Intracellular Research Articles

Development of bioassay platforms for biopharmaceuticals using Jurkat-CAR cells by AICD

The assessment of bioactivity for therapeutic antibody release assay poses challenges, particularly when targeting immune checkpoints. An in vitro bioassay platform was developed using the chimeric antigen receptor on Jurkat cells (Jurkat-CAR) to analyze antibodies targeting immune checkpoints, such as CD47/SIRPα, VEGF/VEGFR1, PD-1/PD-L1, and CD70/CD27. For CD47/SIRPα, the platform involved a Jurkat-CAR cell line expressing the chimeric SIRPα receptor (CarSIRPα). CarSIRPα was created by sequentially fusing the SIRPα extracellular region with the CD8α hinge region, the transmembrane (TM) and intracellular (IC) domains of CD28, and the intracellular signaling domain of CD3ζ. The resulting Jurkat-CarSIRPα cells can undergo “activation-induced cell death (AICD)” upon incubation with purified or cellular CD47, as evidenced by the upregulation of CD69, IL-2, and IFN-γ. Similar results also appeared in Jurkat CarVEGFR1, Jurkat CarPD1 and Jurkat CARCD27 cells. These cells are perfectly utilized for the bioactivity analysis of therapeutic antibody. Our study indicates that the established in vitro assay platform based on Jurkat-CAR has been confirmed repeatedly and has shown robust reproducibility; thus, this platform can be used for screening or for release assays of given antibody drugs targeting immune checkpoints.

Abstract LB291: High-plexy intracellular and surface CITE-Seq offers multiomic insights into cell function

Abstract CITE-Seq allows researchers to combine high-plex protein information with whole transcriptome sequencing to gain insights about individual cell states. Due to the harsh fixation and permeabilization required for intracellular (IC) antibody staining, only a few studies have recovered mRNA expression profiles alongside IC protein detection. This experiment utilized a novel IC CITE-Seq assay that enables robust profiling of multiple IC protein targets in combination with mRNA and high-plex surface proteins. In this study, we stimulated human peripheral blood mononuclear cells (PBMCs) alongside a resting control. After stimulation, we stained the surface proteins with a 40-plex BD® AbSeq Panel, including the Immune Discovery Panel and human Single-Cell Multiplexing Kit (SMK). Resting and stimulated cells were pooled, and a portion of the sample pool immediately underwent cell capture as a live control using a BD Rhapsody™ single-cell microwell system. The other portion of the pool was stored in BD® OMICS-Guard Sample Preservation Buffer for either 5 minutes or 24 hours followed by permeabilization, IC staining with a 10-plex IC AbSeq Panel (50-plex total, surface and IC), and cells captured on the the same system. We show IC protein detection for the expected positive targets, including pH2AX, actCaspase-3, and cPARP in the stimulated population, as well as T-Bet, Granzyme B and Helios in the resting lymphocytes. Analysis shows excellent correspondence between IC CITE-Seq protein and transcriptome for T-Bet, Granzyme B and Helios. The gene expression correlation for surface antigen and mRNA between the IC and live control each had R2 values > 0.9. The mRNA sensitivity for the IC sample compared to live control was within whole transcriptome analysis (WTA) assay variation at 86–89% and 95–97% median molecules and genes per cell. Our data showed that the new IC CITE-Seq assay can provide rich single-cell multiomics information including mRNA and surface and intracellular proteins. For Research Use Only. Not for use in diagnostic or therapeutic procedures.BD, the BD Logo and BD Rhapsody are trademarks of Becton, Dickinson and Company or itsaffiliates. © 2023 BD. All rights reserved. NPM-2532 (v1.0) 1023 Citation Format: Tracy L. Campbell, Adam Wright, Ying Wah Lee, Anne Tran, Yan Chen, Joe Olives, Hye-Won Song, Larry Wang. High-plexy intracellular and surface CITE-Seq offers multiomic insights into cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB291.

Reduced White Matter Fiber Density in Patients with Multiple Sclerosis.

