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In this study, we investigated the effects of nucleolin on lipopolysaccharide (LPS)-induced activation of MAPK and NF-KappaB (NF-κB) signaling pathways and secretion of TNF-α, IL-1β and HMGB1 in THP-1 monocytes. Immunofluorescence assay and Western blotting were used to identify the nucleolin expression in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Inactivation of nucleolin was induced by neutralizing antibody against nucleolin. THP-1 monocytes were pretreated with anti-nucleolin antibody for 1 h prior to LPS challenge. The irrelevant IgG group was used as control. Secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1) and activation of MAPK and NF-κB/I-κB signaling pathways were examined to assess the effects of nucleolin on LPS-mediated inflammatory response. Nucleolin existed in cell membrane, cytoplasm and nucleus of THP-1 monocytes. Pretreatment of anti-nucleolin antibody significantly inhibited the LPS-induced secretion of TNF-α, IL-1β and HMGB1. P38, JNK, ERK and NF-κB subunit p65 inhibitors could significantly inhibit the secretion of IL-1β, TNF-α and HMGB1 induced by LPS. Moreover, the phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65) was significantly increased after LPS challenge. In contrast, pretreatment of anti-nucleolin antibody could significantly inhibit the LPS-induced phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). However, the irrelevant IgG, as a negative control, had no effect on LPS-induced secretion of TNF-α and IL-1β and phosphorylation of p38, JNK, ERK and p65 (or nuclear translocation of p65). We demonstrated that nucleolin mediated the LPS-induced activation of MAPK and NF-κB signaling pathways, and regulated the secretion of inflammatory mediators (TNF-α, IL-1β and HMGB1).

Nanoscale metal-organic frameworks (nMOFs) are excellent radiosensitizers for radiotherapy-radiodynamic therapy (RT-RDT). Herein, we report surface modification of a Hf-DBP nMOF for the co-delivery of a hydrophobic small-molecule toll-like receptor 7 agonist, imiquimod (IMD), and a hydrophilic macromolecule, anti-CD47 antibody (αCD47), for macrophage modulation and reversal of immunosuppression in tumors. IMD repolarizes immunosuppressive M2 macrophages to immunostimulatory M1 macrophages, while αCD47 blocks CD47 tumor cell surface marker to promote phagocytosis. Upon X-ray irradiation, IMD@Hf-DBP/αCD47 effectively modulates the immunosuppressive tumor microenvironment and activates innate immunity to orchestrate adaptive immunity when synergized with an anti-PD-L1 immune checkpoint inhibitor, leading to complete eradication of both primary and distant tumors on a bilateral colorectal tumor model. nMOFs thus provide a unique platform to co-deliver multiple immunoadjuvants for macrophage therapy to induce systematic immune responses and superb antitumor efficacy.

Rationale: Regression of atherosclerosis is an important clinical goal; however, the pathways that mediate the resolution of atherosclerotic inflammation and reversal of plaques are poorly understood. Regulatory T cells (Tregs) have been shown to be atheroprotective, yet the numbers of these immunosuppressive cells decrease with disease progression, and whether they contribute to atherosclerosis regression is not known., Objective: We investigated the roles of Tregs in the resolution of atherosclerotic inflammation, tissue remodeling, and plaque contraction during atherosclerosis regression., Methods and Results: Using multiple independent mouse models of atherosclerosis regression, we demonstrate that an increase in plaque Tregs is a common signature of regressing plaques. Single-cell RNA-sequencing of plaque immune cells revealed that unlike Tregs from progressing plaques that expressed markers of natural Tregs derived from the thymus, Tregs in regressing plaques lacked Nrp1 expression, suggesting that they are induced in the periphery during lipid-lowering therapy. To test whether Tregs are required for resolution of atherosclerotic inflammation and plaque regression, Tregs were depleted using CD25 monoclonal antibody in atherosclerotic mice during apolipoprotein B antisense oligonucleotide-mediated lipid lowering. Morphometric analyses revealed that Treg depletion blocked plaque remodeling and contraction, and impaired hallmarks of inflammation resolution, including dampening of the T helper 1 response, alternative activation of macrophages, efferocytosis, and upregulation of specialized proresolving lipid mediators., Conclusions: Our data establish essential roles for Tregs in resolving atherosclerotic cardiovascular disease and provide mechanistic insight into the pathways governing plaque remodeling and regression of disease.

Triple-negative breast cancer (TNBC) lacks a well-defined molecular target and is associated with poorer outcomes compared to other breast cancer subtypes. Programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) blockade therapy shows a 10% to 20% response rate in TNBC patients. Our previous studies show that PD-L1 proteins are heavily glycosylated in TNBC, and the glycosylation plays an important role in the PD-L1 protein's stability and immunosuppressive function. However, a strategy for PD-L1 deglycosylation in TNBC is poorly defined. Here we found that a saccharide analog, 2-deoxy- d-glucose (2-DG), inhibits glycosylation of PD-L1 and its immunosuppressive function by combining with EGFR inhibitor, gefitinib. Interestingly, 2-DG/gefitinib-induced deglycosylation of PD-L1 decreased the expression level of PD-L1 protein as well as its binding with PD-1. However, there was no significant decrease in 4-1BB expression and its binding with 4-1BBL by 2-DG/gefitinib. Furthermore, we demonstrated that the combination treatment of 2-DG/gefitinib and 4-1BB antibody enhances antitumor immunity in TNBC syngeneic murine models. Together, our results suggest a new immunotherapeutic strategy to enhance antitumor immunity by PD-L1 deglycosylation and 4-1BB stimulation in TNBC., (© 2020 Wiley Periodicals LLC.)

Background: CKD is associated with increased oxidative stress that correlates with occurrence of cardiovascular events. Modifications induced by increased oxidative stress particularly affect circulating lipoproteins such as HDL that exhibit antiatheromatous and antithrombotic properties in vitro ., Methods: To explore the specific role of oxidative modifications of HDL in CKD and their effect on the platelet-targeting antiaggregant properties of HDL, we used a CKD (5/6 nephrectomy) rabbit model. For ex vivo assessment of the antiaggregant properties of HDL, we collected blood samples from 15 healthy volunteers, 25 patients on hemodialysis, and 20 on peritoneal dialysis. We analyzed malondialdehyde, 4-hydroxynonenal (HNE), and 4-hydroxy-2-hexenal protein adduct levels. Platelet aggregation and activation were assessed by aggregometry, thromboxane B2 assay, or FACS. We modified HDL from controls by incubating it overnight at 37°C with 100 µ M of HNE., Results: HDL from CKD rabbits and patients on hemodialysis had HNE adducts. The percentage of platelet aggregation or activation induced by collagen was significantly higher when platelets were incubated with HDL from CKD rabbit and hemodialysis groups than with HDL from the control group. In both rabbits and humans, platelet aggregation and activation were significantly higher in the presence of HNE-modified HDL than with HDL from their respective controls. Incubation of platelets with a blocking antibody directed against CD36 or with a pharmacologic inhibitor of SRC kinases restored the antiaggregative phenotype in the presence of HDL from CKD rabbits, patients on hemodialysis and peritoneal dialysis, and HNE-modified HDL., Conclusions: HDL from CKD rabbits and patients on hemodialysis exhibited an impaired ability to inhibit platelet aggregation, suggesting that altered HDL properties may contribute to the increased cardiovascular risk in this population., (Copyright © 2020 by the American Society of Nephrology.)

