Bispecific Antibody Designed for Targeted NK Cell Activation and Functional Assessment for Biomedical Applications.

Natural killer (NK) cells serve as key innate effectors and their activity has been considered a prognostic biomarker in diverse human diseases. Currently, NK cell functional assays have several problems primarily related to adequate preparation, labeling, or treatment of target cells, which are cumbersome and often hamper consistent sensitivity for NK cells. Here, bispecific antibodies (BsAb's) targeting NKG2D and 2B4 receptors, whose combination mounts selective cytotoxicity and IFN-γ production of NK cells, are developed as acellular, consistent, and easy-to-use strategies for assessing NK cell functions. These NK cell activator BsAb's (NKABs) are constructed in symmetric dual bivalent formats with different interdomain spacings [immunoglobulin G (IgG)-single-chain variable fragment (scFv) and dual-variable domain (DVD)-Ig] and kappa constant (Cκ)-scFv format linking two scFv's with a Cκ domain. These NKABs are specific and superior to a combination of monospecific antibodies for NK cell activation. NKAB elicits both direct cytotoxicity and IFN-γ production via integration of NKG2D and 2B4 signals. Moreover, stimulation with NKAB IgG-scFv and Cκ-scFv reveals defective NK cell functions in X-linked lymphoproliferative disease involving 2B4 dysfunction in NK cells and multiple myeloma in peripheral blood mononuclear cells and whole blood, respectively. Hence, this work provides a proof of concept that NKAB facilitates the reliable and comprehensive measurement of NK cell function in clinical settings for diagnostic and prognostic purposes.

Multifunctional Microparticles with Stimulation and Sensing Capabilities for Facile NK Cell Activity Assay.

Natural killer (NK) cells are a subset of innate lymphoid cells playing an important role in immune surveillance and early defense against infection and cancer. They recognize and directly kill infected or transformed cells. At the same time, they produce various cytokines and chemokines to regulate other immune cells. NK cell activity can be a useful marker for health screenings because impaired NK cell functions may indicate a more susceptible environment for infection or tumor development. Currently, most NK cell activity assays are focused on measuring either cytokine secretion, in particular, interferon γ (IFN-γ), or cytotoxicity against target cells such as K562, thus only providing partial information on NK cell activity. In order to develop a comprehensive test for measuring NK cell function, cytotoxicity and cytokine secretion ability should be measured simultaneously. In addition, current NK cell assays are performed by stimulating NK cells with cocktails of cytokines, antibody-coated beads, or live target cells. In this study, we developed multifunctional microparticles for NK cell activity assay (MNAs) that allow simultaneous stimulation and sensing various NK cell activities, including cytokine secretion and cytotoxicity. The surfaces of MNAs are decorated with multiple functional biomolecules, including antibodies that stimulate NK cells by engaging NK cell activating receptors, antibodies that can capture cytokines secreted by NK cells, and a peptide sensor that reacts with granzyme B, a key molecule released by NK cells for cytotoxicity. The performances of MNAs are assessed using flow cytometry and live cell imaging. NK cell activity is measured by simply mixing MNAs with NK cells and performing flow cytometry, and the results are comparable to those measured by standard NK cell activity assays.

Cycle stability of conversion-type iron fluoride lithium battery cathode at elevated temperatures in polymer electrolyte composites.

Metal fluoride conversion cathodes offer a pathway towards developing lower-cost Li-ion batteries. Unfortunately, such cathodes suffer from extremely poor performance at elevated temperatures, which may prevent their use in large-scale energy storage applications. Here we report that replacing commonly used organic electrolytes with solid polymer electrolytes may overcome this hurdle. We demonstrate long-cycle stability for over 300 cycles at 50 °C attained in high-capacity (>450 mAh g-1) FeF2 cathodes. The absence of liquid solvents reduced electrolyte decomposition, while mechanical properties of the solid polymer electrolyte enhanced cathode structural stability. Our findings suggest that the formation of an elastic, thin and homogeneous cathode electrolyte interphase layer on active particles is a key for stable performance. The successful operation of metal fluorides at elevated temperatures opens a new avenue for their practical applications and future successful commercialization.