Introduction: Improved understanding of multiple sclerosis (MS) symptomatology, disease mechanisms, and clinical effectiveness can be achieved by investigating microstructural damage. The aim was to gain deeper insights into changes in white matter (WM) tracts in MS patients. Methods: Diffusion magnetic resonance imaging-based tractography was utilized to segment WM tracts into regions of interest for further quantitative analysis. However, tractography is susceptible to false-positive findings, reducing its specificity and clinical feasibility. To address these limitations, the Convex Optimization Modeling for Microstructure Informed Tractography (COMMIT) technique was used. COMMIT was used to derive measures of intracellular compartment (IC) and isotropic compartments from multishell diffusion data of 40 healthy controls (HCs) and 40 MS patients. Results: The analysis revealed a widespread pattern of significantly decreased IC values in MS patients compared with HCs across 61,581 voxels (pFWE < 0.05, threshold-free cluster enhancement [TFCE] corrected). Similar WM structures studied using the fractional anisotropy (FA) value also showed a reduction in FA among MS patients compared with HCs across 57,304 voxels (pFWE < 0.05, TFCE corrected). Out of the 61,581 voxels exhibiting lower IC, a substantial overlap of 47,251 voxels (76.72%) also demonstrated lower FA in MS patients compared with HCs. Discussion: The data suggested that lower IC values contributed to the explanation of FA reductions. In addition, IC showed promising potential for evaluating microstructural abnormalities in WM in MS, potentially being more sensitive than the frequently used FA value.

Effects of Lactobacillus rhamnosus DSM7133 on Intestinal Porcine Epithelial Cells.

Antimicrobial resistance is one of the biggest health challenges nowadays. Probiotics are promising candidates as feed additives contributing to the health of the gastrointestinal tract. The beneficial effect of probiotics is species/strain specific; the potential benefits need to be individually assessed for each probiotic strain or species. We established a co-culture model, in which gastrointestinal infection was modeled using Escherichia coli (E. coli) and Salmonella enterica serovar Typhimurium (S. enterica serovar Typhimurium). Using intestinal porcine epithelial cells (IPEC-J2), the effects of pre-, co-, and post-treatment with Lactobacillus (L.) rhamnosus on the barrier function, intracellular (IC) reactive oxygen species (ROS) production, proinflammatory cytokine (IL-6 and IL-8) response, and adhesion inhibition were tested. E. coli- and S. Typhimurium-induced barrier impairment and increased ROS production could be counteracted using L. rhamnosus (p < 0.01). S. Typhimurium-induced IL-6 production was reduced via pre-treatment (p < 0.05) and post-treatment (p < 0.01); increased IL-8 secretion was decreased via pre-, co-, and post-treatment (p < 0.01) with L. rhamnosus. L. rhamnosus demonstrated significant inhibition of adhesion for both S. Typhimurium (p < 0.001) and E. coli (p < 0.001 in both pre-treatment and post-treatment; p < 0.05 in co-treatment). This study makes a substantial contribution to the understanding of the specific benefits of L. rhamnosus. Our findings can serve as a basis for further in vivo studies carried out in pigs and humans.

Twin Molecular Docking for Intra Cellular Electric Field Sensing and Modulation - A Literature Review - A New Concept in Therapeutics

Twin Molecular Docking for Intra Cellular Electric Field Sensing and Modulation - A Literature Review - A New Concept in Therapeutics

Doravirine and Rilpivirine Intra Cellular Accumulation in the Clinical Setting

Background: Doravirine (DOR) and Rilpivirine (RPV) are the NNRTIs currently most used in the clinical setting, in dual and triple drug regimens (2DR and 3DR). Intracellular (IC) Pharmacokinetics (PK) of these drugs has not been fully elucidated. Our aim was to compare plasma PK and IC accumulation in real-life experienced patients (pts). Methods: Pts on DOR- and RPV-including Antiretroviral (ARV) regimen were considered. DOR and RPV plasma and IC (PBMCs) concentrations were measured at 12 (37%) (T12) and 24 ± 4 hours (63%) (T24) after last drug intake by means of UHPLC-MSMS validated methods. Results: 90 pts (65% on 3DR and 35% on 2DR) were included: 52% on DOR- and 48% on RPV-containing ARV. RPV IC/plasma ratio was significantly higher than DOR IC/plasma ratio: 6.034 (4.878-7.186) vs. 1.479 (1.256-1.702) (p=0.001) independently from timing T12 (p=0.003) and T24 (p&lt;0.001). RPV in 3DR resulted to have higher plasma and IC accumulation compared to 2DR. Linear and significative correlations between DOR and RPV plasma and IC concentrations were found (+0.749, p&lt;0.001 and +0.733, p&lt;0.001). No significative correlation between overall DOR and RPV PK and creatinine, BMI or age or difference by gender was found. Conclusion: RPV proved to accumulate in PBMCs at a higher degree as compared to DOR: RPV and DOR IC levels were 498% and 50% higher than in plasma. RPV showed an IC PBMC/plasma ratio 3-fold higher than DOR. Potential explanation could rely on the higher lipophilicity of RPV. Clinical significance of these data needs to be investigated in further studies.