T-cell receptor (TCR) ligation-mediated protein phosphorylation regulates the activation, cellular responses, and fates of T cells. Here, we used time-resolved high-resolution phosphoproteomics to identify, quantify, and characterize the phosphorylation dynamics of thousands of phosphorylation sites in primary T cells during the first 10 min after TCR stimulation. Bioinformatic analysis of the data revealed a coherent orchestration of biological processes underlying T-cell activation. In particular, functional modules associated with cytoskeletal remodeling, transcription, translation, and metabolic processes were mobilized within seconds after TCR engagement. Among proteins whose phosphorylation was regulated by TCR stimulation, we demonstrated, using a fast-track gene inactivation approach in primary lymphocytes, that the ITSN2 adaptor protein regulated T-cell effector functions. This resource, called LymphoAtlas, represents an integrated pipeline to further decipher the organization of the signaling network encoding T-cell activation. LymphoAtlas is accessible to the community at: https://bmm-lab.github.io/LymphoAtlas., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Deletion of TGFβ inducible early gene-1 (TIEG) in mice results in an osteopenic phenotype that exists only in female animals. Molecular analyses on female TIEG knockout (KO) mouse bones identified increased expression of sclerostin, an effect that was confirmed at the protein level in serum. Sclerostin antibody (Scl-Ab) therapy has been shown to elicit bone beneficial effects in multiple animal model systems and human clinical trials. For these reasons, we hypothesized that Scl-Ab therapy would reverse the low bone mass phenotype of female TIEG KO mice. In this study, wildtype (WT) and TIEG KO female mice were randomized to either vehicle control (Veh, n = 12/group) or Scl-Ab therapy (10 mg/kg, 1×/wk, s.c.; n = 12/group) and treated for 6 weeks. Following treatment, bone imaging analyses revealed that Scl-Ab therapy significantly increased cancellous and cortical bone in the femur of both WT and TIEG KO mice. Similar effects also occurred in the vertebra of both WT and TIEG KO animals. Additionally, histomorphometric analyses revealed that Scl-Ab therapy resulted in increased osteoblast perimeter/bone perimeter in both WT and TIEG KO animals, with a concomitant increase in P1NP, a serum marker of bone formation. In contrast, osteoclast perimeter/bone perimeter and CTX-1 serum levels were unaffected by Scl-Ab therapy, irrespective of mouse genotype. Overall, our findings demonstrate that Scl-Ab therapy elicits potent bone-forming effects in both WT and TIEG KO mice and effectively increases bone mass in female TIEG KO mice., (© 2020 Wiley Periodicals, Inc.)

Purpose: To evaluate whether antitumor immunity is enhanced by combining radiofrequency (RF) ablation and anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) therapy and to evaluate its effect on untreated tumors., Materials and Methods: First, 40 mice with tumors established in the bilateral flanks were randomly divided into 4 groups: the control group, the RF ablation-alone group, the anti-CTLA-4-alone group, and the RF ablation + anti-CTLA-4 group. In each group, 8 mice were used for untreated tumor evaluation and survival observation, and another 2 mice were killed for histopathologic study. Then, a rechallenge test was performed in another 32 mice to determine whether systemic antitumor immunity was established., Results: Although the volume of the untreated tumors continued to increase until the end of the observation in all groups, tumor growth rates in the RF ablation + anti-CTLA-4 group were significantly smaller than tumor growth rates in the other 3 groups (all P < .05). The overall survival time of mice in the RF ablation + anti-CTLA-4 group was significantly longer than that of mice in the other 3 groups (all P < .05). Histopathologic studies of the untreated tumors showed more CD4-and CD8+ lymphocyte infiltration in mice from the RF ablation + anti-CTLA-4 group than in mice from the other 3 groups (all P < .05). After a tumor rechallenge, tumor rejection was apparent in 75% of the mice in the RF ablation + anti-CTLA-4 group, in 25% of the mice in the RF ablation group, and in 0% of the mice in the control and anti-CTLA-4 groups., Conclusions: This study demonstrated that RF ablation-induced systemic antitumor immunity was enhanced by the combined use of anti-CTLA-4 therapy in a multi-subcutaneous murine hepatoma model., (Copyright © 2020 SIR. Published by Elsevier Inc. All rights reserved.)

There are limited options for targeted therapies for colorectal cancer (CRC). Anti-EGFR therapy is limited to CRC without KRAS mutations. Even worse, most of CRC are refractory to currently immune checkpoint blockade. DKK2, which is upregulated in CRC, was recently found to suppress host immune responses, and its blockage effectively impeded tumor progression in benign genetic CRC models in our previous study. Here, our recent study demonstrated that in human CRC tumor samples expressing high levels of DKK2, DKK2 blockade caused stronger activation of tumor infiltrating CD8 + T cells in ex vivo culture. Intriguingly, we observed a correlation of high DKK2 expression with increased lymph node metastasis prevalence in these CRC patients as well. Furthermore, in a mouse genetic CRC model with mutations in APC and KRAS, which more closely mimics advanced human CRC, we confirmed the tumor inhibitory effect of DKK2 blockade, which significantly retarded tumor progression and extended survival, with increased immune effector cell activation and reduced angiogenesis. Based on this, we performed a combined administration of DKK2 blockade with sub-optimal anti-VEGFR treatment and observed a synergetic effect on suppressing tumor angiogenesis and progression, as well as extending survival, better than those of every single therapy. Thus, this study provides further evidence for the potential therapeutic application of DKK2 blockade in the clinical treatment of human CRC., (Copyright © 2020 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as "leaky" hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels., Methods: We employed the antibody to treat Cx30 A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca 2+ imaging and ATP release assay in vitro., Findings: Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca 2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels., Interpretation: Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30 A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. FUND: Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200., Competing Interests: Declaration of Competing Interest Drs. F. Mammano, G. Yang and F. Zonta report a patent: “Fully human antibody specifically inhibiting connexin 26”, Inventors: Qu Z, Yang G, Mammano F, Zonta F, International application number: PCT/CN2016/109847, pending to ShanghaiTech University; and a patent: “Composition and Methods to treat Ectodermal Dysplasia 2, Clouston Type”, Inventors: Mammano F, Yang G, Zonta F, International Application No.: PCT/CN2019/088689, International Filing Date: 2019-05-28, pending to ShanghaiTech University. All other Authors have nothing to declare., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Eosinophils are important multifunctional granulocytes. When studying eosinophil function and its contribution to diseases, mouse models are often used. Mouse eosinophils selectively express sialic acid-binding immunoglobulin-like lectin (Siglec)-F. Its closest functional paralog on human eosinophils is Siglec-8. These Siglecs are being used to target eosinophils when exploring their mechanistic roles in disease and for potential therapeutic benefit. In order to facilitate preclinical studies of human Siglec-8, we developed transgenic mouse strains expressing human Siglec-8 only on the surface of eosinophils with or without endogenous Siglec-F and have begun characterizing various cellular functions in vitro and in vivo. Eosinophils from Siglec-8+ mice, with or without Siglec-F, responded to Siglec-8 antibody engagement in vitro by up-regulating surface CD11b, whereas Siglec-F antibody had no such effect. Engagement of Siglec-F or Siglec-8 with respective antibodies in vitro resulted in only modest increases in cell death. Administration of rat Siglec-F antibodies to mice led to a significant decrease in Siglec-F surface expression on eosinophils due to internalization, and thus appeared to decrease eosinophil numbers based on Siglec-F+ cells, but with proper gaiting strategies did not in fact result in significant eosinophil depletion. In marked contrast, administration of mouse Siglec-8 antibodies rapidly and effectively depleted eosinophils from blood and spleens of mice, but an F(ab') 2 version did not, indicating an Fc-mediated mechanism for eosinophil depletion in vivo. Siglec-8 expressing mice with or without endogenous Siglec-F will be useful to study Siglec-8-based therapeutics, and may be a preferred approach when acute or chronic eosinophil depletion is needed., (©2020 Society for Leukocyte Biology.)

