood. In CD4 T-cells, we found that a clinically relevant concentration of HCQ inhibited the mitochondrial antioxidant system triggered by TCR crosslinking, leading to increased mitochondrial superoxide, impaired activation-induced autophagic flux, and reduced proliferation of CD4 T-cells. In antigen-presenting cells, HCQ also reduced constitutive activation of the endo-lysosomal protease legumain and toll-like receptor 9, thereby reducing cytokine production, but it had little apparent impact on constitutive antigen processing and peptide presentation. HCQ's effects did not require endo-lysosomal pH change, nor impaired autophagosome-lysosome fusion. We explored the clinical relevance of these findings in patients with celiac disease-a prototypic CD4 T-cell-mediated disease-and found that HCQ limits ex vivo antigen-specific T cell responses. We report a T-cell-intrinsic immunomodulatory effect from HCQ and suggest potential re-purposing of HCQ for celiac disease.

Axial variation in flexural stiffness of plant stem segments: measurement methods and the influence of measurement uncertainty.

BACKGROUND: Flexural three-point bending tests are useful for characterizing the mechanical properties of plant stems. These tests can be performed with minimal sample preparation, thus allowing tests to be performed relatively quickly. The best-practice for such tests involves long spans with supports and load placed at nodes. This approach typically provides only one flexural stiffness measurement per specimen. However, by combining flexural tests with analytic equations, it is possible to solve for the mechanical characteristics of individual stem segments. RESULTS: A method is presented for using flexural tests to obtain estimates of flexural stiffness of individual segments. This method pairs physical test data with analytic models to obtain a system of equations. The solution of this system of equations provides values of flexural stiffness for individual stalk segments. Uncertainty in the solved values for flexural stiffness were found to be strongly dependent upon measurement errors. Row-wise scaling of the system of equations reduced the influence of measurement error. Of many possible test combinations, the most advantageous set of tests for performing these measurements were identified. Relationships between measurement uncertainty and solution uncertainty were provided for two different testing methods. CONCLUSIONS: The methods presented in this paper can be used to measure the axial variation in flexural stiffness of plant stem segments. However, care must be taken to account for the influence of measurement error as the individual segment method amplifies measurement error. An alternative method involving aggregate flexural stiffness values does not amplify measurement error, but provides lower spatial resolution.