1698. Pharmacokinetics/pharmacodynamics of Epetraborole, a Novel Bacterial Leucyl-tRNA Synthetase Inhibitor, and High Intracellular Penetration in the Intracellular Hollow Fiber System Model of Mycobacterium avium Complex Lung Disease

Abstract Background Epetraborole (EBO) is a boron-containing, oral inhibitor of bacterial leucyl-tRNA synthetase, an essential enzyme in protein synthesis; EBO demonstrates potent activity against nontuberculous mycobacteria. Dose-effect studies in the intracellular hollow fiber system model of Mycobacterium avium complex (MAC) lung disease, or HFS-MAC, have demonstrated that EBO is highly bactericidal. In this study we used dose fractionation in the HFS-MAC system to find the best pharmacokinetic/ pharmacodynamic (PK/PD) index that was associated with EBO microbial kill and acquired microbial resistance (AMR). Methods A dose-fractionation design was used to dose 30 HFS-MAC units. ED20, ED50 and ED80 doses were administered as one of 4 dose schedules of twice daily, once daily, twice weekly, or once weekly at a half-life of 10.4h. Total MAC burden and EBO-resistant MAC burden were determined every 7 days by culturing HFS-MAC contents on Middlebrook 7H10 agar. For drug concentrations, the central compartment of each HFS-MAC unit was sampled throughout the 28 days, and after the final dose, intracellular (IC) PK in the MAC-infected monocytes were established. EBO PK/PD index parameters including 0-168h area under the concentrations-time curve (AUC0-168), peak concentration, or % of time concentration persists above the intracellular MIC (%TMIC), which was 0.5 mg/L, were modeled versus total MAC burden using the inhibitory sigmoid maximal effect (Emax) model. The PK/PD index was chosen using Akaike Information Criteria (AIC). For AMR, EBO PK/PD index exposure value versus EBO-resistant MAC burden were modeled using a quadratic function, and the best model was chosen using AIC. Results The measured EBO concentrations demonstrated PKs shown in Figure 1. The median EBO IC versus extracellular (IC/EC) elimination rate constants were 0.018h-1 versus 0.061h-1 (p&amp;lt; 0.0001), which means slower IC EBO elimination, leading to IC/EC to AUC ratio of 11. Based on AIC scores in Table 1, the PK/PD driver for microbial kill was unequivocally AUC on each sampling day. For AMR the PK/PD driver was also AUC. Table 1.Corrected Akaike Information Criteria (AIC) Scores for PK/PD Indicesa-The intracellular EBO MIC was 0.5 mg/LFigure 1.Epetraborole concentrations achieved in the HFS-MAC during the 168-hour dosing interval. Epetraborole concentrations were measured for each of the four dosing schedules, and are shown together with compartmental pharmacokinetics modeling during the 168-hour dosing interval. A. Twice a day dosing. B. Once a day dosing. C. Once every 3.5 days. D. Once every 7 days. Conclusion The PK/PD index best associated with EBO microbial kill and AMR was AUC. Disclosures Shruti Athale, PhD, Praedicare Inc.: Stocks/Bonds Moti Chapagain, MD, PhD, Praedicare Inc.: Stocks/Bonds Jotam Pasipanodya, MD, PhD, Praedicare Inc.: Employee MRK Alley, PhD, ABBOTT LABS: Stocks/Bonds|ABBVIE: Stocks/Bonds|AN2 Therapeutics: Author on epetraborole patent|AN2 Therapeutics: Salary|AN2 Therapeutics: Ownership Interest|AVANOS MED INC: Stocks/Bonds|NABRIVA THERAPEUTICS PLC: Stocks/Bonds|NOVARTIS AG: Stocks/Bonds Tawanda Gumbo, MD, Hydronium Biopharma: Ownership Interest|Hydronium Biopharma: Stocks/Bonds|Praedicare Africa Pvt Ltd: Board Member|Praedicare Africa Pvt Ltd: Ownership Interest|Praedicare Africa Pvt Ltd: Stocks/Bonds|Praedicare Inc.: Patents|Praedicare Inc.: Ownership Interest|Praedicare Inc.: Stocks/Bonds.