Ricin, a highly lethal plant-derived toxin, is a potential biological threat agent due to its high availability, ease of production and the lack of approved medical countermeasures for post-exposure treatment. To date, no specific ricin receptors were identified. Here we show for the first time, that the low density lipoprotein receptor-related protein-1 (LRP1) is a major target molecule for binding of ricin. Pretreating HEK293 acetylcholinesterase-producer cells with either anti-LRP1 antibodies or with Receptor-Associated Protein (a natural LRP1 antagonist), or using siRNA to knock-down LRP1 expression resulted in a marked reduction in their sensitivity towards ricin. Binding assays further demonstrated that ricin bound exclusively to the cluster II binding domain of LRP1, via the ricin B subunit. Ricin binding to the cluster II binding domain of LRP1 was significantly reduced by an anti-ricin monoclonal antibody, which confers high-level protection to ricin pulmonary-exposed mice. Finally, we tested the contribution of LRP1 receptor to ricin intoxication of lung cells derived from mice. Treating these cells with anti-LRP1 antibody prior to ricin exposure, prevented their intoxication. Taken together, our findings clearly demonstrate that the LRP1 receptor plays an important role in ricin-induced pulmonary intoxications.

Co-stimulatory T-cell inhibitors are used in the treatment of rheumatoid arthritis and to prevent rejection of renal transplants. Inhibitors of the intereukin (IL-17) cytokine are indicated for psoriasis, psoriatic arthritis and ankylosing spondylitis and anti- IL-23 drugs for psoriasis. Serious infections occur in 4.2% to 25.0% of co-stimulatory inhibitors and 1.0% to 2.0% with IL-17 or IL-23 inhibitors. Underlying disease, steroid dose greater than 7.5 to 10.0 mg, and comorbidities influence risk in individual patients. Opportunistic infections or reactivation of tuberculosis are rare., Competing Interests: Disclosure The author received funding from Stanford University and Hospitals and Pfizer for a study in Patient Centered Care in Rheumatoid Arthritis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

There are 2 populations of T lymphocytes, αβ T and γδ T cells, that can be distinguished by the expression of either an αβ TCR or a γδ TCR, respectively. Pairing of the Ag binding heterodimer, which consists of TCR-α/TCR-β (TCRαβ) or TCR-γ/TCR-δ (TCRγδ), with proteins of the CD3 complex forms the complete αβ or γδ TCR. Despite some similarities in the structure of TCRαβ and TCRγδ and the shared subunits of the CD3 complex, the 2 receptors differ in important aspects. These include the assembly geometry of the complex, the glycosylation pattern, the plasma membrane organization, as well as the accessibility of signaling motifs in the CD3 intracellular tails. These differences are reflected in the different demands and outcomes of ligand-induced signaling. It was shown that exposure of the proline-rich sequence (PRS) in CD3ε occurs with all activating αβ TCR ligands and is required to induce αβ TCR signaling. In sharp contrast, CD3ε PRS exposure was not induced by binding of those ligands to the γδ TCR that have been studied. Further, signaling by the γδ TCR occurs independently of CD3ε PRS exposure. Interestingly, it can be enhanced by anti-CD3ε Ab-induced enforcement of CD3ε PRS exposure. This review contrasts these two similar, but different immune receptors., (©2020 Society for Leukocyte Biology.)

Therapeutic antibodies have revolutionized modern medicine. At present, antibodies are successfully used for treatment of diverse human diseases, ranging from cancer to viral infections. All clinically approved antibodies rely on highly specific recognition of their target antigen. Antigen-binding promiscuity, binding to autoantigens, and propensity for self-binding (homophilic interaction) are highly undesirable characteristics of antibody drug candidates. Nevertheless, the immune system of all healthy individuals constantly produces and uses large quantities of antibodies that can be classified as inappropriate for development as drugs. Here, I provide arguments that antibodies with 'aberrant' properties have therapeutic potential. They could be useful in certain complex pathological conditions, thus enriching our armamentarium for treatment of human diseases., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Purpose: The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met., Methods: A phage library, with a functional size of 5.5 × 10 10 individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques., Results: Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (K aff : 0.3 × 10 9  M -1 ), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%)., Conclusion: Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells.

Introduction: Cluster headache (CH) is the most common trigeminal autonomic cephalalgia with a significant need for novel treatment options. While the use of most of the acute CH medications is supported by clinical trials and based on a pathophysiological concept for the generation of pain, the scientific evidence for preventive CH medications is very limited., Areas Covered: This article reviews acute and preventive substances for the pharmacological treatment of CH with a focus on the mode of action of these drugs. We also summarized the clinical trial evidence and discuss future research directions., Expert Opinion: Recommendations for current pharmacological CH therapies, in particular for CH prevention, are often based on small open label studies with inconclusive results. Larger trials are missing. A shared pathophysiological mechanism of action of these preventatives does not exist. Future studies with CGRP(R) antibodies and novel substances with specific actions are needed and will thereby help to understanding the pathophysiology of CH.

