Neuroinflammatory disease disrupts the blood-CNS barrier via crosstalk between proinflammatory and endothelial-to-mesenchymal-transition signaling.

Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1ß-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1ß-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.

Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

Development of accurate disease models and discovery of immune-modulating drugs is challenged by the immune systems highly interconnected and context-dependent nature. Here we apply deep-learning-driven analysis of cellular morphology to develop a scalable "phenomics" platform and demonstrate its ability to identify dose-dependent, high-dimensional relationships among and between immunomodulators, toxins, pathogens, genetic perturbations, and small and large molecules at scale. High-throughput screening on this platform demonstrates rapid identification and triage of hits for TGF-{beta}- and TNF--driven phenotypes. We deploy the platform to develop phenotypic models of active SARS-CoV-2 infection and of COVID-19-associated cytokine storm, surfacing compounds with demonstrated clinical benefit and identifying several new candidates for drug repurposing. The presented library of images, deep learning features, and compound screening data from immune profiling and COVID-19 screens serves as a deep resource for immune biology and cellular-model drug discovery with immediate impact on the COVID-19 pandemic.

Accuracy and Vividness in Motor Imagery Ability: Differences between Children and Young Adults.

Motor imagery (MI) refers to the imagination of a motor task without actual movement execution. The purpose of this study was to compare MI accuracy and vividness, and motor proficiency between children (n = 101; 7-12 years) and young adults (n = 140; 18-25 years). Results indicated that young adults were significantly more accurate and rated their MI significantly more vivid than children. For MI accuracy, between-subject effects showed that young adults had higher scores than children on three of the four subscales and the action subscale significantly predicted motor proficiency. These findings indicate that MI ability continues to develop into adulthood.

Identification of potential treatments for COVID-19 through artificial intelligence-enabled phenomic analysis of human cells infected with SARS-CoV-2

To identify potential therapeutic stop-gaps for SARS-CoV-2, we evaluated a library of 1,670 approved and reference compounds in an unbiased, cellular image-based screen for their ability to suppress the broad impacts of the SARS-CoV-2 virus on phenomic profiles of human renal cortical epithelial cells using deep learning. In our assay, remdesivir is the only antiviral tested with strong efficacy, neither chloroquine nor hydroxychloroquine have any beneficial effect in this human cell model, and a small number of compounds not currently being pursued clinically for SARS-CoV-2 have efficacy. We observed weak but beneficial class effects of {beta}-blockers, mTOR/PI3K inhibitors and Vitamin D analogues and a mild amplification of the viral phenotype with {beta}-agonists.

Rapid single B cell antibody discovery using nanopens and structured light.

Accelerated development of monoclonal antibody (mAb) tool reagents is an essential requirement for the successful advancement of therapeutic antibodies in today's fast-paced and competitive drug development marketplace. Here, we describe a direct, flexible, and rapid nanofluidic optoelectronic single B lymphocyte antibody screening technique (NanOBlast) applied to the generation of anti-idiotypic reagent antibodies. Selectively enriched, antigen-experienced murine antibody secreting cells (ASCs) were harvested from spleen and lymph nodes. Subsequently, secreted mAbs from individually isolated, single ASCs were screened directly using a novel, integrated, high-content culture, and assay platform capable of manipulating living cells within microfluidic chip nanopens using structured light. Single-cell polymerase chain reaction-based molecular recovery on select anti-idiotypic ASCs followed by recombinant IgG expression and enzyme-linked immunosorbent assay (ELISA) characterization resulted in the recovery and identification of a diverse and high-affinity panel of anti-idiotypic reagent mAbs. Combinatorial ELISA screening identified both capture and detection mAbs, and enabled the development of a sensitive and highly specific ligand binding assay capable of quantifying free therapeutic IgG molecules directly from human patient serum, thereby facilitating important drug development decision-making. The ASC import, screening, and export discovery workflow on the chip was completed within 5 h, while the overall discovery workflow from immunization to recombinantly expressed IgG was completed in under 60 days.

Differences in accuracy and vividness of motor imagery in children with and without Developmental Coordination Disorder.