Treatment Outcome of Autoimmune Associated Epilepsy (AAE) vs Acute Symptomatic Seizures in Autoimmune Encephalitis (ASSAE)–A Single Center Experience

Objective To assess the seizure treatment outcome in autoimmune encephalitis (AE). Background Seizures due to AE etiology are increasingly recognized. Timely confirmation of autoimmune etiology may lead to improved outcomes by allowing for earlier immunotherapy, which is more effective than anti-seizure medication (ASM). Currently, seizures/epilepsy of autoimmune etiology can be subcategorized as autoimmune associated epilepsy or acute symptomatic seizures in autoimmune encephalitis with varied response to immunotherapy. Design/Methods This was a retrospective single center study including patients diagnosed with AE, with a minimum follow-up of 12 months after disease onset in alive cases. Results 28 out of 31 patients were analyzed (3 without seizure were excluded). 12 were found to have autoantibodies (Abs) against cell membrane (CM) protein (3 anti-NMDAR, 4 anti-GABAbR, 4 anti-LGI-1, 1 anti-GABAaR); 4 with non-specific Abs (low titer of anti-GAD, VGCC); 11 with no Abs identified; 3 were with intracellular (IC) Abs (MOG, Hu, GFAP). All patients received immunotherapy and ASM treatment. Among the patients with CM-Abs, 10 of the 12 remained seizure free, 7 were successfully weaned off ASM. Among those with nonspecific/no Abs, only 4 of the 15 patients (26.7%) remained seizure free. All 3 patients with intracellular Abs did poorly, and only one patient with anti-MOG-Abs survived with drug resistant epilepsy (DRE). Within the group of CM-Abs, 2 patients (16.7%) with anti-GABAbR-Abs developed DRE, compared to over 70% of patients in group with nonspecific/no Abs or IC-Abs. Upon further investigation of DRE patients, several features were observed: 1) poor response to immunotherapy, 2) sustained abnormal brain MRI T2/FLAIR signal, 3) persistent focal epileptiform features and frequent ictal patterns in EEGs. Conclusions Patients with CM-Abs likely suffer ASSAE with favorable long-term outcome. Patients without identified auto-Abs or IC-Abs likely develop AAE and consequently develop DRE. Further research focusing on biomarkers predicting AAE and DRE is needed for treatment guidance.

Luteolin and Chrysin Could Prevent E. coli Lipopolysaccharide-Ochratoxin A Combination-Caused Inflammation and Oxidative Stress in In Vitro Porcine Intestinal Model.

Simple SummaryEnvironmental stressors, such as mycotoxins and enteropathogenic bacteria, can exert a detrimental effect on the gastrointestinal tract of the swine on contaminated corn-based feed which leads to the development of retarded growth; weight loss, thus, contributes greatly to lowered meat productivity. Plant-derived compounds such as flavonoid-type chrysin and luteolin were investigated in porcine enterocytes to evaluate their beneficial properties in gut damage. Toxic effects of ochratoxin A mycotoxin and bacterial outer membrane component, lipopolysaccharide could not be alleviated by the applied flavonoids even in combinations. However, luteolin and chrysin could prevent the excessive inflammatory responses by lowering cytokines’ production elicited by mycotoxin and bacterial toxin. Due to their antioxidant properties, these flavonoids were effective in compensating redox homeostasis tipped by combined exposure of ochratoxin A and inflammatory lipopolysaccharide. The porcine intestinal epithelial cell model applied by us was proven to be suitable for studying the harmful effects of the toxins and at the same time to evaluate the protective effects of flavonoids without living animal sacrifices. This study also suggests that mycotoxin-weakened intestinal function might result in the development of enteropathogen-caused secondary infections.Ochratoxin A (OTA) and lipopolysaccharide (LPS) intake can cause gastrointestinal disorders. Polyphenolic chrysin (CHR) and luteolin (LUT) display anti-inflammatory and antioxidant properties. Porcine intestinal epithelial (jejunal) IPEC-J2 cells were treated with OTA (1 µM, 5 µM and 20 µM), E. coli LPS (10 µg/mL), CHR (1 µM) and LUT (8.7 µM) alone and in their combinations. Cell viabilities (MTS assay) and extracellular (EC) hydrogen-peroxide (H2O2) production (Amplex red method) were evaluated. Intracellular (IC) reactive oxygen species (ROS) were assessed using a 2′-7′dichlorodihydrofluorescein diacetate (DCFH-DA) procedure. ELISA assay was used to evaluate IL-6 and IL-8 secretion. OTA decreased cell viabilities (p < 0.001) which could not be alleviated by LUT or CHR (p > 0.05); however, EC H2O2 production was successfully suppressed by LUT in IPEC-J2 cells (p < 0.001). OTA with LPS elevated the IC ROS which was counteracted by CHR and LUT (p < 0.001). IL-6 and IL-8 secretion was elevated by LPS + OTA (p < 0.001) which could be inhibited by LUT (p < 0.01 for IL-6; p < 0.001 for IL-8). Based on our results, CHR and LUT exerted beneficial effects on IC ROS levels and on cytokine secretion (LUT) in vitro; thus, they might be used as dietary and feed supplements to avoid OTA- and LPS-related health risks.

The role of the different CD3γ domains in TCR expression and signaling.