Conventional antibodies used for cancer therapies can only target the extracellular epitopes of tumor-associated antigens (TAAs); however, they struggle to enter cancer cells to interact with intracellular TAAs. Intrabodies are the engineered single-chain antibodies that can be transferred to the living cells or expressed within the cells and target the intracellular TAAs, owing to their nanosizes, enhanced motility and specific and potent binding affinities for the TAAs. Intrabody-based technology is a supplement to the current gene silencing technologies that can regulate a variety of biochemical processes and cellular functions and could be applicable for clinical treatment of tumors. Here, we review the development and current status of the intrabodies for the targeted treatment of cancers., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Obesity is a risk factor for type 2 diabetes (T2D); however, not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from donors with T2D and non-T2D (ND), especially obese donors (BMI ≥30 kg/m 2 ). Islets from obese donors with T2D had reduced insulin secretion, decreased β-cell exocytosis, and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis, and reduced granule docking. This was accompanied by reduced expression of the exocytotic proteins SNAP25, STXBP1, and VAMP2, likely because CD36 induced downregulation of the insulin receptor substrate (IRS) proteins, suppressed the insulin-signaling phosphatidylinositol 3-kinase/AKT pathway, and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human β-cell line EndoC-βH1 increased IRS1 and exocytotic protein levels, improved granule docking, and enhanced insulin secretion. Our results demonstrate that β-cells from obese donors with T2D have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit β-cell function in T2D associated with obesity., (© 2020 by the American Diabetes Association.)

Background: Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). We demonstrated that MBG induces fibrosis via mechanism involving inhibition of Fli1, a nuclear transcription factor and a negative regulator of collagen-1 synthesis. We hypothesized that PE blockade of increased MBG with antibody would lessen the fibrosis of umbilical arteries and lower the blood pressure in rats with PE., Methods: We tested 36 pregnant Sprague-Dawley rats in which 12 were made hypertensive by 1.8% Na supplementation (days 6-19 of gestation), 12 pregnant rats served controls. At day 19, PE rats received one intraperitoneal injection of polyclonal anti-MBG-4 antibody (0.5 ug/ml) for 4 hours., Results: PE was associated with higher blood pressure (117 ± 2 vs. 107 ± 2 mm Hg; P < 0.01), plasma MBG levels (1.54 ± 0.34 vs. 0.49 ± 0.11 nmol/L; P < 0.01), protein excretion (26 vs. 12 mg/24 hours), sFlt-1 (3-fold), decrease in Fli1 (7-fold) and increase in collagen-1 in aorta (4-fold) vs. control rats (all P < 0.01). In 12 rats treated with polyclonal anti-MBG-4 antibody blood pressure dropped (93 ± 3 mm Hg) and Fli1 was decreased much less (2-fold; P < 0.01 vs. nontreated rats)., Conclusions: These results demonstrate that in experimental PE elevated MBG level is implicated in umbilical fibrosis via suppression of Fli1., (© American Journal of Hypertension, Ltd 2019. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

BRAF mutations occur in approximately 50% of melanoma patients. The mutated BRAF kinase continuously activates the mitogen-activated protein kinase (MAPK) pathway to promote cell growth and proliferation. Vemurafenib as a specific BRAF inhibitor can significantly prolong progression-free survival in melanoma patients. However, most patients developed resistance to Vemurafenib after 6 months. The mechanism of drug resistance is not yet fully understood. In this study, we found that proteins secreted by drug-resistant cells protect sensitive cells from Vemurafenib. By RNA-seq, we compared differentially expressed genes between resistant and sensitive cells. We demonstrated that drug-resistant cells secrete more IL-6 protein than sensitive cells. For the first time, we found that IL-6 expressed by drug-resistant cells consists of the following transcripts: IL6-201, IL6-202 and IL6-205. We confirmed that it is the IL6-202 and IL6-205 transcripts that confer drug resistance to Vemurafenib by reactivating the MAPK pathway while IL6-201 is not responsible for the resistance in A375 melanoma cells. Neutralizing IL-6 significantly increased the sensitivity of drug-resistant cells to Vemurafenib. Overall, these results reveal a new mechanism of drug resistance in melanoma., Competing Interests: Declaration of competing interest The authors declare that the research article titled “Transcripts 202 and 205 of IL-6 confer resistance to Vemurafenib by reactivating the MAPK pathway in BRAF(V600E) mutant melanoma cells” does not have competing financial interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Notch signaling provides an important cue in the mammalian developmental process. It is a key player in T cell development and function. Notch ligands such as Delta-like ligands (DLL) 1, 3, 4, and JAG1, 2 can impact Notch signaling positively or negatively, by trans -activation or cis -inhibition. Trans and cis interactions are receptor-ligand interaction on two adjacent cells and interaction on the same cell, respectively. The former sends an activation signal and the later, a signal for inhibition of Notch. However, earlier reports suggested that Notch is activated in the absence of Notch ligand-expressing APCs in a purified population of CD4 T cells. Thus, the role of ligands in Notch activation, in a purified population of CD4 T cells, remains obscure. In this study, we demonstrate that mature CD4 T cells are capable of expressing Notch ligands on their surface very early upon activation with soluble antibodies against CD3 and CD28. Moreover, signaling solely through CD28 induces Notch ligand expression and CD3 signaling inhibits ligand expression, in contrast to Notch which is induced by CD3 signaling. Additionally, by using decoys, mimicking the Notch extracellular domain, we demonstrated that DLL1, DLL4, and JAG1, expressed on the T cells, can cis -interact with the Notch receptor and inhibit activation of Notch. Thus, our data indicate a novel mechanism of the regulation of Notch ligand expression on CD4 T cells and its impact on activated Notch., (Copyright © 2020 Mitra, Shanthalingam, Sherman, Singh, Canakci, Torres, Lawlor, Ran, Golde, Miele, Thayumanavan, Minter and Osborne.)

An anti-EGFR nanobody was labeled at the C-terminus with a lysosome-sorting NPGY (Asn-Pro-Gly-Tyr) motif via sortase-mediated ligation to enhance the engagement of the clathrin-mediated endocytosis. The synergistic effects of NPGY motif and nona-arginine peptide were found to induce robust internalization and lysosomal trafficking, which in turn improved anti-tumor activity of an antibody-drug conjugate.