Progressive Impairment of NK Cell Cytotoxic Degranulation Is Associated With TGF-ß1 Deregulation and Disease Progression in Pancreatic Cancer.

Natural killer (NK) cells are key effectors in cancer immunosurveillance and can be used as a prognostic biomarker in diverse cancers. Nonetheless, the role of NK cells in pancreatic cancer (PC) remains elusive, given conflicting data on their association with disease prognosis. In this study, using conventional K562 target cells and complementary engineered target cells providing defined and synergistic stimulation for NK cell activation, a correlation between impaired NK cell cytotoxic degranulation and PC progression was determined. Peripheral blood mononuclear cells (PBMCs) from 31 patients with newly diagnosed PC, 24 patients with non-malignant tumors, and 37 healthy controls were analyzed by flow cytometry. The frequency, phenotype, and effector functions of the NK cells were evaluated, and correlations between NK cell functions and disease stage and prognosis were analyzed. The results demonstrated that effector functions, but not frequency, of NK cells was progressively decreased on a per-cell basis during PC progression. Impaired cytotoxic degranulation, but not IFN-γ production, was associated with clinical features indicating disease progression, such as high serum CA19-9 and high-grade tumors. Significantly, this impairment correlated with cancer recurrence and mortality in a prospective analysis. Furthermore, the impaired cytotoxic degranulation was unrelated to NKG2D downregulation but was associated with increased circulating and tumor-associated TGF-ß1 expression. Thus, NK cell cytotoxic activity was associated with PC progression and may be a favorable biomarker with predictive and prognostic value in PC.

Direct potentiation of NK cell cytotoxicity by 8-azaguanine with potential antineoplastic activity.

This study identified 8-azaguanine (8-AG) as a novel immunomodulatory drug (IMiD) through a high-throughput screen of the Preswick Chemical Library in a model of human NK cell cytotoxicity against blood cancer cells. 8-AG, originally developed as an antineoplastic agent, significantly increased the cytotoxicity of NK cells and was superior in this activity to previously known IMiDs, such as fluoxetine and amphotericin B, identified from the same library. IFN-γ expression was also slightly increased by 8-AG. Mechanistically, 8-AG increased conjugate formation between NK and target cells and subsequent cytolytic granule polarization, but not calcium mobilization, regulation of activating receptors, or expression of perforin or granzyme B. Thus, the antineoplastic activity of 8-AG should be re-evaluated in light of this novel potentiating effect on NK cells.

Ginsenoside F1 Promotes Cytotoxic Activity of NK Cells via Insulin-Like Growth Factor-1-Dependent Mechanism.

Ginsenosides are the principal active components of ginseng and are considered attractive candidates for combination cancer therapy because they can kill tumors and have favorable safety profiles. However, the overall benefit of ginsenosides remains unclear, particularly in cancer immunosurveillance, considering the controversial results showing repression or promotion of immune responses. Here we identify a potentiating role of ginsenoside F1 (G-F1) in cancer surveillance by natural killer (NK) cells. Among 15 different ginsenosides, G-F1 most potently enhanced NK cell cytotoxicity in response to diverse activating receptors and cancer cells. G-F1 also improved cancer surveillance in mouse models of lymphoma clearance and metastatic melanoma that rely on NK cell activity. G-F1-treated NK cells exhibited elevated cytotoxic potential such as upregulation of cytotoxic mediators and of activation signals upon stimulation. NK cell potentiation by G-F1 was antagonized by insulin-like growth factor (IGF)-1 blockade and recapitulated by IGF-1 treatment, suggesting the involvement of IGF-1. Thus, our results suggest that G-F1 enhances NK cell function and may have chemotherapeutic potential in NK cell-based immunotherapy. We anticipate our results to be a starting point for further comprehensive studies of ginsenosides in the immune cells mediating cancer surveillance and the development of putative therapeutics.

Mechanisms of Transformation of Bulk Aluminum-Lithium Alloys to Aluminum Metal-Organic Nanowires.