The CD3 subunits of the T-cell antigen receptor (TCR) play a central role in regulation of surface TCR expression levels. Humans who lack CD3γ (γ—) show reduced surface TCR expression levels and abolished phorbol ester (PMA)-induced TCR down-regulation. The response to PMA is mediated by a double leucine motif in the intracellular (IC) domain of CD3γ. However, the molecular cause of the reduced TCR surface expression in γ— lymphocytes is still not known. We used retroviral vectors carrying wild type CD3γ or CD3δ or the following chimeras (EC-extracellular, TM-transmembrane and IC): δECγTMγIC (δγγ for short), γγδ, γδδ and γγ-. Expression of γγγ, γγδ, γδδ or γγ- in the γ— T cell line JGN, which lacks surface TCR, demonstrated that cell surface TCR levels in JGN were dependent on the EC domain of CD3γ and could not be replaced by the one of CD3δ. In JGN and primary γ— patient T cells, the tested chimeras confirmed that the response to PMA maps to the IC domain of CD3γ. Since protein homology explains these results better than domain structure, we conclude that CD3γ contributes conformational cues that improve surface TCR expression, likely at the assembly or membrane transport steps. In JGN cells all chimeric TCRs were signalling competent. However, an IC domain at CD3γ was required for TCR-induced IL-2 and TNF-α production and CD69 expression, indicating that a TCR without a CD3γ IC domain has altered signalling capabilities.

Carbonic anhydrase IX proteoglycan-like and intracellular domains mediate pulmonary microvascular endothelial cell repair and angiogenesis.

The lungs of patients with acute respiratory distress syndrome (ARDS) have hyperpermeable capillaries that must undergo repair in an acidic microenvironment. Pulmonary microvascular endothelial cells (PMVECs) have an acid-resistant phenotype, in part due to carbonic anhydrase IX (CA IX). CA IX also facilitates PMVEC repair by promoting aerobic glycolysis, migration, and network formation. Molecular mechanisms of how CA IX performs such a wide range of functions are unknown. CA IX is composed of four domains known as the proteoglycan-like (PG), catalytic (CA), transmembrane (TM), and intracellular (IC) domains. We hypothesized that the PG and CA domains mediate PMVEC pH homeostasis and repair, and the IC domain regulates aerobic glycolysis and PI3k/Akt signaling. The functions of each CA IX domain were investigated using PMVEC cell lines that express either a full-length CA IX protein or a CA IX protein harboring a domain deletion. We found that the PG domain promotes intracellular pH homeostasis, migration, and network formation. The CA and IC domains mediate Akt activation but negatively regulate aerobic glycolysis. The IC domain also supports migration while inhibiting network formation. Finally, we show that exposure to acidosis suppresses aerobic glycolysis and migration, even though intracellular pH is maintained in PMVECs. Thus, we report that 1) the PG and IC domains mediate PMVEC migration and network formation, 2) the CA and IC domains support PI3K/Akt signaling, and 3) acidosis impairs PMVEC metabolism and migration independent of intracellular pH homeostasis.

Characteristics of Chromophoric Dissolved Organic Matter (CDOM) Produced by Heterotrophic Bacteria Isolated from Aquaculture Systems

Heterotrophic bacteria (HB) play an important role in aquatic ecosystems as recyclers of dissolved organic matter (DOM). The objective of this study was to characterize the spectral characteristics of intracellular (IC), and extracellular (EC) compounds produced by 12 HB isolated from two aquaculture systems. Microorganisms belonging to the genera Bacillus, Paenibacillus, and Psychrobacillus were identified by analysis of the 16S ribosomal gene. Aliquots of bacterial culture were centrifugated every hour (1st to 7th) to obtain the EC compounds. The pellet was ultrasound-lysed to obtain the IC compounds. Excitation-emission matrices were used in combination with parallel factor analysis (PARAFAC) to characterize the fluorescent components of DOM (FDOM). PARAFAC indicated two protein-like components and two humic-like components in both cell spaces. At the IC, B. macquariensis showed a high fluorescence index (FI), probably associated with fulvic acid, quinones, or ketones. Psychrobacillus insolitus showed an inverse correlation between spectral slopes S275–295 and S350–400 in the EC and IC fractions, which may indicate differential release of low and high molecular weight molecules in these two fractions. The opposite occurred with B. licheniformis and P. alvei. The origin of FDOM in HB is an important finding of this work. The most significant amount of protein-like substances was produced at the IC level, with the humic- and fulvic-type at the EC. The main finding of this work is the evidence of differential production of humic-type or protein-type FDOM production by HB species from marine and freshwater aquaculture systems in their intracellular and extracellular fractions, as well different relative molecular weight. For aquaculture, these findings suggest that some bacterial species show promise in supplying essential amino acids to growing organisms, and others play a major role in nutrient exchange and the global carbon cycle.