Prolonged therapy, drug toxicity, noncompliance, immune suppression, and alarming emergence of drug resistance necessitate the search for therapeutic vaccine strategies for tuberculosis (TB). Such strategies ought to elicit not only IFN-γ, but polyfunctional response including TNF-α, which is essential for protective granuloma formation. Here, we investigated the impact of PD-1 inhibition in facilitating protective polyfunctional T cells (PFTs), bacillary clearance, and disease resolution. We have observed PD-1 inhibition preferentially rescued the suppressed PFTs in active tuberculosis patients. In addition, polyfunctional cytokine milieu favored apoptosis of infected MDMs over necrosis with markedly reduced bacillary growth (≪CFU) in our in vitro monocyte-derived macrophages (MDMs) infection model. Furthermore, the animal study revealed a significant decline in the bacterial burden in the lungs and spleen of infected mice after in vivo administration of α-PD-1 along with antitubercular treatment. Our findings suggest that rescuing polyfunctional immune response by PD-1 inhibition works synergistically with antituberculosis chemotherapy to confer improved control over bacillary growth and dissemination. In summary, our data strongly indicate the therapeutic potential of α-PD-1 as adjunct immunotherapy that can rejuvenate suppressed host immunity and enhance the efficacy of candidate therapeutic vaccine(s)., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti-claudin (CLDN)-4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF-7 (luminal subtype) and MDA-468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)-induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA-468 also showed the same level of antitumor activity and survival as MCF-7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Natural Killer (NK) cell responses are regulated by a variety of different surface receptors. While we can determine the overall positive or negative effect of a given receptor on NK cell functions, investigating NK cell regulation in a quantitative way is challenging. To quantitatively investigate individual receptors for their effect on NK cell activation, we chose to functionalize latex beads that have approximately the same size as lymphocytes with defined amounts of specific antibodies directed against distinct activating receptors. This enabled us to investigate NK cell reactivity in a defined, clean, and controllable system. Only CD16 and NKp30 could activate the degranulation of resting human NK cells. CD16, NKG2D, NKp30, NKp44, and NKp46 were able to activate cultured NK cells. NK cell activation resulted in the induction of polyfunctional cells that degranulated and produced IFN-γ and MIP-1β. Interestingly, polyfunctional NK cells were only induced by triggering ITAM-coupled receptors. NKp44 showed a very sensitive response pattern, where a small increase in receptor stimulation caused maximal NK cell activity. In contrast, stimulation of 2B4 induced very little NK cell degranulation, while providing sufficient signal for NK cell adhesion. Our data demonstrate that activating receptors differ in their effectiveness to stimulate NK cells., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intranasal administration of antibodies to glutamate in a dose of 250 μg/kg for two weeks facilitated spatial learning and memory formation in the Morris water maze in aging C57BL/6 mice. In animals treated with glutamate antibodies, the content of serotonin and dopamine metabolites 3-MT and HVA in the hippocampus decreased, but no changes in the metabolism of neurotransmitter acids were revealed. In the prefrontal cortex, dopamine level decreased and the content of its metabolite DOPAC increased; in parallel, an increase in excitatory and inhibitory amino acids (aspartic acid, glutamate, glycine, taurine, and GABA) was observed.

Background Aims: Key obstacles in human iNKT cell translational research and immunotherapy include the lack of robust protocols for dependable expansion of human iNKT cells and the paucity of data on phenotypes in post-expanded cells., Methods: We delineate expansion methods using interleukin (IL)-2, IL-7 and allogeneic feeder cells and anti-CD2/CD3/CD28 stimulation by which to dependably augment Th2 polarization and direct cytotoxicity of human peripheral blood CD3 + Vα24 + Vβ11 + iNKT cells., Results: Gene and protein expression profiling demonstrated augmented Th2 cytokine secretion (IL-4, IL-5, IL-13) in expanded iNKT cells stimulated with anti-CD2/CD3/CD28 antibodies. Cytotoxic effector molecules including granzyme B were increased in expanded iNKT cells after CD2/CD3/CD28 stimulation. Direct cytotoxicity assays using unstimulated expanded iNKT cell effectors revealed α-galactosyl ceramide (α-GalCer)-dependent killing of the T-ALL cell line Jurkat. Moreover, CD2/CD3/CD28 stimulation of expanded iNKT cells augmented their (α-GalCer-independent) killing of Jurkat cells. Co-culture of expanded iNKT cells with stimulated responder cells confirmed contact-dependent inhibition of activated CD4 + and CD8 + responder T cells., Discussion: These data establish a robust protocol to expand and novel pathways to enhance Th2 cytokine secretion and direct cytotoxicity in human iNKT cells, findings with direct implications for autoimmunity, vaccine augmentation and anti-infective immunity, cancer immunotherapy and transplantation., (Copyright © 2020 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Normothermic machine perfusion (NMP) is an emerging modality for kidney preservation prior to transplantation. NMP may allow directed pharmacomodulation of renal ischemia-reperfusion injury (IRI) without the need for systemic donor/recipient therapies. Three proven anti-IRI agents not in widespread clinical use, CD47-blocking antibody (αCD47Ab), soluble complement receptor 1 (sCR1), and recombinant thrombomodulin (rTM), were compared in a murine model of kidney IRI. The most effective agent was then utilized in a custom NMP circuit for the treatment of isolated porcine kidneys, ascertaining the impact of the drug on perfusion and IRI-related parameters. αCD47Ab conferred the greatest protection against IRI in mice after 24 hours. αCD47Ab was therefore chosen as the candidate agent for addition to the NMP circuit. CD47 receptor binding was demonstrated by immunofluorescence. Renal perfusion/flow improved with CD47 blockade, with a corresponding reduction in oxidative stress and histologic damage compared to untreated NMP kidneys. Tubular and glomerular functional parameters were not significantly impacted by αCD47Ab treatment during NMP. In a murine renal IRI model, αCD47Ab was confirmed as a superior anti-IRI agent compared to therapies targeting other pathways. NMP enabled effective, direct delivery of this drug to porcine kidneys, although further efficacy needs to be proven in the transplantation setting.

Plasmodium falciparum merozoite invasion into erythrocytes is an essential step of the blood-stage cycle, survival of parasites, and malaria pathogenesis. P. falciparum merozoite Rh5 interacting protein (PfRipr) forms a complex with Rh5 and CyRPA in sequential molecular events leading to erythrocyte invasion. Recently we described PfRipr as a conserved protein that induces strain-transcending growth inhibitory antibodies in in vitro assays. However, being a large and complex protein of 1086 amino acids (aa) with 87 cysteine residues, PfRipr is difficult to express in conventional expression systems towards vaccine development. In this study we sought to identify the most potent region of PfRipr that could be developed to overcome difficulties related to protein expression, as well as to elucidate the invasion inhibitory mechanism of anti-PfRipr antibodies. Using the wheat germ cell-free system, Ecto- PfRipr and truncates of approximately 200 aa were expressed as soluble proteins. We demonstrate that antibodies against PfRipr truncate 5 (PfRipr&#95;5: C 720 -D 934 ), a region within the PfRipr C-terminal EGF-like domains, potently inhibit merozoite invasion. Furthermore, the antibodies strongly block PfRipr/Rh5 interaction, as well as that between PfRipr and its erythrocyte-surface receptor, SEMA7A. Taken together, PfRipr&#95;5 is a potential candidate for further development as a blood-stage malaria vaccine.