Fabrication and applications of lightweight, high load-bearing, thermally stable composite materials would benefit greatly from leveraging the high mechanical strength of ceramic nanowires (NWs) over conventional particles or micrometer-scale fibers. However, conventional synthesis routes to produce NWs are rather expensive. Recently we discovered a novel method to directly convert certain bulk bimetallic alloys to metal-organic NWs at ambient temperature and pressure. This method was demonstrated by a facile transformation of polycrystalline aluminum-lithium (AlLi) alloy particles to aluminum alkoxide NWs, which can be further transformed to mechanically robust aluminum oxide (Al2O3) NWs. However, the transformation mechanisms have not been clearly understood. Here, we conducted advanced materials characterization (via electron microscopy and nuclear magnetic resonance spectroscopies) and chemo-mechanical modeling to elucidate key physical and chemical mechanisms responsible for NWs formation. We further demonstrated that the content of Li metal in the AlLi alloy could be reduced to about 4 wt % without compromising the success of the NWs synthesis. This new mechanistic understanding may open new avenues for large-scale, low-cost manufacturing of NWs and nanofibers for a broad range of composites and flexible ceramic membranes.

Development of Anti-Insect Microencapsulated Polypropylene Films Using a Large Scale Film Coating System.

Films containing microencapsulated cinnamon oil (CO) were developed using a large-scale production system to protect against the Indian meal moth (Plodia interpunctella). CO at concentrations of 0%, 0.8%, or 1.7% (w/w ink mixture) was microencapsulated with polyvinyl alcohol. The microencapsulated CO emulsion was mixed with ink (47% or 59%, w/w) and thinner (20% or 25%, w/w) and coated on polypropylene (PP) films. The PP film was then laminated with a low-density polyethylene (LDPE) film on the coated side. The film with microencapsulated CO at 1.7% repelled P. interpunctella most effectively. Microencapsulation did not negatively affect insect repelling activity. The release rate of cinnamaldehyde, an active repellent, was lower when CO was microencapsulated than that in the absence of microencapsulation. Thermogravimetric analysis exhibited that microencapsulation prevented the volatilization of CO. The tensile strength, percentage elongation at break, elastic modulus, and water vapor permeability of the films indicated that microencapsulation did not affect the tensile and moisture barrier properties (P > 0.05). The results of this study suggest that effective films for the prevention of Indian meal moth invasion can be produced by the microencapsulation of CO using a large-scale film production system. PRACTICAL APPLICATION: Low-density polyethylene-laminated polypropylene films printed with ink incorporating microencapsulated cinnamon oil using a large-scale film production system effectively repelled Indian meal moth larvae. Without altering the tensile and moisture barrier properties of the film, microencapsulation resulted in the release of an active repellent for extended periods with a high thermal stability of cinnamon oil, enabling commercial film production at high temperatures. This anti-insect film system may have applications to other food-packaging films that use the same ink-printing platform.

Indian meal moth (Plodia interpunctella)-resistant food packaging film development using microencapsulated cinnamon oil.

UNLABELLED: Insect-resistant laminate films containing microencapsulated cinnamon oil (CO) were developed to protect food products from the Indian meal moth (Plodia interpunctella). CO microencapsulated with polyvinyl alcohol was incorporated with a printing ink and the ink mixture was applied to a low-density polyethylene (LDPE) film as an ink coating. The coated LDPE surface was laminated with a polypropylene film. The laminate film impeded the invasion of moth larvae and repelled the larvae. The periods of time during which cinnamaldehyde level in the film remained above a minimum repelling concentration, predicted from the concentration profile, were 21, 21, and 10 d for cookies, chocolate, and caramel, respectively. Coating with microencapsulated ink did not alter the tensile or barrier properties of the laminate film. Microencapsulation effectively prevented volatilization of CO. The laminate film can be produced by modern film manufacturing lines and applied to protect food from Indian meal moth damage. PRACTICAL APPLICATION: The LDPE-PP laminate film developed using microencapsulated cinnamon oil was effective to protect the model foods from the invasion of Indian meal moth larvae. The microencapsulated ink coating did not significantly change the tensile and barrier properties of the LDPE-PP laminate film, implying that replacement of the uncoated with coated laminate would not be an issue with current packaging equipment. The films showed the potential to be produced in commercial film production lines that usually involve high temperatures because of the improved thermal stability of cinnamon oil due to microencapsulation. The microencapsulated system may be extended to other food-packaging films for which the same ink-printing platform is used.