Factors Influencing the Intracellular Concentrations of the Sofosbuvir Metabolite GS-331007 (in PBMCs) at 30 Days of Therapy.

Sofosbuvir (SOF) is an HCV NS5B polymerase inhibitor, and GS-331007 is its major metabolite. The aim of this study was to investigate whether clinical and pharmacological factors could influence GS-331007 intracellular (IC) concentrations in peripheral blood mononuclear cells (PBMCs) associated with a sustained virological response in patients treated with SOF and ribavirin (RBV). Drug levels were analyzed using liquid chromatography at different days of therapy, whereas variants in genes encoding transporters and nuclear factors were investigated using real-time PCR. This study enrolled 245 patients treated with SOF; 245 samples were analyzed for pharmacogenetics and 50 were analyzed for IC pharmacokinetics. The GS-331007 IC concentration at 30 days was associated with its plasma concentration determinate at 30, 60 and 90 days of SOF-therapy and with daclatasvir concentrations at 7 days of therapy. No genetic polymorphism affected IC exposure. In linear multivariate analysis, ledipasvir treatment, baseline albumin and estimated glomerular filtration rate were significant predictors of IC exposure. This study presents data on an IC evaluation in a cohort of patients treated with SOF, also considering pharmacogenetics. These results could be useful for regions where SOF–RBV treatment is considered the standard of care; moreover, they could further deepen the knowledge of IC exposure for similar drugs in the future.

Analysis of brain structural connectivity networks and white matter integrity in patients with mild cognitive impairment

AbstractBackgroundMild cognitive impairment (MCI) is considered a prodromal phase of Alzheimer’s disease (AD) and patients with MCI convert to AD at a rate of about 10–15% per year. Because of this high conversion rate to AD, studies of MCI may yield insight into early pathological changes in AD. Here, we used a novel approach to analyze structural connectivity and white matter (WM) integrity using fractional anisotropy (FA), intra‐cellular (IC), extra‐cellular (EC), and isotropic (ISO) maps between patients with MCI and healthy controls (HC).MethodThirty‐two HC (age mean ± S.D. = 69 ± 6 years; 22 females) and 20 MCI (71 ± 9 years; 7 females) were included in this study. All subjects completed the Mini‐Mental State Exam (MMSE) and the Montreal Cognitive Assessment (MoCA). Multi‐shell DTI were download from ADNI (http://adni.loni.usc.edu/). Pre‐processing was performed by Mrtrix3, FSL, and ANTs. Tractography was performed with 5 million seeds using the iFOD2 algorithm. Connectome was generated from the Desikan‐Killiany parcellation file and was subsequently filtered using the COMMIT2 algorithm. A three‐compartment Stick‐Zeppelin‐Ball model was used to create the IC, the EC, and the ISO compartments, respectively. Statistical analyses (ANCOVA) were performed by connectomestats (Mrtrix3), randomise (FLS), and by an in‐house Matlab script for Spearman’s correlations.ResultThe two groups did differ in MMSE and MoCA (Mann–Whitney U test: Z=2.031; p=0.021; Z=1.683; p=0.046, respectively). Differences between HC and MCI were found principally in the fornix for FA and in several WM areas for IC and ISO (Figure 1, Table 1). Connectometry analysis found differences between groups in two distinct tracts: Tract#1 between the left‐lateraloccipital and the left‐insula (t=4.25, p=0.023) and Tract#2 between the left‐middletemporal and the left‐parsopercularis (t=3.34, p=0.013) (Figure 2). Figure 3 and Table 2 show the results for the correlations between DTI metrics and the neurological scores.ConclusionIn this study, by using a multi‐shell diffusion MRI acquisition and robust processing of DTI data, we detected alterations in WM integrity and structural connectivity in MCI subjects. Our results demonstrate the potential of these advanced diffusion MRI methods as biomarkers to distinguish MCI and HC.

Carbonic Anhydrase IX and Hypoxia Promote Rat Pulmonary Endothelial Cell Survival during Infection.

Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. CA (carbonic anhydrase) IX is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics, and angiogenesis. We hypothesized that CA IX is important for PMVEC survival and that CA IX expression and release from PMVECs are increased during infection. Although the plasma concentration of CA IX was unchanged in human and rat pneumonia, there was a trend toward increasing CA IX in the bronchoalveolar fluid of mechanically ventilated critically ill patients with pneumonia and a significant increase in CA IX in the lung tissue lysates of pneumonia rats. To investigate the functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. By using these cells, we found that infection promotes intracellular (IC) expression, release, and MMP (metalloproteinase)-mediated extracellular cleavage of CA IX in PMVECs. IC domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX IC domain promoted cell death after infection, suggesting that the IC domain has an important role in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report 1) that CA IX increases in the lungs of pneumonia rats and 2) that the CA IX IC domain and hypoxia promote PMVEC survival during infection.