Arteriogenesis is a process by which a pre-existing arterioarterial anastomosis develops into a functional collateral network following an arterial occlusion. Alternatively activated macrophages polarized by IL10 have been described to promote collateral growth. This study investigates the effect of different levels of IL10 on hind-limb reperfusion and the distribution of perivascular macrophage activation types in mice after femoral artery ligation (FAL). IL10 and anti-IL10 were administered before FAL and the arteriogenic response was measured by Laser-Doppler-Imaging perioperatively, after 3, 7, and 14 d. Reperfusion recovery was accelerated when treated with IL10 and impaired with anti-IL10. Furthermore, symptoms of ischemia on ligated hind-limbs had the highest incidence after application of anti-IL10. Perivascular macrophages were immunohistologically phenotyped using CD163 and CD68 in adductor muscle segments. The proportion of alternatively activated macrophages (CD163 + /CD68 + ) in relation to classically activated macrophages (CD163 - /CD68 + ) observed was the highest when treated with IL10 and suppressed with anti-IL10. This study underlines the proarteriogenic response with increased levels of IL10 and demonstrates an in-vivo alteration of macrophage activation types in the perivascular bed of growing collaterals.

Scope: Epigallocatechin gallate (EGCG), an active polyphenol in green tea, exhibits various physiological effects, including activation of low-density lipoprotein receptors (LDLR). The previous studies have suggested that EGCG activates LDLR via extracellular signal-regulated kinase (ERK) pathway in HepG2 cells. However, the detailed molecular mechanism remains unclear. Recently, 67 kDa laminin receptor (67LR) is identified as a receptor for EGCG. Therefore, this study aims to determine whether 67LR is involved in the mechanism of LDLR activation by EGCG., Methods and Results: EGCG induces upregulation of LDLR when 67LR is knocked down in HepG2 cells. Similar effect is observed after the cells are treated with 67LR monoclonal antibody. The loss of antiallergic effect following 67LR siRNA knockdown and 67LR antibody treatment confirms the results since the antiallergic effect of EGCG is known to be mediated by 67LR., Conclusion: EGCG activates LDLR expression via 67LR-independent pathway in HepG2 cells., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Introduction : Outcomes in metastatic colorectal cancer are improving, due to the tailoring of therapy enabled by better understanding of clinical behavior according to molecular subtype. Areas covered : A review of the literature and recent conference presentations was undertaken on the topic of systemic treatment of metastatic colorectal cancer. This review summarizes expert discussion of the current evidence for therapies in metastatic colorectal cancer (mCRC) based on molecular subgrouping. Expert opinion : EGFR-targeted and VEGF-targeted antibodies are now routinely incorporated into treatment strategies for mCRC. EGFR-targeted antibodies are restricted to patients with extended RAS wild-type profiles, with evidence that they should be further restricted to patients with left-sided tumors. Clinically distinct treatment pathways based on tumor RAS, BRAF, HER2 and MMR status, are now clinically applicable. Evidence suggests therapy for additional subgroups will soon be defined; the most advanced being for patients with KRAS G12 C mutation and gene TRK fusion defects.

Background: Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction due to blood transfusions. The pathophysiology of this complication is still not widely elucidated by the scientific community, especially regarding the direct role of blood platelets within the cellular mechanism responsible for the development of TRALI., Study Design and Methods: In this study, a mouse model was used to induce the development of antibody-mediated acute lung injury through injections of lipopolysaccharide and an anti-major histocompatibility complex Class I antibody. BALB/c mice were pretreated with an anti-GPIbα antibody, which induces platelet depletion, or ML354, a protease receptor 4 pathway inhibitor, 30 minutes before TRALI induction., Results: Depletion of platelets before TRALI induction appeared to reduce the severity of TRALI without completely inhibiting its development. Also, inhibition of platelet activation by ML354 did not prevent the onset of TRALI. Finally, the stimuli used for TRALI induction also triggered specific platelet activation upon ex vivo stimulation., Conclusions: This study suggests that blood platelets are not critically required for TRALI induction, although they are to some extent involved in its pathophysiology., (© 2020 AABB.)

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

Hybridoma and phage display are two powerful technologies for isolating target-specific monoclonal antibodies based on the binding. However, for complex membrane proteins, such as G protein-coupled receptors (GPCRs), binding-based screening rarely results in functional antibodies. Here we describe a function-based high-throughput screening method for quickly identifying antibody antagonists and agonists against GPCRs by combining glycosylphosphatidylinositol-anchored antibody cell display with β-arrestin recruitment-based cell sorting and screening. This method links antibody genotype with phenotype and is applicable to all GPCR targets. We validated this method by identifying a panel of antibody antagonists and an antibody agonist to the human apelin receptor from an immune antibody repertoire. In contrast, we obtained only neutral binders and antibody antagonists from the same repertoire by phage display, suggesting that the new approach described here is more efficient than traditional methods in isolating functional antibodies. This new method may create a new paradigm in antibody drug discovery.

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

Previous studies demonstrated spontaneous type 2 airway inflammation with eosinophilia in juvenile Scnn1b (sodium channel, non-voltage-gated 1, β-subunit)-transgenic ( Scnn1b -Tg) mice with muco-obstructive lung disease. IL-1 receptor (IL-1R) signaling has been implicated in allergen-driven airway disease; however, its role in eosinophilic inflammation in muco-obstructive lung disease remains unknown. In this study, we examined the role of IL-1R signaling in the development of airway eosinophilia and type 2 inflammation in juvenile Scnn1b -Tg mice. We determined effects of genetic deletion of Il1r1 (IL-1 receptor type I) on eosinophil counts, transcript levels of key type 2 cytokines, markers of eosinophil activation and apoptosis, and tissue morphology in lungs of Scnn1b -Tg mice at different time points during neonatal development. Furthermore, we measured endothelial surface expression of intercellular adhesion molecule 1 (ICAM-1), an integrin involved in eosinophil transendothelial migration, and determined effects of eosinophil depletion using an anti-IL-5 antibody on lung morphology. Lack of IL-1R reduced airway eosinophilia and structural lung damage, but it did not reduce concentrations of type 2 cytokines and associated eosinophil activation in Scnn1b -Tg mice. Structural lung damage in Scnn1b -Tg mice was also reduced by eosinophil depletion. Lack of IL-1R was associated with reduced expression of ICAM-1 on lung endothelial cells and reduced eosinophil counts in lungs from Scnn1b -Tg mice. We conclude that IL-1R signaling is implicated in airway eosinophilia independent of type 2 cytokines in juvenile Scnn1b -Tg mice. Our data suggest that IL-1R signaling may be relevant in the pathogenesis of eosinophilic airway inflammation in muco-obstructive lung diseases, which may be mediated in part by ICAM-1-dependent transmigration of eosinophils into the lungs.