Motor imagery (MI) provides a unique window on the integrity of movement representation. Studies have shown that children with Developmental Coordination Disorder (DCD) experience problems with tasks thought to rely on an internal model of movements. Therefore, the purpose of this study was to compare MI accuracy and MI vividness between typically developing (TD) and children with DCD. Ninety-three children with ages between 7 and 12 years (TD: n = 51; DCD: n = 42) were tested with the Movement Imagery Questionnaire (MIQ-c) to assess MI vividness and the Florida Praxis Imagery Questionnaire (FPIQ) to assess MI accuracy. To compare differences between the groups for each assessment and in the subscales, two separate general linear model analyses were conducted: A 2 × 3 (Group [TD, DCD] × Subscales [internal visual imagery, external visual imagery, kinesthetic imagery]) for MI vividness and a 2 × 4 (Group [TD, DCD] × Subscales [position, object, kinesthetic, action]) for MI accuracy. Results indicated that children with DCD scored significantly lower (p < .05) on MI accuracy than TD children, but there were no significant differences between the groups on MI vividness. Additionally, there were significant differences in the subscales for both measurements of MI. Specifically, results showed lower scores overall for the kinesthetic subscale. These findings indicate that the MI deficit seen in children with DCD is probably associated with MI accuracy, not MI vividness. These results suggest the need of further exploration into specific measurements of MI in children with DCD.

Flow Cytometry-Based Epitope Binning Using Competitive Binding Profiles for the Characterization of Monoclonal Antibodies against Cellular and Soluble Protein Targets.

A key step in the therapeutic antibody drug discovery process is early identification of diverse candidate molecules. Information comparing antibody binding epitopes can be used to classify antibodies within a large panel, guiding rational lead molecule selection. We describe a novel epitope binning method utilizing high-throughput flow cytometry (HTFC) that leverages cellular barcoding or spectrally distinct beads to multiplex samples to characterize antibodies raised against cell membrane receptor or soluble protein targets. With no requirement for sample purification or direct labeling, the method is suited for early characterization of antibody candidates. This method generates competitive binding profiles of each antibody against a defined set of known or unknown reference antibodies for binding to epitopes of an antigen. Antibodies with closely related competitive binding profiles indicate similar epitopes and are classified in the same bin. These large, high-throughput, multiplexed experiments can yield epitope bins or clusters for the entire antibody panel, from which a conceptual map of the epitope space for each antibody can be created. Combining this valuable epitope information with other data, such as functional activity, sequence, and selectivity of binding to orthologs and paralogs, enables us to advance the best epitope-diverse candidates for further development.

High-throughput screening of hybridoma supernatants using multiplexed fluorescent cell barcoding on live cells.

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules.

Does efavirenz replacement improve neurological function in treated HIV infection?

OBJECTIVES: The contribution of specific antiretroviral drugs to cognitive function in HIV-infected people remains poorly understood. Efavirenz (EFV) may plausibly cause cognitive impairment. The objective of this study was therefore to determine whether chronic EFV therapy is a modifier of neurocognitive and neurometabolic function in the setting of suppressive highly active antiretroviral therapy. METHODS: We performed an open-label phase IV controlled trial. Adult subjects who were stable on suppressive EFV therapy for at least 6 months were switched to ritonavir-boosted lopinavir (LPV/r) with no change in the nucleoside reverse transcriptase inhibitor (NRTI) backbone. The following parameters were assessed before and 10 weeks after therapy switch: cognitive function (by CogState® computerized battery); brain metabolites (by proton magnetic resonance spectroscopy); brain activity [by attentional processing task-based functional magnetic resonance imaging]; and sleep quantity and quality [by sleep diary, Pittsburgh Sleep Quality Index (PSQI) and Epworth Sleepiness Scale]. RESULTS: Sixteen subjects completed the study. Despite most subjects (81%) self-reporting memory problems at baseline, cognitive function, brain metabolites, and brain activity showed no change at 10 weeks after switch. Sleep quality improved on switch off EFV [mean PSQI (standard deviation): EFV, 8.5 (6.5); LPV/r, 5.8 (5.5); mean difference -0.4; 95% confidence interval -6.0 to -0.7]. CONCLUSIONS: This is the first study to assess the effects of chronic EFV therapy on neurological function in a controlled setting. We conclude that EFV withdrawal is unlikely to result in significant modification of neurocognitive function in otherwise stable HIV-infected people.

Autologous tolerogenic dendritic cells for rheumatoid and inflammatory arthritis.