Psychomotor impairments and therapeutic implications revealed by a mutation associated with infantile Parkinsonism-Dystonia.

Parkinson disease (PD) is a progressive, neurodegenerative disorder affecting over 6.1 million people worldwide. Although the cause of PD remains unclear, studies of highly penetrant mutations identified in early-onset familial parkinsonism have contributed to our understanding of the molecular mechanisms underlying disease pathology. Dopamine (DA) transporter (DAT) deficiency syndrome (DTDS) is a distinct type of infantile parkinsonism-dystonia that shares key clinical features with PD, including motor deficits (progressive bradykinesia, tremor, hypomimia) and altered DA neurotransmission. Here, we define structural, functional, and behavioral consequences of a Cys substitution at R445 in human DAT (hDAT R445C), identified in a patient with DTDS. We found that this R445 substitution disrupts a phylogenetically conserved intracellular (IC) network of interactions that compromise the hDAT IC gate. This is demonstrated by both Rosetta molecular modeling and fine-grained simulations using hDAT R445C, as well as EPR analysis and X-ray crystallography of the bacterial homolog leucine transporter. Notably, the disruption of this IC network of interactions supported a channel-like intermediate of hDAT and compromised hDAT function. We demonstrate that Drosophila melanogaster expressing hDAT R445C show impaired hDAT activity, which is associated with DA dysfunction in isolated brains and with abnormal behaviors monitored at high-speed time resolution. We show that hDAT R445C Drosophila exhibit motor deficits, lack of motor coordination (i.e. flight coordination) and phenotypic heterogeneity in these behaviors that is typically associated with DTDS and PD. These behaviors are linked with altered dopaminergic signaling stemming from loss of DA neurons and decreased DA availability. We rescued flight coordination with chloroquine, a lysosomal inhibitor that enhanced DAT expression in a heterologous expression system. Together, these studies shed some light on how a DTDS-linked DAT mutation underlies DA dysfunction and, possibly, clinical phenotypes shared by DTDS and PD.

CD40 intracellular TRAF binding domains modulate B cell activation and experimental autoimmune encephalomyelitis development

Abstract CD40 is a costimulatory surface protein expressed on multiple immune cells, including B cells, where it is required for their full activation. Upon interacting with CD40 ligand, CD40 initiates downstream signaling that culminates in activation of naïve B cells. Previous work from our lab revealed that cell autonomous expression of CD40 on B cells regulates their activity, including germinal center development and antibody class switching. Intracellular (IC) domains of the CD40 protein are crucial for signaling. Several of these domains are known to be associated with TNF receptor associated factor proteins (TRAFs) which facilitate further downstream signaling. These include a TRAF6 binding domain and a TRAF2/3 binding domain. We analyzed the CD40 IC domain requirements for CD40 signaling in B cells and for induction of experimental autoimmune encephalomyelitis (EAE). To do so, multiple mouse strains were generated with elimination of the putative TRAF6 or TRAF2/3 domains by CRISPR technology. Whereas eliminating the putative TRAF6 binding site (Domain 1) had little to no effect on B cell viability and in vitro activation by anti-CD40 antibody, eliminating the putative TRAF2/3 binding site (Domain 2) impaired both these functions. Similarly, generation of high affinity IgG in vivo was also dependent on Domain 2 but not Domain 1. In contrast, both Domain 1 and Domain 2 were required for full development of EAE. Interestingly, mixed BM chimeras revealed that the ameliorating effect of CD40 mutations on EAE was mediated by hematopoietic derived non-B cells. Our results indicate that the CD40 TRAF2/3 binding Domain 2 is critical for CD40 dependent B cell function, while both Domain 1 and Domain 2 expression by other APCs function in EAE induction.

Flow Cytometric Methods for the Detection of Intracellular Signaling Proteins and Transcription Factors Reveal Heterogeneity in Differentiating Human B Cell Subsets.