Systemic lupus erythematosus is a chronic inflammatory disease which involves multiple organs. Self-specific B and T cells play a main role in the pathogenesis of lupus and have been defined as a logical target for selective therapy. The protein annexin A1 (ANX A1) is a modulator of the immune system involving many cell types. An abnormal expression of ANX A1 was found on activated B and T cells during autoimmunity, suggesting its importance as a potential therapeutic target. We hypothesize that it may be possible to down-regulate the activity of autoreactive T and B cells from lupus patients in a humanized immunodeficient mouse model by treating them with an antibody against ANX A1. When cultured in the presence of anti-ANX A1, peripheral blood mononuclear cells (PBMC) from lupus patients showed a decreased number of immunoglobulin (Ig)G anti-dsDNA antibody-secreting plasma cells, decreased T cell proliferation and expression of activation markers and increased B and T cell apoptosis. We employed a humanized model of SLE by transferring PBMCs from lupus patients to immunodeficient non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. The humanized animals presented autoantibodies, proteinuria and immunoglobulin deposition in the renal glomeruli. Treatment of these NOD-SCID mice with an anti-ANX A1 antibody prevented appearance of anti-DNA antibodies and proteinuria, while the phosphate-buffered saline (PBS)-injected animals had high levels after the transfer. The treatment reduced the levels of autoantibodies to several autoantigens, lupus-associated cytokines and disease symptoms., (© 2019 British Society for Immunology.)

Glycosylation is a common post-translational modification that occurs during the production of antibodies. Glycans attached to antibodies play an important role in the pharmacokinetics, efficacy, and safety of therapeutic antibodies. In the modern antibody industry, it is important to adjust and control glycosylation modifications. The formation of specific sugar structures via glycosylation engineering is constantly evolving. This review summarizes the recent progress in glycosylation modifications, as well as the major discoveries and current understanding of the mechanisms involved, to provide new ideas for the research and development of therapeutic antibodies.

Over the last decade, antibodies have become an important component in the arsenal of cancer therapeutics. High-specificity, low off-target effects, desirable pharmacokinetics and high success rate are a few of the many attributes that make antibodies amenable for development as drugs. To design antibodies for successful clinical applications, however, it is critical to have an understanding of their structure, functions, mechanisms of action and pharmacokinetic/pharmacodynamic properties. This review highlights some of these key aspects, as well as certain limitations encountered, with monoclonal antibody therapy. Further, we discuss rational combination therapies for clinical applications, some of which could help overcome the limitations.

While monoclonal antibodies are the fastest-growing class of therapeutic agents, we lack a method that can directly quantify the on- and off-target binding affinities of newly developed therapeutic antibodies in crude cell lysates. As a result, some therapeutic antibody candidates could have a moderate on-target binding affinity but a high off-target binding affinity, which not only gives a reduced efficacy but triggers unwanted side effects. Here, we report a single-molecule counting method that precisely quantifies antibody-bound receptors, free receptors, and unbound antibodies in crude cell lysates, termed digital receptor occupancy assay (DRO). Compared to the traditional flow cytometry-based binding assay, DRO assay enables direct and digital quantification of the three molecular species in solution without the additional antibodies for competitive binding. When characterizing the therapeutic antibody, cetuximab, using DRO assay, we found the on-target binding ratio to be 65% and the binding constant ( K d ) to be 2.4 nM, while the off-target binding causes the binding constant to decrease by 0.3 nM. Other than cultured cells, the DRO assay can be performed on tumor mouse xenograft models. Thus, DRO is a simple and highly quantitative method for cell-based antibody binding analysis which can be broadly applied to screen and validate new therapeutic antibodies.

Correct selection of the cell division axis is important for cell differentiation, tissue and organ morphogenesis, and homeostasis. Both integrins, which mediate interactions with extracellular matrix (ECM) components such as fibronectin, and cadherins, which mediate interactions between cells, are implicated in the determination of spindle orientation. We found that both cadherin- and integrin-based adhesion resulted in cell divisions parallel to the attachment plane and elicited identical spindle responses to spatial adhesive cues. This suggests that adhesion topology provides purely mechanical spatial cues that are independent of the molecular nature of the interaction or signaling from adhesion complexes. We also demonstrated that cortical integrin activation was indispensable for correct spindle orientation on both cadherin and fibronectin substrates. These data suggest that spindle orientation responses to adhesion topology are primarily a result of force anisotropy on the cell cortex and show that integrins play a central role in this process that is distinct from their role in cell-ECM interactions., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Natural killer (NK) cells are innate lymphocytes that directly kill tumor and pathogen-infected cells upon activation by cytokines and NK cell receptors (NKRs) without previous sensitization. It is known that cell metabolism affects the differentiation and effector functions of immune cells. For instance, interleukin-2 and interleukin-15 treatment increases glycolysis and oxidative phosphorylation (OXPHOS) in NK cells to support their effector functions. However, little is known about the metabolic reprogramming of human NK cells upon their activation by NKRs. In this study, we investigated the metabolism of NK cells stimulated via NKRs. We found that NK cells upregulated glycolysis and OXPHOS in response to anti-CD16 antibody or NKG2D ligand engagement. Inhibition of either glycolysis or OXPHOS impaired NK cell production of interferon-γ. Interestingly, inhibition of glycolysis but not OXPHOS decreased NK cell killing and dampened NK cell degranulation and Fas ligand expression, suggesting that glycolysis is more critical for NKR-activated cell cytotoxicity. Thus, our study provides insight into understanding the metabolic requirements underlying different effector functions of human NK cells., (Copyright © 2020 Wang, Guan, Wang, Chai, Xu and Lam.)

During a time span of just a few years, the CD38 antibody, daratumumab, has been established as one of the most important new drugs for the treatment of multiple myeloma, both in the relapsed/refractory setting and, more recently, as a first-line treatment. Although much is known about the pleiotropic modes of action of daratumumab, we are still not sure how to use it in an optimal manner. Daratumumab targets CD38 on myeloma cells and a high level of CD38 expression facilitates complement-mediated cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Since the expression of CD38 by myeloma cells is downregulated during treatment with daratumumab, it may seem reasonable to introduce a wash-out period and retreat with daratumumab at a later time point when CD38 expression has recovered in order to gain the maximum benefit of daratumumab's capacity to kill myeloma cells by CDC, ADCC and ADCP. In other aspects, CD38 seems to serve as a survival factor for myeloma cells by facilitating protective myeloma cell-stromal-cell interactions, contributing to the formation of nanotubes that transfer mitochondria from the stromal cells to myeloma cells, boosting myeloma cell proliferation and survival and by generation of immunosuppressive adenosine in the bone marrow microenvironment. In addition, continuous exposure to daratumumab may keep immune suppressor cells at a low level, which boosts the anti-tumor activity of T-cells. In fact, one may speculate if in the early phase of treatment of a myeloma patient, the debulking effects of daratumumab achieved by CDC, ADCC and ADCP are more important while at a later stage, reprogramming of the patient's own immune system and certain metabolic effects may take over and become more essential. This duality may be reflected by what we often observe when we watch the slope of the M-protein from myeloma patients responding to daratumumab: A rapid initial drop followed by a slow decline of the M-protein during several months or even years. Ongoing and future clinical trials will teach us how to use daratumumab in an optimal way., Competing Interests: The authors declare no conflict of interest.