OBJECTIVES: To assess the safety of intra-articular (IA) autologous tolerogenic dendritic cells (tolDC) in patients with inflammatory arthritis and an inflamed knee; to assess the feasibility and acceptability of the approach and to assess potential effects on local and systemic disease activities. METHODS: An unblinded, randomised, controlled, dose escalation Phase I trial. TolDC were differentiated from CD14+ monocytes and loaded with autologous synovial fluid as a source of autoantigens. Cohorts of three participants received 1×106, 3×106 or 10×106 tolDC arthroscopically following saline irrigation of an inflamed (target) knee. Control participants received saline irrigation only. Primary outcome was flare of disease in the target knee within 5 days of treatment. Feasibility was assessed by successful tolDC manufacture and acceptability via patient questionnaire. Potential effects on disease activity were assessed by arthroscopic synovitis score, disease activity score (DAS)28 and Health Assessment Questionnaire (HAQ). Immunomodulatory effects were sought in peripheral blood. RESULTS: There were no target knee flares within 5 days of treatment. At day 14, arthroscopic synovitis was present in all participants except for one who received 10×106 tolDC; a further participant in this cohort declined day 14 arthroscopy because symptoms had remitted; both remained stable throughout 91 days of observation. There were no trends in DAS28 or HAQ score or consistent immunomodulatory effects in peripheral blood. 9 of 10 manufactured products met quality control release criteria; acceptability of the protocol by participants was high. CONCLUSION: IA tolDC therapy appears safe, feasible and acceptable. Knee symptoms stabilised in two patients who received 10×106 tolDC but no systemic clinical or immunomodulatory effects were detectable. TRIAL REGISTRATION NUMBER: NCT01352858.

Evaluation of recombinant monoclonal antibody SVmab1 binding to Na V1.7 target sequences and block of human Na V1.7 currents.

Identification of small and large molecule pain therapeutics that target the genetically validated voltage-gated sodium channel Na V1.7 is a challenging endeavor under vigorous pursuit. The monoclonal antibody SVmab1 was recently published to bind the Na V1.7 DII voltage sensor domain and block human Na V1.7 sodium currents in heterologous cells. We produced purified SVmab1 protein based on publically available sequence information, and evaluated its activity in a battery of binding and functional assays. Herein, we report that our recombinant SVmAb1 does not bind peptide immunogen or purified Na V1.7 DII voltage sensor domain via ELISA, and does not bind Na V1.7 in live HEK293, U-2 OS, and CHO-K1 cells via FACS. Whole cell manual patch clamp electrophysiology protocols interrogating diverse Na V1.7 gating states in HEK293 cells, revealed that recombinant SVmab1 does not block Na V1.7 currents to an extent greater than observed with an isotype matched control antibody. Collectively, our results show that recombinant SVmab1 monoclonal antibody does not bind Na V1.7 target sequences or specifically inhibit Na V1.7 current.

Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes.

In morphological profiling, quantitative data are extracted from microscopy images of cells to identify biologically relevant similarities and differences among samples based on these profiles. This protocol describes the design and execution of experiments using Cell Painting, which is a morphological profiling assay that multiplexes six fluorescent dyes, imaged in five channels, to reveal eight broadly relevant cellular components or organelles. Cells are plated in multiwell plates, perturbed with the treatments to be tested, stained, fixed, and imaged on a high-throughput microscope. Next, an automated image analysis software identifies individual cells and measures ∼1,500 morphological features (various measures of size, shape, texture, intensity, and so on) to produce a rich profile that is suitable for the detection of subtle phenotypes. Profiles of cell populations treated with different experimental perturbations can be compared to suit many goals, such as identifying the phenotypic impact of chemical or genetic perturbations, grouping compounds and/or genes into functional pathways, and identifying signatures of disease. Cell culture and image acquisition takes 2 weeks; feature extraction and data analysis take an additional 1-2 weeks.

Discovery and bio-optimization of human antibody therapeutics using the XenoMouse® transgenic mouse platform.

Since the late 1990s, the use of transgenic animal platforms has transformed the discovery of fully human therapeutic monoclonal antibodies. The first approved therapy derived from a transgenic platform--the epidermal growth factor receptor antagonist panitumumab to treat advanced colorectal cancer--was developed using XenoMouse(®) technology. Since its approval in 2006, the science of discovering and developing therapeutic monoclonal antibodies derived from the XenoMouse(®) platform has advanced considerably. The emerging array of antibody therapeutics developed using transgenic technologies is expected to include antibodies and antibody fragments with novel mechanisms of action and extreme potencies. In addition to these impressive functional properties, these antibodies will be designed to have superior biophysical properties that enable highly efficient large-scale manufacturing methods. Achieving these new heights in antibody drug discovery will ultimately bring better medicines to patients. Here, we review best practices for the discovery and bio-optimization of monoclonal antibodies that fit functional design goals and meet high manufacturing standards.