The flow cytometric detection of intracellular (IC) signaling proteins and transcription factors (TFs) will help to elucidate the regulation of B cell survival, proliferation and differentiation. However, the simultaneous detection of signaling proteins or TFs with membrane markers (MMs) can be challenging, as the required fixation and permeabilization procedures can affect the functionality of conjugated antibodies. Here, a phosphoflow method is presented for the detection of activated NF-κB p65 and phosphorylated STAT1, STAT3, STAT5 and STAT6, together with the B cell differentiation MMs CD19, CD27 and CD38. Additionally, a TF-flow method is presented that allows the detection of the B cell TFs PAX5, c-MYC, BCL6 and AID and antibody-secreting cell (ASC) TFs BLIMP1 and XBP-1s, together with MMs. Applying these methods on in vitro-induced human B cell differentiation cultures showed significantly different steady-state levels, and responses to stimulation, of phosphorylated signaling proteins in CD27-expressing B cell and ASC populations. The TF-flow protocol and Uniform Manifold Approximation and Projection (UMAP) analysis revealed heterogeneity in TF expression within stimulated CD27- or CD38-expressing B cell subsets. The methods presented here allow for the sensitive analysis of STAT, NF-κB p65 signaling and TFs, together with B cell differentiation MMs, at single-cell resolution. This will aid the further investigation of B cell responses in both health and disease.

Fusion of a Novel Native Signal Peptide Enhanced the Secretion and Solubility of Bioactive Human Interferon Gamma Glycoproteins in Nicotiana benthamiana Using the Bamboo Mosaic Virus-Based Expression System.

Plant viruses may serve as expression vectors for the efficient production of pharmaceutical proteins in plants. However, the downstream processing and post-translational modifications of the target proteins remain the major challenges. We have previously developed an expression system derived from Bamboo mosaic virus (BaMV), designated pKB19, and demonstrated its applicability for the production of human mature interferon gamma (mIFNγ) in Nicotiana benthamiana. In this study, we aimed to enhance the yields of soluble and secreted mIFNγ through the incorporation of various plant-derived signal peptides. Furthermore, we analyzed the glycosylation patterns and the biological activity of the mIFNγ expressed by the improved pKB19 expression system in N. benthamiana. The results revealed that the fusion of a native N. benthamiana extensin secretory signal (SSExt) to the N-terminal of mIFNγ (designated SSExt mIFNγ) led to the highest accumulation level of protein in intracellular (IC) or apoplast washing fluid (AWF) fractions of N. benthamiana leaf tissues. The addition of 10 units of ‘Ser-Pro’ motifs of hydroxyproline-O-glycosylated peptides (HypGPs) at the C-terminal end of SSExt mIFNγ (designated SSExt mIFNγ(SP)10) increased the solubility to nearly 2.7- and 1.5-fold higher than those of mIFNγ and SSExt mIFNγ, respectively. The purified soluble SSExt mIFNγ(SP)10 protein was glycosylated with abundant complex-type N-glycan attached to residues N56 and N128, and exhibited biological activity against Sindbis virus and Influenza virus replication in human cell culture systems. In addition, suspension cell cultures were established from transgenic N. benthamiana, which produced secreted SSExt mIFNγ(SP)10 protein feasible for downstream processing. These results demonstrate the applicability of the BaMV-based vector systems as a useful alternative for the production of therapeutic proteins, through the incorporation of appropriate fusion tags.

MnDPDP: Contrast Agent for Imaging and Protection of Viable Tissue.

The semistable chelate manganese (Mn) dipyridoxyl diphosphate (MnDPDP, mangafodipir), previously used as an intravenous (i.v.) contrast agent (Teslascan™, GE Healthcare) for Mn-ion-enhanced MRI (MEMRI), should be reappraised for clinical use but now as a diagnostic drug with cytoprotective properties. Approved for imaging of the liver and pancreas, MnDPDP enhances contrast also in other targets such as the heart, kidney, glandular tissue, and potentially retina and brain. Transmetallation releases paramagnetic Mn2+ for cellular uptake in competition with calcium (Ca2+), and intracellular (IC) macromolecular Mn2+ adducts lower myocardial T1 to midway between native values and values obtained with gadolinium (Gd3+). What is essential is that T1 mapping and, to a lesser degree, T1 weighted imaging enable quantification of viability at a cellular or even molecular level. IC Mn2+ retention for hours provides delayed imaging as another advantage. Examples in humans include quantitative imaging of cardiomyocyte remodeling and of Ca2+ channel activity, capabilities beyond the scope of Gd3+ based or native MRI. In addition, MnDPDP and the metabolite Mn dipyridoxyl diethyl-diamine (MnPLED) act as catalytic antioxidants enabling prevention and treatment of oxidative stress caused by tissue injury and inflammation. Tested applications in humans include protection of normal cells during chemotherapy of cancer and, potentially, of ischemic tissues during reperfusion. Theragnostic use combining therapy with delayed imaging remains to be explored. This review updates MnDPDP and its clinical potential with emphasis on the working mode of an exquisite chelate in the diagnosis of heart disease and in the treatment of oxidative stress.