Persistent neural activity has been observed in vivo during working memory tasks, and supports short-term (up to tens of seconds) retention of information. While synaptic and intrinsic cellular mechanisms of persistent firing have been proposed, underlying cellular mechanisms are not yet fully understood. In vitro experiments have shown that individual neurons in the hippocampus and other working memory related areas support persistent firing through intrinsic cellular mechanisms that involve the transient receptor potential canonical (TRPC) channels. Recent behavioral studies demonstrating the involvement of TRPC channels on working memory make the hypothesis that TRPC driven persistent firing supports working memory a very attractive one. However, this view has been challenged by recent findings that persistent firing in vitro is unchanged in TRPC knock out (KO) mice. To assess the involvement of TRPC channels further, we tested novel and highly specific TRPC channel blockers in cholinergically induced persistent firing in mice CA1 pyramidal cells for the first time. The application of the TRPC4 blocker ML204, TRPC5 blocker clemizole hydrochloride, and TRPC4 and 5 blocker Pico145, all significantly inhibited persistent firing. In addition, intracellular application of TRPC4 and TRPC5 antibodies significantly reduced persistent firing. Taken together these results indicate that TRPC4 and 5 channels support persistent firing in CA1 pyramidal neurons. Finally, we discuss possible scenarios causing these controversial observations on the role of TRPC channels in persistent firing., Competing Interests: The authors declare no conflict of interest.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important etiology of pneumonia. Interleukin (IL)-9 is a T helper 9 (Th9) cytokine and participates in the pathogenesis of infectious diseases. Here, we investigated the role of IL-9 by using an MRSA pneumonia animal model. The BALB/c mice underwent nasal inhalation with an ST239 MRSA strain to establish the mouse model of MRSA pneumonia, and a subset of mice were intravenously injected with IL-9 neutralizing antibody or immunoglobulin (Ig) G. At 3 and 8 days postinfection, the peripheral blood, bronchioalveolar lavage fluid (BALF), and lung tissues were collected. The frequencies of Th9 cells and the levels of cytokines in peripheral blood, BALF, and lung tissues were determined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. The colony counts of MRSA in BALF and lung tissue were detected. The lung pathological changes were examined using hematoxylin and eosin staining. Data from flow cytometry, qRT-PCR, and ELISA showed that MRSA-infected mice exhibited higher frequency of Th9 cells and higher IL-9 mRNA and protein levels in the peripheral blood, BALF, and lung tissues of mice. In contrast, the neutralization of IL-9 abrogated MRSA inoculation-induced Th9 cell generation and IL-9 production in BALF and lung tissues. Furthermore, bacterial counting and histological examination showed that the numbers of bacteria in BALF and lungs and the lung pathological scores induced by MRSA inoculation were attenuated by the neutralization of IL-9. Moreover, cell counting and ELISA results demonstrated that IL-9 neutralization diminished the MRSA inoculation-induced count of neutrophils and macrophages and levels of pro-inflammatory cytokines in BALF. Collectively, IL-9 neutralization attenuated inflammation of MRSA pneumonia by regulating Th9/IL-9 expression., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Nanosized metal-organic frameworks (nMOFs) have shown great promise as high-capacity carriers for a variety of applications. For biomedicine, numerous nMOFs have been proposed that can transport virtually any molecular drug, can finely tune their payload release profile, etc. However, perspectives of their applications for the targeted drug delivery remain relatively unclear. So far, only a few works have reported specific cell targeting by nMOFs exclusively through small ligands such as folic acid or RGD peptides. Here we show feasibility of targeted drug delivery to specific cancer cells in vitro with nMOFs functionalized with such universal tool as an antibody. We demonstrate ca. 120 nm magnetic core/MOFs shell nanoagents loaded with doxorubicin/daunorubicin and coupled with an antibody though a hydrophilic carbohydrate interface. We show that carboxymethyl-dextran coating of nMOFs allows extensive loading of the drug molecules (up to 15.7 mg/g), offers their sustained release in physiological media and preserves antibody specificity. Reliable performance of the agents is illustrated with trastuzumab-guided selective targeting and killing of HER2/neu-positive breast cancer cells in vitro. The approach expands the scope of nMOF applications and can serve as a platform for the development of potent theranostic nanoagents. STATEMENT OF SIGNIFICANCE: The unique combination of exceptional drug capacity and controlled release, biodegradability and low toxicity makes nanosized metal-organic frameworks (nMOFs) nearly ideal drug vehicles for various biomedical applications. Unfortunately, the prospective of nMOF applications for the targeted drug delivery is still unclear since only a few examples have been reported for nMOF cell targeting, exclusively for small ligands. In this work, we fill the important gap and demonstrate nanoagent that can specifically kill target cancer cells via drug delivery based on recognition of HER2/neu cell surface receptors by such universal and specific tool as antibodies. The proposed approach is universal and can be adapted for specific biomedical tasks using antibodies of any specificity and nMOFs of a various composition., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

As a neurological disorder, epilepsy has affected over 65 million people all over the world because of the unforeseeable seizures it might cause. However, in-depth understandings of the pathogenesis of epilepsy and effective treatments for the disease are still lacked. Recent discoveries suggest that autophagy, as an endogenous self-cleansing pathway in mammals, might be involved in the onset of epilepsy. Our study assumes that a non-histone DNA binding protein, high mobility group box-1 (HMGB1), formerly considered as a crucial inflammatory factor, may mediate the autophagy of neurons in epileptic mouse brain. To verify this hypothesis, pilocarpine induced epilepsy mouse model was constructed. The mice were treated with HMGB1 antibody for 4 weeks after the initial epileptic seizure. Behavioral test results suggested a recovery of learning ability and memory in epileptic mice when treated with HMGB1 antibody. Pathological changes in hippocampus were inspected under microscopes and hippocampus damages caused by seizures in mouse with epilepsy such as increased intracellular space were alleviated by HMGB1 antibody treatment. Moreover, the expressions of the proteins involved in autophagy pathways were detected by immunofluorescence staining and western blot. microtubule-associated protein 1A/1B-light chain 3 (LC3), Beclin 1, autophagy protein-5 (ATG5), and ATG7 levels were significantly decreased by HMGB1 antibody while the level of p62 was increased. TdT-mediated dUTP Nick-End Labeling (TUNEL) illustrated that cell apoptosis induced by seizures in hippocampus was mitigated by HMGB1 antibody. In conclusion, we propose that HMGB1 may induce increased autophagy in epilepsy mouse model., Competing Interests: Declaration of Competing Interest We would like to declare on behalf of our co-authors that the work entitled “High mobility group box 1 antibody represses autophagy and alleviates hippocampus damage in pilocarpine-induced mouse epilepsy model” has not been published previously, and not under consideration for publication elsewhere, in whole or in part. There is no interest conflict in this study., (Copyright © 2019 Elsevier GmbH. All rights reserved.)