Dietary Vitamin D and Its Metabolites Non-Genomically Stabilize the Endothelium.

Vitamin D is a known modulator of inflammation. Native dietary vitamin D3 is thought to be bio-inactive, and beneficial vitamin D3 effects are thought to be largely mediated by the metabolite 1,25(OH)2D3. Reduced serum levels of the most commonly measured precursor metabolite, 25(OH)D3, is linked to an increased risk of multiple inflammatory diseases, including: cardiovascular disease, arthritis, multiple sclerosis, and sepsis. Common to all of these diseases is the disruption of endothelial stability and an enhancement of vascular leak. We previously performed an unbiased chemical suppressor screen on a genetic model of vascular instability, and identified cholecalciferol (D3, dietary Vitamin D3) as a factor that had profound and immediate stabilizing and therapeutic effects in that model. In this manuscript we show that the presumed inactive sterol, D3, is actually a potent and general mediator of endothelial stability at physiologically relevant concentrations. We further demonstrate that this phenomenon is apparent in vitamin D3 metabolites 25(OH)D3 and 1,25(OH)2D3, and that the effects are independent of the canonical transcription-mediated vitamin D pathway. Our data suggests the presence of an alternative signaling modality by which D3 acts directly on endothelial cells to prevent vascular leak. The finding that D3 and its metabolites modulate endothelial stability may help explain the clinical correlations between low serum vitamin D levels and the many human diseases with well-described vascular dysfunction phenotypes.

Strategy for identifying repurposed drugs for the treatment of cerebral cavernous malformation.

BACKGROUND: Cerebral cavernous malformation (CCM) is a hemorrhagic stroke disease affecting up to 0.5% of North Americans that has no approved nonsurgical treatment. A subset of patients have a hereditary form of the disease due primarily to loss-of-function mutations in KRIT1, CCM2, or PDCD10. We sought to identify known drugs that could be repurposed to treat CCM. METHODS AND RESULTS: We developed an unbiased screening platform based on both cellular and animal models of loss of function of CCM2. Our discovery strategy consisted of 4 steps: an automated immunofluorescence and machine-learning-based primary screen of structural phenotypes in human endothelial cells deficient in CCM2, a secondary screen of functional changes in endothelial stability in these same cells, a rapid in vivo tertiary screen of dermal microvascular leak in mice lacking endothelial Ccm2, and finally a quaternary screen of CCM lesion burden in these same mice. We screened 2100 known drugs and bioactive compounds and identified 2 candidates, cholecalciferol (vitamin D3) and tempol (a scavenger of superoxide), for further study. Each drug decreased lesion burden in a mouse model of CCM vascular disease by ≈50%. CONCLUSIONS: By identifying known drugs as potential therapeutics for CCM, we have decreased the time, cost, and risk of bringing treatments to patients. Each drug also prompts additional exploration of biomarkers of CCM disease. We further suggest that the structure-function screening platform presented here may be adapted and scaled to facilitate drug discovery for diverse loss-of-function genetic vascular disease.

Lack of CCM1 induces hypersprouting and impairs response to flow.

Cerebral cavernous malformation (CCM) is a disease of vascular malformations known to be caused by mutations in one of three genes: CCM1, CCM2 or CCM3. Despite several studies, the mechanism of CCM lesion onset remains unclear. Using a Ccm1 knockout mouse model, we studied the morphogenesis of early lesion formation in the retina in order to provide insight into potential mechanisms. We demonstrate that lesions develop in a stereotypic location and pattern, preceded by endothelial hypersprouting as confirmed in a zebrafish model of disease. The vascular defects seen with loss of Ccm1 suggest a defect in endothelial flow response. Taken together, these results suggest new mechanisms of early CCM disease pathogenesis and provide a framework for further study.

ARF6 inhibition stabilizes the vasculature and enhances survival during endotoxic shock.

The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia.

AAP principles concerning retail-based clinics.

The American Academy of Pediatrics views retail-based clinics (RBCs) as an inappropriate source of primary care for pediatric patients, as they fragment medical care and are detrimental to the medical home concept of longitudinal and coordinated care. This statement updates the original 2006 American Academy of Pediatrics statement on RBCs, which flatly opposed these sites as appropriate for pediatric care, discussing the shift in RBC focus and comparing attributes of RBCs with those of the pediatric medical home.

Interleukin receptor activates a MYD88-ARNO-ARF6 cascade to disrupt vascular stability.

The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1ß (IL-1ß), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1ß on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1ß signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.